the study of genetic diversity in relation to the...
TRANSCRIPT
i
THE STUDY OF GENETIC DIVERSITY IN RELATION TO THE LEAVES’
MORPHOLOGICAL AND BIOCHEMICAL ADAPTATIONS OF
MULBERRY GENOTYPES GROWN IN MUZAFFARABAD AZAD JAMMU
AND KASHMIR
Ammara Munir
(Regd. No. 2012-umdb-13345)
Session 2012- 2015
Department of Biotechnology
Faculty of Sciences
University Of Azad Jammu And Kashmir, Muzaffarabad, Pakistan.
ii
THE STUDY OF GENETIC DIVERSITY IN RELATION TO THE LEAVES’
MORPHOLOGICAL AND BIOCHEMICAL ADAPTATIONS OF
MULBERRY GENOTYPES GROWN IN MUZAFFARABAD AZAD JAMMU
AND KASHMIR
by
AmmaraMunir
(Regd. No. 2012-umdb-13345)
A Thesis
submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in
Biotechnology
Session 2012- 2015
Department of Biotechnology
Faculty of Sciences
University Of Azad Jammu And Kashmir, Muzaffarabad, Pakistan
iii
iii
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DEDICATION
This humble effort is
sincerely dedicated to my
loving husband, respectable
parents and my kids
Maham and Rayyan
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CONTENTS Page
TABLE OF CONTENTS v-viii
LIST OF FIGURES ix
LIST OF TABLES x
LIST OF ABBREVIATIONS xi-xii
ACKNOWLEDGMENT xiii-xiv
ABSTRACT xv-xvi
1 INTRODUCTION 1-4
2 REVIEW OF LITERATURE 5-16
2.1 GENERAL 5
2.2 STATUS OF MORUS 5
2.3 IMPORTANT NUTRIENTS 6
2.4 ROLE OF LEAVES 7
2.5 MEDICAL SIGNIFICANCE 8
2.6 USES AS A FUEL 11
2.7 GENETIC DIVERSITY 11
2.8 ROLE OF DEVELOPMENTAL GENES 15
3 MATERIAL AND METHODS 17-31
3.1 STUDY AREA 17
3.2 MORPHOLOGICAL ATTRIBUTES 18
3.3 BIOCHEMICAL ANALYSIS 19
3.3.1 Determination of Protein 19
3.3.2 Determination of Carbohydrates 19
3.3.3 Pigment Analysis 20
vi
3.3.4 Mineral Analysis 20
3.3.5 Determination of Anti-oxidant Activity by DPPH
Assay
20
3.3.6 Phenolics 21
3.4 MOLECULAR CHARACTERIZATION 21
3.4.1 Collection of Plants and Storage 21
3.4.2 Preparation of Plant Material 22
3.4.3 CTAB Preparation 22
3.4. 4. DNA Confirmation 22
4.4.5 DNA Extraction Protocol 22
3.4.6 Polymerase Chain Reaction (PCR) 23
3.4.7 Primers Dilutions 23
3.4.8 PCR Conditions 23
3.5 DATA SCORING 25
3.6 STATISTICAL ANALYSES OF DATA 26
3.8 CHARACTERIZATION OF CUC2 GENES 26
3.8.1 Morus Species and Strains 26
3.8.2 Genomic DNA Isolation 26
3.8.3 Reagents 26
3.8.4Protocol 27
3.8.5 Amplification of CUC2 Gene Sequences 28
3.8.6 Cloning of PCR Products, Identification of
Recombinant Colonies, and DNA Sequencing
29
3.8.7 DNA Sequence Analysis 30
4 RESULTS AND DISSCUSSIONS 32-69
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4.1 LEAF MORPHOLOGY 32
4.1.1The Qualitative Traits of Leaf 32
4.1.2 Quantitative Aspects 33
4.1.3 Correlation between the Morphological Traits of
Mulberry Accessions
36
4.1.4 Cluster and Principal Component Analysis of
Morphological Parameters
37
4.2 BIOCHEMICAL ANALYSIS 42
4.2.1 Measurement of Carbohydrate Content 42
4.2.2 Measurement of Protein Content 44
4.3 PIGMENTS 47
4.3.1 Chlorophyll a 47
4.3.2 Chlorophyll b 48
4.3.3 Carotenoids 50
4.3.4 Anthocyanins 51
4.3.5 Determination of Phenolic 53
4.3.6 Antioxidant Potential 54
4.3.7 Determination of Mineral Content 56
4.3.8 Potassium 56
4.3.9 Sodium 57
4.4 THE MORUS CUC2 GENES 59
4.4.1Phylogenetic Analysis 62
4.4.2 Evolutionary Divergence among Sequences 64
4.4.3 Estimate of Nucleotide Composition 64
4.4.4Tajima’s Neutrality Values 65
viii
4.5 MOLECULAR CHARACTERIZATION 68
4.5.1 Statistical Tools Used in Analysis 68
4.5.2 Simple Sequence Repeats (SSR) 68
4.5.4 Genetic Dissimilarity Coefficient 69
SUMMARY 75-76
LITERATURE CITED 77-101
ANNEXURE 1 102-
105
ANNEXURE 2 106-
107
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LIST OF FIGURES
Figure
No.
Page
1 Map depicting the territory of Muzaffarabad 17
2 Dendrogram showing hierarchical cluster analysis of
morphological traits of 30 Mulberry accession
38
3 Principal Component analysis based on morphological
characters of 30 Mulberry accession
39
4 Graphical representation of carbohydrate content in mulberry
accessions
44
5 Graph showing proteins in mulberry accessions 47
6 Graph showing chlorophyll a content in different mulberry
accessions
48
7 Graphical presentation of chlorophyll b content in mulberry
accessions
49
8 Carotenoids in mulberry accessions 51
9 Anthocyanin content of mulberry accessions 52
10 Showing total phenolic content of mulberry accessions 54
11 Showing IC50 values of Mulberry accessions 55
12 Representing potassium found in different accessions 57
13 Showing sodium concentrations in Morus accessions 58
14 Gene structure of Arabidopsis thaliana and morusnotabilis 60
15 The amino acid sequence showing the conserved No Apical
Meristem (NAM) domain
62
x
16 Showing molecular Phylogenetic analysis by Maximum
Likelihood method between the eight Morus strains
63
17 Evolutionary divergence among eight strains of Morus 64
18 DNA of thirty accessions 68
19 Principal component analyses for Morus traits 70
20 PCA for ISSR 72
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LIST OF TABLES
Table No. Page
1 List of Mulberry accessions used in the study 18
2 List of SSR markers 24
3 List of ISSR markers 25
4 PCR components for the amplification of primers 25
5 Qualitative characterization of Mulberry accessions 34
6 Means of five morphological traits of thirty mulberry
accessions
35
7 Pearson correlation coefficient matrix for qualitative variables
of thirty accessions
37
8 Showing eigenvalue and % variance of five quantitative
parameters of mulberry traits
39
9 Biochemical constitutions of Mulberry accessions 43
10 Maximum identity between CUC2 a, CUC2 c and CUC2d
genes
59
11 Exon and intron lengths in Morus CUC2c genes 61
12 Nucleotide composition of sequences 65
13 Values of Tajima’s test 65
14 Grouping of accession on the basis of SSR analysis 69
15 Showing Analysis of Molecular Variance (SSR) 70
16 Grouping of accessions of ISSR analysis 71
17 AMOVA showing difference in ISSR 72
18 Markers (ISSR) along with its total bands and polymorphism 73
xii
LIST OF ABBRIVATION
AFLP
ANOVA
AMOVA
BLAST
Bp
CUC
CTAB
DIA
DPPH
DNA
dNTPs
DB
EDTA
EST
Fw
FAO
GAE
HPLC
ISSR
IPTG
MEGA
mM
MCL
NAM
Amplified Fragment Length Polymorphism
Analysis of variance
Analysis of molecular variance
Basic Local Alignment Search Tool
base pair
Cup Shaped Cotyledon
Cetyltrimethyl ammonium bromide
Diaphorase
2,2-diphenyl-1-picrylhydrazyl
Deoxyribonucleic acid
Deoxy nucleotide Triphosphates
Data Base
Ethylene diamine tetra acetic acid
Per oxidase
Fresh weight
Food and Agriculture Organization
Gallic acid equivalent
high pressure liquid chromatography
Inter-Sequence simple Repeat Polymorphism
Isopropyl β-D-1-thiogalactopyranoside
Molecular Evolutionary Genetics
AnalysismilliMolar
Maximum composite likelihood
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NCBI
NaCl
PCR
PPO
PCA
RAPD
rpm
SSR
TAE
TA
TBE
TE
Taq Pol
µL
X-GAL
No apical meristem
National center for biotechnology information
Sodium chloride
Polymerase chain reaction
polyphenol oxidase
principal component analysis
Random amplification of polymorphic DNA
Revolution per minute
Simple Sequence Repeat Polymorphism
Tris-base acidic acid and EDTA
Taq-polymerase amplified
Tris borate-EDTA
Tris- EDTA
Taq polymerase
Micro litter
5-bromo-4-chloro-3-indolyl-β-D
galactopyranoside
xiv
ACKNOWLEDGEMENT
Words are bound and knowledge is limited to praise ALMIGHTY ALLAH, the
Lord of the world, the Omnipotent, the Beneficent, Who gave me the requisite potential
and diligence for the successful accomplishment of this task. Special praise to
HAZRAT MUHAMMAD (PBUH), who is ever lasting source of knowledge for the
whole mankind.
I am grateful for the financial assistance by Punjab Government (fee
reimbursement scheme) and Higher Education Commission (HEC) Islamabad Pakistan,
under the IRSIP PhD Fellowship Scheme.
I’m appreciative to my husband, my parents, and my in laws for all their love,
prayers, sacrifices, sympathies, guidance and encouragement which served as “beacon
of hope” all along my work. I offer my richest and heartiest gratitude to them. My kids
who suffered a lot during my Ph.D. duration.
I confess here that thesis would not turn into reality without the principal
contribution, affection, untiring help, invigorating encouragement and moral support of
my affectionate supervisor Prof. Dr. Syed Abdul Majid. He is a supervisor with
distinction and I have never such a beautiful soul. I am happy to be under his
supervision and I pray to God for his healthy life.
Here, I would like to express my gratitude to Chairman Department of
Biotechnology, Dr. Basharat Ahmad, and all my respected teachers of the
Biotechnology Department specially, Syed Dilnawaz Ahmad Garzedi (ex
vicechancellor), Dr. Ghazanfar Ali, Dr. Shaukat Ali, Dr. Rehanakausar, Dr. Syed
Rizwan Abbas, Dr. HamayunShaheen, Dr. SaiqaAndleeb, Dr. RizwanTaj and DrZahid
for their direction, meaningful suggestion and helpful attitude during the course of this
degree. I am extremely gratefulto Dr. Tariq, Dr. Wasim Akhtar, Dr. Illyas Ahmed, from
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Quaid-e-Azam University, Islamabad for their cooperation. I feel extremely privileged
for their cooperation, valuable suggestion, sympathetic attitude, words of
encouragement and advice over the course of my research.
I do not have words at my command to express my heartiest thanks, gratitude and
profoundtomy external supervisors, June Bowman Nasrallah, my lab technician Tiffany
Stauderman at Cornell University New York, Ithaca, USA. Their visionary research
activities made my investigations more fruitful and I hope it will reshape my upcoming
research directions. I really have no words to express my cordial and sincere
thankfulness to my friends Anila, Asia, Maryam, Khadija, Ghazala, Sidra, Ainulbatool,
Sadia, Tasleem,Zeeshan and Maria. I can’t forget the love, cooperation and colorful
moments with my friends during my Ph.D.
In addition to this I am really thank full to Sericulture Department for their full
support during my sample collection and provision of information. I cannot forget all
those peoples who provide me spiritual and moral support and they always make a silent
prayer for me. I have only one sentence for all of you, I love you all and your love led
me every step to fruition. I would like to acknowledge all the clerical staff and lab staff
at all research institute and Universities for their help and good behavior. May the
Almighty Allah shower his blessing to all those who assisted me at different stages
during my academic career.
Ammara Munir
xvi
Abstract
The present study was conducted on different accessions of Moraceae to
evaluate the genetic variation in them on the basis of morphological and biochemical
attributes as well as by using molecular markers (SSR and ISSR). The developmental
genes cup shaped cotyledons (CUC2) were also studied in order to confirm their
functional status. For morphological, biochemical and molecular studies mulberry fresh
leaves were taken and all the parameters were taken in replicates. Morphologically
leaves of the majority of the accessions had light green color, acute tip, and soft surface
along with cordate shaped leaves. The others had lobed shape, dark color, doubly
serrate or dentate, acuminate and coarse leaves. There were significant differences in
means of lamina length, lamina width and leaf area index. In biochemical analysis, the
carbohydrates content was the highest in Korean subni (3.7626±0.204 cm) followed by
Morus nigra(3.728±0.1795 cm), Morus latefolia (3.643±0.2458 cm) and P.F.I
(3.568±0.3057 cm). Punjab II (1.704±0.1695 cm) showed the lowest carbohydrates
content. The protein contents of Kanmasi Japan (1.7007±0.0049 cm) were higher as
compared to Punjab-II-punjab (1.4030±0.0455 cm). The higher values of carbohydrate
and protein content revealed that the leaves had the more palatability for silkworms.
Lun-40-punjab showed the highest total phenolic content. Phenolic play a key role in
traditional medicines. The maximum IC50 value was of Morus indica(172.21 mg/g)
followed by Morus alba(152.7 mg/g) and PFI (122.4 mg/g). Punjab II Punjab (11.6),
Kanmasi Japan (11.5) showed the minimum and same amount of anti-oxidant activity.
One of the developmental genes CUC2 was observed in seven Morus strains.
This strategy produced good-quality sequences, which were analyzed and compared to
each other and to theCUC2c sequence of the reference M. notabilis strain.The
sequencing results showed that the mean distance present in the overall sequence pairs
xvii
was 0.017. Molecular diversity between thirty accessions was checked through DNA
markers i.e. SSR and ISSRs by the use of different softwares (NTSYS PC 2.2, Minitab,
Statista, R) The SSRs produced 15 polymorphic bands out of 25 (60%) SS18 and
MULSTR1 showed best results with the highest PIC and hundred percent
polymorphism while the least was shown by SS04. It was also obvious that pic of SSR
was somehow less than ISSR. The analysis of molecular variance (AMOVA) gave P
value of 0.6434. The ISSR markers showed more polymorphism with more PIC value.
The P value obtained from the results was 0.1089, while CV was 35.7. Our diversity
results proved that all group were somewhat distant.
1
Chapter 1
INTRODUCTION
Mulberry is a plant that is grown for silkworm rearing. It is the exclusive food for
the silkworm, which during its larval life is reared for silk production. Mulberry forms the
basic food material for silkworms. Genus Morus is found commonly in Asia, Europe, North
America, South America, and Africa. It is widely cultured in Eastern, Central, and Southern
Asia for making silk. Mulberry has the ability to nurture in a varied climate, geography and
soil forms. These are found all over the world from tropical to subarctic and altitudes well
above the sea level (4000 m). Usually they are found at higher places like Himalaya
mountains, as in northern areas (Elmaci and Altug, 2002; Darias-Martin et al., 2003;
Arabshahi-Delouee and Urooj, 2007; Ercisli and Orhan, 2007). It is local to Nepal,
Pakistan, China, Japan, Barma, and India (Anonymous, 2001).
The synthetic organization and nutritious possibilities of some mulberry species
have been considered around the world (Gerasopoulos and Stavroulakis, 1997; Darias-
Martin et al., 2003; Ercisli and Orhan, 2007). The fruits are full of phenolic contents like
anthocyanin, carotenoid and flavonoids (Sass-Kiss, 2005; Zadernowski et al.,2005; Cieslik
et al., 2006; Lin and Tang, 2007). Hardly any types of mulberry were assessed for its
consumable fruits (M. alba, Morus nigra, M.indica, Morus laevigata) and timber
(M.serrata and M.laevigata) (Hou, 2003). In some republics, the trees and the fruits are
named by Egyptian-inferred labels as tut, or shah toot (unrivaled). The one with large white
fruit is a cross breed frequently found in Pakistan and profoundly refreshing delicious
organic product which is eaten crisp and also dried.
The Pakistan populace fundamentally relies upon ranger service, domesticated
animals and farming for its subsistence. The standard per capita pay is 847 US dollars,
2
joblessness rate ranges from 6.0 to 6.5 percent for each annum territory under development
is around 166432 hectares. It is estimated that 13 percent of total geographical region is
rain fed. Eighty four percent of people bear small land reforms. The major harvests are
rice, wheat, maize and the beans, beats, oil seeds, and vegetable are also produced in
appropriate amounts. Among fruits pears, walnuts, apricots and apple are mostly produced
(Govt. AJ & K, 2006).
Mulberry natural item is by and large seen as a nutritious substance and it can be
eaten normally or comprehensively used as a piece of the formation of wine, natural item
crush, stick and canned support (Ning et al., 2005). White mulberry leaf tea experiments
on mouse provide excellent results for nervousness, wretchedness, climbing movement and
warm reactions (Yadav et al., 2008).
Pakistan is a rich source of pharmaceutical plants with different plant territories. It
has four species and many varieties in Pakistan. Mulberry is a quite beneficial medicinal
plant for humans as well as for animals too. AJK is rich in dense forests having a variety
of medicinal plants. Genetic diversity of Mulberry in AJK is still unknown; therefore it is
important to evaluate the level of diversity using DNA markers. No previous studies have
been done on mulberry in AJK. The focus of this was also on checking the functionality of
CUC2 genes in the Morus species.
Mulberry leaves has been used in Chinese medicine for a long period of time for
curing many diseases. Its fruit is used in making of jams, jellies, and cough syrups. There
are a lot of reports on increasing use of these precious plants by the native people (Qureshi
et al., 2007; Mahmood et al., 2011a; b; c).Therefore, leaves are more important for
researchers. The utmost key appeal for mulberry upbringing is its Seri cultural value in
3
Middle East and Europe, while Japan they are assessed as a produce which increase the
high land farming (Chitra and Padjama, 2002).
Mulberry leaf supports the development of the silkworm hatchlings, being viewed
as a total esteem supplement. The selection of mulberry assortments depends on the
ingredients present in it. They are rich source of proteins, carotenoids, lipids, carbohydrate,
anthocyanin, etc. The leaves are used in medicine in China and also help in relaxing mucus.
They are also utilized for the treatment of illness like fever, itching eyes, headaches, sore
throats and dizziness. The fruit sap is energizer and act as a mouth wash too. The juice is
purging and tonic. Tooth ache is cured by its bark and used as purgative (Anonymous,
2001).
Production of mulberry leaves on scientific lines is essential for organizing
sericulture on sound economic lines. It is estimated that one metric ton of mulberry leaves
is necessary for the rearing of silkworms emerging out of one case of eggs which will yield
about 25 kg to 30 kg of cocoons of high quality. Mulberry tree can grow in a variety of
climatic conditions (Tuigong, et al., 2015).
Hereditary portrayal of germplasm assets is vital for their successful administration
and effective use, particularly class like mulberry having accessible germplasm displays
such phenotypic decent variety with no data about its hereditary base. Numerous sub-
atomic marker systems have been effectively utilized as a part of distinguishing proof and
hereditary decent variety investigation in mulberry, for example, RAPD (Xiang et al.,
1995); RFLP (Zhao and Pam, 2004), SSR (Aggarwal et al., 2004), ISSR (Zhao et al., 2007)
and AFLP (Sharma et al., 2000). It is important that the latest studies showed the use
4
of morphological characters or molecular makers (Chatti et al., 2004; Hedfi et al., 2003;
Saddoud et al., 2005, 2007; Salhi-Hannachi et al., 2004, 2005, 2006).
Sericulture has the potential of poverty eradication and economic empowerment
especially youth in Pakistan because it is a labour intensive venture. Silk production has
the potential of serving as a supplement to the textile industry in Pakistan due to the
dwindling cotton production.
Keeping in view the importance of mulberry leaves in our area, this Ph.D. study is
conducted on the basis of these objectives
To study the morphological characters of mulberry varieties
To assess correlations in morphological characters in mulberry
To identify the nutritionally superior varieties by biochemical analysis
To perform genetic characterization of mulberry varieties using molecular markers
To analyze the Morus orthologues of CUC2 genes
5
Chapter 2
REVIEW OF LITERATURE
2.1 GENERAL
The plants having the medicinal constituents are integral part of earning for the poor
people throughout the globe. There were about 32000 species of higher plants (Prance, 2001)
and among them 10 percent are of medicinal value (Shinwari, 2010). The peers had been
using the plants for treatment of many diseases, which has been transferred from them to
their new generations (Bhardwaj and Gakhar, 2005). Some plants are reaped for their basic
elements as were used in Unani and Eastern (medicines) (Juden, 2003). Some organizations
are there for applied use of these medicinal plants. There are few educational institutes
where they are studying practical implications of medicinal plants (Gilani and Atta-ur-
Rahman, 2005).
2.2 Status of Morus
The mulberry family (Moraceae) covers 37genera and nearly 1100 species spread
over humid and mild areas in the world. It unveils a complex range of floral architecture;
productive systems and pollination patterns that give base for taxonomical distribution. It
is small to average size, monoecious, perennial, and wind fertilizedtree (Kafkas et al.
2008).
Mulberry is the common name having four main species viz alba, nigra and rubra
of Morus genera. Many hybrids and related species are also found. White one is initiated
from Central and Eastern China Mulberry is the most vital product plant in sericulture since
it is utilized for silkworm (Bombyx mori) raising. Mulberries are a gathering of little trees
or bushes having a place with the family Moraceae, found in the calm and
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subtropical districts of the northern half of the globe. In China, there are around
3,000 mulberry varieties. As indicated by conventional morphological arrangement, they
are gathered into 15 species and 4 assortments (Pan, 2000).
Phenotypic plasticity lies at the intersection of a variety of disciplines like ecology,
physiology, developmental morphology, genetics and evolution. Phenotypic characters are
regularly utilized for the assessment of plant genotypes in reproducing programs The
plasticity (phenotypic) is found in many different areas as hereditary, evolutionary studies,
physiological and morphological aspects (Mace et al., 2010). The associations between
different morphological and agronomical characters are needed to be encountered. It will
help out the breeders to design suitable approaches for getting looked-for trends (Hébert
et al., 1994).
2.3 Important Nutrients
Jadhav et al. (2000) explored the value of applying fertilizers which when applied
to the mulberry trees helped increasing the content like proteins and carbs that further
enhances the production of cocoon and silk. He also revealed that the nutrients affect the
ultimate product as well as the increment in body size and silkworm growth.
It was observed that the leaves of this nutritious plant are rich in b-carotene as
contrasted with green verdant vegetables, like spinach, fenugreek leaves and amaranth. On
the other hand, high amount of iron was also observed in the leaves of mulberry. Similarly,
the amount of calcium recorded in mulberry was higher as compared to spinach, amaranth,
mustard and fenugreek leaves. It also contains defensive supplements such as, calcium,
iron, zinc and ascorbic acid. These outcomes recommend that mulberry is a big source of
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7
nutrients for the world's expanding population (Colding and Pinstrup-Anderson, 2000;
Babu 2000).
The researchers pinpoint products of specific mulberries (white, black, whitish long
fruit, and dark) for revealing their basic make up materials, ultimate minerals, and anti-
oxidant abilities. So these fruits are collected by the people of northern areas of Pakistan.
The key dietary parts (lipids, proteins, strands, starches, and aggregate sugar) were found
in appropriate range with great value. Considerable variations were found in complete dry
mass, pH, (%) and citrus extract. A small amount of Vitamin B2 plus B3 was seen in fruits.
Adequate amounts of citric acid, phenolic materials, alkaloids were observed in every fruit.
Raw materials are found in direction as Fe>Zn>Ni. The radical scavenging activity shows
a direct relation with increasing phenolic constituents of the particular fruit. As a result,
mulberry fruits were found as a potential source of nourishment and common cancer
prevention agents (Imran et al., 2010).
In the history of China, the mulberry fruit is used as medicinal food for a long time.
Its fruit pigment is natural dye for food processing on commercial scale (Qin et al., 2010).
It has been improved for yield and quality of leaf. Globally, mulberry is used as feed for
silkworm, and also valued for its fruit, as a delicious vegetable, for landscaping, for its
medicinal properties and also as animal feed (Zepeda, 1991).
2.4 Role of Leaves
The mulberry leaves are about 70-90 percent exceptionally suitable and eatable for
70-90 percent animals eating herbs. About 15-28 percent of proteins are available in
youthful shoots and foliage parts in different assortments. The foundation of this enduring
search is through stakes or seed, and it is gathered by leaf picking or cutting entire branches
8
8
or stems. Yields rely upon assortment, area (month to month temperature, sun oriented
radiation, and precipitation), plant thickness, manure application and collecting method,
however as far as edible supplements, mulberry delivers more than most conventional
scrounges. The leaves can be utilized as supplements trading concentrates for dairy cows,
as the fundamental sustain for goats, sheep and rabbits, and as in fixing in mono gastric
eating methodologies (Sánchez, 2000).
Mulberry leaf supports the development of the silkworm hatchlings, being viewed
as a total esteem supplement, and due to its nutritious status. Its nutritious importance is
learned by its biochemical components (Bapu, 2000). Henceforth the present examination
was attempted to think about its biochemical parameters in leaves and roots of different
genotypes of mulberry. According to Kumar and Chauhan (2011) the AR-12 mulberry
contains most astounding biochemical components. The mulberry leaves are used as food
for silkworms. It is sometimes used as a fodder for cattle and as a vegetable in some parts
of world. Morus alba contain many natural antioxidants and is supposed to be a good
source of diet. Many researchers found the plants very useful. Humans are benefited by the
plants in so many ways so as medicine. Its use initiated in early ages, may be from the era
when a person firstly got a sickness about three thousand years ago, some people may have
idea to utilize the plants as a remedy for his sickness. Hence, the plant usage from medical
point of view is not a new stuff for curing health issues. A new track to research on phyto-
pharmacolgy has been given in this paper (Bagachi et al., 2013).
2.5 Medicinal Significance
The testing of anti-oxidants in a sample is done by a simple way given by (Mujeeb
et al., 2011) by 2, 2-diphenyl-1-picrylhydrazyl. The endowment of hydrogen is done when
9
9
the DPPH radical is scavenged by antioxidants which are strengthened by the creation of
non-radical end product. Extracts of Morus alba leaf showed liver defensive movement.
Liv.52 was used as a standard medicine that delivered noteworthy fluctuations in biological
aspects through the action of CCL4. Pretreatment with alcoholic and watery concentrates
essentially kept the biochemical and histological changes activated by CCl4 in the liver.
The above investigation demonstrates that the alcoholic and watery concentrates had
hepatoprotective actions(Hogade et al., 2010).
Saini et al. (2012) studied the anti-proliferative and polyphenolic properties of
Ficus species. It was observed that anticancer agent were more in F. palmata as compared
to others. In Ficus palmate fruits, the most flavonoid and phenolic components and their
cancer prevention agents are moderately slow Leaves of F. miceocarpa are rich source of
triterpenoids having various medicinal importance like anti-ulcer, antifungal, lipid
lowering, anti-diabetic and anti-bacterial activity (Higa et al., 1996). Its leaves are utilized
as a part of growth, tumors, and dermatitis (Kuo and Li, 1997; Yogesh et al., 2014).
Leaves of mulberry are rich in C, mulberrofuran G and albanol B, and all of them
exhibited high antibacterial activity with MIC’s (Sohn et al., 2004). Different components
of mulberry, for example, chloroform had high antimicrobial activity against Bacillus
subtilis, and portions separated with acidic corrosive against Staphylococcus aureus and
Escherichia coli (Kim et al., 1993). Its kuwanon C, sanggenon B and D, bioactive particles
have solid antimicrobial activity against Staphylococcus aureus, Streptococcus faecalis,
Mycobacterium smegmatis and a few molds species (Nomura et al., 1988).
In past studies mulberry organic products have been accounted for to display an
assortment of natural exercises: they shield liver from harm, reinforce the joints, encourage
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10
the release of urine and bring down circulatory strain and have diuretic, anthelmintic,
expectorant, hypoglycaemic, emetic and odontalgic (Zhishen et al., 1999); anti-thrombotic
(Yamamoto et al., 2006); antioxidant (Naderi et al., 2004; Hassimotto et al., 2005);
antimicrobial (Takasugi et al., 1979); anti-inflammatory (Kim and Park, 2006); and
neuroprotective effects (Kang et al., 2006). These activities are generated by phenolic,
flavonoids (Ercisli and Orhan, 2007; Lin and Tang, 2007) and anthocyanins (Lee et al.,
2004).
In the study of Yamashita(1990), quantitative changes of biochemical components
like, carbohydrates, adenine nucleotides and amino acids in the stems of mulberry trees
were followed from spring autumn. The ATP contents of stems increased before bud break,
whereas the sucrose contents and carbohydrates from stem decreased. Similarly, proline
contents of stems increased from time to time until its leaf shedding. Kiran et al. (2011)
took a shot at element content investigation of plants varieties. A low measure of Cd and
Pb were found. The wild consumable leafy foods play a significant role as a natural
medications as a homegrown treatments and in pharmaceutical industry (Khan et al., 2011).
Rehman et al. (2014) also estimated the biochemical components of mulberry like, lipids,
glycerides, and fatty acids. The total lipids were fractionated into three major lipid groups,
glycolipids, neutral lipids and phospholipids.
Anthocyanins are the compound segments that give the extraordinary shading to
many foods grown from the ground, for example, blueberries, red cabbages and purple
sweet potatoes. Epidemiological examinations have shown that the direct utilization of
anthocyanin items, for example, red wine or bilberry extricate is related with a lower danger
of cardiovascular malady and change of visual capacities. As of late, there is expanding
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11
intriguing in the pharmaceutical capacity of anthocyanin. This survey condenses current
learning on the different sub-atomic confirmations of disease chemoprevention by
anthocyanin. These instruments can be subdivided into the accompanying viewpoints: 1)
the anti-oxidation 2) the sub-atomic components engaged with hostile to carcinogenesis,
3) the sub-atomic systems associated with the apoptosis enlistment of tumor cells. At last,
the bioavailability and structure-movement relationship of anthocyanin are likewise
outlined (Hou, 2003).
2.6 Wood usage as a Fuel
Woods use a fuel is the main reason for the deforestation in the target area. People
cut it and use it for domestic purposes in cold weathers as it is prolonged and tough.
Dalbergia and Pinus highly endangered reported woods. Shinwari and Khan gave a same
story of many kinds that are highly pressurized for over-use. The Suleman Mountains also
face the same problem of wood shortage because of the large wood consumption in winters.
So there should be the Substitute of energy boosting the maintenance of Flora and also the
use of present assets for replantation. Practical and enchanting management events ought
to be grounded on ethnobotanical studies of the territory, as it gives base for upkeep and
public progress doings (Shinwari and Khan, 2000).
Biell, and Jaroszewska (2017) suggested that sodium and potassium present in
leaves of certain berries may be a good source of minerals important for bone health and
helpful in releasing hypertension. The functional properties of food are associated with the
content of fibre in the form of non-digestible carbohydrates, on which symbiotic bacteria
feed in the large intestine (Dhingra et al., 2011; Fuller et al., 2016).
2.7 Genetic Diversity
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Ipek et al. (2012) demonstrated that, DNA-based markers have been utilized
broadly on account of their many focal points over conventional biochemical and
morphological markers. Many examinations have demonstrated that atomic markers are
helpful in portraying the hereditary connections among firmly related mulberry genotypes
and cultivars. Accordingly, in the present examination, polymer chain response based DNA
fingerprinting systems were utilized to explore the hereditary connections among mulberry
genotypes developing in various agro-climatic areas of Turkey (Ipek et al., 2012).
Wangari et al., (2013) examined the hereditary decent variety between and inside
2 species of mulberry incorporate Morus alba and Morus indica. Five individuals for every
species were genotyped with 13 straightforward arrangement rehash (SSR) markers. It was
obvious from this investigation that the mulberry increases did not bunch on the premise
of their land beginning, and neither as indicated by the gathering of species they fall into.
The investigation demonstrated a strong connection between the two species and in this
manner, mulberry change should target inspecting a bigger number of people inside species
as opposed to among species.
Bhattacharya and Ranade (2001) studied that the genotyping of mulberry was only
characterizes by microsatellite markers (like RAPD). Information of genetic characteristics
of germplasm is essential for effective management of mulberry in which phenotypic
diversity is high but no information about its genetic base. These microsatellite markers
showed the higher polymorphism in Morus species. These genetic markers are very
efficient for the characterization of germplasm and molecular systematics of mulberry.
The Morus species are the main source of plant food for silkworms. Mulberry is
widely found all over the world including Asia, America, Europe and China for silk
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production. It is very important for efficient consumption of resources to categorize its
genomics. In case of mulberry, there is high phenotypic divergence and more studies are
required to get information about its genetic base. There are many markers like SSR,
RAPD, AFLP and SRAP for studying the genetic diversity and credentials of mulberry
(Shabir et al., 2010).
Wangari et al. 2013 led an investigation on significance and protection of
extraordinary mulberry assortments. Their point was to examine the hereditary assorted
variety among the two mulberry types of Kenya including Morus alba and Morus indica
by sing SSR markers. They utilized thirteen SSR markers and found around 35
polymorphic groups. A high level of varietal change (95%) was found inside the species
and five percent between the species (Wangari et al., 2013)
Vijayan (2005) explained the progressive use of molecular markers in plant genetic
studies as these are polymorphic and more stable. ISSR is among one of the simplest PCR
based markers and is more reproducible dominant marker like RAPD. Because of this
property these are widely used in finger printing, genetic mapping, MAS, population
structure analysis and variety identification etc. in Morus spp. These markers are widely
used for phylogenetic relationship among cultivated varieties for identification of
taxonomic positions of genotypes. As ISSR are one of the economical marker systems that
give high polymorphism with high efficacy, that’s why they could be used in future
mulberry genome analysis (Vijayan, 2005).
Nine isozyme designs were concentrated to derive the level of hereditary decent
variety and between connections among 14 types of mulberry utilized as a part of
reproducing programs. Esterase, polyphenol oxidase and diaphorase carried 22 alleles and
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12 isozyme loci. The settling energy of isozymes was not as much as that of molecular
markers, consequently these markers ought to be utilized for phylogenetic examinations in
mulberry (Ananda Rao et al., 2011). The present examination was attempted with
hereditarily more solid DNA markers to ponder the hereditary relationship among eleven
chose genotypes of mulberry gathered from Japan, India and Italy.Utilizing ten RAPD and
ten ISSR preliminaries, 60.75 percent and 74.13 percent of DNA polymorphism could be
recognized among these genotypes. The hereditary likeness among the genotypes differed
from 0.73 to 0.89 when pooled information from ISSR and RAPD were utilized for
UPGMA investigation. The phonogram got from the entire informational collection by
utilizing UPGMA, assembled every one of the genotypes into two particular groups. In one
bunch every one of the genotypes from India and China were gathered while the other
gathering included the Japanese, Italian and the single genotype from Philippines.
However, the unmistakable gathering of Japanese genotypes with the Indian ones offers a
plausibility of using them in hereditary change of M. alba genotypes of India (Srivastava
et al., 2004; Sattayasai et al., 2008).
The researches have shown that the leaves are antibacterial, astringent, diaphoretic,
hypoglycemic, odontalgic and ophthalmic (Duke and Ayensu, 1985). The leaves are
collected in the late autumn and can be used fresh but generally they were dried. They
should be used internally in the treatment of nose bleeds, colds, eye infections and
influenza, (Bown, 1995). Markers are important to plant reproducers as the base line of
hereditary data on crops and for use in subsidiary choice of qualities to which the markers
are connected. In the great reproducing approach, the markers were constantly the
unmistakable morphological and other phenotypic characters, and the raisers consumed
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extensive exertion and time in refining the crosses as the tight linkage or relationship of
the coveted characters with the undeniable phenotypic characters was never unequivocally
settled. Besides, roundabout determination for a quality utilizing such morphological
markers was not down to earth because of (1) a scarcity of reasonable markers, (2) the
unfortunate pleiotropic impacts of numerous morphological markers on plant phenotype,
and (3) the powerlessness to score different morphological mutant attributes in a solitary
isolating populace. With the headway in atomic science, the utilization of sub-atomic
markers in plant rearing has turned out to be exceptionally typical and has offered ascend
to "sub-atomic reproducing". Sub-atomic rearing includes principally "quality labeling",
trailed by "marker-helped choice" of wanted qualities or genomes. Quality labeling alludes
to the ID of existing DNA or the presentation of new DNA that can work as a tag or mark
for the quality of intrigue. All together for the DNA groupings to be monitored as a tag,
essential requirements exist. This audit additionally compresses the accomplishments in
quality labeling that have been made in the course of the last 7 to 8 years (Ranade et al.,
2001).
Study of plants ethnobotanical inventory allows drawing some possible conclusions
about the impacts of their uses near future needs and planning for their development and
conservation (Saghir et al., 2001;Ishtiaq et al., 2006; Ajaib et al., 2010). Information about
importance of medicinal plants does not appear to be same among the native people and
men have more information of these plants as compared to women (Hamayun, 2003).
It was found that small farmers are the vital role players in conservation of
traditional varieties. They collected potato roots from markets and used RAPD and ISSR’s
to estimate the genetic variation. They concluded correlation coefficient of 0.80 for RAPD
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and 0.89 for ISSRs (Moulin et al., 2012). Conserved genotypes documentation,
categorization and estimation for genetic upgrading programs are very necessary
(Gonçalves et al., 2008; Laurentin, 2009; Sudré et al., 2010).
2.8 Role of Developmental Genes
The leaf appearance of “Ryoumenguwa” a modified Morus alba, has cup shaped
two or more blades on one petiole. It has been under investigation for four years for
clarifying the leaf developmental changes and leaf internal changes. The leaf types are
single, multi and cup shaped bladed. Single type is similar to wild one “Ichibei” whereas
the cup shaped are like the first one. About (45.6%) of leaves during the evaluation time,
the foremost type of leaf was single flat plus one extra blade expanded further in the mid
in the shoots. The 2nd leading kind was cup type blade. Fused vein arrangements are another
character and forty two percent of all leaves have the same kind. Bulging was another extra
ordinary character in leaves (3.35%). The cellular analysis showed that the Ryoumenguwa
had mostly dorsoventrally polarity (Feng et al., 2004).
The fundamental body manufacturing of higher plants is set up amid
embryogenesis; nonetheless, not at all like the circumstance in creatures, practically the
whole grown-up plant is delivered post embryonically from two meristems arranged at
inverse finishes of the incipient organism (Hülskamp et al., 1994).The fundamental body
engineering of higher plants is set up amid embryogenesis; nonetheless, not at all like the
circumstance in creatures, practically the whole grown-up plant is delivered post
embryonically from two meristems arranged at inverse finishes of the incipient organism
(Hülskamp et al., 1994). Foundation of root-shoot meristems amid embryogenesis is
significant for legitimate plant improvement and in this way ought to be firmly managed.
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The formative procedure through which cell bunches in the incipient organism procure
particular formative destinies as each comparative site has named design development
(Jürgens et al., 1991; Barton and Poethig, 1993).
The expansion of example through the local control of cell multiplication
proportions ensues embryogenesis as well as is an intermittent instrument amid plant
advancement. For example, botanical organs emerge in concentric whorls from the flower
meristem as lumps of cells isolated by limit districts with a low rate of cell expansion
(Furner, 1996). Multiplying leaf primordia are isolated from the shoot meristem by slower
multiplying limit locales (Callos and Medford, 1994), and trichomes emerge through very
expanded multiplication of individual leaf epidermal cells contrasted and the encompassing
cells (Hülskamp et al., 1994).A few qualities have been distinguished that are
communicated particularly in organ or meristem limits. The Arabidopsis cup-shaped
cotyledon 1&2 (Takada et al., 2001; Aida et al., 1997) and the petunia no apical
meristem(Souer et al., 1996) qualities are communicated in limits between vegetal organ
primordia and in the limit among the cotyledons. Changes in these qualities cause
surrenders in the foundation of a few limits, bringing about organ combinations. This
reality recommends that limits are effectively settled and kept up (Duval et al., 2002). The
above literature concluded that mulberry leaves are of great meaning so, its use as a
traditional medicine is not neglect able.
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Chapter 3
MATERIALS AND METHODS
3.1 STUDY AREA
This study was conducted in Muzaffarabad, Azad Kashmir (Figure 3.1). The
thirty accessions were provided by Sericulture Department Patika as shown in Table 1.
These accessions were reconfirmed from National Herbarium of Pakistan, Quaid-e-Azam
University, and Islamabad. The morphological data was collected during the months May
and June, 2015.
Figure 3.1 Map depicting the territory of Muzaffarabad
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Table 3.1: List of Mulberry accessions used in the study
Serial No. Accessions Location
1 Punjab II punjab Patika sericulture dept.
2 Gumgi Srilanka Patika sericulture dept.
3 Japan ealy Patika sericulture dept.
4 Pak I punjab Patika sericulture dept.
5 Punjab I Patika sericulture dept.
6 Morus latefolia Srilanka Patika sericulture dept.
7 Morus latefolia Late Patika sericulture dept.
8 Husang china Patika sericulture dept.
9 PFI Patika sericulture dept.
10 Kanmasi Japan Patika sericulture dept.
11 Punjab II punjab Patika sericulture dept.
12 Lun-40-punjab Patika sericulture dept.
13 Korean subni Patika sericulture dept.
14 Gumgi korean early Patika sericulture dept.
15 Morus latefolia early Patika sericulture dept.
16 Morus latefolia Patika sericulture dept.
17 Morus alba I Patika sericulture dept.
18 Kanmasi Japan early Patika sericulture dept.
19 Pak II Punjab Patika sericulture dept.
20 Husang china early Patika sericulture dept.
21 Husang china late Patika sericulture dept.
22 NARC local Patika sericulture dept.
23 Gumgi korean late Patika sericulture dept.
24 Punjab II Patika sericulture dept.
25 Morus Indica Patika,whole AJK
26 Morus alba Patika,whole AJK
27 Morus macorura Patika,whole AJK
28 Morus nigra Patika,whole AJK
29 Morus serrata Patika,whole AJK
30 Japan late Patika sericulture dept.
3.2 MORPHOLOGICAL ATTRIBUTES
Morphological measurements were taken at the Sericulture Research Center Patika
and surroundings of Muzaffarabad Azad Jammu and Kashmir. Three replicates were taken
from each accession. Ten parameters were measured for data analysis. The qualitative traits
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were leaf color, shape, margins, tip and base. While the quantitative aspects were lamina
length, lamina width, petiole length, petiole width and leaf area index. The parameters were
measured in centimeters. The parameters were recorded when the leaves were in fully
expanded state.
The leaf base was taken as a fix point and length was recorded up to the tip. The
widest part was taken to measure its breadth. Petiole was cut from the base and then
measured its length. Area index of leaf was calculated by using Turner’s formula i.e. length
× width ×0.75(Turner, 1974). These measurements followed Adolkar et al., (2007) and
Food and Agriculture organization.
3.3 BIOCHEMICAL ANALYSIS
In biochemical analyses following parameters were done:
3.3.1 Estimation of Protein
It was quantitatively detected by the method of Lowry (1951). For protein content
estimation, Bradford method (Bradford, 1976) was used with modifications. For extraction
of proteins, 0.25 g of leaf sample was ground in china motor with 5.5 mL phosphate buffer
(pH 7.6). The homogenate was centrifuged at 8000 rpm for 20 min. 0.1 mL supernatant
and 2.9 mL of bio reagent were added to make final volume up to 3 mL and stirred
immediately. Absorption was recorded at wavelength of 595nm after 25 min by UV Visible
spectrophotometer. Bovine serum albumin was used for its standardization.
3.3.2 Determination of Carbohydrate
Ranganna et al.(1983) method (with minor modification) was used for
determination of carbohydrate contents in all selected mulberry varieties. For this purpose,
2.0 g leaves were taken from mulberry samples and crushed with the help of motor and
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pestle. The slurry was centrifuged for 12 minutes at 6000 rpm. 0.5 mL of clear supernatant
and 0.50 mL of pure water were gathered in glass tube and for blue color 4 ml of an throne
reagent was added. For estimating carbohydrate contents, the absorbance of green color
was measured at wavelength of 625 nm with the help of UV- Visible Spectrophotometer
Model T60 UV by PG Instruments. The carbohydrate contents were calculated by standard
sugar solutions method.
3.3.3 Pigment Analysis
For pigment estimation 0.1g sample was homogenized with 80 percent acetone with
the help of mortar and pestle and centrifugation was done. Then, optical density was
measured at different wavelengths. For anthocyanins, methanol/HCL/water were instead
of 80 percent acetone (Sims and Gamon, 2002).
3.3.4 Mineral Analysis
Sodium and potassium were analyzed by taking their leaves and boil in 10 mL per
chloric acid for about 35 minutes. To this double distilled water was used to make it one
liter. Potassium and sodium contents were assayed with the help photometer (Flame) and
NaOH and KOH were used for getting standard curve. Results were taken in µg (Szabo-
Nagy et al., 1992).
3.3.5 Determination of Anti-oxidant Activity by DPPH Assay
The method given by Hatano et al., (1998) was used. The DPPH solution was made
0.0008 g of DPPH was added to 10 mL of ethanol. For checking antioxidant activity,
solution in different concentration was made. Total volume of mixture was 1500µl in which
control solution was made which contained 1000 µL ethanol and 500 µL DPPH
respectively. The experiment was conducted on the plant extracts. There were 5 treatments.
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DPPH solution concentration was same for all 5 treatments. But the concentration of
ethanol n sample extract were changed in each treatments, the range of ethanol was made
(925,700, 475, 250, and 25 µL) respectively and plant extracts were made with different
concentration (75, 300, 525, 750and 975 µL) respectively. The shaken fusion was placed
in absence of light for thirty min. and then observed absorbance at 517 nm. The capacity
to scavenge the DPPH radical was calculated using the following equation:
% inhibition = (Controlled reaction Absorbance - Absorbance of sample)/absorbance of
control) -100
3.3.6 Phenolics
1 g of dried sample was taken and made slurry with the help of (1000µl*10) warm
D.W. the slurry was filtered by using watt man filter paper. Solutions were made for
estimation of phenolic compounds in plant extract. Following solutions were made with
known concentrations. 10 percent Folin-Ciocalteau’s reagent was made (1 mL of Folin-
Ciocalteau’s in 9mL of Distil H2O), 2.50 mL of it was added to test sample. 2.5 mL of 7.50
percent Na2CO3 soln. was made (7.5 g of sodium nitrate was put into 100 mL distil Water).
0.5 mL of the plant extract was made. Test solution was consisting of 500 µL of sample,
2500 µL FC and 2500 µL of sodium carbonate was added into test tube. And put into
incubator for forty five minutes at 46°C, reading were noted at 765nm Singleton et al.
(1999).
3.4 MOLECULAR CHARACTERIZATIONS
Mulberry leaves were used for this purpose.
3.4.1 Collection of Plants and Storage
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The Mulberry assortments were gathered from Sericulture office Patikah,
Muzaffarabad. Every assortment was distinguished by Herbarium of Pakistan, Quaid e
Azam University Islamabad. The assortments are given in the table underneath, the plant
leaves was saved in fixed zipper packs with the silica gel (Fisher substance L-12167) at -
20 ºC for an additional use.
3.4.2 Preparation of Plant Material
The leaves was cut into little pieces with a cleaned scissor already washed with 70
percent ethanol and distil water and permitted to dry. CTAB method of Richards (1997)
was followed for extracting DNA.
3.4.3 Preparation CTAB Buffer
CTAB buffer was made of 20 mM (EDTA, pH 8.0), 100 mM (Tris HCl, pH 8.0),
1.4 M (NaCl) and at the end added 2 % mercepto-ethanol in it.
3.4.4 Extraction Protocol of DNA
Cetyl Trimethylammonium bromide was used to extract DNA. Sample was
crumpled in crusher in liquid nitrogen by using pre heated (60°C) by addition of 2000µL
buffer and mercaptoethanol about 15µL. The mixture was then moved to a 1.5mL
Eppendorf and put into the water steam bath which was already set on 65 °C. The
Eppendorf tubes were spin for ten minutes at 12,000 revolutions per minute and the clear
upper most layers was collected in a separate Eppendorf. Then an equal amount of
chloroform iso-amyalcohol (24:1) was added to the Eppendorf containing the supernatant.
After this the Eppendorf was gently inverted 3-4 times. For removing proteins, the whole
process was done repeatedly for five to six times. This results in a clear in a dull yellow to
white supernatant and to this an equal amount of isopropanol was added and sample was
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left at -20°C for overnight. Next day the Eppendorf containing suspended DNA is spun at
13,000 rpm for 12 min and isopropanol was thrown away from it and 70% ethanol was
used to wash away pelleted DNA and then allowed to air dry. Nuclease free water was
added to the pellet and let it be dissolved thoroughly and put it at -20 °C for further analysis.
3.4.5 DNA Confirmation
To assure the presence or absence of genomic DNA, gel electrophoresis was done
by the use of extracted DNA (5 μL) and bromophenol blue dye (2 μL) dye. For staining
purpose ethidium bromide was used and gel was seen under ultra violet by Dolphin Doc
Plus gel documentation system.
3.4.6 Polymerase Chain Reaction (PCR)
PCR reactions were done for 49 markers and detail is given in the table below. The
reaction mixture was prepared for a total volume of 25 µL containing 2.5µL Taq buffer, 3
µL Mgcl2, 1 µL DNTPs, 3µL DNA, 1.5µL marker, 0.5µL Taq polymerase and 13.5µL
nuclease free water.
3.4.7 Dilution of Primers
The dilution of primers was done by the advice of manufacturer. The amount of
primer in each vile was 25 Pico moles per micro liter.
3.4.8 PCR Optimization
It was done by the followings:
1. Initial denaturation was at 94 °C for 7 minutes
2. Thirty five cycles of denaturation at 94 °C for 1 minute
3. Annealing was done according to the markers for one minute
4. One minute extension at 72 °C
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5. Do the last extension for ten minute at 72 °C
Table 3.2: List of SSR markers
Locus Repeat Motif Primers Annealing
Temperatur
es
Allele
size (Bp)
Referenc
e
SS04 (TG)27 F: CGAGGGAGGGATGAGGAGC
R: CACATTCATGCACCCTCCTATA
61 180-200
Aggarwal
et al.
(2004)
SS05 (CA)5CC(CA)
27
F:TCCAGCAAAGATGTGACAAAAGTT
R: TTGCCTTCCCGATTATGCTG
61 270-310
SS09 (CA)56 F: AGAACCCTTCCGCCCTATG
R: CCTTGGCGTAGGCAAAGTTG
58 300-400
SS18 (CA)27 F: TCTTCGCCCGTTGTTCGC
R:AGCAATTTTCTTCAACTCACCTTCT
61 280-370
MulSTR1 (GTT)6+(GTT)
4
F:GCCGTGTACCAGTGGAGTTTGCA
R:TGACCGTTTCTTCCACTTACCTAATG
69 180-300
MulSTR2 (GTT)11 F: CGTGGGGCTTAGGCTGAGTAGAGG
R:CACCACCACTACTTCCTCTCTTCCA
G
72 260
MulSTR5 (CAA)8 F: CCCCCTGCAATGCCCTCTTTC
R:TGGGCGAGGCAGGGAAGATTC
66 180-200
MulSTR6 (GT)15 F:TCCTTAGGTTTTTGGGGTCTGTTTAT
R:CCTCATTCTCCTTTCACTTATTGTTG
68 250-290
MulSTR4 (GAA)6 F:GGTCAAGCGCTCCAGAGAAAAG
R:CACCACCACTACTTCTCTTCTTCCAG
65 110-150
SS01 (CA)30 F: CGGTCACGCCTTCTTCTCC
R: TGACCGAGAAATGAGGAAGGAG
60 115-180
Table 3.3: List of ISSR markers
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Marker
name
Sequence Annealing
Temperatures
Reference
UBC 820 GTGGTGTGTGTGTGTGTC 54
(Kalpana et al., 2012)
UBC 828 TGT GTG TGT GTG TGT GA 54
UBC 826 ACACACACACACACACAC 52
UBC 827 ACACACACACACACACAG 52
UBC 841 GAGAGAGAGAGAGAGAYC 54
UBC 858 ATGATGATGATGATGATG 52
UBC 864 ATGATGATGATGATGATG 45
UBC 809 AGAGAGAGAGAGAGAG 49
IS05 GTGTGTGTGTGTCC 42 (Wolfe et al., 1998)
IS17 TGTGTGTGTGTGTGTGGT 52
IS21 GTCGTCGTCGTCGTCGTC 60
Table 3.4: PCR components for the amplification of primers
Reagents Concentrations Volume(µl)
Taq buffer 10X 2.5
MgCl₂ 25Mm 2.50
DNTPs 2.5 Mm 1
DNA 2.50
Forward primer 32 pmoles 1
Reverse primer 32 pmoles 1
Taq DNA Polymerase 5 U 1
Nano Pure Water 13.5
Total volume 25
Using 2 percent agarose gel made in 0.5 X TAE buffer, PCR product was
confirmed. Band size was assured by using 1 kilobytes ladder along PCR results.
3.6 DATA SCORING
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Data was scored on the basis of presence (1) or absence (0) of isomorphic or
polymorphic bands.
3.7 STATISTICAL ANALYSES OF DATA
Data was analyzed by using different programs. GraphPad PRISM 6.0 were used
to analyze morphological, biochemical and molecular data. For morphology LSD,
correlation, and Duncan’s multiple range test was done by MSTATC. Principal component
analysis was done by PAST. Biochemical analyses include GRAPHPAD PRISM 6.0.
Diversity was evaluated by the use of NTSYS-pc and Minitab and R. Dendrogram was
made on likeness index basis by software NTSYS 2.20 by Rohlf (1997).
3.8 CHARACTERIZATION OF CUC2 GENES
3.8.1 Morus Species and Strains
Morus latefolia (syn alba), Morus serrata, Morus nigra, and Morus indica were
used in the study. Specific strains of Morus alba(Narc and lun-4-Punjab), Morus serrata
(Morus latefolia), Morus nigra (Husang china) and Morus Indica were used in this study.
Total seven strains were used. Among these strain, Morus serrata and Morus nigra were
selected as a wild plants from the surrounding of study area while Narc, Lun-40-Punjab,
Morus Latefolia, Husang China, and Morus Indica were obtained from Sericulture
department Muzaffarabad.
3.8.2 Genomic DNA Extraction
It was done from leaves according to Richard et al. (1997).
3.8.3 Reagents
Cetyl trimethylammonium bromide (CTAB) buffer (freshly prepared) pH 8
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50 mM ethylene diamine tetra acetic acid (EDTA)
100 mM Tris HCl
1.5 M Sodium Chloride (NaCl)
2% ß-mercaptoethanol (typically used for plants having high polyphenol content)
Chloroform:isoamylalcohol (24:1)
Isopropanol, -20 °C
70% ethanol, -20 °C
Nuclease-free water
3.8.4 Protocol
Fresh and young mulberry leaves were collected from the field and were brought
to the laboratory in liquid nitrogen. The samples were ground in special microfuge grinding
tubes with the help of grinding pestles. To each ground sample 1.5 mL of pre-heated CTAB
buffer was added and 15 µL of ß-merceptoethanol (final concentration: 2%) and incubated
for 65 minutes on hot plate already set at 65°C. Centrifugation was done at 11,500 rpm
for 17 minutes. The upper layer was shifted to a fresh Eppendorf and then mixed with equal
concentration of chloroform and isoamyl alcohol (24:1). After thorough mixing by
inverting the tubes 3-4 times, the tubes were centrifuged and supernatant was collected.
Extraction with the chloroform- isoamyl alcohol solution was repeated 5-6 times to remove
proteins, resulting in a clear colorless or slightly colored supernatant. DNA precipitation
was performed by adding an equal amount of chilled iso-propanol and incubation at -20ºC
for overnight and after which the DNA was pelleted by centrifugation at twelve thousand
revolutions per min for 13 minutes. Upper layer was thrown and the pellet was eroded with
70% ethanol and allowed to dry in an incubator set at 37 °C. Then 50µL nuclease-free
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water was added to the tubes and DNA was allowed to dissolve on ice, and kept at -20 °C
for upcoming use.
3.8.5 Amplification of CUC2 Gene Sequences
Morus CUC2 sequences were amplified using the Polymerase Chain Reaction
(PCR). For each of the three CUC2 genes in the Morus genome, forward (f) and reverse (r)
primers were designed based on the three CUC2 gene sequences available on the Morus
Genome Database (Morus DB) website (http://morus.swu. edu.cn/morus), using the
PrimerSelect program in the DNASTAR Lasergene 14 software
(https://www.dnastar.com).
For each of CUC2c and CUC2d, one primer set was designed to amplify a fragment
that spanned the entire gene, which is expected to be 1750 base pair and 1357 bp in length,
respectively. The primer sequences are:
For CUC2c: cuc2c230f: GTACAAATTCAAAGTATCAAAGGTGGTG)
cuc2c1980r: AAAATTAAAAGCCAAAAACAGTTATAGATC)
For CUC2d: cuc2d605f: GAGAAAAAGCCTTCAAAAACAAACGG)
cuc2d1962r: CAATTAACCCCTGAATACAAGTCATCCC)
For the longer CUC2a/b, two primer sets were required to amplify two overlapping
fragments that spanned the entire gene, a 1548-bp N-terminal fragment and a 1513-bp C-
terminal fragment. The primer sequences are:
For the N-terminal fragment,
cuc2a305f: TTGAAGTTTGAACTAGAACGCAAATCG
cuc2a1853r: CGAATCCACTAATCAAACGCTAATGC
For the C-terminal fragment,
31
31
cuc2a1825f: GGAGCATTAGCGTTTGATTAGTGGAT
cuc2a3338r: GCTTAGCAATGTCTCTGAAATGTCCC
PCR was done in a 25 µL tube, which contained 0.5-2.5 µLMorus DNA, 2.5 µL
Thermo pol buffer, and 0.25 µL Taq polymerase (New England Bio-labs, Ipswich, M.A,
U.S.A) 2.5 µL mM dNTP mixture, 0.5 µL of each of the two primers (10 µM) and 16.75
µL nuclease-free water. Amplification was performed by the same procedure used before.
The products size was confirmed as before in the section 3.4.8.
3.8.6 Cloning of PCR products, Identification of Recombinant Colonies, and DNA
Sequencing
For cloning, the PCR products obtained from two PCR reactions were gel purified
through electrophoresis on one percent agarose gel, the band containing the amplified DNA
fragments were excised from the gel, the DNA turned into extracted out from the agarose
using the QIAquick®Gel, Extraction kit (Qiagen Sciences, Germantown, united states) and
resuspended in 30 µl of nuclease-free water. The refined PCR yields were ligated to the
pGEM®-T Easy vector (Promega Corporation, Wisconsin, USA) by TA cloning agreeing
with the producer’s guidelines: the ligation mix consisted of 2X Rapid Ligation Buffer
(five micro liter), 50 ng pGEM®-T Easy Vector (one micro liter), PCR product (3 µL), T4
DNA Ligase (1 µL), and nuclease-free water up to a final volume of 10 µL, and was
incubated overnight at 4 °C in order to obtain the maximum number of transformants.
For bacterial transformation, 2 µL of the ligation mix were used to transform high-
efficiency competent cells E. coli JM109 competent cells (Promega, Cat. No. L2001; supE
hsdD5thiD(lac-proAB) D(srl-recA) (306:Tn10) (tetr ) F’[traD36 proAB+ lacIq lacZDM15
d]) as follows:
32
32
A tube containing 50 µl of JM109 cells was taken from the -80ºC and defrosted on
ice. To these cells, added 2 µL ligtion reaction, and were tapped very gently to blend the
contents. The tubes were placed on ice for 20 minutes, heat shocked for 45 seconds at 42
ºC, and returned to ice for 2 minutes. After addition of 950 µL LB, the tubes were
incubated for 1.5 hours in a shaker set at 37 ºC and 150 rpm. To select for transformed
colonies using blue-white colony selection, 100 µL of the transformation mix was put on
LB agar medium having hundred micro gram per milli liter ampicillin onto which 50 µl of
X-gal (2% in dimethyl formamide) and 10 µL of a 10 mM IPTG solution were spread. The
plate was incubated at 37ºC overnight and several white colonies, which, unlike the blue
colonies, are likely to contain the inserted PCR product, were picked onto a fresh
LB+ampicillin to generate a “master” plate.
Colony PCR was performed to identify the white colonies that contained the desired
insert. Bacterial cells (either picked directly from the master plate or taken from overnight
cultures) were subjected to PCR using the T7 and SP6 primers, which are complementary
to vector sequences that flank the TA cloning site. The 25µl PCR reaction mix contained
2.5 µL 10x Thermopol buffer, 2 µL 2.5mM dNTPs, 0.5 µL T7 primer, 0.5µl SP6 primer,
14.25 µL milliQ water, and 0.25 µL Taq polymerase. PCR conditions were as described
for CUC2 gene-specific primers.
Colonies that produced a PCR product of the expected size were then grown by
culturing overnight in LB + ampicillin liquid medium. Plasmid DNA was purified from
these cells using the QIAprep® SpinMini prep kit (Qiagen, Cat. No. 27106), and 1µg of
plasmid DNA was submitted to the Cornell University Institute of Biotechnology for
33
33
sequencing using the T7 and SP6 primers, and as necessary, using the CUC2 gene-specific
primers listed above.
3.8.7 DNA Sequence Analysis
Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi)
searches were performed to confirm that the amplified fragments were indeed CUC2
sequences. Overlapping (DNA) sequences were then investigated using the DNASTAR
Lasergene 14 suite of software. For each sample, a consensus sequence spanning the entire
length of the amplified fragment was generated using SeqMan Pro. The consensus
sequence was then analyzed in SeqBuilder, translated into an amino-acid sequence, and the
derived amino-acid sequences were aligned using MegAlign. The phylogenetic tree was
made by Mega 7 software (Kumar et al., 2016).
34
Chapter 4
RESULTS AND DISCUSSIONS
Thirty accessions of mulberry were selected to examine the genetic variation of
Morus found in Azad Jammu and Kashmir. Following findings were observed in the
mulberry leaves’ morphology, biochemical and genetic analysis.
4.1 LEAF MORPHOLOGY
Many plant species possess an assortment of differentiating natural surroundings
inside a restricted range and accordingly plants must manage these differentiating
ecological conditions. Instruments by which species categories, may possess a wide living
space run incorporate reversible changes to ecological conditions, for example,
supplements, H2O, saltiness, temperature fluctuations and that effects the trees to attain
great phenotypic versatility (Williamson et al., 1995; Fukui et al., 2000; Vijayan,
2009).Phenotypic plasticity is an essential way of natural heterogeneity (Guo et al., 2007).
Morphological estimations were taken at the Sericulture Research Center Patika
and surroundings of Muzaffarabad Azad Jammu and Kashmir. The mulberry accessions
include both cultivated and wild assortments of mulberry. Three replicates were taken from
each accession. Ten parameters were measured for information investigation.
4.1.1The Qualitative Traits of Leaf
Leaf color
It was fresh green, light green or dark green.
Leaf Shape
It was found that majority of the accessions had cordate shaped leaves and minority
had lobate shaped leaves.
35
Margins
Leaf margins were serrate, doubly serrate or dentate.
Tip
It was found to be acute and acuminate.
Base
It was cordate and truncate.
4.1.2 Quantitative Aspects
The Table 4.1 showed that the mulberry accessions were slightly different in
morphology. Most of them were fresh green in color. Pak I Punjab, Morus Latefolia,
Kanmasi Japan early, Punjab II and Morus serrata were dark green in color. The tip shape
was diverse i.e. acuminate to acute. The leaves shape was very diverse even on the same
branch of a tree the leaves were different with different margins. Some of them had serrated
margins and others had dentate margins.
The results of Least significant coefficient cleared that the lamina and petiole
length, lamina and petiole width, and leaf area were, were considerably dissimilar across
the thirty accession of Mulberry leaf.
Table 4.1: Qualitative characterization of Mulberry accessions
Accessions Leaf Color Shape Tip Base Margins Surface
Punjab II punjab Fresh green Cordate Accuminate Cordate Serrate Coarse
36
Gumgi Srilanka Fresh green Cordate Accuminate Cordate Serrate Soft
Japan ealy Fresh green Cordate Accuminte Cordate Serrate Soft
Pak I punjab dark green Cordate Accumunate Truncate Serrate Soft
Punjab I Fresh green Cordate Accuminate Cordate Doubly serrate Soft
Morus latefolia Srilanka Light green Cordate Acute Cordate Dentate Soft
Morus latefolia Late Light green Lobate Accuminate Cordate Serrate Soft
Husang china Light green Cordate Acute Cordate Serrate Soft
PFI Light green Cordate Acute Cordate Serrate Soft
Kanmasi Japan Light green Cordate Acute Cordate Serrate Soft
Kanmasi Japan late Light green Cordate Acute Cordate Serrate Soft
Lun-40-punjab Light green Cordate Acute Cordate Serrate Soft
Korean subni Light green Cordate Acute Cordate Serrate Soft
Gumgi korean early Light green Cordate Acute Cordate Serrate Soft
Morus latefolia early Light green Cordate Acute Cordate Serrate Soft
Morus latefolia Dark green Cordate Acute Cordate Serrate Soft
Morus alba I Light green Cordate Acute Cordate Serrate Soft
Kanmasi Japan early Dark green Cordate Acute Cordate Serrate Coarse
Pak II Punjab Light green Cordate Acute Cordate Serrate Coarse
Husang china early Light green Cordate Acute Cordate Serrate Coarse
Husang china late Light green Cordate Acute Cordate Serrate Soft
NARC local Light green Cordate Acute Cordate Serrate Soft
Gumgi korean late Light green Cordate Acute Cordate Serrate Soft
Punjab II Dark green Cordate Acute Cordate Serrate coarse
Morus Indica Light green Lobate Accuminate Cordate Serrate Soft
Morus alba Light green Cordate Accuminate Cordate Serrate Soft
Morus macorura Light green Cordate Accuminate Cordate Dentate Soft
Morus nigra Light green Cordate Accuminate Cordate Serrate Soft
Morus serrate Dark green Cordate Accuminate Cordate Serrate Soft
Japan late Light green Cordate Accuminate Cordate Serrate Soft
Table 4.2: Means of five morphological traits of thirty mulberry accessions
37
Accessions Lamina
length
Lamina
width Petiole length
Petiole
width
Leaf area
index
Punjab II Punjab 10.77DEFGH 9.700EFGHI 2.333CDE 0.2333CDE 79.47EFG
Gumgi Srilanka 10.83DEFGH 8.767FGHIJ 3.733ABCD 0.2000DEF 71.61EFG
Japan early 9.533EFGH 7.700JK 3.333ABCDE 0.2333CDE 57.83FG
Pak I punjab 10.33DEFGH 7.567JK 2.200DE 0.1000 G 58.69FG
Punjab I 10.17DEFGH 7.733JK 3.367ABCDE 0.2333CDE 58.92FG
Morus latefolia
Srilanka 9.900DEFGH 8.167IJ 2.767BCDE 0.2000DEF 65.64EFG
Morus latefolia Late 16.63AB 11.50BCD 3.500ABCDE 0.3667 A 146.8 AB
Husang China 10.03DEFGH 9.467EFGHI 3.300ABCDE 0.2000DEF
71.42
EFG
P.F.I 10.30DEFGH 8.233HIJ 2.633BCDE 0.1333FG
67.31
EFG
Kanmasi Japan 12.33CDEF 10.33DEF 4.033AB 0.2333CDE
99.04
CDEF
Kanmasi Japan late 12.63CDE 9.833EFGH 3.033ABCDE 0.2333CDE
92.99
DEF
Lun-40-punjab 9.667EFGH 8.633GHIJ 2.267DE 0.2000DEF
63.59
EFG
Korean subni 13.03CD 10.97CDE 3.967ABC 0.3333AB
108.2
BCDE
Gumgi korean early 14.77BC 12.63B 3.400ABCDE 0.3333AB 140.6 BC
Morus latefolia
early 11.40DEFG 8.433GHIJ 3.033ABCDE 0.2000DEF
73.10
EFG
Morus latefolia 11.17DEFG 9.900EFG 3.000ABCDE 0.3000ABC
82.11
EFG
Morus alba I 9.667EFGH 8.767FGHIJ 3.133ABCDE 0.1667EFG
74.68
EFG
Kanmasi Japan early 7.933H 8.433GHIJ 2.900ABCDE 0.2000DEF
61.55
EFG
Pak II Punjab 14.60BC 5.833L 3.233ABCDE 0.1333FG 34.98 G
Husang china early 12.00CDEFG 12.03BC 2.567BCDE 0.2667BCD
132.7
BCD
Husang china late 14.8BC 9.700EFGHI 3.300ABCDE 0.2333CDE
87.80
DEF
NARC local 10.33DEFGH 8.833FGHIJ 3.367ABCDE 0.2333CDE
72.84
EFG
Gumgi korean late 11.00DEFGH 9.000FGHIJ 2.067E 0.2333CDE
74.75
EFG
Punjab II 19.6A 15.37A 3.667ABCDE 0.3667A 193.6 A
Morus Indica 9.900DEFGH 6.333KL 3.567ABCDE 0.1000G 53.97 FG
Morus alba 9.000GH 8.867FGHIJ 3.333ABCDE 0.1000G 59.91 FG
Morus macorura 10.87DEFGH 10.57CDE 3.467ABCDE 0.1000G 88.03DEF
38
Morus nigra 9.833EFGH 8.433GHIJ 4.133AB 0.1333FG
65.16
EFG
Morus serrata 10.63DEFGH 7.700JK 4.500A 0.2000DEF
61.33
EFG
Japan late 9.267EFG 8.633GHIJ 3.367ABCDE 0.1000G 60.37FG
Values with same letter showed no difference in LSD
The maximum lamina length was observed in accession Punjab II, Morus latefolia
Late and Pak II Punjab (19.6cm), (16.63cm), and (14.60cm) respectively while accession
Kanmasi Japan early has minimum lamina length (7.933cm). Lamina width varied across
all the accessions, and ranged from 5-15cm. Accession Punjab II had the highest lamina
width (15.37cm), and accession Pak II Punjab had the lowest lamina width (5.833cm).
Highest petiole length was noted in accessions Morus serrata, Morus nigra, Kanmasi Japan
and Korean subni respectively. Petiole width ranged from 0.366-0.1000 cm. Punjab II and
Morus latefolia late had the highest petiole width (0.3667cm) followed by Korean subni
and Gumgi korean early (0.3333cm). Pak I punjab had the lowest petiole width followed
by Morus Indica, Morus alba, Morus macorura and Japan late. Leaf area ranged from
193.6-34.9cm, Punjab II had the highest leaf area followed by Morus latefolia Late and
Gumgi korean early while shortest leaf area Pak II Punjab, Morus indica, Morus alba and
Japan late respectively.
Same work was done by Gray (1990) that supports the plasticity in leaf and fruit
characteristics. Mulberry is found in wide natural distribution that encourages the
indication that flexibility is found in these types. The quantitative characters varied across
different environments as well as among different varieties. Kitajima et al., (1997) reported
phenotypic plasticity of leaf characters resulting in the leaf polymorphism in plants. The
study carried out by Pandey and Nager (2002) clarified that it’s the genetic make-up that
39
enables plants to cope with the environmental conditions and can manage themselves
according to their surrounding conditions specially that affect the morphology.
4.1.3 Correlation between the Morphological Traits of Mulberry Accessions
It was revealed from the Table 4.3 that a positive and significant correlation was
seen in lamina length and lamina width (r=0.70) and leaf area index (r= 0.762) while non-
significant negative correlation was found in petiole length (r=-0.199) and petiole width (-
0.162).Only the correlation between leaf area index and lamina length (0.76٭) and between
leaf area index and petiole length (0.993٭٭) was significant. Lamina width is positively
linked to the lamina length (r=0.702), petiole width (r=0.384) and leaf area index
(r=0.993٭٭). Petiole length is negatively and not substantially associated to lamina length
(r=-0.199), lamina width (r=-0.30) and leaf area index (r=-0.108).
Table 4.3: Pearson correlation coefficient matrix for qualitative variables of thirty
accessions
Characters Lamina
length
Lamina
width
Petiole
length
Petiole
width
Leaf area
index
Lamina length 1 0.702 -0.199 -0.162 0.762
Lamina width 0.702 1 -0.30 0.384 0.993٭٭
Petiole length -0.199 -0.30 1 0.355 -0.108
Petiole width -0.162 0.384 0.355 1 0.132
Leaf area
index
1 0.312 0.108- ٭٭0.993 ٭0.76
4.1.4 Cluster and Principal Component Analysis of Morphological Parameters
It was obvious from the cluster analysis that the genotypes that were showing
similarity with each other form two clusters (Figure 4.1). These two clusters were divided
40
into sister groups. The first cluster was sub divided in four sub clusters. While the 2nd one
made two more clusters. The first cluster had two sub clusters; one was Punjab II Punjab
and the other one was further sub divided into a cluster showing that Morus latefolia,
Gumgi Korean were identical and Husang china was showing some diversity.
The second cluster contains Pak II Punjab as a main group and the other was sub
clustered into four sister clusters. The varieties which were in the same cluster showed
similarity to one another.
41
Figure 4.1. Dendrogram showing hierarchical cluster analysis of morphological traits of 30
Mulberry accession
Through cluster analysis, two clusters could be formed from the genotypes that
were having similarity. These clusters were divided into sister group.
42
Figure 4.2 Principal Component analysis based on morphological characters of 30
Mulberry accession
42
Table 4.4: Showing Eigen value and % variance of five quantitative parameters of
mulberry traits
Eigen value % variance
1 1108.44 99.922
2 0.471282 0.042484
3 0.320634 0.028904
4 0.0727931 0.006562
5 0.00242718 0.0002188
The principal component analysis was carried out to check the relationship between
variables and the results are presented in the Figure 2.
Morus spp. is cultivated below various climatic situations starting from temperate
to tropical (Kafkas et al., 2008). Plants as mulberry possess the capability to withstand a
broad range of environmental thrushes. Randomly taken 30 mulberry accessions had been
investigated in this study. All accessions showed varied response in leaf morphology. The
means of all the traits according to its LSD had significant difference. The environmental
changes are not only responsible for changing the population structure based on its genetic
makeup to survive in the new environment and the level of each individual to alter its
phenotype according to its environment. Same outputs were conveyed via (Karst and
Lechowicz, 2007) where they found variances in flexibility in accessions according to
ecological influences.
Plants activities are being affected by many constraints in vicinity and they have to
respond to the surrounding scenarios. Wherein vegetation acquires a excessive degree of
phenotypic plasticity (Fukui, 2000; Amakawa et al. 2000; Vijayan 2009).Phenotypic
plasticity is consequently an imperative approach through this character plantlets
43
correspond surroundings hetero-genety (Guo et al., 2007). It is found in the study that
Gumgi Korean early, Pak I punjab, Japan late, Morus serrata are the varieties that show
the phenotypic plasticity as they were more obvious difference in their leaf morphology.
Phenotypic credits are imperative to think about the morphological characters in
leaf yield. To assess the plant genotypes, the plant raisers utilize the phenotypic characters
to examine those (Mace et al., 2010). Morphological portrayal of mulberry had been
utilized for instance a device for inspection of hereditary connections between various
assortments that will be productive in its enhancement (Tikader, 1997; Adolkar et al.,
2007). Mulberry is found in an extensive range of distribution and it is believed to have a
considerable measure of versatility in its species. Along these lines, the leaf characters like
lamina length, width, petiole length, width and stature changes as nature changes (Gray,
1990) also revealed the plasticity in mulberry fruit and leaf characters. While, Kitajima et
al., (1997) examined the topical shade trees and found that there seasons likewise influence
the size and stomata functioning.
Phenotypic plasticity causes differential leaf forms. Jones and Corlett (1992)
worked on the Polygonium amphibium L., Polygonaceae in various environments and came
with a conclusion that there was a high phenotypic plasticity there in leaf characters.
Pandey and Nagar (2002) found that leaves can be different depending on their growth
adaptations. Therefore it is pointed out that the ability to withstand different environmental
scenarios, natural phenotypic differences is at top role. Singh and Singh (2003) had the
same output in Saccharum officinarum L. officinarian.Reduction in developmental aspects,
leaf zone and progression was seen in water deficient crops Jones & Colbert (1992).
44
Drought is responsible for minimizing shoot vicinity, and varies mulberry morphological
features (Susheelamma et al., 2000; Mujeeb et al., 2004).
In this study, the evaluation of difference confirmed an extensive range of variant
and great distinction (P ≤ 0.05) in lamina length, width and petiole length, and width and
leaf area.
Similar consequences had been stated by Tikader & Kamble (2008) describing
noteworthy variations in mulberry progression and yield traits. Mulberry phenotype
difference was also presented by these researchers (Thangavelu et al., 2000; Tikader &
Rao, 2002). This type of overall result reported by Ogunbodede and Ajibade (2001) that
genetic and environment effect on leaves’ qualitative features used for its identity. A plant
can also be recognized by its newly developed leaves, shoots (colorations) (Adolkar et al.,
2007).
On the basis of hierarchical clustering, three groups were obvious from the
dendrogram. Pak II punjab, Lun-40-punjab, Morus Indica, PFI, Morus latefolia
Srilanka,Japan late, Morus alba clustered. Similarly, Punjab II Punjab, Morus latefolia
Late, Gumgi korean late form tight cluster while the other accessions made cluster
separately. Hence, we can say that the topographical base is not having prominent role
(Tikader and Roy, 2002; Tikader et al., 2003).
Cluster analysis based on morphology was studied by (Sharma et al. 2000) testified
that based on morphology dissimilarities in accession they form cluster. Principal
component analysis for relationship among different accessions showed most of the
variance explained by bipolt charting. According to first and second components,
component near to origin has higher positive coefficient with respect to the desirability of
45
high level of these indicators. Lamina length showed about 99.92% variance, and the least
variance was shown by the leaf area index i.e., 0.0002188.
4.2 BIOCHEMICAL ANALYSIS
Data was subjected to statistical analysis by using computer software GraphPad
Prism 6. Means and standard deviations were given here along with the tables and graphs.
4.2.1 Measurement of Carbohydrate Content
The quantitative analysis of various nutritional parameters of mulberry leaves is
shown by tables and graphs. The carbohydrate content in mulberry leaves of young plant
was determined and is the highest in Korean subni (3.7626±0.2040 mg/g) followed by
Morus nigra (3.728±0.1795 mg/g), Morus latefolia (3.643±0.2458 mg/g) and P.F.I
(3.568±0.3057 mg/g). The lowest carbohydrate content was found in Morus serrata
(1.8743±0.39763 mg/g), Gumgi Korean early (1.8046±0.1550 mg/g) and Punjab II
(1.704±0.1695 mg/g).
Table 4.5: Biochemical constitutions of Mulberry accessions
Varieties Carbohydra
tes
Proteins Chlorophyll
A
Chlorophyll
B
Carotenoids Anthocyani
ns
Punjab-II-
Punjab
3.36±0.21 1.40±0.04
6
0.028±0.000 0.045±0.000 0.083±0.000 0.06±0.002
Gumgi
Srilanka
3.55±0.10 1.66±0.00
2
0.026±0.000 0.031±0.000 0.122±0.000 0.02±0.004
Japan ealy 3.63±0.11 1.63±0.00
6
0.022±0.000 0.014±0.000 0.114±0.000 0.04±0.004
Pak-I-Punjab 2.54±0.49 1.60±0.00
3
0.034±0.000 0.044±0.000 0.096±0.000 0.10±0.010
Punjab-I 2.63±0.27 1.60±0.03 0.029±0.000 0.017±0.000 0.094±0.000 0.08±0.002
Morus latefolia
Srilanka
2.71±0.20 1.23±0.03 0.030±0.000 0.017±0.000 0.081±0.000 0.07±0.002
Morus latefolia
Late
2.76±0.21 1.51±0.01
7
0.029±0.000 0.020±0.000 0.109±0.000 0.05±0.003
46
Husang China 2.76±0.16 1.45±0.00
3
0.035±0.000 0.017±0.000 0.041±0.000 0.01±0.004
P.F.I 3.66±0.31 1.57±0.00
3
0.029±0.000 0.017±0.002 0.083±0.000 0.10±0.003
Kanmasi Japan 2.70±0.24 1.70±0.00
5
0.013±0.000 0.021±0.000 0.036±0.005 0.06±0.010
Kanmasi Japan
late
2.58±0.22 1.41±0.00
4
0.031±0.000 0.018±0.000 0.073±0.000 0.08±0.002
Lun-40-punjab 2.67±0.25 1.61±0.00
2
0.030±0.000 0.017±0.000 0.156±0.000 0.08±0.004
Korean subni 3.76±0.20 1.52±0.00
1
0.032±0.000 0.011±0.000 0.092±0.000 0.05±0.003
Gumgi korean
early
1.80±0.15 1.61±0.00
2
0.031±0.000 0.019±0.000 0.124±0.000 0.07±0.002
Morus latefolia
early
2.58±0.10 1.56±0.00
2
0.032±0.000 0.036±0.000 0.097±0.000 0.05±0.005
Morus latefolia 3.64±0.25 1.60±0.06
8
0.031±0.000 0.034±0.000 0.095±0.001 0.07±0.002
Morus alba I 3.00±0.55 1.66±0.00
5
0.029±0.000 0.021±0.000 0.076±0.000 0.06±0.004
Kanmasi Japan
early
2.49±0.55 1.58±0.00
5
0.027±0.000 0.012±0.000 0.036±0.000 0.09±0.010
Pak-II-Punjab 2.33±1 1.34±0.01
7
0.026±0.004 0.023±0.000 0.133±0.000 0.03±0.004
Husang China
early
3.59±0.35 1.63±0.02
3
0.030±0.000 0.039±0.000 0.115±0.000 0.02±0.002
Husang China
late
3.61±0.42 1.61±0.00
2
0.020±0.000 0.014±0.000 0.158±0.000 0.02±0.002
NARC local 3.47±0.32 1.60±0.00
7
0.029±0.000 0.027±0.000 0.103±0.000 0.04±0.001
Gumgi korean
late
3.49±0.25 1.52±0.01
7
0.029±0.000 0.017±0.000 0.136±0.000 0.07±0.004
Punjab-II 1.70±0.17 1.48±0.01
1
0.032±0.000 0.016±0.002 0.115±0.000 0.02±0.004
Morus Indica 3.07±0.78 1.48±0.02
2
0.025±0.000 0.017±0.000 0.108±0.000 0.06±0.002
Morus alba 3.31±0.68 1.40±0.00
6
0.029±0.000 0.026±0.000 0.042±0.000 0.05±0.002
Morus
macroura
2.76±0.36 1.43±0.00
9
0.029±0.000 0.012±0.001 0.091±0.000 0.08±0.001
47
Morus nigra 3.73±0.18 1.66±0.00
4
0.026±0.004 0.013±0.000 0.197±0.000 0.06±0.002
Morus serrata 1.87±0.40 1.55±0.02
0
0.033±0.002 0.017±0.000 0.082±0.000 0.01±0.003
Japan late 2.47±0.08 1.59±0.00
9
0.026±0.003 0.038±0.000 0.081±0.000 0.07±0.004
The results of Srivastava and Elangovan (2011) were similar to our findings. They
assessed leaf biological conformations in S.36,Vishwa, S.1635, S.799, and M.5 and
concluded that S.1635 had maximum carbohydrate content. Purohit and Kumar (1996) also
found maximum total carbs content material in S-1635 cultivar (22.83%). Iqbal et al.,
(2010) said overall sugary material in Pakistan to be found as 21.1633-34.77 percent
mulberries fruits grown in Pakistan whilst the current experiment has approximately
0.3730-0.655 mg/g.
The carbohydrate contents in specific mulberry culmination showed a tremendous
variation amongst unique mulberry genotypes. Some fruiting mulberry types; S_36 had
max carbs while BR_2 had min. The variations in mulberry sorts in regard of leaves overall
fats content material in leaves are especially good sized (P>0.010) (Kumar and Chauhan,
2011). Plants sugars can be used as a feeding material for silkworm and had rebuilding
ability (Freeze, 1998). These are foremost additives liable for growing silkworms,
upgrading and silk business. The silk moths make use of sugars as supply of power for the
making of lipids and amino acids. Horie, (1995) emphasised that amongst 20 examined
sugars, Sucrose intensely inspired the eating performance. Nutritive values of proteins are
very critical as silkworm larvae make use of the leaf nitrogenous matter for their growth
and development and synthesis of silk protein. Sujathamma and Dandin (2000) determined
maximum proteins in T-G, sugars in O.P.H-three and overall chlorophyll in T.R-4. Patil
48
etal. (2001) estimated bio-chemical ingredients’ in S/36, Vishwa., S/1635, S/799, and M/5
and found that S/1635 had maximum carbs.
Figure 4.3: Graphical representation of carbohydrate content in mulberry accessions
4.2.2 Measurement of Protein Content
No significant difference was found in protein content of different accessions. The highest
amount of proteins was found in Kanmasi Japan (1.7007±0.0049), Morus
alba(1.6647±0.0045mg/mL), Morus nigra (1.6640±0.0044mg/mL) and Gumgi Srilanka
(1.6633±0.0015mg/mL). Meanwhile the minimum protein content was in Husang china
A c c e s s io n s
Car
bohy
drat
es m
g/g
f.w
Pu
nja
b I
I p
un
jab
Gu
mg
i S
rila
nk
a
Ja
pa
n e
aly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
rus
la
tefo
lia
Sri
lan
ka
Mo
rus
la
tefo
lia
La
te
hu
sa
ng
ch
ina
PF
I
Ka
nm
as
i J
ap
an
Ka
nm
as
i J
ap
an
la
te
Lu
n-4
0-p
un
jab
Ko
rea
n s
ub
ni
Gu
mg
i k
ore
an
ea
rly
Mo
rus
la
tefo
lia
ea
rly
Mo
rus
la
tefo
lia
Mo
rus
alb
a I
Ka
nm
as
i J
ap
an
ea
rly
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
rus
In
dic
a
Mo
rus
alb
a
Mo
rus
ma
co
rura
Mo
rus
nig
ra
Mo
rus
se
rra
ta
Ja
pa
n l
ate
0
1
2
3
4
5
*** ***
49
(1.4487±0.0031mg/mL), Kanmasi Japan late (1.4100±0.0036mg/mL) and Punjab-II-
punjab (1.4030±0.0455mg/mL).
The results in the current study revealed that leaves of target accession are good source of
nutrients. Our results coincide with the other studies showing sugar concentrations in black
mulberry fruits are between 6-9 percent. The protein concentrations in black mulberry
fruits ranged between 7.66-12.93 percent according to other authors (Martin, 1995; Machii
et al., 2000; Kitahara et al., 2002; Ghosh et al., 2003; Bamikole et al., 2005; Srivastava et
al., 2006).The reason for differences may be the variation in varieties and ecological zones.
Thomsen et al. (1991) and Freeze (1998) emphasized that protein is the main
component of our living things. The presence of high protein content in plants showed that
increased food value or protein based bioactive components. Mulberries can live better in
sub-tropical and tropical regions, full of nutrients, mainly produced for its leaves, and as
forage too. They are wealthy in protein (15-35 percent),P: 2.420, Ca: 0.230 to 0.970
percent, without or in minor traces of unhealthy compounds (Omar et al., 1999; Saddul et
al., 2004; Srivastava et al., 2006). The presence of beta carotene in leaves could be a very
good agent of egg yolk color, as it is altered to Vit A and Xanthophyll in animals (Moller
et al., 2000).
The investigations by (Gupta et al 2005; Maisuthisakul et al., 2008) confirmed
that the varieties were in the order of increasing protein content in leaves as M. rubra > M.
nigra > M. albaand serve as a good source of proteins than other vegetables (leafy).
50
Figure 4.4: Graph showing proteins in mulberry accessions
4.3 Pigments
Pigments are imparting a great contribution to the photosynthesis, enhances food
production, washing out the bad effects of carbon dioxide from the environment, and on
wider scale they are also used in medical technology and environmental cleanup (Vermaas,
2000).
4.3.1 Chlorophyll A
The chlorophyll A content is the highest in Husang China (0.347±0.0002), Pak-I-
Punjab(0.0336±0.0001), Morus Serrata (0.0331±0.0016µg/mol) and Punjab II
(0.0317±0.0002). Two varieties, Korean subbni (0.0315± 0.0004) and Morus latefolia
early (0.0315±0.0001) have not any significant difference in their mean values. While the
Pro
tein
s m
g/g
of
F.W
.
Pu
nja
b I
I p
un
jab
Gu
mg
i S
rila
nk
a
Jap
an
ea
ly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
rus
la
tefo
lia
Sri
lan
ka
Mo
rus
la
tefo
lia
La
te
hu
sa
ng
ch
ina
PF
I
Ka
nm
as
i Ja
pa
n
Ka
nm
as
i Ja
pa
n l
ate
Lu
n-4
0-p
un
jab
Ko
rea
n s
ub
ni
Gu
mg
i k
ore
an
ea
rly
Mo
rus
la
tefo
lia
ea
rly
Mo
rus
la
tefo
lia
Mo
rus
alb
a I
Ka
nm
as
i Ja
pa
n e
arl
y
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
rus
In
dic
a
Mo
rus
alb
a
Mo
rus
ma
co
rura
Mo
rus
nig
ra
Mo
rus
se
rra
ta
Jap
an
la
te
0 .0
0 .5
1.0
1.5
2.0
A c c e s s io n s
51
least values are found in Husang China late (0.0205± 0.0004) and Kanmasi japan (0.0125
±0.0004).
Figure 4.5: Graph showing chlorophyll a content in different mulberry accession
Ch
loro
ph
yll
a
mo
l/m
l
Pu
nja
b I
I p
un
jab
Gu
mg
i S
rila
nk
a
Ja
pa
n e
aly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
rus
la
tefo
lia
Sri
lan
ka
Mo
rus
la
tefo
lia
La
te
hu
sa
ng
ch
ina
PF
I
Ka
nm
as
i J
ap
an
Ka
nm
as
i J
ap
an
la
te
Lu
n-4
0-p
un
jab
Ko
rea
n s
ub
ni
Gu
mg
i k
ore
an
ea
rly
Mo
rus
la
tefo
lia
ea
rly
Mo
rus
la
tefo
lia
Mo
rus
alb
a I
Ka
nm
as
i J
ap
an
ea
rly
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
rus
In
dic
a
Mo
rus
alb
a
Mo
rus
ma
co
rura
Mo
rus
nig
ra
Mo
rus
se
rra
ta
Ja
pa
n l
ate
0 .00
0.01
0.02
0.03
0.04
A c c e s s io n s
52
4.3.2 Chlorophyll B
Figure 4.6: Graphical presentation of chlorophyll b content in mulberry accessions
The highest chlorophyll b content was found in Pak-I-Punjab (0.0437±0.0001)
followed by Husang China early (0.0387±0.0000), Japan late (0.0376±0.0032) and Morus
latefolia early (0.0365±0.0001). Narc local (0.0275±0.0002), Morus alba
(0.0258±0.0004), and Pak-II-Punjab (0.0234±0.0044) showed a little bit difference. While
Ch
loro
ph
yll
b
mo
l/m
l
Pu
nja
b I
I p
un
jab
Gu
mg
i S
ril
an
ka
Ja
pa
n e
aly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
ru
s l
ate
fo
lia
Sril
an
ka
Mo
ru
s l
ate
fo
lia
La
te
hu
sa
ng
ch
ina
PF
I
Ka
nm
as
i J
ap
an
Ka
nm
as
i J
ap
an
la
te
Lu
n-4
0-p
un
jab
Ko
re
an
su
bn
i
Gu
mg
i k
ore
an
ea
rly
Mo
ru
s l
ate
fo
lia
ea
rly
Mo
ru
s l
ate
fo
lia
Mo
ru
s a
lba
I
Ka
nm
as
i J
ap
an
ea
rly
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
ru
s I
nd
ica
Mo
ru
s a
lba
Mo
ru
s m
ac
oru
ra
Mo
ru
s n
igra
Mo
ru
s s
erra
ta
Ja
pa
n l
ate
0 .00
0.01
0.02
0.03
0.04
0.05
A c c e s s io n s
53
the least chl b content was found in Morus Macroura (0.0121±0.0002), Kanmasi Japan
early (0.0116±0.0004) and Korean subni (0.0106±0.0004). The consequences confirmed
that there had been normally sorts of carotenoids (lutein and beta-carotene) inside the silk
gland and cocoon shell, a bit violaxanthin was revealed in silk gland, and the pigment
discovered in the blood changed into particularly lutein in all kinds of silkworm examined.
Chlorophyll ‘a’ and ‘b’ had no longer consumed inside the yellow-red series of silkworm.
This scheme used to hit upon noticeable dyes mentioned might be utilized to reproduce
novel colorings of shells and to increase and make use of the colorings observed in
mulberry.
Chlorophyll is one of the anti-oxidant compounds found in the green leaf parts of
the plants. Usually it is found in the green leaves, stems, roots and flowers. Usually
chlorophyll a concentration is 2-3 times greater than the chlorophyll b concentration. My
results showed some similarity with the research of Buavaroon et al. (2001).
4.3.3 Carotenoids
There are two types of carotenoids found in the gland (silk) and cocoon shell i.e.,
lutein and β-carotene.Carotenoid show a role in regulating temperature in addition to
photosynthesis, so the yellow-to-crimson regions of the leaf will likely have starch content
material decrease than the inexperienced regions but better than the regions pigmented via
anthocyanins (Deming-Adams and Adams, 1996).
The carotenoid content in Morus nigra was the highest i.e., 0.197 ±0.0 and the least
carotenoid content was in Kanmasi Japan early (0.034 ± 0.00) as well as in Morus alba
(0.0423±0.0002). According Kumar and Chauhan, (2011) the carotenoids in unique
varieties were greater in S.54 i.e. 0.1950 miligram per gram, and S.146 had 0.0190. A
54
comparable trend became clear for Total Carotenoids Content (TCC). The differences in
their readings are very noteworthy (P>0.01)
Ca
roti
no
ids
mo
l/m
l
Pu
nja
b I
I p
un
jab
Gu
mg
i S
rila
nk
a
Ja
pa
n e
aly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
rus
la
tefo
lia
Sri
lan
ka
Mo
rus
la
tefo
lia
La
te
hu
sa
ng
ch
ina
PF
I
Ka
nm
as
i J
ap
an
Ka
nm
as
i J
ap
an
la
te
Lu
n-4
0-p
un
jab
Ko
rea
n s
ub
ni
Gu
mg
i k
ore
an
ea
rly
Mo
rus
la
tefo
lia
ea
rly
Mo
rus
la
tefo
lia
Mo
rus
alb
a I
Ka
nm
as
i J
ap
an
ea
rly
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
rus
In
dic
a
Mo
rus
alb
a
Mo
rus
ma
co
rura
Mo
rus
nig
ra
Mo
rus
se
rra
ta
Ja
pa
n l
ate
0 . 0 0
0 .0 5
0 .1 0
0 .1 5
0 .2 0
0 .2 5
A c c e s s io n s
55
Figure 4.7 Carotenoids in mulberry accessions
4.3.4 Anthocyanins
Anthocyanin content relies upon on climate, place of cultivation, and is especially better in
sunny climates (Matus et al., 2009). Anthocyanin content is the highest in PFI
(0.1006±0.0002), Pak-I- Punjab (0.0952±0.0002), followed by Kanmasi japan early
(0.0926±0.0002), and Morus macroura (0.0823±0.0002). Gumgi Srilanka
(0.0153±0.0002), Morus serrata (0.0139±0.002) (0.0139±0.0002 and Husang China
(0.0116±0.0003) have the minimum amount of anthocyanins.
Ca
roti
no
ids
mo
l/m
l
Pu
nja
b I
I p
un
jab
Gu
mg
i S
rila
nk
a
Ja
pa
n e
aly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
rus
la
tefo
lia
Sri
lan
ka
Mo
rus
la
tefo
lia
La
te
hu
sa
ng
ch
ina
PF
I
Ka
nm
as
i J
ap
an
Ka
nm
as
i J
ap
an
la
te
Lu
n-4
0-p
un
jab
Ko
rea
n s
ub
ni
Gu
mg
i k
ore
an
ea
rly
Mo
rus
la
tefo
lia
ea
rly
Mo
rus
la
tefo
lia
Mo
rus
alb
a I
Ka
nm
as
i J
ap
an
ea
rly
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
rus
In
dic
a
Mo
rus
alb
a
Mo
rus
ma
co
rura
Mo
rus
nig
ra
Mo
rus
se
rra
ta
Ja
pa
n l
ate
0 . 0 0
0 .0 5
0 .1 0
0 .1 5
0 .2 0
0 .2 5
A c c e s s io n s
56
Figure 4.8: Anthocyanin content of mulberry accessions
Our results are not in line with Xiuming Liu et al. (2004), they observations in case
of mulberries fruit were 147.680 to 2725mL/L. They have crucial effects on human
wellbeing. Bae et al. (2007) endorses the presence of anthocyanin (anti-oxidant) value of
mulberry fruitlings. They have a considerable quantity of nutritionally valued secondary
metabolites. The antioxidant role of anthocyanins is attributed to their ordinary shape,
namely the oxonium ion within the C ring. Anthocyanins antioxidant were attributed to
aglycone moiety, sugar remains are at position 3, the oxidation state of the C ring (Wang
et al., 1999). Lee and Wicker (1991) suggested Cyanidin-3-glucoside and cyanidin-3-
rutinoside as imperative contents.
an
tho
cyan
ins
mo
l/m
l
Pu
nja
b I
I p
un
jab
Gu
mg
i S
rila
nk
a
Jap
an
ea
ly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
rus
la
tefo
lia
Sri
lan
ka
Mo
rus
la
tefo
lia
La
te
hu
sa
ng
ch
ina
PF
I
Ka
nm
as
i Ja
pa
n
Ka
nm
as
i Ja
pa
n l
ate
Lu
n-4
0-p
un
jab
Ko
rea
n s
ub
ni
Gu
mg
i k
ore
an
ea
rly
Mo
rus
la
tefo
lia
ea
rly
Mo
rus
la
tefo
lia
Mo
rus
alb
a I
Ka
nm
as
i Ja
pa
n e
arl
y
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
rus
In
dic
a
Mo
rus
alb
a
Mo
rus
ma
co
rura
Mo
rus
nig
ra
Mo
rus
se
rra
ta
Jap
an
la
te
0 .0 0
0 .0 5
0 .1 0
A c c e s s io n s
57
4.3.5 Determination of Phenolic
Phytochemicals rich-mulberry eliminates related show great kind of shielding and
therapeutics part in the pathogenic infection. The shielding influence is undoubtedly
because of the existence of phenolic mixes in mulberry. In pharmacology the targeted plant
has phenols particularly anthocyanins and flavonoids (Chen et al., 2006).A few others extra
phenolic mixes have been diagnosed in polyphenol-wealthy concentrate from Mulberry,
for instance, Catechin, Gallic acid derivates, protocatechuic acid, Gallo-catechin, gallate
epicatechin and Naringenin (Chan et al., 2010).
The highest phenolic content was found in Kanmasi japan late, followed by Husang
china and Japan early. The lowest content was in Japan late. Phenolic content of mulberry
leaves of thirty accessions were significantly different which shows that they had
difference from one another. The different TPC values of accessions were shown in Figure
4.9. Our results match with these results as these had a considerable difference in
values; M. alba, 1650±12.25 had maximum total phenols, while the minimum content was
noted in M. nigra, (880±7.20). M. laevigata with white large fruit and M. laevigata (large
black fruit) had prominent variability in TPC. Chinese origin mulberries showed
1515.9±5.70 (Lin and Tang, 2007) and Turkish origin mulberries had 181–1422.0 mg
GAE/100 g fw (Ercisli and Orhan, 2007), which are similar with the present study. In any
case, the lower measure of aggregate phenols was accounted for by (Okwu, 2005), and
(Khan et al., 2006). The variety of phenol mixes in organic products relies upon many
elements, for example, level of development at gather, hereditary contrasts, and natural
conditions amid natural product improvement. In red-hued fruits, phenols increment amid
the last maturing stage, because of the maximal aggregation of anthocyanins and flavonols
58
(Zadernowski et al., 2005). Phenols show extensive range of natural events (Al-Bayati
and Al-Mola, 2008) and it concluded mulberry fruits as a perfect source of these natural
constituents.
Figure 4.9: Showing total Phenolic content of mulberry accessions
4.3.6 Antioxidant potential
Prior research has shown the strong anti-oxidant activity in extracts of mulberry
(Zhang et al., 2008). Normally, strong hydroxyl and DPPH activities are found in extract
of unique fragments of Toot (Khan et al., 2013;Katsube et al., 2006). Due to the fact that
A c c e s s io n s
To
tal
Ph
en
oli
c C
on
ten
ts
Pu
nja
b I
I p
un
jab
Gu
mg
i S
rila
nk
a
Ja
pa
n e
aly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
rus l
ate
folia
Sri
lan
ka
Mo
rus l
ate
folia
La
te
hu
sa
ng
ch
ina
PF
I
Ka
nm
asi
Ja
pa
n
Ka
nm
asi
Ja
pa
n l
ate
Lu
n-4
0-p
un
jab
Ko
rea
n s
ub
ni
Gu
mg
i k
ore
an
ea
rly
Mo
rus l
ate
folia
ea
rly
Mo
rus l
ate
folia
Mo
rus a
lba
I
Ka
nm
asi
Ja
pa
n e
arl
y
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
rus I
nd
ica
Mo
rus a
lba
Mo
rus m
ac
oru
ra
Mo
rus n
igra
Mo
rus s
err
ata
Ja
pa
n l
ate
0
1
2
3
4
5
59
structural functions of phenolic compounds are liable for the anti-oxidant pastime, the
antioxidant activities of the examined mulberry leaves can be associated with their overall
phenol content and the underlying mechanism advised as explaining the neuroprotective
effect of mulberries polyphenolic compounds may be mainly summarized as scavenging
intracellular ROS and inhibition of LDL oxidation. Doi et al., (2001) suggested the
inhibition of oxidational alteration of human and rabbit, as DPPH was scavenged by 1-
butaol.
IC
50
va
lue
s
Pu
nja
b I
I p
un
jab
Gu
mg
i S
rila
nk
a
Ja
pa
n e
aly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
ru
s l
ate
folia
Srila
nk
a
Mo
ru
s l
ate
folia
La
te
hu
sa
ng
ch
ina
PF
I
Ka
nm
asi
Ja
pa
n
Ka
nm
asi
Ja
pa
n l
ate
Lu
n-4
0-p
un
jab
Ko
re
an
su
bn
i
Gu
mg
i k
ore
an
ea
rly
Mo
ru
s l
ate
folia
ea
rly
Mo
ru
s l
ate
folia
Mo
ru
s a
lba
I
Ka
nm
asi
Ja
pa
n e
arly
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
ru
s I
nd
ica
Mo
ru
s a
lba
Mo
ru
s m
ac
oru
ra
Mo
ru
s n
igra
Mo
ru
s s
erra
ta
Ja
pa
n l
ate
0
5 0
1 0 0
1 5 0
2 0 0
A c c e s s io n s
60
Figure 4.10: Showing IC50 values of Mulberry accessions indicating the maximum and
minimum significant and non-significant data
The maximum IC50value was of Morus indica (172.2 µg/mL) followed by Morus alba
(152.7 µg/mL) and PFI (122.3 µg/mL). Punjab II Punjab, Kanmasi Japan and NARC
showed the minimum and same amount of anti-oxidant activity. Khan et al. (2013) found
the high regression and correlation values for phenolic and antioxidant promises of the
extracts, therefore the toot (Mulberry) can scavenge more effectively. As a result it acts as
a good source of plant derived medicines.
4.3.7 Determination of Mineral Content
Minerals are found in fruit as well as leaves of mulberry plants. The main minerals of
mulberry leaves are K followed by Na, P and Mg.
4.3.8 Potassium
Potassium concentration was the highest in Morus alba, Punjab II and Morus
macroura and found the lowest concentration in Morus latefolia Srilanka. Our results
conflicts with that of (Ercisli and Orhan, 2007) they showed the mineral concentrations for
the fruits and the mean values of K, Na, P, Mg, Ca, Fe, Mn, Zn and Cu were 1041 mg/100g,
277 mg/100g, 170 mg/100g, 128 mg/100g, 32 mg/100g, 7.07 mg/100g, 5.63 mg/100g, 3.02
mg/100g, and 0.34 mg/100g, respectively. They also found higher P, K, Ca and Mn
contents. These differences may be due to ecological factors, growing conditions and
genetic factors. Mineral nutrition of plant is controlled by environment, soil and plant
factors (Marschner, 2011). A research conducted on mulberry fruits in Antalya by Özdemir
and Topuz (1998) stated that the main minerals of black mulberry fruits were K, Ca, P and
Mg.. Since the uptake of nutrients from the soil is genetically controlled, plant species and
61
varieties show different response to nutrients evenwhen they are grown in the same
conditions (Ershadi and Talaie, 2000;Erdal and Baydar, 2005; Küçükyumuk, 2007).
Figure 4.11: Representing potassium found in different accessions
4.3.9 Sodium
Sodium was the highest in concentration in Kanmasi Japan leaves followed by
Punjab II and Morus serrata. The concentration of sodium was found significantly different
among different mulberry accessions. In the study conducted by Kitahara et al. (2002), it
was pointed out that the mineral patters of leaf samples mostly consist of Ca, K, and Mg.
po
tass
ium
(m
g/g
)
Pu
nja
b I
I p
un
jab
Gu
mg
i S
ril
an
ka
Ja
pa
n e
aly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
ru
s l
ate
foli
a S
ril
an
ka
Mo
ru
s l
ate
foli
a L
ate
hu
sa
ng
ch
ina
PF
I
Ka
nm
as
i J
ap
an
Ka
nm
as
i J
ap
an
la
te
Lu
n-
40
-p
un
jab
Ko
re
an
su
bn
i
Gu
mg
i k
ore
an
ea
rly
Mo
ru
s l
ate
foli
a e
arly
Mo
ru
s l
ate
foli
a
Mo
ru
s a
lba
I
Ka
nm
as
i J
ap
an
ea
rly
Pa
k I
I p
un
jab
Hu
sa
ng
ch
ina
ea
rly
Hu
sa
ng
ch
ina
la
te
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
ru
s I
nd
ica
Mo
ru
s a
lba
Mo
ru
s m
ac
oru
ra
Mo
ru
s n
igra
Mo
ru
s s
erra
ta
Ja
pa
n l
ate
0
5 0
1 0 0
A c c e s s io n s
62
The mineral composition of leaves depended no longer only at the kinds, but also on the
growing conditions, including soil and geographical situations on this examine, Na was
most important, followed by way of Ca, k, Mg, P, Mn, Fe, Zn and Cu. The leaf nutrient
composition of decided on mulberry trees cultivars became 0.28 percent of P, 1.68 percent
of k and 0.15 percent of Na. The mulberry tree appears to be additionally an excellent
fodder in view that its nutrient contents are higher than the ones of alfalfa which had been
from 0.2 to 0.35 percent for P, 1.2 to 2.3 percent for k and 0.06 to 0.23 percent for Na
(Mauriès, 2003). The blackberry foliage had nice interplay with oats hay on fiber
digestibility, aspect which alfalfa lacks. This shows that mulberry timber can be hired as
sources of fodder or protein (Doran et al., 2007).
63
Figure 4.12: Showing sodium concentrations in Morus accessions
Black mulberry fruits which are cultivated in Isparta than other regions have rich
nutrition elements. For this reason they are important raw material for food technology
production (jam, marmalade, paste, pulp, jelly, juice, pekmez, etc.). Their leaves have also
rich nutrition elements like their fruits. Leaves can be used for animal nutrition, tea
production and they can be used for various purposes. Kara dut leaves which are cultivated
in Isparta region are very important for the reasons above. However, in order to produce
So
diu
m (
mg
/g)
Pu
nja
b I
I p
un
jab
Gu
mg
i S
rila
nk
a
Jap
an
ea
ly
Pa
k I
pu
nja
b
Pu
nja
b I
Mo
rus
late
folia
Sri
lan
ka
Mo
rus
late
folia
La
te
hu
san
g c
hin
a
PF
I
Ka
nm
asi
Ja
pa
n
Ka
nm
asi
Ja
pa
n l
ate
Lu
n-4
0-p
un
jab
Ko
rea
n s
ub
ni
Gu
mg
i k
ore
an
ea
rly
Mo
rus
late
folia
ea
rly
Mo
rus
late
folia
Mo
rus
alb
a I
Ka
nm
asi
Ja
pa
n e
arl
y
Pa
k I
I p
un
jab
Hu
san
g c
hin
a e
arl
y
Hu
san
g c
hin
a l
ate
NA
RC
lo
ca
l
Gu
mg
i k
ore
an
la
te
Pu
nja
b I
I
Mo
rus
Ind
ica
Mo
rus
alb
a
Mo
rus
ma
co
rura
Mo
rus
nig
ra
Mo
rus
serr
ata
Jap
an
la
te
0
2
4
6
A c c e s s io n s
64
certain crops from black mulberry industrially, relevant researches on this purpose should
be conducted and orchards with appropriate varieties should be set up.
4.4 THE MORUS CUC2 GENES
A draft genome sequence of the Morus notabilis C.K.Schneid. (Chuansang) was
published in 2013 and is available at the Morus genome wide database website (MorusDB;
http://morus.swu.edu.cn/morusdb/). A search of MorusDB with the Arabidopsis thaliana
CUC2 gene (AtCUC2; At5g53950) identified three distinct gene sequences, designated
CUC2a/b (scaffold 692), CUC2c (scaffold 1197), and CUC2d (scaffold 897), which
exhibit, respectively, 70%, 78% and 77% sequence identity with AtCUC2. Also, the
similarity between the three cuc2 genes is also shown in the below table.
Table 4.6: Maximum identity between CUC2 a, CUC2 c and CUC2d genes
Percent Identity
CUC2c CUC2a CUC2d
CUC2c - 51.9 65.7
CUC2a 78.7 - 52.2
CUC2d 46.3 77.4 -
The above table shows that the maximum percent identity was found between CUC2a and
CUC2c and minimum in CUC2c and CUC2d. In addition to this sequence similarity, the
Morus CUC2 genes and the AtCUC2 gene have similar overall structures, and consist of
three exons interrupted by two introns, as shown in Figure 4.13. The primary difference
between the two species is in the lengths of the exons and introns (Table 4.7).
65
Figure 4.13 Gene structures of Arabidopsis thaliana and Morus notabilisshowingthe
location of primers on the gene. http://morus.swu.edu.cn/cgi-
bin/gb2/gbrowse_details/mulberry?ref=scaffold 1147
The above figure showed the CUC2 gene structure of Arabidopsis thaliana along
with Morus notabilis. According to this gene structure, there were three exons and two
introns in the M. notabilis CUC2 gene and the position of primers were shown on the gene
length.
Table 4.7 Exon and intron lengths in Morus CUC2c genes
Accession Exon 1 (bp) Intron 1 (bp) Exon 2 (bp) Intron 2 (bp) Exon 3 (bp)
M.notabilis 185 121 278 124 605
MI 185 122 278 140 605
ML 185 121 278 124 605
HC 185 119 278 128 609
MN 185 125 278 128 609
66
MS 185 126 278 128 609
N 185 127 278 130 605
LUN 40 187 124 278 131 605
The M. notabilis CUC2 genomic sequences were used to design oligonucleotide
primers for amplification of the corresponding genomic sequences from seven strains
belonging to four Morus species, namely M. indica, M. latefolia, M. nigra, and M. alba
(see Materials and Methods). Only the CUC2c primers gave consistent amplification and
produced PCR products of the expected size in all samples analyzed. However, several
attempts at direct sequencing of gel-purified CUC2c PCR products either failed or
produced sequences of poor quality, possibly because the primers were annealing to other
genomic sequences. Consequently, a strategy of cloning the PCR products into the
pGEM®-T Easy vector prior to sequencing of cloned inserts was employed, as described
in materials and methods. This strategy produced good-quality sequences, which were
analyzed and compared to each other and to the CUC2c sequence of the reference M.
notabilis strain.
Figure 4.14: The amino acid sequence showed the conserved No Apical Meristem (NAM)
domain
67
67
The NAM domain is shown in the box. The asterisk showed the changes in the
amino acid sequences of Morus species compared with the Morus notabilis. We have found
protein sequences and there are only a few amino acids that were changes in all the
sequences as compared with Morus notabilis sequence.
4.5 PHYLOGENETIC ANALYSIS
Maximum Likelihood method based on the Tamura-Nei model was used to find out
the evolutionary history (Tamura and Nei, 1993). The highest log likelihood (-1830.93)
tree is shown. Initially Neighbor-Join and BioNJ algorithms are used for getting the initial
trees. The matrix of pairwise distances estimated using the Maximum Composite
Likelihood approach.
Figure 4.15: Showing molecular Phylogenetic analysis by Maximum Likelihood method
between the eight Morus strains
68
The tree is drawn to scale, with branch lengths measured in the number of
substitutions per site. Evolutionary analyses were conducted in MEGA7. According to the
above phylogenetic tree there were two main groups. The one only had Lun 40 punjab
showed 0.000 percent similarity to any other strain. While the other group was sub divided
into two sub groups. A phylogenetic tree was also constructed for the examine traces and
it was determined that there have been predominant clusters. First contained the Morus
Latefolia (Ml) in the separate clade and confirmed 100% similarity with the Morus
notabilis. The second one cluster was divided into sub-clusters and four further sub clusters
on the idea of similarities. Morus indica, Husang china and Morus latefolia and Morus
notabilis stood in one group.
4.6 EVOLUTIONARY DIVERGENCE AMONG SEQUENCES
Figure 4.16: Evolutionary divergence among eight strains of Morus
In order to find out genetic diversification amongst selected mulberry traits,
analyses had been carried out using the maximum Composite likelihood model (Tamura et
al., 2004) for CUC2 gene. The analysis involved eight nucleotide sequences and all the
positions containing gaps and missing information had been removed. The analysis was
done in MEGA7 (Tamura et al., 2011). In figure 4.16 evolutionary divergences amongst
sequences is shown. Estimates of evolutionary divergence values vary from a decrease
69
restriction of 0.034 to 0.012.The mean distance found in overall sequence pairs was 0.019
which showed low levels of diversity in selected Morus varieties.
4.7 ESTIMATE OF NUCLEOTIDE COMPOSITION
The genetic relationship between the nucleotides sequences of considered strains
were obtained by using MEGA 7 for cuc2 gene. It was found that the average of T (U) was
25.5, C was 21.0, A was 31.6, G was 21.9 and the overall aggregate of nucleotides was
1072.6. Showing that A was the highest and C was the lowest (Table 4.8)
Table 4.8 Nucleotide composition of sequences
T(U) C A G Total
Lun 40 Punjab 25.5 22.1 30.5 21.8 1071.0
Morus indica 25.3 20.7 32.2 21.9 1073.0
Morus latefolia 25.6 20.4 31.9 22.1 1071.0
Morus notabilis.seq 25.7 20.3 31.9 22.1 1074.0
Morus serrata 25.5 20.7 31.9 21.9 1074.0
Narc 25.7 20.8 31.9 21.6 1074.0
Morus nigra 25.5 22.1 30.5 21.8 1071.0
Husang china 25.3 20.7 32.2 21.9 1073.0
Avg. 25.5 21.0 31.6 21.9 1072.6
4.8 TAJIMA’S NEUTRALITY VALUES
To estimate the nucleotide diversity of CUC2 gene among selected Morus strains,
Tajima’s neutrality test was performed which showed nucleotide diversity for all eight
strains (Table 4.9). The data scores indicated a low diversity among sampled Pakistani
Morus varieties on this basis of neutrality values of Tajima’s test.
70
Table 4.9: Values for Tajima’s test
M S ps Ɵ Π D
8 47 0.043925 0.016941 0.018858 0.608134
Abbreviations: m= number of sequences, S= Number of segregating sites, Ps= sim, Ɵ =
Ps/a1, π= nucleotide diversity and D is the Tajima test statistics
While the proteins in NAC family are concerned in numerous procedures (Olsen et
al., 2005), mostly a few of them had been characterized. One of the pleasant characterized
features of them is defining organs throughout early, flowery and somatic development
(Souer et al., 1996; Aida et al., 1997; Vroemen et al., 2003; Mitsuda et al., 2005). They
are additionally concerned with signaling of abscisic acid and auxin (Xie et al., 2000; Aida
et al., 2002) and plant responses to abiotic and biotic strain (Xie et al., 1999; He et al.,
2005; Hu et al., 2006). Newest stories told the NAC, genes also are concerned in law of
senescence (Guo and Gan, 2006; Uauy et al., 2006). But, no NAC gene has been
pronounced to be concerned in lateral shoot branching so far.
Arabidopsis leaves are simple with small serrations on their margins. Whilst CUC2
mutants produce leaves with smooth margins, flowers with extended CUC2 expression due
to faulty miR164 regulation display deeper and larger serrations than the wild kind
(Nikovics et al., 2006). CUC3 mutants additionally display reduced serrations, at the same
time as CUC1, which is not always expressed in leaves, performs no position in
Arabidopsis leaf development. While CUC2 acts early on with the onset of teeth, CUC3 is
concept to act simplest at later levels to maintain tooth outgrowth (Hasson et al., 2011).
Apparently, chimeric constructs, wherein the CUC2 promoter drives the expression of
CUC1 rescue regular leaf serration in CUC2 mutants, additionally set off leaflet formation
in genetic backgrounds missing miR164.
71
Those consequences display that, despite the fact that CUC1 isn't expressed in
developing leaves, the CUC1 protein is partly feature best friend interchangeable with
CUC2.
In species with compound leaves the feature of NAM/CUC3 genes is extended to
specify the boundaries amongst leaflets. Really, the genes are expressed at the constraints
of leaflet primordia, and their inactivation results in fused and less leaflets (Berger et al.,
2009; Blein et al., 2008; Cheng et al., 2012; Wang et al., 2013). Alternatively, tomato
plants expressing the advantage of function miR164-resistant allele Gob4-d produce deeply
lobed leaflets (Berger et al., 2009). This information are suggestive of a conservation of
the mechanisms controlling boundary specification most of the apex and leaf primordia
with one-of-a-kind architectures. It targeted on NAM/CUC3 mutant phenotypes and
highlighted the proper expression styles of these genes in enhancement. NAM/CUC3 genes
are expressed in narrow and discontinuous domain names, frequently restricted to a few
cells on the boundary among outgrowing systems. Regulation of this expression pattern is
crucial for correct organ development as CUC overexpression results in extreme
phenotypes (Hibara et al., 2006; Laufs et al., 2004). CUC2 uniformly expressed throughout
the leaf margin in preference to its discrete expression pattern on the tooth sinuses, a
smooth leaf margin is shaped in vicinity of the typical serrated shape (Bilsborough et al.,
2011). Live imaging experiments endorse that CUC2 expression is down regulated in
tissues where convergent PIN1 polarities’ are predicted to gather excessive auxin tiers
(Heisler et al., 2005). Communally, these upshots counsel that CUC2 expression inside the
SAM is inhibited via PIN1-generated auxin activity maxima. CUC2 genes redundantly
promote axillary meristem formation (Raman et al., 2008). Two articles propose that an
72
auxin minimal is required for axillary meristem formations in Arabidopsis and tomato
(Wang et al., 2014). Even though this has no longer been examined, this auxin minimal
should permit CUC expression therefore inducing axillary meristem formation. Thus, in
Arabidopsis, MIR164A regulates the extent of CUC2 expression, which in flip governs the
extent of leaf serration.
Apparently, quantitative trait locus (QTL) mapping has discovered a unmarried
nucleotide polymorphism in MIR164A miRNA* which modifies MI-R164A biogenesis
and considerably reduces its accumulation (Todesco et al., 2012). Leaf improvement
constitutes a first rate version to take a look at cell parameters managed with the aid of
CUC genes. Through analogy with cellular mechanisms going on at lateral organ primordia
barriers, CUC2 has been recommended to restriction boom of sinuses on the leaf margin
(Nikovics et al., 2006). In comparison, CUC2 promotes teeth outgrowth via a non-mobile-
self-reliant pathway regarding auxin (Bilsborough et al., 2011; Kawamura et al., 2010).
Those opposing outcomes spotlight the fact that CUCs manage cellular proliferation in
exclusive approaches to permit differential growth.
4.9 MOLECULAR CHARACTERIZATION
Genomic DNA of thirty accessions was extracted by CTAB method given by Richards
(1997) (Figure4.17).
Figure 4.17: DNA of sampled varities
Genomic DNA of thirty accessions were accrued from Muzaffarabad was extracted with
the aid of CTAB technique Richards (1997) (Figure 4.17). The best of extracted DNA
quantity were checked with the aid of running it on 1.5 percent gel. Concentration of
73
DNA was decided with the aid of spectrophotometer. The ten SSR and twelve ISSR
markers detail along with PCR conditions were elaborated in methodology section. PCR
results of all markers used are given in the annexure 1.
4.9.1 Statistical Tools Used in Analysis
The data was in 0 and 1 form and were analyzed by the following software were
utilized in analysis Bio conductor implementation in R, NTSYS Version 2.2, Minitab 17,
Statistic and Statista.
4.9.2 Simple Sequence Repeats (SSR)
Table 4.10: Grouping of accession on the basis of SSR analysis
Analysis of selected material on the basis of ISSR revealed two main groups A and
B separated from each other at 16 genetic distance (appendix 1) Group A was having two
cluster A-1 and A-2 separated from each other at genetic distance 9 with former having
Groups Cluster Accession in each cluster
A A-1 5, 6, 2, 1, 3, 4
A-2 7, 8, 9, 10, 11, 12, 13, 14
B B-1 25, 23, 24, 29, 30, 28, 26, 27
B-2 21, 22, 18, 19, 20, 17, 15, 16
74
accessions 5, 6, 2, 1, 3 and 4 while later constituted of accessions 7, 8, 9, 10, 11, 12, 13 and
14.
Group B was also further divided in to two cluster B-1 and B-2 with genetic
distance of 9.5. Cluster B-1 was having the highest number of accessions than group B-2.
Cluster B-1 contained 25, 23, 24, 29, 30, 28, 26 and 27accessions while cluster B-2 was
having 21, 22, 18, 19, 20, 17, 15 and 16 accessions.More over percent polymorphism was
also less when compared with later. Highest numbers of bands were recorded by SS18 with
a total of 7 bands along with 100 % polymorphism. PIC was also highest for this marker.
While marker SS09, MulSTR2, MulSTR5, SS05, Mul6 and MulSTR4 had no
polymorphism. Moreover PIC values of SSR are also a bit less than ISSR but that too is of
whole marker rather than its allele’s frequency.
4.9.3 Genetic Dissimilarity Coefficient
Comparing the accession by genetic dissimilarity coefficient (gdc) revealed that
highest level of dissimilarity existed in accession 1 and 30 i.e 29.66 (annexure 2) genetic
dissimilarity coefficient). While, there was 28.67 gdc between accession 1 and 29.
Accession 2 and 30 were third highest gdc with 28.66 gdc level. Similarly the lowest gdc
of all accession was in 4 with 5 and 8 with 9 (1.44 gdc level). Second lowest gdc i.e 1.76
was observed in the accessions 13 with 14, 26 with 27, 27 with 28 and 29 with 30.
Accessions 5 with 6, 9 with 10 and 28 with 29 were having third lowest gdc of 2.03 in all
the accessions.
Table 4.11: Analysis of Molecular Variance (SSR)
AMOVA
SSD MSD Df sigma2 P.value
SSR -18.28462 -18.284615 1 1.1308 0.6436
Error 107.55128 3.841117 28 3.8411
75
Total 89.26667 3.078161 29
CV 19.57
AMOVA were same as ISSR as they showed non-significant banding patterns of
all the markers as compare to each other’s. We were able to find genetic diversity but non
significance can be due to close relation of markers and not considering alleles of each
marker gene. More over P value of SSR is much higher than ISSR making ISSR first
choice SSR.
Figure 4.18: Principal Component analyses for Morus traits
PCA analysis of SSR revealed diversification of accession along with random
pattern moreover they were coordination shows that these accession will have same least
genetic base (similarity) if compared to ISSR genetic. In linkage tree there will be linked
far away than ISSR.
76
Table 4.13: Grouping of accession on the basis of ISSR analysis
Groups Cluster Accession in each cluster
A
A-1 19,20,23,21,22
A-2 29,30,24,25,28,26,27
B
B-1 16, 17, 18, 11, 12, 15, 13, 14
B-2 7, 10, 8, 9, 6, 4, 5, 3, 1, 2
Analysis of selected material on the basis of ISSR revealed two main groups A and
B separated from each other at 15.5 genetic distances (annexure 2 dendrogram). Group A
was having two cluster A-1 and A-2 separated from each other at genetic distance 6 with
former having accessions 19, 20, 23, 21 and 22 while later constituted of accessions 29,
30, 24, 25, 28, 26 and 27.
Group B was also further divided in to two cluster B-1 and B-2 with genetic
distance of 9. Cluster B-1 was having highest number of accessions than group B-2.
Cluster B-1 contained 16, 17, 18, 11, 12, 15, 13, and 14 accessions while cluster B-2 was
having 7, 10, 8, 9, 6, 4, 5, 3, 1, and 2 accessions.
4.9.4 Genetic dissimilarity coefficient (ISSR)
Comparing the accession by genetic dissimilarity coefficient (gdc) revealed that
highest level of dissimilarity existed in accession 1 and 30 i.e,29.76 (appendix 1 genetic
dissimilarity coefficient). While there was 28.77 gdc between accession 1 and 29.
Accession 2 and 30 were third highest gdc with 28.73 gdc level. Similarly the lowest gdc
of all accession was in 13 and 14 (2.85 gdc level). Second lowest gdc i.e. 3.02 was observed
in the accessions 26 and 27. Accessions 29 and 30 were having third lowest gdc of 3.34 in
all the accessions.
Table 4.13: Amova showing differences in ISSR
77
AMOVA
SSD MSD Df sigma2 P.value
G -6702.29 6702.287 1 231.39 0.1089
Error 2674.385 396.0745 32 396.07
Total 5972.098 180.9727 33
CV 35.7
AMOVA showed non-significant banding patterns of all the markers as compare to
each other’s. We were able to find genetic diversity but non significance can be due to
close relation of markers and not considering alleles of each marker gene
Figure 4.19: PCA for ISSR
78
PCA analysis revealed diversification of accession but the dimension was not in
random pattern moreover they were a bit of chance that they will have same genetic base
if compared linkage tree on ISSR bases.
Table 4.14: Markers (ISSR), its total band and polymorphism
S. No MARKER TOTAL
BANDS
POLYMORPHIC % POLYMORPHISM PIC
1 UBC 827 6 6 100 0.991
2 UBC 820 5 5 100 0.992
3 UBC 841 4 4 100 0.993
4 UBC 858 5 5 100 0.990
5 UBC 828 5 1 20 0.974
6 IS 15 7 7 100 0.992
7 IS 21 6 6 100 0.991
8 UBC 809 8 8 100 0.995
9 UBC 864 7 7 100 0.992
10 UBC 826 5 5 100 0.992
11 IS 17 4 4 100 0.996
ISSR results showed that all the marker were highly polymorphic with high pic
value. This is because of more number of bands per marker which forced to presence in
almost all the accessions. Highest number of bands were observed in IS 15 (7 bands) along
with high % polymorphism but pic was not so high for it. Highest PIC was observed in IS
17 i.e. 0.996. Marker UBC 828 was least polymorphic of all with 20 %. More over PIC
can’t be related with % polymorphism as it’s only based on just marker presence but not
its alleles.
Results given by Galvan et al. (2003) showed that there was a prominent difference
among the Ficus ecotypes. He proposed ISSR are better than RAPD phylogeny
79
study.Similar reflection was from Kafkas et al. (2009) results in hazel nut where the
UPGMA dendrogram made 2 main groups and three by PCA.
The present study revealed high overall genetic diversity and a significant amount
of differentiation. So it appears that genetic diversity is maintained because migration and
genetic differentiation both are present and balancing each other. It was revealed from
Andrews (2010) that balanced collection instead of steering selection sustains the
polymorphism in an environment. Instead of all this M alba sustain extra ordinary diversity
than the same studies on other trees (Suarez-Montes et al. 2011; Melendez-Ackerman et
al. 2005). Tika khan et al., (2014) showed same results in their study for Berberis species.
80
SUMMARY
The morphological data showed that the accessions are different in certain features. There
was a difference in qualitative as well as quantitative characters. Different morphological
and biochemical parameters were studied along with DNA based markers for checking the
diversity. According to morphology among the leaves of thirty accessions majority had
light green color, acute tip, and soft surface having cordate shaped leaves. The others had
lobed shape, dark color, doubly serrate or dentate, acuminate and coarse leaves. There were
significant differences in means of lamina length, lamina width and leaf area index. In
biochemical analysis, the carbohydrates content was the highest in Korean subni followed
by Morus nigra and Morus latefolia. The greater values of starch and protein content
indicated that the leaves had the more palatability for silkworms. The highest total phenolic
content was in Lun 40 punjab. Phenolic play a key role in traditional medicines. CUC2
gene study was also done which conferred there were some changes in the amino acid
sequences responsible for different leaf shapes. Further it was suggested to use more
primers for studying this gene for making better results. Molecular diversity between thirty
accessions was checked through DNA markers i.e. SSR and ISSRs by the use of different
software. The SSR produced 15 polymorphic bands out of 25 (60%) SS18 and MULSTR1
showed best results with highest PIC and 100 percent polymorphism while the least was
shown by SS04. It was also obvious that PIC of SSR was somehow less than ISSR. The
AMOVA gave P value of 0.6434. The UPGMA dendrogram divided samples in to two
main groups. ISSR showed all the markers were highly polymorphic with more pic. IS17
had highest polymorphism and UBC with least 20 percent polymorphism. P value was
81
0.1089 for this while CV was 35.7. Our diversity results proved that all group were
somewhat distant.
82
CONCLUSIONS AND RECOMMENDATIONS
This mulberry has been studied for the first time in AJK and yet the diversity was found in
cultivated and wild species. Genetic distance was very high making these accession totally
apart but that too cannot be accurate as number of marker studied are not too much as
required for the genetic diversity analysis. PIC value of the selected marker is very high as
the present work was on diversity so whole marker presence was kept in touch rather than
each of the alleles PIC (need more accuracy if using). Number of markers must be increased
to the required minimum level for diversity. More over trait specific markers must be
included for accuracy. Experiment must be repeated several times to reduce errors. Data
must also be compared with world to see the origin of the accessions. More over search for
more number of indigenous accessions are required as 30 accessions are way too much less
for diversity yet it can give the base line for future as its first of its type. If possible they
must be comparison with wild relative for evaluation studies. More over if all the alleles
are identified with accuracy then PIC of each the markers (sub-units) must be used
otherwise whole markers must be considered as selection or analysis criteria. Apart from
it our study highly suggests that these markers (both SSR and ISSR) must be used in genetic
diversity detection sets of mulberry plant as they were efficient in detecting diversity.
Inclusion of these in whole sets of primers will help breeders to develop genetic relation
for developing variety or other analysis.
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Annexure 1
Gels representing the results of different markers
UBC 827
Ubc 841
113
UBC 841
UBC 858
UBC 826
IS15
114
IS17
UBC 854
UBC 864
UBC 826
SS04
115
SS18
MUL STR6
SS09
MULSTR 2
MULSTR1
116
MULSTR5
SS05
ANNEXURE II
117
SSR dendrogram
DENDROGRAM FOR ISSR
Punjab II punjabGumgi SrilankaJapan ealyPak I punjabPunjab I Morus latefolia SrilankaMorus latefolia LateHusang chinaPFI Kanmasi JapanKanmasi Japan lateLun-40-punjabKorean subniGumgi korean earlyMorus latefolia earlyMorus latefoliaMorus alba IKanmasi Japan earlyPak II punjabHusang china earlyHusang china lateNARC localGumgi korean latePunjab II Morus IndicaMorus albaMorus macoruraMorus nigra
Punjab II punjab
Gumgi Srilanka1.73727
Japan ealy 0.85692 1.91704
Pak I punjab 0.77508 1.5702 0.84513
Punjab I 0.97314 1.18366 0.98843 1.01336
Morus latefolia Srilanka0.87455 1.54139 1.16976 0.93444 1.08378
Morus latefolia Late1.29784 1.72397 0.99958 0.9633 0.94265 1.22112
Husang china1.11718 1.31349 1.20788 1.05606 0.73582 0.92354 1.16221
PFI 0.95254 1.30407 0.84376 0.78468 0.52422 1.05907 0.82013 0.97693
Kanmasi Japan1.08529 1.71623 1.09522 0.74477 1.2355 1.44965 1.3045 1.411 0.96
Kanmasi Japan late0.75868 1.61988 0.97514 0.83648 1.12597 0.97335 1.38413 1.2156 0.89 0.83257
Lun-40-punjab1.32616 1.60102 1.21303 0.84841 1.12094 1.34882 0.68424 1.24792 0.92 1.08137 1.24125
Korean subni 1.2462 1.5722 1.1632 0.97493 0.97215 1.40357 0.64708 1.36935 0.84 1.13593 1.31801 0.57768
Gumgi korean early1.46182 1.57626 1.18315 1.1557 1.11737 1.55027 0.86589 1.51561 1 1.25773 1.424 0.85522 0.53267
Morus latefolia early1.28015 1.63379 1.06333 1.13467 0.96691 1.48068 1.3806 1.09579 0.96 1.00368 1.12394 1.41052 1.44352 1.43811
Morus latefolia1.01344 1.50708 0.85908 0.9506 0.86805 1.36662 1.22415 1.1187 0.89 1.11446 1.11137 1.15836 1.05883 0.98678 1.03799
Morus alba I 1.05306 1.44898 1.32752 0.8435 1.02435 0.94586 1.25769 0.76996 0.94 1.10857 0.95837 1.11712 1.3714 1.64229 1.13773 1.3466
Kanmasi Japan early1.11884 1.18564 1.04924 0.9343 0.69079 1.07097 1.06284 0.93256 0.45 1.14152 0.88977 1.00321 1.09517 1.1848 0.9817 0.95817 0.86593
Pak II punjab 1.05475 1.50023 0.97971 1.04823 0.84721 1.23033 1.31728 0.76097 0.93 1.14372 0.92212 1.22251 1.34623 1.38393 0.75367 0.75779 0.97328 0.80202
Husang china early1.15 1.33374 1.20107 1.35769 0.61831 1.20456 1.39079 0.71094 1.03 1.60847 1.36259 1.57797 1.46553 1.50387 1.14567 0.95948 1.29228 1.05079 0.85653
Husang china late1.08054 1.39331 1.20764 1.13639 0.78965 1.20113 1.14421 1.11205 0.8 1.41359 1.10804 1.02413 1.01279 1.20179 1.30898 1.00011 1.10804 0.66354 0.96071 1.11692
NARC local 1.44697 1.5152 1.31088 1.2986 0.92159 1.57001 1.03191 1.16367 0.95 1.41354 1.38674 0.83939 0.88769 1.00516 1.30258 0.96652 1.30975 0.89922 0.95135 1.22124 0.73099
Gumgi korean late1.19096 1.18172 1.48324 1.17425 0.91409 1.1474 1.15013 1.15577 1 1.47873 1.35772 1.11093 0.89103 1.08184 1.62746 1.11884 1.28315 1.09628 1.34851 1.16045 0.96172 1.08572
Punjab II 1.39652 1.24019 1.50007 1.21611 0.8899 1.16592 0.90519 1.12707 0.9 1.53987 1.52668 1.13644 0.97859 1.23084 1.63553 1.44313 1.23108 1.10867 1.54161 1.2775 1.23633 1.29354 0.75972
Morus Indica 1.45823 1.2455 1.51504 1.11718 0.9715 1.36927 1.03754 1.12422 0.93 1.5128 1.54652 0.95078 1.08137 1.36748 1.52644 1.37096 1.03992 0.91534 1.38667 1.40523 0.97358 1.0935 1.10346 0.9002
Morus alba 1.63847 1.3976 1.65006 1.34567 1.12271 1.42331 1.15794 1.15794 1.07 1.73364 1.6571 1.14378 1.34491 1.62108 1.6648 1.60439 1.07922 0.97498 1.47827 1.49204 1.10012 1.20315 1.31471 1.00337 0.43203
Morus macorura1.6257 1.54838 1.63599 1.24319 1.21433 1.53431 1.02367 1.26231 1.11 1.56144 1.62352 0.78445 1.08131 1.41985 1.67022 1.51828 1.09339 1.06806 1.45624 1.64198 1.07117 0.98284 1.23087 1.07262 0.52033 0.54675
Morus nigra 1.50686 1.29842 1.47115 1.15821 0.97577 1.3246 0.93036 1.10956 0.86 1.36108 1.34545 0.75728 0.94046 1.11927 1.28525 1.3104 0.99585 0.74888 1.18175 1.40816 0.84776 0.7933 1.04929 1.00955 0.79059 0.86927 0.71989
Morus serrata 1.5782 1.44911 1.4403 1.33099 1.11674 1.72762 1.27064 1.41295 0.88 1.20663 1.30001 1.0272 1.11575 1.24532 1.36245 1.22067 1.30998 0.86132 1.19127 1.50576 1.11248 0.84501 1.33629 1.32159 1.1654 1.21581 1.08301 1.01347
Japan late 1.41325 1.35244 1.23954 1.19414 0.88437 1.52495 1.10654 1.26594 0.6 1.16765 1.17095 0.9946 1.03202 1.15249 1.184 1.11144 1.18648 0.58845 1.07569 1.32041 0.91546 0.81272 1.24378 1.17233 1.01152 1.06082 1.03589 0.87368
118