genetic characterization of mtdna from saraiki …

GENETIC CHARACTERIZATION OF mtDNA

FROM SARAIKI POPULATION OF

PAKISTAN

SIKANDAR HAYAT

DEPARTMENT OF ZOOLOGY

UNIVERSITY OF THE PUNJAB

LAHORE, PAKISTAN

(2014)

GENETIC CHARACTERIZATION OF mtDNA

FROM SARAIKI POPULATION OF

PAKISTAN

A THESIS SUBMITTED TO

THE UNIVERSITY OF THE PUNJAB

IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS

FOR THE DEGREE OF DOCTOR OF

PHILOSOPHY

IN

ZOOLOGY

BY

SIKANDAR HAYAT

SUPERVISOR:

PROF. DR. TANVEER AKHTAR

DEPARTMENT OF ZOOLOGY

UNIVERSITY OF THE PUNJAB

LAHORE, PAKISTAN

(2014)

DEDICATED

TO

MY LOVING FATHER

WHOSE LOVE IS MORE PRECIOUS,

THAN PEARLS AND DIAMONDS

BY THE VIRTUE OF WHOSE PRAYS,

HAD BEEN ABLE TO REACH

AT THIS HIGH POSITION

MY SWEET MOTHER

WHO IS HEAVEN FOR ME

WHOSE HANDS ARE ALWAYS

RAISED FOR MY WELL-BEING

EVEN AT THIS MOMENT OF TIME

MY BELOVED SWEET

BROTHERS&SISTERS

WHO ARE THE WORLDS FOR ME,

WHOSE LOVE ENCOURAGED ME

AT EVERY STEP FOR MY HIGHER

IDEALS OF LIFE

CONTENTS

Title Page #

ACKNOWLEDGEMENTS i

SUMMARY iv

1. INTRODUCTION AND LITERATURE REVIEW 1

1.1 MITOCHONDRIA 4

1.2 MITOCHONDRIAL MORPHOLOGY 5

1.3 NUCLEAR AND MITOCHONDRIAL DNA 7

1.4 mtDNA GLOBAL PHYLOGENY 10

1.5 THE IMPORTANT CHARACTERISTICS OF HUMAN 10

MITICHONDRIAL DNA

1.6 SIGNIFICANCE OF mtDNA 18

1.7 mtDNA POLYMORPHISMS 20

2. MATERIALS AND METHODS 24

2.1 POPULATION STUDY 24

2.2 Blood SAMPLES COLLECTION 24

2.3 DNA EXTRACTION 27

2.4 PRIMER DSIGNING 27

2.5 PCR AMPLIFICATION OF THE mtDNA 28

2.6 GEL ELECTROPHORESIS 29

2.7 PURIFICATION OF PCR PRODUCTS 30

2.8 mtDNA SEQUENCING 30

2.9 SEQUENCING ANALYSIS 31

2.10 STATISTICAL ANALYSIS 31

2.11 PHYLOGENETIC ANALYSIS 32

3. RESULTS 34

4. DISCUSSION 79

5. REFRENCES 86

LIST OF FIGURES

Figure #

Title Page #

1.1

Internal structural features of the mitochondria.

6

1.2 Genes, rRNA and tRNA coded in Human mitochondrial DNA.

8

1.3 Schematic diagram of mtDNA control region.

9

1.4 Mitochondrial DNA, Schematic diagram of the 16.6-kb, circular,

double-stranded mtDNA molecule.

12

1.5 Phylogenetic tree of major languages in Pakistan. 15

2.1-A Consent form for Saraiki donor‟s record in national language

from Pakistan.

25

2.1-B

2.2

2.3

3.1

Consent form for Saraiki donor‟s record from Pakistan.

Thermal cycling profile for mtDNA control region amplification

of Saraiki population from Pakistan.

Summary of Research work plan for the Saraiki population

mtDNA control region analysis from Pakistan.

Map of Pakistan showing study area of Saraiki Population.

26

29

33

38

3.2 Showing sampling areas along with numbers of samples from

the Saraiki donors of Pakistan. 39

3.3 Showing total samples including males and females of Saraiki

population from Pakistan.

39

3.4 Showing donors of different age groups (18-60 years) along with

number of samples from Saraiki population of Pakistan.

40

3.5 Consanguinity % age of parents in Saraiki population of 85

samples collected from different areas of Pakistan.

40

3.6 Agarose gel electrophoresis showing the bands of extracted

genomic DNA of different quantity.

44

3.7 Agarose gel electrophoresis showing the bands of extracted

genomic DNA of different quantity.

44

3.8 PCR product size of the samples, 010, 011, 012, 013,014 loaded

on 2% agarose gel.

45

3.9 PCR product size of the samples, 015, 016, 019, 020,021 loaded

on 2% agarose gel.

45

3.10 Sequence showing haplotype variations in sample SS-054.

46


51


56


60


64

3.15 Different Haplogroups frequency % age found in 85 Saraiki

population sample from different areas of Pakistan.

68

3.16 Comparison of genetic diversity among Saraiki population and

others various ethnic groups of Pakistan.

69

3.17 Dendrogram showing genetic affinities among various Saraiki 78

samples from different areas of Pakistan.

LIST OF TABLES

Table #

Title Page #

1.1

Characteristics comparison between mitochondrial DNA and

nuclear DNA.

13

2.1 Primers used for PCR amplification and sequencing of mtDNA

control region. 27

2.2 Cycling conditions for mtDNA control region amplification of

Saraiki population from Pakistan.

28

3.1

3.2

Showing consent form summarized data of Saraiki population

from Pakistan.

Occurrence and distribution of nucleotide substitutions, deletions

and insertions in the hypervariable regions I, II and III of

mitochondrial DNA control region from unrelated Saraiki persons

of Pakistan.

34

41

3.3 Estimated haplotypes and haplogroups in Saraiki population from

different areas of Pakistan.

69

3.4 Haplogroups (%) frequency in Saraiki population from Pakistan.

74

3.5 Population genetics parameters of Saraiki population from

different areas of Pakistan

75

ABBREVIATIONS/ UNIT ABBREVIATIONS

ºC Degree Centigrade

EDTA Ethylene diamine tetra acetic acid

Fig. Figure

KPk Khyber Pakhtunkhawa

ATP Adenosine tri phosphate

nDNA Nuclear DNA

mtDNA Mitochondrial DNA

DNA Deoxyribo Nucleic Acid

L-strand Light strand

H-strand Heavy Strand

D-loop Displacement loop

STR Short tandem repeat

bp Base pair

SNP Single nucleotide polymorphism

RFLPs Restriction Fragment Length Polymorphisms

PCR Polymerase chain reaction

HVR Hyper Variable region

pM Paicomol

µl Micro liter

MgCl2 Magnesium Chloride

mM Milli mole

V Voltage

U.V Ultra violet

Rpm Revolution per minute

-ve Negative

CEMB Center of Excellence in Molecular Biology

M. Garh Muzaffar Garh

D. I. Khan Dera Ismail Khan

N Hyplotypes numbers

HG haplogroup

HGT Haplogroup Type

SA South Asian

WEA West Eurasian

SEA South East Asian

EEA East Eurasian

WA West Asian

SWA South West Asia

EA East Asia

AF Africa

Mg Muzaffar Garh

Ly Layyah

Bk Bhakar

Mi Mianwali

DIK Dera Ismail Khan

UK Umar kot

GC Guanine-Cytosine

KYA Thousand Years Ago

MCL Maximum Composite Likelihood

rCRS revised Cambridge Reference Sequence

A Adenine

C Cytosine

G Guanine

T Thymine

HVI Hypervariable region I

HVII Hypervariable region II

HVIII Hypervariable region III

i

ACKNOWLEDGEMENTS

I indebted to Almighty Allah, the propitious, the benevolent and sovereign whose

blessing and glory flourished my thoughts and thrived my ambitions, giving me talented

teachers, affectionate parents, sweet brothers, sweet sisters and unique friends. Trembling

lips and wet eyes praise for Holy Prophet Muhammad (S.A.W) for enlightening our

conscience with the essence of faith in Allah, converging all His kindness and mercy upon

him.

If there were dreams to sell, marry and sad to tell and crier rings the bell, what would

you buy, I will say that “University Charming Days”. Actually it is impossible, but it shows

my blind love to this institution, which is homeland of knowledge, wisdom and intellectually.

I love my Alma–Mater with the soul of heart, because it is just like the lap of mother. I am

proud of being student of this university.

My research work was accomplished under enthusiastic guidance, sympathetic

attitude, encouragements, inexhaustible inspiration and enlightened supervision of

Prof. Dr. Tanveer Akhtar. I offer my heartiest gratitude to my caring and kind supervisor

for her untiring help, sagacious suggestions, step-to-step guidance and close supervision

during the conduct of these investigations and in preparation of this manuscript. Thanks are

also to Prof. Dr. Muhammad Akhtar Chairman of Zoology Department University of the

Punjab, Lahore for continuous constructive criticism, co-operation and sympathetic attitude.

I am grateful to all my teachers of my academic career, who provide me their

shoulder to rise above and there will be shareholder of each and every success of my life. I

will be failing in my duties if do not extend my zealous thanks to Mr. Muhammad Hassan

Siddiqi who helped me a lot in practical work and provide me moral support in the

ii

preparation of this manuscript. I am thankful to Dr. Muhammad Ayub Director

General Fisheries, Punjab and Malik Muhammad Ramzan, Deputy Director Fisheries, who

provide me moral support in the preparation of this manuscript. My cordial thanks goes to

Dr. Allah Rakha, Dr. Muhammad Akram Tariq and Dr. Abdul Razaq for their guidance

and help in data analysis.

Friends are the assets of life especially when together also express my gratitude to

Muhammad Arif Bhutta, Syed Aoun Raza, Muhammad Tayyab, Ghulam Hussain,

Muhammad Naeem Haider, Muhammad Sajid and Ghazanfar Abbas for their sincere

help and inspiring cooperation. I would like to take this opportunity to extend my thanks

from the core of my heart to my fellows Sana Shahbaz and Afia Muhammad Akram

without their cooperation and encouragement; I would never fulfill my work. I am thankful

to lab. Attendant Mr. Ghulam Abbas Anjum who helped me much during practical work.

I am unable to express my deepest gratitude towards my affectionate Uncle

Mian Muhammad Iqbal for the strenuous efforts done by him to enable me for developing

higher ideas of life for his patience and prayers he made for my success.

I find a great desire to mention cordial thanks to my wife for whom I regret to have

no better word than „Thanks a lot” for her sincere help and everlasting cooperation. My

acknowledgement will remain incomplete if don‟t acknowledge my sisters whose love is

more precious than diamonds and pearls. Nothing is completed for me without expressing my

deepest love, obligation and sincerity to my brothers Mian Ghulam Abbas,

Qari Khizzar Hayat and Mian Zulfaqar Ali for their moral support, who has always

motivated me to carry myself through the noble ideals of life. I am also thankful to all those

who pray and help me in the completion of this research work.

iii

Finally, I will like to pay my sincere thanks to my adorable and affectionate Father

and Mother for their financial, moral support and valuable assistance in my academic

pursuits. Here I can say without reservation that whatever I am, it is because of prayers of my

beloved parents. Their unfathomable love, prayers and help, both moral and financial, have

rendered me sound environment during my studies.

Sikandar Hayat

iv

SUMMARY

The analysis of mitochondrial DNA (mtDNA) control region was carried in 85

unrelated Saraiki males and females of different age groups inhabiting the different areas of

Pakistan. DNA was extracted from blood preserved in EDTA vacutainers. Hypervariable

regions I, II and III (HVRI, HVRII & HVRIII) were PCR amplified and sequenced.

Sequencing results were aligned and compared with revised Cambridge Reference Sequence

(rCRS) which showed the presence of total 63 different haplotypes, 58 of them were unique

and 05 were common haplotypes in more than one individual. The most common haplotype

observed was (W6) with a frequency 12.9% of population samples. The Saraiki population

was detected with genetic diversity 0.9570 and 0.9458 power of discrimination. High

molecular diversity and low match probabilities confirmed the value of control region data in

the investigation of maternal genetic lineages in Saraiki population. The Saraiki individuals

showed similarities in their mtDNA lineage composition which may be the result of

endogamy. This study aimed to investigate the sequences of mitochondrial DNA (mtDNA)

control region in Saraiki population and contribute in extension of forensic mtDNA reference

data for Saraiki population in Pakistan. Moreover, this research will develop the knowledge

about Saraiki population in understanding of geographic and linguistic factors on population

diversity and structure in this region which will provide a base for future work in this field.

1. INTRODUCTION AND LITERATURE REVIEW

Pakistan came into its existence in 14 August 1947 and this area already has been

remained very important in the human history. Moreover, Pakistan is located in that area

which has been the way of numerous invaders and those have took part in the cultural and

linguistic diversity found in this country. Pakistan is located, near Afghanistan, Iran in the

west, India in the East and Arabian Sea in south, having latitude and longitude 33.6667oN

and 73.1667oE. Geographically Pakistan is divided into various regions on the basis of

physical characteristics. Those areas are Northern and Western mountainous areas, Salt hilly

areas, Potohari regions, upper and lower part of River Indus.

According to this distribution, the Northern Mountains are spread to Pakistan‟s northern

territory, Kashmir and Gilgit. While the western areas divide Khyber Pakhtunkhwa (KPK)

and Balochistan. The Potohari region and Salt ranges are part of the Punjab province.

Pakistani population is divided into more than 18 ethnic and 60 linguistic groups on the basis

of their origins and languages (Grimes, 1992; Larik, 2000).

The upper part of River Indus is the part of Punjab and is known by the Saraiki belt.

Anthropologists have distinct the existence of Saraiki Civilization near to the Indus valley on

the Western side and Harrapa Civilization on its Eastern side near about 40,000 years ago.

Similar to most of the other civilizations of the Indus Valley, the Saraiki culture represents

historic pre-Aryan people of a Semite origin (Shackle, 1977). In Pakistan, most of the ethnic

groups have their prehistoric basis but language is one of the important characteristics

markers. Every population differs from other population on the basis of language,

civilization, physical appearance and genetic characteristics. Based on the social structure,

people are divided into various tribes and caste. So, the Saraiki ethnic group distinguishes

1

itself on the basis of Saraiki language. In addition to this, consanguineous marriages are the

customs of Saraiki peoples which make them an important subject of this study.

In Pakistan Saraiki area extended from central Punjab to adjoining areas of Sindh,

Khayber Pakhtunkhwa (KPK) and Balochistan. In Punjab the word Saraiki was first time

known in 1960's earlier to that it was known as Lehnda or Multani. Saraiki is mostly spoken

in those districts which are located in South Punjab. Saraiki language is spoken by the some

population of KPK district (Dera Ismail Khan) and in some areas of province Sindh and

Balochistan. Majority of Pakistanis from southern districts of Punjab speak Saraiki as a first

language.

The human progression begins with the introduction of genus Homo about 2.5 -1.5

million years ago whose early sign was found in Africa (Klien, 1989). With the passage of

time, various species of the genus Homo were identified including H. ergaster, H. erectus,

and the H. floresiensis (Swisher et al., 1994; Gabunia and Vekua, 1995; Brown et al.,

2004), all these species now have been disappeared with the entry of modern H. sapiens,

which is the fully developed species of human being that was appeared about 100,000 years

ago in East Africa (Klien, 1989). The scientific society believes that modern humans came

in Africa and many other waves of migrations help to explain their way out of Africa (Luis et

al., 2004; Macaulay et al., 2005). Fossils and archaeological record explained that scattering

of modern humans became possible at that time when weather conditions become suitable for

life. The discovery of 125,000 old pieces in Eritrea`s Red Sea coast suggested that people

from the Horn of Africa moved across the Arabian headland to the south part of the Red Sea

(Walter et al., 2000). From Arabia, people migrated towards West, East and reached Western

Europe and Siberia and East Asia about 39 KYA. These migrations of people resulted in the

2

development of several different populations and various races of modern humans that are

differentiated on the base of differences in their physical characteristics, culture and

language. In addition to fossil and archaeological evidence in the favour of African origin for

modern humans, molecular genetics records are also in the favour of this idea (Batzer et al.,

1996; Tattersall, 1997; Shiver et al., 1997; Scozzari et al., 1988; Stringer and Andrew, 1988).

The social divisions are very inflexible and have been remained with no disturbance

by increasing urbanization and cultural changes in the different societies of modern human

(Tamang and Thangaraj, 2012). Pakistan lies on the postulated shoreline which was

followed by modern humans during coming out of Africa and therefore it is recognized as

one of the first region that was occupied by the humans (Qamar et al., 1999). Pakistani

population diversity is measurable on the basis of civilization, languages, religions and

genetic lineages. On the basis of customs and languages Pakistani population has been

divided into various18 ethnic groups having different origins, among which endogamy is

generally accomplished (Ayub et al., 2009). The endogamy of populations generates a high

degree of genetic discrimination (Murci et al., 2004). In order to achieve the best and

resourceful knowledge for forensic purposes it requires that on regional or ethnic basis a

reliable data should be developed.

Pakistani culture is generally organized in the tribal form, which is more common in

the rural areas of Pakistan than in the cities. Normally, the tribe names are inherent and do

not change after marriage. However, females do not pass their tribe name to their children.

Generally speaking, in Pakistan marriages within families are frequently followed by ethnic

and religious association and relationships. Cousin marriage is accomplished in the rural

parts of the population but is limited to the Muslims (Tadmouri et al., 2009).

3

In many human populations from Pakistan the mtDNA control region remained

almost unknown due to insufficient data available from those populations. Due to this factor

a best quality data of mtDNA control region for forensic applications has not been developed

yet. So, missing of best quality mtDNA is challenging the country by the presence of many

various human groups with discrete genetic diversity level (Bobillo et al., 2010).

1.1 MITOCHONDRIA

Mitochondria are the most important organelles found in cytoplasm of eukaryotic

cells and play fundamental role in the respiration of the cells (Bandelt et al., 2006). Enzymes

within the outer and inner membranes of the mitochondria help in converting materials into

adenosine triphosphate (ATP) which is fuel for the metabolic activities of the cell (Davidson,

2010; Reece et al., 2011). The mitochondrion is the place in which the final stages of aerobic

respiration take place. This complex system empowers cells to use aerobic respiration to

generate approximately 15 times more ATP than anaerobic respiration (Davidson, 2010).

The mitochondrial genome is made up of approximately 17,000 base pairs (Butler,

2009). The main characteristic of mitochondria relevant to this study is that they possess their

own genome and contains highly informative polymorphic sites (Reljanovic et al., 2012).

These semi-autonomous organelles also possess the important RNA machinery for

mitochondrial DNA translation and transcription (Scheffler, 2008). Even though

mitochondria cannot survive independently of the cell in which they live but mitochondria

can replicate their own DNA autonomously of nuclear DNA (Hartl and Jones, 2005). Beside

the energy formation, mitochondria are very important machinery in signaling of calcium and

metabolism regulation within the cells (Giezen and Tovar, 2005).

4

All eukaryotic cells contain at least some mitochondria which act as energy producing

organelle (WU et al., 2013). These mitochondria possess their own DNA, having important

proteins for oxidation systems (Smeitink et al., 2001). As all eukaryotic cells have the

mitochondria so, those species which possess the mitochodiria can be easily forensically

investigated on the basis of mtDNA analysis. On account of mtDNA analysis maximum

information are obtained from analyzed samples for forensic purpose.

Human cells have a different number of mitochondria, depending upon the metabolic

activities of the particular cells (Davidson, 2010) and typically the copy number of

mitochondrial DNA is 100-10000 copies/cell (Malyarchuk and Rogozin, 2004). Some cells

like that of ascidian spermatozoa contain one mitochondrion whereas humans, heart and

muscle cells contain thousands of mitochondria and the tissues that are high in respiratory

activity have mitochondria with a greater amount of folding which increase the surface area

for respiration (Hahn and Voth, 1994).

1.2 MITOCHONDRIAL MORPHOLOGY

The animal mitochondrion‟s structure is very complex (Fig.1.1) due to its vital

function in energy production. It has two membranes that separate into four distinct sections

in which each membrane-bound section is able to generate ATP (Mcbride et al., 2006). The

two membranes divide the organelle into a narrow inner membrane space and a large internal

matrix (Davidson, 2010). The outer membrane of the mitochondrion contains channel

proteins while the inner mitochondrial membrane consists of widespread folding called

cristae (Davidson, 2010). The inward folds of the cristae increases the surface area available

for enzymes concerned in cellular respiration (Davidson, 2010).

5

Fig. 1.1: Internal structural features of the animal mitochondria (Davidson, 2010).

Mitochondria can be found in a variety of different morphologies in mammals

(Okamoto and Shaw, 2005). They range in shape from long tubules inter connected to one

another to small separate spheres (Okamoto and Shaw, 2005). Mitochondria also range in

size between approximately 1 to 10 micrometers but are known to vary in both size and

morphology regularly (Davidson, 2010).

The mtDNA, because of its circular structure and location inside the cell is more rigid

and strong than the nuclear DNA (nDNA), it is shown that mtDNA is secure from

degradation even when exposed to prolonged environmental conditions while such properties

have not been observed in the case of nDNA. That is why mtDNA is present even in older

and degraded samples. Hence, this property has made mtDNA more valuable than nDNA in

the field of forensic sciences.

6

1.3 NUCLEAR AND MITOCHONDRIAL DNA

Multicellular organisms grow by slow process of progressive change that begins with

the fusion of very specialized cells an egg and a sperm. As the result of egg and sperm union

a zygote develops containing genetic information (nDNA) inherited from both parents that

articulate the fetus development (Gilbert, 2006). As the zygote divides mitotically to produce

all cells of the body, nuclear genes are not lost or changed and the genome of each cell is

equal to that of every other cell. Nuclear DNA is enveloped into individual chromosomes

having total twenty three pairs in normal cells. In every eukaryotic cell, its chromosome

possess a very long DNA molecule consisting of many genes which are known as the unit of

heritage (Richly and Leister, 2004).

Mitochondrial DNA is known as major source of important proteins in outside of the

eukaryotic cells nucleus. The human mtDNA was sequenced in 1981 first time by Anderson

(Hoong and Lek, 2005). As human mtDNA is double stranded so both of these strands are

differentiated from each other by nucleotides composition having heavy strand (H-strand)

and light strand (L-strand) (Fig. 1.2). Heavy strand is rich in guanine while light strand is

rich in cytosine. mtDNA has 16,569 bp in length (Ketmaier and Bernardini, 2005) and

possess many copies in human cells having high mutations, lacking recombination and

maternal heritage (Tsutsumi et al., 2006). Due to these properties of mtDNA, it has become

very easy to get maximum information about the ancient DNA by its analysis for certain

forensic cases (Witas and Zawicki, 2004; Tsutsumi et al., 2006). mtDNA has many copies

and according to some scientist that a cell contains100-10,000 copies of mtDNA on the basis

of energy demand by the cell, greater the demand of energy larger will be the numbers of

7

copies of mtDNA. Mitochondrial DNA molecule contains 37 genes; out of 37 genes 28 are

present on heavy strand while 9 genes are present on light strand (Andrews et al., 1999).

Fig. 1.2: Genes, rRNA and tRNA coded in human mitochondrial DNA (Childs, 2003).

Large numbers of the mtDNA molecules are present in each cell of the organisms.

These molecules have different ranges in different cells for example a single oocyte cell

contains about 50,000 molecules while sperm cell contains only few hundred mtDNA

molecules. Normally epithelial cells are used for forensic cases and these cells commonly

contain 5000 mtDNA molecules (Bogenhagen and Clayton, 1974). This property of mtDNA

molecule has made it a vital and sensitive molecule for the detection of very low amount of

the DNA present in a sample or the detection of DNA from those samples which have been

badly degraded. In any individual mtDNA is derived from its mother because during

fertilization only small portion of sperm enter into the egg cell (Manfredi et al., 1997). As

mitochondria are present in the tail of sperm cell and during fertilization only head of sperm

cell incorporate into egg cell and tail remain outside that is why new organism obtained

mtDNA from its mother. So, mitochondrial DNA analysis is used to detect the maternal

inheritance in any generation (Gill et al., 1994). In some individuals mtDNA mutation rate is

8

much higher than that of nuclear DNA (Schriner et al., 2000). Due to this mutation a

population shows significant diversity which can be helpful to know the evolutionary history

of the population, that is why nuclear and mitochondrial mutation rates for the human lineage

have been deeply discussed (Scally and Durbin, 2012).

Mammalian mitochondrial DNA is divided into two different replication origins. The

origin of the heavy strand is inside the D-loop of mtDNA and light strand originates within a

cluster of five tRNA genes nearly opposite of the D-loop. The single focus of current forensic

typing is the D-loop. The D-loop consists of about 1100 base pairs of non coding DNA and is

generally known as the hypervariable region. The hypervariable region is further divided into

three segments. Hypervariable region I (HVR1) spans nucleotide positions 16024-16365;

hypervariable region II (HVR2) span nucleotide positions 73-340 and hypervariable region

III (HVR3) spans nucleotide positions 438-576. HVRI and HVR2 are usually targeted

whereas HVR3 is hardly study in forensic case work (Fig.1.3). The hypervariable region

mutate at a rate of 10 to 17 times more than marked areas of the nuclear genome (Bar et al.,

2000).

Fig.1.3 Schematic diagram of human mtDNA control region (Rakha et al., 2011).

9

The distinguishing character of human mitochondrial DNA (mtDNA) is that it

possesses a lot of information for population and evolutionary genetics studies

(Cavelier et al., 2000). Another important and main character is the maternal inheritance

(Giles et al., 1980). Mitochondrial DNA (mtDNA) has huge potential in forensic genetics,

allowing recognition of genetic material. Single Nucleotide polymorphism SNPs have a

number of characteristics that make it unique for forensic analysis (Bento et al., 2009).

1.4 mtDNA GLOBAL PHYLOGENY

Human maternal lineages have been classified into different haplogroups. Basal

haplogroups reveal continent specificity (Richards et al., 2000; Herrnstadt et al., 2002). The

possible root of human mtDNA tree is between haplogroups L0 and L1 separating the

phylogenetic tree into two basic clades (Chen et al., 2000; Salas et al., 2002; Salas et al.,

2004; Kivisild et al., 2004). Only L3 expand from Africa in the form M and N haplogroups,

giving rise to Eurasian variations and majority of western Eurasians are identified by N

haplogroup sub-clade (Finnila et al., 2001; Herrnstadt et al., 2002; Palanichamy et al., 2004).

It has been seen that M and N haplogroups almost equally contribute in the identification of

eastern Eurasian (Schurr and Wallace, 2002; Yao et al., 2002).

1.5 THE IMPORTANT CHARACTERISTICS OF HUMAN mtDNA

In human population mtDNA has been used in large number of fields like evolution,

anthropology, history, inheritance and in many forensic cases. With the knowledge of

mtDNA analysis inheritance history creation and verification has increased rapidly in the

field of forensic science. Due to this improvement every individual want to get more

knowledge about his ancestors and believe that mtDNA analysis will be very helpful in

giving the maximum information about the inheritance and in reorganization/identification of

unknown suspect (Chaitanya et al., 2014). mtDNA also play a key role to get the

10

information about maternal inheritance. As Mitochondrial DNA is present in high numbers in

the cells as compared to nuclear DNA so it has become very easy to find it in greatly

degraded samples for forensic analysis (Adachi et al., 2014).

The basic aim of this study is to get knowledge about control region of human

mitochondrial DNA.

Followings are the important basic properties of human mtDNA (Scheffler, 2008).

• Human mtDNA molecule has small number of proteins and RNAs which very important

for mitochondrion functions and activities.

• mtDNA is double stranded molecule having large numbers of genes which ply key role in

the vital functions of the cell.

• mtDNA is only transferred from mother.

• It has no recombination and replicates quickly.

Mitochondria have a small circular genome (Fig. 1.4) which is made up of

approximately 17,000 base pairs (Butler, 2009). The main characteristic of mitochondria

relevant to this study is that they possess their own genome. High concentrations of mtDNA

are found in bones, teeth and hair which are very useful for forensic analysis. mtDNA is

circular in shape which means there are no free ends for exonuclease activity to use to

degrade the genome. It has been observed that mtDNA is widely used to know a specie

characters and phylogeny. Overall, mtDNA is forensically very small molecule with a high

mutation rate and conserved gene (Gray, 1989).

11

Fig. 1.4: Mitochondrial DNA, Schematic diagram of the 16.6-kb, circular, double-stranded

mtDNA molecule and the outer circle represents the heavy strand and the inner circle the

light strand. The genes encoding the mitochondrial RC: MTND1–6, MTCOI–II, MTATP6

and MTCYB (Andrews et al., 1999).

Mitochondrial DNA is similar to nuclear DNA in a few ways but different in some

ways (Table 1.1). One major difference between nDNA and mtDNA is their inheritance

pattern within families. The genome of an individual consists of equal contributions of

chromosomes from both the maternal and paternal germ lines while mtDNA is inherited only

from the maternal line (Taanman, 1999). Displacement loop or D-Loop of mtDNA has been

extensively studied for its potential use in forensic applications. The D-Loop has also been

referred to as the control region because it regulates the gene products produced by the

coding regions of the mitochondrial genome (Parson and Coble, 2001).

12

TABLE 1.1: Characteristics comparison between mitochondrial DNA and nuclear DNA

(Alonso et al., 2004).

Mitochondrial DNA Nuclear DNA

Close, Circular molecule Linear molecule

16, 569 base pairs in size 3 billion base pairs in length

1100 base pair non coding region A large portion of genome non coding

Maternaly inherited Biparental inherited

No recombination in mtDNA Recombination in nDNA

50 to many thousand copy per cell Two copies per cell

The D-Loop is also a region of polymorphism within the mitochondrial genome due

to high frequency of base changes (Bini et al., 2003). These highly polymorphic regions have

been called hypervariable regions and have been the focus of forensic DNA typing for

recognition purposes (Garritsen, 2001). Polymorphisms of the hypervariable regions in the

D-Loop structure have also been important in anthropological studies on the historic origins

and migratory patterns of early human populations (Pliss, 2007). Since mtDNA is inherited

matrilinearly, specific mtDNA haplotypes have also been useful in determining an

individual‟s ethnic origins (Herrnstadt et al., 2002).

With the improvement of knowledge in the field of molecular biology and molecular

genetics it became very easy to support the evidences about the evolution of modern human

from anthropological and archeological records (Ingman et al., 2000; Macaulay et al., 2005).

In recent times with the great development of genetic markers it has become very easy to test

diverse hypotheses about the genetic history and evolution of human populations (Rogers and

Jorde, 1995; Hammer et al., 1998; Templeton, 2002; Hebsgaard et al., 2007). Particularly,

13

Anderson (1981) revolutionized the molecular genetics by completing the 16,569 base pairs

sequencing of human mitochondrial genome due to which it has become possible to get

knowledge about the time of origin and time of dispersal of human population out of Africa

to other various continents (Ingman et al., 2000; Macaulay et al., 2005).

How humans occupied the earth is one of very important indescribable story of

human race (Goebel, 2007)? Mitochondrial DNA is a key tool in order to know the

prehistory of human population. From the Phylogenetic tree analysis it becomes obvious that

modern humans have expanded out from Africa (Watson et al., 1997; Ingman et al., 2000).

According to some scientist the sub-Saharan Africa along the India has been remained the

migratory rote of modern human expansion from Africa (Forster and Matsumura, 2005;

Macaulay et al., 2005) and then it migrate to North Africa and Europe (Olivieri et al., 2006).

But still modern human expansion from Africa is still unclear due to lack of strong molecular

evidences about human history.

Genetic and paleoanthropological evidences expose that human population expanded

on account of geographic improvement that began approximately 60,000 years ago in Africa

and gradually started occupying all those regions of the Earth that were suitable for

habitation. Genetic data obtained from current humans recommend that due to continuous

loss of genetic diversity today expansion of human become possible. This great human

expansion from Africa can help to improve the evolutionary knowledge of modern human

(Brenna et al., 2012).

There is a significant similarity in the linguistic hierarchy (Fig. 1.5) which confirms

the Darwin‟s assumptions that if someone knew the human biological hierarchy, he can know

14

about his languages (Darwin, 1860). Molecular Genetic evidences pointed out that all the

modern human population has common ancestry (Li and Durbin, 2011).

Fig. 1.5: Phylogenetic tree of major languages (Desmet et al., 2012).

With passage of time Different genetic adaptations were made by the modern human

when their ancestors came out of Africa (Ingman et al., 2000). A fossil was found about

50,000 years ago across the Eurasia whose morphology was similar to that of present day

humans (Fu et al., 2013). Different environmental factors are also affecting the human

genetics in different ways. With the change of environment many genetic adaptations have

been occurred in human population. As human came out of Africa he has to face numbers of

environmental challenges. In recent times, through genetic analysis it has came to know that

15

large numbers of the genes have been changed in East Asian populations extensively

(Xu et al., 2009).

Many hypothesis support the anatomically single origin for modern humans

(Cann et al., 1987; Underhill and Kivisild, 2007). In order to know the maternal common

ancestor of modern human, phylogenetic data has played important role in this regard

(Mellars, 2006; Torroni et al., 2006). But still in spite of phylogenetic data improvement,

there are no strong evidences about the distribution of Homo sapiens population (Mellars,

2006; Hawks, 2007). According to some scientists the mtDNA molecules in human

population are same at the time of birth (Taylor and Turnbull, 2005) and each mitochondrion

contains an average of five copies of its genomic DNA. There are hundreds to thousands of

mtDNA molecules within a single cell (Kavlick et al., 2011).

Human mitochondrial DNA has been generally used to get the knowledge about its

evolution, migration and population history (Tambets et al., 2004). Human mtDNA is

inherited from mother and shows a minute rate of recombination. Due to this low rate of

recombination it has been widely used to improve the knowledge about the population

migration globally (Torroni et al., 2006). Mitochondrial DNA (mtDNA) variation has been

verified to be the most powerful genetic marker for investigating gene pools and tracing

maternal genetic similarities (Cavalli-Sforza and Feldman, 2003; Malyarchuk et al., 2008).

Due to great improvement in mtDNA analysis for forensic purpose now it has become

possible that on the base of this analysis missing and criminal persons can be easily identified

when short tandem repeat technique is not giving any information about those persons

(Chaitanya et al., 2014).

16

mtDNA analysis has been used effectively in order to determine the family

relationships when there is a gap of several generations between ancestor and living person

(Gill et al., 1994; Weichhold et al., 1998). The maternal inheritance of animal‟s mtDNA is

almost worldwide and highly concentrated (Rawi et al., 2011; Luca and Farrell, 2012). An

individual genome consists of equal contributions of chromosomes from both the maternal

and paternal germ lines while mtDNA is inherited solely from the maternal line (Taanman,

1999). mtDNA is only inherited from mother and this property of mtDNA has many

important applications in the fields of evolution anthropology, population history, genetics,

genealogy and forensic science (Shriver and Kittles, 2004; Taylor and Turnbull, 2005;

Blansit, 2006; Torroni et al., 2006; Kayser, 2007; Underhill and Kivisild, 2007).

All modern humans have a certain types of mtDNA. These types of mtDNA are

related with the SNP pattern within the mitochondrial genome and are called haplogroups.

Each person belongs to a definite haplogroup and a particular haplogroup can exhibit a

person‟s common female specific place of origin (Torroni et al., 2006). Haplogroup analysis

is also used to trace population relocation patterns (Santos, 2004).

mtDNA macrohaplogroups have been distributed broadly worldwide in different

regions on the basis of distinctive characteristics of that particular region. In this regard L is

primarily haplogroup found in Africa which shows the largest diversity of mtDNA

haplogroups. After L haplogroup, M and N haplogroups show highest diversity in South

Asia especially in India (Basu et al., 2003; Metspalu et al., 2004; Palanichamy et al., 2004;

Sun et al., 2006; Thangaraj et al., 2006; Chaubey et al., 2007). From this, it is clear that after

dispersal of human from Africa, India played important role in human evolution. Both M

17

and N haplogroups are found in high frequency in India but among those haplogroups M is

more common as compared to N haplogroup.

Basically mitochondrial DNA haplogroups are divided into three major haplogroups

which are L, M and N whose distribution is very distinctive. Among these three major

haplogroups L haplogroup is oldest and is restricted to African populations. L haplogroup has

sub-clade like L0, L4, L6, L3 and L7 and with the passage of time L3 haplogroup came out

of Africa in the form of M and N haplogroup about 60,000 years ago (Mishmar et al., 2003).

Later on these haplogroups introduced in South East Asia, Australia and South Asia.

According to some scientist N haplogroup has been found in West Asia, East Asia, Australia,

Europe and South Asia (Salas et al., 2002; Mishmar et al., 2003; Kong et al., 2003).

In the beginning human mtDNA study was based on restriction fragment length

polymorphisms (RFLPs) (Cann et al., 1987; Scozzari et al., 1988), but with the passage of

time as polymerase chain reaction (PCR) and sequencing techniques developed, scientist

started both these techniques to study human mtDNA instead of RFLP analysis (Torroni et

al., 1996; Macaulay et al., 1999).

1.6 SIGNIFICANCE OF mtDNA

The highly recombination nature of mtDNA is very important in the analysis of

forensic casework, like identification of missing individuals and mother inheritance due to

which it has become possible to identifying suspects by mtDNA analysis, when STR analysis

cannot provide required information (Chaitanya et al., 2014).

Sequencing of mtDNA offers many advantages in forensic science applications. One

advantage is the abundance of mtDNA within the cell. There are 500 to 2,000 copies of

mtDNA in each human cell as compared to only two copies of nuclear DNA in most cells

18

and some cells do not contain nuclear DNA at all (Parson and Coble, 2001). mtDNA analysis

data is very useful for species identification (Wells and Stevens, 2008). Thus, in a small

biological sample recovered from a crime scene, there will be excess quantity of mtDNA for

analysis as compared to nDNA. So, mtDNA is beneficial for those cases in which the

extracted DNA sample is very small (Kavlick et al., 2011). Moreover, some tissues in the

body that do not contain significant amounts of nDNA but contain sufficient amount of

mtDNA useful for forensic analysis. For example, hair shaft essentially lacks nDNA but

contains considerable amounts of mtDNA. Hair is a common type of forensic evidence. In

addition, finger and toe nails naturally lack nDNA but contain mtDNA (Saferstein, 2007).

Difference and variations in mitochondrial DNA provide major information in order

to get significant knowledge about human evolution and these information play a key role in

forensic cases. Usually, human mitochondrial DNA possesses diverse features such as single

nucleotide polymorphism, hypervariable regions and short tandem repeat (Lee et al., 2009)

which have made it more valuable. mtDNA is divided into two main sections, one of them is

large coding region responsible for the construction of various biological molecules involved

in the energy production for the cell and other is a small 1.2 kilobase pair fragment which is

called the control region. This control region is highly polymorphic and has different

hypervariable regions. These hypervariable regions have been broadly used in forensic

investigations (Parson et al., 1998).

The control a region is polymorphic within the mitochondrial genome due to high

frequency of base substitutions (Bini et al., 2003) and mtgenome in it are enriched in

sequence variation due to a higher mutation rate which allows researchers to create a mtDNA

profile (Kohnemann et al., 2008). This control region is also responsible for the defined

19

binding of several nuclear encoded proteins that regulate mtDNA replication and

transcription (Falkenberg et al., 2007).

Mitochondrial control region DNA pattern have been recommended to express the

geographic origin as well as the migrations history of the populations (Irwin et al., 2008; Van

Oven and Kayser, 2008; Saunier et al., 2009; Zgonjanin et al., 2010). The hyper variable

regions are very helpful in forensic for identification purposes (Garritsen et al., 2001).

Polymorphisms of the hypervariable regions in the D-Loop structure have also been

important in anthropological studies on the historic origins and migratory patterns of early

human populations (Pliss, 2007). Since mtDNA is inherited matrilinearly, specific mtDNA

haplotypes have also been useful in determining an individual‟s ethnic origins. So, control

region is very important and is used in forensic cases and population genetics (Barbosa et al.,

2008). Thus mtDNA haplogroup typing has become a basic instrument for the study of

human evolution and ancestry (Lee et al., 2013).

1.7 mtDNA POLYMORPHISMS

In humans 99.9 % of the genome is identical and out of 99.9% only 0.1-2.0% of the

DNA sequence displays variations. These variations cause genotypic and phenotypic

differences between individuals. These variations arise due to polymorphisms which are non-

pathogenic changes that exist at significant frequencies. Different types of polymorphism

have been exposed in the coding as well as in non-coding regions of the human genome

which not only untie our evolutionary past but to determine our biological future. One of this

polymorphism single nucleotide polymorphism (SNPs) is most common polymorphism in

the human genome and it includes single base substitutions, deletions and insertions. The

base substitutions are categorized into two groups namely transitions and transversions.

During transition purine is replaced by a purine (A ↔ G) or a pyrimidine by a pyrimidine

20

(C ↔ T). While in the case of transversion a purine is replaced by a pyrimidine. According to

Collins and Jukes (1994) the transition mutation mostly occurs in the mammalian genome as

compared to transversions mutations. According to the single nucleotide polymorphism

database more than 6 million SNPs lie within genes (Serre and Hodson, 2006). SNPs were

the first generation of polymorphic genetic markers. The use of SNPs was realized late

in1970‟s with the improvement of restriction fragment length polymorphism (RFLP)

(Roberts and Murray, 1976).

Polymorphisms are changes which occurred in nucleotides sequences of DNA control

region. Among different causes of polymorphisms in mtDNA one of them is lacking of

protective histone proteins. In support of mtDNA typing for forensic analysis, extensive

population research has shown that the haplotype of SNPs in the D-Loop are well correlated

with an individual‟s specific ethnicity (Pliss, 2007). This highly polymorphic nature of

mtDNA has made it more valuable and significant for biological evidence in forensic

casework (Maruyama et al., 2013). Mitochondrial DNA polymorphism has proved very

important in getting molecular genetics sketches of world populations as well as for the

explanation of the human evolutionary history and past migrations (Bermisheva et al., 2003).

The totally maternal inheritance, the absence of recombination and the high rate of mutation

in the control region mainly in hypervariable region I, II and III are the important properties

of mitochondrial DNA (Ge et al., 2010). In D-loop of mtDNA mutation occurs at high

frequency as compared other regions of mtDNA (Michikawa et al., 1992).

Analyses of the polymorphisms have enabled the identification of haplogroups and

construction of phylogenetic trees to observe the close association of different populations

(Nagai et al., 2003; Tanaka et al., 2004; Asari et al., 2007; Mabuchi et al., 2007; Sekiguchi et

21

al., 2008; Nohira et al., 2010). Comparison of mtDNA polymorphisms among various ethnic

groups has been important in the identification of different populations. The presence of

different mtDNA genome in a cell is known as heteropasmy and it was firstly identified

during mitochondrial diseases study (Holt et al., 1988; Wallace et al., 1988). Different

studies revealed that heteroplasmy is not present only in defective individuals but it is also

present in normal individuals which can be easily observed in healthy samples (Santos et al.,

2005; Santos et al., 2008; Irwin et al., 2009). It has been observed that heteroplasmy levels

may be different between tissues (Grzybowski et al., 2003: Irwin et al., 2009; Goto et al.,

2011) and populations (Irwin et al., 2009). Due to its peculiar properties, heteroplasmy is

also used as genetic markers for forensics applications. At the time of oogenesis some

blockages occurred as result of which heteroplasmic alleles frequency vary from generations

to generations and not remain same. To know about the alterations in allele frequencies at

heteroplasmic sites, it has become very important to explore the dynamics of maternal

mtDNA transmission (Goto et al., 2011).

The unique property of heteroplasmy is that it explores the presence of two or more

unique mtDNA types within a single cell (Paneto et al., 2007). Researchers now consider

both length heteroplasmy and sequence heteroplasmy as prospects (Li et al., 2010). In

mitochondrial genome the pattern of heteroplasmy variations is not clear still, making

explanation of particular difficulty for forensic experts (Budowle et al., 2003; Naue et al.,

2011). Knowledge of human mitochondrial heteroplasmy level has become very significance

in the number of fields (Ramos et al., 2013).

The variations occurring in mitochondria variations play vital role in knowing the

human origin and aging process (Taylor and Turnbull, 2005). These variations occurring in

22

mitochondrial DNA cause the development of diverse mitochondrial databases having

information related to human evolution and genetics (Kogelnik et al., 1996). Usually

hypervariable regions are analyzed for forensic and evolutionary purposes due to the high

polymorphic rate (Fridman et al., 2011).

To understand human origin and its evolution, nucleotide variations in mtDNA are

the chief subjects in this regard (Lell and Wallace, 2000). Particular nucleotide variants

perform a key role in maternal biogeographic ancestry (Kayser and Knijff, 2011). The most

polymorphic region of mtDNA is its control region which contains 3 hyper variable regions

HVR1, HVR2 and HRV3 (Bogenhagen and David, 1974). Besides HVR1 and HVR2, the

HVR3 provide extra information for haplotype purpose (Haslindawaty et al., 2010). The

investigation of the polymorphisms located in mtDNA genome allows tracing back the

evolutionary process in populations history and analysis of hypervariable region in the non-

coding region or D-Loop, allowed to detect specific characteristic of the populations (Bandelt

et al., 2003).

Considering the huge historical demographic patterns and present day structure of

Saraiki population, characterization of the genetic variation in Saraiki population will be

important in the establishment of representative forensic reference population databases for

the region. In present study mitochondrial DNA control region data of Saraiki population

from Pakistan has been generated for the forensic guidelines as mtDNA reference data using

the global mtDNA phylogeny background. This is the first study to evaluate the power of

discrimination of 85 unrelated Saraiki individuals with the simultaneous analysis of

hypervariable regions from Pakistan to aid human identification in forensic cases and to

investigate the sequences of mitochondrial DNA control region in the population.

23

2. MATERIALS AND METHODS

2.1 POPULATION STUDY

Total 85 blood samples were collected from unrelated healthy males and females

belonging to Saraiki ethnic group from different areas of Pakistan. Out of 85 samples 75

were males and 10 were females belonging to different age groups. Information of donors

regarding family name, gender, age, ethnicity, birthplace of mother and father, consanguinity

of parents and their first language was recorded in consent form (Fig. 2.1-A, 2.1-B).

2.2 BLOOD SAMPLES COLLECTION

About 3ml blood sample was drawn by vein puncture with syringe from healthy

Saraiki males and females volunteers of Pakistan. For this purpose paramedic staff was

involved after approval from ethical committee of University of the Punjab Lahore. This

blood was collected in EDTA vacutainers. Information‟s were collected from all the

participants after the explaining the aims and procedures of the study. After the blood

collection it was store immediately into freezer at low temperature to protect the DNA from

degradation. Then this stored blood was brought to Zoology Department, Parasitolgy

Laboratory in University of the Punjab, Lahore for further process.

24

Fig. 2.1-A: Consent form for Saraiki donor‟s record in national language from

Pakistan.

25

Fig. 2.1-B: Consent form for Saraiki donor‟s record from Pakistan.

26

2.3 DNA EXTRACTION

mtDNA was extracted from collected samples of blood using QIAamp DNA Mini Kit

(Qiagen, Hilden, Germany, Cat#51306) according to company protocol. The DNA quality

was determined by loading it on 0.8% agarose gel along with 100bp DNA marker through

gel electrophoreses. Before loading, the sample was mixed with 6x loading dye (Thermo

Scientific, USA). The extracted DNA was incubated at 70oC for I hour to avoid degradation

by nucleases then it was stored at -40oC in ultra low freezer for further process. For the

quantification of extracted genomic DNA Nano Drop™ 1000 Spectrophotometer (Thermo

Scientific, Wilmington, DE) was used (Sambrook and Russell, 2001).

2.4 PRIMER DSIGNING

Two primers for mtDNA control region amplification and sequencing were designed

using web-based primer designing (http://forensic.yonsei.ac.kr/protocol/mtDNA-CR.pdf).

Primers of required sequences were synthesized from Gene LinkTM USA (Table 2.1) to a

concentration of 100 pM/µl and were diluted to10 pM/µl for further process. These primers

were optimized for optimum reaction condition of temperature, buffer and dNTPs.

TABLE 2.1: Primers used for PCR amplification and sequencing of mtDNA control region.

Sr. No. Primers Name

Primers Sequences (5’→3’)

TM

(oC)

1

Amplification and

sequencing primer-

F15975

CTCCACCATTAGCACCCAAA

56.0

6

Amplification and

sequencing primer-

R635

GATGTGAGCCCGTCTAAACA

55.6

TM, Melting Temperature; F, Forward; R, Reverse

27

2.5 PCR AMPLIFICATION OF mtDNA

The mtDNA control region was amplified by PCR using F15975 and R635 as

forward and reverse primer respectively in thermo cycler Applied Biosystems 2720 USA.

PCR reaction volume was 50μl containing 1-2 ng of genomic DNA, 3μl of 10pM

concentration of each forward and reverse primer, 5μl of 10 X PCR buffers (Thermo

Scientific, USA), 8μl MgCl2 25mM (Thermo Scientific, USA), 5μl dNTPs 7.5mM (Thermo

Scientific, USA, #R0181), 500U taq polymerase (5U/μl concentration) (Thermo Scientific,

USA, #EP0402) 1μl and 23μl dH2O (Vandegrift, 2010). Thermal cycling steps were

carefully optimized keeping in view melting temperature and percentage of GC contents used

in PCR primers. PCR protocol that was performed involved 35 cycling steps and different

annealing temperature and duration of time according to the primers and PCR product is

summarized in (Table 2.2) (Fig. 2.2).

TABLE 2.2: Cycling conditions for mtDNA control region amplification of Saraiki


Cycling condition Temperature (°C) Duration

Pre- denaturation 95°C 11 minutes

Denaturation 95oC

30 Second

Annealing 56°C 30 Second

Annealing 72°C 90 Second

Extension 72°C 7 minutes

28

Fig. 2.2: Thermal cycling profile for mtDNA control region amplification of Saraiki


2.6 GEL ELECTROPHORESIS

After the completion of PCR reaction gel electrophoreses was carried out. Put 1gm of

agarose in a flask and final volume in flask was brought to 50ml by pouring the 0.5X TAE

buffer. The flask was heated in the microwave oven until the agarose was dissolved and the

solution in flask appears clear. After cooling the flask to 50°C, 1μl of ethidium bromide

(Sigma-Aldrich, St. Louis, USA) was added and flask was swirled gently before casting the

gel and set for 30 minutes. Once the gel was casted 5μl of PCR product and 2μl 6x loading

dye (Thermo Scientific, USA, #R0611) was added to the wells in the gel. In addition to this

5μl of 100bp ladder (Thermo Scientific, USA, #SM0323) was loaded as a DNA marker. The

gel was run at 100 V for 45 minutes using Power Pac (Bio-Rad) until it covered the 2/3 of the

distance. For required results gel was observed under ultra violet (U.V) light using and

Photographs were taken under UV transilluminator using (Photo Doc-It™ Imaging System,

UK) (Sambrook and Russell, 2001).

29

2.7 PURIFICATION OF PCR PRODUCTS

After the successful PCR reactions, purification of PCR product was carried out using

Gene JET PCR Purification Kit #K0702 (Thermo Scientific USA) for sequencing. For this

purpose binding buffer was added in PCR products at the ratio of 1:1 by volume in this way a

solution of 800μl was prepared. Then this prepared solution was transferred in the Gene JET

purification column. Centrifuged it for 50 second at 10000 rpm using Sigma 2-16PK

refrigerated laboratory Centrifuge and discarded the flow through. Then added 700μl of

Wash Buffer to the purification column, centrifuged for 50 second at 10000 rpm. Discarded

the filtrate and put the purification column again into collection tube. Centrifuged empty

Gene JET purification column for 1 minute to completely remove any residual wash buffer

and it was essential because the presence of residual ethanol in the DNA sample may inhibit

subsequent reactions. Then, the Gene JET purification column was shifted to a clean micro

centrifuge tube. After that, added 50μL of Elution Buffer in the center of the Gene JET

purification column and centrifuge it for 1 minute at 10000 rpm. Remove Gene JET

purification column and store the purified DNA at -20°C.

2.8 mtDNA SEQUENCING

Mitochondrial DNA sequences are very significant because mtDNA sequence is

related with the geographic origin of the individuals (Maruyama et al., 2009). mtDNA

sequences covering the entire control region (16024-576) was analyzed according to

Irwin et al., 2007. Sequencing was carried out using Big Dye Terminator Cycle Sequencing

v3.1 Kit (Applied Biosystems; Carlsbad, CA, USA) according to the protocol as well as

Sequencing facilities were availed from Center of Excellence in Molecular Biology (CEMB)

Lahore, Pakistan and some other commercial facilities (www.base-asia.com) were also

30

utilized for cost effectiveness. Sequencing reactions were performed using the same set of

forward F15975 and reverse R635 primers separately as for PCR amplification (Chen et al.,

2008).

2.9 SEQUENCING ANALYSIS

In order to know about mtDNA sequence data, I analyzed mitochondrial sequence

variations in Saraiki ethnic group. Investigations were based on HVRI, HVRII and HVRIII in

order to determine the mitochondrial haplogroups distribution and haplotype variations. The

nucleotide positions 16024-576 were analyzed for this purpose. Then aligned data was

compared with revised Cambridge Reference Sequence (Bar et al., 2000). Genious software

(Version 7.1.5, Biomatters Ltd, New Zealand) was used for this alignment and on the basis of

mtDNA control region polymorphisms, haplogroups were assigned (van Oven and Kayser,

2009). The mutations in sequences were thoroughly reviewed to verify their existence using

Geneious software (Drummond et al., 2009). All the samples were sequenced bi-

directionally. Two researchers checked the sequences for verification and individual profile

of each sample was generated. Mito tool and HaploGrep were used to access the quality of

mtDNA data (Fan and Yao, 2011; Kloss-Brandstatter et al., 2011) and for the assignment of

the haplogroups to the donors. In addition to mito tool and HaploGrep, mtDNA software was

also used to access the quality of mtDNA data. PhyloTree Build 16

(http://www.phylotree.org) (van Oven et al., 2008) as classification tree was also used to

assess the quality of mtDNA data (Fan and Yao, 2011, Yang et al., 2013).

2.10 STATISTICAL ANALYSIS

Polymorphic patterns in Saraiki population were performed using Genious software

(Version 7.1.5, Biomatters Ltd, New Zealand). The differences and comparison between

31

mtDNA control regions polymorphism in Saraiki population and other previous studies in

this region were analyzed through statistical analysis. Haplotype diversity and random match

probability was calculated and power of discrimination was statistically evaluated according

to (Tajima, 1989).

2.11 PHYLOGENETIC ANALYSIS

Different methods like minimum spanning, neighbor-joining, maximum likelihood

and maximum parsimony are used for construction of a phylogenetic tree for the mtDNA

sequences. All these methods were characterized by the organized scientific community.

Evolutionary analyses were also carried out in MEGA6 (Tamura et al., 2013). In present

study evolutionary history was analyzed using the Maximum Likelihood method based on

the model presented by Jukes (Jukes and Cantor, 1969).

Plan of this research work is summarized in (Fig. 2.3).

32

Fig. 2.3: Summary of Research work plan for the Saraiki population mtDNA control region

analysis from Pakistan.

Population Blood Sampling

DNA Extraction

DNA Quantification

mt DNA Optimization/Amplification

Complete Control Region Sequencing

Alignment of sequences and Comparison with reference mtDNA

Comparative Analysis with other mtDNA data bases

Phylogenetic Analysis

33

3. RESULTS

Present study was conducted to seek the relationships of Saraiki population from

Pakistan with other populations based on mtDNA sequence data for forensic purpose. For

this study 85 samples of blood were collected from healthy unrelated Saraiki people living in

various areas of Pakistan (Fig. 3.1) and the number of samples collected from those areas had

shown graphically (Fig. 3.2). Out of 85 individuals male samples were 75(88.24%) and

10(11.76%) were females samples (Fig. 3.3). All of these donors were of different age group

ranging in age from 18-60 years (Fig. 3.4). The consanguinity (%) of donors was also

calculated (Fig. 3.5) and the demographic data of the Saraiki donors was summarized in

(Table 3.1).

TABLE 3.1: Showing consent form summarized data of Saraiki population from Pakistan.

Sample

ID

Gen.

Ethnic

Group

Age

(Yrs)

Place of

Birth

Consangui-

nity of

Parents

Mother's

Place of

Birth

Father's

Place of

Birth

Mother

Language

SS-001 M Saraiki 21 Layyah Within caste Layyah Layyah Saraiki

SS-002 M Saraiki 29 Layyah Out of Caste Layyah Layyah Saraiki



SS-005 F Saraiki 24 Layyah Mamozad Layyah Layyah Saraiki



SS-008 F Saraiki 24 Layyah Out of Caste Layyah Layyah Saraiki


34




SS-013 M Saraiki 32

Layyah Khalazad Layyah Layyah Saraiki

SS-014 M Saraiki 23

Layyah Mamonzad Layyah Layyah Saraiki

SS-015 M Saraiki 60

M.Garh Phhopizad M.Garh M.Garh Saraiki

SS-016 M Saraiki 28

M.Garh Mamonzad M.Garh M.Garh Saraiki

SS-017 M Saraiki 31

M.Garh Khalazad M.Garh M.Garh Saraiki

SS-018 M Saraiki 41

M.Garh Mamonzad M.Garh M.Garh Saraiki

SS-019 M Saraiki 24 M.Garh Chachazad M.Garh M.Garh Saraiki

SS-020 M Saraiki 37 M.Garh Within caste M.Garh M.Garh Saraiki

SS-021 F Saraiki 22 M.Garh Mamonzad M.Garh M.Garh Saraiki

SS-022 M Saraiki 34 M.Garh Within caste M.Garh M.Garh

Saraiki

SS-023 M Saraiki 32 M.Garh Mamonzad M.Garh M.Garh Saraiki


Saraiki


Saraiki

SS-026

F Saraiki 19 M.Garh Mamonzad M.Garh M.Garh Saraiki

SS-027

F Saraiki 18 M.Garh Mamonzad M.Garh M.Garh Saraiki

SS-028

F Saraiki 19 M.Garh Chachazad M.Garh M.Garh Saraiki


Saraiki

SS-030 M Saraiki 30

M.Garh Chachazad M.Garh M.Garh Saraiki


Saraiki

SS-032

M Saraiki 50 M.Garh Mamonzad M.Garh M.Garh Saraiki

35


Saraiki


Saraiki

SS-035 M Saraiki 26 Mianwali Within caste Mianwali Mianwali

Saraiki

SS-036 M Saraiki 24 Mianwali Within caste Mianwali Mianwali Saraiki


Saraiki

SS-038 M Saraiki 23 Layyah Within caste Layyah Layyah

Saraiki


Saraiki

SS-040

F Saraiki 31 Bhakar Out of caste Bhakar Bhakar Saraiki

SS-041

F Saraiki 33 Bhakar Within caste Bhakar Bhakar Saraiki

SS-042 M Saraiki 50 Bhakar Within caste Bhakar Bhakar Saraiki

SS-043 M Saraiki 45 Bhakar Within caste Bhakar Bhakar

Saraiki


Saraiki


Saraiki


Saraiki

SS-047 M Saraiki 55 Layyah Out of caste Layyah Layyah Saraiki

SS-048 M Saraiki 35 D.I.Khan

Chachazad D.I.Khan D.I.Khan Saraiki


Within caste D.I.Khan D.I.Khan Saraiki

SS-050 M Saraiki 38 D.I.Khan Within caste D.I.Khan D.I.Khan Saraiki

SS-051 M Saraiki 18 D.I.Khan Chachazad D.I.Khan D.I.Khan Saraiki


Saraiki

SS-053

M Saraiki 26 Mianwali Chachazad Mianwali Mianwali Saraiki


Out of caste D.I.Khan D.I.Khan Saraiki

36


Saraiki

SS-056 M Saraiki 38 Umar

koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Phhopizad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Phhopizad Umar

koat

Umar

Koat

Saraiki


koat

Khalazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Chachazad Umar

koat

Umar

Koat

Saraiki


koat

Out of caste Umar

koat

Umar

Koat

Saraiki


koat

within caste Umar

koat

Umar

Koat

Saraiki


koat

Khalazad Umar

koat

Umar

Koat

Saraiki


koat

within caste Umar

koat

Umar

Koat

Saraiki

SS-075 Male Saraiki 20 Umar

koat

Phhopizad Umar

koat

Umar

Koat

Saraiki


koat

Mamonzad Umar

koat

Umar

Koat

Saraiki

37


koat

Chachazad Umar

koat

Umar

Koat

Saraiki

SS-078 Male Saraiki 45 D.I.Khan Chachazad D.I.Khan D.I.Khan Saraiki

SS-079 Male Saraiki 30 D.I.Khan within caste D.I.Khan D.I.Khan Saraiki

SS-080

Male Saraiki 35 D.I.Khan within caste D.I.Khan D.I.Khan Saraiki

SS-081 Male Saraiki 30 D.I.Khan within caste D.I.Khan D.I.Khan Saraiki

SS-082 Male Saraiki 30 D.I.Khan Chachazad D.I.Khan D.I.Khan Saraiki


koat

Mamonzad Umar

koat

Umar

Koat

Saraiki


koat

within caste Umar

koat

Umar

koat

Saraiki


koat

within caste Umar

koat

Umar

koat

Saraiki

M. Garh, Muzaffar Garh; D. I. Khan, Dera Ismail Khan; Khalazad, 1st Maternal cousin;

Chachazad, 1st Paternal cousin; Mamonzad, 2

nd Maternal Cousin; Phhopizad, 2

nd Paternal

Cousin; Gen. Gender; M, Male; F, Female; Yrs, Years

Fig. 3.1: Map of Pakistan showing study areas of Saraiki population.

38

M. Garh= Muzaffar Garh; D. I. Khan= Dera Ismail Khan

Fig. 3.2: Showing sampling areas along with numbers of samples from the Saraiki donors of

Pakistan.

Fig. 3.3: Showing total samples including males and females of Saraiki population from

Pakistan.

39

Fig. 3.4: Showing donors of different age groups (18-60 years) along with number of

samples from Saraiki population of Pakistan.

Fig. 3.5: Consanguinity % age of parents in Saraiki population of 85 samples collected from


40

During this study the nucleotide sequences of the entire mtDNA control regions

(nt 16024-16569 and nt 1-576) were determined in 85 unrelated individuals from Saraiki

population after DNA extraction and PCR amplification. Presence of extracted DNA was

confirmed by the bands appeared on gel (Fig. 3.6-3.7). The PCR amplification products size

was also observed by gel electrophoresis and it was 1122bp (Fig. 3.8-3.9).

After the PCR amplification of the samples, the PCR products were subjected to

sequencing in order to observe the mutations in Saraiki population with reference to revised

Cambridge Reference Sequence (rCRS) (Fig. 3.10-3.14). The observed mutations in this

population samples compared to rCRS were, transition (75.58%) transversion (4.67%),

insertion (15.56%) and deletion (4.17%). The mutation types for each hypervariable region

were described in (Table 3.2).

TABLE 3.2: Occurrence and distribution of nucleotide substitutions, deletions and insertions

in the hypervariable regions I, II and III of mitochondrial DNA control region from unrelated

Saraiki persons of Pakistan.

Mutation type HV1 HVII HVIII

Substitutions

Number

of

positions

Total

number of

mutations

Number

of

positions

Total

number of

mutations

Number

of

positions

Total

number of

mutations

Transitions

A-G 7 39 9 203 2 2

G-A 7 34 2 13 2 4

C-T 24 122 6 42 6 17

T-C 19 144 10 92 4 31

Total 57 339 27 350 14 54

41

Transversions

A–C 5 14 0 0 2 2

G–C 0 0 0 0 1 1

A–T 4 18 0 0 0 0

G–T 0 0 0 0 0 0

C–A 1 3 0 0 1 2

C–G 0 0 1 1 1 1

T–A 1 2 1 2 0 0

T–G 0 0 0 0 0 0

Total 11 37 2 3 5 6

Insertions

T 0 0 0 0 4 6

C 3 3 5 135 0 0

2CA 0 0 0 0 2 5

2C 0 0 1 4 0 0

Total 3 3 6 139 6 11

Deletion

A 0 0 0 0 2 18

C 0 0 1 1 4 21

G 0 0 0 0 1 1

Total 0 0 1 1 7 40

A, adenine; G, guanine; C, cytosine; T, thymine; HVI, hypervariable region I; HVII,

hypervariable region II; HVIII, hypervariable region III

42

During this study total 983 polymorphic sites were detected in Saraiki population

data. Overall genetic diversity was high (0.9570) with 63 different haplotypes found in 85

individuals with high Power of discrimination (0.9458). Out of 63 haplotypes, 58 of them

were unique and 5 haplotypes were shared by more than one individual. Different

haplogroups having various haplotypes from the different Saraiki areas of Pakistan were

identified during this study (Table 3.3) and their %age frequency of was also calculated

(Table 3.4).

The commonly observed haplogroups in this population were West Asian haplogroup

W6 and East Eurasian haplogroup M5c1, each of these constitute the 12.9% and 11.7% of the

population respectively. The South Asian haplogroups have clear dominance (29.4%)

including U2b2 (9.4%), M2a1a (1.1%), R9 (1.1%), M4 (1.1%), U2c'd (2.3), U2+152 (1.1%),

M18a (2.3%), HV2a (1.1%), R31 (1.1), U8c (1.1%), U4a2a (1.1%), M30+16234 (1.1%) and

relevant subgroups.

The second major types of haplogroups were West Eurasian and South Asian, which

cover the 20% population including U7a (8.2%), U7 (7.0%), U2a1a (3.5%) and relevant

subgroups. East Eurasian and South Asian haplogroup also represented by the 20%

population including M5c1 (11.7%), M5a2a1a (3.5%), M5 (2.3%), L3e'i'k'x (2.3%), M5b2

(1.1%) and relevant subgroups.

Third most prevalent type of haplogroup was West Asian to claim the 16.4%

population including I (1.1%), W6 (12.9%), X2 (1.1%), X2d (1.1%). West Eurasian

haplogroups cover 3.5% population including, R2 (2.3%) and H2a2a1g (1.1%). Combining

HVR1, HVR2 and HVR3, the genetic diversity was found 0.9570 (Table 3.5) (Fig. 3.12)

thus providing a high level of identification.

43

Fig. 3.6: Agarose gel electrophoresis showing the bands of extracted genomic DNA of

different quantity along with 100 bp DNA marker (M) and (-ve) negative control while 001,

002, 003, 004, 005 are samples ID.

Fig. 3.7: Agarose gel electrophoresis showing the bands of extracted genomic DNA of

different quantity along with 100 bp DNA marker (M) and (-ve) negative control while 053,

054, 055, 056, 057 are samples ID.

44

Figure 3.8: PCR product size of the samples, 010, 011, 012, 013,014 loaded on 2% agarose

gel, (–ve) is negative control, (M) is 100bp DNA marker.

Figure 3.9: PCR product size of the samples, 015, 016, 019, 020,021 loaded on 2% agarose

gel, (–ve) is negative control, (M) is100bp DNA marker.

45

Figure 3.10: Sequence showing haplotype variations in sample SS-054.

50

Figure 3.11: Showing haplotype variations with reference to rCRS in sample SS-035.

.

56

Figure 3.12: Showing haplotype variations with reference to rCRS in sample SS-010.

59

Figure 3.13: Showing haplotypes variation with reference to rCRS in sample SS-011.

64

Figure 3.14: Showing haplotype variation with reference to rCRS in sample SS-012.

67

Fig. 3.15: Different Haplogroups frequency % age found in 85 Saraiki population sample

from different areas of Pakistan.

68

Fig. 3.16: Comparison of genetic diversity among Saraiki population and others various

ethnic groups of Pakistan (Murci et al., 2004; Rakha et al., 2011).

Table 3.3: Estimated haplotypes and haplogroups in Saraiki population from different areas

of Pakistan.

Sample

I.D

N Area HG HG T Haplotypes

SS-001

03

Ly

U2b2

SA

16051G 16209C 16239T 16352C

16353T 16362C 73G 146C 152C

189G 234G 263G 315.1C

513.1CACA

SS -002

01

Ly

U2b2

SA

16051G 16209C 16239T 16352C

16353T 16362C 73G 146C 152C

189G 234G 263G 315.1C

SS-003

01

Ly N10a

SEA

16129A 16183C 16184.1C 16189C

16210T 16223T 16266.1C 16362C

73G 185A 189G 263G 315.1C

69

SS-004

01

Ly

D4j1b2

SEA 16129A 16182.1C 16183C 16189C

16223T 16362C 73G 185A 189G

263G 315.1C 489C

SS-005

01

Ly

M5

EEA/SA

16129A 16183C 16223T 73G 263G

315.1C 489C

SS-006

01

Ly

U2b2

SA

16051G 16209C 16239T 16352C

16353T 16362C 73G 146C 152C

189G 234G 263G 315.1C

517.1CACA

SS-007

01

Ly

U2a1a

WEA/SA

16051G 16154C 16206C 16230G

16311C 16325C 16519C 73G 263G

309.1C 315.1C

SS-008

01

Ly

U7a

WEA/SA

16309G 16318T 16519C 16527T

73G 151T 152C 263G 309.1C

315.1C 515d 516d

SS-009

01

Ly

M2a1a

SA

16223T 16270T 16319A 16352C

16519C 73G 195C 204C 263G

315.1C 447G 489C

SS-010

01

Ly

R9

SA

16129A 16304C 16519C 73G 152C

234G 263G 309.1C 315.1C 516d

517d

SS-011

01

Ly

U2a1a

WEA/SA

16051G 16154C 16206C 16230G

16311C 16519C 73G 195C 263G

309.1CC 312.1C

SS-012

01

Ly

I

WA

16129A 16223T 16311C 16391A

16519C 73G 199C 204C 250C

263G 309.1C 315.1C 454.1T

SS-013

01

Mg X2

WA

16189C 16323T 16519C 73G 153G

195C 263G 309.1C 315.1C

SS-014

01

Mg M30+16234 SA

16093C 16223T 16234T 16274A

16325C 16519C 73G 195A 263G

309.1C 315.1C 489C 514d 515d

SS-015

01

Mg U7

WEA/SA

16309G 16318T 16519C 73G 152C

263G 309.1CC 315.1C 515d 516d

525T

SS-016

10

Mg;

Ly

M5c1

EEA/SA

16129A 16223T 16519C 73G 150T

263G 309.1C 315.1C 333C 489C

575T

SS-017

01

Mg

U7a

WEA/SA

16318T 16519C 73G 151T 152C

263G 309.1C 315.1C 514d 515d

SS-018

01

Mg U7

WEA/SA

16092C 16183C 16309G 16318T

16519C 73G 152C 263G 315.1C

525T

70

SS-019

01

Mg U7

WEA/SA

16309G 16318T 16519C 73G 152C

263G 309.1CC 315.1C 514d 515d

525T

SS-020

01

Mg L3e'i'k'x EEA/SA

16129A 16223T 16519C 73G 150T

263G 309.1CC 315.1C

SS-021

01

Mg L3e'i'k'x

EEA/SA

16129A 16223T 16519C 73G 150T

263G 309.1C 315.1C

SS-022

01

Mg M5a2a1a

EEA/SA

16129A 16218T 16223T 16264T

16265C 16319A 16519C 73G 263G

309.1C 315.1C 489C

SS -023

01

Mg X2d

WA

16183C 16189C 16210C 16223T

16278T 16311C 16519C 73G 195C

263G 315.1C

SS-024

01

Mg U2

WEA/SA

16051G 16172C 16184T 73G 263G

309.1C 315.1C

SS -025

01

Mi U2

WEA/SA

16051G 16172C 16184T 73G 263G

309.1C 315.1C 517d 518d

SS-026

01

Mi U7a

WEA/SA

16069T 16274A 16318T 16519C

73G 151T 152C 263G 315.1C 514d

515d

SS-027

01

Mi M5a2a1a

EEA/SA

16129A 16223T 16265C 16344T

16519C 73G 263G 315.1C 374G

489C

SS-028

01

Ly U2b2

SA

16051G 16209C 16239T 16278T

16352C 16353T 64d 73G 146C

152C 234G 263G 310.1C

SS-029

01

Mi U2+152

SA

16051G 16129A 16247G 16254G

73G 150T 152C 263G 309.1C

315.1C 456T

SS-030

01

Bk M18a

SA

16134T 16223T 16318C 16519C

73G 93G 194T 246C 263G 315.1C

489C

SS-031

01

Bk M18a

SA

16134T 16323T 16318C 16519C

73G 93G 194T 246C 263G 315.1C

489C

SS-032 01 Bk U2b2 SA 16051G 16209C 16239T 16352C

16353T 73G 146C 151T 152C

234G 263G 315.1C

SS-033

01

Bk U2b2

SA 16051G 16209C 16239T 16352C

16353T 73G 146C 152C 234G

263G 315.1C

71

SS-034

01

Ly HV2a

SA

16214T 16217C 16335G 16519C

73G 152C 246C 263G 309.1C

315.1C 514G 527T 574C 576C

SS-035

01

Ly U2b2

SA

16051G 16209C 16239T 16352C

16353T 16362C 73G 146C 152C

189G 234G 263G 315.1C 320G

513.1CACA

SS-036

01

DIK M3a1+204 SA/AF

16223T 16311C 16519C 73G 204C

217C 263G 309.1C 315.1C 482C

489C

SS-037

01

DIK R31

SA

16129A 16213A 16362C 16519C

16525G 73G 263G 309.1C 315.1C

SS-038

01

DIK U2b2

SA

16051G 16209C 16239T 16352C

16353T 73G 146C 234G 263G

315.1C

SS-039

01

DIK

U2a1a

WEA/SA

16029A 16051G 16154C 16206C

16230G 16311C 16326C 16519C

73G 263G 309.1C 315.1C 569.1T

SS -040

01

Mi

H2a+152

16311

SWA

16311C 152C 263G 309.1C 315.1C

513A

SS-041

01

Mi J1b1b

EA

16069T 16126C 16145A 16186T

16263.1A 16519C 73G 263G 271T

295T 309.1C 315.1C 462T 489C

508G 514d 515d

SS-042

01

DIK U7a

WEA/SA

16309G 16318T 16519C 73G 151T

152C 263G 309.1C 315.1C 516d

517d

SS-043

01

Mi

W6

WA

16192T 16223T 16292T 16519C

73G 152C 189G 194T 195C 204C

207A 263G 309.1C 315.1C

SS-044

01

Uk M4

SA

16145A 16176T 16223T 16261T

16266T 16291T 16311C 16519C

73G 263G 315.1C 489C

SS-045

10

Uk,

DIk

W6

WA

16192T 16223T 16266T 16292T

16325C 16519C 73G 189G 194T

195C 204C 207A 263G 309.1C

315.1C

SS-046

01

Uk U2c'd

SA

16051G 16126C 16178C 16179T

16234T 16247G 73G 146C 152C

263G 315.1C 570d

72

SS-047

01

Uk U2c'd

SA

16051G 16234T 16247G 16254G

16311C 16519C 73G 150T 152C

263G 315.1C 513d 514d

SS-048

01

Uk U7a

WEA/SA

16309G 16318C 16519C 73G 151T

152C 263G 309.1C 315.1C 514d

515d

SS-049

01

Uk

U7a

WEA/SA

16309G 16318T 16519C 73G 151T

152C 263G 309.1C 315.1C 515d

516d

SS-050

01

Uk U8c

SA

16168A 16309C 16318C 16519C

73G 151T 152C 263G 309.1C

315.1C

SS-051

01

Uk M5a2a1a

SA

16129A 16223T 16265C 16362C

16519C 73G 263G 315.1C 489C

516A

SS-052

01

Uk

R2

WEA

16071T 16111T 16278T 16311C

16519C 73G 150T 152C 263G

315.1C

SS-053

01

Uk D4a

EA

16129A 16223T 16362C 16519C

73G 152C 263G 315.1C 489C

516A

SS-054

01

Uk H2a2a

SWA

16243C 16519C 153G 200G 263G

309.1C 315.1C

SS -055

01

Uk

U7a

WEA/SA

16168A 16309G 16318C 16519C

73G 151T 152C 263G 309.1C

315.1C

SS -056

01

Uk R2

WEA

16071T 16111T 16519C 73G 150T

152C 263G 315.1C

SS -057

01

Uk U4a2a

SA

16519C 73G 195C 263G 310C

SS -058

01

DIK M5b2

EEA/SA

16048A 16129A 16223T 16519C

73G 263G 309.1C 315.1C 489C

509A 517d 518d

SS-059

02

Uk D4j1a EA

16086C 16189C 16223T 16266T

73G 263G 309.1C 315.1C 489C

SS-060

01

DIK

M30

SA

16223T 16519C 73G 152C 195A

263G 309.1C 315.1C 489C 514d

515d 569.1T

SS-061

01

Uk M5

EEA/SA

16029A 16037T 16129A 16223T

16519C 73G 263G 315.1C 489C

544.1T 550C 564C 569.1T 571T

73

SS-062

01

Uk U7

WEA/SA

16309G 16318T 16519C 73G 152C

263G 268T 309.1C 315.1C 499A

SS-063

01

Uk U7

WEA/SA

16264T 16309G 16318T 16519C

73G 152C 263G 309.1C 315.1C

499A 514d 515d

SS-064

01 Uk U7

WEA/SA

16264T 16309G 16318T 16519C

73G 152C 263G 309.1C 315.1C

499A 514d 515d

Bold Italic characters in the table highlight those nucleotides that were used to define

haplogroups in the population.

N, Hyplotypes numbers; HG, haplogroup; HGT, Haplogroup Type; SA, South Asian; WEA,

West Eurasian; SEA, South East Asian; EEA, East Eurasian; WA, West Asian; SWA, South

West Asia; EA, East Asia; AF, Africa; Mg, Muzaffar garh,; Ly, Layyah; Bk, Bhakar; Mi, Mianwali;

DIK, Dera Ismail Khan; UK, Umar kot

TABLE 3.4: Haplogroups (%) frequency in Saraiki population from Pakistan.

Haplogroup Sample Frequency

(%)

Haplogroup Sample Frequency

(%)

U2b2

08 9.4 H2a2a

01 1.1

N10a

01 1.1 L3e'i'k'x 02 2.3

D4j1b2

01 1.1 M5a2a1a 03 3.5

M5

02 2.3 X2d

01 1.1

U2a1a

03 3.5 U2

01 1.1

U7a

07 8.2 H2a2a1g

01 1.1

M2a1a 01 1.1 U2+152 01

1.1

R9

01 1.1 M18a

02 2.3

I

01 1.1 HV2a

01 1.1

X2

01 1.1 M3a1+204 01 1.1

M30+16234 01 1.1 R31 01 1.1

74

U7 06 7.0 H2a+152

16311

01

1.1

M5c1

10 11.7 J1b1b 01

1.1

W6

11 12.9 M4

01 1.1

U2c'd

02 2.3 U8c

02 2.3

R2

02 2.3 U4a2a

01 1.1

M5b2

01 1.1 D4j1a 02 2.3

M30

01 1.1 D4a

01 1.1

TABLE 3.5: Population genetics parameters of Saraiki population from different areas of

Pakistan.

Total no. of the samples 85

Haplotypes 63 (05 Shared)

Polymorphic sites 140

Random match probability 0.0542

Power of discrimination 0.9458

Genetic diversity 0.9570

This study characterize mtDNA control region data using the global mtDNA

phylogeny background (Parson and Bandelt, 2007; van Oven and Kayser, 2009), from the

Saraiki population and established forensic guidelines for mtDNA reference data generation

of the population.

CLUSTER ANALYSIS

A dendrogram was constructed of Saraiki population from Pakistan using maximum

likehood method to assess the genetic affinities among the individuals of Saraiki population

(Fig. 3.16). The bootstrap consent tree roundabout from the 100 replicates was taken to

75

characterize the history of evolution from analyzed texa (Felsenstein, 1985). Initial tree for

basic evolution research was constructed by applying the Neighbor-Joining method to a

model of pair wise distances estimation using the Maximum Composite Likelihood method.

In order to model evolutionary rate differences among sites (5 categories (+G, parameter =

0.3762)) a discrete Gamma distribution was used. Total 557 positions were present in the

final dataset.

Dendrogram presented a picture of genetic relationship and variability among the

individuals of the population. According to dendrogram this population was grouped into

eleven clades. In the dendrogram it was observed that generally all the Saraiki individuals

showed genetically close association among each other‟s. The clade I includes samples 001,

003, 007, 008, 009, 013, 35, 49, 50, 54, 56, 71, 23, 39 and 60. The analysis of this clade

showed that samples 001, 007; 003, 008; 009, 013; 54, 56 and 60, 23 showed genetically

common behavior among each other‟s while 35, 39, 50, 49 and 71 showed some distance

from other individuals of this clade. Clade II consists of samples number 010, 017, 019, 24,

47, 51, 52, 57, 61, 31, 32, 40, 42, 58 and 63. In this clade samples number 057, 58; 47, 31;

51, 52; 63,42 and 010, 017 showed genetic similarities while 019, 32, 61 and 24 had some

distance from other individual of this clade and it was also observed that 40 is very close to

63 and 42 as compared to other outlier individuals of this clade. Clade III includes samples

015, 34, 66, 37 and 62. In this clade 66, 37 had similar genetic make-up while 015, 034

samples were separated from 127 to show different genetic behavior. Clade IV consists of

011, 016, 36, 55, 38, 44, 43, 41, 46 and 64 samples. According to this clade observation 46,

55; 43, 44; and 41, 64 each showed similar genetic structure. It has also been observed that

41 and 64 showed different genetic from 38 sample while 011, 016, 36 were at some distance

76

from other members of this clade. Clade V include two samples 002 and 012 both of these

individuals expressed very closed genetic relationship. In clade VI 33 and 48 samples were

very similar while 006 was separate from 004 and 005 samples which were close to each

other‟s. In this clade analysis it was also observed that sample 45 showed distinct behavior

from other five samples. The clade VII include samples 14, 25 and 26 in which 14 and 26

samples were genetically very close to each other‟s while sample number 25 was at some

distance. The samples ID 018 and 59 of clade VIII genetically expressed very close

relationship and same expression had been shown by clades IX and XI samples but in clade

X, 22 and 30 showed close phylogenetic relationship while sample 27 showed separate

genetic behavior.

77

Fig. 3.17: Dendrogram showing genetic affinities among various Saraiki samples from


78

4. DISCUSSION

Present day Pakistan is bounded by Iran and Afghanistan in west, India in east and

China in north. This study established the haplotype diversity of Saraiki population and

makes a distinction between Saraiki individuals living in different areas of Pakistan. Saraiki

people are divided into distinct haplogroups and lineages. Results of present investigation

conducted on 85 blood samples collected from unrelated healthy males and females of

different age groups. It was observed that different cultural groups were present in every

society and their origin was known from various records like fossils and archaeology records

(Ariffin et al., 2007). Saraiki population in Pakistan is ethnically and linguistically different

from other populations. In order to understand the genetic structure of Saraiki population,

this study was conducted because still no study has been accompanied in Saraiki population

from Pakistan. For this purpose mtDNA has been widely used to study the human migration,

evolution, ancestor identification and forensics cases (Pakendorf and Stoneking, 2005;

Ariffin et al., 2007).

This was the first study which published entire mtDNA control region sequences of

Sariki population from Pakistan. The most important properties of mtDNA D-loop that

formulate it more valuable for human identification and its evolutionary studies include the

large number of copies, maternal inheritance and very fast rate of development (Pereira et al.,

2010). mtDNA mutation rate is several times high as compared nuclear gene (Ingman et al.,

2000). On account of these and some other properties mitochondrial DNA could be used in

forensic investigations in the Asia (Hoong and Lek, 2005). Mitochondrial DNA D-loop

which is known as control region is very important for forensic cases and population study in

the region (Barbosa et al., 2008).

79

mtDNA sequence analysis in Saraiki population from Pakistan showed high level of

genetic diversity (0.957) from Kalashi (0.851), Parsi (0.950) and Brahui (0.952) populations.

By comparing among above populations it was concluded that the observed high genetic

diversity in Saraiki population as compared to others population be due to cultural practices

and strong endogamy in those populations as compared to Saraiki population (Ghosh et al.,

2011). In the same way some other populations Pathan (0.993), Baluch (0.974), Hazara

(0.992), Hunza Burusho (0.980), Makrani (0.975), Pakistani Karachi (0.992) and Sindhi

(0.992) from Pakistan showed high genetic diversity than that of kalashi, Brahui and Saraiki

populations (Murci et al., 2004; Rakha et al., 2011) because they may be less endogamous as

compared to Kalashi and Saraiki populations. During this study genetic structure, genetic

variations and genetic distances were investigated among different samples of the population

on base of haplotypes data (Adams et al., 2008).

During the present study, it was observed that the South Asian haplogroups had clear

dominance having 29.4% frequency including U2b2 9.4% and some other relevant sub

groups. U haplogroup is the second most haplogroup found in India and is known as a

complex mtDNA lineage with an age of 51000-67000 years. It has been observed that sub-

clade U2 of U haplogroup is most common in South Asia (Metspalu et al., 2004) and is less

common in West Asia and Europe (Maji et al., 2008). This indicates that Saraiki population

is more close to South Asian haplogroup as compared to others haplogroup.

The second major types of haplogroups in the present study were West Eurasian and

South Asian, which cover the 20% population including U7a 8.2%, U7 7.0% and relevant

subgroups. The presence of the haplogroup U7 in other populations indicates that Saraiki

population has some association with those populations. U7 is Eastern and Indian haplogroup

80

(Murci et al., 2004). I was observed that in many European populations haplogroup U7 was

absent (Richards et al., 2000) showing the limited association of European populations with

Asian populations. It has been noted that in West Asia U7 frequency is 4% Near East and

10% in Iran while in South Asia its frequency is 12% in Gujarat and in whole India its

frequency remained 2% (Metspalu et al., 2004). While during present study its frequency

was noted up to 7% (Sikandar et al., 2014). Haplogroup U7 has a sub clade U7a whose

frequency in Sri has been found up to 13.3% (Ranaweera et al., 2014) while in Makrani

population from Pakistan its frequency has been found up to 6% (Siddiqi et al., 2014), this

low frequency indicate that Makrani population is more endogamous. It has been observed

that haplogroup U7a is found nearly in all those populations where the U7 haplogroup has

been found. It has also been observed that these variations in haplogroups frequency are

based upon geographic distribution (Rosser et al., 2000).

East Eurasian and South Asian haplogroups are also represented by the 20%

population in present study including M5c1 11.7% and relevant subgroups. Traces of gene

flows from the Indian subcontinent to Pakistan had been reported and it was confirmed by

the presence of sub-haplogroup M5C1 about which it is thought that it was originated

probably in central India and from India it spread out to its eastern and western regions

(Chandrasekar et al., 2009). The Asian mtDNA phylogeny is subdivided into two major

haplogroups one of them is haplogroup M and other is N haplogroup. The most common

haplogrou in Indian population is M haplogrou and is rare outside Southern Asia

(Kalaydjieva et al., 2005). The higher distribution of haplogroups M and N all over the world

indicates that when humans migrate out of Africa both M and N haplogroups were part of the

that colonization (Macaulay et al., 2005). There are different hypothesis about the origin of

81

M haplogroup. Some scientists claimed that m haplogroup was originated from South West

Asian and followed back migration to Africa (Kivisild et al. 2003) while others other

scientists believe in African ancestry for M haplogroup (Murci et al., 1999).

Third most prevalent type of haplogroup in present study was West Asian which

claims the 16.4% population including W6 12.9%. Although haplogroup W is not found in

high frequency in European populations but at low frequency it is common in that population

(Richards et al., 2000). Haplogroup W6 was found to be 5.3% in Georgia (Olivieri et al.,

2013) while during present study its frequency was found 12.9% (Sikandar et al., 2014). W6

haplogroup is also found in German individuals which belonging to the Corded Ware culture

(Adler, 2012). It has been observed that variations in the haplogroup are due to geographic

differences (Rosser et al., 2000).

Results of present study show that West Eurasian haplogroups cover 3.5% population

including, R2 2.3% and H2a2a1g 1.1%. R2 Haplogroup is abundantly present in southern

Pakistan because its frequency in Kalashi and Makrani population has been calculated 6.3%

and 6% respectively (Siddiqi et al., 2014). It has also been observed that haplogroup R2 was

also present in India population while its low frequency was recorded in some regions like,

Near East, Iran, Central Asia and Arabian Peninsula (Murci et al., 2004; Metspalu et al.,

2004; Al-Abri et al., 2012). The presence of R2 haplogroup in European population is

limited (Bermisheva et al., 2002). Collectively U7, W and R2 comprise about 14% of the

mother gene pool in India (Metspalu et al., 2004)) and while in Iran and Pakistan it was 13%

and 10% respectively but it declined in the Central Asia and reached upto 5% (Metspalu et

al., 2004). Moreover in Western India, haplogrou R2 was found with the frequency of 9.5%

(Khurana et al., 2014). But this R2 haplogroup frequency gradually decreases gradually as it

82

goes on toward the western part of India and then its frequency become insignificant in

Europe. This low frequency of clade R2 in Saraiki population is due geographic distribution.

J1b1b is one sub clade of haplogroup J and its frequency was 1.1% in present study which

confirms that Saraiki population has some association with, North Africa, Western Asia,

Europe and Central Asia (Karafet et al., 2008). The frequency of haplogroup D4a in Japanese

samples was found to be 7.3% (Tanaka et al., 2004) while in Pakistan its frequency in Saraiki

population has been noted 1.1% which indicate some association between these two

populations. mtDNA haplogroups diversity depends more upon geographic distribution of

the population (Mogentale-Profizi et al., 2001).

Results of present study confirm that all individuals of Saraiki population from

Pakistan show common maternal lineages although significant regional differences are

present. However, the fact that most of the closest phylogenetic branches of haplogroups of

Saraiki population mtDNAs have also been found in Indian population which suggests that

the Saraiki mtDNAs have close association with them. It has been pointed out that the

frequency and diversity of a haplogroups is indication of possible common origin of that

particular haplogroup (Barbujani, 2000).

This study had carried out the first extensive analysis of mtDNA diversity within

Saraiki population of Pakistan. This study allowed comparing the Saraiki population

diversity with that previously reported populations in Pakistan, to investigate differences

within Pakistani populations and to evaluate the population histories. In a worldwide

comparison, Saraiki population mostly clusters around a South Asian samples. It was

observed that maximum Pakistani populations clustered with South Asian and Middle

Eastern populations (Qamar et al., 2002). Mitochondrial DNA sequences analysis exposed

83

the very high level of genetic diversity in the Saraiki population which can be compared with

the others population from South Asia and West Eurasia. The Saraiki populations studied

exhibit similar mtDNA lineage composition with many haplogroups. Comparisons of

mtDNA haplogroup frequencies between the Saraiki populations of different areas revealed

that the genetic structure of the Saraiki people was very similar which may be the result of

endogamy.

In this study it has been found that South Asian haplogroup was dominant over others

haplogroups in Saraiki population so the unique geographical location of Saraiki poplulation

among West Eurasian and South East Asian may be due to heterogonous mitochondrial DNA

haplogroups make-up. The results of this study point out strong genetic affinities of Saraiki

population with other populations. Mitochondrial DNA analysis will be helpful for forensic

cases and genetic study.

CONCLUSION

Overall, this study provides broad mtDNA variations survey of Saraiki population

from Pakistan.

The molecular analysis of the Saraiki population is providing better knowledge of its

ancestry and diversity from other population on the basis of haplogroup distribution.

This study also emphasized the importance of gene migration between various

populations.

It also highlights the genetic affiliation of the Saraiki individuals with each other from


84

It has been calculated that this study will be valuable for creating a useful data of

Saraiki people inhabiting in different areas of Pakistan and will hand out as a

background for forensics data base of different Pakistani populations.

ACKNOWLEDGMENTS: Financial assistance provided by the Vice Chancellor‟s Lump

sum grant (2010-2011) is gratefully acknowledged.

85

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