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INTRODUCTION

Sweet corn is a variety in the Zea mays species, namely var. rugosa (Bonaf),

convar. Saccharata (Sturt. ), (after SĂVULESCU �i ZAHARIDI, 1957) and differs from

the normal corn by the presence of one or more mutant genes that affect carbohydrate

metabolism in the endosperm.

In our country, the research related to the improvement of sweet corn began at

I.C.C.P.T. Fundulea, in 1957 (MURE�AN et al., 1966). Sweet corn is mentioned in the

paper called “Corn – monographic study” published by SĂVULESCU and ZAHARIDE

in 1957. The first sweet corn hybrid obtained in Romania at Fundulea was the simple

Delicious hybrid, approved in 1969.

In 1971, the program for developing the inbred lines and early sweet corn hybrids

was initiated at the Turda Agricultural Research Station, and in 1988 the trilinear Dulcin

hybrid was approved (CĂBULEA et al., 1994; HA� et al., 1994; HA�, 2002a). The

valuable sugary lines are currently found as parental forms of the Dulcin, Prima, Estival

and Deliciul Verii approved hybrids (HA� and CĂBULEA, 1998, HA�, V. et al., 1999).

The identification of the inbred lines polymorphism with the help of the DNA level

polymorphism is extremely useful thanks to the fact that the obtained result reflects

directly the genotype and it is not influenced by the environmental conditions (LODHI et

al., 1994, POP et al., 2003).

Based on the above findings, in the presented PhD thesis we decided to take further

the national research mentioned above, by showing the variability of the sweet corn

inbred lines, not only at the phenotype level but also at the molecular level and to

establish the statistical links between these two types of expressions. Such links could be

used to initiate a selection program assisted by molecular markers of the lines that

compose the commercial hybrids of the sweet corn.

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1. RESEARCH OBJECTIVES, MATERIAL AND METHOD

1.1. GOALS OF RESEARCHES

Any improvement program proposes to obtain a new genetic variability that can be

subsequently used by selection to obtain new genotypes that are more advanced.

The production spore, in what concerns the sweet corn, represents a modest gain

compared to the spore realized with normal corn, according to TRACY (1990b), due to

the narrower genetic base of sweet corn, which limits the possibilities of the genetic gain

for the production.

The main purpose of our research was to characterize and establish the

phylogenetic relationships among the 18 inbred lines and the 49 sweet corn hybrids, using

the RAPD methodology, and comparing the results obtained by hybridization formulas

developed on the base of lines and commercial hybrids tests through conventional and

unconventional methods (molecular markers).

The research initiated by us is probably the first attempt to identify the variability

of sweet corn through the RADP method, in Romania.

1.2. THE GOALS OF THE RESEARCH AFFERENT TO THE PHD THESIS

In the initiated research we proposed that, through the experiences made on 18

inbred lines and 49 sweet corn hybrids, to identify and select a suitable biological material

for obtaining advanced combinations, involving expressions in the hybrids of the most

valuable characters of the parental lines.

Given the fundamental benchmarks knowledge regarding the importance and

relevance of the research on phenotypic and genetic diversity of inbred lines and simple

hybrids, at the analysis methods, the objectives pursued in this paper are presented as

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follows, on two categories of characterizing the sweet corn inbred lines and hybrids:

phenotypic and molecular levels.

At the phenotypic level we pursued the following:

the characterization of inbred sweet corn lines on the basis of their

“per se” value and also on the basis of the phenotypic differentiation

index;

the evaluation of the genetic diversity of the sweet corn inbred lines

quantitative characters, parental forms of the studied hybrids on the

basis of general and specific combining capacity;

the determination of the genetic diversity degree between parental

forms by the intensity level of sweet corn hybrids heterosis;

the indication of the environmental conditions influences on the

genetic mechanisms involved in the expression of the characters in

what concerns the sweet corn.

For the molecular characterization of the studied inbred lines and hybrids of sweet

corn we headed the research towards the following aspects:

the establishment of the DNA extraction protocol using the RAPD

technique (Random Amplified Polymorphyc DNA) to identify

genetic polymorphism in vivo at 18 sweet corn lines and 49 simple

hybrids;

the identification of the primers able to determine the molecular level

of polymorphism between the inbred lines and the analyzed hybrids;

the detection of the genetic diversity and the relatedness degree

between the 18 inbred lines and also among the studied sweet corn

hybrids, by DNA fingerprint obtained with the RAPD technique.

We also aim to highlight the type of connection between the general and specific

combining ability calculated at the phenotypical level on one hand and the genetic

fingerprint of the sweet corn inbred lines on the other hand.

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2. MATERIALS AND WORKING METHODS

2.1. THE BIOLOGICAL MATERIAL

The biological material used in the research was represented by 18 sweet corn

inbred lines (T152 su, T193A su, T209 su, T339R su, LC153 su, LC154 su, TD101 su,

T345R su, T346 su, TA25 su, TA26 su, TA27 su, TA28 su, TA22 su, TA21 su, T233 su,

T244 su, TC209 su), parental forms, and 49 simple hybrids made in a cyclic m x n crosses

(factorial) system for quantitative genetic research, the tester lines being T233 su, T244 su

and TC209 su.

The biological material for DNA isolation was represented by the embryos of the

genotypes of the sweet corn mentioned above.

To supplement the square balanced grid there were also introduced in the tests four

more sweet corn simple hybrids, three of them being approved and included in “The

Official List of the Varieties and Hybrids” (Prima, Deliciul verii and Delicios) and one of

them being a simple sweet corn hybrid, the maternal form of the Estival hybrid; the

component line of the SW87 su (the 19th line), was also analyzed using the RAPD

technique.

For amplification were used 20 decamer primers (UBC 281, UBC 241, UBC 245,

UBC 561, UBC 286, UBC 599, OPB08, OPB09, OPB10,OPB11OPB17,OPAB 11,OPX

O3, AB 11, OPE 14, OPAL 20,OPB03, OPB18, OPC14, OPD19). The origin of the

primers was different, so that the first 6 nucleotide primers are produced by the University

of British Columbia (UBC), 14 by Mycroshinth (OPAB, OPA, OPB, OPE, OPC, OPD,

OPAL, OPX, AB).

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2.2. THE WORKING METHOD

2.2.1 The organiazation of the experiences

The research regarding the genetic determinism and also the study of the effects of

the general and specific transmission capacity of some characters of specific interest for

the sweet corn were made using the experimental data from a series of three cyclic

systems, comprising 45 hybrids.

In addition other four sweet corn (approved) simple hybrids were introduced in the

research (Prima, Deliciul verii, Delicious and the mother form of the Estival hybrid), to

complete the setting system (49 variants = 7 x 7 and (7+1)/2 repetitions) in the field, but

also for their use to compare the rest of the system hybrids to reflect a genetic progress.

Forms of parental inbred lines were also tested in 2005 and 2006, in a randomized

block experience in a single location (Turda), in four repetitions. Testing hybrids was

done, too, during the same two years: 2005 and 2006.

2.2.2. Observations and analysis

The observations and analysis carried out on experimental variants in 2005 and

2006 focused on a number of 25 characters. Measurements were made at physiological

maturity (approximately 21 days from the onset of the stigmata) in packs of 10 plants and

10 ears of each parcel plot repetition, totaling 40 plants and 40 ears/option (simple

hybrid)/year: 

• vegetative characters of the plant: plant height, the height of the main

ears insertion, the number of leaves/plant, number of branches on

panicle, the length of the leaf of the main ear, the width of the leaf of

the main ear, number of shoots/plant;

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• ear characters: ear weight, husk weight, ear length, husk length,

length of bracts, number of grain rows/ear, depth, the thickness of the

rachides, cone-shape index;

• physiological characteristics: early vegetative vigor, leaf area.

2.2.3. The methodology of the rapd analysis used in evidence at the molecular level

variability in sweet corn

2.2.3.1. DNA extraction methods

For the RAPD analysis of the DNA of sweet corn (rich in carbohydrates), the

extraction was attempted by two methods: the extraction from embryos and from

endosperm. There were tested two types of protocols, then the chosen DNA extraction

protocol was optimized through which was obtained a higher purity. The biological

material for the DNAs isolation was represented by the grain embryos from the sweet

corn of the biological material described above.

To obtain a high quality DNA that can be used in the RAPD’s technique was used

the protocol described by LODHI et al., 1994, as amended (POP et al., 2003).

2.2.3.2. DNA quantification

After the extraction of DNA the samples were quantified spectophotometric using

the BioPhotometer Eppendorf apparatus.

2.2.3.3. Amplification RAPD protocol

The amplification for the samples was done using the Eppendorf Mastercycler

Gradient programmed as follows:

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3 minutes at 95oC – predenaturation, followed by 45 cycles in the next

temperature profile:

1 minute at 93oC – denaturation;

1 minute at 34oC – primers fixing;

1 minute at 72oC – extension;

final extension: 10 minutes at 72oC.

2.2.3.4. Image analysis

The analysis of the gels was done using the Total Lab TL 100 program, yielding

the binary matrix. The binary matrix was then used to calculate the genetic distances. For

the calculation and for achieving the dendrogram we used the RAPDistance program,

using the appliance of the Nei Li index and the UPGMA method (Un-weighted Pair

Group Method with Arithmetic Mean) for the genetic distances respectively Neighbor

Joining Tree method for the dendrograms.

2.2.4. Statistical methods used to reveal phenotypic and genetic variability

in sweet corn

To asses the diversity of the 18 inbred lines involved in the crosses system tested at

SCDA Turda, the phenotypic differentiation index (IDF) was calculated according to

HERERT and VINCOURT (1985).

where Xm and Xn are the averages of the characters of the m and n parental lines.

At the quantitative characters level the genetic diversity was estimated based on the

general combining ability (CGC) and the specific combining ability (CSC), the

characteristics of each line, of each interaction between the lines and also for a cyclic

crosses (factorial) system.

The estimation of the polygenic diversity was realized:

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- at the level of the homozygous locus by calculating the additive genes effects:

,

where

Xm/n = the average of the m or n parent;

.

- at the level of the in and between allele interactions by calculating the unadditive genes effects (ŝmn); ), where

ŝmn = the genetic effect of the interaction between the genes of the two

parents;

.

2.2.5. Statistic methods used to reveal variability at the molecular level

The interpretation of the obtained gels was made through the statistical analysis of

the images. The molecular markers are treated as binary variables, the data processing

taking into account:

- the analysis of multiple variables is used, so forming the matrix of similarity or

difference between the possible pairs of individual or operational taxonomic

units;

- two similar individuals simultaneously have the minimum value of the distance

and maximum value of the similarity;

- distance and similarity are inversely proportional;

- the similarity is estimated taking into account the number of coincidences.

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3. RESULTS AND DISCUSSIONS

3.1. THE PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF THE

STUDIED SWEET CORN INBRED LINES

3.1.1. The phenotypic expression of the characteristics of sweet corn inbred lines

The calculated average values of the characters of interest for the improvement of

the sweet corn covers the biometrization for a period of two years (2005 and 2006), period

during which the experiences were carried out. These values restore the “per se” value of

the character of the studied inbred lines. The phenotypic characterization of the sweet

corn parental forms inbred lines studied at SCDA Turda is shown in Table 1 by

calculating the average value of the biometrization of 6 characters for the plant and 12

characters for the ear.

The market presents the key requirements related to the cobs appearance. Thus, for

fresh consumption, is preferred the corn with at least 16 rows of grains, deep and straight

lines, the ear measuring between 25 and 30 cm, covered entirely with grains and with a

cylindrical form (TRACY, 1994).

In the table mentioned above it can be noticed that between the studied sweet corn

inbred lines exist some differences statistically assured regarding each character

separately.

Plant height was expressed by values between 97.40 cm (TA27 su) and 155.53 cm

(T244 su). The insertion of the main ear was situated at heights between 16.21 cm (TA27

su) and 53.62 cm (T152 su).

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Table 1

Agronomic assessments of sweet corn inbred lines studied (average values Turda 2005 and 2006)

Linia

Line

Înălţime plantă

Plant height

(cm)

Înălţime inserţie ştiulete

Height ear insertion

(cm)

Număr ramificaţii la

panicul

Branch of tassel no.

Greutate ştiulete fără pănuşi

Dehusked ear

weight (g)

Lungime ştiulete

Ear lenght

(cm) T152su 143,62 *** 53,62 *** 5,51 ooo 87,93 15,88*** T193Asu 147,26 *** 50,78 *** 9,75 oo 80,75 12,65 T209su 123,35 36,49 8,48 ooo 87,11 13,50 T339Rsu 137,87 *** 42,66 10,05 o 87,95 16,90*** LC153su 117,39 ooo 44,18 36,21 *** 80,60 11,47 ooo LC154su 120,48 oo 41,23 17,70 *** 71,87o 13,40 TD101su 128,68 38,99 22,56 *** 82,70 16,44*** T345Rsu 122,00 o 41,58 9,79 oo 89,96 13,46 T346su 152,70 *** 51,59 *** 10,48 81,69 16,19*** TA25su 106,27 ooo 32,33 o 13,97 * 90,48 12,12 oo TA26su 131,38 * 35,97 10,73 78,32 12,04 oo TA27su 97,40 ooo 16,21 ooo 7,42 ooo 90,06 14,53 TA28su 108,11 ooo 29,48 ooo 6,16 ooo 115,78*** 11,38 ooo TA22su 134,51 ** 36,99 8,86 ooo 89,79 12,21 oo TA21su 104,44 ooo 29,41 ooo 13,36 61,65ooo 11,61 ooo T233su 110,77 ooo 30,86 oo 9,48 oo 87,27 12,71 T244su 155,53 *** 47,44 ** 8,84 ooo 90,03 13,35 TC209su 141,19 *** 41,16 9,81 oo 91,56 16,07*** Media 126,83 38,94 12,18 85,86 13,66 DL/LSD 5% 4,52 5,46 1,72 11,9 1,03 DL/LSD 1% 6,01 7,24 2,29 15,8 1,37 DL/LSD 0,1% 7,74 9,34 2,95 20,36 1,76

 

The number of branches at the panicle is a character with a fairly large amplitude

from 5.51 (T152 su) to 36.21 (LC153 su).

Dehusked ear weight, very important production data, showed changes in the

average values between 61.65 grams (TA21 su) and 115.78 grams (TA 28 su). The ears

size, character important both for production and for the commercial aspect, showed

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changes in what concerns the average values calculated for the two years of study, (Table

1), from 11.38 cm (TA 28 su) up to 16.90 (T339R su).

The sweet corn for fresh consumption must present small and profound grains

(TRACY, 1994). Grain size, as observed in Figure 1, varies from 0.5 cm (LC153 su) to

0.76 cm (T346 su).

Figure 1. Grain thickness for 18 sweet corn parental inbred lines (average values

Turda, 2005 and 2006)

Sweet corn for fresh consumption must present small and deep grains (TRACY,

1994). At the 18 parental lines studied, the average values recorded over the two years go

from 0.40 cm (T152 su) to 0.89 cm (TA27 su, TA28 su).

By calculating the phenotypic differentiation index (IDF) (HERBERT and

VINCOURT, 1985) the average values of the characters of two genotypes (parental lines)

can be compared (Figure 2 and 3).

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Figure 2. Phenotype differentiation index (PDI) for plant characteristics of 18

sweet corn inbred lines (2005 and 2006)

Figure 3. Phenotype differentiation parameters for ear characteristics of 18 sweet

corn inbred lines (2005 and 2006) The high value of this calculated index reveals the differentiation degree between

the lines while the lower values show the similarity degree.

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As a result, looking at the plants characters, the T152 su inbred line is very

different from the other three inbred lines used in the crosses system as a maternal form

(T233 su, T244 su and TC209 su) through the higher values of the phenotypic

differentiation index ( 54.9, 82.1 and respectively 71). The most close lines regarding

from a phenotypic point of view resulted to be the TA28 su with T233 su (6.8), T346 su

with T244 su (8.1) and TA22 su with TC209 su (9.0).

The calculated phenotypic differentiation index for the studied sweet corn

characters of the inbred lines are presented in Figure 4. Thus, from this calculation it can

be seen that the higher phenotypic differentiation degree at the level of the ears characters

occurred between line TA21 su and T244 su (62.6), the same as between line T346su and

T233su (58.1), and respectively TC209 su (50.2). Very similar in terms of the ears

characters proved to be the T345su and TC209 su (6.1) lines, T339R su and T244 su (8.1)

the same as LC153 su and T233su (8.4).

3.1.2. The characterization of the lines using the general combining capacity

(GCC)

Genetic diversity at the level of the quantitative characters can be estimated based

on the effects of the general combining capacities that are characteristic to the inbred lines

of the sweet corn. The differences between the compared lines reflect the differences at

the level of the homozygous locus with cumulative effects.

Between the three sweet corn inbred lines used in the crosses as maternal forms,

there resulted the fact that they can be used as favorable gene sources, having the capacity

to transmit positive additive characters as follows:

• production capacity, represented by the weight of the ear is best transmitted

additive positively from the line “su” TC209 (ĝ =17.19), while the T224 and T233

lines transmit additive negatively this character;

• the height of the plant is transmitted additive positive by the TC209 (ĝ=3.21) sweet

line, while lower height can be transmitted by the T244 (ĝ=1.39) sweet line.

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Of the three sweet corn inbred lines, used as paternal lines we can say that:

• the height of the plant is transmitted additive positive from the T339R (16.26),

T346 (14.34), T152 (7.09), TA22 (6.12) and T209 (5.30) sweet lines, while the

lower height can be transmitted by the LC153, T193A and T345R sweet lines;

• the inbred TD101su (4.39), LC153su (4.06), LC154su (3.25) lines have a general

ability of transmitting a panicle with a high number of branches, while the TA21,

T339R, TA25 and T345R sweet lines transmit to the hybrids a lower number of

panicle branches.

With the results for the additive genetic effects on the characteristics of the sweet

corn ear could be found that:

• for transmission at the additive level of the production character expressed by the

ear weight, there can be observed the LC153 (18.66), T339R (12.76) and

TA25(6.37), while the TA28 (5.51) and T346 (2.53) lines transmit a much lower

weight for the ear in progeny;

• the length character of the ear is transmitted additive positive by the T209su (2.24),

T339Rsu (1.40) and TD101su (1.34), while the T346 (0.40), LC154 (0.39) and

T152 (0.38) sweet lines transmit a much shorter ear at the hybrids.

3.1.3. Line characterization with the aid of specific combination capacity (SCC)

Gene interaction both intra and interalelic were determined quantitatively trough

the effects of specific combination capacity (ŝmn) calculated in cyclic cross breeding

system. In order to quantitatively express the level of intra and interalelice genetic

differentiation, the sum of these specific effects was calculated for the analyzed

characteristics (Σŝij). This way the differences were identified between inbred lines of

sweet corn at unaditive genetically level, both for the transmission of the plants

characteristics and the ears.

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The obvious differences for (Σŝij ), for plant characteristics, were manifested by

T233su line in combination with T193Asu (32.79) (the most obvious differences at

unaditive genetic level), TA26su (4.87) and T152su (4.25).

T244 sweet corn inbred line is different at unaditive genetic level from line

TA27su (11.9), but also from line LC154 su (9.68), TA21 su (9.42), LC153su (8.88),

TA22su (8.61), TA28su (8.58), T339Rsu (8.12), TA26su (4.67). The closest, for the sweet

line T244, at intra and interalelic for plant characteristics was proven to be line T193Asu

(-49.30).

Towards the inbred sweet corn line TC209, from the perspective of intra and

interalelice genetic action, according to (Σŝij ), there are differences at the unaditive

transmission levels of the plant characteristics for the sweet sugary type lines: T193A

(16.51), T345R (7.85), TD101 (6.52), TA22 (5.21), T209 (3.71).

The genetic analysis at the unadditive level for the three sweet corn inbred lines,

maternal forms from the factorial crosses system reflect the fact that the most pronounced

differentiations for the values of the calculated effects sum, for the ears characters are

manifested by:

line T233su in combination with T193Asu (105.91);

line T244su in combination with lines T339Rsu (37.64), TA27su (37.07),

TA22su (23.85), TA21su (19.35), TA28su (15.14);

line TC209su in combination with T193Asu (42.84), TA25su (20.62),

LC154su (16.21), TA22su (16.72), T345Rsu (9.72).

 

3.1.4. THE EXPRESSION OF HETEROSIS IN SWEET CORN HYBRIDS

THAT ARE STUDIED AT ARDS TURDA

For the weight of the ear, in average over the experimental system, for the parental

lines, there was registered an average weight for the ear, 85.86 grams, while for the hybrid

combinations it is 168.45 grams. The amplitude of the values recorded for the hybrids is

comprised between 92.4 grams (T244su x T193Asu) and 206.69 grams for the (TC209su

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x LC153su) hybrid, while in the case of the lines at which values are comprised between

61.65 grams and 115.78 grams. In all cases, hybrid combinations go above the average of

parental forms. However there are cases when the differences between the average value

of the parents and hybrids is relatively reduced (T244su x T193Asu, 85.39 respectively

92.4). As a consequence, heterosis is also low manifested.

One explanation is that the lines, parental forms of the simple hybrids, are obtained

by self-pollination and selection from simple hybrids. Thus, these lines have in them

some polygenic blocks belonging to different groups, which gives a value of the “per se”

character but there also exists a high probability of a lack of differentiation (HAS, 1999).

The heterosis of the production with the higher percentage values was recorded in

general at the hybrids derived from TC209su: TC209su x LC153su (140.10%), TC209su

x TA26su (127.35%), TC209su x TA27su (125.14%), but also at the hybrids that had as a

maternal form the sugary T233 (T233su x T346su – 118.09%; T233su x LC153su –

117.33%, T233su x T3339Rsu – 101.34%) line or the T244su (T244su x LC153su -

108.5%; T244su x T339Rsu – 106.8%; T244su x TA26su – 103.03%).

The superior values of the heterosis were also recorded in what concerned the

depth of the grain (14.53% and 138.57%).

3.1.5. The expression of some production characters of the genotypes in relation with

the environental conditions at the sweet corn simple hybrids studied at ARDS Turda

The data regarding the influence of the hybrid, of the year upon the weight of the

ear were statistically processed. From the analysis resulted that both of the experimental

factors (year, genotype), the same as the simple or double interactions between them had a

significant influence upon the ears weight, the weight of the husk, ear length, number of

grain rows/ear.

As we can observe in the above image (Figure 4), husk weight is more influenced

by the climatic conditions, the year having a much bigger influence in the manifestation

of this character.

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The length of the ear is an important character in what concerns the production but

also the appearance is very important for marketing.

Figure 4. Factor rate (%) involved in ear weight and husks weight expression in

the 49 hybrids studied in 2005 and 2006

The influence of the factors and the simple and double interactions between them

is very important. In Figure 5 we can observe that the weight of factor B (genotype) is the

biggest (61%), which sows that the years had a lower influence in what concerns this

character (the length of the ear).

Figure 5. Factor rate (%) involved in ear length and kernel rows number /ear

expression in the 49 hybrids studied in 2005 and 2006

In the case of the number of grain rows/ear the genotype had the greatest share of

influence (84%), in comparison with the year (7%) or the interaction of these two factors

(3%).

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Majority share in genotype contribution when it comes to expressing the studied

characters shows the fact that the sweet corn hybrids have a relatively stable behavior, in

different environment conditions, in what concerns the studied characters and genotypes

(HA�, 1999).

3.1.6. Characterization of inbred lines based on rapd analysis

3.1.6.1. Results regarding the extraction and quantification of DNA

After the extraction of the DNA quantification were made at the Eppendorf

BioPhotometer Spectrophotometer at a wavelength of A260/A280.

In what concerns the analyzed corn lines the studies show that the best results, in

terms of quantity and purity of the DNA, are obtained from embryos by applying the

modified Lodhi extraction protocol.

Regarding the amount of DNA obtained, the highest values were recorded when

using as biological material the embryos, by using the first extraction protocol (modified

Lodhi). Thus from the embryos were obtained quantities like 7893 – 12000 ng/μl, while

from the endosperm the values were lower, respectively 3450-4370 ng/μl. In this case the

purity values were comprised between 1.5 and 2.

The extraction made from the endosperm by using the protocol after the Barry

Miller method was able to obtain a DNA with a purity comprised between 1.45 – 1.5, and

at the extraction made from the embryos the purity of the DNA ranged from 1.2 to 1.7.

Therefore, at all the other genotypes the DNA extraction was made only from

embryos by using the protocol created by LODHI et al., as amended (POP et al., 2003).

The amount of DNA was adequate (1275 ng/μl and 10593 ng/μl), since for the

amplification were used 50 ng. The purity of the DNA was also ranged between 1.53 and

2.01.

The DNA extraction was made, at the sweet corn inbred lines, from embryos, in

table number 20 being presented the numerical results on the quantity of DNA and its

purity.

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3.1.6.2. Results Regarding the Reaction Products Amplification and Electrophoresis

To amplify the samples were used from the 20 primers that gave polymorphic

bands for DNA extraction only 6 of them (OPB10, OPAB11, UBC241UBC 286, OPB 17,

AB 11).

The amplification of the samples was made in three repetitions for each primer.

The interpretation of the obtained gels was made by the statistical analysis of

images. In this analysis were included only the bands that had a high luminous intensity.

The standard DNA used consists of 40 fragments between 100 and 4000 pb. After

comparison with the standard DNA we could determine the size of each amplified

fragment.

 Figure 6. Amplicons obtained using UBC286 primer, 1–T346; 2- TA21; 3–TC209; 4–TA25; 5-TA28; 6–T339R; 7–T193A; 8–T209; 9–TD101; 10-TA26, 11–SW87; 12-T152; 13–T233, 14–T345R, 15–T244, 16–LC153; 17-TA22, 18–LC154, 19-TA27.

Primers that revealed polymorphism between the genotypes are: OPB 10 with 7

polymorphic bands, OPAB 11 with 9 polymorphic bands, UBC 241 with 10 polymorphic

bands, UBC 286 with 12 polymorphic bands, OPB 17 with 8 polymorphic bands and AB

11 with 10 polymorphic bands.

Most polymorphic bands were obtained with the primer UBC 286 (Figure 6). The

fragments resulting from amplification with the primers had the length between 200 and

2000 pb, most having between 300 and 1200 bp.

From the figures presented it is clear that all the studied sweet corn inbred lines

have considerable differences of genetic structure, revealed by RAPD markers.

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Smaller the calculated genetic distances, the inbred lines are more closely related

(genetically closer). As a consequence, the TA27su and TD101su lines present the

greatest degree of genetic proximity, having the smallest genetic distance (0.02),

according to the molecular analysis.

The same, the LC153 and TA22 sweet lines are very remote phylogenetically,

having the highest genetically distance calculated using the molecular RAPD markers

(0.32). Also great genetically distances are recorded with this markers between the LC153

and SW87 (0,3) sweet lines, T233(0,3), TC209 (0,28), T193A (0,27) and between LC154

and TA22 (0,28), SW87 and T233 (0,26).

In the dendogram (Figure 7) is shown that the LC153su and LC154su lines form a

separate group in comparison with the other studied inbred lines, the greatest phylogenetic

distance being recorded by taking into account the TA22su, T346su and TC209su lines.

The fact that the TA25su, TA26su and TA27su lines are found in the same group

of the dendrogram, and the genetic distances are small, merely confirm the accuracy of

the experimental results that we obtained, this sweet corn inbred lines being of common

origin. Furthermore, analyzing the origin of the lines, we find the truth of the molecular

analysis, the TA26su and TA27su lines being closer in the same group, especially since

their origin (genealogy) is closer.

It can be argued, based on the results discussed in this chapter, that the RAPD

analysis represents a sensible identification tool, molecularly speaking, for identifying the

variability between the sweet corn inbred lines.

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0.1

TA22

T346

TC209

T209

TA28

TA21

TA25

T193A

T339R

TA27

TA26

TD101

SV87

T152

T345R

T233

T244

LC153

LC154

Figure 7. Dendrogram of the sweet corn inbred lines tested

3.1.7. Marker – assisted efficiency of sweet corn inbred lines

In order to illustrate the marker – assisted selection of the sweet corn inbred lines

were calculated the linear regression coefficients of genetic distances between parental

lines and the performances of the hybrids obtained from these lines.

It started from the idea that a significant regression coefficient indicates a link of

real growth /decrease between the values of the genetic distances and the performances of

the hybrids for the studied characters.

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When testing the specific combining ability, RAPD markers – assisted selection

proved effective for the elements of productivity in sweet corn ( ear weight, ear length and

number of grains/row) and less or totally ineffective for plant height, number of rows/ear

and grain depth. Clearly, according to the used testers, there must be identified the RAPD

markers that have the maximum effectiveness in the markers – assisted selection, for all

the characters of interest in improving this variety of corn.

 

4. CONCLUSSIONS

Based on the obtained results some conclusions and recommendations on specific

issues can be shown, regarding the variability of the analyzed inbred lines that are

important for the improvement process of the sweet corn.

1. For the vegetative characters of the plant (plant height, height of main ears

insertion, number of panicle branches and number of shoots per plant) with

genetic effects that can be additive transmitted there can be observed the

following lines: T244, TC209, T339R, LC153, LC154, T346 and T345R.

2. Lines that can transmit genetic additive effects for the vegetative characters of

the ear (ear weight ear length, husk weight, ear diameter, number of grain rows

per ear, grain depth) are: T339R, LC153, T346, TA27, TA28.

3. The genetic intra and interalelic interactions were determined quantitatively by

the effects of the specific combining ability, so that the most pronounced

differences at the genetic unadditive level are shown by the following lines:

• T233 in combination with T193A , TA26 şi T152; 

• T244 in combination with TA27, LC154, TA21, LC153, TA22, TA28, T339R, 

TA26; 

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• TC209 in combination with T193A, T345R, TD101, TA22, T209. 

4. In the study conducted on 49 simple direct hybrids, high values of the heterosis

were recorded for the ear weight for hybrid combinations: TC209 x LC153

(140.10%), TC209 x TA26 (127.35%), TC209 x TA27 (125.14%) the same for

the combinations: T244 x LC153 (108.5%); T244 x T339R (106.8%); T244 x

TA26 (103.03%).

5. Choosing the primers proved to be quite difficult, only 6 of the 20 primers

showing polymorphism between genotypes.

6. The largest genetic distances were registered between the inbred lines of

different origins (LC153 and TA22), while the small genetic distances show a

more close relationship between these lines (TA27 with TD101, LC153 with

LA154, but also TA26 with TA27), the RAPD analysis being in generally in

accordance with the data regarding the origins of the analyzed material.

7. For the elements that are important for the productivity of the sweet corn, like

the ear weight, ear length and number of grains/row, RAPD markers – assisted

selection can successfully replace the testing of the specific combining capacity

for parental lines. This statement is sustained with positive results, statistically

assured, of the linear regression coefficients between the genetic distances

between the parental lines and the performances of the hybrids obtained from

these lines, for the characters of interest.

8. Based on these results it can be concluded that molecular markers – assisted

selection cannot efficiently replace the testing of the general combining

capacity. First, the testers have different capacities to highlight the value of the

tested inbred lines, and second, CGC testing is an effective way of eliminating

from the following steps the lines which in generally have a poor combining

capacity.