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Teză de doctorat
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.