Journal of Plant Sciences 2016; 4(6): 153-164 http://www.sciencepublishinggroup.com/j/jps doi: 10.11648/j.jps.20160406.14 ISSN: 2331-0723 (Print); ISSN: 2331-0731 (Online) Genetic Diversity Analysis and DNA Fingerprinting of Mungbean (Vigna radiata L.) Genotypes Using SSR Markers Md. Rezwan Molla 1 , Iftekhar Ahmed 1 , Md. Motiar Rohman 2 , Md. Amjad Hossain 1 , Md. Aziz Zilani Chowdhury 3 1 Molecular Biology Laboratory, Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh 2 Molecular Breeding Laboratory, Plant Breeding Division, BARI, Gazipur, Bangladesh 3 Crops Division, Bangladesh Agricultural Research Council (BARC), Farmgate, Dhaka, Bangladesh Email address: [email protected] (M. R. Molla), [email protected] (I. Ahmed), [email protected] (M. M. Rohman), [email protected] (M. A. Hossain), [email protected] (M. A. Z. Chowdhury) To cite this article: Md. Rezwan Molla, Iftekhar Ahmed, Md. Motiar Rohman, Md. Amjad Hossain, Md. Aziz Zilani Chowdhury. Genetic Diversity Analysis and DNA Fingerprinting of Mungbean (Vigna radiata L.) Genotypes Using SSR Markers. Journal of Plant Sciences. Vol. 4, No. 6, 2016, pp. 153-164. doi: 10.11648/j.jps.20160406.14 Received: October 13, 2016; Accepted: October 31, 2016; Published: November 23, 2016 Abstract: Microsatellite combines several features of an ultimate molecular marker and they are used increasingly in various plant genetic studies and applications. Characterization of mungbean genotypes on the basis of DNA fingerprinting has become an efficient tool to link genotypic variation. This work is reporting the utilization of a small set of five previously developed mungbean microsatellite (SSR) markers for the identification and discrimination of six HYVs and 36 landraces. All five microsatellite markers were found to be polymorphic. Variation was found in number of alleles, allele frequency, observed and expected heterozygosity. Using five primers across 42 genotypes a total of 20 alleles with an average number of 4 alleles per locus were found of which GBssr-MB91 showed highest number of alleles (6) (size ranging from 135 to 152 bp) followed by 4 alleles (from 160 to 176 bp and 175 to 195 bp) and 3 alleles (from 264 to 282 bp and 283 to 304 bp) were detected at the loci LR7322B, LR7323A, LR7323B and GBssr-MB77, respectively. The narrow genetic base could be one of the reasons for the low yield of polymorphic markers in the study. The primer GBssr-MB91 also yielded highest number of PIC value (0.803). Genetic differentiation (Fst) values were found in the ranges 0.443 to 0.747 with an average of 0.686 and gene flow (Nm) values ranged from 0.085 to 0.314 with an average of 0.237. Over all Nei’s genetic distance value (D) obdervedfrom nil to 2.706 among 861accessions pair resulting as a means of permutation combination of 42 mungbean genotypes. The UPGMA dendogram based on Nei’s genetic distance separated the genotypes, BARI mung-1 and BD6906 from other 40 genotype. Out of 42 genotypes, 36 genotypes were identified with at least one and/or combination of 4 primers. Keywords: DNA Fingerprinting, Genetic Diversity, Microsatellite (SSR) Marker, Mungbean, Polymorphism 1. Introduction Mungbean (Vignaradiata L. Wilczek) also known as green-gram belongs to subgenus Ceratotropis is an important legume food crop in south and Southeast Asia where 80% of the world’s mungbean. It is an important crop among the palatable pulses in Bangladesh. This crop provides protein- rich food, restores and maintains the soil fertility by fixing atmospheric nitrogen, and also fits well in different cropping systems. However, the average yield of mungbean has to be as low as 670 kg/ha [1]. There are many reasons for such low yield [2]. The low productivity of this crop can be attributed to narrow genetic base (resulting in low yield potential land susceptibility to biotic and abiotic stresses) and lack of suitable plant types for different cropping situations [2, 3]. Variability is the touch stone to a breeder to evolve high yielding varieties through selection. The assessment of genetic variation is a major concern of plant breeders and population genetics. Availability of sufficient variation required for the production of new varieties that are aimed
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Journal of Plant Sciences 2016; 4(6): 153-164
http://www.sciencepublishinggroup.com/j/jps
doi: 10.11648/j.jps.20160406.14
ISSN: 2331-0723 (Print); ISSN: 2331-0731 (Online)
Genetic Diversity Analysis and DNA Fingerprinting of Mungbean (Vigna radiata L.) Genotypes Using SSR Markers
Md. Rezwan Molla1, Iftekhar Ahmed
1, Md. Motiar Rohman
2, Md. Amjad Hossain
1,
Md. Aziz Zilani Chowdhury3
1Molecular Biology Laboratory, Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh 2Molecular Breeding Laboratory, Plant Breeding Division, BARI, Gazipur, Bangladesh 3Crops Division, Bangladesh Agricultural Research Council (BARC), Farmgate, Dhaka, Bangladesh
Hercules, CA, USA) electrophoresis system. A pre-run of the
gel for 30 mins at 120 W was followed by a final run at 60W
and 50ºC upon loading of denatured PCR products for a
specified period of time depending on the size of amplified
DNA fragment (usually 1 hour for 100 bp). A molecular
weight marker DNA (100 bp DNA ladder, Biobasic, Canada)
was loaded on either side of the gel. After completion of
electrophoresis, the DNA fragments were visualized
following the Promega (Madison, WI) silver-staining
protocol.
2.6. Scoring and Analysis of Microsatellite Data
The bands representing particular alleles at the
microsatellite loci were scored manually and designated the
bands as A, B, C, etc. from the top to the bottom of the gel by
three experienced scientists individually. The genotypes of
different individuals were hypothetically scored as AA, BB,
CC, etc. for homozygous or as AB, AC, BC etc. for
heterozygous. A single genotypic data matrix was constructed
for all loci. Polymorphism Information Content (PIC) was
computed by deducting sum of square values for all the
frequencies of different alleles produced by a single marker
locus from one using the formula: PIC=1- ΣXi2, Where, Xi is
the frequency of the i-th allele of a particular locus.
PIC provides an estimate of the discriminatory power of a
marker by taking into account, not only the number of alleles
that are expressed, but also the relative frequencies of those
Journal of Plant Sciences 2016; 4(6): 153-164 156
alleles. PIC values range from 0 (monomorphic) to 1 (very
high discriminative, with many alleles in equal frequencies).
The software DNA FRAG version 3.03 was used to estimate
allelic length [26]. Expected (He) and observed heterozygosity
(Ho) were also calculated as per [27, 18] formula and with the
help of POPGENE (version 1.32) [28] computer package
program. Estimation of Nei’s genetic distance values (D) [27]
and construction of UPGMA (Unweighted Pair Group Method
of Arithmetic Means) dendrogram was constructed using the
software POPGENE (version 1.32) [28].
3. Results and Discussion
All forty two mungbean genotypes were successfully
amplified with the five microsatellite primer pairs (LR7322B,
LR7323A, LR7323B, GBssr-MB91 and GBssr-MB77).
Based on previous results of Kumar et al. [29] and Gwag et
al. [30], primer pairs referred to as loci and DNA bands as
alleles. All five microsatellite markers were found to be
polymorphic, revealing a total of 20 alleles with an average
number of 4 alleles per locus were found in the present study.
The narrow genetic base could be one of the reasons for the
low yield of polymorphic markers in the study. At the GBssr-
MB91 locus showed highest number of observed alleles (6)
among the 42 mungbean genotypes ranging in size from 135
to 152 bp. Likewise, 4 alleles (size ranging from 160 to 176
bp and 175 to 195 bp) and 3 alleles (from 264 to 282 bp and
283 to 304 bp) were detected at the loci LR7322B,
LR7323A, LR7323B and GBssr-MB77 respectively in
descending order (Table 2) and the effective number of allele
was also highest (5.079) for GBssr-MB91 (Table 2). Narrow
genetic base has been among the mungbean accessionsin this
study. Three to five alleles size ranging from 171 to 285 bp
were obtained by Kumar et al. [29] while conducting
isolation of microsatellite markers in mungbean, Vigna,
although some variation occurred might be due to mutation
of dinucleotide repeat units which could also be indicative of
varietal differences. Allele frequency ranged from 0.063 to
0.563 observed in the present study. DNA banding patterns
were generated by the primer pairs in 42 mungbean
genotypes are shown in Figure 1.
Figure 1. Microsatellite profiles of 42 mungbean genotypes at locus GBssr-MB91 and LR7323A; M: molecular wt. marker (100 bp DNA ladder); Lane 01:
BARI mung-1; ; Lane 02: BARI mung-2; Lane 03: BARI mung-3; Lane 04: BARI mung-4; Lane 05: BARI mung-50; Lane 6: BARI mung-6; Lane 07:
Sonamug, Lane 08: BD6874; Lane 09: BD6875; Lane 10: BD6876; Lane 11: BD6877; Lane 12: BD6878; Lane 13: BD6879; Lane 14: BD6880; Lane 15:
BD6881; Lane 16: BD6882; Lane 17: BD6884; Lane 18: BD6885; Lane 19: BD6886; Lane 20: BD6887; Lane 21: BD6888; Lane 22: BD6889; Lane 23:
BD6890; Lane 24: BD6891; Lane 25: BD6892; Lane 26: BD6893; Lane 27: BD6894; Lane 28: BD6895; Lane 29: BD6896; Lane 30: BD6897; Lane 31:
BD6898; Lane 32: BD6899; Lane 33: BD6900; Lane 34: BD6901; Lane 35: BD6902; Lane 36: BD6903; Lane 37: BD6904; Lane 38: BD6905; Lane 39:
BD6906; Lane 40: BD6907; Lane 41: BD6908; Lane 42: BD6909.
157 Md. Rezwan Molla et al.: Genetic Diversity Analysis and DNA Fingerprinting of Mungbean (Vigna radiata L.)
Genotypes Using SSR Markers
The PIC values, which are reflection of allele diversity,
provide an estimate of the discriminating power of a marker
by taking into account not only the number of alleles at a
locus, but also relative frequencies of these alleles. The PIC
values are dependent on the genetic diversity of the cultivars
chosen and this investigation had a high proportion of
traditional varieties which would have the effect of
increasing the PIC values. It is important to indicate that the
selection by breeders have increased the frequency of the
alleles or allelic combination with favorable effects at the
expense of the others, eventually eliminating many of them
[31]. The markers and their allele size along with their
frequencies and PIC values have been shown in the Table 2.
The PIC values for five primers obtained in the present study
varied from 0.538 for LR7323B to 0.803 for GBssr-MB91,
with an average PIC value of 0.637 (Table 2). Among the
markers used in this study GBssr-MB91 and LR7323A
showed higher PIC values than the LR7322B, GBssr-
MB77and LR7323B. Lower PIC value may be the result of
closely related genotypes and higher PIC values might be the
result of diverse genotypes. The number of alleles amplified
by a primer and its PIC values also depends upon the repeat
number and the repeat sequence of the microsatellite
sequences [32, 33, 22]. These observations are in agreement
with those of Kumar et al. [29] and Gwaget al. [30] whose
showed that (AG) and (GA) repeats yield higher number of
alleles and higher PIC values. GBssr-MB91 and LR7323A
having (GA)n repeat were two most informative
microsatellite markers for this set of germplasm, as they
yielded 5 alleles. For GBssr-MB91 [(AG)34(GA)14] and
LR7323A [(GA)13], 6 and 4 alleles were observed and
average PIC values were 0.803 and 0.645, respectively which
were not unusual based on repeat number and the repeat
motif to that observed in previous study [29, 30]. Table 2. Size and frequency of alleles and diversity index at five SSR loci across 42 mungbean genotypes.
Locus Repeat Motif Sequence of primers (5´-3´) Allele sizes (bp) Allele frequency PIC
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