Cross Species Amplification of Adzukibean Derived Microsatellite Loci and Diversity Analysis in Greengram and Related Vigna Species  

M. Sathya , P. Jayamani
Department of Pulses, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, 641 003, Tamil Nadu, India
Author    Correspondence author
Molecular Plant Breeding, 2013, Vol. 4, No. 11   doi: 10.5376/mpb.2013.04.0011
Received: 01 Feb., 2013    Accepted: 07 Feb., 2013    Published: 20 Feb., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Sathya et al., 2013, Cross Species Amplification of Adzukibean Derived Microsatellite Loci and Diversity Analysis in Greengram And Related Vigna Species, Molecular Plant Breeding, Vol.4, No.11 89-95 (doi: 10.5376/mpb.2013.04.0011)

Abstract

Greengram or mungbean (Vigna radiata (L.) Wilczek) is a well known grain legume in Asian countries. Among the different DNA markers, microsatellite or simple sequence repeats (SSRs) are the markers of choice for various genetic studies due to co-dominant nature, loci specificity and high reproducibility. In the present study, a set of thirty-five microsatellite primer pairs derived from adzukibean (Vigna angularis (Willd.) Ohwi & Ohashi) were used to assess the transferability and tested for their ability to amplify microsatellite loci in greengram and related Vigna species. Of the thirty five microsatellite markers, thirty-two were successfully amplified across the thirty six genotypes and twenty eight were polymorphic. A total of 83 microsatellite alleles were generated with an average of 2.96 alleles per locus. Number of alleles ranged from two to five. Dendrogram formed based on UPGMA, 36 genotypes were grouped into five clusters. Similarly, the neighbour-joining tree developed based on weighted average for dissimilarity matrix grouped 36 genotypes into five groups. The finding suggests that adzukibean derived microsatellite markers are highly informative and could be used to improve the greengram at molecular level.

Keywords
Adzukibean; Cross species amplification; Genetic diversity; Greengram; Microsatellite markers

Greengram or mungbean (Vigna radiata (L.) Wilczek) is one of the important pulse crops grown in India, belongs to the subgenus Ceratotropis in the genus Vigna and is a self pollinating diploid grain legume (2n=22) with a genome size of 560 Mb (Arumuganathan and Earle, 1991). With its high protein content (22%~28%), greengram is a major source of dietary protein for the predominantly vegetarian population of India.The area undergreengram in India is around 3.8 million hectares with a production of 1.0 million tonnes (Anon., 2010).

Grain legumes are the most important crops in the world next to cereals, since they provide one-third of the dietary protein for human consumption. These crops belong to the family Fabaceae. The Vigna is one of the most important genus in the Fabaceae and it contains 100 to 150 species mainly found in Asia and Africa. This genus is divided into seven sub-genera (Verdcourt, 1970; Marechel et al., 1978). Two of these sub-genera viz., Vigna and Ceratotropis contain the most important cultivated species. The sub genus Vigna includes two cultivated viz., V. unguiculata and V. subterrarea and is widely distributed in Afica. The sub-genus Ceratotropis includes seven domesticated crops viz., V. radiata, V. mungo, V. aconitifolia, V. umbellata, V. angularis, V. trilobata and V. reflxo-pilosa which are mainly distributed in Asia. Hence the species of sub-genus Ceratotropisis called as Asian Vigna species. Productivity of the Asian Vigna has been poor owing to various reasons such as lack of genetic variability, susceptibility to various pest and diseases, poor harvest index and absence of suitable ideotypes for different cropping system. The transfer of agrono- mically important traits to the cultivars by conventional breeding method is quite laborious and time consuming, and almost impractical when the trait governed by polygenes. 
In recent years, molecular marker technology has greatly accelerated the breeding programs for the improvement of various crops. Different molecular markers have been used for the molecular analysis of grain legumes (Gupta and Gopalakrishna, 2008). DNA based markers such as Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), and Simple Sequ- ence Repeats (SSRs) or Microsatellite are used in molecular analysis of Vigna species. Among different DNA markers, microsatellite (or) Simple Sequence Repeats (SSRs) are the marker of choice for various genetic studies because, which detect sequence variation in the hypervariable region of tandem repeats of 2~4 bp, are a powerful tool for genome analysis because of their co-dominant nature, loci specificity and high reproducibility (Tautz and Renz, 1984). Microsatellite markers have been used for genome mapping and genetic diversity studies in many crop plants (Ford et al., 2002; Blair et al., 2006; Sangiri et al., 2007). To date, only few reports available on isolation and development of microsatellite markers in greengram. Therefore available SSR makers from other Vigna species should be validated for their transferability and utility in those species in which they are few or unavailable. In the present study, a set of 35 microsatellite primer pairs derived from adzukibean were used to assess the transferability and tested for their ability to amplify microsatellite loci in greengram and different species of Asian Vigna.
1 Results and Discussion
Of the 35 microsatellite markers used, 32 primer pairs showed amplification across the 36 genotypes (18 cultivated genotypes, 9 advanced breeding lines of greengram and nine 9 species of Vigna). Out of 32 primer pairs, 28 were found to be polymorphic and four primers found to be monomorphic (CEGD 091, CEDG 173, CEDG 186 and CEDG 245). The 28 polymorphic primer pairs generated a total of 83 alleles and number of alleles per locus ranged from two to nine with an average of 2.96 alleles per locus (Table 1). The PIC value for microsatellite loci ranged from 0.057 to 0.641 with an average of 0.266.


Table 1 List of SSR primers used and the level of polymorphism detected

Based on the dendrogram from UPGMA, the 36 genotypes were grouped into five clusters (Figure 1). Among the five clusters, cluster I was the largest with 27 genotypes followed by cluster IV with three genotypes. Cluster I had seven sub-clusters. Sub cluster Ia contain six genotypes, sub-cluster Ib had one genotype, sub-cluster Ic had two genotypes, sub-cluster Id had four genotypes, sub-cluster Ie had two genotypes, sub-cluster If had 11 genotypes and sub-cluster Ig had one genotype. Cluster II had wild species namely TMV(Mb) 1 and V. acontifolia; cluster III had V. radiata var. sublobata/1 and V. radiata var. sublobata/2; cluster IV with wild species V. trilobata/1, V. trilobata/2 and V. trilobata/3; cluster V had V. umbellata and V. glabrescence.


Figure 1 Dendrogram of 36 greengram genotypes based on SSR marker data

The neighbour-joining tree developed based on weighted average for dissimilarity matrix grouped the 36 genotypes into five groups (Figure 2). All the cultivated genotypes clustered under group I. Wild species TMV(Mb) 1, V. acontifolia (G2), V. radiata var. sublobata/1, V. radiata var. sublobata/2 (G3), V. trilobata/1, V. trilobata/2, V. trilobata/3 (G4), V. umbellata, V. glabresence (G5), were distinctly separated from cultivated genotypes and represented in distinct groups.


Figure 2 Neighbour-joining tree of 36 greengram genotypes based on SSR marker data

The ability to use the same microsatellite primer in different plant species depends on the sequence conservation in the primer binding sites flanking the microsatellite loci and stability of these sequences during evolution (Choumane et al., 2000; Decroocq et al., 2003). Microsatellite primer pairs used in the current study originated from adzukibean. Of the 35 primer pairs, 32 showed amplification across 36 genotypes 28 showed polymorphism. Previously, Chaitieng et al (2006) had observed that about 67% of adzukibean microsatellite markers gave ampli- fication in blackgram and 26% were polymorphic between cultivated and wild blackgram genotypes. This indicates the conservation of microsatellite sequence between two species during evolution. The conservation of microsatellite sequences also observed across other legumes (Choumane et al., 2000; Ford et al., 2002; Phansak et al., 2005). The transferability of microsatellite markers across species may increase their utility and decrease the development cost. The microsatellite markers conserved between species also serve as valuable tool for comparative mapping studies (Dirlewanger et al., 2004; Yu et al., 2004; Gupta et al., 2008).
In the present study, 28 polymorphic microsatellite markers produced 83 alleles with an average of 2.96 alleles per locus. The results were comparable with those reported earlier by Gwag et al (2010) in greengram. In the present investigation, allele size varied from 90~350 bp which was in close agreement with allele size reported by Souframanien and Gopalakrishna (2009) in blackgram for adzukibean derived SSR markers. The highest PIC value indicated that informativeness of the primer pairs. Hence, the primer pairs CEDA AG 002, CEDG 117, CEDG 149 and CEDG 244 are considered to be worth in future studies in the field of taxonomical and genetic resource management.
The primer pairs viz., CEDG 044 and CEDG 048 could differentiate cultivated genotypes from wild species and also showed polymorphism among cultivated genotypes. Primer pairs CEDG 008, CEDG 010, CEDG 015, CEDG 037, CEDG 43, CEDG 050, CEDG 068, CEDG 086, CEDG 092, CEDG 097, CEDG 181, CEDG 225 and CEDG 244 could differentiate cultivated genotypes from wild species and showed polymorphism among wild species. Primer pairs CEDA AG 002, CEDG 115, CEDG 117, CEDG 133 and CEDG 149 showed polymorphism among cultivated genotypes as well as among wild species.
Primer pairs CEDG 008, CEDG 044, CEDG 068, CEDG 092, CEDG 097, CEDG 037, CEDG 143 could be used to identify hybrids between V. radiata×V. umbellata. The primer pairs viz., CEDG 010 and CEDG 048 could be used to identify hybrids between V. radiata×V. trilobata; CEDG 043 for V. radiata×V. acontifolia; CEDG 050 for V. radiata×V. glabrescens; CEDG 173 for V. radiata×V. sublobata. Primer pair CEDG 015 showed polymorphism among the accessions V. trilobata. The primer pairs CEDG 086, CEDG 143 and CEDG 186 produced unique alleles in Vigna umbellata; CEDG 071 in Vigna radiata var. sublobata accessions; CEDG 271 in Vigna glabrescence; CEDG 068 in TMV(Mb) 1 (Vigna acontifolia) and CEDG 118 in Vigna trilobata/1 and Vigna trilobata/2.
Dendrogram formed based on unweighted pair group method with arithmetic average (UPGMA), the 36 genotypes were grouped into five clusters. Among five clusters, cluster I had the highest number of genotypes (27) followed by cluster IV (3). All the cultivated genotypes were grouped under the cluster I and cluster I had seven sub-clusters, among seven sub-clusters, sub-cluster VI had eleven genotypes followed by sub-cluster I had six genotypes. TMV(Mb) 1(cultivated-Vigna acontifolia) and V. acontifolia (wild) which is small drought resistant annual trailing herb comes under cluster II;V. radiata var. sublobata/1 and V. radiata var.sublobata/2 is a wild progenitor of cultivated greengram formed separate cluster III; V. trilobata/1, V. trilobata/2 and V. trilobata/3 which are resistant to drought formed cluster IV and cluster V had wild species in the tertiary gene pool viz., V. umbellata and V. glabrescence. Gupta and Gopalakrishna (2009) and Souframanien and Gopalakrishna (2009) by using SSR markers, 20 and 29 blackgram genotypes, respectively were grouped into five clusters. Genotypes from the distinct clusters could be used in the hybridization programme to get high yielding cultivars. Attempts were made to cross wild species included in the present study with cultivated greengram. This would enable the breeders to diversify the genetic base and to transfer important traits to cultivated genome.
The neighbour-joining tree developed based on weighted average for dissimilarity matrix grouped the 36 genotypes into five groups. All the cultivated genotypes clustered under group I and group I had seven sub-groups, among seven sub-groups, sub-group I had eight genotypes followed by sub-group six having seven genotypes, group II had wild species namely TMV(Mb) 1 and V. acontifolia; group III had V. radiata var. sublobata/1 and V. radiata var. sublobata/2; group IV with wild species V.trilobata/1, V. trilobata/2 and V. trilobata/3; group V had wild species V. umbellata and V. glabrescence. The genotype COGG10-09 formed a separate sub-cluster in cluster I of UPGMA analysis but it formed a sub-group along with other cultivated genotypes in the neighbour-joining method. All the cultivated genotypes grouped separately in both the analyses. However, there was a variation in the composition of genotypes in the sub-groups of the two methods of analysis. This study demonstrated that the adzukibean microsatellite markers are highly polymorphic and informative and can be success- fully used to genome analysis in greengram and in related Vigna species. Results indicated that sufficient variability is present in the greengram genotypes and could be helpful in the selection of suitable parents for breeding purpose and gene mapping studies.
2 Materials and Methods
2.1 Plant materials and DNA extraction
The material for this study consisted of 36 genotypes including eighteen cultivated, nine advanced breeding lines of greengram (Vigna radiata) and nine wild species of Vigna (Table 2). The genotypes were obtained from Department of Pulses, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore. The young leaves of the 15 days old plants were collected and the DNA was extracted by CTAB (mini-prep) method. The extracted DNA was purified for the RNA contamination by RNAase treatment. Purified DNA were then quantified and quality assessed by agarose gel-electrophoresis. Based on intensity of the bands, DNA was diluted to appropriate concentration for the use in molecular analysis.


Table 2 List of genotypes used for genetic diversity studies using SSR markers

2.2 Microsatellite analysis
Thirty-five adzukibean (Vigna angularis) micro- satellite primer pairs (Wang et al., 2004; Han et al., 2005; Chaitieng et al., 2006) were used in the study. SSR amplification reaction were carried out in a volume of 15 µL containing 50 ng of genomic DNA,1.5 µL of Tris with 15 Mm of Mgcl2, 10 Mm of dNTPs, 25Mm of MgCl2, 5 µm of (forward and reverse) primer , 0.5 unit of Taq DNA polymerase. The amplification was performed in master cycler gradient PCR (eppendorf). Amplification conditions were, initial denaturation at 94ºC for 3 minutes followed by 35 cycle of denaturation at 94ºC for 45 seconds, annealing at 45~60ºC for 1 minute (depending upon the primer) and extension 72ºC for 1 minute and final extension at 72ºC for 10 minutes. PCR products were subjected to electrophoresis in 3% agarose at 100 V for 3 hours in Electrophoresis system-GeNeiTM in 1X Tris-borate-EDTA buffer. The gel was observed under Gel Documentation System (Gel Stan).
2.3 Statistical analysis
The gels were scored and represented by their allele sizes as allelic data. Using the DARwin 5.0 software package (Perrier and Jacquemond-Collet., 2005), a simple matching dissimilarity index was calculated from the allele-size data set with 100 bootstraps and this matrix was then subjected to UPGMA and Neighbour-Joining analysis.Cluster analysis was performed using UPGMA strategy to obtain a dendro- gram. Neighbour-joining tree was also developed based on weighted average for dissimilarity matrix. Poly- morphic Information Content (PIC) values were calculated for SSR markers in order to characterize the capacity of each primer to reveal or detect polymorphic loci among the genotypes. PIC value was calculated using the formula PIC=1-∑pi2, where, pi is the frequency of the ‘ith allele (Smith et al., 1997).
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