Development and characterization of a new set of genomic microsatellite markers in rice bean (Vigna umbellata (Thunb.) Ohwi and Ohashi) and their utilization in genetic diversity analysis of collections from North East India.

Banshanlang Iangrai,D Evanoreen Ann Khongwir,Emica Mary Gatphoh,Nikhil Kumar Chrungoo,Gratify Pale,Arunava Pattanayak,Alpana Das,Manoj Prasad

doi:10.1371/journal.pone.0179801

Banshanlang Iangrai, D Evanoreen Ann Khongwir + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0179801

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Abstract

Rice bean [Vigna umbellate (Thumb.) Ohwi and Ohashi] is an underutilized crop believed to be domesticated in the Myanmar-Thailand region of Asia. In India, rice bean is mainly cultivated in the North-Eastern Hills, which is a hotspot for biological diversity. A 5' anchored PCR was used to develop microsatellite markers in rice bean. Twenty-eight specific primer pairs were designed and employed to characterize sixty five ricebean accessions collected from North East India. A total of 179 alleles were amplified with an average of 6.393 alleles per locus. The gene diversity was high (mean 0.534) in the accessions collected from Darjeeling, Nagaland and Manipur, which are bordering areas with East Nepal and Myanmar, respectively. Exceptionally high outcrossing rate was observed in the entire population. Population structure analysis identified three distinct clusters in which accessions collected from areas bordering Myanmar and East Nepal grouped separately. Using a combination of STRUCTURE and Principal Coordinate Analysis, relative affinity of the intermediate accessions could be established. However, differences in allelic counts among populations were non-significant. The results showed that there is a high level of genetic diversity within the accessions, with high outcrossing rate.

Highlights

DNA sample concentration was determined using a NanoDrop2000 (Thermo Fisher Corporation Inc.), and the DNA samples were diluted to 10 ng/μl prior to polymerase chain reaction (PCR) amplification
DNA sequencing of the clones indicated the presence of a total of 124 microsatellite repeats (Table 2)
The study demonstrated that 5’ anchored PCR-based enrichment method is a fast and reliable method for developing new SSR markers, and for a better understanding of the nature of the admixtures identified by STRUCTURE software, principal co-ordinate analysis (PCoA) is a valuable statistical tool

Summary

Introduction

Rice bean [Vigna umbellate (Thunb) Ohwi and Ohashi] is a less utilized grain legume cultivated mainly in Nepal, Bhutan, North East India up to Myanmar, Southern China, Northern. In the North Eastern Hill Region of India (Arunachal Pradesh, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim, and Tripura) rice bean is grown predominantly under rainfed conditions in a mixed farming system, under shifting cultivation or in kitchen gardens and backyards. It is grown on a limited scale in the eastern and western peninsular tracts of India. The most comprehensive study so far with a very wide range of cultivated and wild rice bean accessions is by Tian et al [1] They studied 472 accessions (cultivated and wild) from 16 Asian countries and reported high gene diversity (>0.5) in the South and South East Asian accessions. The 5’-anchored polymerase chain reaction [28,29] is one of the simplest and fast methodsfor developing SSR enriched genomic libraries and subsequent isolation of microsatellites that does not require substantial investment

Materials and methods

38 BKSB-182

Results and discussion