Legume Research

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Legume Research, volume 46 issue 10 (october 2023) : 1405-1409

Development of Genome-wide Simple Sequence Repeat Markers from Whole-genome Sequence of Mungbean (Vigna radiata)

Kanimoli Mathivathana Mayalagu1,2, Karthikeyan Adhimoolam3, Jagadeeshselvam Nallathambi1, Veera Ranjani Rajagopalan1, Madhumitha Balasubramanian1,4,*, Madiha Natchi Samu Shihabdeen1, Senthil Natesan5, Raveendran Muthurajan1, Sudha Manickam1
1Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
2Department of Plant Breeding and Genetics, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai-625 104, Tamil Nadu, India.
3Department of Biotechnology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai-625 104, Tamil Nadu, India.
4Department of Agriculture, School of Agriculture and Biosciences, Karunya Institute of Technology and Sciences, Coimbatore-641 114, Tamil Nadu, India.
5Department of Plant Molecular Biology and Bioinformatics, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
  • Submitted19-06-2020|

  • Accepted03-05-2021|

  • First Online 31-08-2021|

  • doi 10.18805/LR-4444

Cite article:- Mayalagu Mathivathana Kanimoli, Adhimoolam Karthikeyan, Nallathambi Jagadeeshselvam, Rajagopalan Ranjani Veera, Balasubramanian Madhumitha, Shihabdeen Samu Natchi Madiha, Natesan Senthil, Muthurajan Raveendran, Manickam Sudha (2023). Development of Genome-wide Simple Sequence Repeat Markers from Whole-genome Sequence of Mungbean (Vigna radiata) . Legume Research. 46(10): 1405-1409. doi: 10.18805/LR-4444.
Mungbean is an important pulse crop and it is mainly cultivated in Asia for human consumption. Its small genome and diploid nature make it a well-suited model organism among legume crops. Thus, cost-effective, reliable and highly polymorphic molecular markers distributing the whole genome are needed for diversity, mapping and functional genomics studies in this model species. The whole-genome sequence of mungbean was obtained and used as a source of identification of simple sequence repeats (SSR). The sequence reads were aligned and SSRs detection was performed using the Phobos plugin tandem repeat finder in the Geneious software program. A total of 12 mungbean genotypes were selected to validate the newly developed SSR markers. In the present study, a total of 12, 49,774 and 11, 86, 386 perfect and imperfect SSR repeats were identified from the mungbean genome. The tri-repeats were the most abundant (26.10%), followed by hexa (20.82%), penta (20.45%), tetra (17.65%) and di-repeats (14.95%). We designed 1330 SSR primers based on the genomic sequence of flanking perfect SSRs (Di and tri-repeats). Among them, 50 SSR primers uniformly distributed across the 11 mungbean chromosomes were selected and used to validate 12 mungbean genotypes. The newly developed genomic SSR markers generated in the present study are a valuable genomic resource for the mungbean breeding programs.
Mungbean (Vigna radiata) is an important grain legume crop in Asian agriculture, particularly in India and is becoming popular in Asia’s contiguous areas. It is well suited to many cropping systems and constitutes an essential source of cereal-based diets worldwide, covering more than six million hectares per annum (Karthikeyan et al., 2014). Mungbean originated in India and is predominantly grown in Asian countries such as China, India, Pakistan and Thailand. India is the world’s largest producer and the total area covered under mungbean in India was 3.44 million ha with a total production of 1.78 million tonnes in 2019-2020. Mungbean seed has 24% digestible protein with low flatulence and is rich in vitamin A, iron, calcium and zinc. It is a drought-resistant crop suitable for dryland farming and predominantly used as an intercrop with other crops. Diverse uses of mungbean make it a more widely desired crop plant and are rapidly increasing its demand. Therefore, it is necessary to improve traits with economic value, including yield potential, protein content and biotic and abiotic stress resistance, to enhance mungbean potential as a crop (Nair et al., 2019). Different breeding strategies have been used to improve the economically important traits in the mungbean. However, the success is limited due to the precision in breeding programs. Advances in sequencing technology have provided a powerful set of genetic tools which can facilitate the establishment of genomics-based breeding programs. Plant geneticists believe that molecular markers are useful genomics tools in plant breeding programs to make the selection more efficient. With the advent of molecular markers, selection decisions were made by integrating information from molecular markers and phenotypic data through marker-assisted selection (MAS) (Fujino et al., 2019).

At present, simple sequence repeats (SSR) and single nucleotide polymorphism (SNPs) play an important role in many types of genetic analyses, including the construction of linkage maps, QTL mapping, diversity assessment of germplasm and identification of molecular markers for MAS in different crops. However, only a few hundred mungbean SSR markers have been developed for QTL mapping and linkage maps associated with important traits like resistance to abiotic and biotic stress and yield (Kitsanachandee et al., 2013; Tangphatsornruang et al., 2009; Kaewwongwal et al., 2017). Owing to its small genome size and evolutionary divergence, the mungbean is a potentially important model plant among the legumes. The publishing of the whole genome sequence (WGS) in 2014 (Kang et al., 2014) has accelerated mungbean research in diverse ways. The availability of WGS provides information not only for the identification of genes, study of the evolution of species, genome structure but also is an ideal resource for the genome-wide identification of SSRs.

With this backdrop, we have developed a new set of SSR markers for augmenting the genomic resources especially, to assist the genetic and molecular dissection of mungbean genes that encode traits with economic value, including quantitative traits. This was achieved by using the following procedures: (i) identification of the SSRs from mungbean WGS; (ii) design of primer pairs in regions flanking the SSR motifs from WGS; (ii) experimental validation of representative SSR markers using fragment analysis among 12 mungbean genotypes.

SSR identification and primer design

The mungbean WGS (Vr 1.0), which is publicly available at https://legumeinfo.org/gbrowsevigra1.0 (Verified 28th Feb 2018), was obtained and used as a source of identification of SSRs with di-, tri- and tetranucleotide repeats. Geneious software (Kearse et al., 2012) was used to screen the mungbean genome sequences. The parameter used for SSRs identification in this study was from 2-8 bp motifs and mononucleotide was not considered due to the difficulty of distinguishing real SSRs from sequencing or assembly error. DNA sequences were searched for perfect and compound or imperfect SSRs, with a basic motif for 2-8 bp. Di-, tri- and tetranucleotide SSRs with a repeat number of five or greater were identified. The presence of repeat regions (SSRs) in the mungbean genome and their positions were determined through the Phobos plugin available in the Geneious software. The results from the PHOBOS output were filtered to identify only the perfectly matching SSRs. Primer premier 3.0 (PREMIER Biosoft International, Palo Alto, California USA) (Rozen and Skaletsky, 2000) was used to design primer pairs from the genomic sequence flanking perfect SSRs only.

Plant genetic materials and genomic DNA isolation

 The seeds of the 12 mungbean genotypes used in this study were obtained from the National Pulses Research Center, Tamil Nadu Agricultural University, Vamban, Tamil Nadu and India. The genomic DNA was isolated from 10-day-old young leaves of mungbean genotypes using the cetyltrimethyl ammonium bromide (CTAB) method (Sudha, 2009). The quantity and quality of the genomic DNA were confirmed using UV Bio-spectrophotometer (Eppendorf, Germany) and agarose gel (0.8%). The final concentration of all the samples was adjusted to 50 ng/µl Biospectrophotometer).
PCR analysis and validation of selected SSRs
A total of 50 SSR markers, at least four SSR markers per chromosome, covering the eleven mungbean chromosomes were selected and used for validation in mungbean genotypes. The PCR analysis for SSR markers and gel electrophoresis conducted following the methodology adopted by Karthikeyan et al., (2012) and Sudha et al., (2013). Briefly, PCR was conducted in a total volume of 10 µl, including 1 µl of genomic DNA, 0.5 µl of the forward primer and 0.5 µl of reverse primer (Eurofins Genomics India, Bangalore, India), 7.0 µl 1X master mix (Amplicon, Denmark) and 1 µl of ddH2O. Amplification was carried out in a thermal cycler (Eppendorf, Germany) programmed to run the following temperature profile: 3 min at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at 55-60°C and 1 min at 72°C. The final elongation step was extended to 10 min at 72°C and finally maintained at 4°C. The five microliters of PCR products were separated by Agarose gel (3%) electrophoresis in 1X TBE bufier with 120 V for 2.0 h and the amplified bands were visualized in the gel documentation system (Gel Doc XR + Imaging system, BIO-RAD, USA). The general scheme for developing SSR markers from WGS and their validation in mungbean genotypes presented in Fig 1.

Fig 1: Schematic representation for the development and validation of mungbean SSR markers

The Mungbean crop is valued for its nutritional and agronomic benefits. However, mungbean breeding programs have stagnated due to limited funding from various funding agencies and research organizations. Thus, more breeding programs integration with genomic tools have to be planned for crop improvement. Molecular markers are powerful genomics tools to study the genetic variation and identify the genomic regions/ genes associated with a specific trait in plants. Among the various marker systems, SSR was the best choice for various applications, primarily in marker-assisted breeding (MAB) programs. Compare to other markers, SSR markers are reliable and relatively inexpensive molecular markers. Because they use PCR, are codominant and have high levels of allelic diversity. Unfortunately, the number of markers from mungbean on a public platform is very limited. Primarily, random markers and SSR markers from closely related species, including adzuki bean and soybean were used in mungbean genetic studies (Isemura et al., 2012; Sudha et al., 2013; Kitsanachandee et al., 2013). The first version of the mungbean genome sequence was recently published and it is a game-changer to mungbean molecular breeding programs. It facilitated to development of genome-wide markers and cost-effective genotyping platforms. The availability of WGS of mungbean offers the chance to develop genome-wide SSRs.

 Discovery and mining of genomic SSR loci from WGS have had fruitful applications in many plant species, including soybean (Song et al., 2010), peanut (Lu et al., 2019), castor bean (Tan et al., 2014). In the present study, a total of 12, 49,774 and 11, 86, 386 perfect and imperfect SSR repeats with di-, tri-, tetra-, penta and hexanucleotide repeats equal to, or longer than 8, 5, 4, 3 and 2 repeat units were identified from the mungbean genome. This study examined the frequency and distribution of SSRs with motifs of 2–8 bp long and the least lengths of 18 bp in the mungbean genome. The standard we used was based on the point that polymorphism levels and mutation rate correlate positively with the number of repeat units. As a result, a higher polymorphic ratio is expected for these SSR markers developed in this study. Frequency analysis of various nucleotide repeats in mungbean shown that tri-repeats were the most abundant (26.10%), followed by hexa (20.82%), penta (20.45%), tetra (17.65%) and di-repeats (14.95%). There were 186839 di-repeats, 326232 tri-repeats, 220922 tetra-repeats, 255544 penta-repeats and 260237 hexa-repeats (Fig 2 and Table 1). These results agree with the reports of Katti et al., 2001; Lawson and Zhang, 2006; Bhandawat et al., 2016, who described that tri repeats were more abundant in various crops.

Fig 2: Distribution of different nucleotide repeats in the mungbean genome.

Table 1: Distribution of different nucleotide repeats in the mungbean genome.

Among the tri-repeats, (ATT) n is the most abundant (44.8%), followed by (AAG) n (23.6%), (AAC) n (13.1%), (AGG) n (4.1%), (ACT) n (3.9%), (AGT) n (3.8%), (ACC) n (3.8%), (ACG) n (1.0%), (AGC) n (1.0%) and (CCG) n (0.9%). Of the dinucleotide motifs, (AT) n is the most abundant (58.5%), followed by (AG) n (24.2%) and (AC) n (17.2%). The (GC) n motif is the least frequent (0.1%) dinucleotide in the genome. This result is consistent with other studies indicating that genomic SSRs with GC-rich repeats are rare in dicot species (Wang et al., 1994; Tangphatsornruang et al., 2009). Among the di repeats, the AT was the most abundant repeats. AT repeats have been reported most abundant dinucleotide repeats in many crops (Cardle et al., 2000; Morgante et al., 2002). AAT and AAAT repeats were abundant for tri and tetranucleotide repeats, respectively. ATT/AAT has been reported to be the most common tri-nucleotide motif in other crops, including soybean (Cregan et al., 1999), groundnut (Ferguson et al., 2004) and chickpea (Lichtenzveig et al., 2005). 

SSR repeat regions in the mungbean genome and their positions were determined by the Phobos plugin available in the Geneious software. The results from the PHOBOS output were filtered to identify only the perfectly matching SSRs. Moreover, Phobos does not interact directly with Primer 3, but Phobos were used through Geneious. The results of the loci search in Phobos can be easily piped to Primer 3. We designed a set of primer pairs based on the genomic sequence of flanking perfect SSRs (Di and tri-repeats) (Table 2) only and validated a subset of primer pairs. A total of 1330 SSR primers were designed. Among them, 50 SSR primers uniformly distributed across the 11 mungbean chromosomes were selected and used to validate 12 mungbean genotypes originated from India (Fig 3). Initially, these primer pairs’ effectiveness was detected in mung bean genotype VRM (Gg) 1 and then tested in other genotypes. Eventually, 10 of 50 SSR primer pairs were identified as polymorphic. It shows that newly developed SSR primers had good amplification efficiency and were the potential to analyse the genetic diversity in mungbean accessions. The proportions of effectiveness and polymorphism were lower than had been observed in similar studies in mungbean and its relatives Wang et al., 2015; Chen et al., 2015; Pratap et al., 2016. The low polymorphism may be due to the low diversity of samples used, given that the 12 genotypes were all from India. A study of SSR and other molecular markers in the Indian mungbean similarly showed limited diversity (Singh et al., 2014; Gupta et al., 2014; Kaur et al., 2018). The low diversity in Vigna germplasm has been reported for several Vigna species, including mungbean, urdbean, ricebean and adzuki bean, except when the tested samples were selected wide geographical region or wild genotypes were included. In summary, the novel genomic SSR markers generated in the present study are a valuable genomic resource for the mungbean research community. These newly developed SSR markers will help assess genetic diversity, fill the gaps of current maps, QTL fine mapping and map-based cloning of genes of interest in mungbean.

Table 2: Details of perfect SSR repeats in the mungbean genome.

Fig 3: PCR amplification of SSR marker Vr_SSR_03_ 07 on mungbean genotypes.

This work was financially supported through grants from the Department of Biotechnology, Government of India (GOI) (BT/PR5095/AGR/2/847/2012, Phase: II, (Dt 27-5-2015), the Science and Engineering Research Board (SERB) to SM, Department of Science and Technology (DST), GOI, NPDF fellowship program (PDF/2016/003676) to KA.

  1. Bhandawat, A., Singh, G., Raina, A.S., Kaur, J., Sharma, R.K. (2016). Development of genic SSR marker resource from RNA- Seq data in Dendrocalamus latiflorus. J. Plant Biochem Biotechnol. 25(2): 179-190.

  2. Cardle, L., Ramsay, L., Milbourne, D., Macaulay, M., Marshall, D. and Waugh, R. (2000). Computational and experimental characterization of physically clustered simple sequence repeats in plants. Genetics. 156(2): 847-854.

  3. Chen, H., Qiao, L., Wang, L., Blair, M.W. and Cheng, X. (2015). Assessment of genetic diversity and population structure of mung bean (Vigna radiata) germplasm using EST- based and genomic SSR markers. Gene. 566(2): 175-183.

  4. Cregan, P.B., Jarvik, T., Bush, A.L., Shoemaker, R.C., Lark, K.G., Kahler, A.L. and Specht, J.E. (1999). An integrated genetic linkage map of the soybean genome. Crop Science. 39(5): 1464-1490.

  5. Ferguson, M.E., Burow, M.D., Schulze, S.R., Bramel, P.J., Paterson, A.H., Kresovich, S. and Mitchell, S. (2004). Microsatellite identification and characterization in peanut (A. hypogaea L.). Theoretical and Applied Genetics. 108(6): 1064-1070.

  6. Fujino, K., Hirayama, Y., Kaji, R. (2019). Marker-assisted selection in rice breeding programs in Hokkaido. Breed Science. 69(3): 383-392.

  7. Gupta, S.K., Bansal, R. and Gopalakrishna, T. (2014). Development and characterization of genic SSR markers for mungbean [Vigna radiata (L.) Wilczek]. Euphytica. 195(2): 245-258.

  8. Gwag, J-G., Dixit, A., Park, Y-J., Ma, K-H., Kwon, S-J., Cho, G-T., Lee, G-A., Lee, S-Y., Kang, H-K. and Lee, S-H. (2010). Assessment of genetic diversity and population structure in mungbean. Genes and Genomics 32: 299-308.

  9. Isemura, T., Kaga, A., Tabata, S., Somta, P., Srinives, P., Shimizu, T., Jo, U., Vaughan, D.A., Tomooka, N. (2012). Construction of a genetic linkage map and genetic analysis of domestication related traits in mungbean (Vigna radiata). PLoS One. 7(8): e41304. 

  10. Kaur, G., Joshi, A., Jain, D. (2018). Marker-assisted evaluation of Genetic Diversity in Mungbean [Vigna radiata (L.) Wilcezk] genotypes. Brazilian Archives of Biology and Technology Vol.61, e18160613.

  11. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P., and Drummond, A. (2012). Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics (Oxford, England), 28(12): 1647-1649. 

  12. Kaewwongwal, A., Chen, J., Somta, P., Kongjaimun, A., Yimram, T., Chen, X., et al. (2017). Novel Alleles of Two Tightly Linked Genes Encoding Polygalacturonase-Inhibiting Proteins (VrPGIP1 and VrPGIP2) Associated with the Br Locus that Confer Bruchid (Callosobruchus spp.) Resistance to Mungbean (Vigna radiata) Accession. V2709. Front. Plant Sci. 8: 1692.

  13. Kang, Y.J., Kim, S.K., Kim, M.Y., Lestari, P., Kim, K.H., Ha, B.K. and Shim, S. (2014). Genome sequence of mungbean and insights into evolution within Vigna species. Nature communications. 5: 5443.

  14. Karthikeyan, A., Sudha, M., Nagarajan, P., Pandiyan, M., Muthurajan, R., Natesan, S., Kathithachalam, A. (2012). Using SSR marker to identify the MYMV resistance gene in mungbean [Vigna radiata (L.) Wilczek]. Rom. J. Biol. Plant Biol. 57: 105-113. 

  15. Karthikeyan, A., Shobhana, V.G., Sudha, M., Raveendran, M., Senthil, N., Pandiyan, M. and Nagarajan, P. (2014). Mungbean yellow mosaic virus (MYMV): a threat to green gram (Vigna radiata) production in Asia. International journal of pest management. 60(4): 314-324.

  16. Katti, M.V., Ranjekar, P.K, Gupta, V.S. (2001). Differential distribution of simple sequence repeats in eukaryotic genome sequences. Mol Biol Evol 18: 1161-1167

  17. Kitsanachandee, R., Somta, P., Chatchawankanphanich, O., Akhtar, K.P., Shah, T.M., Nair, R.M., Bains, T.S., Sirari, A., Kaur, L. and Srinives, P. (2013). Detection of quantitative trait loci for mungbean yellow mosaic India virus (MYMIV) resistance in mungbean [Vigna radiata (L.) Wilczek] in India and Pakistan. Breed Sci. 63: 367-373.

  18. Lawson, M.J., Zhang, L. (2006). Distinct patterns of SSR distribution in the Arabidopsis thaliana and rice genomes. Genome Biol. 7(R14): 3.

  19. Lichtenzveig, J., Scheuring, C., Dodge, J., Abbo, S. and Zhang, H. B. (2005). Construction of BAC and BIBAC libraries and their applications for generation of SSR markers for genome analysis of chickpea, Cicer arietinum L. Theoretical and Applied Genetics. 110(3): 492-510.

  20. Lu, Q., Hong, Y., Li, S., Liu, H., Li, H., Zhang, J., Lan, H., Liu, H., Li, X., Wen, S., Zhou, G., Varshney, R. K., Jiang, H., Chen, X., and Liang, X. (2019). Genome-wide identification of microsatellite markers from cultivated peanut (Arachis hypogaea L.). BMC genomics. 20(1): 799. 

  21. Morgante, M., Hanafey, M. and Powell, W. (2002). Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes. Nature genetics. 30(2): 194-200.

  22. Nair, R.M, Pandey, A.K., War, A.R., Hanumantharao, B., Shwe, T., Alam, A.K.M.M., Pratap, A., Malik, S.R., Karimi. R., Mbeyagala, E.K., Douglas, C.A., Rane, J. and Schafleitner, R. (2019). Biotic and abiotic constraints in mungbean production-progress in genetic improvement. Front. Plant Sci. 10:1340.

  23. Pratap, A., Gupta, S., Tomar, R., Malviya, N., Maurya, R., Pandey, V.R., Mehandi, S., Singh, N.P. (2016). Cross-genera amplification of informative microsatellite markers from common bean and scarlet runner bean for assessment of genetic diversity in mungbean (Vigna radiata). Plant Breed. 135: 499-505. 

  24. Rozen, S. and Skaletsky, H. (2000). Primer 3 on the WWW for general users and for biologist programmers. In Bioinformatics methods and protocols. Humana Press, Totowa, NJ. (pp. 365-386).

  25. Singh, A., Dikshit, H.K., Jain, N., Singh, D. and Yadav, R.N. (2014). Efficiency of SSR, ISSR and RAPD markers in molecular characterization of mungbean and other Vigna species. India J. Biotech. 13: 81-8.

  26. Song, Q., Jia, G., Zhu, Y., Grant, D., Nelson, R.T., Hwang, E-Y., Hyten, D.L., Cregan, P.B. (2010). Abundance of SSR motifs and development of candidate polymorphic SSR markers (BARCSOYSSR_1. 0) in soybean. Crop Sci 50: 1950-1960.

  27. Sudha, M. (2009). DNA isolation protocol for Vigna radiata with free of phenolics. Nat Protoc. 167.

  28. Sudha, M., Anusuya, P., Mahadev, N.G., Karthikeyan, A., Nagarajan, P., Raveendran, M., Senthil, N., Pandiyan, M., Angappan, K., Balasubramanian, P. (2013). Molecular studies on mungbean [Vigna radiata (L.) Wilczek] and ricebean [Vigna umbellata (Thunb.)] interspecific hybridization for Mungbean Yellow Mosaic Virus resistance and development of species-specific SCAR marker for ricebean. Arch. Phytopathol. Plant Prot. 46: 503-517.

  29. Tan, M., Wu, K., Wang, L. et al. (2014). Developing and characterising Ricinus communis SSR markers by data mining of whole- genome sequences. Mol Breeding. 34: 893-904. 

  30. Tangphatsornruang, S., Somta, P., Uthaipaisanwong, P., Chanprasert, J., Sangsrakru D., Seehalak, W., Sommanas, W., Tragoonrung S. and Srinives P. (2009). Characterization of microsatellites and gene contents from genome shotgun sequences of mungbean [Vigna radiata (L.) Wilczek] BMC Plant Biology. 9: 137.

  31. Wang, Z., Weber, J.L., Zhong, G. and Tanksley, S.D. (1994). Survey of plant short tandem DNA repeats. Theor Appl Genet. 88(1): 1-6.

  32. Wang, L.X., Chen, H.L., Bai, P., Wu, J.X., Wang, S.H., Blair, M.W. and Cheng, X.Z. (2015). The transferability and polymorphism of mung bean SSR markers in rice bean germplasm. Molecular breeding. 35(2): 77. 

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