VEGETOS: An International Journal of Plant ResearchOnline ISSN: 2229-4473
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Research Article, Vegetos Vol: 29 Issue: 3

Identification of Conserved Orthologous Set Markers in Cultivated Vigna radiata (L.) Wilczek

Rajappa JJ1*, Umdale SD2, Kole PR2 and Bhat VK2
1Division of Natural Resource Management (Agroforestry), ICAR Research Complex for NEH Region, Umiam (Barapani), Meghalaya-793 103, India
2Division of Plant Genomic Resources, National Bureau of Plant Genetic Resources, Pusa campus, New Delhi-110012, India
Corresponding author : Rajappa JJ
Division of Natural Resource Management (Agroforestry), ICAR Research Complex for NEH Region, Umiam (Barapani), Meghalaya-793 103, India
Tel: +91-8414881520
Fax: +420 385 310 356
E-mail: [email protected]
Received: May 29, 2016 Accepted: July 18, 2016 Published: July 25, 2016
Citation: Rajappa JJ, Umdale SD, Kole PR, Bhat VK (2016) Identification of Conserved Orthologous Set Markers in Cultivated Vigna radiata (L.)Wilczek. Vegetos 29:3. doi: 10.5958/2229-4473.2016.00066.5

Abstract

Identification of Conserved Orthologous Set Markers in Cultivated Vigna radiata (L.) Wilczek

With an aim to develop widely applicable conserved gene markers to identify and tag orthologous genes from related Vigna species to reveal phylogenetic relationships and the nature of genes conserved in genus Vigna across the evolution, low copy nuclear Conserved Ortholog Set (COS) genes were tested to explore the interspecific genetic relationship of Vigna radiata (L.) Wilczek with other species such as V. mungo (L.), V. umbellata Thunb., V. angularis Wild and V. unguiculata (L.) Walp. To detect COS regions, computational approach was followed utilizing the available expressed sequence tags (EST) database of Vigna radiata and its related species. The ESTs were processed to eliminate sequence repeats, contaminants and low-complexity sequences. Upon alignment with Soybean genome, only high quality ESTs were grouped into clusters from which consensus sequences representing putative genes are generated for each Vigna species. From 6443 ESTs, 2550 contigs were acquired to develop 230 primer pairs to amplify conserved orthologous sequences across Vigna species. Among 14 primer pairs used for validation with genotypes of V. radiata, 3 gave double bands (paralogs) and 9 produced single bands indicating single copy orthologs. This infers that DNA sequences identified by this comparative genomics approach would be of great use in analyzing genomic information of related, unexplored crop genomes to augment gene discovery and plant breeding in other legumes.

Keywords: Comparative genomics; Conserved orthologous set (COS) markers; ESTs; Vigna

Keywords

Comparative genomics; Conserved orthologous set (COS) markers; ESTs; Vigna

Introduction

Legumes provide protein-rich food for a large part of the world’s population hence the research on legumes is essential for the establishment of extensive genetic and genomic resources, which can accelerate the discovery of critical genes. Cultivated Vigna species are an important protein source in countries where people have limited access to food rich in protein [1]. Globally, Vigna species are cultivated as three main types such as Asian beans, African beans and American Vigna. The Asian Vigna is: moth bean (V. aconitifolia (Jacq.) Marechal), adzuki bean (V. angularis (Willd.) Ohwi and Ohashi), black gram (V. mungo L.), mungbean (V. radiata L.), rice bean (V. umbellata Thunb.); two African beans; bambara ground nut (V. subterranean L.) and cowpea (Vigna unguiculata (L.) Walp). One of the prerequisites for increasing yield of these legumes is the study on comparative genomics for better understanding of genome structure [2] which is essential for the establishment of extensive genetic and genomic resources to accelerate the discovery of critical to genes for crop improvement. Genetic improvement of Vigna species through conventional breeding has been slow due lack of exploitable genetic variability within the cultivated germplasm and also due to limited gene pool and sexual-incompatibility with wild and related species, the reservoir of desirable genes. Genome research in mungbean is still far behind the other major legume crops such as soybean, cowpea, and common bean, or even their relative but less important, adzuki bean. The genome study in mungbean and related Vigna species has been made possible by using genetic markers from other related legumes, and this trend will continue since only limited genetic resources are available for further study in this crop. The utility of marker assisted selections in improvement of Vigna species is limited largely due to limited marker polymorphism within the species and hence there is a scope to look for diversity in related crop species. Efforts are being made to develop high-throughput markers with greater resolution [3]. A high degree of similarity in the nucleotide sequences among green gram and other Vigna species reported in earlier studies by performing comparative genome analysis using DNA markers [4,5]. To alleviate the use of the wide genetic diversity present in wild relatives and landraces of crops, more information is needed on the organization and structure of their genes and genomes. Molecular markers linked to loci with important effects can facilitate the introgression of those traits into adapted germplasm. Agriculturally important traits captured during domestication are often coded by very limited number of loci with major phenotypic effects. These loci possibly have putative orthologous counterparts in other species [6] and therefore molecular markers, such as Conserved Orthologus Set (COS) markers are of great use in comparing genomic information between the phylogenetically related species within the genus or family. They are extremely useful for the analysis of genome evolution among closely and distantly related species within Leguminosae family. For a given group of species, a COS is formed by identifying a gene from each species that is orthologous to all other genes in the set. These markers are apparent single-copy evolutionary conserved genes in two or more species that share common ancestry (are orthologous) [7]. Information from the comparative genomic analyses within the genus Vigna will elucidate the genetics of domestication and enable the isolation of novel genes for use in breeding of mungbean germplasm in particular and Vigna in general. Generation of molecular level genetic data in Vigna has led to search for ways and means to utilize the existing plant genetic resources to support breeding challenges aiming at gainful applications in crop improvement. Molecular markers such as RAPD, AFLP [8] RFLP, ISSR [9,10], SSRs [11] and sequence tagged microsatellite site [12] have been used in mungbean to test their usefulness in genetic diversity among cultivars. During domestication, species of Vigna have got the agriculturally important traits coded by very limited number of loci with major phenotypic effects. It is common to find that these loci have putative orthologous counterparts in other species within the same genus and therefore molecular markers, such as Conserved Orthologous Set (COS) markers, are powerful in comparing genomic information across species. Orthologous genes are called so as they are related by common ancestry sharing the same function or activity and acquire homologous relationship across the speciation event.
COS sequences were first described by Tanksley et al. with identification of a subset of plant genes that have remained relatively stable in both sequence and copy number since the radiation of flowering plants from their last common ancestors [7]. This is the landmark work to enunciate utility of COS sequences in comparative genomics and phylogenetic studies. In a further refinement, Wu et al. [13] identified and annotated a large set of conserved, singlecopy, putatively orthologous genes using a set of approaches of computational and phylogenetic algorithms to demonstrate the use of these new ortholog resources to elucidate issues related to comparative genomics, molecular systematics, and gene evolution studies in the euasterid clade. Mingai Li [14] developed widely applicable COS markers (pCOS) for phylogenetic reconstructions at low taxonomic level and found that these markers are highly informative in phylogenetic reconstruction of congeneric species. An understanding of conservation of genome structure among legume species is a prerequisite, to use existing wide genetic diversity present in landraces and wild relatives of legumes [2]. Since the last three decades, we have seen large advancement in linking plant genomes through comparative genetic maps, especially for species belonging to the same family [15]. In the present study, computational approach of finding COS markers in Vigna species, we have used ESTs for finding conserved regions as molecular markers constructed from ESTs since they are contained within an exon region of genes that are actually expressed. The present study was undertaken with the objectives of mining the large EST collections of Vigna species to identify nonredundant ESTs, design primer pairs across all four species for putative COS markers across the Vigna species and finally to test some of these markers in V. radiata. To define conserved genic regions between V. radiata and other related Vigna species with soybean genome (which is the most sequenced and is also a legume) orthologous groups that share among them were identified. Validation is done using some of the newly designed primers from contigs of Vigna species and proven their amplification in V.radiata.
We demonstrate here the use of this new Ortholog resource to shed light on issues related to comparative genomics, molecular systematics and gene evolution studies in the legumes especially in Vigna genus. The present study was undertaken with the objectives of mining the large EST collections of Vigna species to identify nonredundant ESTs, design primer pairs across all four species for putative COS markers across the Vigna species and finally to test some of these markers in V. radiata. To define conserved genic regions between V. radiata and other related Vigna species with soybean genome (which is the most sequenced and is also a legume) orthologous groups that share among them were identified.

Materials and Methods

EST datasets
The ESTs of all Vigna species in the study were searched and retrieved from NCBI (ftp://ftp.ncbi.nih.gov/blast/db/). Also the genome sequence data set of Soybean was downloaded and used as reference genome for identifying conserved genomic regions in Vigna species. EST
Processing and assembly
The repetitive and ambiguous sequences in the downloaded ESTs were first trimmed. Subsequently, ESTs with sequences <30 bp were omitted from the final data set. The processed EST sequence files were combined and assembled into contigs using the CAP3 program at both high and low stringency levels. The steps followed while EST analyses [16] are depicted in Figure 1.
Figure 1: EST processing and Analysis.
Primer designing
The Conserved Primer 2.0 pipeline was implemented and the command line made it possible to design intron-flanking primer pairs or marker candidates for polymorphism discovery in a highthroughput manner and to use any genome size of the model species and any number of the ESTs as inputs without memory and speed restrictions. Processed ESTs are input for designing primers in batch (Tables 1-3) and got custom synthesized.
Table 1: Statistics of EST analysis for Vigna spp.
Table 2: List of conserved primers designed from contigs obtained using available ESTs of Vigna species and used for amplification in V.radiata genotypes.
Table 3: List of Conserved markers identified in the study.
Pant materials and DNA Isolation
Genomic DNA was isolated using the CTAB isolation method from young leaves collected from plants of 40 mungbean varieties. DNA quantity is estimated by Nanodrop method and quality by running 2uL of genomic DNA solution mixed with 1 μL loading buffer on a 1% agarose gel. The DNA was then diluted to a concentration of 10ng per uL for PCR amplification.
PCR Amplification and marker analysis
To test the feasibility of using Vigna COS markers in mungbean, genomic DNA fragments from 40 varieties were amplified using 14 COS markers. PCRs were conducted in a 25-ml reaction volume each reaction consisted of 10 mM Tris-HCl (pH 9.0 at room temperature), 1.5 mM MgCl2, 100 mM each of dNTPs, 0.1 mM each primer, 10 ng of genomic DNA template, and 1unit of Taq DNA polymerase. Reactions were heated at 940C for 4 min followed by 35 cycles of 1 min at 940C, 1 min at specific annealing temperature for each primer pairs and a 1-min extension at 720C. Final reactions were extended at 720C for 5 min. Amplification was performed in a programmable thermal controller. Following the amplification reactions, the PCR products were separated on 1.8% agarose gel and visualized using ethedium bromide staining (Figure 2). Successful amplification of COS markers are mentioned in Table 2.
Figure 2: Representative patterns of DNA Amplification of single copy genes in V. radiata.

Results and Discussion

Approximately 6443 ESTs were processed and analyzed for the searching of COS in Vigna genus for redundancy minimization and assembling of sequences. The non-redundant ESTs analysed (Figure 1) were used for the development of specific intron-based markers. Soybean genomic sequence database as reference is used to predict intron positions in the EST sequences and then designed a pair of primers flanking the intron position. The Multiple sequence alignments, with the Soybean genomic sequence inferred intron position, facilitate design of PCR primers that anneal to conserved exon sequences and amplify across more diverged introns. A query EST was considered to be homologous to a subject-coding sequence only if there were at least 100 bp overlapping and 80% similarity between them. Only high-quality ESTs are grouped into clusters based on sequence similarity and assembly of clusters which would represent a putative gene, likewise consensus sequences representing putative genes are generated for each Vigna species in the study. We found four contigs in Vigna radiata from 829 processed ESTs, 33 contigs for V. mungo from 299 processed ESTs, 268 contigs out of 3006 ESTs of V. umbellata, and 175 contigs from 2309 ESTs belonging to V. unguiculata (Table 1). These high-quality ESTs are grouped into ‘clusters’ based on sequence similarity. The maximum informative consensus sequences generated by assembling these clusters represent a putative gene. The output of all Vigna EST processing pipeline is the list of putative genes belonging to respective Vigna species providing list of 230 conserved primers. Of these, 14 markers were used to validate the COS markers generated with amplification of genomic DNA from 40 genotypes of Vigna radiata in Indian collections.
The successful amplification revealed the mixed type of result such as single band and multiple bands. To confirm the amplification, repeated reactions were carried for multiple/double banded amplicons and there was no change in such reactions even after changing the required components and thermal cycling conditions of PCR reaction. Three primer pairs (CSD-48FR, CA908739-54FR and umbellata_contig16-59FR) produced double or multiple bands which indicates that they probably multi gene loci and hence were not considered. Primer–genotype combinations that gave what appeared to be single bands on agarose gels were noted which are likely to be noted as single copy genes are considered as orthologs. Primers that produced single bands with genotypes of V. radiata varieties were 53- FR, 56-FR, 58-FR, 60-FR, AGA, SP, CGGCS and PK (Figure 2). Of these, the primer pair 57-FR was derived from contig derived from ESTs belonging to Vigna mungo, whereas 58-FR and 60-FR were from contig of V. umbellata. This infers that the there is a possibility of these kind of markers derived from available genomic information (ESTs) of related species can amplify in V.radiata. Finally we have found that amplification of single copy genes attained were from 11 primer pairs (Figure 2).
Molecular (COS) markers in comparative genomic studies within Vigna
Though much progress has been made in the genomics of Vigna species, yet it is still far behind that in other grain legumes like common bean and soybean. Most of the cultivated Vigna species have a narrow genetic base resulting in limited marker polymorphism within the germplasm. Due to this major limitation, most of the genetic linkage maps in Vigna species have been constructed using inter-specific or inter-sub specific crosses to increase the level of polymorphism. The use of COS markers as a starting point for marker development was motivated by their expected low copy number in the genomes of various legume species for which genomic information is not abundant. A major challenge for comparative legume genomics is to translate information gained from model species into improvements in crop legumes. The complexity of that challenge may well be defined by the structural and functional similarities and dissimilarities among these very fascinating genomes. Agriculturally important traits captured during domestication are often coded by very limited number of loci with major phenotypic effects. It is common to find that these loci have putative orthologous counterparts (Orthologous Set markers) in other species and therefore such molecular markers are powerful in comparing genomic information across species.
The present study aimed at identifying the COS markers through computational approach and validating some of them in wet-lab screening lead us to use of this new ortholog resource can shed light on issues related to comparative genomics, molecular systematics, and gene evolution studies in the Vigna genus. COS markers thus selected can further be taken for characterization to test their applicability for phylogenetic studies in Asiatic Vigna species as COS markers are evolutionary conserved single-copy genes of great use in constructing syntenic genetic maps among species. The COS markers reported here will be useful for comparative mapping at the family level and may help to establish the syntenic relationship between genomes of different Vigna species, allowing a picture of chromosome evolution. In this study, a set of 230 COS markers were identified using ESTs of four Vigna species and some amplified in V.radiata can serve as anchor markers for a syntenic map of other Vigna species. This study forms the basis for a number of significant outcome for genomics of Vigna in general: (1) the genic markers developed here may be used across Vigna species to determine patterns of chromosomal evolution, as argued previously for markers with defined utility [17], and to characterize syntenic relationships between V. radiata and other related species under cultivation; (2) with the aid of shared anchor markers, the Vigna map created may be integrated with all existing legume maps containing various important domestication traits; (3) the high levels of syntenic relationships if detected between these species will enable the future identification of tightly linked markers for direct marker-assisted trait selection and future mapbased isolation of candidate genes.
COS markers conserved in legume species can be used in phylogenetic studies and in the identification of conserved noncoding regions or to make comparative maps of major crop species. Although the amount of genetic data available for plants is increasing exponentially, most of the work is being done in just a few species. The identification of a set of such markers common to a variety of species within genus would allow researchers studying less wellcharacterized plant species to take advantage of gains being made in legume species such as Medicago, soybean and other model or reference plant species. Because ESTs give an idea of the genes expressed in an organism, and because EST data are abundant for a variety of species, ESTs are ideal starting material for the identification of genes conserved among species.

Acknowledgment

The authors are thankful to the Director, NBPGR, New Delhi, for providing the facilities for the research work. The scholarship provided to the author1 by the Indian Council of Agricultural Research, New Delhi, India, for pursuing Ph.D. research from Indian Agricultural Research Institute, New Delhi is gratefully acknowledged.

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