Pea (Pisum sativum L.) is an important grain legume grown in temperate regions of the world because its seeds are a cheap and rich source of protein and contribute to the nutritional quality of human and animal diets. Marker-assisted selection (MAS) for agronomic traits such as yield, quality, and tolerance to abiotic and biotic stresses is not widely applied in pea due to unavailability of a reference pea genome and the limited number of molecular markers for tagging of agronomically important genes in pea improvement programs (Jain et al., 2012; Smýkal et al., 2012). Pea expressed sequence tag (EST) sequences ( http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html) are valuable tools for developing breeder-friendly markers from coding regions of genes and have been used in the past to develop a modest number of simple sequence repeat (SSR) markers in pea (Xu et al., 2012; Mishra et al., 2012; DeCaire et al., 2012; Zhuang et al., 2013). Genomic resources of the sequenced model legume Medicago truncatula Gaertn. ( http://gbrowse.jcvi.org/cgi-bin/gbrowse/medicago/) also offer a wealth of information for developing EST-derived genic markers in closely related species using a comparative genomics approach (Smýkal et al., 2012). Genic markers developed in this study using the conserved sequences between the two legumes are valuable because they can add density to gene-rich linkage maps of pea, establish macro- or microsynteny between M. truncatula and pea, and have higher chances of transferability between closely related species. This information can help in identifying markers that are tightly linked to the genes of interest or candidate gene/quantitative trait locus for agronomic traits.
Investigation of conserved regions in different studies has provided strong evidence for sequence correlations between M. truncatula and pea (Choi et al., 2004a; Aubert et al., 2006; Bordat et al., 2011). This information can be used to develop genic markers based on sequence homology between the related species. Choi et al. (2004b) developed EST-based introntargeted primers after aligning M. truncatula ESTs with the homologous genomic sequences of Arabidopsis (DC.) Heynh. and used them to construct a genetic map of M. truncatula. The basic assumption for this strategy is that introns or noncoding regions contain more DNA polymorphism than exons or coding regions (Brauner et al., 2002). A similar strategy—one that allows amplification of genomic DNA fragments covering two or more exons and bracketing polymorphic intron regions between those exons—was used in this study to develop pea EST-derived genic markers. Markers developed in this study are also available as cross-species markers within the legume family.
METHODS AND RESULTS
Primers were designed from pea EST sequences having significant similarity (score ≥100; E-value ≤e−50) using the BLASTn search with M. truncatula gene calls from the contig assembly (Mt3.0) of M. truncatula. Approximately 1200 M. truncatula gene calls were searched for presence of introns. One or more introns were present in 510 of the 1200 M. truncatula gene calls and were aligned with the available pea ESTs (n = 18576) in the database. Seventy-seven primers were designed from the pea ESTs having well-conserved sequences with M. truncatula gene calls spanning one or more introns. Primers were designed by importing sequences into Primer-BLAST ( www.ncbi.nlm.nih.gov/tools/primer-blast/) and selecting primers 18–24 bp long with annealing temperatures of 55–65°C. New primers were designed to amplify fragments from 150 to 1200 bp.
Genomic DNA of 16 pea genotypes including widely grown cultivars and plant introduction lines (i.e., Shawnee, Melrose, Medora, Lifter, Radley, PI 179449, Green Arrow, Frolic, A778-26-6, Sparkle, JI73, Bohatyr, ICI12043, PI 240515, PI 103709, PI 169603) was extracted from leaf tissue using a modified cetyltrimethylammonium bromide (CTAB) extraction protocol (Rogers and Bendich, 1985). PCR amplifications were performed in 25-µM reaction mixtures with 50 ng of template DNA, 0.2 µM of each forward and reverse primers, 200 µM dNTPs, 2.5 mM MgCl2, 1× PCR buffer, and 0.5 U Taq DNA polymerase in a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, Carlsbad, California, USA). The PCR profile included an initial denaturation at 95°C for 3 min followed by 35 cycles of 95°C for 1 min, 51–62°C for 50 s (according to the primer's annealing temperature), 72°C for 1 min, and a final extension at 72°C for 10 min. Length polymorphism was viewed with ethidium bromide in 8% Polyacrylamide gels run in a Mega-Gel high-throughput electrophoresis system for 5 h at 250 V (C.B.S. Scientific, San Diego, California, USA). If length polymorphism was not detected, PCR products were digested with restriction enzymes (New England BioLabs, Ipswich, Massachusetts, USA) to generate cleaved amplified polymorphic sequence (CAPS) markers and separated on 2% agarose to detect polymorphism. Amplified fragments were run with a 25/100-bp DNA ladder (Bioneer, Alameda, California, USA) and analyzed for fragment size using AlphaView Stand Alone analysis software version 3.4 (ProteinSimple, Santa Clara, California, USA). Each EST-derived genic marker was considered polymorphic when the PCR band pattern of one of the 16 pea genotypes was different from the others with regard to size or CAPS polymorphism ( Appendix S1 (APPSD13000026_Appendix S1.docx)). Different polymorphic fragments for a particular locus were considered as different alleles. Seventy-five primer pairs resulted in successful PCR amplification in which 66% (42 primer pairs) were monomorphic and 44% (33 primer pairs) were polymorphic among the 16 pea genotypes, which are parents of several pea mapping populations being used to map different disease resistance loci. The segregation analysis using these polymorphic markers has been conducted in a large number of mapping populations developed from crossing of these genotypes as parents (data not shown). All the primers generated a clear fragment pattern, with PCR products ranging in size from 150 to 1200 bp with two to three alleles per marker. Table 1 summarizes the forward and reverse primer sequence, size range of the original fragment (bp), annealing temperature, M. truncatula gene call, and the equivalent pea EST GenBank accession number. These EST-derived genic markers are codominant, highly reproducible, and easy to score. PCR products among the 16 pea genotypes were analyzed for allele number, observed heterozygosity (Ho), expected heterozygosity (He) or gene diversity, and polymorphic information content (PIC) using PowerMarker version 3.25 (Liu and Muse, 2005) (Table 2). Ho and He values ranged from 0.0000 to 0.0625 and from 0.0377 to 0.6391, respectively. The PIC ranged from 0.0370 to 0.5659 with an average of 0.2708. Twenty-four EST-derived genic markers were tested in two lentil (Lens culinaris Medik.) genotypes, and PCR amplification of 12 markers determined the transferability of these markers in related genera ( Appendix S2 (APPSD1300026_Appendix S2.docx)). This lends support from other studies on transferability of cross-species markers based on conserved sequences (Phan et al., 2006). Cross-species transferability of EST-derived genic markers is due to the conserved nature of primers picked up from coding sequences. More detailed polymorphism analysis and linkage analysis using mapping populations will establish connections between the genetic and genomic information of the closely related species.
CONCLUSIONS
The current study identifies and characterizes new EST-derived genic markers based on comparative mapping between pea and M. truncatula. Thirty-three polymorphic and 42 monomorphic primer sequences were described in this study. These EST-derived genic markers were mined from conserved M. truncatula gene sequences; therefore, they can be used to anchor genomic regions between pea and M. truncatula and possibly among other members of the legume family. These markers show polymorphism among 16 pea genotypes that include parents of several pea mapping populations being used to map different disease resistance loci. These molecular markers will be useful to develop gene-rich linkage maps and to tag genes for agronomically important traits. In addition, amplification of these markers in lentil demonstrates the transferability of these markers across related species.
TABLE 2.
Results of 33 polymorphic EST-derived genic loci screened in 16 genotypes of Pisum sativum.
