Computational protocol: Isolation and characterization of novel EST-derived genic markers in Pisum sativum (Fabaceae)1

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Protocol publication

[…] 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 ( 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 (). PCR amplifications were performed in 25-μL 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). 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. 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 () (). 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). This lends support from other studies on transferability of cross-species markers based on conserved sequences (). 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. […]

Pipeline specifications

Software tools BLASTN, Primer-BLAST, PowerMarker
Application qPCR
Organisms Pisum sativum, Medicago truncatula, Lens culinaris