Development and characterization of expressed sequence tag-derived simple sequence repeat markers in tropical forage legume Stylosanthes guianensis (Aubl.) Sw.

Most species of Stylosanthes (stylo) genus are important tropical pasture legumes with very versatile, widely adapted, and productive characteristics. These legumes are commercially used in various agricultural systems in many tropical and subtropical regions. However, the few molecular markers for the stylo species limit their genetic improvement. In this study, 36,558 expressed sequence tags (ESTs) have been de novo assembled using Illumina paired-end sequencing in Stylosanthes guianensis (Aubl.) Sw. to develop simple sequence repeat (SSR) markers. We searched these ESTs for SSRs and identified 4115 SSR loci from 3643 ESTs (9.96 %). Dinucleotide and trinucleotide repeat motifs were the most abundant types (30.50 and 50.33 %, respectively), whereas tetranucleotide, pentanucleotide, and hexanucleotide motifs represented <10 % of all SSRs. The motif AG/CT was the most abundant, accounting for 21.7 % of all SSRs. Moreover, 2008 SSR markers were developed using 1873 SSR-containing unigenes. A total of 115 EST-SSR markers located in the coding region were amplified using polymerase chain reaction to detect 29 S.guianensis accessions. Of these 115 markers, 96 produced reliable bands with expected sizes, and 81 markers were polymorphic, with 2–6 alleles among the 29 accessions. Analysis of the genetic diversity of all 29 accessions revealed similarity coefficients that ranged from 0.528 to 0.983. The EST-SSR markers developed in this study represent the first large-scale development of SSR markers for S.guianensis. These SSR markers will provide a valuable resource for genetic diversity studies, cultivar fingerprinting, construction of genetic maps, identification of quantitative trait loci for important traits, and molecular marker-assisted selection breeding in stylo species.