Primer development and functional classification of EST-SSR markers in Ulmus species

At present, the available SSR markers of elm (Ulmus) have been seriously deficient, which can not meet the needs of molecular marker-assisted breeding and evaluation of germplasm resources. In this study, transcriptome data of Ulmus pumila were used as materials to develop EST-SSR markers of elms. EST-SSR markers were also classified according to gene function for the first time. In this study, 8288 perfect and 569 compound SSR loci were detected from 17,624 EST sequences (36,609,384 bp). Most of the perfect SSR sequences were based on short sequence lengths of 10–22 bp. The most common SSR repeat units were A/T (3330, 40.18%), AG/CT (1211, 14.61%), and AAG/CTT (568, 6.85%). All EST-SSR loci were function classified based on the Cluster of Orthologous Groups (COG) database. There were substantial differences in the type and proportion of SSR repeat units among the functional classes. Most EST-SSR loci were located in gene-coding regions. Ninety pairs were randomly selected and tested for validation using polymerase chain reaction amplification. Forty-nine primers were verified with clear bands. The number of alleles per locus (Na) ranged from 2 to 6; the number of effective alleles (Ne) ranged from 1.061 to 3.261; the observational heterozygosity (Ho) and expected heterozygosity (He) ranged from 0 to 0.968 and 0.112 to 0.651, respectively. The clustering pattern is consistent with traditional taxonomy that provides a molecular basis for the classification of elm. There were differences in the effective amplification and percentage of polymorphic loci among different functional SSR loci. The analysis of 880 primer combinations showed that markers within a single COG group were less effective than across groups. This study clarified the importance of SSR primer selection for clustering results. This study developed a large number of SSR markers in elm, providing effective markers for molecular marker-assisted breeding, genetic mapping, and other related research.