Development of EST-SSR Markers Linked to Flowering Candidate Genes in L. Based on RNA Sequencing.

L. is an important cold-season grass with excellent cold and drought tolerance, good palatability, and nutrition. Flowering time is a key trait that affects forage and seed yield. Development of EST-SSR (expressed sequence tag simple sequence repeat) markers based on flowering genes contributes to the improvement of flowering traits. In the study, we detected 155 candidate genes related to flowering traits from 10,591 unigenes via transcriptome sequencing in early- and late-flowering genotypes. These candidate genes were mainly involved in the photoperiodic pathway, vernalization pathway, central integrator, and gibberellin pathway. A total of 125 candidate gene-based EST-SSRs were developed. Further, 15 polymorphic EST-SSRs closely associated to 13 candidate genes were used for genetic diversity and population structure analysis among 20 accessions, including two contrasting panels (early-flowering and late-flowering). Among them, primer 28366, designed from heading date 3a (), effectively distinguished early- and late-flowering genotypes using a specifically amplified band of 175 bp. The polymorphic information content (PIC) value ranged from 0.12 to 0.48, with an average of 0.25. The unweighted pair group method analysis (UPGMA) cluster and structure analysis showed that the 20 genotypes with similar flowering times tended to group together. These newly developed EST-SSR markers have the potential to be used for molecular markers assisted selection and germplasm evaluation of flowering traits in .