Transcriptome-Based Development of EST-SSR Molecular Markers and Fingerprint Construction of Trifolium Species

The genus Trifolium comprises numerous species that serve as globally important forage and ornamental crops. However, phenotypic difference between species were difficult to define in many cases because of the wide range of diversity caused by primary polymorphism. To effectively identify and differentiate Trifolium species, a total of 5288 candidate EST-SSR molecular markers were developed based on Trifolium repens transcriptome sequencing results, and 132 EST-SSRs that produced clear, reproducible, and highly polymorphic bands were verified after random selection and initial screening. Finally, 202 different bands were amplified by the 28 pairs of SSR primers, and variety identification and DNA fingerprinting were constructed for 16 Trifolium varieties mainly cultivated in China. The polymorphism information index (PIC) ranged from 0.117 to 0.432, with an average of 0.311. Cluster analysis and principal component analysis demonstrated that white clover clustered into a separate group, suggesting a relatively distant genetic relationship with the other 12 Trifolium materials. The DNA fingerprint map of Trifolium species constructed using highly polymorphic markers can effectively distinguish 16 different Trifolium materials. Notably, these markers developed from T. repens show high interspecific transferability, providing a powerful tool for further dissecting genetic diversity within the Trifolium genus, accelerating marker-assisted breeding programs, and reconstructing species domestication trajectories.