Characterization and Development of EST-SSR Markers from Transcriptome Sequences of Chrysanthemum (Chrysanthemum x morifolium Ramat.)

Simple sequence repeat (SSR) markers are valuable for genetic and breeding applications, but SSR resources for the ornamental genus chrysanthemum (Chrysanthemum x morifolium Ramat.) are still limited. Expressed sequence tags (ESTs) are sources of SSRs that represent an opportunity to develop SSRs to accelerate molecular breeding in chrysanthemum. In total, 4661 SSR loci were identified from 3823 SSR-containing unigenes in the chrysanthemum transcriptome with an average of one SSR per 6.98 kb. Of these SSR sequences, trinucleotide repeats (30.0%) predominated, followed by dinucleotide repeats (17.9%). In total, 1584 primer pairs were subsequently synthesized. By screening the parents and six individuals of the F-1 progeny, 831 (52.5%) valid EST-SSR markers were identified, of which 361 (43.4%) were polymorphic. The annotation of unigenes containing polymorphic SSRs indicated that 330 (93.5%) demonstrated significant homology to other plant protein sequences. Twenty-five polymorphic EST-SSR markers were further selected for transferability analysis and exhibited 93% amplification in six Ajania species and six other Chrysanthemum species. Based on genotyping of the 59 samples, neighbor-joining analysis revealed six phylogenetic groupings, which was confirmed by population structure analysis and principal component analysis (PCA). Phylogenetic relationships among the 59 samples revealed by SSRs were highly consistent with the traditional taxonomic classification of Chrysanthemum and Ajania. The polymorphism information content (PIC) values ranged from 0.29 to 0.86, with a mean of 0.67, indicating high levels of informativeness. This research reveals the SSR distribution characteristics of chrysanthemum and provides a large number of new EST-SSR markers for further genetic diversity studies, genetic mapping, and molecular marker-assisted selection breeding for chrysanthemum.