Divergence and relationships of <i>Cymbidium tortisepalum</i> and its kindred germplasms by EST-SSR markers

Molecular marker-based identification of Chinese orchids among species, genera, and hybrids before flowering has important breeding and genetic research value. In this study, the <i>Cymbidium tortisepalum</i> germplasm ‘Lianbansu’ was chosen for transcriptome sequencing, yielding 9,485 putative EST-SSRs discovered from 28,235 unigenes. Then, 192 primers were randomly selected from the designed primers, and 38 pairs of high-quality primers were screened out through experiments. These high-quality markers were used to examine the genetic diversity, population structure, and evolutionary history of 51 orchid germplasms. The number of alleles ranged from 3 to 12, with an average of 6. The observed and expected heterozygosity with an average of 0.4662 and 0.5973. The first two principal coordinates detected 39.66% of the total variation. It was found that the <i>ΔK</i>-value had one peak (When K = 2). Several germplasms of <i>C. tortisepalum</i>, <i>Cymbidium sinense</i>, <i>Cymbidium hybridum</i>, and some interspecific or intergeneric hybrids and unidentified germplasms could be effectively distinguished using these markers. In conclusion, a set of EST-SSR markers with outstanding transferability was created in this study that can be utilised for hybrid parent tracing and germplasm identification of orchids before flowering.