Abstract Owing to the lack of Cymbidium ensifolium genomic resource, the development and application of molecular markers have been limited. In order to obtain more efficient markers, we sequenced C. ensifolium transcriptome deeply to search simple sequence repeat (SSR). Here, high-quality reads were assembled into 101 423 unigenes. The unigenes were used to explore genic-SSR derived from gene (genic-SSR), which resulted in 17 793 genic-SSRs. The location of the SSR was estimated according to the open reading frame (ORF) and untranslated region (UTR) within the unigenes. A total of 3 065 genic-SSRs was located within 5'UTR；5 235 was within 3'UTR; 2 907 was within coding region; 6 586 was undetermined. A total of 80 was chosen from genic-SSRs sets, based on their motif, size and location. The primers were designed for 80 genic-SSRs using software "PRIMER 3.0", and then tested for their amplification. Of 80 genic-SSR primers, 61 succeeded in PCR amplification and 39 showed polymorphism among 12 C. ensifolium accessions. Among 39 polymorphic markers, the polymorphism information content (PIC) averaged 0.348, ranging from 0.141 to 0.746. The 39 polymorphic markers identified 108 alleles among 12 C. ensifolium accessions, and the allelic number was 2.77 per locus, ranging from 2 to 6. The SSR26 was the most polymorphic marker, with PIC of 0.746 and 6 alleles. The Nei genetic distance was estimated for each pair of the 12 C. ensifolium accessions, which ranged from 0.010 to 0.475, with an average 0.260. In addition, unigenes containing genic-SSR were annotated on the basis of BLAST similarity searches. The unigenes containing the 61 genic-SSRs were searched against non-redundant (Nr), gene ontology database (GO), eukaryotic orthologous groups (KOGs) and kyoto encyclopedia of genes and genomes (KEGG) database, respectively. Most of them were annotated as crucial genes that were associated with important biological function. There were 52 unigenes hit Nr sequences, of which 38 were classified into 25 GO terms, 18 were assigned into 9 KOG categories, 15 were involved in 14 KEGG pathways. Thus, the 61 genic-SSRs having PCR product were also endowed with the corresponding biological information. These polymorphic genic-SSRs enriched the types of C. ensifolium molecular markers. They are very useful to analyze genetic diversity and reveal population structure in C. ensifolium. They could be also used to explore gene with linkage mapping and population genetics-based approaches, such as association mapping. The annotation of genic-SSR is extremely helpful to understand associations between these markers and the interesting traits. All of the information will in turn accelerate research on genomics, and functional genomics of C. ensifolium.
