Transcriptome sequencing and development of EST-SSR markers in Pinus dabeshanensis, an endangered conifer endemic to China

Pinus dabeshanensis Cheng et Law (P. dabeshanensis) is an endangered endemic occurring along the border between Anhui Province and Hubei Province (China). This species is known from only four natural populations. However, few genomic studies have been conducted on this species. In the present study, a cDNA library of P. dabeshanensis needles was sequenced using Illumina HiSeq™ 2000 paired-end sequencing technology. By mining 42,248 non-redundant unigenes, 1966 expressed sequence tag–simple sequence repeats (EST-SSRs) derived from 1835 unigenes were identified. The most abundant repeat motif was trinucleotide (41.6 %), followed by dinucleotide (23.25 %), mononucleotide (21.41 %), hexanucleotide (7.83 %), pentanucleotide (4.43 %) and tetranucleotide repeats (1.53 %). The top tri- and dinucleotide motifs included AGC/CTG and AT/AT, respectively. A total of 20,194 unigenes were assigned to three main GO categories with 153,373 GO terms. A KOG classification showed that 11,604 unigenes and 732 SSR-containing unigenes were assigned to 25 and 23 possible functional categories, respectively. Among the 1966 EST-SSRs, 431 primer pairs were successfully designed. After selecting 82 of these pairs at random for further validation, 44 pairs amplified unambiguous bands, 19 of which were polymorphic among 24 individuals of P. dabeshanensis. A total of 57 alleles were identified, varying from 2 to 5 alleles per locus with an average of 3.0. The observed and expected heterozygosity per locus ranged from 0 to 0.958 and from 0.082 to 0.759, respectively. The PIC values ranged from 0.077 to 0.707 with an average of 0.380. The generation of such large-scale sequence data in the present study provides a valuable resource for gene discovery and molecular marker development. Such markers will be robust tools for genomic research and breeding of P. dabeshanensis.