Transcriptome Analysis and Identification of a Female-Specific SSR Marker in Pistacia chinensis Based on Illumina Paired-End RNA Sequencing

Abstract

Pistacia chinensis Bunge (P. chinensis), a dioecious plant species, has been widely found in China. The female P. chinensis plants are more important than male plants in agricultural production, as their seeds can serve as an ideal feedstock for biodiesel. However, the sex of P. chinensis plants is hard to distinguish during the seedling stage due to the scarcity of available transcriptomic and genomic information. In this work, Illumina paired-end RNA sequencing assay was conducted to unravel the transcriptomic profiles of female and male P. chinensis flower buds. In total, 50,925,088 and 51,470,578 clean reads were obtained from the female and male cDNA libraries, respectively. After quality checks and de novo assembly, a total of 83,370 unigenes with a mean length of 1.3 kb were screened. Overall, 64,539 unigenes (77.48%) could be matched in at least one of the NR, NT, Swiss-Prot, COG, KEGG, and GO databases, 71 of which were putatively related to the floral development of P. chinensis. Additionally, 21,662 simple sequence repeat (SSR) motifs were identified in 17,028 unigenes of P. chinensis, and the mononucleotide motif was the most dominant type of repeats (52.59%) in P. chinensis, followed by dinucleotide (22.29%), trinucleotide (20.15%). The most abundant repeats were AG/CT (13.97%), followed by AAC/GTT (6.75%) and AT/TA (6.10%). Based on these SSR, 983 EST-SSR primers were designed, 151 of which were randomly chosen for validation. Of these validated EST-SSR markers, 25 SSR markers were found to be polymorphic between male and female plants. One SSR marker, namelyPCSSR55, displayed excellent specificity in female plants, which could clearly distinguish between male and female P. chinensis. Altogether, our findings not only reveal that the EST-SSR marker is extremely effective in distinguishing between male and female P. chinensis but also provide a solid framework for sex determination of plant seedlings.