Abstract: [Objective] Chinese fir (Cunninghamia lanceolata) is an important timber species distributed mainly in southern China. Current genetic analyses of this species lag behind other conifer species due to the limitation of available molecular markers. Accordingly, transcriptome sequence data were used to improve the efficiency of SSR development for the species. [Method]Utilizing Chinese fir transcriptome sequences from the Sequence Read Archive (SRA) database of NCBI. CLC and CMiB software were used to assemble sequence reads, to mine SSRs and design PCR amplicon primers for contigs that contained SSRs. Four universal fluorescent labeling primers and multiplex PCR were used to accomplish genotyping for polymorphic loci. [Result]De novo assembly produced 35633 contigs, the total length was 31.5 Mb, of which mini-and max-contigs were 155 bp and 23794 bp, respectively, with an average length of 884 bp. In total, 2156 SSRs were identified distributed in 1822(5.11%) contigs, with threshold repeat numbers of 6, 5, 4, 3 and 2 for di-, tri-, tetra-, penta-and hexa-SSRs, respectively. 256 contigs contained one or more SSRs, and the numbers of compound SSR contigs was 118. The average SSR density was 68.4 SSRs·Mb^-1. The most common SSR types were tri-SSRs (41.7%), followed by hexa-(29.8%), penta-(12.7%), di-(11.1%) and tetra-(4.7%). EST-SSR markers based on the 1822 SSR-containing contigs were developed, of which 1582 contigs could design primer pairs. Of the 35 primer pairs designed, 29 produced clear PCR fragment patterns with one or two bands. Polymorphic genotypes were obtained for 28 loci (80%) with the number of alleles per locus ranging from 3 to 12 for the 16 studied individuals. The average PIC value was 0.573, which indicates that the identified EST-SSR markers have a high degree of polymorphism. Principal Coordinates Analysis (PCA) showed that these EST-SSR loci can be used for identifying the provenances, even individuals of Chinese fir. [Conclusion] Combined SSRs mining and multiplex-PCR methods, we established the flow chart of EST-SSR markers development from transcriptome sequences of Chinese fir, and developed 28 polymorphic EST-SSR loci. These markers have been used in our ongoing analysis of genetic diversity in Chinese fir. Compared with traditional methods of SSR markers development, our method significantly improved PCR efficiency and dramatically reduced project costs. The new technologies will promote molecular genetics studies in Chinese fir, and also provide a basis for SSR marker development in other species.

Key words: Cunninghamia lanceolata, microsatellite markers, EST-SSR, transcriptome sequences, de novo assembly