Development of SSR markers based on transcriptome of Camellia japonica and analysis of genetic relationship

Objective Develop the EST-SSR markers based on transcriptome sequencing and apply these markers to analyze the genetic relationships of Camellia germplasms. Method Using 50 518 Unigenes obtained from the transcriptome sequencing of C. ‘Red Leaved Black Magic’ leaves as background data, SSR markers were searched out, and polymorphic primers were then designed and screened to construct the UPGMA evolutionary tree for 8 Camellia germplasms. Result 13 197 SSR sites were founded out. The occurrence frequency and the distribution frequency were estimated to be 19.52% and 26.12%, respectively, with the average distribution distance of 4.33 kb. Among the 6 types of SSR repeat motifs, mono-nucleotide, di-nucleotide and tri-nucleotide were the frequent dominant motifs, accounting for 48.420%、34.917% and 15.473%, respectively. The types of motifs with high occurrence frequency included A/T, AG/CT and AT/TA, accounting for 79.61% of the total SSR loci; AAG/CTT, ACC/GGT, AAT/ATT were in the majority among the tri-nucleotides; 10 974 pairs of primers were designed, and 73 pairs of primers were finally used to amplify effectively based on 90 pairs selected randomly, resulting in the rate of effective amplification of 81.11%; 29 primers showed some amplification polymorphism, accounting for 39.73%. The average number of effective alleles was estimated to be 3.264 with a total of 72 alleles. The mean values of observed heterozygosity (H_o) and expected heterozygosity (H_e) were measured to 0.208 and 0.638 respectively, as well as the average polymorphic information content (PIC) of 0.496; genetic relationship and cluster analysis indicated that eight Camellia germplasms could be classified into four categories at a similarity coefficient of 0.58: wild C. japonica in group Ⅰ; C. ‘Black Opal’, C. ‘Red Leaved Black Magic’ and C. ‘Night Rider’ together in group Ⅱ; C. japonica ‘Black Magic’ and C. japonica ‘Jinhua Meinǖ’ in group Ⅲ and group Ⅳ respectively. Conclusion The result showed that Camellia transcriptome Unigenes can be used as an effective source for SSR markers exploiting, which lays a theoretical foundation for studies on genetic diversity, identification of genetic relationship and marker-assisted breeding of Camellia.