Development Of EST-SSR Markers And Genetic Diversity Analysis Of Tea-oil Camellia In Southern Jiangxi

Tea-oil camellia（Camellia oleifera Abel.）is a perennial evergreen shrub or tree.It is native to China and considered as an important woody oil economic crop with long cultivation history and wide cultivation area.Some excellent clones have been successfully cultivated in China,such as the"Ganzhouyou series"from the Ganzhou Institute of Forestry Sciences in Jiangxi.However,the genetic background of the clones is relatively limited,and the morphological characteristics between the seedlings and seeds are difficult to determine which seriously affects the promotion of tea-oil camellia in southern Jiangxi products and the overall process of seeding tea oil in China.To fill these research gaps,this study we sequenced the transcriptome of tea-oil camellia in southern Jiangxi,SSR molecular markers with polymorphismwere developed,the SSR-PCR reaction system was also optimized to analyse the genetic diversity of 19 strains of tea-oil camellia in southern Jiangxi,and fingerprint map was constructed for molecular identification.The main results are as follows:Transcriptome sequencing was performed on C.oleifera‘GLS Ganzhouyou 4’,C.oleifera‘Ganzhouyou 9’,C.oleifera‘Ganzhouyou 23’,C.oleifera var.monosperma,C.vietnamensis,C.yuhsienensis,showed a total of 38.43 Gb of data.The total length,average length,N50 and GC content are 115,860,769 bp,963 bp,1,569 bp and 42.10%,respectively.A total of 120,202 Unigenes were obtained and following blast with records in databases,these unigenes were identified as 89,522 Nr,91,640 Nt,59,200 Swissf Prot,63,239 KEGG,61,777 KOG,57,869 Pfam,66,370 GO;64,399 CDS were detected,and a total of 50,519 SSR locis were detected and distributed in 34,550 Unigenes by MISA software.The occurrence frequency of SSR markers was 42.03%,the average distance was 2.29 kb,and the motif types are abundant,mainly dinucleotide repeats.The SSR-PCR reaction system of tea-oil camellia in southern Jiangxi was optimized by orthogonal design.The optimal SSR-PCR reaction system was 20μL total system composed of 9μL 2×Taq PCR Master mix,7μL dd H₂O,1.5μL each of the forward and reverse primers and 1μL DNA template.Premier 3 was used to design 29,122 primer pairs according to SSR locis,and 201 pairs were screened.There were 86 pairs of SSR primers were successfully amplified by agarose gel electrophoresis,and 21 pairs of primers were further screened by capillary electrophoresis and shown stable amplification results and good polymorphism.Using the developed 21 pairs of EST-SSR molecular markers,a total of 265 germplasms of 19 strains of tea-oil camellia in southern Jiangxi were analyzed for genetic diversity,genetic differentiation,genetic structure and construction of DNA fingerprints.Overall,the genetic diversity of tea-oil camellia in southern Jiangxi is rich,with moderate genetic differentiation among most strains and close genetic relationships among several strains.Through AMOVA analysis,the genetic variation was found to mostly occurred within population.Furthermore,the genetic structure which was based on Bayesian model analysis divided the 19 tea-oil camellia strains into three categories,which were not completely divided according to strain and the degree of hybridization between tea-oil camellia strains in southern Jiangxi was high.UPGMA clustering analysis showed that C.oleifera‘Ganzhouyou16’（S16）and other tea-oil strains in southern Jiangxi could be divided into two groups with a genetic distance of 0.12.The fingerprint of 19 tea-oil camellia strains in southern Jiangxi was constructed using the primer combination method,and results showed that the tea-oil strains could be separated.In conclusion,21 EST-SSR molecular markers were developed in this study,which provided abundant marker sources for future related studies.Genetic diversity of tea-oil camellia in southern Jiangxi was effectively analyzed using EST-SSR molecular markers,and corresponding fingerprints were constructed,which provided molecular basis for accurate and rapid identification of tea-oil camellia in southern Jiangxi.It is helpful to accelerate the process of molecular marker-assisted selection breeding of tea-oil camellia in southern Jiangxi and its further development and utilization.