Development Of SSR Markers And Construction Of Genetic Linkage Map In Tea Plant

Tea plant{Camellia sinensis (L.) O. Kuntze), which origined from China, have been cultivated for more than2,000years and now it have become one of the most important beverage crops all over the world. Some achievements in the research area of molecular biology of tea plant have been made in the past two decades. One important aspect for that is the application of DNA molecular markers for genetic diversity analysis, phylogenetic study and linkage map construction in tea plant. For further putting forward these researches and facilitating QTLs mapping and MSA breeding in this important crop, more SSR markers and a linkage map based on a new mapping population were developed in this study. The main results were listed as follow:1. Three batches of transcriptome sequences based on next generation sequencing approaches of tea plant were collected and searched for the presence of SSR loci. Difference frequencies for mono-and di-nucleotides SSR loci in the three transcriptome were found. In the～52.6Mb assembled sequences,17,517SSR with repeat motif of1-6bp and at least15bp in total length were identified. For the development of SSR marker5,337PCR primer-pairs were designed and565of them were identified experimentally. As a result,279new polymor-phic SSR markers of tea plant were developed.2. To assist the selection of mapping parents,72newly developed SSR markers were employed to analysis the heterozygosity and relationship of24clonal tea cultivars. The highest heterozygosities were observed in Fuandabaicha (72.5%) and Fudingdabaicha (68.1%), while the lowest was Changyebaihao (only37.5%). Dendrogram drew with the information provided by the72markers divided the24clonal tea cultivars into two groups:seven from Yunnan province and others. This result was consistent with the morphologic taxology. Given five candidate parents, these results supported using Long-jin43×Fudingdabaicha, Baihaozao or Wuniuzao as genetic mapping parent couples.3. A population consisting of188Fl individuals derived from a cross between Longjin43and Baihaozao were used for the construction of tea plant genetic linkage map.137SSR markers developed in this study and other18from references were genotyped in this population. During genotyping,15F1individuals were excluded for they showed high frequencies of unexpected alleles. Linkage maps containing136SSR markers in total were constructed with JoinMap4.0via two approaches described by Tavassolian et al.(2010)."One-step method" resulted in16linkage groups which span909.7cM with an average interval of8.35cM, while the results for "two step method" were17,947.3cM and8.85cM, respectively.