Development Of Genomic SSR Molecular Fingerprinting In Camellia Sinensis And Application In The Identification Of Commodity Tea Cultivar

DNA fingerprinting represents the genomic DNA sequence differences between individual or population by using some molecular markers techniques,such as RAPD、AFLP、SSR、SNP etc,the genotype was expressed at the DNA level with good stability,high accuracy and convenient.DNA fingerprinting has been widely used in the identification and purity of varieties,protection and promotion of new cultivars,the relationship among varieties in tea germplasm resources.In recent years,there are many reports about DNA fingerprinting of tea clonal cultivars,but most of DNA fingerprinting was constructed by using EST-SSR,compared with the genomic SSR(gSSR),the EST-SSR marker has low polymorphism,While gSSR can be uniformly throughout the genome,it is necessary to construct the DNA fingerprinting of tea germplasm resources by using gSSR markers.The study was based on SSR molecular marker technology and selected DNA sequences from whole genome of shuchazao cultivar,we designed and screened the genomic SSR primers.Finally,five SSR loci were further recommened as a core marker set for fingerprinting of 80 cultivars.the identification technology of tea varieties was settled,which offered a practical,quick and reliable pipeline for protection and promotion of the clonal tea cultivar.In this study gSSR molecular marker screening methods and the main results were as follows:(1)Some reports had shown that shorter repeat motifs in the genome of tea cultivar appears more frequent than longer repeat motifs,Based on the result of genome of shuchazao variety,we selected DNA sequences which contain the number of repeat motifs from 2 to 5 bp,finally we designed 415 primer pairs.(2)We selected the DNA of shuchazao tea cultivar as the template for PCR amplification,415 of primer pairs were chosen randomly to synthesize,and 306 primer pairs succeeded in PCR amplification.the amplification which had a single clear band was sequenced,the results of 181 primer pairs were consistent with target sequences.181 pairs of primers were randomly picked up for the second screening in other 6 tea cultivars,of which 131 pairs of primers exhibit polymorphism were obtained by PAGE.(3)We selected 36 pairs of primers with high polymorphism and clear bands from 131 primers after third screening in other 80 tea cultivars as the primers to construct DNA fingerprinting.A data set consisting of the genotypes of 80 tea cultivars at 36 SSR loci was created,a total of 535 alleles were detected,allele number varied from 7 to 20 per marker(average 14.9),and the polymorphic information content(PIC)ranged from 0.71 to 0.92(average 0.86).according to different combinations of 36 primers fingerprint identification probability(PI and PIsibs value),We selected five markers as core marker which constrcted the fingerprinting of 80 cultivars of C.sinensis.(4)It was possible to fully discriminate all 80 cultivars which contains 23 cultivars of C.assamica and 57 cultivars of C.sinensis by a combination of five markers,according to a data set consisting of the genotypes of 80 tea cultivars at 5 SSR loci was created,and then we made the DNA fingerprinting QR codes which contains DNA fingerprinting,variety name and marker information of 80 cultivars by software.the identification technology of tea varieties was settled,which applied to the identification of “Huangkui” and “Shuchazao” cultivar,we made a unique fingerprint identification card for each other,it is very important for us to protect and promote the elite clonal tea cultivar.