| Single nucleotide polymorphism(SNP)markers developed by Expressed Sequence Tags(EST)have many advantages,such as high abundance,high polymorphism,and is suitable for high throughput analysis.EST-SNP not only can be used to evaluate the genetic diversity of germplasm resources,to construct genetic maps,breed selection,evolution and population diversity,and is directly related to functional genes,which can facilitate the analysis of genetic traits and functional gene cloning.Tea[Camellia sinensis(L.)P Kuntze]is one of the world,s three major non-alcoholic beverages,and an important economically crop in China.Beverage plant Coffee(Coffea L.)is grown in tropical and subtropical Asia,Africa and Latin American countries,and coffee is one of the most widely consumed non-alcoholic beverages,representing a multibillion-dollar global industry.In this thesis,mainly using EST-SNP molecular marker technology,the tea and coffee were used as the research object,and the development and application of SNP molecular markers were studied.The obtained results are as follows:1.Using ESTs and nucleotide sequences of Camilla sinensis from public databases NCBI,1786 single nucleotide SNP markers were developed.96 SNP markers were screened and verified by 40 tea varieties from China.Based on the validation result,60 SNP markers with polymorphism were screened out.Based on genotyping results,the principal coordinate analysis divided 40 tea varieties into three clusters,but the Bayesian cluster analysis was divided into two clusters.Fujian varieties were assigned to East China in Bayesian cluster analysis.These SNP markers,in combination with a high-throughput genotyping protocol,effectively established and verified specific DNA fingerprints for tea germplasm resources.These SNP markers can accurate and efficient genotype identification,and will be an effective tool for tea germplasm management and breeding.2.Authentication by SNP-based DNA methods is important for protection and value enhancement of good-quality brands.A total of 14 commercial tea products including green,black and oolong tea were used as testing materials.Based on profiles of 60 SNP markers,genetic identity of each tea sample unambiguously identified by multi-locus matching and ordination analysis.The results of the principal coordinate analysis showed that there were significant diversity among the tea varieties used in the commercial tea,and there was a significant difference between the Assam tea and the Chinese tea,and proved that Japanese tea is cultivated from the seeds of Chinese tea varieties.This protocol allows tea products to be traced through the production chain back to the original variety.The technique is robust and cost-effective,demonstrating significant potential for practical application.3.Fujian white tea from Fujian province,China,is one of the specialty tea products made from local varieties.Adulteration in this product has been pervasive in market,it is of practical significance to develop a method for authentication of the botanical origin of Fujian white tea.A total of 125 single leaf samples,representing 10 commercial white tea products purchased from domestic and international markets,35 reference white tea varieties and processed products from Fujian,China and 30 reference tea varieties from South China and other Asian countries,were analyzed using a panel of 192 Single Nucleotide Polymorphism(SNP)markers.Based on SNP profiles of a single tea bud or leaf,the fraudulent products accounted for six out of the ten commercial white tea products tested and were unambiguously differentiated.This method established a link between an individual tea leaf of a particular whito tea product,with authentic Fujian white tea,and it can be potentially applied to other tea products,especially for the protection of geographical indications protection products and geographical indications products.4.Using EST of coffee from public databases,we developed 7538 single nucleotide polymorphism(SNP)markers and selected 180 for validation using 25 C.arabica and C.canephora accessions from Puerto Rico.Based on the validation result,we designated a panel of 55 SNP markers that are polymorphic across the two species.This panel enabled the differentiation of all tested accession of C.canephora,which account for 79.2%of the total polymorphism in the samples.Only 21.8%of the polymorphic SNPs were detected in the 12 C.arabica cultivars,which,nonetheless,were able to unambiguously differentiate the 12 Arabica cultivars:into ten unique genotypes,including two mutant groups.Several local Puerto Rican cultivars,including’Limani’,’Fronton’ and ’TARS 18087’,showed substantial genetic difference from the other common Arabica cultivars,such as ’Catuai’,’Borbon’ and ’Mundo Nuevo’.In this study,55 stable SNP markers that areintra-and interspecific polymorphisms were used as genotyping tools,thus can serve as a genotyping tool to assist cofifee germplasm management,propagation of planting material,and coffee varietal authentication. |
