Development Of DUS Test Guidelines And Genetic Diversity For Ziziphus Mauritiana

Ziziphus mauritiana(Rhamnaceae),known as India jujube,Ber or Taiwan green jujube.In recent years,despite the high speed increasing of the planting areas,consumption numbers,as well as new cultivars,it is difficult to protect the rights and interests of breeders because of the lack of an effective mechanism for the protection of germplasm resources management.Therefore,it is urgent to develop guidelines for the conduct of tests for the distinctness,uniformity and stability(DUS)for Z.mauritiana to protect the intellectual property rights of new varieties.On the other hand,the study of genetic diversity of Z.mauritiana is mostly confined to local areas or countries,and lack of in-depth and extensive research.For this reason,the study of genetic diversity of Z.mauritiana in major global distribution areas is of great significance to the conservation of its management,origin and evolution and breeding.In this study,we studied the germplasm resources of Z.mauritiana by field investigation and literature review,and constructed a database of known cultivars of Z.mauritiana.We developed guidelines for the conduct of tests for DUS of Z.mauritiana based on phenotype distribution of cultivars in China.In addition,we developed polymorphic SSR markers based on high-throughput sequencing data of Z.mauritiana.Finally,we constructed fingerprinting and analyzed genetic diversity of 117 Z.mauritiana accessions from China,Vietnam,Pakistan and Myanmar.The main results were as follows:(1)A total of 140 Z.mauritiana cultivars were investigated,49 of them were field surveys and 91 were consulted through literature.And 57 phenotypic characters were investigated,including 12 qualitative characters,21 pseudo-qualitative characters and 24 quantitative characters.The phenotypic characters of 49 Z.mauritiana cultivars in China were investigated and analyzed.The diversity index H of the descriptive characters was between 0.000 and 1.641,with a mean of 0.706.The coefficient of variation of quantitative characters ranged from 10.99% to 61.80%,among them,the coefficient of variation of fruit size was the largest,and the coefficient of variation of fruit length/fruit width was the smallest.The correlation analysis of 17 quantitative characters showed that the majority have a positive correlation,and principal component analysis showed that there were 5 principal components whose eigenvalue > 1,and the cumulative contribution rate was 80.95%.R-type clustering results showed that most phenotypic characters were independent,and some characters were highly correlated with each other.The Q-type clustering result classified 49 cultivars into three groups,and the classification results were consistent with the phenotypic changes.(2)Based on the TGP 5-11,14 documents and analysis of phenotypic characters,47 test traits and 18 standard cultivars in the DUS test guideline of Z.mauritiana were determined.Among the 47 test traits,10 were qualitative characteristics,16 were pseudo-qualitative characteristics and 21 were quantitative characteristics,accounting for 21.3%,34.0% and 44.7%,respectively.Eighteen characters were mandatory characteristics and 14 characters were graphical characteristics,accounting for 38.3% and 36.2%,respectively.(3)Based on high-throughput sequencing,55 nSSR and 2 cpSSR loci were developed.Among 55 nSSR loci,including 16 EST-SSR that polymorphism ratio of 16.0%,39 G-SSR that polymorphism ratio of 48.8%,and the cp SSR polymorphism ratio was 4%.(4)We chose 14 nSSR that high polymorphism and good stability to study genetic diversity and construct DNA fingerprint for 117 accessions of Z.mauritiana.Among 14 nSSR markers,a total of 137 alleles were detected among 117 accessions with a mean of 9.79.The allele size ranged from 71 to 364 bp.The diversity index H was 3.028 and genetic similarity was 0.11~1.00 for 117 accessions.Based on UPGMA phylogenetic tree,all the accessions can be divided into eight groups at a similarity coefficient of 0.48.In general,the classification is basically consistent with geographical origin.(5)SSR analysis suggested that 98 accessions had 3 alleles and 12 accessions had 4 alleles at certain loci among 117 Z.mauritiana accessions.‘Myanmar No.5' was first determined to be tetraploid with a basic chromosome number of X=12 using the root tip squash method,and was selected as a reference for following flow cytometry analysis with 18 accessions.By flow cytometry,there were 7,7,3 and 1 accessions that were tetraploid,hexaploid,pentaploid and octaploid,respectively.The tetraploid,hexaploidy,pentaploid and octaploid accessions accounted for 38.9%,38.9%,16.7% and 5.6%,respectively.(6)Among 2 cpSSR markers,5 alleles were detected among 117 accessions with a mean of 2.5.All 117 accessions could be assigned to 5 chloroplast haplotypes,haplotype C was dominant haplotype that made up the largest proportion(76%,89 accessions)of all accessions and presented in all four countries.Cluster analysis suggested Haplotype E was far from the other haplotypes.Network analysis illustrated haplotype C intermediated B,D,and E with a one-step mononucleotide mutation of the SSR motif.Haplotype B and D were intermediated with haplotype A in one-step mutation.