Development Of Chloroplast SSR Markers And Studies On Genetic Diversity Of Zizipiius Jujuba

Chinese jujube (Ziziphus jujuba Mill.) is native in China, belonging to the genus ofZiziphus in Rhamnaceae. Jujube is one of important economic tree species for its nutrient-enriched fruit and the resources of Chinese medicine. Jujube is rich in its germplasm,elucidating its genetic diversity and the relationship between jujube and sour jujube (Z.jujuba var. spinosa), which will help us conserve the germplasm better. To track thisproblem, researchers have performed a few studies using the molecular markers from nuleargenome, however, molecular markers, such as cpSSR, was still not used to evaluate thegenetic structure of jujube germplasm based on the cytoplasmic inheritance. cpSSR cancharacterized its genetic structure by revealing the composition of chlorotype in jujubegermplasm, and it improve us understanding the geographic origin of the jujube. Therefore,it is very useful to develop cpSSR markers, and then to study the genetic structure anddiversty.The main contents and results of this study were as follows:(1) High-salt low-pH method and a modified high-salt low-pH method were utilized forchloroplast and cpDNA isolation. The results showed: the value of OD260nm/OD280nmofcpDNA isolated by the original method was1.28, therefore, it can’t meet the requirements ofsubsequent chloroplast genomes sequencing. However, the yields of cpDNA extraction by themodified method was dramatically higher than that by the routine method, and the extractedcpDNA of mature leaves by this improved method was integral, high-quality and pure. Inaddition, the value of OD260nm/OD280nmwas1.8to2.0, and degradation was nearly free. Thus,the quality of cpDNA extracted by this modified high-salt low-pH method can meet therequirements of subsequent chloroplast genomes sequencing.(2) According to the whole genome sequencing datas of Z. jujuba, the reads belonging tochloroplast genome were extracted by blasting all reads to the reference choloroplast genomeof Vitis vinifera.Then, those extracted reads were assembled by SOAP, and then a longscafford (151,963bp) was obtained. The assembled genome was then annotated, which wascomposed of four contents: long single copy region, short single copy region and twoinverted repeat regions, and134genes totally. (3) We designed15and28specific primer sets on the gene-coding regions and inter-gene regions to evaluate the accuracy of assembled results. The PCR and sequencing by ABI3730XL revealed the high accuracy (98%) of assembling in the gene-coding region, but thenon-conding area was about92%.(4) According to sequencing results,11jujube cultivars and4wild-types were selected toevaluated its polymorphism by PCR and polyacryamide gel electrophoresis. In total,9fragments showed polymorphism and6of those were selectd for further work.(5) To evaluate the chlorotype diversity of jujube and sour jujube, we collected72jujubecultivars,23sour jujbe wild-types and one Z. mauritiana Lam. The PCR were performedusing the specific fluorescence marked primer and electrophoresised on the ABI3730XLgenetic analyzer. The row data were treated by Genemapper and manual edited, and then thefingerprint data were obtained. In total,20alleles from6cpSSR locus were checked out in96samples, the average number of effect (Ne) alleles was1.065～1.769, diversityinformation (I) was0.159～0.1747。 In general, diversity and unbiased diversity were0.061～0.435and0.062～0.439. The diversity indice of sour jujube (Ne=1.619, I=0.483,h=0.303) were relatively higher than jujube (Ne=1.393, I=0.437, h=0.256), and the Neigenetic identity between jujube and sour jujube was0.970. AMOVA analysis revealed thevariance in population was95%, which was higher than the variance between population(5%).(6) The fingerprints data was then transformed into0/1matrix data and subjected tocluster analysis by NTSYS. Cluster analysis revealed that96samples were composed of7chlorotypes. Z. mauritiana was separated as one branch. Jujube and sour jujube were notclustered into two branch, however, they shared two chlorotypes, ct1and ct4. ct2and ct3haplotypes only belong to jujube, and on the contrary, ct5and ct6haplotypes were unique tosour jujube.In conclusion, jujube and sour jujube showed same genetic background for morecommon chlorotypes and thus the significant genetic differentiation did not occur. Itindicates that jujube and sour jujube have very close genetic relationship, and also confirmedthat the evolution from sour jujube to jujube was by several lines.