Studies On The Genetic Diversity And Relationship Of Jasminum Sambac (l.) Aiton Cultivars By ISSR Analysis

Jasminum sambac(L.)Atiton is sweetclover Branch Jasminum,which originated in Persian Gulf.and its variety resource is very abundant.The utilization and classification of Jasmine germplasm resources were adopted by the botany classification system of linnaean or Engler for a long time. There are arguements on the genetic relationship and classification among the native varieties of Jasmine for many years,which had made grate trouble in breeding new variety and utilizating resource.As some species,cultivars or strains are much similar in the appearance and morphological characters, while their relationships of heredity and relative were very different. Therefore,in this study we analyzed the classification and genetic relationship among 189 cultivars of Jasmine by ISSR-PCR.The main result of this research is as following:1.Three DNA extraction methods of CTAB,SDS and modified CTAB were studied.The results showed that the modified CTAB method was the best to extract Jasmine genomic DNA based on the purity and yield of DNA. The products(bands)were clear and undegradation by agarose gel electrophoresis.The values of A₂₆₀/A₂₈₀were between 1.81 and 1.84 detected by U/V spectrophotometer.This method could get good purity DNA and was qualified to apply to ISSR-PCR analysis.2.The different PCR programs and the ingredients of ISSR reaction system such as Taq DNA Polymerase,dNTP,DNA and primers were screened by different concentrations.Suitable reaction systems and programs of the ISSR analysis for Jasmine genomic DNA were designed.The following was the 20μL reaction system with DNA template 75ng,Primer 5μmol,rTaq polymerase 1 U,dNTP 4 mmol,buffer(including Mg²⁺)2ul.The procedure was that predenature at 94℃for 5min,denature at 94℃for 1min,anneal at X℃for 1min,elongate at 72℃for 2min,cycling number at 40,holding at 72℃for 10min,and keeping final products at 4℃.3.10 ISSR primers screened from 100 ISSR primers were utilized for the amplification reaction of Jasmine template DNA by PCR reaction.A total of 70 bands were obtained by amplification of the polymorphic primers of 30 cuttings,among which 34 bands were found to be polymorphic.The percentage of polymorphic bands was 48.57%.A total of 70 bands were obtained by amplification of the polymorphic primers of 135 seedings,among which 42 bands were found to be polymorphic.The percentage of polymorphic bands was 60%.A total of 66 bands were obtained by amplification of the polymorphic primers of 24 seedings,among which 44 polymorphic bands were found to be polymorphic.The percentage of polymorphic bands was 65.7%.Every primer could amplify 7 bands averagely, and the size of band amplified was between 100bp and 2000bp.4.The dendrogram was obtained by the UPGMA clustering method.24 Jasmine cultivars could be clustered into two groups according to the dendrogram.Of there,one group has only a variety,that is with more buds. The other was a big group,which could be divided into four sub-groups,the first subgroup has the same shape of leaf,the second subgroup owns the character of a few branches and buding late,the third subgroup with few branch and buding late,the fourthly subgroup with few branch and buds.5.All DNA template can be effectively analyzed by ISSR method,which is not influenced by environmental conditions.It was quite reliable that the genetic diversity and relationship of 189 Jasmine cultivars were analyzed by ISSR method at the molecular level.ISSR-PCR analysis is a relatively new technology,which is very simple,reliable and highly effective in studying the Jasmine germplasm resources.And it could provide some valuable information for parent combinations on the hybridization breeding at the molecular level.