Studies On The Genetic Diversity And Relationship Of Longan Cultivars By ISSR Analysis

Longan (Dimocarpus longan Lour.) is originated in south China, which is one of thespecial local fruits, and its variety resource is very abundant. The utilization andclassification of longan germplasm resources were adopted the botany classificationsystem of linnaean or Engler for a long time. There are arguements on the geneticrelationship and classification among the native varieties of Longan for many years,which had made grate trouble in breeding new variety and utilizating resource, as somespecies, cuitivars or strains are much similar in the appearance and morphologicalcharacters, while their relationships of heredity and relative were very different.Therefor, in this study we analyzed the classification and genetic relationship among 60cultivars of longan by ISSR-PCR. The main result of this research as follows: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 longangenomic DNA based on the purity and yield of DNA. The products (bands) were clearand degradation in gel electrophoresis. The values of A₂₆₀/A₂₈₀ were between 1.81-1.84detected by U/V spectrophotometer. This method could get good purity DNA and wasqualified to apply to ISSR—PCR.2. The different PCR program and the ingredients in ISSR reaction system such asTag DNA Polymerase, dNTP, DNA and primers were screened by differentconcentration. Suitable reaction system and program of the ISSR analysis for longangenomic DNA was setted. The following was the 20μL reaction system with DNAtemplate 75ng, Primer 5μmol, rTaq polymerase 1 U, dNTP 4 mmol, Tris-HCl (pH 8.3) 200μmol. The program was that predenature in 94℃for 5min, denature in 94℃for1min, renature in 36℃for 1min, elongated in 72℃for 2min, holding in 72℃for 10min,cycling number in 40, and final products kept at 4℃.3. 12 ISSR primers which screened from 100 ISSR primers were used for theAmplification reaction to 60 longan material by PCR. A total of 119 bands were obtainedby amplification of the polymorphic primers, among which 52 bands were commonbands, and 67 polymorphic bands were found to be polymorphic. The percentage ofpolymorphic bands was 56.30%. Every primer could amplify averagely 10 bands, andthe size of band amplified was between 100bp and 3000bp.4. The dendrogram obtained by the UPGMA clustering method. 60 longan cultivarscould be clustered into two groups on the dendrogram. Of there, the first group has onlya variety, that is longli [Dimocarpus confinis (How et Ho) H. S. Lour]. The other wereclustered into a big group, which could be divided into seven sub-groups, the firstsubgroup consisted of two cuitivars such as Dongbi and NO. 2 of unknown seeding, thesecond subgroup consisted of Lidongben cuitivar, the third subgroup consisted of threecultivars, such as Wulongling, Jiuyewu and Jiayuan, the fourthly subgroup consisted oftwo cultivars, such as Linglong and Zhaobailu, the fifth subgroup consisted of twelvecultivars, such as Chezhan, Guixiang and Xiangmi et. al, the sixth subgroup consisted oftwenty-seven cultivars, such as Dawuyuan, Daguangyan and Chuliang et. al, the seventhsubgroup consisted of twelve cultivars, such as Qingshanshuigui, Hongwuben andCaohuiben et. al.5. All DNA sequences can be directly analysed by ISSR method, which is no failedunder the influence of environment condition. The results are very reliable that thegenetic diversity and relationship of 60 Longan cultivars are analysed by ISSR methodon the mmolecular level. It is a new technology, which is very effective, simple andreliable in studing on the longan germplasm resources. And it could provide informationof value in parent combination at Molecular Level on the hybridization breeding.