| Longan is a subtropical and luxury fruit in South China. Scientists have done a lot of work in classification and identification of Longan. However, there still exist some of inconsistencies in the classification of species, which is not conducive to the development of Longan. In recent years, some new lines have been found in Fujian Province, their genetic relationship need to be studied further. In addition, a number of seed-wilted Longan have been found in Fujian Province, there have been not any studies about genetic relationship of these plants. It is necessary to use the ISSR technology to evaluate the classification of Longan genetic diversity. Meanwhile new breeding lines and some seed-wilted Longan will be identificatied in this researsh. The main results of the research are as follows:1．Improved the method of extracting DNA from Longan. Some secondary metabolites such as polyphenols, polysaccharides and pigments were rich in the leaves of Longan, which were easy to form complex with DNA . The complex was insoluble and could inhibit the activity of Taq enzyme and then effect the PCR amplification. The method of extracting DNA from Longan have been Improved in this researsh: adding the step of acetone in the method of improved CTAB. To remove the beed rich polysaccharides, DNA can be added to the tenth volume of ethanol 1/30th size NaAc. ,ice bath for one hour, adding the same volume of chloroform, 1000g/m centrifugal 10min,thus re-precipitation of DNAThe method were effective to avoid the interference from the secondary metabolites, accordingly improved the quanlity and quantity of DNA extracted from leaves of Longan.2．Optimize the ISSR-PCR system of Longan. Consulting the general program of ISSR reation , after iterative experiments , the optimal amplificatin program for Longan ISSR-PCR was established as follow: Predenaturalization at 94℃for 5min; DNA was amplified for 41 cycles, each cycle includes denaturation for 1min at 94℃and annealing for 1min at 52℃and elongation for 90s at 72℃, and stored at 4℃. The ISSR amplification system was a 20μL reaction mixture containing 25ng DNA, 2．5 mmol/L Mg, 100μmol/L dNTP2μL, 10×Buffer2．5μL , primer0．2μmol/L, 1U Taq DNApolymerse.3．ISSR analysis of Longan genetic resources. 51 accessions of Longan genetic resources were analyzed by ISSR with 12 primers. A total of 155 DNA bands were amplified. On the average, one primer amplified 12．9 sites, the number of the band amplified by each primer ranged from 10(UBC840) tol6(UBC873) . The polymorphic degree was up to 95．4%. The results showed polymorphism of Longan germplasm was high. The genetic comparibility among all the breeds was calculated by euclidean distance method (ED) , the cultivars of Longan was analysed by UPGMA, dendrogram genetic comparibility was established. When D=0．55, the 51 genetic resources of Longan can be divided into two types (type of Longan from Chinese and type of Longan from Thailan); when D=0．37, the 51 genetic resources of Longan can be divided into four types (type of Longan from Thailan, type of Honghezi, type of Chuliang and Dongbi, type of Fuyan and Wulongling); when the D = 0．27, the 46 genetic resources of Longan can be divided into eight types .4．Found the ISSR marker linked to the seed-abortive character of Longan. Bulked segregation analysis(BSA) strategy was employed to identify ISSR markers linked to the seed-abortive characte of Longan. A total of 12 oligonucletide primers were screened on the DNA of fertile and sterile bulks. Primer UBC842(5' GAGAGAGAGAGAGAGAYG3' ) gived repeatable polymorphism between the two bulks. DNA from individual plants of each bulks and their sister lines were then used as templates for screening with the primer UBC842．DNA polymorphism generated by the primer UBC842 were verified linking with the seed-abortive characteof Longan.
|
PDF Full Text Request