Establishment Of ISSR Reaction System And Analysis Of Genetic Diversity Of Brassica Campestris L. Var. Rosularis

Brassica campestris L. ssp. chinensis var. rosularis Tsen belongs to Cruciferae Brassica and is native to China, and is one of important vegetables early grown in the Yangtze river basin. Because of its rich nutrition and unique flavor,it is widely cultivated all over the country.Because of the feature such as various morphology，st rong adaptability about Brassica campestris L.var cultivars,wide distribution and complex genetic background, it is very difficult to research on the resource survey of Brassica campestris L.var varieties, species classification and identification, diversity analysis,which limits the process of genetic improvement. This study uses ISSR molecular marker technique to analyze48savoy germplasms by single factor gradient and orthogonal design experiments.1. For optimizing ISSR-PCR reaction system of Brassica campestris L． var． rosularis，single factor gradient and orthogonal design experiments were conducted．An ideally ISSR-PCR reaction system was established，namely20μL reaction system containing template DNA30ng，0.50μmol L^-1p rimer，0.25mmol L^-1d NTP，1.0mmol L^-1Mg²⁺ and Taq DNA polymerase1.0U．The optimal PCR amplification program was:3min at94℃for predenaturation，followed by35cycles of30sec at94℃for denaturation，1min at50℃for annealing，90sec at72℃for extension，finally extension at72℃for7min and holding the samples at4℃．2. Using5primer and8savoy established system for stability test, results showed that the system program is stable and reliable, and can be used in savoy molecular identification and genetic diversity analysis.3. Screened9primers from the60ISSR random primers which can amplify clear and specific ands. With the analyzation of48savoy germplasms’ genetic diversity, there are85polymorphic bands in103bands, apolymorphic rate of82.52%.Each primer amplified11.4polymorphic bands on average. The similar coefficient was0.5922-0.9709with an average of0.7827, The results show the abundant polymorphism.4. Analysis by UPGMA cluster analysis method ISSR markers with48savoy, divided them into four categories varieties,in the dice similarity coefficient of0.802,by NTSYS pc2.10e. The first category including34varieties，‘Man dijin’、‘Jingxuan huangxinwu’ and so on.The second category including5varieties，‘Huiwu2hao’、‘Liuwei wutacai2hao’ and so on. The third category including ‘Changzhou xiaobaye’ and ‘Shanghai xiaobaye’. The fourth category including7varieties，‘Wenling zhoupiwu’、 ‘Baxia xiaojia’ so on.