苹果S-SAP分子标记体系建立及应用

Establishment and Application of S-SAP System in Malus domestica

【目的】建立和优化苹果S-SAP分子标记体系,探讨应用S-SAP技术区分元帅芽变的可能性,为从DNA分子水平上对苹果芽变鉴定和利用奠定基础。【方法】利用富士、寒富和嘎啦初步建立S-SAP分析体系;根据谱带量和多态性等对32对引物组合进行筛选;从DNA酶切、PCR扩增、银染方法等角度对影响S-SAP反应的因子进行优化;利用6对多态性引物,对8个元帅芽变进行S-SAP分析,采用NTsys-pc2软件的SIMQUAL程序计算相似系数,UPGAM方法进行循环同化阶层聚类分析,并通过Treeplot程序生成聚类图。【结果】优化的苹果S-SAP分析体系为,改良CTAB法提取基因组总DNA;MseⅠ和PstⅠ双酶切基因组总DNA;Fermentas公司的TaqDNA聚合酶进行PCR扩增;6%变性聚丙烯酰胺凝胶分离选择性扩增产物;改良弱碱法银染检测差异片段。筛选出6个适宜苹果品种分析的S-SAP引物。发现LTRP1/Mtcc引物组合在元帅芽变分析中具有多态性,矮红、红星与新元帅等具有多态性位点,供试芽变资源的遗传相似系数在0.88—0.98之间,以相似系数0.93为标准,8个元帅芽变资源可分为4类。【结论】建立了基于Ty1-copia组反转录转座子的苹果S-SAP标记体系,筛选出了6个适宜苹果品种分析的S-SAP引物,利用LTRP1/Mtcc引物组合成功区分了元帅芽变。

[ Objective ] This study aimed at establishing and optimizing S-SAP system of apple, exploring the possibility of using S-SAP system to distinguish apple bud sports, lay a foundation for distinguishing and utilizing of apple bud sports at DNA molecular level. [Method] The primary S-SAP system was established using Fuji, Hanfu and Gala. Thirty-two primer combinations were screened according to band number variability and polymorphism. The effects of DNA restriction enzyme digestion, PCR amplification and silver stained method on S-SAP analysis of apple were studied. Eight Delicious bud sports were analyzed by S-SAP with six primer combinations. The SIMQUAL program of NTsys-pc2 software was adopted to calculate similarity coefficient. The cluster analysis was performed using UPGAM method, and the dendrogram was generated by Treeplot program. [Result] The optimum S-SAP for apple was as follows： Total DNA extracted with modified CTAB method. The total genome DNA was cut using Mse I/Pst I double digests. The Taq DNA polymerase from Fermentas was used to perform PCR amplification. The selective PCR products were separated by 6% denatured polyacrylamide gel and the different fragments were detected by modified mild alkali method. Six S-SAP primer combinations that are suitable for apple cultivar were screened, and the LTRP1/Mtcc primer combination presented polymorphic bands in Red Spur Delicious, Starking Delicious and Xinyuanshuai. The similarity coefficient of the analyzed materials ranged from 0.88 to 0.98. Eight Delicious bud sports could be grouped into four distinct families based on similarity of 0.93 [Conclusion] Tyl-copia-based S-SAP system was established and six S-SAP primer combinations that are suitable for apple cultivar were screened, and the Delicious bud sports could be differentiated by LTRP1/Mtcc primer combination.