应用SSR荧光标记法构建22个柚类品种的分子身份证  

Establishment of molecular identity of 22 pomelo varieties using fluorescent labeled SSR markers

作　　者：吴仕蔓 娄兵海 陈传武 唐艳 邓崇岭 武晓晓 WU Shiman;LOU Binghai;CHEN Chuanwu;TANG Yan;DENG Chongling;WU Xiaoxiao(College of Life Science,Guangxi Normal University,Guilin 541004,Guangxi,China;Guangxi Laboratory of Germplasm Innovation and Utilization of Specialty Commercial Crops in North Guangxi/Guangxi Citrus Breeding and Cultivation Research Center of Engineering Technology/Guangxi Academy of Specialty Crops,Guilin 541004,Guangxi,China)

机构地区：[1]广西师范大学生命科学学院,广西桂林541004 [2]广西桂北特色经济作物种质创新与利用实验室·广西柑橘育种与栽培工程技术研究中心·广西特色作物研究院,广西桂林541004

出　　处：《果树学报》2023年第4期605-614,共10页Journal of Fruit Science

基　　金：国家重点研发计划项目(2019YFD1001402-HX01);广西科技重大专项(桂科AA22068092);国家现代农业产业技术体系广西创新团队柑橘首席专家岗位项目(nycytxgxcxtd-2021-05-01);广西农业科技自筹经费项目(Z202090);广西特色作物试验总站(TS202101)。

摘　　要：【目的】应用荧光SSR分子标记构建柚类种质分子身份证,用于品种资源鉴定。【方法】收集22份柚类材料,筛选SSR荧光标记引物,采用荧光毛细管电泳检测技术分析扩增条带分子质量和等位基因,获得相应的扩增带型,从而对每份柚材料进行标记。采用个位阿拉伯数字和小写英文字母对不同等位基因分子质量大小进行编码,再按照引物扩增带型数由大到小顺序将各位点赋值编码的数字串联排序,构建供试样品的分子身份证。【结果】筛选出4对多态性较好的引物,使用这4对引物共检测到49个扩增带型和41个SSR等位位点,平均每对引物的有关带型数12.25个、位点数10.25个。【结论】通过扩增带型分析,构建了22份柚类种质的分子身份证,包括12条信息独特的身份证。遗传聚类与分子身份证分析结果一致,为柚类品种鉴定和保护提供了重要依据。【Objective】Pomelo is a collection of perennial fruit trees that belong to one of the three basic species of citrus genus in Rutaceae.It is inferred that they are originated in Southeast Asia,or in South China where they have been cultivated for 3000 years.In China,there are a myriad of pomelo germplasm resources likely ascribed to the monembryony whereby pomelo plants are prone to genetically vary during long-term breeding,selection,and cultivation.Relatively low genetic diversity but high morphological variation makes traditional morphology identification among hundreds of pomelo germplasms and varieties difficult.The molecular technique at multiple levels can be applied to improve the accuracy of the identification.SSR is informative,codominantly inherited,highly polymorphic,easily utilized,but not affected by environments and phytomorphological characteristics.The identification of the size of target gene DNA fragment by capillary electrophoresis with primers of 5’-end fluorescence labeling(FAM,HEX,TAMRA)is accurate to even 1 bp,with reliable and stable detection performance.The molecular ID codes of 22 pomelo germplasm resources were established using SSR markers.【Methods】The 22 germplasm resources were collected from the germplasm repository of Guangxi Academy of Specialty Crops, and orchards of Quanzhou county, Guilin city, and Gaoming farm, Nanningcity, Guangxi. According to literature reports, 21 pairs of primers with high polymorphism andgood repeatability were synthesized, labeled with three kinds of fluorescence, and used for PCR amplification.The genomic DNA was extracted by magnetic bead method genomic DNA kit, and the purity,concentration and integrity of the extracts were assessed by NanoDrop and agarose gel electrophoresis.A 10 μL PCR system was adopted, including 5.0 μL of 2 × Taq PCR Master Mix, 0.5 μL of each of forwardand reverse primers (10 mol ·L^(-1)), 0.5 μL genomic DNA (about 20 ng · μL^(-1)), and 3.0 μL of ultrapurewater. The PCR procedure was performed as follows: an

关 键 词：柚类 种质资源 SSR荧光标记 分子身份证 

分 类 号：S666.3[农业科学—果树学]