| 柑橘品种鉴定的SSR标记开发和指纹图谱库构建 |
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| 引用本文： | 李益,马先锋,唐浩,李娜,江东,龙桂友,李大志,牛英,韩瑞玺,邓子牛.柑橘品种鉴定的SSR标记开发和指纹图谱库构建[J].中国农业科学,2018,51(15):2969-2979. |
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| 作者姓名： | 李益 马先锋 唐浩 李娜 江东 龙桂友 李大志 牛英 韩瑞玺 邓子牛 |
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| 作者单位： | 湖南农业大学园艺园林学院;农业部科技发展中心;中国农业科学院柑桔研究所;广西特色作物研究院 |
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| 基金项目： | 国家自然科学基金（31720103915）、2018年农产品质量安全监管专项（111721301092361007）、广西柑橘新品种选育及无病毒苗木繁殖研究特聘专家岗位 |
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| 摘 要： | 【目的】筛选出一套SSR核心引物,用于构建柑橘品种的DNA指纹图谱库,为柑橘种苗认证、品种登记和植物新品种保护提供技术支撑。【方法】以柑橘属8个主要栽培种类为试材进行PCR扩增,扩增产物经琼脂糖凝胶电泳检测,筛选出多态性丰富、扩增条带稳定的引物对部分参试品种进行PCR扩增,扩增产物通过变性聚丙烯酰胺凝胶电泳进行进一步的引物筛选;筛选得到的引物进行荧光标记,并选用部分柑橘品种进行PCR扩增,扩增产物利用基因分析仪检测,分别计算各引物的PIC值、等位基因数目,选取一套多态性丰富的适合进行荧光毛细管电泳检测的SSR引物组合,进而对所有参试品种进行分析,构建柑橘品种的DNA指纹图谱库,同时利用筛选的SSR标记对参试品种进行鉴定。【结果】初期以柑橘属8个种的代表品种(太田椪柑、沙田柚、沅江酸橙、大红甜橙、邓肯葡萄柚、枸橼、费米耐劳柠檬和墨西哥来檬)为材料,用362对SSR引物进行PCR扩增,扩增产物经4%琼脂糖凝胶电泳检测,初步筛选出80对种间多态性高、扩增稳定的SSR引物;进一步从8个种类里选择不同类型的64个柑橘品种,用上述筛选出的80对SSR引物进行PCR扩增,扩增产物经6%变性聚丙烯酰胺凝胶电泳检测,再筛选出60对品种类型间多态性高、扩增稳定、条带清晰的SSR引物,并分别进行荧光标记;另以168份柑橘品种为材料对上述60对SSR引物进行PCR扩增,经荧光毛细管电泳检测,依据各引物的PIC值、等位基因数目、峰图读取难易程度、扩增稳定性及染色体位置等,最终筛选出21对SSR引物作为指纹图谱库构建的核心引物。为消除不同仪器、不同试验批次等引起的误差,为21对SSR引物各主要等位变异选择相应的参照品种。根据各引物标记的荧光颜色及扩增片段大小进行合理组合,21对SSR引物可分成5组进行多重电泳,共采集500份柑橘品种的DNA指纹。利用SSR标记,有111份品种仅用1对引物即可鉴别,86份品种用2对引物组合即可鉴别,24份品种用3对引物组合即可鉴别,另外279份品种虽无法单独一一鉴别,但可分为87个小组,组内品种无法区分,组间品种易鉴别。【结论】利用筛选出的21对SSR核心引物,构建了包含500份柑橘品种的DNA指纹图谱库,筛选出部分品种的特异标记,可以鉴别221份柑橘品种和87个品种组合,构建了基于毛细管电泳平台的柑橘SSR分子标记品种鉴定体系。
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| 关 键 词： | 柑橘 SSR 毛细管电泳 品种鉴定 DNA指纹图谱库 /> |
| 收稿时间： | 2018-01-19 |
SSR Markers Screening for Identification of Citrus Cultivar and Construction of DNA Fingerprinting Library |
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| LI Yi,MA XianFeng,TANG Hao,LI Na,JIANG Dong,LONG GuiYou,LI DaZhi,NIU Ying,HAN RuiXi,DENG ZiNiu.SSR Markers Screening for Identification of Citrus Cultivar and Construction of DNA Fingerprinting Library[J].Scientia Agricultura Sinica,2018,51(15):2969-2979. |
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| Authors: | LI Yi MA XianFeng TANG Hao LI Na JIANG Dong LONG GuiYou LI DaZhi NIU Ying HAN RuiXi DENG ZiNiu |
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| Institution: | 1.College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128;2.Development Center for Science and Technology, Ministry of Agriculture, Beijing 100122;3. Institute of Citrus Research, Chinese Academy of Agricultural Sciences, Chongqing 400712;4.Guangxi Academy of Specialty Crops, Guilin 541004, Guangxi |
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| Abstract: | 【Objective】The purpose of the present work aims at the construction of DNA fingerprint library of citrus cultivar by using a series of SSR core primers in order to provide references for citrus nursery plant identification, cultivars registration and plant variety protection. 【Method】SSR primers were primarily screened by analyzing 8 mainly cultivated species belonging to Citrus while the PCR products were separated by agarose gel electrophoresis, the primers that generated high polymorphic and stable PCR products were chosen for further PCR amplification with more citrus cultivars, and the PCR products were separated by polyacrylamide gel electrophoresis. the further screened primers were labeled with fluorescent and used for amplification of the all tested cultivars. The PCR products were detected by fluorescence capillary electrophoresis and the primers suitable for fluorescent capillary electrophoresis were screened according to their PIC, number of alleles identified, and employed to construct the citrus cultivar DNA fingerprinting library, and the selected primers were used to identify the tested cultivars.【Result】Firstly, 80 pairs of polymorphic and stable primers were chosen from 362 SSR primers by 4% agarose gel electrophoresis with representative cultivars of 8 species of Citrus genus(including Ota ponkan, Shatian pummelo, Yuanjiang soar orange, Dahong sweet orange, Duncan grapefruit, citron, Femminello lemon, Mexico lime); Secondly, 60 pairs of SSR primers with high polymorphism, stability and clear amplification products were further selected from the above 80 pairs of primers by 6% polyacrylamide gel electrophoresis with 64 citrus cultivars and labeled with fluorescent dyes; Thirdly, 21 pairs of SSR core primers were verified for the construction of fingerprint library from above 60 pairs of primers by capillary electrophoresis with 168 citrus cultivars on the basis of PIC, the allele number, peak reading difficulty, amplification stability and chromosome location. The corresponding reference cultivars were selected for 21 pairs of primers to eliminate the errors caused by different instruments and experimental batches. Through the multiple electrophoresis with the 5 combination groups of 21 primers according to the color and size of amplified fragment, we established 500 citrus cultivars fingerprinting library. Among the tested cultivars, 111 were identified by using only one pair of primers, 86 with the combination of two pairs of primers; and 24 with the combination of 3 pairs of primers, the remaining 279 cultivars were not able to be identified individually, they could, however, be divided into 87 groups, between which it was clearly distinguished but within which the cultivars were indistinguishable.【Conclusion】The DNA fingerprint library containing 500 citrus cultivars was constructed by using 21 pairs of screened SSR core primers, the specific markers for some cultivars were selected; 221 cultivars and 87 cultivar groups were identified; and a citrus cultivar identification system based on SSR fluorescence labeling was constructed. |
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| Keywords: | Citrus SSR capillary electrophoresis variety identification DNA fingerprints |
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