SSR标记在辣椒DUS测试中的应用研究
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摘要
为了评价SSR分子标记在辣椒(Capsicum annuum L.)品种特异性、一致性、稳定性(DUS)测试中的应用潜力,采用聚丙烯酰胺凝胶电泳技术和毛细管电泳技术,对辣椒SSR引物进行筛选,采用入选的30对核心引物,构建130份不同来源品种的指纹图谱。30对引物共检测到等位基因163个,每对引物等位基因变幅为2～15,平均等位基因数为5.43,多态信息量(PIC)的变异范围为0.17～0.76,平均为0.43。聚类分析表明,该套引物可以将所有参试品种区分开来,遗传基础相近的品种优先聚在一起。130个品种的遗传距离为0.01～0.91。有20对品种遗传距离小于0.10,其中3对品种有1个位点的差异,采用差异位点数判断品种特异性更适合目前辣椒品种现状。采用10对引物对品种0313963-4进行了一致性检测,综合一致性比率R值为96.5%,低于DUS测试中对表型性状一致性的要求。因此SSR标记直接用于辣椒DUS判定还需进一步研究,但是可用于近似品种筛选。
To evaluate the application of SSR markers in pepper DUS testing,here we employed thirty pairs of SSR primers in fingerprinting analysis of 130 varieties.A total of 163 polymorphic alleles were detected by30 primer pairs,which ranged from 2 to 15 with a mean value of 5.43.The polymorphism information content(PIC)ranged from 0.17 to 0.76,with the average of 0.43.These genotypes can be clarified using the polymorphic information.These pepper cultivars with close genetic background were clustered.The genetic distance of the 130 cultivars ranged from 0.01 to 0.91.There were 20 pairs of cultivars whose genetic distance was less than 0.10,among which 3 pairs had one locus difference.It was more suitable to judge the distinctness of pepper varieties by using the number of difference locus.Ten pairs of primers were used to detect the uniformity of the variety0313963-4 and the comprehensive uniformity ratio R value was 96.5,which was lower than the requirement of phenotypic trait in DUS test.Therefore,SSR markers directly used in pepper DUS determination still need further study,but it can be used in the selection of similar varieties.
To evaluate the application of SSR markers in pepper DUS testing,here we employed thirty pairs of SSR primers in fingerprinting analysis of 130 varieties.A total of 163 polymorphic alleles were detected by30 primer pairs,which ranged from 2 to 15 with a mean value of 5.43.The polymorphism information content(PIC)ranged from 0.17 to 0.76,with the average of 0.43.These genotypes can be clarified using the polymorphic information.These pepper cultivars with close genetic background were clustered.The genetic distance of the 130 cultivars ranged from 0.01 to 0.91.There were 20 pairs of cultivars whose genetic distance was less than 0.10,among which 3 pairs had one locus difference.It was more suitable to judge the distinctness of pepper varieties by using the number of difference locus.Ten pairs of primers were used to detect the uniformity of the variety0313963-4 and the comprehensive uniformity ratio R value was 96.5,which was lower than the requirement of phenotypic trait in DUS test.Therefore,SSR markers directly used in pepper DUS determination still need further study,but it can be used in the selection of similar varieties.
引文
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[20]Portis E,Nagy I,Sasva′ri Z,Sta′gel A,Barchi L,Lanteri S.The design of Capsicum spp.SSR assays via analysis of in silico DNA sequence,and their potential utility for genetic mapping.Plant Science,2007,172:640-648
[21]Minamiyama Y,Tsuro M,Hirai M.An SSR-based linkage map of Capsicum annuum.Molecular Breeding,2006,18:157-169
[22]Lee J M,Nahm S H,Kim Y M,Kim B D.Characterization and molecular genetic mapping of microsatellite loci in pepper.Theoretical and Applied Genetics,2004,108:619-627
[23]Yi G,Lee J M,Lee S,Choi D,Kim B D.Exploitation of pepper EST-SSRs and an SSR-based linkage map.Theoretical and Applied Genetics,2006,114:113-130
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[2]赖运平,张浙峰,王丽容,王莉花,余毅.薏苡新品种特异性、一致性和稳定性测试指南研制.植物遗传资源学报,2016,17(4):690-695Lai Y P,Zhang Z F,Wang L R,Wang L H,Yu Y.Development of distinctness,uniformity and stability test guidelines for new varieties of job′s tears.Journal of Plant Genetic Resources,2016,17(4):690-695
[3]唐浩,余汉勇,张新明,魏兴华.水稻新品种测试的标准品种DNA指纹图谱多样性分析.植物遗传资源学报,2015,16(1):100-106Tang H,Yu H Y,Zhang X M,Wei X H.Analysis on the diversity of DNA fingerprinting of the example varieties used for DUS test of rice new varieties.Journal of Plant Genetic Resources,2015,16(1):100-106
[4]UPOV.Guidelines for DNA-profiling:Molecular markers selection and database construction.Geneva:Office of the Union,2010,1-13
[5]UPOV.Possible use of molecular markers in the examination of distinctness,uniformity and stability.Geneva:Office of the Union,2011,1-8
[6]Singh R K,Sharma R K,Singh A K,Singh V P,Singh NK,Tiwari S P,Mohapatra T.Suitability of mapped sequence tagged microsatellite site markers for establishing distinctness,uniformity and stability in aromatic rice.Euphytica,2004,135(2):135-143
[7]张金渝,张建华,杨晓洪,金航,米艳华,肖植文,孔令明,肖卿.玉米DUS测试标准品种的SSR分子指纹图谱的构建.玉米科学,2006,14(4):47-52Zhang J Y,Zhang J H,Yang X H,Jing H,Mi Y H,Xiao ZW,Kong L M,Xiao Q.Building of SSR fingerprinting map of standard varieties on maize in DUS testing.Journal of Maize Sciences,2006,14(4):47-52
[8]王凤格,易红梅,赵久然,孙世贤,杨国航,任洁,王璐.DNA指纹技术在玉米区域试验品种真实性及一致性检测中的应用.分子植物育种,2016,14(2):456-461Wang F G,Yi H M,Zhao J R,Sun S X,Yang G H,Ren J,Wang L.The application of DNA fingerprint technology in maize varieties′authenticity and consistency Identification in maize regional test.Molecular Plant Breeding,2016,14(2):456-461
[9]匡猛,杨伟华,许红霞,王延琴,周大云,冯新爱.中国棉花主栽品种DNA指纹图谱构建及SSR标记遗传多样性分析.中国农业科学,2011,44(1):32-36Kuang M,Yang W H,Xu H X,Wang Y Q,Zhou D Y,Feng XA.Construction of DNA fingerprinting and analysis of genetic diversity with SSR markers for cotton major cultivars in China.Scientia Agricultura Sinica,2011,44(1):32-36
[10]陆光远,伍晓明,张冬晓,刘凤兰,陈碧云,高桂珍,许鲲.SSR标记分析国家油菜区试品种的特异性和一致性.中国农业科学,2008,41(1):32-42Lu G Y,Wu X M,Zhang D X,Liu F L,Chen B Y,Gao G Z,Xu K.SSR-based evaluation of distinctness and uniformity of rapeseed(Brassica napus L.)varieties under Chinese national official field tests.Scientia Agricultura Sinica,2008,41(1):32-42
[11]赖运平,张浙峰,王丽容,何巧林,黄维藻,张新明,堵苑苑,余毅.利用SSR标记筛选DUS测试中甘蓝型油菜近似品种.分子植物育种,2013,11(2):174-184Lai Y P,Zhang Z F,Wang L R,He Q L,Huang W Z,Zhang X M,Du Y Y,Yu Y.Screening similar varieties of rapeseed(Brassica napus L.)in DUS testing using SSR markers.Molecular Plant Breeding,2013,11(2):174-184
[12]李贝贝,姜建福,张颖,樊秀彩,孙海生,张国海,刘崇怀.葡萄品种DNA指纹数据库的构建及遗传多样性分析.植物遗传资源学报,2018,19(2):338-350Li B B,Jiang J F,Zhang Y,Fan X C,Sun H S,Zhang G H,Liu C H.Construction of DNA fingerprinting and analysis of genetic diversity for grape cultivars.Journal of Plant Genetic Resources,2018,19(2):338-350
[13]黄丹娟,马建强,陈亮.茶树PVP申请品种的SSR分子标记鉴定和系谱关系分析.茶叶科学,2016,36(1):68-76Huang D J,Ma J Q,Chen L.SSR identification and pedigree analysis of PVP application cultivars in tea plant.Journal of Tea Science,2016,36(1):68-76
[14]王立新,李宏博,廖琴,邱军,常利芳,刘丽华,任立平,高新欢,赵昌平.利用分子标记筛查小麦相似品种(系).作物学报,2010,36(9):1490-1497Wang L X,Li H B,Liao Q,Qiu J,Chang L F,Liu L H,Ren LP,Gao X H,Zhao C P.Screening wheat varieties with genetic similarity using molecular markers.Acta Agronomica Sinica,2010,36(9):1490-1497
[15]张晗,姚凤霞,刘永杰,宋卫华,许金芳,王东建,李汝玉.EST-SSR标记在冬小麦品种DUS测试中的应用.麦类作物学报,2010,30(5):801-806Zhang H,Yao F X,Liu Y J,Song W H,Xu J F,Wang D J,Li R Y.Application of EST-SSRS to distinctness test of new varieties.Journal of Triticeae Crops,2010,30(5):801-806
[16]郑永胜,张晗,王东建,孙加梅,王雪梅,段丽丽,李华,王玮,李汝玉.基于荧光检测技术的小麦品种SSR鉴定体系的建立.中国农业科学,2014,47(19):3725-3735Zheng Y S,Zhang H,Wang D J,Sun J M,Wang X M,Duan L L,Li H,Wang W,Li R Y.Development of a wheat variety identification system based on fluorescently labeled SSRmarkers.Scientia Agricultura Sinica,2014,47(19):3725-3735
[17]王立新,常利芳,李宏博,季伟,刘丽华,赵昌平.小麦区试品系DUS测试的分子标记.作物学报,2010,36(7):1114-1125Wang L X,Chang L F,Li H B,Ji W,Liu L H,Zhao CP.Molecular markers for estimating distinctness,uniformity and stability of wheat cultivars of regional trials.Acta Agronomica Sinica,2010,36(7):1114-1125
[18]黄婷,马啸,张新全,张新跃,张瑞珍,符开欣.多花黑麦草DUS测定中SSR标记品种鉴定比较分析.中国农业科学,2015,48(2):381-389Huang T,Ma X,Zhang X Q,Zhang X Y,Zhang R Z,Fu KX.Comparation of SSR molecular markers analysis of Annual Ryegrass varieties in DUS testing.Scientia Agricultura Sinica,2015,48(2):381-389
[19]周春阳,谢立波,李景富,周宇.辣椒叶片基因组DNA提取方法的研究.辣椒杂志,2011(3):35-37Zhou C Y,Xie L B,Li J F,Zhou Y.The extraction of genomic DNA from hot pepper.Journal of China Capsicum,2011(3):35-37
[20]Portis E,Nagy I,Sasva′ri Z,Sta′gel A,Barchi L,Lanteri S.The design of Capsicum spp.SSR assays via analysis of in silico DNA sequence,and their potential utility for genetic mapping.Plant Science,2007,172:640-648
[21]Minamiyama Y,Tsuro M,Hirai M.An SSR-based linkage map of Capsicum annuum.Molecular Breeding,2006,18:157-169
[22]Lee J M,Nahm S H,Kim Y M,Kim B D.Characterization and molecular genetic mapping of microsatellite loci in pepper.Theoretical and Applied Genetics,2004,108:619-627
[23]Yi G,Lee J M,Lee S,Choi D,Kim B D.Exploitation of pepper EST-SSRs and an SSR-based linkage map.Theoretical and Applied Genetics,2006,114:113-130
[24]Kwon Y S,Lee J M,Yi G B,Yi S I,Kim K M,Soh E H,Bae K M,Park E K,Song I H,Kim B D.Use of SSR markers to complement tests of distinctiveness,uniformity,and stability(DUS)of pepper(Capsicum annuum L.)varieties.Molecules and Cells,2005,19(3):428-435
[25]王凤格,赵久然,戴景瑞,王璐,易红梅,郭景伦,孙世贤,廖琴,杨国航.利用SSR标记进行玉米品种一致性检测研究.分子植物育种,2007,5(1):95-104Wang F G,Zhao J R,Dai J R,Wang L,Yi H M,Guo J L,Sun S X,Liao Q,Yang G H.Uniformity analysis of maize varieties by a set of SSR markers.Molecular Plant Breeding,2007,5(1):95-104
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