红花EST-SSR分子标记开发与初步验证
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摘要
旨在开发红花EST-SSR标记,为红花分子辅助育种和种质资源评价提供有力工具.先对红花转录组进行测序,选用MISA软件筛选鉴定SSR位点.在云南红花(YH)转录组中,共获得46 016个SSR位点,分布频率为32.09%,平均每3.11 kb有一个SSR.云南红花SSR位点以二,三核苷酸重复基序为主,其优势重复基序分别为AG/CT(44%)和AAG/CTT(24%).SSR位点重复次数相差较大,其中重复为5次比率最高,SSR位点数为9 369(26.63%),其次为6次(8 148, 23.16%).同时,利用Primer3.0进行引物设计,随机筛选27对引物,在60份不同来源红花种质中进行多态性验证,获得12对具有多态性稳定的引物,多态性引物比率为44.4%.结果表明,基于红花转录组序列开发SSR标记是可行的,开发的标记具有稳定扩增性,丰富多态性等优点,开发的SSR标记将有助于红花功能基因挖掘,遗传连锁图谱构建和遗传多样性分析.
The aim of this study was to develop EST-SSR molecular markers, providing a powerful tool for molecular marker assisted breeding and germplasm resource assessment for safflower.Based on the sequencing of safflower transcriptome, the SSR loci were searched by MISA software, and the polymorphism of some SSR primers were identified. In the YH safflower transcriptome, a total of 46 016 SSRs were identified through software analysis, at a frequency of 32.09%, with one SSR locus occurring in per 3.11 kb. Among the repeat types identified, dinucleotide and trinucleotide were the dominant repeat types, representing AG/CT and AAG/CTT, accounting 44% and 24%, respectively. SSR repeat frequencies were largely different, and the largest number of repeats was 5, accounting for 26.63% of the total repeat types, followed by 6 repeats(23.16%). Meanwhile, 27 primer pairs were randomly selected and synthesized, which were verified by PCR amplification of genomic DNA from 60 safflower samples. The results showed that 12 primer pairs produced expected bands. The ratio of polymorphic primers was 44.4%. In conclusion, the final results indicated that it was feasible to develop SSR markers based on transcriptome sequencing of safflower. The developed markers have the advantages of steady amplification and high polymorphism. The developed SSR markers will contribute to functional gene mining, genetic linkage mapping and genetic diversity analysis of safflower.
The aim of this study was to develop EST-SSR molecular markers, providing a powerful tool for molecular marker assisted breeding and germplasm resource assessment for safflower.Based on the sequencing of safflower transcriptome, the SSR loci were searched by MISA software, and the polymorphism of some SSR primers were identified. In the YH safflower transcriptome, a total of 46 016 SSRs were identified through software analysis, at a frequency of 32.09%, with one SSR locus occurring in per 3.11 kb. Among the repeat types identified, dinucleotide and trinucleotide were the dominant repeat types, representing AG/CT and AAG/CTT, accounting 44% and 24%, respectively. SSR repeat frequencies were largely different, and the largest number of repeats was 5, accounting for 26.63% of the total repeat types, followed by 6 repeats(23.16%). Meanwhile, 27 primer pairs were randomly selected and synthesized, which were verified by PCR amplification of genomic DNA from 60 safflower samples. The results showed that 12 primer pairs produced expected bands. The ratio of polymorphic primers was 44.4%. In conclusion, the final results indicated that it was feasible to develop SSR markers based on transcriptome sequencing of safflower. The developed markers have the advantages of steady amplification and high polymorphism. The developed SSR markers will contribute to functional gene mining, genetic linkage mapping and genetic diversity analysis of safflower.
引文
[1]王岩军.红花的组织培养及基因工程研究进展[J].新疆农业科学,2008(S1):237-241.
[2] 江磊,李刚,岳帅,等.11个红花品种遗传多样性与亲缘关系的SRAP分析[J].中国油料作物学报,2013,35(5):546.
[3] 杨玉霞,吴卫,郑有良.红花研究进展[J].四川农业大学学报,2004,22(4):365-369.
[4] PEARL S A, BURKE J M. Genetic diversity inCarthamus tinctorius (Asteraceae; safflower), an underutilized oilseed crop[J]. American Journal of Botany, 2014,101(10):1640-1650.
[5] 唐小慧,任超翔,陈江,等.利用SSR分子标记分析我国红花主要栽培品种遗传多样性[J].中药材,2017(12):2800-2804.
[6] 黄国庆,郭加沅,肖国樱.SSR标记在水稻遗传育种中的应用[J].江西农业学报,2007,19(4):20-22.
[7] 李新海,傅骏骅.利用SSR标记研究玉米自交系的遗传变异[J].中国农业科学,2000,33(2):1-9.
[8] 朱振东,贾继增.小麦SSR标记的发展及应用[J].遗传,2003,25(3):355-360.
[9] 许盛宝.小麦SSR标记遗传图谱构建与株高和抽穗期的QTL定位[D].乌鲁木齐:新疆农业大学,2005.
[10]李媛媛,沈金雄,王同华,等.利用SRAP、SSR和AFLP标记构建甘蓝型油菜遗传连锁图谱[J].中国农业科学,2007,40(6):1118-1126.
[11] 匡猛,杨伟华,许红霞,等.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[J].中国农业科学,2011,44(1):20-27.
[12] 宋海斌,崔喜波,马鸿艳,等.基于SSR标记的甜瓜品种(系)DNA指纹图谱库的构建[J].中国农业科学,2012,45(13):2676-2689.
[13] 张鹏,张海洋,郭旺珍,等.以SRAP和EST-SSR标记分析芝麻种质资源的遗传多样性[J].作物学报,2007,33(10):1696-1702.
[14] 陈燕萍,陈美霞,徐建堂,等.圆果黄麻成熟叶片总DNA提取及SRAP扩增体系的建立与优化[J].福建农林大学学报(自然版),2011,40(5):461-466.
[15] Grabherr M G, Haas B J, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology, 2011,29(7):644.
[16] 时小东,朱学慧,盛玉珍,等.基于转录组序列的楠木SSR分子标记开发[J].林业科学,2016,52(11):71-78.
[17] 乔婷婷,马春雷,周炎花,等.浙江省茶树地方品种与选育品种遗传多样性和群体结构的EST-SSR分析[J].作物学报,2010,36(5):744-753.
[18] 柯涛,毛晗,董彩华,等.芝麻EST-SSR引物的开发与应用[J].中国油料作物学报,2013,35(1):43-47.
[19] ZHUANG X, MCPHEE K E, CORAM T E, et al. Development and characterization of 37 novel EST-SSR markers inPisum sativum (Fabaceae)[J]. Applications in Plant Sciences, 2013,1(1):38-51.
[20] 张振,张含国,莫迟,等.红松转录组SSR分析及EST-SSR标记开发[J].林业科学,2015,51(8):114-120.
[21] DUTTA S, KUMAWAT G, SINGH B P, et al. Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan,(L.) Millspaugh] [J]. BMC Plant Biology, 2011,11(1):17.
[22] XU Y, MA R C, XIE H, et al. Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region[J]. Genome, 2004,47(6):1091-1104.
[23] CHEN C, ZHOU P, CHOI Y A, et al. Mining and characterizing microsatellites from citrus ESTs[J]. Theoretical & Applied Genetics, 2006,112(7):1248-1257.
[24] 甘霖,覃碧,刘实忠,等.巴西橡胶树转录组中SSR位点的信息分析[J].广东农业科学,2014,41(16):142-146.
[25] LI D, ZHI D, BI Q, et al. De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.)[J]. Bmc Genomics, 2012,13(1):192.
[26] 陶爱芬,陈婉婷,祁建民,等.基于转录组测序的黄麻SSR分子标记开发与初步验证[J].中国农业大学学报,2018(6):24-32.
[27] 陆云峰,杨安娜,张俊红,等.紫楠转录组EST-SSR标记开发及通用性分析[J].农业生物技术学报,2018,26(6):1014-1024.
[28] WANG Z, FANG B, CHEN J, et al. De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweetpotato (Ipomoea batatas)[J]. Bmc Genomics, 2010,11(1):726.
[29] MORGANTE M, HANAFEY M, POWELL W. Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes[J]. Nature Genetics, 2002,30(2):194-200.
[30] 潜宗伟,陈海丽,崔彦玲,等.菠菜转录组SSR位点分析及其分子标记的开发[J].农业生物技术学报,2016(11):1688-1697.
[31] 李炎林,杨星星,张家银,等.南方红豆杉转录组SSR挖掘及分子标记的研究[J]. 园艺学报,2014,41(4):735-745.
[2] 江磊,李刚,岳帅,等.11个红花品种遗传多样性与亲缘关系的SRAP分析[J].中国油料作物学报,2013,35(5):546.
[3] 杨玉霞,吴卫,郑有良.红花研究进展[J].四川农业大学学报,2004,22(4):365-369.
[4] PEARL S A, BURKE J M. Genetic diversity inCarthamus tinctorius (Asteraceae; safflower), an underutilized oilseed crop[J]. American Journal of Botany, 2014,101(10):1640-1650.
[5] 唐小慧,任超翔,陈江,等.利用SSR分子标记分析我国红花主要栽培品种遗传多样性[J].中药材,2017(12):2800-2804.
[6] 黄国庆,郭加沅,肖国樱.SSR标记在水稻遗传育种中的应用[J].江西农业学报,2007,19(4):20-22.
[7] 李新海,傅骏骅.利用SSR标记研究玉米自交系的遗传变异[J].中国农业科学,2000,33(2):1-9.
[8] 朱振东,贾继增.小麦SSR标记的发展及应用[J].遗传,2003,25(3):355-360.
[9] 许盛宝.小麦SSR标记遗传图谱构建与株高和抽穗期的QTL定位[D].乌鲁木齐:新疆农业大学,2005.
[10]李媛媛,沈金雄,王同华,等.利用SRAP、SSR和AFLP标记构建甘蓝型油菜遗传连锁图谱[J].中国农业科学,2007,40(6):1118-1126.
[11] 匡猛,杨伟华,许红霞,等.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[J].中国农业科学,2011,44(1):20-27.
[12] 宋海斌,崔喜波,马鸿艳,等.基于SSR标记的甜瓜品种(系)DNA指纹图谱库的构建[J].中国农业科学,2012,45(13):2676-2689.
[13] 张鹏,张海洋,郭旺珍,等.以SRAP和EST-SSR标记分析芝麻种质资源的遗传多样性[J].作物学报,2007,33(10):1696-1702.
[14] 陈燕萍,陈美霞,徐建堂,等.圆果黄麻成熟叶片总DNA提取及SRAP扩增体系的建立与优化[J].福建农林大学学报(自然版),2011,40(5):461-466.
[15] Grabherr M G, Haas B J, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology, 2011,29(7):644.
[16] 时小东,朱学慧,盛玉珍,等.基于转录组序列的楠木SSR分子标记开发[J].林业科学,2016,52(11):71-78.
[17] 乔婷婷,马春雷,周炎花,等.浙江省茶树地方品种与选育品种遗传多样性和群体结构的EST-SSR分析[J].作物学报,2010,36(5):744-753.
[18] 柯涛,毛晗,董彩华,等.芝麻EST-SSR引物的开发与应用[J].中国油料作物学报,2013,35(1):43-47.
[19] ZHUANG X, MCPHEE K E, CORAM T E, et al. Development and characterization of 37 novel EST-SSR markers inPisum sativum (Fabaceae)[J]. Applications in Plant Sciences, 2013,1(1):38-51.
[20] 张振,张含国,莫迟,等.红松转录组SSR分析及EST-SSR标记开发[J].林业科学,2015,51(8):114-120.
[21] DUTTA S, KUMAWAT G, SINGH B P, et al. Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan,(L.) Millspaugh] [J]. BMC Plant Biology, 2011,11(1):17.
[22] XU Y, MA R C, XIE H, et al. Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region[J]. Genome, 2004,47(6):1091-1104.
[23] CHEN C, ZHOU P, CHOI Y A, et al. Mining and characterizing microsatellites from citrus ESTs[J]. Theoretical & Applied Genetics, 2006,112(7):1248-1257.
[24] 甘霖,覃碧,刘实忠,等.巴西橡胶树转录组中SSR位点的信息分析[J].广东农业科学,2014,41(16):142-146.
[25] LI D, ZHI D, BI Q, et al. De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.)[J]. Bmc Genomics, 2012,13(1):192.
[26] 陶爱芬,陈婉婷,祁建民,等.基于转录组测序的黄麻SSR分子标记开发与初步验证[J].中国农业大学学报,2018(6):24-32.
[27] 陆云峰,杨安娜,张俊红,等.紫楠转录组EST-SSR标记开发及通用性分析[J].农业生物技术学报,2018,26(6):1014-1024.
[28] WANG Z, FANG B, CHEN J, et al. De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweetpotato (Ipomoea batatas)[J]. Bmc Genomics, 2010,11(1):726.
[29] MORGANTE M, HANAFEY M, POWELL W. Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes[J]. Nature Genetics, 2002,30(2):194-200.
[30] 潜宗伟,陈海丽,崔彦玲,等.菠菜转录组SSR位点分析及其分子标记的开发[J].农业生物技术学报,2016(11):1688-1697.
[31] 李炎林,杨星星,张家银,等.南方红豆杉转录组SSR挖掘及分子标记的研究[J]. 园艺学报,2014,41(4):735-745.