花椰菜转录组SSR位点分析及其分子标记开发
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摘要
【目的】开发适合花椰菜的SSR分子标记,为花椰菜品种遗传关系鉴定、遗传图谱绘制等提供候选标记。【方法】以24份花椰菜材料为研究对象,通过MISA软件对其转录组SSR位点信息进行分析,设计SSR引物,对这些引物进行PCR扩增,开发高多态性花椰菜的SSR分子标记;利用差异条带对不同花椰菜材料的亲缘关系进行分析。【结果】从转录组数据中得到66 450条Unigene基因,包含10 715个SSR位点,发生频率为12.09%,平均分布距离为5.9kb。SSR位点中优势类型为二核苷酸重复基序,出现频率占总SSR的51.16%;其次是三核苷酸,占总SSR的47.37%。重复序列基序共49种,其中出现频率较高的重复基序主要为AG/CT、GA/TC和AAG/CTT。有1 164个长度≥20bp的SSR位点,获得具有潜在高多态性的SSR引物6 119对。随机设计的40对引物中有31对引物能进行有效扩增,其中有17对引物在24份花椰菜品种中表现出多态性。UPGMA分析显示,在遗传距离为0.625时,17对多态性引物可将24份花椰菜分为3类。【结论】开发的花椰菜SSR标记类型丰富,出现频率高,具有较高的可用性。
【Objective】SSR molecular markers were developed for genetic diversity analysis and genetic mapping construction in cauliflower.【Method】In this study,24 cauliflower materials were used to analyze SSR loci of transcription group by MISA software.SSR primers were designed and amplified by PCR to develop SSR markers of highly polymorphic cauliflower.The genetic relationships of different cauliflower materials were analyzed by using different bands.【Result】A total of 66 450 Unigenes were obtained from transcriptional data and then 10 715 simple sequence repeats(SSR)loci were identified with occurring frequency of 12.09%and mean distribution distance of 5.9 kb.Dinucleotide repeat was the main type of SSR loci(51.16%),followed by trinucleotide repeat motif(47.37%).Forty-nine repeat motifs were identified,and the most abundant motifs were AG/CT,GA/TC and AAG/CTT.There were 1 164 SSR loci with length larger than 20 bp.A total of 6 119 pairs of SSR primers with the potential to produce polymorphism were designed and 31 primers were randomly selected for PCR amplification using 24 loofah materials,of which 17 primers showed polymorphism.According to the UPGMA analysis,24 plants were divided into 3 groups at the genetic distance of 0.625.【Conclusion】The large amount of SSR markers obtained can be widely used.
【Objective】SSR molecular markers were developed for genetic diversity analysis and genetic mapping construction in cauliflower.【Method】In this study,24 cauliflower materials were used to analyze SSR loci of transcription group by MISA software.SSR primers were designed and amplified by PCR to develop SSR markers of highly polymorphic cauliflower.The genetic relationships of different cauliflower materials were analyzed by using different bands.【Result】A total of 66 450 Unigenes were obtained from transcriptional data and then 10 715 simple sequence repeats(SSR)loci were identified with occurring frequency of 12.09%and mean distribution distance of 5.9 kb.Dinucleotide repeat was the main type of SSR loci(51.16%),followed by trinucleotide repeat motif(47.37%).Forty-nine repeat motifs were identified,and the most abundant motifs were AG/CT,GA/TC and AAG/CTT.There were 1 164 SSR loci with length larger than 20 bp.A total of 6 119 pairs of SSR primers with the potential to produce polymorphism were designed and 31 primers were randomly selected for PCR amplification using 24 loofah materials,of which 17 primers showed polymorphism.According to the UPGMA analysis,24 plants were divided into 3 groups at the genetic distance of 0.625.【Conclusion】The large amount of SSR markers obtained can be widely used.
引文
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[2]朱世杨,张小玲,罗天宽,等.花椰菜种质资源萌发期耐盐性综合评价[J].核农学报,2012,26(2):380-390.Zhu S Y,Zhang X L,Luo T K,et al.A comprehensive evaluation of salt tolerance in caulilfower(Brassica oleracea L.var.botrytis L.)during germination stage[J].Journal of Nuclear Agricultural Sciences,2012,26(2):380-390.
[3]李文萍,林俊城,黄科.全球花椰菜生产与贸易现状分析[J].中国蔬菜,2014(9):5-10.Li W P,Lin J C,Huang K.Analysis about present status of global cauliflower production and its trade[J].China Vegetables,2014(9):5-10.
[4]李志琪.应用AFLP标记技术研究甘蓝类蔬菜遗传多样性及亲缘关系[D].北京:中国农业科学院,2003.Li Z Q.Studies on genetic diversity and relationship of Brassica oleracea L.var.botrytis L.using AFLP markers[D].Beijing:Chinese Academy of Agricultural Sciences,2003.
[5]林珲.花椰菜种质资源形态标记和RAPD标记研究[D].福州:福建农林大学,2007.Lin H.Study in cauliflower germplasm using morphological markers and RAPD markers[D].Fuzhou:Fujian Agriculture and Forestry University,2007.
[6]田源.甘蓝类蔬菜遗传多样性及亲缘关系RAPD、SSR分析[D].哈尔滨:东北农业大学,2007.Tian Y.Gentic diversity and relationship analusis of Brassica oleracea L.by RAPD SSR[D].Harbin:Northeast Agricultural University,2007.
[7]马二磊,王燕,刘莉,等.松花型花椰菜主要品种鉴定的分子标记分析[J].植物遗传资源学报,2011,11(5):621-624.Ma E L,Wang Y,Liu L,et al.Cultivar identification in the loose curd cauliflower with molecular markers[J].Journal of Plant Genetic Resources,2011,11(5):621-624.
[8]楼珏,张小玲,罗天宽,等.利用SSR和SRAP标记分析花椰菜自交系的遗传多样性[J].分子植物育种,2015,13(3):605-614.Lou J,Zhang X L,Luo T K,et al.Analysis of the genetic diversity in cauliflower inbred lines by SSR and SRAP markers[J].Molecular Plant Breeding,2015,13(3):605-614.
[9]姚雪琴,李光庆,谢祝捷,等.长三角区域花椰菜遗传多样性及与其近缘种亲缘关系的SRAP分析[J].上海农业学报,2016,32(5):22-27.Yao X Q,Li G Q,Xie Z J,et al.Genetic diversity of cauliflower in Yangtze River Delta region and SRAP analysis of the genetic relationship with its related species[J].Acta Agriculturae Shanghai,2016,32(5):22-27.
[10]王冬梅.甘蓝类作物亲缘关系的SSR分析[D].北京:中国农业科学院,2011.Wang D M.Analysis of genetic relationships of Brassica oleracea based on SSR markers[D].Beijing:Chinese Academy of Agricultural Sciences,2011.
[11]Tuler A C,Carrijo T T,Nóia L R,et al.SSR markers:a tool for species identification in Psidium(Myrtaceae)[J].Molecular Biolology Report,2015,42(11):1501-1513.
[12]Zhang H,Wei L,Miao H,et al.Development and validation of genic-SSR markers in sesame by RNA-seq[J].BMC Genomics,2012(13):316.
[13]Ding Q,Li J,Wang F,et al.Characterization and development of EST-SSRs by deep transcriptome sequencing in Chinese cabbage(Brassica rapaL.ssp.pekinensis)[J].Int J Genomics,2015,9:1-11.
[14]Zhai L,Xu L,Wang Y,et al.Novel and useful genic-SSRmarkers from de novo transcriptome sequencing of radish(Raphanus sativus L.)[J].Molecular Breeding,2014,33(3):611-624.
[15]刘峰,王运生,田雪亮,等.辣椒转录组SSR挖掘及其多态性分析[J].园艺学报,2012,39(1):168-174.Liu F,Wang Y S,Tian X L,et al.SSR mining in pepper(Capsicum annuum L.)transcriptome and the polymorphism analysis[J].Acta Horticulturae Sinica,2012,39(1):168-174.
[16]魏明明,陈钰辉,刘富中,等.基于转录组测序的茄子SSR标记开发[J].植物遗传资源学报,2016,17(6):1082-1091.Wei M M,Chen Y H,Liu F Z,et al.Development of SSRmarkers for eggplant with tanscriptome sequencing data[J].Journal of Plant Genetic Resources,2016,17(6):1082-1091.
[17]Wu T Q,Luo S B,Wang R,et al.The first illumina-based de novo transcriptome sequencing and analysis of pumpkin(Cucurbita moschata Duch)and SSR marker development[J].Molecular Breeding,2014,34(3):1437-1447.
[18]李荣华,王直亮,陈静芳,等.菜薹转录组中SSR信息与可用性分析[J].园艺学报,2016,43(9):1816-1824.Li R H,Wang Z L,Chen J F,et al.Analysis of SSR information in transcriptome and their usability in flowering Chinese cabbage[J].Acta Horticulturae Sinica,2014,43(9):1816-1824.
[19]李满堂,张仕林,邓鹏,等.洋葱转录组SSR信息分析及其多态性研究[J].园艺学报,2015,42(6):1103-1111.Li M T,Zhang S L,Deng P,et al.Analysis on SSR information in transcriptome of onion and the polymorphism[J].Acta Horticulturae Sinica,2015,42(6):1103-1111.
[20]潜宗伟,陈海丽,崔彦玲.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,2016,24(11):1688-1697.Qian Z W,Chen H L,Cui Y L.Analysis of the SSR loci and development of molecular markers in Brassica oleracea transcriptome[J].Journal of Agricultural Biotechnology,2016,24(11):1688-1697.
[21]李翠婷,张广辉,马春花,等.野三七转录组中SSR位点信息分析及其多态性研究[J].中草药,2014,45(5):1468-1472.Li C T,Zhang G H,Ma C H,et al.Analysis on SSR loci information intranscriptome of Panax vienamensis var.fuscidiscus and its polymorphism[J].Chinese Traditional and Herbal Drugs,2014,45(5):1468-1472.
[22]李珊,周天华,赵桂仿,等.马蹄香表达序列标签资源的SSR信息分析[J].中草药,2010,41(3):464-468.Li S,Zhou T H,Zhao G F,et al.Data mining for simple sequence repeats in expressed sequence tags fromSaruma henryi Oliv.[J].Chinese Traditional and Herbal Drugs,2010,41(3):464-468.
[23]Singh H,Deshmukh R K,Singh A,et al.Highly variable SSRmarkers suitable for rice genotyping using agarose gels[J].Molecular Breeding,2010,25(2):359-364.
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