基于RAD-seq技术开发灵宝杜鹃多态性SSR标记
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摘要
灵宝杜鹃(Rhododendron henanense subsp. lingbaoense)隶属杜鹃花科(Ericaceae)杜鹃属,是河南特有物种,因分布范围狭窄、自然更新能力差等原因,种群规模日益缩小,对其进行系统研究与保护日益迫切。为开发用于灵宝杜鹃保护遗传学研究的简单重复序列(simple sequence repeat, SSR)分子标记,本研究采用RAD-seq (restriction-site associated DNA sequencing)技术对灵宝杜鹃进行简化基因组测序,并进行SSR搜索与引物设计。在组装的120 970个基因序列中共检测到6 107个SSR位点,二核苷酸基序类型所占比例最高,数量为3 848个,占位点总数的63.01%;其次是三核苷酸,有765个,占位点总数的12.53%。利用Primer Premier 3.0软件对这些SSR位点成功设计出3 843对引物。随机选取合成100对引物,在20个灵宝杜鹃个体中进行验证,有87对可扩增出目的条带。利用Cervus 3.0软件,对挑选出的15对多态性较好的SSR位点进行分析,多态位点信息含量介于0.463～0.893之间,平均值为0.649;其中,高度多态位点有13个,未检测出哈迪-温伯格平衡偏离的位点有15个。本研究为开展基于灵宝杜鹃及杜鹃属植物SSR分子标记的相关研究提供了数据支持。
Rhododendron henanense subsp. lingbaoense belongs to Ericaceae, Rhododendron, and is a rare and endemic species in Henan Province. Because of its narrow distribution and poor natural regeneration, the population size has been shrinking, and systematic research and protection has become increasingly urgent. In order to develop SSR(simple sequence repeat) molecular markers for conservation genetics research on R.henanense subsp. lingbaoense, simplified genome sequencing by restriction-site associated DNA sequencing(RAD-seq), SSR search and primer design were performed. A total of 6 107 SSR loci were detected in 120 970 assembled sequences. Among them, the proportion of di-nucleotide motifs was the highest, accounting for63.01% of the total loci; followed by tri-nucleotides with 765, accounting for 12.53%. 3 843 pairs of primers were successfully designed for these SSR loci using software Primer Premier 3.0. A total of 100 pairs ofprimers were randomly selected and verified with 20 individuals of R. henanense subsp. lingbaoense, and 87 were able to amplify the target bands. Fifteen pairs of polymorphic SSR loci were further analyzed using Cervus 3.0, and the polymorphic information content ranged from 0.463 to 0.893 with an average of 0.649.Among them, there were 13 highly polymorphic loci, no deviation from Hardy-Weinberg equilibrium was detected in the 15 loci. Above results could provide data support for the study of SSR markers in R. henanense subsp. lingbaoense and Rhododendron.
Rhododendron henanense subsp. lingbaoense belongs to Ericaceae, Rhododendron, and is a rare and endemic species in Henan Province. Because of its narrow distribution and poor natural regeneration, the population size has been shrinking, and systematic research and protection has become increasingly urgent. In order to develop SSR(simple sequence repeat) molecular markers for conservation genetics research on R.henanense subsp. lingbaoense, simplified genome sequencing by restriction-site associated DNA sequencing(RAD-seq), SSR search and primer design were performed. A total of 6 107 SSR loci were detected in 120 970 assembled sequences. Among them, the proportion of di-nucleotide motifs was the highest, accounting for63.01% of the total loci; followed by tri-nucleotides with 765, accounting for 12.53%. 3 843 pairs of primers were successfully designed for these SSR loci using software Primer Premier 3.0. A total of 100 pairs ofprimers were randomly selected and verified with 20 individuals of R. henanense subsp. lingbaoense, and 87 were able to amplify the target bands. Fifteen pairs of polymorphic SSR loci were further analyzed using Cervus 3.0, and the polymorphic information content ranged from 0.463 to 0.893 with an average of 0.649.Among them, there were 13 highly polymorphic loci, no deviation from Hardy-Weinberg equilibrium was detected in the 15 loci. Above results could provide data support for the study of SSR markers in R. henanense subsp. lingbaoense and Rhododendron.
引文
方文培.1983.中国杜鹃花属的新分类群[J].植物分类学报,21(4):457-469.(Fang W P.1983.New taxa of the genus Rhododendron from China[J].Acta Phytotaxonomica Sinica,21(4):457-459.)
高贤明,史淑兰.1995.河南省珍稀濒危植物观赏特性的评价[J].武汉植物学研究,13(1):87-94.(Gao X M,Shi SL.1995.Evaluations on the ornamental characteristic of the rare and threatened plants in Henan[J].Journal of Wuhan Botanical Research,13(1):87-94.)
韩军旺,张莹,袁志良,叶永忠.2008.河南小秦岭国家级自然保护区野生灵宝杜鹃的引种及开发[J].安徽农业科学,36(34):14954-14955.(Han J W,Zhang Y,Yuan Z L,Ye Y Z.2008.Study on the introduction and development of wild Rhododendron lingbaoense in Xiaoqinling National Nature Reserve of Henan[J].Journal of Anhui Agriculture Science,36(34):14954-14955.)
李海洋,李荣华,夏岩石,等.2018.基于RAD-seq数据开发烟草多态性SSR标记[J].中国烟草科学,39(1):1-9.(Li H Y,Li R H,Xia Y S,et al.2018.Development of polymorphic SSR markers in tobacco based on RAD Sequencing[J].Chinese Tobacco Science,39(1):1-9.)
李美芹,潘叶羽,钱萍仙,等.2016.杜鹃花EST-SSR标记的开发及遗传多样性分析[J].植物生理学报,52(3):356-364.(Li M Q,Pan Y Y,Qian P X,et al.2016.Development of EST-SSR primers for azalea and genetic analysis of cultivars[J].Plant Physiology Journal,52(3):356-364.)
李太强,刘雄芳,万友名,等.2017.滇东南濒危植物长梗杜鹃转录组微卫星特征分析[J].林业科学研究,30(4)533-541.(Li T Q,Liu X F,Wan Y M,et al.2017.Characteristic analysis of microsatellites in the transcriptome of Rhododendron longipedicellatum,an endangered species endemic to southeastern Yunnan,China[J].Fores Research,30(4):533-541.)
马宏,李太强,刘雄芳,等.2017.杜鹃属植物保护生物学研究进展[J].世界林业研究,30(4):13-17.(Ma H,Li TQ,Liu X F,et al.2017.Research progress in conservation biology of Rhododendron[J].World Forestry Research,30(4):13-17.)
王久利,朱明星,徐明行,等.2017.基于RAD-seq技术的异型花SSR信息分析[J].植物研究,37(3):447-452(Wang J L,Zhu M X,Xu M X,et al.2017.Analysis on SSR in Sinoswertia tetraptera base on RAD-seq[J].Bulletin of Botanical Research,37(3):447-452.)
王洋坤,胡艳,张天真.2013.RAD-seq技术在基因组研究中的现状及展望[J].遗传,36(1):41-49.(Wang Y K,Hu Y,Zhang T Z.2013.Current status and perspective of RAD-seq in genomic research[J].Hereditas(Beijing),36(1):41-49.)
翁梅,孙静,叶永忠.2012.河南省重点保护植物优先保护等级划分研究[J].河南农业科学,41(1):129-133.(Weng M,Sun J,Ye Y Z.2012.Study of conservation priority classes of key protected plant in Henan[J].Journal of Henan Agricultural Sciences,41(1):129-133.)
杨世鹏,孙雪梅,王丽慧,等.2018.基于菊芋转录组的SSR分子标记开发及鉴定[J].分子植物育种,16(2):484-492.(Yang A P,Sun X M,Wang L H,et al.2018.Development and identification of SSR markers based on transcriptome of Helianthus tuberosus L.[J].Molecular Plant Breeding,16(2):484-492.)
朱海生,黄丽芳,王彬,等.2018.美洲南瓜转录组SSR信息分析及其分子标记开发[J].中国细胞生物学杂志,40(1):99-107.(Zhu H S,Huang L F,Wang B,et al.2018.Analysis on SSR information in transcriptome and development of molecular markers in summer squash[J].Chinese Journal of Cell Biology,40(1):99-107.)
Catchen J M,Amores A,Hohenlohe P,et al.2011.Stacks:Building and genotyping loci de novo from short-read sequences[J].G3-Genes Genome and Genetic,1(3):171-182.
Li T,Liu X,Li Z,et al.2018.Study on reproductive biology of Rhododendron longipedicellatum:A newly discovered and special threatened plant surviving in limestone habitat in southeast Yunnan,China[J].Frontiers in Plant Science,9(1):33.
Liang J Y,You C X,Guo S S,et al.2016.Chemical constituents of the essential oil extracted from Rhododendron thymifolium and their insecticidal activities against Liposcelis bostrychophila or Tribolium castaneum[J].Industrial Crops and Products,79(1):267-273.
Ma Y,Nielsen J,Chamberlain D F,et al.2014.The conservation of Rhododendrons is of greater urgency than has been previously acknowledged in China[J].Biodiversity Conservation,23(12):3149-3154.
Untergasser A,Cutcutache I,Koressaar T,et al.2012.Primer3-new capabilities and interfaces[J].Nucleic Acids Research,40(15):e115.
Willing E M,Hoffmann M,Klein J D,et al.2011.Paired-end RAD-seq for de novo assembly and marker design without available reference[J].Bioinformatics,27(16):2187-2193.
高贤明,史淑兰.1995.河南省珍稀濒危植物观赏特性的评价[J].武汉植物学研究,13(1):87-94.(Gao X M,Shi SL.1995.Evaluations on the ornamental characteristic of the rare and threatened plants in Henan[J].Journal of Wuhan Botanical Research,13(1):87-94.)
韩军旺,张莹,袁志良,叶永忠.2008.河南小秦岭国家级自然保护区野生灵宝杜鹃的引种及开发[J].安徽农业科学,36(34):14954-14955.(Han J W,Zhang Y,Yuan Z L,Ye Y Z.2008.Study on the introduction and development of wild Rhododendron lingbaoense in Xiaoqinling National Nature Reserve of Henan[J].Journal of Anhui Agriculture Science,36(34):14954-14955.)
李海洋,李荣华,夏岩石,等.2018.基于RAD-seq数据开发烟草多态性SSR标记[J].中国烟草科学,39(1):1-9.(Li H Y,Li R H,Xia Y S,et al.2018.Development of polymorphic SSR markers in tobacco based on RAD Sequencing[J].Chinese Tobacco Science,39(1):1-9.)
李美芹,潘叶羽,钱萍仙,等.2016.杜鹃花EST-SSR标记的开发及遗传多样性分析[J].植物生理学报,52(3):356-364.(Li M Q,Pan Y Y,Qian P X,et al.2016.Development of EST-SSR primers for azalea and genetic analysis of cultivars[J].Plant Physiology Journal,52(3):356-364.)
李太强,刘雄芳,万友名,等.2017.滇东南濒危植物长梗杜鹃转录组微卫星特征分析[J].林业科学研究,30(4)533-541.(Li T Q,Liu X F,Wan Y M,et al.2017.Characteristic analysis of microsatellites in the transcriptome of Rhododendron longipedicellatum,an endangered species endemic to southeastern Yunnan,China[J].Fores Research,30(4):533-541.)
马宏,李太强,刘雄芳,等.2017.杜鹃属植物保护生物学研究进展[J].世界林业研究,30(4):13-17.(Ma H,Li TQ,Liu X F,et al.2017.Research progress in conservation biology of Rhododendron[J].World Forestry Research,30(4):13-17.)
王久利,朱明星,徐明行,等.2017.基于RAD-seq技术的异型花SSR信息分析[J].植物研究,37(3):447-452(Wang J L,Zhu M X,Xu M X,et al.2017.Analysis on SSR in Sinoswertia tetraptera base on RAD-seq[J].Bulletin of Botanical Research,37(3):447-452.)
王洋坤,胡艳,张天真.2013.RAD-seq技术在基因组研究中的现状及展望[J].遗传,36(1):41-49.(Wang Y K,Hu Y,Zhang T Z.2013.Current status and perspective of RAD-seq in genomic research[J].Hereditas(Beijing),36(1):41-49.)
翁梅,孙静,叶永忠.2012.河南省重点保护植物优先保护等级划分研究[J].河南农业科学,41(1):129-133.(Weng M,Sun J,Ye Y Z.2012.Study of conservation priority classes of key protected plant in Henan[J].Journal of Henan Agricultural Sciences,41(1):129-133.)
杨世鹏,孙雪梅,王丽慧,等.2018.基于菊芋转录组的SSR分子标记开发及鉴定[J].分子植物育种,16(2):484-492.(Yang A P,Sun X M,Wang L H,et al.2018.Development and identification of SSR markers based on transcriptome of Helianthus tuberosus L.[J].Molecular Plant Breeding,16(2):484-492.)
朱海生,黄丽芳,王彬,等.2018.美洲南瓜转录组SSR信息分析及其分子标记开发[J].中国细胞生物学杂志,40(1):99-107.(Zhu H S,Huang L F,Wang B,et al.2018.Analysis on SSR information in transcriptome and development of molecular markers in summer squash[J].Chinese Journal of Cell Biology,40(1):99-107.)
Catchen J M,Amores A,Hohenlohe P,et al.2011.Stacks:Building and genotyping loci de novo from short-read sequences[J].G3-Genes Genome and Genetic,1(3):171-182.
Li T,Liu X,Li Z,et al.2018.Study on reproductive biology of Rhododendron longipedicellatum:A newly discovered and special threatened plant surviving in limestone habitat in southeast Yunnan,China[J].Frontiers in Plant Science,9(1):33.
Liang J Y,You C X,Guo S S,et al.2016.Chemical constituents of the essential oil extracted from Rhododendron thymifolium and their insecticidal activities against Liposcelis bostrychophila or Tribolium castaneum[J].Industrial Crops and Products,79(1):267-273.
Ma Y,Nielsen J,Chamberlain D F,et al.2014.The conservation of Rhododendrons is of greater urgency than has been previously acknowledged in China[J].Biodiversity Conservation,23(12):3149-3154.
Untergasser A,Cutcutache I,Koressaar T,et al.2012.Primer3-new capabilities and interfaces[J].Nucleic Acids Research,40(15):e115.
Willing E M,Hoffmann M,Klein J D,et al.2011.Paired-end RAD-seq for de novo assembly and marker design without available reference[J].Bioinformatics,27(16):2187-2193.