龙眼EST-SSR标记开发及无患子科5个属种质遗传多样性分析
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摘要
基于龙眼品种‘香脆’果实的转录组测序数据库,开发龙眼EST-SSR引物,评价其在无患子、红毛丹、荔枝、龙荔等4个近缘属植物上的通用性,并进行无患子科5个属55份种质的遗传多样性分析。在13 934条龙眼Unigene中搜索到SSR位点6 683个,SSR发生频率为47.96%,平均分布距离为4.48 kb;单、二和三核苷酸重复是主要的SSR类型,分别占52.78%、26.25%和20.19%;设计出4 670对SSR引物,随机合成185对不同重复类型的SSR引物进行验证,有效扩增率为78.92%(146对),其中多态性引物占34.25%(50对),条带多态率50%～100%;龙眼多态性引物在无患子属、韶子属、荔枝属、龙荔属等4个近缘属上的转移率分别为38.30%、38.30%、68.09%和74.47%。UPGMA聚类分析显示,55份材料在遗传相似系数0.561处明显地划分为龙眼、龙荔、荔枝、无患子和红毛丹5个大类。
Based on the transcriptome sequencing data of flesh of a new longan cultivar‘Xiangcui',this study developed the novel EST-derived SSR markers to access their applicability on the 4 relative plants(Sapindus mukorossi,Rambutan,Litchi chinensis and Dimocarpus confinis)and then explored the genetic relationship among the 55 germplasms of 5 genera of Sapindaceae family. The results showed that a total of 6 683 SSR loci were mined from 13 934 unigenes with a frequence of 47.96% and mean distance of 4.48 kb. The most abundant type of repeat motif was single nucleotide(52.78%),followed by dinucleotide(26.25%)and trinucleotide(20.19%). 4 670 pairs of SSR primers were designed and 185 pairs of primers with different repeat motifs were randomly selected for further verification. Of all,the effective PCR success rate shared 78.92%(146 pairs),polymorphism rate shared 34.25%(50 pairs),polymorphic band rate ranged from 50% to 100%. And the rate of longan polymorphic primers transferring to S. mukorossi Gaertn.,N. lappaceum Linn.,L. chinensis Sonn. and D. confinis H.S.Lo were 38.30%,38.30%,68.09% and 74.47%,respectively. Furthermore,with the genetic similarity coefficient of 0.561,UPGMA clustering analysis divided the 55 germplasms into 5 groups,including longan,longli,litchi,Sapindus,and Nephelium.
Based on the transcriptome sequencing data of flesh of a new longan cultivar‘Xiangcui',this study developed the novel EST-derived SSR markers to access their applicability on the 4 relative plants(Sapindus mukorossi,Rambutan,Litchi chinensis and Dimocarpus confinis)and then explored the genetic relationship among the 55 germplasms of 5 genera of Sapindaceae family. The results showed that a total of 6 683 SSR loci were mined from 13 934 unigenes with a frequence of 47.96% and mean distance of 4.48 kb. The most abundant type of repeat motif was single nucleotide(52.78%),followed by dinucleotide(26.25%)and trinucleotide(20.19%). 4 670 pairs of SSR primers were designed and 185 pairs of primers with different repeat motifs were randomly selected for further verification. Of all,the effective PCR success rate shared 78.92%(146 pairs),polymorphism rate shared 34.25%(50 pairs),polymorphic band rate ranged from 50% to 100%. And the rate of longan polymorphic primers transferring to S. mukorossi Gaertn.,N. lappaceum Linn.,L. chinensis Sonn. and D. confinis H.S.Lo were 38.30%,38.30%,68.09% and 74.47%,respectively. Furthermore,with the genetic similarity coefficient of 0.561,UPGMA clustering analysis divided the 55 germplasms into 5 groups,including longan,longli,litchi,Sapindus,and Nephelium.
引文
Chen Xiu-ping,Jiang Fan,Jiang Ji-mou,Hu Wen-shun,Zheng Shao-quan.2016.Morphological characterization of Dimocarpus confinis.Southeast Horticulture,(5):12-17.(in Chinese)陈秀萍，姜帆，蒋际谋，胡文舜，郑少泉.2016.龙荔种质资源形态特征鉴定.东南园艺，(5):12-17.
Decroocq V,Fave M G,Hagen L,Bordenave L,Decroocq S.2003.Development and transferability of apricot and grape EST microsatellite markers across taxa.Theoretical and Applied Genetics,106:912-922.
Fang Zhi-zhen,Ye Xin-fu,Zhou Dan-rong,Jiang Cui-cui,Pan Shao-lin.2016.Analysis on SSR information in‘Furongli’plum transcriptome and development of molecular markers in Prunus salicina Lindl.Journal of Fruit Science,33(4):416-424.(in Chinese)方智振，叶新福，周丹蓉，姜翠翠，潘少霖.2016.‘芙蓉李’转录组SSR信息分析与分子标记开发.果树学报，33(4):416-424.
Fu Jia-xin.2010.Development,application of microsatellite markers and superior offspring selection of litchi and longan[Ph.D.Dissertation].Guangzhou:South China Agricultural University.(in Chinese)傅嘉欣.2010.荔枝龙眼微卫星标记研发、育种应用及优异杂种单株选育[博士论文].广州：华南农业大学.
Gao Hui-ying,Jiang Fan,Chen Xiu-ping,Zheng Shao-quan.2007.Random amplification polymorphic DNA(RAPD)analysis of the typical longan genotypes from different origins.Journal of Fujian Forestry Science and Technology,34(1):67-71.(in Chinese)高慧颖，姜帆，陈秀萍，郑少泉.2007.不同地域的代表性基因型龙眼RAPD分析.福建林业科技，34(1):67-71.
Guo Dong-liang,Wang Jing,Han Dong-mei,Pan Xue-wen,Li Jian-guang.2018.Analysis on simple sequence repeat(SSR)information in apical transcriptome and development of molecular markers in Dimocarpus longan.Plant Physiology Journal,54(5):863-871.(in Chinese)郭栋梁，王静，韩冬梅，潘学文，李建光.2018.龙眼顶芽转录组简单重复序列（SSR）标记信息分析及分子标记开发.植物生理学报，54(5):863-871.
Hong Shinan.2015.Development and application of SSR primers based on the transcriptome in Dimocarpus longan Lour.[M.D.Dissertation].Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)洪仕南.2015.基于龙眼转录组的SSR引物开发与应用研究[硕士论文].福州：福建农林大学.
Hu Wen-shun,Huang Ai-ping,Jiang Fan,Jiang Ji-mou,Chen Xiu-ping,Zheng Shao-quan.2015.Identification and genetic diversity of reciprocal hybrids in longan(Dimocarpus longan)by SSR.Acta Horticulturae Sinica,42(10):1899-1908.(in Chinese)胡文舜，黄爱萍，姜帆，蒋际谋，陈秀萍，郑少泉.2015.龙眼正反交后代的SSR鉴定及遗传多样性分析.园艺学报，42(10):1899-1908.
Jiang D,Zhou G Y,Hong Q B.2006.Analysis of microsatellites in citrus unigenes.Acta Genetica Sinica,33:345-353.
Jiang Fan,Gao Hui-ying,Chen Xiu-ping,Zheng Shao-quan.2008.Analysis of ITS sequences of rDNA in Dimocarpus plants.Journal of Fruit Science,25(2):262-268.(in Chinese)姜帆，高慧颖，陈秀萍，郑少泉.2008.龙眼属rDNA的ITS序列分析.果树学报，25(2):262-268.
Li X Y,Xu H X,Feng J J,Chen J W.2014.Development and application of genic simple sequence sepeat markers from the transcriptome of loquat.J Amer Soc Hort Sci,139(5):507-517.
Lin T X,Yi L,Ishiki K.2005.Genetic diversity of Dimocarpus longan in China revealed by AFLP markers and partial rbcl gene sequences.Scientia Horticulturae,3:489-498.
Liu Yu-lin,Li Wei,Dong Shu-bin,Mu Xian-yun,Zhang Zhi-xiang.2017.Characterization of SSRs and development of EST-SSR markers in transcriptome of Xanthoceras sorbifolia Bunge.Journal of Northwest A&F University(Nat Sci Ed),45(3):89-95.(in Chinese)刘玉林，李伟，董树斌，沐先运，张志翔.2017.文冠果转录组SSR特征分析及EST-SSR标记开发.西北农林科技大学学报（自然科学版），45(3):89-95.
Luo Xianrui,Chen Dezhao.1985.Flora Reipublicae Popularis Sinicae:Tomus 47(1):Sapindaceae.Beijing:Science Press:27-31.(in Chinese)罗献瑞，陈德昭.1985.中国植物志.第47卷，第1分册：无患子科.北京：科学出版社：27-31.
Mahar K S,Palni L M S,Ranade S A,Pande V,Rana T S.2017.Molecular analyses of genetic variation and phylogenetic relationship in Indian soap nut(Sapindus L.)and closely related taxa of the family Sapindaceae.Meta Gene,13:50-56.
Morgante M,Hanafey M,Powell W.2002.Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes.Nature Genetics,30(2):194-200.
Pan Li-mei,Zhu Jian-hua,Qin Xian-quan,Peng Hong-xiang,Huang Feng-zhu,Lu Mei-ying.2011.Genetic diversity analysis of Dimocarpus confinis in Guangxi by intersimple sequence repeats(ISSRs).Journal of Fruit Science,28(5):814-819.(in Chinese)潘丽梅，朱建华，秦献泉，彭宏祥，黄凤珠，卢美英.2011.广西龙荔遗传多样性的ISSR分析.果树学报，28(5):814-819.
Qiu wu-ling.1996.Fruit flora of China﹒Longan.Beijing:China Forestry Publishing Press.(in Chinese)邱武陵.1996.中国果树志﹒龙眼.北京：中国林业出版社.
Ramchiary N,Nguyen V D,Li X,Hong C P,Dhandapani V,Choi S R,Yu G,Piao Z Y,Lim Y P.2011.Genic microsatellite markers in Brassica rapa:development,characterization,mapping,and their utility in other cultivated and wild Brassica relatives.DNA Res,18:305-320.
Su Wei-qiang,Huang Hai-bin,Lu Yu-ying,Liu Rong-guang.1993.Analysis genetic relationship of D.confinis,longan and Litchi(cv.)based on the peroxidase and isoenzyme.Guangxi Agrieultural Sciences,(4):158-159.(in Chinese)苏伟强，黄海滨，陆玉英，刘荣光.1993.龙荔与龙眼荔枝过氧化物酶同工酶分析及亲缘关系研究初报.广西农业科学，(4):158-159.
Sun Qing-ming,Ma Wen-chao,Ma Shuai-peng,Zhao Jun-sheng,Bai Li-jun,Chen Jie-zhen,Cai Chang-he,Xiang Xu,Ou Liang-xi.2011.Characteristics of SSRs derived from ESTs and development of EST-SSR markers in litch(Litchi chinensis Sonn.).Scientia Agricultura Sinica,44(19):4037-4049.(in Chinese)孙清明，马文朝，马帅鹏，赵俊生，白丽军，陈洁珍，蔡长河，向旭，欧良喜.2011.荔枝EST资源的SSR信息分析及EST-SSR标记开发.中国农业科学，44(19):4037-4049.
van Buren R,Li J G,Zee F,Zhu J H,Liu C M,Arumuganathan A K,Ming R.2011.Longli is not a hybrid of longan and lychee as revealed by genome size analysis and trichome morphology.Tropical Plant Biol,4:228-236.
Vendramin E,Dettori M T,Giovinazzi J,Micali S,Quarta R,Verde J.2007.A set of ES-SSRs isolated from peach fruit transeriptome and their transportability across Prunus species.Molecular Ecology Notes,7:307-310.
Wang L,Zhao S,Gu C,Zhou Y,Zhou H,Ma J,Cheng J,Han Y.2013.Deep RNA-Seq uncovers the peach transcriptome landscape.Plant Mol Biol,83:365-377.
Wei Xiu-qing,Xu Ling,Zhang Xi-juan,Yu Dong,Chen Zhi-feng,Zhang Li-mei,Chen Chang-zhong,Xu Jia-hui.2018.Analysis on SSR information in transcriptome and development of molecular markers in wax apple.Acta Horticulturae Sinica,45(3):541-551.(in Chinese)魏秀清，许玲，章希娟，余东，陈志峰，张丽梅，陈长忠，许家辉.2018.莲雾转录组SSR信息分析及其分子标记开发.园艺学报，45(3):541-551.
Xiao J,Zhao J,Liu M,Liu P,Dai L,Zhao Z.2015.Genome-wide characterization of simple sequence repeat(SSR)loci in Chinese Jujube and Jujube SSR primer transferability.PLoS ONE,10(5):e0127812.doi:10.1371/journal.pone.0127812.
Yan Xiu-qin,Lu Min,An Hua-ming.2015.Analysis on SSR information in transcriptome and development of molecular markers in Rosa roxburghii.Acta Horticulturae Sinica,42(2):341-349.(in Chinese)鄢秀芹，鲁敏，安华明.2015.刺梨转录组SSR信息分析及其分子标记开发.园艺学报，42(2):341-349.
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Zhao Neng,Yuan Xiao-long,Miao Fu-jun,Wang Yi,Chen Wei,Li Jiang,Wu Tao,Wang Juan.2017.Development of SSR molecular markers based on transcriptome data of Pinus kesiya var.langbianensis.Biotechnology Bulletin,33(5):71-77.(in Chinese)赵能，原晓龙，缪福俊，王毅，陈伟，李江，吴涛，王娟.2017.思茅松转录组SSR分析及标记开发.生物技术通报，33(5):71-77.
Decroocq V,Fave M G,Hagen L,Bordenave L,Decroocq S.2003.Development and transferability of apricot and grape EST microsatellite markers across taxa.Theoretical and Applied Genetics,106:912-922.
Fang Zhi-zhen,Ye Xin-fu,Zhou Dan-rong,Jiang Cui-cui,Pan Shao-lin.2016.Analysis on SSR information in‘Furongli’plum transcriptome and development of molecular markers in Prunus salicina Lindl.Journal of Fruit Science,33(4):416-424.(in Chinese)方智振，叶新福，周丹蓉，姜翠翠，潘少霖.2016.‘芙蓉李’转录组SSR信息分析与分子标记开发.果树学报，33(4):416-424.
Fu Jia-xin.2010.Development,application of microsatellite markers and superior offspring selection of litchi and longan[Ph.D.Dissertation].Guangzhou:South China Agricultural University.(in Chinese)傅嘉欣.2010.荔枝龙眼微卫星标记研发、育种应用及优异杂种单株选育[博士论文].广州：华南农业大学.
Gao Hui-ying,Jiang Fan,Chen Xiu-ping,Zheng Shao-quan.2007.Random amplification polymorphic DNA(RAPD)analysis of the typical longan genotypes from different origins.Journal of Fujian Forestry Science and Technology,34(1):67-71.(in Chinese)高慧颖，姜帆，陈秀萍，郑少泉.2007.不同地域的代表性基因型龙眼RAPD分析.福建林业科技，34(1):67-71.
Guo Dong-liang,Wang Jing,Han Dong-mei,Pan Xue-wen,Li Jian-guang.2018.Analysis on simple sequence repeat(SSR)information in apical transcriptome and development of molecular markers in Dimocarpus longan.Plant Physiology Journal,54(5):863-871.(in Chinese)郭栋梁，王静，韩冬梅，潘学文，李建光.2018.龙眼顶芽转录组简单重复序列（SSR）标记信息分析及分子标记开发.植物生理学报，54(5):863-871.
Hong Shinan.2015.Development and application of SSR primers based on the transcriptome in Dimocarpus longan Lour.[M.D.Dissertation].Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)洪仕南.2015.基于龙眼转录组的SSR引物开发与应用研究[硕士论文].福州：福建农林大学.
Hu Wen-shun,Huang Ai-ping,Jiang Fan,Jiang Ji-mou,Chen Xiu-ping,Zheng Shao-quan.2015.Identification and genetic diversity of reciprocal hybrids in longan(Dimocarpus longan)by SSR.Acta Horticulturae Sinica,42(10):1899-1908.(in Chinese)胡文舜，黄爱萍，姜帆，蒋际谋，陈秀萍，郑少泉.2015.龙眼正反交后代的SSR鉴定及遗传多样性分析.园艺学报，42(10):1899-1908.
Jiang D,Zhou G Y,Hong Q B.2006.Analysis of microsatellites in citrus unigenes.Acta Genetica Sinica,33:345-353.
Jiang Fan,Gao Hui-ying,Chen Xiu-ping,Zheng Shao-quan.2008.Analysis of ITS sequences of rDNA in Dimocarpus plants.Journal of Fruit Science,25(2):262-268.(in Chinese)姜帆，高慧颖，陈秀萍，郑少泉.2008.龙眼属rDNA的ITS序列分析.果树学报，25(2):262-268.
Li X Y,Xu H X,Feng J J,Chen J W.2014.Development and application of genic simple sequence sepeat markers from the transcriptome of loquat.J Amer Soc Hort Sci,139(5):507-517.
Lin T X,Yi L,Ishiki K.2005.Genetic diversity of Dimocarpus longan in China revealed by AFLP markers and partial rbcl gene sequences.Scientia Horticulturae,3:489-498.
Liu Yu-lin,Li Wei,Dong Shu-bin,Mu Xian-yun,Zhang Zhi-xiang.2017.Characterization of SSRs and development of EST-SSR markers in transcriptome of Xanthoceras sorbifolia Bunge.Journal of Northwest A&F University(Nat Sci Ed),45(3):89-95.(in Chinese)刘玉林，李伟，董树斌，沐先运，张志翔.2017.文冠果转录组SSR特征分析及EST-SSR标记开发.西北农林科技大学学报（自然科学版），45(3):89-95.
Luo Xianrui,Chen Dezhao.1985.Flora Reipublicae Popularis Sinicae:Tomus 47(1):Sapindaceae.Beijing:Science Press:27-31.(in Chinese)罗献瑞，陈德昭.1985.中国植物志.第47卷，第1分册：无患子科.北京：科学出版社：27-31.
Mahar K S,Palni L M S,Ranade S A,Pande V,Rana T S.2017.Molecular analyses of genetic variation and phylogenetic relationship in Indian soap nut(Sapindus L.)and closely related taxa of the family Sapindaceae.Meta Gene,13:50-56.
Morgante M,Hanafey M,Powell W.2002.Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes.Nature Genetics,30(2):194-200.
Pan Li-mei,Zhu Jian-hua,Qin Xian-quan,Peng Hong-xiang,Huang Feng-zhu,Lu Mei-ying.2011.Genetic diversity analysis of Dimocarpus confinis in Guangxi by intersimple sequence repeats(ISSRs).Journal of Fruit Science,28(5):814-819.(in Chinese)潘丽梅，朱建华，秦献泉，彭宏祥，黄凤珠，卢美英.2011.广西龙荔遗传多样性的ISSR分析.果树学报，28(5):814-819.
Qiu wu-ling.1996.Fruit flora of China﹒Longan.Beijing:China Forestry Publishing Press.(in Chinese)邱武陵.1996.中国果树志﹒龙眼.北京：中国林业出版社.
Ramchiary N,Nguyen V D,Li X,Hong C P,Dhandapani V,Choi S R,Yu G,Piao Z Y,Lim Y P.2011.Genic microsatellite markers in Brassica rapa:development,characterization,mapping,and their utility in other cultivated and wild Brassica relatives.DNA Res,18:305-320.
Su Wei-qiang,Huang Hai-bin,Lu Yu-ying,Liu Rong-guang.1993.Analysis genetic relationship of D.confinis,longan and Litchi(cv.)based on the peroxidase and isoenzyme.Guangxi Agrieultural Sciences,(4):158-159.(in Chinese)苏伟强，黄海滨，陆玉英，刘荣光.1993.龙荔与龙眼荔枝过氧化物酶同工酶分析及亲缘关系研究初报.广西农业科学，(4):158-159.
Sun Qing-ming,Ma Wen-chao,Ma Shuai-peng,Zhao Jun-sheng,Bai Li-jun,Chen Jie-zhen,Cai Chang-he,Xiang Xu,Ou Liang-xi.2011.Characteristics of SSRs derived from ESTs and development of EST-SSR markers in litch(Litchi chinensis Sonn.).Scientia Agricultura Sinica,44(19):4037-4049.(in Chinese)孙清明，马文朝，马帅鹏，赵俊生，白丽军，陈洁珍，蔡长河，向旭，欧良喜.2011.荔枝EST资源的SSR信息分析及EST-SSR标记开发.中国农业科学，44(19):4037-4049.
van Buren R,Li J G,Zee F,Zhu J H,Liu C M,Arumuganathan A K,Ming R.2011.Longli is not a hybrid of longan and lychee as revealed by genome size analysis and trichome morphology.Tropical Plant Biol,4:228-236.
Vendramin E,Dettori M T,Giovinazzi J,Micali S,Quarta R,Verde J.2007.A set of ES-SSRs isolated from peach fruit transeriptome and their transportability across Prunus species.Molecular Ecology Notes,7:307-310.
Wang L,Zhao S,Gu C,Zhou Y,Zhou H,Ma J,Cheng J,Han Y.2013.Deep RNA-Seq uncovers the peach transcriptome landscape.Plant Mol Biol,83:365-377.
Wei Xiu-qing,Xu Ling,Zhang Xi-juan,Yu Dong,Chen Zhi-feng,Zhang Li-mei,Chen Chang-zhong,Xu Jia-hui.2018.Analysis on SSR information in transcriptome and development of molecular markers in wax apple.Acta Horticulturae Sinica,45(3):541-551.(in Chinese)魏秀清，许玲，章希娟，余东，陈志峰，张丽梅，陈长忠，许家辉.2018.莲雾转录组SSR信息分析及其分子标记开发.园艺学报，45(3):541-551.
Xiao J,Zhao J,Liu M,Liu P,Dai L,Zhao Z.2015.Genome-wide characterization of simple sequence repeat(SSR)loci in Chinese Jujube and Jujube SSR primer transferability.PLoS ONE,10(5):e0127812.doi:10.1371/journal.pone.0127812.
Yan Xiu-qin,Lu Min,An Hua-ming.2015.Analysis on SSR information in transcriptome and development of molecular markers in Rosa roxburghii.Acta Horticulturae Sinica,42(2):341-349.(in Chinese)鄢秀芹，鲁敏，安华明.2015.刺梨转录组SSR信息分析及其分子标记开发.园艺学报，42(2):341-349.
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