中国南瓜转录组SSR信息分析及其分子标记开发
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摘要
该文对中国南瓜(Cucurbita moschata Duch)开展转录组测序分析,共获得61 882条Unigene,利用MISA软件搜索1 Kb以上的12 526条Unigene,共检测出5 407个SSR位点,分布于4 348条Unigene中,出现频率为43.17%,平均分布距离为4.76 Kb。优势重复基序为单核苷酸、二核苷酸和三核苷酸,分别占总SSR的47.92%、24.45%和19.66%。二核苷酸重复基序中以GA/TC为优势重复基序,三核苷酸重复基序以GAA/TTC为主。利用Primer 3.0共设计出6 489对SSR引物。从92对有效扩增引物中随机选择30对引物,对30个中国南瓜种质进行多态性验证分析,其中18对(占60%)引物表现稳定可重复的多态性。利用UPGMA作图,可将30份供试材料分为2类。利用中国南瓜转录组数据进行SSR标记开发能获得较高频率的SSR位点,且类型丰富,为中国南瓜遗传多样性分析和遗传图谱构建提供更丰富可靠的标记选择。
In this study, 61 882 unigenes were obtained by transcriptome sequencing analysis from Cucurbita moschata Duch. 12 526 unigenes above 1 Kb pairs were found by MISA software, and a total of 5 407 SSR locis were identified that were distributed in 4 348 unigenes. The frequency of these SSR locis was 43.17%, and the mean distribution distance of loci was 4.76 Kb. Mean while, the major repeat motifs were mononucleotide, dinucleotide and trinucleotide, which accounted for 47.92%, 24.45% and 19.66%, respectively. Furthermore, the GA/TC were the predominant dinucleotide repeat motifs, and the GAA/TTC were the predominant trinucleotide repeat motifs.6 489 pairs of SSR primers were designed by Primer 3.0, then 30 pairs of primers were randomly selected from92 pairs of effective amplification primers for the polymorphism analysis of 30 Cucurbita moschata germplasms,and 18 out of 30 SSR primers showed stable and reproducible polymorphism. According to the UPGMA mapping results, 30 Cucurbita moschata samples were divided into 2 categories. Using transcriptome data of Cucurbita moschata to develop SSR markers can obtain higher frequencies of SSR locis and the types of them were rich. It provides more abundant and reliable marker selection for genetic diversity analysis and genetic map construction of Cucurbita moschata.
In this study, 61 882 unigenes were obtained by transcriptome sequencing analysis from Cucurbita moschata Duch. 12 526 unigenes above 1 Kb pairs were found by MISA software, and a total of 5 407 SSR locis were identified that were distributed in 4 348 unigenes. The frequency of these SSR locis was 43.17%, and the mean distribution distance of loci was 4.76 Kb. Mean while, the major repeat motifs were mononucleotide, dinucleotide and trinucleotide, which accounted for 47.92%, 24.45% and 19.66%, respectively. Furthermore, the GA/TC were the predominant dinucleotide repeat motifs, and the GAA/TTC were the predominant trinucleotide repeat motifs.6 489 pairs of SSR primers were designed by Primer 3.0, then 30 pairs of primers were randomly selected from92 pairs of effective amplification primers for the polymorphism analysis of 30 Cucurbita moschata germplasms,and 18 out of 30 SSR primers showed stable and reproducible polymorphism. According to the UPGMA mapping results, 30 Cucurbita moschata samples were divided into 2 categories. Using transcriptome data of Cucurbita moschata to develop SSR markers can obtain higher frequencies of SSR locis and the types of them were rich. It provides more abundant and reliable marker selection for genetic diversity analysis and genetic map construction of Cucurbita moschata.
引文
1李新峥,杨鹏鸣,刘振威,杜晓华.中国南瓜主要性状遗传特性的研究.华南农业大学学报(Li Xinzheng,Yang Pengming,Liu Zhenwei,Du Xiaohua.Study on genetic character of Cultivars moschata main traits.Journal of South China Agricultural University)2011;32(1):7-10.
2郑道君,云天海,张治礼,邓长智,谢良商.中国南瓜海南农家品种资源遗传多样性和亲缘关系研究.核农学报(Zheng Daojun,Yun Tianhai,Zhang Zhili,Deng Changzhi,Xie Liangshang.Study on genetic diversity and relationship for the Hainan island landraces of Cucurbita moschata.Journal of Nuclear Agricul Tural Sciences)2016;30(5):869-77.
3褚盼盼,向长萍,张称心,刘成平.中国南瓜种质资源农艺性状与RAPD标记分析.核农学报(Chu Panpan,Xiang Changping,Zhang Chenxin,Liu Chengping.Genetic diversity of Cucurbita moschata genotypes revealed by RAPD markers and Agronomic traits.Journal of Nuclear Agricul Tural Sciences)2007;21(5):441-6.
4云天海,郑道君,谢良商,邓长智.中国南瓜海南农家品种间的遗传特异性分析和DNA指纹图谱构建,植物遗传资源学报(Yun Tianhai,Zheng Daojun,Xie Liangshang,Deng Changzhi.Analysis of genetic specificity and DNA fingerprinting establishment for the Hainan island landraces of Cucurbita moschata.Journal of plant genetic resources)2013;14(4):675-85.
5 Maria M,Pico B,Fernandez de Cordova P.Molecular diversity of a germplasm collection of squash(Cucurbita moschata)determined by SRAP and AFLP markers.Crop Science 2004;44:653-66.
6 Kalia RK,Rar MK,Kalia S,Singh R,Dhawan AK.Microsatellite markers:An overview of the recent progress in plants.Euphytica 2011;177(3):309-34.
7 Kumar B,Kumar U,Yadav HK.Identification of EST-SSRs and molecular diversity analysis in Mentha piperita.Crop J 2015;3(4):335-42.
8 Daniela P,Rosa LB,Caroline MC.Genetic diversity of Cucurbita pepo,C.argyrosperma and C.ficifolia accessions evaluated through microsatellite markers.Horticultura Brasileira 2013;31:361-8.
9赵树琪,梁冰,李黎贝,庞朝友,宿俊吉,宋美珍,等.基于SSR标记的陆地棉早熟相关种质遗传多样性分析.植物遗传资源学报(Zhao Shuqi,Liang Bing,Li Libei,Pang Chaoyou,Su Junji,Song Meizhen,et al.Genetic diversity analysis of early-maturity related germplasm in upland cotton(Gossypium hirsutum L.)based on SSR markers.Journal of Plant Genetic Resources)2016;17(4):599-606.
10李丽,张万清,刘玲,吴萍,马连平.SSR标记对甜瓜品种纯度和真实性的鉴定.分子植物育种(Li Li,Zhang Wanqing,Liu Ling,Wu Ping,Ma Lianping.Application of SSR markers in melon hybrid purity test and variety specificity identification.Molecular Plant Breeding)2015;13(11):2522-30.
11石星星,纪小红,张磊,吕茜茜,刘亚婷,王玉刚,等.利用SSR标记鉴定结球甘蓝杂交种真实性及纯度.分子植物育种(Shi Xingxing,Ji Xiaohong,Zhang Lei,LüQianqian,Liu Yating,Wang Yugang,et al.Authenticity and purity of heading cabbage hybrids identified by SSR markers.Molecular Plant Breeding)2015;13(2):331-7.
12高天翔,蔡宇良,冯瑛,赵晓军.中国樱桃14个自然居群遗传多样性和遗传结构的SSR评价.园艺学报(Gao Tianxiang,Cai Yuliang,Feng Ying,Zhao Xiaojun.Genetic diversity and denetic structure of Prunus pseudocerasus populations from China as revealed by SSR markers.Acta Horticulturae Sinica)2016;43(6):1148-56.
13江锡兵,汤丹,龚榜初,赖俊声.基于SSR标记的板栗地方品种遗传多样性与关联分析.园艺学报(Jiang Xibing,Tang Dan,Gong Bangchu,Lai Junsheng.Genetic diversity and association analysis of local cultivars of Chinese chestnut based on SSRmarkers.Acta Horticulturae Sinica)2015;42(12):2478-88.
14王迎儿,邢乃林,应泉盛,张慧波,黄芸萍,王毓洪.南瓜种质资源遗传多样性的SSR分析.浙江农业科学(Wang Yinger,Xing Nailin,Ying Quansheng,Zhang Huibo,Wang Yuhong.SSRanalysis of genetic diversity of pumpkin germplasm resources.Journal of zhejiang agricultural sciences)2017;58(7):1161-5.
15王瑞,吴廷全,钟玉娟,黄河勋.95份南瓜种质资源亲缘关系的SSR分析.中国农学通报(Wang Rui,Wu Tingquan,Zhong Yujuan,Huang Hexun.Genetic relationship analysis of 95 accessions of squash germplasms by SSR markers.Chinese Agricultural Science Bulletin)2016;32(34):135-42.
16向成钢,王艳玲,张雪梅,王长林,尹玲,王迎杰.SSR标记在南瓜亲缘关系分析中的应用.北方园艺(Xiang Chenggang,Wang Yanling,Zhang Xuemei,Wang Changlin,Yin Ling,Wang Yingjie.Application of SSR markers in genetic relationships analysis of Cucurbita.Northern Horticulture)2013;15:104-10.
17王洋洋,单文琪,徐文龙,崔崇士,屈淑平.印度南瓜转录组SSR信息分析及其多态性研究.园艺学报(Wang Yangyang,Shan Wenqi,Xu Wenlong,Cui Chongshi,Qu Shuping.Analysis on SSR information in transcriptome and the polymorphism of Cucurbita maxima.Acta Horticulturae Sinica)2016;43(3):578-86.
18朱海生,张前荣,刘建汀,陈敏氡,王彬,薛珠政,等.丝瓜转录组SSR位点分析及其分子标记开发.中国细胞生物学学报(Zhu Haisheng,Zhang Qianrong,Liu Jianting,Chen Mindong,Wang Bin,Xue Zhuzheng,et al.Analysis on SSR Loci in transcriptome and development of molecular markers in Luffa cylindrical.Chinese Journal of Cell Biology)2016;38(9):1118-27.
19刘峰,王运生,田雪亮,茆振川,邹学校,谢丙炎.辣椒转录组SSR挖掘及其多态性分析.园艺学报(Liu Feng,Wang Yunsheng,Tian Xueliang,Mao Zhenchuang,Zhou Xuexiao,Xie Bingyan.SSR mining in pepper(Capsicum annuum L.)transcriptome and the polymorphism analysis.Acta Horticulturae Sinica)2012;39(1):168-74.
20朱海生,黄丽芳,王彬,刘建汀,叶新如,陈敏氡.美洲南瓜转录组SSR信息分析及其分子标记开发.中国细胞生物学学报(Zhu Haisheng,Huang Linfang,Wang Bin,Liu Jianting,Ye Xinru,Chen Mindong,et al.Analysis on SSR information in transcriptome and development of molecular markers in summer squash.Chinese Journal of Cell Biology)2018;40(1):99-107.
21 Zhai L,Xu L,Wang Y,Cheng H,Chen Y,Gong Y,Liu L.Novel and useful genic-SSR markers from de novo transcriptome sequencing of radish(Raphanus sativus L.).Mol Breed 2014;33(3):611-24.
22方智振,叶新福,周丹蓉,姜翠翠,潘少霖.‘芙蓉李’转录组SSR信息分析与分子标记开发.果树学报(Fang Zhizhen,Ye Xinfu,Zhou Danrong,Jiang Cuicui,Pan Shaolin.Analysis on SSR information in‘Furongli’plum transcriptome and development of molecular markers in Prunus salicina Lindl.Journal of Fruit Science)2016;33(4):416-24.
23 Varshney RK,Graner A,Sorrells ME.Genic microsatellite markers in plants:Features and applications.Trends Biotechnol 2005;23(1):48-55.
24 Jiang D,Zhong GY,Hong QB.Analysis of microsatellites in citrus unigenes.Acta Genetica Sinica 2006;33(4):345-53.
25孙佳莹,辛大伟,单彩云,刘春燕,胡国华,陈庆山.SSR标记开发软件SSR MINING1.0的编制.中国农业科学(Sun Jiaying,Xin Dawei,Shan Caiyun,Liu Chunyan,Hu Guohua,Chen Qingshan.Programming for SSR marker developing software SSRMINING 1.0.Scientia Agricultura Sinica)2008;41(10):3336-42.
26杨曦.鲫鱼(Carassius auratus)表达序列标签资源的SSR构成与分布分析.生物技术通报(Yang Xi.Analysis of simple sequence repeats in expressed sequence tags from Carassius auratus.Biotechnology Bulletin)2012;11:139-43.
27 Xiang X,Zhang Z,Wang Z,Zhang X,Wu G.Transcriptome sequencing and development of EST-SSR markers in Pinus dabeshanensis,an endangered conifer endemic to China.Mol Breeding 2015;35:158.
28李满堂,张仕林,邓鹏,侯喜林,王建军.洋葱转录组SSR信息分析及其多态性研究.园艺学报(Li Mantang,Zhang Shilin,Deng Peng,Hou Xilin,Wang Jianjun.Analysis on SSR information in transcriptome of onion and the polymorphism.Acta Horticulturae Sinica)2015;42(6):1103-11.
2郑道君,云天海,张治礼,邓长智,谢良商.中国南瓜海南农家品种资源遗传多样性和亲缘关系研究.核农学报(Zheng Daojun,Yun Tianhai,Zhang Zhili,Deng Changzhi,Xie Liangshang.Study on genetic diversity and relationship for the Hainan island landraces of Cucurbita moschata.Journal of Nuclear Agricul Tural Sciences)2016;30(5):869-77.
3褚盼盼,向长萍,张称心,刘成平.中国南瓜种质资源农艺性状与RAPD标记分析.核农学报(Chu Panpan,Xiang Changping,Zhang Chenxin,Liu Chengping.Genetic diversity of Cucurbita moschata genotypes revealed by RAPD markers and Agronomic traits.Journal of Nuclear Agricul Tural Sciences)2007;21(5):441-6.
4云天海,郑道君,谢良商,邓长智.中国南瓜海南农家品种间的遗传特异性分析和DNA指纹图谱构建,植物遗传资源学报(Yun Tianhai,Zheng Daojun,Xie Liangshang,Deng Changzhi.Analysis of genetic specificity and DNA fingerprinting establishment for the Hainan island landraces of Cucurbita moschata.Journal of plant genetic resources)2013;14(4):675-85.
5 Maria M,Pico B,Fernandez de Cordova P.Molecular diversity of a germplasm collection of squash(Cucurbita moschata)determined by SRAP and AFLP markers.Crop Science 2004;44:653-66.
6 Kalia RK,Rar MK,Kalia S,Singh R,Dhawan AK.Microsatellite markers:An overview of the recent progress in plants.Euphytica 2011;177(3):309-34.
7 Kumar B,Kumar U,Yadav HK.Identification of EST-SSRs and molecular diversity analysis in Mentha piperita.Crop J 2015;3(4):335-42.
8 Daniela P,Rosa LB,Caroline MC.Genetic diversity of Cucurbita pepo,C.argyrosperma and C.ficifolia accessions evaluated through microsatellite markers.Horticultura Brasileira 2013;31:361-8.
9赵树琪,梁冰,李黎贝,庞朝友,宿俊吉,宋美珍,等.基于SSR标记的陆地棉早熟相关种质遗传多样性分析.植物遗传资源学报(Zhao Shuqi,Liang Bing,Li Libei,Pang Chaoyou,Su Junji,Song Meizhen,et al.Genetic diversity analysis of early-maturity related germplasm in upland cotton(Gossypium hirsutum L.)based on SSR markers.Journal of Plant Genetic Resources)2016;17(4):599-606.
10李丽,张万清,刘玲,吴萍,马连平.SSR标记对甜瓜品种纯度和真实性的鉴定.分子植物育种(Li Li,Zhang Wanqing,Liu Ling,Wu Ping,Ma Lianping.Application of SSR markers in melon hybrid purity test and variety specificity identification.Molecular Plant Breeding)2015;13(11):2522-30.
11石星星,纪小红,张磊,吕茜茜,刘亚婷,王玉刚,等.利用SSR标记鉴定结球甘蓝杂交种真实性及纯度.分子植物育种(Shi Xingxing,Ji Xiaohong,Zhang Lei,LüQianqian,Liu Yating,Wang Yugang,et al.Authenticity and purity of heading cabbage hybrids identified by SSR markers.Molecular Plant Breeding)2015;13(2):331-7.
12高天翔,蔡宇良,冯瑛,赵晓军.中国樱桃14个自然居群遗传多样性和遗传结构的SSR评价.园艺学报(Gao Tianxiang,Cai Yuliang,Feng Ying,Zhao Xiaojun.Genetic diversity and denetic structure of Prunus pseudocerasus populations from China as revealed by SSR markers.Acta Horticulturae Sinica)2016;43(6):1148-56.
13江锡兵,汤丹,龚榜初,赖俊声.基于SSR标记的板栗地方品种遗传多样性与关联分析.园艺学报(Jiang Xibing,Tang Dan,Gong Bangchu,Lai Junsheng.Genetic diversity and association analysis of local cultivars of Chinese chestnut based on SSRmarkers.Acta Horticulturae Sinica)2015;42(12):2478-88.
14王迎儿,邢乃林,应泉盛,张慧波,黄芸萍,王毓洪.南瓜种质资源遗传多样性的SSR分析.浙江农业科学(Wang Yinger,Xing Nailin,Ying Quansheng,Zhang Huibo,Wang Yuhong.SSRanalysis of genetic diversity of pumpkin germplasm resources.Journal of zhejiang agricultural sciences)2017;58(7):1161-5.
15王瑞,吴廷全,钟玉娟,黄河勋.95份南瓜种质资源亲缘关系的SSR分析.中国农学通报(Wang Rui,Wu Tingquan,Zhong Yujuan,Huang Hexun.Genetic relationship analysis of 95 accessions of squash germplasms by SSR markers.Chinese Agricultural Science Bulletin)2016;32(34):135-42.
16向成钢,王艳玲,张雪梅,王长林,尹玲,王迎杰.SSR标记在南瓜亲缘关系分析中的应用.北方园艺(Xiang Chenggang,Wang Yanling,Zhang Xuemei,Wang Changlin,Yin Ling,Wang Yingjie.Application of SSR markers in genetic relationships analysis of Cucurbita.Northern Horticulture)2013;15:104-10.
17王洋洋,单文琪,徐文龙,崔崇士,屈淑平.印度南瓜转录组SSR信息分析及其多态性研究.园艺学报(Wang Yangyang,Shan Wenqi,Xu Wenlong,Cui Chongshi,Qu Shuping.Analysis on SSR information in transcriptome and the polymorphism of Cucurbita maxima.Acta Horticulturae Sinica)2016;43(3):578-86.
18朱海生,张前荣,刘建汀,陈敏氡,王彬,薛珠政,等.丝瓜转录组SSR位点分析及其分子标记开发.中国细胞生物学学报(Zhu Haisheng,Zhang Qianrong,Liu Jianting,Chen Mindong,Wang Bin,Xue Zhuzheng,et al.Analysis on SSR Loci in transcriptome and development of molecular markers in Luffa cylindrical.Chinese Journal of Cell Biology)2016;38(9):1118-27.
19刘峰,王运生,田雪亮,茆振川,邹学校,谢丙炎.辣椒转录组SSR挖掘及其多态性分析.园艺学报(Liu Feng,Wang Yunsheng,Tian Xueliang,Mao Zhenchuang,Zhou Xuexiao,Xie Bingyan.SSR mining in pepper(Capsicum annuum L.)transcriptome and the polymorphism analysis.Acta Horticulturae Sinica)2012;39(1):168-74.
20朱海生,黄丽芳,王彬,刘建汀,叶新如,陈敏氡.美洲南瓜转录组SSR信息分析及其分子标记开发.中国细胞生物学学报(Zhu Haisheng,Huang Linfang,Wang Bin,Liu Jianting,Ye Xinru,Chen Mindong,et al.Analysis on SSR information in transcriptome and development of molecular markers in summer squash.Chinese Journal of Cell Biology)2018;40(1):99-107.
21 Zhai L,Xu L,Wang Y,Cheng H,Chen Y,Gong Y,Liu L.Novel and useful genic-SSR markers from de novo transcriptome sequencing of radish(Raphanus sativus L.).Mol Breed 2014;33(3):611-24.
22方智振,叶新福,周丹蓉,姜翠翠,潘少霖.‘芙蓉李’转录组SSR信息分析与分子标记开发.果树学报(Fang Zhizhen,Ye Xinfu,Zhou Danrong,Jiang Cuicui,Pan Shaolin.Analysis on SSR information in‘Furongli’plum transcriptome and development of molecular markers in Prunus salicina Lindl.Journal of Fruit Science)2016;33(4):416-24.
23 Varshney RK,Graner A,Sorrells ME.Genic microsatellite markers in plants:Features and applications.Trends Biotechnol 2005;23(1):48-55.
24 Jiang D,Zhong GY,Hong QB.Analysis of microsatellites in citrus unigenes.Acta Genetica Sinica 2006;33(4):345-53.
25孙佳莹,辛大伟,单彩云,刘春燕,胡国华,陈庆山.SSR标记开发软件SSR MINING1.0的编制.中国农业科学(Sun Jiaying,Xin Dawei,Shan Caiyun,Liu Chunyan,Hu Guohua,Chen Qingshan.Programming for SSR marker developing software SSRMINING 1.0.Scientia Agricultura Sinica)2008;41(10):3336-42.
26杨曦.鲫鱼(Carassius auratus)表达序列标签资源的SSR构成与分布分析.生物技术通报(Yang Xi.Analysis of simple sequence repeats in expressed sequence tags from Carassius auratus.Biotechnology Bulletin)2012;11:139-43.
27 Xiang X,Zhang Z,Wang Z,Zhang X,Wu G.Transcriptome sequencing and development of EST-SSR markers in Pinus dabeshanensis,an endangered conifer endemic to China.Mol Breeding 2015;35:158.
28李满堂,张仕林,邓鹏,侯喜林,王建军.洋葱转录组SSR信息分析及其多态性研究.园艺学报(Li Mantang,Zhang Shilin,Deng Peng,Hou Xilin,Wang Jianjun.Analysis on SSR information in transcriptome of onion and the polymorphism.Acta Horticulturae Sinica)2015;42(6):1103-11.
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