基于SSR标记的杨树新品种鉴定及核心引物筛选
|
摘要
【目的】利用经过长期筛选的11对SSR引物对50份杨树新品种(系)进行扩增,探究11对引物的品种鉴定能力和核心引物的筛选依据,为杨树新品种的鉴定、育种工作和核心引物的筛选工作奠定基础。【方法】使用毛细管电泳技术对扩增结果进行检测,计算等位重复序列数、Shannon信息指数和引物多态性信息指数等。按0/1矩阵记录扩增条带的有无,并通过非加权组平均法(UPGMA)进行聚类分析。计算单个引物和11对引物组合的遗传相似系数,分析遗传相似系数之间的相关性,剔除相关性较低的引物,再对剩余的引物组合进行聚类分析。【结果】11对引物共扩增出122个DNA片段,平均每对引物扩增的等位重复序列数为11.091个;不同引物PIC值的变化范围是0.530～0.908,平均值为0.803。11对引物的聚类结果显示,当支距为0.40时,参试样品可以分为2个大类;当支距为0.37时,可分为4个亚类,聚类结果与品种(系)的谱系来源基本吻合。在11对现有引物的基础上,通过分析单个引物和11对引物的遗传相似系数之间的相关性,优化得到9对核心引物。相关性分析和聚类分析表明,优化后的9对引物具有高效鉴定能力和亲缘关系聚类效果。【结论】本研究证实SSR分子标记可以有效鉴定杨树新品种,并较好反映品种之间的亲缘关系;同时,利用遗传相似系数的相关性可优化现有的引物,为杨树的育种及核心引物的筛选工作提供了参考。
[Objective] Eleven pairs of SSR primers being screened for a long time were used to amplify 50 new Populus varieties(clone) in order to explore the cultivar identification ability of SSR markers and the screening basis of core primers, which lays a foundation for the identification, breeding and screening of core primers for new Populus varieties. [Method] Capillary electrophoresis was used to detect the amplified fragments. The number of allelic repeat sequences, Shannon information index, and primer polymorphism information index were calculated based on the electrophoresis. The 0/1 matrix which recorded the absence and the presence of the amplification bands was used for cluster analysis by the unweighted pair-group method with arithmetic means(UPGMA). The genetic similarity coefficients of a single primer and 11 pairs of primer combinations were calculated, and the correlation between the genetic similarity coefficients was analyzed. The primers with low correlation were removed, and then the remaining primers were clustered.[Result] The results showed that a total of 122 DNA fragments were amplified by 11 pairs of primers, and the average number of allelic repeat sequences amplified by each pair of primers was 11.091. The PIC of different primers varied from 0.530 to 0.908, with an average value of 0.803. The clustering results of 11 pairs of primers showed that when the branch distance was 0.40, the samples could be divided into 2 categories; when the branch distance was 0.37, it could be divided into 4 subcategories and the clustering results were basically consistent with the pedigree sources of the new varieties(clone). On the basis of the existing primers, 9 pairs of core primers were obtained by analyzing the correlation of the genetic similarity coefficients of individual primers and 11 pairs of primers, which has high-efficiency identification ability and clustering effect of genetic relationship. [Conclusion] This study confirmed that SSR molecular markers could effectively identify new poplar varieties and better reflect the relationship between varieties.Meanwhile, the existing primers could be optimized by the correlation of genetic similarity coefficient,which will provide a reference for poplar breeding and the selection of core primers.
[Objective] Eleven pairs of SSR primers being screened for a long time were used to amplify 50 new Populus varieties(clone) in order to explore the cultivar identification ability of SSR markers and the screening basis of core primers, which lays a foundation for the identification, breeding and screening of core primers for new Populus varieties. [Method] Capillary electrophoresis was used to detect the amplified fragments. The number of allelic repeat sequences, Shannon information index, and primer polymorphism information index were calculated based on the electrophoresis. The 0/1 matrix which recorded the absence and the presence of the amplification bands was used for cluster analysis by the unweighted pair-group method with arithmetic means(UPGMA). The genetic similarity coefficients of a single primer and 11 pairs of primer combinations were calculated, and the correlation between the genetic similarity coefficients was analyzed. The primers with low correlation were removed, and then the remaining primers were clustered.[Result] The results showed that a total of 122 DNA fragments were amplified by 11 pairs of primers, and the average number of allelic repeat sequences amplified by each pair of primers was 11.091. The PIC of different primers varied from 0.530 to 0.908, with an average value of 0.803. The clustering results of 11 pairs of primers showed that when the branch distance was 0.40, the samples could be divided into 2 categories; when the branch distance was 0.37, it could be divided into 4 subcategories and the clustering results were basically consistent with the pedigree sources of the new varieties(clone). On the basis of the existing primers, 9 pairs of core primers were obtained by analyzing the correlation of the genetic similarity coefficients of individual primers and 11 pairs of primers, which has high-efficiency identification ability and clustering effect of genetic relationship. [Conclusion] This study confirmed that SSR molecular markers could effectively identify new poplar varieties and better reflect the relationship between varieties.Meanwhile, the existing primers could be optimized by the correlation of genetic similarity coefficient,which will provide a reference for poplar breeding and the selection of core primers.
引文
[1]张忠涛,孙乐智.我国的杨树资源与开发利用[J].林业建设,2001(5):21-24.Zhang Z T,Sun L Z.Poplar resource and utilization in China[J].Forestry Construction,2001(5):21-24.
[2]何承忠,张志毅,安新民,等.我国杨树育种现状及其展望[J].西南林学院学报,2006,26(4):86-89.He C Z,Zhang Z Y,An X M,et al.Present situation and prospect to poplar breeding in China[J].Journal of Southwest Forestry College,2006,26(4):86-89.
[3]栾鹖慧,苏晓华,张冰玉.杨属(Populus L.)种质资源遗传学评价研究进展[J].植物学报,2011,46(5):586-595.Luan H H,Su X H,Zhang B Y.Research progress in genetic evaluation of Populus L.germplasm resources[J].Chinese Bulletin of Botany,2011,46(5):586-595.
[4]潘惠新.杨树新品种选育研究[J].林业科技开发,2002,16(3):3-4.Pan H X.Research on selective breeding of new cultivars for poplar[J].Forest Research,2002,16(3):3-4.
[5]张鹤,张文庆,赵敬东.DNA分子标记在植物新品种保护中的应用现状及发展前景[J].安徽农业科学,2009,37(34):16897-16899.Zhang H,Zhang W Q,Zhao J D.Application status and development of DNA molecular marker in protection of new plant variety[J].Journal of Anhui Agricultural Sciences,2009,37(34):16897-16899.
[6]张德强,张志毅,杨凯.杨树分子标记研究进展[J].北京林业大学学报,2000,22(6):79-84.Zhang D Q,Zhang Z Y,Yang K.Advances of molecular marker researches in poplar[J].Journal of Beijing Forestry University,2000,22(6):79-84.
[7]王和勇,乔爱民,陈敏,等.植物遗传标记的发展及应用[J].仲恺农业技术学院学报,2000,13(4):58-64.Wang H Y,Qiao A M,Chen M,et al.The development and application of plant genetic markers[J].Journal of Zhongkai Agrotechnical College,2000,13(4):58-64.
[8]李柱刚,崔崇士,马荣才,等.遗传标记在植物上的发展与应用[J].东北农业大学学报,2001,32(4):396-401.Li Z G,Cui C S,Ma R C,et al.Development and application of genetic markers in plants[J].Journal of Northeast Agricultural University,2001,32(4):396-401.
[9]Castiglione S,Wang G,Damiani G,et al.RAPD fingerprints for identification and for taxonomic studies of elite poplar(Populus spp.)clones[J].Theoretical&Applied Genetics,1993,87(1-2):54-59.
[10]尹佟明,孙晔,易能君,等.美洲黑杨无性系AFLP指纹分析[J].植物学报,1998,40(8):778-780.Yin T M,Sun Y,Yi N J,et al.Genome fingerprinting analysis in Populus deltoids[J].Acta Botanica Sinica,1998,40(8):778-780.
[11]崔国惠,倪中福,刘志勇,等.小麦杂种优势群研究Ⅲ普通小麦和斯卑尔脱小麦微卫星分子标记遗传差异的研究[J].农业生物技术学报,1999,7(4):333-338.Cui G H,Ni Z F,Liu Z Y,et al.Study on wheat heterotic groupⅢgenetic diversity revealed by microsatellite marker in wheat(Triticum aestivum L.)and spelt(Triticum spelta L.)[J].Journal of Agricultural Biotechnology,1999,7(4):333-338.
[12]卫尊征,张金凤,张德强,等.白、青杨派间杂交幼胚培养及杂种子代的分子鉴定[J].北京林业大学学报,2008,30(5):73-77.Wei Z Z,Zhang J F,Zhang D Q,et al.Immature ovule culture of inter-sectional hybrids between(Populus tomentosa×P.bolleana)and P.simonii and molecular identification of the hybrid filial generation[J].Journal of Beijing Forestry University,2008,30(5):73-77.
[13]姚俊修,毛秀红,李善文,等.基于荧光SSR标记的白杨派种质资源遗传多样性研究[J].北京林业大学学报,2018,40(6):92-100.Yao J X,Mao X H,Li S W,et al.Genetic diversity of germplasm resources of Leuce based on SSR fluorescent marker[J].Journal of Beijing Forestry University,2018,40(6):92-100.
[14]韩志校,张军,左力辉,等.基于SSR和ITS标记的杨树多态性及聚类分析[J].中国农业科技导报,2017,19(7):36-43.Han Z X,Zhang J,Zuo L H,et al.Populus polymorphism and cluster analysis based on SSR and ITS markers[J].Journal of Agricultural Science and Technology,2017,19(7):36-43.
[15]梁海永,刘彩霞,杨敏生,等.杨树品种的SSR分析及鉴定[J].河北农业大学学报,2005,28(4):27-31.Liang H Y,Liu C X,Yang M S,et al.Simple sequence repeat(SSR)analysis and identify of different cultivars in Populus L.[J].Journal of Agricultural University of Hebei,2005,28(4):27-31.
[16]王辉,杨敏生,朱建峰.利用SSR对杨属部分种及杂种的分析鉴定[J].东北林业大学学报,2008,36(12):4-6.Wang H,Yang M S,Zhu J F.SSR analysis of some species and hybrids in Populus[J].Journal of Northeast Agricultural University,2008,36(12):4-6.
[17]赵双菁,刘莹莹,魏敏静,等.标记引物数量对白桦遗传多样性的影响[J].防护林科技,2016(4):6-10,26.Zhao S J,Liu Y Y,Wei M J,et al.Effects of number of labeling primers on genetic diversity of Betula platyphylla[J].Protection Forest Science and Technology,2016(4):6-10,26.
[18]苏龙,徐志健,乔卫华,等.广西药用野生稻遗传多样性分析及SSR引物数量对遗传多样性结果的影响研究[J].植物遗传资源学报,2017,18(4):603-610.Su L,Xu Z J,Qiao W H,et al.Study on genetic diversity of Oryza officinalis in Guangxi Province and the effect of the number of SSR primers on genetic diversity analysis[J].Journal of Plant Genetic Resources,2017,18(4):603-610.
[19]Allen G C,Flores-Vergara M A,Krasynanski S,et al.A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide[J].Nature Protocols,2006,1(5):2320-2325.
[20]Peakall R,Smouse P E.GenAlEx 6.5:genetic analysis in Excel.population genetic software for teaching and research:an update[J].Bioinformatics,2012,28(28):2537-2539.
[21]Kalinowski S T,Taper M L,Marshall T C.Revising how the computer program cervus accommodates genotyping error increases success in paternity assignment[J].Molecular Ecology,2010,16(5):1099-1106.
[22]李亚玲,韩国民,何沙娥,等.基于DNA分子标记数据构建系统进化树的新策略[J].生物信息学,2008,6(4):168-170.Li Y L,Han G M,He S E,et al.A new strategy for construction of phylogenetic tree based on DNA molecular mark data[J].China Journal of Bioinformatics,2008,6(4):168-170.
[23]Botstein D.A theory of modular evolution for bacteriophages[J].Annals of the New York Academy of Sciences,2010,354(1):484-491.
[24]刘峰,冯雪梅,钟文,等.适合棉花品种鉴定的SSR核心引物的筛选[J].分子植物育种,2009,7(6):1160-1168.Liu F,Feng X M,Zhong W,et al.Screening of SSR core primer pairs for identificating cotton cultivar[J].Molecular Plant Breeding,2009,7(6):1160-1168.
[25]贾会霞,姬慧娟,胡建军,等.杨树新品种的SSR指纹图谱构建和倍性检测[J].林业科学,2015,51(2):69-79.Jia H X,Ji H J,Hu J J,et al.Fingerprints of SSR markers and ploidy detection for new Populus varieties[J].Scientia Silvae Sinicae,2015,51(2):69-79.
[26]高伟,王坤波,刘方,等.SSR引物及多态性位点数对陆地棉野生种系聚类结果的影响[J].植物遗传资源学报,2013,14(2):237-242.Gao W,Wang K B,Liu F,et al.Effection of the quantity of SSRprimer and allele on cluster analysis of Gossypium hirsutum Linn races[J].Journal of Plant Genetic Resources,2013,14(2):237-242.
[27]王彪,常汝镇,陶莉,等.分析中国栽培大豆遗传多样性所需SSR引物的数目[J].分子植物育种,2003,1(1):82-88.Wang B,Chang R Z,Tao L,et al.Identification of SSR primer numbers for analyzing genetic diversity of chinese soybean cultivated soybean[J].Molecular Plant Breeding,2003,1(1):82-88.
[28]李瑾.基于两种毛细管电泳系统检测呼吸道病毒的多重PCR技术的建立及应用[D].北京:中国疾病预防控制中心,2012.Li J.Development of two novel multiplex RT-PCR assays for simultaneous detection of 16 human respiratory virus types/subtypes based on capillary electrophoresis[D].Beijing:Chinese Center for Disease Control and Prevention,2012.
[29]肖璐.7种猪病毒性疫病QIAxcel及Bio-Plex检测方法的建立与初步应用[D].成都:四川农业大学,2017.Xiao L.Simultaneous typing of seven porcine virus pathogens by QIAxcel and Bio-Plex and its preliminary application[D].Chengdu:Sichuan Agricultural University,2017.
[2]何承忠,张志毅,安新民,等.我国杨树育种现状及其展望[J].西南林学院学报,2006,26(4):86-89.He C Z,Zhang Z Y,An X M,et al.Present situation and prospect to poplar breeding in China[J].Journal of Southwest Forestry College,2006,26(4):86-89.
[3]栾鹖慧,苏晓华,张冰玉.杨属(Populus L.)种质资源遗传学评价研究进展[J].植物学报,2011,46(5):586-595.Luan H H,Su X H,Zhang B Y.Research progress in genetic evaluation of Populus L.germplasm resources[J].Chinese Bulletin of Botany,2011,46(5):586-595.
[4]潘惠新.杨树新品种选育研究[J].林业科技开发,2002,16(3):3-4.Pan H X.Research on selective breeding of new cultivars for poplar[J].Forest Research,2002,16(3):3-4.
[5]张鹤,张文庆,赵敬东.DNA分子标记在植物新品种保护中的应用现状及发展前景[J].安徽农业科学,2009,37(34):16897-16899.Zhang H,Zhang W Q,Zhao J D.Application status and development of DNA molecular marker in protection of new plant variety[J].Journal of Anhui Agricultural Sciences,2009,37(34):16897-16899.
[6]张德强,张志毅,杨凯.杨树分子标记研究进展[J].北京林业大学学报,2000,22(6):79-84.Zhang D Q,Zhang Z Y,Yang K.Advances of molecular marker researches in poplar[J].Journal of Beijing Forestry University,2000,22(6):79-84.
[7]王和勇,乔爱民,陈敏,等.植物遗传标记的发展及应用[J].仲恺农业技术学院学报,2000,13(4):58-64.Wang H Y,Qiao A M,Chen M,et al.The development and application of plant genetic markers[J].Journal of Zhongkai Agrotechnical College,2000,13(4):58-64.
[8]李柱刚,崔崇士,马荣才,等.遗传标记在植物上的发展与应用[J].东北农业大学学报,2001,32(4):396-401.Li Z G,Cui C S,Ma R C,et al.Development and application of genetic markers in plants[J].Journal of Northeast Agricultural University,2001,32(4):396-401.
[9]Castiglione S,Wang G,Damiani G,et al.RAPD fingerprints for identification and for taxonomic studies of elite poplar(Populus spp.)clones[J].Theoretical&Applied Genetics,1993,87(1-2):54-59.
[10]尹佟明,孙晔,易能君,等.美洲黑杨无性系AFLP指纹分析[J].植物学报,1998,40(8):778-780.Yin T M,Sun Y,Yi N J,et al.Genome fingerprinting analysis in Populus deltoids[J].Acta Botanica Sinica,1998,40(8):778-780.
[11]崔国惠,倪中福,刘志勇,等.小麦杂种优势群研究Ⅲ普通小麦和斯卑尔脱小麦微卫星分子标记遗传差异的研究[J].农业生物技术学报,1999,7(4):333-338.Cui G H,Ni Z F,Liu Z Y,et al.Study on wheat heterotic groupⅢgenetic diversity revealed by microsatellite marker in wheat(Triticum aestivum L.)and spelt(Triticum spelta L.)[J].Journal of Agricultural Biotechnology,1999,7(4):333-338.
[12]卫尊征,张金凤,张德强,等.白、青杨派间杂交幼胚培养及杂种子代的分子鉴定[J].北京林业大学学报,2008,30(5):73-77.Wei Z Z,Zhang J F,Zhang D Q,et al.Immature ovule culture of inter-sectional hybrids between(Populus tomentosa×P.bolleana)and P.simonii and molecular identification of the hybrid filial generation[J].Journal of Beijing Forestry University,2008,30(5):73-77.
[13]姚俊修,毛秀红,李善文,等.基于荧光SSR标记的白杨派种质资源遗传多样性研究[J].北京林业大学学报,2018,40(6):92-100.Yao J X,Mao X H,Li S W,et al.Genetic diversity of germplasm resources of Leuce based on SSR fluorescent marker[J].Journal of Beijing Forestry University,2018,40(6):92-100.
[14]韩志校,张军,左力辉,等.基于SSR和ITS标记的杨树多态性及聚类分析[J].中国农业科技导报,2017,19(7):36-43.Han Z X,Zhang J,Zuo L H,et al.Populus polymorphism and cluster analysis based on SSR and ITS markers[J].Journal of Agricultural Science and Technology,2017,19(7):36-43.
[15]梁海永,刘彩霞,杨敏生,等.杨树品种的SSR分析及鉴定[J].河北农业大学学报,2005,28(4):27-31.Liang H Y,Liu C X,Yang M S,et al.Simple sequence repeat(SSR)analysis and identify of different cultivars in Populus L.[J].Journal of Agricultural University of Hebei,2005,28(4):27-31.
[16]王辉,杨敏生,朱建峰.利用SSR对杨属部分种及杂种的分析鉴定[J].东北林业大学学报,2008,36(12):4-6.Wang H,Yang M S,Zhu J F.SSR analysis of some species and hybrids in Populus[J].Journal of Northeast Agricultural University,2008,36(12):4-6.
[17]赵双菁,刘莹莹,魏敏静,等.标记引物数量对白桦遗传多样性的影响[J].防护林科技,2016(4):6-10,26.Zhao S J,Liu Y Y,Wei M J,et al.Effects of number of labeling primers on genetic diversity of Betula platyphylla[J].Protection Forest Science and Technology,2016(4):6-10,26.
[18]苏龙,徐志健,乔卫华,等.广西药用野生稻遗传多样性分析及SSR引物数量对遗传多样性结果的影响研究[J].植物遗传资源学报,2017,18(4):603-610.Su L,Xu Z J,Qiao W H,et al.Study on genetic diversity of Oryza officinalis in Guangxi Province and the effect of the number of SSR primers on genetic diversity analysis[J].Journal of Plant Genetic Resources,2017,18(4):603-610.
[19]Allen G C,Flores-Vergara M A,Krasynanski S,et al.A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide[J].Nature Protocols,2006,1(5):2320-2325.
[20]Peakall R,Smouse P E.GenAlEx 6.5:genetic analysis in Excel.population genetic software for teaching and research:an update[J].Bioinformatics,2012,28(28):2537-2539.
[21]Kalinowski S T,Taper M L,Marshall T C.Revising how the computer program cervus accommodates genotyping error increases success in paternity assignment[J].Molecular Ecology,2010,16(5):1099-1106.
[22]李亚玲,韩国民,何沙娥,等.基于DNA分子标记数据构建系统进化树的新策略[J].生物信息学,2008,6(4):168-170.Li Y L,Han G M,He S E,et al.A new strategy for construction of phylogenetic tree based on DNA molecular mark data[J].China Journal of Bioinformatics,2008,6(4):168-170.
[23]Botstein D.A theory of modular evolution for bacteriophages[J].Annals of the New York Academy of Sciences,2010,354(1):484-491.
[24]刘峰,冯雪梅,钟文,等.适合棉花品种鉴定的SSR核心引物的筛选[J].分子植物育种,2009,7(6):1160-1168.Liu F,Feng X M,Zhong W,et al.Screening of SSR core primer pairs for identificating cotton cultivar[J].Molecular Plant Breeding,2009,7(6):1160-1168.
[25]贾会霞,姬慧娟,胡建军,等.杨树新品种的SSR指纹图谱构建和倍性检测[J].林业科学,2015,51(2):69-79.Jia H X,Ji H J,Hu J J,et al.Fingerprints of SSR markers and ploidy detection for new Populus varieties[J].Scientia Silvae Sinicae,2015,51(2):69-79.
[26]高伟,王坤波,刘方,等.SSR引物及多态性位点数对陆地棉野生种系聚类结果的影响[J].植物遗传资源学报,2013,14(2):237-242.Gao W,Wang K B,Liu F,et al.Effection of the quantity of SSRprimer and allele on cluster analysis of Gossypium hirsutum Linn races[J].Journal of Plant Genetic Resources,2013,14(2):237-242.
[27]王彪,常汝镇,陶莉,等.分析中国栽培大豆遗传多样性所需SSR引物的数目[J].分子植物育种,2003,1(1):82-88.Wang B,Chang R Z,Tao L,et al.Identification of SSR primer numbers for analyzing genetic diversity of chinese soybean cultivated soybean[J].Molecular Plant Breeding,2003,1(1):82-88.
[28]李瑾.基于两种毛细管电泳系统检测呼吸道病毒的多重PCR技术的建立及应用[D].北京:中国疾病预防控制中心,2012.Li J.Development of two novel multiplex RT-PCR assays for simultaneous detection of 16 human respiratory virus types/subtypes based on capillary electrophoresis[D].Beijing:Chinese Center for Disease Control and Prevention,2012.
[29]肖璐.7种猪病毒性疫病QIAxcel及Bio-Plex检测方法的建立与初步应用[D].成都:四川农业大学,2017.Xiao L.Simultaneous typing of seven porcine virus pathogens by QIAxcel and Bio-Plex and its preliminary application[D].Chengdu:Sichuan Agricultural University,2017.