内蒙古中部地区小叶锦鸡儿遗传多样性研究
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摘要
小叶锦鸡儿(Caragana microphylla Lam.)是豆科锦鸡儿属的重要植物,是生长在半干旱、干旱、沙漠化地区重要物种之一。在我国北方地区具有丰富的野生资源,极具有开发利用潜力。
本试验在全面调查我国野生小叶锦鸡儿分布的基础上,从内蒙古中部地区选取了7个小叶锦鸡儿居群,从每个居群筛选了30份样本作为试验材料,研究了野生小叶锦鸡儿种质资源的遗传基础及其差异,从居群形态学水平和DNA分子水平上揭示了其遗传多样性。从而为小叶锦鸡儿野生种质资源的有效保护、开发利用、核心种质的构建和优异新品种的筛选提供理论依据。主要研究结果如下:
1.表型多样性的研究结果:
小叶锦鸡儿11个表型性状无论在居群内还是居群间无不表现出显著(P＜0.01)或极显著(P＜0.001)差异,变异系数较大,变幅为3.34%～31.72%,平均变异系数为14.74%;11个表型性状的表型分化系数Vst为16.9%(按方差分量计算)(16.3%,按多样性指数计算),即居群内表型变异占了83.1%,表明居群内的表型变异是小叶锦鸡儿遗传变异的主要来源,居群内的多样性高于居群间的;从总体性状看来,内蒙古中部地区小叶锦鸡儿居群各分布区的表型多样性程度不同,依次为西乌旗居群(H′=1.8442)＞正镶白旗居群(H′=1.8285)＞四子王旗居群(H′=1.8019)＞察哈尔右翼后旗居群(H′=1.7911)＞镶黄旗居群(H′=1.7631)＞化德居群(H′=1.7250)＞多伦居群(H′=1.7236);对11个表型性状进行相关性分析,性状间都表现出了显著或极显著差异,与地理环境生态因子之间的偏相关分析表明,表型性状的差异与地理环境生态因子具有较高的相关性;主成分结果显示,千粒重、荚果长、荚果柄长、关节长这四个性状累积贡献率达到了94.32%,是造成小叶锦鸡儿表型变异的主要因素;经聚类分析,7个小叶锦鸡儿居群可以分为三大类,Mantel's test发现表型欧式距离与地理距离相关性不是很高。
2.ISSR分子标记研究结果:
利用SDS方法提取了较高质量的小叶锦鸡儿种子DNA,并建立了小叶锦鸡儿良好的ISSR反应体系:反应总体积为25ul、其中含75ngDNA、0.6umol/L引物、0.4mmol/LdNTPs、2.5mmol/L Mg~(2+)、1.5U Taq酶、10×PCRbuffer(10mmol/L Tris-HCl pH8.3,50mmol/L KCl,0.001%gelatin)和反应程序:预变性5min 94℃,然后45个cycles为94℃45s、53℃1min、72℃1.5min,最后72℃延伸10min,之后4℃保存。ISSR分析结果表明,18个引物对168份野生材料进行扩增,共获得269个位点,平均每个引物大约扩增出14.94个位点,扩增片段一般都在280bp～1500bp之间,多态性位点为177个,多态性达到66.27%。小叶锦鸡儿无论在种水平(H=0.2849,I=0.4208)还是居群水平(H=0.2280,I=0.3413)上都表现出较高的遗传多样性;基因分化系数(Gst)为19.98%,说明小叶锦鸡儿遗传多样性主要存在于居群内,占总遗传多样性的80.02%;居群间存在基因流Nm=2.0025;其分子多样性参数与地理环境生态因子相关性较低;聚类结果表明,7个小叶锦鸡儿居群可以分为三大类,经Mantel's test发现遗传距离与地理位置距离相关性不是很高。
3.对小叶锦鸡儿表型与分子水平上的数据进行耦合,发现二者能够很好的吻合,相关性很高,并且能够更好的阐明小叶锦鸡儿具有较高水平的遗传多样性。
Caragana microphylla Lam.is an important species in the semi-arid,drought and desert regions.It has a potential development in North China where there are rich wildlife resources.
The experiment is based on a thorough investigation on the ecological distribution of C.microphylla Lam.,choosing C.microphylla Lam.from seven regions in central Inner Mongolia.30 samples from each region are chosen as the experimental material in order to research systematicly on the genetic foundation and variation of their germplasm. Meanwhile,their genetic diversity is revealed by systematic studies on different levels of morphology and DNA molecules.It is very helpful to provide theory for effective protection and development of wild germplasm resources as well as construction of core germplasm.Furtherly it lays a foundation for selecting and exploiting excellent Caragana breed.The main research results are as following:
1.Phenotypic diversity of the findings:
Eleven phenotypic traits of C.microphylla Lam.among or within are discussed in analyzing characters such as apical leaves,pods and seed.Analysis of variance for all characters are marked(P<0.01) or extremely marked(P<0.001),with large coefficient of variation,from 3.34%～31.72%.The average is 14.74%.The eleven phenotypic differentiation(Vst =16.9%at variance components;16.3%,according to the diversity index) shows that the variation within the population(83.1%) is slightly higher than that among others populations.In general,the phenotypic variation of C.microphylla Lam.in the central Inner Mongolia is different,in turn as Xiwuqi >Zhengxiangbaiqi>Siziwangqi >Chaha'eryouyihouqi > Xianghuangqi > Huade > Duolun in the relative analysis of the eleven phenotypic traits,there are marked or extremely marked difference among most traits;in the partial correlative analysis of geographically ecological genes,differences of phenotypic traits are highly related to geographically ecological genes.The result from the main components shows that cumulative contributions' rate of 1000-seed weight,pod length,pod stipule length and articulation length reach 94.32 percent.It is the main source for the phenotypical variation according to the PCA;Based on the cluster analysis,the seven C.microphylla Lain.populations can be divided into three categories by phenotypic traits,the correlation between phenotypic Continental distance and geographical distance is not very close according to Mantel's test.
2.ISSR molecular marker research findings:
C.microphylla Lam.'s DNA of high quality is distilled by SDS method,which accords with ISSR analysis.Meanwhile a good ISSR reaction system of C.microphylla Lam.is established:PCR is 25ul,with75ngDNA,0.6umol / L primer,0.4 mmol / L dNTPs, 2.5mmol / L Mg~(2+),1.5U Taq enzyme,10×PCRbuffer(10mmol / L Tris-HCl pH8.3, 50mmol / L KCl,0.001%gelatin),and response procedures:pre-5 rain denaturation at 94℃,and then 45 cycles 94℃45s,and 53℃1min,72℃1.5min,the final extension of 10 min at 72℃,after 4℃.The results of ISSR analysis shows that the genetic diversity among 7 C.microphylla Lam.Accessions in population is tested by ISSR,168 wild materials are amplified by 18 ISSR primers,as a result 269 bands are gained.Each primer generated 14.94 polymorphic bands,From280bp to 1500bp,177(66.27%) bands are found to be polymorphic.Higher genetic diversity is both observed at species level(H = 0.2849,I = 0.4208) and population level(H = 0.2280,I = 0.3413).Its correlation with genetic diversity parameters and geographically ecological genes is not so close.Based on genetic differentiation(Gst) 19.98 percent,most of genetic variation of C.microphylla Lam.is within population(80.02%).The gene among populations Nm=2.0025.Base on the cluster analysis shows that seven C.microphylla Lam.populations can be divided into three categories,Mantel's test found that the correlation between genetic distance and geographical distance correlation is not very close.
3.The data couples on Phenotypic and molecular levels of C.microphylla Lam.,which is well coincided and suggests C.microphylla Lam.has a higher level of genetic diversity.
本试验在全面调查我国野生小叶锦鸡儿分布的基础上,从内蒙古中部地区选取了7个小叶锦鸡儿居群,从每个居群筛选了30份样本作为试验材料,研究了野生小叶锦鸡儿种质资源的遗传基础及其差异,从居群形态学水平和DNA分子水平上揭示了其遗传多样性。从而为小叶锦鸡儿野生种质资源的有效保护、开发利用、核心种质的构建和优异新品种的筛选提供理论依据。主要研究结果如下:
1.表型多样性的研究结果:
小叶锦鸡儿11个表型性状无论在居群内还是居群间无不表现出显著(P＜0.01)或极显著(P＜0.001)差异,变异系数较大,变幅为3.34%～31.72%,平均变异系数为14.74%;11个表型性状的表型分化系数Vst为16.9%(按方差分量计算)(16.3%,按多样性指数计算),即居群内表型变异占了83.1%,表明居群内的表型变异是小叶锦鸡儿遗传变异的主要来源,居群内的多样性高于居群间的;从总体性状看来,内蒙古中部地区小叶锦鸡儿居群各分布区的表型多样性程度不同,依次为西乌旗居群(H′=1.8442)＞正镶白旗居群(H′=1.8285)＞四子王旗居群(H′=1.8019)＞察哈尔右翼后旗居群(H′=1.7911)＞镶黄旗居群(H′=1.7631)＞化德居群(H′=1.7250)＞多伦居群(H′=1.7236);对11个表型性状进行相关性分析,性状间都表现出了显著或极显著差异,与地理环境生态因子之间的偏相关分析表明,表型性状的差异与地理环境生态因子具有较高的相关性;主成分结果显示,千粒重、荚果长、荚果柄长、关节长这四个性状累积贡献率达到了94.32%,是造成小叶锦鸡儿表型变异的主要因素;经聚类分析,7个小叶锦鸡儿居群可以分为三大类,Mantel's test发现表型欧式距离与地理距离相关性不是很高。
2.ISSR分子标记研究结果:
利用SDS方法提取了较高质量的小叶锦鸡儿种子DNA,并建立了小叶锦鸡儿良好的ISSR反应体系:反应总体积为25ul、其中含75ngDNA、0.6umol/L引物、0.4mmol/LdNTPs、2.5mmol/L Mg~(2+)、1.5U Taq酶、10×PCRbuffer(10mmol/L Tris-HCl pH8.3,50mmol/L KCl,0.001%gelatin)和反应程序:预变性5min 94℃,然后45个cycles为94℃45s、53℃1min、72℃1.5min,最后72℃延伸10min,之后4℃保存。ISSR分析结果表明,18个引物对168份野生材料进行扩增,共获得269个位点,平均每个引物大约扩增出14.94个位点,扩增片段一般都在280bp～1500bp之间,多态性位点为177个,多态性达到66.27%。小叶锦鸡儿无论在种水平(H=0.2849,I=0.4208)还是居群水平(H=0.2280,I=0.3413)上都表现出较高的遗传多样性;基因分化系数(Gst)为19.98%,说明小叶锦鸡儿遗传多样性主要存在于居群内,占总遗传多样性的80.02%;居群间存在基因流Nm=2.0025;其分子多样性参数与地理环境生态因子相关性较低;聚类结果表明,7个小叶锦鸡儿居群可以分为三大类,经Mantel's test发现遗传距离与地理位置距离相关性不是很高。
3.对小叶锦鸡儿表型与分子水平上的数据进行耦合,发现二者能够很好的吻合,相关性很高,并且能够更好的阐明小叶锦鸡儿具有较高水平的遗传多样性。
Caragana microphylla Lam.is an important species in the semi-arid,drought and desert regions.It has a potential development in North China where there are rich wildlife resources.
The experiment is based on a thorough investigation on the ecological distribution of C.microphylla Lam.,choosing C.microphylla Lam.from seven regions in central Inner Mongolia.30 samples from each region are chosen as the experimental material in order to research systematicly on the genetic foundation and variation of their germplasm. Meanwhile,their genetic diversity is revealed by systematic studies on different levels of morphology and DNA molecules.It is very helpful to provide theory for effective protection and development of wild germplasm resources as well as construction of core germplasm.Furtherly it lays a foundation for selecting and exploiting excellent Caragana breed.The main research results are as following:
1.Phenotypic diversity of the findings:
Eleven phenotypic traits of C.microphylla Lam.among or within are discussed in analyzing characters such as apical leaves,pods and seed.Analysis of variance for all characters are marked(P<0.01) or extremely marked(P<0.001),with large coefficient of variation,from 3.34%～31.72%.The average is 14.74%.The eleven phenotypic differentiation(Vst =16.9%at variance components;16.3%,according to the diversity index) shows that the variation within the population(83.1%) is slightly higher than that among others populations.In general,the phenotypic variation of C.microphylla Lam.in the central Inner Mongolia is different,in turn as Xiwuqi >Zhengxiangbaiqi>Siziwangqi >Chaha'eryouyihouqi > Xianghuangqi > Huade > Duolun in the relative analysis of the eleven phenotypic traits,there are marked or extremely marked difference among most traits;in the partial correlative analysis of geographically ecological genes,differences of phenotypic traits are highly related to geographically ecological genes.The result from the main components shows that cumulative contributions' rate of 1000-seed weight,pod length,pod stipule length and articulation length reach 94.32 percent.It is the main source for the phenotypical variation according to the PCA;Based on the cluster analysis,the seven C.microphylla Lain.populations can be divided into three categories by phenotypic traits,the correlation between phenotypic Continental distance and geographical distance is not very close according to Mantel's test.
2.ISSR molecular marker research findings:
C.microphylla Lam.'s DNA of high quality is distilled by SDS method,which accords with ISSR analysis.Meanwhile a good ISSR reaction system of C.microphylla Lam.is established:PCR is 25ul,with75ngDNA,0.6umol / L primer,0.4 mmol / L dNTPs, 2.5mmol / L Mg~(2+),1.5U Taq enzyme,10×PCRbuffer(10mmol / L Tris-HCl pH8.3, 50mmol / L KCl,0.001%gelatin),and response procedures:pre-5 rain denaturation at 94℃,and then 45 cycles 94℃45s,and 53℃1min,72℃1.5min,the final extension of 10 min at 72℃,after 4℃.The results of ISSR analysis shows that the genetic diversity among 7 C.microphylla Lam.Accessions in population is tested by ISSR,168 wild materials are amplified by 18 ISSR primers,as a result 269 bands are gained.Each primer generated 14.94 polymorphic bands,From280bp to 1500bp,177(66.27%) bands are found to be polymorphic.Higher genetic diversity is both observed at species level(H = 0.2849,I = 0.4208) and population level(H = 0.2280,I = 0.3413).Its correlation with genetic diversity parameters and geographically ecological genes is not so close.Based on genetic differentiation(Gst) 19.98 percent,most of genetic variation of C.microphylla Lam.is within population(80.02%).The gene among populations Nm=2.0025.Base on the cluster analysis shows that seven C.microphylla Lam.populations can be divided into three categories,Mantel's test found that the correlation between genetic distance and geographical distance correlation is not very close.
3.The data couples on Phenotypic and molecular levels of C.microphylla Lam.,which is well coincided and suggests C.microphylla Lam.has a higher level of genetic diversity.
引文
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