白花树天然群体遗传多样性与油脂特性研究
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摘要
国槐(Sophora japonica L.),小到中等乔木,是我国优良的材用、药用、食用和观赏树种。国槐自然分布于中国、朝鲜和日本,广泛引种栽培于世界其他各地。
本研究在国槐浙江杭州、湖北孝感、陕西西安、陕西咸阳、北京、河南濮阳和山东泰安等7个群体中,各随机抽取130个单株作为采种样株,对其种子的长、宽、长宽比、表面积、体积和千粒重等6个性状作了观测分析,采用巢氏设计进行了方差分析,相关性和聚类等统计分析,分析不同性状的表型变异及规律,试图为国槐遗传多样性研究提供量化指标,为国槐遗传资源评价、保存和利用提供依据。以北京市丰台苗圃基地收集保存的国槐品种和无性系为试验材料,旨在探讨国槐DNA的提取和SRAP-PCR的最佳反应体系,为进一步开展国槐遗传资源的SRAP分析,在分子水平上鉴别品种与无性系及其亲缘关系提供科学依据。
主要研究结果如下：
1国槐种子的形态性状存在不同程度的变异,变异系数范围为5.24%-14.57%；6个表型性状在群体间和群体内都达到了极显著水平；6个性状的平均表型分化系数为53.11%,表明群体间遗传变异略高于群体内；种子形态性状与地理纬度和经度的相关程度较低。利用分析结果显示7个国槐群体可以分为3类：1)泰安群体；2)湖北孝感和北京群体；3)浙江杭州,西安,咸阳和河南濮阳聚为一类。研究发现,国槐种子没有明显的地理变异规律,可能是长期地人为栽培造成的,也可能是由于采样的不全面或不连续性造成的。
2采用正交试验确定了国槐的最佳SRAP-PCR反应体系(20 uL):Taq聚合酶1.5 U,dNTPs 0.30 mmol/L,模板DNA 90 ng, Mg2+2.0 mmol/L,引物0.3 umol/L,ddH2O补至20 uL。
3从120对引物组合中,筛选出12对PCR产物条带清晰、多态性高的引物组合,进行国槐品种与无性系的亲缘关系的分子识别研究。12对引物PCR扩增的条带大多集中在100bp-2000bp之间。在所获得的430条扩增条带,415条具有多态性位点,多态性位点比率为96.60%,其中4对引物的多态性达到了100%。试验结果还表明,SRAP能检测国槐品种及无性系的多个遗传位点。采用Em9-Me12引物组合可以很好的区分国槐的不同品种及无性系,并建立了国槐的SRAP指纹图谱。
4国槐品种和无性系的遗传相似系数(SM)为0.68-0.89,平均相似系数为0.73,说明国槐品种和无性系间的相似性比较高,遗传基础比较窄。基于遗传相似系数SM,按非加权配对算术平均法对国槐品种和无性系进行聚类分析,在相似系数0.76处,将其分为4类,第二类为国槐4号和国槐35号；第三类为蝴蝶槐(S.japonica var.oligophylla Franch);第四类为龙爪槐(S.japonica var. pendula Loud.),曹州2号(S.japonica 'Caozhou 2')和金叶国槐(S.japonica f.flavi-rameus);其余为第一类,第一类又可以分为两个亚组；蝴蝶槐(S.japonica var.oligophylla Franch)单独聚为第三类,表明不同叶型间的差距比较大。
Sophora japonica L. is a tree species with timber, medicinal, edible and ornamental values. This species is naturally distributed in China, Korea and Japan, and widely planted in many parts of the world.
Seed morphology was examined in this study.130 families in 7 populations of S. japonica were sampled. Seed length, seed width, the ratio of seed width to length, seed surface area, seed volume and 1000-seed's weight were measured in order to find out genetic variations and variation patterns. It was expected that research results would provide scientific bases for the assessment at genetic resources and strategy for conservation and utilizition of S. japonica. Phenotypic variation among and within populations were analyzed by ANOVA and correlation analysis. Genetic materials of varieties and clones of S. japonica were used for DNA extraction and the best SRAP-PCR reaction system selection for further development of the SRAP analysis. It was intended in this study that a theoretical basis to show genetic relationships at molecular level.
The main findings and conclusions are as follows:
1 Genetic variations were, to a certain extent, found in phenotypic properties of seed, coefficient of variation was ranged from 5.24% to 14.57%. There were significant differences in seed morphological characteristics among and within populations. The average differentiation coefficient among populations was 53.11%, which was higher than that within population. No significant correlation was found in geographic variations of seed with latitude and longitude. It was indicated in cluster analysis that 7 populations of S. japonica were divided into 3 groups:1) Taian groups; 2) Hubei and Beijing groups; 3) Zhejiang, Xi'an, Xianyang and Henan populations. It was found that there was no any significant geographical variations in seed morpholgy, which suggested that introduction and cultivation in wide range of area or discontinuous sampling would be responsible for the phenomenon.
2 Orthogonal experimental design was applied to establish the optimal reaction system for SRAP-PCR (20 uL):Taq polymerase 1.5 U, dNTPs 0.30 mmol/L, the template DNA 90 ng, Mg2+2.0 mmol/L, primer 0.3 umol/L, ddH2O fill to 20 uL.
3 There were 12 primer combinations that PCR product bands were clear, highly polymorphic selected from 120 primer combinations, which were applied in genetic relationship and molecular identification study of varieties and clones of S. japonica. The rang of PCR amplification of 12 primer combinations are mostly concentrated in bands between 100bp-2000bp. Of the total of 430 bands amplified,415 were polymorphic loci. The percentage of polymorphic fragments were 96.60%, of which 4 pairs of primers polymorphism were 100%. It was showed in the results that multiple genetic loci of varieties and clones could be detected with SRAP markers. Primers combination Em9-Me12 could be used to distinguish different varieties of S. japonica. Rich and clear SRAP fingerprints were also obtained.
4 Levels of genetic similarity in coefficients (SM) were calculated and a dendrogram was constructed by the unweighted pair group method of arithmetic average (UPGMA). SM value was in the range of 0.68-0.89, with an average similarity coefficient of 0.73, indicating the similarity between varieties and clones of S. japonica were relatively high, which means the genetic base was relatively narrow. Based on the similarity coefficients of 0.76, varieties and clones were clustered into four categories. The second category was consisted of 4 and 35 of S. japonica; the fourth category inclued S. japonica var. pendula Loud. (LZ), S. japonica 'Caozhou 2'(C2) and S. japonica f. flavi-rameus (JY). The rests were clustered into the first category, which could be divided into two subgroups. S. japonica var.oligophylla Franch. itself was clustered in the third category, which indicated that there were large differences in different shapes of leaves.
本研究在国槐浙江杭州、湖北孝感、陕西西安、陕西咸阳、北京、河南濮阳和山东泰安等7个群体中,各随机抽取130个单株作为采种样株,对其种子的长、宽、长宽比、表面积、体积和千粒重等6个性状作了观测分析,采用巢氏设计进行了方差分析,相关性和聚类等统计分析,分析不同性状的表型变异及规律,试图为国槐遗传多样性研究提供量化指标,为国槐遗传资源评价、保存和利用提供依据。以北京市丰台苗圃基地收集保存的国槐品种和无性系为试验材料,旨在探讨国槐DNA的提取和SRAP-PCR的最佳反应体系,为进一步开展国槐遗传资源的SRAP分析,在分子水平上鉴别品种与无性系及其亲缘关系提供科学依据。
主要研究结果如下：
1国槐种子的形态性状存在不同程度的变异,变异系数范围为5.24%-14.57%；6个表型性状在群体间和群体内都达到了极显著水平；6个性状的平均表型分化系数为53.11%,表明群体间遗传变异略高于群体内；种子形态性状与地理纬度和经度的相关程度较低。利用分析结果显示7个国槐群体可以分为3类：1)泰安群体；2)湖北孝感和北京群体；3)浙江杭州,西安,咸阳和河南濮阳聚为一类。研究发现,国槐种子没有明显的地理变异规律,可能是长期地人为栽培造成的,也可能是由于采样的不全面或不连续性造成的。
2采用正交试验确定了国槐的最佳SRAP-PCR反应体系(20 uL):Taq聚合酶1.5 U,dNTPs 0.30 mmol/L,模板DNA 90 ng, Mg2+2.0 mmol/L,引物0.3 umol/L,ddH2O补至20 uL。
3从120对引物组合中,筛选出12对PCR产物条带清晰、多态性高的引物组合,进行国槐品种与无性系的亲缘关系的分子识别研究。12对引物PCR扩增的条带大多集中在100bp-2000bp之间。在所获得的430条扩增条带,415条具有多态性位点,多态性位点比率为96.60%,其中4对引物的多态性达到了100%。试验结果还表明,SRAP能检测国槐品种及无性系的多个遗传位点。采用Em9-Me12引物组合可以很好的区分国槐的不同品种及无性系,并建立了国槐的SRAP指纹图谱。
4国槐品种和无性系的遗传相似系数(SM)为0.68-0.89,平均相似系数为0.73,说明国槐品种和无性系间的相似性比较高,遗传基础比较窄。基于遗传相似系数SM,按非加权配对算术平均法对国槐品种和无性系进行聚类分析,在相似系数0.76处,将其分为4类,第二类为国槐4号和国槐35号；第三类为蝴蝶槐(S.japonica var.oligophylla Franch);第四类为龙爪槐(S.japonica var. pendula Loud.),曹州2号(S.japonica 'Caozhou 2')和金叶国槐(S.japonica f.flavi-rameus);其余为第一类,第一类又可以分为两个亚组；蝴蝶槐(S.japonica var.oligophylla Franch)单独聚为第三类,表明不同叶型间的差距比较大。
Sophora japonica L. is a tree species with timber, medicinal, edible and ornamental values. This species is naturally distributed in China, Korea and Japan, and widely planted in many parts of the world.
Seed morphology was examined in this study.130 families in 7 populations of S. japonica were sampled. Seed length, seed width, the ratio of seed width to length, seed surface area, seed volume and 1000-seed's weight were measured in order to find out genetic variations and variation patterns. It was expected that research results would provide scientific bases for the assessment at genetic resources and strategy for conservation and utilizition of S. japonica. Phenotypic variation among and within populations were analyzed by ANOVA and correlation analysis. Genetic materials of varieties and clones of S. japonica were used for DNA extraction and the best SRAP-PCR reaction system selection for further development of the SRAP analysis. It was intended in this study that a theoretical basis to show genetic relationships at molecular level.
The main findings and conclusions are as follows:
1 Genetic variations were, to a certain extent, found in phenotypic properties of seed, coefficient of variation was ranged from 5.24% to 14.57%. There were significant differences in seed morphological characteristics among and within populations. The average differentiation coefficient among populations was 53.11%, which was higher than that within population. No significant correlation was found in geographic variations of seed with latitude and longitude. It was indicated in cluster analysis that 7 populations of S. japonica were divided into 3 groups:1) Taian groups; 2) Hubei and Beijing groups; 3) Zhejiang, Xi'an, Xianyang and Henan populations. It was found that there was no any significant geographical variations in seed morpholgy, which suggested that introduction and cultivation in wide range of area or discontinuous sampling would be responsible for the phenomenon.
2 Orthogonal experimental design was applied to establish the optimal reaction system for SRAP-PCR (20 uL):Taq polymerase 1.5 U, dNTPs 0.30 mmol/L, the template DNA 90 ng, Mg2+2.0 mmol/L, primer 0.3 umol/L, ddH2O fill to 20 uL.
3 There were 12 primer combinations that PCR product bands were clear, highly polymorphic selected from 120 primer combinations, which were applied in genetic relationship and molecular identification study of varieties and clones of S. japonica. The rang of PCR amplification of 12 primer combinations are mostly concentrated in bands between 100bp-2000bp. Of the total of 430 bands amplified,415 were polymorphic loci. The percentage of polymorphic fragments were 96.60%, of which 4 pairs of primers polymorphism were 100%. It was showed in the results that multiple genetic loci of varieties and clones could be detected with SRAP markers. Primers combination Em9-Me12 could be used to distinguish different varieties of S. japonica. Rich and clear SRAP fingerprints were also obtained.
4 Levels of genetic similarity in coefficients (SM) were calculated and a dendrogram was constructed by the unweighted pair group method of arithmetic average (UPGMA). SM value was in the range of 0.68-0.89, with an average similarity coefficient of 0.73, indicating the similarity between varieties and clones of S. japonica were relatively high, which means the genetic base was relatively narrow. Based on the similarity coefficients of 0.76, varieties and clones were clustered into four categories. The second category was consisted of 4 and 35 of S. japonica; the fourth category inclued S. japonica var. pendula Loud. (LZ), S. japonica 'Caozhou 2'(C2) and S. japonica f. flavi-rameus (JY). The rests were clustered into the first category, which could be divided into two subgroups. S. japonica var.oligophylla Franch. itself was clustered in the third category, which indicated that there were large differences in different shapes of leaves.
引文
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