光皮桦分子标记的开发及其在遗传多样性研究中的应用
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摘要
利用白桦的表达序列标签(Expressed Sequence Tags, ESTs)开发光皮桦(Betula luminifera H.Wink.)特异分子标记,然后利用这些引物评价光皮桦种子园的遗传多样性,并以它们的遗传距离为依据构建了光皮桦核心种质。主要结论如下:
1.筛选了109对候选的扩增共有序列遗传标记(Amplified Consensus Genetic Markers, ACGM),其中105对在白桦中可以扩增出稳定而清晰的PCR产物;然后利用来自不同种源的光皮桦Lin’an5和Sichuan4筛选这105对引物,结果95对至少在一种光皮桦中有稳定的扩增产物。
2.筛选出80个基于保守序列的EST-SSR (Simple Sequence Repeat, SSR)候选位点,其中59对在白桦中可以获得稳定而清晰的PCR产物;然后利用Lin’an5和Sichuan4筛选这59对引物,结果28对至少在一种光皮桦中有稳定的扩增产物。
3.利用本研究开发的光皮桦ACGM分子标记对来自不同种源的62份光皮桦遗传多样性进行评价。结果表明该群体的Nei遗传多样性指数( Nei-H)和多态位点百分数(Nei-P%)分别为0.2138和54.74%。居群中安徽(pop9)居群的观测等位基因数(Na)、有效等位基因数(Ne)、Nei-H和Nei-P%最小,分别为1.1022、1.0723、0.0423和10.22%。临安居群(pop1)的Na、Ne、Nei-H和Nei-P%最大,分别为1.4307、1.3125、0.1741和43.07%。
4.利用本研究开发的光皮桦ACGM分子标记对光皮桦种子园中62份样品所代表的519份光皮桦资源的遗传多样性进行分析,结果筛选出36份核心种质。t检验结果表明,核心种质的Na、Ne、Nei-H和Nei-P%在概率0.01水平上与初始种质差异不显著,说明核心种质能很好的代替初始种质。
Special molecular markers were developed for Betula luminifera using Birch ESTs (Expression sequence tags) data. We then used these markers to evaluate the genetic diversity of B.luminifera germplasm. Based on their genetic distance, B.luminifera core germplasm was constructed. The main conclusions were as follows:
1.Based on birch EST data, primers for 109 ACGM candidate loci were developed and tested in birch. Of these, 105 yielded single, stable and clear PCR products. We then tested the utility of those 105 markers in B. luminifera. The results showed 95 yielded stable and clear PCR products for at least one B. luminifera genotype of Lin’an 5 and Sichuan 4.
2. Based on birch EST data, primers for 80 EST-SSR candidate loci were developed and tested in birch. Of these, 59 yielded single, stable and clear PCR products.We then tested the utility of those 59 markers in B.luminifera. The results showed 28 yielded stable and clear PCR products for at least one B.luminifera genotype of Lin’an 5 and Sichuan 4.
3. The genetic diversity of 62 samples from B. luminifera germplasm was evaluated using ACGM markers. Nei gene diversity index and percentage of polymorphic loci of the samples were calculated, the values are 0.2138 and 54.74%, respectively. The observed number of alleles, effective number of alleles, Nei gene diversity index and percentage of polymorphic loci in Anhui (pop9) are the lowest, values are 1.1022, 0.0423, 1.0723 and 10.22%, respectively; whereas the highest in Linan (pop1), values are 1.4307, 1.3125, 0.1741 and 43.07%, respectively.
4. The genetic diversity of 62 B.luminifera germplasm resources, representing 519 samples, was evaluated using ACGM markers. Of these 62 samples, 36 were selected as core collection. Results of t-test showed that the observed number of alleles, effective number of alleles, Nei gene diversity and Shannon information index of core collection had no significant differences with initial germplasm on 0.01 level. These results demonstrated that the core collection can represent initial germplasm resources.
1.筛选了109对候选的扩增共有序列遗传标记(Amplified Consensus Genetic Markers, ACGM),其中105对在白桦中可以扩增出稳定而清晰的PCR产物;然后利用来自不同种源的光皮桦Lin’an5和Sichuan4筛选这105对引物,结果95对至少在一种光皮桦中有稳定的扩增产物。
2.筛选出80个基于保守序列的EST-SSR (Simple Sequence Repeat, SSR)候选位点,其中59对在白桦中可以获得稳定而清晰的PCR产物;然后利用Lin’an5和Sichuan4筛选这59对引物,结果28对至少在一种光皮桦中有稳定的扩增产物。
3.利用本研究开发的光皮桦ACGM分子标记对来自不同种源的62份光皮桦遗传多样性进行评价。结果表明该群体的Nei遗传多样性指数( Nei-H)和多态位点百分数(Nei-P%)分别为0.2138和54.74%。居群中安徽(pop9)居群的观测等位基因数(Na)、有效等位基因数(Ne)、Nei-H和Nei-P%最小,分别为1.1022、1.0723、0.0423和10.22%。临安居群(pop1)的Na、Ne、Nei-H和Nei-P%最大,分别为1.4307、1.3125、0.1741和43.07%。
4.利用本研究开发的光皮桦ACGM分子标记对光皮桦种子园中62份样品所代表的519份光皮桦资源的遗传多样性进行分析,结果筛选出36份核心种质。t检验结果表明,核心种质的Na、Ne、Nei-H和Nei-P%在概率0.01水平上与初始种质差异不显著,说明核心种质能很好的代替初始种质。
Special molecular markers were developed for Betula luminifera using Birch ESTs (Expression sequence tags) data. We then used these markers to evaluate the genetic diversity of B.luminifera germplasm. Based on their genetic distance, B.luminifera core germplasm was constructed. The main conclusions were as follows:
1.Based on birch EST data, primers for 109 ACGM candidate loci were developed and tested in birch. Of these, 105 yielded single, stable and clear PCR products. We then tested the utility of those 105 markers in B. luminifera. The results showed 95 yielded stable and clear PCR products for at least one B. luminifera genotype of Lin’an 5 and Sichuan 4.
2. Based on birch EST data, primers for 80 EST-SSR candidate loci were developed and tested in birch. Of these, 59 yielded single, stable and clear PCR products.We then tested the utility of those 59 markers in B.luminifera. The results showed 28 yielded stable and clear PCR products for at least one B.luminifera genotype of Lin’an 5 and Sichuan 4.
3. The genetic diversity of 62 samples from B. luminifera germplasm was evaluated using ACGM markers. Nei gene diversity index and percentage of polymorphic loci of the samples were calculated, the values are 0.2138 and 54.74%, respectively. The observed number of alleles, effective number of alleles, Nei gene diversity index and percentage of polymorphic loci in Anhui (pop9) are the lowest, values are 1.1022, 0.0423, 1.0723 and 10.22%, respectively; whereas the highest in Linan (pop1), values are 1.4307, 1.3125, 0.1741 and 43.07%, respectively.
4. The genetic diversity of 62 B.luminifera germplasm resources, representing 519 samples, was evaluated using ACGM markers. Of these 62 samples, 36 were selected as core collection. Results of t-test showed that the observed number of alleles, effective number of alleles, Nei gene diversity and Shannon information index of core collection had no significant differences with initial germplasm on 0.01 level. These results demonstrated that the core collection can represent initial germplasm resources.
引文
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