松毛虫对环境适应性的基因分析
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摘要
本文运用AFLP分子标记的方法,对不同温度、湿度下的3种松毛虫种群和2种林分类型下的油松毛虫种群进行了遗传多样性和遗传结构的研究,并找到3种松毛虫与温度、湿度相关的特异性条带,以及松毛虫与不同林分类型相关的特异性条带,并进行克隆、测序和序列分析。研究结果如下:
①本试验以油松毛虫为材料,建立了适合松毛虫基因组DNA研究的AFLP体系。用优化的AFLP反应体系,以油松毛虫、赤松毛虫和马尾松毛虫为材料筛选引物,从81对引物组合中筛选出10对多态性高,重复性好的引物组合。
②油松毛虫、赤松毛虫和马尾松毛虫中存在于种群内的基因多样度HS为0.0895,不同种群间遗传分化系数GST为0.7623,即种群间的遗传变异占种群总的遗传多样性的76.23%,小部分变异存在于种群内(23.77%)。不同松毛虫种群之间的基因流Nm为0.1559,说明种群之间基因交流不强烈,无法有效抵消遗传漂变所引起的种群分化。聚类结果表明,油松毛虫与马尾松毛虫相聚为一类,在与赤松毛虫聚在一起。不同林分类型中油松毛虫种群的总基因多样度HT为0.3550,其中存在于种群内的基因多样度HS为0.2722,不同种群间遗传分化系数GST为0.2332,即种群间的遗传变异占种群总的遗传多样性的23.32%,大部分变异存在于种群内(76.68%),说明油松毛虫具有丰富的遗传多样性,种群之间基因交流强烈,种群间的分化程度较低。
③分别构建了油松毛虫、赤松毛虫、马尾松毛虫和混交林中油松毛虫的基因组DNA混合池,采用81对Pst I /Taq I引物组合,对4个基因池进行AFLP扩增,获得了松毛虫与环境有关的2个分子标记MW和SC。在引物P1-T4中,观察到了1条马尾松毛虫所特有的多态性DNA片段,标记为MW,56bp;在P7-T9引物组合中,又观察到了1条赤松毛虫所特有的多态性DNA片段,标记为SC, 124bp。对以上2个标记进行了验证,结果证明这2个标记的准确率达100%。将2个特异性DNA片段克隆到T-Easy载体上,进行测序。通过BLAST和序列比对分析发现,这些序列是新的未见报道的序列。
In order to find differentially expressed DNA-AFLP fragments of pine caterpillars which related to different temperature and humidity and different stand types, by use of AFLP technique, the study examined the genetic structure and diversity among the 3 natural populations of the Chinese pine caterpillar (Dendrolimus tabulaeformis Tsai et Liu, Dendrolimus spectabilis Butler and Dendrolimus punctatus (Walker)) and in the different stand types 2 natural populations of Dendrolimus tabulaeformis Tsai et Liu. The differentially expressed DNA fragments were cloned and sequenced.
①the genomic DNA of Dendrolimus tabulaeformis Tsai et Liu was taken as materials,this test developed a set of optimized response system of amplified fragment length polymorphism. With Dendrolimus tabulaeformis Tsai et Liu, Dendrolimus spectabilis Butler and Dendrolimus punctatus (Walker) as material, ten pairs of informative primers were identified from eighty-one pairs primers by using the optimization of AFLP reaction system.
②Genetic diversities of Dendrolimus tabulaeformis Tsai et Liu, Dendrolimus spectabilis Butler and Dendrolimus punctatus (Walker): At population level, gene diversity with in populations (Hs) was 0.0895; coefficient of population differentiation (Gst) was 0.7623. Genetic variation among populations accounted for the genetic diversity of the total population of 76.23%, few portions of variation exist in the population (23.77%). Pine dendrolimus different gene flow between populations Nm (0.1559) on the exchange of genes between populations is not strong, can not be effectively offset by genetic drift caused by population differentiation. Clustering results show that Dendrolimus tabulaeformis Tsai et Liu and Dendrolimus punctatus (Walker) together with a category, with the Dendrolimus spectabilis Butler together. Genetic diversities of Dendrolimus tabulaeformis Tsai et Liu in the different stand types: At population level, total gene diversity (Ht) was 0.3550, gene diversity with in populations (Hs) was 0.2722, coefficient of population differentiation (Gst) was 0.2332. The research result shows the genetic diversity of Dendrolimus tabulaeformis Tsai et Liu is very abundant, but there is relatively low inter-population genetic differentiation while most resided within the different populations and gene flow between populations of strong.
③Dendrolimus tabulaeformis Tsai et Liu, Dendrolimus spectabilis Butler , Dendrolimus punctatus (Walker) and Dendrolimus tabulaeformis Tsai et Liu of stand types bolting bulk were construct respectively. DNA-AFLP analysis was carried out in four bulks by 81 pairs of AFLP primer combinations. Two different bands-MW and SC were obtained. A specific DNA fragment polymorphism of Dendrolimus punctatus (Walker) was observed , marked MW, 56bp in P1-T4 primers. A specific DNA fragment polymorphism of Dendrolimus spectabilis Butler was observed, marked SC, 124bp in P7-T9 primers. These two markers were tested to prove that the two markers with 100% accuracy. To two specific DNA fragment was cloned into T-Easy vector.Two differentially expressed DNA fragments were sequenced. Sequence and BLAST analysis indicated that these sequences are not reported.
①本试验以油松毛虫为材料,建立了适合松毛虫基因组DNA研究的AFLP体系。用优化的AFLP反应体系,以油松毛虫、赤松毛虫和马尾松毛虫为材料筛选引物,从81对引物组合中筛选出10对多态性高,重复性好的引物组合。
②油松毛虫、赤松毛虫和马尾松毛虫中存在于种群内的基因多样度HS为0.0895,不同种群间遗传分化系数GST为0.7623,即种群间的遗传变异占种群总的遗传多样性的76.23%,小部分变异存在于种群内(23.77%)。不同松毛虫种群之间的基因流Nm为0.1559,说明种群之间基因交流不强烈,无法有效抵消遗传漂变所引起的种群分化。聚类结果表明,油松毛虫与马尾松毛虫相聚为一类,在与赤松毛虫聚在一起。不同林分类型中油松毛虫种群的总基因多样度HT为0.3550,其中存在于种群内的基因多样度HS为0.2722,不同种群间遗传分化系数GST为0.2332,即种群间的遗传变异占种群总的遗传多样性的23.32%,大部分变异存在于种群内(76.68%),说明油松毛虫具有丰富的遗传多样性,种群之间基因交流强烈,种群间的分化程度较低。
③分别构建了油松毛虫、赤松毛虫、马尾松毛虫和混交林中油松毛虫的基因组DNA混合池,采用81对Pst I /Taq I引物组合,对4个基因池进行AFLP扩增,获得了松毛虫与环境有关的2个分子标记MW和SC。在引物P1-T4中,观察到了1条马尾松毛虫所特有的多态性DNA片段,标记为MW,56bp;在P7-T9引物组合中,又观察到了1条赤松毛虫所特有的多态性DNA片段,标记为SC, 124bp。对以上2个标记进行了验证,结果证明这2个标记的准确率达100%。将2个特异性DNA片段克隆到T-Easy载体上,进行测序。通过BLAST和序列比对分析发现,这些序列是新的未见报道的序列。
In order to find differentially expressed DNA-AFLP fragments of pine caterpillars which related to different temperature and humidity and different stand types, by use of AFLP technique, the study examined the genetic structure and diversity among the 3 natural populations of the Chinese pine caterpillar (Dendrolimus tabulaeformis Tsai et Liu, Dendrolimus spectabilis Butler and Dendrolimus punctatus (Walker)) and in the different stand types 2 natural populations of Dendrolimus tabulaeformis Tsai et Liu. The differentially expressed DNA fragments were cloned and sequenced.
①the genomic DNA of Dendrolimus tabulaeformis Tsai et Liu was taken as materials,this test developed a set of optimized response system of amplified fragment length polymorphism. With Dendrolimus tabulaeformis Tsai et Liu, Dendrolimus spectabilis Butler and Dendrolimus punctatus (Walker) as material, ten pairs of informative primers were identified from eighty-one pairs primers by using the optimization of AFLP reaction system.
②Genetic diversities of Dendrolimus tabulaeformis Tsai et Liu, Dendrolimus spectabilis Butler and Dendrolimus punctatus (Walker): At population level, gene diversity with in populations (Hs) was 0.0895; coefficient of population differentiation (Gst) was 0.7623. Genetic variation among populations accounted for the genetic diversity of the total population of 76.23%, few portions of variation exist in the population (23.77%). Pine dendrolimus different gene flow between populations Nm (0.1559) on the exchange of genes between populations is not strong, can not be effectively offset by genetic drift caused by population differentiation. Clustering results show that Dendrolimus tabulaeformis Tsai et Liu and Dendrolimus punctatus (Walker) together with a category, with the Dendrolimus spectabilis Butler together. Genetic diversities of Dendrolimus tabulaeformis Tsai et Liu in the different stand types: At population level, total gene diversity (Ht) was 0.3550, gene diversity with in populations (Hs) was 0.2722, coefficient of population differentiation (Gst) was 0.2332. The research result shows the genetic diversity of Dendrolimus tabulaeformis Tsai et Liu is very abundant, but there is relatively low inter-population genetic differentiation while most resided within the different populations and gene flow between populations of strong.
③Dendrolimus tabulaeformis Tsai et Liu, Dendrolimus spectabilis Butler , Dendrolimus punctatus (Walker) and Dendrolimus tabulaeformis Tsai et Liu of stand types bolting bulk were construct respectively. DNA-AFLP analysis was carried out in four bulks by 81 pairs of AFLP primer combinations. Two different bands-MW and SC were obtained. A specific DNA fragment polymorphism of Dendrolimus punctatus (Walker) was observed , marked MW, 56bp in P1-T4 primers. A specific DNA fragment polymorphism of Dendrolimus spectabilis Butler was observed, marked SC, 124bp in P7-T9 primers. These two markers were tested to prove that the two markers with 100% accuracy. To two specific DNA fragment was cloned into T-Easy vector.Two differentially expressed DNA fragments were sequenced. Sequence and BLAST analysis indicated that these sequences are not reported.
引文
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