主要梨品种细胞质遗传多态性分析
摘要
梨属于蔷薇科的梨亚科,品种很多,长期以来在分类上存在很多的问题。本论文的目的是研究主要梨品种细胞质遗传多态性。采用PCR-RFLP方法,对提取出的总DNA用10对叶绿体通用引物进行扩增,对PCR产物用限制性内切酶AluⅠ, HaeⅢ, HinfⅠ, Hin6Ⅰ, RsaⅠ, MvaⅠ和TaqI进行酶切,对19种梨(包括新疆梨系统、白梨系统、西洋梨系统、秋子梨系统、杜梨、沙梨系统)的叶绿体基因组trnS-trnfM非编码区进行克隆、测序。应用DPS v7.05和DNAMAN、DNAStar、ClustalX-1.83、PHYLIP-3.68软件进行分析。通过序列比对,再进行聚类分析,最后依据所得结果确定所测分子序列的亲缘关系,构建系统进化树。
结果显示：10对引物中只有7对(cp01,cp 02,cp 03,cp 04,cp 06,cp 09,cp 10)能在梨属植物上扩增出一条特异性谱带,这说明梨属植物叶绿体基因组序列十分保守,3个引物对(cp05, cp07, cp08)不能在梨属植物上扩增出谱带。931份引物对/酶切组合中,cp09/MvaⅠ, cp03/Hin6Ⅰ的酶切位点有显著差异。
对梨属植物的cpDNA trnS-trnfM区域进行克隆、测序,所得的序列长度为：库尔勒香梨和鸭梨的序列最长(1642bp),苹果梨、早酥梨、慈梨、象牙、翠伏的序列最短(1272bp)。
用DNAMAN软件对序列进行比对分析：库尔勒香梨与白梨系统的同源性为：85.01%,与新疆梨系统的同源性为：78.60%,与西洋梨系统的同源性为：78.28%,与沙梨系统的同源性为：77.47%,与秋子梨系统的同源性为：77.91%。
根据ClustalX软件完全比对的结果,用PHYLIP-3.68软件的邻接法对cpDNA trnS-trnfM区域序列变异位点构建系统进化树。黑酸梨和京白聚为一类,伏茄和身不知聚为一类,冬巴和新世纪聚为一类。库尔勒香梨和新疆句句梨、金川雪梨、京白、黑酸梨聚为一类。
本实验的主要结论：库尔勒香梨与苹果梨,鸭梨,早酥,慈梨,砀山梨,金川雪梨,新疆句句梨,锦丰的平均距离系数较小,与其他梨的平均距离系数较大。
早酥是苹果梨和身不知杂交出来的品种。早酥与苹果梨的同源性为：99.45%,与身不知的同源性为：97.76%。锦丰是苹果梨和慈梨杂交出来的品种。锦丰与苹果梨的同源性为：78.76%,锦丰与慈梨的同源性为：78.71%。和父本相比,后代和母本的同源性较高。库尔勒香梨在与18种梨的序列比对中,和砀山梨的同源性最高为：98.36%。库尔勒香梨与白梨系统和新疆梨系统的亲缘关系较近,与沙梨系统和秋子梨系统的亲缘关系较远。
因此,本实验的意义是结合分子生物学和生物信息学对19个梨品种进行细胞质分析,搞清白梨系统、新疆梨系统、西洋梨系统、杜梨、沙梨系统和秋子梨系统的胞质基因的遗传多样性。以期为研究梨属植物亲缘关系提供进一步的遗传学证据。
There were many species of Pyrus. Because many interspecific hybrids were fertile,there were so many questions on Pyrus relationships for a long time.This experiment researched on the genetic diversity of nineteen cultivars in Pyrus derived from cpDNA PCR-RFLP.Ten universal primer pairs of chloroplast genome were used to amplify cpDNA non-coding regions in nineteen Pyrus species. The PCR products were digested by seven restriction enzymes. To clone and sequence the cpDNA non-coding regions in nineteen Pyrus species. DPS v7.05 was used to account Jaccard, the dendrogram was constructed by UPGMA. DNAMAN、DNAStar、ClustalX-1.83 and PHYLIP-3.68 were used to build the system tree.According to the sequences,we could make sure the relationship among these Pyrus.
The result showed that seven primer pairs(cp01, cp02, cp03, cp 04, cp06, cp09, cp 10)generated special bands from Pyrus. The combination have 931 between primer pairs and restriction enzymes.The fragements digested of cp09/MvaI, cp03/Hin6I were different.
The trnS-trnfM region in chloroplast genome were cloned and sequenced in nineteen species of Pyrus. The nucleotide sequences of Korla pear and Yali were longest(1642bp),and the shorest sequences(1272bp) were Pingguoli,Zaosuli,Cili,Xiangya,Cuifu.
The nucleotide sequences were aligned with DNAMAN software. The identity of the Korla pear and P.bretschneideri Rhed. was 85.01%, the identity of the Korla pear and P.sinkiangensis Yu was 78.60%, the identity of the Korla pear and P.communis L.was 78.28%, the identity of the Korla pear and P.pyrifolia (Burm.) Nakai was 77.47%, the identity of the Korla pear and Pyrus ussuriensis Maxim. was 77.91%.
According to the aligment, the trnS-trnfM region in chloroplast genome were built in neigorbor way of the PHYLIP-3.68 software.According the phylogenetic tree,Heisuanli and Jingbaili were group,Fuqie and Shenbuzhi were group,Dongba and Xinshiji were group, the Korla pear,Jujuli,Jinchuanxueli, Heisuanli and Jingbaili were group.
The conclusions were as follows:the average distance coefficient of the Korla pear with Yali,Dangshanli,Pingguoli,Cili,Jinchuanxueli,Jinfeng and Jujuli were smaller; that of Korla pear with the other cultivars were bigger. the Korla pear and P.sinkiangensis Yii had nearer relationships. the Korla pear and P.pyrifolia (Burm.) Nakai had farer relationships.
Zaosu was hybridized by Pingguoli and Shenbuzhi. The identity of the Zaosu and Pingguoli was 99.45%, The identity of the Zaosu and Shenbuzhi was 97.76%. Jinfeng was hybridized by Pingguoli and Cili. The identity of the Jinfeng and Pingguoli was 78.76%, The identity of the Jinfeng and Cili was 78.71%.Contrasted to the male parent,the offsprings were closed with the female parent. The identity of the Korla pear and the Dangshanli were 98.36%,which were hingest in the another Pyrus species.Hence, the Korla pear was closed with P.bretschneideri Rhed.and P.sinkiangensis Yu.
Therefore,the meaning of the paper was able to analyse the relationships with biology and bioinformatics.We were able to study the polymorphism in Pyrus.The evidents were provided to research the relationships of the Pyrus.
结果显示：10对引物中只有7对(cp01,cp 02,cp 03,cp 04,cp 06,cp 09,cp 10)能在梨属植物上扩增出一条特异性谱带,这说明梨属植物叶绿体基因组序列十分保守,3个引物对(cp05, cp07, cp08)不能在梨属植物上扩增出谱带。931份引物对/酶切组合中,cp09/MvaⅠ, cp03/Hin6Ⅰ的酶切位点有显著差异。
对梨属植物的cpDNA trnS-trnfM区域进行克隆、测序,所得的序列长度为：库尔勒香梨和鸭梨的序列最长(1642bp),苹果梨、早酥梨、慈梨、象牙、翠伏的序列最短(1272bp)。
用DNAMAN软件对序列进行比对分析：库尔勒香梨与白梨系统的同源性为：85.01%,与新疆梨系统的同源性为：78.60%,与西洋梨系统的同源性为：78.28%,与沙梨系统的同源性为：77.47%,与秋子梨系统的同源性为：77.91%。
根据ClustalX软件完全比对的结果,用PHYLIP-3.68软件的邻接法对cpDNA trnS-trnfM区域序列变异位点构建系统进化树。黑酸梨和京白聚为一类,伏茄和身不知聚为一类,冬巴和新世纪聚为一类。库尔勒香梨和新疆句句梨、金川雪梨、京白、黑酸梨聚为一类。
本实验的主要结论：库尔勒香梨与苹果梨,鸭梨,早酥,慈梨,砀山梨,金川雪梨,新疆句句梨,锦丰的平均距离系数较小,与其他梨的平均距离系数较大。
早酥是苹果梨和身不知杂交出来的品种。早酥与苹果梨的同源性为：99.45%,与身不知的同源性为：97.76%。锦丰是苹果梨和慈梨杂交出来的品种。锦丰与苹果梨的同源性为：78.76%,锦丰与慈梨的同源性为：78.71%。和父本相比,后代和母本的同源性较高。库尔勒香梨在与18种梨的序列比对中,和砀山梨的同源性最高为：98.36%。库尔勒香梨与白梨系统和新疆梨系统的亲缘关系较近,与沙梨系统和秋子梨系统的亲缘关系较远。
因此,本实验的意义是结合分子生物学和生物信息学对19个梨品种进行细胞质分析,搞清白梨系统、新疆梨系统、西洋梨系统、杜梨、沙梨系统和秋子梨系统的胞质基因的遗传多样性。以期为研究梨属植物亲缘关系提供进一步的遗传学证据。
There were many species of Pyrus. Because many interspecific hybrids were fertile,there were so many questions on Pyrus relationships for a long time.This experiment researched on the genetic diversity of nineteen cultivars in Pyrus derived from cpDNA PCR-RFLP.Ten universal primer pairs of chloroplast genome were used to amplify cpDNA non-coding regions in nineteen Pyrus species. The PCR products were digested by seven restriction enzymes. To clone and sequence the cpDNA non-coding regions in nineteen Pyrus species. DPS v7.05 was used to account Jaccard, the dendrogram was constructed by UPGMA. DNAMAN、DNAStar、ClustalX-1.83 and PHYLIP-3.68 were used to build the system tree.According to the sequences,we could make sure the relationship among these Pyrus.
The result showed that seven primer pairs(cp01, cp02, cp03, cp 04, cp06, cp09, cp 10)generated special bands from Pyrus. The combination have 931 between primer pairs and restriction enzymes.The fragements digested of cp09/MvaI, cp03/Hin6I were different.
The trnS-trnfM region in chloroplast genome were cloned and sequenced in nineteen species of Pyrus. The nucleotide sequences of Korla pear and Yali were longest(1642bp),and the shorest sequences(1272bp) were Pingguoli,Zaosuli,Cili,Xiangya,Cuifu.
The nucleotide sequences were aligned with DNAMAN software. The identity of the Korla pear and P.bretschneideri Rhed. was 85.01%, the identity of the Korla pear and P.sinkiangensis Yu was 78.60%, the identity of the Korla pear and P.communis L.was 78.28%, the identity of the Korla pear and P.pyrifolia (Burm.) Nakai was 77.47%, the identity of the Korla pear and Pyrus ussuriensis Maxim. was 77.91%.
According to the aligment, the trnS-trnfM region in chloroplast genome were built in neigorbor way of the PHYLIP-3.68 software.According the phylogenetic tree,Heisuanli and Jingbaili were group,Fuqie and Shenbuzhi were group,Dongba and Xinshiji were group, the Korla pear,Jujuli,Jinchuanxueli, Heisuanli and Jingbaili were group.
The conclusions were as follows:the average distance coefficient of the Korla pear with Yali,Dangshanli,Pingguoli,Cili,Jinchuanxueli,Jinfeng and Jujuli were smaller; that of Korla pear with the other cultivars were bigger. the Korla pear and P.sinkiangensis Yii had nearer relationships. the Korla pear and P.pyrifolia (Burm.) Nakai had farer relationships.
Zaosu was hybridized by Pingguoli and Shenbuzhi. The identity of the Zaosu and Pingguoli was 99.45%, The identity of the Zaosu and Shenbuzhi was 97.76%. Jinfeng was hybridized by Pingguoli and Cili. The identity of the Jinfeng and Pingguoli was 78.76%, The identity of the Jinfeng and Cili was 78.71%.Contrasted to the male parent,the offsprings were closed with the female parent. The identity of the Korla pear and the Dangshanli were 98.36%,which were hingest in the another Pyrus species.Hence, the Korla pear was closed with P.bretschneideri Rhed.and P.sinkiangensis Yu.
Therefore,the meaning of the paper was able to analyse the relationships with biology and bioinformatics.We were able to study the polymorphism in Pyrus.The evidents were provided to research the relationships of the Pyrus.
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