中国西北部地区蜜蜂形态特征和线粒体DNA多样性研究
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摘要
本研究采集我国西北部新疆伊犁河谷地区新疆黑蜂81群,样品来自于定地饲养25年以上的蜂场;甘肃东北部与宁夏南部山区东方蜜蜂85群,样品来自于土法定地饲养蜂场。全部样本均来自不同的气候类型和地理环境。运用蜜蜂形态指标测定技术并结合针对东方蜜蜂特有形态特征而配置的测定标准,对全部采集样品的41个形态特征指标进行测定。同时扩增全部样品mtDNA COⅠ-COⅡ基因中tRNAleu-COⅡ间非编码区及COⅡ5’端基因,运用限制性内切酶DraⅠ酶切分析并测序。运用SAS 6.12统计系统、SPSS 13.0统计系统对形态学数据进行基本统计量分析、主成分分析、判别分析和聚类分析。运用Bioedit 7.0和MEGA 3.0对测序所得样品序列进行序列比对分析和构建系统发生树。分析结果如下:
1.新疆黑蜂形态学分析可以得到两个类群:A类群与欧洲黑蜂聚为一类,属于形态学分类的M谱系;B类群与新近发现的哈萨克蜂聚为一类,属于形态学分类的C谱系。
2.新疆黑蜂分子生物学分析共检测到6种西方蜜蜂单元型,这6种单元型可以分为三个大类群:C类群可以归为形态学分类中的M谱系,包括了4个西方蜜蜂单元型,各单元型间存在碱基缺失、替换;D类群可以归为形态学的C谱系,包含有一种单元型;E类群属于形态学分类中的M谱系,包含有一个单元型。
3.通过对新疆黑蜂形态学与分子生物学的分析发现:B类群与D类群同属于形态学分析中的C谱系,与意大利蜂和哈萨克蜂类似,所属新疆黑蜂种群基本一致(有5群差异)。A类群、C类群和E类群同属于形态学分析中的M谱系,所属蜂群基本一致(有5群差异)。
4.东方蜜蜂形态学分析可以得到两个类群:F类群来自甘肃东北部平原、丘陵地带;H类群来自宁夏南部六盘山山区。在与不同气候类型的地区的东方蜜蜂比较中,F类群与H类群聚为一类,和其他地区不同气候地理环境下的东方蜜蜂分离。
5.东方蜜蜂分子生物学分析共检测到限制性内切酶DraⅠ酶切产物类型三种,通过测序分析得到11种mtDNA tRNAleu - COⅡ间非编码区单元型,其中4种单元型是首次发现。将11种单元型与亚洲东方蜜蜂相同区域单元型进行比对,并进行系统发生树分析,所得结果基本一致,即所得单元型属于亚洲大陆型,与亚洲半岛型和海岛型明显分离。
81 Apis mellifera honeybee colonies from Yili River Valley of Xinjiang province of China and 85 Apis cerana honeybee coloies from northeast of Gansu and south of Ningxia were identified using both morphological morphometric analysis and mitochondrial DNA tRNAleu- COⅡgene. 41 morphological characters were analysized for each individual bee, and in total 15 honeybee workers were employed for each sample. Variance analysis, principal component analysis, discriminant analysis and cluster analysis were performed using SAS 6.12. The mitochondrial DNA tRNAleu- COⅡgene were amplified by primer pair E2/H2, both DraⅠdisgested and sequencing were performed.
Morphological characters multivariate statistical analysis of Apis mellifera from Xinjiang revealed two distinct morphoclusters of bee: (1) B colony was cluster with the Apis mellifera mellifera, and A colony cluster with the newly discovered subspecies Apis mellifera pomonella.
Mitochondrial DNA sequence analysis of Apis mellifera from Xinjiang were revealed 6 different haplotypes were found in the tRNAleu- COⅡgene. This 6 haplotypes could cluster the 3 large type, C type clusted like the Apis mellifera anatolica, include the 4 haplotypes, and have a little gene mutation. D type clusted with the Apis mellifera ligustica, the E type culsted like the Apis mellifera mellifera, only have one sample.
Synthesized the conclusion of above, we could know the A colony and D type are the same Apis mellifera honeybee samples. The B colony and D type / E type have the same Apis mellifera honeybee samples.
Morphological characters multivariate statistical analysis of Apis cerana from the northeast of Gsnsu and south of Ningxia, could revealed two distanct morphoclusters of bee, F colony was cluster all honeybee come from the plant and low hill region of northeast of Gansu, H colony was cluster from the Liupanshan Mountain in the south of Ningxia. This two place have different climate and geographical type.
The PCR products of Apis cerana were digested with DraⅠand three restricton pattens were revealed.Through sequenced and 11 different haplotypes were found in the non-coding region between tRNAleu and COⅡ.The sequence of the haplotypes were 96-97bp long.The sequences found in the study were compared with those which hand been described before.The alignment results and the phylogenetic trees showed that the honeybee Apis cerana adjacent countries had similar mtDNA sequences and they all falled into the Asian mainland group.
1.新疆黑蜂形态学分析可以得到两个类群:A类群与欧洲黑蜂聚为一类,属于形态学分类的M谱系;B类群与新近发现的哈萨克蜂聚为一类,属于形态学分类的C谱系。
2.新疆黑蜂分子生物学分析共检测到6种西方蜜蜂单元型,这6种单元型可以分为三个大类群:C类群可以归为形态学分类中的M谱系,包括了4个西方蜜蜂单元型,各单元型间存在碱基缺失、替换;D类群可以归为形态学的C谱系,包含有一种单元型;E类群属于形态学分类中的M谱系,包含有一个单元型。
3.通过对新疆黑蜂形态学与分子生物学的分析发现:B类群与D类群同属于形态学分析中的C谱系,与意大利蜂和哈萨克蜂类似,所属新疆黑蜂种群基本一致(有5群差异)。A类群、C类群和E类群同属于形态学分析中的M谱系,所属蜂群基本一致(有5群差异)。
4.东方蜜蜂形态学分析可以得到两个类群:F类群来自甘肃东北部平原、丘陵地带;H类群来自宁夏南部六盘山山区。在与不同气候类型的地区的东方蜜蜂比较中,F类群与H类群聚为一类,和其他地区不同气候地理环境下的东方蜜蜂分离。
5.东方蜜蜂分子生物学分析共检测到限制性内切酶DraⅠ酶切产物类型三种,通过测序分析得到11种mtDNA tRNAleu - COⅡ间非编码区单元型,其中4种单元型是首次发现。将11种单元型与亚洲东方蜜蜂相同区域单元型进行比对,并进行系统发生树分析,所得结果基本一致,即所得单元型属于亚洲大陆型,与亚洲半岛型和海岛型明显分离。
81 Apis mellifera honeybee colonies from Yili River Valley of Xinjiang province of China and 85 Apis cerana honeybee coloies from northeast of Gansu and south of Ningxia were identified using both morphological morphometric analysis and mitochondrial DNA tRNAleu- COⅡgene. 41 morphological characters were analysized for each individual bee, and in total 15 honeybee workers were employed for each sample. Variance analysis, principal component analysis, discriminant analysis and cluster analysis were performed using SAS 6.12. The mitochondrial DNA tRNAleu- COⅡgene were amplified by primer pair E2/H2, both DraⅠdisgested and sequencing were performed.
Morphological characters multivariate statistical analysis of Apis mellifera from Xinjiang revealed two distinct morphoclusters of bee: (1) B colony was cluster with the Apis mellifera mellifera, and A colony cluster with the newly discovered subspecies Apis mellifera pomonella.
Mitochondrial DNA sequence analysis of Apis mellifera from Xinjiang were revealed 6 different haplotypes were found in the tRNAleu- COⅡgene. This 6 haplotypes could cluster the 3 large type, C type clusted like the Apis mellifera anatolica, include the 4 haplotypes, and have a little gene mutation. D type clusted with the Apis mellifera ligustica, the E type culsted like the Apis mellifera mellifera, only have one sample.
Synthesized the conclusion of above, we could know the A colony and D type are the same Apis mellifera honeybee samples. The B colony and D type / E type have the same Apis mellifera honeybee samples.
Morphological characters multivariate statistical analysis of Apis cerana from the northeast of Gsnsu and south of Ningxia, could revealed two distanct morphoclusters of bee, F colony was cluster all honeybee come from the plant and low hill region of northeast of Gansu, H colony was cluster from the Liupanshan Mountain in the south of Ningxia. This two place have different climate and geographical type.
The PCR products of Apis cerana were digested with DraⅠand three restricton pattens were revealed.Through sequenced and 11 different haplotypes were found in the non-coding region between tRNAleu and COⅡ.The sequence of the haplotypes were 96-97bp long.The sequences found in the study were compared with those which hand been described before.The alignment results and the phylogenetic trees showed that the honeybee Apis cerana adjacent countries had similar mtDNA sequences and they all falled into the Asian mainland group.
引文
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