利用COⅠ基因序列和杂交实验分析鉴定桨角蚜小蜂属的本地种群

Identification of naturally occurring Eretmocerus parasitoids by CO Ⅰsequence analysis and crossing test

桨角蚜小蜂体型小,个体差异小,传统的形态学鉴定方法难以快速准确鉴定。本研究以海氏桨角蚜小蜂,古桥桨角蚜小蜂及两个本地桨角蚜小蜂未知种群为研究对象,利用线粒体DNA细胞色素C氧化酶亚基I(mitochondrial cytochrome oxidase subunit I gene,mt DNA COⅠ)基因序列分析和相似种种间杂交方法,鉴定了桨角蚜小蜂属的两个本地种群(河北廊坊和新疆吐鲁番种群)。通过对靶标片段剪辑序列的测序及比对分析,以最大简约法(Maximum parsimony,MP)和最大似然法(Maximum likelihood,ML)分别构建系统发育树,并以Kimura双参数模型计算种内和种间遗传距离,进而利用种群间杂交手段分析相似种间生殖隔离情况。结果显示:桨角蚜小蜂廊坊种群与古桥桨角蚜小蜂聚为一支,两者的种群内遗传距离分别为0.0029和0.0173,种群间遗传距离为0.011;桨角蚜小蜂新疆种群和海氏桨角蚜小蜂聚为一支,两者的种内遗传距离分别为0.0023和0.000,种间遗传距离0.024,进一步的种间杂交试验表明,新疆种群与海氏桨角蚜小蜂间无生殖隔离现象,证明两种群属于同一物种。桨角蚜小蜂属新疆吐鲁番种与海氏桨角蚜小蜂可能属于同一物种。结合COⅠ基因序列分析和种间杂交试验来共同鉴定未知桨角蚜小蜂不仅可以提高鉴定准确度,更能一定程度上弥补分子鉴定手段可能存在的缺陷。

Eretmocerus parasitoids are of a parasitoid genus with small size and tiny difference,which makes them hard to be identified quickly and accurately by traditional morphological identification methods. The mitochondrial cytochrome c oxidase subunit I（ CO Ⅰ） genes analysis and crossing test between two similar populations were conducted for identifying two Eretmocerus species or populations.The CO Ⅰ genes of 4 Eretmocerus species or populations（ Eretmocerus hayati,Eretmocerus furuhashii,Eretmocerus sp. XJ,Eretmocerus sp. LF） were amplified using universal primers and sequenced. The phylogenetic trees were respectively established by Maximum parsimony（ MP） and Maximum likelihood（ ML） method. The intra- and inter- species genetic distances were calculated with MEGA 5. 0 software using the Kimura 2- Parameter model. Furthermore,a crossing test between two similar populations wasconducted to confirm the reproductive compatible. The results indicated that Er. furuhashii and Er. sp.LF were assembled into the same branch. Meanwhile,the intra- specific genetic distances of two populations were 0. 0029 and 0. 0173,but the inter- specific genetic distance of them was 0. 011.Similarly,Er. hayati and Er. sp. XJ were assembled into the same branch. Meanwhile,the intra-specific genetic distances of two populations were 0. 0023 and 0. 000,but the inter- specific genetic distance of them was 0. 024. Furthermore,the results of crossing test between Er. hayati and Er. sp. XJ suggested that there was no significant reproductive incompatible. That is to say,they were same species.In conclusion,the combination COⅠ sequence analysis with crossing test for identifying similarly small insect species not only improved the accuracy of identification,but also provided an effective complement to COⅠ sequence analysis. This research could shade a light on how to identify of other tiny insects to some extent.