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中国新发现芙新姬小蜂孤雌产雌品系的分子鉴定及其内共生菌Rickettsia的检测

杨月梅1,2 轩景丽2 叶福宇2 郭建洋2 杨利艳1 刘万学2

(1.山西师范大学生命科学学院, 山西临汾 041004)
(2.中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室, 北京 100193)

【摘要】【目的】芙新姬小蜂Neochrysocharis formosa (Westwood) 是世界上蔬菜潜叶蝇的优势寄生蜂。2015年6月, 我们在北京发现该寄生蜂存在孤雌产雌品系 (thelytokous strain) , 为在中国首次发现。由于芙新姬小蜂孤雌产雌品系和两性品系 (arrhenotokous strain) 难于从形态上进行区分, 本研究旨在从分子水平对这两种品系进行鉴定, 为孤雌产雌品系的后续研究和应用奠定基础。【方法】以芙新姬小蜂孤雌产雌品系TH-China和两性品系AR-China的室内饲养种群为研究对象, 分别扩增芙新姬小蜂两种品系的核糖体基因 (28S r DNA和ITS-1基因) 和线粒体COⅠ基因序列, 比较两种品系的遗传分化;采用Rickettsia特异性引物NforRick1/NforRick2扩增其16S r DNA, 克隆测序并进行BLAST比对;检测在TH-China卵巢管、幼虫、蛹和成虫, 以及AR-China成虫中是否含有诱导形成孤雌产雌特性的内共生菌Rickettsia。【结果】分别以引物LCO1490/HCO2198和COⅠ1/COⅠ2扩增的两段COⅠ基因序列为分子标记建立的系统发育树表明, 芙新姬小蜂两种品系分别聚为2个分支, 品系间的遗传距离分别为0.039和0.023;以ITS-1基因为分子标记时, 两种品系间有21个差异位点, 遗传距离为0.008;以28S r DNA为分子标记时, 两种品系间无差异位点。从TH-China的Rickettsia中扩增的16S r DNA序列与日本报道的芙新姬小蜂孤雌产雌品系内共生菌Rickettsia sp.的16S r DNA序列 (Gen Bank登录号:AB185963) 100%一致。芙新姬小蜂TH-China卵巢管、幼虫、蛹和成虫中均含有Rickettsia, 而AR-China成虫不含Rickettsia, 推测Rickettsia是孤雌产雌特性的形成原因。【结论】线粒体COⅠ基因可将芙新姬小蜂孤雌产雌品系和两性品系分为2个明显的遗传支系, 而核糖体基因28S r DNA和ITS-1基因在两种品系间没有遗传分化或不能形成2个遗传分化支系。

【关键词】 芙新姬小蜂;孤雌产雌品系;两性品系;分子鉴定;Rickettsia;COⅠ;ITS-1;28S rDNA;

【DOI】

【基金资助】 北京市自然科学基金项目 (6142016) ; 国家重点研发计划项目 (2016YFC1201200) ; 国家自然科学基金项目 (31372005) ;

Molecular identification of the thelytokous strain of Neochrysocharis formosa (Hymenoptera: Eulophidae) newly found in China and detection of its endosymbiont Rickettsia

YANG Yue-Mei1,2 XUAN Jing-Li2 YE Fu-Yu2 GUO Jian-Yang2 YANG Li-Yan1 LIU Wan-Xue2

(1.Shanxi Normal University, College of Life Science, Linfen, Shanxi , China 041004)
(2.State Key Laboratory for Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing , China 100193)

【Abstract】【Aim】The eulophid wasp Neochrysocharis formosa (Westwood) is an important dominant parasitoid against vegetable agromyzid leafminers all over the world. In June 2015, we found the thelytokous strain of N. formosa in Beijing, which was its first discovery in China. It is difficult to distinguish thelytokous strain from arrhenotokous strain because of no distinct morphological differentiation. In this study molecular methods were adopted to identify these two strains so as to provide a basis for future research and application of the thelytokous strain of N. formosa. 【Methods】The genetic differentiation between the thelytokous strain TH-China and the arrhenotokous strain AR-China of N. formosa reared for several generations indoor was analyzed by multiple molecular markers (COⅠgene, ITS-1 gene and 28S rDNA). The 16S rDNA sequence was amplified from the endosymbiont Rickettsia of the two strains using the specific primers NforRick1/NforRick2, sequenced and blasted against NCBI database. The existence of Rickettsia which can induce thelytoky was detected in ovarioles, larvae, pupae and adults of TH-China, and adults of AR-China. 【Results】Phylogenetic trees constructed based on two fragments of COⅠgene amplified with the primer pairs LCO1490/HCO2198 and COⅠ1/COⅠ2 showed that the thelytokous and arrhenotokous strains of N. formosa diverged into two distinct clades and their genetic distance is 0.039 and 0.023, respectively. Using the ITS-1 gene and 28S rDNA as the molecular markers, there are 21 and 0 discrepant sites between the two strains, respectively, and the genetic distance of the two strains based on ITS-1 gene is 0. 008. The 16S rDNA sequence amplified from Rickettsia in TH-China strain has 100% identity with that from Rickettsia in the thelytokous strain in Japan (Gen Bank accession no.: AB185963) . Rickettsia was detected in ovarioles, larvae, pupae and adults of the thelytokous strain, but absent in adults of the arrhenotokous strain, suggesting that Rickettsia might contribute to the thelytoky. 【Conclusion】The thelytokous and arrhenotokous strains of N. formosa are easily differentiated by COⅠgene sequences, while no genetic variation is found based on 28S rDNA and almost no genetic divergence based on ITS-1 gene.

【Keywords】 Neochrysocharis formosa; thelytokous strain; arrhenotokous strain; molecular identification; Rickettsia; COⅠ; ITS-1; 28S rDNA;

【DOI】

【Funds】 Supported by the Beijing Municipal Natural Science Foundation (6142016); National Key Research and Development Program of China (2016YFC1201200); National Natural Science Foundation of China (31372005);

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This Article

ISSN:0454-6296

CN:11-1832/Q

Vol 60, No. 05, Pages 582-593

May 2017

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Abstract

  • 1 Materials and methods
  • 2 Results
  • 3 Discussion
  • References