DNA条形码技术在草地天敌昆虫分类鉴定中的应用
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摘要
利用DNA条形码技术对收集到的8种草地螟寄生性天敌昆虫线粒体DNA细胞色素C氧化酶亚基Ⅰ(mtDNA COⅠ)的部分序列进行测定,通过比对分析,以邻接法(NJ)和最大简约法(MP)构建天敌昆虫系统发育树,以Kimura双参数模型计算天敌昆虫种内及种间的遗传距离。结果发现:与Genbank中寄生蝇及寄生蜂序列进行对比、剪切,共得到584bp的寄生蝇条形码基因序列49条、625bp的寄生蜂条形码基因序列45条。其中,49条寄生蝇的平均碱基含量中AT含量为71.1%,大于GC含量;45条寄生蜂的平均碱基含量中AT含量为74.0%,大于GC含量;均表现出AT偏向性。4种寄生蝇种内平均遗传距离为0.0015,种间平均遗传距离为0.1137;4种寄生蜂种内平均遗传距离为0.0015,种间平均遗传距离为0.3010,两类天敌昆虫的种间遗传距离均远大于种内遗传距离;聚类分析显示每一种天敌对应一个分支。说明基于COⅠ基因的条形码技术可以用于草地螟寄生性天敌昆虫的鉴别。
Loxostege sticticalis Linnaeus is a kind of worldwide pests of husbandry and there are many natural enemies in nature. The purpose of this study is to clarify the identify of the parasitic natural enemy insects of L. sticticalis. We used DNA barcoding technology to determine the partial sequences of mitochondrial cytochrome coxidase subunit Ⅰ gene(CO Ⅰ) of 8 species of parasitic natural enemies of L. sticticalis. Based on the comparison analysis, we use neighbor-joining(NJ) and maximum parsimony(MP) to construct phylogenetic tree and Kimura's two-parameter model to calculate the intraspecific and interspecific genetic distance of enemy insects. The results indicated that by comparing and shearing the sequences of parasitic flies and parasitic wasps in Genbank, a total of 94 bar code gene sequences of parasitic flies of 584 bp and 625 bp of parasitic wasps were obtained; The average AT content of 49 parasitic flies was 71.1%, higher than that of GC content(28.9%); The average AT content of 45 parasitic wasps was 74.0%, which was higher than GC content(26%), all of them showed AT bias. The average intraspecific genetic distance of four tachinids was 0.0015, average interspecific genetic distance was 0.1137, the Average intra-specific genetic distance of four parasitoids was 0.0015, average inter-specific genetic distance was 0.3010, the genetic distance between species is much larger than those within species; Cluster analysis showed that there was a branch for each natural enemy. The barcoding technology based on CO Ⅰ gene can be used for the identification of parasitic natural enemies of L. sticticalis.
Loxostege sticticalis Linnaeus is a kind of worldwide pests of husbandry and there are many natural enemies in nature. The purpose of this study is to clarify the identify of the parasitic natural enemy insects of L. sticticalis. We used DNA barcoding technology to determine the partial sequences of mitochondrial cytochrome coxidase subunit Ⅰ gene(CO Ⅰ) of 8 species of parasitic natural enemies of L. sticticalis. Based on the comparison analysis, we use neighbor-joining(NJ) and maximum parsimony(MP) to construct phylogenetic tree and Kimura's two-parameter model to calculate the intraspecific and interspecific genetic distance of enemy insects. The results indicated that by comparing and shearing the sequences of parasitic flies and parasitic wasps in Genbank, a total of 94 bar code gene sequences of parasitic flies of 584 bp and 625 bp of parasitic wasps were obtained; The average AT content of 49 parasitic flies was 71.1%, higher than that of GC content(28.9%); The average AT content of 45 parasitic wasps was 74.0%, which was higher than GC content(26%), all of them showed AT bias. The average intraspecific genetic distance of four tachinids was 0.0015, average interspecific genetic distance was 0.1137, the Average intra-specific genetic distance of four parasitoids was 0.0015, average inter-specific genetic distance was 0.3010, the genetic distance between species is much larger than those within species; Cluster analysis showed that there was a branch for each natural enemy. The barcoding technology based on CO Ⅰ gene can be used for the identification of parasitic natural enemies of L. sticticalis.
引文
[1] 曹卫菊,罗礼智,徐建祥.我国草地螟的迁飞规律及途径[J].昆虫知识,2006,43(3):279-283.Cao Weiju,Luo Lizhi,Xu Jiangxiang.The migratory behavior and pathways of meadow moth,Loxostege sticticalis in China[J].Chinese Bulletin of Entomology,2006,43(3):279-283.
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[3] 陈晓,姜玉英,孟正平,等.极端气候成为我国草地螟暴发周期终结的重要因子[J].昆虫学报,2016,59(12):1363-1375.Chen Xiao,Jiang Yuying,Meng Zhengping,et al.Extreme climate has become an important factor causing the termination of outbreak periods of Loxost sticticalis(Lepidoptera:Pyralidae) in China[J].Acta Entomologica Sinica,2016,59(12):1363-1375.
[4] 张跃进,姜玉英,江幸福.我国草地螟关键控制技术研究进展[J].中国植保导刊,2008,28(5):15-19.Zhang Yuejin,Jiang Yuying,Jiang Xingfu.Advances on the key control techniques of Loxostege sticticalis in China[J].China Plant Protection,2008,28(5):15-19.
[5] Mikhal'Tsov V P,Khitsova L N.Extent of infestation of beet webworm by some species of tachinids (Diptera,Tachinidae) as an index of their range//Skarlato O A.Systematics of diptera(Insecta):Ecological and morphological principles[C].New Delhi:Oxonian Press,1985:95-96.
[6] 崔万里.草地螟生物学特性的观察[J].昆虫知识,1992(5):289-292.Cui Wanli.Observation on biological characteristics of Loxostege sticticalis[J].Entomological Knowledge,1992(5):289-292.
[7] Malysh J M,Kazartsev I A,Frolov A N,et al.Molecular detection of Cotesia vestalis(Hymenoptera:Braconidae) in the beet webworm Loxostege sticticalis L.(Lepidoptera:Crambidae) [J].Journal of Applied Entomology,2015,140(3):232-235.
[8] 徐林波,刘爱萍,王慧.草地螟的生物学特性及室内毒力测定研究[J].草业科学,2007,24(9):83-85.Xu Linbo,Liu Aiping,Wang Hui.Studies on biological characteristic and virulence test of Loxostege sticticalis[J].Pratacultural Science,2007,24(9):83-85.
[9] Simmonds F J.The biology of parasites of Loxostege sticticalis L.in North America.IV.Cryptus inornatus Pratt(Ichneumonidae,Cryptinae)[J].Proceedings of the Royal Society A,1948,23(1):71-79.
[10] Hebert P D N,Cywinska A,Ball S L,et al.Biological identifications through DNA barcodes[J].Proceedings of the Royal Society of Biological Sciences,2003,270(1512):313-321.
[11] Hebert P D N,Penton E H,Burns J M,et al.Ten species in one:DNA barcoding reveals cryptic species in theneotroical skipper butterfly Astraptes fulgerator[J].Proceedings of the National Academy of Sciences of the United States of America,2004a,101 (41):14812-14817.
[12] Brown W M,George M,Wilson A C.Rapid evolution of animal mitochondrial DNA[J].Proceedings of National Academy of Sciences of the United States of America,1979,76(4):1967-1971.
[13] Taberlet P,Coissac E,Pompanon F,et al.Power and limitations of the chloroplast trn L (UAA) intron for plant DNA barcoding[J].Nucleic Acids Research,2007,35(3):e14.
[14] Hajibabaei M,Janzen D H,Burns J M,et al.DNA barcodes distinguish species of tropical Lepidoptera[J].Proceedings of National Academy of Sciences of the United States of America,2006,103(4):968-971.
[15] 王剑峰,乔格侠.DNA条形编码在蚜虫类昆虫中的应用[J].动物分类学报,2007,32(1):153-159.Wang Jianfeng,Qiao Gexia.Application of DNA barcoding to Aphidinea(Insecta:Hemiptera) studies[J].Acta Zootaxonomica Sinica,2007,32(1):153-159.
[16] Buahom N,李志红,吴佳教,等.基于 DNA条形码技术的泰国番石榴中实蝇幼虫分子鉴定研究[J].植物检疫,2011,25(1):49-52.Buahom N,Li Zhihong,Wu Jiajiao,et al.Molecular identification of fruit fly larvae from Thailand based on DNA barcoding[J].Plant Quarantine,2011,25(1):49-52.
[17] Karimi J,Hassani-kakhki M,Awal M M.Identifying thrips (Insecta:Thysanoptera) using DNA barcodes[J].Journal of Cell and Molecular Research,2010,2(1):35-41.
[18] 尤欢,周力兵,邓裕亮,等.云南边境地区果实蝇属种类DNA条形码鉴定[J].昆虫学报,2014,57(11):1343-1350.You Huan,Zhou Libing,Deng Yuliang,et al.Identification of Bactrocera species from the border areas of Yunnan province,southwestern China using DNA barcoding(Diptera:Tephritidae)[J].Acta Entomologica Sinica,2014,57(11):1343-1350.
[19] 乔玮娜,万方浩,张爱兵,等.DNA条形码技术在田间常见蓟马种类识别中的应用[J].昆虫学报,2012,55(3):344-356.Qiao Weina,Wan Fanghao,Zhang Aibing,et al.Application of DNA barcoding technology for species identification of common thrips(Insecta:Thysanoptera) in China[J].Acta Entomologica Sinica,2012,55(3):344-356.
[20] Kumar S,Tamura K,Nei M.MEGA3:Integrated software for molecular evolutionary genetics analysis and sequence alignment[J].Briefings in Bioinformatics,2004,5(2):150-163.
[21] 李晓叶,钱路,陈茂华.DNA条形码技术在几种苹果园鳞翅目害虫鉴定中的应用[J].西北农业学报,2015,24(5):141-147.Li Xiaoye,Qian Lu,Chen Maohua.Application of DNA barcoding technology to identification of some Lepidopera pests in apple orchards[J].Acta Agriculturae Boreali-occidentalis Sinica,2015,24(5):141-147.
[22] Behere G T,Tay W T,Russell D A,et al.Molecular markers to discriminate among four pest species of Helicoverpa(Lepidoptera:Noctuidae)[J].Bulletin of Entomological Research,2008,98(6):599-603.
[23] 杨明禄,肖丽,韩旭,等.DNA条形码技术在花黄斑蜂鉴定中的应用[J].中国农学通报,2015,31(32):22-25.Yang Minglu,Xiao Li,Han Xu,et al.Application of DNA barcoding in classification of Anthidium florentinum(Hymenoptera:Megachilidae) [J].Chinese Agricultural Science Bulletin,2015,31(32):22-25.
[24] 胡泽章,孙猛,吕兵,等.DNA条形码技术在小花蝽属昆虫分类鉴定中的应用[J].中国生物防治学报,2017,33(4):487-495.Hu Zezhang,Sun Meng,Lv Bing,et al.Application of DNA barcoding technique for species identification of Orius Wolff(Heteroptera:Anthocoridae)[J].Chinese Journal of Biological Control,2017,33(4):487-495.
[25] Hebert P D N,Gregory T.The promise of DNA barcoding for taxonomy[J].Systematic Biology,2005,54(5):852-859.
[26] Aliabadian M,Kaboli M,Nijman V,et al.Molecular identification of birds:performance of distance-based DNA barcoding in three genes to delimit parapatric species[J].PLoS ONE,2009,4(1):e4119.
[2] 罗礼智,李光博,曹雅忠.草地螟第3个猖獗为害周期已经来临[J].植物保护,1996,22(5):50-51.Luo Lizhi,Li Guangbo,Cao Yazhong.Third rampant cycles of Loxostege sticticalis have arrived[J].Plant Protection,1996,22(5):50-51.
[3] 陈晓,姜玉英,孟正平,等.极端气候成为我国草地螟暴发周期终结的重要因子[J].昆虫学报,2016,59(12):1363-1375.Chen Xiao,Jiang Yuying,Meng Zhengping,et al.Extreme climate has become an important factor causing the termination of outbreak periods of Loxost sticticalis(Lepidoptera:Pyralidae) in China[J].Acta Entomologica Sinica,2016,59(12):1363-1375.
[4] 张跃进,姜玉英,江幸福.我国草地螟关键控制技术研究进展[J].中国植保导刊,2008,28(5):15-19.Zhang Yuejin,Jiang Yuying,Jiang Xingfu.Advances on the key control techniques of Loxostege sticticalis in China[J].China Plant Protection,2008,28(5):15-19.
[5] Mikhal'Tsov V P,Khitsova L N.Extent of infestation of beet webworm by some species of tachinids (Diptera,Tachinidae) as an index of their range//Skarlato O A.Systematics of diptera(Insecta):Ecological and morphological principles[C].New Delhi:Oxonian Press,1985:95-96.
[6] 崔万里.草地螟生物学特性的观察[J].昆虫知识,1992(5):289-292.Cui Wanli.Observation on biological characteristics of Loxostege sticticalis[J].Entomological Knowledge,1992(5):289-292.
[7] Malysh J M,Kazartsev I A,Frolov A N,et al.Molecular detection of Cotesia vestalis(Hymenoptera:Braconidae) in the beet webworm Loxostege sticticalis L.(Lepidoptera:Crambidae) [J].Journal of Applied Entomology,2015,140(3):232-235.
[8] 徐林波,刘爱萍,王慧.草地螟的生物学特性及室内毒力测定研究[J].草业科学,2007,24(9):83-85.Xu Linbo,Liu Aiping,Wang Hui.Studies on biological characteristic and virulence test of Loxostege sticticalis[J].Pratacultural Science,2007,24(9):83-85.
[9] Simmonds F J.The biology of parasites of Loxostege sticticalis L.in North America.IV.Cryptus inornatus Pratt(Ichneumonidae,Cryptinae)[J].Proceedings of the Royal Society A,1948,23(1):71-79.
[10] Hebert P D N,Cywinska A,Ball S L,et al.Biological identifications through DNA barcodes[J].Proceedings of the Royal Society of Biological Sciences,2003,270(1512):313-321.
[11] Hebert P D N,Penton E H,Burns J M,et al.Ten species in one:DNA barcoding reveals cryptic species in theneotroical skipper butterfly Astraptes fulgerator[J].Proceedings of the National Academy of Sciences of the United States of America,2004a,101 (41):14812-14817.
[12] Brown W M,George M,Wilson A C.Rapid evolution of animal mitochondrial DNA[J].Proceedings of National Academy of Sciences of the United States of America,1979,76(4):1967-1971.
[13] Taberlet P,Coissac E,Pompanon F,et al.Power and limitations of the chloroplast trn L (UAA) intron for plant DNA barcoding[J].Nucleic Acids Research,2007,35(3):e14.
[14] Hajibabaei M,Janzen D H,Burns J M,et al.DNA barcodes distinguish species of tropical Lepidoptera[J].Proceedings of National Academy of Sciences of the United States of America,2006,103(4):968-971.
[15] 王剑峰,乔格侠.DNA条形编码在蚜虫类昆虫中的应用[J].动物分类学报,2007,32(1):153-159.Wang Jianfeng,Qiao Gexia.Application of DNA barcoding to Aphidinea(Insecta:Hemiptera) studies[J].Acta Zootaxonomica Sinica,2007,32(1):153-159.
[16] Buahom N,李志红,吴佳教,等.基于 DNA条形码技术的泰国番石榴中实蝇幼虫分子鉴定研究[J].植物检疫,2011,25(1):49-52.Buahom N,Li Zhihong,Wu Jiajiao,et al.Molecular identification of fruit fly larvae from Thailand based on DNA barcoding[J].Plant Quarantine,2011,25(1):49-52.
[17] Karimi J,Hassani-kakhki M,Awal M M.Identifying thrips (Insecta:Thysanoptera) using DNA barcodes[J].Journal of Cell and Molecular Research,2010,2(1):35-41.
[18] 尤欢,周力兵,邓裕亮,等.云南边境地区果实蝇属种类DNA条形码鉴定[J].昆虫学报,2014,57(11):1343-1350.You Huan,Zhou Libing,Deng Yuliang,et al.Identification of Bactrocera species from the border areas of Yunnan province,southwestern China using DNA barcoding(Diptera:Tephritidae)[J].Acta Entomologica Sinica,2014,57(11):1343-1350.
[19] 乔玮娜,万方浩,张爱兵,等.DNA条形码技术在田间常见蓟马种类识别中的应用[J].昆虫学报,2012,55(3):344-356.Qiao Weina,Wan Fanghao,Zhang Aibing,et al.Application of DNA barcoding technology for species identification of common thrips(Insecta:Thysanoptera) in China[J].Acta Entomologica Sinica,2012,55(3):344-356.
[20] Kumar S,Tamura K,Nei M.MEGA3:Integrated software for molecular evolutionary genetics analysis and sequence alignment[J].Briefings in Bioinformatics,2004,5(2):150-163.
[21] 李晓叶,钱路,陈茂华.DNA条形码技术在几种苹果园鳞翅目害虫鉴定中的应用[J].西北农业学报,2015,24(5):141-147.Li Xiaoye,Qian Lu,Chen Maohua.Application of DNA barcoding technology to identification of some Lepidopera pests in apple orchards[J].Acta Agriculturae Boreali-occidentalis Sinica,2015,24(5):141-147.
[22] Behere G T,Tay W T,Russell D A,et al.Molecular markers to discriminate among four pest species of Helicoverpa(Lepidoptera:Noctuidae)[J].Bulletin of Entomological Research,2008,98(6):599-603.
[23] 杨明禄,肖丽,韩旭,等.DNA条形码技术在花黄斑蜂鉴定中的应用[J].中国农学通报,2015,31(32):22-25.Yang Minglu,Xiao Li,Han Xu,et al.Application of DNA barcoding in classification of Anthidium florentinum(Hymenoptera:Megachilidae) [J].Chinese Agricultural Science Bulletin,2015,31(32):22-25.
[24] 胡泽章,孙猛,吕兵,等.DNA条形码技术在小花蝽属昆虫分类鉴定中的应用[J].中国生物防治学报,2017,33(4):487-495.Hu Zezhang,Sun Meng,Lv Bing,et al.Application of DNA barcoding technique for species identification of Orius Wolff(Heteroptera:Anthocoridae)[J].Chinese Journal of Biological Control,2017,33(4):487-495.
[25] Hebert P D N,Gregory T.The promise of DNA barcoding for taxonomy[J].Systematic Biology,2005,54(5):852-859.
[26] Aliabadian M,Kaboli M,Nijman V,et al.Molecular identification of birds:performance of distance-based DNA barcoding in three genes to delimit parapatric species[J].PLoS ONE,2009,4(1):e4119.