DNA条形码揭示日本纺织娘(Mecopoda niponensis)个体内和个体间的序列变异

摘要/Abstract

摘要： 采用DNA条形码通用引物对分布于我国的两种纺织娘14个个体进行了扩增与测定。结果显示，6个纺织娘Mecopoda elongata个体均可由PCR产物直接测序法获得良好测序结果，序列长度均为658 bp，且不存在碱基插入/缺失。而8个日本纺织娘M. niponensis个体PCR法直接测序结果峰图杂乱，软件无法识别，后经克隆法测定的85条克隆序列长度为580-662 bp，其中，20条为明显的线粒体假基因(Nuclear sequences of mitochondrial origin，numts)，包括4条存在终止密码子，15条存在碱基插入/缺失，1条存在读码框摆动；38条为疑似numts(含≥2个非同义替换位点)。利用Kimura 2-parameter(K2P)模型计算遗传距离发现，两种纺织娘之间的遗传距离为0.077，纺织娘及日本纺织娘个体间的遗传距离分别为0-0.008及0.030-0.051，而日本纺织娘个体内克隆间的遗传距离则分别为0-0.143(含numts)及0-0.083(不含numts)。邻接法系统发育树聚类结果显示，纺织娘形成一个单系分支，自举值为99，而日本纺织娘的COI和numts序列则聚类形成多个分支，提示这些numts序列可能是线粒体COI多次向核内转移的结果。

关键词: 纺织娘, 线粒体DNA, DNA条形码, 线粒体假基因, 异质性

Abstract: The mitochondrial COI barcoding region of 14 individuals from two Mecopoda species with Folmer primers were amplified. All M. elongata COI fragments were 658 bp in length, without stop codons or indels. Due to the serious sequence ambiguities caused by direct sequencing, 85 clones were sequenced from eight M. niponensis individuals, with sequence lengths ranging from 580 to 662 bp. Among these, 20 of the clones were distinct nuclear sequences of mitochondrial origin, numts(four clones with stop codons, 15 clones with indels, one clone was frameshift), and 38 clones contained point mutations that caused two or more amino acid sequence differences as compared with the orthologs. The Kimura 2-parameter(K2P)model was adopted to calculate genetic distance, and the results indicated that the genetic distances among M. elongata individuals varied from 0 to 0.008. Among M. niponensis, intra-individual clones ranged from 0 to 0.143(inclusive of numts)or 0 to 0.083(exclusive of numts), and the inter-individual distance ranged from 0.030 to 0.051(exclusive of numts). The genetic distance between the two Mecopoda species was 0.077. Moreover, the neighbor-joining(NJ)phylogenetic tree based on the Kimura 2-parameter distances indicated that all the M. elongata individuals grouped together with high confidence(99%), and the real COI sequences and numts of M. niponensis clustered into several groups. These results indicate that numerous independent nuclear integration events in M. niponensis were scattered at various time points, which suggests a continuous process of numt generation.

Key words: Mecopoda, NtDNA, DNA barcoding, Numts Heteroplasmy

周志军, 尚娜, 常岩林, 石福明. DNA条形码揭示日本纺织娘(Mecopoda niponensis)个体内和个体间的序列变异[J]. 生物技术通报, 2014, 0(5): 129-136.

Zhou Zhijun, Shang Na, Chang Yanlin, Shi Fuming. DNA Barcodes Reveal Intra-and Inter-Individual Sequence Variation in the Bush Cricket Mecopoda niponensis(Orthoptera：Tettigoniidae)[J]. Biotechnology Bulletin, 2014, 0(5): 129-136.

参考文献

[1] Hebert PDN, Cywinska A, Ball SL, et al. Biological identifications through DNA barcodes[J]. P Roy Soc B-Biol Sci, 2003, 270(1512)：313-321.
[2] 常虹, 郝德君, 肖荣堂, 等. 基于线粒体COI基因的齿小蠹属昆虫DNA条形码研究[J]. 昆虫学报, 2012, 55(9)：1075-1081.
[3] 程磊, 常玉梅, 鲁翠云, 等.鲫属鱼类DNA 条码及种与亚种划分[J]. 动物学研究, 2012, 33(5)：463-472.
[4] Greenstone MH, Rowley DL, Heimbach U, et al. Barcoding generalist predators by polymerase chain reaction：carabids and spiders[J]. Mol Ecol, 2005, 14(10)：3247-3266.
[5] Hebert PDN, Penton EH, Burns JM, et al. Ten species in one：DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator[J]. PNAS, 2004, 101(41)：14812-14817.
[6] Hebert PDN, Stoeckle MY, Zemlak TS, et al. Identification of Birds through DNA Barcodes[J]. PloS Bio, 2004, 2(10)：e312.
[7] Kerr KCR, Stoeckle MY, Dove CJ, et al. Comprehensive DNA barcode coverage of North American birds[J]. Mol Ecol Notes, 2007, 7(4)：535-543.
[8] Kumar NP, Rajavel AR, Natarajan R, et al. DNA barcodes can distinguish species of Indian mosquitoes(Diptera：Culicidae)[J]. J Med Entomol, 2007, 44(1)：1-7.
[9] 马英, 李海龙, 亮鲁, 等. DNA条形码技术在青海海东地区小型兽类鉴定中的应用[J]. 生物多样性, 2012, 20(2)：193-198.
[10] Smith MA, Wood DM, Janzen DH, et al. DNA barcodes affirm that 16 species of apparently generalist tropical parasitoid flies(Diptera, Tachinidae)are not all generalists[J]. PNAS, 2007, 104(12)：4967-4972.
[11] 杨聪慧, 韩辉林, 迟美妍, 等. DNA条形码技术在北京百花山地区夜蛾科物种鉴定中的应用[J]. 昆虫学报, 2012, 55(9)：1082-1092.
[12] Meyer A, Kocher TD, Basasibwaki P, et al. Monophyletic origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA sequences[J]. Nature, 1990, 347(6293)：550-553.
[13] Moritz C, Cicero C. DNA barcoding：promise and pitfalls[J]. PloS Biol, 2004, 2(10)：e354.
[14] Meyer CP, Paulay G. DNA barcoding：error rates based on comprehensive sampling[J]. PloS Biol, 2005, 3(12)：e422.
[15] Elias M, Hill RI, Willmott KR, et al. Limited performance of DNA barcoding in a diverse community of tropical butterflies[J]. P Roy Soc B-Biol Sci, 2007, 274(1627)：2881-2889.
[16] Lopez JV, Yuhki N, Masuda R, et al. Numt, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat[J]. J Mol Evol, 1994, 39(2)：174-190.
[17] Bensasson D, Zhang DX, Hewitt GM. Frequent assimilation of mitochondrial DNA by grasshopper nuclear genomes[J]. Mol Biol Evol, 2000, 17(3)：406-415.
[18] Lu XM, Fu YX, Zhang YP. Evolution of mitochondrial cytochrome B pseudogene in genus Nycticebus[J]. Mol Biol Evol, 2002, 19(12)：2337-2341.
[19] Kolokotronis SO, Macphee RD, Greenwood AD. Detection of mitochondrial insertions in the nucleus(NuMts)of Pleistocene and modern muskoxen[J]. BMC Evol Biol, 2007, 7：67.
[20] Pamilo P, Viljakainen L, Vihavainen A. Exceptionally high density of NUMTs in the honeybee genome[J]. Mol Biol Evol, 2007, 24(6)：1340-1346.
[21] Black IV, WC, Bernhardt SA. Abundant nuclear copies of mitochondrial origin(NUMTs)in the Aedes aegypti genome[J]. Insect Mol Biol, 2009, 18(6)：705-713.
[22] Thalmann O, Hebler J, Poinar HN, et al. Unreliable mtDNA data due to nuclear insertions：a cautionary tale from analysis of humans and other great apes[J]. Mol Ecol, 2004, 13(2)：321-335.
[23] Song H, Buhay JE, Whiting MF, et al. Many species in one：DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified[J]. PNAS, 2008, 105(36)：13486-13491.
[24] Moulton MJ, Song H, Whiting MF. Assessing the effects of primer specificity on eliminating numt coamplification in DNA barcoding：a case study from Orthoptera(Arthropoda：Insecta)[J]. Mol Ecol Resour, 2010, 10(4)：615-627.
[25] Jin XB, Xia KL. An index catalogue of Chinese Tettigonioidea(Orthoptera：Grylloptera)[J]. Journal of Orthoptera Research, 1994, 3：15-41.
[26] 周志军, 杨明茹, 常岩林, 等.两种纺织娘线粒体基因组的比较分析[J]. 昆虫学报, 2013, 56(4)：408-418.
[27] Folmer O, Black M, Hoeh W, et al. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[J]. Mol Mar Biol Biotechnol, 1994, 3(5)：294-299.
[28] Burland TG. DNASTAR’s Lasergene sequence analysis software[J]. Methods Mol Biol, 2000, 132：71-91.
[29] Thompson JD, Gibson TJ, Plewniak F, et al. The CLUSTAL_X windows interface：flexible strategies for multiple sequence alignment aided by quality analysis tools[J]. Nucleic Acids Res, 1997, 25(24)：4876-4882.
[30] Tamura K, Peterson D, Peterson N, et al. MEGA5：molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Mol Biol Evol, 2011, 28(10)：2731-2739.
[31] Cheng S, Higuchi R, Stoneking M. Complete mitochondrial genome amplification[J]. Nat Genet, 1994, 7(3)：350-351.
[32] Collura RV, Auerbach MR, Stewart CB. A quick direct method that can differentiate expressed mitochondrial genes from their nuclear pseudogenes[J]. Curr Biol, 1996, 6：1337-1339.
[33] Sorenson MD, Quinn TW. Numts：a challenge for avian systematics and population biology[J]. The Auk, 1998, 115(1)：214-221.
[34] 王继文.动物线粒体假基因的识别及其在进化生物学中的应用[J]. 动物学杂志, 2004, 39(3)：103-108.
[35] Chung WK, Steiper ME. Mitochondrial COII introgression into the nuclear genome of Gorilla gorilla[J]. Int J Primatol, 2008, 29(5)：1341-1353.
[36] Zhang DX, Godfrey MH. Nuclear integrations：challenges for mitochondrial DNA markers[J]. TREE, 1996, 11(6)：247-251.