苜蓿滑刃线虫线粒体基因组及其系统发育研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0628

摘要/Abstract

摘要：

苜蓿滑刃线虫（Aphelenchoides medicagus）是一种兼性植物寄生线虫,可在多种真菌上完成生活史,同时对大豆和苜蓿具有弱寄生性。本研究采用Illumina平台对苜蓿滑刃线虫进行低覆盖全基因组测序,从获得的全基因组序列中提取线粒体基因,并利用“种子”序列进行组装;同时对其中富含AT的非编码部分进行PCR扩增与Sanger测序,将扩增与组装的结果进行拼接,共获得14 411 bp的线粒体基因组。基因注释结果显示,苜蓿滑刃线虫共有蛋白编码基因12个,tRNA基因22个,rRNA基因2个,其基因构成和排列与贝西滑刃线虫（A. besseyi）、松材线虫（Bursaphelenchus xylophilus）及拟松材线虫（B. mucronatus）完全相同。基于氨基酸序列的系统发育显示,苜蓿滑刃线虫与贝西滑刃线虫互为姐妹群,且与松材线虫、拟松材线虫处在高度支持的滑刃线虫科单系中。本研究表明,低覆盖全基因组测序法可以成功组装出线粒体基因组的大部分序列,证明了利用该方法获取线虫线粒体全基因组序列的可行性。

关键词: 苜蓿滑刃线虫, 滑刃线虫科, 线粒体基因组, Illumina平台, 低覆盖全基因组测序

Abstract:

Aphelenchoides medicagus is a facultative plant-parasitic nematode that may complete its life cycles on various fungi,meanwhile it has weak parasitism on soybean and alfalfa. In presented study,Illumina platform was used to sequence A. medicagus genome in low coverage. Assembly was made by extracting mitochondrial reads and subsequently assembled by the seed sequence. For the AT rich non-coding region,amplification was made using PCR and sequenced by Sanger method. The jointed result from the assembled and amplified sequences revealed the mitochondrial genome of A. medicagus consisted of 14 411 bp nucleotides,including 12 protein coding genes,22 tRNA and 2 rRNA genes after gene annotation. These genes are arranged in same way as those in A. besseyi,Bursaphelenchus xylophilus,and B. mucronatus. The phylogeny analysis using amino acid sequences showed that A. medicagus and A. besseyi clustered together as a sister group,which together with B. xylophilus and B. mucronatus to form a highly supported monophyletic Aphelenchoididae clade. This study demonstrates that low-coverage whole-genome sequencing method can be used to assemble the most of mitochondrial genome,and thus support this technique to be a feasible tool in mitochondrial genome sequencing.

Key words: Aphelenchoides medicagus, Aphelenchoididae, mitochondrial genome, Illumina platform, low-coverage whole-genome sequencing

薛清, 杜虹锐, 薛会英, 王译浩, 王暄, 李红梅. 苜蓿滑刃线虫线粒体基因组及其系统发育研究[J]. 生物技术通报, 2021, 37(7): 98-106.

XUE Qing, DU Hong-rui, XUE Hui-ying, WANG Yi-hao, WANG Xuan, LI Hong-mei. Mitochondrial Genome and Phylogeny of Aphelenchoides medicagus[J]. Biotechnology Bulletin, 2021, 37(7): 98-106.

图/表 5

参考文献 32

[1]	Sánchez-Monge A,Flores L,Salazar L,et al.An updated list of the plants associated with plant-parasitic Aphelenchoides(Nematoda:Aphelenchoididae)and its implications for plant-parasitism within this genus[J].Zootaxa,2015,4013(2):207-224. doi: 10.11646/zootaxa.4013.2.3 pmid: 26623893
[2]	Hunt DJ.Aphelenchida, Longidoridae and Trichodoridae:Their Systematics and Bionomics[M].UK:CAB International, Wallingford,1993.
[3]	McCuiston JL,Hudson LC,Subbotin SA,et al.Conventional and PCR detection of Aphelenchoides fragariae in diverse ornamental host plant species[J].J Nematol,2007,39(4):343-355. pmid: 19259510
[4]	Jones JT,Haegeman A,Danchin EGJ,et al.Top 10 plant-parasitic nematodes in molecular plant pathology[J].Mol Plant Pathol,2013,14(9):946-961. doi: 10.1111/mpp.12057 pmid: 23809086
[5]	Wang Z,Bert W,Gu JF,et al.Aphelenchoides medicagus n. sp. (Tylenchina:Aphelenchoididae)found in Medicago sativa imported into China from the USA[J].Nematology,2019,21(7):709-723. doi: 10.1163/15685411-00003247 URL
[6]	王珍,李冉,李红梅,等.不同真菌和植物种类对苜蓿滑刃线虫繁殖的影响[J].植物保护,2021,47(1):97-102.
	Wang Z,Li R,Li HM,et al.Effects of different fungi and plant species on reproduction of Aphelenchoides medicagus[J].Plant Prot,2021,47(1):97-102.
[7]	Holterman M,Holovachov O,van den Elsen S,et al.Small subunit ribosomal DNA-based phylogeny of basal Chromadoria(Nematoda)suggests that transitions from marine to terrestrial habitats(and vice versa)require relatively simple adaptations[J].Mol Phylogenetics Evol,2008,48(2):758-763. doi: 10.1016/j.ympev.2008.04.033 URL
[8]	Sultana T,Kim J,Lee SH,et al.Comparative analysis of complete mitochondrial genome sequences confirms independent origins of plant-parasitic nematodes[J].BMC Evol Biol,2013,13:12. doi: 10.1186/1471-2148-13-12 pmid: 23331769
[9]	Hegedusova E,Brejova B,Tomaska L,et al.Mitochondrial genome of the basidiomycetous yeast Jaminaea angkorensis[J].Curr Genet,2014,60(1):49-59. doi: 10.1007/s00294-013-0410-1 pmid: 24071901
[10]	Kern EMA,Kim T,Park JK.The mitochondrial genome in nematode phylogenetics[J].Front Ecol Evol,2020,8:250. DOI:10.3389/fevo,2020.00250. doi: 10.3389/fevo,2020.00250 URL
[11]	Sun LH,Zhuo K,Wang HH,et al.The complete mitochondrial genome of Aphelenchoides besseyi(Nematoda:Aphelenchoididae), the first sequenced representative of the subfamily Aphelenchoidinae[J].Nematology,2014,16(10):1167-1180. doi: 10.1163/15685411-00002844 URL
[12]	Humphreys-Pereira DA,Elling AA.Mitochondrial genomes of Meloidogyne chitwoodi and M. incognita(Nematoda:Tylenchina):comparative analysis, gene order and phylogenetic relationships with other nematodes[J].Mol Biochem Parasitol,2014,194(1/2):20-32. doi: 10.1016/j.molbiopara.2014.04.003 URL
[13]	Kim T,Lee Y,Kil HJ,et al.The mitochondrial genome of Acrobeloides varius(Cephalobomorpha)confirms non-monophyly of Tylenchina(Nematoda)[J].PeerJ,2020,8:e9108. doi: 10.7717/peerj.9108 URL
[14]	Ye F,Samuels DC,Clark T,et al.High-throughput sequencing in mitochondrial DNA research[J].Mitochondrion,2014,17:157-163. doi: 10.1016/j.mito.2014.05.004 URL
[15]	Phillips WS,Brown AMV,Howe DK,et al.The mitochondrial genome of Globodera ellingtonae is composed of two circles with segregated gene content and differential copy numbers[J].BMC Genom,2016,17:706. doi: 10.1186/s12864-016-3047-x URL
[16]	Ma XY,Agudelo P,Richards VP,et al.The complete mitochondrial genome of the Columbia lance nematode, Hoplolaimus columbus, a major agricultural pathogen in North America[J].Parasites Vectors,2020,13(1):321. doi: 10.1186/s13071-020-04187-y URL
[17]	Kanzaki N,Futai K.A PCR primer set for determination of phylogenetic relationships of Bursaphelenchus species within the Xylophilus group[J].Nematology,2002,4(1):35-41. doi: 10.1163/156854102760082186 URL
[18]	Chen S,Zhou Y,Chen Y,et al.Fastp:an ultra-fast all-in-one FASTQ preprocessor[J].Bioinformatics,2018,34(17):i884-i890. doi: 10.1093/bioinformatics/bty560 URL
[19]	Sedlazeck FJ,Rescheneder P,von Haeseler A.NextGenMap:fast and accurate read mapping in highly polymorphic genomes[J].Bioinformatics,2013,29(21):2790-2791. doi: 10.1093/bioinformatics/btt468 pmid: 23975764
[20]	Li H,Handsaker B,Wysoker A,et al.The sequence alignment/map format and SAMtools[J].Bioinformatics,2009,25(16):2078-2079. doi: 10.1093/bioinformatics/btp352 URL
[21]	Meng G,Li Y,Yang C,et al.MitoZ:a toolkit for animal mitochondrial genome assembly, annotation and visualization[J].Nucleic Acids Res,2019,47(11):e63. doi: 10.1093/nar/gkz173 URL
[22]	Bernt M,Donath A,Jühling F,et al.MITOS:improved de novo metazoan mitochondrial genome annotation[J].Mol Phylogenet Evol,2013,69(2):313-319. doi: 10.1016/j.ympev.2012.08.023 URL
[23]	Jühling F,Pütz J,Bernt M,et al.Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements[J].Nucleic Acids Res,2012,40(7):2833-2845. doi: 10.1093/nar/gkr1131 pmid: 22139921
[24]	Kerpedjiev P,Hammer S,Hofacker IL.Forna(force-directed RNA):simple and effective online RNA secondary structure diagrams[J].Bioinformatics,2015,31(20):3377-3379. doi: 10.1093/bioinformatics/btv372 pmid: 26099263
[25]	Kuraku S,Zmasek CM,Nishimura O,et al.aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity[J].Nucleic Acids Res,2013,41(web server issue):W22-W28. doi: 10.1093/nar/gkt389 URL
[26]	Lefort V,Longueville JE,Gascuel O.SMS:smart model selection in PhyML[J].Mol Biol Evol,2017,34(9):2422-2424. doi: 10.1093/molbev/msx149 URL
[27]	Miller MA,Pfeiffer W,Schwartz T.Creating the CIPRES Science Gateway for inference of large phylogenetic trees[C]//2010 Gateway Computing Environments Workshop(GCE). NJ:IEEE,2010:1-8.
[28]	Stamatakis A.RAxML version 8:a tool for phylogenetic analysis and post-analysis of large phylogenies[J].Bioinformatics,2014,30(9):1312-1313. doi: 10.1093/bioinformatics/btu033 URL
[29]	Zhang ZQ.Animal biodiversity:an outline of higher-level classification and survey of taxonomic richness(COVER)[J].Zootaxa,2011,3148(1):3. doi: 10.11646/zootaxa.3148.1 URL
[30]	Bert W,Leliaert F,Vierstraete AR,et al.Molecular phylogeny of the Tylenchina and evolution of the female gonoduct(Nematoda:Rhabditida)[J].Mol Phylogenet Evol,2008,48(2):728-744. doi: 10.1016/j.ympev.2008.04.011 pmid: 18502668
[31]	Gómez-Rodríguez C,Crampton-Platt A,Timmermans MJTN,et al.Validating the power of mitochondrial metagenomics for community ecology and phylogenetics of complex assemblages[J].Methods Ecol Evol,2015,6(8):883-894. doi: 10.1111/mee3.2015.6.issue-8 URL
[32]	Crampton-Platt A,Timmermans MJ,Gimmel ML,et al.Soup to tree:the phylogeny of beetles inferred by mitochondrial metagenomics of a Bornean rainforest sample[J].Mol Biol Evol,2015,32(9):2302-2316. doi: 10.1093/molbev/msv111 pmid: 25957318

基因名 Gene name	起始位点 Start	终止位点 Stop	长度 Length/bp	基因间隔 Intergenic space	起始/终止密码子 Start/stop codons
cox1	955	2535	1 581	0	ATT/TAA
trnC（gca）	2534	2587	54	-2
trnM（cat）	2588	2641	54	0
trnD（gtc）	2642	2696	55	0
trnG（tcc）	2697	2751	55	0
cox2	2761	3450	690	9	ATT/TAA
trnH（gtg）	3450	3503	54	-1
rrnL	3505	4415	911	1
nad3	4423	4758	336	7	ATA/TAA
nad5	4842	6347	1 506	83	ATT/TAG
trnA（tgc）	6346	6399	54	-2
trnP（tgg）	6400	6453	54	0
trnV（tac）	6453	6508	56	-1
nad6	6521	6949	429	12	ATT/TAA
nad4L	6952	7180	229	2	TTG/T
trnW（tca）	7181	7236	56	0
trnE（ttc）	7243	7296	54	6
rrnS	7297	7963	667	0
trnS2（tga）	7966	8019	54	2
trnY（gta）	8021	8074	54	1
nad1	8066	8944	879	-9	ATG/TAA
atp6	8944	9543	600	-1	ATT/TAA
trnK（ttt）	9542	9598	57	-2
trnL2（taa）	9599	9653	55	0
trnS1（tct）	9654	9706	53	0
nad2	9720	10536	817	13	ATA/T
trnI（gat）	10546	10604	59	9
trnR（acg）	10601	10654	54	-4
trnQ（ttg）	10655	10708	54	0
trnF（gaa）	10709	10762	54	0
cob	10760	11884	1 125	-3	ATA/TAA
trnL1（tag）	11880	11936	57	-5
cox3	11942	12691	750	5	ATT/TAG
trnN（gtt）	12692	12745	54	0
trnT（tgt）	12746	12800	55	0
nad4	12807	14033	1 227	6	ATA/TAA

基因名 Gene name	起始位点 Start	终止位点 Stop	长度 Length/bp	基因间隔 Intergenic space	起始/终止密码子 Start/stop codons
cox1	955	2535	1 581	0	ATT/TAA
trnC（gca）	2534	2587	54	-2
trnM（cat）	2588	2641	54	0
trnD（gtc）	2642	2696	55	0
trnG（tcc）	2697	2751	55	0
cox2	2761	3450	690	9	ATT/TAA
trnH（gtg）	3450	3503	54	-1
rrnL	3505	4415	911	1
nad3	4423	4758	336	7	ATA/TAA
nad5	4842	6347	1 506	83	ATT/TAG
trnA（tgc）	6346	6399	54	-2
trnP（tgg）	6400	6453	54	0
trnV（tac）	6453	6508	56	-1
nad6	6521	6949	429	12	ATT/TAA
nad4L	6952	7180	229	2	TTG/T
trnW（tca）	7181	7236	56	0
trnE（ttc）	7243	7296	54	6
rrnS	7297	7963	667	0
trnS2（tga）	7966	8019	54	2
trnY（gta）	8021	8074	54	1
nad1	8066	8944	879	-9	ATG/TAA
atp6	8944	9543	600	-1	ATT/TAA
trnK（ttt）	9542	9598	57	-2
trnL2（taa）	9599	9653	55	0
trnS1（tct）	9654	9706	53	0
nad2	9720	10536	817	13	ATA/T
trnI（gat）	10546	10604	59	9
trnR（acg）	10601	10654	54	-4
trnQ（ttg）	10655	10708	54	0
trnF（gaa）	10709	10762	54	0
cob	10760	11884	1 125	-3	ATA/TAA
trnL1（tag）	11880	11936	57	-5
cox3	11942	12691	750	5	ATT/TAG
trnN（gtt）	12692	12745	54	0
trnT（tgt）	12746	12800	55	0
nad4	12807	14033	1 227	6	ATA/TAA