长臂缨鲆核糖体RNA基因序列多态性特征分析
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摘要
为了解鲽形目Pleuronectiformes鲆科Bothidae长臂缨鲆Crossorhombuskobensis(Jordan&Starks,1906)核糖体RNA基因的序列多态性特征,本研究共获得该鱼类包括18S、5.8S、ITS1和ITS2全长及28S部分序列的128条克隆序列。经序列比对、聚类分析以及重组检测,结果显示5.8S (158bp)无长度变异,而其他4个基因片段则表现出较高的长度多态性,并可分为不同序列类型:18S (1856～1893 bp)有4种序列类型A、B、C和R; 28S (967～974bp)和ITS1 (407～505bp)均有3种类型A、B和R;ITS2(423～447bp)存在2种类型A和B。此外5个基因片段在碱基组成中均表现出GC偏倚,并且ITS2(71.14%)>ITS1(65.37%)>28S(62.22%)>5.8S(57.67%)>18S(54.95%)。对具有不同序列类型的18S、28S和ITS进行真、假基因推断时,通常的判别特征不足以提供有力依据,因此增加了与4种鲆科近缘鱼类长冠羊舌鲆Arnoglossus macrolophus、青缨鲆Crossorhombus azureus、大鳞短额鲆Engyprosopongrandisquama以及冠毛鲆Lophonectes gallus相应基因片段的比对。各基因片段的插入/缺失以及特异性碱基差异位点比对结果显示:18S和28S的短序列类型A与4种鲆科鱼类序列一致,而其他序列类型则不同; ITS1序列类型A与4种鲆科鱼类在类型B的缺失位点均无缺失,因此推测18S、28S和ITS1的A类型为真基因,而其他类型为假基因。ITS2的A和B类型在差异位点上与4个鲆科鱼类不存在一致性,没有足够的依据对两个类型做出真、假基因的推断。长臂缨鲆核糖体RNA基因中, 5.8S序列最为保守遵循协同进化的方式,而其他4个基因片段为非协同进化的方式。
To better understand the polymorphism characteristics of ribosomal RNA genes of Crossorhombus kobensis(Jordan & Starks, 1906) from Bothidae, Pleuronectiformes, a total of 128 clone sequences were obtained, including full-length sequences of 18 S, ITS1, 5.8S, and ITS2 and partial fragments of 28 S. After sequence alignments, clustering analyses and recombination detection, the results showed that only 5.8S(158 bp) had no length variation, while the other four gene fragments showed high length polymorphism and resulted in several distinct types: 18S(1856-1893 bp) with four types of Types A, B, C, and R; 28S(967-974) and ITS1(407-505 bp) both had three types of Types A, B and R; ITS2(423-447 bp) had two types of Types A and B. All five gene fragments showed GC-bias, and ITS2(71.14%) > ITS1(65.37%) > 28S(62.22%) > 5.8S(57.67%) > 18S(54.95%). The current characteristics criteria were not sufficient to provide strong evidence for the inference of functional gene or pseudogene of 18 S, 28 S and ITS sequences. Therefore, comparison with each of corresponding gene fragment of four affinis species from family Bothidae was conducted, Arnoglossus macrolophus, Crossorhombus azureus, Engyprosopon grandisquama, and Lophonectes gallus. The alignment showed that the indels and differential sites of Type A sequences of both 18 S and 28 S were the similar as those of the four species; and Type A of ITS1, as well as the four species, had no fragment deletion at the missing loci of Type B. Therefore, Type A sequences of 18 S, 28 S and ITS1 were speculated as functional genes, while the other types were putative pseudogenes. As for ITS2, the divergence loci of Type A and Type B compared to each of the four species had no consistency, and there was no evidence to infer the status of ITS2. In this study, 5.8S rDNA is the most conserved gene, suggesting a concerted evolution, while non-concerted evolution was confirmed in other four genes because of high intra-individual polymorphism.
To better understand the polymorphism characteristics of ribosomal RNA genes of Crossorhombus kobensis(Jordan & Starks, 1906) from Bothidae, Pleuronectiformes, a total of 128 clone sequences were obtained, including full-length sequences of 18 S, ITS1, 5.8S, and ITS2 and partial fragments of 28 S. After sequence alignments, clustering analyses and recombination detection, the results showed that only 5.8S(158 bp) had no length variation, while the other four gene fragments showed high length polymorphism and resulted in several distinct types: 18S(1856-1893 bp) with four types of Types A, B, C, and R; 28S(967-974) and ITS1(407-505 bp) both had three types of Types A, B and R; ITS2(423-447 bp) had two types of Types A and B. All five gene fragments showed GC-bias, and ITS2(71.14%) > ITS1(65.37%) > 28S(62.22%) > 5.8S(57.67%) > 18S(54.95%). The current characteristics criteria were not sufficient to provide strong evidence for the inference of functional gene or pseudogene of 18 S, 28 S and ITS sequences. Therefore, comparison with each of corresponding gene fragment of four affinis species from family Bothidae was conducted, Arnoglossus macrolophus, Crossorhombus azureus, Engyprosopon grandisquama, and Lophonectes gallus. The alignment showed that the indels and differential sites of Type A sequences of both 18 S and 28 S were the similar as those of the four species; and Type A of ITS1, as well as the four species, had no fragment deletion at the missing loci of Type B. Therefore, Type A sequences of 18 S, 28 S and ITS1 were speculated as functional genes, while the other types were putative pseudogenes. As for ITS2, the divergence loci of Type A and Type B compared to each of the four species had no consistency, and there was no evidence to infer the status of ITS2. In this study, 5.8S rDNA is the most conserved gene, suggesting a concerted evolution, while non-concerted evolution was confirmed in other four genes because of high intra-individual polymorphism.
引文
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KUMAR R,SINGH M,KUSHWAHA B,et al,2013.Molecular characterization of major and minor rDNA repeats and genetic variability assessment in different species of mahseer found in North India[J].Gene,527(1):248-258.
LARKIN M A,BLACKSHIELDS G,BROWN N P,et al,2007.Clustal W and clustal X version 2.0[J].Bioinformatics,23(21):2947-2948.
LIAO D,1999.Concerted evolution:Molecular mechanism and biological implications[J].American Journal of Human Genetics,64(1):24-30.
LIBRADO P,ROZAS J,2009.Dna SP v5:a software for comprehensive analysis of DNA polymorphism data[J].Bioinformatics,25(11):1451-1452.
MáRQUEZ L M,MILLER D J,MACKENZIE J B,et al,2003.Pseudogenes contribute to the extreme diversity of nuclear ribosomal DNA in the hard coral Acropora[J].Molecular Biology and Evolution,20(7):1077-1086.
MARTIN D P,MURRELL B,GOLDEN M,et al,2015.RDP4:Detection and analysis of recombination patterns in virus genomes[J].Virus Evolution,1(1):vev003.
MEYER A,TODT C,MIKKELSEN N T,et al,2010.Fast evolving18S r RNA sequences from Solenogastres(Mollusca)resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity[J].BMC Evolutionary Biology,10:70.
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NELSON J S,2006.Fishes of the world[M].4th ed.New York:John Wiley and Sons Inc.
P??BO S,IRWIN D M,WILSON A C,1990.DNA damage promotes jumping between templates during enzymatic amplification[J].Journal of Biological Chemistry,265(8):4718-4721.
PéREZ M,VIEITES J M,PRESA P,2005.ITS1-r DNA-based methodology to identify world-wide hake species of the Genus Merluccius[J].Journal of Agricultural and Food Chemistry,53(13):5239-5247.
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XIAO LONGQIAN,M?LLER M,ZHU HUA,2010.High nrDNAITS polymorphism in the ancient extant seed plant Cycas:Incomplete concerted evolution and the origin of pseudogenes[J].Molecular Phylogenetics and Evolution,55(1):168-177.
XIAO LONGQIAN,M?LLER M,2015.Nuclear ribosomal ITSfunctional paralogs resolve the phylogenetic relationships of a late-miocene radiation cycad Cycas(Cycadaceae)[J].PLo SOne,10(1):e0117971.
XU JIANPENG,ZHANG QUANQI,XU XIAOFEI,et al,2009.Intragenomic variability and pseudogenes of ribosomal DNAin Stone flounder Kareius bicoloratus[J].Molecular Phylogenetics and Evolution,52(1):157-166.
XU JING,XU YING,YONEZAWA T,et al,2015.Polymorphism and evolution of ribosomal DNA in tea(Camellia sinensis,Theaceae)[J].Molecular Phylogenetics and Evolution,89:63-72.
YAO HUI,SONG JINGYUAN,LIU CHANG,et al,2010.Use of ITS2 region as the universal DNA barcode for plants and animals[J].PLoS One,5(10):e13102.
ZURIAGA M A,MAS-COMA S,BARGUES M D,2015.Anuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease[J].Memórias Do Instituto Oswaldo Cruz,110(3):353-362.
龚理,2016.鳎亚目(Soleoidei)鱼类核糖体RNA基因序列特征分析及其在系统研究中的适用性[D].北京:中国科学院大学.GONG LI,2016.Sequence analyses of the ribosomal RNAgene in Soleoidei and its application in phylogenetic inference[D].Beijing:University of Chinese Academy of Sciences(in Chinese with English abstract).
龚理,时伟,杨敏,等,2017.5种鳎科鱼类核糖体ITS1序列比较[J].水产学报,41(3):321-329.GONG LI,SHI WEI,YANG MIN,et al,2017.Comparative analysis of the first internal transcribed spacer of ribosomal DNA in five Soleidae species[J].Journal of Fisheries of China,41(3):321-329(in Chinese with English abstract).
司李真,武宝生,孔晓瑜,等,2017.11种鲈形目鱼类的核糖体基因GC含量及其与硬骨鱼类的特征比较[J].中国水产科学,24(4):657-668.SI LIZHEN,WU BAOSHENG,KONGXIAOYU,et al,2017.Analysis of the GC content of ribosomal genes of 11 species of Perci-formes and comparison with other teleostean fishes[J].Journal of Fishery Sciences of China,24(4):657-668(in Chinese with English abstract).
áLVAREZ I,WENDEL J F,2003.Ribosomal ITS sequences and plant phylogenetic inference[J].Molecular Phylogenetics and Evolution,29(3):417-434.
BAILEY C D,CARR T G,HARRIS S A,et al,2003.Characterization of angiosperm nr DNA polymorphism,paralogy,and pseudogenes[J].Molecular Phylogenetics and Evolution,29(3):435-455.
DABERT M,DASTYCH H,HOHBERG K,et al,2014.Phylogenetic position of the enigmatic clawless eutardigrade genus Apodibius Dastych,1983(Tardigrada),based on 18Sand 28S r RNA sequence data from its type species A.confusus[J].Molecular Phylogenetics and Evolution,70:70-75.
ELDER JR J F,TURNER B J,1995.Concerted evolution of repetitive DNA sequences in eukaryotes[J].Quarterly Review of Biology,70(3):297-320.
GONG LI,SHI WEI,YANG MIN,et al,2016a.Long duplication of18S ribosomal DNA in Cynoglossus lineolatus(Pleuronectiformes:Cynoglossidae):novel molecular evidence for unequal crossing over model[J].Acta Oceanologica Sinica,35(12):38-50.
GONG LI,SHI WEI,YANG MIN,et al,2016b.Non-concerted evolution in ribosomal ITS2 sequence in Cynoglossus zanzibarensis(Pleuronectiformes:Cynoglossidae)[J].Biochemical Systematics and Ecology,66:181-187.
GONG LI,SHI WEI,YANG MIN,et al,2018a.Characterization of18S-ITS1-5.8S r DNA in eleven species in Soleidae:implications for phylogenetic analysis[J].Hydrobiologia,819(1):161-175.
GONG LI,SHI WEI,YANG MIN,et al,2018b.Marked intra-genomic variation and pseudogenes in the ITS1-5.8S-ITS2 r DNA of Symphurus plagiusa(Pleuronectiformes:Cynoglossidae)[J].Animal Biology,68(4):353-365.
HALL T A,1999.Bio Edit:a user-friendly biological sequence alignment editor and analysis program for Windows95/98/NT[J].Nucleic Acids Symposium Series,41:95-98.
HILLIS D M,DIXON M T,1991.Ribosomal DNA:molecular evolution and phylogenetic inference[J].Quarterly Review of Biology,66(4):411-453.
KELLER I,CHINTAUAN-MARQUIER I C,VELTSOS P,et al,2006.Ribosomal DNA in the grasshopper Podisma pedestris:Escape from concerted evolution[J].Genetics,174(2):863-874.
KRIEGER J,HETT A K,FUERST P A,et al,2006.Unusual intraindividual variation of the nuclear 18S r RNA gene is widespread within the acipenseridae[J].Journal of Heredity,97(3):218-225.
KUMAR R,SINGH M,KUSHWAHA B,et al,2013.Molecular characterization of major and minor rDNA repeats and genetic variability assessment in different species of mahseer found in North India[J].Gene,527(1):248-258.
LARKIN M A,BLACKSHIELDS G,BROWN N P,et al,2007.Clustal W and clustal X version 2.0[J].Bioinformatics,23(21):2947-2948.
LIAO D,1999.Concerted evolution:Molecular mechanism and biological implications[J].American Journal of Human Genetics,64(1):24-30.
LIBRADO P,ROZAS J,2009.Dna SP v5:a software for comprehensive analysis of DNA polymorphism data[J].Bioinformatics,25(11):1451-1452.
MáRQUEZ L M,MILLER D J,MACKENZIE J B,et al,2003.Pseudogenes contribute to the extreme diversity of nuclear ribosomal DNA in the hard coral Acropora[J].Molecular Biology and Evolution,20(7):1077-1086.
MARTIN D P,MURRELL B,GOLDEN M,et al,2015.RDP4:Detection and analysis of recombination patterns in virus genomes[J].Virus Evolution,1(1):vev003.
MEYER A,TODT C,MIKKELSEN N T,et al,2010.Fast evolving18S r RNA sequences from Solenogastres(Mollusca)resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity[J].BMC Evolutionary Biology,10:70.
MIGHELL A J,SMITH N R,ROBINSON P A,et al,2000.Vertebrate pseudogenes[J].FEBS Letters,468(2-3):109-114.
NELSON J S,2006.Fishes of the world[M].4th ed.New York:John Wiley and Sons Inc.
P??BO S,IRWIN D M,WILSON A C,1990.DNA damage promotes jumping between templates during enzymatic amplification[J].Journal of Biological Chemistry,265(8):4718-4721.
PéREZ M,VIEITES J M,PRESA P,2005.ITS1-r DNA-based methodology to identify world-wide hake species of the Genus Merluccius[J].Journal of Agricultural and Food Chemistry,53(13):5239-5247.
TAMURA K,STECHER G,PETERSON D,et al,2013.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,30(12):2725-2729.
XIAO LONGQIAN,M?LLER M,ZHU HUA,2010.High nrDNAITS polymorphism in the ancient extant seed plant Cycas:Incomplete concerted evolution and the origin of pseudogenes[J].Molecular Phylogenetics and Evolution,55(1):168-177.
XIAO LONGQIAN,M?LLER M,2015.Nuclear ribosomal ITSfunctional paralogs resolve the phylogenetic relationships of a late-miocene radiation cycad Cycas(Cycadaceae)[J].PLo SOne,10(1):e0117971.
XU JIANPENG,ZHANG QUANQI,XU XIAOFEI,et al,2009.Intragenomic variability and pseudogenes of ribosomal DNAin Stone flounder Kareius bicoloratus[J].Molecular Phylogenetics and Evolution,52(1):157-166.
XU JING,XU YING,YONEZAWA T,et al,2015.Polymorphism and evolution of ribosomal DNA in tea(Camellia sinensis,Theaceae)[J].Molecular Phylogenetics and Evolution,89:63-72.
YAO HUI,SONG JINGYUAN,LIU CHANG,et al,2010.Use of ITS2 region as the universal DNA barcode for plants and animals[J].PLoS One,5(10):e13102.
ZURIAGA M A,MAS-COMA S,BARGUES M D,2015.Anuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease[J].Memórias Do Instituto Oswaldo Cruz,110(3):353-362.