鲽科10种鱼类核糖体ITS2序列比较分析
|
摘要
为了解鲽科鱼类ITS2序列的多态性特征,本研究获得了鲽科10种鱼类310条ITS2序列,长度在419～486 bp之间。种内序列长度差异最小的为粒鲽(Clidoderma asperrima)(419～420 bp)和太平洋拟庸鲽(Hippoglossoides elassodon)(419～420 bp),其次为北岩鲽(Lepidopsetta polyxystra)(447～452 bp)和刺黄盖鲽(Limanda aspera)(457～463 bp),松木高眼鲽(Cleisthenes pinetorum)(452～462 bp)和圆斑星鲽(Verasper variegatus)(465～479 bp)的种内序列差异分别为10 bp和14 bp;其余4种鱼类根据长度差异(14～32 bp)分型为长(A型)、短(B型)序列类型,同时检测存在重组类型(R型),其中长度差异最大的是钝吻黄盖鲽(Pseudopleuronectes yokohamae)(454～486 bp),其次为尖吻黄盖鲽(P. herzensteini)(433～458 bp)、虫鲽(Eopsetta grigorjewi)(420～439 bp)、星突江鲽(Platichthys stellatus)(466～480 bp)。通过双参数模型(K2P)计算遗传距离可见,种内遗传距离多集中于0.002～0.027之间,仅星突江鲽和尖吻黄盖鲽因类型差异导致较高数值(0.043和0.053);不同物种间遗传距离在0.046～0.180之间。10种鱼类ITS2的GC含量为63.95%～70.16%;9种鱼类的二级结构均为具有5个分支(HelixⅠ～Ⅴ)的闭合环状结构,仅圆斑星鲽中由于存在HelixⅣ变异形成HelixⅣ-a和HelixⅣ-b而具有6个分支。基于ITS2构建的鲽科10种鱼类的系统进化树显示,不同种鱼类的克隆序列均单独聚支。序列的多态性特征分析表明,在具有不同序列类型的虫鲽、星突江鲽、尖吻黄盖鲽和钝吻黄盖鲽4种鲽科鱼类中,ITS2以非协同进化的方式存在,而其他6种鱼类为协同进化;虽然存在种间K2P遗传距离小于种内的个例,但ITS2在属间不同物种的区分上具有适用性。本研究结果丰富了鲽形目鱼类的ITS2数据,也将为鱼类的核糖体RNA序列多态性的研究提供科学依据。
The main objective of this study was to better understand the characteristics of ITS2 sequence polymorphism in Pleuronectidae species. We cloned and sequenced ITS2 fragments in 10 species of Pleuronectidae, and then performed further analyses, including analysis of polymorphic statistics, calculation of Kimura-2-parameter(K2 P) genetic distances, prediction of secondary structure and minimum free energy, and determination of recombinants. Finally, the phylogenetic relationship among different species was constructed by Maximum likelihood(ML) and Bayesian Inference(BI) method with Bothus myriaster as outgroup species. A total of 310 sequences were obtained from 10 species of Pleuronectidae. Among 6 of 10 species, Clidoderma asperrima(419﹣420 bp) and Hippoglossoides elassodon(419﹣420 bp) have the lowest variation in length, comparing to Lepidopsetta polyxystra(447﹣452 bp) and Limanda aspera(457﹣463 bp) with 5 bp and 6 bp variations, and Cleisthenes pinetorum(452﹣462 bp) and Verasper variegatus(465﹣479 bp) with 10 bp and 14 bp variations. For other four species, the biggest length variation of each species is 14﹣32 bp, resulting in longer, shorter or recombinant sequence types that are classified as types A, B and R. The biggest length variation occurs in Pseudopleuronectes yokohamae(454﹣486 bp), then Ps. herzensteini(433﹣458 bp), Eopsetta grigorjewi(420﹣439 bp), and Platichthys stellatus(466﹣480 bp)(Table 1). The K2 P genetic distances of intra-species ranges from 0.002 to 0.027, except the high values in Pl. stellatus and Ps. herzensteini, up to 0.043 and 0.053, respectively; while the genetic distance of inter-species ranges from 0.046 to 0.180(Table 2). The GC contents of ITS2 in the 10 species range from 63.95% to 70.16%(Table 1). The secondary structure has a uniform loop structure with five branches HelixⅠ﹣Ⅴ, except for V. variegatus with Helix Ⅳ-a and Ⅳ-b(Fig. 2). The phylogenetic trees constructed based on ITS2 sequences of 10 Pleuronectidae species show that the different clones of each species are clustered together(Fig. 3). According to the ITS2 sequence polymorphism characteristics, these four species Ps. yokohamae, Ps. herzensteini, E. grigorjewi, and Pl. stellatus with different types may be evolved in non-concerted evolution, while the other six species in concerted evolution. Although the K2 P genetic distance of intra-species has overlap with that of inter-species, ITS2 is considered to be applicable for species identification among these species. The results of this study will enrich the ITS2 data of flatfish, and further provide a scientific reference for the researches on ribosomal RNA polymorphism of teleostean fishes.
The main objective of this study was to better understand the characteristics of ITS2 sequence polymorphism in Pleuronectidae species. We cloned and sequenced ITS2 fragments in 10 species of Pleuronectidae, and then performed further analyses, including analysis of polymorphic statistics, calculation of Kimura-2-parameter(K2 P) genetic distances, prediction of secondary structure and minimum free energy, and determination of recombinants. Finally, the phylogenetic relationship among different species was constructed by Maximum likelihood(ML) and Bayesian Inference(BI) method with Bothus myriaster as outgroup species. A total of 310 sequences were obtained from 10 species of Pleuronectidae. Among 6 of 10 species, Clidoderma asperrima(419﹣420 bp) and Hippoglossoides elassodon(419﹣420 bp) have the lowest variation in length, comparing to Lepidopsetta polyxystra(447﹣452 bp) and Limanda aspera(457﹣463 bp) with 5 bp and 6 bp variations, and Cleisthenes pinetorum(452﹣462 bp) and Verasper variegatus(465﹣479 bp) with 10 bp and 14 bp variations. For other four species, the biggest length variation of each species is 14﹣32 bp, resulting in longer, shorter or recombinant sequence types that are classified as types A, B and R. The biggest length variation occurs in Pseudopleuronectes yokohamae(454﹣486 bp), then Ps. herzensteini(433﹣458 bp), Eopsetta grigorjewi(420﹣439 bp), and Platichthys stellatus(466﹣480 bp)(Table 1). The K2 P genetic distances of intra-species ranges from 0.002 to 0.027, except the high values in Pl. stellatus and Ps. herzensteini, up to 0.043 and 0.053, respectively; while the genetic distance of inter-species ranges from 0.046 to 0.180(Table 2). The GC contents of ITS2 in the 10 species range from 63.95% to 70.16%(Table 1). The secondary structure has a uniform loop structure with five branches HelixⅠ﹣Ⅴ, except for V. variegatus with Helix Ⅳ-a and Ⅳ-b(Fig. 2). The phylogenetic trees constructed based on ITS2 sequences of 10 Pleuronectidae species show that the different clones of each species are clustered together(Fig. 3). According to the ITS2 sequence polymorphism characteristics, these four species Ps. yokohamae, Ps. herzensteini, E. grigorjewi, and Pl. stellatus with different types may be evolved in non-concerted evolution, while the other six species in concerted evolution. Although the K2 P genetic distance of intra-species has overlap with that of inter-species, ITS2 is considered to be applicable for species identification among these species. The results of this study will enrich the ITS2 data of flatfish, and further provide a scientific reference for the researches on ribosomal RNA polymorphism of teleostean fishes.
引文
Bailey C D,Carr T G,Harris S A,et al.2003.Characterization of angiosperm nrDNA polymorphism,paralogy,and pseudogenes.Molecular Phylogenetics and Evolution,29(3):435-455.
Coleman A W.2007.Pan-eukaryote ITS2 homologies revealed by RNA secondary structure.Nucleic Acids Research,35(10):3322-3329.
Coleman A W.2015.Nuclear rRNA transcript processing versus internal transcribed spacer secondary structure.Trends in Genetics,31(3):157-163.
Darty K,Denise A,Ponty Y.2009.VARNA:Interactive drawing and editing of the RNA secondary structure.Bioinformatics,25(15):1974-1975.
Edger P P,Tang M,Bird K A,et al.2014.Secondary structure snalyses of the nuclear rRNA internal transcribed spacers and sssessment of its phylogenetic utility across the Brassicaceae(Mustards).PLoS One,9(7):e101341.
Fromm L,Falk S,Flemming D,et al.2017.Reconstitution of the complete pathway of ITS2 processing at the pre-ribosome.Nature Communications,8(1):1787.
Gerbi S A.1986.The evolution of eukaryotic ribosomal DNA.Biosystems,19(4):247-258.
Gong L,Shi W,Yang M,et al.2016.Non-concerted evolution in ribosomal ITS2 sequence in Cynoglossus zanzibarensis(Pleuronectiformes:Cynoglossidae).Biochemical Systematics and Ecology,66:181-187.
Gong L,Shi W,Yang M,et al.2018.Marked intra-genomic variation and pseudogenes in the ITS1-5.8S-ITS2 rDNA of Symphurus plagiusa(Pleuronectiformes:Cynoglossidae).Animal Biology,64(4):353-365.
Hall T A.1999.BioEdit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.Nucleic Acids Symposium Series,41(41):95-98.
Joseph N,Krauskopf E,Vera M I,et al.1999.Ribosomal internal transcribed spacer 2(ITS2)exhibits a common core of secondary structure in vertebrates and yeast.Nucleic Acids Research,27(23):4533-4540.
Kitade Y,Ootsuka S,Iitsuka O,et al.2003.Effect of DMSO on PCRof Porphyra yezoensis(Rhodophyta)gene.Journal of Applied Phycology,15(6):555-557.
Kryukov K,Sumiyama K,Ikeo K,et al.2012.A new database(GCD)on genome composition for eukaryote and prokaryote genome sequences and their initial analyses.Genome Biology and Evolution,4(4):501-512.
Larkin M A,Blackshields G,Brown N P,et al.2007.Clustal W and Clustal X version 2.0.Bioinformatics,23(21):2947-2948.
Li Y,Jiao L,Yao Y J.2013.Non-concerted ITS evolution in fungi,as revealed from the important medicinal fungus Ophiocordyceps sinensis.Molecular Phylogenetics and Evolution,68(2):373-379.
Liao D Q.1999.Concerted evolution:Molecular mechanism and biological implications.American Journal of Human Genetics,64(1):24-30.
Librado P,Rozas J.2009.DnaSP v5:a software for comprehensive analysis of DNA polymorphism data.Bioinformatics,25(11):1451-1452.
Marinho M,Junqueira A,Azeredo-Espin A.2011.Evaluation of the internal transcribed spacer 2(ITS2)as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies(Diptera:Calliphoridae).Genetica,139(9):1189-1207.
Marinho M,Junqueira A,Paulo D F,et al.2012.Molecular phylogenetics of Oestroidea(Diptera:Calyptratae)with emphasis on Calliphoridae:Insights into the inter-familial relationships and additional evidence for paraphyly among blowflies.Molecular Phylogenetics and Evolution,65(3):840-854.
Martin D P,Murrell B,Golden M,et al.2015.RDP4:Detection and analysis of recombination patterns in virus genomes.Virus Evolution,1(1):vev003.
Redmond N E,Mc Cormack G P.2009.Ribosomal internal transcribed spacer regions are not suitable for intra-or inter-specific phylogeny reconstruction in haplosclerid sponges(Porifera:Demospongiae).Journal of the Marine Biological Association of the United Kingdom,89(6):1251-1256.
Schoch C L,Seifert K A,Huhndorf S,et al.2012.Nuclear ribosomal internal transcribed spacer(ITS)region as a universal DNAbarcode marker for Fungi.Proceedings of the National Academy of Sciences of the United States of America,109(16):6241-6246.
Schultz J,Wolf M.2009.ITS2 sequence-structure analysis in phylogenetics:A how-to manual for molecular systematics.Molecular Phylogenetics and Evolution,52(2):520-523.
Shapoval N A,Lukhtanov V A.2015.Intragenomic variations of multicopy ITS2 marker in Agrodiaetus blue butterflies(Lepidoptera,Lycaenidae).Comparative Cytogenetics,9(4):483-497.
Tamura K,Stecher G,Peterson D,et al.2013.MEGA6:molecular evolutionary genetics analysis version 6.0.Molecular Biology And Evolution,30(12):2725-2729.
Xu J,Zhang Q,Xu X.2009.Intragenomic variability and pseudogenes of ribosomal DNA in Stone flounder.Molecular Phylogenetics and Evolution,52(1):157-166.
Yao H,Song J Y,Liu C,et al.2010.Use of ITS2 region as the universal DNA barcode for plants and animals.PLoS One,5(10):e13102.
Zheng X,Cai D,Yao L,et al.2008.Non-concerted ITS evolution,early origin and phylogenetic utility of ITS pseudogenes in Pyrus.Molecular Phylogenetics and Evolution,48(3):892-903.
李倩,闫淑珍,陈双林.2015.绒泡菌目黏菌的ITS1-5.8S-ITS2二级结构的比较分析.菌物学报,34(2):235-245.
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.中国水产科学,24(4):657-668.
袁万安.2010.核糖体转录间隔子2应用于鱼类种属的鉴别.遗传,32(4):369-374.
Coleman A W.2007.Pan-eukaryote ITS2 homologies revealed by RNA secondary structure.Nucleic Acids Research,35(10):3322-3329.
Coleman A W.2015.Nuclear rRNA transcript processing versus internal transcribed spacer secondary structure.Trends in Genetics,31(3):157-163.
Darty K,Denise A,Ponty Y.2009.VARNA:Interactive drawing and editing of the RNA secondary structure.Bioinformatics,25(15):1974-1975.
Edger P P,Tang M,Bird K A,et al.2014.Secondary structure snalyses of the nuclear rRNA internal transcribed spacers and sssessment of its phylogenetic utility across the Brassicaceae(Mustards).PLoS One,9(7):e101341.
Fromm L,Falk S,Flemming D,et al.2017.Reconstitution of the complete pathway of ITS2 processing at the pre-ribosome.Nature Communications,8(1):1787.
Gerbi S A.1986.The evolution of eukaryotic ribosomal DNA.Biosystems,19(4):247-258.
Gong L,Shi W,Yang M,et al.2016.Non-concerted evolution in ribosomal ITS2 sequence in Cynoglossus zanzibarensis(Pleuronectiformes:Cynoglossidae).Biochemical Systematics and Ecology,66:181-187.
Gong L,Shi W,Yang M,et al.2018.Marked intra-genomic variation and pseudogenes in the ITS1-5.8S-ITS2 rDNA of Symphurus plagiusa(Pleuronectiformes:Cynoglossidae).Animal Biology,64(4):353-365.
Hall T A.1999.BioEdit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.Nucleic Acids Symposium Series,41(41):95-98.
Joseph N,Krauskopf E,Vera M I,et al.1999.Ribosomal internal transcribed spacer 2(ITS2)exhibits a common core of secondary structure in vertebrates and yeast.Nucleic Acids Research,27(23):4533-4540.
Kitade Y,Ootsuka S,Iitsuka O,et al.2003.Effect of DMSO on PCRof Porphyra yezoensis(Rhodophyta)gene.Journal of Applied Phycology,15(6):555-557.
Kryukov K,Sumiyama K,Ikeo K,et al.2012.A new database(GCD)on genome composition for eukaryote and prokaryote genome sequences and their initial analyses.Genome Biology and Evolution,4(4):501-512.
Larkin M A,Blackshields G,Brown N P,et al.2007.Clustal W and Clustal X version 2.0.Bioinformatics,23(21):2947-2948.
Li Y,Jiao L,Yao Y J.2013.Non-concerted ITS evolution in fungi,as revealed from the important medicinal fungus Ophiocordyceps sinensis.Molecular Phylogenetics and Evolution,68(2):373-379.
Liao D Q.1999.Concerted evolution:Molecular mechanism and biological implications.American Journal of Human Genetics,64(1):24-30.
Librado P,Rozas J.2009.DnaSP v5:a software for comprehensive analysis of DNA polymorphism data.Bioinformatics,25(11):1451-1452.
Marinho M,Junqueira A,Azeredo-Espin A.2011.Evaluation of the internal transcribed spacer 2(ITS2)as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies(Diptera:Calliphoridae).Genetica,139(9):1189-1207.
Marinho M,Junqueira A,Paulo D F,et al.2012.Molecular phylogenetics of Oestroidea(Diptera:Calyptratae)with emphasis on Calliphoridae:Insights into the inter-familial relationships and additional evidence for paraphyly among blowflies.Molecular Phylogenetics and Evolution,65(3):840-854.
Martin D P,Murrell B,Golden M,et al.2015.RDP4:Detection and analysis of recombination patterns in virus genomes.Virus Evolution,1(1):vev003.
Redmond N E,Mc Cormack G P.2009.Ribosomal internal transcribed spacer regions are not suitable for intra-or inter-specific phylogeny reconstruction in haplosclerid sponges(Porifera:Demospongiae).Journal of the Marine Biological Association of the United Kingdom,89(6):1251-1256.
Schoch C L,Seifert K A,Huhndorf S,et al.2012.Nuclear ribosomal internal transcribed spacer(ITS)region as a universal DNAbarcode marker for Fungi.Proceedings of the National Academy of Sciences of the United States of America,109(16):6241-6246.
Schultz J,Wolf M.2009.ITS2 sequence-structure analysis in phylogenetics:A how-to manual for molecular systematics.Molecular Phylogenetics and Evolution,52(2):520-523.
Shapoval N A,Lukhtanov V A.2015.Intragenomic variations of multicopy ITS2 marker in Agrodiaetus blue butterflies(Lepidoptera,Lycaenidae).Comparative Cytogenetics,9(4):483-497.
Tamura K,Stecher G,Peterson D,et al.2013.MEGA6:molecular evolutionary genetics analysis version 6.0.Molecular Biology And Evolution,30(12):2725-2729.
Xu J,Zhang Q,Xu X.2009.Intragenomic variability and pseudogenes of ribosomal DNA in Stone flounder.Molecular Phylogenetics and Evolution,52(1):157-166.
Yao H,Song J Y,Liu C,et al.2010.Use of ITS2 region as the universal DNA barcode for plants and animals.PLoS One,5(10):e13102.
Zheng X,Cai D,Yao L,et al.2008.Non-concerted ITS evolution,early origin and phylogenetic utility of ITS pseudogenes in Pyrus.Molecular Phylogenetics and Evolution,48(3):892-903.
李倩,闫淑珍,陈双林.2015.绒泡菌目黏菌的ITS1-5.8S-ITS2二级结构的比较分析.菌物学报,34(2):235-245.
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.中国水产科学,24(4):657-668.
袁万安.2010.核糖体转录间隔子2应用于鱼类种属的鉴别.遗传,32(4):369-374.