长臂缨鲆核糖体RNA基因序列多态性特征分析

doi:10.11978/2018038

热带海洋学报 ›› 2019, Vol. 38 ›› Issue (1): 55-66.doi: 10.11978/2018038CSTR: 32234.14.2018038

长臂缨鲆核糖体RNA基因序列多态性特征分析

杨敏^1,²(), 孔晓瑜¹(), 时伟¹, 龚理^1,³

1. 中国科学院热带海洋生物资源与生态重点实验室, 中国科学院南海海洋研究所, 广东广州 510301
2. 中国科学院大学, 北京 100049
3. 浙江海洋大学, 海洋科学与技术学院, 国家海洋设施养殖工程技术研究中心, 浙江舟山 316022

收稿日期:2018-04-09 修回日期:2018-06-07 出版日期:2019-01-16 发布日期:2019-01-16
作者简介:
作者简介：杨敏 (1989—), 女, 山东省日照市人, 博士研究生, 从事鱼类分类及系统进化研究。E-mail: minyang@scsio.ac.cn
基金资助:
国家自然科学基金项目(31272273)

Analysis of polymorphism characteristics of ribosomal RNA genes in Crossorhombus kobensis (Pleuronectiformes: Bothidae)

Min YANG^1,²(), Xiaoyu KONG¹(), Wei SHI¹, Li GONG¹

1. Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
3. National Engineering Research Center for Facilitated Marine Aquaculture, Marine Science and Technology College, Zhejiang Ocean University, Zhoushan, 316022, China

Received:2018-04-09 Revised:2018-06-07 Online:2019-01-16 Published:2019-01-16
Supported by:
National Natural Science Foundation of China (31272273)

摘要/Abstract

摘要：

为了解鲽形目Pleuronectiformes鲆科Bothidae长臂缨鲆Crossorhombus kobensis (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 (423~447 bp)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Engyprosopon grandisquama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。

关键词: 核糖体RNA基因, 长臂缨鲆, 多态性, 假基因, 非协同进化, 重组

Abstract:

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 18S, ITS1, 5.8S, and ITS2 and partial fragments of 28S. 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 18S, 28S 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 18S and 28S 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 18S, 28S 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.

Key words: ribosomal RNA gene, Crossorhombus kobensis, polymorphism, pseudogene, non-concerted evolution, recombination

杨敏, 孔晓瑜, 时伟, 龚理. 长臂缨鲆核糖体RNA基因序列多态性特征分析[J]. 热带海洋学报, 2019, 38(1): 55-66.

Min YANG, Xiaoyu KONG, Wei SHI, Li GONG. Analysis of polymorphism characteristics of ribosomal RNA genes in Crossorhombus kobensis (Pleuronectiformes: Bothidae)[J]. Journal of Tropical Oceanography, 2019, 38(1): 55-66.

图/表 6

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参考文献 34

[1]	龚理, 徐晖, 李军, 等, 2015. 褐牙鲆(♀)、夏鲆(♂)及其杂交子一代的ITS1序列特征分析[J]. 中国水产科学, 22(1): 17-23.
	GONG LI, XU HUI, LI JUN, et al, 2015. Characterization of the first internal transcribed spacer of ribosomal DNA in Paralichthys olivaceus (♀) and P. dentatus (♂) hybrids[J]. Journal of Fishery Sciences of China, 22(1): 17-23 (in Chinese with English abstract).
[2]	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 RNA gene in Soleoidei and its application in phylogenetic inference[D]. Beijing:University of Chinese Academy of Sciences (in Chinese with English abstract).
[3]	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).
[4]	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, KONG XIAOYU, 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).
[5]	ÁLVAREZ I, WENDEL J F, 2003. Ribosomal ITS sequences and plant phylogenetic inference[J]. Molecular Phylogenetics and Evolution, 29(3): 417-434.
[6]	BAILEY C D, CARR T G, HARRIS S A, et al, 2003. Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes[J]. Molecular Phylogenetics and Evolution, 29(3): 435-455.
[7]	DABERT M, DASTYCH H, HOHBERG K, et al, 2014. Phylogenetic position of the enigmatic clawless eutardigrade genus Apodibius Dastych, 1983 (Tardigrada), based on 18S and 28S rRNA sequence data from its type species A. confusus[J]. Molecular Phylogenetics and Evolution, 70: 70-75.
[8]	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.
[9]	GONG LI, SHI WEI, YANG MIN, et al, 2016a. Long duplication of 18S ribosomal DNA in Cynoglossus lineolatus (Pleuronectiformes: Cynoglossidae): novel molecular evidence for unequal crossing over model[J]. Acta Oceanologica Sinica, 35(12): 38-50.
[10]	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.
[11]	GONG LI, SHI WEI, YANG MIN, et al, 2018a. Characterization of 18S-ITS1-5.8S rDNA in eleven species in Soleidae: implications for phylogenetic analysis[J]. Hydrobiologia, 819(1): 161-175.
[12]	GONG LI, SHI WEI, YANG MIN, et al, 2018b. Marked intra-genomic variation and pseudogenes in the ITS1-5.8S-ITS2 rDNA of Symphurus plagiusa (Pleuronectiformes: Cynoglossidae)[J]. Animal Biology, 68(4): 353-365.
[13]	HALL T A, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J]. Nucleic Acids Symposium Series, 41: 95-98.
[14]	HILLIS D M, DIXON M T, 1991. Ribosomal DNA: molecular evolution and phylogenetic inference[J]. Quarterly Review of Biology, 66(4): 411-453.
[15]	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.
[16]	KRIEGER J, HETT A K, FUERST P A, et al, 2006. Unusual intraindividual variation of the nuclear 18S rRNA gene is widespread within the acipenseridae[J]. Journal of Heredity, 97(3): 218-225.
[17]	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.
[18]	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.
[19]	LIAO D, 1999. Concerted evolution: Molecular mechanism and biological implications[J]. American Journal of Human Genetics, 64(1): 24-30.
[20]	LIBRADO P, ROZAS J, 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data[J]. Bioinformatics, 25(11): 1451-1452.
[21]	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.
[22]	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.
[23]	MEYER A, TODT C, MIKKELSEN N T, et al, 2010. Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity[J]. BMC Evolutionary Biology, 10: 70.
[24]	MIGHELL A J, SMITH N R, ROBINSON P A, et al, 2000. Vertebrate pseudogenes[J]. FEBS Letters, 468(2-3): 109-114.
[25]	NELSON J S, 2006. Fishes of the world[M]. 4th ed. New York: John Wiley and Sons Inc.
[26]	PÄÄBO 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.
[27]	PÉREZ M, VIEITES J M, PRESA P, 2005. ITS1-rDNA-based methodology to identify world-wide hake species of the Genus Merluccius[J]. Journal of Agricultural and Food Chemistry, 53(13): 5239-5247.
[28]	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.
[29]	XIAO LONGQIAN, MÖLLER M, ZHU HUA, 2010. High nrDNA ITS 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.
[30]	XIAO LONGQIAN, MÖLLER M, 2015. Nuclear ribosomal ITS functional paralogs resolve the phylogenetic relationships of a late-miocene radiation cycad Cycas (Cycadaceae)[J]. PLoS One, 10(1): e0117971.
[31]	XU JIANPENG, ZHANG QUANQI, XU XIAOFEI, et al, 2009. Intragenomic variability and pseudogenes of ribosomal DNA in Stone flounder Kareius bicoloratus[J]. Molecular Phylogenetics and Evolution, 52(1): 157-166.
[32]	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.
[33]	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.
[34]	ZURIAGA M A, MAS-COMA S, BARGUES M D, 2015. A nuclear 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.

基因	正/反向引物及序列(5'-3')		长臂缨鲆 C. kobensis	长冠羊舌鮃 A. macrolophus	青缨鲆 C. azureus	大鳞短额鲆 E. grandisquama	冠毛鲆 L. gallus
18S	Z-18S-10	5'-TCTGGTTGATTCTGCCAGTAG-3'	MH031723—MH031736	MH173821	MH173822	MH173823	MH173824
18S	F-18S-1874	5'-ATGATCCTTCCGCAGGTTCA-3'	MH031723—MH031736	MH173821	MH173822	MH173823	MH173824
ITS1-5.8S	Z-18S-1720	5ʹ-TCGCTACTACCGATTGGATGGTTTA-3’	MH036892—MH036912	MH179078	MH179079	MH179080	MH179081
ITS1-5.8S	F-5.8S-150	5'-AAGCGACCCTCAGACAGGCGTAG-3'	MH036892—MH036912	MH179078	MH179079	MH179080	MH179081
5.8S-ITS2	Z-5.8S-10	5'-CAACTCTTAGCGGTGGATCA-3'	MH036913—MH036924	MH179074	MH179075	MH179076	MH179077
5.8S-ITS2	F-28S-100	5'-GCTCTTCCCTCTTCACTCG-3'	MH036913—MH036924	MH179074	MH179075	MH179076	MH179077
ITSZ	Z-18S-1720	5ʹ-TCGCTACTACCGATTGGATGGTTTA-3’	MH036925—MH036936	—	—	—	—
ITSZ	F-28S-100	5'-GCTCTTCCCTCTTCACTCG-3'	MH036925—MH036936	—	—	—	—
28S*	Z-28S-900	5'-CCCACCCCGACCCGTCTTGAA-3'	MH031711—MH031722	MH173825	MH173826	MH173827	MH173828
28S*	F-28S-1800	5'-GACTGACCCATGTTCAACTGCTGT-3'	MH031711—MH031722	MH173825	MH173826	MH173827	MH173828

基因类型		克隆数	长度范围 /bp	保守位点	变异位点	简约位点	单碱基突变	单倍型	单倍型多样性	核苷酸多样度	平均核苷酸差异数	GC含量 /%	最小自由能 /(kcal·mol^-1)
18S	A	6	1856~1858	1829	28	0	28	6	1	0.00503	9.333	54.74~54.98	-715.1~-722.5
	B	2	1892~1893	1880	12	—	—	2	1	0.00634	12	55.18~55.20	-729.5~-730.4
	C	2	1877~1878	1868	9	—	—	2	1	0.00479	9	54.84~54.90	-715.8~-721.9
	R	4	1861~1865	1837	30	3	27	4	1	0.00836	15.5	54.83~55.16	-706.6~-722.7
	合计	14	1856~1893	1814	79	7	72	14	1	0.00734	13.462	54.74~55.20	-706.6~-730.4
ITS1	A	23	500~505	488	22	7	15	21	0.9921	0.00736	3.605	64.81~65.81	-223.6~-234.8
	B	6	407~408	403	5	0	5	4	0.8	0.0041	1.667	65.93~66.67	-190.2~-194.0
	R	4	430~463	417	13	0	13	4	1	0.01485	6	63.26~66.74	-193.9~-211.0
	合计	33	407~505	475	38	19	19	29	0.9905	0.01484	5.89	63.26~66.74	-190.2~-234.8
5.8S	合计	45	158	150	8	0	8	8	0.2899	0.00225	0.356	56.96~58.86	-58.2~-62.4
ITS2	A	9	439~447	438	8	1	7	8	0.9722	0.005	2.194	71.14~71.81	-208.8~-220.2
	B	15	423~429	418	12	6	6	11	0.9048	0.00648	2.743	71.56~71.96	-202.1~-216.8
	合计	24	423~447	430	20	8	12	19	0.9601	0.00797	3.862	71.14~71.96	-202.1~-220.2
28S	A	7	967~968	958	10	0	10	6	0.9524	0.00295	2.857	61.98~62.29	-398.3~-405.9
	B	4	973~974	972	2	0	2	3	0.8333	0.00103	1	62.22~62.38	-400.6~-403.9
	R	1	972	—	—	—	—	1	1	—	—	62.24	-403.5
	合计	12	967~974	962	12	1	11	10	0.9697	0.00246	2.379	61.98~62.38	-398.3~-405.9

长臂缨鲆核糖体RNA基因序列多态性特征分析

Analysis of polymorphism characteristics of ribosomal RNA genes in Crossorhombus kobensis (Pleuronectiformes: Bothidae)

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