长臂缨鲆核糖体RNA基因序列多态性特征分析
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
收稿日期: 2018-04-09
要求修回日期: 2018-06-07
网络出版日期: 2019-01-16
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)
Received date: 2018-04-09
Request revised date: 2018-06-07
Online published: 2019-01-16
Supported by
National Natural Science Foundation of China (31272273)
Copyright
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。
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 . DOI: 10.11978/2018038
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.
Tab. 1 PCR primers information for different ribosomal RNA gene fragments and Genbank accessions表1 核糖体RNA基因片段PCR引物序列及Genbank序列号 |
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 |
F-18S-1874 | 5'-ATGATCCTTCCGCAGGTTCA-3' | ||||||
ITS1-5.8S | Z-18S-1720 | 5ʹ-TCGCTACTACCGATTGGATGGTTTA-3’ | MH036892—MH036912 | MH179078 | MH179079 | MH179080 | MH179081 |
F-5.8S-150 | 5'-AAGCGACCCTCAGACAGGCGTAG-3' | ||||||
5.8S-ITS2 | Z-5.8S-10 | 5'-CAACTCTTAGCGGTGGATCA-3' | MH036913—MH036924 | MH179074 | MH179075 | MH179076 | MH179077 |
F-28S-100 | 5'-GCTCTTCCCTCTTCACTCG-3' | ||||||
ITSZ | Z-18S-1720 | 5ʹ-TCGCTACTACCGATTGGATGGTTTA-3’ | MH036925—MH036936 | — | — | — | — |
F-28S-100 | 5'-GCTCTTCCCTCTTCACTCG-3' | ||||||
28S* | Z-28S-900 | 5'-CCCACCCCGACCCGTCTTGAA-3' | MH031711—MH031722 | MH173825 | MH173826 | MH173827 | MH173828 |
F-28S-1800 | 5'-GACTGACCCATGTTCAACTGCTGT-3' |
注: ITSZ包括ITS1、5.8S和ITS2; *表示不是全长序列; “—”表述无此序列。 |
Fig. 1 Comparison of different types of four ribosomal RNA gene fragments from Crossorhombus kobensis with four species from family Bothidae. a) Three types of 18S rDNA; * showed the indel sites and differential sites among Types A, B and C; b) two types of 28S rDNA; * showed the indel sites in Type A and Type B; c) two types of ITS1 sequences; the grey shadow presents deleted fragments in Type B; d) two types of ITS2 sequences; the loci with underlines were microsatellites. A. mac presented A. macrolophus; C. azu presented C. azureus; E. gra presented E. grandisquama; L. gal presented L. gallus图1 长臂缨鲆4个核糖体RNA基因不同序列类型与鲆科4种鱼类序列比对 |
Tab. 2 Information of ribosomal RNA gene sequences in Crossorhombus kobensis genome表2 长臂缨鲆核糖体RNA基因序列特征信息统计 |
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 |
注: “—”表示此处无数值。 |
Fig. 2 The cluster trees constructed based on 18S rDNA (a), 28S rDNA (b), ITS1 (c), and ITS2 (d) sequences in Crossorhombus kobensis by neighbor-joining method. Numbers on internal branches indicate support values (≥50%)图2 长臂缨鲆18S rDNA(a)、28S rDNA(b)、ITS1(c)以及ITS2(d)序列NJ聚类分析分支节点位置数字为置信值 (≥50%) |
Fig. 3 The recombination patterns of ribosomal RNA genes in C. kobensis. a) Four recombination types of 18S rDNA; b) one recombination type of 28S rDNA; c) three recombination types of ITS1. Type A was presented by black; Type B was presented by dark dot; Type C was presented by vertical lines图3 长臂缨鲆核糖体RNA基因不同类型序列间发生重组 |
The authors have declared that no competing interests exist.
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[4] |
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[5] |
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[6] |
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[7] |
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[8] |
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[9] |
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[10] |
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[11] |
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[12] |
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[13] |
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[14] |
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[15] |
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[16] |
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[17] |
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[18] |
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[19] |
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[20] |
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[21] |
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[22] |
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[23] |
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[24] |
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[25] |
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[26] |
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[27] |
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[28] |
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[29] |
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[30] |
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[31] |
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[32] |
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[33] |
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[34] |
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