Journal of Tropical Oceanography >
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
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.
YANG Min , KONG Xiaoyu , SHI Wei , GONG Li . Analysis of polymorphism characteristics of ribosomal RNA genes in Crossorhombus kobensis (Pleuronectiformes: Bothidae)[J]. Journal of Tropical Oceanography, 2019 , 38(1) : 55 -66 . DOI: 10.11978/2018038
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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