3种色型仿刺参线粒体和核糖体序列片段遗传结构比较研究

doi:10.6040/j.issn.1671-9352.0.2020.231

摘要/Abstract

摘要：

仿刺参作为我国重要的海水养殖物种,良种选育是目前研究的重点,其中体色是重要的考虑因素之一。为了比较3种不同体色仿刺参的遗传结构,采用PCR扩增与测序技术对我国3种体色(青色、白色和紫色)仿刺参线粒体16S rRNA和COⅠ与核糖体18S rRNA-ITS-28S rRNA序列片段进行分析,揭示其遗传结构差异及系统发育关系。结果表明, 16S rRNA、COⅠ和18S rRNA-ITS-28S rRNA序列长度分别为812~830 bp、877~915 bp、1 536~1 572 bp。16S rRNA、COⅠ和18S rRNA-ITS-28S rRNA序列在青色、紫色和白色仿刺参中分别检测到单倍型个数分别为4/5/5、5/3/4和4/4/3, COⅠ序列多态性位点数目所比例最大, 16S rRNA序列最小。3种色型仿刺参16S rRNA、COⅠ和18S rRNA-ITS-28S rRNA序列遗传距离数值均比较小,范围分别为0~0.014 7、0~0.021 2和0~0.010 3,没有达到种的分化水平。以紫海胆为外群,基于COⅠ序列构建系统发育树,结果表明白刺参单独聚为一支,但是紫色和青色仿刺参不同个体间相互交叉聚集,无法通过发育树区分。

关键词: 仿刺参, 体色, 16S rRNA, COⅠ, 18S rRNA-ITS-28S rRNA, 遗传结构

Abstract:

The sea cucumber Apostichopus japonicus is an important marine aquatic species in China. Selecting good breeding is a major issue for sea cucumber aquaculture, and body color is chosen as an important consideration. In order to explore the genetic structure and phylogenetic relationship of sea cucumber populations with different body color characteristics, 16S rRNA, COⅠ and 18S rRNA-ITS-28S rRNA sequences were amplified and sequenced. The results showed that the lengths of 16S rRNA, COⅠ and 18S rRNA-ITS-28S rRNA sequences were 812-830 bp, 877-915 bp and 1 536-1 572 bp, respectively. In the green, white and purple morphs of the sea cucumber, 4/5/5, 5/3/4, and 4/4/3 haplotypes were detected in 16S rRNA, COⅠ and 18S rRNA-ITS-28S rRNA sequences, respectively. The number of polymorphic loci in the COⅠ sequences was the highest, and that of 16S rRNA sequences was the smallest. The genetic distances of 16S rRNA, COⅠ and 18S rRNA-ITS-28S rRNA sequences among three color morphs were both small, ranging from 0-0.014 7, 0-0.021 2, and 0-0.010 3, respectively, and they did not reach the species differentiation level. The phylogenetic tree was constructed based on the COⅠ sequences, and Strongylocentrotus purpuratus was chose as the outgroup. The results showed that the white sea cucumbers were clustered into one branch, the purple and green sea cucumbers were cross-aggregated with each other and could not be distinguished.

Key words: Apostichopus japonicus, body color, 16S rRNA, COⅠ, 18S rRNA-ITS-28S rRNA, genetic structure

中图分类号:

S917.4

郭战胜,王振,侯旭光,张海涛. 3种色型仿刺参线粒体和核糖体序列片段遗传结构比较研究[J]. 《山东大学学报(理学版)》, 2020, 55(11): 1-7.

Zhan-sheng GUO,Zhen WANG,Xu-guang HOU,Hai-tao ZHANG. Comparative study on genetic structure of three color variants of the sea cucumber (Apostichopus japonicus) based on mitochondrial and ribosomal genes[J]. JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2020, 55(11): 1-7.

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引物名称	序列(5′-3′)	退火温度/℃
16S-F	CGCCGTTTATCAAAAACAT	50
16S-R	CCGGTCTGAACTCAGATCACG	50
16S-COⅠ-F	RAGTTTTGGTTGGGGCAACCGTG	60
16S-COⅠ-F	CCCTCTTTCAACTCCTGCAGAG	60
COⅠ-F	ATAATGATAGGAGGRTTTGG	46
COⅠ-R	GCTCGTGTRTCTACRTCCAT	46
ITS-F	ACGAGATGGAGCAATAACAGG	53
ITS-R	AAATCCAGCGGGTAATCTCAT	53

序列名称	物种名称	序列长度/bp	序列碱基组成/%
序列名称	物种名称	序列长度/bp	T	C	A	G	A+T
	B1—B5	812~828	27.11	20.15	32.67	20.08	59.78
16S rRNA	W1—W5	826~829	27.50	20.40	32.17	19.94	59.67
	P1—P5	826~830	27.67	20.26	32.19	19.87	59.86
	平均值	824.20	27.43	20.27	32.34	19.96	59.77
	B1—B5	877~913	31.45	21.03	27.48	20.04	58.93
COⅠ	W1—W5	898~915	31.45	20.71	27.89	19.94	59.34
	P1—P5	889~911	31.69	20.79	27.51	20.01	59.20
	平均值	902.40	31.53	20.84	27.63	20.00	59.16
18S	B1—B5	1 557~1 572	19.43	30.52	18.83	31.22	38.26
rRNA-ITS-28S	W1—W5	1 547~1 566	19.42	30.42	18.78	31.37	38.20
rRNA	P1—P5	1 536~1 566	19.35	30.37	18.78	31.50	38.13
	平均值	1 557.40	19.40	30.44	18.80	31.36	38.20

序列片段	样本	单倍型数H	单倍型多样性Hd	多态性位点数S	平均核苷酸差异数K	核苷酸多样性指数Pi
16S rRNA	B1—B5	4	0.900	3	1.400	0.001 73
	P1—P5	5	1.000	12	6.200	0.007 53
	W1—W5	5	1.000	8	4.500	0.005 45
	总体	6	0.705	7	1.333	0.001 65
COⅠ	B1—B5	5	1.000	13	6.400	0.007 31
	P1—P5	3	0.800	11	5.200	0.005 85
	W1—W5	4	0.900	4	2.100	0.002 34
	总体	9	0.886	21	4.990	0.005 75
ITS	B1—B5	4	0.900	9	3.800	0.002 45
	P1—P5	4	0.900	3	1.400	0.000 91
	W1—W5	3	0.700	5	2.200	0.001 43
	总体	8	0.848	15	2.610	0.001 71

	B1	B2	B3	B4	B5	P1	P2	P3	P4	P5	W1	W2	W3	W4	W5
B1
B2	5.0/9.2/1.3
B3	2.5/11.3/0.0	2.5/4.6/0.0
B4	1.2/4.5/0.6	3.7/10.4/0.6	1.2/12.4/0.6
B5	2.5/6.7/4.5	6.2/9.2/4.5	6.2/10.1/4.5	2.5/7.8/5.2
P1	2.5/5.7/5.1	3.7/5.7/5.1	0.0/3.4/5.1	1.2/5.7/5.8	8.5/3.4/6.5
P2	1.2/4.5/0.7	4.9/10.4/0.7	1.2/10.1/0.7	0.0/6.7/1.3	7.3/17.9/5.2	2.4/4.5/0.7
P3	2.5/10.1/5.2	8.7/17.4/5.8	8.7/18.1/4.5	4.9/11.2/5.2	11.0/20.1/5.9	14.7/11.4/1.3	12.2/19.0/0.7
P4	1.2/3.4/5.8	6.2/10.4/6.5	4.9/10.1/5.2	2.5/4.5/5.8	8.5/9.0/5.2	9.8/4.5/1.9	7.3/3.0/1.3	7.3/12.4/0.6
P5	2.5/9.0/0.7	4.9/5.7/0.7	3.7/7.8/0.7	3.7/10.1/1.3	7.3/7.8/6.5	7.3/0.0/2.6	8.5/9.0/0.0	11.0/15.8/2.6	3.6/7.9/3.3
W1	1.2/4.5/3.2	6.2/11.5/3.2	4.9/11.3/3.2	2.5/5.6/3.9	6.1/5.6/7.2	8.5/5.7/5.8	6.1/6.7/2.6	8.5/13.5/5.9	3.6/3.4/6.5	6.1/9.0/2.6
W2	1.2/6.7/1.3	4.9/11.5/1.3	4.9/12.4/1.3	1.2/7.8/1.9	3.6/16.6/5.2	9.8/5.7/3.2	7.3/12.2/0.0	6.1/20.0/1.9	2.4/6.7/2.6	4.9/6.7/0.7	1.2/3.3/3.2
W3	1.2/6.7/9.6	4.9/11.5/9.7	1.2/12.4/9.6	1.2/9.0/10.3	8.5/13.3/10.4	2.4/5.7/8.4	0.0/12.2/1.3	13.4/21.2/1.9	7.3/6.7/2.6	8.5/10.1/3.3	6.1/3.3/5.2	7.3/11.0/7.1
W4	1.2/5.6/0.6	4.9/11.5/0.6	3.7/11.3/0.6	1.2/6.7/1.3	4.9/11.1/4.5	7.3/5.7/2.6	6.1/11.1/0.0	8.5/16.7/0.7	2.4/4.5/1.3	1.2/10.1/0.0	3.6/1.1/2.6	2.4/8.8/0.0	6.1/3.3/5.8
W5	1.2/5.6/0.6	4.9/11.5/0.6	4.9/11.3/0.6	1.2/6.7/1.3	3.6/14.4/5.2	9.8/5.7/3.2	8.5/12.2/0.0	9.8/16.7/1.3	7.3/4.5/1.9	6.1/10.1/0.0	4.9/1.1/2.6	2.4/9.9/0.0	9.7/9.9/5.8	3.6/4.4/0.0

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