九孔鲍MSTN基因cSNP多态性及与生长性状关联分析

doi:10.11978/2018053

摘要/Abstract

摘要：

九孔鲍(Haliotis diversicolor supertexta)是中国南方具有较高经济价值的养殖贝类。肌肉生长抑制素(Myostatin, MSTN)是转化生长因子β超家族(TGF-β)中参与动物肌肉生长的重要调控因子, 因此MSTN基因是动物遗传改良的重要候选基因。为探究九孔鲍MSTN基因的多态性及其生长相关性, 实验采用PCR产物直接测序方法对九孔鲍MSTN基因进行单核苷酸多态性(SNP)筛选, 对MSTN基因SNP与体质量、壳长、壳宽进行关联性分析, 运用实时荧光定量PCR (qRT-PCR)技术检测九孔鲍MSTN基因不同基因型的表达水平。结果显示: 在九孔鲍MSTN基因中共筛选出9个SNP位点。SNP位点与生长性状关联分析发现, 九孔鲍MSTN基因编码区SNP位点g909C>T发生C/T同义突变与九孔鲍体质量、壳长、壳宽显著相关。TT基因型九孔鲍的体质量显著高于CC基因型和TC基因型个体; TT基因型的壳长、壳宽显著高于CC基因型(p<0.05)。通过qRT-PCR检测显示, 在九孔鲍MSTN基因SNP位点g909C>T的3种基因型中, TC和CC基因型的基因表达量显著高于TT基因型(p<0.05)。研究结果表明MSTN基因SNP位点g909C>T可作为九孔鲍标记辅助选择育种重要候选标记。

关键词: 九孔鲍, MSTN, 单核苷酸多态性, 生长性状, 关联分析

Abstract:

Myostatin is an important member of the transforming growth factor (TGF) family that functions to regulate muscle development and growth in animals. It has become one of the most important target genes for genetic improvement of the production of farmed animals. The purpose of this study was to analyze correlation between single nucleotide polymorphisms of the myostatin gene and growth traits of the Haliotis diversicolor supertexta, which is an important aquaculture shellfish. The single nucleotide polymorphisms of the myostatin gene (Hs-MSTN) of H. diversicolor supertexta were identified by direct sequencing of PCR products, and association analysis of SNPs with H. diversicolor supertexta growth traits and Hs-MSTN expression level. Nine SNPs were identified in cDNA of Hs-MSTN, including four synonymous mutations. The associations of these SNPs were analyzed with H. diversicolor supertexta growth traits, including shell length, shell height, and body weight. The g909C>T SNP locus was found to be associated with shell length, shell height, and body weight. The TT type H. diversicolor supertexta showed significantly higher traits values than those of TC and CC types in body weight, and the TT type H. diversicolor supertexta also showed significantly higher traits values than CC type in shell length and shell height (p<0.05). On the contrary, Hs-MSTN transcripts in the musle of those of TC and CC types significantly more than in that of TT type (p<0.05). The results suggested that Hs-MSTN might regulate the addutor musle growth negatively, and SNP g909C>T could be used as a candidate marker for slective breeding of H. diversicolor supertexta.

Key words: Haliotis diversicolor supertexta, MSTN, SNP, growth traits, association analysis

中图分类号:

P735.542

艾加林, 栗志民, 刘建勇, 申玉春. 九孔鲍MSTN基因cSNP多态性及与生长性状关联分析[J]. 热带海洋学报, 2019, 38(2): 78-85.

Jialin AI, Zhimin LI, Jianyong LIU, Yuchun SHEN. Single nucleotide polymorphisms of myostation gene and its association with growth traits in Haliotis diversicolor supertexta[J]. Journal of Tropical Oceanography, 2019, 38(2): 78-85.

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引物	引物序列(5°—3°)	温度/℃	位置
SNP-1F	GTCAGGTGCGTAGCAAAC	57	173~1003bp
SNP-1R	AGATGATGATGTCGGTTGT	57	173~1003bp
SNP-2F	AGCCTCTTGTTCATTCCTACT	52	1871~2657bp
SNP-2R	ACACCCTTATCTGCTATACCTT	52	1871~2657bp
β-actin-F	TGCCCATCTACGAAGGTTACGCC	60	荧光定量内参(200bp)
β-actin-R	GCTGCTGTGGACATCTCCTGCTC	60	荧光定量内参(200bp)
MSTN-RF	GTGACTCGAATGGACACGGA	60	荧光定量(114bp)
MSTN-RR	GCATCTGACCGCGATACCAT	60	荧光定量(114bp)

位点	等位基因	PIC	N_e	H_e	H_o	HWE p值
5′UTR g252G>A	G(0.6176)/A(0.3824)	0.3608	1.8951	0.4751	0.4353	0.4367
ORF g414A>C g558C>A g909C>T g960A>T	A(0.7882)/C(0.2118) C(0.8000)/A(0.2000) C(0.6905)/T(0.3095) A(0.8780)/T(0.1220)	0.2781 0.2668 0.3360 0.1913	1.5011 1.4706 1.7465 1.2725	0.3358 0.3219 0.4300 0.2155	0.3059 0.2588 0.3810 0.2195	0.4056 0.0673 0.2919 0.8620
3′UTR g2148G>A g2164A>G g2420C>G g2520C>G	G(0.7706)/A(0.2294) A(0.6118)/G(0.3882) C(0.7353)/G(0.2647) C(0.6824)/G(0.3176)	0.2911 0.3621 0.3135 0.3395	1.5469 1.9048 1.6374 1.7652	0.3557 0.4778 0.3916 0.4361	0.3412 0.4471 0.2941 0.3294	0.7074 0.5509 0.0205(p<0.05) 0.2524

位点	基因型	基因型频率	体质量/g	壳长/mm	壳宽/mm
5′UTR
g252G>A	AG(37)	0.41	11.62±4.14	48.76±6.00	30.31±3.64
	AA(13)	0.14	9.42±2.94	45.07±5.25	28.35±2.89
	GG(40)	0.45	11.41±4.47	47.57±7.23	29.42±4.94
ORF
g414A>C	AC(28)	0.31	11.59±4.60	48.35±6.72	30.17±4.00
	AA(56)	0.62	10.79±3.98	47.02±6.63	29.18±4.41
	CC(6)	0.07	13.32±4.14	51.07±5.12	31.40±3.63
g558C>A	AC(23)	0.26	12.49±4.57	49.84±6.34	30.84±3.91
	AA(6)	0.07	11.35±4.48	48.32±5.27	30.15±3.69
	CC(61)	0.68	10.71±3.99	46.83±6.71	29.13±4.38
g909C>T	TC(35)	0.39	11.48±3.34^b	48.57±5.03^ab	30.29±3.03^ab
	CC(43)	0.48	10.26±4.10^b	45.79±6.94^b	28.45±4.69^b
	TT(11)	0.12	14.00±6.00^a	52.05±7.65^a	31.97±4.79^a
g960A>T	TA(17)	0.20	11.47±3.35	48.96±5.25	30.76±2.96
	AA(68)	0.79	11.19±4.46	47.38±7.01	29.54±4.22
	TT(1)	0.01	15.68	53.32	31.83
3′UTR
g2148G>A	AG(29)	0.30	11.85±4.34	48.59±6.39	30.33±4.66
	GG(59)	0.61	10.54±3.98	46.63±6.52	29.03±3.95
	AA(8)	0.08	11.27±3.02	48.33±5.12	30.02±2.93
g2164A>G	AG(46)	0.47	11.49±4.44	48.10±7.31	29.89±4.96
	AA(36)	0.37	10.62±3.81	46.49±5.57	28.92±3.28
	GG(16)	0.16	10.92±3.72	47.66±5.52	30.02±3.31
g2420C>G	CG(27)	0.28	11.99±4.40	48.77±6.51	30.48±4.75
	CC(58)	0.60	10.54±4.02	46.61±6.58	29.02±3.99
	GG(11)	0.11	10.99±2.9	47.9±4.69	29.67±2.77
g2520C>G	GC(34)	0.35	11.00±4.78	47.1±8.01	29.15±5.47
	CC(48)	0.50	11.14±3.76	47.64±5.47	29.71±3.28
	GG(14)	0.15	10.51±3.06	47.07±5.15	29.67±2.92