Association analysis of SNPs polymorphisms in mstn gene of Paralichthys olivaceus with growth traits
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摘要:
为研究牙鲆 (Paralichthys olivaceus) 肌肉生长抑制基因 (mstn) 多态性，开发其分子辅助育种新标记，采用重测序方法对120尾牙鲆 (3个双克隆杂交家系：60 尾；3个雌核发育家系：60 尾) 的mstn基因进行单核苷酸多态性 (Single nucleotide polymorphism, SNP) 位点筛选，并对不同基因型个体的生长性状进行多重比较及SNP标记验证。结果显示，共检测到7个SNPs位点，均为颠换型突变；外显子区3个 (Exonl 1: G2063A; Exonl 3: C3883T, C4009A)，为同义突变；内含子区3个 (Intron 1: A2444T; Intron 2: T3816C, A3832C)；3'端非编码区1个 (3'UTR: C4564A)。SNPs与生长性状关联分析结果表明，A3832C和C4564A标记位点与牙鲆体质量、体长、体高等生长性状显著相关 (P<0.05)，其中A3832C 位点AA基因型和C4564A位点AC基因型在生长上表现出优势，A3832C位点CC和C4564A位点AA、CC基因型在生长上表现出劣势，其他5个SNPs位点对牙鲆生长性状影响不显著 (P>0.05)。将A3832C和C4564A标记位点在牙鲆生长快速群体 (F: 60 尾) 和生长缓慢群体 (S: 60 尾) 中验证，验证结果与多重比对结果一致，表明2个SNPs位点具有较高的稳定性。综上所述，研究筛选出了牙鲆mstn基因中与生长性状显著相关的A3832C和C4564A两个标记位点，为牙鲆分子辅助育种提供了理论参考。
Abstract:In order to study the polymorphism of mstn gene in Japanese flounder (Paralichthys olivaceus) and develop new markers for its molecular assisted breeding, we srceened 120 individuals of Japanese flounder (Three double clone hybrid families: 60, three Gynogenesis families: 60) mstn genes for single nucleotide polymorphisms (SNP), and conducted multiple comparisons and SNPs markers validation on the growth traits with different genotypes. The results detected seven SNPs loci, all of which were transposition type mutation; three Exon regions (Exonl 1: G2063A; Exonl 3: C3883T, C4009A) were synonymous mutations; three Intron regions (Intron I: A2444T; Intron II: T3816C, A3832C); and one non Coding region at 3' end (3' UTR: C4564A). The correlation analysis between SNPs and growth traits show that A3832C and C4564A were significantly correlated with growth traits such as body mass, body length and body height (P<0.05). Among them, A3832C AA genotype and C4564A AC genotype showed advantages in growth; A3832C CC, C4564A AA and CC genotype showed disadvantages in growth, while the other SNPs loci had no significant impact on growth traits (P>0.05). A3832C and C4564A markers were validated in fast-growing population (F: 60) and slow-growing population (S: 60), and the validation results are consistent with the results of multiple alignments, indicating that the two SNPs loci have high stability. In summary, this study screened two marker loci (A3832C, C4564A) in the mstn gene of Japanese flounder, which are significantly related to growth traits, providing theoretical references for its molecular assisted breeding.
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Keywords:
- Paralichthys olivaceus /
- mstn gene /
- SNPs /
- Growth traits
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图 1 牙鲆mstn基因SPNs的连锁不平衡分析
注：方块中数字表示SNPs之间的连锁程度 (R2)，D' 值越高方块颜色越深。
Figure 1. Linkage disequilibrium analysis of SNPs in mstn gene of P. olivaceus
Note: The number in the block represents the linkage degree (R2) between SNPs; the higher the D' value is, the darker the color of the block is.
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图 2 A3832C位点不同基因型对应生长性状分布
注：性状参数中间的线为中位线。
Figure 2. Distribution of growth traits corresponding to different genotypes at A3832C locus
Note: The line in the middle of the trait parameter is the median line.
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图 3 C4564A位点不同基因型对应生长性状分布
注：性状参数中间的线和点分别为中位线和平均值。
Figure 3. Distribution of growth traits corresponding to different genotypes at C4564A locus
Note: The lines and points in the middle of the trait parameters are the median and mean values, respectively.
表 1 测序用引物序列
Table 1 Primer sequences used for sequencing
引物
Primer序列 (5'—3')
Primer sequence (5'–3')扩增区域
Product position退火温度
Temperature/℃产物大小
Product size/bpmstn1 F: AGTCCTGTATGATTGAAACTG 1513—3317 55 1 800 R: TGAAACCTCTGGAGGCCTGAAG mstn2 F: GTGAATAGTGTGGGTGCATGC 3157—4263 56 1 102 R: ACGCTCTCCGTCCCAACTCAAG mstn3 F: CATGCACTTGCAGAAGTATCC 4064—5530 55 1 456 R: CAGCAAACAAGCTAGAAACTT 
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表 2 牙鲆mstn基因的SNPs引物及延伸引物信息
Table 2 SNPs primer and extended primer information of mstn gene of P. olivaceus
引物
Primer序列 (5'—3')
Primer sequence (5'–3')多态性
Polymorphism延伸引物
Extended primer方向
Direction产物
Production大小
Size/bpSNP 1 F: TGTCTCACATTGTGCTCTATC
R: CTTCATTCGCAGTTTGCTCAG[G/A] TGCGACCAAGAGACGCACCATCA 正向 AG 23 SNP 2 F: CCACAGAGACAATCATGATGAT
R: TGTACTGATTCCTGTACAATC[A/T] TTTTTTTTTTATCTAGCGTCTCCCGTGCGCTC 反向 AT 32 SNP 3 F: CTGTGGTTAACCAACCGTCAG
R: CTTCTGCAAGTGCATGTACTC[T/C] GACTGCTGTCATCCCACTCT 正向 CT 20 A3832C F: CTGTGGTTAACCAACCGTCAG
R: CTTCTGCAAGTGCATGTACTC[A/C] TTTTATGAATGGTTGCTGAA
ATGACAAAAATGAAAT反向 GT 36 SNP 5 F: CTGTGGTTAACCAACCGTCAG
R: CTTCTGCAAGTGCATGTACTC[C/T] CTCTCCTGACTCGCTTTGGGCC 反向 AG 22 SNP 6 F: CTGTGGTTAACCAACCGTCAG
R: CTTCTGCAAGTGCATGTACTC[A/G] TTTTTTTTAATAGTTGGCCTTGTAGCGCTT 反向 CT 30 C4564A F: ATGCACACATGCTGGACGTTG
R: AGCTTAGCGAAACGTGTATTAG[C/A] CGTATAAATGGAGAGAACAC
GGTAACGATGGAGAAAAAAG反向 GT 40
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表 3 牙鲆数量性状的正态分布检验
Table 3 Test of normal distribution of quantitative traits of P. olivaceus
性状
Trait平均值±标准差
$ \overline { x}\pm s$最大值
Max.最小值
Min.偏度
Skewness峰度
Kurtosis体质量 Body mass/g 422.02±212.94 950.30 117.40 0.396 −1.101 全长 Total length/mm 343.89±58.49 483.43 228.03 0.292 −0.801 体长 Body length/mm 300.03±55.37 436.88 194.53 0.312 −0.842 头长 Head length/mm 82.56±12.94 118.12 56.59 0.37 −0.692 体高 Body height/mm 107.14±3.98 179.80 15.58 −1.029 0.032 尾柄长Tail shank length/mm 40.99±2.21 103.12 15.23 1.558 1.042 尾柄高 Tail shank height/mm 27.87±0.49 39.29 16.25 0.213 −0.94
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表 4 牙鲆mstn基因位点信息
Table 4 Information on mstn gene loci in P. olivaceus
序号
No.SNP 位点
SNP site位置
Mutation area/bp类别
Mutation type所在区域
Region1 G2063A 2 063 G转化为A Exonl 1 2 A2444T 2 444 A转化为T Introl 1 3 T3816C 3 816 T转化为C Introl 2 4 A3832C 3 832 A转化为C Introl 2 5 C3883T 3 883 C转化为T Exonl 3 6 C4009A 4 009 C转化为A Exonl 3 7 C4564A 4 564 C转化为A 3'UTR
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表 5 牙鲆mstn基因中SNP位点的等位基因和基因型频率
Table 5 Allele and genotype frequencies of SNP loci in mstn gene of P. olivaceus
位点
Site等位基因
Allele等位基因频率
Allele frequency基因型
Genotype个体数
Number基因型频率
Genotype frequency哈温平衡
Hardy-weinbergA3832C A
C0.74
0.26AA 69 0.58 χ2=16.09
P=0.000321AC 40 0.33 CC 11 0.09 C4564A A
C0.35
0.65AA 3 0.03 χ2=37.69
P=6.53×10−9AC 78 0.64 CC 39 0.33
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表 6 牙鲆mstn基因的2个SNPs标记位点不同基因型个体生长性状的多重比较
Table 6 Multiple comparisons of growth traits among individuals with different genotypes at two SNPs marker loci of mstn gene of P. olivaceus
位点
Site基因型
Genotype样本数
n/尾体质量
Body mass/g体长
Body length/mm体高
Body height/mm头长
Head length/mm尾柄长
Tail shank
length/mm尾柄高
Tail shank
height/mmA3832C AA 69 467.25±211.16a 310.73±55.66a 114.75±41.34a 85.03±13.43a 39.95±20.41a 28.88±5.58a AC 40 393.70±216.69b 295.25±54.00a 100.20±47.31ab 81.70±11.69ab 44.46±28.80a 27.47±4.92a CC 11 225.77±66.07c 250.30±21.00b 84.75±33.95b 70.17±4.62b 34.99±28.90a 23.02±2.92b C4564A AA 3 223.80±97.85a 243.45±59.42a 78.88±53.58a 68.8±12.0a 41.71±27.21a 22.73±5.92a AC 78 498.68±210.44b 318.21±54.85b 115.10±45.45b 87.09±12.76b 43.78±24.30a 29.76±5.35b CC 39 279.57±136.42a 268.02±36.55a 93.41±35.15a 74.56±8.03a 35.38±24.25a 24.48±3.26a 注：不同小写字母表示不同基因型生长性状存在差异显著 (P<0.05)。 Note: Different lowercase letters represent significant differences in growth traits of different genotypes (P<0.05).
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表 7 生长快速 (F: 60尾) 和生长缓慢 (S: 60尾) 牙鲆的生长性状表型数据
Table 7 Phenotypic data of growth traits in P. olivaceus with fast growth (F: 60 individuals) and slow growth (S: 60 individuals)
家系
Family体质量
Body mass/g体长
Body length/cm体高
Body height/cm头长
Head length/cm尾柄长
Tail shank length/cm尾柄高
Tail shank height/cmF 605.66±133.19a 34.53±3.66a 13.86±1.46a 9.62±0.95a 3.52±0.61a 3.21±0.38a S 235.54±74.10b 25.48±2.53b 7.57±3.98b 7.25±0.63b 2.29±0.39b 2.36±0.27b 注：不同小写字母表示不同群体牙鲆生长性状间差异显著。 Note: Different lowercase letters represent significant differences in growth traits among different populations of P. olivaceus.
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表 8 牙鲆mstn基因SNPs标记 (A3832C、C4564A) 验证
Table 8 Verification of SNPs markers (A3832C, C4564A) for mstn gene of P. olivaceus
SNP标记 基因型
GenotypeF S G-test P A3832C AA 43 17 40.557 P<0.001 AC 17 14 CC 0 29 C4564A AA 0 3 42.462 P<0.001 AC 56 22 CC 4 35
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