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Polymorphism in CgTyr1 gene and its association with shell color traits in the Pacific oyster (Crassostrea gigas)

  • Corresponding author: Qi LI, qili66@ouc.edu.cn
  • Received Date: 2018-12-03
    Accepted Date: 2019-02-25
  • The Pacific oyster Crassostrea gigas is cultured worldwide due to the advantages of rapid growth and good adaptability, and has become one of the most commercially important bivalve species. In our successively selective breeding, three strains of C. gigas with black, white and gold shell color traits have been developed. Tyrosinase (Tyr) is known as one of the most important enzymes in the regulation and production of melanin in animals. In this study, exons in the C. gigas TYR gene (CgTyr1) were sequenced. Mutations of the CgTyr1 gene and its association with shell color were analyzed in the three shell color strains of C. gigas. A total of 23 single nucleotide polymorphisms (SNP) were detected using single-strand conformation polymorphism (SSCP) and sequencing analysis, of which 11 SNP loci had highly significant differences between the three shell-color strains. In the SNP loci with significant difference, mutation c.591C/T, c.632G/A and c.1155T/C are non-synonymous which lead to amino acid changes Ala122Val, Gly136Ser and Phe310Ser. For further analysis, 11 SNP loci with a highly significant difference were selected for haplotype construction. One specific haplotype for every shell color strain was constructed and confirmed in the validating group. The mutations and haplotypes that are strongly associated with the shell color phenotypes in this study could be useful in understanding the molecular mechanism of pigmentation, and potentially applied to marker-assisted selection breeding programs for C. gigas.
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Polymorphism in CgTyr1 gene and its association with shell color traits in the Pacific oyster (Crassostrea gigas)

    Corresponding author: Qi LI, qili66@ouc.edu.cn
  • 1. Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China
  • 2. Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao National Laboratory for Marine Science and Technology, Qingdao    266237, China

Abstract: The Pacific oyster Crassostrea gigas is cultured worldwide due to the advantages of rapid growth and good adaptability, and has become one of the most commercially important bivalve species. In our successively selective breeding, three strains of C. gigas with black, white and gold shell color traits have been developed. Tyrosinase (Tyr) is known as one of the most important enzymes in the regulation and production of melanin in animals. In this study, exons in the C. gigas TYR gene (CgTyr1) were sequenced. Mutations of the CgTyr1 gene and its association with shell color were analyzed in the three shell color strains of C. gigas. A total of 23 single nucleotide polymorphisms (SNP) were detected using single-strand conformation polymorphism (SSCP) and sequencing analysis, of which 11 SNP loci had highly significant differences between the three shell-color strains. In the SNP loci with significant difference, mutation c.591C/T, c.632G/A and c.1155T/C are non-synonymous which lead to amino acid changes Ala122Val, Gly136Ser and Phe310Ser. For further analysis, 11 SNP loci with a highly significant difference were selected for haplotype construction. One specific haplotype for every shell color strain was constructed and confirmed in the validating group. The mutations and haplotypes that are strongly associated with the shell color phenotypes in this study could be useful in understanding the molecular mechanism of pigmentation, and potentially applied to marker-assisted selection breeding programs for C. gigas.

  • 在自然界中,软体动物表现出高度的壳色多态性,绚丽多彩的颜色和花纹深受人们的喜爱[1]。在消费行为上,由于不同壳色的贝类会对消费者产生不同程度的吸引,会影响消费者做出不同的消费选择[2-3],从而使得人们偏爱的品种体现出较高的经济价值。长牡蛎(Crassostrea gigas)是一种广泛分布的双壳贝类,同时也是世界上养殖产量最高的水生动物[4]。经过连续的人工选育,长牡蛎壳黑、壳白和壳金3种壳色品种(系)被成功构建[5]。长牡蛎的壳色是一种可稳定遗传的性状,近年来已有学者对不同壳色长牡蛎的遗传参数评估[6-7]、壳色相关AFLP鉴定[8]以及转录组学[9-10]方面开展了研究,但关于酪氨酸酶基因与长牡蛎壳色性状的关联性尚未见报道。

    黑色素是一种较为常见的动物色素。尽管其合成通路尚未完全被人所知,但是酪氨酸酶被认为是软体动物中黑色素合成调控的关键酶之一[11-13]。酪氨酸酶是一种含铜蛋白质,在黑色素的合成中参与3个不同的催化反应:将酪氨酸羟化形成L-多巴,进而氧化L-多巴形成多巴醌,然后从2个不同的途径分别合成真黑素和褐黑素[14-17]。在长牡蛎基因组中至少存在26个酪氨酸酶相关基因,其中2个酪氨酸酶基因(CgTyr1和CgTyr2)已被成功克隆[16-17]。在分子水平上,单核苷酸多态性(SNP)是最为常见的一种可遗传突变。由于其在基因组上大量稳定存在、共显性遗传和随机分布的特点,已被广泛应用在遗传学研究中[18]。目前,在青鳉(Oryzias latipes)和黄颡鱼(Tachysurus fulvidraco)中,已发现酪氨酸酶基因的突变与色素沉积存在关联性[19-20]

    为探究酪氨酸酶基因与长牡蛎壳色的关联性,本研究以壳黑、壳白和壳金3种长牡蛎壳色群体(图1)为对象,采用单链构象多态性(single-strand conformation polymorphism,SSCP)和测序方法对CgTyr1基因进行了SNP分型筛选,并以此构建单倍型,分析其与壳色变异的相关性,旨在为今后分子标记辅助长牡蛎壳色选育提供参考资料。

    Figure 1.  C. gigas with three shell colors

1.   材料与方法
  • 2016年4月于山东乳山养殖海域采集2015年构建的壳黑、壳白和壳金3种壳色长牡蛎第5代选育群体。将采集到的所有壳色长牡蛎样品随机分为筛选组和验证组,最终得到筛选组:壳黑(BS)71粒、壳白(WS)54粒、壳金(GS)80粒;验证组:壳黑、壳白和壳金各10粒。采用苯酚氯仿法提取长牡蛎闭壳肌基因组DNA[21]。提取的DNA使用Nanodrop测定浓度,将浓度稀释至100 ng/μL,置于4 °C保存待用。

  • 根据CgTyr1基因cDNA序列(GenBank accession no. NM_001305297.1),使用Primer Premier 5设计引物。为尽可能多的检测SNP位点,针对其4个外显子,共设计10对引物,覆盖基因外显子的91.2%(表1)。使用LASERGENE 7软件对测序片段进行拼接和比对。

    引物
    primer
    序列
    sequence(5′→3′)
    长度/bp
    length
    退火温度/°C
    annealing temperature
    P1-1 AGTCTTGCCGACATCTTT 284 50
    AAGCAGGAACCTGGAGA
    P1-2 AAGCAGGAACCTGGAGA 110 50
    ATGAAGCCTTACATAGTCG
    P2-1 TGATTTCATCCAATTACTTATCTTT 274 56
    GGGCGGGTGTTCAATATAG
    P3-1 TTATCATTTGATCCCTCCT 254 50
    AAGTTTTAATGGAGTGGTTT
    P4-1 CTGGAATCAGAGCCTTGC 236 56
    TGGGTTGATGCCGTTAG
    P4-2 GTCAAAGAGCTAACGGCAT 210 56
    AAACAAGGAACGGGCTAC
    P4-3 CCAGGTCCAGCAGAAGAG 207 56
    GAATGTAAGCCCAAGCATC
    P4-4 AAGCCCTACCAGAATACCTT 267 56
    GAGTCCATTAGACTGCACGA
    P4-5 TACGCAGGCAAATACAAAG 294 56
    TGGTTCCATCTCCCTAACA

    Table 1.  Primer sets used for analysis of SNPs in the CgTyr1 gene in C. gigas

    PCR反应为10 μL体系,其中模板DNA 100 ng,1× PCR buffer,dNTP混合液0.2 mmol/L,引物1 μmol/L,MgCl2 2 mmol/L和Taq DNA聚合酶0.25 U (TaKaRa)。PCR反应条件为94 °C预变性3 min;35个循环:94 °C变性20 s,退火10 s,72 °C延伸20 s;72 °C延伸5 min。在扩增后的PCR产物中加入等体积的变性剂(98%去离子甲酰胺、0.25%溴酚蓝、0.25%二甲苯菁、10 mmol/L EDTA),置于PCR仪上98 °C变性5 min后立即冰浴5 min,防止复性。

    采用浓度为10%的非变性聚丙烯酰胺进行凝胶电泳。取10 μL变性产物点样,先在300 V电压室温条件下预电泳10 min,待变性产物完全进入凝胶后,将电泳槽移入4 °C冰箱内,并将电压调至150 V继续电泳10 h。采用银染法进行染色。

  • 根据电泳条带的不同类型,为尽可能减少误差,在每种壳色及引物的每块胶板上,挑选典型个体进行测序,得到相应带型对应的碱基序列。

  • 采用卡方检验对检测到的SNP位点进行显著性分析。使用Popgene计算显著差异SNP位点的观测杂合度((Ho)、期望杂合度(He)、有效等位基因数(Ne)和多态性信息含量(PIC)。使用SHEsis (http://analysis.bio-x.cn/myAnalysis.php)[22-23]分析哈迪温伯格平衡,构建单倍型,并分析相关位点的连锁不平衡(LD)。

2.   结果
  • 通过PCR-SSCP方法,对不同群体的CgTyr1基因的外显子进行扩增,获得了相应基因片段的不同带型。挑选典型带型进行测序,得到每个个体的相应序列(图2)。通过壳黑、壳白和壳金长牡蛎群体之间的相互比对,共发现23个SNP位点。为进一步探究CgTyr1基因上SNP位点与不同壳色性状的相关性,筛选11个与壳色性状存在极显著关联(P<1×10-6)的SNP位点进行后续分析(附表1)。每个SNP位点均以该基因mRNA序列的第一个碱基到该突变位点的碱基数来命名。

    Figure 2.  Part of SSCP band patterns and sequence

    外显子
    region
    SNP位点
    mutation
    壳黑群体
    black strain (n=71)
    壳白群体
    white strain (n=54)
    壳金群体
    gold strain (n=80)
    P
    P value
    exon 1 c.367A/C 基因型
    genotype
    数量(频率)
    no. (Freq.)
    数量(频率)
    no. (Freq.)
    数量(频率)
    no. (Freq.)
    AA 62(0.873) 25(0.463) 78(0.975) 1.29E-13
    AC 9(0.127) 29(0.537) 0(0.000)
    CC 0(0.000) 0(0.000) 2(0.025)
    HW 0.325(0.569) 7.276(0.007) 80.00(0.000)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    A 0.937 0.731 0.975 Ile
    C 0.063 0.269 0.025 Ile
    c.466C/T 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    CT 21(0.296) 3(0.056) 0(0.000) 3.19E-08
    TT 50(0.704) 51(0.944) 80(1.000)
    HW 2.138(0.544) 0.044(0.997) 0.000(1.000)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    C 0.148 0.028 0 Thr
    T 0.852 0.972 1 Thr
    c.475C/G 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    CC 0(0.000) 5(0.093) 2(0.025) 5.66E-15
    CG 9(0.127) 29(0.537) 1(0.013)
    GG 62(0.873) 20(0.370) 77(0.963)
    HW 0.325(0.569) 1.450(0.229) 50.38(0.000)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    C 0.063 0.361 0.031 Val
    G 0.937 0.639 0.969 Val
    c.591C/T* 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    CC 69(0.972) 41(0.759) 80(1.000) 2.06E-07
    CT 2(0.028) 13(0.241) 0(0.000)
    HW 0.014(0.904) 1.011(0.315) 0.000(1.000)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    C 0.986 0.880 1 Ala
    T 0.014 0.120 0 Val
    c.632A/G* 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    AA 0(0.000) 6(0.111) 0(0.000) 7.26E-18
    AG 43(0.606) 26(0.481) 1(0.013)
    GG 28(0.394) 22(0.407) 79(0.988)

    Table 1.  Genetic polymorphisms in CgTyr1 gene in C. gigas

    在与壳色性状存在极显著关联的SNP位点中,突变位点c.367A/C、c.466C/T、c.475C/G、c.591C/T和c.632A/G位于外显子1,突变位点c.724A/G位于外显子2,突变位点c.850C/T位于外显子3,突变位点c.1155C/T、c.1207C/T、c.1231A/C和c.1289A/C位于外显子4。通过对SNP突变所导致氨基酸多态性的分析,发现位点c.591C/T、c.632A/G和c.1155C/T属于非同义SNP突变位点,分别导致Ala122Val、Gly136Ser和Phe310Ser的氨基酸突变。

  • 通过3种壳色长牡蛎群体CgTyr1基因序列的比较,筛选出的11个与壳色性状存在极显著相关SNP位点(P<1×10−6)的Ho、He、Ne和PIC表2。可以看出,壳黑长牡蛎群体存在9个SNP位点、壳白长牡蛎群体存在8个SNP位点、壳金长牡蛎群体中存在5个SNP位点。

    群体
    strain
    位点
    locus
    观测杂合度
    Ho
    期望杂合度
    He
    有效等位基因数
    Ne
    多态性信
    息含量
    PIC
    壳黑群体
    black strain
    c.367A/C 0.126 0.119 1.134 0.112
    c.466C/T 0.295 0.253 1.337 0.220
    c.475C/G 0.126 0.119 1.134 0.111
    c.591C/T 0.028 0.028 1.020 0.027
    c.632A/G 0.605 0.425 1.730 0.333
    c.850C/T 0.394 0.467 1.866 0.356
    c.1207C/T 0.408 0.327 1.481 0.272
    c.1231A/C 0.408 0.343 1.518 0.283
    c.1289A/C 0.422 0.335 1.500 0.278
    壳白群体
    white strain
    c.367A/C 0.537 0.396 1.647 0.315
    c.466C/T 0.056 0.054 1.057 0.052
    c.475C/G 0.537 0.465 1.856 0.354
    c.591C/T 0.241 0.213 1.268 0.189
    c.632A/G 0.482 0.460 1.838 0.352
    c.724A/G 0.130 0.213 1.268 0.189
    c.850C/T 0 0.327 1.480 0.271
    c.1155C/T 0 0.369 1.576 0.298
    壳金群体
    gold strain
    c.367A/C 0 0.049 1.051 0.048
    c.475C/G 0.013 0.061 1.064 0.059
    c.632A/G 0.013 0.013 1.013 0.012
    c.724A/G 0.300 0.364 1.568 0.297
    c.850C/T 0.350 0.351 1.536 0.288

    Table 2.  Polymorphic parameters of 11 SNP loci of CgTyr1 gene in three shell color strains

    在壳黑长牡蛎群体中,突变位点c.367A/C、c.466C/T、c.475C/G和c.591C/T属于低度多态性位点(PIC<0.25),其他突变位点均为中度多态性位点(0.5>PIC>0.25)。在壳白长牡蛎群体中,突变位点c.466C/T、c.591C/T和c.724A/G属于低度多态位点(PIC<0.25),其他突变位点均为中度多态性位点(0.5>PIC>0.25)。在壳金群体中,突变位点c.367A/C、c.475C/G和c.632A/G属于低度多态性位点(PIC<0.25),其他突变位点均为中度多态性位点(0.5>PIC>0.25)。

  • 分别选择壳黑、壳白和壳金长牡蛎群体中与壳色性状存在极显著关联的SNP位点,分析关联位点间的连锁不平衡(表3表4表5)。在壳黑群体中,突变位点c.850C/T与其他突变位点均存在强连锁不平衡(D′>0.75)。在壳金群体中,CgTyr1基因上5个突变位点均表现出强连锁不平衡(D′>0.75)。

    壳黑群体
    black strain
    c.367A/Cc.466C/Tc.475C/Gc.591C/Tc.632A/Gc.850C/Tc.1207C/Tc.1231A/Cc.1289A/C
    c.367A/C 0.65 1 0.431 0.477 0.987 0.554 0.014 0.041
    c.466C/T 0.005 0.65 1 0.611 0.999 0.247 0.323 0.320
    c.475C/G 1 0.005 0.431 0.477 0.987 0.554 0.014 0.041
    c.591C/T 0.039 0.002 0.039 1 1 0.153 0.110 0.132
    c.632A/G 0.035 0.149 0.035 0.006 1 0.881 0.835 0.886
    c.850C/T 0.038 0.100 0.038 0.025 0.251 0.999 0.822 0.999
    c.1207C/T 0.005 0.041 0.005 0.001 0.458 0.148 1 1
    c.1231A/C 0 0.065 0 0.001 0.449 0.109 0.919 1
    c.1289A/C 0 0.066 0 0.001 0.484 0.155 0.958 0.959
    注:在每种壳色群体中,对角线上方为D′,对角线下方为R2,下同
    Notes: the figure above the diagonal represents D′, the figure below the diagonal represents R2, the same below

    Table 3.  Linkage disequilibrium analysis of 11 SNP loci of CgTyr1 gene in black shell color strain

    壳白群体
    white strain
    c.367A/Cc.466C/Tc.475C/Gc.591C/Tc.632A/Gc.724A/Gc.850C/Tc.1155C/T
    c.367A/C 1 0.935 0.003 0.065 0.183 0.006 0.427
    c.466C/T 0.01 1 1 0.995 0.570 1 0.122
    c.475C/G 0.568 0.016 1 0.644 0.045 0.075 0.278
    c.591C/T 0 0.004 0.242 1 0.361 0.227 0.291
    c.632A/G 0.003 0.015 0.127 0.074 1 0.225 0.169
    c.724A/G 0.002 0.068 0 0.002 0.074 0.622 0.041
    c.850C/T 0 0.007 0.003 0.028 0.007 0.014 0.042
    c.1155C/T 0.021 0.001 0.043 0.036 0.017 0 0.001

    Table 4.  Linkage disequilibrium analysis of 8 SNP loci of CgTyr1 gene in white shell color strain

    壳金群体
    gold strain
    c.367A/Cc.475C/Gc.632A/Gc.724A/Gc.850C/T
    c.367A/C 1 1 1 1
    c.475C/G 0.795 1 0.990 0.990
    c.632A/G 0 0 1 0.998
    c.724A/G 0.008 0.010 0.002 1
    c.850C/T 0.007 0.009 0.022 0.090

    Table 5.  Linkage disequilibrium analysis of 5 SNP loci of CgTyr1 gene in gold shell color strain

    利用3种壳色长牡蛎群体中表现出与壳色存在极显著关联的SNP位点进行单倍型构建,成功为每种壳色群体构建出1种单倍型,并且每种单倍型在相应群体中的频率,与其在其他壳色群体中的表达存在极显著差异(P<1×10−6)(表6)。

    组别
    group
    单倍型
    haplotype
    单倍型序列
    sequence
    壳黑群体(频率)
    black strain (freq.)
    壳白群体(频率)
    white strain (freq.)
    壳金群体(频率)
    gold strain (freq.)
    P
    P value
    构建组
    construction group
    HB A T G C A A T T T C A 18.36 (0.129) 0.00 (0.000) 0.00 (0.000) 1.09E-07
    HW A T G C G A T T C C C 0.00 (0.000) 15.50 (0.144) 0.00 (0.000) 2.87E-09
    HG A T G C G C T T C A C 0.00 (0.000) 2.50 (0.023) 37.00 (0.231) 2.65E-10
    验证组
    validation group
    HB A T G C A A T T T C A 4.00 (0.200) 0.00 (0.000) 0.00 (0.000) 0.029
    HW A T G C G A T T C C C 0.00 (0.000) 3.50 (0.175) 0.00 (0.000) 0.043
    HG A T G C G C T T C A C 0.00 (0.000) 0.50 (0.025) 5.00 (0.250) 0.028

    Table 6.  Haplotype analysis of 11 SNP loci of CgTyr1 gene in three shell color strains

  • 为进一步验证所构建的单倍型是否具有普遍适用性,同样使用PCR-SSCP方法在验证组中针对CgTyr1基因进行扩增分型和测序,得到每个个体的碱基序列。选取上述11个表现出极显著关联(P<1×10−6)的SNP位点(表2),构建单倍型。验证组的单倍型构建结果表明,单倍型haplotype black(HB)、haplotype white(HW)和haplotype gold(HG)分别为壳黑、壳白和壳金长牡蛎群体的优势单倍型(P<0.05)(表6)。

3.   讨论
  • 已有研究表明,海洋软体动物的基因组中存在较高的杂合度。长牡蛎作为一种重要的水产养殖动物,其表现出的高度多态性意味着在基因组中存在大量的SNP位点。有学者认为,在编码区平均每60个碱基中就会出现1个SNP位点,而在非编码区平均每40个碱基就可能存在1个SNP位点[24]。长牡蛎全基因组测序表明其基因的多态性水平为2.3%,而人类基因组的多态性水平仅有0.09%[25]。在本研究所扩增的CgTyr1基因外显子上,共计发现了23个SNP位点,平均的SNP位点出现频率约为1.3%。黑色素是广泛存在于动物中的一种重要色素。酪氨酸酶作为关键酶,参与合成真黑素和褐黑素。目前,在长牡蛎中2个酪氨酸酶相关基因已被克隆(CgTyr1基因和CgTyr2基因),均在色素合成中承担重要角色。CgTyr1基因被发现从长牡蛎的担轮幼虫时期开始表达,位于担轮幼虫的非钙化壳上,并且表达仅仅表现在绞合部和贝壳边缘,而在贝壳的中心部位没有检测到其表达,表明其可能在幼虫的贝壳形成过程中起到关键作用[16]。同样,CgTyr2基因在内套膜边缘检测到高度表达,表明其可能参与角质层的分泌和着色过程[17]。已有研究表明,三角帆蚌(Hyriopsis cummingii)HcTyr基因和HcTyp-1基因存在多个SNP位点,与三角帆蚌内壳色和珍珠层颜色性状存在关联[26-27]

    在水产动物中,已有不少学者针对TYR基因突变和颜色性状的关联性开展研究。斑马鱼(Danio rerio)酪氨酸酶基因的等位基因i导致在眼睛和表皮的黑色素沉积[27]。在该基因中,共有4个等位基因(i1i4i5i6)被发现[28]。其中,等位基因i6可能由于其存在245个碱基的缺失而表现出与白化性状高度关联[29]。研究表明,向非洲爪蟾(Xenopus tropicalis)胚胎中注射酪氨酸酶基因的向导RNA(sgRNA)和Cas9 mRNA可导致个体出现明显的白化性状。在牡蛎中,CgTyr1基因上同样可能存在类似的等位基因,控制和影响不同的壳色性状,这需要今后利用基因编辑技术进一步验证。

    本研究中,在长牡蛎CgTyr1基因上共发现了11个SNP位点与壳色性状存在极显著相关(P<1×10−6),其中有8个SNP位点是同义突变,并不改变其编码氨基酸的类型。但是,在基因的翻译过程中,tRNA与简并密码子相结合的偏好并不是随机的,同义SNP突变可能改变或降低基因的翻译效率[30-31]。已有研究表明,同义SNP突变可能改变mRNA的空间折叠和稳定性,进而影响其功能。所以,本研究在构建单倍型时,将8个同义SNP位点纳入考量范围,与3个非同义SNP一同构建单倍型,具有较高的可信度。

    连锁不平衡分析显示,与壳色性状存在极显著相关(P<1×10−6)的11个SNP位点存在不同程度的连锁遗传现象。这种在CgTyr1基因上的多重突变(multiple mutation)可能是导致壳色多态性的原因。相比于单个SNP突变位点与性状的关联分析,单倍型分析可以评估多重突变对表型所带来的细微影响。单倍型分析为二倍体生物进化研究提供了一种更为有效的手段,并在鉴定复杂遗传性状的候选基因中起到了重要作用[23]。探究一个基因与某个性状的关系,关键是找到与这个性状存在关联性的单倍型。本研究为每一种壳色群体都建立了1种单倍型,并且每种单倍型在相应群体中的频率,与其在其他壳色群体中的表达存在极显著差异(P<1×10−6),这表明CgTyr1基因是导致长牡蛎个体表现出不同壳色的重要基因。

    综上所述,本研究筛查了壳白、壳黑和壳金3种长牡蛎群体CgTyr1基因的SNP位点,共发现23个SNP突变,其中11个SNP位点与壳色性状存在极显著关联(P<1×10−6)。在这11个SNP位点中,突变位点c.591C/T、c.632A/G、c.1155C/T属于非同义SNP位点,分别导致Ala122Val、Gly136Ser和Phe310Ser的氨基酸突变,其余突变位点均为同义SNP位点。基于与壳色性状存在极显著关联(P<1×10−6)的11个SNP位点,实验为每一种壳色群体都构建了1种单倍型,并在验证组中得到了确认。研究结果为长牡蛎壳色品系的标记辅助育种提供了重要基础资料。

    ·续附表1·
    外显子
    region
    SNP位点
    mutation
    壳黑群体
    black strain (n=71)
    壳白群体
    white strain (n=54)
    壳金群体
    gold strain (n=80)
    P
    P value
    HW 13.39(0.000) 0.167(0.683) 0.003(0.955)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    A 0.303 0.352 0.006 Gly
    G 0.697 0.648 0.994 Ser
    exon 2 c.724A/G 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    AA 71(1.000) 44(0.815 49(0.613) 3.39E-07
    AC 0(0.000) 7(0.130) 24(0.300)
    CC 0(0.000) 3(0.056) 7(0.087)
    HW 0.000(1.000) 8.123(0.004) 2.358(0.125)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    A 1 0.880 0.762 Pro
    C 0 0.120 0.237 Pro
    exon 3 c.850C/T 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    CC 12(0.169) 11(0.204) 4(0.050) 7.81E-07
    CT 28(0.394) 0(0.000) 28(0.350)
    TT 31(0.437) 43(0.796) 48(0.600)
    HW 1.607(0.205) 54.00(0.000) 0.001(0.974)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    C 0.366 0.204 0.225 Ser
    T 0.634 0.796 0.775 Ser
    exon 4 c.1155C/T* 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    CC 0(0.000) 13(0.241) 0(0.000) 3.73E-09
    TT 71(1.000) 41(0.759) 80(1.000)
    HW 0.000(1.000) 54.00(0.000) 0.000(1.000)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    C 0 0.241 0 Phe
    T 1 0.759 1 Ser
    c.1207C/T 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    CC 42(0.592) 54(1.000) 80(1.000) 1.43E-14
    CT 29(0.408) 0(0.000) 0(0.000)
    HW 4.676(0.031) 0.000(1.000) 0.000(1.000)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    C 0.796 1 1 Asn
    T 0.204 0 0 Asn
    ·续附表1·
    外显子
    region
    SNP位点
    mutation
    壳黑群体
    black strain (n=71)
    壳白群体
    white strain (n=54)
    壳金群体
    gold strain (n=80)
    P
    P value
    c.1231A/C 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    AA 41(0.577) 0(0.000) 80(1.000) 1.87E-28
    AC 29(0.408) 54(1.000) 0(0.000)
    CC 1(0.014) 0(0.000) 0(0.000)
    HW 2.748(0.097) 54.00(0.000) 0.000(1.000)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    A 0.782 0.500 1 Gly
    C 0.218 0.500 0 Gly
    c.1289A/C 基因型
    genotype
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    数量(频率)
    no. (freq.)
    AC 30(0.423) 0(0.000) 0(0.000) 3.96E-15
    CC 41(0.577) 54(1.000) 160(1.000)
    HW 5.094(0.024) 0.000(1.000) 0.000(1.000)
    等位基因
    allele
    频率
    freq.
    频率
    freq.
    频率
    freq.
    氨基酸类型
    amino acid
    A 0.211 0 0 Arg
    C 0.789 1 1 Arg
    注:HW表示哈利温伯格平衡检验;*表示该位点为非同义SNP突变位点
    Notes: HW represents Hardy-Weinberg test; * represents nsSNP locus
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