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ScHsc70 gene SNP markers screening and correlation analysis for high temperature resistance of Sinonovacula constricta new variety “Shenzhe NO. 1”

  • To obtain single nucleotide polymorphism (SNP) markers of heat shock protein 70 (ScHsc70) gene related to heat tolerance traits in Sinonovacula constricta, the full-length sequence of ScHsc70 gene was cloned by direct sequencing. The results showed that the ScHsc 70 gene was 4 048 bp, including 6 introns and 7 exons, and the coding region was 1 950 bp. The high temperature resistance group and control group from a new variety of razor clam were established six potential SNPs Rs1 (g.588 C > T), Rs2 (g.840 C > T), Rs3 (g.885 T > A), Rs4 (g.1233 A > G), Rs5 (g.1467 T > G), Rs6 (g.1482 T > C) were screened from the exon region of ScHSc70 gene by PCR product direct sequencing. SNPs genotyping was performed on the high temperature tolerant population and the control population of the new variety " Shenzhe No.1” by Sanger sequencing. The results of genetic diversity analysis showed that the polymorphism information content (PIC) of the two populations was between 0.111 2 and 0.371 8, and the average polymorphism information content of the control population was 0.267 0, which is higher than that of the high temperature resistant populations (0.236 5). Association analysis results showed that the genotype frequency and allele frequency of Rs1, Rs3 and Rs4 were significantly different between control group and high temperature tolerant group. Haploid linkage disequilibrium analysis showed that SNPs markers of ScHsc70 gene could form 2 haploid blocks and 7 haplotypes, among which CCT haplotypes were significantly correlated with high temperature tolerance traits. It was found that Rs2 and Rs3 were in a linkage state (r2 = 0.86, LOD = 25.56, Dx = 1.0), and could be used as SNP markers for the Genetic breeding of S. constricta for high temperature tolerance. In summary, Rs1, Rs3, Rs4 and haplotype CCT can be used as candidate auxiliary molecular markers for high temperature tolerance breeding of razor clam, which lays a theoretical foundation for the subsequent functional verification of resistance-related SNPs.
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ScHsc70 gene SNP markers screening and correlation analysis for high temperature resistance of Sinonovacula constricta new variety “Shenzhe NO. 1”

    Corresponding author: Jiale LI, jlli@shou.edu.cn
    Corresponding author: Heding SHEN, hdshen@shou.edu.cn
  • Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding Shanghai, Shanghai Ocean University, Shanghai    201306, China

Abstract: To obtain single nucleotide polymorphism (SNP) markers of heat shock protein 70 (ScHsc70) gene related to heat tolerance traits in Sinonovacula constricta, the full-length sequence of ScHsc70 gene was cloned by direct sequencing. The results showed that the ScHsc 70 gene was 4 048 bp, including 6 introns and 7 exons, and the coding region was 1 950 bp. The high temperature resistance group and control group from a new variety of razor clam were established six potential SNPs Rs1 (g.588 C > T), Rs2 (g.840 C > T), Rs3 (g.885 T > A), Rs4 (g.1233 A > G), Rs5 (g.1467 T > G), Rs6 (g.1482 T > C) were screened from the exon region of ScHSc70 gene by PCR product direct sequencing. SNPs genotyping was performed on the high temperature tolerant population and the control population of the new variety " Shenzhe No.1” by Sanger sequencing. The results of genetic diversity analysis showed that the polymorphism information content (PIC) of the two populations was between 0.111 2 and 0.371 8, and the average polymorphism information content of the control population was 0.267 0, which is higher than that of the high temperature resistant populations (0.236 5). Association analysis results showed that the genotype frequency and allele frequency of Rs1, Rs3 and Rs4 were significantly different between control group and high temperature tolerant group. Haploid linkage disequilibrium analysis showed that SNPs markers of ScHsc70 gene could form 2 haploid blocks and 7 haplotypes, among which CCT haplotypes were significantly correlated with high temperature tolerance traits. It was found that Rs2 and Rs3 were in a linkage state (r2 = 0.86, LOD = 25.56, Dx = 1.0), and could be used as SNP markers for the Genetic breeding of S. constricta for high temperature tolerance. In summary, Rs1, Rs3, Rs4 and haplotype CCT can be used as candidate auxiliary molecular markers for high temperature tolerance breeding of razor clam, which lays a theoretical foundation for the subsequent functional verification of resistance-related SNPs.

  • 缢蛏(Sinonovacula constricta),俗称蛏子、蜻子,属软体动物门(Mollusca)、瓣鳃纲(Lamellibranchia)、异齿亚纲(Heterodonta)、帘蛤目(Veneroida)、竹蛏科(Solenidae)、缢蛏属(Sinonovacula)[1],主要分布于西太平洋沿海的中国和日本。蛏子作为我国沿海地区的主要经济贝类,从北部的辽宁至南部广东沿海均有分布,是我国四大海水养殖贝类之一,养殖历史悠久。在我国浙江、福建、山东、江苏、辽宁、广东和广西沿海均已开展养殖[2-3]

    温度因子影响着海洋无脊椎动物生长和发育,温度与疾病暴发有直接的相关性[4]。在一定的温度范围内,大多数海洋贝类的代谢率会随着温度的升高而升高,当超过其承受最高温范围后,会引起贝类生理功能的紊乱[5],严重时会使贝类大量死亡[6-7],特别在夏季持续降雨、极度高温等极端环境下,高密度养殖的缢蛏会大面积死亡。近年来,随着海洋环境的恶化,野生缢蛏资源锐减;加上连续多代的人工繁育,造成缢蛏种质资源快速退化,遗传多样性不断下降,抗逆性减弱。因此,研究贝类抗热机制,筛选与贝类抗热相关功能基因并进行分子标记开发,培育耐高温贝类新品种至关重要。

    热休克蛋白(HSP)是在正常生长和发育中起重要作用的一类蛋白质,可以帮助生物体调节应激反应[8-10]。当生物体受到温度、盐度、缺氧、重金属、病毒和细菌感染的胁迫时,HSP表达水平显著增加,以提高生物体抗应激水平,从而维持细胞的稳态和存活[11-13]。关于热休克蛋白70 (HSP70) 在水产贝类抗热性和抗其他胁迫方面的重要作用,已经在许多海洋双壳类物种中进行了相关研究,如香港巨牡蛎(Crassostrea hongkongensis)[14],美洲牡蛎(C. virginica)[15],欧洲牡蛎(Ostrea edulis)[16],地中海贻贝(Mediterranean mussel)[17],扇贝(Argopecten irradians)[18-19],文蛤(Meretrix meretrix)[20-21] 和缢蛏(Sinonovacula constricta)[22]。单核苷酸多态性(single nucleotide polymorphism, SNP)被广范应用于遗传连锁图谱构建、关联分析、分子标记辅助育种(MAS)和品种鉴定等方面,具有巨大的应用前景[23-25]。目前在水产动物中大多利用生长及免疫相关基因分别进行生长和抗病关联分析的研究,如已有斑马鱼(Denio rerio)[26]、鲤(Cyprinus carpio)[27]、文蛤[28]、缢蛏[29]和凡纳滨对虾(Litopenaeus vannamei)[30]等相关基因的SNPs的筛选,为水产动物MAS奠定了基础。

    缢蛏ScHSc70基因SNPs的筛选与耐高温性状的关联分析未见相关报道,本团队在前期研究中已成功克隆了缢蛏ScHsc70 基因序列 (登录号:JF748730)。通过构建耐高温群体和对照组群体,以ScHsc70 基因作为候选基因,通过直接测序法克隆ScHsc70 基因的DNA全长序列,比对确定外显子和内含子区域。采用PCR产物直接测序法筛查ScHsc70 外显子中潜在的SNPs,然后利用Sanger法对2个群体中相应SNPs进行基因分型,通过与耐高温性状的关联分析,获得与耐高温性状相关的SNPs和单倍型,为缢蛏的分子标记辅助育种提供有效的候选分子标记。

    • 缢蛏“申浙1号”养殖新品种是以国内6个自然群体为基础群体,采用传统的群体选育方法,以生长快、存活率高为主要选育指标,经过连续5代选育获得的[31]。用于筛选SNPs和关联分析的缢蛏新品种均采自浙江宁海养殖基地。9月龄时,随机选取600个缢蛏个体,低温包装带回实验室暂养。

    • 将取回后的缢蛏暂养1周后,分成编号为1和2的两组,分别养殖在0.6 m × 0.8 m × 0.5 m的塑料箱中,1组260个个体于常温 (32 ± 0.5) °C下养殖;2组260个个体于(35.5 ± 0.5) °C 恒温养殖,海水比重为1.019±0.001。每天分早晚2次定时投喂牟氏角毛藻(Chaetoceros muelleri)和湛江等鞭金藻(1sochrysis zhanjangensis)的混合液,72 h后对存活个体进行随机取样,2组存活个体视为耐高温群体,1组存活个体作为对照组。

    • 采用TIANamp Marine Animals DNA Kit (TIANGEN) 试剂盒,各提取120个缢蛏耐高温群体个体和对照组群体个体外套膜基因组DNA,实验方法参照试剂盒说明书。提取的DNA经1%琼脂糖凝胶电泳检测其完整性,并用超微量分光光度计(D2000)检测其浓度和纯度,-20 °C保存备用。

    • 根据NCBI数据库中缢蛏ScHsc70基因序列(登录号:JF748730),利用Primer premer 5.0设计3对特异性引物(表1),3对引物均由生工生物工程(上海)股份有限公司合成,以上述提取的DNA为模板,采用PCR技术分别扩增ScHsc70基因片段。PCR扩增反应体系为30 μL:2×Taq PCR master Mix 15 μL,上下游引物(10 μmol/L)各 0.9 μL,DNA模版 1.2 μL,ddH2O 12 μL。扩增程序:94 °C 5 min;94 °C 30 s,57 °C 1 min,72 °C 1 min,35个循环;72 °C 10 min。琼脂糖凝胶电泳检测到PCR产物具有特异性条带后,将不同片段大小的特异性条带分别进行割胶回收、纯化后与载体pMD19-T连接,转化至大肠杆菌感受态细胞,阳性单克隆送至生工生物工程(上海)股份有限公司测序,用BioEdit软件进行序列比对与拼接。

      引物名
      primer
      引物序列 (5'-3')
      primer sequence (5'-3')
      用途
      usage
      退火温度/°C
      annealing temperature
      HSP70-F1 TCAGGGAAACAGGACCAC DNA序列克隆 54.4
      HSP70-R1 TACGCATACGACGCACAG DNA序列克隆 55.2
      HSP70-F2 TTCGACTTAGGAGGCGGTAC DNA序列克隆 57.1
      HSP70-R2 CTGGAGCAACTTCTGGATCTTC DNA序列克隆 58.3
      HSP70-F3 TGCGTCGTCTGCGTATTGC DNA序列克隆 59.0
      HSP70-R3 ACCCTCCAGTTCCTTCTGCTT DNA序列克隆 58.6
      HSP-E04-F TGATGTCACAGATTTGTTGCG 第4外显子克隆 57.0
      HSP-E04-R GGTGGAAACTGCGAAAATGTG
      HSP-E05-F CGTCCTTCCTTGGATGTCTGAACC 第5外显子克隆 57.0
      HSP-E05-R TTACATTCATTAGCTCACCAAAGT
      HSP-E06-F GCAATGATAGATTGGCTCTGTTTG 第6外显子克隆 57.0
      HSP-E06-R GTTGTAAAACTACACCACAGATGG

      Table 1.  The primer of ScHsc70 gene of S. constricta

    • 从构建的耐高温群体和对照组群体中各随机选取20个缢蛏基因组DNA,利用引物(表1)扩增ScHsc70基因的部分外显子片段,PCR产物经大肠杆菌感受态细胞连接转化后送生工生物工程(上海)股份有限公司进行测序,用DNAman软件比对分析序列及寻找SNPs位点。

    • 上海迈浦生物科技有限公司采用Sanger测序法,成功检测107个对照组个体和111个耐高温个体的SNPs基因分型,使用测序仪(3730 xl)进行测序,用DNA Sequencing analysis软件分析测序结果,用Sequencing Analysis 5.2.0软件进行解读,并用Sequencher 5.1软件包进行比对分析。

    • 采用POPGENE version 1.32软件计算有效等位基因数(Ne)、观测杂合度(Ho)和期望杂合度(He)等遗传多样性参数;Chi-square检验估计群体Hardy-Weinberg平衡偏离;用SPSS 20.0软件进行卡方(χ2)检验,分析各SNP位点与缢蛏耐高温性状的相关性,连锁不平衡分析采用Haploview 4.2软件进行,P<0.05为显著性差异。

    2.   结果
    • 序列总长度为4 048 bp,编码区1 950 bp,6个内含子,7个外显子,第4外显子最长554 bp,第3外显子最短为153 bp,内含子长度在237~494 bp之间,内含子符合GT-AG剪接规则(登录号:MK140678)。经序列比对发现6个候选SNPs位点,且都在第4~5外显子中,以起始密码子TAA第1个的碱基开始编号,以突变位置和碱基突变类型进行命名: Rs1(g. 588 C>T)、Rs2(g. 840 C>T)、Rs3(g. 885 T>A)、Rs4(g. 1233 A>G)、Rs5(g. 1467 T>G)、Rs6(g. 1482 T>C) (表2)。其中4个转换,2个颠换,6个SNPs位点中氨基酸没有发生改变。

      名称
      name
      外显子区域
      exon region
      碱基突变类型
      base mutation type
      氨基酸突变类型
      amino acid mutation type
      Rs1 4 转换 亮氨酸(L)-亮氨酸(L)
      Rs2 4 转换 丙氨酸(A)-丙氨酸(A)
      Rs3 4 颠换 苏氨酸(T)-苏氨酸(T)
      Rs4 5 转换 苏氨酸(T)-苏氨酸(T)
      Rs5 5 颠换 丝胺酸(S)-丝胺酸(S)
      Rs6 5 转换 丝胺酸(S)-丝胺酸(S)

      Table 2.  The 6 SNPs loci mutation information of ScHsc70 gene in S. constricta

    • 利用重测序方法对“申浙1号”缢蛏对照群体和耐高温群体进行SNPs的基因分型。共获得218个有效个体,对照组107个,耐高温群体111个。在对照组群体中,多态性信息含量(PIC)的范围为0.196 2~0.371 8,其中Rs1和Rs4属于中度多态(0.25<PIC<0.5),其余为低度多态(PIC<0.25);在耐高温群体中,多态性信息含量(PIC)的范围0.111 2~0.351 9,其中Rs1、Rs4和Rs6属于中度多态(0.25<PIC<0.5),其余为低度多态(PIC<0.25)。χ2检验表明,在对照组群体中,除Rs3和Rs5不符合Hardy-Weinberg平衡外(P<0.05),其余都符合Hardy-Weinberg平衡(P>0.05);在耐高温群体中,除Rs5不符合Hardy-Weinberg平衡外(P<0.05),其余都符合Hardy-Weinberg平衡(P>0.05)。

    • 采用SPSS20.0软件分析ScHsc70基因的6个SNPs在对照群体和耐热性群体中的基因型分布频率、等位基因频率、卡方检验及其与高温抗性的相关性。结果显示,仅Rs1、Rs3和Rs4的基因型频率和等位基因频率在对照组和耐高温群体之间存在显著性差异(P<0.05),表明这3个位点与耐高温性状显著相关(P<0.05) (表4);利用Haploview 4.2软件中的Four Gamete Rule计算方法对ScHsc70基因的SNPs进行连锁不平衡分析(图1),6个SNPs可形成2个连锁不平衡单倍块和7种单倍型,其中仅有CCT单倍型与耐高温性状显著相关(P<0.05)(表5)。此外,本实验发现Rs2和Rs3处于连锁状态(r2=0.86,LOD=25.56,Dx=1.0),可以作为缢蛏耐高温遗传育种的SNP标签(图1)。

      位点
      locus
      基因型(N)
      genotype
      基因型频率 genotype frequency χ2
      P
      等位基因
      allele
      等位基因频率 allele frequency χ2
      P
      对照组群体
      control group
      耐高温群体
      resistant group
      对照组群体
      control group
      耐高温群体
      resistant group
      Rs1 CC (81) 37/0.346 44/0.396 6.420 C 179/0.579 147(0.662) 3.170
      TT (28) 20/0.187 8/0.072 0.040 T 149/0.421 75(0.338) 0.006
      TC (109) 50/0.467 59/0.532
      Rs2 TT (17) 4/0.037 1/0.009 5.764 T 29(0.136) 14(0.937) 6.434
      CC (173) 82/0.766 98/0.883 0.056 C 185(0.864) 208(0.063) 0.011
      TC (25) 21/0.196 12/0.108 1
      Rs3 TT (167) 80/0.748 95/0.856 6.181 A 33(0.154) 17(0.077) 6.468
      AA (11) 6/0.056 1/0.009 0.045 T 181(0.846) 205(0.923) 0.011
      TA (37) 21/0.196 15/0.135
      Rs4 AA (32) 22/0.206 9/0.081 7.150 A 95(0.444) 78(0.351) 4.235
      GG (72) 33/0.308 42/0.378 0.028 G 117(0.556) 144(0.649) 0.040
      GA (110) 51/0.477 60/0.541
      Rs5 TT (166) 84/0.785 83/0.748 0.778 G 25(0.126) 25(0.149) 0.000
      GG (6) 3/0.028 5/0.045 0.678 T 187(0.874) 186(0.851) 0.986
      TG (42) 19/0.178 23/0.207
      Rs6 TT (4) 1/0.009 3/0.027 1.114 T 34(0.159) 41(0.815) 0.509
      CC (147) 74/0.692 73/0.658 0.573 C 180(0.841) 181(0.185) 0.475
      TC (64) 32/0.299 35/0.315

      Table 4.  The SNPs statistical analysis of S. constricta ScHsc70 gene in control group and resistant group

      Figure 1.  Linkage disequilibrium map showing the pair-wise LD between the SNPs of ScHsc70 gene in S. constricta

      单倍块
      double block
      单倍型
      haplotype
      分布频率
      distribution frequency
      耐高温群体频率
      resistant group frequency
      对照组群体频率
      control group frequency
      χ2 P
      1 CCT 0.134 63 /0.572 45 /0.425 4.710 0.030
      TCT 0.148 38 /0.338 45/ 0.421 1.414 0.234
      CTA 0.199 9 /0.077 15 /0.136 1.943 0.163
      2 GTC 0.097 35 /0.315 30 /0.277 0.318 0.573
      ATC 0.105 39 /0.351 47 /0.438 1.762 0.184
      GGC 0.090 21 /0.185 13 /0.120 1.897 0.168
      GTT 0.067 17 /0.149 17 /0.159 0.014 0.907

      Table 5.  Association analysis of ScHsc70 haplotypes with resistance to high temperature for S. constricta

    3.   讨论
    • 在植物信号传导途径中,环境改变如高温、高盐和细菌侵染会导致胞质内蛋白质变性,变性的蛋白质聚集,进而引起热休克蛋白70/90和热激转录因子HsfA/B的积累,热激蛋白使变性的蛋白质复性,具有分子伴侣的功能,有助于高温下保持和恢复蛋白质的活性结构[32]

      HSP70 作为应激保护蛋白,高温刺激下可诱导机体的HSP合成增加,使生物体抵抗应激物的侵害,从而维持细胞的稳态和存活[11]。菲律宾蛤仔(Ruditapes Philippinarum)[33]在高温热胁迫12 h后体内HSP70 达到最高峰,可能促进异常蛋白的恢复。Nakano等[34]对潮间杜父鱼研究表明,当机体内HSP70储存较多时机体具有更大的热耐受能力,能够适应更大的温差变化。淡水海绵(Ephydatia fluviatilis)[35]受亚致死热刺激后,表现出较强的抵抗力。冯冰冰等[22]在缢蛏弧菌感染实验发现其肝脏中ScHsc70 mRNA水平在一段时间后会大量表达。贝类的高温耐受性是一个非常复杂的过程,高温性状是由多个基因控制,贝类耐热性分子机制尚未可知,本实验中以ScHsc70作为缢蛏耐高温选育的候选基因进行两个群体间SNPs与耐高温性状的关联性分析,为后续的抗逆性相关SNPs筛选提供理论基础。

    • SNP标记是鉴定数量性状基因座位、发现致病基因[24]的一种有效方法,通过不同表现型与SNPs间的相关性进行分子标记辅助选择,可以提高筛选效率。徐田军等[36]在牙鲆(Paralichthys olivaceus)野生群体MHC-DAA基因中筛选出与抗病相关的等位基因。大多数SNPs位于基因组的非编码区,对蛋白质无直接影响,这一类SNPs作为遗传标记在群体遗传和生物进化研究中有着很重要的作用[37-38]。Halushka等[39]根据对75个基因的检测结果推测人类基因组中大约存在100万个SNPs位点,50万个在非编码区,20~40万个在编码区而分布在编码区的非同义突变只有2.4~4万个。Weber等[40]认为编码区的SNPs位点具有更高的遗传稳定性。

      本研究中,克隆得到ScHsc70基因的全长4 048 bp,包含7个外显子和6个内含子,在7个外显子中初步筛选出6个潜在SNP位点(表2),分布密度为1/494 bp。长牡蛎(C. gigas) 外显子编码区SNPs的分布密度为1/60 bp[41],缢蛏生长因子受体结合蛋白2基因外显子上SNPs的分布密度为1/65 bp[42],说明不同物种、同一物种不同基因外显子上SNPs的分布密度不同。另外,6个SNPs中4个变异位点为转换,2个变异位点为颠换,4个转换变异类型中C→T变异最多,可能是因为CpG二核苷酸上的胞嘧啶残基是人类基因组中最易发生突变的位点,其中大多数是甲基化的,可自发地脱去氨基而形成胸腺嘧啶[43]

    • 本实验以新品种“申浙1号”为基础群体,通过高温胁迫构建耐高温群体,与对照组间的存活率存在显著性差异,说明温度胁迫对受试个体产生区分度,群体遗传结构可能会发生变化。耐高温群体和对照组群体6个SNPs基因分型中,所有位点多态性信息含量位于0.111 2~0.371 8之间,对照群体的平均多态信息含量高于耐高温群体的多态信息含量,很大原因是经过耐高温实验导致的。经过连续多代的人工选择,多态信息含量出现多个低度多态位点(PIC<0.25)[44],有一定程度的下降。本实验中获得的3个SNPs与耐高温性状显著相关,可以作为耐高温遗传育种的候选分子标记辅助育种。

      在对照组群体中,Rs3和Rs5不符合Hardy-Weinberg平衡(P<0.05),在耐高温群体中,Rs5不符合Hardy-Weinberg平衡(P<0.05)(表3),两个群体遗传多样性发生轻微的变化,而且部分SNPs基因型频率和等位基因频率发生差异(表4),这可能是由于经过第一代耐高温选育,与抗热性相关的SNPs的基因型和等位基因在选育过程中被定向选择导致的。在抗性群体的构建过程中,SNPs偏离Hardy-Weinberg平衡现象的出现,可以作为与抗性相关的初步推断[45]

      名称 name 对照组群体 (107个)  control group 耐高温群体(111个)  resistant groups
      locus Ne He Ho PIC H-W Ne He Ho PIC H-W
      Rs1 1.950 8 0.487 4 0.467 3 0.368 6 0.634 7 1.809 6 0.449 4 0.531 5 0.347 3 0.052 9
      Rs2 1.306 0 0.234 3 0.196 3 0.206 8 0.080 7 1.113 4 0.118 7 0.108 1 0.111 2 0.330 0
      Rs3 1.352 9 0.260 9 0.196 3 0.226 8 0.008 5 1.164 7 0.142 1 0.135 1 0.131 5 0.623 1
      Rs4 1.975 2 0.493 7 0.476 6 0.371 8 0.684 5 1.837 6 0.457 9 0.540 5 0.351 9 0.055 8
      Rs5 1.282 9 0.220 5 0.177 6 0.196 2 0.036 8 1.338 9 0.254 2 0.207 2 0.221 0 0.048 2
      Rs6 1.364 8 0.267 3 0.299 1 0.231 6 0.233 3 1.430 9 0.302 5 0.315 3 0.255 8 0.652 3
      mean 1.538 8 0.327 4 0.302 2 0.267 0 0.279 7 1.449 2 0.287 5 0.306 3 0.236 5 0.293 7
      注:Ne 代表有效等位基因数,He代表期望杂合度,Ho代表观测杂合度,PIC代表多态信息含量,H-W代表Hard-Weinberg平衡;P<0.05表示差异性显著
      Notes: Ne represents effective number of alleles, He represents expected heterozygosity, Ho represents observed heterozygosity, PIC represents polymorphism information content, H-W represents Hardy-Weinberg equilibrium; P<0.05 is considered to be statistically significant

      Table 3.  The genetic polymorphism information of 6 SNPs loci in control groups and resistant groups from S. constricta

      连锁不平衡(linkage disequilibrium, LD)是一条染色体上2个等位基因间的非随机相关。理论上,LD的强度与2个SNP间的距离有关,距离越小,发生重组的机会越小,LD越强,反之亦然。r2和Dx来度量LD的强弱[46],当Dx> 0.33,r2 > 0.1认为是有意义的连锁不平衡[47]。在研究SNPs与疾病的相关性时,运用单倍型连锁不平衡分析与抗病性的关联分析更能说明问题[48]。本研究通过连锁不平衡分析获得一个单倍型(CCT)与耐高温性状显著相关(P<0.05),同时发现一个SNP标签,可以作为耐高温新品种培育的候选分子标记。

      SNP已经广泛应用于水产养殖育种进程中,目前多以鱼类研究为主,且主要集中在与生长[49]、抗性[26-27]相关等领域;海洋贝类中多集中在生长[29]相关领域,未见与抗逆性相关的报道。从高产到高品质、抗逆性是水产种业发展的趋势,分子标记辅助育种(MAS)是培育高品质、抗逆性新品种的有效手段。关于贝类抗逆性相关的候选基因比较少,后期应构建精密的遗传连锁图谱,加强全基因组学、基因组选择育种和功能基因组学的研究,寻找与抗性相关的候选基因,借助分子标记辅助育种技术快速高效的培育优良抗逆性新品种。

      孟德龙为同等贡献第一作者。

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