栉孔扇贝抗鳗弧菌感染性状候选基因的多态性研究

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	栉孔扇贝抗鳗弧菌感染性状候选基因的多态性研究
其他题名	The Polymorphisms of candidate Genes Associated with Susceptibility/Resistance of Zhikong Scallop (Chlamys farreri) to Listonella anguillarum
	李凌
学位类型	博士
	2009-05-28
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	栉孔扇贝 Cflysg Cfc1qdc Cflitaf 多态性疾病抗性疾病敏感性 Pcr-rflp Bi-pasa Pcr
摘要	栉孔扇贝（Chlamys farreri）是我国北方地区主要的养殖贝类之一，曾为沿海各省带来巨大的经济效益。但自1997年以来，陆续爆发的病害问题给扇贝养殖业造成了巨大的经济损失，严重影响了该产业的健康发展。目前认为培育抗病性强的扇贝优良品种是解决病害问题的根本途径。由于传统的育种方法费时费力，无法满足对良种的迫切需求，因此有必要通过分子手段加快抗病品种的培育步伐。标记辅助育种（marker assisted selection，MAS）是成功应用于动物育种中的分子手段之一，但由于缺乏与抗病性状相关的标记，MAS目前还无法在软体动物中得到应用。因此，寻找与抗病性状相关的分子标记是在软体动物中发展MAS的关键。本研究利用鳗弧菌（Listonella anguillarum）对栉孔扇贝进行攻毒感染实验，初步得到敏感群体和抗病群体后采用PCR、PCR-RFLP、Bi-PASA PCR等方法研究了CfLysG、CfC1qDC和CfLITAF基因多态性及其与栉孔扇贝对鳗弧菌抗性的关系。研究发现，栉孔扇贝CfLysG的基因序列中共有104个单核苷酸多态性（SNP）位点和29个插入/缺失（I/D）多态性位点。有17个多态性位点位于启动子区域，选择其中的-753 I/D、-391A/G和-284I/D多态性进行检测，发现这三个位点的基因型在敏感群体和抗病群体中的分布均符合Hardy-Weinberg平衡（P>0.05）。其中-753 ID基因型和-284 ID基因在抗病群体中的频率高于在敏感群体中的频率，但两者之间无显著性差异（P>0.05）。-391 AG基因型在抗病群体中的频率显著高于敏感群体（P=0.007），表明-391 AG基因型与栉孔扇贝对鳗弧菌的抗性显著相关。为验证这一相关性，对-391位点不同基因型的扇贝进行攻毒感染实验。统计发现，具有-391 AA基因型的扇贝累计死亡率显著高于具有-391 AG基因型的扇贝（P=0.001），进一步证实了CfLysG基因-391 AG基因型与栉孔扇贝对鳗弧菌的抗性显著相关。CfLysG基因的外显子共有3处SNP，其中仅第三外显子上的+3473 A/C为非同义突变。统计分析表明，+3473位点不同基因型在敏感群体中的分布频率符合Hardy-Weinberg平衡（P>0.05），而在抗病群体中则偏离Hardy-Weinberg平衡（P<0.01）。+3473 AA基因型在抗病群体中的频率显著高于在敏感群体中的频率（P=0.022），表明+3473 AA基因型与栉孔扇贝对鳗弧菌的抗性显著相关。CfLysG基因第1内含子存在+96 I/D和+487 I/D两处大片段的I/D多态性。统计发现，这两个位点的基因型在敏感群体和抗病群体中的分布频率均符合Hardy-Weinberg 平衡（P>0.05）。其中+96 DD基因型和+487 ID基因型在抗病群体中的频率均略高于在敏感群体中的频率，但两者之间无显著性差异（P>0.05）。表明这两个位点的多态性与栉孔扇贝对鳗弧菌的抗性无显著相关性。对CfLysG基因各多态性位点的统计分析表明，各位点之间存在不同程度的连锁不平衡，提示有单体型的存在。对19种频率>1%的单体型在敏感群体及抗病群体中的频率进行分析，发现-753 I/-391 G/-284 I/+96 I/+487 D/+3473 A单体型在抗病群体中的频率显著高于敏感群体（P=0.044），表明该单体型与栉孔扇贝对鳗弧菌的抗性显著相关。在栉孔扇贝CfC1qDC基因cDNA序列上共发现14处SNP。对+423 T/C多态性与栉孔扇贝对鳗弧菌抗性的关系进行了分析。统计发现，+423位点各基因型在敏感群体和抗病群体中的分布均符合Hardy-Weinberg平衡（P>0.05）。+423 TT基因型在抗病群体中的频率显著高于在敏感群体中的频率（P=0.005），表明+423 TT基因型与栉孔扇贝对鳗弧菌的抗性显著相关。在栉孔扇贝CfLITAF基因cDNA序列中共发现3处SNP及1处I/D多态性。对+145 I/D多态性进行研究，发现所有敏感个体及抗病个体中均同时存在+145 位点所有等位基因，表明+145位点多态性与栉孔扇贝对鳗弧菌的抗性不相关。以上研究表明，栉孔扇贝CfLysG基因-391 AG基因型、+3473 AA基因型、-753 I/-391 G/-284 I/+96 I/+487 D/+3473 A单体型以及CfC1qDC基因+423 TT基因型与栉孔扇贝对鳗弧菌的抗性显著相关，提示它们可作为与栉孔扇贝抗病相关的候选分子标记应用于贝类抗病育种中，为贝类的标记辅助育种提供参考。此外，抗病相关分子标记的发现还有利于加深对扇贝发病机理的理解，并有助于发掘预防及治疗贝类疾病的新方法。
其他摘要	Zhikong scallop (Chlamys farreri) is one of the economic scallop species cultivated widely in North China and contributes enormously to the economic development of coastal provinces. Since the summer of 1997, frequent diseases have caused catastrophic economic losses to scallop aquaculture which resulted in the production decreasing drastically. Cultivating new strains of scallops with enhanced resistance to diseases is now considered to be one of the basic and ultimate solutions to disease control. Since the traditional breeding techniques are usually costly and time-consuming, and could not fulfill the urgent requirement for thoroughbred, it is necessary to accelerate and improve selective breeding of resistance strains in virtue of molecular methods. Marker assisted selection (MAS) is one of the molecular methods successfully applied in animal breeding. However, MAS is currently far from practice in mollusks as the lack of markers associated with quantitative traits. Identification of markers associated with resistance to pathogens is necessary for the development of MAS in mollusks. In this study, Zhikong scallops were classified into susceptible and resistant stocks according to the survival time after L. anguillarum challenge. The nucleotide sequence polymorphisms in CfLysG, CfC1qDC and CfLITAF genes from Zhikong scallop were investigated to explore their associations with susceptibility/resistance to L. anguillarum by PCR, PCR-RFLP and Bi-PASA PCR methods. One hundred and four sites of single nucleotide polymorphisms (SNPs) and twenty nine sites of insert/deletion (I/D) polymorphisms were identified in CfLysG gene. Seventeen polymorphisms located in its promoter region. Three of them, -753 I/D polymorphism, -391 A/G SNP and -284 I/D polymorphism were selected to analyze their distributions in susceptible and resistant stocks. Statistical analysis revealed that the genotypic frequencies of all alleles were in Hardy-Weinberg equilibrium (HWE) (P>0.05) at loci -753, -391 and -284 in both stocks. Among them, the -753 ID genotype and -284 ID genotype were more prevalent in resistant stock than those in susceptible stock, but there were no significant differences in the frequency distributions between these two stocks (P>0.05). In contrast, the frequency of -391 AG genotype in resistant stock was significantly higher than that in susceptible stock (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L. anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 AA genotype was significantly higher than those with -391 AG genotype (P=0.001), which further validate the significant association between -391 AG genotype and the resistance of Zhikong scallop to L. anguillarum. Three SNPs were found in the exons of CfLysG gene, among which +3473 A/C in exon III was a non-synonymous SNP. Statistical analysis revealed that the genotypic frequencies of alleles at locus +3473 were in HWE (P>0.05) in susceptible stock, while not in HWE (P<0.01) in resistant stock. The frequency of +3473 AA genotype in resistant stock was significantly higher than that in susceptible stock, indicating its significant association with the resistance of Zhikong scallop to L. anguillarum. Two large fragment I/D polymorphisms, +96 I/D polymorphism and +487 I/D polymorphism, were identified in intron I of CfLysG gene. The genotypic frequencies of all alleles were in HWE (P>0.05) at loci +96 and +487 in both stocks. The +96 DD genotype and +487 ID genotyp were more prevalent in resistant stock than those in susceptible stock, but there were no significant differences in the frequency distributions between these two stocks (P>0.05). The results suggested that these two polymorphisms were not significantly associated with the resistance of Zhikong scallop to L. anguillarum. Linkage disequilibrium was found between the multiple loci of CfLysG gene, indicating the existence of haplotypes. Nineteen haplotypes with frequency above 1% were identified. Among them, the frequency of -753 I/-391 G/-284 I/+96 I/+487 D/+3473 A haplotype in resistcant stock was significantly higher than that in susceptible stock (P=0.044), indicating its significant association with the resistance of Zhikong scallop to L. anguillarum. Fourteen SNPs were identified in CfC1qDC gene, and the association between +423 T/C SNP and the resistance of Zhikong scallop to L. anguillarum was investigated. The genotypic frequency of each allele was in HWE (P>0.05) at locus +423 in both stocks. The +423 TT genotype was significantly more prevalent in resistant stock than that in susceptible stock (P=0.005), suggesting its significant association with the resistance of Zhikong scallop to L. anguillarum. Three SNPs and one I/D polymorphism were found in CfLITAF gene. The association between +145 I/D polymorphism and the resistance of Zhikong scallop to L. anguillarum was investigated. All alles in locus +145 could be found in each susceptible and resistant individual, suggesting that there was no association between +145 I/D polymorphism and the resistance of Zhikong scallop to L. anguillarum. These results above suggested that, the -391 AG genotype, +3473 AA genotype and -753 I/-391 G/-284 I/+96 I/+487 D/+3473 A haplotype of CfLysG gene and +423 TT genotype of CfC1qDC gene were significantly associated with the resistance of Zhikong scallop to L. anguillarum, and could be potential markers applied in future selection of Zhikong scallop with enhanced disease resistance. These results would hopefully provide reference for MAS in mollusks. Furthermore, identification of markers associated with disease could also improve the understanding of pathogenesis and aid in the development of preventive and therapeutic measures.
页数	107
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/437
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	李凌. 栉孔扇贝抗鳗弧菌感染性状候选基因的多态性研究[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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