中国科学院海洋研究所机构知识库

凡纳滨对虾是我国乃至世界的主导对虾养殖品种，在对虾养殖业中占有十分重要的地位，然而随着养殖环境的恶化，病害频发问题已经成为限制产业健康可持续发展的最大障碍。由带有毒性质粒的副溶血弧菌引发的急性肝胰腺坏死综合征（AHPNS）自2013年爆发以来，给对虾养殖业造成了巨大经济损失。解决病害问题的根本途径是培育抗病品种，分子育种被认为是加快抗病品种培育的最有效途径，而鉴定抗病性状的关键基因及分子标记是开展分子育种的基础。本论文旨在建立一种适用于凡纳滨对虾等水产经济物种的高通量候选基因关联分析方法，并应用于抗病标记的筛选。本研究首次将三代靶向测序技术应用于对虾候选基因的分型，通过对对虾抗菌免疫相关的重要基因ALFs和PI3K进行精确基因分型，以及与抗弧菌性状进行关联分析，不仅证实了高通量基因分型技术的可行性与准确性，也发掘了一批与抗弧菌性状显著相关的SNP位点，可用于指导对虾抗弧菌性状的遗传选育。主要进展如下：

1、对虾候选基因高通量分型技术的建立：依据构建PacBio SMRT三代测序文库所需的样品要求，采用两步法PCR的方式进行扩增子制备，通过灰度法衡量扩增子DNA浓度进行等摩尔混样建库，构建了与三代测序结果更加适配的数据分析流程。进一步通过数据拆分、参考基因组比对、变异调用、单倍型构建与二次变异调用等流程，获得了2,416,000条测序reads，共1387个多态性位点，平均突变频率67 SNPs/kb，InDel类型突变有332个，并鉴定了9个结构变异（> 50 bp）。通过测序数据质量分析发现，99.89%的扩增子的位点测序质量在Q30以上，平均测序深度达22,000X以上，均满足了变异挖掘的要求，同时说明了测序数据的准确性和该基因分型策略与流程的可信度。

2、对虾ALFs基因内与抗副溶血弧菌性状相关SNP标记的筛选：以本实验室培育的“广泰1号”多家系种质库个体为材料进行了副溶血弧菌感染实验，取最初死亡的96尾凡纳滨对虾作为敏感群体，最后存活的96尾虾作为抗性群体，对候选基因LvALF1、2、4、5、6、7以及LvALF8在实验群体内进行扩增，并通过三代测序技术对候选基因进行了基因分型，开展抗弧菌性状相关位点的发掘。在以上7个候选基因中，共得到984个可信度高的多态性位点（MAF > 0.01）, 通过关联分析，在LvALF1、LvALF6、LvALF7及LvALF8中分别发现了1个、39个、40个及11个与抗弧菌性状显著相关的SNP标记（p < 0.05）,其中候选基因LvALF6 的位点ALF6-2_1277_A/G 、ALF6-2_1291_A/AG 及ALF6-2_3065_A/T，以及LvALF7的位点ALF7_3203_A/G与抗弧菌性状极显著相关（p < 0.01）。对所有标记进行单倍型预测，发现LvALF6的单倍型Block5（ATG/CCG）、LvALF7的单倍型Block1（AC）以及LvALF8的单倍型Block1（CGC）等共25个单倍型均与抗弧菌性状显著相关（p < 0.05）。

3、对虾PI3K基因内与抗副溶血弧菌性状相关SNP标记的筛选：使用三代测序技术对重要免疫基因LvPI3K的基因组序列（7378 bp）开展了变异挖掘和与抗弧菌性状的关联分析，共发掘到91个可信度高的SNP位点，通过关联分析鉴定到21个与抗弧菌性状显著相关的标记（p < 0.05），以及5个与抗弧菌性状显著相关的单倍型，其中位点PI3K_5366_T/C和PI3K_2205_GC/G与抗弧菌性状极显著相关（p < 0.01）。进一步利用Sanger测序在验证群体中证实了三代靶向测序技术分型结果的可靠性。

总之，本论文所建立的基于三代测序的靶向分型方法为凡纳滨对虾等水产动物提供了一种高效、低成本的基因分型方法，所发掘的抗弧菌性状相关位点对开展凡纳滨对虾抗弧菌性状分子育种具有一定指导意义。

The Pacific white shrimp (Litopenaeus vannamei) is the dominant aquaculture shrimp species in China, even in the world, which occupies an important position in the shrimp industry. However, diseases have become the biggest obstacle to the development of shrimp aquaculture due to the environmental deterioration. The Acute hepatopancreatic necrosis syndrome (AHPNS) caused by Vibrio parahaemolyticus with toxic plasmid, has brought huge economic losses to the shrimp aquaculture industry since its first outbreak in 2013. Molecular breeding is considered as the most effective way to speed up the cultivation of disease-resistant broodstocks. Clarifying the development of disease resistance traits and discovery of disease-related markers are the basis of molecular breeding. The main purpose of this study is to establish a high-throughput candidate gene association analysis method which is suitable for Litopenaeus vannamei and other aquaculture species. This is the first report of applying third-generation target sequencing technology for high-throughput genotyping of candidate genes in shrimp. Through accurate genotyping and association analysis of the immune related genes, including ALFs and PI3K, the feasibility and accuracy of this high-throughput genotyping technique were verified. Furthermore, the markers identified by this approach will be very useful for the marker assisted breeding of disease resistance in Litopenaeus vannamei. The research progresses are as follows:

1、The establishment of high-throughput genotyping technique：Based on the criteria of constructing the PacBio SMRT library, a two-step PCR approach was used to preparate the amplicons, and the DNA concentrations of amplicons were measured by grayscale method in order to mix the samples in equimolar content. Our laboratory has constructed a data analysis pipeline which was more compatible with the results of the third-generation sequencing results. Through the process of demultiplexing, reference genome blast, variation calling, haplotype construction, secondary variation calling and so on, a total of 2,416,000 reads had been obtained, and 1387 mutation sites were obtained. The average mutation frequency in all candidate genes is around 67 SNPs/kb. Besides, a total of 332 Indels and 9 SVs(> 50bp) were also identified. The sequencing quality at 99.89% sites was above Q30 and the average sequencing depth was 22,000X, which means that the sequencing quality was qualified for the requirements of variation calling, and it also demonstrated the high accuracy of this genotyping method.

2、Screening of markers in ALFs gene associated with the resistance trait of shrimp against Vibrio : Infection experiment against Vibrio was carried out for several families from the new variety “GuangTai No. 1” bred by our laboratory to prepare the experimental materials. The first 96 dead shrimp were selected as the sensitive population, and the last 96 survived shrimp were collected as the resistant population. Candidate genes LvALF1、2、4、5、6、7 and LvALF8 were amplified in the experimental population. All candidate genes were genotyped by three-generation sequencing technique and the associated sites of shrimp against Vibrio were discovered by association analysis. A total of 984 credible polymorphic sites (MAF > 0.01) were obtained. Through the association analysis approach, 1 SNP、39 SNPs、40 SNPs and 11 SNPs were found to be significantly(p < 0.05) associated with the resistance trait of shrimp against Vibrio in LvALF1、LvALF6、LvALF7 and LvALF8, respectively. Among them, the sites ALF6-2_1277_A/G 、ALF6-2_1291_A/AG 、ALF6-2_3065_A/T and ALF7_3203_A/G were significantly (p < 0.01) associated with the resistance trait of shrimp to Vibrio. Based on the haplotype prediction of all markers, it was found that 28 haplotypes, such as Block5(ATG / CCG) in LvALF6, Block1(AC) in LvALF7 and Block1(CGC) in LvALF8, were significantly (p < 0.05) associated with the resistance trait of shrimp to Vibrio.

3、Screening of markers in PI3K gene associated with the resistance trait of shrimp against Vibrio: PI3K is one of the important genes in antibacterial immunity in shrimp. Genotyping and association analysis had been done based on the third-generation sequencing technique for the LvPI3K genome sequence. A total of 91 polymorphic sites were obtained. Using the association analysis approach, 21 makers and 5 haplotypes showed significant associations with the resistance trait of shrimp against Vibrio had been found. Besides, the sites PI3K_5366_T/C and PI3K_2205_GC/G showed very significant associations with the resistance trait of shrimp against Vibrio. Besides, the result of Sanger sequencing verified the accuracy of the third-generation sequencing.

In summary, the approach established in this paper provides an efficient genotyping method with low cost for aquaculture species, and the identified markers will be useful for the marker assisted breeding of disease resistance in Litopenaeus vannamei.