Abstract：Nucleotide binding and oligomerization domain 1 gene (NOD1) plays important role in fish innate immunity. In order to obtain the SNPs associated with the disease resistance in Nile tilapia (Oreochromis niloticus), genome sequence NOD1 (GenBank No. MF479261) was cloned. Through cloning sequencing or direct sequencing of PCR products, 44 SNPs were obtained from 39 individuals of 20 Nile tilapia parents family. By directly sequencing or SNaPshot method, 83 individuals from susceptible group (SG) and 83 individuals from resistance group (RG) of offspring were used to analyze polymorphisms and genetic parameter. Popgen 32 was used to calculate the genetic parameter and polymorphisms of the NOD1 SNPs in susceptible group and resistance group. The results showed that 24 SNPs were genotyped successfully. The polymorphism information content (PIC) value of the 24 SNPs of NOD1 gene ranged from 0.09 to 0.37, which suggested that all 24 SNPs were low or moderate polymorphism. The correlation between the NOD1 SNPs and the trait of S.agalactiae resistance was analyzed. The results showed that SNP5 (G-2284C) was significantly associated with S. agalactiae susceptible trait (P＜0.05), SNP13 (G-8956C) were significantly associated with S. agalactiae resistant trait (P＜0.05). SNP12 (G-8955C) was significantly associated with S. agalactiae resistant/susceptible trait (P＜0.05). The linkage disequilibrium analysis and prediction of haplotypes showed that all the 24 NOD1 SNPs formed 5 haplotype blocks and 16 haplotypes. The haplotypes H4-2 (AC) was significantly associated with S. agalactiae resistant trait. These results indicate that the SNP site SNP5, SNP12, SNP13 and the haplotypes H4-2 of NOD1 gene can be candidate molecular markers for molecular marker assisted selection. This study provides data base for breeding resistant strains of Nile tilapia.
Bi D, Gao Y, Chu Q, et al. 2017. NOD1 is the innate immune receptor for iE-DAP and can activate NF-κB pathway in teleost fish [J]. Developmental & Comparative Immunology, 76: 238-246. Carneiro L A M, Travassos L H, Girardin S E. 2007. Nod-like receptors in innate immunity and inflammatory diseases [J]. Annals of medicine, 39(8): 581-593. Chang M, Wang T, Nie P, et al. 2011. Cloning of two rainbow trout nucleotide-binding oligomerization domain containing 2 (NOD2) splice variants and functional characterization of the NOD2 effector domains [J]. Fish & shellfish immunology, 30(1): 118-127. Cock J, Gitterle T, Salazar M, et al. 2009. Breeding for disease resistance of Penaeid shrimps [J]. Aquaculture, 286(1-2): 1-11. Fu G H, Bai Z Y, Xia J H, et al. 2014c. Characterization of the LECT2 gene and its associations with resistance to the big belly disease in Asian seabass [J] Fish Shellfish Immunology, 37(1):131-138. Fu G H, Wang Z Y, Xia J H, et al. 2014a. The MCP-8 gene and its possible association with resistance to Streptococcus agalactiae in tilapia. Fish & Shellfish Immunology, 40(1): 331-336. Fu G H, Wang Z Y, Xia J H, et al. 2014b. The LBP Gene and Its Association with Resistance to Aeromonas hydrophila in Tilapia. Internal Journal Molecular Science, 15(12:, 22028-22041. Gao F Y, Pang J C, Lu M X, et al. 2018. Molecular characterization, expression and functional analysis of NOD1, NOD2 and NLRC3 in Nile tilapia (Oreochromis niloticus). Fish and Shellfish Immunology, 73:207–219. Hubert S, Bussey J T, Higgins B, et al. 2009. Development of single nucleotide polymorphism markers for Atlantic cod (Gadus morhua) using expressed sequences [J]. Aquaculture, 296(1-2): 7-14. Kim E J, Lee J R, Chung W C, et al. 2013. Association between genetic polymorphisms of NOD 1 and Helicobacter pylori-induced gastric mucosal inflammation in healthy Korean population [J]. Helicobacter, 18(2):143-50. doi: 10.1111/hel.12020. Kimchi-Sarfaty C, Oh J M, Kim I W, et al. 2007. A “silent” polymorphism in the MDR1 gene changes substrate specificity [J].Science, 315 (5811): 525-528. Laing K J, Purcell M K, Winton J R, et al. 2008. A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish [J]. BMC evolutionary biology, 8(1): 42-56. Li J, Chu Q, Xu T. 2016. A genome-wide survey of expansive NLR-C subfamily in miiuy croaker and characterization of the NLR-B30. 2 genes [J]. Developmental & Comparative Immunology, 61: 116-125. Li J, Gao Y, Xu T. 2015. Comparative genomic and evolution of vertebrate NOD1 and NOD2 genes and their immune response in miiuy croaker [J]. Fish & shellfish immunology, 46(2): 387-397. Li L, Rui W, Liang W, et al.2013. Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China [J]. Veterinary Microbiology, 167(3-4): 719-724. Li S, Chen X, Hao G, et al. 2016. Identification and characterization of a novel NOD-like receptor family CARD domain containing 3 gene in response to extracellular ATP stimulation and its role in regulating LPS-induced innate immune response in Japanese flounder (Paralichthys olivaceus) head kidney macrophages[J]. Fish & shellfish immunology, 50: 79-90. Liu L, Li Y W, He R Z, et al. 2014. Outbreak of Streptococcus agalactiae infection in barcoo grunter, Scortum barcoo (McCulloch &Waite), in an intensive fish farm in China [J]. Journal of Fish Disease, 37(12): 1067-1072. Millar D S, Horan M, Chuzhanova N A, et al. 2010. Characterisation of a functional intronic polymorphism in the human growth hormone (GH1) gene, Hum. Genomics, 4 (5): 289-301. Ogura Y, Sutterwala F S, Flavell R A. 2006.The inflammasome: first line of the immune response to cell stress [J]. Cell, 126(4): 659-662. Pant S D, Verschoor C P, Schenkel F S, et al. 2011. Bovine PGLYRP1 polymorphisms and their association with resistance to Mycobacterium avium ssp. Paratuberculosis[J]. Anim. Genet. 42 (4): 354-360. Rajendran K V, Zhang J, Liu S, et al. 2012. Pathogen recognition receptors in channel catfish: I. Identification, phylogeny and expression of NOD-like receptors [J]. Developmental & Comparative Immunology, 37(1): 77-86. Robinson N, Goddard M, Hayes B. 2008. Use of gene expression data for predicting continuous phenotypes for animal production and breeding [J]. Animal, 2(10): 1413-1420. Robinson N, Hayes B. 2008. Modelling the use of gene expression profiles with selective breeding for improved disease resistance in Atlantic salmon (Salmo salar) [J]. Aquaculture, 285(1-4): 38-46. Shen Y B., Fu G H., Liu F., et al. 2015. Characterization of the duodenase-1 gene and its associations with resistance to Streptococuus agalactiae in hybrid tilapia (Oreochromis spp.)[J]. Fish & Shellfish Immunology, 45(2): 717-724. Unajak S, Santos M D, Hikima J, et al. 2011. Molecular characterization, expression and functional analysis of a nuclear oligomerization domain proteins subfamily C (NLRC) in Japanese flounder (Paralichthys olivaceus) [J]. Fish & shellfish immunology, 31(2): 202-211. Wan Q, Su J, Chen X, et al. 2013b. Genomic sequence comparison, promoter activity, SNP detection of RIG-I gene and association with resistance/susceptibility to grass carp reovirus in grass carp (Ctenopharyngodon idella) [J]. Developmental & Comparative Immunology, 39(4): 333-342. Wan Q, Su J, Chen X, et al. 2013d. Gene-based polymorphisms, genomic organization of interferon-β promoter stimulator 1 (IPS-1) gene and association study with the natural resistance to grass carp reovirus in grass carp Ctenopharyngodon idella [J]. Developmental & Comparative Immunology, 41(4): 756-765. Wan Q, Su J, Wang L, et al. 2012. A 15 nucleotide deletion mutation in coding region of the RIG-I lowers grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus [J]. Fish & shellfish immunology, 33(2): 442-447. Wan Q, Su J, Wang L, et al. 2013a. Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene [J]. Gene, 516(2): 320-327. Wan Q, Wang L, Su J, et al. 2013c. Genetic structure, polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella [J]. Gene, 521(1): 166-175. Wang L, Su J, Peng L, et al. 2011. Genomic structure of grass carp Mx2 and the association of its polymorphisms with susceptibility/resistance to grass carp reovirus [J]. Molecular immunology, 49(1-2): 359-366. Wang L, Su J, Yang C, et al. 2012. Genomic organization, promoter activity of grass carp MDA5 and the association of its polymorphisms with susceptibility/resistance to grass carp reovirus [J]. Molecular immunology, 50(4): 236-243. Wilmanski J M, Petnicki‐Ocwieja T, Kobayashi K S. 2008. NLR proteins: integral members of innate immunity and mediators of inflammatory diseases [J]. Journal of leukocyte biology, 83(1): 13-30. Yue G H. 2014. Recent advances of genome mapping and marker-assisted selection in aquaculture [J] Fish and Fisheries. 15(3): 376-396. Zhu L Y, Nie L, Zhu G, et al. 2013. Advances in research of fish immune-relevant genes: A comparative overview of innate and adaptive immunity in teleosts [J]. Development Comparative Immunology, 39(1-2): 39–62. 高风英，卢迈新，曹建萌等. 2018. 尼罗罗非鱼IPS-1基因SNP位点的筛选及其与链球菌抗性的关联分析[J]. 农业生物技术学报，26(1): 20~33. (Gao F Y, Lu M X, Cao J M, et al. 2018. Screening of IPS-1 Gene SNPs and Their Association with Resistance to the Infection of Streptococcus agalactiae in Oreochromis niloticus [J]. Journal of Agricultural Biotechnology, 26(1): 20~33.DOI: 10.3969/j.issn.1674-7968.2018.01.003 刘福平,白俊杰. 2008. 单核苷酸多态性及其在水产动物遗传育种中的应用[J].中国水产科学, 15 (4): 704-712. (Liu F P, Bai J J. Single nucleotide polymorphisms and its application in genetic breeding of aquatic animals [J]. Journal of Fishery Sciences of China, 15 (4): 704-712. ) 刘浩，白俊杰，李胜杰等. 2016. 大口黑鲈ghrelin 基因SNPs的筛选及与生长性状关联性分析[J]. 水产学报，40(4): 521-527. (Liu H, Bai J J, Li S J, et al. 2016. SNPs detection of ghrelin gene and its association with growth traits in largemouth bass (Micropterus salmoides) [J]. Journal of Fisheries of China, 40(4):521-527.) 卢迈新，黎炯，叶星，等. 2010. 广东与海南养殖罗非鱼无乳链球菌的分离、鉴定与特性分析[J].微生物学通报, 37(5): 766-774. ( (Lu M X, Li J, Ye X, et al. 2010. Identification and characterizations of Streptococcus agalactia isolated from Tilapia cultured in Guangdong and Hainan provices[J]. Microbiology China, 37(5): 766-774.) 卢迈新.罗非鱼链球菌病研究进展[J].南方水产, 2010, 6(1): 75-79. (Lu M X. 2010. Review of research on Streptococcosis in tilapia [J]. South China Fisheries Science, 6 (1): 75-79.) 孙效文 等编著. 2010. 鱼类分子育种学[M].海洋出版社, 北京. P-13. (Sun X W et al. 2010. Molecular breeding of Fish. Ocean Press. Beijing. P-13.)
