WHOLE GENOME MAPPING OF DISEASE RESISTANCE/SUSCEPTIBILITY-ASSOCIATED SNPS IN CATFISH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1005404
• Grant No.: 2015-67015-22975
• Project No.: ALA016-4-14048
• Proposal No.: 2014-06252
• Program Code: A1221
• Project Start Date: Jan 15, 2015
• Project End Date: Jan 14, 2018
• Grant Year: 2015

Project Director
Liu, J.

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
Fisheries

Non Technical Summary
Catfish is teh primary aquaculture species in teh United States. The catfish industry accounts for over 60% of all US aquaculture production. In recent years, however, it has encountered unprecedented challenges due to devastating diseases of ESC and columnaris, high feed prices, and fierce international compettition. Currently, there is no effective disease control measures. Genetic improvement of disease resistance has proven to be the most effective and reliable way for disease control in aquaculture. Therefore, our long-term goal is to enhance catfish stocks with good disease resistance traits to support a sustainable and profitable aquaculture industry. To reach this long-term goal, genetics of disease resistance must be understood, especially the QTLs controlling the resistance. Detailed linkage maps with QTL information for disease resistance will then be used to improve brood stocks by marker-assisted selection, or introgression of valuable disease resistance QTLs from both channel catfish and blue catfish. This project is designed to fill these gaps by fulfilling the following two specific objectives: 1). Genome wide scan of QTLs conferring resistance to ESC and columnaris using F2 and F4 fish using the 250K catfish SNP array; and 2). Fine QTL analysis by genotyping a large number of F2 and F4 individuals using evenly-spaced markers from mapped QTL regions. We have generated the whole genome sequence and identified millions of SNPs, developed the 250K SNP arrays, and generated large resource families that are best suited for QTL analysis for disease resistance. Briefly, Backcross progenies will be used for mapping the disease resistance genes using SNP markers on the 250K SNP panel. Fish will be challenged by bacterial pathogens. After disease challenge, disease development will be monitored real time. When fish is about to lose balance, their timing of death will be recorded and symptoms of the diseases will also be recorded. Their blood will be drawn for the preparation of genomic DNA. Their genotypes will be determined and the correlation genotypes with phenotypes will be determined by mapping the disease resistance genes. Once mapped, the disease resistance loci will be used to select for resistant catfish.

Animal Health Component

100%

Research Effort Categories

Basic

40%

Applied

60%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3710	1080	50%
311	3710	1040	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3710 - Catfish;

Field Of Science
1080 - Genetics; 1040 - Molecular biology;

Keywords

catfish

qtl

snp

aquaculture

genome

Goals / Objectives
In recent years, however, catfish industryhas encountered unprecedented challenges due to devastating diseases of ESC and columnaris, for which there are currently no effective control measures. Genetic improvement of disease resistance has proven to be the most effective and reliable way for disease control in aquaculture. Our long-term goal is to enhance catfish stocks with good disease resistance traits to support a sustainable and profitable aquaculture industry. To reach this long-term goal, genetics of disease resistance must be understood, especially the QTLs controlling the resistance. Detailed linkage maps with QTL information for disease resistance will then be used to improve brood stocks by marker-assisted selection, or introgression of valuable disease resistance QTLs from both channel catfish and blue catfish. This project is designed to fill these gaps by fulfilling the following two specific objectives: 1). Genome wide scan of QTLs conferring resistance to ESC and columnaris using F2 and F4 fish using the 250K catfish SNP array; and 2). Fine QTL analysis by genotyping a large number of F2 and F4 individuals using evenly-spaced markers from mapped QTL regions.

Project Methods
In this project, we have two objectives. First, QTLs mapping of resistance to ESC and columnaris using F2 fish and F4 (for ESC disease only because ESC disease resistance was selected at each generation) fish using the 250K catfish SNP chip.Briefly, Backcross progenies will be used for mapping the disease resistance genes using SNP markers on teh 250K SNP panel. Fish will be challenged by immersion using bacterial pathogens Edwardsiella ictaluri and Flavobacterium columnare. After disease challenge, disease development will be monitored real time. When fish is about to lose balance, their timing of death will be recorded and symptoms of the diseases will also be recorded. Their blood will be drawn for the preparation of genomic DNA. Their genotypes will be determined using SNP markers that we developed. When the genotypes and disease resistance phenotype is known, GWAS or QTL analysis will be conducted using SVS software packages. In the second objective, we will conduct Fine QTL analysis by genotyping a large number of F2 and F4 (for ESC only) fish using evenly spaced markers from mapped QTL regions. SNP markers will be selected from local region at a high density, at least one SNP per 4-5 kb DNA, and they will be genotyped using the MassArray technology with approximately 2,000 fish. This would allow fine QTL mapping of the disease resistance QTL to a small region.

Progress 01/15/15 to 01/14/18

Outputs
Target Audience:Aquaculture industry, aquaculture research community, animal genetics community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project allowed training of six graduate students. How have the results been disseminated to communities of interest?All the results were reported in pAG conference as well as World Aquaculture Society meetings. The results have been published as soon as they became available. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have exceeded thegoals we proposed for this project. We mapped QTL for columnaris in F2 populations, and mapped QTL for ESC disease resistance in F4, F4, and channel catfish populations. Specifically, for columnaris, to identify genes associated with columnaris resistance, we performed a genome-wide association study (GWAS) using the catfish 250k SNP array with 340 backcross progenies derived from crossing female channel catfish (Ictalurus punctatus) with male F1 hybrid catfish (female channel catfish I. punctatus × male blue catfish I. furcatus). A genomic region on chromosome 7 was found to be significantly associated with columnaris resistance. Within this region, five have known functions in immunity, including pik3r3b, cyld-like, adcyap1r1, adcyap1r1-like, and mast2. In addition, 3 additional suggestively associated QTL regions were identified on chromosomes 7, 12, and 14. The resistant genotypes on the QTLof chromosomes 7 and 12 were found to be homozygous with both alleles being derived from channel catfish. The paralogs of the candidate genes in the suggestively associated QTL of chromosome 12 were found on the QTLs of chromosome 7. Many candidate genes on the four associated regions are involved in PI3K pathway that is known to be required by many bacteria for efficient entry into the host. The GWAS revealed four QTLs associated with columnaris resistance in catfish. Strikingly, the candidate genes may be arranged as functional hubs; the candidate genes within the associated QTLs on chromosomes 7 and 12 are not only co-localized, but also functionally related, with many of them being involved in the PI3K signal transduction pathway, suggesting its importance for columnaris resistance. For ESC disease resistance studies, we initially conducted GWAS analysis using F4 backcross progenies.In this study, 500 individuals of the fourth generation of backcross progenies of hybrid catfish were used to conduct genome wide association study (GWAS) for ESC resistance. A genomic region on linkage group 1 was found significantly associated with ESC disease resistance. Within this QTL, 16 genes have known functions in immunity, including wwp1, inhba, fzd8, klf6, nck1, agtr1b, racgap1, nlrp12, trpc1, acbd5a, abi1, abi2, apbb1ip, myo3a, stat2, nlrc3. Among the 16 genes, 8 genes or their paralogs were differently expressed after ESC challenge. The nck1 was identified nearby the most significantly associated SNP, and it was significantly upregulated in channel catfish intestine after ESC challenge. In addition to the significant QTL, two suggestively associated QTL for ESC resistance were identified on linkage group 12 and 16. The nlrc3 duplicates were identified within all of the three QTL. Nck1 may function as a linker between surface exposed ligand and host cell receptors for E. ictaluri internalization. Nlrc3 may play an important role in activation of innate immune response following E. ictaluri infection in catfish. We specifically characterized the NCK1 gene and its expression:Adaptor proteins non-catalytic region of tyrosine kinase (NCK) and Abelson interactor (ABI) are crucial for disease response. NCK1 was identified to be a candidate gene for enteric septicemia of catfish (ESC) disease resistance, and was speculated to play similar roles during ESC and enteropathogenic Escherichia coli (EPEC) pathogenicity. ABI1 was reported as a positional candidate gene for bacterial cold water disease (BCWD) resistance in rainbow trout. In this study, three NCK genes and six ABI genes were identified in the channel catfish (Ictalurus punctatus) genome and blue catfish (I. furcatus) transcriptome, and annotated by domain structures, phylogenetic and syntenic analyses. Their expression patterns were examined in the intestine and liver of catfish after challenge with Edwardsiella ictaluri. In the intestine, NCK1, ABI2a, ABI2b, ABI3a were differentially expressed after E. ictaluri infection. In the liver, NCK2a, NCK2b, ABI1b, ABI2a, ABI2b were significantly upregulated in ESC susceptible fish. In general, the NCK and ABI genes, with exception of ABI3a gene and NCK1 gene, were expressed at higher levels in susceptible fish after infection than in control fish, but were expressed at lower levels in resistant fish than in the control fish. Taken together, these results support the notion that NCK and ABI genes are involved in disease processes facilitating pathogenesis of the E. ictaluri bacteria. After this analysis, we further determined QTL in F2 fish to make sure that all the QTL were included in the F4 because only few fish were used as founders for the production of F3 and F4 fish.In this study, we conducted GWAS analysis to locate genomic regions associated with ESC resistance by selectively genotyping F2 backcross families with the catfish 690K SNP arrays. Two genomic regions on LG1 and LG23 were determined to be associated with ESC resistance as revealed by a mixed linear model and family-based association test. A number of genes within these QTL regions have known functions in immunity, making them potential candidates as disease resistance genes. For instance, seven genes on LG1 (nck1, agtr1, trpc1, abi1, apbb1ip, actr3b, and vav3) and three genes on LG23 (mrc1l, prkcq, and gata3) were involved in immune-related functions. These genes mainly function in signaling pathways of phagocytosis and T-cell activation, suggesting their roles in disease resistance. This study demonstrated the power of GWAS analysis for the identification of QTL in the hybrid system. We previously reported one QTL in LG1 using fourth generation of backcross families, which was validated here in the F2 backcross families, suggesting that this QTL is operating in various populations of a broad genetic background, making it useful for application in marker-assisted selection. Further, we determined QTL in channel catfish to determine the complexities of ESC resistance QTL.In this study, three significant quantitative trait locus (QTL) located on LG1 and LG26, and three suggestive QTL located on LG1, LG3, and LG22, respectively, were identified to be associated with ESC resistance by genome-wide association study (GWAS) and the 690K catfish SNP arrays.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fu Q, Yang Y, Li C, Zeng Q, Zhou T, Li N, Liu Y, Liu S, Li D, Liu ZJ. 2017. The CC and CXC chemokine receptors in channel catfish (Ictalurus punctatus) and their involvement in disease and hypoxia responses. Developmental and Comparative Immunology 77: 241-251.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fu Q, Yang Y, Li C, Zeng Q, Zhou T, Li N, Liu Y, Li Y, Wang X, Liu S, Li D, Liu ZJ. 2017. The chemokinome superfamily: II. The 64 CC chemokines in channel catfish and their involvement in disease and hypoxia responses. Developmental and Comparative Immunology 73: 97-108.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Geng X, Liu S, Yuan Z, Jiang Y, Zhi D, and Liu ZJ. 2017. A genome wide association study reveals that genes with functions for bone development are associated with body conformation in catfish. Marine Biotechnology, DOI 10.1007/s10126-017-9775-3
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang X, Liu S, Yang Y, Fu Q, Abebe A, Liu ZJ. 2017. Identification of NF-?B related genes in channel catfish and their expression profiles in mucosal tissues after columnaris bacterial infection. Developmental and Comparative Immunology 70: 27-38.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhou T, Liu S, Geng X, Jin Y, Jiang C, Bao L, Yao J, Zhang Y, Zhang J, Sun L, Wang X, Li N, Tan S, Liu ZJ. 2017. GWAS analysis of QTL for enteric septicemia of catfish and their involved genes suggest evolutionary conservation of a molecular mechanism of disease resistance. Molecular Genetics and Genomics 292: 231-242. DOI 10.1007/s00438-016-1269-x
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhou T, Li N, Liu S, Jin Y, Fu Q, Gao S, Wang X, Liu ZJ. 2017. The NCK and ABI adaptor genes in catfish and their involvement in ESC disease responses. Developmental and Comparative Immunology 73: 119-123.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fu Q, Zeng Q, Li Y, Yang Y, Li C, Zhou T, Li N, Liu S, Yao J, Jiang C, Li D, Liu ZJ. 2017. The chemokinome superfamily in channel catfish: I. CXC subfamily and their involvement in disease defense and hypoxia responses. Fish and Shellfish Immunology 60: 380-390.
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Suxu Tan, Tao Zhou, Wenwen Wang, Yulin Jin, Xiaozhu Wang, Xin Geng, Dongya Gao, Rex Dunham, and Zhanjiang Liu. GWAS analysis of QTL for resistance against Edwardsiella ictaluri in F2 interspecific hybrid catfish. Submitted to Molecular Genetics and Genomics.
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Huitong Shi, Xiaozhu Wang, Tao Zhou, Chenglong Wu, Shikai Liu, Lisui Bao, Ning Li, Yujia Yang , Zihao Yuan, Yulin Jin, Suxu Tan, Wenwen Wang, Xiaoxiao Zhong, Guyu Qin, Dongya Gao, Rex Dunham, Zhanjiang Liu. Identification of QTL and candidate genes associated with the resistance for enteric septicemia of catfish using genome-wide association analysis in channel catfish. Submitted to Molecular Genetics and Genomics.

Progress 01/15/16 to 01/14/17

Outputs
Target Audience:Aquaculture industry, aquaculture research community, and aquaculture genetics, genomics and breeding community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project allowed training of three graduate students, and additional several students in the collaborative efforts. How have the results been disseminated to communities of interest?The results were reported promptly through meeting presentations (world aquaculture society, PAG, and other venues), and publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Catfish is the primary aquaculture species in the United States. Its production is threatened by serious disease problems. Among several major diseases, columnaris disease is the most frequently occurring disease and considered as the second most important disease affecting the catfish industry, but little is known about the genetic basis for columnaris resistance in catfish. In this study, we performed a genome-wide association study (GWAS) using the catfish 250k SNP array with backcross progenies derived from crossing female channel catfish (Ictalurus punctatus) with male F1 hybrid catfish. A genomic region on chromosome 7 was found to be significantly associated with columnaris resistance. The resistant genotypes were found to be homozygous with both alleles being derived from channel catfish. The disease resistance QTL was narrowed down to a region of approximately 620 Kb. Within this region, 10 genes were identified, of which five have known functions in immunity. These genes included pik3r3b, cyld-like, adcyap1r1, adcyap1r1-like, and mast2. In addition, 3 additional suggestively associated QTL regions were identified on chromosomes 7, 12, and 14. Strikingly, the candidate genes may be arranged as functional hubs; the candidate genes within the major QTL on chromosome 7, as well as those within the suggestive QTLs on chromosome 12 and chromosome 14 are not only co-localized, but also functionally related, with many of them being involved in the PI3K signal transduction pathway that is known to be required by many bacteria for efficient entry into the host, suggesting its importance for columnaris resistance. These finding suggest that genes are arranged in "functional hubs" that may work in a coordinated fashion. Because columnaris is the most significant disease for aquaculture, this work provides foundation for the understanding of disease resistance against columnaris. Genotyping for ESC resistance has been completed, and QTL analysis is under way. Enteric septicemia of catfish (ESC), caused by the bacterial pathogen Edwardsiella ictaluri, is the leading disease problem for the catfish industry. It causes tens of millions of dollars of economic losses each year. Channel catfish (Ictalurus punctatus) is generally susceptible to the disease, while blue catfish (I. furcatus) is generally resistant. Therefore, the hybrid catfish is a great model system to study ESC resistance because of the strong phenotypic contrast between channel catfish and blue catfish. In this study, 500 individuals of the fourth generation of backcross progenies of hybrid catfish were used to conduct genome wide association study (GWAS) for ESC resistance. A genomic region on linkage group 1 was found significantly associated with ESC disease resistance. Within this QTL, 16 genes have known functions in immunity, including wwp1, inhba, fzd8, klf6, nck1, agtr1b, racgap1, nlrp12, trpc1, acbd5a, abi1, abi2, apbb1ip, myo3a, stat2, nlrc3. Among the 16 genes, 8 genes or their paralogs were differently expressed after ESC challenge. The nck1 was identified nearby the most significantly associated SNP, and it was significantly upregulated in channel catfish intestine after ESC challenge. In addition to the significant QTL, two suggestively associated QTL for ESC resistance were identified on linkage group 12 and 16. The nlrc3 duplicates were identified within all of the three QTL. Nck1 may function as a linker between surface exposed ligand and host cell receptors for E. ictaluri internalization. Nlrc3 may play an important role in activation of innate immune response following E. ictaluri infection in catfish.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Zhou T, Liu S, Geng X, Jin Y, Jiang C, Bao L, Yao J, Zhang Y, Zhang J, Sun L, Wang X, Li N, Tan S, Liu ZJ. 2017. QTLs for ESC disease resistance of catfish as revealed by a genome wide association study. Molecular Genetics and Genomics, in revision.
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Zhou T, Li N, Liu S, Jin Y, Fu Q, Gao S, Wang X, Liu ZJ. 2017. Channel catfish nck and abi adaptor genes and their involvement in ESC disease responses. Fish & Shellfish Immunology, submitted.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Tan S, Yao J, Zhou T, Liu ZJ. 2016. Identification, annotation and expression analysis of 29 Rho GTPase genes from channel catfish (Ictalurus punctatus) after bacterial infections. Developmental and Comparative Immunology, 10.1016/j.dci.2016.10.005.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yang Y, Fu Q, Zhou T, Li Y, Liu S, Zeng Q, Wang X, Jin Y, Qin Z, Dunham R, Liu ZJ. 2016. Analysis of apolipoprotein genes and their involvement in disease response of channel catfish after bacterial infection. Developmental and Comparative Immunology, in press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yuan Z, Liu S, Yao J, Zeng Q, Liu ZJ. 2016. Expression of Bcl-2 genes in channel catfish after bacterial infection and hypoxia stress. Developmental and Comparative Immunology 65: 79-90.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Li Z, Yao J, Xie Y, Geng X, Liu ZJ. 2016. Phosphoinositide 3-kinase family in channel catfish and their regulated expression after bacterial infection. Fish & Shellfish Immunology 49:364-373.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Fu Q, Li Y, Yang Y, Li C, Yao J, Zeng Q, Qin Z, Li Dao, Liu ZJ. 2016. Septin genes in channel catfish (Ictalurus punctatus) and their involvement in disease defense responses. Fish and Shellfish Immunology 49:110-121.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Song L, Li C, Xie Y, Liu S, Zhang J, Yao J, Jiang C, Li Y, Liu ZJ. 2016. Genome-wide identification of Hsp70 genes in channel catfish and their regulated expression after bacterial infection. Fish and Shellfish Immunology 49:154-162.

Progress 01/15/15 to 01/14/16

Outputs
Target Audience:Aquaculture community and animal genome and genetics community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project allowed training of two Ph.D. students in the areas of genomics and quantitative genetics. How have the results been disseminated to communities of interest?The results have been and are to be presented in various meetings including the International Symposium on Aquaculture Genetics, International Plant and Animal Genome Conference (PAG), and World Aquaculture Society Conferences. The Results of columnaris resistance QTL work has been published in BMC Genomics. What do you plan to do during the next reporting period to accomplish the goals?In the coming year, we plan to conduct GWAS analysis on ESC resistance using F2 and F4 fish populations. When possible, we will also characterize the genes involved in the disease resistance pathways.

Impacts
What was accomplished under these goals? Catfish is the primary aquaculture species in the United States. Its production is threatened by serious disease problems. Among several major diseases, columnaris disease is the most frequently occurring disease and considered as the second most important disease affecting the catfish industry, but little is known about the genetic basis for columnaris resistance in catfish. In this study, we performed a genome-wide association study (GWAS) using the catfish 250k SNP array with backcross progenies derived from crossing female channel catfish (Ictalurus punctatus) with male F1 hybrid catfish. A genomic region on chromosome 7 was found to be significantly associated with columnaris resistance. The resistant genotypes were found to be homozygous with both alleles being derived from channel catfish. The disease resistance QTL was narrowed down to a region of approximately 620 Kb. Within this region, 10 genes were identified, of which five have known functions in immunity. These genes included pik3r3b, cyld-like, adcyap1r1, adcyap1r1-like, and mast2. In addition, 3 additional suggestively associated QTL regions were identified on chromosomes 7, 12, and 14. Strikingly, the candidate genes may be arranged as functional hubs; the candidate genes within the major QTL on chromosome 7, as well as those within the suggestive QTLs on chromosome 12 and chromosome 14 are not only co-localized, but also functionally related, with many of them being involved in the PI3K signal transduction pathway that is known to be required by many bacteria for efficient entry into the host, suggesting its importance for columnaris resistance. These finding suggest that genes are arranged in "functional hubs" that may work in a coordinated fashion. Because columnaris is the most significant disease for aquaculture, this work provides foundation for the understanding of disease resistance against columnaris. Genotyping for ESC resistance has been completed, and QTL analysis is under way.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Jiang C, Zhang J, Yao J, Liu S, Li Y, Song L, Li C, Wang X, Liu ZJ. 2015. Complement regulatory protein genes in channel catfish and their involvement in disease defense response. Developmental and Comparative Immunology 53: 33-41.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Tian Y, Yao J, Liu S, Jiang C, Zhang J, Li Y, Liu ZJ. 2015. Identification and expression analysis of 26 oncogenes of the receptor tyrosine kinase family in channel catfish after bacterial infection and hypoxic stress. Comparative Biochemistry and Physiology Part D, Genomics and Proteomics 14: 16-25.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Geng X, Sha J, Liu S, Bao L, Zhang J, Wang R, Yao J, Li C, Feng J, Sun F, Sun L, Jiang C, Dunham R, Zhi D, Liu ZJ. 2015. A genome-wide association study in catfish reveals the presence of functional hubs of related genes within QTLs for columnaris disease resistance. BMC Genomics 16: 196. DOI 10.1186/s12864-015-1409-4.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Sun L, Liu S, Bao L, Li Y, Feng J, Liu ZJ. 2015. Claudin multigene family in channel catfish and their expression profiles in response to bacterial infection and hypoxia as revealed by meta-analysis of RNA-Seq datasets. Comparative Biochemistry and Physiology, Part D, Genomics and Proteomics 13: 60-69.