Genetic mapping of Koi herpesvirus resistance (KHVR) in Mirror carp (Cyprinus carpio) revealed genes and molecular mechanisms of disease resistance

Journal Pre-proof Genetic mapping of Koi herpesvirus resistance (KHVR) in Mirror carp (Cyprinus carpio) revealed genes and molecular mechanisms of disease resistance

Zhiying Jia, Lin Chen, Yanlong Ge, Shengwen Li, Wenzhu Peng, Chitao Li, Yuyong Zhang, Xuesong Hu, Zhixiong Zhou, Lianyu Shi, Peng Xu PII:

S0044-8486(19)32237-9

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734850

Reference:

AQUA 734850

To appear in:

aquaculture

Received date:

7 September 2019

Revised date:

10 December 2019

Accepted date:

11 December 2019

Please cite this article as: Z. Jia, L. Chen, Y. Ge, et al., Genetic mapping of Koi herpesvirus resistance (KHVR) in Mirror carp (Cyprinus carpio) revealed genes and molecular mechanisms of disease resistance, aquaculture (2019), https://doi.org/10.1016/ j.aquaculture.2019.734850

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© 2019 Published by Elsevier.

Journal Pre-proof

Genetic mapping of Koi herpesvirus resistance (KHVR) in Mirror carp (Cyprinus carpio) revealed genes and molecular mechanisms of disease resistance Zhiying Jiaa†, Lin Chenb†, Yanlong Ge a , Shengwen Lia ,Wenzhu Pengb, Chitao Lia , Yuyong Zhanga , Xuesong Hua , Zhixiong Zhoub, Lianyu Shia* , Peng Xub,c* a

Heilongjiang River Fisheries Research Institute, CAFS, Harbin, China; Key

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Laboratory of Aquatic Genomics, Ministry of Agriculture, CAFS, Beijing, China b

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State Key Laboratory of Marine Environmental Science, College of Ocean and Earth

c

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Sciences, Xiamen University, Xiamen 361102, China

Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for

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Marine Science and Technology, Qingdao, 266071, China †

Corresponding author:

Lianyu Shi

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*

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These authors contributed equally to this work.

Peng Xu

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E-mail: [email protected]. Tel.:86-0451-84861311(O);

E-mail: [email protected]. Tel.:86-592-2880812(O)

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ABSTRACT Koi herpesvirus is a double-stranded DNA virus that causes fatal disease and mortality in koi and common carp (Cyprinus carpio) of all ages. Koi herpesvirus disease has been added to the list of notifiable diseases of the World Organisation for Animal Health. The genetic basis of fish resistance to Koi herpesvirus remains little understood, and thus, it cannot be used in genetic breeding programs to produce high-resistance carp

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strains. Dissection of the molecular mechanism of resistance could help prevent or

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reduce losses due to Koi herpesvirus disease. Common carp is a widely distributed

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species cultured all around the world. Its allotetraploidy and high economic and environmental value have attracted a great deal of interest in its genetics and genomics.

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Here, we use both QTL mapping and GWAS in Mirror carp subjected to Koi

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herpesvirus infection to detect potential loci and genes related to resistance to this virus. Many immune-related genes were identified around the significant QTLs or

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SNPs, including tnfa, hif1a, galectin-8, rootletin, and palladin. GO and KEGG analyses were performed based on the union of the candidates. Of particular interest

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were the Herpes simplex infection pathway and mTOR signaling pathway, which play

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potential roles in the immune response. Using publicly available transcriptome data from Koi herpesvirus infected carp, the expression patterns of candidate genes were obtained, and the results indicated potential roles of homoeolog functional divergence in immune response in the allotetraploid common carp. Overall, our study of the genetic architecture of carp Koi herpesvirus resistance provides many valuable candidate genes, which could be considered in the marker-assisted selection of common carp strains with high resistance to Koi herpesvirus.

Keywords: common carp, QTL, GWAS, KHV, Koi herpesvirus

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Journal Pre-proof 1. Introduction Koi herpesvirus (KHV), also known as Cyprinid herpesvirus-3 (CyHV-3), is a double-stranded DNA (dsDNA) virus. The genome of KHV is approximately 295 kbp, the largest reported to date for this family (Aoki, et al., 2007). KHV causes disease and mortality in koi and common carp (Cyprinus carpio) of all ages. The first known occurrence of KHV disease (KHVD) dates to 1996, but the first major outbreaks were recorded in Israel in 1998 (Haenen, et al., 2004). Subsequently, the virus was reported

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in Japan (Sano, et al., 2004), Taiwan (Tu, et al., 2004), throughout Europe (Bergmann, et al., 2006; Haenen, et al., 2004) and in most regions around the world. Its spread is

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mainly due to the global fish trade, especially international ornamental koi shows, and

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causes a major threat to carp production; consequently, it has been added to the list of notifiable diseases of the World Organisation for Animal Health (OIE).

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KHV can exhibit latent infection, maintaining its DNA in fish hosts for an extended period in the absence of productive infection, and can then be reactivated when

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environmental conditions such as temperature stress are favorable for its growth (Eide,

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et al., 2011). KHVD often occurs when the water temperature is between 15 °C and

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25 °C (Gilad, et al., 2003; Toffan, et al., 2019). It is highly contagious and extremely virulent, with a mortality rate of 80%-100%. Cohabitation experiments indicate that some carp species, such as goldfish, common bream (Abramis brama), silver carp and grass carp, can carry KHV asymptomatically and transmit it to wild carp (Kempter, et al., 2012). Fish infected with KHV show several gross pathological signs, including interstitial nephritis and gill necrosis as well as petechial hemorrhages in the liver, and the massive production of mucus on the gills can lead to gasping behavior by fish on the surface of ponds (Amin, et al., 2018). The inflammation and severe dysfunction of osmoregulation in the gill, gut and kidney are particular contributors to death in the case of acute disease progression (Negenborn, et al., 2015). Ways to minimize the damage and improve sustainable production include vaccines development and 3

Journal Pre-proof breeding of disease-resistant strains. Many vaccines have been proved high protection of fish challenged with KHV, including ORF149 DNA vaccine loaded onto SWCNTs with 81.9% protection(Hu, et al., 2019) And the pAct/GP-based DNA vaccine (Nuryati, et al., 2010), have become a mainstream technology for the prevention and control of aquatic diseases worldwide. In contrast to susceptible fish, viral loads in resistant fish were found to be significantly lower, and resistant ones suffer lesser damages and could easily recover from the disease, suggesting that resistant fish have a mechanism

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to control virus replication (Adamek, et al., 2019; Tadmor-Levi, et al., 2017).

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Therefore, a major and increasingly important strategy for disease control and for the

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desires of sustainable aquaculture development is selective breeding programs to produce stocks with improved resistance to certain pathogens, exploiting naturally

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occurring genetic variation (heritability) for resistance in farmed aquaculture

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populations due to the significant heritability of disease resistance (Gjedrem, 2015; Houston, 2017; Yáñez, et al., 2014). Traditional breeding has made some progress in

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the development of fish disease resistance. Hybrids of goldfish and common carp show partial susceptibility to KHV infection, and their mortality rate is lower (Hedrick,

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et al., 2006). KHV resistance from the feral strain “Amur Sassan” was successfully

generation

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introgressed into two susceptible cultured strains up to the first backcross (BC1) through

family-based

selection

(Tadmor-Levi,

et

al.,

2017).

Marker-assisted selection demonstrates high efficacy without the time-consuming and labor-intensive methods that limit traditional breeding programs. However, marker-assisted selection must be based on effective loci, which must be identified via genetic dissection. The downregulation of mucin mRNA expression in the gills and gut of common carp during infection with pathogenic viruses could increase the susceptibility of virus- infected carp to secondary bacterial infection (Adamek, et al., 2017). ACP, AKP, GSH, T-AOC, LZM, and IgM were activated in response to the infection of KHV, especially AKP, and these could act as indicators of KHV resistance in common carp (Jia, et al., 2018). A possible interaction between carp 4

Journal Pre-proof IL-12 and IL-10 may be a response to the course of KHV infection (Sunarto and McColl, 2015). RAD-seq-based GWAS analysis of common carp Koi herpesvirus resistance (KHVR) identified the trim25 gene on chromosome 33 as a promising positional and functional candidate containing a putative premature stop mutation (Palaiokostas, et al., 2018). However, in-depth study of KHVR is still in the early stages. Notably, successful examples of disease control using marker-assisted breeding have been reported in aquaculture species; for example, the use of epithelial

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cadherin, which was identified as the major determinant of the resistance of Atlantic

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salmon individuals to infectious pancreatic necrosis virus (IPNV), has led to a 75%

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decrease in the number of IPN outbreaks in the salmon farming industry (Moen, et al., 2015).

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Common carp (Cyprinus carpio) is one of the most important aquaculture species and

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is widely distributed from Asia to Europe. Due to its economic and environmental importance and allopolyploid genome, great efforts have been made to examine the

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genetics and genomics of common carp over the past decade, including a comparative

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exome study of C. carpio and D. rerio (Henkel, et al., 2012), a complete genome assembly (Xu, et al., 2014b) and an examination of allotetraploid genome evolution

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(Xu, et al., 2018). Because of the development of sequencing technology, large numbers of genetic markers have flooded into genetic studies, especially SNP markers, which were used for developing the common carp 250 K SNP array (Xu, et al., 2014a). Genetic linkage maps are suitable for genome-wide dissection, and at least one version has been published for many important aquaculture species, such as catfish (Li, et al., 2014), tilapia (Liu, et al., 2013), Atlantic salmon (Gonen, et al., 2014), and common carp (Peng, et al., 2016). All these publicly available resources provide a platform for further studies of evolutionary genomics, ecology, physiology, immunology, and the genetic mapping of economic traits. In this study, we aimed to unravel the causative variation underlying KHVR in Mirror 5

Journal Pre-proof carp. First, a high-density genetic map was constructed based on a full-sib family. Afterward, we conducted QTL mapping to identify KHVR-related genetic loci or QTLs. A genome-wide association study was also utilized to better explain the genetic architecture of Mirror carp resistance to KHV. For candidate annotation, the latest common carp reference genome in the NCBI SRA (accession number PRJNA510861) was screened to identify possible genes surrounding the significant SNPs and regions. Furthermore, GO and KEGG pathway analysis was performed to

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increase our understanding of the potential molecular mechanisms underlying KHVR.

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The expression patterns of candidate genes were calculated based on the publicly

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available RNA-seq data from carp KHV infection to better explain the regulatory mechanisms involved. Considering all the above, the results of this study have great

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value for future marker-assisted selection of enhanced resistance to KHV in common

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carp.

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2.1 Ethics statement

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2. Materials and methods

All experiments involving the handling and treatment of fish were approved by the

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Animal Care and Use committee at Heilongjiang River Fisheries Research Institute. 2.2 Experimental fish, KHV infection, and sample collection The current study is based on fish from the selective breeding of Mirror carp infected by KHV in Kuandian Research Base of Heilongjiang River Research Institute in Liaoning, China. In generation G1, eight families of fish with survival rates higher than that of the control were collected after KHV infection. Eventually, one male and one female from a family with a moderate survival rate of 40.4% were used to construct a G2 family. Approximately 150,000 individuals were hatched. Once the fish reached 4 to 5 cm in body length, 50,000 fish were placed in a net at a density of 61.7 individuals/m3 . Before the challenge test, these animals were acclimatized for 6

Journal Pre-proof more than forty days and fed with a commercial carp feed twice a day (1.5% body weight). Water quality parameters were measured and monitored (water temperature of 20 ± 1℃; pH 7.0). The infection of the fish was conducted as described by Dixon (Dixon, et al., 2009) via cohabitation with KHV- infected carp. Generally, KHV-infected carp from Kuandian Research Base with convincing features of disease were used as infection sources. The infected fish for cohabitation were then introduced into the tank

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containing the test carp families at the rate of 1:10 of the test fish. Water temperature

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was set at 23℃ during infection, which was proved to result in high mortality (Gilad,

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et al., 2003). The dying fish with the clinical signs of gasping movements in shallow water, gill necrosis and sunken eyes were recorded on a daily basis. During the

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disease outbreak, blood samples were taken from dying fish and stored in liquid

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nitrogen. At the end of the disease phase, blood samples were collected from the survivors. The virus was detected in the fish samples by PCR amplification of the

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ORF72, capsid triplex subunit 2 (Adamek, et al., 2013; Davison, et al., 2013) to

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ensure that the fish analyzed were infected with KHV (Figure S1). 2.3 DNA extraction, genotyping, and quality control

(Qiagen,

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Genomic DNA was extracted from blood using the DNeasy 96 Blood & Tissue Kit Shanghai,

China).

After

quantification

by

a

Nanodrop

1000

spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and integrity examination by agarose gel electrophoresis, DNA samples were stored at -20°C for further experiments. The DNA samples used for genotyping were diluted to a final concentration of 50 ng/μL and genotyped at GeneSeek (Lincoln, NE, USA) using the common carp 250 K SNP array (Xu, et al., 2014b). The Affymetrix CEL files were analyzed using Affymetrix Genotyping Console software (version 4.0) for quality control, and SNP calling was performed based on the Affymetrix Axiom GT1 algorithm. The dish value was set as default, and SNPs with a call rate greater than 95% 7

Journal Pre-proof were collected for further analysis. The CHP files generated from Affymetrix Genotyping Console were then extracted and converted to Ped/Map format for further analysis. All individuals with missing genotypes >5% and SNPs with missing genotypes >5% and minor allele frequency <5% were removed using PLINK software. In- house Perl scripts were used for further data cleaning. Only the SNPs that were heterozygous in at least one parent and conformed to Mendelian inheritance were used for further linkage analysis. A total of 179 individuals (94 decedents and 85 survivors)

2.4 Linkage map construction and QTL mapping

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and 52,226 high-quality SNPs were retained for further analysis.

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After genotyping quality control and polymorphism detection in the mapping family,

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we adopted stricter criteria on the data filtration. The SNPs were divided into three categories according to their segregation patterns: 1:1 segregation in only the female

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parent, 1:1 segregation in only the male parent and 1:2:1 segregation in both parents. Genetic linkage maps were constructed using JoinMap version 4.1 with a CP

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population type, which is designed to handle F1 population data.

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QTL mapping was initially performed for the KHVR trait using the MapQTL 6

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software package with “multiple QTL model (MQM) mapping” algorithms. LOD score significance thresholds were calculated by permutation test (n=1,000). QTLs with LOD scores exceeding the chromosome-wide LOD threshold of P < 0.01 were considered significant, and the chromosome-wide LOD threshold of P<0.05 was considered suggestive. Furthermore, QTL analysis was performed via a remote web server, the GridQTL portal (http://gridqt1.cap.ed.ac.uk/) (Seaton, et al., 2006), which implements a linear regression methodology, considering the linkage phase between markers according to pedigree information. 2.5 Genome-wide association study Accounting for population substructure can lower false positive rates and increase power (Purcell, et al., 2007). Therefore, a centered relatedness matrix in PLINK was 8

Journal Pre-proof drawn using the SNPs after quality control to test for potential population structure among the surveyed fish individuals. The first and second dimensions were plotted using the R package (Figure S2). Association analysis between genotypes and the KHVR trait was originally performed using the PLINK case-control model. The genomic inflation factor (λ, based on median chisq) was 1 for the KHVR trait calculated in PLINK, suggesting an even population structure. The Bonferroni criterion (0.05/number of SNPs) provides an extremely

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stringent threshold, considering that GWAS is hypothesis generating (Wang, et al.,

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2012; Yang, et al., 2011). The threshold of significance was set at 4.0. GEMMA

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implements the Genome-wide Efficient Mixed Model Association algorithm and fits the relatedness matrix, phenotype and genotype into a univariate linear mixed model

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(Zhou, Stephens, 2012), which has demonstrated great potential in previous GWAS

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programs (Chen, et al., 2018; Kong, et al., 2019). In this study, GEMMA was additionally used to perform GWAS for the KHVR trait with the same data set for

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comparison. The proportion of variance in the phenotypes explained (PVE or “chip heritability”) was 0.29 as estimated by GEMMA. Finally, 52,043 SNPs remained in the

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analysis. The Wald frequentist approach was chosen to test the significance of the SNPs.

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Furthermore, Manhattan plots based on the - log10 (P-value) and a QQ plot displaying the expected and observed P-value were drawn using R packages. 2.6 Candidate Genes and Mechanisms Integrating the results of both QTL mapping and GWAS, loci and genomic regions that were significantly or suggestively correlated with the trait were identified, and candidate genes around these loci or regions were extracted from the latest version of the common carp reference genome (NCBI Sequence Read Archive under BioProject accession PRJNA510861) at 50 K intervals up- or downstream. GO and KEGG analyses of the candidates were performed using the online tools KEGG

(http://www.genome.jp/kegg/pathway.html)

and

OmicShare 9

Journal Pre-proof (http://www.omicshare.com/tools/Home/Soft/pathwaygsea) to better understand the genetic mechanisms active during disease resistance. The raw RNA-seq data of anterior kidney samples under KHV infection were obtained from previously published study (Neave, et al., 2017) in NCBI Sequence Read Archive under BioProject accession number PRJNA314552. Quality control of raw reads was performed using Trimmomatic and FastQC software. The high-quality mRNA reads were mapped to the latest version of the common carp genome using

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HISAT2. Successfully mapped reads were assembled using Cufflinks v.2.2.1 and Cuffmerge. Reads per kilobase of transcript per million mapped reads (RPKM) values

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were calculated as expression profiles.

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3.1 Mortality rate and sample structure

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3. Results and discussion

After 45 days in the net, fish began to die. The duration was approximately one month,

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and the outbreak process occurred in ten days (Figure 1). In total, 7116 individuals

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died, yielding a survival rate of 67.6%, higher than that of Marianske Lazne scaly

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carp (Piačková, et al., 2013). 3.2 Linkage analysis

The genetic linkage map of Mirror carp contained 32,160 SNP markers in 23,936 loci with a total length of 19,425 cM and an average marker interval of 0.60 cM (Figure 2). A considerable number of genetic linkage maps have been published in the last few years, including many important aquaculture species, such as catfish (Li, et al., 2014), tilapia (Liu, et al., 2013), Atlantic salmon (Gonen, et al., 2014), and common carp. The first genetic linkage map of common carp was constructed based on gene markers, RAPD markers and microsatellites in 2004, and this 272- marker-based map was used for mapping a locus associated with cold tolerance (Sun, Liang, 2004). However, the 161-microsatellite- marker-based genetic linkage map of common carp could not 10

Journal Pre-proof identify an accurate group number (Zhang, et al., 2011). The same year, another map with 186 microsatellites and 241 SNPs was construc ted and used for comparative genome analysis (Zheng, et al., 2011). All the above maps and others not mentioned here were restricted with respect to marker density, and their ability to identify trait-related loci was limited. Compared with previously published versions of common carp linkage maps, this one was undoubtedly superior and facilitated the fine mapping of important traits. The genetic linkage map used in this study is comparable to the

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linkage map of Yellow River carp with the highest known density (Peng, et al., 2016),

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which included 28,194 SNP markers with a total length of 14,146 cM and an average

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marker interval of 0.38 cM. There are 3,966 more SNPs in the map of Mirror carp than in that of Yellow River carp. The two maps shared 6,526 SNPs, exhibiting high

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similarity and collinearity (Figure 3), which demonstrated the high accuracy of the

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Mirror carp genetic map.

In this study, we took advantage of the common carp 250K SNP genotyping array,

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collected high-throughput genotyping data accurately and efficiently from a Mirror

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carp mapping family, and constructed a high-density and high-resolution genetic map of Mirror carp. SNP markers are the most abundant type of markers in the genome and

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have a high level of polymorphism compared with other genetic markers, making them ideal for high-density linkage map construction. However, in the past, it was a considerable challenge to develop a sufficient number of SNP markers and conduct cost-effective genotyping in a relatively large mapping population, especially in many nonmodel aquaculture species. With advances in sequencing technologies, an increasing number of reference genomes have been completely assembled, and various sequencing-based SNP genotyping technologies have been developed, providing rapid and cost-effective high-throughput SNP genotyping platforms for linkage mapping. Recently, a high-density genetic linkage map was constructed based on Affymetrix Axiom SNP genotyping data for channel catfish, presenting the highest marker density with high accuracy among linkage maps (Li, et al., 2014). The high-density genetic 11

Journal Pre-proof linkage map of Mirror carp provides a valuable resource for genome assembly, and comparative genomic studies also permitted reliable identification of important economic traits. The comprehensively improved genome assembly of Mirror carp was also crucial for the identification of trait-related genes and loci. 3.3 QTLs and SNPs associated with KHVR in Mirror carp A total of three QTL regions were identified significantly, and one was suggestive of being associated with KHVR, according to MapQTL 6. The four QTL regions

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contained five SNPs distributed on four LGs, LG17, LG33, LG39, and LG43 (Figure 4 and Table 1). The most significant QTL, KHVR17, located on LG17 at 128.551–

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129.701 cM, presented the highest LOD value of 4.40, explaining 10.1% of the total

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phenotypic variation (PVE).

QTLs in GridQTL were identified separately based on the sire and dam. Six SNPs in

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GridQTL showed significant association with KHVR, including a s hared SNP, SP014932, in QTL KHVR43 from MapQTL (Table 2). Two additional linkage groups,

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LG41 and LG44, also contained SNPs that exceeded the significance threshold.

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GridQTL was previously applied in the QTL mapping of growth, meat quality and

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breast meat yield traits in turkey (Aslam, et al., 2011) and sex-associated QTLs in turbot (Scophthalmus maximus) (Hermida, et al., 2013) and was used in the replication and refinement of a QTL influencing milk protein percentage on ovine chromosome 3 (García‐Gámez, et al., 2012). Here, the results of GridQTL seemed to differ from those of MapQTL 6, which could be due to the differing strengths and weaknesses of these programs in QTL mapping. Therefore, comparative QTL mapping may work better for causal loci or gene identification in genetic studies. In the GWAS, PLINK and GEMMA showed high similarity (Figure 4, Figure S3 and Table S2), although none of the SNPs exceeded the genome-wide Bonferroni threshold. Empirical data suggest that most traits of interest for animal production (e.g., growth and disease resistance) are underpinned by a polygenic genetic 12

Journal Pre-proof architecture (de los Campos, et al., 2013). Accordingly, genetic mapping programs focusing on such traits often fail to identify loci or genes significantly associated with these traits. In aquaculture species, where large full-sibling family sizes are typically available, the genetic mapping of such polygenic traits using a large sample size would be more promising. Single- family-based QTL analysis shows excellent performance in identifying rare alleles associated with candidate traits. GWAS using a large sample size and marker

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size could strongly support the causal loci of economic traits. The combination of QTL

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mapping and GWAS in this study provides a comprehensive understanding of the

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genetic architecture of KHV resistance traits.

3.4 Candidate genes and potential molecular mechanisms associated with KHVR of

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Mirror carp

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To further identify potential causative genes underlying KHVR, we screened the reference genome and collected protein-coding genes from the QTL (Table 3 and

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Figure 5) and GWAS (Table 4) regions. Of particular interest, many of the candidates

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were involved in disease response. Some of the genes were identified from both QTL mapping and GWAS, including galectin-8 (lgals8) and rootletin (crocc). Galectin 8, a

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cytosolic lectin, plays many roles in diverse immune cell processes, including pathogen recognition, shaping the course of adaptive immune responses and fine-tuning the inflammatory response (Rabinovich, Toscano, 2009). Galectin-8 can determine how B cells sense and extract tethered antigens and thereby modulate B cell responses in vivo (Obino, et al., 2018). Rootletin is a coiled-coil protein, a structural component of the ciliary rootlet, which links the base of the cilium to the cell body, indicating a role in structural assurance and signal mediation (Akiyama, et al., 2017), which might be involved in the KHV infection process. Located in KHVR39, the cell surface amine oxidase could directly control lymphocyte migration (Salmi, et al., 2001). The KHVR43 contained NCK-interacting protein with SH3 13

Journal Pre-proof domain (Nckipsd), involved in receptor- interacting proteins transduce signals that are required for cell-cell junction assembly, cell morphology, and barrier function (Rahimi, 2017). Nckipsd is a cytoskeletal protein binding to Palladin (the candidate from GWAS), and required for the organization of normal actin cytoskeleton, which is important for cell morphology, motility, and cell adhesion (Rönty, et al., 2007). Tumor necrosis factor alpha (Tnfa) is a strong candidate in KHVR39-2. The expression of tnfa was altered during zebrafish development and acute inflammation, as shown by a

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comparison of the mecp2-null zebrafish with the wild type (van der Vaart, et al.,

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2017). The absence of tumor necrosis factor (TNF) causes lethal infection by

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Leishmania major in mice (Hu, et al., 2018). Rho-associated kinases Rock1 and Rock2 are downstream targets of the small GTPases involved in cell adhesion and

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motility, proliferation and apoptosis (Hartmann, et al., 2015). Protein phosphatase 1A

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(Ppm1a) and Ppm1b act as IKKβ phosphatases, playing critical roles in controlling inflammation, immune response, and antiapoptotic responses (Sun, et al., 2009).

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Depletion of ppm1a in mammalian cells enhances TGFβ signaling, which controls diverse pathogenesis of diseases, including cancer and autoimmune diseases (Lin, et

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al., 2006). RNA polymerase II subunit A C-terminal domain phosphatase SSU72

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(Ssu72) is also related to the inflammatory response. Ssu72 overexpression in T-cell- mediated immunity has potential utility for the treatment of autoimmune arthritis (Lee, et al., 2017).

Candidates from GWAS also exhibited high correlations with the immune response and disease process. Low PIN2/TERF1-interacting telomerase inhibitor 1 (PINX1) expression was associated with disease-specific survival (Shi, et al., 2015). Neuromedin-U (Nmu) promotes mast-cell- mediated inflammation (Moriyama, et al., 2005), and NMU-NMUR1 neuronal signaling provides a selective mec hanism through which the innate immune system coordinates to promote rapid type 2 cytokine responses that can induce antimicrobial, inflammatory and tissue-protective type 2 responses at mucosal sites (Klose, et al., 2017). Palladin contains 14

Journal Pre-proof immunoglobulin- like domains, and high levels of palladin expression occur in wounded tissue and contribute to the invasive behavior of metastatic cells (Goicoechea, et al., 2009; Goicoechea, et al., 2008). To better understand the genetic mechanisms of KHV resistance, GO and KEGG pathway analysis was performed based on the union of candidates. Remarkably, some vital functional categories were involved, such as response to stimulus (GO:0050896), cell communication (GO:0007154), cellular metabolic process (GO:0044237), and the

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Herpes simplex infection (HSV, KO05168) pathway and mammalian target of rapamycin (mTOR, KO04150) signaling pathway, which were potentially required and

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critical for the survival of Mirror carp under KHV infection (Tables S3-S4, Figures S4-S5). The mTOR signaling pathway serves as a central regulator of cell metabolism,

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growth, proliferation and survival, and it is activated during various cellular processes

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(e.g., tumor formation and angiogenesis, insulin resistance and T-lymphocyte activation) and is deregulated in human diseases such as cancer and type 2 diabetes

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(Laplante, Sabatini, 2009). Herpes simplex virus (HSV) is a DNA virus of the alpha

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herpesvirus subfamily. During HSV infection, multiple mechanisms of pathogen recognition are active. Mast-cell-derived TNF and IL-6 can protect mice from

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HSV-induced mortality (Rui, et al., 2013). Toll- like receptor 2 (TLR2) is involved in the activation of the host response, playing important roles in the inflammatory response and the immunopathology of HSV infection (Kurt-Jones, et al., 2004). Common carp is an allotetraploid potentially derived from hybridization between two distinct diploid species and is an informative system for analyzing the effects of recent polyploidy on gene expression under stress. The presence of homoeologs has been proposed to confer adaptive plasticity, for example, through neo functionalization or tissue-specific expression (Ramírez-González, et al., 2018). Understanding the coordination of homoeologs and identifying the mechanisms associated with KHV resistance should help define strategies to improve trait biology in common carp. Here, 15

Journal Pre-proof we collected transcriptome data from common carp under KHV infection to determine the similarities and differences between candidate homoeolog expression levels under KHV infection. We analyzed the latest version of the common carp genome for the presence of duplicated candidate genes and calculated the expression levels of individual genes. Under normal conditions, the homoeologous gene of amine oxidase (mao) in subgenome B has strikingly higher expression than its paralog. In the acute infection process, both mao genes were downregulated in response to KHV,

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similar to the palladin gene (Figure 6 and Table S2). However, only the papoa gene

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identified in this study responded to KHV, with its homoeolog and the total

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expression level balanced; this phenomenon could represent dosage-sensitive regulation to ensure optimal RNA and protein supplies and maintain normal biological

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processes (Xu, et al., 2018) or so-called homoeolog functional divergence. Although

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two copies of the tnfa gene were identified in the common carp genome, only one showed expression in the published data, where it was upregulated in response to

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KHV; the other may be silenced. Altogether, homoeolog expression bias may play a

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4. Conclusions

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role in Mirror carp KHV resistance with various regulation patterns.

Here, we constructed a high-density genetic map for Mirror carp and performed QTL mapping to identify KHVR-related genetic loci or QTLs. GWAS was also utilized as a complementary approach. Not surprisingly, many immune-related genes were identified around the significant QTLs or SNPs, including tnfa, hif1a, galectin-8, rootletin, palladin. GO and KEGG pathway analysis according to the union of candidate genes revealed the important role of the Herpes simplex infection pathway and mTOR signaling pathway in the disease response. The expression patterns of the candidate genes suggested their potential roles and homoeolog functional divergence in the immune response. Our study concentrating on the genetic architecture of carp KHV resistance provides many valuable genomic candidates, which could be 16

Journal Pre-proof considered for marker-assisted selection of high KHV resistance in common carp strains. Declaration of interest The authors declare that there is no conflict of interest. Acknowledgements This work was supported by the Central- level Non-profit Scientific Research Institutes

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Special Funds (grant number HSY201701Z); China Agriculture Research System (grant number CARS-45-06); Special Financial Resources Protection of the Ministry of

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Agriculture (grant number 2130135) and Heilongjiang Province Leading Talent

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Program.

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Author contributions

Peng Xu conceived and supervised the study. Lianyu Shi and Zhiying Jia supervised

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the samples. Zhiying Jia conducted the selection of the fish, DNA extraction. Yanlong

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Ge, Zhiying Jia and Shengwen Li conducted the KHV infection experiment and collected blood samples. Chitao Li and Xuesong Hu performed family propagation and

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fish culture. Lin Chen analyzed the data and drafted the manuscript. Wenzhu Peng and Zhixiong Zhou constructed the genetic linkage map of Mirror carp. Peng Xu, Lin Chen and Zhiying Jia revised the manuscript. All authors have read and approved the manuscript. References Adamek, M., Hazerli, D., Matras, M., et al., 2017. Viral infections in common carp lead to a disturbance of mucin expression in mucosal tissues. Fish and Shellfish Immunology. 71, 353-358. Adamek, M., Syakuri, H., Harris, S., et al., 2013. Cyprin id herpesvirus 3 infection disrupts the skin barrier of common carp (Cyprinus carpio L.). Veterinary microbiology. 162, 456-470. Adamek, M., Matras, M., Dawson, A., et al., 2019. Type I interferon responses of common carp strains with different levels of resistance to koi herpesvirus disease during infection with CyHV-3 o r SVCV. Fish & shellfish immunology. 87, 809-819. 17

Journal Pre-proof Akiyama, T., Inoko, A., Kaji, Y., et al., 2017. SHG-specificity o f cellular rootletin filaments enables naïve imaging with universal conservation. Scientific reports. 7, 39967. Amin, M ., Adrianti, D.N., Las mika, N.L.A., et al., 2018. Detection of koi herpesvirus in healthy co mmon carps, Cyprinus carpio L. VirusDisease. 29, 445-452. Aoki, T., Hirono, I., Kurokawa, K., et al., 2007. Geno me sequences of three ko i herpesvirus isolates representing the expanding distribution of an emerg ing disease threatening koi and co mmon carp worldwide. Journal of virology. 81, 5058-5065. Aslam, M.L., Bastiaansen, J.W., Crooijmans, R.P., et al., 2011. Whole geno me QTL mapping for g rowth, meat quality and breast meat yield traits in turkey. BMC genetics. 12, 61. Berg mann, S., Kempter, J., Sadowski, J., et al., 2006. First detection, confirmat ion and isolation of koi herpesvirus

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Journal Pre-proof Figure legends Fig. 1. Cumulative mortality rate of Mirror carp after KHV challenge. Fish began to die after being introduced into the net. Fig. 2. Genetic linkage map of Mirror carp. The map contains 32,160 SNP markers and has a total length of 19,425 cM. Fig. 3. Linkage map comparison between Mirror carp and Yellow River carp. This diagram was constructed using the 6,526 shared SNPs in the maps of Mirror carp and Yellow River carp. The X-axis represents the linkage groups of Mirror carp, and the

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Y-axis represents the linkage groups of Yellow River carp. Fig. 4. QTL mapping and GWAS of KHVR in Mirror carp. QTL mapping (upper plot) was conducted with MapQTL software, and GWAS (lower plot) was conducted

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using PLINK software. The results from the two approaches showed high consistency.

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Fig. 5. QTL regions and candidate genes for KHVR in Mirror carp. The dashed line indicates the genome-wide significance threshold. The vertical dashed lines

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indicate the QTL regions that harbor candidate genes. Fig. 6. Candidate gene expression and homoeolog functional divergence under

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KHV infection. Green triangles indicate candidates identified in this study. A and B

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of the homoeologs.

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represent the subgenome of common carp, and sum means the total expression levels

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Journal Pre-proof Table 1 Genomic regions associated with KHVR in Mirror carp based on MapQTL 6. The significance threshold was calculated by permutation test. QTL name KHVR17 KHVR33 KHVR39 KHVR43

Chromosome 17 33 39 43

CI (cM) 128.551-129.701 401.827-403.569 41.327-42.627 223.023-226.744

No. of SNPs 1 1 1 2

Nearest marker LOD SP016983 4.4 SP031280 3.75 SP015580 4.02 SP014932 4.25

Permutation* 4.2 3.2 3.7 3.9

Note: * represents the chromosome-wide significance LOD threshold of P < 0.01,

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except that KHVR33 met the chromosome-wide significance LOD threshold of P <

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0.05.

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Exp% 10.1 8.7 8.4 9.9

Journal Pre-proof Table 2 Genomic regions associated with KHVR in Mirror carp based on GridQTL. The QTL effect was scaled by the standard deviation of the trait. The chromosome-wide F-statistics for P < 0.05 and <0.01 were determined by permutation test.

Dam Sire & Dam Sire Dam

Effect (s.e.) 0.81 (0.18) 0.51 (0.16) -0.69 (0.21) 0.48 (0.15) 0.48 (0.16) -0.46 (0.15)

F_value 20.3 10.28 11 9.87 9.48 9.12

P_0.05 P_0.01 7.03 10.69 6.64 8.47 6.88 10.35 8.25 11.71 8.71 11.79 6.9 10.43

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Closest marker SP015664 SP015670 SP002206 SP014953 SP014932 SP010596

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Position (cM) 104 113 254 249 224 320

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Chromosome 39 39 41 43 43 44

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QTL name KHVR39-2 KHVR39-3 KHVR41 KHVR43-2 KHVR43-3 KHVR44

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Journal Pre-proof Table 3 Summary of candidate genes from QTL for KHVR in Mirror carp. LG

Gene name

Annotation

KHVR17 KHVR33

17 33

KHVR39

39

mao manea bach2 lgals8 ube2j1 tnfa rock2 henmt ppm1a crocc mfap2 slc25a34 fbxo42 nckipsd ssu72

Amine oxidase Glycoprotein endo-alpha-12-mannosidase Transcription regulator protein BACH2 Galectin-8 Ubiquitin-conjugating enzyme E2 J1 Tumor necrosis factor-3 alpha Rho-associated protein kinase 2 RNA 2'-O-methyltransferase Protein phosphatase 1A Rootletin/Ciliary rootlet coiled-coil Microfibrillar-associated protein 2 Solute carrier family 25 member 34 F-box only protein 42 NCK-interacting protein with SH3 domain RNA polymerase II subunit A C-terminal domain phosphatase SSU72 Rootletin

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KHVR43-3

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KHVR41 KHVR43

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KHVR39-2 KHVR39-3

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Journal Pre-proof Table 4 Candidate genes from GWAS for KHVR in Mirror carp. LG

Position (bp)

P-value

Nearby gene

Annotation

SP183584 SP015620

39 39

3366692 9129278

1.87E-05 3.07E-05

papoa pinx1

SP015636 SP015626 SP183931

39 39 39

10272651 9947437 6619125

4.02E-05 4.08E-05 4.73E-05

syne2 tf3b ralgps2

SP014932 SP015658 SP015649 SP015660 SP183584 SP015579 SP015603 SP015603 SP015626 SP015586

43 39 39 39 39 39 39 39 39 39

154352 11097481 11131173 11281201 3366692 3638247 7666258 7666258 9947437 4673575

6.47E-05 8.77E-05 8.77E-05 9.20E-05 1.87E-05 2.10E-05 3.07E-05 3.07E-05 4.08E-05 4.33E-05

crocc slain2 palld sh3rf1 lgals8 snw1 lpp3 dab1 xrcc3 tagap

SP184466 SP015623

39 39

10450906 9676610

4.99E-05 6.41E-05

Poly(A) polymerase alpha PIN2/TERF1-interacting telomerase inhibitor 1 Nesprin-2 Transcription factor IIIB 90 kDa subunit Ras-specific guanine nucleotide-releasing factor RalGPS2 Rootletin SLAIN motif-containing protein 2 Palladin E3 ubiquitin-protein ligase SH3RF1 Galectin-8 SNW domain-containing protein 1 Lipid phosphate phosphohydrolase 3 Disabled homolog 1 DNA repair protein XRCC3 T-cell activation Rho GTPase-activating protein Neuromedin-U Hypoxia-inducible factor 1-alpha

nmu hif1a

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SNP ID

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Journal Pre-proof

Highlights •

A family-based QTL mapping and GWAS were performed using 250 K SNP array of Koi herpesvirus challenged Mirror carp.

•

Immune-related genes were identified around the significant QTLs or SNPs, including tnfa, hif1a, galectin-8, rootletin and palladin.

•

Herpes simplex infection pathway and mTOR signaling pathway play potential

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Homoeolog functional divergence was found in the allotetraploid Mirror carp

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during Koi herpesvirus infection.

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roles in the immune response.

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Journal Pre-proof Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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