Candidate genes mapping to QTL regions
A number of SNPs in contigs with homology to genes of known immune function were found to either be the SNPs, or map closely (within 5 cM distance) to SNPs, with suggestive (P <0.01 before Bonferroni correction) or significant (P <0.05 after Bonferroni correction) associations with hours of survival and/or dead or alive traits after challenge with A. hydrophila (Tables 4, 5 and 6).
The ubiquitin-protein ligases or E3 enzymes (e3 ubiquitin ligase 1110314_603 on LG7, ubiquitination factor e4b isoform 2 117051_67 on LG10 and ubiquitin-conjugating enzyme e2 c 2465_218 on LG21, Table 6) are a diverse group of enzymes that, as part of an enzyme cascade, attach ubiquitin to a lysine on the target protein resulting in poly- or mono-ubiquination which targets specific protein substrates for degradation by the proteasome (proteolysis) . More than 500 distinct E3 enzymes have been found in mammals. Silencing of ubiquitin ligase associated proteins has been shown to affect disease resistance in plants . A SNP coding for e3 ubiquitin ligase occurs at the same map position around 23.4 cM on LG7 as SNP 62374_157 which is significantly associated with hours of survival (FASTA P <0.05 and GRAMMA P <0.01 after Bonferroni correction Additional file 1, Table 4).
The proteasome is a large complex which catalyses the degradation of ubiquitinated proteins, a process requiring ATP to unfold and translocate the substrate into the core of the proteasome for proteolysis . The architecture of the proteasome ensures that only those molecules which are targeted for degradation are affected and the proteolytic enzymes at the core of the proteasome cleave peptide bonds with broad specificity. With degradation of intracellular proteins by the proteasome, some of the by-products are transported to the endoplasmic reticulum where they bind to major histocompatibility class I molecules and result in antibody production . Variation in the proteasome subunit genes (eg. variation in the proteasome subunit beta type-6 precursor) which affects the structure and function of the proteasome could therefore have downstream effects on cellular immunity.
Lymphocyte-specific protein tyrosine kinase (SNP 134389_297 on LG 3, Table 6) is highly expressed in the thymus, initiates tyrosine phosphorylation cascade in T-cells and plays a crucial role in T-cell maturation, signalling and hence immunity [23–25]. The basic mechanisms that regulate expression of this gene have been shown to be highly conserved between teleost fish and mammals .
The major histocompatibility class I antigen (MHC I, 111876_59 on LG5, Table 6) alerts the immune system to the presence of foreign material inside a cell. MHC I presenting proteins (HLS’s) occur on the cell surface. The MHC II interacting molecule CD4 communicates with T-cell receptors, and it is MHC II (133571_269 on LG 18, Table 6) that is known to mainly fight bacterial pathogens , although MHC I has evolved MHC II type functionality in some fish species such as Atlantic cod Gadus morhua. Three MHC class I alleles have been found to be associated with improved resistance and four MHC class II alleles were found to be associated with increased susceptibility of Atlantic salmon to Aeromonas salmonicida infection . Fixed allele frequency differences were detected for several MHC I SNPs, including SNP 111876_59 which mapped 3.4 cM from the QTL detected on LG5, between samples from lines of rohu that were selected for resistance or susceptibility to A. hydrophila. More than 5-fold up- or down-regulation of MHC I transcripts was also detected in the resistant line fish using mRNA-seq and differential expression was confirmed for one transcript (contig 88601) in the skin, gill and intestine using RT qPCR .
The highly variable alpha chain of the T cell receptor (110434_333 on LG15, Table 6) occurs on the surface of T lymphocytes, and along with the beta chain, recognises antigens bound to MHC molecules. Two c alpha chain molecules have been detected in common carp (possibly as a result of tetraploidisation) . A. hydrophila has been found to significantly increase the expression of beta chain T cell antigen receptors in Nile tilapia peripheral blood leukocytes grown in culture . Activation of invariant natural killer T cells, with an invariant T-cell antigen receptor alpha chain, have been proposed as attractive targets for developing new vaccines for infectious diseases because of their ability to recognise glycolipid antigens from pathogenic bacteria including Streptococcus pneumonia.
Heat shock 70 kDa (HSP70 or mortalin, 52577_884 on LG18, Table 6) binds to antigen presenting cells via toll-like receptors and leads to the secretion of pro-inflammatory cytokines and broad immunostimulation . HSP70 acts as an intracellular chaperone, which stabilises proteins, giving them a possible role in general stress tolerance. This protein plays a role in cell proliferation, stress response and maintenance of the mitochondria. Seven contigs with homology to HSP70 were up-regulated more than 3-fold (median 4.89) in resistant compared to susceptible line rohu . Heat shock protein 105/110 (53470_163 on LG1, Table 6) is a member of the HSP70 family of molecular chaperones which functionally relates to heat shock cognate protein 70 (HSC70) and HSP90, and is known to prevent the aggregation of denatured proteins in cells under severe stress . HSP60 (132709_550 on LG14, Table 6), like HSP70, is believed to play an important role in the control of the immune response .
A number of genes with putative functions affecting mucous secretions (mucin-5b precursor 115737_104 on LG 16, mucin 2 17842_95, mucin 2 53178_329 and mucin subtype tracheobronchial 69593_98 on LG13, Table 6) were associated with QTL of significant or suggestive association with the two traits. Two rohu contigs with homology to the mucin-5b precursor gene [detected by 9] were on average 3.8 times more highly expressed in resistant line than susceptible line fish (interquartile range 1.28, Figure 4). Five contigs with homology to mucin 2 [detected by 9] were on average 2.29 more highly expressed in resistant line than susceptible line rohu (interquartile range 0.362). High molecular weight glycoprotein polymers called mucins are found on the outer body surfaces and intestine of fish. These glycoproteins form a highly viscous gel that protects the epithelium from microbial and other disturbances. Common carp increase the amount and total glycosylation of high molecular weight glycoproteins in the skin in response to increased bacterial loads [36, 37]. Twenty-six contigs with homology to zona pellucida glycoprotein have been found to show two- to seven-fold higher expression (median 4.13, interquartile range 2.45) in resistant compared to susceptible line rohu . Choriogenin is another high molecular weight glycoprotein and was found to be 3.5 times more highly expressed in resistant compared to susceptible line rohu . Higher expression of these glycoprotein genes could result in the secretion of greater quantities of glycoproteins, including mucin, on the skin and gut surface leading to greater protection and readiness against bacterial disease.
Serum lectins (c-type lectin receptor c 53025_556 on LG5, Table 6) are found in the mucus and have been shown to agglutinate with and alter the viability and pathogenicity of Gram-negative bacteria including A. hydrophila[38–41]. Seven-fold higher expression of the serum lectin isoform 1 precursor gene was detected in resistant line than susceptible line rohu, and alternative isoforms of galactoside-binding soluble lectin 9 in resistant and susceptible line rohu were detected by . Along with a higher production of mucin, higher production of lectins found in the mucus of the skin and gut could lead to a greater preparedness to combat and resist infection by bacterial pathogens. Cluster of differentiation 22 (CD22 antigen 31265_40 on LG8, Tables 4 and 5) is a lectin that is found on the surface of mature B cells and prevents over activation of the immune system . CD22 is a negative regulator of antigen receptor signaling in B cells. In mice CD22 is down-regulated on wild-type B-1 cells in response to LPS .
Tributyltin binding protein (111569_63 on LG 19, P <0.05 after Bonferroni correction for FASTA and GRAMMA tests for hours of survival and FASTA, GRAMMA and ASSOC tests for dead or alive, Tables 4 and 5) is a glycoprotein (possible lipocalin) that is believed to be involved in the transportation, detoxification and excretion of xenobiotic compounds such as tributyltin in the blood of Japanese flounder . Tributyltin-binding protein is excreted from the body of Japanese flounder via the skin mucus. Tributyltin-binding protein is up-expressed more than four fold in the spleen of turbot 3 days after challenge with Aeromonas salmonicida.
Complement protein component c7 (87896_2052 on LG6, Table 5) plays an important role in the membrane attack system of the innate and adaptive immune response by serving as a membrane anchor, facilitating the formation of pores in the plasma membrane of target cells . In a condition known as complement component 7 deficiency, human patients are more susceptible to recurrent infections, particularly to bacterial diseases such as caused by meningococcal infection .
Other genes with putative immune function with SNPs of interest included pore forming protein (perforin 1 113696_50 on LG 14, P <0.05 after Bonferroni correction for the GRAMMA test of hours of survival, Table 4) which is a key molecule involved in T-cell and natural killer-cell-mediated cytolysis, inserting itself into the target membrane forming a pore which allows cytolytic proteins to enter the cell and trigger it to self-destruct , dipeptidylpeptidase 7 (554_399 on LG22, Table 4) which suppresses apoptosis of resting lymphocytes  and immunity related gtpase e4 (134666_118 on LG 7, Table 6) which is one of a family of proteins that are activated as part of an early immune response (induced by interferon) and that localises to and disrupts the phagocytic vacuole during infection. Large temporal changes in perforin gene expression post-infection were detected by quantitative real-time PCR in spleen (up to + 20 fold at 12 h post-infection, Figure 5a) and gill tissue (9, 11, 8 and 7 fold at 3 h, 24 h, 48 h and 7 days post-infection, respectively, Figure 5c). Along with linkage and genome-wide association evidence for a QTL mapping to the perforin gene region on LG14 (Tables 3, 4 and 5), these patterns suggest that differential expression of the perforin gene itself could play an important role in the immune defense of L. rohita against A. hydrophila infection, and that polymorphisms affecting the expression of this gene during the time course of infection could influence disease resistance.
Comparison of traits and tests
In all cases except one, the position of QTL mapped using half-sib regression analysis co-located within 10 cM of a nominally significant SNP in the GWAS analysis for the two traits. In two cases on LG15 at 29 cM and LG23 at 0 cM, the QTL peak from the linkage analysis (Table 3) co-located to the same position as significant SNPs in the GWAS analysis for the hours of survival trait (Table 4). There was good correspondence between the results for the ASSOC, FASTA and QFAM GWAS test results (most suggestive/significant results were detected by >1 GWAS test). As the challenge tests were performed for the different families over different total time frames it was not valid to compare hours of survival between families using the “total” option in qfam. The concordance between the findings for the hours of survival and dead or alive trait analyses was fairly high. Overall, many of the same regions (on linkage groups 1, 4, 5, 7, 10, 14, 15, 19, 20, 21, 23 and 24) contained SNPs with suggestive or significant associations for both the hours of survival and dead or alive traits (Tables 4 and 5), heritability estimates for both traits were similar (0.05 and 0.07 respectively) and the genetic correlation between the two traits was high and positive (0.79), indicating that the same underlying genetic mechanisms may be affecting these traits.
As the heritability of hours of survival and dead or alive post-challenge with A. hydrophila is low (similar levels of heritability for A. hydrophila resistance have been found for common carp, 0.04)  this is likely to be a polygenic trait influenced by the small additive effects of many genes and by the environment. Polymorphisms affecting the regulation of expression or amino acid structure of the proteins expressed by the immune genes highlighted in this study could be the type of causative mutations contributing to overall A. hydrophila resistance in L. rohita. It is not possible to identify the causative mutations from the results of this current study, but the genes and linkage group regions highlighted here provide clues that will direct the focus of future investigations, and provide potentially useful loci for marker assisted selection to improve A. hydrophila resistance in this and related species.