HSMI DISEASE RESISTANCE IN SALMONIDS
20200190584 · 2020-06-18
Inventors
- Jacob Seilø Torgersen (Ås, NO)
- Torkjel Bruheim (Trondheim, NO)
- Vibeke Evenstad Emilsen (Ranheim, NO)
- Thomas Moen (Aas, NO)
- Nina Santi (Trondheim, NO)
Cpc classification
Y02A40/81
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C12Q1/6883
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to methods of predicting resistance to heart and skeletal muscle inflammation in salmonids, the method comprising determining the alleles present at a DNA polymorphism in the salmonid and predicting whether or not the salmonid is resistant to heart and skeletal muscle inflammation based on the determination of the alleles. The invention also relates to related methods of detecting, in a sample from a salmonid, the alleles present at a DNA polymorphism associated with resistance to heart and skeletal muscle inflammation, methods for obtaining an indication of risk of a salmonid developing heart and skeletal muscle inflammation, uses of such DNA polymorphisms, and methods of detecting, in a sample from a salmonid, one or more salmonid gene variants.
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. A method of detecting resistance to heart and skeletal muscle inflammation in a salmonid, the method comprising: detecting, in a sample from the salmonid, alleles present at a DNA polymorphism associated with resistance to heart and skeletal muscle inflammation, wherein the presence of the DNA polymorphism is indicative of the salmonid being resistant to heart and skeletal muscle inflammation.
7. The method of claim 6, wherein detecting the alleles present at a DNA polymorphism comprises sequence analysis of the sample.
8. A method of detecting, in a sample from a salmonid, one or more salmonid gene variants in the locus defined by position 78,364,536 bp to 91,273,924 bp in the Atlantic salmon genome; and assaying the sample to detect the one or more salmonid gene variants, the one or more salmonid gene variants selected from one or more of the DNA polymorphisms listed in Tables 1 to 6.
9. The method of claim 8, wherein the DNA polymorphism is located on chromosome 3 or 14.
10. (canceled)
11. The method of claim 8, wherein the DNA polymorphism is DNA polymorphism No. 1 or 9, or both.
12. The method of claim 8, wherein the DNA polymorphism is selected from any one or more of polymorphism Nos. 1, 2, 7 and 9, and any combination thereof.
13. The method of claim 8, wherein the DNA polymorphism is selected from any one or more of polymorphism Nos. 1, 2, 7, 9, 13, 14, 15 and 16, and any combination thereof.
14. The method of claim 8, wherein the salmonid is an Atlantic salmon (i.e. Salmo salar), rainbow trout (i.e. Oncorhynchus mykiss), or coho salmon (i.e. Oncorhynchus kisutch).
15. The method of claim 8, wherein the method is for predicting PRV infection.
16. The method of claim 8, further comprising selecting the salmonid for use as broodstock, wherein the salmonid is selected, based on the detected one or more gene variants.
17. A method of producing offspring that have a higher than normal chance of having resistance to heart and skeletal muscle inflammation, the method comprising: detecting, in a sample from a salmonid, alleles present at a DNA polymorphism associated with resistance to heart and skeletal muscle inflammation, wherein the presence of the DNA polymorphism is indicative of the salmonid being resistant to heart and skeletal muscle inflammation; and using the salmonid being resistant to heart and skeletal muscle inflammation to produce offspring.
18. The method of claim 17, wherein producing offspring comprises providing eggs from the salmonid for fertilization based on the presence of the detected DNA polymorphism.
19. The method of claim 17, wherein producing offspring comprises fertilization of eggs from the salmonid based on the presence of the detected DNA polymorphism.
20. (canceled)
21. The method of claim 7, wherein the DNA polymorphism is selected from any one or more of polymorphism Nos. 1, 2, 7, 9, 13, 14, 15 and 16, and any combination thereof.
Description
[0055] The present invention will now be described by way of example with reference to the accompanying figures, in which:
[0056]
[0057]
[0058]
1. CHALLENGE TRIAL
[0059] The challenge trial was conducted as a cohabitant challenge at VESO Vikan (Namsos, Norway). A total of 1351 Atlantic salmon (Salmo salar) smolts of an average weight of 83 grams were included in the test. The smolts were recruited form 507 families in the AquaGen breeding nucleus. Each smolt had been tagged with Passive Integrated Transponder (PIT) tags prior to testing. Three hundred and four smolts were used as shedders. The shedders were injected with blood cells infected with PRV virus (from a clinical outbreak of HSMI in Nord-Trrndelag, Norway, in 2012). The AquaGen breeding nucleus of Atlantic salmon constitute a closed population of Atlantic salmon, having been selectively been bred for traits desirable for aquaculture production (fast growth rate, late sexual maturation, resistance to particular diseases) for twelve generations. The base population consisted of wild Atlantic salmon collected from a large collection of Norwegian rivers (Gjedrem et al. 1991).
[0060] The challenge was performed in sea water with a fish density of 40-60 kg/m.sup.3 and at a temperature of 12 C.1 C. Mortalities were registered daily until the test was terminated at 10 weeks post challenge. At this point all test fish (N=887) were registered and sampled: From each fish a heart biopsy was taken and stored on RNAlater for subsequent quantitative PCR. From 240 fish, heart and skeletal muscle biopsies were taken and fixed in 10% buffered formalin for subsequent histopathology.
2. HISTOPATHOLOGY
[0061] Formalin-fixed samples were prepared for histological examinations by standard paraffin wax techniques and stained with haematoxylin and eosin (H&E stain).
[0062] Sections of cardiac and skeletal muscle tissue from individual fish were classified histologically based on the presence of mononuclear leukocyte infiltration and muscular degeneration and necrosis. The atrium, epicardium, compact and spongy layers of the ventricle and the endocardium were examined and evaluated. The findings were graded from 0-3 according to the following criteria:
TABLE-US-00001 Score Description 0 No pathological findings 1 Few focal lesions, slightly increased number of leukocytes 2 Several distinct lesions and moderate increase in numbers of leukocytes 3 Multifocal to confluent lesions and severe increase in number of leukocytes
[0063] Histopathology scores are directly indicative of tissue damages caused by the disease. Thus, histopathology scores can be used as indicators of an individual's disease status and hence, in the context of a controlled challenge test, of an individual's resistance to the disease to which they were challenge-tested.
3. REVERSE TRANSCRIPTION QUANTITATIVE PCR (RT-QPCR)
[0064] In order to provide an alternative (less expensive) phenotype analysis for HSMI infection or resistance, expression (transcription) levels of the Atlantic salmon cluster of differentiation 8 (cd8) gene was measured using reverse transcription quantitative PCR. The gene product of the gene, Cd8, is a marker for cytoxic T-cells. Cytoxic T-cells are, on their side, markers of viral infection. RT-qPCR for cd8 was performed as follows:
[0065] Total RNA was purified with a RNeasy 96 Universal Tissue 8000 Kit
[0066] using a Qiagen Biorobot Universal. Duplex real time PCR was performed using primers targeted against the Atlantic salmon cd8 alpha chain (forward primer: 5TCGTGCAAAGTGGGAAAGGT 3, reverse primer: 5 GGTGCCCAAACGATCAAATC 3, TaqMan probe: 5 FAM-ACCCTACTGCATCCTG-MGB 3) and the housekeeping gene elf1a (forward primer: 5-TGC CCC TCC AGG ATG TCT AC-3, reverse primer: 5-CAC GGC CCA CAG GTA CTG-3, TaqMan probe: VIC 5-CCA ATA CCG CCG ATT TT-3 MGB). Amplification was performed using QuantiTect Probe RT-PCR kit (Qiagen) on a Rotor-Gene Q 2 channel Real Time PCR Machine, 40 cycles of 95 C. 15 sec and 60 C. 60 sec.
4. GENOTYPING
[0067] All fish were genotyped using a custom AxiomSNP genotyping array from Affymetrix (San Diego, Calif., USA), and that formed a SNP-chip containing 56,177 SNPs. Genotyping was done according to the Axiom 2.0 Assay Manual Workflow User Guide (http://media.affymetrix.com/support/downloads/manuals/axiom_2_assay_manual_workflow_prepguide.pdf). Genotype calling was done using the Affymetrix Power Tools programs (http://www.affymetrix.com/estore/partners_programs/programs/developer/tools/po10 wertools.affx), according to best practices recommendations from Affymetrix (http://media.affymetrix.com/support/downloads/manuals/axiom_best_practice_sup plement_user_quide.pdf). Samples displaying poor genotype quality metrics were culled according to the best practices recommendations from Affymetrix.
[0068] The SNP-chip was proprietary of AquaGen, and contained SNPs evenly distributed across the Atlantic salmon genome.
5. GENOME-WIDE ASSOCIATION STUDY (GWAS)
[0069] The genotype data were used for performing a genome-wide association study (GWAS). Each individual SNP was assayed for its association to two phenotypes: histopathology scores and concentrations of cd8 mRNA as measured using RT-qPCR (this phenotype will be referred to as cd8-qPCR). Both phenotypes were used as indicators of morbidity and/or mortality. After culling of individuals with poor genotype quality, 779 individuals remained that had both genotypes and phenotypes (histopathology scores, cd8-qPCR) connected to them. Each of the 56,177 genotyped SNPs were tested individually for their association to histopathology scores, cd8-qPCR, using a linear mixed model. When the phenotype was histopathology score, the linear mixed model for each SNP was
y=mean+animal+geno+error
[0070] where y=histopathology score for the animal in question; mean=the overall mean across all animals; animal=the additive genetic value of the animal in question (random effect); geno=genotype at the SNP for the animal in question (random regression; coded as 0, 1, and 2 for one homozygous genotype, the heterozygous genotype, and the other homozygous genotype, respectively); error=residual error.
[0071] When the phenotype was cd8-qPCR, the linear mixed model for SNP i was
y=mean+plate+animal+geno+error
[0072] where y=histopathology score; mean=the overall mean across all animals; plate=fixed effect of the sample plate on which the RNA sample corresponding to the animal in question was located; animal=the additive genetic value of the animal in question (random effect); geno=genotype at the SNP for the animal in question (random regression; coded as 0, 1, and 2 for one homozygous genotype, the heterozygous genotype, and the other homozygous genotype, respectively); error=residual error.
[0073] The linear mixed model equations were solved using the software DMU. The likelihood ratio test (LRT) statistic was used, equal to two times the natural logarithm of the ratio H1/H0, where H1 and H0 are the likelihoods under the alternative hypothesis (model incorporates geno) and the null hypothesis (model does not incorporate geno), respectively.
[0074] The GWAS results revealed that histopathology scores are largely explained by a quantitative trait locus (QTL) located on Atlantic salmon chromosome 3 (
[0075] The GWAS results further revealed that cd8-qPCR values are largely explained by a quantitative trait locus (QTL) located on Atlantic salmon chromosome 3 (
[0076] Since the QTL for histopathology score and the QTL for cd-qPCR are both located on chromosome 3, it is reasonable to hypothesise that the two QTL are in fact one and the same QTL, i.e. they are both caused by one and the same underlying causative mutation, mapping to a specific location on chromosome 3. This expectation was backed up by the observed, high (in absolute value) genetic correlation between the two traits within the data set (Pearson correlation coefficientstandard error=0.910.16).
[0077] Table 1 contains DNA polymorphisms located on Atlantic salmon chromosome 3 that were found to be associated strongly to histopathology score and to cd8-qPCR. The table contains the positions of these DNA polymorphisms, the p-values of their LRT statistics, and the identity of the high-resistance and low-resistance alleles pertaining to each DNA polymorphism. The positions in Table 1 are relative to the published version of Atlantic salmon chromosome 3 (GenBank identifier NC_027302.1). The high-resistance alleles in Table 1 are the alleles that correlate to low histopathology scores and high cd8-qPCR values. Conversely, the low-resistance alleles in Table 1 correlate to high histopathology scores and low cd8-qPCR values. High histopathology scores signify large extent of tissue damage. Low cd8-cPCR signify high concentrations of cd8 mRNA. High histopathology scores and high concentrations of cd8 mRNA are signs of viral infection.
[0078] As can be seen from
[0079] The DNA polymorphisms in Table 2 have low minor allele frequency (0.10 or smaller), meaning that one of the alleles at the quantitative trait locus on chromosome 14 has low frequency within the studied population. This is a likely reason why the quantitative trait locus on chromosome 14 did not turn up as highly (genome-wide) significant when histopathology score was used as trait; only 240 animals were phenotyped for histopathology score, and few of these 240 animals harboured the rare allele at the quantitative trait locus on chromosome 14 (hence there was very little statistical power to detect the quantitative trait locus on chromosome 14 when histopathology score was used as trait). In fact, the DNA polymorphisms in Table 2 are associated with histopathology score (P<0.05), but they are not genome-wide significant, i.e. they are not significant after multiple testing has been taken into account.
[0080] The GWAS was performed using a SNP-chip containing 56,177 DNA polymorphisms. These are just a small fraction of all the DNA polymorphisms that exist in the Atlantic salmon genome. In order to identify additional DNA polymorphism associated to histopathology scores and/or cd8-qPCR, we used the following approach: The parents (hereafter referred to as the parents) of the individuals that had been put through the HSMI test (the offspring) were genotyped on an Affymetrix Axiom SNP-chip containing 745,999 DNA polymorphisms distributed across the Atlantic salmon genome. The DNA polymorphisms on this chip had earlier been identified by AquaGen researchers on the basis of Illumina whole-genome sequencing of 28 individual Atlantic salmon from AquaGen. The parental genotypes (29,879 DNA polymorphisms on chromosome 3) were combined with offspring genotypes (2,278 polymorphisms on chromosome 3, all of which were part of the larger (29,879 polymorphisms) subset), in order to impute genotypes on all 29,879 DNA polymorphisms onto the offspring. This was done using the software Flmpute, using default settings. The pedigree linking the two generations was used in the Flmpute analysis, for improved accuracy. The same procedure of imputation was employed for DNA polymorphisms on chromosome 14. On chromosome 14, 25,659 DNA putative DNA polymorphisms found in the parents were imputed onto the offspring, exploiting 2,359 DNA polymorphisms that were found in both sets of animals. Following imputation of DNA polymorphisms on chromosome 3 and 14, the imputed data sets were used in GWAS analysis, testing associations to both histopathology scores and cd8-qPCR, using the GWAS approach described above.
[0081] On chromosome 3 (GenBank identifier NC_027302.1), imputed DNA polymorphisms strongly correlated to histopathology scores were primarily found in the area stretching from position 78,364,536 bp to 91,273,924 bp (
[0082] All DNA polymorphisms in Table 3 are capable of predicting resistance to heart and skeletal muscle infection. The DNA polymorphisms were identified in GWAS for histopathology score and/or cd8-qPCR, using imputed data. DNA polymorphisms associated to histopathology score and/or cd8-qPCR are not listed in Table 3 if they were already listed in Table 1.
[0083] On chromosome 14, imputed DNA polymorphisms strongly correlated to cd8-PCR values were primarily found in the area stretching from position 43,552,599 to 57,552,882 relative to the published chromosome sequence (GenBank identifier NC_027313.1). The dbSNP identifiers, positions, and high-resistance/low-resistance alleles of the DNA polymorphisms most strongly associated to cd8-qPCR scores are to be found in Table 4.
[0084] All DNA polymorphisms in Table 4 are capable of predicting resistance to heart and skeletal muscle infection. The DNA polymorphisms were identified in GWAS for cd8-qPCR, using imputed data. DNA polymorphisms associated to cd8-qPCR are not listed in Table 3 if they were already listed in Table 1.
[0085] In order to identify additional DNA polymorphisms associated to HSMI, the Inventors devised and implemented the following approach: 99 random Atlantic salmon originating from the same population as the parents and the offspring, were whole-genome sequenced on Illumina HighSeq 2000, producing paired-end reads to an average genome coverage of 18 (range 8 to 32, assuming a genome size of 3.0 billion base pairs). The reads were aligned to the reference sequence of chromosome 3 or 14 of Atlantic salmon (GenBank identifiers NC_027302.1 or NC_027313.1).) using BWA mem version 0.7.10-r789 (Li and Durbin 2009). SNPs and short indels were identified using Freebayes version 0.9.15-1 (Garrison and Marth 2012); to filter away low-quality variants, using run-time parameters-use-mapping-quality and -min-mapping-quality 1, in addition to vcffilter-f QUAL>20. The SNP-detection process also returned genotypes on the 99 animals, for all identified DNA polymorphisms. SNPs and short indels were annotated using snpEff version 4.0e (Cingolani et al. 2012). The snpEff annotation database was based on the CIGENE annotation version 2.0 (Lien et al., submitted). DNA polymorphisms in sufficiently strong LD (r.sup.2>0.5) with the best (most strongly associated to heart and skeletal muscle inflammation) DNA polymorphisms on chromosome 3 or chromosome 14 (more precisely, to DNA polymorphism ss1868447536 and/or ss1868368511 from chromosome 3 (Table 1) and DNA polymorphism ss1868307106 from chromosome 14 (Table 2)) were identified by running the computer program PLINK v1.9 (Chang et al. 2015) (options --r2 -Id-snp rs159406379 --chr-set 29 --no-xy --Id-window 999999999 --Id-window-kb 500). The list of such DNA polymorphisms was mapped against the annotation database, producing a list of DNA polymorphism that are 1) associated to resistance to heart and skeletal muscle inflammation and 2) putative functional according to the SnpEff annotation.
[0086] Table 5 contains a list of DNA polymorphisms derived using this LD-based approach. The table contains chromosome, positions and the identify of resistance- and non-resistance alleles of DNA polymorphisms, in addition to the names of the genes wherein the DNA polymorphisms reside and the annotated function of the DNA polymorphisms within these genes.
[0087] Table 6 contains the DNA sequences of all DNA polymorphisms listed in Tables 1,2, 3, 4, and 5.
TABLE-US-00002 TABLE 1 DNA polymorphisms capable of predicting resistance to heart and skeletal muscle inflammation in Atlantic salmon. Ssid = identifier of the DNA polymorphism within the GenBank dbSNP database; chr/GenBank ID = chromosome number of the DNA polymorphism within the Atlantic salmon genome, and the GenBank ID of the chromosome sequence; pos = position of the DNA polymorphism within the GenBank sequence; res. allele and non-res. allele = identity of alleles conferring resistance and non-resistance to heart and skeletal muscle inflammation, respectively; p-value histopat. and p-value cd8-qPCR = p-value from tests for association between DNA polymorphisms and histopathology scores and cd8-qPCR values, respectively. non- DNA Chr/GenBank res. res. p-value p-value pol # Ssid ID pos allele allele histopat. cd8-qPCR 1 ss1868447536 3/NC_027302.1 81438207 A G 1.86E13 7.38E08 2 ss1868822487 3/NC_027302.1 90860718 A C 6.57E12 4.99E07 3 ss1868354611 3/NC_027302.1 80185895 G A 2.02E10 9.25E05 4 ss1868795339 3/NC_027302.1 81446760 A G 3.68E09 0.00015859 5 ss1868047992 3/NC_027302.1 70308540 C T 4.03E07 5.18E05 6 ss1868331125 3/NC_027302.1 80980820 T C 7.45E07 5.12E05 7 ss1868368511 3/NC_027302.1 90967849 G A 0.00018551 2.34E08 8 ss1868396576 3/NC_027302.1 79186351 A G 0.03444835 1.77E07
TABLE-US-00003 TABLE 2 DNA polymorphisms capable of predicting resistance to heart and skeletal muscle inflammation in Atlantic salmon. Ssid = identifier of the DNA polymorphism within the GenBank dbSNP database; chr/GenBank ID = chromosome number of the DNA polymorphism within the Atlantic salmon genome, and the GenBank ID of the chromosome sequence; pos = position of the DNA polymorphism within the GenBank sequence; res. allele and non-res. allele = identity of alleles conferring resistance and non-resistance to heart and skeletal muscle inflammation, respectively; p-value cd8-qPCR = p-value from tests for association between DNA polymorphisms and cd8-qPCR values. DNA res. non-res. p-value pol # Ssid Chr/GenBank ID pos allele allele cd8-qPCR 9 ss1868307106 14/NC_027313.1 54098314 G A 8.63E08 10 ss1868764075 14/NC_027313.1 53722865 A G 1.41E07 11 ss1867927751 14/NC_027313.1 56768896 A G 2.50E07 12 ss1868318796 14/NC_027313.1 53376433 A G 7.58E07
TABLE-US-00004 TABLE 3 DNA polymorphisms capable of predicting resistance to heart and skeletal muscle inflammation in Atlantic salmon. Ssid = identifier of the DNA polymorphism within the GenBank dbSNP database; chr/GenBank ID = chromosome number of the DNA polymorphism within the Atlantic salmon genome, and the GenBank ID of the chromosome sequence; pos = position of the DNA polymorphism within the GenBank sequence; res. allele and non-res. allele = identity of alleles conferring resistance and non-resistance to heart and skeletal muscle inflammation, respectively; p-value histopat. = p-value from tests for association between DNA polymorphisms and histopathology scores. DNA res. non-res. p-value pol # Ssid Chr/GenBank ID pos allele Allele histopat. 13 ss1868538659 3/NC_027302.1 89441560 T G 6.75E14 14 ss1868161997 3/NC_027302.1 83185840 T C 1.81E13 15 ss1868435260 3/NC_027302.1 91273924 G A 2.05E13 16 ss1868124696 3/NC_027302.1 81717100 G A 2.45E13 17 ss1868447536 3/NC_027302.1 81438207 A G 2.73E13 18 ss1868365111 3/NC_027302.1 90342957 T G 3.31E13 19 ss1868638847 3/NC_027302.1 81578538 A C 4.49E13 20 ss1868822487 3/NC_027302.1 90860718 A C 1.56E12 21 ss1868065680 3/NC_027302.1 83391479 A C 3.51E12 22 ss1868469043 3/NC_027302.1 84674335 G A 5.41E12 23 ss1868846411 3/NC_027302.1 81998536 A G 5.53E12 24 ss1868822542 3/NC_027302.1 85851618 G A 7.12E12 25 ss1868354611 3/NC_027302.1 80185895 G A 1.25E10 26 ss1868256346 3/NC_027302.1 78364536 C T 1.27E10 27 ss1868658490 3/NC_027302.1 89046115 A G 2.41E10 28 ss1868596935 3/NC_027302.1 89147285 C T 2.41E10 29 ss1868707552 3/NC_027302.1 88964614 A G 3.84E10 30 ss1868609032 3/NC_027302.1 80186731 A G 4.79E10 31 ss1868016481 3/NC_027302.1 81964975 T C 7.39E10 32 ss1868225037 3/NC_027302.1 81446554 A G 1.39E09 33 ss1868795339 3/NC_027302.1 81446760 A G 1.43E09 34 ss1867950637 3/NC_027302.1 81434272 C T 1.97E09 35 ss1868512714 3/NC_027302.1 81435622 T C 1.97E09 36 ss1868391605 3/NC_027302.1 89376084 T C 2.23E09 37 ss1868833692 3/NC_027302.1 84407555 G A 5.30E09 38 ss1868833185 3/NC _027302.1 84235339 T C 7.33E09 39 ss1867959638 3/NC_027302.1 84243207 G A 7.33E09 40 ss1868803581 3/NC_027302.1 87252679 A G 8.17E09 41 ss1868417386 3/NC_027302.1 59788304 C T 8.44E09 42 ss1868147018 3/NC_027302.1 70020249 G A 2.43E06 43 ss1868368511 3/NC_027302.1 91016402 C T 1.22E05
TABLE-US-00005 TABLE 4 DNA polymorphisms capable of predicting resistance to heart and skeletal muscle inflammation in Atlantic salmon. Ssid = identifier of the DNA polymorphism within the GenBank dbSNP database; chr/GenBank ID = chromosome number of the DNA polymorphism within the Atlantic salmon genome, and the GenBank ID of the chromosome sequence; pos = position of the DNA polymorphism within the GenBank sequence; res. allele and non-res. allele = identity of alleles conferring resistance and non-resistance to heart and skeletal muscle inflammation, respectively; p-value cd8-qPCR = p-value from tests for association between DNA polymorphisms and cd8-qPCR values. DNA res. non-res. p-value pol # Ssid Chr/GenBank ID pos allele allele cd8-qPCR 44 ss1868139598 14/NC_027313.1 51294667 C T 2.84E08 45 ss1868332325 14/NC_027313.1 51406871 G A 2.84E08 46 ss1868178221 14/NC_027313.1 43552599 G T 3.93E08 47 ss1868319115 14/NC_027313.1 53447794 G A 5.12E08 48 ss1868572240 14/NC_027313.1 53448484 G A 5.12E08 49 ss1868640625 14/NC_027313.1 53322659 C A 5.12E08 50 ss1868798676 14/NC_027313.1 53215491 G A 7.25E08 51 ss1868380723 14/NC_027313.1 57552882 C G 9.79E08 52 ss1868247261 14/NC_027313.1 45948549 G A 9.79E08 53 ss1868437423 14/NC_027313.1 49052367 G A 9.79E08 54 ss1868651852 14/NC_027313.1 52370012 T C 9.79E08 55 ss1868188039 14/NC_027313.1 53279960 G T 9.79E08 56 ss1868081927 14/NC_027313.1 53291831 A G 9.79E08 57 ss1868083595 14/NC_027313.1 53299815 G T 9.79E08
TABLE-US-00006 TABLE 5 Functional DNA polymorphisms found be the Inventors to be associated to heart and skeletal muscle inflammation. The identifiers of the DNA polymorphisms are a combination of a short name for the Atlantic salmon chromosome wherein they reside (ssa03 = chromosome 3 or ssa14 = chromosome 14), followed by underscore and the position of the DNA polymorphism within the published sequence of the chromosomes (NC_ 027302.1 for chromosome 3 and NC_027313.1 for chromosome 14); res. allele and non-res. allele = identity of the alleles conferring resistance and non-resistance to heart and skeletal muscle inflammation, respectively. DNA pol res. non-res. # identifier Chr/GenBank ID pos allele allele 58 ssa03_90078096 3/NC_027302.1 90078096 G T 59 ssa03_81619243 3/NC_027302.1 81619243 C T 60 ssa03_81620999 3/NC_027302.1 81620999 G A 61 ssa03_84674335 3/NC_027302.1 84674335 G A 62 ssa03_81618268 3/NC_027302,1 81618268 G A 63 ssa03_84675406 3/NC_027302.1 84675406 T A
TABLE-US-00007 TABLE6 SequencesoftheDNApolymorphismsoftheInvention.DNAsequencesareorientedinthesame direction(sameDNAstrand)asthepublishedgenomereferenceforAtlanticsalmon(accession numberGCA_000233375.4).ThetwoallelesofeachDNApolymorphismaregivenwithinbrackets. Thedeletionalleleofinsertion-deletionDNApolymorphismsareannotatedas.SEQIDres. allele=sequencenumberwithintheSequenceListingsFilecorrespondingtotheresistance alleleoftheDNApolymorphisminquestion;SEQIDnon-res.allele=sequencenumberwithinthe SequenceListingsFilecorrespondingtothenon-resistancealleleoftheDNApolymorphismin question.Allsequencesarepresented5to3relativetosequenceGCA_000233375.4 DNA SEQID SEQID POL RES. NON-RES. # ALLELE ALLELE SEQUENCE 1 1 64 CCTATTGAGAAACCAGTTGGGTCATTGCTTACTGA[G/A]AAACCAGTAGGGACATTACCTATTGAGTAACCAGT 2 2 65 CAGAATGCCATCCTGAAGGATCTTGTGCTACAGTT[C/A]CTACTGAGAATAGTTTCCCGCCGCCTACTGGACAG 3 3 66 AGGTTAATGATTACCAGGTTAGCATGCGTGAGCAA[A/G]GCATTCACTACTCATGCGCTGGGAGAGCAGAAAAT 4 4 67 CCATCTATTTGATAGTCCTTTCCATATTTGATAGA[G/A]ACATATGCCCTAGGTGAATTTACACATTTTAAGCA 5 5 68 TTTTTCATTGCCATGCCAATTGATCTGAGTCTCCA[T/C]GTTGTCATTGCAGCAAGAGGTTGCACAGTACAGAA 6 6 69 GGTCACATGGCCTTTGAATAAACAGCAAAATACAT[C/T]CTCTCATGTACAAATCTTCCATCAGCCAAACGCTT 7 7 70 CACGGGGGGCCAGTACAAAAAAAAATGCATGAAAT[G/A]AAATGGATGAAATGTATGAATTCACTACTGTAAGT 8 8 71 ACATGAAACGAGGGGTTAATATGATAATAAGAAGG[G/A]AGTTTTAATCTATAACACACCTCGTTTATTCTCCT 9 9 72 AAAACTATACATTTTGGTGGAAGGCAAAATCTTAA[G/A]CTCATTTGAGCTAAAACTCGCAAATATATTGGCTG 10 10 73 AAGCGGTTCGATGAGTCATTCTGGATCCAAGCCTT[A/G]TATGAGAAACTCACAGCTGCAACTCTAAGCCAAAC 11 11 74 CTGGACAGGCTCTTTGGTAGTGTTCGTGCTCTGTG[A/G]TGACTGTTCATCTTCAGCCTGGCTTGGCTGGTCTG 12 12 75 TTTATATCTCTTGGCGGTTATTAGTTACATTCTTT[G/A]TAAAATGATAATAGAGCACAATATTCACTTGGCTA 13 13 76 TCAAACGACATGACGTAACAGGAACTACAGTAGAG[G/T]AATCTTGTCTCTGGCCTCTCAGAAATAGGTTAAGT 14 14 77 ATGGAAAATGCAGCCTCATCACAGTCCACTCCAGA[C/T]ACCTTCCCTAACGGCAGATTTCATGAGACATATCT 15 15 78 TAAATGCATGTGACCTACTACACCCTCCATTGACC[A/G]CTCCTGTAATTATATCCAGCCAGGTCAGTCCTTAG 16 16 79 TACACTAGCATATGAAACCACGTGACAAAGAATGA[A/G]AAGGTGCACTCTTTAGATCATCAACAAAAAGATGT 17 17 80 CCTATTGAGAAACCAGTTGGGTCATTGCTTACTGA[G/A]AAACCAGTAGGGACATTACCTATTGAGTAACCAGT 18 18 81 TGTTGACAGAGTCAGGTGCATGGCAGGTGACATAC[G/T]ATTGCTCCTTCAATGCCTATATCGTATGTGAGATA 19 19 82 CAAAAGCCACATCAACACAACATGAAGGCTAGATA[C/A]AGATATAGTGAAACAGAATCTTACAGTTTCCATGT 20 20 83 CAGAATGCCATCCTGAAGGATCTTGTGCTACAGTT[C/A]CTACTGAGAATAGTTTCCCGCCGCCTACTGGACAG 21 21 84 TCTGTTGATGTCTATATTTGGCTAATATTCTTTTT[C/A]ATTTTCTGTTTCAGAGTGGGCAGTTTGACTATGAC 22 22 85 AGCTAGAGTTGTTGGTGACAGTAAAGGAGAAAGAG[A/G]GGACCGGCCAGCCATGTGTCAGCAATTGAGAAATG 23 23 86 AATATGCGTGTCACAGTTCCCATTTCTGAGGCTGA[G/A]AGATAAACAGGCAGAGATAAACAGTCACAATGGTT 24 24 87 TCCCAGGTGGCACTGATAGATGAGTACACATTCCC[A/G]GTAGGATCAATGTTTGTTCGCACCCTGCTATTCTT 25 25 88 AGGTTAATGATTACCAGGTTAGCATGCGTGAGCAA[A/G]GCATTCACTACTCATGCGCTGGGAGAGCAGAAAAT 26 26 89 GCGCTCGCCATAGTCTCCTCATTGTGTAACTATAA[T/C]CATGGAGAAATATGTGCAACAACACAGTTGAGACT 27 27 90 CCCGTCTGTCAGTCATTCCATGTTCGGTGTCATTA[G/A]CTGAGGGCTGAAGCACGGACGTTGAACAGAAGCTC 28 28 91 TAAAATGGCAGTTAAGTCAGCTTGTATATTGGGTC[T/C]AAAGATAACAGTTAATGTGCTGAGGCTTGATAAAC 29 29 92 ACTCTGCCCAAACACACACTCTCTGTCTCTGCCAG[G/A]TGATCCAGCCCCCTGCTCACGCTACATCTTCTCGC 30 30 93 TAATCTACAGCCAATGCAAGTACATTCTGTCGACC[G/A]AAGAGCTCTGGCCATATCAGCCCTGTTATGACCAT 31 31 94 TACACGGTACACTTGCTGCACTATATAAAAACACT[C/T]GGGTCCACAGCGGGAGAAGAAAGTCACGGCTTTAA 32 32 95 CCAGTTGGGACATTTCCTATTGAGAAACCAGTCGG[G/A]ATATTACCTGTTGAGAAACCAGTTGGGACATTACC 33 33 96 CCATCTATTTGATAGTCCTTTCCATATTTGATAGA[G/A]ACATATGCCCTAGGTGAATTTACACATTTTAAGCA 34 34 97 TCGGGACGTTGCCTATTGAGAAACTAATTGGGATA[T/C]TGCCTATTGAGAAGCAAGTTGGGACATTGCCCATT 35 35 98 TTGAGAAACAAGTTGGGATATTGCCCGTTGAGATG[C/T]TAGTTGGGACATTGCCCATTGAGAAACCAGTCGGG 36 36 99 TGAGCGTAGTATGACAGAGTGGTAGTTAGTTGTCT[C/T]ACTGACCCTCCGTAGCTGGTTGGTGAAGAACAGGG 37 37 100 CTAGTTCCCACTTCACAGGAGGGACATTTAAAAAT[A/G]CATGCAAGTGATATGTAAATATGAGTAAAAGGGTT 38 38 101 CAGTACATACAAAACTGTAGCCAAGAATGCATTTC[C/T]GTAAGCTTGAATTACTATCATATCAGTCTTTACAG 39 39 102 TTCAACAGATGCCTTTAGCCCAAGCTGATGAAACG[A/G]ATACAGATGCCATCATTGGGGCAGATTAAACATGT 40 40 103 TCAGTTTTCTCAAGTGTCTGTCTGACCGTGTCGTT[G/A]TCAAACCAGTTCCAGATCGCAGGCCTTGGAAGCCG 41 41 104 CAACAGCCTAGCCCAAGGCTGTCCAACCCCGTTCC[T/C]GGAGAGCTAACATCAGCCCGCTGCCTGGCTAGTGT 42 42 105 CCTCCCCCAATGCCAAATGCCTCATATCTGTTGGA[A/G]CCTCCACAGAGAATGGCATCTGAAAAACATGTTTA 43 43 106 GCAGCACTGATGAAGGCATTGAAGCCTTTTGTTAA[C/T]ATTTTGAGCATGGGTTTAATTAAGTACATTTTTGC 44 44 107 TCCTCTTCAAATTGGACACAAGTCTTCGTCCTCAT[C/T]TCTCTCTGCATCTTCATTTTCCTCCTTCTCTGAAT 45 45 108 GGTTGTTTCCCAAACATAAATTGATATGGAGAGTC[G/A]TGTCATTTCGTGTCATTTCTTGTGCAATTATATGC 46 46 109 ATGGCAGCAAGTAGGGTGGACGAAATGGAGGACAT[T/G]GAGAAAAAGCTGGTGAAGCAGCAGCTGTGCTGGAA 47 47 110 CAAACATCAAATATCCGTGCTAAATGAAATGGCAA[G/A]CTAACATAAATTAGCTTGATCGTTTAATAAGTACT 48 48 111 AGGCACCATAAGACTCCAAGAGGACACTAACAGCT[G/A]GTGAAAAACATGGACCTGGGTCCTGTTATCCTTGT 49 49 112 GGTTCGGGGGAGCTGTGTTACCGTGCACTGTCTAT[C/A]TCAGATTGCTAAATAATTACCATGACTGCTTCTGA 50 50 113 AACAGCAGTGCCAGAGAAGGAACTTTACATTTAAT[G/A]TAATTCTGAGTCATTAACATTTGGCTGGACGGTGT 51 51 114 AGGGTATAGATGGGCTCATGACCATGACCTCCACC[C/G]TTACCTTTACCGCCTCCCTCCGCCATCACGGGCTG 52 52 115 TGAAAAGGCGTAGGAGAGAGAAGCGAAGAGACAGA[G/A]AAAATACGCTAATATCAGGTGTTGTTCTGAGTTTC 53 53 116 CAACAGGAGATAAACCCCTCTACTGTGCAGCAGGG[G/A]AGAACAGATATTCACAATCAGGGCTGCAATACAGA 54 54 117 CTGTCTGTCCACTAAATCACTGGATATATATATGA[C/T]GGGCTGACGGGACAGGACAGGGAGACATATGAGGG 55 55 118 TTCCATGATGTAACTTAAGATGCCCCACTGTGTAG[G/T]TGGAAAGAGTGATGAGATGTGGGCAAGAAAAGTAG 56 56 119 TAAAATCATGGGGACGGTCTCTGAGAAAATGCACT[A/G]AACAAACCAATACTATTTTTTTTTTCATATTGCAA 57 57 120 ACTAGAAATTATGCAAGCAGATTGACTTAATACCA[G/T]TCTGGCCTATAGGGTTGTTTTTTTATTCAGACATG 58 58 121 AGCCTCCAGGTTGGTCTCAGGGTTTAAGACCTACC[T/G]TTCATTCTGAATAGCCTCCAGGTTGGCACGGCTGT 59 59 122 GCTGGCCTGCTTCTCCTCGTCCTCATCTGGCTGTG[T/C]GCAGCGGAGTGTGTAAGTAGCCTAATCCACCTGAA 60 60 123 AGCCAAGAAAAAAACTGCAATTTTATTAATAGTTT[A/G]AAAGAAGCTCCATAGTTCATGAAAACATTGTTTTC 61 61 124 AGCTAGAGTTGTTGGTGACAGTAAAGGAGAAAGAG[A/G]GGACCGGCCAGCCATGTGTCAGCAATTGAGAAATG 62 62 125 CGGCTGTACAAAGATCGTCTGAAGGTCACAGGCGG[A/G]TTGAACTCTGACCTGCTCAACGTGACCATAGCCCA 63 63 126 GCACTTGTTAGCCTAGCCTAGTTTAGCCTAGCCTC[A/T]GAGATAGATGCTTGTGGATTTCCCTCAGGCAGAAT
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