Genome-wide association study identifying immune markers in Qingyuan partridge chickens infected with avian Leukosis virus subgroup J

Abstract

In poultry, Avian Leukosis Virus (ALV) causes high mortality and significant eradication costs, with no effective vaccines, hindering industry development. Improving resistance to ALV-J offers both economic and ecological value. The Qingyuan Partridge Chickens is a high-quality, possesses certain disease resistance capabilities. This study explored resistance molecular markers in Qingyuan Partridge Chickens through cell experiments, viral infection, and genomic resequencing. Results showed that interferon-alpha (IFN-α) expression significantly suppressed the ALV-J duplicated, with notable immune response differences between sexes. Genome-wide association study (GWAS) identified 62 single nucleotide polymorphisms (SNPs) linked to immune responses, cloacal viral positivity, and tissue indices. Selection for the C allele at chromosome 13 positions 8654609-8654610, the G allele at chromosome 9 position 21398106, and the G-T-C haplotype at chromosome 3 positions 59308643-59309366, while selecting against the T and A alleles at chromosome 1 positions 114656880 and 69131730, will effectively enhance immune function and reduce viral susceptibility. Gene Ontology analysis highlighted enrichments in biological processes like glial cell differentiation, cellular components such as side of membrane, and molecular functions like hydrolase activity. Quantitative PCR showed significant expression differences across immune organs in five genes, including IFN-α, interferon β (IFN-β), dual specificity phosphatase 1 (DUSP1), sosondowah ankyrin repeat domain family member C (SOWAHC), and dual specificity phosphatase 10 (DUSP10). These findings provide a genetic basis for breeding ALV-J-resistant Qingyuan Partridge Chickens through marker-assisted selection.

Keywords: Avian Leukosis Virus (ALV); IFN-α; Qingyuan Partridge chickens; genome-wide association study (GWAS).

Fig. 1

Relative expression levels of IFN-α after interference with IFN-α gene. sh1-4 represent fragments 1 to 4, and shNC is the control group.

Fig. 2

(A) The relative expression of Env after transfection with overexpressed IFN-α. (B) Interference with the relative expression of Env after transfection with IFN-α. (*) indicates significant difference (P < 0.05), (**) indicates highly significant difference (P < 0.01).

Fig. 3

(A) S/P values of p27 in cell supernatants after IFN-α overexpression. (B) S/P values of p27 in cell supernatants after IFN-α interference. (*) indicates significant difference (P < 0.05), (**) indicates highly significant difference (P < 0.01).

Fig. 4

Organ indices changes in Qingyuan Partridge chickens following ALV-J challenge. (A) Comparison of organ indices between ALV-J infected and control group of hens. (B) Comparison of organ indices between ALV-J infected and control group of roosters. (C) Comparison of organ indices between hens and roosters in the ALV-J infected group. (D) Comparison of organ indices between hens and roosters in the control group. (*) means significantly different (P <0.05). (**) means the difference extremely significant (P <0.01).

Fig. 5

Plasma immune factor levels in Qingyuan Partridge chickens following ALV-J challenge. (A) Comparison of plasma immune factor levels between ALV-J infected and control hens. (B) Comparison of plasma immune factor levels between ALV-J infected and control rooster chickens. (C) Comparison of plasma immune factor levels between rooster and hens in the ALV-J infected group. (D) Comparison of plasma immune factor levels between rooster and hens in the control group. (*) means significantly different (P < 0.05). (**) means the difference extremely significant (P < 0.01).

Fig. 6

Tissue sections of the spleen, thymus and bursa of fabricius from the control group and infected group of hens (40X).

Fig. 7

Correlation of immune traits after ALV-J infection.

Fig. 8

Manhattan and Q-Q plots visualizing SNP additive effects for cloacal viral positivity, IFN-α, IFN-β, IL-6, and the indices of the bursa of Fabricius, spleen, thymus, and pancreas. The X-axis represents the chromosomal position of each SNP, and the Y-axis shows the –log₁₀(P) value. The genome-wide significance threshold (P < 3.93×10⁻⁹) and suggestive significance threshold (P < 7.87×10⁻⁸) are indicated.

Fig. 9

Linkage disequilibrium (LD) structure and haplotype association analyses of ALV-J–related genomic regions. (A) Chromosome 1: LD heatmap and haplotype–phenotype associations for CHR1-BLOCK15 and CHR1-BLOCK7. (B) Chromosome 3: LD heatmap and haplotype–phenotype associations for CHR3-BLOCK31.

Fig. 10

GO annotation analysis for candidate genes.

Fig. 11

The relative expression of IFN-α (A), IFN-β (B), DUSP1 (C), AJAP1 (D), SOWAHC (E), and DUSP10 (F) genes. Data represent mean ± SD of six biological replicates per group (n = 6). Statistical significance was evaluated using two-way ANOVA. (*) means significantly different (P < 0.05). (**) means the difference is extremely significant (P < 0.01).