Purpose To investigate the expression differences and polymorphisms of the natural resistance-associated macrophage protein 1 (NRAMP1) gene in various tissues of Wuding chickens, and to analyze their association with resistance to pullorum disease, providing effective candidate molecular markers for disease-resistance breeding in chickens.
Methods Molecular diagnostic techniques were employed to identify the Salmonella Pullorum infection status of individual Wuding chickens, which were categorized into an infected group and an uninfected group. The mRNA expression levels of NRAMP1 gene in thymus and spleen of both groups were detected using qRT-PCR. Single nucleotide polymorphisms (SNPs) in the coding region of NRAMP1 gene were screened via direct sequencing of PCR products. The correlation between these SNP loci and resistance to pullorum disease was analyzed to identify functional SNPs.
Results Molecular diagnosis revealed that the positive and negative rates of pullorum disease in Wuding chickens were 42.1% and 57.9%, respectively. qRT-PCR analysis showed that the mRNA expression levels of NRAMP1 gene in thymus and spleen of the uninfected group were extremely significantly higher than those in the infected group (P<0.01). A total of eight SNP loci were detected across exon 2, exon 8, and exon 9 of NRAMP1 gene, among which c.163A>G (exon 2), c.668G>A (exon 8), and c.919G>A (exon 9) were missense mutations. Association analysis indicated there were extremely significant differences in genotype frequency at the c.163A>G locus between the infected and uninfected groups (P<0.01).
Conclusion The expression level of NRAMP1 gene is closely related to resistance to pullorum disease. Furthermore, the c.163A>G locus on exon 2 may be a key functional mutation affecting resistance and could serve as a potential molecular marker for disease-resistance breeding in chickens.
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