The main objective of this study was to analyze the possible polymorphisms in 17 primer fragments of six candidate genes of the keratin family, including KRT27 (exons 3 and 8, introns 2 and 5), KRT31 (intron 2), KRT36 (exons 1, 2, and 3), KRT38 (exon 3, introns 2 and 7), KRT81 (exons 1 and 2, introns 1 and 3), KRT85 (exon 3, intron 2) in CMXT using the polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods.
On the basis of the sequences of the KRT27, KRT31, KRT36, KRT38, KRT81, and KRT85 genes obtained from the National Center for Biotechnology Information GenBank database (GenBank accession numbers AC_000176, AC_ 000176, AC_000176, NC_00176, AC_000162, AC_000162, respectively), 17 pairs of PCR primers (P1-P17, Table 1) were designed to amplify different PCR products including exon3, exon8, intervening sequence 2 (IVS2), IVS5 of KRT27; IVS2 of KRT31; exon2, exon3 of KRT36; exon3, exon7, IVS2 of KRT38; exon1, exon2, IVS1, IVS3 of KRT81; and exon3, IVS2 of KRT85.
Where, [Y.sub.ijn] is the observed value of the trait, [mu] is the overall population mean, gI is the KRT36 gene effect, gII is the KRT38 gene effect, gIII is the KRT85 gene effect, [q.sub.j] is the group effect, and [e.sub.ijn] is the random error.
In this study, PCR-SSCP and DNA sequencing methods were used to identify polymorphisms in the sheep KRT27, KRT31, KRT36, KRT38, KRT81, and KRT85 genes.
In the P8 fragment of KRT36 gene, three unique SSCP banding patterns were observed (named CC, CD, and DD, Figure 1B).
Association of the KRT31, KRT36, KRT38, and KRT85 genes polymorphisms with wool traits in CMXT sheep
Therefore, the sheep KRT31, KRT36, KRT38, and KRT85 genes had significant effects on wool traits, suggesting these are potential candidate genes for wool traits in MAS.
Association analysis of combined genotypes of KRT36, KRT38, and KRT85 genes with wool traits in CMXT sheep
Based on the analysis of the combined genotypes of KRT36, KRT38, and KRT85, a total of 26 superior combined genotypes were found in the analyzed population, among which the combined genotype DD-GG-II was dominant.
In the present study, PCR-SSCP and genomic DNA sequencing methods were used to screen for genetic variation in different exons and their flanking regions within sheep KRT27, KRT31, KRT36, KRT38, KRT81, and KRT85 genes.