Zhou 2016

Introduction to KAP6 Gene Family

• KAP6 is part of the high glycine-tyrosine keratin-associated protein (HGT-KAP) family.

• This family is believed to encompass multiple genes, and the study identifies new members in sheep.

Identification of New KAP6 Genes

• A KRTAP6 coding sequence was utilized to search the Ovine Genome (v3.1).

• Five homologous regions (R1–R5) were detected, each containing an open reading frame (ORF).

• Regions R1–R5 showed high similarity to sheep skin Expressed Sequence Tags (ESTs).

• Phylogenetic analysis classified R1–R5 with KAP6 sequences from various species, separate from other HGT-KAPs.

Characteristics of Regions

• R1: Similar to characterized KRTAP6-1.

• R2–R5: New sequences, distinct from known KAPs.

• PCR primers confirmed the presence of these genes, producing amplicons in 96 sheep.

• SNP Variations:

  • KRTAP6-2: 5 SNPs observed.

  • KRTAP6-3: 3 SNPs and a 45-bp insertion/deletion detected.

  • KRTAP6-4: 3 SNPs.

  • KRTAP6-5: 5 SNPs and an 18-bp insertion/deletion.

• Allele frequencies varied significantly between Merino and Romney breeds.

Role of Keratin-Associated Proteins (KAPs)

• KAPs are crucial for wool and hair fibre structures, forming a semi-rigid matrix for keratin filaments.

• Compositions mainly consist of cysteine, glycine, and tyrosine.

• KAPs are categorized into three groups:

  • High Sulfur (HS) KAPs

  • Ultra-High Sulfur (UHS) KAPs

  • High Glycine-Tyrosine (HGT) KAPs

• Presence in wool varies, influencing fibre properties and characteristics.

Variation and Polymorphism in KAP6 Genes

• Study aimed to explore variations in newly identified KAP6 genes.

• Four KAP6 genes identified showed polymorphism across different breed samples.

• KRTAP6 genes established have similar coding sequences but differ significantly in flanking regions, impacting gene expression.

Phylogenetic Analysis and Comparisons

• R1–R5 genes clustered closely with known KAP6 sequences from various species.

• Suggested that KAP6 gene family is more diverse in sheep than in other species, such as humans (three members).

Experimental Validation

• PCR experiments confirmed the presence of four new KAP6 genes using region-specific primers.

• SSCP analysis identified multiple allele patterns, showing variant sequences:

  • R2: six alleles with five SNPs (all synonymous).

  • R3: five alleles, including a 45-bp insertion/deletion.

  • R4: three alleles, one non-synonymous SNP.

  • R5: identified six alleles with specific insertions/deletions.

Genetic Variation Between Breeds

• Significant genetic variance identified between NZ Romney and Merino sheep

• KRTAP6-3: allele B prevalent in Merino, absent in NZ Romney.

• KRTAP6-4: allele C common in NZ Romney, rare in Merino.

• KRTAP6-5: allele D found more frequently in Merino than NZ Romney.

Discussion and Implications

• Discovery of four new KAP6 genes increases known family members from one to five.

• This supports previous studies indicating multiple KAP6 genes exist in sheep.

• Identified genes may link to wool traits and potential genetic markers for breeding.

• More extensive studies needed to define gene impact on wool characteristics.

Methodology Overview

• Research adhered to Animal Welfare legislation for sheep blood collection.

• 96 sheep samples analyzed (48 Merino, 48 NZ Romney).

• Genomic DNA isolated and analyzed through established protocols, including PCR amplification and SSCP for heterozygote detection.

• Allelic variants sequenced for genetic variability assessment.

Conclusion

• Revealed a substantial diversity in KAP6 genes with implications for understanding wool genetics and improvement through breeding strategies.