Identification Of New Ovine KRTAP Family Genes And Their Effect On Wool Traits

Wool keratin-associated proteins(KAPs)are a structural component of the wool fibre,which plays a role in defining the properties of the wool fibre.The KRTAP family genes encode these proteins.In humans 88 functional KRTAP family genes have been identified to date,while in sheep the number is only 27.This research is taken Merino × Southdown lambs and New Zealand(NZ)Romney lambs as the research object,KRTAP family genes that already identified from other species as reference,genomic analysis,homology search and evolutionary analysis will be used to preliminary screening of suspected genes from ovine genome database.Then amplify sequences of suspected genes and determine whether they are new KRTAP family genes or not.PCR-SSCP and sequencing method will be used for detecting SNPs.Comparison of genetic variation differences among two types of wool sheep populations.Then association analysis will be conducted between SNPs and wool traits to reveal the possible genetic contribution of KRTAP family genes on the wool growth,and to determine the feasibility of new genes as molecular marker candidate gene.1.There were total of five new members of KRTAPs were identified from sheep: KRTAP22-1,KRTAP22-2,KRTAP21-1,KRTAP21-2 and KRTAP26-1.These new genes are located on the oving chromosome 1,and between KRTAP8-2 and KRTAP24-1.2.PCR-SSCP was used to screen for variation in KRTAP22-1 in 390 Merino × Southdown-cross lambs and 75 New Zealand(NZ)Romney sheep.Three PCR-SSCP banding patterns were detected and DNA sequencing revealed that the banding patterns represented three different nucleotide sequences(A–C).Two single nucleotide polymorphisms(SNPs)were identified in these sequences.Variant B was most common with a frequency of 81.3% in NZ Romney sheep,while in the Merino × Southdown-cross lambs A was more common with a frequency of 51.8%.The presence of B was found to be associated with increased wool yield and decreased mean fiber curvature(MFC).Sheep of genotype BB or AB had a higher wool yield than those of genotype AA.These results suggest that ovine KRTAP22-1 variation may be useful when developing breeding programs based on increasing wool yield,or decreasing wool curvature.3.No mutations in ovine KRTAP22-2 were detected in the Merino × Southdown-cross lambs and New Zealand Romney lambs.Three alleles were detected in caprine KRTAP22-2 on four goat breeds(Chaida Black goats,the Ziwuling Black goat,the Hexi Cashmere goat and the Inner Mongolia cashmere goat).Three SNPs and a 6bp deletion were identified between these sequences.One of SNPs was a non-synonymous mutation,which resulting in a mutation between arginine and glycine.C has a 6 bp insert,and an addition of 2 amino acids(arginine and cysteine).AA and AB are dominant genotypes.A is the dominant allele in these goat breeds.The big difference on gene variation in KRTAP22-2 may result from the different selection on the gene between the two species.4.In the 363 Merino × Southdown-cross lambs,seven PCR-SSCP banding patterns were detected in the KRTAP21-1 and DNA sequencing revealed that the banding patterns represented six different nucleotide sequences(A-F).Six SNPs were identified between these sequences,and three of them were non-synonymous.In the 363 samples,the correlation analysis only conducted between AA and AC,which were the most common genotypes.The results showed that,only Yield was slightly different but not significant between AA and AC.The effect of other alleles on wool traits remains to be further studied.5.In the 389 Merino × Southdown-cross lambs,five PCR-SSCP banding patterns were detected in the KRTAP21-2 and DNA sequencing revealed that the banding patterns represented five different nucleotide sequences(A-E).Four SNPs were identified between these sequences,and one of them was a non-synonymous mutation.The gene variation of KRTAP21-2 gene had effect on the MSL,FDSD and PF.Lambs with genotype AC had larger MSL than that of CE(P<0.05).Lambs with genotype CE had higher FDSD(P<0.05)than Lambs with genotype CC and BC.The genotype of PF was higher than that of CC and BC(P<0.05).In the ‘single-variant' model,the presence of allele E would result a decrease in MSL.In the ‘multi-variant' model analysis,the effect of allele E on MSL persisted(P <0.05).The SNPs of allele E was located coding region,and the presence of the allele E would reduce the MSL of the wool.The biggest effect on this gene on the wool traits was MSL.When developing breeding programs based on increasing MSL,KRTAP21-2 gene can be used as a molecular marker gene.6.Four PCR-SSCP banding patterns were detected in KRTAP26-1.DNA sequencing revealed that these patterns represented four nucleotide sequences(A-D).Seven single nucleotide polymorphisms(SNPs)were identified between these sequences.All of these SNPs were located in the coding region,and two of them were non-synonymous substitutions.The frequency of the variants in 383 Merino × Southdown-cross sheep and 94 NZ Romney sheep were different,which in the former breed were: A: 49.6%,B: 25.7%,C: 23.0% and D: 1.7%,while in the another sheep breed were: A: 40.4%,B: 47.3%,C: 0.5% and D: 11.7%.The association of variation in KRTAP26-1 with variation in wool traits was investigated in 383 MerinoSouthdown cross sheep.The effects of D on wool traits were not investigated as it was found at a very low frequency.A,B and C were found to be associated with a number of wool traits,but the strongest associations were with four traits: MFD,FDSD,MSL and PF.The presence of B was associated with an increase in MFD,mean fibre curvature(MFC)and PF.The presence of C was found to be associated with an increase in wool Yield and MSL;and a decrease in MFD,FDSD and PF.Sheep of genotypes AB and BB were of higher MFD,than those of genotypes AC and BC(P < 0.01).Genotypes AA,AB and BB had a higher FDSD than AC and BC(P< 0.05).Genotypes AC and BC had MSL over 6% higher than AB and BB(P < 0.05).In terms of PF,AB and BB had higher PF compared to AC and BC(P < 0.01).These results may be useful in the future development of breeding programs based on decreasing wool diameter and diameter standard deviation,or increasing wool length.7.In the 383 Merino × Southdown-cross lambs and 48 NZ Romney lambs,seven PCR-SSCP banding patterns were detected in the KRTAP6-3 and DNA sequencing revealed that the banding patterns represented seven different nucleotide sequences(A-G).Six SNPs and two 45 bp deletion were identified between these sequences.Sheep with genotype AB had the highest MFD,FDSD and PF than those of the genotype AA and AG(P < 0.001).Sheep with genotype AA had higher MFD,FDSD and PF than those of genotype AG(P < 0.001).The MFD,FDSD and PF of sheep with G decreased significantly,and the reduction of the three traits was in line with the wool market demand.Therefore,KRTAP6-3 can be used as a molecular marker gene when taken above three traits as breeding object.In summary,the variations of ovine KRTAP22-1,KRTAP21-2,KRTAP26-1 and KRTAP6-3 were association with wool traits and had the potential as a candidate gene for molecular markers of wool traits.