Identification Of Ovine Kreatin Genes And Correlation On Wool Traits

In order to satisfy the international wool market demands,nowadays world sheep industry improve wool quality through breeding and improving existing sheep breeds.Improving sheep wool traits in molecular level can accelerate the breeding process.Gene Marker is used to screen and label the target genes.Keratin Associated Protein?KAP?and Keratin Intermediate Filament Protein?KIF?are the major structural proteins that make up wool fibers,these two protins are encoded by KRTs and KRTAPs,respectively.Studies have found that these variations and expression of the two keratin families are related to the presence of wool traits.Candidate gene methods can be used to identify potential genetic markers associated with wool traits,what includes measurement of wool yield?Yield?,mean staple length?MSL?,mean staple strength?MSS?,mean fiber diameter?MFD?,fiber diameter standard deviation?FDSD?,coefficient of variation of fiber diameter?CVFD?,coarse edge measurement?CEM: the percentage of fibers that are 10 microns greater than the average mean micron?,mean fiber curvature?MFC?,and prickle factor?PF: the percentage of fibers of diameter greater than 30 microns?,clean fleece weight?CFW?.At present,the research about these two gene families in sheep needs more supplements and improvements.The research content of this thesis focuses on five Keratin Associated Protein?KAP?genes?KRTAP6-1,KRTAP8-1,KRTAP15-1,KRTAP20-2,KRTAP27-1?and one Keratin Intermediate Filament?KIF?gene?KRT81?.The New Zealand Romney,Merino,and Merino × Southern Down were used as the research objects.The sequences of KRTAPs and KRTs of other species were used as reference.Genomic analysis,source search and evolutionary analysis were used to screen suspected genes for sheep wool traits,analyzing and identifying new sheep genes by gene amplification and sequencing;using PCR-SSCP and sequencing technology to screen new gene SNPs,comparing the differences of genetic variation among different taxa and analyzing the correlation between SNPs and wool traits.The main results are as follows:?1?Four alleles?A,B,E and F,there is no new alleles were found?were detected in 357 New Zealand Romney sheep KRTAP6-1.The presence of A was correlated with higher CVFD,CEM and MFD,sheep with AA was higher than AB and BB,?P<0.05?.If we want to improve CVFD,CEM and MFD of sheep,KRTAP6-1 can be used as a molecular marker gene.?2?Correlation analysis was conducted on KRTAP8-1gene polomorphisms and wool traits of 406 New Zealand Romney sheep.A total of 5 alleles?A-E?were found,and the sheep with genotype of AD and BD were significantly higher than sheep with genotype AA in GFW,CFW and MFD.Sheep with AB,AC?P<0.001?;Sheep with genotype of AD and BD were significantly lower than other genotypes in PF?P<0.05?.The results show that sheep KRTAP8-1 can be used as a potential molecular marker to increase hair production.?3?Four alleles?A-D?of KRTAP15-1 were detected in 573 sheep,and four SNPs were found in the coding region,which includs 3 non-synonymous variations.The presence of allele B was associated with decreasing in Yeild?P<0.05?,while the presence of allele C was associated with increasing in Yield?P<0.05?.The study found that the presence of allele B was associated with a decrease in sheep's wool yield,and this correlation was not affected when other variants in GLMM were corrected;C was also associated with lower FDSD?P<0.05?,and the MFD and PF of wool were decreased.The P values were 0.098 and 0.08,respectively.Sheep with genotype of AC had a higher wool yield than sheep with genotype AA or AB.This show that KRTAP15-1 can be used as a potential molecular marker to increase the wool yield.?4?Based on human KRTAP20-1?NM₁81615?as model gene sequence to design primers,assume the sheep KRTAP20-1 was subjected to PCR amplification.Polyacrylamide gel electrophoresis revealed eight alleles?A-H?.The amplified sequences were highly homologous to human KRTAP20-1,but different from other gene sequences,indicating that the amplified sequence is sheep KRTAP20-1.And located on the sheep chromosome 1,belonging to the high glycine-tyrosine protein?HGT-KAP?.A 12-bp insertion/deletion and 6 SNPs were found in the sequence analysis;the presence of allele A was associated with a decrease in MFD,FDSD,and PF?P<0.001?;The presence of C is associated with decreasing in GFW and Yield?P<0.05?.The variation of KRTAP20-1 affects wool Yield and MFD,which can be used as a molecular marker for character improvement of wool.?5?Primer was designed based on the human KRTAP27-1 coding sequence?AB₀96937?,and the putative KRTAP27-1 sequence was amplified in sheep.Four alleles were detected in 549 New Zealand Romney sheep?A-D?,a total of 6 genotypes?AA,AB,AC,BB,BC and BD?;sequencing results is highly homologous with human KRTAP27-1,then can determine the amplification sequence is sheep KRTAP27-1,which is located on sheep chromosome 1,belong to high-sulfur protein?HS-KAP?.Five single nucleotide polymorphisms?SNPs?were detected,of them,two SNPs were non-synonymous causing amino acid changes in P.Asp24 Glu and P.Ser148Pro at c.72C/A and c.442T/G.The presence of allele B was associated with an increase in GFW,CFW,and MSL?P<0.05?.The results show that KRTAP27-1 can be used as a potential molecular marker for sheep wool production.?6?Primer was designed based on the sequence of sheep KRT81?NM₀02281?.PCR-SSCP detection was performed in 402 New Zealand Romney sheep.Three alleles?A,B and C?and a 24-bp insertion/deletion found in the region.Correlation analysis with wool phenotypic traits showed that the presence of C was associated with increasing in GFW and CFW?P<0.05?;the presence of A had a tendency to decrease sheep GFW?P=0.061<0.2?;The presence of B was associated with decreasing in CFW of New Zealand Romney sheep?P<0.05?and decreasing in Yield?P=0.067<0.2?.The results indicate that sheep KRT81 can be used as a candidate gene for improving wool production.Conclusion: In this thesis,we identified two new members of the KAPs family,they were KRTAP20-1 and KRTAP27-1,respectively.Variations in sheep KRTAP6-1,KRTAP8-1,KRTAP15-1,KRTAP20-1,KRTAP27-1 and KRT81 were found to be associated with wool traits.These six sheep keratin genes can be used as candidate genes for molecular markers of wool traits.