Polymorphisms In Sheep DSG4 Gene And Their Associations With Lengths And Numbers Of Crimps Of Wool Plaits

Desmoglein 4 gene(DSG4) was discovered in human and mice in 2003.The encoded protein DSG4 is a key mediator of keratinocyte cell adhesion in the hair follicle,where it coordinates the transition from proliferation to differentiation.Since its discovery,a number of mutaions in DSG4 gene have been found to induce hypotrichosis in human, mice and rats,suggesting it may be a potential candidate gene in the search for genes which affect wool traits in sheep.The purpose of this study was to screen for polymorphisms in the sheep DSG4 gene,and to explore its possible association with wool traits in fur sheep breeds.By PCR-SSCP method,ten pairs of PCR primers were designed to amplify and screen partial 5'UTR and 3'UTR,complete exon 1,exon 2,exon 4,exon 6,exon 7,nearly complete exon 3,exon 5,exon 16 and partial intron regions adjacent to those exons of DSG4 gene in 248 fur sheep samples including Tan,Han Small-tailed and their crossbreds. As a result,nine pairs of primers of SSCP analysis showed polymorphisms;thereby 22 alleles were identified and sequenced to confirm mutations.We identified 20 SNPs and a TTG ins/del in sheep DSG4 gene,in which one each SNP locates at 5'UTR and 3'UTR,13 SNPs at coding sequence with five of them leading to amino acid substitutions,and five SNPs at introns.The TTG ins/del that locates at exon 16 leads to an amino acid substitution Ser/Iso and also a Gly ins/del.Interestingly,five SNPs and TTG ins/del at exon 16 and one SNP at 3'UTR were in complete linkage disequilibrium in these sheep populations,forming two alleles which were defined as X and Y.Moreover,ten coding haplotypes were then constructed based on the 13 coding polymorphisms.Using SAS GLM procedure,the effects of polymorphisms on the lengths and numbers of crimps of wool plaits in Tan sheep and their crossbreds were analyzed.The results indicated that polymorphisms at exon 16 and 3'UTR had a significant effect on wool traits(P＜0.01).In the crossbreds,homozygous individuals YY genotype had a significantly higher least squares mean than genotypes XX or XY on lengths of scapular wool plaits at birth(P＜0.01),numbers of crimps of scapular wool plaits at birth(P＜0.01), numbers of crimps of scapular wool plaits at weaning(P＜0.01 ).Genotype YY also had significantly higher numbers of crimps of rump wool plaits at weaning than genotype XX (P＜0.05),and also higher than genotype XY(P＞0.05).However.as for Tan sheep,there were no significant differences between these three genotypes on all the above traits, though genotype YY had a slightly higher but not significant least squares mean than genotype XX or XY(P＞0.05).It is worth to notice that the rich variations at DSG4 gene may offer opportunities for the development of genetic markers associated with wool traits in other sheep breeds.A total of 457 sheep samples including seven local Chinese breeds,Suffolk and Polled Dorset were scanned for C ins/del that locates at 5'UTR by PCR-RFLP using the restriction enzyme EcoO109 I.The frequency of allele and homozygotes of the C deletion in all the nine breeds was very low.The bioinformatics analysis indicated that the C ins/del caused changes in the numbers and kinds of transcription factors binding to the 5'UTR, suggesting the C deletion likely to have some effect on the expression of DSG4 gene in the hair follicle of sheep.In addition,a total of 787 sheep samples including eight local Chinese breeds,Suffolk and Polled Dorset,four breeds each from Iran and Pakistan were scanned for alleles X and Y.As a result,all the variants were still in complete linkage disequilibrium in all 18 sheep breeds,which demonstrated that exon 16 could be very conservative and therefore alleles X and Y may have originated from different ancestral populations.Interestingly,alleles X and Y were found in all breeds from China and Iran and three breeds from Pakistan, whereas allele Y was not present in Suffolk,Polled Dorset and only one of the Pakistan breeds.Based on the difference in distribution of mitochondrial DNA lineages among Asian and European sheep(e.g.lineage C is extensively present in Asian sheep),we postulate that the allele Y of DSG4 gene probably originated from the ancestral populations of lineage C.