| Hulunbuir short-tailed sheep is one of the breeds of mutton sheep growing in Hulunbuir area of China,and the short and fleshy tail is the unique symbol of this breed.In recent years,with the improvement of people's living standard and the concern of cardiovascular disease,the fat of sheep tail has been neglected gradually,and breeding of short fat tail sheep has become the current breeding trend.The Hulunbuir short-tailed sheep has been widely concerned because of its short tail phenotype.Based on the short tail form of the sheep,the causes of the short tail phenotype were studied from the aspects of morphology,genome,molecular biology and protein function.We think that for the Hulunbuir short-tailed sheep,the structure of the tail vertebrae is one of the reasons for this short tail phenotype.For this reason,we first analyze the causes by observing the tail characteristics and the tail vertebrae,and then make a comparison between the Barag sheep with the same living environment and the other phenotype except the tail phenotype,and then select the candidate genes related to the short tail phenotype.After obtaining the candidate genes,the related verification experiments were carried out to investigate the formation mechanism of the sheep short tail phenotype.The results are as follows:1.Through direct observation,it is found that there are three types of tail types in the Hulunbuir short-tailed sheep population with different lengths,however,the length of three types of tail was shorter than that of other sheep;By taking X-rays and making specimens of tail vertebrae,it was found that the tail of Hulunbuir short-tailed sheep was smaller in bone size and quantity than the tail of normal sheep,and there were deformed tail vertebrae in Hulunbuir short-tailed sheep.2.Barag sheep,which resemble Hulunbuir short-tailed sheep but have a normal tail morphology,served as controls,to carry out genome resequencing analysis.A total of 72,101,346 SNP loci were obtained after comparison with the reference genome.The selective sweep of population heterozygosity(Hp)and genetic fixation indext(Fst)was used to find seven candidate genomic regions covering a genomic length of 940 kb and contained nine genes.It was found that there were T gene associated with spinal development,and the c.G334 T mutation of T gene was validated.3.Functional study of transcription factor Brachyury protein encoded by T gene.Prokaryotic expression obtained transcription factor Brachyury protein and mutant protein,and its binding DNA fragment was used as a probe to detect protein function.It is found that T gene c.G334 T mutation can cause the change of amino acid,which leads to the change of the structure of Brachyury T domain,which affects the transcription efficiency of transcription factor Brachyury.4.Aiming at the c.G334 T mutation of T gene in sheep,the sg RNA of covering mutation sites and ss DNA for homologous repair were designed,and the mouse model of the same mutation was made by using CRISPR/Cas9.The results showed that the new T gene c.G334 T mutation could lead to short tail phenotype in mice,and there were different lengths in the tail,which was the same as that in Hunlunbuir short-tailed sheep population.In this study,the molecular mechanism of the short tail phenotype of Hunlunbuir short-tailed sheep was studied,and a new c.G334 T mutation was found in the T gene related to development of the spine,and the mutation played a key role in the formation of the short tail phenotype.This will lay a foundation for the study of the formation mechanism of sheep short tail phenotype and the short tail phenotype of other animals and provide a theoretical basis for the molecular breeding of short fat tail sheep in the future. |
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