Method For Identification Of The Thoracolumbar Number Of Ujumuqin Sheep And Genome-wide Association Analysis Of Multiple Spine Trait

Multiple spines are important morphological traits existing in the Mongolian sheep group.Animal spine includes neck,chest,waist,sacral and caudal vertebrae,but in multiple spine studies usually the chest and lumbar spine.The number of normal sheep spines is 19(T13L6),the number of multiple spinal sheep is 20(T13L7 or T14L6),and some reach 21(T14L7),where T represents the thoracic spine,L represents the lumbar spine and numbers indicate the number of vertebrae.The method of determining the individual spine number of sheep is often verified only by freehand touch or slaughter.The ability to determine the spine number of Ujumuqin sheep in the living state is of great value for the screening of multiple spine sheep and the selection of new strains.This test is the first to establish Fixed mount combined with digital X-ray photography(Digital Radiography,DR)imaging sheep spine detection method,and using this living detection method,in October 2020,576 adult sheep were determined and in2021,525 lambs still determined,the ratio of multiple spinal sheep in the sheep farm was determined.At the same time,3 lambs with 21 spines(T14L7)were selected,their semen was collected,and 265 ewes with different spinal phenotypes were artificially fertilized,and the spines of born lambs were tested in April 2021.The spine phenotype of offspring lambs was counted,and the number and proportion of offspring of different spine phenotypes between T14L7 ram and ewes of various spine types were preliminarily studied to determine the combination type of the intersection.A total of 96 sheep were selected from four spinal phenotypes,collected DNA for high-throughput sequencing,genome-wide association analysis,selected genes related to multi-spine traits;selected five single nucleotide(SNP)mutation genes in the analysis results,designed primers for PCR amplification and one-generation forward sequencing to verify the accuracy of high-throughput sequencing.Lay the foundation for revealing the mechanism of multiple spine formation.The results showed that there were 576 adult sheep in 2020,and had four spinal phenotypes.Multiple spinal sheep accounted for 95.7%.Among them,25 were T13L6,accounting for 4.3%,343 T13L7,accounting for 59.6%,177 T14L6,accounting for 30.7%;31 T14L7,accounting for 5.4%.In 2021 lambs were born in 225 lambs,the spine phenotype was still 4,accounting for 97.1%,15 T13L6,2.9%,282 T13L7,53.7%,190T14L6,36.2%,38 T14L7,7.2%.Of the 265 ewes tested in the artificial insemination test,There were 11 ewes with the T13L6 phenotype,Its offspring had two T13L6 spine phenotypes,For 18.2%,The rest are multispine,For 81.8%;There were 148 ewes with the T13L7 phenotype,Its offspring had 1 T13L6 spine phenotype,For 0.7%,Multiple spine phenotypes accounted for 99.3%;There were 94 ewes with the T14L6 phenotype,Its offspring had 1 T13L6 spine phenotype,For 1.1%,The rest were multispinal sheep,Accounting for 98.9%;There were12 ewes with the T14L7 phenotype,Its produced offspring without normal spinal sheep,all were multispinal sheep.So it is 100%.In artificial insemination ewes,multispinal phenotype sheep account for 99.7% of the population.The above four mating schemes produce a large proportion of multispinal sheep,which can be used to establish multispinal Ujumuqin sheep strains.The results of the genome-wide association analysis indicate that the following:When the Manhattan plot threshold is 4: comparing T13L7 and T13L6 group to get 9 significant SNP sites,When annotated to only the three known genes,The BRK1,FANCD20 S,and RBFOX1 genes were identified.Although the T14L6 and T13L6 comparison groups showed 174 significant SNP sites,they were not annotated for spine development.When the threshold was 3.5: 2562 significant SNP sites were obtained in the T13L7 and T13L6 comparison groups,annotated to the SATB2,SMAD9,and HOXB3 genes involved in cartilage development.Comparing the T14L6 and T13L6,6,447 significant SNP loci,annotated to LEO1,MEIS2,CDK6 genes involved in the regulation of bone marrow cell differentiation,and C2CD3,WDPCP,ROR2 genes involved in embryonic morphological development.When the threshold is 3: 10551 significant SNP sites are obtained in T13L7 and T13L6,annotated to ABL1,ITGAV,TMEM119 genes of osteocyte proliferation,and BRK1,ERCC2,FOXP1,RCN1,MGAT1 genes involved in embryonic development;In PCR sequencing-validated SNP site consensus trials with high-throughput sequencing,the validated site consensus rate averaged 98%.We may draw some conclusions:The established Baoding frame and DR imaging technology is highly accurate in detecting the sheep spine number.It can be applied to production practice;The basic strain of multiple spinal Ujumuqin sheep was initially established in the sheep farm,and the proportion of multiple spinal sheep reached more than 95%;T14L6,T13L7 ewes and T14L7 rams are more suitable for establishing stable lines of sheep.The results of genome-wide association analysis indicate that genes such as BRK1,ERCC2,FOXP1,RCN1,and MGAT1 involved in embryonic development may be involved in multispine formation.