Identification Of The Causative Mutation Underlying The QTL For Vertebral Numbers On Pig Chromosome7

The number of vertebrate is an economically important trait that affects carcass lengthand meat production in pigs. As a large-animal model for human medicine, deciphering themolecular basis of the number of vertebral variation on pig will benefit researches onspondylodynia in humans.To understand the genetic mechanism of phenotypic variation in pig vertebral number,researchers have detected two genome-wide significant QTLs on Sus scrofachromosomes(SSC)1and7. It has been shown that NR6A1c.134G>A is a promisingcandidate causal variation underlying the major QTL on SSC1. More recently, VRTN hasbeen proposed to be the candidate causative gene for the major QTL on SSC7,and9polymorphic sites within the VRTN gene are candidate causal mutations.In this study, we collected samples from western commercial population(n=608),erhualian(n=322) and "laiwu"(n=329) pigs. All these animals were recorded for vertebralnumbers. Genome-wide association studies using porcine60K SNP chips identified themost significant SNPs at the same area of SSC7. The P value, a SNP of MARC0038565inwestern commercial pigs, is1.08x10-52which is far stonger than the genome-widesignificant level of10-6.To dissect the genetic basis of the major locus, we further collected samples fromWhite Duroc×Chinese Erhualian intercross F2population(n=1029), sutai pig(n=435)andChinese Erhualian×tongcheng pigs(n=61) which are3experimental populations ofChinese and Western origins, These animals were genotyped using60K DNA chips.Genome-wide association studies consistently identified the locus across the3populationsand mapped the locus to a947-Kb region on SSC7. An identical-by-descent sharing assayrefined the locus to a100-Kb segment that harbors only two genes including VRTN andSYNDIG1L. Of them, VRTN has been proposed as a strong candidate of the major locus inWestern modern breeds.Further, we resequenced the VRTN gene using DNA samples of35parental animalswith known QTL genotypes by progeny testing. Concordance tests revealed4candidatecausal variants as their genotypes showed the perfect segregation with QTL genotypes ofthe tested animals. An integrative analysis of evolutional constraints and functionalelements supported two VRTN variants in a complete linkage disequilibrium phase as themost likely causal mutations. The promising variants significantly affect the number ofthoracic vertebrae (one vertebra) in large scale outbred animals, and are segregating at rather high frequencies in Western pigs and at relatively low frequencies in a number ofChinese breeds. Altogether, we show that VRTN variants are significantly associated withthe number of thoracic vertebrae in both Chinese and Western pigs.To verify the causality of the two candidate QTNs, We constructed four plasmidscontaining the two QTNs. We recombined the plasmid with the PGL4.20luciferasereporter vector and then transfected the vector into human embryonic kidney293T cells.We used the multifunctional Eliasa to detect the luciferase signal strength in the fourvectors, We found that the luciferase signal strength of homozygote mutant vector(VRTN-CC, ins/ins) is threefold higher than the others. It is thus supposed that Both of twosites have effect on the development of somite.The finding advances our understanding of the genetic architecture of the vertebralnumber in pigs. Furthermore, our finding is of economical importance as it provides arobust breeding tool for the improvement of vertebral number and meat production in bothChinese indigenous pigs and Western present-day commercial pigs, and also benefitresearches on spondylodynia in humans.