Molecular Characterization,polymorphism And Association Withlitter Size Of Porcine MLH1 And KLK7 Gene

Yunnan local pig breeds are rich and unique.These established the genetics foundation of Yunnan pig production.Low litter size is an important limited factor for the development of Yunnan pig production.Related researches have demonstrated that the molecular mechanism of pig litter size is sophisticated,and many genes and no-coding RNAs are involved in.At present the genetics mechanism research of low litter size of Yunnan local pig breeds is only limited to DNA level.Mut L homolog 1,colon cancer,nonpolyposis type 2(MLH1)and Kallikrein-related peptidase 7(KLK7)are two important reproduction related genes.The aim of this work was to clone the full-length porcine MLH1 and KLK7 m RNA sequences,search for polymorphism and perform association analyses with the litter size in the Large White and Landrace pig breeds.Results as below:1.Mut L homolog 1,colon cancer,nonpolyposis type 2(MLH1)is a reproduction related gene.In this study,the full-length c DNA sequence of porcine MLH1 gene was cloned through rapid amplification of c DNA ends(RACE)method.The porcine MLH1 gene encodes a protein of 757 amino acids which shares high homology with the MLH1 of five species: chiru(96%),sheep(95%),cattle(93%),southern-white-rhinoceros(92%)and goat(92%).This novel porcine gene was assigned to Gene ID:100337665.Phylogenetic analysis revealed that the porcine MLH1 gene has a closer genetic relationship with the MLH1 gene of southern-white-rhinoceros.PCR-Hha I-RFLP was established to detect GU373696:c.395 G>A substitution of porcine MLH1 gene and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus.Association of this SNP with litter size traits was assessed in Large White(n=200)and Landrace(n=200)pig populations,and result demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows(P<0.01).Our results suggest that MLH1 is not only a tumor related gene but also a reproduction related gene.This established the primary foundation for further insight into this novel pig gene.2.Kallikrein-related peptidase 7(KLK7)is an another reproduction related gene.In this study,we cloned the full-length coding sequence of porcine KLK7 gene through RT-PCR.Sequence analysis of this gene revealed that the pig KLK7 gene encodes a protein of 257 amino acids which has high homology with the KLK7 protein of six species:polar bear(95%),Weddell seal(94%),dog(92%),Pacific walrus(95%),domestic cat(92%)and Amur tiger(91%).This gene is structured in five exons and four introns as revealed by computer-assisted analysis.Phylogenetic analysis revealed that the pig KLK7 gene has a closer genetic relationship with the KLK7 gene of dog.Computer-assisted analysis indicated that porcine KLK7 gene is structured in five exons and four introns.PCR-Alu I-RFLP was established to detect the GU373714:c.390 C>T substitution of porcine KLK7 gene coding sequence and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus.Association of this SNP with litter size traits was assessed in Large White(n=200)and Landrace(n=200)pig populations,and results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows(P<0.05).This indicates that this polymorphic locus of porcine KLK7 gene is a valuable marker deserved to be applied to the marker assistant selection(MAS)in pig breeding.