Fine Mapping And Genetic Identification Of The Gene Encoding The Enterotoxigenic Escherichia Coli F41 Receptor

Enterotoxigenic Eschoerchia coli (ETEC) is a main pathogen causing diarrhea and edema in neonatal and post-weaned piglets. ETEC have five main serologic variants including F4、F41、F5、F6 and F18. ETEC F41 is one of the major serologic variants causing diarrhea in piglets by adhesion with the specific receptor of epithelial cells of small intestine, releasing enterotoxin and forcing large volume of electrolyte and water entering into gut cavity, resulting in diarrhea and edema.In a previous study, we performed a whole genome scan to detect QTL for ETEC F41 receptor using a large scale White Duroc×Erhualian intercross resource population with 183 microsatellite markers covering the entire genome. A major locus for ETEC F41 receptor was mapped to a 6.04-Mb region between SW2509 and S0301 on pig chromosome 4 (SSC4). Basing on the previous study, additional 7 microsatellite markers in the QTL region on chromosome 4 were genotyped across the three-generation pedigree comprising of 19 founders,68 F1 and 816 F2 animals. The ETEC F41 receptor locus was then refined to a region of 2.42 Mb between SW2509 and KVL2754 on SSC4. This result paved an important road to identify the ETEC F41 receptor gene.According to the human-pig comparative genomic map, there are 21 annotated genes in the refined region. Six positional candidate genes including ST3GAL1. TMEM71、ASAP1、NDRG1、ZFAT、KCNQ3 were subjected to expression analysis in the small intestinal tissues with semi-quantitative RT-PCR. The results indicated that all these genes were expressed in jejunum. According to the biological characteristics of the ETEC F41 receptor, ST3GAL1 was selected as one of candidate genes for further study. The 1,637-bp full-length cDNA of porcine ST3GLAL1 was isolated by 5’RACE and 3’RACE assays, which encodes a 343-amino acids protein. A total of 62 polymorphisms including 2 missense mutations (ST3GAL1c.284T>A and ST3GALIc.661A>G),11 synonymous variants and 49 intronic polymorphisms were identified by comparative sequencing using genomic DNA of four founder animals. Of the 62 polymorphisms, ST3GAL1 c.284T>A (in exon 1), c.477C>T (in exon 2) and c.661A>G (in exon 3) were genotyped on all animals of the White Duroc×Erhualian resource population. Transmission disequilibrium test showed that all alleles and haplotypes except for c.477C>T had strong association with susceptibility to ETEC F41 (P< 0.01). Furthermore, ST3GAL1 c.284T>A and c.661A>G were genotyped in 287 purebred outbred piglets from three Western commercial breeds and Chinese indigenous breeds. Case-control analysis showed that both the two polymorphisms were not associated with ETEC F41 adhesion phenotypes. The results indicated that different association of ST3GAL1 with ETEC F41 adhesion phenotypes in deferent populations was likely caused by population heterogeneity, alternatively, ST3GAL1 was not a major gene controlling diarrhea caused by ETEC F41 in pigs, it was only a locus having linkage disequilibrium with the resposible gene.To improve the mapping resolution, the White Duroc×Erhualian intercross were genotyped with Illumina porcine 60k DNA chips. Genome-wide association (GWAS) results showed that the most significantly linked locus was H3GA0011673 C>T (P= 1.09E-09) at 6167551 bp on SSC4 followed by ASGA0017715 T>C and ASGA0017733 C＞T. The three polymorphisms were genotyped on 287 outbred pigs. The case-control analysis indicated that ASGA0017733 C＞T was associated with ETEC F41 adhesion phenotypes of Western commercial pigs. The result suggested that ASGA0017733 C>T polymorphic locus probably located in the responsible gene or is an important marker which was in high linkage disqulibrium with the causal gene. These results provided good basis for positional cloning of the gene encoding the ETEC F41 receptor.The ASGA0017733 C>T polymorphism maps to intron 3 of the ZFAT gene. According to the properties of ETEC F41 receptor, ZFAT was selected as a positional candidate gene for furtherly analysis. The 4108-bp cDNA of ZFAT was isolated by RACE assay, which contains a 3393-bp open reading frame encoding a protein of 1130 amino acids. A total of 7 SNPs including one missense mutation and six synonymous variants (c.60C>T, c.630A>C, c.660T>C, c.1077T>C, c.1416T>C, c.2337T>C and c.2935G>C) were detected by comparative sequencing using cDNA of susceptible and resistant animals. All animals of outbred populations were genotyped for the seven ZFAT polymorphisms. Linkage disequilibrium (LD) analysis indicated that strong linkage disequilibrium existed among most of ZFAT polymorphisms. Moreover, LD was higher in Western commercial pigs than that in Chinese indigenous pigs. The results of association analysis showed that only c.2337T>C had significant (P< 0.05) association with susceptibility to ETEC F41 adhesion phenotypes in Chinese native pigs. In comparison, five polymorphisms except for c.630A>C and c.660T>C had association with susceptibility to ETEC F41 in Western commercial pigs (P< 0.05). These closely associated loci (c.60C>T, c.1077T>C, c.1416T>C, c.2337T>C and c.2935G>C) could benefit fine mapping of the gene encoding the ETEC F41 receptor.This study further convinced the major QTL for susceptability to ETEC F41 on pig SSC4 and refined the critical region to the SW2509-KVL2754 interval (5.56 Mb-7.98 Mb). The results shed new insights into the final identification of the ETEC F41 receptor genes and their casuative mutation(s). The novel informatic and significant markers of ST3GAL1 and ZFAT could be used to select disease resitance to ETEC F41 in pigs by marker assisted selection (MAS) schemes.