Ssc15 On The Breeding Candidate Gene Isolation, Identification And Expression Of Differences In Study,

Reproduction is an important economic trait that has great effect on production of domestic animals. The objective of this study was to isolate, identify candidate genes for litter size traits on pig chromosome 15 by using methods of comparative genomics, electronic cloning, RH mapping, sequencing, association study between gene and litter size traits, and expression difference analysis. Expression differences of another 13 genes related to litter size traits were also detected in this study for verifying whether these genes affect litter size traits on mRNA level and elucidating the possible mechanism that they affect litter size traits. The main results are as follows:(1) Pig ESTs were identified through standard BLAST ananlysis by using mRNA sequences of human homologous genes on chromosome 2. Highly conserved pig ESTs were assembled to EST contigs. And primers selected from the contigs were used to isolate part sequences of 21 genes (ACVR1, ARHE, CED-6, CMYA3, COL4A4, DPP4, EPHA4, FLJ11457, GAD1, HAT1, HES6, HSPE1, KCNJ3, KLF7, NFE2L2, NR4A2, NRP2, ORC2L, SH3BP4, SP3 and TRIP12) from Erhualian, Dahuabai, Landrance and Duroc genomic DNA by sequencing. The obtained sequences showed more than 80% (from 82% to 96%) similarity to corresponding human mRNA sequences, and the total length of obtained sequences is 8943bp. The sequences obtained from sequencing were accepted by GenBank, the accession numbers were uninterrupted numbers from AY805665 to AY805748.(2) The INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was employed to determine the precise location of 21 genes isolated above. Statistical analysis revealed that all the 21 genes except ARHE were closely linked to genes or markers with LOD score threshold greater than 8 (threshold of significance) on pig chromosome 15, ARHE was located closely to marker SW2072, with LOD score threshold 6.61. The gene order of the 21 genes on the SSC15 framework RH map is conserved when compared with HSA2 (or MMU2 and MMU1) physical map but with a micro-inversion of DPP4-ACVR1- NR4A2-KCNJ3 as conventionally drawn. The chromosomal rearrangement between HSA2 and SSC15 is likely to extend to SSC15q11-15 and HSA2q11-23, one borderline of chromosomal rearrangement is centromere of HSA2, the other is located on HSA2q23.3 after ARHE gene. All genes except ARHE were mapped within QTLs characterized for ovulation rate, thereinto, KLF7, NRP2 and ORC2L were located closely to 51 cM position, EPHA4, COL4A4 and TRIP12 were proximal to 79 cM position. This improved SSC15 IMpRH map and the comparative analysis will be a great aid in the search for candidate genes underlying the QTLs.(3) Four breeds of pigs (Erhualian, Dahuabai, Landrance and Duroc) with different fertility were applied to screen potential polymorphisms related to reproduction trait based on DNA pooling and sequencing. Nineteen polymorphisms (including 13 SNPs and 6 indels) were found successfully in the fragment of 8943bp on SSC15 with the average rates of one polymorpgism per 470 bp and one SNP per 688 bp.(4) Genotyping 237 individuals from Erhualian, Dahuabai, Landrance and Duroc breeds for seven polymorphic sites of DPP4-1, SH3BP4-2, ORC2L, NRP2, COL4A4-2, COL4A4-3 and TRTP12 in 6 genes by PCR-RFLP were carried out for determining allele frequencies among different pig populations, and also for Linkage disequilibrium (LD) analysis and marker-trait association studies. LD analysis revealed two closely linked sites, ORC2L and NRP2, forming to haplotype block. The results of LD analysis suggested that QTN affecting pig fertility on SSC15 was adjacent to ORC2L and NRP2 or was themselves of ORC2L and NRP2.(5) Marker-trait association study showed that only NRP2 polymorphic site was associated with number of piglets born alive significantly (P=0.0198), and was associated with number of piglets born approaching significantly (P=0.0614). NRP2 was concluded as candidate gene for litter size traits based on the results of association studies, LD studies, allele frequencies studies and RH mapping.(6) K1 (CT) and H4 (IT) were the most advantageous haplotypes for litter size and K3 (TA) was the disadvantageous haplotype for litter size probably based on haplotype combination (K: consited of COL4A4-2 and COL4A4-3; H: consited of ORC2L and NRP2) analysis.(7) Pig ESTs were identified through standard BLAST ananlysis by using mRNA sequences (alternatively splicing: NM₂01266 and NM₀18534) of human NRP2 gene, and 4 and 5 EST contigs were obtained respectively for the two splicings when assembled these highly conserved pig ESTs, the obtained sequences were enough for selecting primers for isolating full cDNA sequence of pig NRP2.(8) Three alternatively splicings homologous to human NM₂01266, NM₀03872 and NM₂01267 were found in pig by using electronic cloning and sequencing.(9) Expression profiles of NRP2 in ruttish Erhualian and Duroc pig were analyzed by real time PCR. NRP2 gene is widely expressed in tissues of pituitary, ovary, uterus, oviduct, heart, liver, spleen, lung, kidney, skin, fat, muscle, brain, intestines and stomach, and expressed more in Erhualian than in Duroc taking all tissues together.(10) It was found greater expression of NRP2-1 and NRP2-2 in pituitary, ovary, uterus and oviduct of ruttish Erhualian than corresponding tissues of ruttish Duroc, which suggested enhancing expression of NRP2 was favorable to increasing litter size. But the difference of NRP2 expression was not significant between two breeds, which suggested the key phase that NRP2 affected litter size was not oestrum probably, or NRP2 could enlarge cell signals and finally affect litter size ascribe that it was the receptor of nerve axon guidance factors.(11) Twelve (IGF1, P450arom, ESR, FSHR, EGF, PAPP-A, DDP4, NRP2, GnRHR, INHA, PRLR, RBP4) of 14 genes detected for expression differences in ruttish Erhualian and Duroc were found expressing greater in Erhualian's uterus than in Durocs', especially RBP4 (relative quantitation=123.35) and ESR (relative quantitation=10.67) showed highly significant differences in two breeds, which suggested enhancing expression of RBP4 and ESR was favorable to early embryonic survival. These results also suggested high litter size of Erhualian was the results of integrated effect of many genes.