Studies On Genes Related With Reproductive Traits In Goats

337 goats from 4 goat breeds (Xiangdong black goat, Nanjiang brown goat, Guizhou blackgoat, Boer goat) were analyzed to look for markers of genes related with reproductive traits by usingmethods of PCR-RFLP, Microsatellite, PCR-SSCP, directly sequencing and sequence analysis. Theresults were gained in the following several aspects:1 Studies on microsatellite markers related with fecundity traitsSelected microsatellite markers at OarAE101, BM143, BMS2508 locus linked with Fec~Bgene related with ovine fecundity traits and another microsatellite marker at OarHH35 locusinfluence on fecundity, the correlation between the producing lamb trait and genetic markerswere studied in Xiangdong black goat, genetic diversity among 4 goat breeds was investigatedby these markers. The results showed as following:1.1 The PIC of all 4 microsatellite locus reached high polymorphism (PIC＞0.73) level in 4goat breeds, and the level of genetic variation within population of Xiangdong black goat wasthe highest and that of Guizhou black goat was the lowest.1.2 The topology tree derived from single linkage cluster analysis showed that there werecommon origins between Xingdong black goat and Nanjiang brown goat.1.3 Related research results between the 4 microsatellite locus and the litter size ofXingdong black goat are as followed :(1) The markers of BMS2508 and OarHH35 from 4 microsatellites with the computationbased on General Linear Model were confirmed to be a major locus significantly associated withlitter size of Xiangdong black goat (P＜0.05), and that of BM143 and OarAE101 had not beendetected the significant influence (P＞0.05).(2) In the BMS2508 locus, there were the significant positive correlation for the allele 145bp , allele 93 bp loci with the litter size of Xiangdong black goat, there were the significantnegative correlation between allele 122 bp and the litter size of Xiangdong black goat, the leastsquares means of genotype 132 bp/145 bp, 93 bp/132 bp for the litter size was 4, 3.23 numberborn / litter respectively, but the least squares means of genotype 122 bp/122 bp for the littersize only had 1 number born / litter.(3) In the BM143 locus, the least squares means of genotype 100 bp/106 bp and 106 bp/112bp for the litter size were 3 number born/litter respectively, there were the significant positiveassociation between the allele 104 bp, 106 bp, 110 bp and the litter size of Xiangdong black goat.The least squares means of all genotype in BM143 locus for the litter size was 2.2451 numberborn/litter.(4) In the OarAE101 locus, the least squares means of genotype 107 bp/111 bp for the littersize was the best highest (4.0 number born/litter), the least squares means of genotype 109bp/109 bp, 107 bp/107 bp, 119 bp/119 bp, 111 bp/111 bp, 125 bp/125 bp for the litter sizewere higher, was 2.67, 2.5, 2.4, 2.33 and 2.25 number born/litter respectively, so the allele 107bp, 109 bp, 111 bp, 119 bp and 125 bp for the litter size in Xiangdong black goat had significantpositive correlation. (5) In the OarHH35 locus, the least squares means of genotype 135 bp/135 bp was thehighest (3.67 number born/litter), thereinto the least squares means of genotype 123 bp/135 bp,125 bp/125 bp had also reached 3 number born/litter, this indicated that allele 135 bp and 125 bpfor the litter size in Xinagdong black goat had significant positive correlation.(6) Carried on the fixed effect of 4 microsatellite locus were merged, the correlationbetween merge genotype and the litter size in Xiangdong black goat showed the least squaresmeans of the 28 merge genotypes had reached significant level (P＜0.05).1.4 Comparison of gene frequency among 4 goat breeds was discovered that gene frequencyof BMS2508-93 bp,OarAE101-119 bp in Xiangdong black goat were higher than the others,which had a positive correlation with litter size.1.5 Comparison of genotypes among 4 goat breeds, 19 kinds of genotypes that onlyappeared in Xiangdong black goat for the litter size and the least squares means of this genotypeswere over 2.1 number born/litter were found.2 Studies on genes related with controlling follicular growth2.1 Cloning and analyzing sequence of 5' transcriptional regulatory region offollicle-stimulating hormone receptor (FSHR) gene in goat(1) Sequence of 5' transcriptional regulatory region of FSHR gene in goat was cloned andsequenced at the first time, the sequence was accepted by GenBank of National Center forBiotechnology Information, National Institutes of Health in USA, the GenBank accessionnumbers was AY765375.(2) Its transcription promoter regions had the characteristic of TATA-promoter on the 5'transcriptional regulatory region of FSHR gene in goat .On the sequence there was a TATA-box,a TATA-like box, a few CAAT-like box and a GC-box, but there was no pyrimidine-rich near thetranscription initiation site.(3) Scanning the upper stream of 5' transcriptional regulatory region of FSHR gene, 4characteristic sequences that might have transcriptional regulatory function were searched, thatwas 2 3' HarfERE, 1 E-box, 1 GC-box.(4) The polymorphism of 5' flanking region studied using restriction endonuclease TaqⅠbyPCR-RFLP, the bases pattern of Xiangdong black goat at that locus was the allele B pattern,which results to the disappearance of the restriction site of TaqⅠ.2.2 Study on cloning and polymorphism of exon of growth differentiation factor 9B(GDF9B) gene(1) All sequence of exon 1 of GDF9B gene was sequenced in goat, two sequences wereaccepted by GenBank of National Center for Biotechnology Information, National Institutes ofHealth in USA, the GenBank accession numbers were AY917128 and AY968810 respectively.(2) Overwhelming majority sequence of exon 2 and partial estron sequence on GDF9 genein goat was sequenced, the sequence was accepted by GenBank of National Center forBiotechnology Information, National Institutes of Health in USA, the GenBank accessionnumbers was AY947812.(3) Homology of exon 1 sequence of GDF9B gene between Xiangdong black goat andAustrian sheep was 99.1%, and that of exon 2 sequence of GDF9B gene was 98.2%, thisindicated to have higher conservative inheritance of the exon sequence on GDF9B gene betweengoat and sheep.(4) Comparison of the same sequence with sheep, the base-variability of estron of GDF9Bgene was higher than that of exon of GDF9B gene in Xiangdong black goat. 2.3 SNP of Growth differentiation factor 9 (GDF9) gene and its relationship with the littersize in 4 goat breeds(1) Applying the powerful PCR-SSCP technique to analyze the polymorphism of GDF9gene in 4 goat breeds, the results were showed, fragments amplified by primer had threegenotypees (AA, AB and BB), mutation gene frequency in Xiangdong black goat was obviouslywanted to be higher than that in Guizhou black goat and Nanjiang brown goat, but that of Boergoat was closest to that of Xingdong black goat. x~2 fittness test showed that fragments amplifiedin Xingdong black goat and Nanjiang brown goat were in a state of Hardy-Weinberg equilibrium,but that in Guizhou black goat and Boer goat were not in a state of Hardy-weinberg equilibrium.(2) We cloned and sequenced fragments amplified of homozygosity (AA,BB), andcomparison with the same sequence in GenBank, it showed that there was one mutation C→A in233 bp of exon 1. We translated this mutual and normal sequences into amino acid, andcompared homology of the amino acid, it showed this base mutation did not cause the change ofthe amino acids.(3) The SAS software GLM process used, the least squares means of genotypes of GDF9gene for the litter size were analyzed, it showed that fragments amplified by primer had nosignificant effects for the litter size in 4 goat breeds (P＞0.05), but the least squares means of thegenotype effects between breeds had very difference.(4) Making use of the SAS software GLM process to consider fixed effect together gene ofGDF9 and breeds for the influence on the litter size, and the least squares means was analyzed,it indicated that fragments amplified between genotypes and the litter size by primer had moresignificant effects on the litter size (P＜0.0001), and that between breeds and the litter size byprimer had more significant effects on the litter size (P＜0.0001). Therefore it showed clearlyGDF9 gene for the influence on the number born/litter in goat was to rise the different functionwith the dissimilarity of the breeds.