Study Of Candidate Genes For Fecundity In Jining Grey Goat

Based on the results of major gene on sheep prolificacy,the relation of prolificacy major gene and prolificacy capability of Jining Grey goat were studied using biology informatics and molecule bio-technology.In the study,GDF9 and BMP15 was cloned and then detected for mutation.The results of the study would provide the theory basis for marker-assisted selection in high prolificacy of Jining Grey goat and prolificacy goat breeding.1.Cloning and sequence analysis of GDF9 gene in goatEight pairs of primers were designed based on GDF9 sequence of sheep to amplify fragment of GDF9 gene in goat genome,then cloned fragments were connected for complete GDF9 gene(Genbank: EF446168)after sequenced.The result of sequence analysis using biology informatics showed that it was 5508bp and was composed of two exons and a 1123bp intron in middle.One exon was 397bp,and the other was 965bp,which coded 453 amino acids.Compared GDF9 gene of goat to sheep and cow, the homology was respectively 97%and 94%,and the coding sequence homology was 99%and 96%, and the amino acid homology was 98.6%and 95.58%.2.The SNPs Analyzing in goat GDF9 gene exon by PCR-SSCPTen pairs of primers were designed according to goat GDF9 gene sequence(Genbank:EF446168) and the mutation in the GDF9 gene exon were detected using PCR-SSCP.The result showed three splice mutations in the coding sequence of GDF9(183bp C→A,719bp C→T,959bp G→A and 1189bp G→A).C183A mutation couldn't change amino acid.C719T mutation caused valine in 240 amino acid to turn into alanine.A959C mutation caused glutamine in 320 amino acid to turn into proline.G1189A mutation caused valine in 397 amino acid to turn into isoleucine.3.The GDF9 gene polymorphism Analysing using LDR and its effect on litter sizeFour mutation points were detected in Jining Grey goats(234),Lubei white goats(90)and Yimeng black goats(84)using LDR.The result throughx~2 analysis showed that the gene frequency of four points in all populations were in Hardy-Weinberg balance except for Lubei white goats in A959C point. Variance analysis for genotype frequency of four points in three goat varieties showed there was no difference for four points in Jining Grey goat and Yimeng black goat,but Jining Grey goat and Lubei white goat was in opposite(P＜0.01).There was obviously difference for Lubei white goat and Yimeng black goat in C183A,C719T and G1189A points except A959C.After the Least squares analysis in SAS software for genotype in four points and litter size,the result showed that there was no difference in C183A points for litter size for Jining Grey goat and Lubei white goat.There was different in C183A points for single litter Yimeng black goat(P＜0.05),that was CC(1.17149)＞AA(1.0897＞AC(1.0578)in C183A.there was no difference in C719T and A959C points for litter size for Jining Grey goat and Lubei white goat and Yimeng black goat.There was no difference in G1189A points for litter size for Jining Grey goat.There was different in G1189A points for Lubei white goat(P＜0.05),that was AG(2.6014)＞GG(2.4416)＞AA(2.1806).There was also difference in G1189A point to litter size of Yimeng black goat(P＜0.01),which was AA(1.4574)＞AG(1.1136)＞GG(1.0781)in genotype.4.Cloning and sequence analysis of BMP15 gene in goatNine pairs of primers were designed based on BMP15 sequence of sheep and cow to amplify fragment of BMP15 gene in goat genome,then cloned fragments were connected to complete goat BMP15 gene(Genbank:EU743938)after sequenced.The result of sequence analysis by biology informatics showed that it was 6648bp and was composed of two exons and a 5309bp introne in middle. One exon was 328bp,and the other was 851bp,which coded 394 amino acids.Compared BMP15 gene of goat to cow,the DNA homology was 90.727%.Compared to sheep and cow,coding sequence homology was 98%for both,and amino acid homology was 98%and 97%.5.the analysis of SNPs in goat BMP15 Exon by F-CSGE(fluorescence-based conformation sensitive gel electrophoresis)One pair primer was designed according to goat BMP15 gene sequence to cover the first exon,and two pair primers were designed to cover the second exon.SNPs of exon was searched using F-CSGE and DNA sequence technology.The result showed that one single nucleotide mutated(A→G)at 901 bp. The 301th amino acid changed from serine to glycine,and there was no other mutation in coding zone.6.The goat BMP15 gene polymorphism Analysing and its effect on litter size using LDRBMP15 gene mutation points were detected in Jining grey goats(234),Lubei white goats(90)and Yimeng black goats(84)using LDR.The three genotypes of BMP15 mutation were all detected in the three goat breeds.The result through x~2 analysis showed that the gene frequency of all populations were in Hardy-Weinberg balance except for Jining grey goats.G was the dominance allele in Jining Grey goats and Lubei white goats,but in Yimeng black goats A was dominance allele.By Least squares analysis in SAS software for genotype role in four points and litter size,the result showed that there was no difference in BMP15 mutation point for litter size for Jining grey goats.There was different for litter size for Lubei white goat(P＜0.05),that was GG(2.5621)＞AG(2.5350)＞AA(2.2257).So the A901G mutation in BMP15 gene was the dominant gene for Lubei white goats,and litter size was more with the G allel.The G allele could be the prolificacy molecular genetic marker. The result was the same to the Yimeng black goat.7.The goat FecB gene polymorphism Analysing and its effect on litter size using LDRFecB gene mutation points were detected in Jining grey goats(234),Lubei white goats(90)and Yimeng black goats(84)using LDR.Three genotypes of FecB mutation were detected in Jining grey goats and Lubei white goats,and A gene(wild type)was the dominance allele.The result throughx~2 analysis showed that the genotype frequency of Jining grey goats was in Hardy-Weinberg balance and Lubei white goat was not in Hardy-Weinberg balance.FecB mutation in Yimeng black goat was not detected.After Least squares analysis in SAS software for FecB genotype role and litter size,the result showed that there was no difference in FecB gene mutation point for litter size for Jining grey goats and Lubei white goats.Though there was not different for litter size for Lubei white goat,the litter size of GG(2.8656)and AG(2.537)were higher than AA(2.4341).Role value of genotype was GG(3.1593)＞AG(2.5634)＞AA(2.5061),and this was similar to Booroola mode.