Study On Cloning, Polymorphisms And Expression Of Genes Related With Seasonal Reproduction Pathways In Goats

Increasing of annual fetuses will greatly benefit for promoting the production efficiency of goats.Jining Grey goat is a year-round estrus breed. The objectives of this study was firstly to detect theexpression pattern of genes related with reproductive seasonality in several tissues of goats, then toanalyze the association between polymorphisms of three candidate genes (TSHB, DIO2and RFRP) andreproductive seasonality in goats. The results will benefit for further understanding in molecularmechanism of seasonal reproduction and decreasing the reproductive seasonality of goats usingmarker-assisted selection.The expression patterns of genes related with reproductive seasonality in goats were studied in24tissues of goats using RT-PCR way. The expression level of these genes in hypothalamus, pituitary,ovary-uterus and/or pineal in Jining Grey goats and Liaoning Cashmere goats were detected usingTaqman probe and SYBR Green RT-qPCR methods. Then, the DNA sequences of the three candidategenes (TSHB, DIO2and RFRP) were cloned and their polymorphisms were detected in several goatbreeds with different reproductive seasonality. The main results were listed as follows:KiSS-1, RFRP, TSHB, AANAT, HIOMT and MT1gene were all mainly expressed in the tissues ofbrain. The expression tissues were exclusive and special for each of the eight genes, respectively, whichmay be consitent with their special function in goats. But DIO2gene was expressed generally in24tissues of two goat breeds studied, which may be predicted to have multiple roles in goats.The expression levels of KiSS-1, RFRP, TSHB, DIO2, AANAT and HIOMT were shown to besignificantly different between seasonal breed of Liaoning Cashmere goat and non-seasonal breed ofJining Grey goat for the important tissues related with goat reproduction. This indicated that these genesmay be responsible for the control of seasonal reproduction in goats. However, the genes DIO3andMT1were shown to have irregular expression patterns in tissues related with reproduction. Furtherstudies are needed to elucidate the functions of these genes in seasonal reproduction of goats.Two DNA fragments of goat TSHB gene were obtained, which were2614bp and1031bp in length,respectively. They comprised about2.5kb5' regulatory region, all of the three exons and two introns.Goat TSHB gene has a coding region of417bp, encoding138amino acids which was predicted to be asecreted protein with a signal peptide of16amino acids. The sequence of TSHB gene is highlyconservative in mammals. In addition, five mutations (C53A,3bp Indel at287-289locus,34bp Indel at584-617locus, A1819C and E2₇2TA) were found in goat TSHB gene and they were shown to be instrong linkage disequilibrium. Notablely, the significantly different genotype distributions existedbetween seasonal and nonseasonal goat breeds. The five closely linked mutations of goat TSHB genemay be associated with reproductive seasonality in goats.A1043bp DNA fragment of goat DIO2gene was obtained and it has high homology (96%) withcorresponding sequence of cattle DIO2gene. Additionally, two SNPs were found in the goat DIO2gene:A273G and A896G, which were located in the exon2and3' flanking sequence, respectively. TheA273G mutation gave rise to amino acid change (Gln103Arg) but with low mutative rate (<0.1) in six goat breeds tested, which should be a new polymorphic locus. For the A896G locus, significantgenotype distribution was shown between the non-seasonal breeds and two seasonal goat breeds exceptfor Liaoning Cashmere goat. And Boer goat was revealed to be the opposite with Saanen goat in bothallelic and genotypic frequencies as well as genotype distribution for the A896G locus. It needs furtherstudies to explore the function of DIO2gene in goats.A4862bp DNA fragment of goat RFRP gene was obtained and the complete CDS of591bpencodes196amino acids, having high homology with those of other mammals. The protein waspredicted to be a secreted protein with a signal peptide of21amino acids. Moreover, two mutations(A712G and T1493C) in5' regulatory region and one mutation (A3438T) in exon2were detected. Thetest of genotype distribution in thirteen goat breeds showed that there was no uniform significantassociation between the three polymorphisms and reproductive seasonality. Interestingly, however, therewas a strong positive correlation (r=0.830, P=0.003) between the G allele frequency of the A712Glocus and average daily sunshine duration in ten local goat breeds, suggesting that RFRP gene hasundergone a selective pressure in sunshine duration and may have indirect relationship withreproductive seasonality in goats. Additionally, no significant difference was found in litter size betweengenotypes for each locus in prolific Jining Grey goats.