| Reproduction is one of important economic traits in sheep production,which can have a direct effect on economic benefit of sheep farm.Since litter size is an important index to evaluate reproductive performance of sheep,it is important to improve litter size for modern sheep production.The litter size is a quantitative trait controlled by minorgene.The selection of livestock through molecular markers linked to quantitative trait loci,and combined with traditional breeding methods,which will greatly improve the efficiency of selection and furtherly improve breeding performance of sheep.Therefore,the current study aimed to analyze gene expression and scan single nucleotide polymorphisms(SNPs)of candidate genes related to reproduction.On the one hand,the real-time PCR was conducted to investigate the expression patterns of 10 genes in heart,liver,spleen,lung,kidney,rumen,duodenum,longissimus dorsi muscle,fat,hypothalamus,pituitary,ovarian tissues of Hu sheep,including NGF,TrkA,KIT,KITLG,LIF,LIFR,NPM1,NCOA1,ADAMTS and NOGGIN.On the other hand,DNA pooling sequencing,restriction fragment length polymorphism(RFLP)technologies and SNPscan were carried out to screen SNPs in all exons and flanking region of above 10 genes,and the correlation of SNPs with litter size in 556 Hu sheep and 444 Small tail Han sheep.The study also analyzed the pyramiding effect of NGF and TrkA genes on litter size by the ways of quantitative genetics to provide a basis for developing polygene pyramiding breeding methods of litter size traits.The main results were as follows:(1)NGF,TrkA,KIT,KITLG,LIF,LIFR,NPM1,NCOA1,ADAMTS1 and NOGGIN m RNA were widely expressed in heart,liver,spleen,lung,kidney,rumen,duodenum,longissimus dorsi muscle,fat,hypothalamus,pituitary and ovary tissues of Hu sheep.The expression of NGF was high in ovary,hypothalamus and heart,while the expression was low in longissimus dorsi muscle,rumen,lung,liver and fat;The expression of TrkA gene was the highest in ovary and lung,and expression was low in longissimus dorsi muscle,rumen,fat,hypothalamus,pituitary gland,spleen,kidney and duodenum;The expression of KITLG gene in heart,ovarian and lung was high,while the expression in the liver,rumen,spleen and longissimus dorsi muscle was the low;The expression of KIT gene was high in the ovary,liver and lung and the expression at spleen,kidney,rumen,duodenum and fat tissue was low as well as the expression at longissimus dorsi muscle was the lowest;The expression of LIF gene was the highest in the ovary,followed by the hypothalamus,pituitary and liver,spleen,lung,kidney,rumen and duodenum intestines,while the expression was lowest in longissimus dorsi muscle and fat tissue;High expression of LIFR genes was in the ovary,heart,pituitary gland,kidney and lung,as well as the low expression at other tissues;NPM1 gene had the high expression in ovary and liver,while the expression was low in longissimus dorsi muscle,fat,pituitary and lung;NCOA1 gene expressed highly in the ovary,pituitary,liver and lung,but the expression levels were the lowest in the longissimus dorsi muscle.ADAMTS1 gene expressed highly in ovary,lung,liver and rumen,and the expression at the longissimus dorsi muscle was the lowest;NOGGIN gene had the highest expression in the ovary,spleen and lung,and showed the lowest expression level in the kidney and longissimus dorsi muscle.The results showed that 10 genes were highly expressed in the ovary,while the expression was low in longissimus dorsi muscle.The results indicated that above genes might be associated with growth and development of ovary in sheep.(2)DNA pooling sequencing was used to scan SNPs of all exons and related flanking region of each gene in this study.A total of 78 SNPs were found in 9 genes except to NOGGIN.3 SNPs were found at KIT gene;6 at KITLG,7 at ADAMTS1,6 SNPs at NCOA1,14 at NPM1,2 at LIF,23 at LIFR,4 at NGF,13 at TrkA.Of which,four were missense mutation,including g.70224398T>A at exon 10 of KIT(Leu→His),g.127756130G>A at exon 9 of ADAMTS1(Met→Val),g.35835474 G> T at exon 7(Ala→Ser),g.35835329 G>A at exon 12(Asp→Ser)of LIFR gene;eight were synonymous mutations,includig g.127751615C>T,g.127753565T>C at exon 2,and g.127753643C>T at exon 5 of ADAMTS1 gene,g.32072394C>T at exon 8 of NCOA1,g.91651197G>A at exon 1 of NGF,g.35835329G>A at exon 6 of LIFR,g.3247689A>T at exon 5 of NPM1;the remaining mutations were located in intron.(3)Restriction fragment length polymorphism(RFLP)technology was used to genotype the mutations of g.91651197G>A in NGF gene,g.105281586C>T and g.105284246G>C in TrkA gene as well as g.124502403C>T and g.124511398T>C in KITLG gene.The correlation analysis of SNPs with litter size of sheep was carried out.The results showed that g.91651197G>A of NGF,g.105281586C>T of TrkA gene and g.124511398T>C were significantly associated with litter size in Hu sheep and Small tail Han sheep(P<0.05),while g.105284246G>C of TrkA was significantly associated with litter size only in Hu sheep(P<0.05).The pyramiding effect of NGF and TrkA gene on litter size indicated that GACTCC genotype combination of the two genes was the optimum combination of genotypes.In conclusion,combination of NGF and TrkA gene affected the litter size of the sheep.(4)After evaluation of above 78 SNPs,62 SNPs out of them can be genotyped by SNPScan method.In Small tail Han sheep population,g.70199073A>G locus of KIT gene,g.35845474C>T,g.3584563T>C and g.35853637T>G of LIFR gene,g.32140565G>A of NCOA1 gene were significantly correlated with litter size(P<0.05).In Hu sheep population,litter size of different genotypes differed significantly(P<0.05)at g.3246266T>G of the NPM1 gene and g.31928230C>T of NCOA1 gene.50 different haplotypes were found at 10 genes.The highest frequency haplotype(F=0.89)was H8 at KITLG gene,which was the dominant haplotype.The lowest frequency of haplotypes was H22 at LIFR and its frequency was 0.0161.In addition,H3,H14,H17,H20,H23,H29,H47 and H48 were significantly related to litter size in sheep(P<0.05). |
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