| To identify the genetic characteristics and to explore usable molecular markers with significant effects on economic important traits for efficient selection and improvement of Qinchuan cattle, PCR-SSCP, PCR-RFLP, DNA sequencing and bioinformatics techniques were applied to detect genetic variations of lipid metaboloism related genes among 787 individuals of seven breeds (Qinchuan, Nanyang, Jiaxian Red, Xianan, Luxi, Angus and Anxi). 4 genes (leptin,MC4R,ApoA1,ApoA4) , 5' untranslation region and 3' region of MC4R and ApoA4 had been analyzed. 101 indivduals among Qinchuan cattle were slaughted and some economic traits, i.e. meat quality and carcass composition, were determinated. Association analyses were carried out to evaluate the effects of genotypes of candidate genes on economic traits of Qinchuan cattle. And molecular markers function was analysed by bioinformation. The objects of this study were to discover the hereditary characteristics and to explore molecular markers with significant effects on economic important traits for efficient selection and improvement of Qinchuan breed. Finally, possible functions of variations identified in these candidiate genes were predicted by bioinformational methods. Results were shown as follows:1. PCR-SSCP and DNA sequencing method were used to study genetic variations of leptin gene exon 2 and exon 3. A 73C>T mutation was identified in exon 2, this SNP was moderate polymorphic in the seven populations; Qinchuan, Luxi, Nanyang, Jiaxian Red were at Hardy-Weinberg equiliabration and Xianan, Angus, Anxi were at Hardy-Weinberg disequiliabration; this SNP was high significantly associated with live weight and backfat thickness (P<0.01), was significantly associated with loin muscle area (P<0.05); CC type and CT type have high live weight and backfat thickness than TT type (P<0.01), CC type has high live weight and backfat thickness than CT type and TT type (P<0.05). This mutation lead Arg change into Cys, which hadα-helix lost in 24 and 146 position,β-sheet lost in 20 and 144 position in 2D structure and had aβ-turn change in 3D structure. The hydrophobicity structure min value change from -2.222 (29th AA) to -1.689 (102nd AA), and the loose degree did not change. The LP2 fragment in exon3 showed AA and AB type, AA has a mutation of 96 C>T, AB has 252C>T, 267T>C and 351C>T mutations. These SNPs were low polymorphic in the seven populations. The seven populations were at Hardy-Weinberg equiliabration in these SNPs. These SNPs were no significantly associated with economic in Qinchuan cattle (P>0.05). The LP3 fragment in exon3 showed no SNP.2. Bioinformatics analysis showed MC4R protein had highly similary strcture in AA sequence in differenet species. The result of DNA pool sequencing showed that 5' region (-293C>G, -193A>T, -192T>G, -129A>G, -84T>C) and exon (1069 C>G) SNPs in cattle MC4R gene. The PCR-RFLP result showed that -293C>G and -129A>G are linkage. Qinchuan, Luxi, Nanyang, Xianan, Jiaxian Red, and Angus populations were moderate polymorphic and were at Hardy-Weinberg equiliabration at -129A>G SNP. Anxi was low polymorphic and was at Hardy-Weinberg disequiliabration at -129A>G SNP. The -129A>G SNP was significantly associated with live weight in Qinchuan cattle (P<0.05), GG type had high live weight than AA type and AG type. All the 7 populations were moderate polymorphic and were at Hardy-Weinberg equiliabration at 1069 C>G SNP. The 1069 C>G SNP was significantly associated with live weight, carcass weight, backfat thickness and marbling scorce in Qinchuan cattle (P<0.05). GG type had high live weight, carcass weight than CC type (P<0.05), GG and CG type had high backfat thickness than CC type (P<0.05), GG type had high marbling scorce than CC type (P<0.05). The 1069 C>G mutation lead Leu change into Val, which hadα-helix lost in 280 position andβ-sheet lost in 281 in Val in 2D structure. The 3D structure, hydrophobicity structure and the loose degree did not change.3. PCR-SSCP and DNA sequencing method were used to detect a 411 C>G in APOA1 gene. This SNP located in intron 2 and was moderate polymorphic in Qinchuan, Luxi, Xianan and Jiaxian Red populations, was low polymorphic in Nanyang, Angus and Anxi populations. Qinchuan cattle were at Hardy-Weinberg disequiliabration at this SNP and other 6 populations were at Hardy-Weinberg equiliabration. The SNP was not significantly associated with economic in Qinchuan cattle (P>0.05). Through 6 random samples sequcening and alignment, 1523 T>G and 1572 C>A mutations were detected. These two SNPs were all located in exon 3 and did not lead to AA change. The 1523 T>G SNP was moderate polymorphic in Luxi, Nanyang and Jiaxian Red populations, was low polymorphic in Qinchuan, Xianan, Angus and Anxi populations. Luxi, Nanyang and Jiaxian Red populations were at Hardy-Weinberg disequiliabration at this SNP and other 4 populations were at Hardy-Weinberg equiliabration. The 1523 T>G SNP was significantly associated with backfat thickness in Qinchuan cattle. GT type had high backfat thickness than TT type (P<0.05). The 1572 C>A SNP was moderate polymorphic in all the 6 populations except Anxi population. Qinchuan and Xianan populations were at Hardy-Weinberg disequiliabration at this SNP and other 5 populations were at Hardy-Weinberg equiliabration. The 1572 C>A SNP was significantly associated with backfat thickness in Qinchuan cattle. CC type had high backfat thickness than AC and AA type (P<0.05).4. cDNA sequences of ApoA4 gene, which contained complete CDS, were e-cloned and identified from fat of Qinchuan cattle. ApoA4 protein had no transmembrane, probably has signal peptide in 20th to 21st AA. The 2D and 3D structure has been prediceted by bioinformatics methods. 6 random samples sequcening and alignment method were used to detect SNPs, no SNP was been found in ApoA4 gene. |
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