Genetic Variation Of MRF Family, MSTN, GHRH, And GHR Genes, And Their Associations With Growth Traits In Chinese Cattle

Genetic variations of MRF family, MSTN, GHRH and GHR genes were detected by PCR-RFLP and PCR-SSCP DNA marker techniques in 521 individuals of five bovine populations (Nanyang cattle, Qinchuan cattle, Jiaxian Red cattle, Chinese Holstein, and Yak), and association analysis were carried out to evaluate the effects of genotypes of polymorphic loci on growth traits of three Chinese cattle populations (Nanyang, Qinchuan and Jiaxian Red ). The objective was to detect the hereditary characteristics and to explore molecular markers with significant effects on economic important traits for efficient selection and improvement of Chinese cattle, and to provide genetic information for construction of molecular marker database, protection and utilization of breed resource of Chinese cattle. The results were as follows:1. Relationship between genetic variation of MSTN gene and growth traits in Nanyang, Qinchuan and Jiaxian Red cattle populations1.1 No polymorphism in MSTN exon 1 and 3 was detected using PCR-SSCP in the five populations. PCR-RFLP tests using restriction endonuclease TaqI were carried out in the 1346 bp fragment of bovine MSTN 5'regulatory region in the five populations. The results showed that no TaqI polymorphism in the five populations. Therefore, this study identified an occurrence of sequencing error in the publishing sequence (Accession No. AF348479) and confirmed the presence of nucleotide A at position -360 (relative to ATG start codon).1.2 PCR-RFLP tests using restriction endonuclease DraI were carried out in the 1346 bp fragment of bovine MSTN 5'regulatory region in the five populations. The results showed the polymorphic PCR-RFLP-DraI in MSTN 5'regulatory region was a bi-allelic loci with allele T and A. The allele frequencies of T/A of Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak populations were 0.955/0.045, 0.973/0.027, 0.972/0.028, 0.918/0.082, 1.000/0.000 respectively. There were three genotypes (TT, TA and AA) in this locus. Yak individuals were all genotype TT, that is, no mutation (T>A) was detected in Yak population. In Nanyang and Qinchuan cattle, individuals with genotype TT and TA were found and no individuals with genotype AA was detected, whereas in Jiaxian Red cattle only one homozygous AA individual was detected. Genotype TT and allele T were dominant in the five populations. The Chi-Square tests showed Nanyang, Qinchuan cattle and Chinese Holstein populations were at Hardy-Weinberg equilibrium, but Jiaxian Red cattle population was at Hardy-Weinberg disequilibrium. Genetic diversity indexes of h/Ne/S/PIC in Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak populations were 0.1505/1.1772/0.2835/0.1345, 0.0864/1.0946/0.1842/0.0826, 0.0545/1.0576/0.1279/0.0530, 0.0523/1.0552/0.1237/0.0501 and 0.0000/1.0000/0.0000/0.0000,so the population genetic polymorphisms from higher to lower were Chinese Holstein, Jiaxina Red, Nanyang, Qinchuan cattle and Yak.1.3 Associations between MSTN-DraI polymorphism and growth traits in Nanyang, Qinchuan and Jiaxian Red cattle were analyzed by least square means (LSM) method. The results showed that age had significant effects on body weight, withers height, body length, heart girth and hucklebone width in the Nanyang cattle (P<0.01), which indicated growth of Nanyang cattle differed significantly at different growth stages and directly correlated with age. Multiple comparison results showed MSTN-DraI genotypes affected hucklebone width of six-month Nanyang cattle and individuals with genotype TT had higher withers height than those with genotype TA (P<0.05).2. Genetic variations of MRF gene family and their associations with growth traits in Nanyang, Qinchuan and Jiaxian Red cattle populations2.1 Four polymorphic loci were detected in four genes (Myf5, MyoD, Myf6, MyoG) of bovine MRF gene family by PCR-RFLP and PCR-SSCP techniques.Three genotypes were detected by PCR-RFLP-TaqI technique in bovine Myf5-1 locus. This polymorphism was caused by a SNP (CAA/CGA) located in intron 2 at position 1948 bp of bovine Myf5 sequence (Accession No. M95684). The allele frequencies of A/B in Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak were 0.173/0.827, 0.242/0.758, 0.248/0.752, 0.262/0.738 and 0.031/0.969. Homozygous AA individual was not detected in Yak population. Allele B was dominant in five populations which were all at Hardy-Weinberg equilibrium. A SNP (A>T) located in bovine Myf5 exon 1 at position 609 bp (Accession No. M95684) was detected by PCR-SSCP and did not change the amino acid sequence. In Nanyang, Qinchuan, Jiaxian Red cattle and Yak population, there were three genotypes and allele A/B frequencies were 0.443/0.557, 0.475/0.525, 0.467/0.533 and 0.199/0.801. The heterozygous genotype frequency was low and allele frequencies of A and B were almost equal in Nanyang, Qinchuan and Jiaxian Red cattle populations. Genotype BB and allele B were dominant in Yak population. Nanyang, Qinchuan, Jiaxian Red cattle and Yak populations were all at Hardy-Weinberg disequilibrium.Two SNP (C>T) were detected in intron 2 of bovine MyoD gene and coding sequence of bovine Myf6 gene respectively. In Myf6 gene the SNP did not cause any change in amino acid sequence. The frequencies of allele A/B at MyoD-intron2 locus in Nanyang, Qinchuan, Jiaxian Red cattle and Chinese Holstein were 0.904/0.096, 0.882/0.118, 0.926/0.074 and 0.980/0.020. The frequencies of allele A/B at Myf6 locus in Nanyang, Qinchuan and Jiaxian Red cattle were 0.983/0.017, 0.995/0.005 and 0.997/0.023. There were two genotypes at the two loci respectively. Individuals of Yak population were all genotype AA, which indicated Yak population was homozygous at the MyoD-intron2 and Myf6 loci. No homozygous BB individual was found and homozygous AA individual was the majority in the others which were all at Hardy-Weinberg equilibrium. Genotype AA and allele A were dominant in the studied populations.Myf5-1-TaqI polymorphism and Myf5-2 SNP were (or closer to) moderate polymorphic and MyoD-intron2 and Myf6 loci were low-polymorphic in Nanyang, Qinchuan and Jiaxian Red cattle. The means of PIC of population at the four loci of Nanyang, Qinchuan and Jiaxian Red cattle were 0.2023, 0.2175 and 0.2123, therefore the genetic polymorphisms from higher to lower were Qinchuan, Jiaxina Red and Nanyang cattle.2.2 Association analysis between the polymorphisms and growth traits of Nanyang, Qinchuan and Jiaxian Red cattle populations were carried out. The results were as follows:For Myf5-1-TaqI polymorphism, eighteen-month Nanyang individuals with genotype AA had higher withers height compared to individuals with genotype AB and BB, and Jiaxian Red cattle with genotype AA had higher hucklebone width than those with genotype AB and BB, whereas Qinchuan cattle with genotype AA had lower withers height and height at hip cross than the other two kinds of individuals (P<0.05).The Myf5-2 SNP had significant effects on weithers height and body length of Nanyang cattle (P<0.05). Homozygous individuals of six-month and twelve-month Nanyang cattle with genotype BB had higher withers height than the other homozygous animals, and homozygous BB animals of six-month Nanyang cattle had higher body length than the other homozygous animals (P<0.05). Compared with homozygous animals in Qinchuan cattle, heterozygous individuals had higher hip width (P<0.05).For MyoD-intron2 SNP, except withers height and height at hip cross, body length, hucklebone width, rump length, heart girth and hip width of heterozygous Qinchuan animals were higher than homozygous animals (P<0.05). For Myf6 SNP, eighteen-month Nanyang cattle with genotype AA had higher hucklebone width when compared with heterozygous animals (P<0.05).3. Association of GHRH exon3-Haeâ…¢polymorphism with growth traits in Nanyang, Qinchuan and Jiaxian Red cattle populations3.1 The polymorphism in exon 1 of bovine GHRH gene was investigated by PCR-RFLP- Haeâ…¢in the five populations. The frequencies of allele A/B in Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak populations were 0.314/0.686, 0.259/0.741, 0.243/0.757, 0.265/0.735 and 1.000/0.000. Yak population was monomorphism in GHRH exon3-Haeâ…¢locus, all animals were genotype AA. Individuals with genotype AB and BB were the majority and allele B was dominant in Nanyang, Qinchuan, Jiaxian Red cattle and Chinese Holstein populations which were all at Hardy-Weinberg equilibrium. Genetic diversity indexes of h/Ne/S/PIC in Nanyang, Qinchuan, Jiaxian Red cattle and Chinese Holstein populations were 0.431/1.757/0.474/0.338, 0.384/1.623/0.559/0.310, 0.368/1.582/0.590/0.300 and 0.390/1.638/0.548/0.314. Since the population PICs at GHRH exon3-Haeâ…¢were intervenient of 0.25 and 0.50, GHRH exon3-Haeâ…¢locus was moderate polymorphic in the five populations. The genetic polymorphisms of five populations from higher to lower were Nanyang, Chinese Holstein, Qinchuan, Jiaxina Red and Yak.3.2 Association analysis and multiple comparisons were carried out between genotypes of GHRH exon3-Haeâ…¢locus and growth traits of Nanyang, Qinchuan, Jiaxian Red cattle. For six-month Nanyang cattle, except heart girth and hucklebone width, body weight, withers heights, body length and average daily gain differed significantly between genotype AB and BB animals (P<0.05). Twenty-four-month Nanyang animals with genotype BB had lower body weight and average daily gain compared with animals with other two genotypes (P<0.05). The effects of GHRH exon3-Haeâ…¢on body length and heart girth reached a significant level in Jiaxain Red cattle, animals with genotype BB were significant lower than those of heterozygous animals (P<0.05).4. Relationship between GHR exon 10 and growth traits in Nanyang, Qinchuan and Jiaxian Red cattle populations4.1 Characterization of the SNP (A>G) located in bovine GHR exon 10 was investigated in the five populations. Allele G/A frequencies of Nanyang, Qinchuan, Jiaxian Red cattle, Chinese Holstein and Yak populations were 0.443/0.557, 0.444/0.556, 0.495/0.505, 0.164/0.836 and 0.114/0.886 respectively. Animals with genotype GG were not detected in Yak population. Genotype AA and allele A were dominant in Chinese Holstein and Yak populations. Genotype frequencies were almost equal in Nanyang, Qinchuan and Jiaxian Red cattle populations. Nanyang, Qinchuan cattle and Yak populations were at Hardy-Weinberg equilibrium, whereas Jiaxian Red cattle and Chinese Holstein were at Hardy-Weinberg disequilibrium. In Nanyang, Qinchuan and Jiaxian Red cattle populations, the SNP was moderate polymorphic (0.25