Polymorphisms in the bovine CIDEC gene are associated with body measurement traits and meat quality traits in Qinchuan cattle.

Previous studies have shown that the cell death-inducing DFF45-like effector-C (CIDEC) gene is involved in lipid storage and energy metabolism, suggesting that it is a potential candidate gene that affects body measurement traits (BMTs) and meat quality traits (MQTs). The aim of this study was to identify polymorphisms of the bovine CIDEC gene and analyze their possible associations with BMTs and MQTs in 531 randomly selected Qinchuan cattle aged between 18 and 24 months. DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism were employed to detect CIDEC single nucleotide polymorphisms (SNPs). We found five SNPs: two in exon 5 (SNP1, g.9815G>A and SNP2, g.9924C>T) and three in the 3'-untranslated region (SNP3, g.13281C>T; SNP4, g.13297A>G; and SNP5, g.13307G>A). SNP1 was a missense mutation that resulted in an arginine to glutamine amino acid change, and exhibited two genotypes (GG and AG). SNP2 was a synonymous mutation that exhibited three genotypes (CC, CT, and TT). SNP3, 4, and 5 were completely linked, and only exhibited two genotypes (CC-AA-GG and CT-AG-GA). We found significant associations between these polymorphisms and BMTs and MQTs (P < 0.05); GG, CT, and CT-AG-GA appeared to be the most beneficial genotypes. Therefore, CIDEC may affect BMTs and MQTs in Qinchuan cattle, and could be used in marker-assisted selection.

Efficient construction of recombinant adenovirus expression vector of the Qinchuan cattle LYRM1 gene.

In this study, we cloned the coding DNA sequence (CDS) region of Qinchuan cattle LYR motif-containing 1 (LYRM1) and constructed a recombinant adenovirus expression vector to examine the function of LYRM1 on the cellular level. Total RNA was extracted from the adipose tissue of Qinchuan cattle, cDNA was obtained by reverse transcription, and polymerase chain reaction was used to amplify the CDS region of the LYRM1 gene. The CDS-containing fragment was inserted into the shuttle vector pAdTrack-CMV to construct pAdTrack-CMV-LYRM1 vector. After linearization of pAdTrack-CMV-LYRM1 and the negative control vector pAdTrack-CMV by restriction endonuclease PmeI, the vectors were transformed into Escherichia coli BJ5183 containing pAdEasy-1 to obtain the recombinant adenovirus vector pAd-LYRM1 and pAd-CMV through homologous recombination. pAd-LYRM1 and pAd-CMV were then digested by PacI and transfected into the 293A cell line. The recombinant adenovirus Ad-LYRM1 and Ad-CMV was obtained at a concentration of 7 x 108 and 1.3 x 109 green fluorescent units/mL, respectively. Preadipocytes derived from Qinchuan cattle were separately infected with Ad-LYRM1 and Ad- CMV. Quantitative real-time polymerase chain reaction demonstrated that the expression of LYRM1 was increased by approximate 28,000-folds after the infection with recombinant adenovirus for 48 h. In conclusion, we successfully cloned the CDS region of the Qinchuan cattle LYRM1 gene, constructed the recombinant adenovirus expression vector, and obtained the adenovirus with high titer, providing valuable materials for studying the function of LYRM1 at the cellular level.

Novel polymorphisms of the PRKAG2 gene and their association with body measurement and meat quality traits in Qinchuan cattle.

Body measurement and meat quality traits play important roles in the evaluation of productivity and economy in cattle, which are influenced by genes and environmental factors. PRKAG2, which encodes the γ2 regulatory subunit of AMPK, is associated with key metabolic pathways in muscle. We detected bovine PRKAG2 gene polymorphisms and analyzed their associations with body measurement and meat quality traits of cattle. DNA samples were taken from 578 Qinchuan cattle aged 18-24 months. DNA sequencing, polymerase chain reaction-restriction fragment length polymorphism, and time-of-flight mass spectrometry were used to detect PRKAG2 single nucleotide polymorphisms (SNPs). Sequence analysis revealed three SNPs in exon 3 (g.95925G>A, g.95973G>C, and g.95992A>G) and one g.96058T>C mutation in intron 3. g.95973G>C, g.95992A>G, and g.96058T>C each showed 3 genotypes: GG, GC, and CC; AA, AG, and GG; and TT, TC, and CC, respectively. In contrast, g.95925G>A only showed 2 genotypes, GG and GA. Analysis showed that g.95925G>A had no effects on body measurement and meat quality traits, whereas the other 3 polymorphisms were significantly associated with some of the body measurement and meat quality traits in the Qinchuan cattle population. It is inferred that the PRKAG2 gene can be used for marker-assisted selection to improve the body measurement and meat quality traits in the Qinchuan cattle population.

Effects of bovine SMO gene polymorphisms on the body measurement and meat quality traits of Qinchuan cattle.

Beef cattle breeding programs focus on improving important economic traits, including growth rates, and meat quantity and quality. Molecular marker-assisted selection based on genetic variation represents a potential method for breeding genetically improved livestock with better economic traits. Smoothened (SMO) protein is a signal transducer that contributes to the regulation of both osteogenesis and adipogenesis through the hedgehog pathway. In this study, we detected polymorphisms in the bovine SMO gene of Qinchuan cattle, and we analyzed their associations with body measurement traits (BMTs) and meat quality traits (MQTs). Using DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism, 3 novel single nucleotide polymorphisms were identified in the SMO gene of 562 cattle: 1 G > C mutation on exon 9 (G21234C) and 2 C > T mutations on exon 11 (C22424T and C22481T). Association analysis showed that polymorphisms on both the G21234C and C22424T loci significantly affected certain BMTs and MQTs (P < 0.05 or P < 0.01), whereas those on the C22481T locus did not (P > 0.05). Therefore, the SMO gene could be used as a candidate gene to alter BMTs and MQTs in Qinchuan cattle or for marker-assisted selection to breed cattle with superior BMTs and MQTs.

New polymorphisms in the novel LYRM1 gene are associated with body measurement and meat quality traits in Qinchuan cattle.

Body measurement and meat quality traits play important roles in the evaluation of productivity in cattle; they are influenced by genetic and environmental factors. Recent studies have shown that LYRM1 is a novel gene related to obesity and may influence fat deposition. We screened for new polymorphisms in the bovine LYRM1 gene and analyzed their association with body measurement and meat quality traits in cattle. DNA samples were obtained from 572 Qinchuan cattle aged from 18 to 24 months. DNA sequencing was used to find the LYRM1 single nucleotide polymorphisms (SNPs). Sequence analysis of LYRM1 revealed four novel SNPs in exon 3: G50A in coding region, C126A, A127T, and T128A in a 3'-untranslated region. G50A, A127T and T128A showed two genotypes: AG and GG, AA and AT, AT and TT, respectively; while C126A showed three genotypes: AA, AC and CC. Analysis showed that these four polymorphisms were significantly associated with body measurement and meat quality traits in the Qinchuan cattle population. We suggest that the LYRM1 gene can be used for marker-assisted selection to improve body measurement and meat quality traits in the Qinchuan cattle population.

Polymorphisms in the delta-like 2 homolog gene and their association with growth and meat-quality traits in Qinchuan cattle.

The delta-like 2 homolog (DLK2) modulates adipogenesis, hematopoiesis, osteogenesis, and other cell-differentiation processes. In the present study, we detected potential polymorphisms in the DLK2 gene in 604 individuals of Qinchuan cattle by using PCR-RFLP and DNA-sequencing methods. Herein, we identified five novel single-nucleotide polymorphisms (SNPs) (g.888G>A, g.910A>G, g.995G>A, g.4321A>G, g.4850A>G) and analyzed their association with measured traits. Four of the five analyzed polymorphisms were associated with at least one of the following traits: body weight (BW), chest depth (CD), chest circumference (CC), back fat thickness (BT), and rib-eye area (REA). To the best of our knowledge, our research is the first to report the association of DLK2 gene polymorphisms with growth and meat quality traits in Qinchuan cattle. In summary, the results of our study suggest that the DLK2 gene can be used as a candidate gene in beef cattle breeding.

SNPs at 3'-UTR of the bovine CDIPT gene associated with Qinchuan cattle meat quality traits.

The CDIPT is crucial to the fatty acid metabolic pathway, intracellular signal transduction and energy metabolism in eukaryotic cells. We detected three SNPs at 3'-untranslated regions (UTR), named 3'-UTR_108 A > G, 3'-UTR_448 G > A and 3'-UTR_477 C > G, of the CDIPT gene in 618 Qinchuan cattle using PCR-RFLP and DNA sequencing methods. At each of the three SNPs, we found three genotypes named as follows: AA, AB, BB (3'-UTR_108 A > G), CC, CD, DD (3'-UTR_448 G > A) and EE, EF, FF (3'-UTR_477 C > G.). Based on association analysis of these SNPs with ultrasound measurement traits, individuals of genotype BB had a significantly larger loin muscle area than genotype AA. Individuals of genotype CC had significantly thicker back fat than individuals of genotype DD. Individuals of genotype EE also had significantly thicker back fat than did individuals of genotype FF. We conclude that these SNPs of the CDIPT gene could be used as molecular markers for selecting and breeding beef cattle with superior body traits, depending on breeding goals.

Novel SNPs in the exon region of bovine DKK4 gene and their association with body measurement traits in Qinchuan cattle.

The aim of this study was to determine whether single nucleotide polymorphisms (SNPs) of bovine Dickkopf homolog 4 (DKK4) are associated with body measurement traits in Qinchuan cattle. By using PCR-SSCP technology and DNA sequencing, we discovered 5 DKK4 SNPs in Qingchuan cattle, including -65G>A and -77G>T in the 5'-untranslated region, 1532C>G and 1533T>C in exon 2, and 2088C>T in exon 3. The sequencing map showed that 1532C>G and 1533T>C were in close linkage disequilibrium and were treated as 1532C>G-1533T>C in this study. Allele frequencies were calculated and analyzed by the chi-square test, which showed that -65G>A and 1532C>G-1533T>C were in Hardy-Weinberg equilibrium (P > 0.05), whereas -77G>T and 2088C>T were not in all 633 tested Qinchuan cattle individuals (P < 0.01). Gene heterozygosity (HE), effective allele number (NE), and polymorphism information content (PIC) were 0.407, 1.686, and 0.324 at -65G>A; 0.472, 1.894, and 0.361 at -77G>T; 0.476, 1.908, and 0.363 at 1532C>G-1533T>C; and 0.218, 1.279, and 0.195 at 2088C>T. We also evaluated the potential association of these SNPs with body measurement traits in all 633 individuals; the results suggest that several SNPs in Qinchuan cattle DKK4 were significantly associated with body length, hip height, rump length, hip width, heart girth, and pin bone width (P < 0.05 and P < 0.01). These results suggest that bovine DKK4 could be used as candidate gene for Qinchuan cattle breeding.

A missense mutant of the PPAR-γ gene associated with carcass and meat quality traits in Chinese cattle breeds.

The peroxisome proliferator-activated receptor gamma (PPAR-γ) is a key molecule in adipocyte differentitation; it transactivates multiple target genes in lipid metabolic pathways. Using PCR-SSCP and DNA sequencing, we evaluated a potential association of an SNP (72472 G﹥T in exon7) of the bovine PPAR-γ gene with carcass and meat quality traits in 660 individuals from five Chinese indigenous cattle breeds, Qinchuan (QC), Luxi (LX), Nanyang (NY), Jiaxian (JX), and Xianan (XN). This 72472 G﹥T mutation identified a missense mutation, Q448H. Two alleles were named C and D. Allele frequencies of PPAR-γ-C/D in the five breeds were 0.7815/0.2185, 0.9/0.1, 0.7442/0.2558, 0.7051/0.2949, and 0.8333/0.1667 for QC, NY, JX, LX, and XN, respectively. Except for the XN breed, all breeds were in Hardy-Weinberg equilibrium at this locus. The polymorphism information content was low for NY and XN (0.16 and 0.24, respectively), while it was moderately high for QC, JX, and LX (0.28, 0.31 and 0.33, respectively). Correlation analysis showed significant association of this missense mutation with carcass length, backfat thickness and water holding capacity in the QC breed. Animals with the genotype CD had significantly greater carcass length than those with genotypes CC and DD, while animals with genotype CC had significantly greater backfat thickness than those with genotypes CD and DD. Animals with genotype CC had lower water holding capacity than those with the genotypes CD and DD. In conclusion, this locus is a candidate for a major quantitative trait locus affecting production traits and could be used for beef breeding selection.

Cloning, expression analysis and sequence prediction of the CCAAT/enhancer-binding protein alpha gene of Qinchuan cattle.

CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor, regulating the differentiation of adipocytes. We cloned the complete open reading frame of C/EBPα gene of Qinchuan cattle and analyzed its protein structures and expression profile in 15 tissues via DNA cloning, sequencing and RT-PCR. Analysis of the putative protein sequences revealed that C/EBPα consists of alpha helices, random coils and a few extended strands. A significant transmembrane structure was observed in amino acid region 233 to 252. A basic leucine zipper domain was also found in amino acid region 277 to 340, which is characteristic of C/EBPs. Homologous comparison with various species indicated that the C/EBPα gene of Qinchuan cattle shares 97, 95, 94, 94, and 93% similarity in amino acid sequences with Sus scrofa, Homo sapiens, Rattus norvegicus, Oryctolagus cuniculus, and Mus musculus, respectively, implying strong sequence conservation of C/EBPα during evolution. RT-PCR revealed that the mRNA expression level of bovine C/EBPα gene in subcutaneous fat is much higher than that in the other 14 tissues, and the relative quantity in fat tissue increases with cattle age.

Identification of ANAPC13 gene polymorphisms associated with body measurement traits in Bos taurus.

Body measurement traits, influenced by genes and environmental factors, play numerous important roles in the value assessment of productivity and economy. There has been some indication that ANAPC13 influences adult height. We used PCR-SSCP and DNA sequencing technology to identify polymorphisms in the ANAPC13 gene. A polymorphism in intron 1 (A > G at base 17) was identified and an additional polymorphic site (C > T at base 42) was also uncovered, which accompanied the previous polymorphism in more than 98% of the subjects. The two novel polymorphisms in exon 1 were assayed and potential associations with body measurement traits were evaluated in 404 individuals. Three genotypes were detected in the study group, named AACC, AGCT and GGTT. Significant differences were observed between genotypes AACC and AGCT for body length, withers height, hip height, hip width, heart girth, pin bone width. However, no associations were found among any genotypes and chest depth. We conclude that polymorphisms and mutations in non-coding regions of the ANAPC13 gene significantly affect body measurement traits.

Two-state stochastic models for memory in ion channels.

AIM: To study quantitatively the memory existing in ion channels. METHODS: Stochastic processes were used to model 2 categories of memory (short-term and long-term) by persisting in the standpoint of two-state, instead of multiple states, but with different transition mechanism. RESULTS: A two-state Markov process with constant transition intensities well fitted the short-term memory and a two-state Markov process within a kind of random environment well fitted the long-term memory. Statistical procedures for parameter estimation were proposed and demonstrated with 2 real examples on the channels of PC12 cells. CONCLUSION: The memory in ion channels can be quantitatively modelled as stochastic process with 2 states.