[Clinicopathological analysis of gastric adenocarcinoma with elevated serum alpha-fetoprotein and enteroblastic differentiation].

Objective: To investigate the immunophenotypic and molecular biological characteristics of patients with elevated serum alpha-fetoprotein (AFP) and enteroblastic differentiated gastric adenocarcinoma (GAED). Methods: The clinicopathological data of 13 patients with elevated serum AFP and GAED admitted to Shanxi Cancer Hospital from 2018 to 2020 were collected. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were used to analyze the immune markers and molecular biological characteristics of the pathological tissues of the patients. Kaplan-Meier method and log rank test were used for survival analysis. Results: Among the 13 patients with GAED, 12 were male and 1 was female, aged 41-70 years, with a median age of 64 years. The lesions were mainly located in the gastric antrum (5 cases) and gastric body (4 cases). IHC results showed that the tumor embryonic protein (AFP, SALL4, GPC3), intestinal epithelial differentiation protein (CDX-2, CD10), and some original intestinal epithelial phenotype markers (OCT3/4, Claudin6) were expressed in the tumor tissues. Combined application of multiple markers can reduce the rate of missed diagnosis. Among the 13 patients, 12 had at least one mutation (1 mutation: 1 case, 2-5 mutations: 3 cases, 6-15 mutations: 8 cases), and 1 case was not detected. The gene with the highest mutation frequency was TP53 (10 cases), and other mutant genes included EPHB1 (3 cases), ATRX (2 cases), EPHA5 (2 cases), GATA3 (2 cases), LRP1B (2 cases) and MAP2K4 (2 cases) were also detected. Three of the 13 patients had structural variations, which were C14orf177-GNAS, AIM1-FGFR3, and EPHA6-ROS1 gene rearrangements. All 13 patients had copy number variation, and 11 patients had copy number variation of more than 2 genes. The common amplification genes were IRS2 (5 cases), PTEN (5 cases), GNAS (4 cases), CCNE1 (3 cases), CEBPA (3 cases), PCK1 (3 cases) and ERBB2 (2 cases). The common deletion genes were SOX2 (5 cases) and MYC (5 cases). Among the 13 patients, 4 died, and 2 of the dead patients had liver metastasis. There were 4 patients with disease-free survival and 5 patients with disease progression, including 3 cases of abdominal metastasis and 2 cases of liver metastasis. The 3-year survival rate of patients was 65.9 %, and the 3-year progression-free survival rate was 30.7 %. Gene LRP1B point mutation was associated with poor prognosis (P＜0.001). There was no significant improvement in the prognosis of patients treated with immunotherapy compared with those treated with chemotherapy alone (P=0.595), but the prognosis of patients treated with postoperative chemotherapy or postoperative chemotherapy plus immunotherapy was better than that of patients treated with surgery alone (P＜0.05). Conclusions: Elevated serum AFP with GAED is a highly invasive tumor with unique molecular characteristics, often accompanied by multiple molecular events. TP53 mutation is the most common type of gene mutation. In addition, some cases are accompanied by HER2 amplification and gene rearrangement.

[Detection of PIK3CA gene mutation and its related prognosis in colorectal cancer based on next-generation sequencing].

Objectives: To investigate the mutation of PIK3CA in colorectal cancer and to analyze their clinicopathological features, and evaluate their role in clinical treatment and prognostication. Methods: A total of 128 paraffin-embbeded tissue samples of colorectal cancer from Shanxi Cancer Hospital from 2018 to 2021 were collected. DNA was extracted from the samples, and next-generation sequencing (NGS) was used to detect PIK3CA mutation. The relationship between PIK3CA mutation, their clinicopathological features, and prognosis were analyzed. Results: Among the 128 colorectal cancer samples, there were 75 males and 53 females; with aged range 32-86 years, median 61.5 years, 27 (21.09%) had PIK3CA mutations. Colorectal cancer with PIK3CA mutation was more likely to occur in male patients (P=0.007), which was related to tumor site (P=0.032), tumor size (P=0.029) and TP53 wild-type (P=0.001). The common site mutations of PIK3CA mostly occurred in tumors with tumor mutation burden≥10 Muts/Mb (P=0.031).PIK3CA mutation had no significant effect on the survival prognosis of patients, but the efficacy of anti-angiogenic therapy was poor in these patients. Conclusions: PIK3CA mutation is a common mutation in colorectal cancer and plays an important role in the occurrence and development of colorectal cancer. PIK3CA mutation may lead to resistance to anti-angiogenic drugs in colorectal cancer, but its impact on survival and prognosis to patients needs further study.

Identification of genetic loci associated with growth traits at weaning in yak through a genome-wide association study.

A multilocus GWAS was performed to explore the genetic architecture of four growth traits in yak. In total, 354 female yaks for which measurements of body weight (BW), withers height (WH), body length (BL) and chest girth (CG) at weaning were available underwent genotyping with the Illumina BovineHD BeadChip (770K). After quality control, we retained 98 688 SNPs and 354 animals for GWAS analysis. We identified seven, 18, seven and nine SNPs (corresponding to seven, 17, seven and eight candidate genes) associated with BW, WH, BL and CG at weaning respectively. Interestingly, most of these candidate genes were reported to be involved in growth-related processes such as muscle formation, lipid deposition, feed efficiency, carcass composition and development of the central and peripheral nervous system. Our results offer novel insight into the molecular architecture underpinning yak growth traits. Further functional analyses are thus warranted to explore the molecular mechanisms whereby these genes affect these traits of interest.

miR-27a controls triacylglycerol synthesis in bovine mammary epithelial cells by targeting peroxisome proliferator-activated receptor gamma.

Growing evidence has revealed that microRNA are central elements in milk fat synthesis in mammary epithelial cells. A negative regulator of adipocyte fat synthesis, miR-27a has been reported to be involved in the regulation of milk fat synthesis in goat mammary epithelial cells; however, the regulatory role of miR-27a in bovine milk fat synthesis remains unclear. In the present study, primary bovine mammary epithelial cells (BMEC) were harvested from mid-lactation cows and cultured in Dulbecco's modified Eagle's medium/F-12 medium with 10% fetal bovine serum, 5 µg/mL of insulin, 1 µg/mL of hydrocortisone, 2 µg/mL of prolactin, 1 µg/mL of progesterone, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. We found that the overexpression of miR-27a significantly suppressed lipid droplet formation and decreased the cellular triacylglycerol (TAG) levels, whereas inhibition of miR-27a resulted in a greater lipid droplet formation and TAG accumulation in BMEC. Meanwhile, overexpression of miR-27a inhibited mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein beta (C/EBPß), perilipin 2 (PLIN2), and fatty acid binding protein 3 (FABP3), whereas miR-27a downregulation increased PPARG, C/EBPß, FABP3, and CCAAT enhancer binding protein alpha (C/EBPα) mRNA expression. Furthermore, Western blot analysis revealed the protein level of PPARG in miR-27a mimic and inhibitor transfection groups to be consistent with the mRNA expression response. Moreover, luciferase reporter assays verified that PPARG was the direct target of miR-27a. In summary, these results indicate that miR-27a has the ability to control TAG synthesis in BMEC via targeting PPARG, suggesting that miR-27a could potentially be used to improve beneficial milk components in dairy cows.

Establishment and application of milk fingerprint by gel filtration chromatography.

Raw milk adulteration frequently occurs in undeveloped countries. It not only reduces the nutritional value of milk, but it is also harmful to consumers. In this paper, we focused on investigating an efficient method for the quality control of raw milk protein. A gel filtration chromatography (GFC) fingerprint method combined with chemometrics was developed for fingerprint analysis of raw milk. To optimize the GFC conditions, milk fat was removed by centrifugation, and GFC analysis was performed on a Superdex 75 10/300GL column (Just Scientific, Shanghai, China) with 0.2 M NaH2PO4-Na2HPO4 buffer (pH 7.0) as the mobile phase. The flow rate was 0.5mL/min, and the detection wavelength was set at 280 nm. Ten batches of 120 raw milk samples were analyzed to establish the GFC fingerprint under optimal conditions. Six major peaks common to the chromatogram of each raw milk sample were selected for fingerprint analysis, and the characteristic peaks were used to establish a standard chromatographic fingerprint. Principal component analysis was then applied to classify GFC information of adulterated milk and raw milk, allowing adulterated samples to be effectively screened out from the raw milk in principal component analysis scores plot. The fingerprint method demonstrates promising features in detecting milk protein adulteration.

Polymorphisms in the LPL gene and their association with growth traits in Jiaxian cattle.

Previous studies showed that the lipoprotein lipase (LPL) gene was involved in metabolism and transport of lipids, suggesting that the LPL is a potential candidate gene affecting growth traits in animals. The aim of this study was to identify polymorphism in the bovine LPL gene and analyze its possible association with growth traits in 218 randomly selected Jiaxian cattle. We used DNA sequencing to identify single nucleotide polymorphisms (SNPs) in the LPL gene. A sequence analysis revealed three SNPs: two in intron 5 (C18306T and C18341T) and one in exon 6 (G18362A). G18362A is a missense mutation leading to a change of the 325th glycine to serine. Based on χ(2) tests, the genotypic distributions of C18306T were in agreement with the Hardy-Weinberg equilibrium (P > 0.05), whereas the other two mutations were not (0.05 > P > 0.01). Association analyses showed that the C18341T SNP was significantly associated with several growth traits (P < 0.01 or P < 0.05), and the G18362A was associated with withers height (P < 0.05). Our results suggest that LPL gene variation may be considered molecular markers for growth traits in Jiaxian cattle.

C/EBPα gene as a genetic marker for beef quality improvement.

Intramuscular fat (IMF) or intramuscular triglycerides are interspersed throughout the skeletal muscles. The IMF, also called marbling, imparts meat with flavor and juiciness and is one of the core criteria for judging carcass value. The quantity of IMF is influenced entirely by genetics. Recently, understanding the underlying genetic bases of IMF has been a focus particularly in the beef industry. In this study, with the deep insights of ameliorating the beef quality by genetic means, the role of the CCAAT/enhancer binding protein alpha (C/EBPα) gene was investigated by over-expressing C/EBPα in bovine muscle stem cells (MSCs) to initiate the adipogenic program. Prior to this, bovine MSCs were isolated and induced to differentiate into adipocytes from cells that were exposed to dexamethasone isobutylmethylxanthine and indomethacin; the presence of insulin and fetal bovine serum was examined. Either ectopic expression of C/EBPα or treatment with dexamethasone and insulin induced the accumulation of fat droplets and the expression of adipogenic induction genes (LPL, PPARγ, C/EBPß, and C/EBPδ). The expression levels of myoblast-related genes (MyoD, Myf5, and Pax7) were also measured to assess the accuracy of the differentiation process. This study provides evidence that the C/EBPα gene is essential for cattle adipose tissue growth and development. Hence, this finding can contribute to improving beef carcass quality.

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.

Identification of single nucleotide polymorphisms of the signal transducer and activator of transcription 3 gene (STAT3) associated with body measurement and carcass quality traits in beef cattle.

Previous studies have shown that the signal transducer and activator of transcription 3 gene (STAT3) is involved in lipid storage and energy metabolism, suggesting that STAT3 is a potential candidate gene that affects body measurement and carcass quality traits in animals. Therefore, the aim of this study was to identify polymorphisms in bovine STAT3 and to analyze their possible associations with body measurement and carcass quality traits in 493 individuals of 2 native Chinese cattle breeds: Qinchuan (N = 371) and Jiaxian cattle (N = 122). DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were employed to detect STAT3 single nucleotide polymorphisms (SNPs). We found 5 SNPs: 1 in an exon (g.65812G>A: exon 16) and 4 in introns (g.43591G>A: 13 intron, g.67492T>G: 19 intron, g.67519T>C: 19 intron, and g.68964G>A: 20 intron). Both g.65812G>A and g.68964G>A were not in Hardy- Weinberg equilibrium (HWE), whereas individual frequencies of each genotype were consistent with HWE for other SNPs in Qinchuan cattle populations. For the Jiaxian cattle, the genotype distributions of the 4 mutations were in HWE except for g.67519T>C. The results indicate that these SNPs have a significant association with some body measurements and carcass quality traits (P < 0.05 or P < 0.01). Therefore, STAT3 might have potential effects on production traits in beef cattle populations and could be used for marker-assisted selection.

Polymorphisms in the SIRT5 gene and their association with body measurement and ultrasound traits in Qinchuan cattle.

Silent information regulator 5 (SIRT5), a member of the Sirtuin family class III nicotinamide adenine dinucleotide-dependent protein deacetylases, plays an important role in metabolic and aging processes in mammals. We identified 4 single-nucleotide polymorphisms (SNPs) (G22010A, G22052A, G22119T, and G22245C) in the 3' untranslated regions of the SIRT5 gene from 572 Qinchuan cattle by sequencing and investigating their association with growth and ultrasound traits. The frequencies of genotype GG and allele G were high at the 4 SNPs. Based on the X(2) test, the genotypic distributions of the 4 SNPs were not in Hardy-Weinberg equilibrium (P < 0.05 or P < 0.01). Association analysis of individual SNPs and haplotype combinations revealed that the 4 loci were significantly associated with some body measurement and ultrasound traits in Qinchuan cattle, and the H1H5 (AG-GA-GG-GG) diplotypes had better performance than other combinations in Qinchuan cattle. Our results demonstrate that SIRT5 may be a candidate for marker-assisted selection in future breeding programs for Qinchuan cattle.

Polymorphisms of the bovine MC3R gene and their associations with body measurement traits and meat quality traits in Qinchuan cattle.

The melanocortin 3 receptor (MC3R) gene, which belongs to the rhodopsin-like family A of the G protein-coupled receptor family, plays a crucial role in feed efficiency and energy homeostasis. The aim of this study was to examine associations between bovine MC3R gene polymorphisms and body measurement traits (BMTs) and meat quality traits (MQTs). We identified three synonymous mutations (T429C, T537C, and T663C) in exon 1 of the MC3R gene in Chinese Qinchuan beef cattle (N = 271) by sequencing. D' and r(2) values revealed that these three SNPs were in strong linkage disequilibrium (LD) (r(2) > 0.33); the T429C and T537C SNPs were in complete LD (D' = 1 and r(2) = 1). Association analyses revealed that the SNPs were significantly associated with BMTs and MQTs in Qinchuan cattle. Individuals with the wild homozygotic genotypes g.TTTT and g.TT had significantly higher values of chest depth, heart girth, back fat thickness, intramuscular fat content, and loin muscle area than the mutant heterozygotic genotypes g.TCTC and g.TC. These results suggest that the MC3R gene affects MQTs in Qinchuan cattle, and that it may be a good candidate gene for marker-assisted selection.

Polymorphisms of the Osteocrin gene and its association with meat quality traits in Qinchuan cattle.

Here, we detected 2 SNPs, A85C and T335C, that were located on the 3rd exon and the 3 untranslated regions of the bovine Osteocrin gene, respectively, using 413 Qinchuan cattle DNA samples. PCR-SSCP and DNA sequencing methods were specifically used. Three genotypes (AA, AC, and CC) were found at A85C; yet, only 2 genotypes (TC and CC) were found at T335C. Association analysis showed that both loci were associated with certain meat quality traits, including back fat thickness and loin muscle area. At the A85C locus, individuals with the CC genotype had greater back fat thickness. In comparison, at the T335C locus, individuals with the TC genotype had greater back fat thickness and a larger loin muscle area. Therefore, these 2 SNPs could be used as genetic markers to enhance Qinchuan cattle breeding programs.

Genetic polymorphisms of the FATP1 gene and their associations with meat quality traits in Chinese Qinchuan cattle.

Fatty acid transport protein 1 (FATP1), an integral membrane protein that facilitates long-chain fatty acid influx, is involved in the genetic network for oleic acid synthesis. The aim of this study was to examine the association of FATP1 polymorphisms with live animal meat quality traits in Chinese Qinchuan cattle. Quantitative real-time PCR analysis demonstrated that FATP1 has a broad tissue distribution in Qinchuan cattle and is highly expressed in longissimus dorsi muscle and back fat. Using direct DNA sequencing of the FATP1 gene in 458 Qinchuan cattle, four single nucleotide polymorphisms (SNPs; g.28265 G>C, g.28381 G>A, g.28470 T>C, and g.28672 G>A) were identified for genotyping within a 671-bp region, including exon 3, intron 3, exon 4, intron 4, and part of exon 5 of the FATP1 gene. Positive effects of genotypes CC (g.28470 T>C locus) and AA (g.28672 G>A locus) on meat quality traits were obtained by association analysis. These results indicate the associations of g.28470 T>C and g.28672 G>A with meat quality traits in Qinchuan cattle. Thus, the FATP1 gene may be used in marker-assisted selection of beef cattle in breeding programs.

Association of CRTC2 gene polymorphisms with growth and meat quality traits of Qinchuan cattle.

Growth and meat quality traits play important roles in the evaluation of cattle productivity and are influenced by genetic and environmental factors. CRTC2 is a recently discovered gene related to obesity that may influence fat deposition. The aim of the current study was to detect polymorphisms of bovine CRTC2 and explore their relationships to growth and meat quality in Qinchuan cattle. Three single nucleotide polymorphisms (SNPs); g.3001 C>T; g.3034 G>A; and g.3467 T>C, were identified from sequencing results of 422 Qinchuan cattle. The genotypic distributions of both g.3034 G>A and g.3467 T>C mutations were in agreement with Hardy-Weinberg equilibrium, (P < 0.05), while the T3001C mutation was not (P > 0.05), based on χ(2) test analysis. The SNPs g.3001 C>T and g.3034 G>A are missense mutations (Ser/Phe and Ser/Thr respectively). Additionally, SNPs g.3034 G>A and g.3467 T>C showed a medium polymorphism level (0.25 < PIC< 0.50), whereas g.3001 C>T showed a low polymorphism level (PIC < 0.25). These three SNPs were significantly associated with several growth and meat quality traits in the Qinchuan cattle population (P < 0.05 or P < 0.01). Collectively, these results demonstrate that CRTC2 is involved in the regulation of cattle growth and meat quality, and suggest that CRTC2 is a potential candidate gene for marker-assisted selection in future breeding development programs for Qinchuan cattle.

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.

Protective effects of ascorbic acid and vitamin E on antioxidant enzyme activity of freeze-thawed semen of Qinchuan bulls.

The aim of this study was to determine the protective effects of the combination of ascorbic acid (Vc) and vitamin E (VE) on antioxidant enzyme activity, sperm motility, viability, and acrosome integrity of Qinchuan bulls after freeze-thaw. In this study, we determined the effects of Vc and VE on the activity of the antioxidant enzyme defense system comprising glutathione peroxidase (GSH-Px), glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD). The combination of Vc and VE had protective effects on sperm motility and viability. With respect to acrosome integrity and the activity of GR and SOD, differences were observed between the experimental groups with added Vc (7 mg/mL) and VE (0.12 IU/mL) and the control group. The activity of GSH-Px in the experimental group (1400 IU/mL Vc and 0.12 IU/mL VE) was not different (P > 0.05) compared with that in the control group, while the activity of CAT showed a significant difference between the 2 groups (P < 0.05). Therefore, we inferred that the combination of Vc (1400 IU/mL) and VE (0.12 IU/mL) protected the sperm quality in the freeze-thaw process.

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.

Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk.

Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 ± 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 µg/µL and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk.

Expression analysis of Gli1 and Gli2 in different tissues and muscle-derived cells of Qinchuan cattle.

The Hedgehog (Hh) signaling pathway regulates the differentiation of many kinds of cells and plays a critical role in many embryonic and postnatal developmental processes. Gli1 and Gli2 are two transcription factors of the Hh signaling pathway. In this study, we used quantitative real-time polymerase chain reaction to detect the relative expression of Gli1 and Gli2 in 13 tissues from three two-year-old purebred Qinchuan cattle, as well as in different cell populations derived from muscle and different stages of myogenic differentiation of myoblasts. The expression levels of Gli1 and Gli2 in muscle were the lowest of the 13 tissues (P < 0.05), and they declined predominantly from preplate (pp)1 to pp6 cells. However, the expression of Gli2 was elevated during myogenic differentiation until the 6th day. We speculated that Hh signaling was negatively activated in myocytes and quiescent myoblasts. The increased expression of Gli1 and Gli2 in the early days of myogenic differentiation suggested that Hh signaling would be activated when the quiescent bovine myoblast was stimulated to initiate myogenic differentiation.

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.