G.B. Luo

Development of an assay for rapid identification of meat from yak and cattle using polymerase chain reaction technique

Yak meat is of good quality with fine texture, high protein and low fat content, and rich in amino acids compared with that of cattle, and it lacks anabolic steroids or other drugs. In general terms, however, the meat yield of yak is relatively low compared with that of the cattle. In order to prevent possible adulteration of yak meat with cattle meat, based on the sequence of mitochondrial 12S rRNA gene, a multiplex PCR-based approach was proposed for rapid identification of the meat from yak and cattle using three primers designed in this work. Through the combinatorial usage of three primers with a single reaction set, two fragments of 290 and 159bp were amplified from the cattle meat DNA, whereas only a fragment of 290bp was obtained from the yak meat DNA. Using the assay described, satisfactory amplification was accomplished in the analysis of raw and heat-treated binary meat mixtures of yak/cattle with a detection limit of 0.1% for cattle meat. The technique is fast and straightforward. It might be a useful tool in the quality control of yak meat and meat products.

Rapid detection of bovine milk in yak milk using a polymerase chain reaction technique

Yak milk contains a greater percentage of protein and has better quality than bovine milk. There has been an increasing focus on yak milk and milk products during the last few years. In the present study, a PCR-based assay was developed for the specific identification of bovine milk in yak milk by designing 3 primers targeting the mitochondrial ND1 gene. The use of 3 primers in a single PCR reaction set yielded 2 amplification fragments of 293 and 190 bp from bovine milk DNA, whereas only 1 amplification fragment of 293 bp was obtained in yak milk DNA. The technique was applied to raw and heat-treated binary mixtures of yak and bovine milks and enabled the specific detection of bovine milk with a detection limit of 0.1%. The assay developed is sensitive, fast, and straightforward, and it might be useful in the quality control of yak milk and milk products.

Technical note: Selection of suitable reference genes for studying gene expression in milk somatic cell of yak (Bos grunniens) during the lactation cycle

Quantitative real-time PCR is the most sensitive technique for gene expression analysis. Data normalization is essential to correct for potential errors incurred in all steps from RNA isolation to PCR amplification. The commonly accepted approach for normalization is the use of reference gene. Until now, no suitable reference genes have been available for data normalization of gene expression in milk somatic cells of lactating yaks across lactation. In the present study, we evaluated the transcriptional stability of 10 candidate reference genes in milk somatic cells of lactating yak, including ACTB, B2M, GAPDH, GTP, MRPL39, PPP1R11, RPS9, RPS15, UXT, and RN18S1. Four genes, RPS9, PPP1R11, UXT, and MRPL39, were identified as being the most stable genes in milk somatic cells of lactating yak. Using the combination of RPS9, PPP1R11, UXT, and MRPL39 as reference genes, we further assessed the relative expression of 4 genes of interest in milk somatic cells of yak across lactation, including ELF5, ABCG2, SREBF2, and DGAT1. Compared with expression in colostrum, the overall transcription levels of ELF5, ABCG2, and SREBF2 in milk were found to be significantly upregulated in early, peak, and late lactation, and significantly downregulated thereafter, before the dry period. A similar pattern was observed in the relative expression of DGAT1, but no significant difference was revealed in its expression in milk from late lactation compared with colostrum. Based on these results, we suggest that the geometric mean of RPS9, PPP1R11, UXT, and MRPL39 can be used for normalization of real-time PCR data in milk somatic cells of lactating yak, if similar experiments are performed.

Differential Expression of microRNAs and their Regulatory Networks in Skin Tissue of Liaoning Cashmere Goat during Hair Follicle Cycles.

ABSTRACT MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that negatively regulate gene expression. Herein, we investigated a selective number of miRNAs for their expression in skin tissue of Liaoning Cashmere goat during hair follicle cycles, and their intracellular regulatory networks were constructed based on bioinformatics analysis. The relative expression of six miRNAs (mir-103-3p, -15b-5p, 17-5p, -200b, -25-3p, and -30c-5p) at anagen phase is significantly higher than that at catagen and/or telogen phases. In comparison to anagen, the relative expression of seven miRNAs (mir-148a-3p, -199a-3p, -199a-5p, -24-3p, -30a-5p, -30e-5p, and -29a-3p) was revealed to be significantly up-regulated at catagen and/or telogen stages. The network analyses of miRNAs indicated those miRNAs investigated might be directly or indirectly involved in several signaling pathways through their target genes. These results provided a foundation for further insight into the roles of these miRNAs in skin tissue of Liaoning Cashmere goat during hair follicle cycles.

Molecular Characterization of Prolactin cDNA and Its Expression Pattern in Skin Tissue of Liaoning Cashmere Goat

The Liaoning Cashmere goat is a local breed found in the Buyun Mountains of the Liaotung Peninsula. It has an estimated population of 1.5 million individuals and is well adapted to the local environment. The goats are white with two horns in both males and females. The main purpose of this breed is the production of cashmere, which is of great economic importance to the local people. The breed is characterized by a high cashmere yield, averaging 0.54 kg for male adults and 0.47 kg for female adults. The diameter of the Liaoning Cashmere fiber, however, is relatively large, compared with that of some other cashmere goat breeds in China (Jin et al. 2011), and the thinner fibers bring a higher price in the market (Li et al. 2005). Breeders are now attempting to lower the fiber diameter of the Liaoning Cashmere goat through modern breeding and genetics technology. Little is known regarding the molecular regulatory mechanism of cashmere growth in this breed. Therefore, it is necessary for us to understand the growth biology of cashmere of

Technical note: Identification of suitable normalizers for microRNA expression analysis in milk somatic cells of the yak (Bos grunniens)

MicroRNA are approximately 18- to 22-nucleotide nonprotein coding molecules that play important roles in the regulation of gene expression at the posttranscriptional level. In the present study, we assessed the suitability of 8 noncoding small RNA as normalizers for microRNA (miR) quantitative analysis in milk somatic cells of lactating yaks, including 3 small nuclear RNA (snRNA; RNU1A, RNU5A, and RNU6B), 3 small nucleolar RNA (snoRNA; SNORA73A, Z30, and SNORA74A), 1 rRNA (5S), and 1 transfer RNA (Met-tRNA). The snRNA RNU1A, RNU5A, and SNORA73A were identified as the most stable references in milk somatic cells of lactating yaks. Also, a minimum of 3 reference RNA (RNU1A, RNU5A, and SNORA73A) were required for the normalization of microRNA expression data in milk somatic cells of the lactating yak. We further evaluated the suitability of the combination of RNU1A, RNU5A, and SNORA73A as reference RNA in milk somatic cells of lactating yaks via detecting the relative expression of miR 16b, miR 21-5p, miR 145, and miR 155 as microRNA of putative interest. In comparison to the colostrum period, on the whole, the expressions of the 4 microRNA were found to be upregulated at an early period and, thereafter, a declining pattern was exhibited from early to final periods in all microRNA investigated. Based on the results from this study, we recommend that the combination of RNU1A, RNU5A, and SNORA73A can be used as normalizers for microRNA quantitative analysis in future longitudinal studies on milk somatic cells of lactating yaks in relation to lactation.

A single nucleotide polymorphism and sequence analysis of CSN1S1 gene promoter region in Chinese Bos grunniens (yak).

The aim of this study was to investigate the polymorphism of the CSN1S1 gene promoter region in 4 Chinese yak breeds, and compare the yak CSN1S1 gene promoter region sequences with other ruminants. A Polymerase Chain Reaction-Single Strand Conformation Polymorphism protocol was developed for rapid genotyping of the yak CSN1S1 gene. One hundred fifty-eight animals from 4 Chinese yak breeds were genotyped at the CSN1S1 locus using the protocol developed. A single nucleotide polymorphism of the CSN1S1 gene promoter region has been identified in all yak breeds investigated. The polymorphism consists of a single nucleotide substitution G→A at position 386 of the CSN1S1 gene promoter region, resulting in two alleles named, respectively, G386 and A386, based on the nucleotide at position 386. The allele G386 was found to be more common in the animals investigated. The corresponding nucleotide sequences in GenBank of yak (having the same nucleotides as allele G386 in this study), bovine, water buffalo, sheep, and goat had similarity of 99.68%, 99.35%, 97.42%, 95.14%, and 94.19%, respectively, with the yak allele A386.

IGF1 mRNA Splicing Variants in Liaoning Cashmere Goat: Identification, Characterization, and Transcriptional Patterns in Skin and Visceral Organs

Insulin-like growth factor I (IGF1) is a member of the insulin superfamily. It performs important roles in the proliferation and differentiation of skin cell and control of hair cycles and is thought to be a potential candidate gene for goat cashmere traits. In this work, we isolated and characterized three kinds of IGF1 mRNA splicing variants from the liver of Liaoning Cashmere goat, and the expression characterization of the IGF1 mRNA splicing variants were investigated in skin and other tissues of Liaoning cashmere goat. The sequencing results indicated that the classes 1w, 1, and 2 of IGF1 cDNAs in Liaoning cashmere goat, each included an open reading frame encoding the IGF1 precursor protein. The deduced amino acid sequences of the three IGF1 precursor proteins differed only in their NH2-terminal leader peptides. Through removal of the signal peptide and extension peptide, the three IGF1 mRNA splicing variants (classes 1w, 1, and 2) resulted in the same mature IGF1 protein in Liaoning cashmere goat. In skin tissue of Liaoning cashmere goat, class 1 and class 2 were detected in all stages of hair follicle cycling, and they had the highest transcription level at anagen, and then early anagen; whereas at telogen both classes 1 and 2 had the lowest expression in mRNA level, but the class 1 appears to be relatively more abundant than class 2 in skin tissue of Liaoning cashmere goat. However, the class 1w transcript was not detected in the skin tissues. Three classes of IGF1 mRNA were transcribed in a variety of tissues, including heart, brain, spleen, lung, kidney, liver, and skeletal muscle, but class 1 IGF1 mRNA was more abundant than classes 1w and 2 in the investigated tissues.