4. Analysis of Pig Vomeronasal Receptor Type 1 (V1R) Promoter Region Reveals
a Common Promoter Motif but Poor CpG Islands
Hunduma Dinka a,b and Minh Thong Leb
aDepartment of Applied Biology, School of Applied Natural Sciences, Adama Science and Technology University, Adama, Ethiopia; bDepartment of Animal Biotechnology, Konkuk University, Seoul, South Korea
ABSTRACT
Promoters are, generally, located immediately upstream of a transcription start site (TSS) and have a variety of regulatory motifs, such as transcription factors (TFs) and CpG islands (CGIs), that participate in the regulation of gene expression. Here analysis of the promoter region for pig vomeronasal receptor type 1 (V1R) was described. In the analysis, TSSs for pig V1R genes was first
identified and five motifs (MV1, MV2, MV3, MV4, and MV5) were found that are shared by at least 50% of the pig V1R promoter input sequences from both strands. Among the five motifs, MV2 was identified as a common promoter motif shared by all (100%) pig V1R promoters. For further analysis, to better characterize and get deeper biological insight associated with MV2, TOMTOM web application was used. MV2 was compared to the known motif databases (such as JASPAR) to see if they are similar to a known regulatory motif (transcription factor). Hence, it was revealed that MV2 serves as the binding site mainly for the BetaBetaAlpha-zinc finger (BTB-ZF) transcription factor gene family to regulate expression of pig V1R genes. Moreover, it was shown that pig V1R promoters are CpG poor, suggesting that their gene expression regulation pattern is in tissue specific manner.
KEYWORDS
CPG island; motif; pig; promoter; transcription factor; vomeronasal receptor type 1 (V1R)
Assessment of RNA Stability in Postmortem Tissue from New-Born Lambs
Fiona McGovern a,b, Tommy Boland a, Marion Ryan b and Torres Sweeneyb
aUCD School of Agriculture and Food Science, University College Dublin, Lyons Research Farm, Newcastle, Dublin, Ireland;
bUCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland
ABSTRACT
The recovery of high quality RNA from postmortem tissue is crucial to gene expression analyses. The acquisition of postmortem tissue has inherent time delays and, hence, understanding the temporal variation in the stability of total RNA is imperative. This experiment aimed: (1 ) to qualitatively and quantitatively assess the integrity of total RNA derived from a range of new-born ovine tissues (liver, spleen, thyroid, skeletal muscle, ileum, and perirenal adipose tissue) which were stored at ambient temperature until extraction at 0, 3, 6, and 9 h postmortem; and (2) to analyze the stability of the reference gene(s) and expression of specific target genes in these tissues. Postmortem sampling time resulted in variable reductions in the relative integrity number (RIN) values across the tissues, ranging from 0.9 to 1.8% in liver, spleen, skeletal muscle, and ileum to 5.7–11.1% in the
thyroid and perirenal adipose tissues, respectively (P < 0.05). In conclusion, tissues with small reductions in RIN value can exhibit disproportionately large differences in the normalization factor used to calculate the target gene expression. Hence, changes in transcript abundance due to RNA degradation are not always sufficiently buffered through normalization with reference genes. The normalization factor should be presented alongside the RIN value in postmortem tissue studies.
KEYWORDS
New-born lamb; ovine; postmortem tissue; RNA stability
Cytoskeleton Genes Expression and Survival Rate Comparison Between
Immature and Mature Yak Oocyte After OPS Vitrification
Xia Wei a, Ye Sijiea, Zeng Weibin b, Xu Qing a, Zheng Jiea and Zi Xiangdong a
aCollege of Life Science and Technology, Southwest University for Nationalities, Chengdu, China; bCollege of Animal Sciences and Technology, Shihezi University, Shihezi, Xinjiang, China
ABSTRACT
This study was conducted to investigate the effect of vitrification on survival rate and cytoskeleton gene expression during yak oocyte maturation. The yak oocytes were incubated for 0 h [germinal vesicle (GV) stage] and in vitro matured for 24 h [metaphase II (MII) stage] to obtain immature and mature oocytes. Survival rate after vitrification were compared between immature and mature yak oocytes and cytoskeleton-related genes [cytokeratin 8 (CK8), β-actin (ACTB), and gap junction
protein, alpha 1 (GJA1 )] were tested by real-time PCR. Our results showed that MII stage survival rate after open pulled straw vitrification (35.60%) is significantly higher than GV stage (25.90%) oocytes. Furthermore, expression of CK8, ACTB, and GJA1 in MII stage oocytes are also significantly higher than GV stage oocytes. In conclusion, our study demonstrated that higher expression of GJA1, CK8, and ACTB in vitrify-warmed MII stage oocytes when compared with GV stage oocytes
and such discrepancy might result in higher survival rate in vitrify-warmed MII stage oocytes.
KEYWORDS
Cytoskeleton genes; survival rate; vitrification; yak oocytes
Draft De Novo Genome Sequence of Agapornis roseicollis for Application
in Avian Breeding
Henriëtte van der Zwan a, Francois van der Westhuizen a, Carina Visserb and Rencia van der Sluisa
aCentre for Human Metabolomics, North-West University, Potchefstroom, North-West, South Africa; bDepartment of Animal
and Wildlife Sciences, University of Pretoria, Pretoria, Gauteng, South Africa
ABSTRACT
In aviculture, lovebirds are considered one of the most popular birds to keep. This African parakeet is known for its range of plumage colors and ease to tame. Plumage variation is the most important price-determining trait of these birds, and also the main selection criterion for breeders. Currently, no genetic screening tests for traits of economic importance or to confirm pedigree data are available for any of the nine lovebird species. As a starting point to develop these tests, the de novo genome of Agapornis roseicollis (rosy-faced lovebird) was sequenced, assembled, and annotated. Sequencing was done on the Illumina HiSeq 2000 platform and the assembly was performed using SOAPdenovo v2.04. The genome was found to be 1.1 Gb in size and 16,044 genes were identified and annotated. This compared well with other previously sequenced avian genomes, such as the chicken, zebra finch, and budgerigar. To assess genome completeness, the number of benchmarking universal single-copy orthologs were identified in the genome. This wascompared to other previously assembled avian genomes and the results indicated that the
genome will be useful in the development of genetic screening tests to aid lovebird breeders in selecting breeding pairs.
KEYWORDS
Avian genomics bioinformatics; whole genome sequencing
Effect of High Hydrostatic Pressure Applied Before Cryopreservation on the
Survival Rate and Quality of Porcine Mesenchymal Stem Cells After Thawing
Joanna Romaneka, Jolanta Opiela a, Daniel Lipiński b and Zdzisław Smorąg a
aDepartment of Animal Reproduction Biotechnology, National Research Institute of Animal Production, Kraków, Poland; bDepartment of Biochemistry and Biotechnology, Poznań University of Life Sciences, Poznań, Poland
ABSTRACT
The aim of the present study was to examine the effects of varied high hydrostatic pressure (HHP) values on survival rate, proliferation rate, cell multipotency (transcript expression of SOX2, C-MYC, and REX1) and apoptosis (expression of phosphatidylserine (PS), SURVIVIN at the RNA level and BAX at the protein level) of porcine mesenchymal stem cells (MSCs). MSCs were isolated from porcine bone marrow and cultured in vitro. Before cryopreservation and storage in liquid nitrogen, MSCs were subjected to HHP at the varied pressures of 20, 30, 40, 50, or 60 MPa for 1 h at 24°C. Immediately after thawing and after 8 days of in vitro culture, cells were subjected to trypan blue staining, cell counting, real-time Polymerase Chain Reaction (PCR), western blotting, and fluorescence microscopy. BAX protein expression was only estimated immediately after HHP to exclusively examine the impact of HHP on apoptosis of MSCs. The viability of MSC subjected to 40, 50, and 60 MPa and estimated immediately after thawing increased significantly (P < 0.001 for 60 MPa and P < 0.05 for 40 and 50 MPa) in comparison to control. The proliferation rate of MSCs subjected to 40 MPa HHP was significantly higher than in the control group (P < 0.02) after 8 days of in vitro culture. After 8 days of in vitro culture, no significant differences were noted in the survival rates, PS exposure, or levels of SOX2, C-MYC, REX1, and SURVIVIN gene expression in all analyzed groups compared to control. In conclusion: 40–60 MPa HHP has a positive impact by improving cell viability in short term. 20–60 MPa HHP does not induce nor decrease apoptosis in MSCs. Fortunately, HHP does not induce differentiation of MSC. Our results calls for further analysis using HHP values higher than 60 MPa.
KEYWORDS
Apoptosis; cryopreservation; HHP; MSCs; pig
Genome-Wide Linkage Analysis Identifies Loci for Testicle and Ovary
Traits in Chickens
Yanfa Sun a,b, Ranran Liu a, Guiping Zhaoa, Maiqing Zheng a, Peng Li a, Li Liu a and Jie Wen a
aInstitute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, P. R. China; bCollege of Life Science,
Longyan University, Longyan, Fujian, P. R. China
ABSTRACT
Development of testes or ovaries is critical to chicken breeders. Understanding the genetic mechanisms influencing the development of the testes and ovaries could enhance selection efforts which target reproductive traits. The linkage analysis was conducted within an F2 population derived from Beijing-You chickens and a commercial broiler line. The results have identified one quantitative trait loci (QTL, designated T1) for bilateral testicular weight (TW) and the percentage of TW to carcass weight, and five QTLs (designated O1–O5) for ovary weight (follicle-free, OW) and the percentage of OW to carcass weight. For the testes traits, QTL T1 is located between 6.55 and 8.56 Mb on GGA13. Especially, the gene gamma-amino butyric acid A receptor, alpha 1 (GABRA1 ) located near the T1 peak. For ovarian traits, QTL O2 was located at 29.31 Mb on GGA7. G proteincoupled receptor 39 (GPR39) present at the O2 peak was expressed at higher levels within the reproductive tract. It is also involved in the regulation of several reproductive functions. Other QTL peaks and the genes’ function in the ovary and testes need to be evaluated. The QTLs and the genes identified in this study could provide valuable information for establishing reproductive traits in chickens, and need further investigation.
KEYWORDS
Chicken; linkage analysis; ovary; QTL; testes
Novel 17-bp Deletion in KDM1B Gene is Significantly Associated with Testis Weight in Male Piglet
Yang Cui a, Tao Hu b, Rui Chen a, Shuai Yu a, Wuzi Dong a, Xiaoyan Lvc and Chuanying Pan a
aCollege of Animal Science and Technology, Northwest A&F University, Yangling, China; bInnovation Experimental College, Northwest A&F University, Yangling, China; cNational Swine Foundation Seed Farm of Ankang Yangchen Modern Agriculture Group Co. Ltd, Ankang, China
ABSTRACT
Lysine-specific demethylase 1B (KDM1B) which plays a crucial role in regulating methylation status at lysine 4 of histone 3 is important for male fertility. The aim of this study was to explore the KDM1B mRNA expression profiles and to identify novel genetic variants of the pig KDM1B gene, as well as to determine the association between these variants and testis measurement traits in male piglets. The KDM1B mRNA expression profiles indicated that this gene widely expressed in all tested organs. In addition, a novel 17-bp deletion (NC_010449.4:g.31142_31159delCATGGATAG TAGTTGCT) within KDM1B gene was found. Notably, this deletion sequence was inconsistent with the prediction by NCBI. Association analysis revealed that the 17-bp indel locus was significantly associated with the testis weight in 40-day-old Large White pigs (P < 0.05). Furthermore, through bioinformatics analysis, transcriptional factor heat shock factor-1 could combine the 17-bp sequence. These results not only extend the genetic variations of the pig KDM1B gene but also contribute to implementing marker-assisted selection in pig breeding.
KEYWORDS
Insertion/deletion (indel); KDM1B gene; pig; testis weight
Novel Muscle-Specific Genes TCAP, TNNI1, and FHL1 in Cattle: SNVs, Linkage
Disequilibrium, Combined Genotypes, Association Analysis of Growth Performance, and Carcass Quality Traits and Expression Studies
Hua Hea,b, Zhi-gang Hu b, Sodnompil Tserennadmid b, Si Chen b and Xiao-lin Liu b
aCollege of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China; bShaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest Agriculture and Forestry University, Yangling, Shaanxi, China
ABSTRACT
TCAP, TNNI1, and FHL1 regulate muscle growth and development. In this study, four single nucleotide variants (SNVs) were discovered in almost all of the exon and intron regions of the TCAP, TNNI1, and FHL1 genes using DNA pooled sequencing, polymerase chain reaction (PCR)-RFLP, and forced-PCR-RFLP methods in 576 cattle. Four SNVs were significantly associated with the growth performance and carcass quality traits of the cattle. In addition, the haplotype, haplotype frequency, and linkage disequilibrium coefficient of three sequence variants were also evaluated in the cattle population. Haplotype analysis demonstrated that eight haplotypes were present in the Qinchuan cattle population and no haplotypes were present in the Chinese Holstein population; haplotype 1 had the highest frequency in the Qinchuan (42.7%) population. Statistical analyses of 12 combined genotypes indicated that some were significantly associated with the growth performance and carcass quality traits of the Qinchuan cattle population. Moreover, the quantitative real-time polymerase chain reaction results demonstrated that the bovine TCAP, TNNI1, and FHL1 genes were exclusively expressed in muscle tissue. These data support the high potentials of the TCAP, TNNI1, and FHL1 as marker genes to improve the growth performance and carcass quality traits of Qinchuan cattle or other animals selection programs.
KEYWORDS
Association analysis; cattle; gene expression; muscle-specific gene; single nucleotide variants
Two New Insertion/Deletion Variants of the PITX2 Gene and their Effects on
Growth Traits in Sheep
Haidong Zhaoa, Shuai Hea, Shuhui Wang a, Yanjiao Zhu a, Hongwei Xu b, Renyun Luoc, Xianyong Lan a, Yong Cai c,d and Xiuzhu Sun a
aCollege of Animal Science and Technology, Northwest A&F University, Yangling, China; bScience Experimental Center, Northwest University for Nationalities, Lanzhou, China; cRuilin Sci-Tech Culture and Breeding Limit Company, Yongjing, China; dCollege of Life Science and Engineering, Northwest University for Nationalities, Lanzhou, China
ABSTRACT
In China, Tong sheep (TS) and Lanzhou fat-tailed sheep (LFTS) are two closely relative endanger breeds for low meat production and low fecundity, finding some marker-assisted selected (MAS) is our first priority for improving their growth traits. For this purpose, Hu sheep (HS) and small-tailed Han sheep (STHS) were compared with two endangered breeds (TS and LFTS). Paired-liked homeodomain transcription factor 2 (PITX2) gene was the important member of PITX family, which could adjust animal growth through hypothalamic–pituitary–adrenal axis. During the past years, insertion/deletion (indel) has become increasingly popular in application as MAS. In this study, two novel indel loci were identified, and five significant differences, including chest width, hip width, chest depth, chest circumference, and body height, were found between different breeds. Interestingly, there was no DD genotype and smaller number of ID genotye. All the ID genotypes were significantly greater than II genotype, which was to say the allele of “D” was dominant variation and its frequency was lower, which demonstrated that it has huge space for selection. Briefly, the two indel were potential and useful DNA markers for selecting excellent individuals in relation to growth traits in sheep.
KEYWORDS
Association; growth traits; indel; PITX2 gene; sheep
