Design and validation of a 90K SNP genotyping assay for the water buffalo (Bubalus bubalis).

BACKGROUND: The availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here. METHODS: The genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K. RESULTS: This 90K "SNP-Chip" was tested in several river buffalo populations and found to have ∼70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production. CONCLUSION: The 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised.

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50).

Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2 n = 50) and the swamp (2 n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1.

Isolation and differentiation potential of an equine amnion-derived stromal cell line.

Stem cells represent an important tool in veterinary therapeutic field such as tissue engineering. In the present study, equine amnion-derived mesenchymal stromal cells were investigated for applications in veterinary science as an alternative source to bone marrow mesenchymal stem cells and adipose stem cells. Amnion stromal cells isolation and characterization protocol is described; the in vitro cell growth rate was calculated by measuring viable cell number over 20 days. The expression of stem cell markers such as Oct-4, Nanog, Sox-2 and CD105 was assessed by retrotranscription quantitative PCR (RT-qPCR) and differentiation into adipocytes, osteocytes and chondrocytes precursors was analyzed by cytochemical staining. This study showed that amnion stromal cells expressing stem cell markers can differentiate into mesoderm lineage and may be an alternative source to mesenchymal stem cells derived from adipose tissue and bone marrow for the use in tissue repair.

Genomic study of the response of chicken to highly pathogenic avian influenza virus.

BACKGROUND: The host mounts an immune response to pathogens, but few data are currently available on the role of host genetics in variation in response to avian influenza (AI). The study presented here investigated the role of the host genetic background in response to in vivo infection with AI virus (AIV). METHODS: Experimental lines of chicken and commercial crosses were experimentally infected intratracheally with 103 EID50/bird of A/Chicken/Italy/13474/99 H7N1 highly pathogenic avian influenza virus (HPAIV). Chickens were genotyped for the Mx polymorphism causing the S631N mutation, and for the Major Histocompatibility Complex (MHC). Whole-genome genotyping was carried out using 60 k Single Nucleotide Polymorphism (SNP) array developed by the poultry Genome-Wide Marker-Assisted Selection Consortium (GWMASC). RESULTS: Variability in response of different chicken lines to the HPAIV infections and some degree of resistance to AI were observed: a statistically significant effect of chicken line on the response to infection was found. There was no association between survival in healthy conditions and polymorphisms at the Mx gene and the MHC-B region. The analysis based on the 60 k SNPs provided a good clustering of the chicken lines, but no specific genetic cluster associated with response to AIV was identified. CONCLUSIONS: Neither the genotype at the Mx gene or MHC-B locus, nor for SNP spanning the whole-genome identified loci involved in variations to response to AIV infection. These results point towards the possibility that either the genetic factors affecting the response of chickens to the H7N1 HPAIV are weak, or relevant alleles were not segregating in the studied populations.

Characterization of maternal antigen that embryos require (MATER/NLRP5) gene and protein in pig somatic tissues and germ cells.

Maternal effect genes produce mRNA or proteins that accumulate in the egg during oogenesis and control the developmental program until embryonic genome activation takes place. NLRP5 (NLR family, Pyrin domain containing 5), also called MATER (Maternal Antigen That Embryos Require) is one of the genes required for normal early embryonic development, although its precise function remains to be elucidated. The aim of the present study was to analyze the NLRP5 gene expression pattern and protein distribution in somatic tissues and germ cells in the pig. Reverse transcription was performed on mRNA from germinal vescicle (GV) oocytes and total RNA from spermatozoa and tissues from different organs. The transcript for NLRP5 gene was identified only in ovaries and oocytes. The presence of NLRP5 protein was detected only in ovaries by western blot analysis and immunohistochemistry.

Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12.

BACKGROUND: Mesenchymal stem cells (MSCs) have been recently investigated for their potential use in regenerative medicine. MSCs, in particular, have great potential, as in various reports they have shown pluripotency for differentiating into many different cell types. However, the ability of MSCs to differentiate into tendon cells in vitro has not been fully investigated. RESULTS: In this study, we show that equine bone marrow mesenchymal stem cells (BM-MSCs), defined by their expression of markers such as Oct4, Sox-2 and Nanog, have the capability to differentiate in tenocytes. These differentiated cells express tendon-related markers including tenomodulin and decorin. Moreover we show that the same BM-MSCs can differentiate in osteocytes, as confirmed by alkaline phosphatase and von Kossa staining. CONCLUSION: As MSCs represent an attractive tool for tendon tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for therapeutic approaches.

Susceptibility of different chicken lines to H7N1 highly pathogenic avian influenza virus and the role of Mx gene polymorphism coding amino acid position 631.

Five chicken lines were experimentally infected with a HPAI H7N1 virus, to examine the variation in susceptibility to infection. Three lines showed high susceptibility to the virus, while two showed some resistance, with 7 out of 20, and 11 out of 15 birds, respectively, remaining healthy and surviving the experimental infection. Genotyping for the G/A polymorphism at position 2032 of Mx cDNA showed that one line was fixed for the G allele, and two were segregating for A and G alleles. Birds in the other two lines were selected to be fixed for the A allele. Statistical analyses indicated that the Mx genotype did not affect the clinical status or the time course of infection after viral inoculation.