Genome-wide identification of copy number variation and association with fat deposition in thin and fat-tailed sheep breeds.

Copy number variants (CNVs) are a type of genetic polymorphism which contribute to phenotypic variation in several species, including livestock. In this study, we used genomic data of 192 animals from 3 Iranian sheep breeds including 96 Baluchi sheep and 47 Lori-Bakhtiari sheep as fat-tailed breeds and 47 Zel sheep as thin-tailed sheep breed genotyped with Illumina OvineSNP50K Beadchip arrays. Also, for association test, 70 samples of Valle del Belice sheep were added to the association test as thin-tailed sheep breed. PennCNV and CNVRuler software were, respectively, used to study the copy number variation and genomic association analyses. We detected 573 and 242 CNVs in the fat and thin tailed breeds, respectively. In terms of CNV regions (CNVRs), these represented 328 and 187 CNVRs that were within or overlapping with 790 known Ovine genes. The CNVRs covered approximately 73.85 Mb of the sheep genome with average length 146.88 kb, and corresponded to 2.6% of the autosomal genome sequence. Five CNVRs were randomly chosen for validation, of which 4 were experimentally confirmed using Real time qPCR. Functional enrichment analysis showed that genes harbouring CNVs in thin-tailed sheep were involved in the adaptive immune response, regulation of reactive oxygen species biosynthetic process and response to starvation. In fat-tailed breeds these genes were involved in cellular protein modification process, regulation of heart rate, intestinal absorption, olfactory receptor activity and ATP binding. Association test identified one copy gained CNVR on chromosomes 6 harbouring two protein-coding genes HGFAC and LRPAP1. Our findings provide information about genomic structural changes and their association to the interested traits including fat deposition and environmental compatibility in sheep.

Immunity and growth improvement of rainbow trout (Oncorhynchus mykiss) fed dietary nettle (Urtica dioica) against experimental challenge with Saprolegnia parasitica.

In this study, effects of nettle (Urtica dioica) on growth, immunity, and gene expressions were examined in rainbow trout after an 8-week feeding period. A total of 264 juvenile rainbow trout (10.72 ± 0.55 g) were selected and stocked randomly in 12 aquaria. Nettle powder was added to the fish feed at three doses, 0.5,1 and 1.5% served as treatments. At the end of 8-week feeding period, the fish were exposed to Saprolegnia parasitica for 3 weeks. Results showed that all treatments fed with nettle diets exhibited significant increases in weight gain and SGR, and decreased FCR compared to the control. Feeding the fish with dietary nettle resulted in significant rises in blood indices and non-specific immunity in comparison with the control. Furthermore, fish fed 0.5% of dietary nettle showed significantly increased expressions of TNF-α, IL-1b, IL-6 and IL-8 genes following 8 weeks of feeding. A significant reduction in mortality rate was observed in the fish treated with 0.5% of nettle compared to the control following challenging with S. parasitica. Our observations indicate that the use of 0.5% nettle powder in rainbow trout diet can improve growth and immunity parameters as well as fish resistance against S. parasitica contamination.

Cloning, expression and characterization of a novel alkaline serine protease gene from native Iranian Bacillus sp.; a producer of protease for use in livestock.

The use of proteases in the last decade has been welcomed in livestock and poultry industries and has led to significant results such as improved feed conversion ratio, weight gain and increased growth performance. In the present study, isolation and identification of a novel alkaline protease from Iranian Bacillus species was performed in order to use in livestock feed. After primary isolation of bacteria from soil samples of rice fields and early detection of bacterial genus, the zymogram plate was performed for evaluation of production extracellular proteases. Of the 11 strains producing protease, one strain that produced more enzymes was selected to continue the work. Characterization of alkaline protease was done using specific enzyme assays. To confirm the genus of isolates as well as to identify the species close to, molecular analysis of 16S rRNA gene sequence was done. After that, bioinformatics analysis carried out in NCBI database for searching bacterial alkaline proteases gene sequences. The primer designed based on gene homology of close species for extraction of alkaline protease gene. The results showed that the enzyme extract had the highest activity at pH 9.0 and 50 °C. The 16S rRNA gene sequence was submitted for the strain called Bacillus sp. RAM on the NCBI site. According to the results of the phylogenetic tree, the bacterium was belonged to Bacillus genus and Bacillus sp. RAM was close to Thuringiensis C405. The isolated alkaline protease gene successfully cloned in pET28a and transferred to the expression host E.coli BL21. The expression of the protease gene was evaluated by SDS-PAGE electrophoresis. The induced recombinant cells expressed the protease and revealed molecular weight band of about 38 kDa. According to the enzyme properties, this alkaline protease can useful for application in animal industry.

Polymorphism detection of promoter region of IFN-γ and IL-2 genes and their association with productive traits in Mazandaran native breeder fowls.

To identify polymorphism in interferon gamma (IFN-γ) and interleukin-2 (IL-2) genes, blood samples were collected from 380 breeder hens of the Mazandaran native fowls breeding station. DNA extraction was performed through a modified saltingout method and fragments of 670 and 659 bp from the promoter regions of IFN-γ and IL-2 genes were amplified by using specific primers, respectively. Following genotyping in the IFN-γ gene using the Tsp509I restriction enzyme, two alleles of A and G with the frequencies of 0.55 and 0.45 and three genotypes of AA, AG and GG were observed with the frequencies of 0.32, 0.46 and 0.22, respectively. For the IL-2 gene, two alleles of A and G were also detected using the MnlI restriction enzyme with the frequencies of 0.58 and 0.42 and three genotypes of AA, AG and GG with the frequencies of 0.33, 0.50 and 0.17, respectively. Statistical analysis revealed significant associations between IL-2 gene single-nucleotide polymorphism and productive traits including the average egg weight (EW) at 345-375 days of age, egg number (EN) at 345-375 days of age and body weight (BW) at 8weeks of age traits (P<0.05). Further, in a mean comparison analysis, there were also significant differences between different genotypes of the IL-2 gene in average EWat 28 and 30weeks of age, in which AG genotypes showed higher performance. Additionally, for the IFN-γ gene, a significant difference was found between the genotypes in average EW at 28 weeks of age trait. Therefore, it can be concluded that the above-mentioned polymorphisms could be considered as the pivotal geneticmakers to improveMazandaran native fowl breeding programmes to achieve the optimum performance in productive traits more efficiently.

A genome scan for quantitative trait loci affecting average daily gain and Kleiber ratio in Baluchi Sheep.

Genomewide association study (GWAS) is an efficient tool for the detection of SNPs and candidate genes in quantitative traits. Growth rate is an important trait for increasing the meat production in sheep. A total of 96 Baluchi sheep were genotyped using Illumina Ovine SNP50 BeadChip to run a GWAS for an average daily gain (ADG) and Kleiber ratio (KR) traits in different periods of age in sheep. Traits included were average daily gain from birth to three months (ADG0-3), from three months to six months (ADG3-6), from six months to nine months (ADG6-9), from nine months to yearling (ADG9-12), from birth to six months (ADG0-6), from three months to nine months (ADG3-9), from three months to yearling (ADG3-12) and corresponding Kleiber ratios (KR0-3, KR3-6, KR6-9, KR9-12, KR0-6, KR3-9 and KR3-12, respectively). A total of 42,243 SNPs passed the quality-control filters and were analysed by PLINK software in a linear mixed model. Two SNPs were identified on two chromosomes at the 5% genomewide significance level for KR(3-9) and KR(6-9). Two candidate genes, namely MAGI1 and ZNF770, were identified correspondingly harbouring and close to these QTL. Also, a total of 21 SNPs were found on chromosomes 2, 3, 5, 6, 7, 10, 17, 19, 20 and 25 at the 5% chromosomewide significance level for ADG and KR traits. Thus, we suggest more studies to discover the causative variants for growth traits in sheep.

Detection of QTL for greasy fleece weight in sheep using a 50 K single nucleotide polymorphism chip.

Genome-wide association studies (GWAS) have introduced an influential tool in the search for quantitative trait loci (QTL) influencing economically important traits in sheep. To identify QTL associated with greasy fleece weight, a GWAS with 50 K single nucleotide polymorphisms (SNPs) was performed in a Baluchi sheep population. Association with greasy fleece weights was tested using the software Plink. The results of our GWAS provided three novel SNP markers and candidate genes associated with greasy fleece weight. A total of three chromosome-wide significant associations were detected for SNP on chromosomes 17 and 20 affecting greasy fleece weight across the four shearing. One of the significant SNP markers was located within ovine known genes namely FAM101A. Further investigation of these identified regions in validation studies will facilitate the identification of strong candidate genes for wool production in sheep.

Cloning and comparative analysis of gene structure in promoter site of alpha-s1 casein gene in Naeinian goat and sheep.

The 5' end or alpha-S1 casein promoter has a significant role in milk protein gene expression. The understanding of the translation process of alpha-S1 casein mutants will provide us an opportunity to make the best selection in livestock providing more proteins in milk. Blood samples were taken from three hundred of Naeinian goats and sheep, and DNA extraction was done using modified salting out method. Polymerase chain reactions (PCR) were carried out using a specific primer pairs for amplification a fragment of 1133 bp from part of 5'-UTR and exon 1 of alpha s1 casein gene. The AluI and HinfI restriction enzyme treatment of all samples provided the same homozygous AA genotype in both species. Subsequently, one sample of each species was selected and cloned, and the final sequences were analyzed by BioEdit, CLC genomic, Mega4 and DNASIS MAX software. Several polymorphisms are recognized between Naeinian goat and sheep that are presented on motif sites. In this research, the interested location, including exon I and a part of 5', was analyzed, and genetic element comparisons were done between Naeinian goat and sheep. The number and location of probable binding sites can have a crucial role as a result of antagonistic and synergistic effects on gene regulation activities.