An updated Axiom buffalo genotyping array map and mapping of cattle quantitative trait loci to the new water buffalo reference genome assembly.

The objectives of this study were to provide the buffalo research community with an updated SNP map for the Axiom Buffalo Genotyping (ABG) array with genomic positions for SNP currently unmapped and to map all cattle QTL from the CattleQTLdb onto the buffalo reference assembly. To update the ABG array map, all SNP probe sequences from the ABG array were re-aligned against the UOA_WB_1 assembly. With the new map, the number of mapped markers increased by approximately 10% and went from 106 778 to 116 708, which reduced the average marker spacing by approximately 2 kb. A comparison of results between signatures of autozygosity study using the ABG and the new map showed that, when the additional markers were used there was an increase in the autozygosity peaks and additional peaks in BBU5 and BBU11 could be identified. After sequence alignment and quality control, 64 650 (UMD3.1) and 76 530 (ARS_UCD1.2) cattle QTL were mapped onto the buffalo genome. The mapping of the bovine QTL database onto the buffalo genome should be useful for genome-wide association studies in buffalo and, given the high homology between the two species, the positions of cattle QTL on the buffalo genome can serve as a stepping stone towards a water buffalo QTL database.

Inbreeding coefficients and runs of homozygosity islands in Brazilian water buffalo.

Characterization of autozygosity is relevant to monitor genetic diversity and manage inbreeding levels in breeding programs. Identification of autozygosity hotspots can unravel genomic regions targeted by selection for economically important traits and can help identify candidate genes for selection. In this study, we estimated the inbreeding levels of a Brazilian population of Murrah buffalo undergoing selection for milk production traits, particularly milk yield. We also studied the distribution of runs of homozygosity (ROH) islands and identified putative genes and quantitative trait loci (QTL) under selection. We genotyped 422 Murrah buffalo for 51,611 SNP; 350 of these had ROH longer than 10 Mb, indicating the occurrence of inbreeding in the last 5 generations. The mean length of the ROH per animal was 4.28 ± 1.85 Mb. Inbreeding coefficients were calculated from the genomic relationship matrix, the pedigree, and the ROH, with estimates varying between 0.242 and 0.035. Inbreeding estimates from the pedigree had a low correlation with the genomic estimates, and estimates from the genomic relationship matrix were much higher than those from the pedigree or the ROH. Signatures of selection were identified in 6 genomic regions, located on chromosomes 1, 2, 3, 5, 16, and 18, encompassing a total of 190 genes and 174 QTL. Many of the genes (e.g., APRT and ACSF3) and QTL identified are related to milk production traits, such as milk yield, milk fat yield and percentage, and milk protein yield and percentage. Other genes are associated with reproduction and immune response traits as well as morphological aspects of the buffalo species. Inbreeding levels in this population are still low but are increasing due to selection and should be managed to avoid future losses due to inbreeding depression. The proximity of genes linked to milk production traits with genes associated with reproduction and immune system traits suggests the need to include these latter genes in the breeding program to avoid negatively affecting them due to selection for production traits.

Genome-wide association study of four composite reproductive traits in Iranian fat-tailed sheep.

Composite reproductive traits are a combination of growth and reproductive traits. They have the advantage of being better attuned to the market drivers since producers are paid on a per kilogram basis and not on a per head basis. In this study, 124 Lori---Bakhtiari ewes were genotyped using the medium-density Illumina Ovine SNP50 array. A genome-wide association study was performed on estimated breeding values of four composite reproductive traits and genetic parameters were also estimated. The traits were litter mean weight at birth, litter mean weight at weaning, total litter weight at birth and total litter weight at weaning. Several suggestive and associated single nucleotide polymorphisms (SNPs) were identified. Neighbouring the top SNPs there were five genes, inhibin ß E subunit (INHBE), inhibin ß C subunit (INHBC), testis expressed 12 (TEX12), ß-carotene oxygenase 2 (BCO2) and WD Repeat Domain 70 (WDR70) identified as possible candidate genes for composite reproductive traits of the Lori-Bakhtiari sheep. These genes are in pathways known to be relevant to fertility and growth characteristics. The results provide new information for the functional annotation of genes associated with fertility traits and add new evidence towards a consensus of quantitative trait loci associated with reproductive traits in sheep.

Genome-wide association study to identify genomic regions affecting prolificacy in Lori-Bakhtiari sheep.

Several causative mutations in candidate genes affecting prolificacy have been detected in various sheep breeds. A genome-wide association study was performed on estimated breeding values for litter size in Lori-Bakhtiari sheep. Prolific ewes with twinning records and others with only singleton records were genotyped using the medium-density Illumina Ovine SNP50 array. Four single nucleotide polymorphisms (SNPs) associated with litter size were identified on chromosomes 3, 6 and 22. The region on sheep chromosome 3 between 75 739 167 and 75 745 152 bp included two significant SNPs (s52383.1 and OAR3_80038014_X.1) in high linkage disequilibrium with each other. The region that surrounds these SNPs contains a novel putative candidate gene: luteinizing hormone/choriogonadotropin receptor (LHCGR), known to be involved in ovarian steroidogenesis and organism-specific biosystem pathways in sheep. Known prolificacy genes BMPR1B, BMP15 and GDF9 were not associated with litter size in Lori-Bakhtiari sheep, suggesting that other biological mechanisms could be responsible for the trait's variation in this breed.

Peripheral cellular and humoral responses to infestation with the cattle tick Rhipicephalus microplus in Santa Gertrudis cattle.

Resistance to cattle tick infestation in single-host ticks is primarily manifested against the larval stage and results in the immature tick failing to attach successfully and obtain a meal. This study was conducted to identify immune responses that characterize the tick-resistant phenotype in cattle. Thirty-five tick-naïve Santa Gertrudis heifers were used in this study, thirty of which were artificially infested for thirteen weeks with tick larvae while five animals remained at a tick-free quarantine property to serve as a control group. Following thirteen weeks of tick infestation, the animals in this trial exhibited highly divergent tick-resistant phenotypes. Blood samples collected throughout the trial were used to measure peripheral immune parameters: haematology, the percentage of cellular subsets comprising the peripheral blood mononuclear cell (PBMC) population, tick-specific IgG1 and IgG2 antibody titres, IgG1 avidity for tick antigens and the ability of PBMC to recognize and proliferate in response to stimulation with tick antigens in vitro. The tick-susceptible cattle developed significantly higher tick-specific IgG1 antibody titres compared to the tick-resistant animals. These results suggest that the heightened antibody response either does not play a role in resistance or might contribute to increased susceptibility to infestation.

Gene expression analysis of blood, liver, and muscle in cattle divergently selected for high and low residual feed intake.

Improving feed efficiency in cattle is important because it increases profitability by reducing costs, and it also shrinks the environmental footprint of cattle production by decreasing manure and greenhouse gas emissions. Residual feed intake (RFI) is 1 measurement of feed efficiency and is the difference between actual and predicted feed intake. Residual feed intake is a complex trait with moderate heritability, but the genes and biological processes associated with its variation still need to be found. We explored the variation in expression of genes using RNA sequencing to find genes whose expression was associated with RFI and then investigated the pathways that are enriched for these genes. In this study, we used samples from growing Angus bulls (muscle and liver tissues) and lactating Holstein cows (liver tissue and white blood cells) divergently selected for low and high RFI. Within each breed-tissue combination, the correlation between the expression of genes and RFI phenotypes, as well as GEBV, was calculated to determine the genes whose expression was correlated with RFI. There were 16,039 genes expressed in more than 25% of samples in 1 or more tissues. The expression of 6,143 genes was significantly associated with RFI phenotypes, and expression of 2,343 genes was significantly associated with GEBV for RFI ( < 0.05) in at least 1 tissue. The genes whose expression was correlated with RFI phenotype (or GEBV) within each breed-tissue combination were enriched for 158 (78) biological processes (Fisher Exact Statistics for gene-enrichment analysis, EASE score < 0.1) and associated with 13 (13) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways ( < 0.05 and fold enrichment > 2). These biological processes were related to regulation of transcription, translation, energy generation, cell cycling, apoptosis, and proteolysis. However, the direction of the correlation between RFI and gene expression in some cases reversed between tissues. For instance, low levels of proteolysis in muscle were associated with high efficiency in growing bulls, but high levels of proteolysis in white blood cells were associated with efficiency of milk production in lactating cows.

Genomewide association analysis of growth traits in Charolais beef cattle.

The objective of this study was to perform a genomewide association study (GWAS) for growth traits in Charolais beef cattle and to identify SNP markers and genes associated with these traits. Our study included 855 animals genotyped using 76,883 SNP from the GeneSeek Genomic Profiler Bovine HD panel. The examined phenotypic data included birth, weaning, and yearling weights as well as pre- and postweaning ADG. After quality control, 68,337 SNP and 823 animals were retained in the analysis. The association analysis was performed using the principal components method via the egscore function of the GenABEL version 1.8-0 package in the R environment. Eighteen SNP located in 13 BTA were associated with growth traits ( < 5 × 10). The most important genes in these genomic regions were (), (), (), (), and ( [angiotensinase C]), due to their relationships with perinatal and postnatal survival, bone growth, cell adhesion, regulation of adipogenesis, and appetite. In conclusion, this study is the first to describe a GWAS conducted in beef cattle in Mexico and represents a basis for further and future research. This study detected new QTL associated with growth traits and identified 5 positional and functional candidate genes that are potentially involved in variations of the analyzed traits. Future analyses of these regions could help to identify useful markers for marker-assisted selection and will contribute to the knowledge of the genetic basis of growth in cattle and be a foundation for genomic predictions in Mexican Charolais cattle.

How many markers are enough? Factors influencing parentage testing in different livestock populations.

Reliability of parentage test panels is usually based on its power to exclude wrong parentage assignments based on allele frequencies. We evaluated the rates of false exclusions and inclusions in parentage assignments, and how these results are affected by allele frequencies, panel sizes and the number of allowed mismatches. We also evaluated the reliability of parentage testing by comparing populations with distinct genetic backgrounds using pure and composite families of cattle and sheep. Allowing for 1% genotype mismatches in true parent-offspring relations provided the best compromise between false-positive and false-negative assignments. Pure breeds needed at least 200-210 single-nucleotide polymorphism (SNP) markers to correctly assign relations, but between 700 and 890 markers to avoid assigning incorrect relationships. Composite breeds needed between 220 (sheep) and 500 (cattle) markers for correct assignment; 680 (cattle) to 4400 (sheep) SNPs were needed to eliminate false-positive assignments. Allowing 0% genotype mismatches decreased false-positive but increased false-negative assignments, whilst a higher threshold of 2% showed the opposite effects. Panels with high minor allele frequencies (0.35-0.45) provided the best chance for correct parentage resolutions requiring fewer markers. Further, we propose that a dynamic threshold would allow adapting to population specific error rates. A comparison to the performance of the official International Society for Animal Genetics SNP panel for cattle and a recently published SNP panel for sheep showed that randomly selected markers performed only slightly worse for the applied parentage test based on opposing homozygotes. This suggests that even with carefully selected panels, only marginal assignment improvements are obtainable for a particular number of SNPs. The main point for improvement is the number of markers used. We recommend using at least 200 SNP markers for parentage testing if the aim is to reduce false-negative results. To fully exclude false positives at least 700 markers are required.

Performance of different SNP panels for parentage testing in two East Asian cattle breeds.

The International Society for Animal Genetics (ISAG) proposed a panel of single nucleotide polymorphisms (SNPs) for parentage testing in cattle (a core panel of 100 SNPs and an additional list of 100 SNPs). However, markers specific to East Asian taurine cattle breeds were not included, and no information is available as to whether the ISAG panel performs adequately for these breeds. We tested ISAG's core (100 SNP) and full (200 SNP) panels on two East Asian taurine breeds: the Korean Hanwoo and the Japanese Wagyu, the latter from the Australian herd. Even though the power of exclusion was high at 0.99 for both ISAG panels, the core panel performed poorly with 3.01% false-positive assignments in the Hanwoo population and 3.57% in the Wagyu. The full ISAG panel identified all sire-offspring relations correctly in both populations with 0.02% of relations wrongly excluded in the Hanwoo population. Based on these results, we created and tested two population-specific marker panels: one for the Wagyu population, which showed no false-positive assignments with either 100 or 200 SNPs, and a second panel for the Hanwoo, which still had some false-positive assignments with 100 SNPs but no false positives using 200 SNPs. In conclusion, for parentage assignment in East Asian cattle breeds, only the full ISAG panel is adequate for parentage testing. If fewer markers should be used, it is advisable to use population-specific markers rather than the ISAG panel.

Genomic architecture of histone 3 lysine 27 trimethylation during late ovine skeletal muscle development.

The ruminant developmental transition from late foetus to lamb is associated with marked changes in skeletal muscle structure and function that reflect programming for new physiological demands following birth. To determine whether epigenetic changes are involved in this transition, we investigated the genomic architecture of the chromatin modification, histone 3 lysine 27 trimethylation (H3K27me3), which typically regulates early life developmental processes; however, its role in later life processes is unclear. Chromatin immunoprecipitation coupled with next-generation sequencing was used to map H3K27me3 nucleosomes in ovine longissimus lumborum skeletal muscle at 100 days of gestation and 12 weeks post-partum. In both states, H3K27me3 modification was associated with genes, transcription start sites and CpG islands and with transcriptional silencing. The H3K27me3 peaks consisted of two major categories, promoter specific and regional, with the latter the dominant feature. Genes encoding homeobox transcription factors regulating early life development and genes involved in neural functions, particularly gated ion channels, were strongly modified by H3K27me3. Gene promoters differentially modified by H3K27me3 in the foetus and lamb were enriched for gated ion channels, which may reflect changes in neuromuscular function. However, most modified genes showed no changes, indicating that H3K27me3 does not have a large role in late muscle maturation. Notably, promyogenic transcription factors were strongly modified with H3K27me3 but showed no differences between the late gestation foetus and lamb, likely reflecting their lack of involvement in the myofibre fusion process occurring in this transition. H3K27me3 is a major architectural feature of the epigenetic landscape of ruminant skeletal muscle, and it comments on gene transcription and gene function in the context of late skeletal muscle development.

Genome-wide detection of signatures of selection in Korean Hanwoo cattle.

The Korean Hanwoo cattle have been intensively selected for production traits, especially high intramuscular fat content. It is believed that ancient crossings between different breeds contributed to forming the Hanwoo, but little is known about the genomic differences and similarities between other cattle breeds and the Hanwoo. In this work, cattle breeds were grouped by origin into four types and used for comparisons: the Europeans (represented by six breeds), zebu (Nelore), African taurine (N'Dama) and Hanwoo. All animals had genotypes for around 680 000 SNPs after quality control of genotypes. Average heterozygosity was lower in Nelore and N'Dama (0.22 and 0.21 respectively) than in Europeans (0.26-0.31, with Shorthorn as outlier at 0.24) and Hanwoo (0.29). Pairwise FST analyses demonstrated that Hanwoo are more related to European cattle than to Nelore, with N'Dama in an intermediate position. This finding was corroborated by principal components and unsupervised hierarchical clustering. Using genome-wide smoothed FST , 55 genomic regions potentially under positive selection in Hanwoo were identified. Among these, 29 were regions also detected in previous studies. Twenty-four regions were exclusive to Hanwoo, and a number of other regions were shared with one or two of the other groups. These regions overlap a number of genes that are related to immune, reproduction and fatty acid metabolism pathways. Further analyses are needed to better characterize the ancestry of the Hanwoo cattle and to define the genes responsible to the identified selection peaks.

Expression of candidate genes for residual feed intake in Angus cattle.

Residual feed intake (RFI) has been adopted in Australia for the purpose of genetic improvement in feed efficiency in beef cattle. RFI is the difference between the observed feed intake of an animal and the predicted feed intake based on its size and growth rate over a test period. Gene expression of eight candidate genes (AHSG, GHR, GSTM1, INHBA, PCDH19, S100A10, SERPINI2 and SOD3), previously identified as differentially expressed between divergent lines of high- and low-RFI animals, was measured in an unselected population of 60 steers from the Angus Society Elite Progeny Test Program using quantitative real-time PCR. Results showed that the levels of gene expression were significantly correlated with RFI. The genes explain around 33.2% of the phenotypic variance in RFI, and prediction equations using the expression data are reasonably accurate estimators of RFI. The association of these genes with economically important traits, such as other feed efficiency-related traits and fat, growth and carcass traits, was investigated as well. The expression of these candidate genes was significantly correlated with feed conversion ratio and daily feed intake, which are highly associated with RFI, suggesting a functional role for these genes in modulating feed utilisation. The expression of these genes did not show any association with average daily gain, eye muscle area and carcass composition.

Heat stress effects on the cumulus cells surrounding the bovine oocyte during maturation: altered matrix metallopeptidase 9 and progesterone production.

When the effects of heat stress are detrimental during maturation, cumulus cells are intimately associated with the oocyte. To determine the extent to which heat stress affects these cells, in this study, transcriptome profiles of the cumulus that surrounded control and heat-stressed oocytes (41 °C during the first 12 h only and then shifted back to 38.5 °C) during in vitro maturation (IVM) were compared using Affymetrix bovine microarrays. The comparison of cumulus-derived profiles revealed a number of transcripts whose levels were increased (n=11) or decreased (n=13) ≥ twofold after heat stress exposure (P<0.01), sufficient to reduce the development of blastocysts by 46.4%. In a separate study, quantitative PCR (qPCR) was used to confirm heat-induced differences in the relative abundances of the transcripts of five different genes (caveolin 1, matrix metallopeptidase 9, FSH receptor, Indian hedgehog homolog, and inducible nitric oxide synthase). Heat stress exposure resulted in >1.7-fold decrease in the protein levels of latent matrix metallopeptidase 9 (proMMP9). Heat-induced reductions in transcript levels were noted at 6 h IVM with reductions in proMMP9 protein levels at 18 h IVM (P=0.0002). Independent of temperature, proMMP9 levels at 24 h IVM were positively correlated with the development rate of blastocysts (R²=0.36; P=0.002). The production of progesterone increased during maturation; heat-induced increases were evident by 12 h IVM (P=0.002). Both MMP9 and progesterone are associated with the developmental competence of the oocyte; thus, it seems plausible for some of the negative consequences of heat stress on the cumulus-oocyte complex to be mediated through heat-induced perturbations occurring in the surrounding cumulus.

Impact of fasting on the rhythmic expression of myogenic and metabolic factors in skeletal muscle of adult mice.

Circadian rhythms and metabolism are tightly integrated, and rhythmic expression of metabolic factors is common in homeostatic processes. We measured the temporal changes in the expression of myogenic regulatory factors and expression and activity level of molecules involved in protein metabolism in skeletal muscles and livers in mice and examined the impact of fasting. Tissues were collected over 24 h (at zeitgeber times ZT1, ZT5, ZT9, ZT13, ZT17, ZT21, and ZT1 the following day) from adult male C57Bl/6J mice that had been either freely fed or fasted for 24 h. In skeletal muscle, there was a robust rise in the mRNA expression of the myogenic regulatory factors MyoD and myogenin during dark hours which was strongly suppressed by fasting. Circadian pattern was observed for mRNA of MuRF1, Akt1, and ribosomal protein S6 in muscles in fed and fasted mice and for Fbxo32 in fed mice. Activity (phosphorylation) levels of Akt(Ser473) displayed temporal regulation in fasted (but not fed) mice and were high at ZT9. Fasting caused significant reductions in phosphorylation for both Akt and S6 in muscles, indicative of inactivation. Hepatic phosphorylated Akt(Ser473) and S6(Ser235/236) proteins did not exhibit daily rhythms. Fasting significantly reduced hepatic Akt(473) phosphorylation compared with fed levels, although (unlike in muscle) it did not affect S6(Ser235/236) phosphorylation. This in vivo circadian study addresses for the first time the signaling activities of key molecules related to protein turnover and their possible cross-regulation of expression of genes related to protein degradation.

Global gene expression profiling reveals genes expressed differentially in cattle with high and low residual feed intake.

Feed efficiency is an economically important trait in beef production. It can be measured as residual feed intake. This is the difference between actual feed intake recorded over a test period and the expected feed intake of an animal based on its size and growth rate. DNA-based marker-assisted selection would help beef breeders to accelerate genetic improvement for feed efficiency by reducing the generation interval and would obviate the high cost of measuring residual feed intake. Although numbers of quantitative trait loci and candidate genes have been identified with the advance of molecular genetics, our understanding of the physiological mechanisms and the nature of genes underlying residual feed intake is limited. The aim of the study was to use global gene expression profiling by microarray to identify genes that are differentially expressed in cattle, using lines genetically selected for low and high residual feed intake, and to uncover candidate genes for residual feed intake. A long-oligo microarray with 24 000 probes was used to profile the liver transcriptome of 44 cattle selected for high or low residual feed intake. One hundred and sixty-one unique genes were identified as being differentially expressed between animals with high and low residual feed intake. These genes were involved in seven gene networks affecting cellular growth and proliferation, cellular assembly and organization, cell signalling, drug metabolism, protein synthesis, lipid metabolism, and carbohydrate metabolism. Analysis of functional data using a transcriptional approach allows a better understanding of the underlying biological processes involved in residual feed intake and also allows the identification of candidate genes for marker-assisted selection.

QTL and gene expression analyses identify genes affecting carcass weight and marbling on BTA14 in Hanwoo (Korean Cattle).

Causal mutations affecting quantitative trait variation can be good targets for marker-assisted selection for carcass traits in beef cattle. In this study, linkage and linkage disequilibrium analysis (LDLA) for four carcass traits was undertaken using 19 markers on bovine chromosome 14. The LDLA analysis detected quantitative trait loci (QTL) for carcass weight (CWT) and eye muscle area (EMA) at the same position at around 50 cM and surrounded by the markers FABP4SNP2774C>G and FABP4_µsat3237. The QTL for marbling (MAR) was identified at the midpoint of markers BMS4513 and RM137 in a 3.5-cM marker interval. The most likely position for a second QTL for CWT was found at the midpoint of tenth marker bracket (FABP4SNP2774C>G and FABP4_µsat3237). For this marker bracket, the total number of haplotypes was 34 with a most common frequency of 0.118. Effects of haplotypes on CWT varied from a -5-kg deviation for haplotype 6 to +8 kg for haplotype 23. To determine which genes contribute to the QTL effect, gene expression analysis was performed in muscle for a wide range of phenotypes. The results demonstrate that two genes, LOC781182 (p = 0.002) and TRPS1 (p = 0.006) were upregulated with increasing CWT and EMA, whereas only LOC614744 (p = 0.04) has a significant effect on intramuscular fat (IMF) content. Two genetic markers detected in FABP4 were the most likely QTL position in this QTL study, but FABP4 did not show a significant effect on both traits (CWT and EMA) in gene expression analysis. We conclude that three genes could be potential causal genes affecting carcass traits CWT, EMA, and IMF in Hanwoo.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of ∼50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (∼100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Genetic mapping of quantitative trait loci for resistance to Haemonchus contortus in sheep.

This paper presents results from a mapping experiment to detect quantitative trait loci (QTL) for resistance to Haemonchus contortus infestation in merino sheep. The primary trait analysed was faecal worm egg count in response to artificial challenge at 6 months of age. In the first stage of the experiment, whole genome linkage analysis was used for broad-scale mapping. The animal resource used was a designed flock comprising 571 individuals from four half-sib families. The average marker spacing was about 20 cM. For the primary trait, 11 QTL (as chromosomal/family combinations) were significant at the 5% chromosome-wide level, with allelic substitution effects of between 0.19 and 0.38 phenotypic standard deviation units. In general, these QTL did not have a significant effect on faecal worm egg count recorded at 13 months of age. In the second stage of the experiment, three promising regions (located on chromosomes 1, 3 and 4) were fine-mapped. This involved typing more closely spaced markers on individuals from the designed flock as well as an additional 495 individuals selected from a related population with a deeper pedigree. Analysis was performed using a linkage disequilibrium-linkage approach, under additive, dominant and multiple QTL models. Of these, the multiple QTL model resulted in the most refined QTL positions, with resolutions of <10 cM achieved for two regions. Because of the moderate size of effect of the QTL, and the apparent age and/or immune status specificity of the QTL, it is suggested that a panel of QTL will be required for significant genetic gains to be achieved within industry via marker-assisted selection.

Sequential microarray to identify timing of molecular responses to Haemonchus contortus infection in sheep.

Anthelmintics are currently the most common method of worm control. The emergence of worms with multiple-drug resistance and issues of residues in the food chain make alternative parasite control measures a priority. To develop improved and sustainable methods for controlling Haemonchus contortus such as genetic selection of resistant sheep, a better understanding of the host-parasite relationship is required. A trial was undertaken using sheep surgically implanted with abomasal fistulas to enable sequential biopsy of the abomasal mucosa during trickle infection with two strains of H. contortus. These were ivermectin-resistant CAVR and ivermectin-sensitive McMaster. From a gross parasitology perspective, this approach enabled the effect of developing immunity to be observed on both the establishment and maturation of two CAVR doses within and between groups. Since the only difference in parasite treatment between the groups was the staggering of the two CAVR doses, microarray results from biopsies taken on the same day in different groups were combined and compared between different biopsy dates to observe differential gene transcription over time. Differential gene transcription was detected by comparing transcription in our array data between different biopsy dates using a low P value screen (P<0.01) and by compiling a list of 82 immunoparasitology-related genes and examining transcription in this list with a higher P value screen (P<0.05). Our microarray data were validated in silico by comparison with intelectin 2, trefoil factor 3, calcium activated chloride channel and mucin 5 from other gene transcription studies and with phenotypic data such as the response by gammadelta T cells and immunoglobulins to H. contortus. The first four genes are involved in non-specific responses to infection and mucosal healing. These were upregulated at the early time points and intelectin 2 remained prominent throughout the trial. As the trial progressed, immunoglobulin genes became strongly upregulated. These included IgCgamma IgG2a heavy chain constant region, IGHE immunoglobulin heavy constant epsilon and IGHM immunoglobulin heavy constant mu.

Microarray analysis of acaricide-inducible gene expression in the southern cattle tick, Rhipicephalus (Boophilus) microplus.

Acaricide-inducible differential gene expression was studied in larvae of Rhipicephalus (Boophilus) microplus using a microarray-based approach. The acaricides used were: coumaphos, permethrin, ivermectin, and amitraz. The microarrays contained over 13 000 probes, having been derived from a previously described R. microplus gene index (BmiGI Version 2; Wang et al., 2007). Relative quantitative reverse transcriptase-PCR, real time PCR, and serial analysis of gene expression data was used to verify microarray data. Among the differentially expressed genes with informative annotation were legumain, glutathione S-transferase, and a putative salivary gland-associated protein.