SNP discovery and population structure analysis in Lassi and Marecha camel breeds using a genotyping by sequencing method.

Pakistani camels have been classified socio-geographically into 20 breeds, but they have not yet been subjected to substantial selective pressures and the genetic basis for these breeds is not understood. However, it should be possible to distinguish them by use of molecular data. This study investigated the genetic diversity and population structure within and between two major Pakistani camel breeds, Marecha and Lassi. As no SNP array is currently available, we first identified 63 619 SNPs using a genotyping by sequencing approach. After quality control, a panel of 36 926 SNPs was used in the analysis. Population structure was investigated with a principal coordinate analysis as well as a cluster analysis using NetView, and multilocus heterozygosity analysis to explore between- and within-breed genetic variation. In addition, between-breed variation was explored using the fixation index, FST . We also compared relationship matrices computed using the VanRaden SNP-based method and a method developed specifically for genotyping by sequencing data. Among the two camel breeds, Lassi showed a lower level of genetic diversity whereas Marecha showed a higher level. As a genotyping platform has not yet been developed for the camel, the SNPs discovered in this study will be useful in future genetic studies in camels.

Using genotyping-by-sequencing to predict gender in animals.

Gender assignment errors are common in some animal species and lead to inaccuracies in downstream analyses. Procedures for detecting gender misassignment are available for array-based SNP data but are still being developed for genotyping-by-sequencing (GBS) data. In this study, we describe a method for using GBS data to predict gender using X and Y chromosomal SNPs. From a set of 1286 X chromosomal and 23 Y chromosomal deer (Cervus sp.) SNPs discovered from GBS sequence reads, a prediction model was built using a training dataset of 422 Red deer and validated using a test dataset of 868 Red deer and Wapiti deer. Prediction was based on the proportion of heterozygous genotypes on the X chromosome and the proportion of non-missing genotypes on the Y chromosome observed in each individual. The concordance between recorded gender and predicted gender was 98.6% in the training dataset and 99.3% in the test dataset. The model identified five individuals across both datasets with incorrect recorded gender and was unable to predict gender for another five individuals. Overall, our method predicted gender with a high degree of accuracy and could be used for quality control in gender assignment datasets or for assigning gender when unrecorded, provided a suitable reference genome is available.

Ovine keratome: identification, localisation and genomic organisation of keratin and keratin-associated proteins.

Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin-associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST-derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome.

An ovine hepatorenal fibrocystic model of a Meckel-like syndrome associated with dysmorphic primary cilia and TMEM67 mutations.

Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.

SOLiD SAGE sequencing shows differential gene expression in jejunal lymph node samples of resistant and susceptible red deer (Cervus elaphus) challenged with Mycobacterium avium subsp. paratuberculosis.

This study compared in vivo lymph node gene expression levels between six young red deer that were either relatively resistant (R) or susceptible (S) to paratuberculosis following experimental challenge with Mycobacterium avium subsp. paratuberculosis. Intestinal lymph nodes were biopsied at 4, 12 and 50 weeks post challenge (pc) and parallel changes in histopathology, immunology and bacterial load monitored. SOLiD SAGE (serial analysis of gene expression) next generation sequencing of biopsied lymph node samples generated a total of 373 million transcript tags 26-28bp in length after filtering. A total of 36,632 unique transcripts were identified and 14,325 of these were able to be annotated. The copy number of each transcript was counted, averaged and compared for R and S animals (R-S). P values and False Discovery Rates (FDR) were calculated for each transcript. Genes differentially upregulated ≥2 fold (FDR<0.5) totalled 9, 40 and 32 in R animals (+ values) and 23, 164 and 47 in S animals (- values) at weeks 4, 12, and 50pc, respectively. Transcripts displaying greatest differential expression between R and S animals at each time point were IFIT2 (189 fold) and S100A8 (-32.7 fold) at week 4, LRR1 (52.7 fold), SERPINF2 (-214.6 fold) at week 12 and CEACAM8 (84.6 fold), and STK31 (-129.5 fold) at week 50, respectively. All 9 genes significantly upregulated at week 4 in R animals relate specifically to host defence and all involve Type I interferon stimulated genes. By contrast genes upregulated in S animals at week 4, relate predominantly to inflammation, but also involve adaptive immune responses, mitochondrial function and apoptosis regulation. At week 12, the genes differentially upregulated in R animals are linked predominantly to regulation of adaptive immunity and mucosal immunity, while many of the genes in S animals are associated with pro-inflammatory interleukins involved with innate and adaptive immunity. These correlated with greater lesion severity and higher MAP numbers in lymph nodes of S animals. By week 50 the number of upregulated genes declined in both groups. A number of genes upregulated in R animals appear to be associated with host resistance and regulation of adaptive immunity, especially CEACAM8. Genes upregulated in S animals involve antigen presentation (ENDOD1) and gut associated immune pathology (HSH2D). In conclusion, gene expression in jejunal lymph nodes of resistant and susceptible deer infer that the resistant phenotype is associated with pathways of adaptive immunity, while susceptibility is linked with upregulated non-protective pro-inflammatory responses, following experimental MAP infection.

Genome-wide DNA methylation analysis: no evidence for stable hemimethylation in the sheep muscle genome.

The importance of maintaining DNA methylation patterns and faithful transmission of these patterns during cell division to ensure appropriate gene expression has been known for many decades now. It has largely been assumed that the symmetrical nature of CpG motifs, the most common site for DNA methylation in mammals, together with the presence of maintenance methylases able to methylate newly synthesised DNA, ensures that there is concordance of methylation on both strands. However, although this assumption is compelling in theory, little experimental evidence exists that either supports or refutes this assumption. Here, we have undertaken a genome-wide single-nucleotide resolution analysis to determine the frequency with which hemimethylated CpG sites exist in sheep muscle tissue. Analysis of multiple independent samples provides strong evidence that stably maintained hemimethylation is a very rare occurrence, at least in this tissue. Given the rarity of stably maintained hemimethylation, next-generation sequencing data from both DNA strands may be carefully combined to increase the accuracy with which DNA methylation can be measured at single-nucleotide resolution.

Identifying chromosomal selection-sweep regions in facial eczema selection-line animals using an ovine 50K-SNP array.

Facial eczema (FE) is a hepato-mycotoxicosis found mainly in New Zealand sheep and cattle. When genetics was found to be a factor in FE susceptibility, resistant and susceptible selection lines of Romney sheep were established to enable further investigations of this disease trait. Using the Illumina OvineSNP50 BeadChip, we conducted a selection-sweep experiment on these FE genetic lines. Two analytical methods were used to detect selection signals, namely the Peddrift test (Dodds & McEwan, 1997) and fixation index FST (Weir & Hill, 2002). Of 50 975 single nucleotide polymorphism (SNP) markers tested, there were three that showed highly significant allele frequency differences between the resistant and susceptible animals (Peddrift nominal P < 0.000001). These SNP loci are located on chromosomes OAR1, OAR11 and OAR12 that coincide precisely with the three highest genomic FST peaks. In addition, there are nine less significant Peddrift SNPs (nominal P ≤ 0.000009) on OAR6 (n = 2), OAR9 (n = 2), OAR12, OAR19 (n = 2), OAR24 and OAR26. In smoothed FST (five-SNP moving average) plots, the five most prominent peaks are on OAR1, OAR6, OAR7, OAR13 and OAR19. Although these smoothed FST peaks do not coincide with the three most significant Peddrift SNP loci, two (on OAR6 and OAR19) overlap with the set of less significant Peddrift SNPs above. Of these 12 Peddrift SNPs and five smoothed FST regions, none is close to the FE candidate genes catalase and ABCG2; however, two on OAR1 and one on OAR13 fall within suggestive quantitative trait locus regions identified in a previous genome screen experiment. The present studies indicated that there are at least eight genomic regions that underwent a selection sweep in the FE lines.

The constituents of Microctonus sp. parasitoid venoms.

Purified RNA transcripts from venom glands dissected from the parasitoid wasp Microctonus hyperodae were copied, cloned and sequenced using traditional dideoxy sequencing methods. Using mass spectrometry analysis of the trypsinised PAGE gel protein bands we identified the RNA transcripts for the 3 most abundant proteins found in the venom and hence obtained their full protein sequence. Other abundant transcripts were also further sequenced. To reduce the effort required to obtain transcript information we dissected venom glands from a second parasitoid, Microctonus aethiopoides (Morocco biotype). The RNA transcripts were purified and reverse transcribed but instead of cloning the cDNA it was directly sequenced using Roche GS20 pyrosequencing. Results from a single GS20 sequencing run provided data similar to that obtained by the traditional methods used in analysing transcripts from M. hyperodae in a fraction of the time and cost. Comparing the transcripts between the two species showed that a similar range of genes are expressed with the putative orthologs of seven of the eight full length genes characterised from M. hyperodae being found in M. aethiopoides. Pyrosequencing should provide a valuable new method for rapidly sampling transcripts from a wide range of specialised insect tissues.