Identification of high utility SNPs for population assignment and traceability purposes in the pig using high-throughput sequencing

The objectives of this study were to develop breed-specific single nucleotide polymorphisms (SNPs) in five pig breeds sequenced with Illumina's Genome Analyzer and to investigate their usefulness for breed assignment purposes. DNA pools were prepared for Duroc, Landrace, Large White, Pietrain and Wild Boar. The total number of animals used for sequencing was 153. SNP discovery was performed by aligning the filtered reads against Build 7 of the pig genome. A total of 313,964 high confidence SNPs were identified and analysed for the presence of breed-specific SNPs (defined in this context as SNPs for which one of the alleles was detected in only one breed). There were 29,146 putative breed-specific SNPs identified, of which 4441 were included in the PorcineSNP60 beadchip. Upon re-examining the genotypes obtained using the beadchip, 193 SNPs were confirmed as being breed specific. These 193 SNPs were subsequently used to assign an additional 490 individuals from the same breeds, using the sequenced individuals as reference populations. In total, four breed assignment tests were performed. Results showed that for all methods tested 99% of the animals were correctly assigned, with an average probability of assignment of at least 99.2%, indicating the high utility of breed-specific markers for breed assignment and traceability. This study provides a blueprint for the way next-generation sequencing technologies can be used for the identification of breed-specific SNPs, as well as evidence that these SNPs may be a powerful tool for breed assignment and traceability of animal products to their breeds of origin.     

Role of endogenous interleukin-12 (IL-12) in induced and spontaneous relapses of experimental autoimmune encephalomyelitis in mice.

Actively induced, chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was studied in SJL/J and in Biozzi ABH mice. In Biozzi ABH mice, relapses occurred spontaneously with high frequency. In SJL/J mice, spontaneous relapses occurred infrequently; however they could be induced reproducibly by reimmunization. In both models, moderately increased levels of serum IL-12(p40) were consistently found shortly before primary attacks, but irregularly at later times. Injections of anti-IL-12 antibody inhibited disease development in both SJL/J and in Biozzi ABH mice. The time window during which treatment needed to be initiated in order to be effective, ranged from before induction until shortly before the symptoms of primary attacks emerged. Such treatment inhibited not only the first attack but also the spontaneous or induced relapses. Most significantly, anti-IL-12 antibody given during remission of primary disease inhibited actively re-induced relapses in SJL/J, but not spontaneous relapses in Biozzi ABH mice. These results indicate that endogenous IL-12 favours EAE development by crucially affecting the active induction process, but that a second burst of IL-12 production may not be necessary for triggering spontaneous relapses.     

Improving the comparative map of SSC2p-q13 by sample sequencing of BAC clones

To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.     

In silico identification and mapping of microsatellite markers on sus scrofa chromosome 4

Marker density of a QTL region on pig chromosome 4 was increased. New microsatellites were identified by in silico mining of BAC-end and genomic shotgun sequences. Among 8,784 BAC-end sequences predicted within the region, 148 microsatellites were identified. In addition, 27,450 CA/TG repeats were identified within the genomic shotgun sequences, of which 157 were most likely located on SSC4q. A selection of 61 new microsatellites was mapped, together with previously mapped markers. The results showed that the human-pig comparative map in combination with BAC-end and genomic sequence resources provides an excellent source for a highly efficient and targeted development of markers.     

On the relationship between an Asian haplotype on chromosome 6 that reduces androstenone levels in boars and the differential expression of SULT2A1 in the testis

Background

Androstenone is one of the major compounds responsible for boar taint, a pronounced urine-like odor produced when cooking boar meat. Several studies have identified quantitative trait loci (QTL) for androstenone level on Sus scrofa chromosome (SSC) 6. For one of the candidate genes in the region SULT2A1, a difference in expression levels in the testis has been shown at the protein and RNA level.

Results

Haplotypes were predicted for the QTL region and their effects were estimated showing that haplotype 1 was consistently related with a lower level, and haplotype 2 with a higher level of androstenone. A recombinant haplotype allowed us to narrow down the QTL region from 3.75 Mbp to 1.94 Mbp. An RNA-seq analysis of the liver and testis revealed six genes that were differentially expressed between homozygotes of haplotypes 1 and 2. Genomic sequences of these differentially expressed genes were checked for variations within potential regulatory regions. We identified one variant located within a CpG island that could affect expression of SULT2A1 gene. An allele-specific expression analysis in the testis did not show differential expression between the alleles of SULT2A1 located on the different haplotypes in heterozygous animals. However a synonymous mutation C166T (SSC6: 49,117,861 bp in Sscrofa 10.2; C/T) was identified within the exon 2 of SULT2A1 for which the haplotype 2 only had the C allele which was higher expressed than the T allele, indicating haplotype-independent allelic-imbalanced expression between the two alleles. A phylogenetic analysis for the 1.94 Mbp region revealed that haplotype 1, associated with low androstenone level, originated from Asia.

Conclusions

Differential expression could be observed for six genes by RNA-seq analysis. No difference in the ratio of C:T expression of SULT2A1 for the haplotypes was found by the allele-specific expression analysis, however, a difference in expression between the C over T allele was found for a variation within SULT2A1, showing that the difference in androstenone levels between the haplotypes is not caused by the SNP in exon 2.     

Localization to chicken Chromosome 5 of a novel locus determining salmonellosis resistance

Clear genetic differences in the susceptibility of chickens to visceral infection by Salmonella have been observed and it has been possible to identify resistant and susceptible lines of inbred chickens. We report here the results of experiments to map directly the gene(s) controlling this trait in chickens by examining crosses between highly susceptible and highly resistant lines. In the mapping panel, a region on chicken Chromosome (Chr) 5 was found to have a large effect on resistance, and this effect was observed in three separate resource populations. Mapping of additional marker loci in the region of the resistance gene further localized it to a region of approximately 2 cM, close to the genes for creatine kinase (CKB) and dynein (DNCH1). This region shows conserved synteny with telomeric regions of human Chr 14 and mouse Chr 12. On the basis of this conserved synteny, this resistance gene seems unlikely to correspond to the previously identified salmonellosis resistance genes Lps (located on mouse Chr 4) or Nos(2) (located on mouse Chr 11). There was no association between Nramp1 and resistance in these crosses, although this gene was shown to contribute to resistance in other crosses. The homologous human and mouse regions at present contain no likely candidate genes for this trait. Thus this appears to be a novel resistance gene, which we designate SAL1.     

Molecular evidence places the swallow bug genus Oeciacus Stål within the bat and bed bug genus Cimex Linnaeus (Heteroptera: Cimicidae)

The genera Cimex Linnaeus and Oeciacus Stål (Heteroptera: Cimicidae) are common haematophagous ectoparasites of bats or birds in the Holarctic region. Both their phylogenetic relationship and the systematics of the entire family previously were based on data from morphology and host relationships. Relationships among nine species of the genus Cimex and three species of the genus Oeciacus were analysed here using two mitochondrial and three nuclear genes. Cimex was shown to be paraphyletic with respect to Oeciacus. Oeciacus is thus proposed as a synonym of Cimex. The characteristic phenotype of Oeciacus results from the specific host association with different species of swallows (Hirundinidae). The morphological characters that have been used as diagnostic for the genera were shown to be valid and can be further used for determination at species level. The present analyses recovered the four traditional morphologically defined species groups of the genus Cimex. However, their relationships were poorly resolved – only the C. hemipterus group showed a well‐supported relationship to the C. pipistrelli group. The molecular differentiation within the Palaearctic C. pipistrelli and the Nearctic C. pilosellus species groups correlates with their karyotype differentiation. Furthermore, the analyses suggest poly‐ or paraphyly of the former genus Oeciacus. Either way this indicates there is a large amount of host‐associated phenotypic convergence in either bat‐ or bird‐associated groups of species. The probability of host choice and subsequent switch in Cimicidae are discussed and possible scenarios of the evolution of host association in species of Cimex are suggested.     

An assessment of European pig diversity using molecular markers: Partitioning of diversity among breeds

Genetic diversity within and between breeds (and lines) of pigs was investigated. The sample comprised 68 European domestic breeds (and lines), including 29 local breeds, 18 varieties of major international breeds, namely Duroc, Hampshire, Landrace, Large White and Piétrain, and 21 commercial lines either purebred or synthetic, to which the Chinese Meishan and a sample of European wild pig were added. On average 46 animals per breed were sampled (range 12–68). The genetic markers were microsatellites (50 loci) and AFLP (amplified fragment length polymorphism, 148 loci). The analysis of diversity showed that the local breeds accounted for 56% of the total European between-breed microsatellite diversity, and slightly less for AFLP, followed by commercial lines and international breeds. Conversely, the group of international breeds contributed most to within-breed diversity, followed by commercial lines and local breeds. Individual breed contributions to the overall European between- and within-breed diversity were estimated. The range in between-breed diversity contributions among the 68 breeds was 0.04–3.94% for microsatellites and 0.24–2.94% for AFLP. The within-breed diversity contributions varied very little for both types of markers, but microsatellite contributions were negatively correlated with the between-breed contributions, so care is needed in balancing the two types of contribution when making conservation decisions. By taking into account the risks of extinction of the 29 local breeds, a cryopreservation potential (priority) was estimated for each of them.     

The imprinted gene DIO3 is a candidate gene for litter size in pigs

Genomic imprinting is an important epigenetic phenomenon, which on the phenotypic level can be detected by the difference between the two heterozygote classes of a gene. Imprinted genes are important in both the development of the placenta and the embryo, and we hypothesized that imprinted genes might be involved in female fertility traits. We therefore performed an association study for imprinted genes related to female fertility traits in two commercial pig populations. For this purpose, 309 SNPs in fifteen evolutionary conserved imprinted regions were genotyped on 689 and 1050 pigs from the two pig populations. A single SNP association study was used to detect additive, dominant and imprinting effects related to four reproduction traits; total number of piglets born, the number of piglets born alive, the total weight of the piglets born and the total weight of the piglets born alive. Several SNPs showed significant (q-value < 0.10) additive and dominant effects and one SNP showed a significant imprinting effect. The SNP with a significant imprinting effect is closely linked to DIO3, a gene involved in thyroid metabolism. The imprinting effect of this SNP explained approximately 1.6% of the phenotypic variance, which corresponded to approximately 15.5% of the additive genetic variance. In the other population, the imprinting effect of this QTL was not significant (q-value > 0.10), but had a similar effect as in the first population. The results of this study indicate a possible association between the imprinted gene DIO3 and female fertility traits in pigs.