Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the MGMT Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set

Due to its cost-efficiency, high resolution melting (HRM) analysis plays an important role in genotyping of candidate single nucleotide polymorphisms (SNPs). Studies indicate that HRM analysis is not only suitable for genotyping individual SNPs, but also allows genotyping of multiple SNPs in one and the same amplicon, although with limited discrimination power. By targeting the three C>T SNPs rs527559815, rs547832288, and rs16906252, located in the promoter of the O6-methylguanine-DNA methyltransferase (MGMT) gene within a distance of 45 bp, we investigated whether the discrimination power can be increased by coupling HRM analysis with pyrosequencing (PSQ). After optimizing polymerase chain reaction (PCR) conditions, PCR products subjected to HRM analysis could directly be used for PSQ. By analyzing oligodeoxynucleotide controls, representing the 36 theoretically possible variant combinations for diploid human cells (8 triple-homozygous, 12 double-homozygous, 12 double-heterozygous and 4 triple-heterozygous combinations), 34 out of the 36 variant combinations could be genotyped unambiguously by combined analysis of HRM and PSQ data, compared to 22 variant combinations by HRM analysis and 16 variant combinations by PSQ. Our approach was successfully applied to genotype stable cell lines of different origin, primary human tumor cell lines from glioma patients, and breast tissue samples.     

Sperm Methylome Profiling Can Discern Fertility Levels in the Porcine Biomedical Model

A combined Genotyping By Sequencing (GBS) and methylated DNA immunoprecipitation (MeDIP) protocol was used to identify—in parallel—genetic variation (Genomic-Wide Association Studies (GWAS) and epigenetic differences of Differentially Methylated Regions (DMR) in the genome of spermatozoa from the porcine animal model. Breeding boars with good semen quality (n = 11) and specific and well-documented differences in fertility (farrowing rate, FR) and prolificacy (litter size, LS) (n = 7) in artificial insemination programs, using combined FR and LS, were categorized as High Fertile (HF, n = 4) or Low Fertile (LF, n = 3), and boars with Unknown Fertility (UF, n = 4) were tested for eventual epigenetical similarity with those fertility-proven. We identified 165,944 Single Nucleotide Polymorphisms (SNPs) that explained 14–15% of variance among selection lines. Between HF and LF individuals (n = 7, 4 HF and 3 LF), we identified 169 SNPs with p ≤ 0.00015, which explained 58% of the variance. For the epigenetic analyses, we considered fertility and period of ejaculate collection (late-summer and mid-autumn). Approximately three times more DMRs were observed in HF than in LF boars across these periods. Interestingly, UF boars were clearly clustered with one of the other HF or LF groups. The highest differences in DMRs between HF and LF experimental groups across the pig genome were located in the chr 3, 9, 13, and 16, with most DMRs being hypermethylated in LF boars. In both HF and LF boars, DMRs were mostly hypermethylated in late-summer compared to mid-autumn. Three overlaps were detected between SNPs (p ≤ 0.0005, n = 1318) and CpG sites within DMRs. In conclusion, fertility levels in breeding males including FR and LS can be discerned using methylome analyses. The findings in this biomedical animal model ought to be applied besides sire selection for andrological diagnosis of idiopathic sub/infertility.     

Short communication: Establishment of a new polymerase chain reaction-sequence-based typing method for genotyping cattle major histocompatibility complex class II DRB3

Sequence-based typing (SBT) is the most comprehensive method for characterizing major histocompatibility complex (MHC) gene polymorphisms. We report here a new PCR-SBT method for genotyping cattle MHC (BoLA) class II DRB3 using the Assign 400ATF ver. 1.0.2.41 software (Conexio Genomics, Fremantle, Australia), which detects alleles in a semiautomated manner. We examined 12 sets of PCR reactions for their ability to amplify BoLA-DRB3 exon 2 and selected an optimal primer set, which used ERB3N-HL031 for first-round PCR and ALL-DRB3B for second-round PCR. Next, we constructed a BoLA-DRB3 allele database using the reference sequences of the Assign 400ATF software and successfully assigned heterozygous samples (including those with deletion alleles) using bidirectional sequencing, unlike our previously described method, which used unidirectional sequencing for detecting of deletion alleles. Next, blood samples of 128 Holstein cattle were used to correlate the results of our modified PCR-SBT method with those of our previously described PCR-SBT method. Each new PCR-SBT result corresponded completely with the DRB3 allele that was genotyped by our previously described PCR-SBT method. Moreover, we confirmed the accuracy of our modified PCR-SBT method by genotyping 7 sire cattle and their 22 calves using Japanese Black cattle. This new method will contribute to high-throughput genotyping of BoLA-DRB3 by sequence-based typing.     

Species diversity and toxigenic potential of Fusarium graminearum complex isolates from maize fields in northwest Argentina

Members of the Fusarium graminearum species complex (Fg complex) are the causal agents of ear rot in maize and Fusarium head blight of wheat and other small grain cereals. The potential of these pathogens to contaminate cereals with trichothecene mycotoxins is a health risk for both humans and animals. A survey of ear rot isolates from maize collected in northwest Argentina recovered 66 isolates belonging to the Fg complex. A multilocus genotyping (MLGT) assay for determination of Fg complex species and trichothecene chemotypes was used to identify 56 of these isolates as F. meridionale and 10 isolates as F. boothii. F. meridionale was fixed for the nivalenol (NIV) chemotype, and all of the F. boothii isolates had the 15-acetyldeoxynivalenol (15ADON) chemotype. The results of genetic diversity analysis based on nine variable number tandem repeat (VNTR) loci supported the hypothesis of genetic isolation between F. meridionale and F. boothii, and provided little evidence of geographic substructure among populations of the dominant pathogen species, F. meridionale. This is the first study to indicate that F. meridionale and F. boothii may play a substantial role in the infection and trichothecene contamination of maize in Argentina. In addition, dominance of the NIV chemotype among Fg complex isolates from Argentina is unprecedented, and of significant concern to food safety and animal production.     

Hot topic: performance of bovine high-density genotyping platforms in Holsteins and Jerseys

Two high-density single nucleotide polymorphism (SNP) genotyping arrays have recently become available for bovine genomic analyses, the Illumina High-Density Bovine BeadChip Array (777,962 SNP) and the Affymetrix Axiom Genome-Wide BOS 1 Array (648,874 SNP). These products each have unique design and chemistry attributes, and the extent of marker overlap and their potential utility for quantitative trait loci fine mapping, detection of copy number variation, and multibreed genomic selection are of significant interest to the cattle community. This is the first study to compare the performance of these 2 arrays. Deoxyribonucleic acid samples from 16 dairy cattle (10 Holstein, 6 Jersey) were used for the comparison. An independent set of DNA samples taken from 46 Jersey cattle and 18 Holstein cattle were used to ascertain the amount of SNP variation accounted by the 16 experimental samples. Data were analyzed with SVS7 software (Golden Helix Inc., Bozeman, MT) to remove SNP having a call rate less than 90%, and linkage disequilibrium pruning was used to remove linked SNP (r² ≥ 0.9). Maximum, average, and median gaps were calculated for each analysis based on genomic position of SNP on the bovine UMD3.1 genome assembly. All samples were successfully genotyped (≥98% SNP genotyped) with both platforms. The average number of genotyped SNP in the Illumina platform was 775,681 and 637,249 for the Affymetrix platform. Based on genomic position, a total of 107,896 SNP were shared between the 2 platforms; however, based on genotype concordance, only 96,031 SNP had complete concordance at these loci. Both Affymetrix BOS 1 and Illumina BovineHD genotyping platforms are well designed and provide high-quality genotypes and similar coverage of informative SNP. Despite fewer total SNP on BOS 1, 19% more SNP remained after linkage disequilibrium pruning, resulting in a smaller gap size (5.2 vs. 6.9 kb) in Holstein and Jersey samples relative to BovineHD. However, only 224,115 Illumina and 241,038 Affymetrix SNP remained following removal of SNP with a minor allele frequency of zero in Holstein and Jersey samples, resulting in an average gap size of 11,887 bp and 11,018 bp, respectively. Combining the 354,348 informative (r² ≥ 0.9), polymorphic (minor allele frequency ≥ 0), unique SNP data from both platforms decreased the average gap size to 7,560 bp. Genome-wide copy number variant analyses were performed using intensity files from both platforms. The BovineHD platform provided an advantage to the copy number variant data compared with the BOS 1 because of the larger number of SNP, higher intensity signals, and lower background effects. The combined use of both platforms significantly improved coverage over either platform alone and decreased the gap size between SNP, providing a valuable tool for fine mapping quantitative trait loci and multibreed animal evaluation.     

Approximating minimum coloring and maximum independent set in dotted interval graphs

Dotted interval graphs are introduced by Aumann et al. as a generalization of interval graphs. We study two optimization problems in dotted interval graphs that find application in high-throughput genotyping. We present improved approximations for minimum coloring and the first approximation for maximum independent set in dotted interval graphs.     

Preliminary virulence genotyping and phylogeny of Escherichia coli from the gut of pigs at slaughtering stage in Brazil

In the current study we screened Escherichia coli from intestine of pigs slaughtered in Mato Grosso, Brazil, for virulence-markers related to human disease. Furthermore, we employed for the first time a phylogenetic assay to explore the association between phylogeny and virulence genotype in E. coli from finished swine. A low prevalence (7.8%) of E. coli harbouring virulence genes was observed. Among the positive isolates, 3.3% could be classified as atypical EPEC, 2.2% as STEC and 2.2% as CDT harbouring E. coli. Virulence genes were not found to co-occur in a strain. Phylogenetic determination of isolates revealed a low prevalence of E. coli lineages related to disease. Therefore, preliminary sampling of 74 pigs indicated that slaughter swine may not be major reservoirs of E. coli capable of causing human disease. In light of the significant association between phylogeny and virulence genotype, we also underscored the phylogenetic grouping of strains as a valuable tool for E. coli surveillance programmes in slaughterhouses.