Androniki Psifidi

A comparison of six methods for genomic DNA extraction suitable for PCR-based genotyping applications using ovine milk samples

Isolation of amplifiable genomic DNA is a prerequisite for the genetic assessment of diseases and disease susceptibility in farm animals. Milk somatic cells are a practical, animal friendly and cost-effective source of genomic DNA in milking ruminants. In this study, six different DNA extraction methods were optimized, evaluated and compared for the isolation of DNA from ovine milk samples. Methods 1 and 2 were direct applications of two commercial kits, Nucleospin((R)) Blood and Nucleospin((R)) Tissue, respectively. Methods 3 and 4 were based on modified protocols of methods 1 and 2, respectively, aiming at increasing DNA recovery and integrity, and eliminating PCR inhibitors. Method 5 was a standard Phenol-Chloroform protocol application and method 6 was based on an in-house developed protocol using silica as the affinity matrix. Spectrophotometer, gel electrophoresis and real-time PCR measurements were used as criteria for evaluating quantity and quality of the extracted DNA. Processing time, intensity of labor and cost for each method were also evaluated. Results suggested that methods 1-4 were considered suitable for molecular downstream applications and performed better than methods 5 and 6. Modifications of protocols 3 and 4 increased the quantity and quality of the extracted DNA from ovine milk samples. Method 3 was proved to be highly efficient and robust for large scale use as demonstrated by its successful application to 1000 individual ovine milk and 50 bulk milk samples.

Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.

Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR.

Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for differences in RTL measurements from samples extracted by different DNA extraction methods within a study.

Characterization of the PRNP gene locus in Chios dairy sheep and its association with milk production and reproduction traits

The objective of this study was to examine the prion protein gene locus (PRNP) in Chios sheep. PRNP is linked with scrapie resistance in small ruminants. Here, its impact on milk production (test-day and total lactation yield) and reproduction (age at first lambing, conception rate at first service, and prolificacy) was assessed. Genotyping at codons 136, 154 and 171 (classical scrapie) and 141 (atypical scrapie) was performed using DNA from milk somatic cells and PCR-RFLP analysis. A total of 1013 Chios ewes raised in 23 flocks were used. This constituted a random sample of the national breeding population. A total of 15 genotypes and 6 alleles linked to codons 136, 154 and 171 were detected. All animals were homozygous for the leucine allele at codon 141. Linear mixed models were used to assess the impact of PRNP genotypes and alleles on milk production and reproduction traits. The TRQ allele, whose association with such traits was assessed for the first time, had an adverse effect on age at first lambing. All other PRNP alleles, including ARR, which is associated with increased resistance to classical scrapie, had no significant effect on the traits studied. No significant associations of the PRNP genotypes with production and reproduction traits were observed. It was concluded that selection for scrapie-resistant sheep is not expected to affect the ongoing breeding programme that aims to enhance the milk yield and reproduction of the Chios breed.

The genomic architecture of resistance to Campylobacter jejuni intestinal colonisation in chickens

BackgroundCampylobacter is the leading cause of foodborne diarrhoeal illness in humans and is mostly acquired from consumption or handling of contaminated poultry meat. In the absence of effective licensed vaccines and inhibitors, selection for chickens with increased resistance to Campylobacter could potentially reduce its subsequent entry into the food chain. Campylobacter intestinal colonisation levels are influenced by the host genetics of the chicken. In the present study, two chicken populations were used to investigate the genetic architecture of avian resistance to colonisation: (i) a back-cross of two White Leghorn derived inbred lines [(61 x N) x N] known to differ in resistance to Campylobacter colonisation and (ii) a 9th generation advanced intercross (61 x N) line.ResultsThe level of colonisation with Campylobacter jejuni following experimental infection was found to be a quantitative trait. A back-cross experiment using 1,243 fully informative single nucleotide polymorphism (SNP) markers revealed quantitative trait loci (QTL) on chromosomes 7, 11 and 14. In the advanced intercross line study, the location of the QTL on chromosome 14 was confirmed and refined and two new QTLs were identified located on chromosomes 4 and 16. Pathway and re-sequencing data analysis of the genes located in the QTL candidate regions identified potential pathways, networks and candidate resistance genes. Finally, gene expression analyses were performed for some of the candidate resistance genes to support the results.ConclusionCampylobacter resistance in chickens is a complex trait, possibly involving the Major Histocompatibility Complex, innate and adaptive immune responses, cadherins and other factors. Two of the QTLs for Campylobacter resistance are co-located with Salmonella resistance loci, indicating that it may be possible to breed simultaneously for enhanced resistance to both zoonoses.

Genome-wide association studies of immune, disease and production traits in indigenous chicken ecotypes

BackgroundThe majority of chickens in sub-Saharan Africa are indigenous ecotypes, well adapted to the local environment and raised in scavenging production systems. Although they are generally resilient to disease challenge, routine vaccination and biosecurity measures are rarely applied and infectious diseases remain a major cause of mortality and reduced productivity. Management and genetic improvement programmes are hampered by lack of routine data recording. Selective breeding based on genomic technologies may provide the means to enhance sustainability. In this study, we investigated the genetic architecture of antibody response to four major infectious diseases [infectious bursal disease (IBDV), Marek’s disease (MDV), fowl typhoid (SG), fowl cholera (PM)] and resistance to Eimeria and cestode parasitism, along with two production traits [body weight and body condition score (BCS)] in two distinct indigenous Ethiopian chicken ecotypes. We conducted variance component analyses, genome-wide association studies, and pathway and selective sweep analyses.ResultsThe large majority of birds was found to have antibody titres for all pathogens and were infected with both parasites, suggesting almost universal exposure. We derived significant moderate to high heritabilities for IBDV, MDV and PM antibody titres, cestodes infestation, body weight and BCS. We identified single nucleotide polymorphisms (SNPs) with genome-wide significance for each trait. Based on these associations, we identified for each trait, pathways, networks and functional gene clusters that include plausible candidate genes. Selective sweep analyses revealed a locus on chromosome 18 associated with viral antibody titres and resistance to Eimeria parasitism that is within a positive selection signal. We found no significant genetic correlations between production, immune and disease traits, implying that selection for altered antibody response and/or disease resistance will not affect production.ConclusionsWe confirmed the presence of genetic variability and identified SNPs significantly associated with immune, disease and production traits in indigenous village chickens. Results underpin the feasibility of concomitant genetic improvement for enhanced antibody response, resistance to parasitism and productivity within and across indigenous chicken ecotypes.

The genomic architecture of mastitis resistance in dairy sheep

BackgroundMastitis is the most prevalent disease in dairy sheep with major economic, hygienic and welfare implications. The disease persists in all dairy sheep production systems despite the implementation of improved management practises. Selective breeding for enhanced mastitis resistance may provide the means to further control the disease. In the present study, we investigated the genetic architecture of four mastitis traits in dairy sheep. Individual animal records for clinical mastitis occurrence and three mastitis indicator traits (milk somatic cell count, total viable bacterial count in milk and the California mastitis test) were collected monthly throughout lactation for 609 ewes of the Greek Chios breed. All animals were genotyped with a custom-made 960-single nucleotide polymorphism (SNP) DNA array based on markers located in quantitative trait loci (QTL) regions for mastitis resistance previously detected in three other distinct dairy sheep populations.ResultsHeritable variation and strong positive genetic correlations were estimated for clinical mastitis occurrence and the three mastitis indicator traits. SNP markers significantly associated with these mastitis traits were confirmed on chromosomes 2, 3, 5, 16 and 19. We identified pathways, molecular interaction networks and functional gene clusters for mastitis resistance. Candidate genes within the detected regions were identified based upon analysis of an ovine transcriptional atlas and transcriptome data derived from milk somatic cells. Relevant candidate genes implicated in innate immunity included SOCS2, CTLA4, C6, C7, C9, PTGER4, DAB2, CARD6, OSMR, PLXNC1, IDH1, ICOS, FYB, and LYFR.ConclusionsThe results confirmed the presence of animal genetic variability in mastitis resistance and identified genomic regions associated with specific mastitis traits in the Chios sheep. The conserved genetic architecture of mastitis resistance between distinct dairy sheep breeds suggests that across-breed selection programmes would be feasible.

Colonization of a commercial broiler line by Campylobacter is under limited genetic control and does not significantly impair performance or intestinal health

ABSTRACT Campylobacter is the leading bacterial cause of foodborne diarrheal illness in humans and source attribution studies unequivocally identify handling or consumption of poultry meat as a key risk factor. Campylobacter colonizes the avian intestines in high numbers and rapidly spreads within flocks. A need therefore exists to devise strategies to reduce Campylobacter populations in poultry flocks. There has been a great deal of research aiming to understand the epidemiology and transmission characteristics of Campylobacter in poultry as a means to reduce carriage rates in poultry and reduce infection in humans. One potential strategy for control is the genetic selection of poultry for increased resistance to colonization by Campylobacter. The potential for genetic control of colonization has been demonstrated in inbred populations following experimental challenge with Campylobacter where quantitative trait loci associated with resistance have been identified. Currently in the literature there is no information of the genetic basis of Campylobacter colonization in commercial broiler lines and it is unknown whether these QTL are found in commercial broiler lines. The aim of this study was to estimate genetic parameters associated with Campylobacter load and genetic correlations with gut health and production traits following natural exposure of broiler chickens to Campylobacter. The results from the analysis show a low but significant heritability estimate (0.095 ± 0.037) for Campylobacter load which indicates a limited genetic basis and that non‐genetic factors have a greater influence on the level of Campylobacter found in the broiler chicken. Furthermore, through examination of macroscopic intestinal health and absorptive capacity, our study indicated that Campylobacter has no detrimental effects on intestinal health and bird growth following natural exposure in the broiler line under study. These data indicate that whilst there is a genetic component to Campylobacter colonization worthy of further investigation, there is a large proportion of phenotypic variance under the influence of non‐genetic effects. As such the control of Campylobacter will require understanding and manipulation of non‐genetic host and environmental factors.

Microbiota composition, gene pool and its expression in Gir cattle (Bos indicus) rumen under different forage diets using metagenomic and metatranscriptomic approaches

Zebu (Bos indicus) is a domestic cattle species originating from the Indian subcontinent and now widely domesticated on several continents. In this study, we were particularly interested in understanding the functionally active rumen microbiota of an important Zebu breed, the Gir, under different dietary regimes. Metagenomic and metatranscriptomic data were compared at various taxonomic levels to elucidate the differential microbial population and its functional dynamics in Gir cattle rumen under different roughage dietary regimes. Different proportions of roughage rather than the type of roughage (dry or green) modulated microbiome composition and the expression of its gene pool. Fibre degrading bacteria (i.e. Clostridium, Ruminococcus, Eubacterium, Butyrivibrio, Bacillus and Roseburia) were higher in the solid fraction of rumen (P<0.01) compared to the liquid fraction, whereas bacteria considered to be utilizers of the degraded product (i.e. Prevotella, Bacteroides, Parabacteroides, Paludibacter and Victivallis) were dominant in the liquid fraction (P<0.05). Likewise, expression of fibre degrading enzymes and related carbohydrate binding modules (CBMs) occurred in the solid fraction. When metagenomic and metatranscriptomic data were compared, it was found that some genera and species were transcriptionally more active, although they were in low abundance, making an important contribution to fibre degradation and its further metabolism in the rumen. This study also identified some of the transcriptionally active genera, such as Caldicellulosiruptor and Paludibacter, whose potential has been less-explored in rumen. Overall, the comparison of metagenomic shotgun and metatranscriptomic sequencing appeared to be a much richer source of information compared to conventional metagenomic analysis.

Bovine telomere dynamics and the association between telomere length and productive lifespan

Average telomere length (TL) in blood cells has been shown to decline with age in a range of vertebrate species, and there is evidence that TL is a heritable trait associated with late-life health and mortality in humans. In non-human mammals, few studies to date have examined lifelong telomere dynamics and no study has estimated the heritability of TL, despite these being important steps towards assessing the potential of TL as a biomarker of productive lifespan and health in livestock species. Here we measured relative leukocyte TL (RLTL) in 1,328 samples from 308 Holstein Friesian dairy cows and in 284 samples from 38 female calves. We found that RLTL declines after birth but remains relatively stable in adult life. We also calculated the first heritability estimates of RLTL in a livestock species which were 0.38 (SE = 0.03) and 0.32 (SE = 0.08) for the cow and the calf dataset, respectively. RLTL measured at the ages of one and five years were positively correlated with productive lifespan (p < 0.05). We conclude that bovine RLTL is a heritable trait, and its association with productive lifespan may be used in breeding programmes aiming to enhance cow longevity.