Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.

Mapping Quantitative Trait Loci for Partial Resistance to Powdery Mildew in an Australian Barley Population

Genomic regions influencing resistance to powdery mildew [Blumeria graminis (DC.) E.O. Speer f. sp. hordei Em. Marchal] were detected in a doubled haploid (DH) barley (Hordeum vulgare L.) population derived from a cross between the breeding line ND24260 and cultivar Flagship when evaluated across four field environments in Australia and Uruguay. Significant quantitative trait loci (OIL) for resistance to B. graminis were detected on six of the seven chromosomes (1H, 2H, 3H, 4H, 5H, and 7H). A QTL with large effect donated by ND24260 mapped to the short arm of chromosome 1H (1 HS) conferring near immunity to B. graminis in Australia but was ineffective in Uruguay. Three OIL donated by Flagship contributed partial resistance to B. graminis and were detected in at least two environments. These OIL were mapped to chromosomes 3H, 4H, and 5H (5HS) accounting for up to 18.6, 3.4, and 8.8% phenotypic variation, respectively. The 5HS QTL contributed partial resistance to B. graminis in all field environments in both Australia and Uruguay and aligned with the genomic region of Rph20, a gene conferring adult plant resistance (APR) to leaf rust (Puccinia hordei Otth), which is found in some cultivars having Vada' or 'Emir' in their parentage. Selection for favorable marker haplotypes within the 3H, 4H, and 5H QTL regions can be performed even in the presence of single (major) gene resistance. Pyramiding such QTL may provide an effective and potentially durable form of resistance to B. graminis.

High resolution mapping of Dense spike-ar (dsp.ar) to the genetic centromere of barley chromosome 7H

Spike density in barley is under the control of several major genes, as documented previously by genetic analysis of a number of morphological mutants. One such class of mutants affects the rachis internode length leading to dense or compact spikes and the underlying genes were designated dense spike (dsp). We previously delimited two introgressed genomic segments on chromosome 3H (21 SNP loci, 35.5 cM) and 7H (17 SNP loci, 20.34 cM) in BW265, a BC7F3 nearly isogenic line (NIL) of cv. Bowman as potentially containing the dense spike mutant locus dsp.ar, by genotyping 1,536 single nucleotide polymorphism (SNP) markers in both BW265 and its recurrent parent. Here, the gene was allocated by high-resolution bi-parental mapping to a 0.37 cM interval between markers SC57808 (Hv_SPL14)-CAPSK06413 residing on the short and long arm at the genetic centromere of chromosome 7H, respectively. This region putatively contains more than 800 genes as deduced by comparison with the collinear regions of barley, rice, sorghum and Brachypodium, Classical map-based isolation of the gene dsp.ar thus will be complicated due to the infavorable relationship of genetic to physical distances at the target locus.

High global diversity of cycloviruses amongst dragonflies

Members of the family Circoviridae, specifically the genus Circovirus, were thought to infect only vertebrates; however, members of a sister group under the same family, the proposed genusCyclovirus, have been detected recently in insects. In an effort to explore the diversity of cycloviruses and better understand the evolution of these novel ssDNA viruses, here we present five cycloviruses isolated from three dragonfly species (Orthetrum sabina, Xanthocnemis zealandica and Rhionaeschna multicolor) collected in Australia, New Zealand and the USA, respectively. The genomes of these five viruses share similar genome structure to other cycloviruses, with a circular ~1.7 kb genome and two major bidirectionally transcribed ORFs. The genomic sequence data gathered during this study were combined with all cyclovirus genomes available in public databases to identify conserved motifs and regulatory elements in the intergenic regions, as well as determine diversity and recombinant regions within their genomes. The genomes reported here represent four different cyclovirus species, three of which are novel. Our results confirm that cycloviruses circulate widely in winged-insect populations; in eight different cyclovirus species identified in dragonflies to date, some of these exhibit a broad geographical distribution. Recombination analysis revealed both intra-and inter-species recombination events amongst cycloviruses, including genomes recovered from disparate sources (e.g. goat meat and human faeces). Similar to other well-characterized circular ssDNA viruses, recombination may play an important role in cyclovirus evolution. © 2013 SGM.

Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

Background Next-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically. Results Progeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations. Conclusion We have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.

New SNPs for population genetic analysis reveal possible cryptic speciation of eastern Australian sea mullet (Mugil cephalus)

Sustainable management of sea mullet (Mugil cephalus) fisheries needs to account for recent observations of regional-scale differentiation. Population genetic analysis is sought to assess the situation of this ecologically and economically important fish species in eastern Australian waters. Here, we report (i) new population genetic markers [single nucleotide polymorphisms (SNPs) and potential microsatellites], (ii) first estimates of spatial genetic differentiation and (iii) prospective power tests for designing more comprehensive studies. Six DNA samples from three sampling regions (North Queensland, South Queensland and central New South Wales) on the eastern coast of Australia were used to prepare restriction site associated DNA (RAD) tag libraries from genomic DNA digested with EcoRI and MseI. A pooled sample of regional RAD tag libraries was sequenced using the Roche GS-FLX Titanium platform. A total of 172837 raw reads (17.4Mbp) were retrieved, 95500 of which were used to discover 1267 SNPs and 1417 microsatellites. A subset of 161 SNPs was validated based on 63 additional DNA samples genotyped using the Sequenom MassArray (iPLEX Gold chemistry). Altogether 92 SNPs (57%) were confirmed, with 40% of these marking fixed variants between northern and southern sampling regions. Our preliminary findings indicate a multispecies fishery stock of M. cephalus in eastern Australian waters, but suggest that strong genetic differentiation occurs north of major fishing grounds. Low potential differentiation within major fishing grounds (e.g. FST=0.0025) can be resolved with a likely power 67% by using standard sample sizes of 50 and validated subsets of available markers.

Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5–6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30 mg semi-engorged ticks fed more reliably, ticks as small as 15 mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10 mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

Association mapping of resistance to Puccinia hordei in Australian barley breeding germplasm

Key message “To find stable resistance using association mapping tools, QTL with major and minor effects on leaf rust reactions were identified in barley breeding lines by assessing seedlings and adult plants.” Abstract Three hundred and sixty (360) elite barley (Hordeum vulgare L.) breeding lines from the Northern Region Barley Breeding Program in Australia were genotyped with 3,244 polymorphic diversity arrays technology markers and the results used to map quantitative trait loci (QTL) conferring a reaction to leaf rust (Puccinia hordei Otth). The F3:5 (Stage 2) lines were derived or sourced from different geographic origins or hubs of international barley breeding ventures representing two breeding cycles (2009 and 2011 trials) and were evaluated across eight environments for infection type at both seedling and adult plant stages. Association mapping was performed using mean scores for disease reaction, accounting for family effects using the eigenvalues from a matrix of genotype correlations. In this study, 15 QTL were detected; 5 QTL co-located with catalogued leaf rust resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. The adult plant resistance gene Rph20 was identified across the majority of environments and pathotypes. The QTL detected in this study offer opportunities for breeding for more durable resistance to leaf rust through pyramiding multiple genomic regions via marker-assisted selection.

Spot form of net blotch resistance in barley is under complex genetic control

Key message: Evaluation of resistance toPyrenophora teresf.maculatain barley breeding populations via association mapping revealed a complex genetic architecture comprising a mixture of major and minor effect genes. Abstract: In the search for stable resistance to spot form of net blotch (Pyrenophora teres f. maculata, SFNB), association mapping was conducted on four independent barley (Hordeum vulgare L.) breeding populations comprising a total of 898 unique elite breeding lines from the Northern Region Barley Breeding Program in Australia for discovery of quantitative trait loci (QTL) influencing resistance at seedling and adult plant growth stages. A total of 29 significant QTL were validated across multiple breeding populations, with 22 conferring resistance at both seedling and adult plant growth stages. The remaining 7 QTL conferred resistance at either seedling (2 QTL) or adult plant (5 QTL) growth stages only. These 29 QTL represented 24 unique genomic regions, of which five were found to co-locate with previously identified QTL for SFNB. The results indicated that SFNB resistance is controlled by a large number of QTL varying in effect size with large effects QTL on chromosome 7H. A large proportion of the QTL acted in the same direction for both seedling and adult responses, suggesting that phenotypic selection for SFNB resistance performed at either growth stage could achieve adequate levels of resistance. However, the accumulation of specific resistance alleles on several chromosomes must be considered in molecular breeding selection strategies.

Phylodynamic evidence of the migration of turnip mosaic potyvirus from Europe to Australia and New Zealand

Turnip mosaic virus (TuMV) is a potyvirus that is transmitted by aphids and infects a wide range of plant species. We investigated the evolution of this pathogen by collecting 32 isolates of TuMV, mostly from Brassicaceae plants, in Australia and New Zealand. We performed a variety of sequence-based phylogenetic and population genetic analyses of the complete genomic sequences and of three non-recombinogenic regions of those sequences. The substitution rates, divergence times and phylogeographical patterns of the virus populations were estimated. Six inter- and seven intralineage recombination-type patterns were found in the genomes of the Australian and New Zealand isolates, and all were novel. Only one recombination-type pattern has been found in both countries. The Australian and New Zealand populations were genetically different, and were different from the European and Asian populations. Our Bayesian coalescent analyses, based on a combination of novel and published sequence data from three nonrecombinogenic protein-encoding regions, showed that TuMV probably started to migrate from Europe to Australia and New Zealand more than 80 years ago, and that distinct populations arose as a result of evolutionary drivers such as recombination. The basal-B2 subpopulation in Australia and New Zealand seems to be older than those of the world-B2 and -B3 populations. To our knowledge, our study presents the first population genetic analysis of TuMV in Australia and New Zealand. We have shown that the time of migration of TuMV correlates well with the establishment of agriculture and migration of Europeans to these countries.

Two distinct classes of QTL determine rust resistance in sorghum

Background: Agriculture is facing enormous challenges to feed a growing population in the face of rapidly evolving pests and pathogens. The rusts, in particular, are a major pathogen of cereal crops with the potential to cause large reductions in yield. Improving stable disease resistance is an on-going major and challenging focus for many plant breeding programs, due to the rapidly evolving nature of the pathogen. Sorghum is a major summer cereal crop that is also a host for a rust pathogen which occurs in almost all sorghum growing areas of the world, causing direct and indirect yield losses in sorghum worldwide, however knowledge about its genetic control is still limited. In order to further investigate this issue, QTL and association mapping methods were implemented to study rust resistance in three bi-parental populations and an association mapping set of elite breeding lines in different environments. Results: In total, 64 significant or highly significant QTL and 21 suggestive rust resistance QTL were identified representing 55 unique genomic regions. Comparisons across populations within the current study and with rust QTL identified previously in both sorghum and maize revealed a high degree of correspondence in QTL location. Negative phenotypic correlations were observed between rust, maturity and height, indicating a trend for both early maturing and shorter genotypes to be more susceptible to rust. Conclusions: The significant amount of QTL co-location across traits, in addition to the consistency in the direction of QTL allele effects, has provided evidence to support pleiotropic QTL action across rust, height, maturity and stay-green, supporting the role of carbon stress in susceptibility to rust. Classical rust resistance QTL regions that did not co-locate with height, maturity or stay-green QTL were found to be significantly enriched for the defence-related NBS-encoding gene family, in contrast to the lack of defence-related gene enrichment in multi-trait effect rust resistance QTL. The distinction of disease resistance QTL hot-spots, enriched with defence-related gene families from QTL which impact on development and partitioning, provides plant breeders with knowledge which will allow for fast-tracking varieties with both durable pathogen resistance and appropriate adaptive traits.

Bayesian statistical analysis of bacterial diversity

Bacteria play an important role in many ecological systems. The molecular characterization of bacteria using either cultivation-dependent or cultivation-independent methods reveals the large scale of bacterial diversity in natural communities, and the vastness of subpopulations within a species or genus. Understanding how bacterial diversity varies across different environments and also within populations should provide insights into many important questions of bacterial evolution and population dynamics. This thesis presents novel statistical methods for analyzing bacterial diversity using widely employed molecular fingerprinting techniques. The first objective of this thesis was to develop Bayesian clustering models to identify bacterial population structures. Bacterial isolates were identified using multilous sequence typing (MLST), and Bayesian clustering models were used to explore the evolutionary relationships among isolates. Our method involves the inference of genetic population structures via an unsupervised clustering framework where the dependence between loci is represented using graphical models. The population dynamics that generate such a population stratification were investigated using a stochastic model, in which homologous recombination between subpopulations can be quantified within a gene flow network. The second part of the thesis focuses on cluster analysis of community compositional data produced by two different cultivation-independent analyses: terminal restriction fragment length polymorphism (T-RFLP) analysis, and fatty acid methyl ester (FAME) analysis. The cluster analysis aims to group bacterial communities that are similar in composition, which is an important step for understanding the overall influences of environmental and ecological perturbations on bacterial diversity. A common feature of T-RFLP and FAME data is zero-inflation, which indicates that the observation of a zero value is much more frequent than would be expected, for example, from a Poisson distribution in the discrete case, or a Gaussian distribution in the continuous case. We provided two strategies for modeling zero-inflation in the clustering framework, which were validated by both synthetic and empirical complex data sets. We show in the thesis that our model that takes into account dependencies between loci in MLST data can produce better clustering results than those methods which assume independent loci. Furthermore, computer algorithms that are efficient in analyzing large scale data were adopted for meeting the increasing computational need. Our method that detects homologous recombination in subpopulations may provide a theoretical criterion for defining bacterial species. The clustering of bacterial community data include T-RFLP and FAME provides an initial effort for discovering the evolutionary dynamics that structure and maintain bacterial diversity in the natural environment.

Development of type I genetic markers from expressed sequence tags in highly polymorphic species

Expressed sequence tag (EST) databases provide a primary source of nuclear DNA sequences for genetic marker development in non-model organisms. To date, the process has been relatively inefficient for several reasons: - 1) priming site polymorphism in the template leads to inferior or erratic amplification; - 2) introns in the target amplicon are too large and/or numerous to allow effective amplification under standard screening conditions, and; - 3) at least occasionally, a PCR primer straddles an exon–intron junction and is unable to bind to genomic DNA template. The first is only a minor issue for species or strains with low heterozygosity but becomes a significant problem for species with high genomic variation, such as marine organisms with extremely large effective population sizes. Problems arising from unanticipated introns are unavoidable but are most pronounced in intron-rich species, such as vertebrates and lophotrochozoans. We present an approach to marker development in the Pacific oyster Crassostrea gigas, a highly polymorphic and intron-rich species, which minimizes these problems, and should be applicable to other non-model species for which EST databases are available. Placement of PCR primers in the 3′ end of coding sequence and 3′ UTR improved PCR success rate from 51% to 97%. Almost all (37 of 39) markers developed for the Pacific oyster were polymorphic in a small test panel of wild and domesticated oysters.

A transcriptomic analysis of the kidney tissue of Tra catfish (pangasianodon hypophthalmus) reared in saline condition: De novo assembly, annotation, SNP discovery

Pangasianodon hypophthalmus is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The current study using Ion Torrent technology generated EST resources from the kidney for Tra catfish reared at a salinity level of 9 ppt. We obtained 2,623,929 reads after trimming and processing with an average length of 104 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 29,940 contigs, and allowing identification of 5,710 putative genes when comppared with NCBI non-redundant database. A large number of single nucleotide polymorphisms (SNPs) were also detected. The sequence collection generated in our study represents the most comprehensive transcriptomic resource for P. hypophthalmus available to date.

Mining Sequential Data – in Search of Segmental Structures

Segmentation is a data mining technique yielding simplified representations of sequences of ordered points. A sequence is divided into some number of homogeneous blocks, and all points within a segment are described by a single value. The focus in this thesis is on piecewise-constant segments, where the most likely description for each segment and the most likely segmentation into some number of blocks can be computed efficiently. Representing sequences as segmentations is useful in, e.g., storage and indexing tasks in sequence databases, and segmentation can be used as a tool in learning about the structure of a given sequence. The discussion in this thesis begins with basic questions related to segmentation analysis, such as choosing the number of segments, and evaluating the obtained segmentations. Standard model selection techniques are shown to perform well for the sequence segmentation task. Segmentation evaluation is proposed with respect to a known segmentation structure. Applying segmentation on certain features of a sequence is shown to yield segmentations that are significantly close to the known underlying structure. Two extensions to the basic segmentation framework are introduced: unimodal segmentation and basis segmentation. The former is concerned with segmentations where the segment descriptions first increase and then decrease, and the latter with the interplay between different dimensions and segments in the sequence. These problems are formally defined and algorithms for solving them are provided and analyzed. Practical applications for segmentation techniques include time series and data stream analysis, text analysis, and biological sequence analysis. In this thesis segmentation applications are demonstrated in analyzing genomic sequences.