Dual culture of atoxigenic and toxigenic strains of Aspergillus flavus to gain insight into repression of aflatoxin biosynthesis and fungal interaction.

Application of atoxigenic strains to compete against toxigenic strains of Aspergillus flavus strains has emerged as one of the practical strategies for reducing aflatoxin contamination in corn, peanut, and tree nuts. The actual mechanism that results in aflatoxin reduction is not fully understood. Real-time RT-PCR and relative quantification of gene expression protocol were applied to elucidate the molecular mechanism. Transcriptional analyses of aflatoxin biosynthetic gene cluster in dual culture of toxigenic and atoxigenic A. flavus strains were carried out. Six targeted genes, aflR, aflJ, omtA, ordA, pksA, and vbs, were downregulated to variable levels depending on paired strains of toxigenic and atoxigenic A. flavus. Consistent with the decreased gene expression levels, the aflatoxin concentrations in dual cultures were reduced significantly in comparison with toxigenic cultures. Fluorescent images showed fungal hyphae in dual culture displayed green fluorescent, and contacts of live hyphae were seen. A coconut agar plate assay was used to show that toxigenic A. flavus colony produced blue fluorescence under long UV exposure, suggesting that aflatoxin is exported outside fungal hyphae. Furthermore, the assay was applied to demonstrate the potential role of thigmo-regulation in fungal interaction.

Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic Aspergillus flavus mixtures in soil.

Aflatoxin B1 is a potent hepatotoxin and carcinogen that poses a serious safety hazard to both humans and animals. Aspergillus flavus is the most common aflatoxin-producing species on corn, cotton, peanuts, and tree nuts. Application of atoxigenic strains to compete against aflatoxigenic strains of A. flavus has emerged as one of the most practical strategies for ameliorating aflatoxin contamination in food. Genes directly involved in aflatoxin biosynthesis are clustered on an 82-kb region of the genome. Three atoxigenic strains (CA12, M34, and AF123) were each paired with each of four aflatoxigenic strains (CA28, CA42, CA90, and M52), inoculated into soil and incubated at 28 °C for 2 weeks and 1 month. TaqMan probes, omtA-FAM, and norA-HEX were designed for developing a droplet digital PCR (ddPCR) assay to analyze the soil population of mixtures of A. flavus strains. DNA was extracted from each soil sample and used for ddPCR assays. The data indicated that competition between atoxigenic and aflatoxigenic was strain dependent. Variation in competitive ability among different strains of A. flavus influenced the population reduction of the aflatoxigenic strain by the atoxigenic strain. Higher ratios of atoxigenic to aflatoxigenic strains increased soil population of atoxigenic strains. This is the first study to demonstrate the utility of ddPCR to quantify mixtures of both atoxigenic and aflatoxigenic A. flavus strains in soil and allows for rapid and accurate determination of population sizes of atoxigenic and aflatoxigenic strains. This method eliminates the need for isolation and identification of individual fungal isolates from experimental soil samples.

First report of an atypical new Aspergillus parasiticus isolates with nucleotide insertion in aflR gene resembling to A. sojae.

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using ß-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B1, B2, G1, and G2 and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153-1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.

Genome-wide DNA-(de)methylation is associated with Noninfectious Bud-failure exhibition in Almond (Prunus dulcis [Mill.] D.A.Webb).

Noninfectious bud-failure (BF) remains a major threat to almond production in California, particularly with the recent rapid expansion of acreage and as more intensive cultural practices and modern cultivars are adopted. BF has been shown to be inherited in both vegetative and sexual progeny, with exhibition related to the age and propagation history of scion clonal sources. These characteristics suggest an epigenetic influence, such as the loss of juvenility mediated by DNA-(de)methylation. Various degrees of BF have been reported among cultivars as well as within sources of clonal propagation of the same cultivar. Genome-wide methylation profiles for different clones within almond genotypes were developed to examine their association with BF levels and association with the chronological time from initial propagation. The degree of BF exhibition was found to be associated with DNA-(de)methylation and clonal age, which suggests that epigenetic changes associated with ageing may be involved in the differential exhibition of BF within and among almond clones. Research is needed to investigate the potential of DNA-(de)methylation status as a predictor for BF as well as for effective strategies to improve clonal selection against age related deterioration. This is the first report of an epigenetic-related disorder threatening a major tree crop.

Distal third humeri fractures treated using the Synthes™ 3.5-mm extra-articular distal humeral locking compression plate: clinical, radiographic and patient outcome scores.

BACKGROUND: Conventional management protocols for distal humeral extra-articular fractures (e.g. conservative, double columnar plating) are often associated with complications. We aimed to describe our experience of using the Synthes™ 3.5-mm extra-articular distal humeral locking compression plate for treatment of extra-articular distal humeral fractures. METHODS: We prospectively studied 23 consecutive patients who underwent fixation, in a tertiary trauma centre, over 2 years. Data, including patient demographics, duration of follow-up, patient satisfaction, visual analogue score (VAS), Oxford Elbow Score, and final outcome on discharge, were collected and analyzed. RESULTS: Of the 23 patients (12 males, 11 females; mean age 47.5 years; range 18 years to 89 years), all fractures united radiologically and clinically after the index procedure, with a mean time to fracture union of 15.7 weeks (range 9 weeks to 34 weeks) and a mean time to discharge of 17.8 weeks (range 13 weeks to 34 weeks). Oxford Elbow Score was 36.5 (range 11 to 48) at 4.6 months postoperatively; at 20 months follow-up, it was 40 (range 14 to 48) and the VAS was 8.5 (range 5 to 10). One patient had radial nerve neuropraxia pre-operatively, and one postoperatively, and both recovered uneventfully 3 months postoperatively. Neither superficial, nor deep infections were observed in this cohort. CONCLUSIONS: The present study reports satisfactory outcome with the usage of the Synthes plate for extra-articular fracture management. It has become the technique of choice in our centre because it provides excellent results.

Heterogeneity in the entire genome for three genotypes of peach [Prunus persica (L.) Batsch] as distinguished from sequence analysis of genomic variants.

BACKGROUND: Peach [Prunus persica (L.) Batsch] is an economically important fruit crop that has become a genetic-genomic model for all Prunus species in the family Rosaceae. A doubled haploid reference genome sequence length of 227.3 Mb, a narrow genetic base contrasted by a wide phenotypic variability, the generation of cultivars through hybridization with subsequent clonal propagation, and the current accessibility of many founder genotypes, as well as the pedigree of modern commercial cultivars make peach a model for the study of inter-cultivar genomic heterogeneity and its shaping by artificial selection. RESULTS: The quantitative genomic differences among the three genotypes studied as genomic variants, included small variants (SNPs and InDels) and structural variants (SV) (duplications, inversions and translocations). The heirloom cultivar 'Georgia Belle' and an almond by peach introgression breeding line 'F8,1-42' are more heterogeneous than is the modern cultivar 'Dr. Davis' when compared to the peach reference genome ('Lovell'). A pair-wise comparison of consensus genome sequences with 'Lovell' showed that 'F8,1-42' and 'Georgia Belle' were more divergent than were 'Dr. Davis' and 'Lovell'. CONCLUSIONS: A novel application of emerging bioinformatics tools to the analysis of ongoing genome sequencing project outputs has led to the identification of a range of genomic variants. Results can be used to delineate the genomic and phenotypic differences among peach genotypes. For crops such as fruit trees, the availability of old cultivars, breeding selections and their pedigrees, make them suitable models for the study of genome shaping by artificial selection. The findings from the study of such genomic variants can then elucidate the control of pomological traits and the characterization of metabolic pathways, thus facilitating the development of protocols for the improvement of Prunus crops.

Application of genomic and quantitative genetic tools to identify candidate resistance genes for brown rot resistance in peach.

The availability of a complete peach genome assembly and three different peach genome sequences created by our group provide new opportunities for application of genomic data and can improve the power of the classical Quantitative Trait Loci (QTL) approaches to identify candidate genes for peach disease resistance. Brown rot caused by Monilinia spp., is the most important fungal disease of stone fruits worldwide. Improved levels of peach fruit rot resistance have been identified in some cultivars and advanced selections developed in the UC Davis and USDA breeding programs. Whole genome sequencing of the Pop-DF parents lead to discovery of high-quality SNP markers for QTL genome scanning in this experimental population. Pop-DF created by crossing a brown rot moderately resistant cultivar 'Dr. Davis' and a brown rot resistant introgression line, 'F8,1-42', derived from an initial almond × peach interspecific hybrid, was evaluated for brown rot resistance in fruit of harvest maturity over three seasons. Using the SNP linkage map of Pop-DF and phenotypic data collected with inoculated fruit, a genome scan for QTL identified several SNP markers associated with brown rot resistance. Two of these QTLs were placed on linkage group 1, covering a large (physical) region on chromosome 1. The genome scan for QTL and SNP effects predicted several candidate genes associated with disease resistance responses in other host-pathogen systems. Two potential candidate genes, ppa011763m and ppa026453m, may be the genes primarily responsible for M. fructicola recognition in peach, activating both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) responses. Our results provide a foundation for further genetic dissection, marker assisted breeding for brown rot resistance, and development of peach cultivars resistant to brown rot.

Effect prediction of identified SNPs linked to fruit quality and chilling injury in peach [Prunus persica (L.) Batsch].

Single nucleotide polymorphisms (SNPs) are a fundamental source of genomic variation. Large SNP panels have been developed for Prunus species. Fruit quality traits are essential peach breeding program objectives since they determine consumer acceptance, fruit consumption, industry trends and cultivar adoption. For many cultivars, these traits are negatively impacted by cold storage, used to extend fruit market life. The major symptoms of chilling injury are lack of flavor, off flavor, mealiness, flesh browning, and flesh bleeding. A set of 1,109 SNPs was mapped previously and 67 were linked with these complex traits. The prediction of the effects associated with these SNPs on downstream products from the 'peach v1.0' genome sequence was carried out. A total of 2,163 effects were detected, 282 effects (non-synonymous, synonymous or stop codon gained) were located in exonic regions (13.04 %) and 294 placed in intronic regions (13.59 %). An extended list of genes and proteins that could be related to these traits was developed. Two SNP markers that explain a high percentage of the observed phenotypic variance, UCD_SNP_1084 and UCD_SNP_46, are associated with zinc finger (C3HC4-type RING finger) family protein and AOX1A (alternative oxidase 1a) protein groups, respectively. In addition, phenotypic variation suggests that the observed polymorphism for SNP UCD_SNP_1084 [A/G] mutation could be a candidate quantitative trait nucleotide affecting quantitative trait loci for mealiness. The interaction and expression of affected proteins could explain the variation observed in each individual and facilitate understanding of gene regulatory networks for fruit quality traits in peach.

Whole genome sequencing of peach (Prunus persica L.) for SNP identification and selection.

BACKGROUND: The application of next generation sequencing technologies and bioinformatic scripts to identify high frequency SNPs distributed throughout the peach genome is described. Three peach genomes were sequenced using Roche 454 and Illumina/Solexa technologies to obtain long contigs for alignment to the draft 'Lovell' peach sequence as well as sufficient depth of coverage for 'in silico' SNP discovery. DESCRIPTION: The sequences were aligned to the 'Lovell' peach genome released April 01, 2010 by the International Peach Genome Initiative (IPGI). 'Dr. Davis', 'F8, 1-42' and 'Georgia Belle' were sequenced to add SNPs segregating in two breeding populations, Pop DF ('Dr. Davis' × 'F8, 1-42') and Pop DG ('Dr. Davis' × 'Georgia Belle'). Roche 454 sequencing produced 980,000 total reads with 236 Mb sequence for 'Dr. Davis' and 735,000 total reads with 172 Mb sequence for 'F8, 1-42'. 84 bp × 84 bp paired end Illumina/Solexa sequences yielded 25.5, 21.4, 25.5 million sequences for 'Dr. Davis', 'F8, 1-42' and 'Georgia Belle', respectively. BWA/SAMtools were used for alignment of raw reads and SNP detection, with custom PERL scripts for SNP filtering. Velvet's Columbus module was used for sequence assembly. Comparison of aligned and overlapping sequences from both Roche 454 and Illumina/Solexa resulted in the selection of 6654 high quality SNPs for 'Dr. Davis' vs. 'F8, 1-42' and 'Georgia Belle', distributed on eight major peach genome scaffolds as defined from the 'Lovell' assembly. CONCLUSION: The eight scaffolds contained about 215-225 Mb of peach genomic sequences with one SNP/~ 40,000 bases. All sequences from Roche 454 and Illumina/Solexa have been submitted to NCBI for public use in the Short Read Archive database. SNPs have been deposited in the NCBI SNP database.

A fruit quality gene map of Prunus.

BACKGROUND: Prunus fruit development, growth, ripening, and senescence includes major biochemical and sensory changes in texture, color, and flavor. The genetic dissection of these complex processes has important applications in crop improvement, to facilitate maximizing and maintaining stone fruit quality from production and processing through to marketing and consumption. Here we present an integrated fruit quality gene map of Prunus containing 133 genes putatively involved in the determination of fruit texture, pigmentation, flavor, and chilling injury resistance. RESULTS: A genetic linkage map of 211 markers was constructed for an intraspecific peach (Prunus persica) progeny population, Pop-DG, derived from a canning peach cultivar 'Dr. Davis' and a fresh market cultivar 'Georgia Belle'. The Pop-DG map covered 818 cM of the peach genome and included three morphological markers, 11 ripening candidate genes, 13 cold-responsive genes, 21 novel EST-SSRs from the ChillPeach database, 58 previously reported SSRs, 40 RAFs, 23 SRAPs, 14 IMAs, and 28 accessory markers from candidate gene amplification. The Pop-DG map was co-linear with the Prunus reference T x E map, with 39 SSR markers in common to align the maps. A further 158 markers were bin-mapped to the reference map: 59 ripening candidate genes, 50 cold-responsive genes, and 50 novel EST-SSRs from ChillPeach, with deduced locations in Pop-DG via comparative mapping. Several candidate genes and EST-SSRs co-located with previously reported major trait loci and quantitative trait loci for chilling injury symptoms in Pop-DG. CONCLUSION: The candidate gene approach combined with bin-mapping and availability of a community-recognized reference genetic map provides an efficient means of locating genes of interest in a target genome. We highlight the co-localization of fruit quality candidate genes with previously reported fruit quality QTLs. The fruit quality gene map developed here is a valuable tool for dissecting the genetic architecture of fruit quality traits in Prunus crops.

Phylogenetics and reticulate evolution in Pistacia (Anacardiaceae).

The systematic position and intrageneric relationships of the economically important Pistacia species (Anacardiaceae) are controversial. The phylogeny of Pistacia was assessed using five data sets: sequences of nuclear ribosomal ITS, the third intron of the nuclear nitrate reductase gene (NIA-i3), and the plastid ndhF, trnL-F and trnC-trnD. Significant discordance was detected among ITS, NIA-i3, and the combined plastid DNA data sets. ITS, NIA-i3, and the combined plastid data sets were analyzed separately using Bayesian and parsimony methods. Both the ITS and the NIA-i3 data sets resolved the relationships among Pistacia species well; however, these two data sets had significant discordance. The ITS phylogeny best reflects the evolutionary relationships among Pistacia species. Lineage sorting of the NIA-i3 alleles may explain the conflicts between the NIA-i3 and the ITS data sets. The combined analysis of three plastid DNA data sets resolved Pistacia species into three major clades, within which only a few subclades were supported. Pistacia was shown to be monophyletic in all three analyses. The previous intrageneric classification was largely inconsistent with the molecular data. Some Pistacia species appear not to be genealogical species, and evidence for reticulate evolution is presented. Pistacia saportae was shown to be a hybrid with P. lentiscus (maternal) and P. terebinthus (paternal) as the parental taxa.

Candidate gene database and transcript map for peach, a model species for fruit trees.

Peach (Prunus persica) is a model species for the Rosaceae, which includes a number of economically important fruit tree species. To develop an extensive Prunus expressed sequence tag (EST) database for identifying and cloning the genes important to fruit and tree development, we generated 9,984 high-quality ESTs from a peach cDNA library of developing fruit mesocarp. After assembly and annotation, a putative peach unigene set consisting of 3,842 ESTs was defined. Gene ontology (GO) classification was assigned based on the annotation of the single "best hit" match against the Swiss-Prot database. No significant homology could be found in the GenBank nr databases for 24.3% of the sequences. Using core markers from the general Prunus genetic map, we anchored bacterial artificial chromosome (BAC) clones on the genetic map, thereby providing a framework for the construction of a physical and transcript map. A transcript map was developed by hybridizing 1,236 ESTs from the putative peach unigene set and an additional 68 peach cDNA clones against the peach BAC library. Hybridizing ESTs to genetically anchored BACs immediately localized 11.2% of the ESTs on the genetic map. ESTs showed a clustering of expressed genes in defined regions of the linkage groups. [The data were built into a regularly updated Genome Database for Rosaceae (GDR), available at (http://www.genome.clemson.edu/gdr/).].