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Allele frequency estimates applied in human DNA profiling are incorporated to polyploids in horticultural plants to use and maintain germplasm effectively. Studies are conducted focusing on disomic and polysomic inheritance patterns of polyploids and an EM-type algorithm is presented to estimate allele frequencies based on them and the method is demonstrated using simulated data.

Pseudomonas syringae pv. actinidiae (Psa) is responsible for bacterial canker of kiwifruit. Biovar 3 of Psa (Psa3) has been causing widespread damage to yellow‐ and green‐fleshed kiwifruit (Actinidia spp.) cultivars in all the major kiwifruit‐producing countries in the world. In some areas, including New Zealand, P. syringae pv. actinidifoliorum (Pfm), another bacterial pathogen of kiwifruit, was initially classified as a low virulence biovar of Psa. Ability to rapidly distinguish between these pathovars is vital to the management of bacterial canker. Whole genome sequencing (WGS) data were used to develop PCR assays to specifically detect Psa3 and Pfm from field‐collected material without the need to culture bacteria. Genomic data from 36 strains of Psa, Pfm or related isolates enabled identification of areas of genomic variation suitable for primer design. The developed assays were tested on 147 non‐target bacterial species including strains likely to be found in kiwifruit orchards. A number of assays did not proceed because although they were able to discriminate between the different Psa biovars and Pfm, they also produced amplicons from other unrelated bacteria. This could have resulted in false positives from environmental samples, and demonstrates the care that is required when applying assays devised for pure cultures to field‐collected samples. The strategy described here for developing assays for distinguishing strains of closely related pathogens could be applied to other diseases with characteristics similar to Psa. [ABSTRACT FROM AUTHOR]

The polymerase chain reaction (PCR)-based technique of random amplification of polymorphic DNA (RAPD) was used to differentiate DNA from species of the genus Mycosphaerella. DNA from two pathogens which cause Sigatoka leafspot diseases of banana, M. fijiensis and M. musicola, and two other Mycosphaerella species which are commonly found on banana, M. musae and M. minima, gave distinct RAPD banding patterns with all PCR primers tested. PCR, using primer RC07, amplified a 1250 bp RAPD fragment from all isolates of M. fijiensis obtained from II geographical origins. This fragment was absent from the other species of Mycosphaerella. In Southern blots of genomic DNA, this band hybridized exclusively to DNA from M. fijiensis, and the pattern of hybridization suggested that it was binding to repeated DNA. A RAPD band amplified with primer PM06 obtained from M. musicola was also found to be species-specific. Southern analysis suggested that the fragment hybridized to a single-copy sequence in the M. musicola genome. Total genomic DNA from M. musicola was found to be a species-specific hybridization probe. Dot—blots confirmed the specificity of these probes, and could be used to identify isolates of Mycosphacrella which cause Sigatoka disease of banana in south-east Asia. [ABSTRACT FROM AUTHOR]

Pseudomonas syringae pv. actinidiae is the etiological agent of kiwifruit canker disease, causing severe economic losses in kiwifruit production areas around the world. Rapid diagnosis, understanding of bacterial virulence, and rate of infection in kiwifruit cultivars are important in applying effective measures of disease control. P. syringae pv. actinidiae load in kiwifruit is currently determined by a labor-intense colony counting method with no high-throughput and specific quantification method being validated. In this work, we used three alternative P. syringae pv. actinidiae quantification methods in two infected kiwifruit cultivars: start of growth time, quantitative PCR (qPCR), and droplet digital PCR (ddPCR). Method performance in each case was compared with the colony counting method. Methods were validated using calibration curves obtained with serial dilutions of P. syringae pv. actinidiae biovar 3 (Psa3) inoculum and standard growth curves obtained from kiwifruit samples infected with Psa3 inoculum. All three alternative methods showed high correlation ( r > 0.85) with the colony counting method. qPCR and ddPCR were very specific, sensitive (5 × 10 2 CFU/cm 2 ), highly correlated to each other ( r = 0.955), and flexible, allowing for sample storage. The inclusion of a kiwifruit biomass marker increased the methods' accuracy. The qPCR method was efficient and allowed for high-throughput processing, and the ddPCR method showed highly accurate results but was more expensive and time consuming. While not ideal for high-throughput processing, ddPCR was useful in developing accurate standard curves for the qPCR method. The combination of the two methods is high-throughput, specific for Psa3 quantification, and useful for research studies (e.g., disease phenotyping and host-pathogen interactions).

The intrinsic disorder is highly abundant in eukaryotic genomes. In the animal kingdom, numerous intrinsically disordered proteins (IDPs) have been characterized, especially in cell signalling and transcription regulation. An intrinsically disordered region often folds in different structures allowing an IDP to recognize and bind different partners at various binding interfaces. In contrast, there have only been a few reports of IDPs from the plant kingdom. Plant-specific GRAS proteins play critical and diverse roles in plant development and signalling and often act as integrators of signals from multiple plant growth regulatory inputs. Using computational and bioinformatics tools, we demonstrate here that the GRAS proteins are intrinsically disordered, thus forming the first functionally required unfoldome in the plant kingdom. Furthermore, the N-terminal domains of GRAS proteins are predicted to contain numerous Molecular Recognition Features (MoRFs), short interaction-prone segments that are located within extended disorder regions and are able to recognize their interacting partners and to undergo disorder-to-order transitions upon binding to these specific partners. Overlapping with the relatively conserved motifs in the N-terminal domains of GRAS proteins, these predicted MoRFs represent the potential protein-protein binding sites and may be involved in molecular recognition during plant development. This study enables us to propose a conceptual framework that guides future experimental approaches to understand structure-function relationships of the entire GRAS family.

Comparative mapping has been the primary approach for analyzing genomes of divergent species because gene linkage is often conserved over evolutionary time, a phenomenon known as synteny. Comparisons are based on computational detection of orthologous sequences between species. Small overlapping oligonucleotide (overgo) probes, consisting of two self-annealing oligonucleotides at the 3'-end, are filled in with labeled deoxynucleotides. Overgo probes are designed against highly conserved regions between two genomes, at regular intervals (30-40 kbp). All methodologies reported to date have used radioactive dATP and dCTP to label overgo probes. We report a nonradioactive labeling protocol to produce digoxigenin-labeled overgo probes for high-throughput screening of the Actinidia chinensis bacterial artificial chromosome library.

A 3.9-kb genomic DNA fragment from the cucurbit pathogen Fusarium solani f. sp. cucurbitae race 2 was cloned. Sequence analysis revealed an open reading frame of 690 nucleotides interrupted by a single 51-bp intron. The nucleotide and predicted amino acid sequences showed 92 and 98% identity, respectively, to those of the cutA gene of the pea pathogen F. solani f. sp. pisi. A gene replacement vector was constructed and used to generate cutA- mutants that were detected with a polymerase chain reaction (PCR) assay. Seventy-one cutA- mutants were identified among the 416 transformants screened. Vector integration was assessed by Southern analysis in 23 of these mutants. PCR and Southern analysis data showed the level of homologous integration was 14%. Disruption of the cutA locus in mutants was confirmed by RNA gel blot hybridization. Neither virulence on Cucurbita maxima cv. Delica at any of six different inoculum concentrations, nor pathogenicity on intact fruit of four different species or cultivars of cucurbit or hypocotyl tissue of C. maxima cv. Crown, was found to be affected by disruption of the cutA gene.

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnlA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA-); both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried at 5' and 3' truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA- transformants. pnlA- transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.

Oligodeoxyribonucleotide primers were designed from conserved amino acid (aa) sequences between pectin lyase D (PNLD) from Aspergillus niger and pectate lyases A and E (PELA/E) from Erwinia chrysanthemi. The polymerase chain reaction (PCR) was used with these primers to amplify genomic DNA from the plant pathogenic fungus Glomerella cingulata. Three different 220-bp fragments with homology to PNL-encoding genes from A. niger, and a 320-bp fragment with homology to PEL-encoding genes from Nicotiana tabacum and E. carotovora were cloned. One of the 220-bp PCR products (designated pnlA) was used as a probe to isolate a PNL-encoding gene from a lambda genomic DNA library prepared from G. cingulata. Nucleotide (nt) sequence data revealed that this gene has seven exons and codes for a putative 380-aa protein. The nt sequence of a cDNA clone, prepared using PCR, confirmed the presence of the six introns. The positions of the introns were different from the sites of the five introns present in the three PNL-encoding genes previously sequenced from A. niger. PNLA was synthesised in yeast by cloning the cDNA into the expression vector, pEMBLYex-4, and enzymatically active protein was secreted into the culture medium. Significantly higher expression was achieved when the context of the start codon, CACCATG, was mutated to CAAAATG, a consensus sequence commonly found in highly expressed yeast genes. The produced protein had an isoelectric point (pI) of 9.4, the same as that for the G. cingulata pnlA product.(ABSTRACT TRUNCATED AT 250 WORDS)