<Emphasis Type="Italic">RB</Emphasis> and <Emphasis Type="Italic">Ph</Emphasis> resistance genes in potato and tomato minimize risk for oospore production in the presence of mating pairs of <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Composed mostly of fungivorous species, the genus Aphelenchoides also comprises 14 plant-parasitic species. The most common and devastating, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis have been reported on more than 900 plant species. The combination of low inter-specific and high intra-specific morphological variability makes morphology-based identification extremely difficult within this genus, and has led to molecular tools being employed to ensure accurate diagnoses. rDNA markers are widely used for the identification of nematodes while the Cytochrome Oxidase I gene (COI) remains relatively unexplored despite its role as the standard barcode for almost all animal groups. To explore its suitability as a diagnostic tool, we studied a fragment of the mtCOI region of the four main plant-parasitic Aphelenchoides within a phylogenetic framework. We generated 69 mtCOI and 123 rDNA sequences of diverse Aphelenchoides taxa; 67 belong to the main plant-parasitic species including the first mtCOI sequence of A. fragariae and the first mtCOI and 28S sequences of A. subtenuis. mtCOI had a similar success rate for PCR amplification. Phylogenetic trees based on the three studied markers are largely in agreement with one another, validating their use for Aphelenchoides diagnosis; additionally, we were able to locate several misidentified sequences of plant-parasitic Aphelenchoides in existing databases. The concatenated analysis from the three markers resulted in a more robust insight into the phylogeny and evolution of Aphelenchoides, revealing that plant-parasitism has evolved independently at least three times within this genus, presumably from fungal-feeding ancestors.     

Development of a Panel of Specific Monoclonal Antibodies to High Molecular Weight Glutenin Subunits and Their Application in Genetic Screening

High molecular weight glutenin subunits (HMW-GS) encoded by different chromosomal loci and alleles (1, 2, 5, 7, 10, and 12) were purified using reversed-phase HPLC from reduced, aqueous propanol extracts of flour from aneuploid or null wheat lines. Unlike previous libraries of monoclonal antibodies developed in our laboratory to SDS-extracted or alkylated HMW-GS, several of the monoclonal antibodies (mAb) developed in this study had a range of specificity patterns for HMW-GS in enzyme-linked immunosorbent assay (ELISA) and on immunoblots. A subset of the mAb bound either x- or y-type HMW-GS but not other gluten proteins, while a few antibodies bound one (mAb 110622, 110421, 140820), or two (mAb 101319, 110804, 140705, 1410460) HMW-GS expressed in each cultivar tested. In most cases, antibodies bound equally to the subunits encoded by different HMW-GS alleles. The more specific antibodies should be useful in research on the quantitative variation of HMW-GS expression and in studies of the role of particular HMW-GS in dough structure. The mAb 101319, which was prepared to subunit 1, bound to HMW-GS 1Bx subunits in ELISA and on immunoblots. This antibody also provided a higher absorbance value in ELISA with extracts of wheat lines expressing the Glu-Ble allele (HMW-GS 20) compared with the Glu-Bli allele (HMW-GS 17+18). Another mAb (110622) detected subunit 2 more strongly than subunit 5 in ELISA and produced a higher signal in immunoblots with subunit 2 even though these subunits are >98.7% homologous in amino acid sequence. An ELISA assay using this antibody was optimized for discrimination of wheat lines with the allelic pairs of subunits 1Dx5-1Dy10 from those with 1Dx2-1Dy12, with the former lines providing stronger dough properties and superior breadmaking quality. The performance of this assay was unaffected by other variations at HMW-GS loci and was demonstrated in sets of biotypes, doubled haploid, and cross-bred breeder's lines.