Genetic Analysis of Victorin Sensitivity and Identification of a Causal Nucleotide-Binding Site Leucine-Rich Repeat Gene in Phaseolus vulgaris.

Cochliobolus victoria, the causal agent of Victoria blight, is pathogenic due to its production of a toxin called victorin. Victorin sensitivity in oats, barley, Brachypodium spp., and Arabidopsis has been associated with nucleotide-binding site leucine-rich repeat (NLR) genes, a class of genes known for conferring disease resistance. In this work, we investigated the sensitivity of Phaseolus vulgaris to victorin. We found that victorin sensivity in Phaseolus vulgaris is a developmentally regulated, quantitative trait. A single quantitative trait locus (QTL) accounted for 34% of the phenotypic variability in victorin sensitivity among Stampede × Red Hawk (S×R) recombinant inbred lines. We cloned two NLR-encoding genes within this QTL and showed one, Phvul05G031200 (PvLOV), confers victorin-dependent cell death when overexpressed in Nicotiana benthamiana. Protein sequences of PvLOV from victorin-sensitive and the victorin-resistant bean parents differ by two amino acids in the leucine-rich repeat region, but both proteins confer victorin-dependent cell death when overexpressed in N. benthamiana.

Expression of drought tolerance genes in tropical upland rice cultivars (Oryza sativa).

Gene expression related to drought response in the leaf tissues of two Brazilian upland cultivars, the drought-tolerant Douradão and the drought-sensitive Primavera, was analyzed. RNA-seq identified 27,618 transcripts in the Douradão cultivar, with 24,090 (87.2%) homologous to the rice database, and 27,221 transcripts in the Primavera cultivar, with 23,663 (86.9%) homologous to the rice database. Gene-expression analysis between control and water-deficient treatments revealed 493 and 1154 differentially expressed genes in Douradão and Primavera cultivars, respectively. Genes exclusively expressed under drought were identified for Douradão, including two genes of particular interest coding for the protein peroxidase precursor, which is involved in three distinct metabolic pathways. Comparisons between the two drought-exposed cultivars revealed 2314 genes were differentially expressed (978 upregulated, 1336 downregulated in Douradão). Six genes distributed across 4 different transcription factor families (bHLH, MYB, NAC, and WRKY) were identified, all of which were upregulated in Douradão compared to Primavera during drought. Most of the genes identified in Douradão activate metabolic pathways responsible for production of secondary metabolites and genes coding for enzymatically active signaling receptors. Quantitative PCR validation showed that most gene expression was in agreement with computational prediction of these transcripts. The transcripts identified here will define molecular markers for identification of Cis-acting elements to search for allelic variants of these genes through analysis of polymorphic SNPs in GenBank accessions of upland rice, aiming to develop cultivars with the best combination of these alleles, resulting in materials with high yield potential in the event of drought during the reproductive phase.

Demographic factors shaped diversity in the two gene pools of wild common bean Phaseolus vulgaris L.

Wild common bean (Phaseolus vulgaris L.) is distributed throughout the Americas from Mexico to northern Argentina. Within this range, the species is divided into two gene pools (Andean and Middle American) along a latitudinal gradient. The diversity of 24 wild common bean genotypes from throughout the geographic range of the species was described by using sequence data from 13 loci. An isolation-migration model was evaluated using a coalescent analysis to estimate multiple demographic parameters. Using a Bayesian approach, Andean and Middle American subpopulations with high percentage of parentages were observed. Over all loci, the Middle American gene pool was more diverse than the Andean gene pool (π(sil)=0.0089 vs 0.0068). The two subpopulations were strongly genetically differentiated over all loci (F(st)=0.29). It is estimated that the two current wild gene pools diverged from a common ancestor ∼111 000 years ago. Subsequently, each gene pool underwent a bottleneck immediately after divergence and lasted ∼40 000 years. The Middle American bottleneck population size was ∼46% of the ancestral population size, whereas the Andean was 26%. Continuous asymmetric gene flow was detected between the two gene pools with a larger number of migrants entering Middle American gene pool from the Andean gene pool. These results suggest that because of the complex population structure associated with the ancestral divergence, subsequent bottlenecks in each gene pool, gene pool-specific domestication and intense selection within each gene pool by breeders; association mapping would best be practised within each common bean gene pool.

A Quantitative Method to Screen Common Bean Plants for Resistance to Bean common mosaic necrosis virus.

ABSTRACT A quantitative method to screen common bean (Phaseolus vulgaris) plants for resistance to Bean common mosaic necrosis virus (BCMNV) is described. Four parameters were assessed in developing the quantitative method: symptoms associated with systemic virus movement, plant vigor, virus titer, and plant dry weight. Based on these parameters, two rating systems (V and VV rating) were established. Plants from 21 recombinant inbred lines (RILs) from a Sierra (susceptible) x Olathe (partially resistant) cross inoculated with the BCMNV-NL-3 K strain were used to evaluate this quantitative approach. In all, 11 RILs exhibited very susceptible reactions and 10 RILs expressed partially resistant reactions, thus fitting a 1:1 susceptible/partially resistant ratio (chi(2) = 0.048, P = 0.827) and suggesting that the response is mediated by a single gene. Using the classical qualitative approach based only on symptom expression, the RILs were difficult to separate into phenotypic groups because of a continuum of responses. By plotting mean percent reduction in either V (based on visual symptoms) or VV (based on visual symptoms and vigor) rating versus enzyme-linked immunosorbent assay (ELISA) absorbance values, RILs could be separated clearly into different phenotypic groups. The utility of this quantitative approach also was evaluated on plants from 12 cultivars or pure lines inoculated with one of three strains of BCMNV. Using the mean VV rating and ELISA absorbance values, significant differences were established not only in cultivar and pure line comparisons but also in virus strain comparisons. This quantitative system should be particularly useful for the evaluation of the independent action of bc genes, the discovery of new genes associated with partial resistance, and assessing virulence of virus strains.

Molecular and phenotypic mapping of genes controlling seed coat pattern and color in common bean (Phaseolus vulgaris L.).

Common bean (Phaseolus vulgaris L.) exhibits a wide variety of seed coat patterns and colors. From a historical perspective, extensive genetic analyses have identified specific genes that control seed coat pattern (T, Z, L, J, Bip, and Ana) and color (P, C, R, J, D, G, B, V, and Rk). Many of these genes exhibit epistatic interactions with other genes, interactions that define the many seed coat patterns and colors observed within the species. To better understand these complex interactions, we began a molecular marker discovery program that previously identified random amplified polymorphic DNA (RAPD) markers linked to many of these genes. We report here the discovery of RAPD markers linked to three additional genes-C, G, and V. These markers, and five RAPD markers we previously discovered linked to other seed coat pattern and color genes, were converted into easily scorable sequence tagged site (STS) markers. We next placed these markers onto a common molecular map shared by the Phaseolus research community and demonstrated a generally wide distribution of the genes throughout the common bean genome. A few previously unrecognized linkages were discovered. The probable existence of multiple genes controlling growth habit in common bean is discussed. Our approach demonstrates the usefulness and feasibility of marker discovery in one population followed by accurate mapping of that marker onto a core, community-wide map.

Crg, a gene required for Ur-3-mediated rust resistance in common bean, maps to a resistance gene analog cluster.

Race-specific resistance to the bean rust pathogen (Uromyces appendiculatus) is provided by a number of loci in common bean (Phaseolus vulgaris). The Ur-3 locus controls hypersensitive resistance (HR) to 44 of the 89 races curated in the United States. To better understand resistance mediated by this locus, we developed new genetic material for analysis. We developed a population of mutagenized seed of cv. Sierra (genotype = Ur-3 ur-4 ur-6) that was screened with a bean rust race that is normally incompatible (HR response) on Ur-3 genotypes. We discovered two mutants of common bean, crg and ur3-delta3, in which uredinia formed on leaves (a compatible interaction) following infection. The F1 generation from a cross of these two mutants expressed the HR response, and the F2 generation segregated in a ratio of 9:7 (HR/uredinia formation). Therefore, the two genes are unlinked. Further genetic analysis determined that the mutation in ur3-delta3 was in the Ur-3 locus, and the mutation in crg was in a newly discovered gene given the symbol Crg (Complements resistance gene). Each mutation was inherited in a recessive manner. Unlike ur3-delta3, crg expressed reduced compatibility to bean rust races 49 and 47 that are normally fully compatible on genotypes, such as Sierra, that are homozygous recessive at the Ur-4 and Ur-6 loci. This suggests a gene mutated in crg is normally a positive compatibility factor for the bean-bean rust interaction. Polymerase chain reaction analysis of crg with primers to common bean resistance gene analogs (RGA) that contain a nucleotide-binding site sequence similar to those found in a number of plant disease resistance genes revealed that crg is missing the SB1 RGA, but not the linked SB3 and SB5 RGAs. Genetic analyses revealed that Crg cosegregates with the SB1 RGA. These results demonstrate that Crg is located near a RGA cluster in the common bean genome.

Disease-resistance related sequences in common bean.

Primers based on a conserved nucleotide binding site (NBS) found in several cloned plant disease resistance genes were used to amplify DNA fragments from the genome of common bean (Phaseolus vulgaris). Cloning and sequence analysis of these fragments uncovered eight unique classes of disease-resistance related sequences. All eight classes contained the conserved kinase 2 motif, and five classes contained the kinase 3a motif. Gene expression was noted for five of the eight classes of sequences. A clone from the SB3 class mapped 17.8 cM from the Ur-6 gene that confers resistance to several races of the bean rust pathogen Uromyces appendiculatus. Linkage mapping identified microclusters of disease-resistance related sequence in common bean, and sequences mapped to four linkage groups in one population. Comparison with similar sequences from soybean (Glycine max) revealed that any one class of common bean disease-resistance related sequences was more identical to a soybean NBS-containing sequence than to the sequence of another common bean class.

Organization and transcription of the gene encoding potato UDP-glucose pyrophosphorylase.

The organization of the gene encoding potato UDP-glucose pyrophosphorylase, one of the key enzymes of carbohydrate metabolic pathway is presented. The gene cloned from cultivar (cv.) Lemhi consists of a 6.6-kb structural and a 1-kb regulatory region. The structural region contains 20 exons and 19 introns. The coding sequence with exception of three bases is identical with the UGPase cDNA previously cloned from Danshaku-Imo cv. [Katsube et al. (1990) UDP-Glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing. J. Biochem. 108, 321-326]. The largest intron contains a tandem repeat consisting of 50 nt core units. A putative polyadenylation site is situated 79 bp downstream of the translation stop codon. A transcription start point (tsp) and a putative TATA-box were located 84 bp and 141 bp upstream of the translation start, respectively. The regulatory region contained general enhancer, suppressor, and regions responsible for tissue specificity of UGPase expression.

A tandemly repeated sequence from the Plasmopara halstedii genome.

A 747-bp tandem repeat element from the genome of the fungus Plasmopara halstedii, the causal agent of downy mildew of sunflower, was cloned and analyzed. The clone can be used as a probe to distinguish races of the pathogen. Sequence analysis of a copy of this element revealed the presence of 103 direct repeats of 6 bp or greater and two potential ORFs. This tandemly repeated element consists of four subunits that may have evolved as a result of several unequal crossing-over events.

Mitochondrial DNA Sequence Divergence among Lycopersicon and Related Solanum Species.

Sequence divergence among the mitochondrial (mt) DNA of nine Lycopersicon and two closely related Solanum species was estimated using the shared fragment method. A portion of each mt genome was highlighted by probing total DNA with a series of plasmid clones containing mt-specific DNA fragments from Lycopersicon pennellii. A total of 660 fragments were compared. As calculated by the shared fragment method, sequence divergence among the mtDNAs ranged from 0.4% for the L. esculentum-L. esculentum var. cerasiforme pair to 2.7% for the Solanum rickii-L. pimpinellifolium and L. cheesmanii-L. chilense pairs. The mtDNA divergence is higher than that reported for Lycopersicon chloroplast (cp) DNA, which indicates that the DNAs of the two plant organelles are evolving at different rates. The percentages of shared fragments were used to construct a phenogram that illustrates the present-day relationships of the mtDNAs. The mtDNA-derived phenogram places L. hirsutum closer to L. esculentum than taxonomic and cpDNA comparisons. Further, the recent assignment of L. pennellii to the genus Lycopersicon is supported by the mtDNA analysis.