Next generation crop models: A modular approach to model early vegetative and reproductive development of the common bean (Phaseolus vulgaris L).

The next generation of gene-based crop models offers the potential of predicting crop vegetative and reproductive development based on genotype and weather data as inputs. Here, we illustrate an approach for developing a dynamic modular gene-based model to simulate changes in main stem node numbers, time to first anthesis, and final node number on the main stem of common bean (Phaseolus vulgaris L.). In the modules, these crop characteristics are functions of relevant genes (quantitative trait loci (QTL)), the environment (E), and QTL × E interactions. The model was based on data from 187 recombinant inbred (RI) genotypes and the two parents grown at five sites (Citra, FL; Palmira, Colombia; Popayan, Colombia; Isabela Puerto Rico; and Prosper, North Dakota). The model consists of three dynamic QTL effect models for node addition rate (NAR, No. d- 1), daily rate of progress from emergence toward flowering (RF), and daily maximum main stem node number (MSNODmax), that were integrated to simulate main stem node number vs. time, and date of first flower using daily time steps. Model evaluation with genotypes not used in model development showed reliable predictions across all sites for time to first anthesis (R2 = 0.75) and main stem node numbers during the linear phase of node addition (R2 = 0.93), while prediction of the final main stem node number was less reliable (R2 = 0.27). The use of mixed-effects models to analyze multi-environment data from a wide range of genotypes holds considerable promise for assisting development of dynamic QTL effect models capable of simulating vegetative and reproductive development.

Integration of common bean ( Phaseolus vulgaris L.) linkage and chromosomal maps.

Fluorescent in situ hybridisation of pooled, closely linked RFLP markers was used to integrate the genetic linkage map and the mitotic chromosome map of the common bean. Pooled RFLP probes showed clear and reproducible signals and allowed the assignment of all linkage groups to the chromosomes of two Phaseolus vulgaris cultivars, Saxa and Calima. Low extension values for signals originating from clustered RFLPs suggest that these clones are physically close to each other and that clusters in the genetic map are not a result of suppression of recombination due to the occurrence of chromosome rearrangements. For linkage group K, clustering of markers could be associated with proximity to centromeres. High variation in the number of 45S rDNA loci was observed among cultivars, suggesting that these terminal sites are highly recombinogenic in common bean.

Identification of putative genes in bean (Phaseolus vulgaris) genomic (Bng) RFLP clones and their conversion to STSs.

A set of 79 previously mapped bean (Phaseolus vulgaris) genomic (Bng) clones were partially sequenced. BLAST database searches detected homologies between 59 of these clones and genes from a variety of plants, especially Arabidopsis thaliana. Some matches in the database to the Bng clones included a putative P-glycoprotein-ABC transporter from Arabidopsis, an early nodulin-binding protein (ENBPI) from Medicago truncatula, a lon-protease protein from spinach, a branched-chain amino-acid aminotransferase from Arabidopsis, and a vacuolar sorting receptor (BP-80) from Pisum sativum. Additional matches were found for genes involved in isoprenoid biosynthesis, sulfur metabolism, proline biosynthesis, and floral development. Sequence tagged site (STSs) were produced for 16 of the clones, 2 of which contain simple sequence repeats (SSRs). Polymorphisms were detected for six of the STSs.

Evidence for a prokaryotic insertion-sequence contamination in eukaryotic sequences registered in different databases.

An insertion-sequence of prokaryotic origin was detected in a genomic clone obtained from a Phaseolus vulgaris bacterial artificial chromosome (BAC) library. This BAC clone, characterized as part of a contig constructed near a virus resistance gene, exhibited restriction fragment length polymorphism with an overlapping clone of the contig. Restriction analysis of DNA obtained from individual colonies of the stock culture indicated the presence of a mixed population of wild-type and insertional mutants. Sequence analysis of both members of the population revealed the presence of IS 10R, an insertion-sequence from Escherichia coli. A BLAST search for IS 10-like sequences detected unexpected homologies with a large number of eukaryotic sequences from Homo sapiens, Arabidopsis thaliana, Drosophila melanogasterand Caenorhabditis elegans. Southern analysis of a random sample of BAC clones failed to detect IS 10 in the BAC DNA. However, prolonged sub-culturing of a set of 15 clones resulted in transposition into the BAC DNA. Eventually, all cultures acquired a 2.3-kb fragment that hybridized strongly with IS 10. Sequence analysis revealed the presence of a preferred site for transposition in the BAC vector. These results indicate that a large number, if not all, of the BAC libraries from different organisms are contaminated with IS 10R. The source of this element has been identified as the DH10B strain of E. coli used as the host for BAC libraries.

Xv4-vrxv4: a new gene-for-gene interaction identified between Xanthomonas campestris pv. vesicatoria race T3 and wild tomato relative Lycopersicon pennellii.

Strains of tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria elicit a hypersensitive response (HR) in leaves of Lycopersicon pennellii LA716. Genetic segregation of the resistance exhibited ratios near 3:1 in F2 populations, which confirmed that a single dominant gene controlled the inheritance of this trait. With the aid of a collection of introgression lines, restriction fragment length polymorphism, and cleaved amplified polymorphic sequence markers, the resistance locus was located on chromosome 3 between TG599 and TG134. An avirulence gene named avrXv4 was also isolated by mobilizing a total of 600 clones from a genomic DNA library of the T3 strain 91-118 into the X. campestris pv. vesicatoria strain ME90, virulent on L. pennellii. One cosmid clone, pXcvT3-60 (29-kb insert), induced HR in resistant plants. The avirulent phenotype of pXcvT3-60 was confirmed by comparing growth rates in planta and electrolyte leakages among transconjugants carrying a mutated or intact clone with the wild-type T3 strain 91-118. A 1.9-kb DNA fragment contained within a 6.8-kb active subclone was sequenced and was determined to carry an open reading frame of 1,077 bp. The predicted AvrXv4 protein exhibits high similarity to members of an emerging new family of bacterial proteins from plant and mammalian pathogens comprising AvrRxv, AvrBsT, YopJ, YopP, AvrA, and YL40.

Method for cloning restriction fragments containing the termini of BAC inserts.

We have developed a method to isolate the termini of BAC clones. The method is based on the two unique NotI sites located approximately 300 bp on either side of the EcoRI cloning site of the BAC vector pECS-BAC4. Our strategy includes the following steps: (i) generation of Southern blots with BAC clones digested with NotI and a second restriction enzyme; (ii) identification of the termini attached to the NotI/EcoRI fragment of the BAC vector via hybridization with a probe derived from sequences located between one NotI site (left or right arm) and the cloning site; (iii) ligation of the doubly digested BAC clone (NotI and the selected second restriction enzyme) with an equally doubly digested cloning plasmid vector; and (iv) confirmation of the clone as a terminus. This strategy has allowed us to begin the construction of a contig near a common bean gene that controls resistance to a group of potyviruses.

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.

A cytoplasmic male sterility-associated mitochondrial peptide in common bean is post-translationally regulated.

Cytoplasmic male sterility in the common bean plant is associated with a dominant mitochondrial mutation designated pvs-or f 239 (for Phaseolus vulgaris sterility sequence open reading frame 239). The sequence is transcribed in both vegetative and reproductive tissues, but the translation product, ORF239, is present only in reproductive tissues. We present evidence to support a model of post-translational regulation of ORF239 expression based on the following observations. In organello translation experiments using purified mitochondria from young seedlings demonstrated accumulation of ORF239 only when a protease inhibitor was included. Proteolytic activity against ORF239 was observed in mitochondrial extracts fractionating with the mitochondrial inner membrane. The DNA sequence encoding a serine-type protease, similar to the lon protease gene of Escherichia coli, was cloned from the Arabidopsis genome. The expression product of this sequence demonstrated proteolytic activity against ORF239 in vitro, with features resembling the activity detected in mitochondrial inner membrane preparations. Antibodies generated against the overexpressed Lon homolog reduced proteolytic activity against ORF239 when added to mitochondrial extracts. Our data suggest that ORF239 was undetected in vegetative tissue due to rapid turnover by at least one mitochondrial protease that acts against ORF239 post-translationally.

Genome conservation among three legume genera detected with DNA markers.

A set of 219 DNA clones derived from mungbean (Vigna radiata), cowpea (V. unguiculata), common bean (Phaseolus vulgaris), and soybean (Glycine max) were used to generate comparative linkage maps among mungbean, common bean, and soybean. The maps allowed an assessment of linkage conservation and collinearity among the three genomes. Mungbean and common bean, both of the subtribe Phaseolinae, exhibited a high degree of linkage conservation and preservation of marker order. Most linkage groups of mungbean consisted of only one or two linkage blocks from common bean (and vice versa). The situation was significantly different with soybean, a member of the subtribe Glycininae. Mungbean and common bean linkage groups were generally mosaics of short soybean linkage blocks, each only a few centimorgans in length. These results suggest that it would be fruitful to join maps of mungbean and common bean, while knowledge of conserved genomic blocks would be useful in increasing marker density in specific genomic regions for all three genera. These comparative maps may also contribute to enhanced understanding of legume evolution.

Genomic localization of tomato genes that control a hypersensitive reaction to Xanthomonas campestris pv. vesicatoria (Doidge) dye.

Xanthomonas campestris pv. vesicatoria causes bacterial spot, one of the most serious diseases of tomatoes. The lycopersicon esculentum accession 'Hawaii 7998' is the only reliable source of resistance to race 1 strains of the pathogen. This resistance is associated with a hypersensitive reaction controlled by multiple nondominant genes. The inoculated area becomes fully necrotic 24 hr after inoculation in 'Hawaii 7998,' whereas full necrosis is observed 5 and 4 days after inoculation in the susceptible species L. pennellii (LA 716) and their F1, respectively. An interspecific backcross population, using 'Hawaii 7998' as the recurrent parent, was analyzed to determine the linkage relationships between the resistance genes and 135 molecular marker loci. The range of responses of the BC1 population included those of the parents. Linkage to a hypersensitive response factor was assessed by comparing the rates of necrosis development between homozygous and heterozygous plants at 8 hr-intervals. Three factors that affect the hypersensitive response of 'Hawaii 7998' were detected. One factor is on the short arm of chromosome I, another on the long arm of chromosome I, and a third on the long arm of chromosome 5. These factors appeared to act independently and to have additive effects.

Pollen fertility restoration by nuclear gene Fr in CMS common bean: an Fr linkage map and the mode of Fr action.

The Fr gene in common bean, Phaseolus vulgaris L., is a unique gene for the study of plant nuclear-mitochondrial interactions because it appears to directly influence plant mitochondrial genome structure, resulting in the restoration of pollen fertility in cytoplasmic male sterile plants. This gene action is distinct from other pollen fertility restoration systems characterized to date. As a first step towards the map-based cloning of this unusual nuclear gene, we identified RAPD markers linked to Fr using bulked segregant analysis of near-isogenic lines. Using DNA gel blot hybridization, we localized the identified RAPD markers to a linkage group on the common bean RFLP map and constructed a linkage map of the Fr region using both RAPD markers and RFLP markers. Analysis of the mode of Fr action with the aid of identified Fr-linked DNA markers indicated that Fr functions in a semidominant fashion, showing dosage effect in controlling the dynamics of a heteroplasmic mitochondrial population. We also present our observations on the developmental distinctions, crucial in the accurate mapping of the Fr gene, between spontaneous cytoplasmic reversion and Fr-driven fertility restoration, two phenomena that are phenotypically indistinguishable.

A molecular marker-based linkage map of Phaseolus vulgaris L.

A seed and flower color marker (P), nine seed protein, nine isozyme and 224 restriction fragment length polymorphism marker loci were used to construct a linkage map of the common bean, Phaseolus vulgaris L. (n = 11). The mapping population consisted of a backcross progeny between the Mesoamerican breeding line 'XR-235-1-1' and the Andean cultivar 'Calima'; the former was used as the recurrent parent. A bean PstI genomic library enriched for single copy sequences (95%) was the source of DNA probes. Sixty percent of the probes tested detected polymorphisms between the parental genotypes with at least one of the four restriction enzymes used here (DraI, EcoRI, EcoRV and HindIII). The computer software Mapmaker was used to determine the linkage relationships and linear order of segregating markers. These markers assorted into 11 linkage groups covering 960 cM of the bean genome. Partial linkage data were used to estimate the total length of the genome at 1200 cM. This estimate and that for the physical size of the genome yield an average ratio of 530 kb/cM. The relatively small size of the genome makes this crop species a good candidate for the isolation of genes via chromosome walking techniques.

Extended map for the phaseolin linkage group ofPhaseolus vulgaris L.

The linkage relationship of 11 bean (Phaseolus vulgaris) seed proteins (including phaseolin), 9 enzyme loci, and theP locus were analyzed in backcross and F2 progenies by use of the software package "Mapmaker." The progenies were obtained by crossing the breeding line 'XR-235-1' and the cultivar 'Calima'. Allelic differences for seed protein loci were detected with SDS-PAGE and those for enzyme loci with starch gel electrophoresis and activity stains. The seed coat color of 'Calima' is a red/beige mottled pattern and that of 'XR-235-1' is white. Segregation at theP locus was followed by recording the phenotype of the BC1S1 and F3 seed. A linkage group comprising ca. 90 cM was detected with the following gene order:Est-2 - 11 -Pha - 8 - (Spe/Spg) - 24 - P - 9 - (Spa/Spv) - 16 -Spba - 22 -Mdh-1. In addition, another linkage group was detected: (Spd/Spf/Sph) - 5 -Spca. Therefore, the seed proteins appear to be organized in clusters in the bean genome.

Linkage between isozyme markers and a locus affecting seed size in Phaseolus vulgaris L.

Backcross and F2 progenies were produced between two bean genotypes, 'XR-235' and 'Calima,' which differ in seed weight by a factor of two. The small-seeded 'XR-235' was used as the pistillate and recurrent parent. These genotypes showed polymorphisms at nine isozyme loci and at the phaseolin locus. Seed size parameters (weight, length, width, and thickness) were determined for each BC1 and F2 individual, i.e., for seeds harvested from 'XR-235' after pollination with F1 and from the F1 after selfing, respectively. A combination of starch gel electrophoresis and enzyme activity staining was used to determine the genotype of each BC1 and F2 individual at the segregating loci. SDS-PAGE and Coomassie blue staining were used to determine geno-type at the phaseolin locus. Tests for independent assortment using two-way contingency and maximum likelihood tables revealed three linkage pairs: Aco-1 - 20 cM - Dia-1; Adh-1 - 2 cM - Got-2; and Est-2 - 11 cM - Pha. Statistical comparisons were made between the means of genotype classes at each segregating locus for all seed size parameters. The results from two independently obtained BC1s and the F2 consistently indicated that the Adh-1-Got-2 segment was linked to a locus that affected seed size and overcame maternal control over seed size. This locus has been designated Ssz-1. This gene exhibited additive gene action and accounted for 30-50% of the seed size difference between the parents.

DNA restriction fragment length polymorphisms correlate with isozyme diversity in Phaseolus vulgaris L.

Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.

Genetic analysis of resistances to races 1 and 2 of Fusarium oxysporum f. sp. lycopersici from the wild tomato Lycopersicon pennellii.

Resistance to race 3 of Fusarium wilt in the wild tomato Lycopersicon pennellii (LA 716) was previously found to be controlled by one major locus, I-3, tightly linked to Got-2 on chromosome 7. This accession was also found to carry resistance to races 1 and 2; a genetic analysis of these resistances is reported in this paper. This analysis proceeded in two steps. First, allelism tests demonstrated that race 1 and 2 resistances carried by L. pennellii were not allelic to the I and I-2 genes originally incorporated into L. esculentum from L. pimpinellifolium. Second, an interspecific backcross with L. pennellii (BC1) was used to determine the mode of inheritance of these new resistances and their chromosomal location by segregation and linkage analysis. BC1 responses to each of the races were determined using progeny tests (BC1S1). BC1S1 plants were inoculated with race 1 or 2 and evaluated after 1 month using a visual disease rating system; mean disease ratings were calculated for each BC1 individual for each race based on the progeny scores. A bimodal frequency distribution of the BC1 mean disease ratings was observed for both races, indicating that one major locus controlled resistance in each case. Statistical comparisons of the mean disease ratings of homozygous versus heterozygous individuals at each of 17 segregating enzyme loci were used to map the resistances to races 1 and 2. Tight linkage was detected between the enzyme locus Got-2 and resistances to both races, as was previously reported for the I-3 locus. Therefore, the Got-2 locus can be used as a selectable marker for resistances to all three races. The relationship of these resistances is discussed in the paper. In addition, as previously reported for race 3, significance was also detected for the chromosome segment marked by Aps-2 on chromosome 8 for both races. Currently many cultivars carry I and I-2 resistances to races 1 and 2. Incorporation of the LA 716 resistances to these two races into cultivars may reduce the likelihood of new race development.

An isozyme marker for resistance to race 3 of Fusarium oxysporum f. sp. lycopersici in tomato.

The inheritance and the linkage relationship of resistance to race 3 of Fusarium oxysporum f. sp. lycopersici derived from Lycopersicon pennellii (LA 716) were analyzed in an interspecific backcross to L. esculentum. Progeny from each backcross (BC1) individual were in-oculated with race 3 and their response was measured according to a visual rating system; progeny responses were used to calculate a mean disease rating for each BC1 individual. The frequency distribution of the disease ratings was bimodal, indicating that resistance to race 3 was controlled by one major locus. Linkage analysis of this locus proceeded in two steps. Initially, disease ratings were compared between homozygotes and heterozygotes at each of 17 segregating marker enzyme loci. Highly significant differences were detected for the chromosome segment marked by the Got-2 locus on chromosome 7. This indicated that Got-2 was linked to the race 3 resistance gene, designated I-3; this gene accounted for the observed bimodal distribution of BC1 disease ratings. In the second step, the genotype of each BC1 individual at the I-3 locus was determined using cluster analysis of the disease ratings; a test for independent assortment between I-3 and Got-2 revealed strong linkage, with an estimated map distance of 2.5 centiMorgans. Additional evidence for this linkage was obtained from the analysis of five breeding lines previously selected for resistance to race 3 solely on the basis of inoculations; all five showed co-selection for Got-2. The Got-2 locus is proposed as a selectable marker to expedite the transfer of race 3 resistance to commercial tomato cultivars.

Localization in the Tomato Genome of DNA Restriction Fragments Containing Sequences Homologous to the rRNA (45s), the Major Chlorophyll a/b Binding Polypeptide and the Ribulose Bisphosphate Carboxylase Genes.

DNA restriction fragments containing sequences homologous to the ribosomal RNA (45s), the major chlorophyll a/b binding polypeptide (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) genes have been localized and mapped in the tomato nuclear genome by linkage analysis. Ribosomal RNA genes map to a single locus, R45s, which resides in a terminal position on the short arm of chromosome 2 and corresponds to the Nucleolar Organizer Region. The size of the 45s repeating unit is estimated to be approximately 9 kb in Lycopersicon esculentum and 11 kb in Lycopersicon pennellii. Five loci were found to contain CAB sequences. Two of the loci, Cab-1 (chromosome 2) and Cab-3 (chromosome 8), together accounted for more than 80% of the hybridization signal. These loci contain more than one CAB structural gene. The other three loci, Cab-2 (chromosome 8), Cab-4 (chromosome 7) and Cab-5 (chromosome 12), each account for <10% of the total signal and may contain only a single copy of the CAB structural sequence. Three loci were found to contain RBCS sequences. Rbcs-2 (chromosome 3) and Rbcs-3 (chromosome 2) were responsible for >80% of the signal, with the remainder being associated with Rbcs-1 (chromosome 2). Rbcs-2 and Rbcs-3 may contain more than one copy of the gene.

Tight linkage between a nuclear male-sterile locus and an enzyme marker in tomato.

The linkage relationship of a nuclear male sterile locus, ms-10, was tested with two enzyme marker loci known to be on the same chromosome (long arm of chromosome 2). The results indicate the gene order is Est-1 - 18 cM - Prx-2 - 1.5 cM - ms-10. The linkage intensity of ms-10 and Prx-2 (1.5 cM) suggests that Prx-2 might provide a selectable marker for male-sterility. In accordance with this idea, the ms-10 allele was placed in cis with a rare-allele of Prx-2 (Prx-2 (1)). Selection on the basis of the codominant Prx-2 (1) allele should allow for more rapid and efficient transfer of the recessive male sterile allele into an array of genetic backgrounds, thus promoting its use in hybrid seed production.

Characterization of a differential low-temperature growth response in two species of Lycopersicon: the plastochron as a tool.

The plastochron model was used to evaluate the differences in the growth response of two Lycopersicon spp. grown under two temperature regimes (25/18 and 12/5°C). Two altitudinal accessions of L. hirsutum Homb. et Bnpl., from low and high altitude, a breeding line of L. esculentum (L.) Mill. and the hybrid between the latter and the high-altitude L. hirsutum were studied. The plastochron (P) values were estimated directly according to the formula of R.O. Erickson and F. Michelini (1957, Am. J. Bot. 44, 297-305), and indirectly through a linear model estimating the exponential rates of leaf elongation (r) and the ln of the plastochron ratios (q). The P values were obtained as P=q/r, and with one exception values obtained with both methods were comparable. Low temperature significantly decreased r in all genotypes, but the extent of this reduction depended on the genotype. The hybrid exhibited the least reduction, followed by the high-elevation L. hirsutum, L. esculentum and the lowelevation L. hirsutum. While the q values of the L. hirsutum accessions were significantly reduced by low temperature, those of L. esculentum and the hybrid were not. With the exception of the low-altitude L. hirsutum, low temperature significantly increased P, however the extent of the increase was significantly greater in L. esculentum. Analysis of temperature dependent changes of r, q and P indicate that L. esculentum extended its P by approximately the same factor its r was reduced. On the other hand, the L. hirsutum accessions increased P to a lesser extent, therefore having the ability to produce, comparatively, more leaves at lower temperatures than the cultivated tomato. The linear model of the plastochron is proposed as a tool for comparative studies of environmental growth responses of different genotypes. Plant size was reduced by low temperature. Considering plant size attained at high temperature as 100%, at low temperature sizes were reduced to 73% for the hybrid, 61% for the high-altitude L. hirsutum, 39% for L. esculentum and 30% for the low-altitude L. hirsutum. The low-temperature regime delayed flowering by two, three and nine plastochrons in the hybrid, the high-altitude L. hirsutum and L. esculentum, respectively, while the low-altitude L. hirsutum did not flower for the duration of the experiment. When artificially pollinated, L. esculentum yielded parthenocarpic fruits, while the high-altitude L. hirsutum and the hybrid produced fruits with viable seeds.