C. Eduardo Vallejos

The Maize Genome Contains a Helitron Insertion

The maize mutation sh2-7527 was isolated in a conventional maize breeding program in the 1970s. Although the mutant contains foreign sequences within the gene, the mutation is not attributable to an interchromosomal exchange or to a chromosomal inversion. Hence, the mutation was caused by an insertion. Sequences at the two Sh2 borders have not been scrambled or mutated, suggesting that the insertion is not caused by a catastrophic reshuffling of the maize genome. The insertion is large, at least 12 kb, and is highly repetitive in maize. As judged by hybridization, sorghum contains only one or a few copies of the element, whereas no hybridization was seen to the Arabidopsis genome. The insertion acts from a distance to alter the splicing of the sh2 pre-mRNA. Three distinct intron-bearing maize genes were found in the insertion. Of most significance, the insertion bears striking similarity to the recently described DNA helicase–bearing transposable elements termed Helitrons. Like Helitrons, the inserted sequence of sh2-7527 is large, lacks terminal repeats, does not duplicate host sequences, and was inserted between a host dinucleotide AT. Like Helitrons, the maize element contains 5′ TC and 3′ CTRR termini as well as two short palindromic sequences near the 3′ terminus that potentially can form a 20-bp hairpin. Although the maize element lacks sequence information for a DNA helicase, it does contain four exons with similarity to a plant DEAD box RNA helicase. A second Helitron insertion was found in the maize genomic database. These data strongly suggest an active Helitron in the present-day maize genome.

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-orf239 (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 organization in dicots. II. Arabidopsis as a ’bridging species’ to resolve genome evolution events among legumes

Abstract Analysis of molecular linkage groups within the soybean (Glycine max L. Merr.) genome reveals many homologous regions, reflecting the ancient polyploidy of soybean. The fragmented arrangement of the duplicated regions suggests that extensive rearrangements, as well as additional duplications, have occurred since the initial polyploidization event. In this study we used comparisons between homoeologous regions in soybean, and the homologous regions in the related diploids Phaseolus vulgaris and Vigna radiata, to elucidate the evolutionary history of the three legume genomes. Our results show that there is not only conservation of large regions of the genomes but that these conserved linkage blocks are also represented twice in the soybean genome. To gain a better understanding of the process of genome evolution in dicots, molecular comparisons have been extended to another well-studied species, Arabidopsis thaliana. Interestingly, the conserved regions we identified in the legume species are also relatively conserved in Arabidopsis. Our results suggest that there is conservation of blocks of DNA between species as distantly related as legumes and brassicas, representing 90 million years of divergence. We also present evidence for an additional, presumably earlier, genome duplication in soybean. These duplicated regions were only recognized by using Arabidopsis as a ’bridging’ species in the genome comparisons.

Genetic and Molecular Characterization of the I Locus of Phaseolus vulgaris

The I locus of the common bean, Phaseolus vulgaris, controls the development of four different phenotypes in response to inoculation with Bean common mosaic virus, Bean common mosaic necrosis virus, several other related potyviruses, and one comovirus. We have generated a high-resolution linkage map around this locus and have aligned it with a physical map constructed with BAC clones. These clones were obtained from a library of the cultivar “Sprite,” which carries the dominant allele at the I locus. We have identified a large cluster of TIR–NBS–LRR sequences associated within this locus, which extends over a distance >425 kb. Bean cultivars from the Andean or Mesoamerican gene pool that contain the dominant allele share the same haplotypes as revealed by gel blot hybridizations with a TIR probe. In contrast, beans with a recessive allele display simpler and variable haplotypes. A survey of wild accessions from Argentina to Mexico showed that this multigene family has expanded significantly during evolution and domestication. RNA gel blot analysis indicated that the TIR family of genes plays a role in the response to inoculations with BCMV or BCMNV.

BAC-end Sequence Analysis and a Draft Physical Map of the Common Bean ( Phaseolus vulgaris L.) Genome

Common bean (Phaseolus vulgaris L.) is a legume that is an important source of dietary protein in developing countries throughout the world. Utilizing the G19833 BAC library for P. vulgaris from Clemson University, 89,017 BAC-end sequences were generated giving 62,588,675 base pairs of genomic sequence covering approximately 9.54% of the genome. Analysis of these sequences in combination with 1,404 shotgun sequences from the cultivar Bat7 revealed that approximately 49.2% of the genome contains repetitive sequence and 29.3% is genic. Compared to other legume BAC-end sequencing projects, it appears that P. vulgaris has higher predicted levels of repetitive sequence, but this may be due to a more intense identification strategy combining both similarity-based matches as well as de novo identification of repeats. In addition, fingerprints for 41,717 BACs were obtained and assembled into a draft physical map consisting of 1,183 clone contigs and 6,385 singletons with ~9x coverage of the genome.

Mapping genes for plant structure, development and evolution: functional mapping meets ontology

One of the fundamental tasks in biology is the identification of genes that control the structure and developmental pattern of complex traits and their responses to the environment during trait development. Functional mapping provides a statistical means for detecting quantitative trait loci (QTLs) that underlie developmental traits, such as growth trajectories, and for testing the interplay between gene action and development. Here we describe how functional mapping and studies of plant ontology can be integrated so as to elucidate the expression mechanisms of QTLs that control plant growth, morphology, development, and adaptation to changing environments. This approach can also be used to construct an evo-devo framework for inferring the evolution of developmental traits.

Punctuated distribution of recombination hotspots and demarcation of pericentromeric regions in Phaseolus vulgaris L.

High density genetic maps are a reliable tool for genetic dissection of complex plant traits. Mapping resolution is often hampered by the variable crossover and non-crossover events occurring across the genome, with pericentromeric regions (pCENR) showing highly suppressed recombination rates. The efficiency of linkage mapping can further be improved by characterizing and understanding the distribution of recombinational activity along individual chromosomes. In order to evaluate the genome wide recombination rate in common beans (Phaseolus vulgaris L.) we developed a SNP-based linkage map using the genotype-by-sequencing approach with a 188 recombinant inbred line family generated from an inter gene pool cross (Andean x Mesoamerican). We identified 1,112 SNPs that were subsequently used to construct a robust linkage map with 11 groups, comprising 513 recombinationally unique marker loci spanning 943 cM (LOD 3.0). Comparative analysis showed that the linkage map spanned >95% of the physical map, indicating that the map is almost saturated. Evaluation of genome-wide recombination rate indicated that at least 45% of the genome is highly recombinationally suppressed, and allowed us to estimate locations of pCENRs. We observed an average recombination rate of 0.25 cM/Mb in pCENRs as compared to the rest of genome that showed 3.72 cM/Mb. However, several hot spots of recombination were also detected with recombination rates reaching as high as 34 cM/Mb. Hotspots were mostly found towards the end of chromosomes, which also happened to be gene-rich regions. Analyzing relationships between linkage and physical map indicated a punctuated distribution of recombinational hot spots across the genome.

Ethylene-induced overproduction of reactive oxygen species is responsible for the development of watersoaking in immature cucumber fruit.

Watersoaking is an ethylene-induced disorder observed in some members of the Cucurbitaceae including cucumber (Cucumis sativus L.), watermelon (Citrullus lanatus Thunb. Matsum and Nakai), and tropical pumpkin (Cucurbita moschata Duch.). Previous studies have found that immature beit-alpha cucumber (cv. Manar) exhibit watersoaking after 6d of continuous exposure to 10 μLL(-1) ethylene in air (21 kPa O(2)). The present study was designed to investigate the early dynamics of ethylene responses in immature cucumber fruit in order to provide insight into the watersoaking triggering mechanism. Changes in respiration, epidermal color, firmness, reactive oxygen species (ROS) production and electrolyte leakage were evaluated as a function of time under different ethylene concentrations and exposure duration. Ethylene concentrations exceeding 10 μLL(-1) did not accelerate changes in any of the evaluated responses. The first detectable change was a significant rise in respiration on day 2, followed by a significant rise in ROS on day 4, and significant degreening, mesocap softening, and increased electrolyte leakage on day 6; the latter responses coincident with incipient watersoaking. Varying the duration of exposure to ethylene indicated that the critical exposure time is between 2 and 4d. Notably, all deleterious responses to ethylene were suppressed under a hypoxic atmosphere. A model is proposed in which ethylene induces a sharp increase in respiration with a concomitant sharp rise in ROS, which the immature fruit is incapable of quenching. The resulting production of excess ROS leads to discoloration and membrane deterioration, leading to the release of cytoplasmic content, rapid softening, and the visual symptom of watersoaking.

Characterization of two recessive genes controlling resistance to all races of bacterial spot in peppers

Bacterial spot, one of the most damaging diseases of pepper, is caused by Xanthomonas euvesicatoria. This pathogen has worldwide distribution and it is particularly devastating in tropical and sub-tropical regions where high temperatures and frequent precipitation provide ideal conditions for disease development. Three dominant resistance genes have been deployed singly and in combination in commercial cultivars, but have been rendered ineffectual by the high mutation rate or deletion of the corresponding cognate effector genes. These genes are missing in race P6, and their absence makes this race virulent on all commercial pepper cultivars. The breeding line ECW12346 is the only source of resistance to race P6 in Capsicum annuum, and displays a non-hypersensitive type of resistance. Characterization of this resistance has identified two recessive genes: bs5 and bs6. Individual analysis of these genes revealed that bs5 confers a greater level of resistance than bs6 at 25°C, but in combination they confer full resistance to P6 indicating at least additive gene action. Tests carried out at 30°C showed that both resistances are compromised to a significant extent, but in combination they provide almost full resistance to race P6 indicating a positive epistatic interaction at high temperatures. A scan of the pepper genome with restriction fragment length polymorphism and AFLP markers led to the identification of a set of AFLP markers for bs5. Allele-specific primers for a PCR-based bs5-marker have been developed to facilitate the genetic manipulation of this gene.

Phaseolus vulgaris — The common bean integration of RFLP and RAPD-based linkage maps

Gregor Mendel (1866) conducted the first genetic analysis of common beans. Mendel studied the inheritance of growth habit, and pod color and shape in a progeny between P. vulgaris and P. nanus (= P. vulgaris, bush type) in order to confirm his findings with peas. Unfortunately, further studies on the inheritance of flower and seed coat color were hampered by his use of interspecific hybrids between P. nanus and P. multiflorus (= P. coccineus), which are now known to yield aberrant ratios. Later, Shaw and Norton (1918) used intraspecific crosses and determined that pigmentation and pigmentation patterns of the seed coat are controlled by multiple independent factors. A few years later Sax (1923) began to identify the multiple components that determine the inheritance of these traits. A single factor was identified as responsible for pigmentation, while two linked factors were identified to control mottling; this appears to be the first report of linkage in beans. Furthermore, Sax (1923) was the first to report linkage between a Mendelian character (seed coat pigmentation) and a QTL (for seed size). Although the common bean was used as experimental material at the inception of genetics, its genetic characterization has lagged behind that of many other crop species.