afila, the origin and nature of a major innovation in the history of pea breeding.

The afila (af) mutation causes the replacement of leaflets by a branched mass of tendrils in the compound leaves of pea - Pisum sativum L. This mutation was first described in 1953, and several reports of spontaneous af mutations and induced mutants with a similar phenotype exist. Despite widespread introgression into breeding material, the nature of af and the origin of the alleles used remain unknown. Here, we combine comparative genomics with reverse genetic approaches to elucidate the genetic determinants of af. We also investigate haplotype diversity using a set of AfAf and afaf cultivars and breeding lines and molecular markers linked to seven consecutive genes. Our results show that deletion of two tandemly arranged genes encoding Q-type Cys(2)His(2) zinc finger transcription factors, PsPALM1a and PsPALM1b, is responsible for the af phenotype in pea. Eight haplotypes were identified in the af-harbouring genomic region on chromosome 2. These haplotypes differ in the size of the deletion, covering more or less genes. Diversity at the af locus is valuable for crop improvement and sheds light on the history of pea breeding for improved standing ability. The results will be used to understand the function of PsPALM1a/b and to transfer the knowledge for innovation in related crops.

An Integrated Linkage Map of Three Recombinant Inbred Populations of Pea (Pisum sativum L.).

Biparental recombinant inbred line (RIL) populations are sets of genetically stable lines and have a simple population structure that facilitates the dissection of the genetics of interesting traits. On the other hand, populations derived from multiparent intercrosses combine both greater diversity and higher numbers of recombination events than RILs. Here, we describe a simple population structure: a three-way recombinant inbred population combination. This structure was easy to produce and was a compromise between biparental and multiparent populations. We show that this structure had advantages when analyzing cultivar crosses, and could achieve a mapping resolution of a few genes.

A reference genome for pea provides insight into legume genome evolution.

We report the first annotated chromosome-level reference genome assembly for pea, Gregor Mendel's original genetic model. Phylogenetics and paleogenomics show genomic rearrangements across legumes and suggest a major role for repetitive elements in pea genome evolution. Compared to other sequenced Leguminosae genomes, the pea genome shows intense gene dynamics, most likely associated with genome size expansion when the Fabeae diverged from its sister tribes. During Pisum evolution, translocation and transposition differentially occurred across lineages. This reference sequence will accelerate our understanding of the molecular basis of agronomically important traits and support crop improvement.

NMR Metabolomics Defining Genetic Variation in Pea Seed Metabolites.

Nuclear magnetic resonance (NMR) spectroscopy profiling was used to provide an unbiased assessment of changes to the metabolite composition of seeds and to define genetic variation for a range of pea seed metabolites. Mature seeds from recombinant inbred lines, derived from three mapping populations for which there is substantial genetic marker linkage information, were grown in two environments/years and analyzed by non-targeted NMR. Adaptive binning of the NMR metabolite data, followed by analysis of quantitative variation among lines for individual bins, identified the main genomic regions determining this metabolic variability and the variability for selected compounds was investigated. Analysis by t-tests identified a set of bins with highly significant associations to genetic map regions, based on probability (p) values that were appreciably lower than those determined for randomized data. The correlation between bins showing high mean absolute deviation and those showing low p-values for marker association provided an indication of the extent to which the genetics of bin variation might be explained by one or a few loci. Variation in compounds related to aromatic amino acids, branched-chain amino acids, sucrose-derived metabolites, secondary metabolites and some unidentified compounds was associated with one or more genetic loci. The combined analysis shows that there are multiple loci throughout the genome that together impact on the abundance of many compounds through a network of interactions, where individual loci may affect more than one compound and vice versa. This work therefore provides a framework for the genetic analysis of the seed metabolome, and the use of genetic marker data in the breeding and selection of seeds for specific seed quality traits and compounds that have high commercial value.

Genetic Variation Controlling Wrinkled Seed Phenotypes in Pisum: How Lucky Was Mendel?

One of the traits studied by Mendel in pea (Pisum sativum L.) was the wrinkled-seeded phenotype, and the molecular basis for a mutation underlying this phenotype was discovered in the 1990s. Although the starch-branching enzyme gene mutation identified at the genetic locus r is most likely to be that in seeds available to Mendel in the mid-1800s, it has remained an open question as to whether or not additional natural mutations in this gene exist within Pisum germplasm collections. Here, we explore this question and show that all but two wrinkled-seeded variants in one such collection correspond to either the mutant allele described previously for the r locus or a mutation at a second genetic locus, rb, affecting the gene encoding the large subunit of Adenosine diphosphoglucose (ADP-glucose) pyrophosphorylase; the molecular basis for the rb mutation is described here. The genetic basis for the phenotype of one (JI 2110) of the two lines which are neither r nor rb has been studied in crosses with a round-seeded variant (JI 281); for which extensive genetic marker data were expected. In marked contrast to the trait studied by Mendel and the rb phenotype; the data suggest that the wrinkled-seeded phenotype in JI 2110 is maternally determined, controlled by two genetic loci, and the extent to which it is manifested is very sensitive to the environment. Metabolite analysis of the cotyledons of JI 2110 revealed a profile for sucrose and sucrose-derived compounds that was more similar to that of wild-type round-seeded, than that of wrinkled-seeded r, pea lines. However, the metabolite profile of the seed coat (testa) of JI 2110 was distinct from that of other round-seeded genotypes tested which, together with analysis of recombinant inbred progeny lines, suggests an explanation for the seed phenotype.

Exploiting a fast neutron mutant genetic resource in Pisum sativum (pea) for functional genomics.

A fast neutron (FN)-mutagenised population was generated in Pisum sativum L. (pea) to enable the identification and isolation of genes underlying traits and processes. Studies of several phenotypic traits have clearly demonstrated the utility of the resource by associating gene deletions with phenotype followed by functional tests exploiting additional mutant sources, from both induced and natural variant germplasm. For forward genetic screens, next generation sequencing methodologies provide an opportunity for identifying genes associated with deletions rapidly and systematically. The application of rapid reverse genetic screens of the fast neutron mutant pea population supports conclusions on the frequency of deletions based on phenotype alone. These studies also suggest that large deletions affecting one or more loci can be non-deleterious to the pea genome, yielding mutants that could not be obtained by other means. Deletion mutants affecting genes associated with seed metabolism and storage are providing unique opportunities to identify the products of complex and related gene families, and to study the downstream consequences of such deletions.

Conserved genetic determinant of motor organ identity in Medicago truncatula and related legumes.

Plants exhibit various kinds of movements that have fascinated scientists and the public for centuries. Physiological studies in plants with the so-called motor organ or pulvinus suggest that cells at opposite sides of the pulvinus mediate leaf or leaflet movements by swelling and shrinking. How motor organ identity is determined is unknown. Using a genetic approach, we isolated a mutant designated elongated petiolule1 (elp1) from Medicago truncatula that fails to fold its leaflets in the dark due to loss of motor organs. Map-based cloning indicated that ELP1 encodes a putative plant-specific LOB domain transcription factor. RNA in situ analysis revealed that ELP1 is expressed in primordial cells that give rise to the motor organ. Ectopic expression of ELP1 resulted in dwarf plants with petioles and rachises reduced in length, and the epidermal cells gained characteristics of motor organ epidermal cells. By identifying ELP1 orthologs from other legume species, namely pea (Pisum sativum) and Lotus japonicus, we show that this motor organ identity is regulated by a conserved molecular mechanism.

Synthesis of an acyltrifluoroborate and its fusion with azides to form amides.

A uniquely stable acyl potassium trifluoroborate, potassium (2-phenylacetyl)trifluoroborate, has been synthesized and isolated. In the presence of an activating Lewis acid, this reagent reacts with azides to form amides in good yields.

High-throughput retrotransposon-based fluorescent markers: improved information content and allele discrimination.

BACKGROUND: Dense genetic maps, together with the efficiency and accuracy of their construction, are integral to genetic studies and marker assisted selection for plant breeding. High-throughput multiplex markers that are robust and reproducible can contribute to both efficiency and accuracy. Multiplex markers are often dominant and so have low information content, this coupled with the pressure to find alternatives to radio-labelling, has led us to adapt the SSAP (sequence specific amplified polymorphism) marker method from a 33P labelling procedure to fluorescently tagged markers analysed from an automated ABI 3730 xl platform. This method is illustrated for multiplexed SSAP markers based on retrotransposon insertions of pea and is applicable for the rapid and efficient generation of markers from genomes where repetitive element sequence information is available for primer design. We cross-reference SSAP markers previously generated using the 33P manual PAGE system to fluorescent peaks, and use these high-throughput fluorescent SSAP markers for further genetic studies in Pisum. RESULTS: The optimal conditions for the fluorescent-labelling method used a triplex set of primers in the PCR. These included a fluorescently labelled specific primer together with its unlabelled counterpart, plus an adapter-based primer with two bases of selection on the 3' end. The introduction of the unlabelled specific primer helped to optimise the fluorescent signal across the range of fragment sizes expected, and eliminated the need for extensive dilutions of PCR amplicons. The software (GeneMarker Version 1.6) used for the high-throughput data analysis provided an assessment of amplicon size in nucleotides, peak areas and fluorescence intensity in a table format, so providing additional information content for each marker. The method has been tested in a small-scale study with 12 pea accessions resulting in 467 polymorphic fluorescent SSAP markers of which 260 were identified as having been mapped previously using the radio-labelling technique. Heterozygous individuals from pea cultivar crosses were identifiable after peak area data analysis using the fluorescent SSAP method. CONCLUSION: As well as developing a rapid, and high-throughput marker method for genetic studies, the fluorescent SSAP system improved the accuracy of amplicon scoring, increased the available marker number, improved allele discrimination, and was sensitive enough to identify heterozygous loci in F1 and F2 progeny, indicating the potential to develop high-throughput codominant SSAPs.

Tendril-less regulates tendril formation in pea leaves.

Tendrils are contact-sensitive, filamentous organs that permit climbing plants to tether to their taller neighbors. Tendrilled legume species are grown as field crops, where the tendrils contribute to the physical support of the crop prior to harvest. The homeotic tendril-less (tl) mutation in garden pea (Pisum sativum), identified almost a century ago, transforms tendrils into leaflets. In this study, we used a systematic marker screen of fast neutron-generated tl deletion mutants to identify Tl as a Class I homeodomain leucine zipper (HDZIP) transcription factor. We confirmed the tendril-less phenotype as loss of function by targeting induced local lesions in genomes (TILLING) in garden pea and by analysis of the tendril-less phenotype of the t mutant in sweet pea (Lathyrus odoratus). The conversion of tendrils into leaflets in both mutants demonstrates that the pea tendril is a modified leaflet, inhibited from completing laminar development by Tl. We provide evidence to show that lamina inhibition requires Unifoliata/LEAFY-mediated Tl expression in organs emerging in the distal region of the leaf primordium. Phylogenetic analyses show that Tl is an unusual Class I HDZIP protein and that tendrils evolved either once or twice in Papilionoid legumes. We suggest that tendrils arose in the Fabeae clade of Papilionoid legumes through acquisition of the Tl gene.

Highly stereoselective synthesis of cis-alkenyl pinacolboronates and potassium cis-alkenyltrifluoroborates via a hydroboration/protodeboronation approach.

A number of alkynyl pinacolboronates bearing various functional groups were synthesized according to literature methods. These were then stereoselectively reduced to the cis-alkenyl pinacolboronates via hydroboration with dicyclohexylborane followed by chemoselective protodeboronation using acetic acid. Treatment with potassium hydrogen fluoride smoothly converted these to the corresponding potassium organotrifluoroborates.

Organotrifluoroborates: protected boronic acids that expand the versatility of the Suzuki coupling reaction.

Organotrifluoroborates represent an alternative to boronic acids, boronate esters, and organoboranes for use in Suzuki-Miyaura and other transition-metal-catalyzed cross-coupling reactions. The trifluoroborate moiety is stable toward numerous reagents that are often problematic for other boron species. Consequently, remote functional groups within the organotrifluoroborates can be manipulated, while retaining the valuable carbon-boron bond.

Linchpin synthons: metalation of aryl bromides bearing a potassium trifluoroborate moiety.

Aryl bromides bearing a potassium trifluoroborate moiety were subjected to lithium-halogen exchange at low temperature using a variety of alkyllithium reagents. A number of different electrophiles were evaluated in their reactions with the aryllithiums produced therein. Under carefully optimized conditions, potassium bromophenyl trifluoroborates afforded good to excellent yields of the corresponding alcohols (64-94% isolated yield) when aldehydes or ketones were used as the electophilic partner. Esters were unfortunately found to be unreactive.

Conservation of Arabidopsis flowering genes in model legumes.

The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have provided a wealth of information about genes and genetic pathways controlling the flowering process, but little is known about the corresponding pathways in legumes. The garden pea (Pisum sativum) has been used for several decades as a model system for physiological genetics of flowering, but the lack of molecular information about pea flowering genes has prevented direct comparison with other systems. To address this problem, we have searched expressed sequence tag and genome sequence databases to identify flowering-gene-related sequences from Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated corresponding sequences from pea by degenerate-primer polymerase chain reaction and library screening. We found that the majority of Arabidopsis flowering genes are represented in pea and in legume sequence databases, although several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appear to have undergone differential expansion, and several important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS C clade, are conspicuously absent. In several cases, pea and Medicago orthologs are shown to map to conserved map positions, emphasizing the closely syntenic relationship between these two species. These results demonstrate the potential benefit of parallel model systems for an understanding of flowering phenology in crop and model legume species.

Estimating genome conservation between crop and model legume species.

Legumes are simultaneously one of the largest families of crop plants and a cornerstone in the biological nitrogen cycle. We combined molecular and phylogenetic analyses to evaluate genome conservation both within and between the two major clades of crop legumes. Genetic mapping of orthologous genes identifies broad conservation of genome macrostructure, especially within the galegoid legumes, while also highlighting inferred chromosomal rearrangements that may underlie the variation in chromosome number between these species. As a complement to comparative genetic mapping, we compared sequenced regions of the model legume Medicago truncatula with those of the diploid Lotus japonicus and the polyploid Glycine max. High conservation was observed between the genomes of M. truncatula and L. japonicus, whereas lower levels of conservation were evident between M. truncatula and G. max. In all cases, conserved genome microstructure was punctuated by significant structural divergence, including frequent insertion/deletion of individual genes or groups of genes and lineage-specific expansion/contraction of gene families. These results suggest that comparative mapping may have considerable utility for basic and applied research in the legumes, although its predictive value is likely to be tempered by phylogenetic distance and genome duplication.

NMR profiling of transgenic peas.

A high throughput proton nuclear magnetic resonance spectroscopy method for the metabolite fingerprinting of plants was applied to genetically modified peas (Pisum sativum) to determine whether biochemical changes, so called 'unintended effects', beyond those intended by incorporation of a transgene, were detectable. Multivariate analysis of 1H NMR (nuclear magnetic resonance) spectra obtained from uniformly grown glasshouse plants revealed differences between the transgenic and control group that exceeded the natural variation of the plants. When a larger data set of six related transgenic lines was analysed, including a null segregant in addition to the wild-type control, multivariate analysis showed that the distribution of metabolites in the transgenics was different from that of the null segregant. However, the profile obtained from the wild-type material was diverse in comparison with both the transgenics and the null segregant, suggesting that the primary cause of the observed differences was that the transformation process selects for a subset of individuals able to undergo the transformation and selection procedures, and that their descendants have a restricted variation in metabolite profile, rather than that the presence of the transgene itself generates these differences.

DETERMINATE and LATE FLOWERING are two TERMINAL FLOWER1/CENTRORADIALIS homologs that control two distinct phases of flowering initiation and development in pea.

Genes in the TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS family are important key regulatory genes involved in the control of flowering time and floral architecture in several different plant species. To understand the functions of TFL1 homologs in pea, we isolated three TFL1 homologs, which we have designated PsTFL1a, PsTFL1b, and PsTFL1c. By genetic mapping and sequencing of mutant alleles, we demonstrate that PsTFL1a corresponds to the DETERMINATE (DET) gene and PsTFL1c corresponds to the LATE FLOWERING (LF) gene. DET acts to maintain the indeterminacy of the apical meristem during flowering, and consistent with this role, DET expression is limited to the shoot apex after floral initiation. LF delays the induction of flowering by lengthening the vegetative phase, and allelic variation at the LF locus is an important component of natural variation for flowering time in pea. The most severe class of alleles flowers early and carries either a deletion of the entire PsTFL1c gene or an amino acid substitution. Other natural and induced alleles for LF, with an intermediate flowering time phenotype, present no changes in the PsTFL1c amino acid sequence but affect LF transcript level in the shoot apex: low LF transcript levels are correlated with early flowering, and high LF transcript levels are correlated with late flowering. Thus, different TFL1 homologs control two distinct aspects of plant development in pea, whereas a single gene, TFL1, performs both functions in Arabidopsis. These results show that different species have evolved different strategies to control key developmental transitions and also that the genetic basis for natural variation in flowering time may differ among plant species.

The Sym35 gene required for root nodule development in pea is an ortholog of Nin from Lotus japonicus.

Comparative phenotypic analysis of pea (Pisum sativum) sym35 mutants and Lotus japonicus nin mutants suggested a similar function for the PsSym35 and LjNin genes in early stages of root nodule formation. Both the pea and L. japonicus mutants are non-nodulating but normal in their arbuscular mycorrhizal association. Both are characterized by excessive root hair curling in response to the bacterial microsymbiont, lack of infection thread initiation, and absence of cortical cell divisions. To investigate the molecular basis for the similarity, we cloned and sequenced the PsNin gene, taking advantage of sequence information from the previously cloned LjNin gene. An RFLP analysis on recombinant inbred lines mapped PsNin to the same chromosome arm as the PsSym35 locus and direct evidence demonstrating that PsNin is the PsSym35 gene was subsequently obtained by cosegregation analysis and sequencing of three independent Pssym35 mutant alleles. L. japonicus and pea root nodules develop through different organogenic pathways, so it was of interest to compare the expression of the two orthologous genes during nodule formation. Overall, a similar developmental regulation of the PsNin and LjNin genes was shown by the transcriptional activation in root nodules of L. japonicus and pea. In the indeterminate pea nodules, PsNin is highly expressed in the meristematic cells of zone I and in the cells of infection zone II, corroborating expression of LjNin in determinate nodule primordia. At the protein level, seven domains, including the putative DNA binding/dimerization RWP-RK motif and the PB1 heterodimerization domain, are conserved between the LjNIN and PsNIN proteins.

Three classes of proteinase inhibitor gene have distinct but overlapping patterns of expression in Pisum sativum plants.

Genes representative of three gene classes encoding proteinase inhibitor proteins, with distinct spatial expression patterns, were isolated and characterized from Pisum. Under standard plant growth conditions, one class is expressed exclusively in seeds, whereas the other two make minor contributions to seed inhibitor proteins but are also expressed in other organs, predominantly in root endodermal and floral reproductive tissues. Two of the gene classes contain few genes and are genetically linked at the Tri locus, whereas the third class displays complex hybridization patterns to genomic DNA and maps to diverse genetic loci. Expression analysis of this last class suggests that only a small number of these genes are expressed. The quantitative effect of the Tri locus on root and floral inhibitor gene expression was examined in near-isogenic lines of pea. The proteins encoded by the three classes are all members of the same family (Bowman-Birk) of enzyme inhibitors but are distinct in terms of overall sequence, active site sequences and inhibitor function.