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We have sequenced the genome of grass pea (Lathyrus sativus), a resilient diploid (2n=14) legume closely related to pea (Pisum sativum). We determined the genome size of the sequenced European accession (LS007) as 6.3 Gbp. We generated two assemblies of this genome, i) EIv1 using Illumina PCR-free paired-end sequencing and assembly followed by long-mate-pair scaffolding and ii) Rbp using Oxford Nanopore Technologies long-read sequencing and assembly followed by polishing with Illumina paired-end data. EIv1 has a total length of 8.12 Gbp (including 1.9 billion Ns) and scaffold N50 59,7 kbp. Annotation has identified 33,819 high confidence genes in the assembly. Rbp has a total length of 6.2 Gbp (with no Ns) and a contig N50 of 155.7 kbp. Gene space assessment using the eukaryote BUSCO database showed completeness scores of 82.8 % and 89.8%, respectively.

Legumes represent the second most important family of crop plants after grasses, accounting for approximately 27% of the world's crop production. Past domestication processes resulted in a high degree of relatedness between modern varieties of crops, leading to a narrower genetic base of cultivated germplasm prone to pests and diseases. Crop wild relatives (CWRs) harbor genetic diversity tested by natural selection in a range of environments. To fully understand and exploit local adaptation in CWR, studies in geographical centers of origin combining ecology, physiology, and genetics are needed. With the advent of modern genomics and computation, combined with systematic phenotyping, it is feasible to revisit wild accessions and landraces and prioritize their use for breeding, providing sources of disease resistances; tolerances of drought, heat, frost, and salinity abiotic stresses; nutrient densities across major and minor elements; and food quality traits. Establishment of hybrid populations with CWRs gives breeders a considerable benefit of a prebreeding tool for identifying and harnessing wild alleles and provides extremely valuable long‐term resources. There is a need of further collecting and both ex situ and in situ conservation of CWR diversity of these taxa in the face of habitat loss and degradation and climate change. In this review, we focus on three legume crops domesticated in the Fertile Crescent, pea, chickpea, and lentil, and summarize the current state and potential of their respective CWR taxa for crop improvement.

Genome-wide association study (GWAS) was conducted to identify loci associated with agronomic (days to flowering, days to maturity, plant height, seed yield and seed weight), seed morphology (shape and dimpling), and seed quality (protein, starch, and fiber concentrations) traits of field pea (Pisum sativum L.). A collection of 135 pea accessions from 23 different breeding programs in Africa (Ethiopia), Asia (India), Australia, Europe (Belarus, Czech Republic, Denmark, France, Lithuania, Netherlands, Russia, Sweden, Ukraine and United Kingdom), and North America (Canada and USA), was used for the GWAS. The accessions were genotyped using genotyping-by-sequencing (GBS). After filtering for a minimum read depth of five, and minor allele frequency of 0.05, 16,877 high quality SNPs were selected to determine marker-trait associations (MTA). The LD decay (LD1/2max,90) across the chromosomes varied from 20 to 80 kb. Population structure analysis grouped the accessions into nine subpopulations. The accessions were evaluated in multi-year, multi-location trials in Olomouc (Czech Republic), Fargo, North Dakota (USA), and Rosthern and Sutherland, Saskatchewan (Canada) from 2013 to 2017. Each trait was phenotyped in at least five location-years. MTAs that were consistent across multiple trials were identified. Chr5LG3_566189651 and Chr5LG3_572899434 for plant height, Chr2LG1_409403647 for lodging resistance, Chr1LG6_57305683 and Chr1LG6_366513463 for grain yield, Chr1LG6_176606388, Chr2LG1_457185, Chr3LG5_234519042 and Chr7LG7_8229439 for seed starch concentration, and Chr3LG5_194530376 for seed protein concentration were identified from different locations and years. This research identified SNP markers associated with important traits in pea that have potential for marker-assisted selection towards rapid cultivar improvement.

The seed-containing pod is the defining structure of plants in the legume family, yet pods exhibit a wide range of morphological variation. Within a species pod characters are likely to be correlated with reproductive strategy, and within cultivated forms will correspond to aspects of yield determination and/or end use. Here variation in pod size, described as pod length: pod width ratio, has been analyzed in pea germplasm represented by 597 accessions. This pod size variation is discussed with respect to population structure and to known classical pod morphology mutants. Variability of the pod length: width ratio can be explained by allelic variation at two genetic loci that may correspond to organ-specific negative regulators of growth.

Here we investigate the plant population genetics of retrotransposon insertion sites in pea to find out whether genetic drift and the neutral theory of molecular evolution can account for their abundance in the pea genome. (1) We asked whether two contrasting types of pea LTR-containing retrotransposons have the frequency and age distributions consistent with the behavior of neutral alleles and whether these parameters can explain the rate of change of genome size in legumes. (2) We used the recently assembled v1a pea genome sequence to obtain data on LTR-LTR divergence from which their age can be estimated. We coupled these data to prior information on the distribution of insertion site alleles. (3) We found that the age and frequency distribution data are consistent with the neutral theory. (4) We concluded that demographic processes are the underlying cause of genome size variation in legumes.

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.

Pea (Pisum sativum) is one of relatively few genetically amenable plant species with compound leaves. Pea leaves have a variety of specialized organs: leaflets, tendrils, pulvini and stipules, which enable the identification of mutations that transform or affect distinct parts of the leaf. Characterization of these mutations offers insights into the development and evolution of novel leaf traits. The previously characterized morphological gene Cochleata, conferring stipule identity, was known to interact with Stipules reduced (St), which conditions stipule size in pea, but the St gene remained unknown. Here we analysed Fast Neutron irradiated pea mutants by restriction site associated DNA sequencing. We identified St as a gene encoding a C2H2 zinc finger transcription factor that is regulated by Cochleata. St regulates both cell division and cell expansion in the stipule. Our approach shows how systematic genome-wide screens can be used successfully for the analysis of traits in species for which whole genome sequences are not available.

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.

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

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.

The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5′ position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3′,5′-hydroxylase (F3′5′H), the enzyme that hydroxylates the 5′ position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3′5′H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3′5′H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3′5′H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.

Mendel's paper 'Versuche über Pflanzen-Hybriden' is the best known in a series of studies published in the late 18th and 19th centuries that built our understanding of the mechanism of inheritance. Mendel investigated the segregation of seven gene characters of pea (Pisum sativum), of which four have been identified. Here, we review what is known about the molecular nature of these genes, which encode enzymes (R and Le), a biochemical regulator (I) and a transcription factor (A). The mutations are: a transposon insertion (r), an amino acid insertion (i), a splice variant (a) and a missense mutation (le-1). The nature of the three remaining uncharacterized characters (green versus yellow pods, inflated versus constricted pods, and axial versus terminal flowers) is discussed.

Rhizobium leguminosarum bv. viciae, which nodulates pea and vetch, makes a mixture of secreted nodulation signals (Nod factors) carrying either a C18:4 or a C18:1 N-linked acyl chain. Mutation of nodE blocks the formation of the C18:4 acyl chain, and nodE mutants, which produce only C18:1-containing Nod factors, are less efficient at nodulating pea. However, there is significant natural variation in the levels of nodulation of different pea cultivars by a nodE mutant of R. leguminosarum bv. viciae. Using recombinant inbred lines from two pea cultivars, one which nodulated relatively well and one very poorly by the nodE mutant, we mapped the nodE-dependent nodulation phenotype to a locus on pea linkage group I. This was close to Sym37 and PsK1, predicted to encode LysM-domain Nod-factor receptor-like proteins; the Sym2 locus that confers Nod-factor-specific nodulation is also in this region. We confirmed the map location using an introgression line carrying this region. Our data indicate that the nodE-dependent nodulation is not determined by the Sym2 locus. We identified several pea lines that are nodulated very poorly by the R. leguminosarum bv. viciae nodE mutant, sequenced the DNA of the predicted LysM-receptor domains of Sym37 and PsK1, and compared the sequences with those derived from pea cultivars that were relatively well nodulated by the nodE mutant. This revealed that one haplotype (encoding six conserved polymorphisms) of Sym37 is associated with very poor nodulation by the nodE mutant. There was no such correlation with polymorphisms at the PsK1 locus. We conclude that the natural variation in nodE-dependent nodulation in pea is most probably determined by the Sym37 haplotype.

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.

Two virus resistance loci on linkage groups II and VI have provided the only sources of natural resistance against Pea seed-borne mosaic virus (PSbMV, Potyviridae) in the important crop plant Pisum sativum L. A combination of parallel approaches was used to collate linked markers, particularly for sbm-1 resistance on linkage group VI. We have identified sequences derived from the genes for the eukaryotic translation initiation factors eIF4E and eIF(iso)4E as being very tightly linked to the resistance gene clusters on linkage groups VI and II, respectively. In particular, no recombinants between sbm-1 and eIF4E were found amongst 500 individuals of an F2 cross between the BC4 resistant line (JI1405) and its recurrent susceptible parent 'Scout'. In a different mapping population, the gene eIF(iso)4E was also shown to be linked to sbm-2 on linkage group II. A parallel cDNA-AFLP comparison of pairs of resistant and susceptible lines also identified an expressed tag marker just 0.7 cM from sbm-1. eIF4E and eIF(iso)4E have been associated with resistance to related viruses in other hosts. This correlation strengthens the use of our markers as valuable tools to assist in breeding multiple virus resistances into peas, and identifies potential targets for resistance gene identification in pea.

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.

SQUAMOSA and APETALA1 are floral meristem identity genes from snapdragon (Antirrhinum majus) and Arabidopsis, respectively. Here, we characterize the floral meristem identity mutation proliferating inflorescence meristem(pim) from pea (Pisum sativum) and show that it corresponds to a defect in the PEAM4 gene, a homolog of SQUAMOSA and APETALA1. ThePEAM4 coding region was deleted in thepim-1 allele, and this deletion cosegregated with thepim-1 mutant phenotype. The pim-2 allele carried a nucleotide substitution at a predicted 5′ splice site that resulted in mis-splicing of pim-2 mRNA. PCR products corresponding to unspliced and exon-skipped mRNA species were observed. The pim-1 and pim-2 mutations delayed floral meristem specification and altered floral morphology significantly but had no observable effect on vegetative development. These floral-specific mutant phenotypes and the restriction ofPIM gene expression to flowers contrast with other known floral meristem genes in pea that additionally affect vegetative development. The identification of PIM provides an opportunity to compare pathways to flowering in species with different inflorescence architectures.

The Arabidopsis genome sequence has given us an inventory of the genes needed to specify a flowering plant. Plants are highly diverse in appearance and the mechanisms whereby this diversity has arisen need explanation. A fundamental question is to what extent diversity arises from remodelling of gene function or relocation of gene pathways, rather than from the gain or loss of genes. Similar types of genetic rewiring may be responsible for both intra- and inter-specific differences in developmental processes. Recent advances in the understanding of shoot, flower and leaf development provide insights to this question.

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.

The partial-shoot theory of the leaf was a controversial hypothesis revived by Arber and supported by her morphological and anatomical studies. This theory highlighted the parallels between leaves and shoots and contrasted with an alternative view that leaves, with their limited growth potential, are completely distinct from shoots. Pea morphological mutants with altered growth potential in their compound leaves are described. The unifoliata mutant has a limited growth potential relative to wild-type;cochleata, afila and insecatus have extended potentials. Characterization of theunifoliata mutation and gene expression patterns indicate that unifoliata is a common factor in pea compound leaf and floral shoot development, and so provides rudimentary support for the idea that some leaves have shoot-like characteristics. Tomato leaves are also considered to lend tentative support. The afila and insecatus leaf forms are described as bipinnate and weakly bipinnate, respectively. These and the tendril-less mutant are potential phenocopies of legume relatives, an idea based on Vavilov's law of homologous series of variation. Arber illustrated, but did not articulate in genetical terms, that morphological variation in structures within an individual plant can be interpreted as reiteration of design. Analogous with Vavilov's view, this can be considered a consequence of the same genetic programme in a different location.

Differences in knotted1-like (knox) gene expression may account for some of the diversity of leaf forms seen in nature. Class 1 knox genes are expressed in the compound leaf primordia of tomato but not in the simple leaf primordia of a range of species examined so far. In order to test the hypothesis that all compound leaves differ from simple leaves in this way, we isolated a class 1 knox cDNA from pea, Pskn1 (Pisum sativum knotted1) and examined its expression pattern. The encoded homeodomain of Pskn1 shares 88% identical residues with KNOTTED1 from maize and an adjacent ELK domain is present. The protein sequence of PSKN1 is 69% identical to TKN2, its nearest related sequence in tomato. Unlike TKn2, Pskn1 was not expressed in newly initiated compound leaves. The expression pattern of Pskn1 resembled those of other class 1 knox genes described in maize and Arabidopsis. Transcripts were detected in the shoot apical meristem and developing vasculature of the vegetative shoot, but expression was not detected in newly initiated and developing compound leaf primordia. The same pattern of expression was observed in the afila mutant, which is characterised by highly ramified compound leaves. Our results suggest that tomato and pea use different developmental processes in the generation of their compound leaves.

The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea.

Summary The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon-based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR-based method for the rapid isolation of LTR sequences of Ty1-copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identified a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers specific for three pea LTRs have been used to reveal polymorphisms associated with the correspond- ing retrotransposons within the Pisum genus.

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.

In wild pea varieties two genes, sym1 and sym2, have been identified that cause resistance to European Rhizobium leguminosarum bv. viciae (Rlv) strains. The sym2 gene has previously been studied in some detail and it was shown that the additional nodulation gene nodX is sufficient to overcome the sym2 controlled nodulation resistance. Here we characterize the sym1 gene. We show that the resistance conferred by sym1 can be overcome by the introduction of nodX in European Rlv strains, indicating that sym1 just as sym2 is involved in Nod factor recognition. Both sym1 and sym2 display a recessive or dominant nature depending on the Rlv strain used for inoculation. Furthermore, introgression lines containing either sym1 or sym2 are able to form nodules with Rlv strain 248 at 26°C, but not at 18°C, indicating that both sym1 and sym2 have a temperature sensitive nature. sym2 was mapped on the pea RFLP map. We found that sym1 maps in the same region of chromosome 1 as sym2. By crossing sym1 and sym2 containing introgression lines we demonstrate that sym1 and sym2 are allelic.

International audience; Retrotransposons are a major agent of genome evolution. Various molecular marker systems have been developed that exploit the ubiquitous nature of these genetic elements and their property of stable integration into dispersed chromosomal loci that are polymorphic within species. The key methods, SSAP, IRAP, REMAP, RBIP, and ISBP, all detect the sites at which the retrotransposon DNA, which is conserved between families of elements, is integrated into the genome. Marker systems exploiting these methods can be easily developed and inexpensively deployed in the absence of extensive genome sequence data. They offer access to the dynamic and polymorphic, nongenic portion of the genome and thereby complement methods, such as gene-derived SNPs, that target primarily the genic fraction.

Retrotransposons are a major agent of genome evolution. Various molecular marker systems have been developed that exploit the ubiquitous nature of these genetic elements and their property of stable integration into dispersed chromosomal loci that are polymorphic within species. The key methods, SSAP, IRAP, REMAP, RBIP, and ISBP, all detect the sites at which the retrotransposon DNA, which is conserved between families of elements, is integrated into the genome. Marker systems exploiting these methods can be easily developed and inexpensively deployed in the absence of extensive genome sequence data. They offer access to the dynamic and polymorphic, nongenic portion of the genome and thereby complement methods, such as gene-derived SNPs, that target primarily the genic fraction.

International audience; Grain legumes contribute significantly to total world food production. Legumes are the primary source of dietary proteins in many developing countries, where protein hunger and malnutrition are widespread. Grain legumes germplasm constitute similar to 15% of the 7.4 M accessions preserved globally. Nearly, 78% of the CGIAR's, 0.217 M accessions, have been characterized, compared to 34% of national genebank collections. Interestingly, limited data on grain quality are available as the primary focus has been on morpho-agronomic traits. Clearly, more resources should be targeted on biochemical evaluation to identify nutritionally rich and genetically diverse germplasm. The formation of core and mini core collections has provided crop breeders with a systematic yet manageable entry point into global germplasm resources. These subsets have been reported for most legumes and have proved useful in identifying new sources of variation. They may however not eliminate the need to evaluate entire collections, particularly for very rare traits. Molecular characterization and association mapping will further aid to insights into the structure of legume diversity and facilitate greater use of collections. The use of high resolution elevational climate models has greatly improved our capacity to characterize plant habitats and species' adaptive responses to stresses. Evidence suggests that there has been increased use of wild relatives as well as new resources resulting from mutagenesis to enhance the genetic base of legume cultigens.

Background: The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea. Methodology/Principal Findings: We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the A and A2 loci in pea. We then used a combination of genetic mapping, fast neutron mutant analysis, allelic diversity, transcript quantification and transient expression complementation studies to confirm the identity of the candidates. Conclusions/Significance: We have identified the pea genes A and A2. A is the factor determining anthocyanin pigmentation in pea that was used by Gregor Mendel 150 years ago in his study of inheritance. The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon, and we have identified a second rare mutant allele. The A2 gene encodes a WD40 protein that is part of an evolutionarily conserved regulatory complex.

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.

Biotin-labelled DNA probes for tandemly repeated sequences have been used in in situ hybridization experiments as chromosome-specific markers in pea. Six of the seven chromosome pairs can be marked at single sites in this way. Translocations from a standard karyotype are revealed as chromosomes that have two hybridization sites, rather than one. By probing a tester set of reciprocal translocation (or interchange) lines, some of the markers can be assigned to chromosomes. The method is rapid and simple and, in the absence of well-resolved chromosome bands, provides a means for clarifying some of the problems in pea cytology.Key words: in situ hybridization, translocations, chromosome markers.

Sequence diversity of 39 dispersed gene loci was analyzed in 48 diverse individuals representative of the genus Pisum. The different genes show large variation in diversity parameters, suggesting widely differing levels of selection and a high overall diversity level for the species. The data set yields a genetic diversity tree whose deep branches, involving wild samples, are preserved in a tree derived from a polymorphic retrotransposon insertions in an identical sample set. Thus, gene regions and intergenic “junk DNA” share a consistent picture for the genomic diversity of Pisum, despite low linkage disequilibrium in wild and landrace germplasm, which might be expected to allow independent evolution of these very different DNA classes. Additional lines of evidence indicate that recombination has shuffled gene haplotypes efficiently within Pisum, despite its high level of inbreeding and widespread geographic distribution. Trees derived from individual gene loci show marked differences from each other, and genetic distance values between sample pairs show high standard deviations. Sequence mosaic analysis of aligned sequences identifies nine loci showing evidence for intragenic recombination. Lastly, phylogenetic network analysis confirms the non-treelike structure of Pisum diversity and indicates the major germplasm classes involved. Overall, these data emphasize the artificiality of simple tree structures for representing genomic sequence variation within Pisum and emphasize the need for fine structure haplotype analysis to accurately define the genetic structure of the species.

The genomes of several legume species contain two Phantastica-like genes. Previous studies on leaf development have found that Phantastica confers leaf blade adaxial identity in plant species with simple leaves and leaflet adaxial identity in pea (Pisum sativum L.), a legume with compound leaves. Previous characterisation of the phantastica mutant of pea, crispa, showed it had radialised leaflets, but stipules were not radialised. This suggested either that mutation of a second redundant gene was required for radialisation of stipules, or, that a null mutation was required. Previously characterised crispa mutants may not have exhibited radialised stipules because they were weak alleles. In this work we show that pea has a second Phantastica-like gene, which lies on a different chromosome to Crispa. The second gene was found to be a pseudogene in several genotypes of pea, therefore it would not have a role in conferring stipule adaxial identity. A new deletion mutant, crispa-4 was identified. The mutant has radialised stipules and leaflets, showing that Crispa confers adaxial identity on both these organs in pea. The nucleotide sequence data reported here are in the EMBL and GenBank Nucleotide Databases under the accession numbers DQ486060 (JI 2822), DQ486061 (JI 15), DQ486062 (JI 281) and DQ486063 (JI 399).

The extensive germplasm resource collections that are now available for major crop plants and their wild relatives will increasingly provide valuable biological and bioinformatics resources for plant physiologists and geneticists to dissect the molecular basis of key traits and to develop highly adapted plant material to sustain future breeding programs. A key to the efficient deployment of these resources is the development of information systems that will enable the collection and storage of biological information for these plant lines to be integrated with the molecular information that is now becoming available through the use of high-throughput genomics and post-genomics technologies. The GERMINATE database has been designed to hold a diverse variety of data types, ranging from molecular to phenotypic, and to allow querying between such data for any plant species. Data are stored in GERMINATE in a technology-independent manner, such that new technologies can be accommodated in the database as they emerge, without modification of the underlying schema. Users can access data in GERMINATE databases either via a lightweight Perl-CGI Web interface or by the more complex Genomic Diversity and Phenotype Connection software. GERMINATE is released under the GNU General Public License and is available at http://germinate.scri.sari.ac.uk/germinate/.

Sequences flanking 73 insertions of the retrotransposon PDR1 have been characterized, together with an additional 270 flanking regions from one side alone, from a diverse collection of Pisum germ plasm. Most of the identified flanking sequences are repetitious DNAs but more than expected (7%) lie within nuclear gene protein-coding regions. The approximate age of 52 of the PDR1 insertions has been determined by measuring sequence divergence among LTR pairs. These data show that PDR1 transpositions occurred within the last 5 MY, with a peak at 1–2.5 MYA. The insertional polymorphism of 68 insertions has been assessed across 47 selected Pisum accessions, representing the diversity of the genus. None of the insertions are fixed, showing that PDR1 insertions can persist in a polymorphic state for millions of years in Pisum. The insertional polymorphism data have been compared with the age estimations to ask what rules control the proliferation of PDR1 insertions in Pisum. Relatively recent insertions (< ∼1.5MYA) tend to be found in small subsets of the Pisum accessions set, “middle-aged” insertions (between ∼1.5 and 2.5 MYA) vary greatly in their occurrence, and older insertions (> ∼2.5 MYA) are mostly found in small subsets of Pisum. Finally, the average age estimate for PDR1 insertions, together with an existing data set for PDR1 retrotransposon SSAP markers, has been used to derive an estimate of the effective population size for Pisum of ∼7.5 × 105.

Pinnate compound leaves have laminae called leaflets distributed at intervals along an axis, the rachis, whereas simple leaves have a single lamina. In simple- and compound-leaved species, the PHANTASTICA (PHAN) gene is required for lamina formation. Antirrhinum majus mutants lacking a functional gene develop abaxialized, bladeless adult leaves. Transgenic downregulation of PHAN in the compound tomato (Solanum lycopersicum) leaf results in an abaxialized rachis without leaflets. The extent of PHAN gene expression was found to be correlated with leaf morphology in diverse compound-leaved species; pinnate leaves had a complete adaxial domain of PHAN gene expression, and peltate leaves had a diminished domain. These previous studies predict the form of a compound-leaved phan mutant to be either peltate or an abaxialized rachis. Here, we characterize crispa, a phan mutant in pea (Pisum sativum), and find that the compound leaf remains pinnate, with individual leaflets abaxialized, rather than the whole leaf. The mutant develops ectopic stipules on the petiole-rachis axis, which are associated with ectopic class 1 KNOTTED1-like homeobox (KNOX) gene expression, showing that the interaction between CRISPA and the KNOX gene PISUM SATIVUM KNOTTED2 specifies stipule boundaries. KNOX and CRISPA gene expression patterns indicate that the mechanism of pea leaf initiation is more like Arabidopsis thaliana than tomato.

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.

*Summary From the characterization of the recessive resistance gene, sbm1, in pea we have identified the eukaryotic translation initiation factor, eIF4E, as a susceptibility factor required for infection with the Potyvirus, Pea seedborne mosaic virus. A functional analysis of the mode of action of the product of the dominant allele revealed a novel function for eIF4E in its support for virus movement from cell-to-cell, in addition to its probable support for viral RNA translation, and hence replication. Different resistance specificities in two independent pea lines were explained by different mutations in eIF4E. On the modelled structure of eIF4E the coding changes were in both cases lying in and around the structural pocket involved in binding the 5¢-m 7 G cap of eukaryotic mRNAs. Protein expression and cap-binding analysis showed that eIF4E encoded by a resistant plant could not bind to m7 G-Sepharose, a result which may point to functional redundancy between eIF4E and the paralogous eIF(iso)4E in resistant peas. These observations, together with related findings for other potyvirus recessive resistances, provide a more complete picture of the potyvirus life cycle.

The genetic structure and evolutionary history of the genus Pisum were studied exploiting our germplasm collection to compare the contribution of different mechanisms to the generation of diversity. We used sequence-specific amplification polymorphism (SSAP) markers to assess insertion site polymorphism generated by a representative of each of the two major groups of LTR-containing retrotransposons, PDR1 (Ty1/copia-like) and Cyclops (Ty3/gypsy-like), together with Pis1, a member of the En/Spm transposon superfamily. The analysis of extended sets of the four main Pisum species, P. fulvum, P. elatius, P. abyssinicum, and P. sativum, together with the reference set, revealed a distinct pattern of the NJ (Neighbor-Joining) tree for each basic lineage, which reflects the different evolutionary history of each species. The SSAP markers showed that Pisum is exceptionally polymorphic for an inbreeding species. The patterns of phylogenetic relationships deduced from different transposable elements were in general agreement. The retrotransposon-derived markers gave a clearer separation of the main lineages than the Pis1 markers and were able to distinguish the truly wild form of P. elatius from the antecedents of P. sativum. There were more species-specific and unique PDR1 markers than Pis1 markers in P. fulvum and P. elatius, pointing to PDR1 activity during speciation and diversification, but the proportion of these markers is low. The overall genetic diversity of Pisum and the extreme polymorphism in all species, except P. abyssinicum, indicate a high contribution of recombination between multiple ancestral lineages compared to transposition within lineages. The two independently domesticated pea species, P. abyssinicum and P. sativum, arose in contrasting ways from the common processes of hybridization, introgression, and selection without associated transpositional activity.

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.

Comparative phenotypic analysis of pea (Pisum sativum) sym35 mutants and Lotus japonicus nin mutants suggested a similar function for thePsSym35 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 PsNinto the same chromosome arm as the PsSym35 locus and direct evidence demonstrating that PsNin is thePsSym35 gene was subsequently obtained by cosegregation analysis and sequencing of three independent Pssym35mutant 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 thePsNin 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.

Summary Two assays based upon PCR detection of a polymorphic PDR1 retrotransposon insertion in Pisum sativum have been developed. Both methods involve PCR with primers derived from the transposon and flanking DNA. The first method uses a dot assay for PCR product detection which could be fully automated for handling thousands of samples. The second method, which is designed to handle lower numbers, requires a single PCR and gel lane per sample. Both methods yield co-dominant markers, with presence and absence of the transposon insertion independently scorable, and both could in principle be applied to any transposable element in any plant species.

A genetic linkage map of Pisum sativum L. was constructed based primarily on RAPD markers that were carefully selected for their reproducibility and scored in a population of 139 recombinant inbred lines (RILs). The mapping population was derived from a cross between a protein-rich dry-seed cultivar ‘Terese’ and an increased branching mutant (K586) obtained from the pea cultivar ‘Torsdag’. The map currently comprises nine linkage groups with two groups comprising only 6 markers (n=7 in pea) and covers 1139 cM. This RAPD-based map has been aligned with the map based on the (JI281×JI399) RILs population that currently includes 355 markers in seven linkage groups covering 1881 cM. The difference in map lengths is discussed. For this alignment 7 RFLPs, 23 RAPD markers, the morphological marker le and the PCR marker corresponding to the gene Uni were used as common markers and scored in both populations.

A trypsin inhibitor locus (Tri) has been mapped close to Vc-2 on Pisum (pea) linkage group 5 using recombinant inbred lines derived from crosses of genotypes showing qualitative variation in seed trypsin inhibitors. F2 seed populations derived from crosses between lines showing qualitative variation in trypsin inhibitors as well as quantitative variation in inhibitor activity showed an association between the segregation of the structural variation and relative activity levels. Clones complementary to Pisum trypsin inhibitor mRNA were used in hybridization analyses which showed that the segregation of protein polymorphisms reflected directly the segregation of polymorphisms associated with the structural genes.

Starch biosynthesis is one of the most important metabolic processes of developing storage organs. The importance of this process in determining seed composition is illustrated by a number of mutations affecting genes in the biosynthetic pathway such as r and rb of pea and some of the high lysine lines of maize and barley. Lesions that limit starch quantity and quality also have effects on the accumulation of lipid protein. To understand the relationship between starch biosynthesis and the production of other storage products we have begun to isolate a number of the genes involved in starch biosynthesis. These genes allow us to define particular mutants biochemically, and by genetic engineering allow us to affect particular steps in starch production in transgenic plants. By analyzing these transgenic plants we hope to develop our understanding of the regulation of starch biosynthesis and its relationship to the determination of seed composition as a whole. We anticipate that these experiments will also produce a number of transgenic plants with modifications in starch quantity and quality.

A cDNA clone, pCD7, was shown to hybrid-select from developing seeds of Pisum sativum L. an mRNA that translated into a polypeptide of apparent Mr 90 000. The translation product was observed only in the earliest stages of embryogenesis and was detected at a developmental stage when virtually all the cotyledon cells are mitotic. Sequence analysis of pCD7 showed it to correspond to a member of the 70 000-Mr heat-shock protein (HSP70) gene family. Transcripts corresponding to pCD7 were detected in different P. sativum organs, with roots apparently showing lower levels of pCD7-homologous RNA than other organs. Hybridizations to P. sativum DNA identified polymorphisms in the genomic DNA corresponding to pCD7 and the segregation of these in selected crosses indicated the existence of at least two genetic loci, one of which mapped to an existing linkage group.

The copy number, genomic arrangement and linkage relationships of two classes of lipoxygenase gene have been investigated in Pisum(pea) lines. Each of the two classes contained two to three members in P. sativum lines. RFLPs associated with genomic fragments containing the 5′ sequences of one gene class permitted its correlation in genetical analyses with a lipoxygenase locus on linkage group 4, which was previously identified through polypeptide variation. Genetical analyses of RFLPs associated with other fragments identified by low- and medium-stringency hybridization to lipoxygenase cDNAs indicate the existence of other unlinked lipoxygenase gene loci.

We describe the cloning of the r (rugosus) locus of pea (Pisum sativum L.), which determines whether the seed is round or wrinkled. Wrinkled (rr) seeds lack one isoform of starch-branching enzyme (SBEI), present in round (RR or Rr) seeds. A major polymorphism in the SBEI gene between near-isogenic RR and rr lines shows 100% cosegregation with the r locus, establishing that the SBEI gene is at the r locus. An aberrant transcript for SBEI is produced in rr embryos. In rr lines the SBEI gene is interrupted by a 0.8 kb insertion that is very similar to the Ac/Ds family of transposable elements from maize. Failure to produce SBEI has complex metabolic consequences on starch, lipid, and protein biosynthesis in the seed.

We have analysed trisomic pea plants by DNA/ DNA hybridization techniques, exploiting the copy number excess of DNA sequences in trisomics, together with a knowledge of their genetics, in order to address problems in the correlation of genetic and cytogenetic information. One such long-standing problem in pea genetics is the assignment of one rDNA locus (Rrn2) to a linkage group on chromosome 7. Our results show that Rrn2 does not reside on the same chromosome as Lg-1 or Vc-2 which we have previously shown to be linked to r, a linkage group 7 specific marker.

The organization of three classes of legumin gene has been studied by exploiting restriction fragment length polymorphisms. Hybridization of cDNA probes, representing different sub-families of legumin gene, to DNA from F2 and F6 plants from selected crosses showed that there was a tight linkage of individual genes within each of two sub-families. One of these sub-families has been mapped to a locus on chromosome 7 close to r, while the other maps to a locus near a on chromosome 1. The results from one of the crosses analyzed indicated that a third class of legumin gene is also linked to a.