Maggie R. Knox

Polymorphism of insertion sites of Ty1-copia class retrotransposons and its use for linkage and diversity analysis in pea.

Abstract A sample of 15 cultivars and 56 Pisum accessions from the JIC germplasm core collection has been studied using a modification of the SSAP (sequence-specific amplification polymorphisms) technique; the specific primer was designed to correspond to the polypurine tract (PPT) of PDR1, a Ty1-copia group retrotransposon of pea. Most of these SSAP products were shown to be PDR1 derived. The PDR1 SSAP markers are more informative than previously studied AFLP or RFLP markers and are distributed throughout the genome. Their pattern of variation makes them ideal for integrating genetic maps derived from related crosses. Data sets obtained with AFLP and PDR1 SSAP markers were used to construct neighbour-joining trees and for principal component analysis. These data sets give greater resolution than hitherto available for the characterisation of variation within Pisum, showing that the genus has three main groups: P. fulvum, P. abyssinicum and all other Pisum spp. P. abyssinicum is not a subgroup of cultivated P. sativum, as was previously thought, but has probably been domesticated independently. Modern cultivars are shown to form a single group within Pisum as a whole.

Comparative analysis of genetic diversity in pea assessed by RFLP- and PCR-based methods.

DNA-based molecular-marker techniques have been proven powerful in genetic diversity estimations. Among them, RFLP was the first and is still the most commonly used in the estimation of genetic diversity of eukaryotic species. The recently developed PCR-based multiple-loci marker techniques, which include RAPD, AFLP, Microsatellite-AFLP and inter-SSR PCR, are playing increasingly important roles in this type of research. Despite the wide application of these techniques, no direct comparison of these methods in the estimation of genetic diversity has been carried out. Here we report a direct comparison of DNA-based RFLP with various PCR-based techniques regarding their informativeness and applicability for genetic diversity analysis. Among ten pea genotypes studied, all the PCR-based methods were much more informative than cDNA-RFLP. Genetic diversity trees were derived from each marker technique, and compared using Mantels test. By this criterion, all trees derived from the various molecular marker techniques, except for the tree derived from inter-SSR PCR, were significantly correlated, suggesting that these PCR-based techniques could replace RFLP in the estimation of genetic diversity. On the basis of this result, AFLP analysis was applied to assess the genetic diversity of a sample of accessions representing the various species and subspecies within the genus Pisum.

Pea Ty1-copia group retrotransposons: transpositional activity and use as markers to study genetic diversity in Pisum.

Abstract The variation in transposition history of different Ty1-copia group LTR retrotransposons in the species lineages of the Pisum genus has been investigated. A heterogeneous population of Ty1-copia elements was isolated by degenerate PCR and two of these (Tps12 and Tps19) were selected on the basis of their copy number and sequence conservation between closely related species for further in-depth study of their transpositional history in Pisum species. The insertional polymorphism of these elements and the previously characterised PDR1 element was studied by sequence-specific amplification polymorphism (SSAP). Each of these elements reveals a unique transpositional history within 55 diverse Pisum accessions. Phylogenetic trees based on the SSAP data show that SSAP markers for individual elements are able to resolve different species lineages within the Pisum genus. Finally, the SSAP data from all of these retrotransposon markers were combined to reveal a detailed picture of the intra and inter-species relationships within Pisum.

PROLIFERATING INFLORESCENCE MERISTEM, a MADS-Box Gene That Regulates Floral Meristem Identity in Pea

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.

Gene-Based Sequence Diversity Analysis of Field Pea (Pisum)

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.

Lablab purpureus—A Crop Lost for Africa?

In recent years, so-called ‘lost crops’ have been appraised in a number of reviews, among them Lablab purpureus in the context of African vegetable species. This crop cannot truly be considered ‘lost’ because worldwide more than 150 common names are applied to it. Based on a comprehensive literature review, this paper aims to put forward four theses, (i) Lablab is one of the most diverse domesticated legume species and has multiple uses. Although its largest agro-morphological diversity occurs in South Asia, its origin appears to be Africa. (ii) Crop improvement in South Asia is based on limited genetic diversity. (iii) The restricted research and development performed in Africa focuses either on improving forage or soil properties mostly through one popular cultivar, Rongai, while the available diversity of lablab in Africa might be under threat of genetic erosion. (iv) Lablab is better adapted to drought than common beans (Phaseolus vulgaris) or cowpea (Vigna unguiculata), both of which have been preferred to lablab in African agricultural production systems. Lablab might offer comparable opportunities for African agriculture in the view of global change. Its wide potential for adaptation throughout eastern and southern Africa is shown with a GIS (geographic information systems) approach.

Natural Variation in Host-Specific Nodulation of Pea Is Associated with a Haplotype of the SYM37 LysM-Type Receptor-Like Kinase

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.

lox1:Ps:2, a Pisum sativum seed lipoxygenase gene.

A11 plants contain LOXs (EC 1.13.11.12), which catalyze the hydroperoxidation of polyunsaturated fatty acids. In vegetative organs, plant LOX genes respond to drought, wounding, pathogen attack, and exposure to jasmonate (for refs. see Melan et al., 1994); metabolism of fatty acid hydroperoxides produced by LOXs can give rise to the plant growth regulators ABA and methyl jasmonate. In soybean seeds, short-chain carbonyl compounds, derived from LOX-produced fatty acid hydroperoxides, are responsible for off-flavors. Plant LOX genes reported in the literature include those for vegetative LOX from Arabidopsis thaliana (Melan et al., 1994), Phaseolus vulgaris (Eiben and Slusarenko, 1994), and Glycine max (Kato et al., 1993) and for seed LOX (LOX-3, Yenofsky et al., 1988; SC514, Shibata et al., 1991) from G. max. We describe here a Pisum sativum LOX gene that corresponds to a seed mRNA that encodes a polypeptide similar in sequence to LOX-2 from soybean seed. We have named the gene loxl:Ps:2, in consultation with the International Society for Plant Molecular Biology Commission on Plant Gene Nomenclature. The 5785 bp of loxl:Ps:2 described in Table I include 1366 bp 5’ to the translation-initiation codon and 884 bp 3’ to the stop codon. Apart from three nucleotide substitutions and one deletion of three nucleotides, the protein coding sequence is identical to that of the pea seed LOX-2 cDNA pPE320 (Ealing and Casey, 1989). Eight introns are in the same positions, relative to the coding sequence of loxl:Ps:Z, as in the soybean LOX-3 gene (Yenofsky et al., 1988). The first intron is relatively large (314 bp, compared to 77-115 bp for the other seven); first introns in the Arabidopsis LOX1, P. vulgaris Loxl, and G. max LOX-3, L4, and SC514 genes are also large, comprising 1242, 1285, 448, 2542, and 624 bp, respectively. The protein sequence predicted by loxl:Ps:2 contains the His, Asn, and Ile residues that are liganded to iron at the active site (see, e.g., Boyington et al., 1993). The loxl:Ps:2 gene has extensive, overlapping tandem repeats between 600 bp and 1.3 kb upstream of the transcription start site (position 1340). There are also several motifs that correspond to known protein-binding sites and several sequences, including AACAAA, an “RY” repeat, and a perfect decanucleotide palindrome, that are thought to be important to the regulation of gene expression in developing seeds.