Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula.

The identification of the molecular polymorphisms giving rise to phenotypic trait variability-both quantitative and qualitative-is a major goal of the present agronomic research. Various approaches such as positional cloning or transposon tagging, as well as the candidate gene strategy have been used to discover the genes underlying this variation in plants. The construction of functional maps, i.e. composed of genes of known function, is an important component of the candidate gene approach. In the present paper we report the development of 63 single nucleotide polymorphism markers and 15 single-stranded conformation polymorphism markers for genes encoding enzymes mainly involved in primary metabolism, and their genetic mapping on a composite map using two pea recombinant inbred line populations. The complete genetic map covers 1,458 cM and comprises 363 loci, including a total of 111 gene-anchored markers: 77 gene-anchored markers described in this study, 7 microsatellites located in gene sequences, 16 flowering time genes, the Tri gene, 5 morphological markers, and 5 other genes. The mean spacing between adjacent markers is 4 cM and 90% of the markers are closer than 10 cM to their neighbours. We also report the genetic mapping of 21 of these genes in Medicago truncatula and add 41 new links between the pea and M. truncatula maps. We discuss the use of this new composite functional map for future candidate gene approaches in pea.

Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.).

This paper aims at providing reliable and cost effective genotyping conditions, level of polymorphism in a range of genotypes and map position of newly developed microsatellite markers in order to promote broad application of these markers as a common set for genetic studies in pea. Optimal PCR conditions were determined for 340 microsatellite markers based on amplification in eight genotypes. Levels of polymorphism were determined for 309 of these markers. Compared to data obtained for other species, levels of polymorphism detected in a panel of eight genotypes were high with a mean number of 3.8 alleles per polymorphic locus and an average PIC value of 0.62, indicating that pea represents a rather polymorphic autogamous species. One of our main objectives was to locate a maximum number of microsatellite markers on the pea genetic map. Data obtained from three different crosses were used to build a composite genetic map of 1,430 cM (Haldane) comprising 239 microsatellite markers. These include 216 anonymous SSRs developed from enriched genomic libraries and 13 SSRs located in genes. The markers are quite evenly distributed throughout the seven linkage groups of the map, with 85% of intervals between the adjacent SSR markers being smaller than 10 cM. There was a good conservation of marker order and linkage group assignment across the three populations. In conclusion, we hope this report will promote wide application of these markers and will allow information obtained by different laboratories worldwide in diverse fields of pea genetics, such as QTL mapping studies and genetic resource surveys, to be easily aligned.

Detection of gene-anchored amplification polymorphism (GAAP) in the vicinity of plant mitochondrial genes.

A simple, semi-automatable method was established for assessing polymorphism in plant mitochondrial genome. A set of 41 mitochondrial markers based on the published Arabidopsis thaliana sequence was developed in Brassicaceae using a gene-anchored amplification polymorphism (GAAP) strategy. PCR primers were selected based on conserved coding regions of mitochondrial genes and used to amplify the corresponding 5' and/or 3' non-coding flanking regions in order to maximise sequence variability between haplotypes. The variations in fragment size were analysed on a LiCor DNA sequencer, but the methodology is compatible with various sequencing systems using denaturing polyacrylamide gels. One advantage of the method is that GAAP products can be directly sequenced (without any cloning steps) through labelled M13 consensus sequences. Mitochondrial GAAP loci gave clear and simple patterns (one or two bands) that were easy to score and highly reproducible. Nearly all mitochondrial loci examined in A. thaliana were conserved within the Brassicaceae family, and half of the primers generated products when DNA from a distant species, Beta vulgaris (Chenopodiaceae), was used as template. The GAAP markers revealed low levels of polymorphism within species but exhibited a high level of polymorphism among genera and families. Our results showed some discrepancies with respect to the published mtDNA sequence of A. thaliana.

Structure and differential expression of two maize ferritin genes in response to iron and abscisic acid.

In plants, synthesis of the iron-storage protein ferritin in response to iron is not regulated at the translational level; this is in contrast to ferritin synthesis in animals. Part of the response is mediated through a transduction pathway which involves the plant hormone abscisic acid. In this work, we report the cloning and sequencing of two maize ferritin genes (ZmFer1 and ZmFer2) coding for members of the two ferritin mRNA subclasses, FM1 and FM2, respectively. Although plant and animal ferritins are closely related proteins, a major difference is observed between the organisation of the genes. Both maize ferritin genes are organised as eight exons and seven introns, the positions of which are identical within the two genes, while animal ferritin genes are interrupted by three introns, at positions different from those found in maize genes. Sequence divergence between the 3' untranslated regions of these genes has allowed the use of specific probes to study the accumulation of FM1 and FM2 transcripts in response to various environmental cues. Such probes have shown that FM1 and FM2 transcripts accumulate with differential kinetics in response to iron; FM1 mRNA accumulate earlier than FM2 mRNA and only FM2 transcripts accumulate in response to exogenous abscisic acid or water stress. Mapping of the transcriptional initiation region of these two genes defined their 5' upstream regions and allowed a sequence comparison of their promoters, which appeared highly divergent. This raises the possibility that the differential accumulation of FM1 and FM2 mRNAs in response to iron, abscisic acid and drought could be due to differential transcription of ZmFer1 and ZmFer2.