Genome-wide analysis identifies gain and loss/change of function within the small multigenic insecticidal Albumin 1 family of Medicago truncatula.

BACKGROUND: Albumin 1b peptides (A1b) are small disulfide-knotted insecticidal peptides produced by Fabaceae (also called Leguminosae). To date, their diversity among this plant family has been essentially investigated through biochemical and PCR-based approaches. The availability of high-quality genomic resources for several fabaceae species, among which the model species Medicago truncatula (Mtr), allowed for a genomic analysis of this protein family aimed at i) deciphering the evolutionary history of A1b proteins and their links with A1b-nodulins that are short non-insecticidal disulfide-bonded peptides involved in root nodule signaling and ii) exploring the functional diversity of A1b for novel bioactive molecules. RESULTS: Investigating the Mtr genome revealed a remarkable expansion, mainly through tandem duplications, of albumin1 (A1) genes, retaining nearly all of the same canonical structure at both gene and protein levels. Phylogenetic analysis revealed that the ancestral molecule was most probably insecticidal giving rise to, among others, A1b-nodulins. Expression meta-analysis revealed that many A1b coding genes are silent and a wide tissue distribution of the A1 transcripts/peptides within plant organs. Evolutionary rate analyses highlighted branches and sites with positive selection signatures, including two sites shown to be critical for insecticidal activity. Seven peptides were chemically synthesized and folded in vitro, then assayed for their biological activity. Among these, AG41 (aka MtrA1013 isoform, encoded by the orphan TA24778 contig.), showed an unexpectedly high insecticidal activity. The study highlights the unique burst of diversity of A1 peptides within the Medicago genus compared to the other taxa for which full-genomes are available: no A1 member in Lotus, or in red clover to date, while only a few are present in chick pea, soybean or pigeon pea genomes. CONCLUSION: The expansion of the A1 family in the Medicago genus is reminiscent of the situation described for another disulfide-rich peptide family, the "Nodule-specific Cysteine-Rich" (NCR), discovered within the same species. The oldest insecticidal A1b toxin was described from the Sophorae, dating the birth of this seed-defense function to more than 58 million years, and making this model of plant/insect toxin/receptor (A1b/insect v-ATPase) one of the oldest known.

PCR identification of durum wheat BAC clones containing genes coding for carotenoid biosynthesis enzymes and their chromosome localization.

Carotenoids are essential components in all plants. Their accumulation in wheat seed determines the endosperm colour, which is an important quality trait in wheat. In this study, we report the isolation of BAC clones containing genes coding for three different enzymes of the carotenoid biosynthesis pathway: phytoene synthase (PSY), phytoene desaturase (PDS), and zeta-carotene desaturase (ZDS). Primers were designed on the basis of wheat ESTs similar to the sequences of these three genes in other species, and used to screen a BAC library from Triticum turgidum var. durum (2n = 28, genomes AABB). Eight, six, and nine 384-well plates containing at least one positive clone were found for PSY, PDS, and ZDS, respectively. BACs selected for each of these genes were then divided in two groups corresponding to the A and B genomes of tetraploid wheat, based on differences in the length of the PCR amplification products, conformation-sensitive gel electrophoresis (CSGE), or cleavage amplification polymorphisms. Positive clones were then assigned to chromosomes using a set of D genome substitution lines in T. turgidum var. durum 'Langdon'. PSY clones were localized on chromosomes 5A and 5B, PDS on chromosomes 4A and 4B, and ZDS on chromosomes 2A and 2B. The strategies used for the PCR screening of large BAC libraries and for the differentiation of BAC clones from different genomes in a polyploid species are discussed.

Sequencing of the Triticum monococcum hardness locus reveals good microcolinearity with rice.

The Hardness ( Ha) locus on chromosome 5D is the main determinant of grain texture in hexaploid wheat. The related genes Puroindoline-a and -b ( Pina-D1, Pinb-D1) and Grain Softness Protein ( Gsp-D1) are tightly linked at this locus. Mutations in the Pina-D1 and Pinb-D1 genes are associated with increased grain hardness. We report here the complete sequence of a 101-kb BAC clone from Triticum monococcum (A(m ) genome) which includes these three genes, and its comparison with the orthologous region in rice. The genes Gsp-A(m) 1, Pina-A(m) 1 and Pinb-A(m) 1 are separated by 37 kb and 32 kb, respectively, and are organized in the same transcriptional orientation. Four additional genes, including a pair of duplicated genes, were identified upstream of Gsp-A(m) 1 within a high-density gene island. These additional genes were found in the same order and orientation, and the same relative distances apart as similar genes previously annotated on rice chromosome 12. An interesting discovery was a small unannotated putative rice gene that was similar to the Gsp-A(m) 1 gene of T. monococcum (65% similarity at the protein level), and that was disposed in the same orientation, and located in the same position relative to the other orthologous genes. The high gene density observed in this BAC (1 gene per 14 kb) was expected for a distal chromosome region, but the level of microcolinearity with rice was higher than that reported in similar distal regions of other wheat chromosomes. Most of the BAC sequence (40%) was represented by repetitive elements, mainly concentrated in regions adjacent to the genes Pina-A(m) 1 and Pinb-A(m) 1. Rearrangements among these repetitive elements might provide an explanation for the frequent deletions observed at this locus in the genomes of the polyploid wheat species.

Construction and characterization of a half million clone BAC library of durum wheat ( Triticum turgidum ssp. durum).

Durum wheat ( Triticum turgidum ssp. durum, 2 n = 4 x = 28, genomes AB) is an economically important cereal used as the raw material to make pasta and semolina. In this paper we present the construction and characterization of a bacterial artificial chromosome (BAC) library of tetraploid durum wheat cv. Langdon. This variety was selected because of the availability of substitution lines that facilitate the assignment of BACs to the A and B genome. The selected Langdon line has a 30-cM segment of chromosome 6BS from T. turgidum ssp. dicoccoides carrying a gene for high grain protein content, the target of a positional cloning effort in our laboratory. A total of 516,096 clones were organized in 1,344 384-well plates and blotted on 28 high-density filters. Ninety-eight percent of these clones had wheat DNA inserts (0.3% chloroplast DNA, 1.4% empty clones and 0.3% empty wells). The average insert size of 500 randomly selected BAC clones was 131 kb, resulting in a coverage of 5.1-fold genome equivalents for each of the two genomes, and a 99.4% probability of recovering any gene from each of the two genomes of durum wheat. Six known copy-number probes were used to validate this theoretical coverage and gave an estimated coverage of 5.8-fold genome equivalents. Screening of the library with 11 probes related to grain storage proteins and starch biosynthesis showed that the library contains several clones for each of these genes, confirming the value of the library in characterizing the organization of these important gene families. In addition, characterization of fingerprints from colinear BACs from the A and B genomes showed a large differentiation between the A and B genomes. This library will be a useful tool for evolutionary studies in one of the best characterized polyploid systems and a source of valuable genes for wheat. Clones and high-density filters can be requested at http://agronomy.ucdavis.edu/Dubcovsky/BAC-library/BAC_Langdon.htm

The Race-Specific Resistance Gene to Powdery Mildew, MlRE, Has a Residual Effect on Adult Plant Resistance of Winter Wheat Line RE714.

ABSTRACT Race-specific resistance genes to powdery mildew have been extensively used in wheat breeding programs, but the complete resistance they provide breaks down when confronted by pathogen isolates with matching virulence. However, when overcome, some race-specific genes have a residual action leading to a reduction of the symptoms. Our objective was to determine if the resistance genes MlRE and Pm4b have a residual effect on adult plant resistance (APR) and on vernalized seedling plant resistance (VPR) in the line RE714. Individuals from two populations (double haploid [DH] and F(3) families) were genotyped for the race-specific genes MlRE and Pm4b and assessed for their resistance under field conditions at the adult plant stage (in 1996 and 1997 for the DH lines and in 1997 for the F(3) families). Vernalized seedlings of the DH population were tested with four powdery mildew isolates. Only the MlRE gene had a significant effect (dominant type) on APR. Neither MlRE nor Pm4b had a significant effect on VPR. The dominant residual effect of the defeated race-specific gene MlRE was a component of APR in the line RE714.