Michel Beckert

Molecular Basis of Evolutionary Events That Shaped the Hardness Locus in Diploid and Polyploid Wheat Species (Triticum and Aegilops)

The Hardness (Ha) locus controls grain hardness in hexaploid wheat (Triticum aestivum) and its relatives (Triticum and Aegilops species) and represents a classical example of a trait whose variation arose from gene loss after polyploidization. In this study, we investigated the molecular basis of the evolutionary events observed at this locus by comparing corresponding sequences of diploid, tertraploid, and hexaploid wheat species (Triticum and Aegilops). Genomic rearrangements, such as transposable element insertions, genomic deletions, duplications, and inversions, were shown to constitute the major differences when the same genomes (i.e., the A, B, or D genomes) were compared between species of different ploidy levels. The comparative analysis allowed us to determine the extent and sequences of the rearranged regions as well as rearrangement breakpoints and sequence motifs at their boundaries, which suggest rearrangement by illegitimate recombination. Among these genomic rearrangements, the previously reported Pina and Pinb genes loss from the Ha locus of polyploid wheat species was caused by a large genomic deletion that probably occurred independently in the A and B genomes. Moreover, the Ha locus in the D genome of hexaploid wheat (T. aestivum) is 29 kb smaller than in the D genome of its diploid progenitor Ae. tauschii, principally because of transposable element insertions and two large deletions caused by illegitimate recombination. Our data suggest that illegitimate DNA recombination, leading to various genomic rearrangements, constitutes one of the major evolutionary mechanisms in wheat species.

Molecular diversity in French bread wheat accessions related to temporal trends and breeding programmes

A set of 41 wheat microsatellite markers (WMS), giving 42 polymorphic loci (two loci on each chromosome), was used to describe genetic diversity in a sample of 559 French bread wheat accessions (landraces and registered varieties) cultivated between 1800 and 2000. A total of 609 alleles were detected. Allele number per locus ranged from 3 to 28, with a mean allele number of 14.5. On the average, about 72% of the total number of alleles were observed with a frequency of less than 5% and were considered to be rare alleles. WMS markers used showed different levels of gene diversity: the highest PIC value occurred in the B genome (0.686) compared to 0.641 and 0.659 for the A and D genomes, respectively. When comparing landraces with registered varieties gathered in seven temporal groups, a cluster analysis based on an Fst matrix provided a clear separation of landraces from the seven variety groups, while a shift was observed between varieties registered before and after 1970. There was a decrease of about 25% in allelic richness between landraces and varieties. In contrast, when considering only registered varieties, changes in diversity related to temporal trends appeared more qualitative than quantitative, except at the end of the 1960s, when a bottleneck might have occurred. New varieties appear to be increasingly similar to each other in relation to allelic composition, while differences between landraces are more and more pronounced over time. Finally, considering a sub-sample of 193 varieties representative of breeding material selected during the twentieth century by the six most important plant breeding companies, few differences in diversity were observed between the different breeding programmes. The observed structure of diversity in French bread wheat collections is discussed in terms of consequences, both for plant breeders and for managers of crop genetic resources.

Down-Regulation of Caffeic Acid O-Methyltransferase in Maize Revisited Using a Transgenic Approach

Transgenic maize (Zea mays) plants were generated with a construct harboring a maize caffeic acidO-methyltransferase (COMT) cDNA in the antisense (AS) orientation under the control of the maize Adh1(alcohol dehydrogenase) promoter. Adh1-driven β-glucuronidase expression was localized in vascular tissues and lignifying sclerenchyma, indicating its suitability in transgenic experiments aimed at modifying lignin content and composition. One line of AS plants, COMT-AS, displayed a significant reduction in COMT activity (15%–30% residual activity) and barely detectable amounts of COMT protein as determined by western-blot analysis. In this line, transgenes were shown to be stably integrated in the genome and transmitted to the progeny. Biochemical analysis of COMT-AS showed: (a) a strong decrease in Klason lignin content at the flowering stage, (b) a decrease in syringyl units, (c) a lowerp-coumaric acid content, and (d) the occurrence of unusual 5-OH guaiacyl units. These results are reminiscent of some characteristics already observed for the maize bm3(brown-midrib3) mutant, as well as for COMT down-regulated dicots. However, as compared with bm3, COMT down-regulation in the COMT-AS line is less severe in that it is restricted to sclerenchyma cells. To our knowledge, this is the first time that an AS strategy has been applied to modify lignin biosynthesis in a grass species.

A worldwide bread wheat core collection arrayed in a 384-well plate.

Bread wheat (Triticum aestivum), one of the world’s major crops, is genetically very diverse. In order to select a representative sample of the worldwide wheat diversity, 3,942 accessions originating from 73 countries were analysed with a set of 38 genomic simple sequence repeat (SSR) markers. The number of alleles at each locus ranged from 7 to 45 with an average of 23.9 alleles per locus. The 908 alleles detected were used together with passport data to select increasingly large sub-samples that maximised both the number of observed alleles at SSR loci and the number of geographical origins. A final core of 372 accessions (372CC) was selected with this M strategy. All the different geographical areas and more than 98% of the allelic diversity at the 38 polymorphic loci were represented in this core. The method used to build the core was validated, by using a second set of independent markers [44 expressed sequence tag (EST)-SSR markers] on a larger sample of 744 accessions: 96.74% of the alleles observed at these loci had already been captured in the 372CC. So maximizing the diversity with a first set of markers also maximised the diversity at a second independent set of locus. To relate the genetic structure of wheat germplasm to its geographical origins, the two sets of markers were used to compute a dissimilarity matrix between geographical groups. Current worldwide wheat diversity is clearly divided according to wheat’s European and Asian origins, whereas the diversity within each geographical group might be the result of the combined effects of adaptation of an initial germplasm to different environmental conditions and specific breeding practices. Seeds from each accession of the 372CC were multiplied and are now available to the scientific community. The genomic DNA of the 372CC, which can be entirely contained in a 384-deep-well storage plate, will be a useful tool for future studies of wheat genetic diversity.

Cloning and characterization of two maize cDNAs encoding Cinnamoyl-CoA Reductase (CCR) and differential expression of the corresponding genes

Cinnamoyl-CoA Reductase (CCR, EC 1.2.1.44) catalyses the first step of the lignin pathway. Two full-length cDNAs identified by sequence analysis as CCR-encoding cDNAs were isolated from a maize root cDNA library. These two cDNAs designated ZmCCR1 and ZmCCR2 exhibit 73% sequence conservation at the nucleotide level for their coding regions and are relatively divergent at their 5′- and 3′-untranslated regions. They both contain a common signature which is thought to be involved in the catalytic site of CCR. Northern blot analysis indicated that ZmCCR2 was expressed at very low levels in roots whereas ZmCCR1 was widely expressed in different organs. The high level of ZmCCR1 gene expression along the stalk suggests that the corresponding enzyme is probably involved in constitutive lignification.

Genetic control of maternal haploidy in maize (Zea mays L.) and selection of haploid inducing lines

SummaryThe effect of genotype on maternal haploid plant production in maize was studied. The frequency of gynogenetic plants when “Stock 6” was used as pollinator varied according to the female parent genotype. No simple relation was observed between genotypic aptitudes for gynogenetic and androgenetic development, which occured after pollination of “W23” plant carrying the “indeterminate gametophyte” gene. Furthermore, the population NS, a favorably responsive genotype to anther culture, does not exhibit exceptional ability for in vivo gynogenesis. The effect of inbreeding and the influence of maternal haploid origin suggest that specific genes control maternal haploid initiation and development. However, gynogenetic development is not limited to a particular genotype. The frequency of maternal haploids may be increased by using specific pollen parents. Attempts were made to select for a high haploidyinducing trait and the present study reports the successful development of lines that can be utilized as pollen parents to induce haploids for experimental purposes and breeding programmes. When an inbred line “WS14”, derived from the cross W23 x Stock 6, was used as pollen parent, 2%–5% maternal haploids were obtained according to the female parent genotype. A high haploidy-inducing potential is a heritable trait and may be controlled by a limited number of genes. Genetic determination of the haploidy-inducing character was examined in relation to the efficiency of the selecting method and the mechanisms involved in the origin of maternal haploids.

Optimization of maize microspore isolation and culture conditions for reliable plant regeneration.

SummaryThe effects of different factors were investigated in the process of isolated microspore culture of Zea mays L., Using donor plants grown in standard conditions and an efficient isolation technology, homogeneous populations of viable microspores at specific developmental stages were obtained and tested in culture. The cytological evolution of the microspores during the first week of culture was monitored using a DNA-specific fluorochrome. It was found that developmental stages of microspores, number of days of pretreatment at 7°C of the tassel, and culture density greatly influenced the number of microspore-derived embryos. Optimal conditions required for embryo and plant production are described.

Molecular and morphological evaluation of doubled-haploid lines in maize. 2. Comparison with single-seed-descent lines

Doubled-haploid (DH) and single-seed-descent (SSD) lines in maize have been compared for quantitatively inherited traits and for RFLP markers. The comparisons of the distributions for agromorphological traits do not allow definite conclusions to be drawn on the similarity of the two reproductive systems. We have used more than 100 RFLP markers to provide a precise description of the parental allele frequency and the recombination fractions. A comparison of two DH populations shows that non-random meiotic reassortment is influenced by differences in the anther culture capacities of the two parental lines. For the DH lines derived from the cross DH5 x DH7, involving two responsive lines in anther culture, the distortion in segregation (P < 0.05) affected less than 20% of the genome with half of the deviations towards each parent. DH lines derived from the cross A188 x DH7, where A188 is a non-responsive line, showed more than twice this level of distortion and an excess of DH7 alleles was found for almost all of the skewed loci. The recombination fractions were homogeneous between the two DH populations for most of the genome. The genome sizes calculated with the DH and the SSD lines derived from the same cross, A188 x DH7, were also similar, which suggests that no selection against recombinant gametes occurs during anther culture. The observed recombination fraction after five meioses (SSD) is on average twice as large as after one meiosis (DH). No difference is observed for recombination fractions greater than 20%. Despite a precise description of the material at the molecular level, it has not been possible to make a definite conclusion as to whether or not the differences in some morphological characters are the consequences of differences in the segregation ratio and/or the recombination frequency. However, the agromorphological evaluation shows a narrow range in differences between the two types of lines and suggests that the use of DH lines is possible in breeding programmes.

Characterization of proteins secreted during maize microspore culture: arabinogalactan proteins (AGPs) stimulate embryo development

To study molecules secreted from cultured plant cells that promote development, maize microspores were transferred into culture and the conditioned media were collected over time and analysed. Electrophoresis indicated that both non-glycosylated and glycosylated proteins including arabinogalactan proteins (AGPs) appeared in the medium and their concentration increased during the time of culture. The development of embryos was correlated with the presence of specific extracellular proteins, using an experimental system based on a tunicamycin inhibition test. In addition, a precise protein analysis was conducted using MALDI-TOF and ESI-MS-MS techniques. These approaches have allowed the identification of 5 other types of proteins: a cell wall invertase, two thaumatin isoforms, one 1-3 beta-glucanase and two chitinase isoforms. Altogether these experiments and results open ways for research aimed at understanding which molecules stimulate embryo formation. Moreover, AGPs may be used to stimulate the development of microspores (pollen embryogenesis) prepared from non-responsive genotypes.

Molecular and morphological evaluation of doubled haploid lines in maize. 1. Homogeneity within DH lines.

The homogeneity of anther culture-derived lines of maize has been evaluated by means of field observations and molecular markers. The homogeneity of the doubled haploid (DH) lines was shown by the absence of segregation for morphological oligogenic traits. The intravariance for polygenic traits for 42 DH and two conventionally derived lines was similar, which confirmed the homozygosity of the DH lines. More than 100 RFLP markers were tested on 189 DH lines derived from two crosses, DH5 x DH7 and A188 x DH7, and 60 single-seed descent (SSD, F6) lines derived from A188 x DH7. The overall rate of heterozygosity for all of the DH lines was approximatively 1% and pertained to 6 lines out of 189, while it was 8.5% for the SSD lines after four selfings. A precise description of the material used suggested that the events which led to this unexpected heterozygozity in DH lines were more likely to have occurred after rather than during the androgenetic process. Nine duplicated pairs of genotypes were found within the DH lines, indicating that a single microspore-derived structure can fragment to give two identical plantlets. Despite the extensive screening with more than 100 markers, only 2 lines showed unexpected banding profiles, and these were probably gametoclonal variants. The use of a direct regeneration system that avoids any callus phase might explain this low frequency of gametoclonal variation.