Pierre Barret

THE TWO GENES HOMOLOGOUS TO ARABIDOPSIS FAE1 CO-SEGREGATE WITH THE TWO LOCI GOVERNING ERUCIC ACID CONTENT IN BRASSICA NAPUS

Abstract KCS (β-keto-acyl-CoA synthase) has been proposed as a candidate gene for explaining the erucic acid level in rapeseed. Degenerate PCR primers corresponding to the FAE1 gene have been designed. Two B. napus genes BN-FAE1.1 and BN-FAE1.2, corresponding to the parental species B. rapa and B. oleracea FAE1 genes, were amplified. Polymorphism was revealed for these two genes by acrylamide electrophoresis of the amplification products. These two genes could then be mapped and a co-segregation of these genes with the E1 and E2 loci controlling erucic acid content was found. Furthermore, mutations observed for one of these genes could explain part of the low erucic trait of the three LEAR types used in this study.

Molecular-mapping analysis in Brassica napus using isozyme, RAPD and RFLP markers on a doubled-haploid progeny

We have undertaken the construction of a Brassica napus genetic map with isozyme (4%), RFLP (26.5%) and RAPD (68%) markers on a 152 lines of a doubled-haploid population. The map covers 1765 cM and comprises 254 markers including three PCR-specific markers and a morphological marker. They are assembled into 19 linkage groups, covering approximatively 71% of the rapeseed genome. Thirty five percent of the studied markers did not segregate according to the expected Mendelian ratio and tended to cluster in eight specific linkage groups. In this paper, the structure of the genetic map is described and the existence of non-Mendelian segregations in linkage analysis as well as the origins of the observed distortions, are discussed. The mapped RFLP loci corresponded to the cDNAs already used to construct B. napus maps. The first results of intraspecific comparative mapping are presented.

A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid

Abstract The synthesis of very long chain fatty acids occurs in the cytoplasm via an elongase complex. A key component of this complex is the β-ketoacyl-CoA synthase, a condensing enzyme which in Arabidopsis is encoded by the FAE1 gene. Two sequences homologous to the FAE1 gene were isolated from a Brassica napus immature embryo cDNA library. The two clones, CE7 and CE8, contain inserts of 1647 bp and 1654 bp, respectively. The CE7 gene encodes a protein of 506 amino acids and the CE8 clone, a protein of 505 amino acids, each having an approximate molecular mass of 56 kDa. The sequences of the two cDNA clones are highly homologous yet distinct, sharing 97% nucleotide identity and 98% identity at the amino acid level. Southern hybridisation showed the rapeseed β-ketoacyl-CoA synthase to be encoded by a small multigene family. Northern hybridisation showed the expression of the rapeseed FAE1 gene(s) to be restricted to the immature embryo. One of the FAE1 genes is tightly linked to the E1 locus, one of two loci controlling erucic acid content in rapeseed. The identity of the second locus, E2, is discussed.

Selection of stable Brassica napus-B. juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). 1. Identification of molecular markers, chromosomal and genomic origin of the introgression

Abstract A scheme of selection combining selfing and backcross was applied to a B. napus line with the blackleg resistance from B. juncea in order to transfer this resistance to a winter oilseed rape variety. Cytogenetic analyses combined with cotyledon blackleg resistance tests at each generation allowed us to obtain a recombinant line showing regular meiotic behavior. The resistance is monogenic and is highly efficient under field conditions. Four-hundred RAPD primers were tested on two segregating populations by bulk segregant analysis. Three markers totally linked to the introgression were identified. The analysis of these markers on both sets of B. napus-B. nigra and B. oleracea-B. nigra addition lines revealed that they are not located on the B4 chromosome of B. nigra, which has already been shown to carry a blackleg resistance gene, but rather on the B8 chromosome. We confirmed that the resistance gene is carried by the B genome of B. juncea. Based on these data, two hypotheses, one involving chromosome rearrangements between the two B genomes of B. nigra and B. juncea, and the other based on a more probable digenic control of the resistance within B. juncea, are discussed.

Characterisation of the radish introgression carrying the Rfo restorer gene for the Ogu-INRA cytoplasmic male sterility in rapeseed (Brassica napus L.)

Abstract Bulked segregant analysis and comparative mapping were applied to identify molecular markers linked to the Rfo restorer gene used for the Ogu-INRA cytoplasmic male-sterility system in rapeseed. These markers were then used to localise the radish introgression on the B. napus genetic map constructed from the cross ‘Darmor.bzh’ x ’Yudal’. The introgression mapped on the DY15 linkage group. From the comparison of this latter group to the linkage group constructed on a F2 progeny segregating for the radish introgression, it was concluded that the introgression had occurred through homoeologous recombination, that it was not distal and that it had replaced a B. napus region of around 50 cM. A QTL involved in aliphatic seed glucosinolate content was located on the DY15 linkage group at a position corresponding to one end of the introgression. The DNA markers identified in this study are being used in map-based cloning of the Rfo gene and in marker-assisted selection.

Identification of RAPD markers linked to the loci controlling erucic acid level in rapeseed

The recent development of the industrial use of rapeseed oil rich in erucic acid has led to increased interest in the improvement of the high-erucic-acid (50–60%) varieties and to research towards genotypes containing a very high erucic acid content. This trait is controlled by two genes with additive effects. The low-erucic-acid trait was relatively easily introduced through backcrosses into various backgrounds because the zero-erucic-acid homozygotes were clearly identified in the segregating populations. To select for high erucic acid level is more difficult because of the partial overlap of the high-erucic-acid homozygous class and the intermediate one, containing heterozygotes. In order to help conventional breeding, RAPD markers were used to map the two genes involved in determining the erucic acid content in a doubled haploid progeny derived from a ‘low x high’ erucic acid F1 hybrid. The two genes were successfully localized in two independent linkage group, through a QTL approach. A close association was found between individual plant genotypes and the erucic acid content of the doubled haploid progeny, and it was shown that the two genes do not contribute uniformly to the C22:1 level. The value of molecular gene mapping of such a trait in a conventional breeding programme is discussed.

Specific molecular marker of the genes controlling linolenic acid content in rapeseed

In rapeseed, which is an agronomically important oilseed, variation in the linolenic acid content of the oil has been obtained through chemical mutagenesis treatment. Conventional breeding of this quantitative trait, however requires specific molecular markers. By means of biochemical experiments, we have established that the induced variation in linolenic acid content is associated with the fad3 gene encoding the microsomal Δ15 desaturase. Using a pair of primers specific to this gene and a doubled haploid progeny derived from a low linolenic x high linolenic acid F1hybrid, we have identified a polymorphism of the fad3 alleles between the low- and the high-linolenic acid genotypes. The structure exon/intron of the fad3 DNA sequence seems to be very similar to that of the Arabidopsis fad3 gene. The choice of the primer pair allows specific amplification of one of the two rapeseed fad3 genes. The value and contribution of specific markers to conventional plant breeding is discussed.

Characterization of backcross generations obtained under field conditions from oilseed rape-wild radish F1 interspecific hybrids: an assessment of transgene dispersal

Abstract Gene flow from glufosinate-resistant transgenic oilseed rape to wild radish was studied over two backcross generations. Under field conditions, seed production from oilseed rape-wild radish F1 hybrids due to pollination by wild radish was always low: on average 0.12 and 0.78 seeds per 100 flowers and per plant, respectively. The cytogenetics of the resulting «BC1» plants can be explained in the main by three different genomic constitutions: either ACRrRr, 2n=37, ACRr, 2n=28 (the same chromosome number as the mother plant), or by the amphidiploid AACCRrRr, 2n=56. The probability of gene exchange through chromosome pairing was high only in plants with 2n=28 or 37 chromosomes. Due to the viability of unreduced or partially reduced female gametes, most of the «BC1» plants (81.9%) were Basta resistant whereas the analysis of oilseed rape specific loci indicated that their transmission varied with the locus. In spite of low male fertility (8.7%), an improvement of the female fertility over the F1 hybrids was observed with an average production of 1.4 and 11 seeds per 100 flowers and per plant, respectively. At the following «BC2» generation, the bar gene transmission (57.2% of Basta-resistant plants) decreased as did the chromosome number, with a majority of plants having between 24 and 27 chromosomes, with 10.5% similar to wild radish (2n=18). The lower the chromosome number, the better the fertility of the «BC2» plants. On average, 7.9 and 229.3 seeds per 100 flowers and per plant were produced. Gene-flow assessment is discussed based on these data.

From Arabidopsis thaliana to Brassica napus: development of amplified consensus genetic markers (ACGM) for construction of a gene map

Abstract.The evolution of genomes can be studied by comparing maps of homologous genes which show changes in nucleic acid sequences and chromosome rearrangements. In this study, we developed a set of 32 amplified consensus gene markers (ACGMs) that amplified gene sequences from Arabidopsis thaliana and Brassica napus. Our methodology, based on PCR, facilitated the rapid sequencing of homologous genes from various species of the same phylogenetic family and the detection of intragenic polymorphism. We found that such polymorphism principally concerned intron sequences and we used it to attribute a Brassica oleracea or Brassica rapa origin to the B. napus sequences and to map 43 rapeseed genes. We confirm that the genetic position of homologous genes varied between B. napus and A. thaliana. ACGMs are a useful tool for genome evolution studies and for the further development of single nucleotide polymorphism suitable for use in genetic mapping and genetic diversity analyses.

Identification of RAPD markers linked to linolenic acid genes in rapessed

SummaryThe inheritance of the low linolenic acid content (derivated from mutant lines) in rapeseed was investigated. Molecular techniques of gene mapping through RAPD markers were applied on a microspore-derived progeny from a high × low linolenic acid F1 hybrid. ‘Bulked segregant analysis’ made it possible to test rapidly number of RAPD primers. Two linkage groups of 6 markers (72.7 cM and 75.6 cM) were determined. Each corresponded to a major QTL which explained 24% and 30.7% of the total phenotypic variation of the linolenic acid content. It was confirmed that two independant mutations were implied in the low linonenic acid content.