New evidence from Sinapis alba L. for ancestral triplication in a crucifer genome

We present clear evidence of ancestral genome triplication in Sinapis alba, a close relative of the cultivated Brassica species. Exceptionally high levels of heterozygosity in the parents of an F1 intercross permitted the mapping of an estimated 87% of all detected restriction fragment length polymorphism (RFLP) loci, with each RFLP probe typically detecting 2 or 3 loci. These duplicated loci were arranged in 8 triplicated homologous linkage blocks and 2 small, duplicated, homologous linkage blocks covering the majority of the S. alba genome. Several large-scale inversions and translocations appear to have rearranged the order of loci within homologous blocks. The role of successive polyploidization events on the evolution of crucifer species is discussed.Key words: polyploidy, yellow mustard, Brassica hirta, genome duplication, hexaploid ancestor, paralogous loci.

Use of RFLP markers for identification of individuals homozygous for resistance to Meloidogyne arenaria in peanut

To increase the efficiency of breeding peanuts resistant to Meloidogyne arenaria, we determined the utility of two RFLP loci linked to a single gene for resistance in identifying individuals putatively homozygous for resistance. DNA was extracted from leaf samples collected from each of 548 individuals from three segregating BC7F2:4 breeding populations (TP293-3-3, TP296-4 and TP301-1-8). The DNA was then digested with Eco RI, and Southern blotted to Hybond-N+ membranes. The membranes were probed with the RFLP probe R2430E, the autoradiographs scored for resistance genotype, then stripped and re-probed with the probe R2545E. Samples from which no data were obtained due to problems in extraction, digestion, or hybridization ranged from a low of 14.4% for TP301-1-8 probed with R2430E to a high of 38.9% for TP296-4-4 probed with R2545E. Locus R2430 identified 27.6, 65.1 and 29.5% of populations TP293-3-3, TP296-4-4 and TP301-1-8, respectively, as being homozygous for resistance. The second locus, R2545E, identified 24.5, 50 and 23.5%, respectively, of these populations as homozygous for resistance. In glasshouse tests of nematode reproduction on progeny of individuals identified as homozygous for resistance based on RFLP patterns, all 15 individuals of each of the 11 single plant progeny lines tested were resistant. Conversely, all progeny from an individual identified as susceptible to M. arenaria based on RFLP patterns supported high levels of nematode reproduction. Utilisation de marqueurs RFLP pour l'identification d'individus homozygotes pour la résistance envers Meloidogyne arenaria chez l'arachide - Pour accroître l'efficacité dans les croisements d'arachides résistantes à Meloidogyne arenaria, nous avons démontré l'utilité de deux loci RFLP liés à un seul gène de résistance en identifiant des individus potentiellement homozygotes pour cette résistance. L'ADN a été extrait d'échantillons de feuilles prélevés sur chacun des 548 individus provenant de trois populations appartenant à des croisements séparant BC7F2:4 (TP293-3-3, TP296-4-4 et TP301-1-8). L'AND été ensuite digéré par ECO RI et transferré par Southern blotting sur des membranes Hybond-N+. Ces membranes ont été sondées grâce à une sonde RFLP (CE243OE), les autoradiographies repérées pour le génotype de résistance, puis dépouillées et sondées à nouveau à l'aide de la sonde R243OE. Les échantillons ne fournissant aucune donnée - cela dß à des problèmes d'extraction, de digestion ou d'hybridisation - représentent de 14,4% pour la population TP-301-1-8 sondée par R2430E à 38,9% pour la population TP296-4-4 sondée par R2545E. Le locus R2430 a identifié 27,6, 65,1 et 29,5% des populations TP293-3-3, TP296-4-4 et TP3O1-1-8, respectivement, comme homozygotes pour la résistance. Le second locus, R2545E, a identifié 24,5, 50 et 23,5%, respectivement, de ces trois populations comme homozygotes pour la résistance. Lors d'expériences en serre concernant la reproduction du nématode sur la descendance d'individus identifiés, sur la base de profils RFLP, comme hymozygotes pour la résistance, chacun des 15 individus de chacune des 11 lignées monoparentales testées se sont montrés résistants. A l'inverse, tous les descendants d'un individu identifié, sur la base des profils RFLP, comme sensible à M. arenaria, permettent un taux élevé de reproduction du nématode.

Genetic variability in a collection of Stagonospora nodorum isolates from Western Australia

Stagonospora nodorum isolates were collected from the Western Australian grain-belt during 1993. These isolates and a subset of isolates taken from a single location were used to assay the level of variation within the pathogen population. The isolates were compared using anonymous nuclear DNA markers. Three low copy-number and a single high copy-number RFLP probe were used to generate polymorphisms. The collection exhibited a high genotypic diversity for the high copy-number probe, a result consistent with the high level of sexual reproduction previously found in the fungal population. The high level of genotypic diversity was consistent with previous international studies. There was no evidence of differentiation between the total collection of isolates and the subset of isolates taken from the single location. Further work needs to be undertaken to determine if the aggressiveness of the pathogen is influenced by the host genotype.

Development of a PCR-based allele-specific assay from an RFLP probe linked to resistance to cereal cyst nematode in wheat

The RFLP locus Xglk605 identified by the probe Tag605 maps to a proximal position on the long arm of wheat chromosome 2B about 7 cM away from a gene conditioning resistance to cereal cyst nematode in the wheat line AUS10894. The clone Tag605 was partially sequenced and the PCR primer set AWP1 was designed. The 292-bp product, which showed no polymorphism between varieties, was cloned and sequenced. A single base difference was found in the sequence of the AWP1 products amplified and cloned from the wheat lines AUS10894 and 'Spear'. PCR primers were designed with 3′ termini that corresponded to the two alleles. A dual-PCR system was developed in which the primer sets AWP2 and AWP3 produced allele-specific amplification. The concentration of the oligonucleotide primers and the sequence of the primer–template mismatches were critical to the success of discriminatory allele amplification. Key words : Triticum aestivum, STS, cereal cyst nematode, RFLP.