Development of a SCAR Marker to Track Canola Resistance Against Blackleg Caused by Leptosphaeria maculans Pathogenicity Group 3.

Blackleg of rapeseed and canola (Brassica napus) is caused by various pathogenicity groups (PG) of Leptosphaeria maculans. The disease occurring in the Canadian prairies for the last two decades was caused by PG2 and was controlled by host resistance. PG3 and PG4 isolates have been found recently in Canada, but there is no resistance available against these pathogenicity groups in commercial Canadian varieties. This study sought to identify canola cultivars that could be used as sources of resistance to PG3 and to develop molecular markers for marker-assisted selection. Resistance to PG3 specifically was found in B. napus 'Dunkeld' and 'Quinta', while B. juncea 'Cutlass' and 'Domo' proved to be resistant to PG2, PG3, and PG4. A set of F2 progeny of 'Westar' (susceptible) × 'Dunkeld' was used to identify genetic markers linked to PG3 resistance. These markers were physically located on a BAC clone from B. rapa subsp. pekinensis containing a homolog to a serine threonine 20 (ste20)-like kinase in Arabidopsis thaliana. Thus, we have developed a sequence characterized amplified region (SCAR) marker available for marker-assisted selection in breeding canola for resistance against blackleg caused by L. maculans PG3. This work has received a provisional patent (serial # 60/977,933 - Oct. 5, 2007).

Genetic Diversity of Gibberella zeae Isolates from Manitoba.

Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight, one of the most important diseases of cereals in the Canadian prairies for the last decade. In 2002, 60 isolates of G. zeae were collected and single spored from naturally infected spikes of wheat from Carman and Winnipeg in Manitoba. These isolates were compared using vegetative compatibility analysis and polymerase chain reaction (PCR)-based sequence related amplified polymorphisms (SRAP). Sixteen vegetative compatibility groups (VCG) were found among the 50 isolates tested. Five VCGs were found in the two locations, five in Carman and six in Winnipeg. Eight SRAP primer pairs amplified 90 polymorphic DNA fragments from 60 isolates and identified 59 distinct haplotypes. Among seven pairs of isolates, each pair from a distinct spike, four had isolates with different VCGs and six comprised different SRAP haplotypes. Principal component analysis and UPGMA separated the dataset into two main groups, each with isolates from both locations. The analysis of molecular variance also revealed that 75 and 20% of the variance was associated with differences among individual isolates and varieties sampled, respectively. Geographic location was not a significant source of variation at P = 0.05 and accounted for only 4% of total variance. A low correlation between VCG and SRAP marker data was detected. This study showed that, although genetic diversity is high among G. zeae isolates, Carman and Winnipeg collections have a similar genetic makeup and are likely part of the same population. The significant proportion of variance accounted by the variety compared with the geographic origin of isolates suggests that seedborne inoculum might have contributed to the genetic diversity within the G. zeae collection under study.

An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens.

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca [Moench] Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the beta-glucuronidase (uidA) reporter gene, was used as binary vector. The highest frequency of transformation (15 transformed tissues g(-1) FW of treated embryogenic tissue) was obtained with 5-d-old tissues grown in liquid medium and co-cultivated with Agrobacterium for 2 d in the same medium but containing 50 microM acetosyringone. Recovery of kanamycin-resistant tissues was improved when tissues were first grown for 10 d on a timentin-containing medium (400 mg l(-1)), to prevent bacterial overgrowth, before application of the selection pressure. After 6 weeks on kanamycin-selection medium, resistant tissues were obtained and showed stable uidA expression. The presence of the transgenes was demonstrated by PCR analysis and their integration into the genome was confirmed by Southern hybridization. Transgenic plants were regenerated from transformed tissues within 4 months after co-culture.

Fine-level genetic structure of white pine blister rust populations.

ABSTRACT The fine-level genetic structure of the white pine blister rust agent, Cronartium ribicola, was investigated by sampling multiple monokaryotic spermogonia directly on cankers in four eastern Canadian white pine (Pinus strobus) plantations and assessing genetic variability, using random amplified polymorphic DNA (RAPD) markers. Ninety-eight percent of the cankers surveyed contained a single DNA haplotype, suggesting spermogonia within cankers are the result of clonal reproduction. A single canker contained two haplotypes that were divided between the upper and lower parts of the canker, suggesting it represented two confluent cankers. In contrast, genotypic diversity was high among cankers. Thirty-seven haplotypes were found among forty-three cankers sampled, and an analysis of molecular variance indicated that 93% (P < 0.001) of the total genetic diversity was attributable to sampling of different cankers, strongly suggesting that multiple infections do not take place in the white pine blister rust pathosystem, i.e., a canker is the result of infection by a single genotype. This result is in contrast with the high level of genetic diversity previously reported among dikaryotic aecidia within cankers and is consistent with the hypothesis that variability in the aecidial stage is the result of outcrossing between resident spermogonia and alien spermatia. The genetic structure of the spermogonial stage, which is the vegetative extension of infection by basidiospores and, therefore, the indirect result of meiosis, was consistent with random mating; the observed genotypic diversity was not significantly different (P > 0.05) from the genotypic diversity expected under the assumption of panmixis. The results indicate that monokaryotic cankers can be genotyped by sampling a single unopened spermogonia per canker.