Molecular genetic studies in Fragaria species : agrobacterium-mediated transformation and fine mapping of the Phytophthora fragariae resistance gene Rpfl.

The fungus Phytophthora fragariae, is able to cause red stele root rot in the strawberry. Symptoms of the disease is discolouration of the stele of the roots, rotting away of the infected roots, dwarfism, wilting, and finally plant death. Chemical control of red stele with soil fumigants reduce the inoculum potential of the fungus in the soil but does not eliminate it completely. Resistant cultivars are looked upon more favourably by growers since the consumer and environmentalist are demanding the decrease of chemical treatments in cultivation. It has previously been demonstrated that short 10 basepair length primers of arbitrary nucleotide sequence can be used to amplify segments of genomic DNA from a variety of species using PCR technology. Polymorphisms among the amplification products are typically detected as DNA segments which amplify from one parent but not from the other and can then be used to construct genetic maps. However, the dominant nature of RAPD markers does not allow the complete determination of between the two classes of homo- and heterzygous genotypes in the segregating population. A molecular map of the diploid strawberry, Fragaria vesca, that consisted of five distinct linkage groups was developed. Two isozymes (PGI and SKDH) and one morphological marker (red fruit colour) were also placed on the map. The red fruit colour locus was shown to be tightly linked to both the SKDH locus and to two RAPD markers. Gene tags identified at the diploid level may help selection procedures for the commercial strawberry. Mapping of the commercial strawberry may possibly be made easier since the genetic homology between the diploid and octoploid species may extend to the linear order of genes on the chromosomes. Classical breeding methods have already been able to incorporate resistances to various diseases in the commercial strawberry. The ability to pyramid resistance genes and accurately screen them is laborious, expensive, and at times problematic, therefore using indirect selection with molecular markers was studied. Recently, a strawberry gene-for-gene model was proposed for red stele resistance. In this study the Rpf1 gene, out of this model, was mapped using RAPD markers. Next, specific primers (SCARs) linked in coupling phase to the gene were developed and the markers were then assessed for their conservation in European and North American genotypes. We are the first to identify molecular markers linked with a commercially interesting trait in the strawberry. We could do so due to the disomic behaviour of the Rpf1 region and the reliable classification into resistance and susceptibility of the individuals of the progeny. Seven RAPD markers for Rpf1 all had distinct presence or absence polymorphisms between the paired bulks. These markers linked to Rpf1 were mapped to within 1.7 -13.9 cM of the gene. RAPD markers are difficult to reproduce and therefore, they preferentially should be converted into sequence characterised amplified region (SCAR) markers. An advantage of SCAR markers is their potential for quick and robust assessment. A SCAR marker was constructed based upon RAPD marker OPO-16C, linked to the susceptibility allele of the Rpf1 gene. Following the cloning and sequencing of this marker we were able to design SCAR primers specific to the alternate resistant allele. Two Rpf1 SCAR primer sets linked to this resistance allele were constructed. These SCAR-R1 primers, when tested on the original F 1 mapping population, amplified the resistant plants only, excluding that of the same recombinants that were detected with the OPO-16 markers. The SCAR markers mapped to the same location as the original RAPD OPO-16C. The molecular markers linked to the Rpf1 gene were shown to be conserved in most of the European and North American genotypes. The RAPDs and SCARs are also good indicators of crossing-over events occurring in the genotypes. Out of 86 genotypes assessed, only five nonconforming genotypes were observed using the RAPD and SCAR primers. This is close to the expected number, dependent on the markers distance from the gene. The linked markers to Rpf1 allowed us to trace this resistance gene by pedigree analysis. Many important breeding stock materials can accurately be assessed with these markers and integrated into a markerassisted breeding programme. These molecular markers may allow the ability to pyramid and screen for the Rpf1 resistance gene more efficiently than present conventional methods. The use of molecular markers for a breeding programme is limited by the number of plants from which DNA can be isolated. Therefore, a DNA mini- preparation method was developed with which a single individual can isolate over 200 samples/day. This DNA isolation technique eliminates time-consuming and non- critical steps such as extensive grinding, ethanol washes, and RNAse treatment. The ability to integrate useful genes directly into genotypes of horticultural interest is one of the final goals of many studies, therefore a reliable transformation and regeneration system needs to be available. The diploid strawberry was used as model system for this research prior to working with the octoploid strawberry due to the less complex nature of its genome. The diploid strawberry was successfully transformed using Agrobacterium- mediated transformation. Primary (RO generation) transformants were vegetatively propagated to produce five RO runner plants which were allowed to self-pollinate to produce the R1 generation seedlings. When RI generation seedlings were tested by GUS histochemical analysis, the segregation data fit a 15:1 ratio (0.5>p>0.25), indicating the independent segregation of two transgene insertion loci. To summarise, the diploid strawberry was first utilised as a model system to construct a molecular map prior to working with the commercial strawberry. In the octoploid strawberry a red stele disease resistance gene, Rpf1 was mapped with seven RAPD markers and highly specific SCAR markers were constructed to this gene. The RAPID and SCAR markers were assessed in 86 genotypes and were shown to be conserved in regard to the Rpf1 region. The diploid strawberry was used as a model system to develop a reliable transformation method to integrate genes back into the plant and stabily passed into the progeny plants. The research presented in this study has opened the door for further work of mapping other genes of interest in the strawberry to help breeders in the future and to reduce pesticide inputs in this crop.