Marker characterisation and validation in breeding programs for rust resistance in sunflowers, Helianthus annuus L.

The purpose of the research described in this thesis was to identify and developnmolecular markers linked to specific genes conferring resistance to the sunflowernfungal pathogen, Puccinia helianthi Schw., to facilitate the production of simflowerngermplasm with pyramided rust resistance genes. To achieve this goal, the researchnrequired the identification of the molecular markers, localisation of the markers to angenetic linkage map to understand the genetic interactions between the genes ofninterest, and to evaluate the markers in a gene pyramiding situation.n F2 populations, segregating for resistance to specific races of simflower rust, werensupplied in which the genetics of the resistance had been characterised. In most cases,nresistance was inherited in a single dominant fashion. Arbitrary primed PCRntechnology (RAPDs and DAFs), together with a pooling strategy (bulked segregantnanalysis), identified 16 DNA markers linked to nine specific rust resistance genes ofnsunflower. Most of the markers were closely linked (l5.0 cM) to the respective rustnresistance genes, while other markers were only associated at a distance of g15 cM.n Seven of the RAPD loci closely linked to the rust resistance genes were converted intonSCAR (Sequence Characterised Amplified Region) loci: SCTO6950, SCX20660,nSCO4950, SCX10400, SCP04300, SCR111500, and SCL20400, representing the rustnresistance genes R1, RAdv, R4/RP1, R2, R5 and RSx53 respectively. Dominantnpolymorphisms were detected for the majority of the SCAR loci amplified. Only thenSCAR markers SCOO4950 and SCL20400 revealed different sized fragments in eachnparent to give co-dominant marker loci.n Co-localisation of the molecular markers to an existing genetic linkage map ofnsunflower demonstrated that two rust resistance gene clusters, on independent linkagengroups, exist: (1) Sunflower linkage group 8 containing the genes R1, R2, and RAH52;nand (2) Sunflower linkage group 13 containing the genes R4, RP1, RAdv, as well asnadditional genes not yet mapped but located to this region by their associations withnother genes within this cluster.n Consistent amplification of the R1 gene marker in lines not previously thought toncontain the R1 gene suggested evidence of a repeat sequence randomly distributednthroughout the sunflower genome, while comparison of the 660bp RAdv markernsequence with known sequences from a gene database, identified significant aminonacid similarity between the marker sequence and the conserved motif sequences,n'CX2CX4HX5C' and 'VLFDSGA', commonly represented in refrofransposons.nEvidence of sequence duplication and the presence of a transposable elementnassociated with rust resistance provides important information about the genomicnregions that contain the resistance genes and the surrounding areas.n Evaluation of the SCAR markers in a wide range of sunflower lines, includingnbreeding lines, rust differentials, inbreds, hybrid cultivars and wild accessions, provednthat the SCARs were successful at detecting individual rust resistance loci. Few linesncontained resistance genes at the R2 and R5 loci, indicating limited use of these lines innbreeding programs even though the resistance exhibited by these genes is veryneffective to the current spectrum of virulent rust races. In contrast, the R4 locus wasnwidely evident in the large selection of germplasm screened, supporting the widenspread usage of this locus in commercial hybrids in Australia in the past decade.n The reliability of the SCAR markers for detecting individual resistance loci withinngene pyramiding experiments was investigated using five crosses, containing differentnpairwise combinations of the rust resistance genes, RAdv, R2, R4, R5 and RSx53, at the F1nstage and in two two-gene crosses (R4//RAdv, and RAdv//RSx53) nat F2 stage. In eachnexperiment, the markers were successful. The markers failed to amplify in only a fewnof the F1 and F2 individuals screened. The dominant nature of most of the SCARnmarkers will limit their usefulness in the gene pyramiding program due to theirninability to differentiate homozygous resistant individuals from heterozygousnindividuals. Further work to identify co-dominant markers, such as SSRs or RFLPsnwill help alleviate this situation.n Research in this thesis contributes a library of molecular markers linked to importantnrust resistance genes, a valuable resource that can be integrated into a sunflower genenpyramiding program to facilitate selection of lines with specific genes. Additionalninformation regarding the genetic linkages between the rust resistance genes, sequencenduplications and refrofransposons located around rust resistance loci contributentowards a better understanding of the possible genetic makeup and evolution of rustnresistance.nnn