Renée Prins

Identification of adult plant resistance to stripe rust in the wheat cultivar Cappelle-Desprez

Following the appearance of stripe rust in South Africa in 1996, efforts have been made to identify new sources of durable resistance. The French cultivar Cappelle-Desprez has long been considered a source of durable, adult plant resistance (APR) to stripe rust. As Cappelle-Desprez contains the seedling resistance genes Yr3a and Yr4a, wheat lines were developed from which Yr3a and Yr4a had been removed, while selecting for Cappelle-Desprez derived APR effective against South African pathotypes of the stripe rust fungus, Puccinia striiformis f. sp. tritici. Line Yr16DH70, adapted to South African wheat growing conditions, was selected and crossed to the stripe rust susceptible cultivar Palmiet to develop a segregating recombinant inbred line mapping population. A major effect QTL, QYr.ufs-2A was identified on the short arm of chromosome 2A derived from Cappelle-Desprez, along with three QTL of smaller effect, QYr.ufs-2D, QYr.ufs-5B and QYr.ufs-6D. QYr.ufs-2D was located within a region on the short arm of chromosome 2D believed to be the location of the stripe rust resistance gene Yr16. An additional minor effect QTL, QYr.ufs-4B, was identified in the cv. Palmiet. An examination of individual RILs carrying single or combinations of each QTL indicated significant resistance effects when QYr.ufs-2A was combined with the three minor QTL from Cappelle-Desprez, and between QYr.ufs-2D and QYr.ufs-5B.

Histopathology and PR‐protein markers provide insight into adult plant resistance to stripe rust of wheat

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a serious disease of wheat. The spring wheat cultivar Kariega expresses complete adult plant resistance to stripe rust, whereas Avocet S is susceptible. In former studies, quantitative trait loci (QTL) analysis of doubled haploid lines derived from a Kariega x Avocet S cross revealed two major QTL (QYr.sgi-7D and QYr.sgi-2B.1) and two minor QTL (QYr.sgi-1A and QYr.sgi-4A.1) responsible for the adult resistance of Kariega in the field. Avocet S contains none of these QTL. In the present study, stripe rust development was compared, by means of fluorescence and confocal laser scanning microscopy, in flag leaves of Kariega, Avocet S and six doubled haploid (DH) lines, containing all four, none or one QTL. Depending on the QTL present, the infection types of the DH lines ranged from resistant to fully susceptible. No differences in fungal growth were observed during the first 5 days post inoculation (dpi), whereas the mean length of the fungal colonies started to differ at 6 dpi. Interestingly, MP 51 carrying QYr.sgi-7D responded with lignification to the fungal growth without restricting it, whereas MP 35 containing QYr.sgi-2B.1 did not show lignified host tissue, but fungal growth was restricted. RT PCR experiments with sequences of pathogenesis-related (PR) proteins resulted in a slightly stronger induction of PR 1, 2 and 5, known markers for the hypersensitive reaction, and peroxidases in MP 51, whereas a second band for chitinases was detected in MP 35 only.

Stem Rust Resistance in a Geographically Diverse Collection of Spring Wheat Lines Collected from Across Africa

Following the emergence of the Ug99 lineage of Puccinia graminis f. sp. tritici (Pgt) a collective international effort has been undertaken to identify new sources of wheat stem rust resistance effective against these races. Analyses were undertaken in a collection of wheat genotypes gathered from across Africa to identify stem rust resistance effective against the Pgt races found in Eastern and Southern Africa. The African wheat collection consisted of historic genotypes collected in Kenya, South Africa, Ethiopia, Sudan, Zambia, Morocco, and Tunisia, and current South African breeding lines. Both Bayesian cluster and principal coordinate analyses placed the wheat lines from Sudan in a distinct group, but indicated a degree of genetic relatedness among the other wheat lines despite originating from countries across Africa. Seedling screens with Pgt race PTKST, pedigree information and marker haplotype analysis confirmed the presence of Sr2, Sr36, Sr24, Sr31, and Lr34/Yr18/Sr57 in a number of the lines. A genome-wide association study (GWAS) undertaken with Diversiry Arrays Technology (DArT) and stem rust (Sr) gene associated markers and Stem Area Infected (SAI) and Reaction Type (RT) field phenotypes, collected from trials carried out across two seasons in Kenya in 2009 and in South Africa in 2011, identified 29 marker-trait associations (MTA). Three MTA were in common between SAI and RT, with the biggest effect MTA being found on chromosome 6AS. Two wheat lines, W1406 and W6979 that exhibited high levels of adult plant stem rust resistance were selected to generate bi-parental mapping populations. Only the MTA on chromosomes 6AS and 3BS, and the locus Lr34/Yr18/Sr57 were confirmed following QTL mapping. Additional stem rust resistance QTL, not detected by the GWAS, were found on chromosomes 2BS, 2DL, 3DL, and 4D.

Development of a Greenhouse Screening Method for Adult Plant Response in Wheat to Stem Rust

Screening for adult plant resistance in wheat to stem rust, caused by Puccinia graminis f. sp. tritici, is generally conducted in field plots. Although such evaluations are successful if managed properly, field ratings are time consuming, expensive, weather dependent, and open to inoculum of unwanted races or other confounding diseases. The objective of this study was to develop a dependable system of screening the response of adult plants to stem rust under greenhouse conditions. A comparison of inoculation methods and incubation environments showed that plants inoculated with urediniospores suspended in water, followed by a 24 h dew period in a plastic chamber constructed in a greenhouse, gave the most consistent results. Measurements of response type, stem rust severity, and frequency in follow-up experiments indicated that the most reliable infection was obtained when plants sprayed with 1.25 mg urediniospores per ml water were incubated in the plastic chamber. Using the optimized protocol, a Kariega × Avocet S doubled haploid population was inoculated with two P. graminis f. sp. tritici races. Depending on the race, composite interval mapping showed flag leaf infection type to be significantly influenced by regions on chromosomes 6A, 6D, and 7D. Stem rust severity and reaction type mapped to chromosomes 6D and/or 6A. The Lr34/Yr18/Sr57 gene derived from Kariega on chromosome 7D affected the rust response on flag leaves but not on stems of greenhouse-grown plants. This study showed that phenotyping and genetic analysis of especially major effect stem rust resistance in adult wheat plants is possible and reproducible under controlled conditions in a greenhouse.

Microsatellite-based DNA fingerprinting of selected grapevine cultivars

Cultivar identification by ampelography is often difficult and is sensitive to environmental conditions, thus it can be problematic to distinguish between closely related cultivars. DNA fingerprinting offers an alternative method that is not influenced by the environment and is relatively easy to perform. However, discriminating between closely related individuals can be problematic if inadequate or insufficient markers are used. Following the estimation of null allele frequencies, an initial set of 35 microsatellite markers was reduced to 20 to generate unique DNA fingerprints for the majority of 111 different grape cultivars and breeding lines. Molecular evidence was utilised to evaluate the accuracy of the reported pedigrees for several cultivars bred in South Africa (SA). The use of markers linked to known downy mildew and powdery mildew resistance loci (Rpv3 and Ren3) provided information regarding the frequency of these resistance loci in the breeding material analysed.

Identification and mapping of net form of net blotch resistance in South African barley

Net form of net blotch (NFNB) caused by the fungus Pyrenophora teres f. teres is an economically important foliar disease of barley (Hordeum vulgare) in southern and eastern Africa. Little attention has been given to disease resistance breeding, and knowledge about the presence of NFNB resistance in breeding lines is limited. Deploying resistance into varieties used in this region is important for future control of the disease. We have identified NFNB disease resistance in existing South African breeders’ lines and have mapped the resistance in line UVC8. Six different trials, three conducted in South Africa and another three in Australia, were used to identify resistance QTL. A major QTL was identified on chromosome 6H having a LOD score of 40.5 and 55% of the phenotypic variance explained. Kompetitive Allele Specific PCR (KASP™) markers were designed for this QTL region. These and microsatellite markers can now be used to routinely select for NFNB resistance.

Development of a low-resolution genome scan marker set for the acceleration of major gene or QTL mapping in wheat

The traditional approach to identify targeted quantitative trait loci (QTL) or genes in a mapping study is to develop partial to comprehensive genetic maps by using a fairly large number of molecular markers. This is a cumbersome and potentially expensive process in which the result is often good coverage of areas with no significant importance. Here we have developed a low-resolution genome scan marker set (LRGS) of 104 microsatellite (simple sequence repeat) markers to identify the geneor QTL-carrying chromosome(s) earlier in a mapping study using a very small number of markers. In doing so, cost and time are reduced. An indication of association or linkage of a marker with the trait of interest, often still below a statistically significant threshold, can then be further investigated by saturating only the putative targeted area with additional markers to produce a high-resolution map of the region potentially housing the gene/QTL. Secondly, the LRGS markers can be used in QTL mapping efforts to provide the necessary chromosome anchoring points for high-throughput marker types with often unknown chromosome positions. This approach, combined with an automated analysis platform, accelerates a mapping effort and reduces the cost associated with the molecular work.

Genome Targeted Introgression of Resistance to African Stem Rust from Aegilops sharonensis into Bread Wheat

A method was designed to target alien homoeologous introgression into a selected genome Stem rust resistant wheat lines were produced accordingly Axiom array DNA analysis confirmed the introgression into the targeted genome