1a.Objectives (from AD-416):
The primary objective of this cooperative research project is to rapidly deploy the rpg4/Rpg5/Rrf1 (rRR) resistance complex into agronomically advanced Midwestern malting barley cultivars.
1b.Approach (from AD-416):
The Rpg5 locus and the region around ARD199 will be sequenced to quickly identify SNPs diagnostic for resistant and susceptible genotypes. A set of molecular markers flanking the rRR resistance complex will be developed so genotyping for the region can be performed by standard PCR or by using high-throughput technology. Marker-assisted selection (using these molecular markers) will then be used to rapidly deploy the rRR resistance complex in barley germplasm. Specifically, resistance will be introduced into the Ug99 susceptible two-rowed (Pinnacle and Conlon) and six-rowed (ND25160) elite malting barley lines. Preliminary disease screening will be conducted in Fargo, with followup screening each year at the BL3 facility in St. Paul and at the stem rust nursery in Kenya.
The cloned rpg4/Rpg5 locus was until recently the only known source of resistance to the virulent stem rust race TTKSK (Ug99) in barley and has not been introduced into U.S. barley varieties leaving production vulnerable if challenged by this race. We are characterizing this important resistance mechanism and have also identified a second source of TTKSK adult plant resistance that will be pyramided with rpg4 in elite malting barley lines adapted to the Midwest growing region.
The thorough characterization of three high-resolution bi-parental populations determined that two tightly linked loci are required for rpg4-mediated resistance against the wheat stem rust races including TTKSK. In the past year we have made exciting new discoveries towards the understanding of the genetic interactions underlying rpg4-mediated resistance. We used post transcriptional gene silencing to show that each gene present at the Rpg5 rye stem rust resistance locus including the Rpg5 gene, a second unrelated NBS-LRR gene (HvRga1) and an actin depolymerization factor (HvAdf3) are all required together for resistance against race QCCJ and presumably TTKSK. Preliminary transcript analysis results have indicated that Rpg5 is upregulated 12 hours post infection with wheat stem rust, indicating that the resistance mechanism is prehaustorially activated. The three tightly linked genes at the Rpg5 locus also act in cooperation with a second tightly linked locus to confer wheat stem rust resistance but the second locus containing the Rrf1 gene is not required for rye stem rust resistance. The most obvious candidate Rrf1 gene was a heat shock protein 70 (HvHSP70.1), but we have used allele and transcript analysis to eliminate HvHSP70.1 as the candidate Rrf1 gene. Further analysis of the region suggests that a second actin depolymerization factor (HvAdf3) is our new Rrf1 candidate gene and validation is underway. We have also used genetic analysis to show that a protein phosphatase 2C gene (HvPP2C) present at the locus acts as a dominant susceptibility factor determining the recessive nature of rpg4-mediated resistance. We have antibodies that cross react with HvPP2C and are currently performing functional analysis studies to determine if HvPP2C inhibits phosphorylation signaling cascades initiated by the Rpg5 kinase domain.
We previously designed molecular markers flanking the genes required for rpg4-mediated resistance and have been using these markers for the rapid deployment of the rpg4 resistance complex. The flanking markers are diagnostic of the transfer of the entire rpg4/Rpg5/Rrf1 (rRR) locus and we will use the markers to identify homozygous rRR positive BC3 F2 backcross lines this year. Marker-assisted selection is being utilized to rapidly deploy the rRR resistance complex into the Ug99 susceptible two-rowed (Pinnacle and Conlon) and six-rowed (ND25160) elite malting barley lines from the breeding program at NDSU. The two-rowed (Harrington X Q21861 #1 BC4F3) and six-rowed (Morex X Q21861 #28 BC4F3) near isogenic lines with genetically defined rRR regions were used as the donor parents and the initial crosses were made in the field in 2010. The F1 seed from redundant crosses were grown in the greenhouse and allowed to self. The F2 seed was collected and 20 F2 plants from 4 F1s of each cross were genotyped with the LRK1 markers and several homozygous rRR positive F2 plants were identified. The homozygous lines were backcrossed to the respective susceptible parent and we have continued this backcrossing scheme and are currently growing the BC3F1 generation in the field and the BC3F2 plants will be screened with our diagnostic molecular markers during the 2012 greenhouse season. We will continue the backcrossing to BC5 then these breeding lines will be provided to regional barley breeders.
This research has had an immediate impact on the rapid deployment of Ug99 resistance into elite two and six-rowed malting barleys adapted to the Midwest. In 2011, we began pyramiding the rRR resistance locus with a new source of adult plant resistance identified in an unimproved Swiss barley landrace designated Sw645. We have developed a recombinant inbred population of the cross Sw645/Harrington and have sent the population to the nursery in Kenya to be evaluated in September 2012. We will also use the Illumina 9K barley chip to genotype the population in order to map the gene to develop molecular markers diagnostic of the Rpg-Sw645 gene to facilitate the pyramiding of this locus with the rRR locus. The three way crosses have been made and we plan to develop the molecular markers for the new gene in 2012.
The applied research funded by this SCA has facilitated the rapid deployment of Ug99 resistance into U.S. barley varieties. The basic research we are conducting to characterize the molecular mechanisms underlying the rpg4/Rpg5 locus will fill current gaps in the knowledge of disease resistance mechanisms. The elucidation of the molecular interactions underlying this locus have also provided knowledge of recessive and temperature dependent resistance and has affirmed the emerging theme that stem rust resistance in barley is a form of pre-haustorial resistance.