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 US barley varieties. This leaves barley production vulnerable if challenged by this highly virulent race of wheat stem rust. We are currently characterizing this important resistance mechanism and have also identified a novel source of TTKSK adult plant resistance, designated RpgSw645, that will be pyramided with the rpg4/Rpg5 locus in elite malting barley lines adapted to the Midwest growing region.
Previous characterization of the rpg4-mediated resistance against the wheat stem rust races including TTKSK identified three genes required for resistance including the rye stem rust resistance gene Rpg5, a second unrelated NBS-LRR gene (HvRga1) and an actin depolymerization factor (HvAdf3) (Wang et al., 2013, MPMI 26(4)/407-18). Alleles of the three genes required for resistance were sequenced and it was determined that polymorphisms in the Rpg5 gene resulting in non-functional proteins are diagnostic of functional rpg4-mediated resistance (Arora et al., Phytopathology, accepted). Thus, the PCR probes specific to the functional Rpg5 alleles are being utilized in the MAS strategy to pyramid resistance in the elite malting barley lines. The three tightly linked genes at the rpg4/Rpg5 locus also act in cooperation with a second tightly linked locus to confer wheat stem rust resistance, but the second locus containing the Rme1 gene is not required for the rye stem rust resistance (in FY12 this gene was referred to as Rrf1, but we have since changed the nomenclature to rpg4 modifier element 1 (Rme1)). This data suggests that although the two pathogens elicit the resistance reaction through a common R-gene (Rpg5) the signaling pathways are somewhat distinct. The distal Rme1 locus has been sequenced using our recently acquired Ion Torrent Personal Genomics Machine (PGM) and the most obvious candidate Rme1 gene was a heat shock protein 70 (HvHSP70.1). In FY12 we reported that the HvHSP70.1 gene was eliminated by candidacy via allele analysis but our recent sequencing revealed a 5’ exon that we missed in the initial low pass sequencing, thus further allele analysis is being performed on the full-length transcript. In FY12 we reported the genetic analysis showing 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 are still conducting the functional analysis studies to determine if HvPP2C inhibits phosphorylation-signaling cascades initiated by the Rpg5 kinase domain.
We are utilizing the Rpg5 specific molecular markers to track rpg4-mediated resistance for the deployment of the rpg4 resistance complex. We were previously utilizing flanking markers diagnostic of the transfer of the entire rpg4/Rpg5/Rme1 (rRR) locus but our recent allele analysis shows that the only polymorphic gene at the locus is the Rpg5 gene, thus we are now only utilizing this marker to track the introgression of the locus. We will use the markers to identify homozygous rRR positive BC5 F2 backcross lines this year. MAS 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 of Dr. Rich Horsley at NDSU. The two-rowed (Harrington X Q21861 #1 BC4F3) and six-rowed (Morex X Q21861 #28 BC4F3) lines with genetically defined rRR regions were used as the donor parents in the initial crosses. The BCnF1 seed from each round of back crossing were grown in the greenhouse and allowed to self. The BCnF2 seed were collected and 20 BCnF2 plants from each backcross were genotyped with the LRK1 markers (Rpg5+ specific markers) and several homozygous rRR positive BCnF2 plants were identified. The homozygous lines were backcrossed to the respective elite susceptible parent and we have continued this backcrossing scheme and are currently growing the BC5F1 generation in the field and the BC5F2 plants will be screened with our diagnostic molecular markers during the 2013 greenhouse season and sent to Kenya for screening in the off season Ug99 nursery. The research has had an impact on the deployment of Ug99 resistance into elite Midwestern malting barley germplasm and the Ug99 resistant prebreeding lines will be provided or made available to the regional barley breeders.
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 initially sent the population to the nursery in Kenya to be evaluated in September 2012. However, the nursery had very low disease pressure and did not provide robust phenotyping data. We resent the population to be evaluated in the off-season nursery in May 2013 with susceptible wheat and barley spreader rows between each entry. We have robust phenotyping data from this nursery. We also acquired an Ion Torrent PGM next generation sequencer in December of 2012 and are utilizing it for genotype-by-sequencing. We have developed a 384 SNP marker panel utilizing the 9K illumina data from the T3 website (http://triticeaetoolbox.org ) that will allow us to rapidly genotype the population at a much reduced cost. We are currently developing the genetic map to characterize the resistance and develop molecular markers diagnostic of the RpgSw645 gene/s to facilitate the pyramiding of this locus or loci with the rRR locus. The three way crosses have been made and we should have the molecular marker/s for RpgSw645 by September. In the greenhouse in 2013 we will begin identifying lines homozygous for rRR and RpgSw645 and start backcrossing to the recurrent elite parents to BC5.
The applied research funded by this SCA has facilitated the deployment of Ug99 resistance into US 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.