Final report for GNC17-245
The North-Central region is the top producer of oats in the United States. However, oat production in this region is constrained by crown rust disease caused by the fungus Puccinia coronata, the most devastating disease of oat worldwide. Epidemics in the recent years have caused production losses of up to 50%. This project aims to minimize these losses by developing new oat germplasm that are resistant to crown rust. As an initial step, crosses of resistant wild relative and susceptible cultivated parents were made to develop eight mapping populations to guide genetic mapping efforts. These populations will be evaluated in field plots for resistance, and SNP markers will be developed to map the locations of genes or loci contributing to this trait. Once candidate genes are identified from different populations, multiple parents will be crossed to pyramid these genes. When advanced, the pyramided lines and associated linked markers will be forwarded to breeders for inclusion in their oat varietal improvement programs. Learning outcomes include knowledge enhancement about sources of resistance and plant immune mechanisms accessible to plant pathologists and breeders. The evaluation plan for these outcomes will be based on the number of seminars or presentations given and number of reads/downloads and citations of published papers. Action outcomes will be the use of this project’s germplasm by breeders to develop new oat varieties and adoption of developed methodologies by members of our scientific community (plant geneticists and molecular biologists). The evaluation plan for the action outcomes will be the actual use of our germplasm in oat breeding programs, and number of reads/downloads and citations of published papers. The long term goal of this project is to increase the overall income of oat growers and boost the production of healthy oat with use of enhanced crown rust resistant varieties.
The learning outcomes from this project include: 1) development of new sources of multi-gene resistance for oat varietal improvement; and 2) information about these new resistance genes/quantitative trait loci (QTLs) and their inheritance pattern, all available to the scientific community for utilization and crop improvement. The target audience for this project is primarily the oat scientific community. This work aims to generate scientific results useful and valuable for the oat industry, particularly in the NCR. Findings from this project also can benefit production in other grains as adult plant resistance can be broad spectrum and the orthology among genes in cereals may allow crop translational applications.
The action outcomes include the use of germplasm generated from this project to breed new oat cultivars; adoption of methods and use of generated data to identify new sources of resistance and better understand the molecular genetic basis of adult plant resistance. Historically, oat cultivars only last a maximum of five years in the field before their resistance to crown rust is broken. The pathogen evolves rapidly and acquires new virulence traits over time. Therefore, there is a constant need to develop new varieties to avoid epidemics and severe economic losses. This project addresses the pre-breeding bottleneck, which is finding novel sources of resistance genes that are easy to cross with the existing cultivars. Ultimately, this project leads to more durable crown rust- resistant cultivars that growers can adopt, which would then help maintain the status of NCR as the top oat producer in the US.
The aim of this project is to identify and pyramid new resistance genes to manage oat crown rust, a prevalent disease in the North Central Region. From 2017 to 2020, I have screened eight mapping populations (OtanaA x CI4706-2, OtanaA x CI9416-2, OtanaA X CI1712-5, Otana X PI189733, OtanaD X PI260616-1, OtanaA X CI8000-4, OtanaA X PI266887-1, OtanaI X PI263412-1, and OtanaA X CI7035-1) together with selections from these populations in the Buckthorn Nursery in St. Paul, MN for crown rust resistance. Using a modified bulk segregant analysis approach (selective genotyping) I selected 12 to 24 highly resistant and 12 to 24 highly susceptible lines from each population. Genotyping of all eight mapping populations was conducted by sending DNA from the lines with extreme phenotypes for Oat SNP Chip genotyping in the USDA Genotyping Lab in Fargo, ND. The resistance phenotype was associated to regions (loci) of the oat genome by counting allele frequencies. These regions potentially contain new resistance alleles that can be used for oat breeding for crown rust resistance. I developed Kompetitive Allele-Specific Primers (KASP) markers for the identified loci for marker-assisted selection and determined the proportion of the phenotype explained by the markers. Further, I have conducted oat crosses to combine some APR loci together so that I can pyramid them into a single line and release it later as a breeding germplasm. To our knowledge, this is the first attempt to combine adult plant resistance genes for rust in oats. With the availability of a public oat reference genome, I was also able to deduce some of the genes that are present in the APR loci, which would help shed a light on how the resistance mechanism works in this pathosystem.
I have presented a poster of my study at the International Congress of Plant Pathology in Boston, MA (2018) and the International Society of Plant-Microbe Interactions Conference in Glasgow, Scotland (2019), which gave me opportunities to communicate my research to international audiences (see attached poster). I was also invited to a departmental seminar at Louisiana State University (2019) to talked about this project. Nazareno_IS-MPMI_Poster_Final
Based on results of the modified bulk segregant analysis, I found a total of 11 APR loci from the eight mapping populations. These loci are located in Mrg03, Mrg04, Mrg05, Mrg06, Mrg11, Mrg12, Mrg15, Mrg18, Mrg20, Mrg21, and Mrg23 linkage groups of the oat genome, and most of them have not been reported previously. I have developed six KASP markers for three QTLs in Mrg21, which can be used for marker-assisted selection and gene pyramiding. I used the same markers to genotype all individuals in the population and compute for the QTL effects using simple interval mapping and found that the three QTLs in Mrg21 explain 11 to 35% of the variation in the crown rust phenotype. The KASP markers for the other loci are still being developed. Meanwhile, to combine some of these APR loci together, I have started making oat crosses since 2017 with parents coming from CI8000-4, PI260616-1, and CI1712-5 mapping populations, together with MNBT1020 and MNBT1021, which are Minnesota oat lines with good crown rust resistance. The progenies from these crosses are already in F6/F7 generation and were tested in the Buckthorn nursery in summer 2020, where a number of lines showed moderate to high resistance to crown rust. In 2019 and 2020, additional parents from CI7035-1, PI266887-1, PI263412, PI189733, CI9416-2, and CI4706-2 populations were added and the progenies from these crosses are now in F3 and F1 generations. Genotyping the advanced lines will ultimately lead to the selection of oat lines with pyramided resistance. There is currently no single resistance gene that can withstand all races of crown rust in the field, so quantitative resistance, as the one that this project is exploiting is viewed to be the best way to achieve durable crown rust resistance. Compared to chemical control, genetic resistance is less costly and deemed to be more sustainable and environment-friendly. Additionally, using the recently-published oat genome, I performed a survey of genes that are closely linked to the markers for APR. Interestingly, I found a putative ortholog of the wheat Yr36 gene in one of the loci in Mrg21. This might suggest that one mechanism of resistance to crown rust is related to high temperature activation and senescence, with plants becoming more resistant in the adult stage and under warmer temperatures. Other genes that are in close proximity to Mrg21 KASP markers include receptor-like kinases, protein phosphatase inhibitor 2, mRNA decapping enzyme, cellulose synthase, universal stress protein, beta-galactosidase, adenine nucleotide transporter BTL1, and auxin transport protein BIG. Most of these genes have been shown by other studies to be involved in basal disease resistance and could be considered potential candidate genes for APR.
The beneficiary for this project is primarily the oat scientific community and later, the growers, when the oat germplasm is released. I have mapped several loci and the development of markers is still ongoing, but once finished, the germplasm should be available to oat breeders for cultivar improvement. Presenting these results in international meetings and seminars is therefore a great outcome for this project, which enabled sharing of the research outputs to scientists around the world. In the meetings, I have interacted with several scientists, researchers, and students from all over the world and talked about my research. I thought it was a great avenue to learn and share experiences with other people in my field. In the same way, presenting a seminar in another university contributed in promoting this project and allowed exchange of ideas. Further, the manuscript that I have written from this project, which would be submitted to Theoretical and Applied Genetics, would also contribute knowledge and help guide breeding efforts for crown rust resistance.
Educational & Outreach Activities
From 2017 to 2020, I have presented my research in the International Congress of Plant Pathology (2018) and the International Society of Plant-Microbe Interactions (IS-MPMI) meeting (2019). I also gave an invited seminar presentation in the Department of Plant Pathology at Louisiana State University in 2019. In addition, I have written a manuscript on mapping some APR loci, which will be submitted for publication this year. However, due to the impacts of the COVID-19 pandemic on travel and research, I was not able to present my research in any scientific conference in 2020.
Our primary output from this project would be the breeding germplasm that we will release to oat breeders as donor for crown rust resistance. Breeders can then use the germplasm for cultivar development, which would eventually be released and planted by growers as cultivars. Therefore, this project basically deals with pre-breeding oat for crown rust resistance. Though it does not have a direct benefit to growers right now, resistant cultivars developed from this project's germplasm will certainly help growers cope up with crown rust in the future. Once all markers for APR loci have been developed, we will be forwarding them to breeders together with the pyramided lines for use in oat improvement.
Oat production has decreased in the US in the past decades and crown rust contributed to the decline somehow. Because of crown rust and other factors (i.e. low profitability), growers are hesitant to plant oats and would rather plant corn or soybean. Since oat is considered as a "health" crop, fungicide spraying is not encouraged, particularly for organically-grown oats. This project aims to address that problem so that growers of oat, organic or not, would not take a lot of losses due to crown rust like the one that happened in Minnesota and South Dakota in 2014 when both states lost 50% and 35% of the yield, respectively, due to crown rust. With the potentially new resistance genes that we are discovering in this study, it would give us better chances in the future to mitigate the impacts of crown rust epidemics.
Our results indicate that there are more sources of crown rust resistance if we just screen and look into more germplasm. Our project aims to develop a sustainable way to manage crown rust and we still think that breeding for disease resistance by combining multiple resistance genes would be the best approach to combat this disease. The more new genes that we discover from resistant donors, the more we realize that there is still genetic diversity in oat that remains to be explored. We plan to exploit these untapped resources and use them for our own advantage to develop lines that are resilient to crown rust. We also realize that the pathogen is always changing, almost every year, as new races are being produced after completing its life cycle in buckthorn (alternate host). This always reminds us not to be complacent and to be careful in deploying resistance genes out in the field in the future.
The availability of a public oat reference genome is also a breakthrough in oat research. Using the genome and transcriptome assemblies, we would be able to pinpoint which genes are responsible for APR using fine mapping in the future. This will pave the way for cloning the very first resistance genes from oat and will give us insights to better understand the mechanism of oat APR resistance.