Location: Cereal Disease Lab2020 Annual Report
1a. Objectives (from AD-416):
Objective 1: Monitor, collect, and characterize U.S. cereal rust pathogens. Sub-objective 1.A. Monitor, collect, and characterize cereal rust pathogen populations in the U.S. for virulence to rust resistance genes in current cultivars. Sub-objective 1.B. Determine levels of genetic variation in Puccinia triticina, P. graminis and P. coronata populations. Sub-objective 1.C. Refine phylogenetics and systematics of P. graminis from Mahonia and other native Berberis spp. in North America. Objective 2: Further develop genomic resources of cereal rust pathogens and identify fungal genes involved in pathogenicity and development. Sub-objective 2.A. Identify effectors of P. graminis f. sp. tritici involved in fungal pathogenicity and host resistance. Sub-objective 2.B. Develop genomic resources and tools for more detailed analysis of P. coronata. Objective 3: Improve host resistance in cereal crops to rust pathogens through investigations in sources and genetics of rust resistance, characterization of various germplasm, and incorporation into adapted germplasm. Sub-objective 3.A. Evaluate wheat, oat and barley germplasm from U.S. breeding programs for rust resistance. Sub-objective 3.B. Identify and characterize new sources of rust resistance in wheat, barley, and oat; and incorporate into adapted germplasm.
1b. Approach (from AD-416):
Cereal rust fungi (Puccinia coronata, P. graminis, and P. triticina) are dynamic leading to constant changes in the U.S. population and erosion of effective rust resistance in cereal crops. In addition, introduction of foreign isolates, such as Ug99, further threaten cereal production. Development of cereal cultivars with effective rust resistance and management strategies of these diseases depend on monitoring, collection, virulence phenotyping, and genotypic characterization of cereal rust pathogen populations. Rust resistant cereal germplasm will be selected by testing wheat, oat, and barley lines from breeding programs throughout the U.S. and other sources for resistance to these pathogens using the prevalent races, and races that have high virulence to rust resistance genes common in released cultivars and breeding lines. Testing with selected isolates of the cereal rust pathogens and host genetics studies will identify the rust resistance genes in breeding lines and germplasm. Advanced germplasm lines with combinations of rust resistance genes will be selected and released for further use in cultivar development. Rust fungi produce a large arsenal of effector proteins in order to infect and colonize the plant host. Genetic and genomic approaches will be used to identify and characterize effector genes from P. graminis and P. coronata.
3. Progress Report:
In the third full year of the project, ARS scientists at St. Paul, Minnesota, made progress in the following objectives: Objective 1: Monitor, collect, and characterize U.S. cereal rust pathogens. Thirty-two races of Puccinia triticina (P. triticina) were identified in samples that were sent to the Cereal Disease Laboratory (CDL). A total of 252 isolates were processed for race identification. Race MNPSD was most common of all isolates. MNPSD was found in soft red winter regions of the southeastern states, and Ohio Valley, in addition to the winter and spring wheat region of the Great Plains. MNPSD and closely related race MPPSD are virulent to the hard red winter wheat SY Monument, widely grown in Kansas and Nebraska. In addition, MNPSD and MPPSD are virulent to genes Lr24, Lr39 and Lr37 in many hard red winter cultivars. Race MBTNB was the most common race in the southeastern states and Ohio Valley. MBTNB is virulent to Lr11, present in the soft red winter wheat cultivars grown in these regions. Race TBBGS was the highest frequency in the hard-red spring region of the northern Great Plains. TBBGS is virulent to Lr21, in many spring wheat cultivars in this region, in addition to Lr39. Virulence to Lr24 and Lr39 are highest in the southern to mid Great Plains region. Virulence to Lr11 and Lr26 is highest in the southeastern states, and virulence to Lr18 was detected at low frequencies in all regions, but most common in the Ohio Valley region. Virulence to Lr2a and Lr21 was highest in Minnesota, South Dakota, and North Dakota. In Oklahoma, losses due to leaf rust were estimated to be 8%, with 3% losses in Texas and Kansas. Losses in other states were at 1% or less. Overall estimated losses in the United States due to leaf rust were 24 million bushels. A total of 120 isolates derived from 81 stem rust samples from wheat and barley were analyzed. Race QFCSC continues to be the dominant race in the region east of Rocky Mountains. Diverse races were identified in stem rust samples collected from barley from the Pacific Northwest, indicating presence of active sexual cycle of stem rust on the alternate host. Rye stem rust was identified from barley stem rust samples collected from California. Eight races of oat stem rust pathogen were identified from 17 samples collected across the United States. TGN continues to be the dominant race. Stem rust sentinel plots were established in southern Texas and southern California to monitor potential incursion of foreign origin that are highly virulent to U.S. wheat, including races in the Ug99 race group. A total of 274 international stem rust samples from 8 countries were analyzed and 433 isolates were derived. Approximately 170 races were detected from these samples. A wide range of novel and significant virulence combinations were detected from these samples. A total of 176 collections of Puccinia coronata (oat crown rust) from various regions of the United States were received. From that sample, 178 individual isolates were purified and tested on oat differential lines carrying various resistance genes. To date, we received 22 collections from various regions of the United States and are in process of characterizing the fungal population diversity. A total of 16 collections of the barley leaf rust pathogen (Puccinia hordei) were received between February and May of 2020. From 10 collections received in 2019, 9 isolates were derived. A total of 94 isolates of Puccinia triticina from different wheat growing regions of the United States collected in 2015-2018 were genotyped with SNP markers at 4,950 loci using the Sequence Based Genotyping technique. The SNP genotypes of these 94 isolates will be combined with genotypes of 94 isolates sequenced and analyzed to determine genotypic relationship between the predominant race groups in the United States. As part of the Delivering Genetic Gain in Wheat Project (DGGW), 29 isolates of P. triticina from Eretria and Jordan were tested for virulence to 20 lines of Thatcher wheat with single genes for leaf rust to designate their virulence phenotypes. The race phenotypes were communicated to DGGW staff. A total of 297 isolates and 503 field samples of Puccinia graminis f. sp. tritici were genotyped from 18 countries. Phylogenetic analysis showed that the isolates from the United States belong to a different lineage than the five previously defined clades (I -V) representing current phenotypic races from Africa, Central Asia, and Europe. United States isolates are sub-divided into 3 groups (VI-A, VI-B and VI-C). Isolates of the race QFCSC, currently the most dominant race in the Great Plains belongs to sub-clade VI-C. This lineage also contains isolates from Europe, Asia, Africa, and South America. Objective 2: Further develop genomic resources of cereal rust pathogens and identify fungal genes involved in pathogenicity and development. Work continued on collecting, purifying, and increasing isolates of P. graminis f. sp. tritici for developing a genetic map and mapping effector genes. Twenty additional F2s were increased and genotyped. In addition, 15 isolates from a natural sexual population in the Pacific Northwest were increased and genotyped. A total of 60 isolates (30 in 1990, 30 in 2015) of P. coronata (Pca) from the United States were sequenced for analysis of genetic diversity and possible sources of pathogenicity. Results indicate that the 2015 population is substantially more virulent than that of 1990. Additionally, there has been a drastic genetic shift in the population from 1990 to 2015. Given that effector-driven evolution plays an important role in gain-of-virulence, several approaches were taken to identify Pca effectors. Our study identified haustorial effector gene candidates that show signatures of selection between the 1990 and 2015 populations, as well as presence and absence variation in predicted effector genes. Notably, a genome-wide association study allowed for the identification of the effector candidate genes. Objective 3: Improve host resistance in cereal crops to rust pathogens through investigations in sources and genetics of rust resistance, characterization of various germplasm, and incorporation into adapted germplasm. Wheat breeding lines from the Uniform Eastern Soft Red Winter wheat nursery, the Uniform Eastern Soft Red Winter Wheat Nursery, the North Regional Performance Nursery, the Southern Regional Performance Nursery, and the Uniform Spring Wheat Nursery were tested for seedling leaf rust resistance with 11 leaf rust races. Leaf rust resistance gene postulations were made for the entries and results communicated to nursery coordinators. Spring wheat breeding lines from public and private breeding programs in Minnesota, South Dakota, North Dakota, and Canada were evaluated for leaf rust resistance in field plots. Results were communicated to respective breeding programs. A total of 1900 elite breeding lines from public and private wheat breeding programs in the United States were screened with 9 domestic and several selected foreign stem rust races at the seedling stage and with 5 domestic races in field stem rust nursery. Resistance genes, especially those effective against Ug99 and other foreign races with significant virulence combinations, were postulated. Data were promptly distributed to collaborators. A total of 450 genetic stocks from other ARS research groups were characterized. Over 370 oat breeding lines from different programs in the United States and Canada were evaluated for crown rust resistance in field plots. An additional 120 advanced oat breeding lines from the regional programs were analyzed for crown rust reaction. We are assessing effectiveness of several adult plant resistance genes in buckthorn for future use to pyramid and develop more durable resistant germplasm. Analysis of 9 different oat recombinant inbred line populations yielded several strong QTL loci explaining from 30% to 50% of phenotypic variance for adult plant crown rust resistance. Easy to use PCR based markers (KASP) have been developed flanking these significant regions. These markers are being further validated to assure their effectiveness for use in a marker-assisted selection scheme. A program to pyramid 4 of these regions is underway and derived lines are being evaluated to identify adult plant resistance genes that are additive in nature before release to the public. Double haploid populations of Linkert x LMPG6 and Linkert x Foremost were evaluated for leaf rust resistance for a third year. Field data from plots will be combined with seedling data from greenhouse tests to map the segregating leaf rust resistance genes using the Illumina 90K SNP wheat chip. Wheat with/without Sr9h-Sr28 linkage block were planted in trials by collaborators in South Dakota. Wheat with/without Sr15-Sr22 linkage block were increased for a generation in the greenhouse and planted as a field increase in the spring. Marker-assisted backcrossing facilitated the generation of fixed BC6F2:3 families with/without 2 different translocations with Sr59. Combining additional Sr genes on chromosome 2B (Sr36, Sr39, Sr40, Sr47, Sr193883) in the background of the homozygous ph1b mutant gene allowed for homoeologous recombination. Recurrent parents used for backcrossing were obtained from the University of Minnesota, South Dakota State University, North Dakota State University, and Montana State University. A total of 1275 wheat lines and 275 barley lines were assessed in Kenya and Ethiopia. Lines included candidate cultivars, advanced and preliminary breeding lines, and mapping populations from USDA-ARS, universities throughout the United States, and private breeding programs.
1. Population genomics of Puccinia triticina in the United States. Wheat leaf rust caused by Puccinia triticina (P. triticina) is an important disease of this crop. In a collection of P. triticina isolates, 188 in total, that were gathered between the years of 2011 to 2018 in the United States, 11 were genotyped using the Genotyping by Sequence technique. Isolates were analyzed by ARS researchers at Saint Paul, Minnesota, using single nucleotide polymorphism (SNP) markers across 4950 loci for a total of 9964 SNP markers. Preliminary results indicate that over 80% of isolates fall into two major groups, North American (NA) 3 and NA5 of SNP genotypes. Isolates in both groups are widely distributed across the wheat growing regions of the Great Plains where hard red winter and spring cultivars are grown and the eastern and southern regions of the United States where soft red winter wheat cultivars are grown. These isolates are virulent to some of the major plant resistance genes present in wheat cultivars grown in the region. Further analysis is needed to confirm the grouping of all isolates into SNP genotype groups followed by characterization of nucleotide diversity, heterozygosity, and similarities and differences in race phenotypes within and between isolates in each group. This analysis is critical in plant gene deployment strategies that mitigate the impact of this pathogen.
2. Virulence changes in Puccinia coronata f. sp. avenae (Pca). Pca populations from 1990 and 2015 showed a drastic shift to broader virulence and whole-genome sequencing demonstrated extensive genetic differentiation between these populations likely as a result of in situ evolution. Substantial genetic variation and linkage disequilibrium decay indicated a strong influence of sexual reproduction in these populations, but ARS researchers at Saint Paul, Minnesota, also observed evidence of long-distance migration within the United States and the expansion of some clonal lineages within a season. A genome wide analysis identified seven pathogen avirulence (Avr) loci associated with virulence phenotypes on 15 Pc resistance genes in oat and provided evidence that some groups of Pc genes recognize the same pathogen effectors. A selective sweep at a single Avr locus is associated with a shift to virulence on these resistance genes in the 2015 population. This analysis points to the need for identification of more plant disease resistance genes and their deployment in oat cultivars.
3. Unique new stem rust populations. ARS researchers located in Saint Paul, Minnesota identified a unique and highly diverse stem rust population in Spain through collaboration with the Global Rust Initiative. Analyses of a large number of isolates have revealed that this population has a large number of novel virulence combinations that are highly unique and potent. For instance, majority of the isolates possess greater virulence profiles than that of Ug99 races, attacking many resistance genes, such as Sr22, Sr33, Sr35, Sr47 and Sr50 being used in breeding for Ug99 resistance. These isolates also process virulence to Sr24 and Sr31, a rare and highly significant virulence combination that was previously known to occur in the Ug99 race group only. Detection of such potent rust populations indicate the continual need for vigilance as well as identification and deployment of new plant resistance genes to combat this pathogen.
4. Oat crown rust virulence in the United States has increased dramatically. ARS researchers at Saint Paul, Minnesota, have identified 60 oat crown rust isolates (30 isolates for 1990 and 30 isolates for 2015) from the United States indicating a significant increase in virulence of this pathogen over the time period. This increase has now made many of the previously used single gene seedling resistance ineffective. There has been a drastic genetic shift in the population from 1990 to 2015, both in terms of virulence and genetic makeup. Both populations show high genetic diversity and admixture, and low levels of differentiation at a regional level. There are reduced levels of linkage disequilibrium and clonality in regions with buckthorn. Utilizing the genomic data, a candidate for a fungal effector gene has been identified from this study displaying a presence/absence variation and evidence of selective sweep. This finding indicates the vulnerability of current oat varieties grown across the United States and the need for additional research to identify more resistance genes to combat this devastating disease.
5. Integration of linkage blocks of Ug99 resistance genes. ARS researchers at St. Paul, Minnesota, have combined multiple linked wheat stem rust resistance genes effective to Ug99 and backcrossed these linkage blocks into conventional wheat germplasm. Resistance genes Sr9h and Sr28 were combined on chromosome arm 2BL whereas genes Sr15 and Sr22 were combined on 7AL. The two linkage blocks were backcrossed into hard red spring wheat cultivars previously released by the University of Minnesota and South Dakota State University. The Ug99-resistance-enhanced lines have been used in the crossing blocks of these two breeding programs for the development of wheat cultivars for the United States with multiple Ug99-resistant genes.
6. Adult plant resistance gene pyramiding for oat crown rust. A total of 11 adult plant resistance (APR) loci have been identified in 9 segregating recombinant inbred oat populations. ARS researchers at St. Paul, Minnesota, have developed Kompetitive Allele-Specific PCR (KASP) markers for regions surrounding each APR loci. Four of these loci, located on oat Mrg6, Mrg11, Mrg20 and Mrg21 linkage maps, have been pyramided into oat cultivars for use as breeding germplasm.
5. Record of Any Impact of Maximized Teleworking Requirement:
Maximized telework hindered the ability to conduct a number of projects related to rust field surveys and sample collections, as well as screening and analysis of germplasm in the later part of the Spring greenhouse season and the extent and breadth of field experiments of 2020. Additionally, due to a shift in focus of various genome centers in response to COVID-19, testing and analysis of much of the molecular work was put on hold. Furthermore, field samples sent to ARS by collaborators has decreased significantly, which was a likely result of reduced travel requirement by their home institutions. Thus, we expect the impact of this maximized telework posture to be significant.
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