Comparative sequencing and SNP marker validation for oat stem rust resistance gene Pg6 in a diverse collection of Avena accessions.

KEY MESSAGE: Comparative sequence analysis was used to design a SNP marker that aided in the identification of new sources of oat stem rust resistance. New races of Puccinia graminis f. sp. avenae (Pga) threaten global oat production. An A. strigosa accession known to carry the broadly effective oat stem rust resistance gene, Pg6, was crossed with two susceptible A. strigosa accessions to generate 198 F2:3 families and 190 F5:6 RILs. The RIL population was used to determine that Pg6 was a single dominant gene located between 475 and 491 Mbp on diploid chromosome AA2 of the A. atlantica genome. This region was further refined by identifying SNPs associated with Pg6 resistance in a panel of previously sequenced A-genome accessions. Twenty-four markers were developed from SNPs that showed perfect association between the Pg6 phenotype and 11 sequenced Avena diploid accessions. These markers were validated in the RILs and F2:3 families, and the markers most closely linked with resistance were tested in a diverse panel of 253 accessions consisting of oat stem rust differentials, all available diploid Avena spp. accessions, and 41 A. vaviloviana accessions from the National Small Grains Collection. One SNP marker located at 483, 439, 497 bp on AA2, designated as AA2_483439497, was perfectly associated with the Pg6 phenotype in Avena strigosa diploids and was within several Kb of a resistance gene analog, RPP13. The marker results and seedling testing against Pga races DBD, KBD, TJS, and TQL enabled the postulation of Pg6 and potential new sources of resistance in the Avena panel. These results will be used to infer Pg6 presence in other germplasm collections and breeding programs and can assist with introgression, gene pyramiding, and cloning of Pg6.

Identification of Winter Habit Bread Wheat Landraces in the National Small Grains Collection with Resistance to Emerging Stem Rust Pathogen Variants.

Wheat stem rust caused by Puccinia graminis f. sp. tritici is a widespread and recurring threat to wheat production. Emerging P. graminis f. sp. tritici variants are rapidly overcoming major gene resistance deployed in wheat cultivars and new sources of race-nonspecific resistance are urgently needed. The National Small Grains Collection (NSGC) contains thousands of wheat landrace accessions that may harbor unique and broadly effective sources of resistance to emerging P. graminis f. sp. tritici variants. All NSGC available facultative and winter-habit bread wheat landraces were tested in a field nursery in St. Paul, Minnesota, against a bulk collection of six common U.S. P. graminis f. sp. tritici races. Infection response and severity data were collected on 9,192 landrace accessions at the soft-dough stage and resistant accessions were derived from single spikes. Derived accessions were tested in St. Paul a second time to confirm resistance and in a field nursery in Njoro, Kenya against emerging races of P. graminis f. sp. tritici with virulence to many known resistance genes including Sr24, Sr31, Sr38, and SrTmp. Accessions resistant in the St. Paul field were also tested at the seedling stage with up to 13 P. graminis f. sp. tritici races, including TTKSK and TKTTF, and with 19 molecular markers linked with known stem rust resistance genes or genes associated with modern breeding practices. Forty-five accessions were resistant in both U.S. and Kenya field nurseries and lacked alleles linked with known stem rust resistance genes. Accessions with either moderate or strong resistance in the U.S. and Kenya field nurseries and with novel seedling resistance will be prioritized for further study.

Genetic characterization and genome-wide association mapping for dwarf bunt resistance in bread wheat accessions from the USDA National Small Grains Collection.

KEY MESSAGE: Dwarf bunt-resistant bread wheat accessions and SNP markers associated with DB resistance identified in this study are valuable resources for characterization and deployment of DB resistance in bread wheat. Dwarf bunt (DB), caused by Tilletia controversa J.G. Kühn, can significantly reduce grain yield and quality on autumn-sown wheat in regions with prolonged snow cover. DB can be managed with the use of resistant cultivars. The objectives of the present study were to characterize DB resistance in a large set of bread wheat accessions from the National Small Grains Collection and use a genome-wide association study approach to identify genetic loci associated with DB resistance. A total of 292 accessions were selected using historical DB resistance data recorded across many trials and years in the Germplasm Resources Information Network (GRIN) and re-tested for DB resistance in replicated field nurseries in Logan, UT, in 2017, 2018, and 2019. Ninety-eight accessions were resistant with DB normalized incidence ≤ 10%, and twenty-eight of these were highly resistant with DB normalized incidence ≤ 1% in both GRIN and the field nurseries. Based on the presence of marker haplotypes of the four published dwarf bunt QTL on 6DS, 6DL, 7AL, and 7DS, highly resistant accessions identified in this study may provide novel resistance and should be further evaluated. This study validated one previously identified QTL on 6DS and identified an additional locus on 6DS. These loci explained 9-15% of the observed phenotypic variation. The resistant accessions and molecular markers identified in the present study may provide valuable resources for characterization and deployment of DB resistance in bread wheat.

Loci and candidate genes controlling root traits in wheat seedlings-a wheat root GWAS.

Two hundred one hexaploid wheat accessions, representing 200 years of selection and breeding history, were sampled from the National Small Grains Collection in Aberdeen, ID, and evaluated for five root traits at the seedling stage. A paper roll-supported hydroponic system was used for seedling growth. Replicated roots samples were analyzed by WinRHIZO. We observed accessions with nearly no branching and accessions with up to 132 cm of branching. Total seminal root length ranged from 70 to 248 cm, a 3.5-fold difference. Next-generation sequencing was used to produce single-nucleotide polymorphism (SNP) markers and genomic libraries that were aligned to the wheat reference genome IWGSCv1 and were called single-nucleotide polymorphism (SNP) markers. After filtering and imputation, a total of 20,881 polymorphic sites were used to perform association mapping in TASSEL. Gene annotations were conducted for identified marker-trait associations (MTAs) with - log10P > 3.5 (p value < 0.003). In total, we identified 63 MTAs with seven for seminal axis root length (SAR), 24 for branching (BR), four for total seminal root length (TSR), eight for root dry matter (RDM), and 20 for root diameter (RD). Putative proteins of interest that we identified include chalcone synthase, aquaporin, and chymotrypsin inhibitor for SAR, MYB transcription factor and peroxidase for BR, zinc fingers and amino acid transporters for RDM, and cinnamoyl-CoA reductase for RD. We evaluated the effects of height-reducing Rht alleles and the 1B/1R translocation event on root traits and found presence of the Rht-B1b allele decreased RDM, while presence of the Rht-D1b allele increased TSR and decreased RD.

New Sources of Adult Plant and Seedling Resistance to Puccinia coronata f. sp. avenae Identified among Avena sativa Accessions From the National Small Grains Collection.

Accessions of cultivated oat (Avena sativa L.) from the United States Department of Agriculture-Agricultural Research Service Small Grains Collection in Aberdeen, ID were characterized for adult plant resistance (APR) and seedling resistance to crown rust, caused by Puccinia coronata f. sp. avenae. Initially, 607 oat accessions with diverse geographic origins were evaluated in field tests in Baton Rouge, LA. Of those, 97 accessions were not fully susceptible and were tested in the field in St. Paul, MN against a diverse P. coronata f. sp. avenae population. Thirty-six accessions that had some level of resistance in both field tests and mean coefficients of infection of ≤20 were further evaluated for APR and seedling resistance. Among these, four accessions (PI 193040, PI 194201, PI 237090, and PI 247930) were resistant to eight P. coronata f. sp. avenae races as seedlings. Twenty-nine accessions had resistance to at least one of the P. coronata f. sp. avenae races. Three accessions (CIav 2272, CIav 3390, and PI 285583) were fully susceptible to all eight P. coronata f. sp. avenae races as seedlings. Further evaluation of the three seedling-susceptible accessions at the flag leaf stage in a growth chamber resulted in moderately susceptible to moderately resistant responses. The resistance sources presented here may contain genes not deployed in elite oat varieties, and may be useful for future crown rust resistance breeding. The adult and seedling resistance found in accessions of the cultivated oat species is especially valuable because it avoids problems associated with the transfer of genes from wild species to cultivated oat.

Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars.

Domesticated crops experience strong human-mediated selection aimed at developing high-yielding varieties adapted to local conditions. To detect regions of the wheat genome subject to selection during improvement, we developed a high-throughput array to interrogate 9,000 gene-associated single-nucleotide polymorphisms (SNP) in a worldwide sample of 2,994 accessions of hexaploid wheat including landraces and modern cultivars. Using a SNP-based diversity map we characterized the impact of crop improvement on genomic and geographic patterns of genetic diversity. We found evidence of a small population bottleneck and extensive use of ancestral variation often traceable to founders of cultivars from diverse geographic regions. Analyzing genetic differentiation among populations and the extent of haplotype sharing, we identified allelic variants subjected to selection during improvement. Selective sweeps were found around genes involved in the regulation of flowering time and phenology. An introgression of a wild relative-derived gene conferring resistance to a fungal pathogen was detected by haplotype-based analysis. Comparing selective sweeps identified in different populations, we show that selection likely acts on distinct targets or multiple functionally equivalent alleles in different portions of the geographic range of wheat. The majority of the selected alleles were present at low frequency in local populations, suggesting either weak selection pressure or temporal variation in the targets of directional selection during breeding probably associated with changing agricultural practices or environmental conditions. The developed SNP chip and map of genetic variation provide a resource for advancing wheat breeding and supporting future population genomic and genome-wide association studies in wheat.

Untapped Sources of Dual Resistance to Hessian Fly and Greenbug in Synthetic Hexaploid Wheats.

The Hessian fly (Hf) and greenbugs (Gb) are major pests of wheat, causing severe economic losses globally. Deploying resistant wheat is the most effective strategy for managing these destructive insects. However, the resistance is not effective against all Hf or Gb biotypes and can impose selection pressure on insects, resulting in the development of virulent biotypes. These challenges must be met through the discovery of new and novel sources of resistance to these pests. Synthetic Hexaploid Wheat (SHW)-developed cultivars are a rich source of resistance against a diverse array of pathogens and pests. In this study, 80 SHW lines were evaluated for their resistance to Hf and Gb under controlled environmental conditions. Of these, a total of 36 SHW lines showed resistance independently to Hf biotype L and Gb biotype E, while 27 lines showed combined resistance to both Hf and Gb. Further, a subset of 10 SHW lines showed resistance to additional Hf biotypes, Great Plains and vH13. The identification of SHW lines resistant to multiple insects and biotypes offers an invaluable resource to breeders who are looking to stack resistance traits to develop elite cultivars as a strategy to alleviate economic impacts upon global wheat production.

A Novel Mutator-Like Transposable Elements With Unusual Structure and Recent Transpositions in Barley (Hordeum vulgare).

Mutator-like transposable elements (MULEs) represent a unique superfamily of DNA transposons as they can capture host genes and cause higher frequency of mutations in some eukaryotes. Despite their essential roles in plant evolution and functional genomics, MULEs are not fully understood yet in many important crops including barley (Hordeum vulgare). In this study, we analyzed the barley genome and identified a new mutator transposon Hvu_Abermu. This transposon is present at extremely high copy number in barley and shows unusual structure as it contains three open reading frames (ORFs) including one ORF (ORF1) encoding mutator transposase protein and one ORF (ORFR) showing opposite transcriptional orientation. We identified homologous sequences of Hvu_Abermu in both monocots and dicots and grouped them into a large mutator family named Abermu. Abermu transposons from different species share significant sequence identity, but they exhibit distinct sequence structures. Unlike the transposase proteins which are highly conserved between Abermu transposons from different organisms, the ORFR-encoded proteins are quite different from distant species. Phylogenetic analysis indicated that Abermu transposons shared closer evolutionary relationships with the maize MuDR transposon than other reported MULEs. We also found phylogenetic incongruence for the Abermu transposons identified in rice and its wild species implying the possibility of horizontal transfer of transposon. Further comparison indicated that over 200 barley genes contain Abermu-related sequences. We analyzed the barley pan genomes and detected polymorphic Hvu_Abermu transposons between the sequenced 23 wild and cultivated barley genomes. Our efforts identified a novel mutator transposon and revealed its recent transposition activity, which may help to develop genetic tools for barley and other crops.

Field resistance to wheat stem rust in durum wheat accessions deposited at the USDA National Small Grains Collection.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a re-emerging disease, posing a significant threat to durum wheat production worldwide. The limited number of stem rust resistance genes in modern cultivars compels us to identify and incorporate new effective genes in durum wheat breeding programs. We evaluated 8,245 spring durum wheat accessions deposited at the USDA National Small Grains Collection (NSGC) for resistance in field stem rust nurseries in Debre Zeit, Ethiopia and St. Paul, MN (USA). A higher level of disease development was observed at the Debre Zeit nursery compared with St. Paul, and the effective alleles of Sr13 in this nursery did not display the level of resistance observed at the St. Paul nursery. Four hundred and ninety-one (∽6%) accessions exhibited resistant to moderately susceptible responses after three field evaluations at Debre Zeit and two at St. Paul. Nearly 70% of these accessions originated from Ethiopia, Mexico, Egypt, and USA. Eight additional countries, namely Portugal, Turkey, Italy, Canada, Chile, Australia, Syria, and Tunisia contributed to 19% of the resistant to moderately susceptible entries. Among the 491 resistant to moderately susceptible accessions, 53.8% (n = 265) were landraces, and 28.4% (n = 139) and 11.4% (n = 55) were breeding lines and cultivars, respectively. Breeding lines and cultivars displayed a higher level and frequency of resistance than the landraces. We concluded that a large number of durum wheat accessions from diverse origins deposited at the NSGC can be exploited for diversifying and improving stem rust resistance in wheat.

Evaluation of Genetic Diversity and Host Resistance to Stem Rust in USDA NSGC Durum Wheat Accessions.

The USDA-ARS National Small Grains Collection (NSGC) maintains germplasm representing global diversity of small grains and their wild relatives. To evaluate the utility of the NSGC durum wheat ( L. ssp. ) accessions, we assessed genetic diversity and linkage disequilibrium (LD) patterns in a durum core subset containing 429 lines with spring growth habit originating from 64 countries worldwide. Genetic diversity estimated using wheat single-nucleotide polymorphism (SNP) markers showed considerable diversity captured in this collection. Average LD decayed over a genetic distance to within 3 cM at = 0.2, with a fast LD decay for markers linked at >5 cM. We evaluated accessions for resistance to wheat stem rust, caused by a fungal pathogen, Pers. Pers. f. sp. Eriks. and E. Henn (), using races from both eastern Africa and North America, at seedling and adult plant stages. Five accessions were identified as resistant to all stem rust pathogen races evaluated. Genome-wide association analysis detected 17 significant associations at the seedling stage with nine likely corresponding to , , and and the remaining potentially being novel genes located on six chromosomes. A higher frequency of resistant accessions was found at the adult plant stage than at the seedling stage. However, few significant associations were detected possibly a result of strong G × E interactions not properly accounted for in the mixed model. Nonetheless, the resistant accessions identified in this study should provide wheat breeders with valuable resources for improving stem rust resistance.

Population Structure and Genotype-Phenotype Associations in a Collection of Oat Landraces and Historic Cultivars.

Population structure and genetic architecture of phenotypic traits in oat (Avena sativa L.) remain relatively under-researched compared to other small grain species. This study explores the historic context of current elite germplasm, including phenotypic and genetic characterization, with a particular focus on identifying under-utilized areas. A diverse panel of cultivated oat accessions was assembled from the USDA National Small Grains Collection to represent a gene pool relatively unaffected by twentieth century breeding activity and unlikely to have been included in recent molecular studies. The panel was genotyped using an oat iSelect 6K beadchip SNP array. The final dataset included 759 unique individuals and 2,715 polymorphic markers. Some population structure was apparent, with the first three principal components accounting for 38.8% of variation and 73% of individuals belonging to one of three clusters. One cluster with high genetic distinctness appears to have been largely overlooked in twentieth century breeding. Classification and phenotype data provided by the Germplasm Resources Information Network were evaluated for their relationship to population structure. Of the structuring variables evaluated, improvement status (cultivar or landrace) was relatively unimportant, indicating that landraces and cultivars included in the panel were all sampled from a similar underlying population. Instead, lemma color and region of origin showed the strongest explanatory power. An exploratory association mapping study of the panel using a subset of 2,588 mapped markers generated novel indications of genomic regions associated with awn frequency, kernels per spikelet, lemma color, and panicle type. Further results supported previous findings of loci associated with barley yellow dwarf virus tolerance, crown rust (caused by Puccinia coronata f. sp. avenae) resistance, days to anthesis, and growth habit (winter/spring). In addition, two novel loci were identified for crown rust resistance.

The USDA barley core collection: genetic diversity, population structure, and potential for genome-wide association studies.

New sources of genetic diversity must be incorporated into plant breeding programs if they are to continue increasing grain yield and quality, and tolerance to abiotic and biotic stresses. Germplasm collections provide a source of genetic and phenotypic diversity, but characterization of these resources is required to increase their utility for breeding programs. We used a barley SNP iSelect platform with 7,842 SNPs to genotype 2,417 barley accessions sampled from the USDA National Small Grains Collection of 33,176 accessions. Most of the accessions in this core collection are categorized as landraces or cultivars/breeding lines and were obtained from more than 100 countries. Both STRUCTURE and principal component analysis identified five major subpopulations within the core collection, mainly differentiated by geographical origin and spike row number (an inflorescence architecture trait). Different patterns of linkage disequilibrium (LD) were found across the barley genome and many regions of high LD contained traits involved in domestication and breeding selection. The genotype data were used to define 'mini-core' sets of accessions capturing the majority of the allelic diversity present in the core collection. These 'mini-core' sets can be used for evaluating traits that are difficult or expensive to score. Genome-wide association studies (GWAS) of 'hull cover', 'spike row number', and 'heading date' demonstrate the utility of the core collection for locating genetic factors determining important phenotypes. The GWAS results were referenced to a new barley consensus map containing 5,665 SNPs. Our results demonstrate that GWAS and high-density SNP genotyping are effective tools for plant breeders interested in accessing genetic diversity in large germplasm collections.