Optimization of Ustilago nuda Inoculum Concentration for Screening Un8-Mediated Loose Smut Resistance in Barley Reveals a Resistance Reaction that Disrupts Seed Germination and Suggests a Role for Abscisic Acid in Disease Development.

Barley loose smut has been effectively controlled for decades through resistance conferred by the Un8 gene. However, evaluation of loose smut reaction using floret inoculation at the standard inoculum concentration is associated with the production of small, discolored seeds in Un8 carriers and susceptible genotypes. Interestingly, Un8 carriers also displayed significantly poorer germination than susceptible genotypes and produce short-lived seedlings following inoculation. To understand these observations, a Un8 carrier (TR11698) and susceptible non-Un8 carrier (CDC Austenson) were assessed for seed traits, Ustilago nuda biomass in the seed, infection rate, and phytohormone profile across a range of lower inoculum concentrations. At lower inoculum concentrations, seed appearance and weight improved in both genotypes, and infection rate increased in CDC Austenson. Pathogen load in the seed was similar in both genotypes and was positively correlated with the CDC Austenson infection rate. No infection was ever observed in TR11698. Significantly, germination rate improved in CDC Austenson, whereas the very low germination rate and short-lived seedlings remained associated with TR11698. It appears that poor seed appearance in both genotypes and low germination rate in the susceptible genotype can be improved by lowering the inoculum concentration. However, the very low germination rates and seedling death associated with the Un8 carrier TR11698 are indicative of Un8-mediated resistance to loose smut. Finally, profiling of 38 phytohormones revealed that larger seeds observed at some inoculum concentrations compared with mock inoculation had higher abscisic acid concentrations. This could represent a pathogen survival strategy by ensuring better growth of the host.

Identification of Putative SNP Markers Associated with Resistance to Egyptian Loose Smut Race(s) in Spring Barley.

Loose smut (LS) disease is a serious problem that affects barley yield. Breeding of resistant cultivars and identifying new genes controlling LS has received very little attention. Therefore, it is important to understand the genetic basis of LS control in order to genetically improve LS resistance. To address this challenge, a set of 57 highly diverse barley genotypes were inoculated with Egyptian loose smut race(s) and the infected seeds/plants were evaluated in two growing seasons. Loose smut resistance (%) was scored on each genotype. High genetic variation was found among all tested genotypes indicating considerable differences in LS resistance that can be used for breeding. The broad-sense heritability (H2) of LS (0.95) was found. Moreover, genotyping-by-sequencing (GBS) was performed on all genotypes and generated in 16,966 SNP markers which were used for genetic association analysis using single-marker analysis. The analysis identified 27 significant SNPs distributed across all seven chromosomes that were associated with LS resistance. One SNP (S6_17854595) was located within the HORVU6Hr1G010050 gene model that encodes a protein kinase domain-containing protein (similar to the Un8 LS resistance gene, which contains two kinase domains). A TaqMan marker (0751D06 F6/R6) for the Un8 gene was tested in the diverse collection. The results indicated that none of the Egyptian genotypes had the Un8 gene. The result of this study provided new information on the genetic control of LS resistance. Moreover, good resistance genotypes were identified and can be used for breeding cultivars with improved resistance to Egyptian LS.

Fine mapping and identification of a candidate gene for the barley Un8 true loose smut resistance gene.

KEY MESSAGE: The candidate gene for the barley Un8 true loose smut resistance gene encodes a deduced protein containing two tandem protein kinase domains. In North America, durable resistance against all known isolates of barley true loose smut, caused by the basidiomycete pathogen Ustilago nuda (Jens.) Rostr. (U. nuda), is under the control of the Un8 resistance gene. Previous genetic studies mapped Un8 to the long arm of chromosome 5 (1HL). Here, a population of 4625 lines segregating for Un8 was used to delimit the Un8 gene to a 0.108 cM interval on chromosome arm 1HL, and assign it to fingerprinted contig 546 of the barley physical map. The minimal tilling path was identified for the Un8 locus using two flanking markers and consisted of two overlapping bacterial artificial chromosomes. One gene located close to a marker co-segregating with Un8 showed high sequence identity to a disease resistance gene containing two kinase domains. Sequence of the candidate gene from the parents of the segregating population, and in an additional 19 barley lines representing a broader spectrum of diversity, showed there was no intron in alleles present in either resistant or susceptible lines, and fifteen amino acid variations unique to the deduced protein sequence in resistant lines differentiated it from the deduced protein sequences in susceptible lines. Some of these variations were present within putative functional domains which may cause a loss of function in the deduced protein sequences within susceptible lines.

Genetic analysis and molecular mapping of a seedling crown rust resistance gene in oat.

KEY MESSAGE: Genetic analysis and genome mapping of a major seedling oat crown rust resistance gene, designated PcKM, are described. The chromosomal location of the PcKM gene was identified and linked markers were validated. Crown rust (Puccinia coronata Corda f. sp. avenae Eriks) is the most important foliar disease of oats and can cause considerable yield loss in the absence of appropriate management practices. Utilization of novel resistant genes is the most effective, economic and environmentally sound approach to control the disease. Crown rust resistance present in the cultivar 'Morton' was evaluated in a population developed from the cross OT3019 × 'Morton' to elucidate the genetic basis of resistance. Crown rust reaction evaluated in field nurseries and greenhouse tests demonstrated that resistance provided by 'Morton' was controlled by a single gene, temporarily designated as PcKM. The gene was initially linked to a random amplified polymorphic DNA band and subsequently converted into a sequence characterized amplified region (SCAR) marker. Genotyping with the PcKM SCAR on the 'Kanota' × 'Ogle' population, used to create the first oat chromosome-anchored linkage map, placed the PcKM gene on chromosome 12D. Consensus map markers present in the same region as the PcKM SCAR were tested on the OT3019 × 'Morton' population and two additional phenotyped populations segregating for PcKM to identify other markers useful for marker-assisted selection. Three markers were perfectly linked to the PcKM phenotype from which TaqMan and KBioscience competitive allele-specific PCR assays were developed and validated on a set of 25 oat lines. The assays correctly identified PcKM carriers. The markers developed in this study will facilitate fine mapping of the PcKM gene and simplify selection for this crown rust resistance.

A major quantitative trait locus conferring adult plant partial resistance to crown rust in oat.

BACKGROUND: Crown rust, caused by Puccinia coronata f. sp. avenae, is the most important disease of oat worldwide. Adult plant resistance (APR), based upon partial resistance, has proven to be a durable rust management strategy in other cereal rust pathosystems. The crown rust APR in the oat line MN841801 has been effective for more than 30 years. The genetic basis of this APR was studied under field conditions in three recombinant inbred line (RIL) populations: 1) AC Assiniboia/MN841801, 2) AC Medallion/MN841801, and 3) Makuru/MN841801. The populations were evaluated for crown rust resistance with the crown rust isolate CR251 (race BRBB) in multiple environments. The 6 K oat and 90 K wheat Illumina Infinium single nucleotide polymorphism (SNP) arrays were used for genotyping the AC Assiniboia/MN841801 population. KASP assays were designed for selected SNPs and genotyped on the other two populations. RESULTS: This study reports a high density genetic linkage map constructed with oat and wheat SNP markers in the AC Assiniboia/MN841801 RIL population. Most wheat SNPs were monomorphic in the oat population. However the polymorphic wheat SNPs could be scored accurately and integrated well into the linkage map. A major quantitative trait locus (QTL) on oat chromosome 14D, designated QPc.crc-14D, explained up to 76% of the APR phenotypic variance. This QTL is flanked by two SNP markers, GMI_GBS_90753 and GMI_ES14_c1439_83. QPc.crc-14D was validated in the populations AC Medallion/MN841801 and Makuru/MN841801. CONCLUSIONS: We report the first APR QTL in oat with a large and consistent effect. QPc.crc-14D was statistically significant in all environments tested in each of the three oat populations. QPc.crc-14D is a suitable candidate for use in marker-assisted breeding and also an excellent target for map-based cloning. This is also the first study to use the 90 K wheat Infinium SNP array on oat for marker development and comparative mapping. The Infinium SNP array is a useful tool for saturating oat maps with markers. Synteny with wheat suggests that QPc.crc-14D is orthologous with the stripe rust APR gene Yr16 in wheat.

New DArT markers for oat provide enhanced map coverage and global germplasm characterization.

BACKGROUND: Genomic discovery in oat and its application to oat improvement have been hindered by a lack of genetic markers common to different genetic maps, and by the difficulty of conducting whole-genome analysis using high-throughput markers. This study was intended to develop, characterize, and apply a large set of oat genetic markers based on Diversity Array Technology (DArT). RESULTS: Approximately 19,000 genomic clones were isolated from complexity-reduced genomic representations of pooled DNA samples from 60 oat varieties of global origin. These were screened on three discovery arrays, with more than 2000 polymorphic markers being identified for use in this study, and approximately 2700 potentially polymorphic markers being identified for use in future studies. DNA sequence was obtained for 2573 clones and assembled into a non-redundant set of 1770 contigs and singletons. Of these, 705 showed highly significant (Expectation < 10E-10) BLAST similarity to gene sequences in public databases. Based on marker scores in 80 recombinant inbred lines, 1010 new DArT markers were used to saturate and improve the 'Kanota' x 'Ogle' genetic map. DArT markers provided map coverage approximately equivalent to existing markers. After binning markers from similar clones, as well as those with 99% scoring similarity, a set of 1295 non-redundant markers was used to analyze genetic diversity in 182 accessions of cultivated oat of worldwide origin. Results of this analysis confirmed that major clusters of oat diversity are related to spring vs. winter type, and to the presence of major breeding programs within geographical regions. Secondary clusters revealed groups that were often related to known pedigree structure. CONCLUSION: These markers will provide a solid basis for future efforts in genomic discovery, comparative mapping, and the generation of an oat consensus map. They will also provide new opportunities for directed breeding of superior oat varieties, and guidance in the maintenance of oat genetic diversity.