Agrobacterium-mediated transfer of the Fusarium graminearum Tri6 gene into barley using mature seed-derived shoot tips as explants.

KEY MESSAGE: We transferred the Tri6 gene into the elite barley GemCraft via new transformation method through shoot organogenesis and identified the rearrangements of transgenes and phenotypic variations in the transgenic plants. Despite its agronomic and economic importance, barley transformation is still very challenging for many elite varieties. In this study, we used direct shoot organogenesis to transform the elite barley cultivar GemCraft with the RNAi constructs containing Tri6 gene of Fusarium graminearum, which causes fusarium head blight (FHB). We isolated 4432 shoot tips and co-cultured these explants with Agrobacterium tumefaciens. A total of 25 independent T0 transgenic plants were generated including 15 events for which transgene-specific PCR amplicons were observed. To further determine the presence of transgenes, the T1 progenies of all 15 T0 plants were analyzed, and the expected PCR products were obtained in 10 T1 lines. Droplet digital (dd) PCR analysis revealed various copy numbers of transgenes in the transgenic plants. We determined the insertion site of transgenes using long-read sequencing data and observed the rearrangements of transgenes. We found phenotypic variations in both T1 and T2 generation plants. FHB disease was evaluated under growth chamber conditions, but no significant differences in disease severity or deoxynivalenol accumulation were observed between two Tri6 transgenic lines and the wildtype. Our results demonstrate the feasibility of the shoot tip transformation and may open the door for applying this system for genetic improvement and gene function research in other barley genotypes.

Mapping of Crown Rust (Puccinia coronata f. sp. avenae) Resistance Gene Pc54 and a Novel Quantitative Trait Locus Effective Against Powdery Mildew (Blumeria graminis f. sp. avenae) in the Oat (Avena sativa) Line Pc54.

The Pc54 oat line carries the crown rust resistance gene Pc54 and an unknown gene effective against powdery mildew. In this study, two recombinant inbred line (RIL) populations were developed to identify the genomic locations of the two genes and produce lists of molecular markers with a potential for marker-assisted selection. The RILs and parents were phenotyped for crown rust and powdery mildew in a controlled environment. They were also genotyped using the 6K Illumina Infinium iSelect oat single nucleotide polymorphism (SNP) chip. Multiple interval mapping placed Pc54 on the linkage group Mrg02 (chromosome 7D) and the novel powdery mildew quantitative trait locus (QTL) QPm.18 on Mrg18 (chromosome 1A) both in mapping and in the validating populations. A total of 9 and 31 significant molecular markers were identified linked with the Pc54 gene and QPm.18, respectively. Reactions to crown rust inoculations have justified separate identities of Pc54 from other genes and QTLs that have previously been reported on Mrg02 except for qPCRFd. Pm3 is the only powdery mildew resistance gene previously mapped on Mrg18. However, the pm3 differential line, Mostyn, was susceptible to the powdery mildew race used in this study, suggesting that Pm3 and QPm.18 are different genes. Determining the chromosomal locations of Pc54 and QPm.18 is helpful for better understanding of the molecular mechanism of resistance to crown rust and powdery mildew in oats. Furthermore, SNPs and single sequence repeats that are closely linked with the genes could be valuable for developing PCR-based molecular markers and facilitating the utilization of these genes in oat breeding programs.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mapping crown rust resistance in the oat diploid accession PI 258731 (Avena strigosa).

Oat crown rust, caused by Puccinia coronata Corda f. sp. avenae Eriks. (Pca), is a major biotic impediment to global oat production. Crown rust resistance has been described in oat diploid species A. strigosa accession PI 258731 and resistance from this accession has been successfully introgressed into hexaploid A. sativa germplasm. The current study focuses on 1) mapping the location of QTL containing resistance and evaluating the number of quantitative trait loci (QTL) conditioning resistance in PI 258731; 2) understanding the relationship between the original genomic location in A. strigosa and the location of the introgression in the A. sativa genome; 3) identifying molecular markers tightly linked with PI 258731 resistance loci that could be used for marker assisted selection and detection of this resistance in diverse A. strigosa accessions. To achieve this, A. strigosa accessions, PI 258731 and PI 573582 were crossed to produce 168 F5:6 recombinant inbred lines (RILs) through single seed descent. Parents and RILs were genotyped with the 6K Illumina SNP array which generated 168 segregating SNPs. Seedling reactions to two isolates of Pca (races TTTG, QTRG) were conditioned by two genes (0.6 cM apart) in this population. Linkage mapping placed these two resistant loci to 7.7 (QTRG) to 8 (TTTG) cM region on LG7. Field reaction data was used for QTL analysis and the results of interval mapping (MIM) revealed a major QTL (QPc.FD-AS-AA4) for field resistance. SNP marker assays were developed and tested in 125 diverse A. strigosa accessions that were rated for crown rust resistance in Baton Rouge, LA and Gainesville, FL and as seedlings against races TTTG and QTRG. Our data proposed SNP marker GMI_ES17_c6425_188 as a candidate for use in marker-assisted selection, in addition to the marker GMI_ES02_c37788_255 suggested by Rine's group, which provides an additional tool in facilitating the utilization of this gene in oat breeding programs.

Mapping and identification of molecular markers for the Pc96 gene conferring resistance to crown rust in oat.

Oat crown rust caused by Puccinia coronata f. sp. avenae P. Syd. & Syd (Pca) is a major constraint to oat (Avena sativa L.) production in many parts of the globe. The objectives of this study were to locate Pc96 on the oat consensus map and to develop SNP markers linked to Pc96 for use in marker-assisted selection. SNP loci linked to the crown rust resistance gene Pc96 were identified by linkage analysis and PACE assays were developed for marker-assisted selection in breeding programs. Pc96 is a race-specific crown rust resistance gene originating from cultivated oat that has been deployed in North American oat breeding programs. Pc96 was mapped in a recombinant inbred line population (n = 122) developed from a cross between the oat crown rust differential known to carry Pc96 and the differential line carrying Pc54. A single resistance locus was identified on chromosome 7D between 48.3 and 91.2 cM. The resistance locus and linked SNPs were validated in two additional biparental populations, Ajay × Pc96 (F2:3, n = 139) and Pc96 × Kasztan (F2:3, n = 168). Based on all populations, the most probable location of the oat crown rust resistance gene Pc96 on the oat consensus map was on chromosome 7D approximately at 87.3 cM. In the Ajay × Pc96 population, a second unlinked resistance gene was contributed by the Pc96 differential line, which mapped to chromosome 6C at 75.5 cM. A haplotype of nine linked SNPs predicted the absence of Pc96 in a diverse group of 144 oat germplasm. SNPs that are closely linked to the Pc96 gene may be beneficial as PCR-based molecular markers in marker-assisted selection.

Genome-Wide Association Study Reveals the Genetic Architecture of Seed Vigor in Oats.

Seed vigor is crucial for crop early establishment in the field and is particularly important for forage crop production. Oat (Avena sativa L.) is a nutritious food crop and also a valuable forage crop. However, little is known about the genetics of seed vigor in oats. To investigate seed vigor-related traits and their genetic architecture in oats, we developed an easy-to-implement image-based phenotyping pipeline and applied it to 650 elite oat lines from the Collaborative Oat Research Enterprise (CORE). Root number, root surface area, and shoot length were measured in two replicates. Variables such as growth rate were derived. Using a genome-wide association (GWA) approach, we identified 34 and 16 unique loci associated with root traits and shoot traits, respectively, which corresponded to 41 and 16 unique SNPs at a false discovery rate < 0.1. Nine root-associated loci were organized into four sets of homeologous regions, while nine shoot-associated loci were organized into three sets of homeologous regions. The context sequences of five trait-associated markers matched to the sequences of rice, Brachypodium and maize (E-value < 10-10), including three markers matched to known gene models with potential involvement in seed vigor. These were a glucuronosyltransferase, a mitochondrial carrier protein domain containing protein, and an iron-sulfur cluster protein. This study presents the first GWA study on oat seed vigor and data of this study can provide guidelines and foundation for further investigations.

Genome-Wide Association Mapping of Crown Rust Resistance in Oat Elite Germplasm.

Oat crown rust, caused by f. sp. , is a major constraint to oat ( L.) production in many parts of the world. In this first comprehensive multienvironment genome-wide association map of oat crown rust, we used 2972 single-nucleotide polymorphisms (SNPs) genotyped on 631 oat lines for association mapping of quantitative trait loci (QTL). Seedling reaction to crown rust in these lines was assessed as infection type (IT) with each of 10 crown rust isolates. Adult plant reaction was assessed in the field in a total of 10 location-years as percentage severity (SV) and as infection reaction (IR) in a 0-to-1 scale. Overall, 29 SNPs on 12 linkage groups were predictive of crown rust reaction in at least one experiment at a genome-wide level of statistical significance. The QTL identified here include those in regions previously shown to be linked with seedling resistance genes , , , , , and and also with adult-plant resistance and adaptation-related QTL. In addition, QTL on linkage groups Mrg03, Mrg08, and Mrg23 were identified in regions not previously associated with crown rust resistance. Evaluation of marker genotypes in a set of crown rust differential lines supported as the identity of . The SNPs with rare alleles associated with lower disease scores may be suitable for use in marker-assisted selection of oat lines for crown rust resistance.

A Consensus Map in Cultivated Hexaploid Oat Reveals Conserved Grass Synteny with Substantial Subgenome Rearrangement.

Hexaploid oat ( L., 2 = 6 = 42) is a member of the Poaceae family and has a large genome (∼12.5 Gb) containing 21 chromosome pairs from three ancestral genomes. Physical rearrangements among parental genomes have hindered the development of linkage maps in this species. The objective of this work was to develop a single high-density consensus linkage map that is representative of the majority of commonly grown oat varieties. Data from a cDNA-derived single-nucleotide polymorphism (SNP) array and genotyping-by-sequencing (GBS) were collected from the progeny of 12 biparental recombinant inbred line populations derived from 19 parents representing oat germplasm cultivated primarily in North America. Linkage groups from all mapping populations were compared to identify 21 clusters of conserved collinearity. Linkage groups within each cluster were then merged into 21 consensus chromosomes, generating a framework consensus map of 7202 markers spanning 2843 cM. An additional 9678 markers were placed on this map with a lower degree of certainty. Assignment to physical chromosomes with high confidence was made for nine chromosomes. Comparison of homeologous regions among oat chromosomes and matches to orthologous regions of rice ( L.) reveal that the hexaploid oat genome has been highly rearranged relative to its ancestral diploid genomes as a result of frequent translocations among chromosomes. Heterogeneous chromosome rearrangements among populations were also evident, probably accounting for the failure of some linkage groups to match the consensus. This work contributes to a further understanding of the organization and evolution of hexaploid grass genomes.