Utilizing Genomics to Characterize the Common Oat Gene Pool-The Story of More Than a Century of Polish Breeding.

This study was undertaken to investigate the diversity and population structure of 487 oat accessions, including breeding lines from the ongoing programs of the three largest Polish breeding companies, along with modern and historical Polish and foreign cultivars. The analysis was based on 7411 DArTseq-derived SNPs distributed among three sub-genomes (A, C, and D). The heterogeneity of the studied material was very low, as only cultivars and advanced breeding lines were examined. Principal component analysis (PCA), principal coordinate analysis (PCoA), and cluster and STRUCTURE analyses found congruent results, which show that most of the examined cultivars and materials from Polish breeding programs formed major gene pools, that only some accessions derived from Strzelce Plant Breeding, and that foreign cultivars were outside of the main group. During the 120 year oat breeding process, only 67 alleles from the old gene pool were lost and replaced by 67 new alleles. The obtained results indicate that no erosion of genetic diversity was observed within the Polish native oat gene pool. Moreover, current oat breeding programs have introduced 673 new alleles into the gene pool relative to historical cultivars. The analysis also showed that most of the changes in relation to historical cultivars occurred within the A sub-genome with emphasis on chromosome 6A. The targeted changes were the rarest in the C sub-genome. This study showed that Polish oat breeding based mainly on traditional breeding methods-although focused on improving traits typical to this crop, i.e., enhancing the grain yield and quality and improving adaptability-did not significantly narrow the oat gene pool and in fact produced cultivars that are not only competitive in the European market but are also reservoirs of new alleles that were not found in the analyzed foreign materials.

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.

Reference genes expression stability in Avena sativa L. during compatible and incompatible interactions with Puccinia graminis.

A reliable qPCR experiment requires the selection of reference genes with a stable level of expression in a given experimental system. This study attempts to determine the reference genes (RGs) for the A. sativa-P. graminis experimental setup. We evaluated nine candidate reference genes in A. sativa (oat line Pg4 and the cultivar Kasztan) during compatible and incompatible interactions with different pathotypes of Puccinia graminis f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). We found that the most appropriate combination of RGs for RT-qPCR data normalization were HNR (heterogeneous nuclear ribonucleoprotein 27C) + EF1A (elongation factor 1-alpha) + EIF4A (eukaryotic initiation factor 4A-3). The worst candidates for normalization in this dataset were CYP (cyclophilin) and TUA (alpha tubulin). Identified reference genes are suitable candidates for the standardization of gene expression studies in the A. sativa-P. graminis interaction system and potentially other related pathogens. To date, this is the first report of RGs selection in this pathosystem.

Validation of reference genes as an internal control for studying Avena sativa-Puccinia coronata interaction by RT-qPCR.

In this study we evaluated eleven candidate reference genes in Avena sativa during compatible and incompatible interactions with two different pathotypes of Puccinia coronata f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). The results obtained confirmed that the combination of two genes would be sufficient for reliable normalization of the expression data. In general, the most stable in the tested plant-pathogen system were HNR (heterogeneous nuclear ribonucleoprotein 27C) and EF1A (elongation factor 1-alpha). ARF (ADP-ribosylation factor) and EIF4A (eukaryotic initiation factor 4A-3) could also be considered as exhibiting high expression stability. CYP (cyclophilin) was shown by all assessment methods to be the worst candidate for normalization in this dataset. To date, this is the first report of reference genes selection in A. sativa-P. coronata interaction system. Identified reference genes enable reliable and comprehensive RT-qPCR analysis of oat gene expression in response to crown rust infection. Understanding the molecular mechanisms involved in the host-pathogen interactions may expand knowledge of durable resistance strategies beneficial to modern oat breeding.

Discovery and Chromosomal Location a Highly Effective Oat Crown Rust Resistance Gene Pc50-5.

Crown rust, caused by Puccinia coronata f. sp. avenae, is one of the most destructive fungal diseases of oat worldwide. Growing disease-resistant oat cultivars is the preferred method of preventing the spread of rust and potential epidemics. The object of the study was Pc50-5, a race-specific seedling crown rust resistant gene, highly effective at all growth stages, selected from the differential line Pc50 (Avena sterilis L. CW 486-1 × Pendek). A comparison of crown rust reaction as well as an allelism test showed the distinctiveness of Pc50-5, whereas the proportions of phenotypes in segregating populations derived from a cross with two crown rust-susceptible Polish oat cultivars, Kasztan × Pc50-5 and Bingo × Pc50-5, confirmed monogenic inheritance of the gene, indicating its usefulness in oat breeding programs. Effective gene introgression depends on reliable gene identification in the early stages of plant development; thus, the aim of the study was to develop molecular markers that are tightly linked to Pc50-5. Segregating populations of Kasztan × Pc50-5 were genotyped using DArTseq technology based on next-generation Illumina short-read sequencing. Markers associated with Pc50-5 were located on chromosome 6A of the current version of the oat reference genome (Avena sativa OT3098 v2, PepsiCo) in the region between 434,234,214 and 440,149,046 bp and subsequently converted to PCR-based SCAR (sequence-characterized amplified region) markers. Furthermore, 5426978_SCAR and 24031809_SCAR co-segregated with the Pc50-5 resistance allele and were mapped to the partial linkage group at 0.6 and 4.0 cM, respectively. The co-dominant 58163643_SCAR marker was the best diagnostic and it was located closest to Pc50-5 at 0.1 cM. The newly discovered, very strong monogenic crown rust resistance may be useful for oat improvement. DArTseq sequences converted into specific PCR markers will be a valuable tool for marker-assisted selection in breeding programs.

Virulence Structure of Puccinia coronata f. sp. avenae and Effectiveness of Pc Resistance Genes in Poland During 2017-2019.

Crown rust caused by Puccinia coronata f. sp. avenae is one of the most destructive diseases of oat, regularly occurring worldwide and leading to significant yield losses. This article characterizes the pathotype structure of P. coronata in Poland and evaluates the potential of crown rust race-specific resistance genes for use in practical breeding conditions in this region. A total of 466 isolates were derived from four locations of intensive oat breeding in Poland in 2017 to 2019, representing P. coronata populations from West, East, South, and Central Poland. Their virulence structure was determined on 35 Pc differential lines in laboratory conditions. In each year and location, high pathotype diversity was observed. In total, 347 (75%) pathotypes were detected. On average P. coronata isolates collected in 2017 and 2018 were virulent to 11% of the oat differentials. In 2019 isolates from East and South of Poland were able to overcome 18.3 and 18.5% of the oat differentials, respectively. There was no isolate virulent against Pc51, Pc52, and Pc91 crown rust resistance genes. P. coronata isolates displayed modest virulence levels, high diversity, and no prevailing pathotype. The information provided here may be helpful for development of resistance breeding strategies and in choosing the most effective major genes for pyramiding into cultivars.

Genetic diversity and population structure of endangered plant species Anagallis foemina Mill. [Lysimachia foemina (Mill.) U. Manns & Anderb.].

Anagallis foemina L. [syn. Lysimachia foemina (Mill.) U. Manns & Anderb.] is an annual, segetal weed from the family Primulaceae, recognized as a very rare and endangered species in many European countries. The rare occurrence of this species is associated with the specificity of the habitats in which it occurs. Knowledge of genetic diversity within and between rare species populations is a crucial step for investigating the causes of extinction as well as developing effective conservation strategies. The current study undertakes the assessment of the genetic variation and population structure of Anagallis foemina L. specimens collected in south-eastern Poland, Volhynian Polesie and West Volhynian Upland based on inter-simple sequence repeats (ISSR) polymorphism. Twenty ISSR primers amplified 374 DNA fragments, of which 79% were polymorphic. The polymorphic information content values ranged from 0.230 to 0.430 with an average of 0.344. An average genetic similarity calculated based on Dice algorithm between all analysed samples was 0.635 (0.28-1.00). The AMOVA study found a significant difference (Φpt = 0.88, P = 0.001) between Anagallis L. genotypes gathered in Volhynian Polesie (VP) and West Volhynian Upland (VU). Analysis indicated, that 89% of the variation existed among groups and 11% within groups. UPGMA analyses grouped A. foemina samples into 2 clearly separated clusters. The plants of the same geographic origin were grouped together. Principal coordinates analysis (PCoA) as well as STRUCTURE also grouped samples in consistence with the collection site, indicating close genetic affinity of plants from the same location. The observed results are typical for fragmented and isolated populations of rare species. Isolation of a small population leads to a decrease in internal genetic variation and to an increase of variation among them. In that case, the conservation of populations from each regional cluster is important to preserve biodiversity.

Chromosomal location of the crown rust resistance gene Pc98 in cultivated oat (Avena sativa L.).

KEY MESSAGE: SNP loci linked to the crown rust resistance gene Pc98 were identified by linkage analysis and KASP assays were developed for marker-assisted selection in breeding programs. Crown rust is among the most damaging diseases of oat and is caused by Puccinia coronata var. avenae f. sp. avenae (Urban and Marková) (Pca). Host resistance is the preferred method to prevent crown rust epidemics. Pc98 is a race-specific, seedling crown rust resistance gene obtained from the wild oat Avena sterilis accession CAV 1979 that is effective at all growth stages of oat. Virulence to Pc98 has been very low in the Pca populations that have been tested. The objectives of this study were to develop SNP markers linked to Pc98 for use in marker-assisted selection and to locate Pc98 on the oat consensus map. The Pc98 gene was mapped using F2:3 populations developed from the crosses Pc98/Bingo and Pc98/Kasztan, where Pc98 is a single-gene line carrying Pc98. Both populations were evaluated in seedling inoculation experiments. Pc98 was mapped relative to Kompetitive Allele-Specific PCR SNP markers in both populations, placing Pc98 on the Mrg20 linkage group of the consensus map. Pc98 was bracketed by two SNP markers GMI_ES22_c3052_382_kom399 and GMI_ES14_lrc18344_662_kom398 in the Pc98/Bingo mapping population with genetic distances of 0.9 cM and 0.3 cM, respectively. Pc98 co-segregated with four SNP markers in the Pc98/Kasztan population, and the closest flanking markers were GMI_DS_LB_6017_kom367 and avgbs2_153634.1.59_kom410 with genetic distances of 0.7 cM and 0.3 cM, respectively. Two SNP loci defined a haplotype that accurately predicted Pc98 status in a diverse group of oat germplasm, which will be valuable for marker-assisted selection of Pc98 in breeding of new oat cultivars.

Identification of molecular markers for the Pc39 gene conferring resistance to crown rust in oat.

KEY MESSAGE: Six new PCR-based markers for the Pc39 crown rust resistance gene in Avena sativa L. were developed. Pc39 was mapped to Mrg11 of the oat consensus map using BLASTn analysis. The aim of this study was the identification of molecular markers for the Pc39 gene in cultivated oat (Avena sativa L.). Pc39 is a major race-specific crown rust resistance gene originally found in an Israeli accession of the wild hexaploid Avena sterilis. The effectiveness of this gene in Europe has decreased in recent years, but is still relatively high and breeding programs would benefit from the availability of molecular markers to aid in its mapping and deployment. The complexity of the oat genome poses a significant obstacle to genetic research. No oat rust resistance genes have yet been cloned, and even the number of relevant molecular markers is very limited. Here, genotyping of a segregating population derived from a cross 'Celer' (Pc39)/STH9210 (susceptible) was conducted using RAPD- and SRAP-PCR-based methods, as well as microarray-based DArT™ and next-generation sequencing DArTseq™ techniques. Markers associated with Pc39 were placed on the hexaploid oat consensus linkage group Mrg11 at 3.7-6.7 cM. Six new PCR-based markers were developed to allow identification of the resistant Pc39 allele. These tightly linked markers will be useful in marker-assisted selection, with the closest, SCAR_3456624, being within 0.37 cM of Pc39. The newly developed markers could find applications in the fine mapping or positional cloning of this gene. Moreover, easy-to-use PCR-based markers linked to Pc39 could facilitate the utilization of this gene in oat breeding programs, especially as a component of crown rust resistance gene pyramids.

Molecular identification and chromosomal localization of new powdery mildew resistance gene Pm11 in oat.

The appropriate selection of various traits in valuable plants is very important for modern plant breeding. Effective resistance to fungal diseases, such as powdery mildew, is an example of such a trait in oats. Marker-assisted selection is an important tool that reduces the time and cost of selection. The aims of the present study were the identification of dominant DArTseq markers associated with a new resistance gene, annotated as Pm11 and derived from Avena sterilis genotype CN113536, and the subsequent conversion of these markers into a PCR-based assay. Among the obtained 30,620 silicoDArT markers, 202 markers were highly associated with resistance in the analysed population. Of these, 71 were selected for potential conversion: 42 specific to resistant and 29 to susceptible individuals. Finally, 40 silicoDArT markers were suitable for primer design. From this pool, five markers, 3 for resistant and 2 for susceptible plants, were selected for product amplification in the expected groups. The developed method, based on 2 selection markers, provides certain identification of resistant and susceptible homozygotes. Also, the use of these markers allowed the determination of heterozygotes in the analysed population. Selected silicoDArT markers were also used for chromosomal localization of new resistance genes. Five out of 71 segregating silicoDArT markers for the Pm11 gene were found on the available consensus genetic map of oat. Five markers were placed on linkage groups corresponding to Mrg12 on the Avena sativa consensus map.

Virulence Structure and Diversity of Puccinia coronata f. sp. avenae P. Syd. & Syd. in Poland During 2013 to 2015.

The crown rust fungus Puccinia coronata f. sp. avenae P. Syd. & Syd. (Pca) attacks cultivated oat and its wild relatives, causing significant losses to the crop worldwide. Although understanding the origin and dynamics of the pathogen's diversity is critical to developing methods for its control, there are little relevant data on Pca virulence diversity in Europe, the global center of oat production. The goal of this study was to analyze the diversity of Pca populations in Poland in 2013 to 2015 based on their ability to overcome currently available host resistance Pc genes. Pca isolate virulence was evaluated on a panel of lines containing 26 major resistance genes of oat. The isolates were able to overcome from 1 to 16 resistance genes each, with most isolates being virulent on five to seven lines. In all years, a very high level of crown rust pathotype diversity was observed, with Simpson and Evenness indices of 0.99. In total, 156 different pathotypes were detected, with no prevalent pathotype in any of the 3 years analyzed. The results showed that the virulence level of P. coronata isolates was relatively low for each year studied (21% on average), most likely owing to the low take up of Pc genes in Polish oat cultivars, meaning that many sources of resistance are still effective against Pca races occurring in Poland. The long-range dispersal of Puccinia spores supported by the availability of wild, weedy, and cultivated Avena species makes it likely that the virulence profile seen in Poland is representative of much of central Europe and beyond.

Detached Leaf Assays for Resistance to Crown Rust Reveal Diversity Within Populations of Avena sterilis.

Crown rust is the most widespread and damaging disease of oat (Avena species). Genetic resistance to the pathogen is the preferred method for crop protection but widespread deployment of limited numbers of major effect genes has promoted the rapid emergence and spread of pathogen races that are able to overcome these genes. Combining genes with even partial resistance may help develop durable cultivars that are less vulnerable to changes in pathogen virulence. Partial resistance is expected to be relatively common in populations of wild species where constant pathogen pressure encourages diversity in host resistance mechanisms, but it may be discarded in conventional screens for major gene resistance. Here, we used a detached leaf assay to detect resistance to the crown rust pathogen, Puccinia coronata Cda. f. sp. avenae, in previously uncharacterized collections of the hexaploid wild oat relative A. sterilis made by the Polish National Centre for Plant Genetic Resources. Many of the accessions were collected in Morocco, the center of diversity for the Avena genus. The detached leaf assessment allowed individual plants to be challenged with multiple pathotypes and their responses compared with 34 known differentials. Broad-spectrum resistance was identified within accession PL 51855, which behaved as a single major locus on crossing to three cultivars. The locus provided resistance to over 50 rust pathotypes, a greater range than seen for any of the known host resistance (Pc) genes. Strong resistance was identified in other accessions, and heterogeneity in response within accessions was common. Several accessions show multiple partial resistance responses that may be of value for developing durable resistance in cultivars. Because the sources of resistance in all but two differential lines were collected outside of Morocco, resistance in all accessions tested here are potentially novel. This study demonstrates that diversity within A. sterilis accessions collected in Morocco could be a very valuable source of resistance to crown rust, and it provides new germplasm for use in resistance breeding programs. Detached leaf assessment provides a valuable first step in the identification of promising candidates in complex gene bank accessions.

Mapping Oat Crown Rust Resistance Gene Pc45 Confirms Association with PcKM.

Molecular mapping of crown rust resistance genes is important to effectively utilize these genes and improve breeding efficiency through marker-assisted selection. Pc45 is a major race-specific crown rust resistance gene initially identified in the wild hexaploid oat Avena sterilis in the early 1970s. This gene was transferred to cultivated oat (Avena sativa) and has been used as a differential for identification of crown rust races since 1974. Previous research identified an association between virulence to Pc45 and PcKM, a crown rust resistance gene in the varieties 'Kame' and 'Morton'. This study was undertaken to reveal the relationship between Pc45 and PcKMPc45 was studied in the crosses 'AC Morgan'/Pc45 and 'Kasztan'/Pc45, where Pc45 is the differential line carrying Pc45 F2 progenies and F2:3 families of both populations were inoculated with the crown rust isolate CR258 (race NTGG) and single gene segregation ratios were observed. SNP markers for PcKM were tested on these populations and linkage maps were generated. In addition, 17 newly developed SNP markers identified from genotyping-by-sequencing (GBS) data were mapped in these two populations, plus another three populations segregating for Pc45 or PcKMPc45 and PcKM mapped to the same location of Mrg08 (chromosome 12D) of the oat chromosome-anchored consensus map. These results strongly suggest that Pc45 and PcKM are the same resistance gene, but allelism (i.e., functionally different alleles of the same gene) or tight linkage (i.e., two tightly linked genes) cannot be ruled out based on the present data.

Characteristics of Resistance to Puccinia coronata f. sp. avenae in Avena fatua.

Crown rust, caused by Puccinia coronata f. sp. avenae, is the most widespread and harmful fungal disease of oat. The best defense against the pathogen is use of cultivars with genetic resistance, which is effective, economic, and an environmentally friendly alternative to chemical control. However, the continuous evolution of the pathogen can rapidly overcome major gene resistance, creating an urgent need to identify new sources. Wild oat accessions have already proven to be valuable donors of many resistance genes, but the weed species Avena fatua remains underexploited. Its abundance across multiple environments and the frequent occurrence of herbicide-resistant populations demonstrate its ready ability to adapt to biotic and abiotic stresses; yet, surprisingly, there are no extensive studies which describe crown rust resistance occurrence in gene bank stocks of A. fatua. In this study, 204 accessions of A. fatua maintained in the collections of the United States Department of Agriculture (USDA) and Polish National Centre for Plant Genetic Resources were evaluated at the seedling stage for crown rust reaction using host-pathogen tests with five highly diverse and virulent races of P. coronata. Of tested genotypes, 85% showed a heterogeneous infection pattern, while 61% were susceptible or moderately susceptible to all races. Of the 79 resistant A. fatua accessions, seedling resistance to at least two P. coronata isolates was recognized within 19 accessions, with 13 displaying a homogeneously resistant phenotype to one or two races. Accessions showing multiple single seedling resistance to three or four isolates were observed. Based on the seedling reaction to isolates used in the study, 18 infection profiles (IP) were determined. Using UPGMA clustering, resistant accessions were divided into six main clusters encompassing samples with similar IPs. Twelve of 18 patterns allowed us to postulate the likely presence of novel crown rust resistance genes, whose origin was predominantly from Kenya or Egypt. Future work will clarify the genetic basis of the resistances observed here, as well as confirm their potential utility in breeding resistant oat cultivars.

Resistance to Puccinia coronata f. sp. avenae in Avena magna, A. murphyi, and A. insularis.

Wild oat tetraploids of the section Pachycarpa have already been proven to be a rich source of useful genes but have largely been unexploited for Puccinia coronata resistance. In this study, accessions of Avena magna, A. murphyi, and A. insularis gathered from European and North American gene banks were evaluated at the seedling stage for crown rust reaction using the host-pathogen test and six highly diverse and virulent P. coronata isolates. Of the 92 Avena accessions analyzed, 58.7% were resistant to at least one crown rust race. In all, 37% of the tested accessions reacted nonuniformly, which indicated their heterogeneity. The highest level of resistance was observed in three of the accessions, one of which was verified by flow cytometry as being hexaploid and two of which were verified as being tetraploids. The infection profiles of 19 accessions corresponded to resistance determined by the genes Pc14, Pc39, Pc40, Pc48, Pc50, Pc54, Pc55, Pc61, Pc67, Pc68, Pc97, Pc101, or Pc104. The patterns of infection of the remaining resistant A. magna and A. murphyi accessions allowed us to postulate the presence of potentially novel crown rust resistance genes.

Avena sterilis L. Genotypes as a Potential Source of Resistance to Oat Powdery Mildew.

The aim of the present study was to identify Avena sterilis genotypes demonstrating a high level of resistance against oat powdery mildew, using host-pathogen tests. The study was conducted on 350 A. sterilis genotypes from different parts of the world. Six single-spore isolates of Blumeria graminis (DC.) f. sp. avenae, which demonstrated different levels of virulence to control lines and cultivars, were used in host-pathogen screening tests. To confirm the resistant response of selected genotypes, 13 other isolates were used. Reactions to the isolates were grouped into three classes: resistant, intermediate, and susceptible. Susceptible cultivars Sam and Fuchs were used as controls to estimate the degree of infection. The results of the screening test showed that 10 genotypes were classified as resistant. The second test based on 13 other isolates revealed that only four of the 10 genotypes were a valuable source of resistance against powdery mildew. The identified genotypes may be used in oat breeding programs to increase the level of resistance against powdery mildew. First, however, further studies aimed at identifying whether this resistance is conditioned by a single gene or combinations of different genes are required.