Identification, introgression, and molecular marker genetic analysis and selection of a highly effective novel oat crown rust resistance from diploid oat, Avena strigosa.

KEY MESSAGE: Oat crown rust is one of the most damaging diseases of oat. We identified a new source of resistance and developed KASP and TaqMan markers for selection in breeding programs. A new highly effective resistance to oat crown rust (Puccinia coronata f. sp. avenae) was identified in the diploid oat Avena strigosa PI 258731 and introgressed into hexaploid cultivated oat. Young plants with this resistance show moderate susceptibility, whereas older plant tissues and adult plants are resistant with no virulent isolates encountered in over 8 years of testing. Resistance was incorporated into hexaploid oat by embryo rescue, colchicine chromosome doubling followed by backcrosses with a hexaploid parent, and selection for stable transmission of resistance. To mitigate flag leaf and panicle chlorosis/necrosis associated with the resistance, crosses were made with derived resistant lines to breeding lines of divergent parentage followed by selection. Subsequently, two F2 sister lines, termed MNBT1020-1 and MNBT1021-1, were identified in which the chlorosis/necrosis was reduced. These two lines performed well in replicated multi-location state trials in 2015 and 2016 out-yielding all cultivar entries. Segregating F2:3 plants resulting from crosses of MNBT lines to susceptible parents were genotyped with the oat 6K SNP array, and SNP loci with close linkage to the resistance were identified. KASP assays generated from linked SNPs showed accurate discrimination of the resistance in derivatives of the resistant MNBT lines crossed to susceptible breeding lines. A TaqMan marker was developed and correctly identified homozygous resistance in over 95% of 379 F4 plants when rust was scored in F4:5 plants in the field. Thus, a novel highly effective resistance and associated molecular markers are available for use in breeding, genetic analysis, and functional studies.

Population Genomics Related to Adaptation in Elite Oat Germplasm.

Six hundred thirty five oat ( L.) lines and 4561 single-nucleotide polymorphism (SNP) loci were used to evaluate population structure, linkage disequilibrium (LD), and genotype-phenotype association with heading date. The first five principal components (PCs) accounted for 25.3% of genetic variation. Neither the eigenvalues of the first 25 PCs nor the cross-validation errors from = 1 to 20 model-based analyses suggested a structured population. However, the PC and = 2 model-based analyses supported clustering of lines on spring oat vs. southern United States origin, accounting for 16% of genetic variation ( < 0.0001). Single-locus -statistic () in the highest 1% of the distribution suggested linkage groups that may be differentiated between the two population subgroups. Population structure and kinship-corrected LD of = 0.10 was observed at an average pairwise distance of 0.44 cM (0.71 and 2.64 cM within spring and southern oat, respectively). On most linkage groups LD decay was slower within southern lines than within the spring lines. A notable exception was found on linkage group Mrg28, where LD decay was substantially slower in the spring subpopulation. It is speculated that this may be caused by a heterogeneous translocation event on this chromosome. Association with heading date was most consistent across location-years on linkage groups Mrg02, Mrg12, Mrg13, and Mrg24.

SNP discovery and chromosome anchoring provide the first physically-anchored hexaploid oat map and reveal synteny with model species.

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology.

BACKGROUND: Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat. RESULTS: Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry. CONCLUSIONS: The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

Addition of individual chromosomes of maize inbreds B73 and Mo17 to oat cultivars Starter and Sun II: maize chromosome retention, transmission, and plant phenotype.

Oat-maize addition (OMA) lines with one, or occasionally more, chromosomes of maize (Zea mays L., 2n = 2x = 20) added to an oat (Avena sativa L., 2n = 6x = 42) genomic background can be produced via embryo rescue from sexual crosses of oat x maize. Self-fertile disomic addition lines of different oat genotypes, mainly cultivar Starter, as recipient for maize chromosomes 1, 2, 3, 4, 5, 6, 7, 9, and the short arm of 10 and a monosomic addition line for chromosome 8, have been reported previously in which the sweet corn hybrid Seneca 60 served as the maize chromosome donor. Here we report the production and characterization of a series of new OMA lines with inbreds B73 and Mo17 as maize chromosome donors and with oat cultivars Starter and Sun II as maize chromosome recipients. Fertile disomic OMA lines were recovered for B73 chromosomes 1, 2, 4, 5, 6, 8, 9, and 10 and Mo17 chromosomes 2, 4, 5, 6, 8, and 10. These lines together with non-fertile (oat x maize) F(1) plants with chromosome 3 and chromosome 7 of Mo17 individually added to Starter oat provide DNA of additions to oat of all ten individual maize chromosomes between the two maize inbreds. The Mo17 chromosome 10 OMA line was the first fertile disomic OMA line obtained carrying a complete chromosome 10. The B73 OMA line for chromosome 1 and the B73 and Mo17 OMA lines for chromosome 8 represent disomic OMA lines with improved fertility and transmission of the addition chromosome compared to earlier Seneca 60 versions. Comparisons among the four oat-maize parental genotype combinations revealed varying parental effects and interactions on frequencies of embryo recovery, embryo germination, F(1) plantlets with maize chromosomes, the specific maize chromosomes retained and transmitted to F(2) progeny, and phenotypes of self-fertile disomic addition plants. As opposed to the previous use of a hybrid Seneca 60 maize stock as donor of the added maize chromosomes, the recovered B73 and Mo17 OMA lines provide predictable genotypes for use as tools in physical mapping of maize DNA sequences, including inter-genic sequences, by simple presence/absence assays. The recovered OMA lines represent unique materials for maize genome analysis, genetic, physiological, and morphological studies, and a possible means to transfer maize traits to oat. Descriptions of these materials can be found at http://agronomy.cfans.umn.edu/Maize_Genomics.html .

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.

Maize centromere mapping: a comparison of physical and genetic strategies.

Centromere positions on 7 maize chromosomes were compared on the basis of data from 4 to 6 mapping techniques per chromosome. Centromere positions were first located relative to molecular markers by means of radiation hybrid lines and centric fission lines recovered from oat-maize chromosome addition lines. These centromere positions were then compared with new data from centric fission lines recovered from maize plants, half-tetrad mapping, and fluorescence in situ hybridizations and to data from earlier studies. Surprisingly, the choice of mapping technique was not the critical determining factor. Instead, on 4 chromosomes, results from all techniques were consistent with a single centromere position. On chromosomes 1, 3, and 6, centromere positions were not consistent even in studies using the same technique. The conflicting centromere map positions on chromosomes 1, 3, and 6 could be explained by pericentric inversions or alternative centromere positions on these chromosomes.

Dissecting the maize genome by using chromosome addition and radiation hybrid lines.

We have developed from crosses of oat (Avena sativa L.) and maize (Zea mays L.) 50 fertile lines that are disomic additions of individual maize chromosomes 1-9 and chromosome 10 as a short-arm telosome. The whole chromosome 10 addition is available only in haploid oat background. Most of the maize chromosome disomic addition lines have regular transmission; however, chromosome 5 showed diminished paternal transmission, and chromosome 10 is transmitted to offspring only as a short-arm telosome. To further dissect the maize genome, we irradiated monosomic additions with gamma rays and recovered radiation hybrid (RH) lines providing low- to medium-resolution mapping for most of the maize chromosomes. For maize chromosome 1, mapping 45 simple-sequence repeat markers delineated 10 groups of RH plants reflecting different chromosome breaks. The present chromosome 1 RH panel dissects this chromosome into eight physical segments defined by the 10 groups of RH lines. Genomic in situ hybridization revealed the physical size of a distal region, which is represented by six of the eight physical segments, as being approximately 20% of the length of the short arm, representing approximately one-third of the genetic chromosome 1 map. The distal approximately 20% of the physical length of the long arm of maize chromosome 1 is represented by a single group of RH lines that spans >23% of the total genetic map. These oat-maize RH lines provide valuable tools for physical mapping of the complex highly duplicated maize genome and for unique studies of inter-specific gene interactions.

Transmission of maize chromosome 9 rearrangements in oat-maize radiation hybrids.

Oat-maize radiation hybrids are oat (Avena sativa L.) plants carrying radiation-induced subchromosome fragments of a given maize (Zea mays L.) chromosome. Since first-generation radiation hybrids contain various maize chromosome rearrangements in a hemizygous condition, variation might be expected in the transmission of these rearrangements to subsequent generations. The transmission and integrity of maize chromosome 9 rearrangements were evaluated in progenies of 30 oat-maize radiation hybrids by using a series of DNA-based markers and by genomic in situ hybridization. Maize chromosome 9 rearrangements were reisolated by self-fertilization in 24 of the 30 radiation hybrid lineages. Normal and deleted versions of maize chromosome 9 were transmitted at similar frequencies of 9.1% and 7.6%, respectively, while intergenomic translocations were transmitted at a significantly higher frequency of 47.6%. Most lines (93%) that inherited a rearrangement had it in the hemizygous condition. Lines with a rearrangement in the homozygous state (7%) were only identified in lineages with intergenomic translocations. Homozygous lines are more desirable from the perspective of stock maintenance, since they may stably transmit a given rearrangement to a subsequent generation. However, their isolation is not strictly required, since hemizygous lines can also be used for genome mapping studies.

A molecular marker map in 'Kanota' x 'Ogle' hexaploid oat (Avena spp.) enhanced by additional markers and a robust framework.

Molecular mapping of cultivated oats was conducted to update the previous reference map constructed using a recombinant inbred (RI) population derived from Avena byzantina C. Koch cv. Kanota x Avena sativa L. cv. Ogle. In the current work, 607 new markers were scored, many on a larger set of RI lines (133 vs. 71) than previously reported. A robust, updated framework map was developed to resolve linkage associations among 286 markers. The remaining 880 markers were placed individually within the most likely framework interval using chi2 tests. This molecular framework incorporates and builds on previous studies, including physical mapping and linkage mapping in additional oat populations. The resulting map provides a common tool for use by oat researchers concerned with structural genomics, functional genomics, and molecular breeding.

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.