New Diversity Arrays Technology (DArT) markers for tetraploid oat (Avena magna Murphy et Terrell) provide the first complete oat linkage map and markers linked to domestication genes from hexaploid A. sativa L.

Nutritional benefits of cultivated oat (Avena sativa L., 2n = 6x = 42, AACCDD) are well recognized; however, seed protein levels are modest and resources for genetic improvement are scarce. The wild tetraploid, A. magna Murphy et Terrell (syn A. maroccana Gdgr., 2n = 4x = 28, CCDD), which contains approximately 31% seed protein, was hybridized with cultivated oat to produce a domesticated A. magna. Wild and cultivated accessions were crossed to generate a recombinant inbred line (RIL) population. Although these materials could be used to develop domesticated, high-protein oat, mapping and quantitative trait loci introgression is hindered by a near absence of genetic markers. Objectives of this study were to develop high-throughput, A. magna-specific markers; generate a genetic linkage map based on the A. magna RIL population; and map genes controlling oat domestication. A Diversity Arrays Technology (DArT) array derived from 10 A. magna genotypes was used to generate 2,688 genome-specific probes. These, with 12,672 additional oat clones, produced 2,349 polymorphic markers, including 498 (21.2%) from A. magna arrays and 1,851 (78.8%) from other Avena libraries. Linkage analysis included 974 DArT markers, 26 microsatellites, 13 SNPs, and 4 phenotypic markers, and resulted in a 14-linkage-group map. Marker-to-marker correlation coefficient analysis allowed classification of shared markers as unique or redundant, and putative linkage-group-to-genome anchoring. Results of this study provide for the first time a collection of high-throughput tetraploid oat markers and a comprehensive map of the genome, providing insights to the genome ancestry of oat and affording a resource for study of oat domestication, gene transfer, and comparative genomics.

Development of oat-based markers from barley and wheat microsatellites.

Although microsatellites are an efficient and reliable genetic marker system, availability is limited in cultivated oat (Avena sativa L.). Previous research has suggested that microsatellites from related species may be adapted to oat. This study investigated the stability of existing oat microsatellites, sequenced polymorphic oat amplicons derived from wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) primers, and redesigned primers to develop oat-based markers. We evaluated 161 published oat microsatellites and identified 9 with polymorphism between mapping parents Ogle1040 and TAM O-301 (OT). We also studied 30 wheat, 1 Aegilops tauschii Coss., and 9 barley primers with reported oat polymorphism. Sixteen primers (1 A. tauschii, 10 wheat, 5 barley) amplified random oat sequences and were used to generate 28 new oat STS markers. Eight primers, 4 each from wheat and barley, amplified oat repetitive motifs, generating 10 new oat SSRs. Four additional SSRs were developed from characterization of thaumatin-like pathogenesis-related protein sequences formerly utilized as the Rast1-4 oat marker. These new markers, along with 9 existing oat SSRs and 6 previously identified disease resistance loci, were mapped in the OT population, joining 3 pairs of linkage groups. Map locations of multiallelic SSRs and disease-resistance QTL interactions suggested possible homoeologous relationships among the oat chromosomes.

Quantitative trait loci in the Ogle/TAM O-301 oat mapping population controlling resistance to Puccinia coronata in the field.

Crown rust is the most damaging disease of cultivated oat (Avena sativa) and genetic resistance is the primary means of controlling the disease. Quantitative trait loci (QTL) with major and minor effects have been identified in Ogle1040 and TAM O-301 (most notably, Pc58 and PcNQMG/LGCG from TAM O-301 and OT-27 from Ogle1040) through single-isolate greenhouse and field tests. To map loci and determine the effectiveness of previously identified QTL against naturally occurring pathogen populations in highly disease-conducive environments, the Ogle/TAM O-301 (OT) recombinant inbred line (RIL) population was grown in Texas and Louisiana over 2 years and in Manitoba, Canada. The genetic region characterized by the Pc58 resistance gene complex, particularly Pc58a, accounted for most of the diseased leaf area (DLA) and infection type (IT) variance in all five experiments. Additionally, the genetic region characterized by PcNQMG/LGCG accounted for a portion of the IT variance in three experiments. Although no QTL was detected on OT-27 in this study, all the markers on this linkage group were associated (P < 0.0001) with reducing both IT and DLA using single-marker analysis. Screening with 25 Puccinia coronata isolates from six different states indicated that Pc58abc and Pc58a were highly effective, while characterization using F(2) populations derived from OT RILs containing the two main genetic regions responsible for crown rust resistance in TAM O-301 (Pc58 and PcNQMG/LGCG) and a minor QTL in Ogle (OT-27) indicated that Pc58a, in combination with a locus in Ogle1040, provided high levels of resistance to natural races in Texas. This study provides new information and key loci in OT mapping population and may be useful for effective control of crown rust in North America.

Qualitative and quantitative trait loci conditioning resistance to Puccinia coronata pathotypes NQMG and LGCG in the oat (Avena sativa L.) cultivars Ogle and TAM O-301.

Mapping disease resistance loci relies on the type and precision of phenotypic measurements. For crown rust of oat, disease severity is commonly assessed based on visual ratings of infection types (IT) and/or diseased leaf area (DLA) of infected plants in the greenhouse or field. These data can be affected by several variables including; (i) non-uniform disease development in the field; (ii) atypical symptom development in the greenhouse; (iii) the presence of multiple pathogenic races or pathotypes in the field, and (iv) rating bias. To overcome these limitations, we mapped crown rust resistance to single isolates in the Ogle/TAM O-301 (OT) recombinant inbred line (RIL) population using detailed measurements of IT, uredinia length (UL) and relative fungal DNA (FDNA) estimates determined by q-PCR. Measurements were taken on OT parents and recombinant inbred lines (RIL) inoculated with Puccinia coronata pathotypes NQMG and LGCG in separate greenhouse and field tests. Qualitative mapping identified an allele conferred by TAM O-301 on linkage group (LG) OT-11, which produced a bleached fleck phenotype to both NQMG and LGCG. Quantitative mapping identified two major quantitative trait loci (QTL) originating from TAM O-301 on LGs OT-11 and OT-32 which reduced UL and FDNA of both isolates in all experiments. Additionally, minor QTLs that reduced UL and FDNA were detected on LGs OT-15 and OT-8, originating from TAM O-301, and on LG OT-27, originating from Ogle. Detailed assessments of the OT population using two pathotypes in both the greenhouse and field provided comprehensive information to effectively map the genes responsible for crown rust resistance in Ogle and TAM O-301 to NQMG and LGCG.

Detached-Leaf Method for Propagating Puccinia coronata and Assessing Crown Rust Resistance in Oat.

The crown rust pathogen Puccinia coronata is an obligate biotroph with wind-disseminated propagules and numerous races. These characteristics make propagation of single-race cultures difficult. Genetic studies using single races in field and greenhouse environments are also problematic because pure cultures can easily become contaminated. In this study, we developed an isolated propagation system for P. coronata and tested its ability to assess host resistance. Oat (Avena sativa) leaf sections (10 cm each) were harvested, disinfested, and suspended in sterile plastic boxes by enclosing 3.5-cm linear sections of each leaf end between 4% agar blocks amended with various chemical constituents. The exposed sections (approximately 3 cm) were inoculated with P. coronata urediniospores suspended in water. Boxes were sealed and incubated in a lighted growth cabinet until the pathogen sporulated. Viable spores were produced on leaves in all treatments, whereas 6-benzylaminopurine (BAP) and kinetin treatments sustained the leaves longer and yielded the most viable spores. Based on these data, the BAP treatment was adopted and used for additional studies. Detached leaves of differential oat cultivars produced the same reactions as whole plants screened under standard conditions in a growth chamber. The proposed detached-leaf system should be useful for the propagation of numerous single-race cultures of P. coronata as well as evaluation of host resistance under highly controlled conditions.

Characterization and mapping of oat crown rust resistance genes using three assessment methods.

ABSTRACT Resistance is the primary means of control for crown rust of oat (Avena sativa L.), caused by Puccinia coronata f. sp. avenae, and better knowledge of the genetics of resistance will enhance resistance breeding. Disease data were generated in the field and greenhouse for parents and recombinant inbred lines of the Ogle/TAM O-301 (OT) oat mapping population using (i) a new quantitative assay that employs quantitative real-time polymerase chain reaction (q-PCR) to estimate fungal growth in the host, (ii) digital image analysis, and (iii) visual ratings. The objectives of this study were to evaluate each assessment method's ability to map a major gene from cv. Ogle and potential quantitative trait loci (QTL) contributed by Ogle and TAM O-301. All three assessment methods identified the major gene in Ogle, which was mapped to linkage group OT6. The resolution produced by q-PCR, however, enabled more precise mapping of the major gene. Quantitative analysis indicated that 64% of the phenotypic variation was accounted for using q-PCR, whereas 41 and 52% were accounted for using visual and digital assessments, respectively. Data generated by q-PCR permitted identification of QTL on linkage groups OT32, accounting for 6% of the phenotypic variation, and OT2, accounting for 4% of the variation. QTL on both OT32 and OT2 were conferred by TAM O-301, one of which (OT2) was indiscernible using data from the visual and digital assessments. The new method of precisely phenotyping crown rust resistance provided a more accurate and thorough means of dissecting resistance in the OT mapping population. Similar methods could be developed and applied to other important cereal rust diseases.

Identification and molecular tagging of a gene from PI 289824 conferring resistance to leaf rust (Puccinia triticina) in wheat.

Host-plant resistance is the most economically viable and environmentally responsible method of control for Puccinia triticina, the causal agent of leaf rust in wheat (Triticum aestivum L.). The identification and utilization of new resistance sources is critical to the continued development of improved cultivars as shifts in pathogen races cause the effectiveness of widely deployed genes to be short lived. The objectives of this research were to identify and tag new leaf rust resistance genes. Forty landraces from Afghanistan and Iran were obtained from the National Plant Germplasm System and evaluated under field conditions at two locations in Texas. PI 289824, a landrace from Iran, was highly resistant under field infection. Further evaluation revealed that PI 289824 is highly resistant to a broad spectrum of leaf rust races, including the currently prevalent races of leaf rust in the Great Plains area of the USA. Eight F1 plants, 176 F2 individuals and 139 F2:3 families of a cross between PI 289824 and T112 (susceptible) were evaluated for resistance to leaf rust at the seedling stage. Genetic analysis indicated resistance in PI 289824 is controlled by a single dominant gene. The AFLP analyses resulted in the identification of a marker (P39 M48-367) linked to resistance. The diagnostic AFLP band was sequenced and that sequence information was used to develop an STS marker (TXW200) linked to the gene at a distance of 2.3 cM. The addition of microsatellite markers allowed the gene to be mapped to the short arm of Chromosome 5B. The only resistance gene to be assigned to Chr 5BS is Lr52. The Lr52 gene was reported to be 16.5 cM distal to Xgwm443 while the gene in PI 289824 mapped 16.7 cM proximal to Xgwm443. Allelism tests are needed to determine the relationship between the gene in PI 289824 and Lr52. If the reported map positions are correct, the gene in PI 289824 is unique.