J. Chong

Identification of a RAPD marker linked to the oat stem rust gene Pg3.

SummaryThe feasibility of identifying molecular markers linked to disease resistance genes in oats was investigated utilizing random primers in conjunction with polymerase chain reaction technology. A pair of near-isogenic oat lines were screened for polymorphic DNA fragments linked to the stem rust resistance gene Pg3. Two primers were identified which amplified DNA fragments that were polymorphic between the lines analyzed. One primer (ACOpR-2) was shown to be completely linked to the Pg3 locus; the other primer was not linked to either the ACOpR-2 or the Pg3 loci. This type of analysis, combined with rapid leaf disc DNA extraction techniques, offers an effective means of identifying useful molecular markers and of applying them to plant breeding selection strategies.

Comparative AFLP mapping in two hexaploid oat populations

Abstract Amplified fragment length polymorphisms (AFLPs) can be used to quickly develop linkage maps in plant species and are especially useful for crops with large genomes like oat (Avena sativa L., 2n=6x=42). High reproducibility and consistency are crucial if AFLP linkage maps are employed for comparative mapping. We mapped AFLP markers in combination with restriction fragment length polymorphism (RFLP) markers in two recombinant inbred populations of hexaploid oat in two laboratories to test the consistency of AFLP markers in a polyploid crop. Eight primer combinations produced 102 and 121 scoreable AFLP markers in the respective populations. In a population from the cross Kanota×Ogle, AFLP markers were placed onto a RFLP reference map consisting of 32 linkage groups. Nineteen linkage groups from another population from the cross Kanota×Marion were assigned to the reference map using AFLP and RFLP markers homologous to those used in the Kanota× Ogle cross. Reproducibility of AFLP assays was high in both laboratories and between laboratories. The AFLP markers were well-distributed across the genome in both populations. Many AFLP markers tended to extend the distance between adjacent RFLP markers in linkage analysis. Of the 27 polymorphic AFLPs common in both populations, 20 mapped to homologous linkage groups, 4 were unlinked in at least one population, and 3 mapped to different linkage groups in the two crosses. We believe that 1 of the 3 markers that mapped to a different linkage group in the two populations mapped to homoeologous linkage groups. The linkage map of hexaploid oat is not yet complete, and genomic rearrangements such as translocations exist among cultivars and are likely to account for the remaining two non-syntenous mapping results. AFLPs provide not only a fast and powerful tool for mapping but could be useful in characterizing genomic structural variations among germplasms in hexaploid oat.

Microsatellite variation in Avena sterilis oat germplasm

The Avena sterilis L. collection in the Plant Gene Resources of Canada (PGRC) consists of 11,235 accessions originating from 27 countries and is an invaluable source of genetic variation for genetic improvement of oats, but it has been inadequately characterized, particularly using molecular techniques. More than 35 accessions have been identified with genes for resistance to oat crown and stem rusts, but little is known about their comparative genetic diversity. This study attempted to characterize a structured sample of 369 accessions representing 26 countries and two specific groups with Puccinia coronataavenae (Pc) and Puccinia graminisavenae (Pg) resistance genes using microsatellite (SSR) markers. Screening of 230 SSR primer pairs developed from other major crop species yielded 26 informative primer pairs for this characterization. These 26 primer pairs were applied to screen all the samples and 125 detected alleles were scored for each accession. Analyses of the SSR data showed the effectiveness of the stratified sampling applied in capturing country-wise SSR variation. The frequencies of polymorphic alleles ranged from 0.01 to 0.99 and averaged 0.28. More than 90% of the SSR variation resided within accessions of a country. Accessions from Greece, Liberia, and Italy were genetically most diverse, while accessions from Egypt, Georgia, Ethiopia, Gibraltar, and Kenya were most distinct. Seven major clusters were identified, each consisting of accessions from multiple countries and specific groups, and these clusters were not well congruent with geographic origins. Accessions with Pc and Pg genes had similar levels of SSR variation, did not appear to cluster together, and were not associated with the other representative accessions. These SSR patterns are significant for understanding the progenitor species of cultivated oat, managing A. sterilis germplasm, and exploring new sources of genes for oat improvement.

Mapping of the oat crown rust resistance gene Pc91

Crown rust is an important disease of oat caused by Puccinia coronata Corda f. sp. avenae Eriks. Crown rust is efficiently and effectively managed through the development of resistant oat varieties. Pc91 is a seedling crown rust resistance gene that is highly effective against the current P. coronata population in North America. The primary objective of this study was to develop DNA markers linked to Pc91 for purposes of marker-assisted selection in oat breeding programs. The Pc91 locus was mapped using a population of F7-derived recombinant inbred lines developed from the cross ‘CDC Sol-Fi’/‘HiFi’ made at the Crop Development Centre, University of Saskatchewan. The population was evaluated for reaction to P. coronata in field nurseries in 2008 and 2009. Pc91 mapped to a linkage group consisting of 44 Diversity Array Technology (DArT) markers. DArTs were successfully converted to sequence characterized amplified region (SCAR) markers. Five robust SCARs were developed from three non-redundant DArTs that co-segregated with Pc91. SCAR markers were developed for different assay systems, such that SCARs are available for agarose gel electrophoresis, capillary electrophoresis, and Taqman single nucleotide polymorphism detection. The SCAR markers accurately postulated the Pc91 status of 23 North American oat breeding lines.

Virulence of oat crown rust [Puccinia coronata f. sp. avenae] in Canada during 2002–2006

Crown rust of oat (Avena sativa) was generally light (trace to 10% severities) in Manitoba and eastern Saskatchewan during 2002–2004 and 2006 and was severe in 2005. By 21 July 2005, fields of “AC Assiniboia” and “Ronald” (with resistance genes Pc38, Pc39, and Pc68) near the buckthorn (Rhamnus cathartica) areas had 40%–80% crown rust severities. In mid-August, severities reaching 60%–100% were commonly observed in oat fields across Manitoba. Using 19 oat crown rust differentials, 59 races were identified in collections of isolates from wild oat (Avena fatua) in the eastern prairie region in 2002, 96 in 2003, 107 in 2004, 105 in 2005, and 76 in 2006. Forty-seven races (66.1% of the isolates) were virulent to Pc68 in 2006 compared with only two (1.2%) in 2002. Increased use of cultivars with Pc68, from 8% of the total area planted to oat in 1998 to >80% in 2006, resulted in a major shift in virulence to Pc68 in the prairie rust population. The frequency of virulence to Pc48 increased from 6.5% in 2002 to 27.0% in 2003. It then decreased to 3.2% in 2006. Isolates with virulence to Pc38 and Pc39 remained prevalent, representing 57.4%–92.7% of the isolates from wild oat during 2002–2006. Virulence to Pc96 ranged from 0.8%–5.7% from 2003–2006. Virulence to Pc91, a gene in “HiFi”, was identified in 2002 and 2003 (one isolate each). Differences in frequencies of virulence to several Pc genes were significant between isolates from wild oat and cultivated oat in some years. An isolate (BRBG-94), virulent to genes Pc68 and Pc94 in the newly released cultivar “Leggett”, was obtained from a line with Pc94 in a rust nursery near Emerson, Manitoba, in 2006.

Incidence and virulence of Puccinia coronata f. sp. avenae in Canada from 1996 to 1998

Crown rust (Puccinia coronata f. sp. avenae) of oat (Avena sativa) was severe in Manitoba in 1996 and 1998 but was relatively mild in 1997. Heavy crown rust infections were also commonly found on wild oat (Avena fatua) in eastern Saskatchewan in 1996 and 1998. Warm weather with frequent dew periods favoured spread of the disease across southern Manitoba in 1996 and 1998. By early to mid-August, moderate to heavy infections (40-90%) were commonly found in fields of susceptible cultivars, such as Robert, Riel, Dumont, AC Preakness, and Jerry. Only trace levels of infections were found on the resistant cultivars AC Assiniboia, AC Medallion, and Triple Crown. From the eastern prairie region (Manitoba and eastern Saskatchewan), 142, 123, and 111 virulence phenotypes were identified, respectively, from 328, 255, and 265 isolates in 1996, 1997, and 1998. From Ontario, 13, 29, and 48 virulence phenotypes were identified, respectively, from 22, 81, and 106 isolates in 1996, 1997, and 1998. Isolates with virulence to both genes Pc38 and Pc39 predominated in Canada during the years from 1996 to 1998. Combined virulence to genes Pc38, Pc39, and Pc68 was found in several isolates from Manitoba in 1998. Virulence to genes Pc48 and Pc96 ranged from 1.8 to 5.8% and virulence to gene Pc68 remained below 1.9% in the eastern prairie region during the years from 1996 to 1998. Virulence to genes Pc58 and Pc59 occurred at low levels in Ontario. Virulence to gene Pc94 was detected for the first time in the prairie region in 1998. Various combinations of genes Pc48, Pc58, Pc59, Pc68, Pc94, and Pc96 would be useful for combining resistance against crown rust in Canada.

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

BackgroundCrown 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.ResultsThis 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.ConclusionsWe 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.

Virulence of Puccinia coronata f. sp. avenae in the Eastern Prairie Region of Canada during 2007–2009

Abstract Unfavourable environmental conditions for crown rust [Puccinia coronata f. sp. avenae] during 2007–2009 resulted in light incidence of crown rust on oat (Avena sativa) in Manitoba and eastern Saskatchewan. The first appearance of crown rust on 11 August in 2008 and on 15 August in 2009 was the latest ever seen in this region over the past three decades. Using 19 oat crown rust differentials, a large number of races were identified from isolates from wild oat each year, and a large proportion of the races were represented by a single isolate. There were significant differences between isolates from wild oat and cultivated oat in frequency of virulence to several genes in some years. Virulence frequency to Pc48, a gene in ‘Triple Crown’, was between 8.2–14.1% in isolates from wild oat and between 11.5–18.2% in isolates from cultivated oat during 2007–2009. Frequency of virulence to Pc68 was between 42.3–45.9% and 70.8–81.8% in isolates from wild oat and cultivated oat, respectively. As cultivars with the Pc38, 39, 68-gene combination were still commonly grown during these years, races with virulence to this gene combination were abundant. These cultivars were gradually replaced by new cultivars with different resistance genes. By 2009, ‘Leggett’ (Pc68, 94) accounted for 17.9% of the total oat hectarage, and ‘HiFi’ (Pc91) accounted for 3.0%. During 2007–2009, virulence to Pc94 was low (≤ 1.8%) or not detected, virulence to Pc91 was found in a single isolate, and virulence to gene temp_pc97 or temp_Pc98 was either low or not detected. The huge increase in frequency of virulence to Pc45 in 2008 and 2009 was most likely a result of isolates with this virulence migrating into the prairie region from the USA.

Identification of single-nucleotide polymorphisms linked to resistance gene Pc68 to crown rust in cultivated oat

A procedure is described for developing single-nucleotide polymorphism (SNP) markers linked to Pc68, a gene conferring resistance to crown rust [Puccinia coronata f. sp. avenae] in many oat (Avena sativa) cultivars currently grown in Canada. Three restriction-fragment length polymorphism (RFLP) markers, located close to the resistance gene Pg9 to stem rust [Puccinia graminis f. sp. avenae] through comparative mapping, were used as sources of DNA-sequence information for SNP identification, since Pc68 is tightly linked or allelic to Pg9. Specific primers designed from the RFLP-marker sequences were used to amplify the target genomic region from recombinant inbred lines with and without Pc68. Putative SNP sites were identified by means of comparative sequence alignment of the polymerase chain reaction (PCR) fragments and were validated by the single-base extension method, a non-gel-based assay for genotyping SNPs. The 774-bp PCR fragment amplified by primers derived from the RFLP marker cdo309 was a sequence-tagged site (STS) marker linked to Pc68, and only the SNPs derived from a region within the STS were linked to Pc68. These SNPs and STS cosegregated in two genetic populations. The map distance between these markers and Pc68 was 4.2 and 6.7 cM (centimorgans), depending on population. The SNP markers identified in the present study can distinguish plants homozygous for Pc68 from heterozygotes, a useful feature for eliminating heterozygous plants in early generations. As SNP markers for other resistance genes or other important traits become available, breeders can benefit from using the technology with high-throughput, automation, and multiplexing capabilities, such as single-base extension assay, in breeding applications, including resistance gene pyramiding.

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

Multienvironment genome‐wide association study of reaction to crown rust in elite oat Oat response to inoculation with 10 well‐characterized Puccinia coronata isolates evaluated Adult plant response to crown rust assessed in 10 location–years Patterns of association compared against genotypes of differential gene stocks QTL placed in the context of current literature