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

Summary The 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.Plant Research Centre publication number: 1443     

Identification of an RAPD marker for the crown rust resistance gene Pc68 in oats.

The feasibility of using bulk segregant analysis to identify molecular markers for disease resistance genes in oats was investigated, utilizing random primers in conjunction with polymerase chain reaction technology. Random primers were screened for the amplification of polymorphic DNA fragments on two pools of genomic DNA isolated from plants that were homozygous for the presence and absence of the crown rust resistance gene Pc68. Ten primers were identified that amplified polymorphic DNA fragments. Of these, one was tightly linked, in repulsion, to the target gene, while the other nine were not linked to this trait. The relatively low cost of polymerase chain reaction technology, coupled with rapid leaf disc genomic DNA extraction techniques should result in the effective use of this linked marker in oat breeding selection programs.     

Chromosome location and allele-specific PCR markers for marker-assisted selection of the oat crown rust resistance gene Pc91

Race-specific seedling resistance genes are the primary means of controlling crown rust of oat caused by Puccinia coronata Corda f. sp. avenae Eriks in Canada. Pc91 is a seedling crown rust resistance gene that is highly effective against the current crown rust population in North America. A number of race-specific resistance genes have been mapped and markers that are closely linked to them have been identified. However, the use of these markers in oat breeding has been limited by the economics of marker-assisted selection (MAS). A crucial step in the successful application of MAS in breeding programs is the development of inexpensive and easy-to-use molecular markers. The primary objective of this study was to develop co-dominant KBioscience competitive allele-specific PCR (KASP) markers linked to Pc91 for deployment in high-throughput MAS in oat breeding programs. The allele-specific marker showed consistent diagnostic polymorphism between the selected 16 North American oat breeding lines. The developed co-dominant marker was also validated on three F₂ populations (AC Morgan × Stainless; SW Betania × Stainless; AC Morgan × CDC Morrison) and one recombinant inbred line population (CDC Sol-Fi × HiFi) segregating for Pc91 using KASP genotyping technology. We recommend the simple, low-cost marker as a powerful tool for pyramiding Pc91 with other effective crown rust resistance loci into a single line. The mapping results indicate that crown rust resistance gene Pc91 resides on the translocated oat chromosome 7C-17A.     

Genetic association of crown rust resistance gene Pc68, storage protein loci, and resistance gene analogues in oats

Segregating F(3) families, derived from a cross between oat cultivar Swan and the putative single gene line PC68, were used to determine the association of seed storage protein loci and resistance gene analogues (RGAs) with the crown rust resistance gene Pc68. SDS-PAGE analysis detected three avenin loci, AveX, AveY, and AveZ, closely linked to Pc68. Their diagnostic alleles are linked in coupling to Pc68 and were also detected in three additional lines carrying Pc68. Another protein locus was linked in repulsion to Pc68. In complementary studies, three wheat RGA clones (W2, W4, and W10) detected restriction fragment length polymorphisms (RFLPs) between homozygous resistant and homozygous susceptible F(3) DNA bulks. Four oat homologues of W2 were cloned and sequenced. RFLPs detected with two of them were mapped using F(3) and F(4) populations. Clone 18 detected a locus, Orga2, linked in repulsion to Pc68. Clone 22 detected several RFLPs including Orga1 (the closest locus to Pc68) and three RGA loci (Orga22-2, Orga22-3, and Orga22-4) loosely linked to Pc68. The diagnostic RFLPs linked in coupling to Pc68 were detected by clone 22 in three additional oat lines carrying Pc68 and have potential utility in investigating and improving crown rust resistance of oat.     

Hypersensitive lignification response as the mechanism of non-host resistance of wheat against oat crown rust

Seven-day-old seedlings of the near-isogenic wheat ( Triticum aestivum L.) lines Prelude and Prelude-Sr5, susceptible and resistant to wheat stem rust, respectively, were inoculated with uredospores of the oat crown rust fungus Puccinia coronata Cda. f. sp. avenae Fraser & Led. Fluorescence microscopy revealed that the majority of colonies developed intercellular infection structures including haustorial mother cells and haustoria after penetration of wheat mesophyll cells. All penetrated cells became necrotic, and exhibited bright yellow autofluorescence. This autofluorescence was not extractable with alkali, and fluorescent cells stained positively with phloroglucinol/HCI, suggesting that hypersensitive cell death was correlated with cellular lignification. Accordingly, the lignin biosynthetic enzymes phenylalanine ammonia-lyase (EC4.3.1.5). 4-coumarate:CoA ligase (EC6.2.1.12), cinnamyl-alcohol dehydrogenase (EC1.1.1.149), and peroxidases (EC1.11.1.7) increased in activity during the expression of resistance. The induced pattern of peroxidase iso/ymes closely resembled that observed for highly incompatible wheat/wheat stem rust interactions. Furthermore, an elieitor was extracted from oat crown rust germlings. which induces lignification when injected into the intercellular space of wheat leaves. This elieitor appears to be functionally similar to that isolated from wheat stem rust germlings. The results suggest that the non-host resistance of wheat to the xenopara-site oat crown rust closely resembles the race/cullivar-speeific resistant mechanism of highly resistant wheat varieties to wheat stem rust.     

Identification of Ug99 stem rust resistance loci in winter wheat germplasm using genome-wide association analysis

The evolution of a new race of stem rust, generally referred to as Ug99, threatens global wheat production because it can overcome widely deployed resistance genes that had been effective for many years. To identify loci conferring resistance to Ug99 in wheat, a genome-wide association study was conducted using 232 winter wheat breeding lines from the International Winter Wheat Improvement Program. Breeding lines were genotyped with diversity array technology, simple sequence repeat and sequence-tagged site markers, and phenotyped at the adult plant stage for resistance to stem rust in the stem rust resistance screening nursery at Njoro, Kenya during 2009-2011. A mixed linear model was used for detecting marker-trait associations. Twelve loci associated with Ug99 resistance were identified including markers linked to known genes Sr2 and Lr34. Other markers were located in the chromosome regions where no Sr genes have been previously reported, including one each on chromosomes 1A, 2B, 4A and 7B, two on chromosome 5B and four on chromosome 6B. The same data were used for investigating epistatic interactions between markers with or without main effects. The marker csSr2 linked to Sr2 interacted with wPt4930 on 6BS and wPt729773 in an unknown location. Another marker, csLV34 linked to Lr34, also interacted with wPt4930 on 6BS and wPt4916 on 2BS. The frequent involvement of wPt4916 on 2BS and wPt4930 on 6BS in interactions with other significant loci on the same or different chromosomes suggested complex genetic control for adult plant resistance to Ug99 in winter wheat germplasm.     

Inheritance of leaf rust and stem rust resistance in 'Roblin' wheat.

The Canadian common wheat (Triticum aestivum L.) cultivar 'Roblin' is resistant to both leaf rust (Puccinia recondita Rob. ex. Desm.) and stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn.). To study the genetics of this resistance, 'Roblin' was crossed with 'Thatcher', a leaf rust susceptible cultivar, and RL6071, a stem rust susceptible line. A set of F6 random lines was developed from each cross. The random lines and the parents were grown in a field rust nursery artificially inoculated with a mixture of P. recondita and P. graminis isolates and scored for rust reaction. The same material was tested with specific races of leaf rust and stem rust. These data indicated that 'Roblin' has Lr1, Lr10, Lr13, and Lr34 for resistance to P. recondita and Sr5, Sr9b, Sr11, and possibly Sr7a and Sr12 for resistance to P. graminis. In a 'Thatcher' background, the presence of Lr34 contributes to improve stem rust resistance, which appears also to occur in 'Roblin'.     

Mapping of the oat crown rust resistance gene <Emphasis Type="Italic">Pc91</Emphasis>

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.     

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat

Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety Oberkulmer was made, and F₂ plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.     

Development of resistance gene analog polymorphism markers for the Yr9 gene resistance to wheat stripe rust.

The Yr9 gene, which confers resistance to stripe rust caused by Puccinia striiformis f.sp. tritici (P. s. tritici) and originated from rye, is present in many wheat cultivars. To develop molecular markers for Yr9, a Yr9 near-isogenic line, near-isogenic lines with nine other Yr genes, and the recurrent wheat parent 'Avocet Susceptible' were evaluated for resistance in the seedling stage to North American P s. tritici races under controlled temperature in the greenhouse. The resistance gene analog polymorphism (RGAP) technique was used to identify molecular markers for Yr9. The BC7:F, and BC7:F3 progeny, which were developed by backcrossing the Yr9 donor wheat cultivar Clement with 'Avocet Susceptible', were evaluated for resistance to stripe rust races. Genomic DNA was extracted from 203 BC7:F2 plants and used for cosegregation analysis. Of 16 RGAP markers confirmed by cosegregation analysis, 4 were coincident with Yr9 and 12 were closely linked to Yr9 with a genetic distance ranging from 1 to 18 cM. Analyses of nullitetrasomic 'Chinese Spring' lines with the codominant RGAP marker Xwgp13 confirmed that the markers and Yr9 were located on chromosome 1B. Six wheat cultivars reported to have 1B/1R wheat-rye translocations and, presumably, Yr9, and two rye cultivars were inoculated with four races of P. s. tritici and tested with 9 of the 16 RGAP markers. Results of these tests indicate that 'Clement', 'Aurora', 'Lovrin 10', 'Lovrin 13', and 'Riebesel 47/51' have Yr9 and that 'Weique' does not have Yr9. The genetic information and molecular markers obtained from this study should be useful in cloning Yr9, in identifying germplasm that may have Yr9, and in using marker-assisted selection for combining Yr9 with other stripe rust resistance genes.     

Physical mapping of the barley stem rust resistance gene rpg4

The barley stem rust resistance gene rpg4 was physically and genetically localized on two overlapping BAC clones covering an estimated 300-kb region of the long arm of barley chromosome 7(5H). Initially, our target was mapped within a 6.0-cM region between the previously described flanking markers MWG740 and ABG391. This region was then saturated by integrating new markers from several existing barley and rice maps and by using BAC libraries of barley cv. Morex and rice cv. Nipponbare. Physical/genetic distances in the vicinity of rpg4 were found to be 1.0 Mb/cM, which is lower than the average for barley (4 Mb/cM) and lower than that determined by translocation breakpoint mapping (1.8 Mb/cM). Synteny at high resolution levels has been established between the region of barley chromosome 7(5H) containing the rpg4 locus and the subtelomeric region of rice chromosome 3 between markers S16474 and E10757. This 1.7-cM segment of the rice genome was covered by two overlapping BAC clones, about 250 kb of total length. In barley the markers S16474 and E10757 genetically delimit rpg4, lying 0.6 cM distal and 0.4 cM proximal to the locus, respectively.     

Rds and Rih mediate hypersensitive cell death independent of gene-for-gene resistance to the oat crown rust pathogen Puccinia coronata f. sp. avenae.

The Pca crown rust resistance cluster in the diploid Avena genus confers gene-for-gene specificity to numerous isolates of Puccinia coronata f. sp. avenae. Recombination breakpoint analysis indicates that specificities conferred by the Pca cluster are controlled by at least five distinct genes, designated Pc81, Pc82, Pc83, Pc84, and Pc85. Avena plants with the appropriate genotype frequently respond to P. coronata by undergoing hypersensitive cell death at the sites of fungal infection. Autofluorescence of host cells in response to P. coronata occurs in plants that develop visible necrotic lesions but not in plants that lack this phenotype. Two newly described, non-Pc loci were shown to control hypersensitive cell death. Rds (resistance-dependent suppressor of cell death) suppresses the hypersensitive response (HR), but not the resistance, mediated by the Pc82 resistance gene. In contrast, Rih (resistance-independent hypersensitive cell death) confers HR in both resistant and susceptible plants. Linkage analysis indicates that Rds is unlinked to the Pca cluster, whereas Rih is tightly linked to it. These results indicate that multiple synchronous pathways affect the development of hypersensitive cell death and that HR is not essential for resistance to crown rust. Further characterization of these genes will clarify the relationship between plant disease resistance and localized hypersensitive cell death.     

Quantitative trait loci for partial resistance to crown rust, Puccinia coronata, in cultivated oat, Avena sativa L.

To facilitate the detection of quantitative trait loci (QTLs) for partial resistance to oat crown rust, Puccinia coronata f. sp. avenae Eriks., a genetic map was generated in a population of 158 F(6)-derived oat recombinant inbred lines from a cross of a partial resistance line MN841801-1 by a susceptible cultivar selection 'Noble-2'. The map, developed using 230 marker loci, mostly restriction fragment length polymorphism and amplified fragment length polymorphism markers, spanned 1,509 cM (Haldane) arranged into 30 linkage groups of 2-18 markers each. Four consistently detected major QTLs for partial rust resistance, Prq1a, Prq1b, Prq2, and Prq7, and three minor QTLs, Prq3, Prq5, and Prq6, were found in tests involving three field and two greenhouse environments. In addition, two major QTLs for flowering time, Ftq1 and Ftq7, and five weaker QTLs, Ftq2, Ftq3, Ftq4, Ftq5, and Ftq6, were revealed. Overlapping of the map segments of Ftq1 and Prq1 and of Ftq7 and Prq7 suggested either linkage between the flowering time QTLs and resistance QTLs or a pleiotropic effect of the Ftq QTLs on rust resistance. Relatively low heritability estimates (0.30) obtained for partial resistance to crown rust in the field indicate a potential value for marker-assisted selection.     

Identification of QTL and association of a phytoene synthase gene with endosperm colour in durum wheat

The yellow colour of durum wheat (Triticum turgidum L. var durum) semolina is due in part to the presence of carotenoid pigments found in the endosperm and is an important end-use quality trait. We hypothesized that variation in the genes coding for phytoene synthase (Psy), a critical enzyme in carotenoid biosynthesis, may partially explain the phenotypic variation in endosperm colour observed among durum cultivars. Using rice sequence information, primers were designed to PCR clone and sequence the Psy genes from Kofa (high colour) and W9262-260D3 (medium colour) durum cultivars. Sequencing confirmed the presence of four Psy genes in each parent, corresponding to a two member gene family designated as Psy1-1, Psy1-2 and Psy2-1 and Psy2-2. A genetic map was constructed using 155 F1-derived doubled haploid lines from the cross W9262-260D3/Kofa with 194 simple sequence repeat and DArT markers. Using Psy1-1 and Psy2-1 allele-specific markers and chromosome mapping, the Psy1 and Psy2 genes were located to the group 7 and 5 chromosomes, respectively. Four quantitative trait loci (QTL) underlying phenotypic variation in endosperm colour were identified on chromosomes 2A, 4B, 6B, and 7B. The Psy1-1 locus co-segregated with the 7B QTL, demonstrating an association of this gene with phenotypic variation for endosperm colour. This work is the first report of mapping Psy genes and supports the role of Psy1-1 in elevated levels of endosperm colour in durum wheat. This gene is a target for the further development of a molecular marker to enhance selection for endosperm colour in durum wheat breeding programs.     

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance.

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.     

Identification and mapping of a leaf rust resistance gene in barley line Q21861.

Barley line Q21861 possesses an incompletely dominant gene (RphQ) for resistance to leaf rust caused by Puccinia hordei. To investigate the allelic and linkage relations between RphQ and other known Rph genes, F2 populations from crosses between Q21861 and donors of Rph1 to Rph14 (except for Rph8) were evaluated for leaf rust reaction at the seedling stage. Results indicate that RphQ is either allelic with or closely linked to the Rph2 locus. A doubled haploid population derived from a cross between Q21861 and SM89010 (a leaf rust susceptible line) was used for molecular mapping of the resistance locus. Bulked segregant analysis was used to identify markers linked to RphQ, using random amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), and sequence tagged sites (STSs). Of 600 decamer primers screened, amplified fragments generated by 9 primers were found to be linked to the RphQ locus; however, only 4 of them were within 10 cM of the target. The RphQ locus was mapped to the centromeric region of chromosome 7, with a linkage distance of 3.5 cM from the RFLP marker CDO749. Rrn2, an RFLP clone from the ribosomal RNA intergenic spacer region, was found to be very closely linked with RphQ, based on bulked segregant analysis. An STS marker, ITS1, derived from Rrn2, was also closely linked (1.6 cM) to RphQ.