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.     

Genetic and physical mapping of Lrk10-like receptor kinase sequences in hexaploid oat (Avena sativa L.).

Oat receptor-like kinase gene sequences, homologous to the Lrk10 gene from wheat (Triticum aestivum L.), were mapped in oat (Avena sativa L.). PCR primers designed from the wheat Lrk10 were used to produce ALrk10 from oat. Two DNA sequences, ALrk1A1 and ALrk4A5, were produced from primers designed from coding and noncoding regions of ALrk10. Their use as RFLP probes indicated that the kinase genes mapped to four loci on different hexaploid oat 'Kanota' x 'Ogle' linkage groups (4_12, 5, 6, and 13) and to a fifth locus unlinked to other markers. Three of these linkage groups contain a region homologous to the short arm of chromosome I of wheat and the fourth contains a region homologous to chromosome 3 of wheat. Analysis with several nullisomics of oat indicated that two of the map locations are on satellite chromosomes. RFLP mapping in a 'Dumont' x 'OT328' population indicated that one map location is closely linked to Pg9, a resistance gene to oat stem rust (Puccinia graminis subsp. avenae). Comparative mapping indicates this to be the region of a presumed cluster of crown rust (Puccinia coronata subsp. avenae) and stem rust resistance genes (Pg3, Pg9, Pc44, Pc46, Pc50, Pc68, Pc95, and PcX). The map position of several RGAs located on KO6 and KO3_38 with respect to Lrk10 and storage protein genes are also reported.     

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.     

Restriction fragment length polymorphisms linked to genes for resistance to crown rust (Puccinia coronata) in near-isogenic lines of hexaploid oat (Avena sativa).

Crown rust, perhaps the most important fungal disease of oat, is caused by Puccinia coronata. An examination of near-isogenic lines (NILs) of hexaploid oat (Avena sativa) was conducted to identify markers linked to genes for resistance to crown rust. These lines were created such that a unique resistance gene is present in each of the two recurrent parent backgrounds. The six NILs of the current study, X434-II, X466-I, and Y345 (recurrent parent C237-89) and D486, D494, and D526 (recurrent parent Lang), thus provide a pair of lines to study each of three resistance genes. Restriction fragment length polymorphisms and resistance loci were mapped using BC1F2 populations. Three markers were found linked to a locus for resistance to crown rust race 203, the closest at 1.9 cM in line D494 and 3.8 cM in line X466-I. In lines D526 and Y345 a marker was placed 1.0 and 1.9 cM, respectively, from the locus conferring resistance to crown rust race 345, and in D486 and X434-II a marker mapped at 8.0 and 10.2 cM from the locus for resistance to rust race 264B.     

High-resolution mapping adjacent to the Pc71 crown-rust resistance locus in hexaploid oat

The D526-derived BC₁F₂ population of hexaploid oat segregates for resistance to crown rust isolate 345. A mapping population consisting of 440 F2 individuals was used to develop a high-resolution RFLP map of the area of the genome where Pc71, the locus conferring this resistance, is located. Twelve RFLP markers have been identified within ca. 6 cM of Pc71, with cosegregating loci Xcdo1502 and Xcdo783 positioned 0.2 cM from the locus. All of the RFLP markers map to the same side of the locus, suggesting either that the Pc71 resistance locus resides at the end of a linkage group, or that there is no detectable heterozygosity on the opposite side. Some degree of microcollinearity between rice and oat is present in this area, as the two markers, Xcdo783 and Xrz69, linked to Pc71 are linked also in rice; however the genetic distance in oat is much less than the genetic distance in rice (6.2 cM and 20 cM, 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.     

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.     

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.     

Four QTLs determine crown rust (Puccinia coronata f. sp. lolii) resistance in a perennial ryegrass (Lolium perenne) population

Crown rust resistance is an important selection criterion in ryegrass breeding. The disease, caused by the biotrophic fungus Puccinia coronata, causes yield losses and reduced quality. In this study, we used linkage mapping and QTL analysis to unravel the genomic organization of crown rust resistance in a Lolium perenne population. The progeny of a pair cross between a susceptible and a resistant plant were analysed for crown rust resistance. A linkage map, consisting of 227 loci (AFLP, SSR, RFLP and STS) and spanning 744 cM, was generated using the two-way pseudo-testcross approach from 252 individuals. QTL analysis revealed four genomic regions involved in crown rust resistance. Two QTLs were located on LG1 (LpPc4 and LpPc2) and two on LG2 (LpPc3 and LpPc1). They explain 12.5, 24.9, 5.5 and 2.6% of phenotypic variance, respectively. An STS marker, showing homology to R genes, maps in the proximity of LpPc2. Further research is, however, necessary to check the presence of functional R genes in this region. Synteny at the QTL level between homologous groups of chromosomes within the Gramineae was observed. LG1 and LG2 show homology with group A and B chromosomes of oat on which crown rust-resistance genes have been identified, and with the group 1 chromosomes of the Triticeae, on which leaf rust-resistance genes have been mapped. These results are of major importance for understanding the molecular background of crown rust resistance in ryegrasses. The identified markers linked to crown rust resistance have the potential for use in marker-assisted breeding.     

Genetic mapping of adult plant leaf rust resistance genes Lr48 and Lr49 in common wheat

Hypersensitive adult plant resistance genes Lr48 and Lr49 were named based on their genetic independence of the known adult plant resistance genes. This study was planned to determine genomic locations of these genes. Recombinant inbred line populations derived from crosses involving CSP44 and VL404, sources of Lr48 and Lr49, respectively, and the susceptible parent WL711, were used to determine the genomic locations of these genes. Bulked segregant analyses were performed using multiplex-ready PCR technology. Lr48 in genotype CSP44 was mapped on chromosome arm 2BS flanked by marker loci Xgwm429b (6.1 cM) and Xbarc7 (7.3 cM) distally and proximally, respectively. Leaf rust resistance gene Lr13, carried by the alternate parent WL711, was proximal to Lr48 and was flanked by Xksm58 (5.1 cM) and Xstm773-2 (8.7 cM). Lr49 was flanked by Xbarc163 (8.1 cM) and Xwmc349 (10.1 cM) on chromosome arm 4BL. The likely presence of the durable leaf rust resistance gene Lr34 in both CSP44 and VL404 was confirmed using the tightly linked marker csLV34. Near-isogenic lines for Lr48 and Lr49 were developed in cultivar Lal Bahadur. Genotypes combining Lr13 and/or Lr34 with Lr48 or Lr49 were identified as potential donor sources for cultivar development programs.     

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     

Linkage mapping of genes controlling resistance to white rust (Albugo candida) in Brassica rapa (syn. campestris) and comparative mapping to Brassica napus and Arabidopsis thaliana.

Genes for resistance to white rust (Albugo candida) in oilseed Brassica rapa were mapped using a recombinant inbred (RI) population and a genetic linkage map consisting of 144 restriction fragment length polymorphism (RFLP) markers and 3 phenotypic markers. Young seedlings were evaluated by inoculating cotyledons with A. candida race 2 (AC2) and race 7 (AC7) and scoring the interaction phenotype (IP) on a 0-9 scale. The IP of each line was nearly identical for the two races and the population showed bimodal distributions, suggesting that a single major gene (or tightly linked genes) controlled resistance to the two races. The IP scores were converted to categorical resistant and susceptible scores, and these data were used to map a single Mendelian gene controlling resistance to both races on linkage group 4 where resistance to race 2 had been mapped previously. A quantitative trait loci (QTL) mapping approach using the IP scores detected the same major resistance locus for both races, plus a second minor QTL effect for AC2 on linkage group 2. These results indicate that either a dominant allele at a single locus (Acal) or two tightly linked loci control seedling resistance to both races of white rust in the biennial turnip rape cultivar Per. The map positions of white rust resistance genes in B. rapa and Brassica napus were compared and the results indicate where additional loci that have not been mapped may be located. Alignment of these maps to the physical map of the Arabidopsis genome identified regions to target for comparative fine mapping using this model organism.     

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.     

来自粗山羊草抗条锈病基因的SSR标记

从粗山羊草[Aegilops tauschii (Coss.) Schmal] Y201中鉴定出1个显性抗小麦条锈病基因, 暂定名为YrY201。应用分离群体分组法(BSA) 筛选到Xgwm273b、Xgwm37和wmc14标记, 与该基因之间的遗传距离分别为11.9、5.8和10.9 cM。根据连锁标记所在小麦微卫星图谱的位置, YrY201被定位在7DL染色体上。分析基因所在染色体的位置及抗病性特征, 认为YrY201是一个新的抗小麦条锈病基因,并可用于分子标记辅助选择。     

Molecular mapping of a gene for stripe rust resistance in spring wheat cultivar IDO377s

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat worldwide. The best strategy to control stripe rust is to grow resistant cultivars. One such cultivar resistant to most races in North America is ‘IDO377s’. To study the genetics of its resistance this spring wheat cultivar was crossed with ‘Avocet Susceptible’ (AvS). Seedlings of the parents, F₂ plants, and F₃ lines were tested under controlled greenhouse conditions with races PST-43 and PST-45 of P. striiformis f. sp. tritici. IDO377s carries a single dominant gene for resistance. Resistance gene analog polymorphism (RGAP) and simple sequence repeat (SSR) techniques were used to identify molecular markers linked to the resistance gene. A total of ten markers were identified, two of which flanked the locus at 4.4 and 5.5 cM. These flanking RGAP markers were located on chromosome 2B with nulli-tetrasomic lines of ‘Chinese Spring’. Their presence in the ditelosomic 2BL line localized them to the long arm. The chromosomal location of the resistance gene was further confirmed with two 2BL-specific SSR markers and a sequence tagged site (STS) marker previously mapped to 2BL. Based on the chromosomal location, reactions to various races of the pathogen and tests of allelism, the IDO377s gene is different from all previously designated genes for stripe rust resistance, and is therefore designated Yr43. A total of 108 wheat breeding lines and cultivars with IDO377s or related cultivars in their parentage were assayed to assess the status of the closest flanking markers and to select lines carrying Yr43. The results showed that the flanking markers were reliable for assisting selection of breeding lines carrying the resistance gene. A linked stripe rust resistance gene, previously identified as YrZak, in cultivar Zak was designated Yr44.     

SNP markers linked to leaf rust and grain mold resistance in sorghum

Grain mold and rust are diseases that can significantly reduce sorghum grain yield. Breeding for resistance to these diseases is hindered by inefficient disease screening. A viable option to greatly improve breeding efficiency is to identify molecular markers or genes linked to the host resistance. In this study, we applied 14,739 single nucleotide polymorphism markers to the sorghum mini core of 242 accessions that had been evaluated for rust resistance in both greenhouse and field and for grain mold in the field for 2 years. Through association mapping we have identified two loci linked to grain mold resistance and five loci linked to rust resistance. Among the two loci linked to grain mold resistance, one contained a homolog of the maize nonhost resistance gene Rxo1. Two of rust-linked loci each contained the rust resistance gene homologous to the maize rust resistance gene Rp1-D which is the B locus (the A locus containing Pu was not linked in this study) and to the wheat rust resistance gene Lr1. The remaining loci contained genes important in other steps of the defense response, such as cyclophilins that mediate resistance response preceding hypersensitive response (HR) and Hin1 directly involved in producing HR. The results from this study will facilitate marker-assisted selection of host resistance to grain mold and rust in sorghum.     

Comparative mapping of homoeologous group 1 regions and genes for resistance to obligate biotrophs in Avena, Hordeum, and Zea mays.

The colinearity of markers linked with resistance loci on linkage group A of diploid oat, on the homoeologous groups in hexaploid oat, on barley chromosome 1H, and on homoeologous maize chromosomes was determined. Thirty-two DNA probes from homoeologous group 1 chromosomes of the Gramineae were tested. Most of the heterologous probes detected polymorphisms that mapped to linkage group A of diploid oat, two linkage groups of hexaploid oat, barley chromosome 1H, and maize chromosomes 3, 6, and 8. Many of these DNA markers appeared to have conserved linkage relationships with resistance and prolamin loci in Avena, Hordeum, and Zea mays. These resistance loci included the Pca crown rust resistance cluster in diploid oat, the R203 crown rust resistance locus in hexaploid oat, the Mla powdery mildew resistance cluster in barley, and the rp3, wsm1, wsm2, mdm1, ht2, and htn1 resistance loci in maize. Prolamin encoding loci included Avn in diploid oat and Hor1 and Hor2 in barley. A high degree of colinearity was revealed among the common RFLP markers on the small chromosome fragments among these homoeologous groups. Key words : disease resistance, colinearity, Gramineae, cereals.     

Identification and characterization of stripe rust resistance gene Yr34 in common wheat

An uncharacterized source of seedling resistance to Puccinia striiformis f.sp. tritici was identified in an advanced wheat breeding line WAWHT2046. Genetic analysis based on a WAWHT2046/Carnamah-derived double haploid (DH) population demonstrated monogenic inheritance of seedling stripe rust resistance in WAWHT2046. The gene controlling stripe rust resistance in line WAWHT2046 was tentatively designated YrWA. The chromosome 5AL located awn inhibitor gene B1, possessed by WAWHT2046, also showed monogenic inheritance when the DH population was scored for the presence and absence of awns. Joint segregation analysis at the B1 and YrWA loci indicated genetic linkage between the two loci. A recombination value of 12.2 cM was computed using Mapmanager. This association located YrWA in the chromosome arm 5AL. Molecular mapping using microsatellite markers placed YrWA distal to B1. All molecular markers mapped proximal to the awn inhibitor locus B1. As no other stripe rust resistance gene is reported to be located in the chromosome arm 5AL, YrWA was permanently designated as Yr34. Yr34 produced an intermediate (23C) seedling infection type and expressed very low stripe rust response (10R-MR) on adult plants in the field, similar to the resistance gene Yr17. In addition to Yr34, this mapping population segregated for three genetically independent adult plant stripe rust resistance genes. The detection of DH lines with completely susceptible response, higher than that shown by the Yr34-lacking parent Carnamah, suggested that both parents contributed adult plant resistance. The use of WAWHT2046 as a parent in breeding programs would also contribute APR in addition to Yr34.     

Detection of quantitative trait loci associated with leaf rust resistance in bread wheat.

Leaf rust, caused by Puccinia recondita Rob. ex Desm., is a common disease in wheat. The objective of this study was to develop molecular markers associated with the quantitative trait loci (QTLs) putatively conferring durable leaf rust resistance in Triticum aestivum L. em. Thell. A population of 77 recombinant inbred lines (RILs) developed from 'Parula' (resistant) and 'Siete Cerros' (moderately susceptible) was used. Bulked segregant analysis was done using random amplified polymorphic DNAs (RAPDs) with DNA enriched for low-copy sequences using hydroxyapatite chromatography. Out of 400 decamer primers screened, 3 RAPD markers were identified between the bulk of the most resistant and the bulk of the most susceptible lines. These were cloned and used as probes on the RILs in Southern hybridizations. Two probes revealed two tightly linked loci. One-way analysis of variance showed that these two loci, and another revealed by the third probe, were linked to QTLs controlling leaf rust resistance based on data taken from 2 years of replicated field trials. Cytogenetic analysis placed the two tightly linked loci on the long arm of chromosome 7B. The third probe detected loci located on the short arms of chromosomes 1B and 1D. It is suggested that the QTL detected on 7BL may well be homoeoallelic to Lr34.     

Isolation and mapping of resistance gene analogs from the Avena strigosa genome

Degenerate primers based on conserved regions of the nucleotide binding site (NBS) domain (encoded by the largest group of cloned plant disease resistance genes) were used to isolate a set of 15 resistance gene analogs (RGA) from the diploid species Avena strigosa Schreb. These were grouped into seven classes on the basis of 60% or greater nucleic acid sequence identity. Representative clones were used for genetic mapping in diploid and hexaploid oats. Two RGAs were mapped at two loci of the linkage group AswBF belonging to the A. strigosa × A. wiestii Steud map, and ten RGAs were mapped at 15 loci in eight linkage groups belonging to the A. byzantina C. Koch cv. Kanota × A. sativa L. cv. Ogle map. A similar approach was used for targeting genes encoding receptor-like kinases. Three different sequences were obtained and mapped to two linkage groups of the hexaploid oat map. Associations were explored between already known disease resistance loci mapped in different populations and the RGAs. Molecular markers previously linked to crown rust and barley yellow dwarf resistance genes or quantitative trait loci were found in the Kanota × Ogle map linked to RGAs at a distance ranging from 0 cM to 20 cM. Homoeologous RGAs were found to be linked to loci either conferring resistance to different isolates of the same pathogen or to different pathogens. This suggests that these RGAs identify genome regions containing resistance gene clusters.