CCN Resistance Research Articles

The dynamics of cereal cyst nematode infection differ between susceptible and resistant barley cultivars and lead to changes in (1,3;1,4)-β-glucan levels and HvCslF gene transcript abundance.

Heterodera avenae (cereal cyst nematode, CCN) infects the roots of barley (Hordeum vulgare) forming syncytial feeding sites. In resistant host plants, relatively few females develop to maturity. Little is known about the physiological and biochemical changes induced during CCN infection. Responses to CCN infection were investigated in resistant (Rha2) and susceptible barley cultivars through histological, compositional and transcriptional analysis. Two phases were identified that influence CCN viability, including feeding site establishment and subsequent cyst maturation. Syncytial development progressed faster in the resistant cultivar Chebec than in the susceptible cultivar Skiff, and was accompanied by changes in cell wall polysaccharide abundance, particularly (1,3;1,4)-β-glucan. Transcriptional profiling identified several glycosyl transferase genes, including CELLULOSE SYNTHASE-LIKE F10 (HvCslF10), which may contribute to differences in polysaccharide abundance between resistant and susceptible cultivars. In barley, Rha2-mediated CCN resistance drives rapid deterioration of CCN feeding sites, specific changes in cell wall-related transcript abundance and changes in cell wall composition. During H.avenae infection, (1,3;1,4)-β-glucan may influence CCN feeding site development by limiting solute flow, similar to (1,3)-β-glucan during dicot cyst nematode infections. Dynamic transcriptional changes in uncharacterized HvCslF genes, possibly involved in (1,3;1,4)-β-glucan synthesis, suggest a role for these genes in the CCN infection process.

Genetic mapping of the Cre8 locus for resistance against cereal cyst nematode (Heterodera avenae Woll.) in wheat

The cereal cyst nematode (CCN, Heterodera avenae Woll.) resistance locus Cre8 on the long arm of chromosome 6B (6BL) of wheat (Triticum aestivum L.) is effective in lowering the nematode population in soil. Identification of reliable and high-throughput molecular markers linked to the Cre8 locus is important for the successful deployment of Cre8-derived resistance in wheat breeding programs. Here, we report the addition of over 600 markers to improve an existing linkage map for the Trident/Molineux wheat population. With the improved map, Cre8 was mapped as a large-effect quantitative trait locus (QTL) near the distal end of 6BL. This QTL explained up to 35 % of the phenotypic variation in CCN resistance. New marker assays were developed for DNA polymorphisms in the Cre8 region. Seven molecular markers closely linked with Cre8 (at 0.9, 2.2 or 5.9 cM from the estimated QTL position) were found to be diagnostic across a panel of wheat cultivars and are recommended for use in marker-assisted selection of the Cre8 resistance locus in wheat breeding. Prospects for the isolation of the causal gene are discussed.

Association mapping for soilborne pathogen resistance in synthetic hexaploid wheat

Soilborne pathogens such as cereal cyst nematode (CCN; Heterodera avenae) and root lesion nematode (Pratylenchus neglectus; PN) cause substantial yield losses in the major cereal-growing regions of the world. Incorporating resistance into wheat cultivars and breeding lines is considered the most cost-effective control measure for reducing nematode populations. To identify loci with molecular markers linked to genes conferring resistance to these pathogens, we employed a genome-wide association approach in which 332 synthetic hexaploid wheat lines previously screened for resistance to CCN and PN were genotyped with 660 Diversity Arrays Technology (DArT) markers. Two sequence-tagged site markers reportedly linked to genes known to confer resistance to CCN were also included in the analysis. Using the mixed linear model corrected for population structure and familial relatedness (Q+K matrices), we were able to confirm previously reported quantitative trait loci (QTL) for resistance to CCN and PN in bi-parental crosses. In addition, we identified other significant markers located in chromosome regions where no CCN and PN resistance genes have been reported. Seventeen DArT marker loci were found to be significantly associated with CCN and twelve to PN resistance. The novel QTL on chromosomes 1D, 4D, 5B, 5D and 7D for resistance to CCN and 4A, 5B and 7B for resistance to PN are suggested to represent new sources of genes which could be deployed in further wheat improvement against these two important root diseases of wheat.

Effective Resources in Wheat and Wheat–Thinopyrum Derivatives for Resistance to Heterodera filipjevi in China

ABSTRACTCereal cyst nematode (CCN) is becoming one of the important soil‐borne pathogens in wheat (Triticum aestivum L.) monocropping or wheat–maize (Zea mays L.)–wheat cropping systems of central China. Heterodera filipjevi (Madzhidov, 1981) Stelter, 1984 was recently recognized as a causal agent of CCN in China, but little information is available on sources of resistance against this nematode species. The present study was initiated to determine the current status of resistance in wheat cultivars against H. filipjevi and to identify effective resources for improvement of CCN resistance in China. A 3‐yr field study of CCN resistance that involved 174 wheat cultivars or wheat–Thinopyrum derivatives was conducted in a wheat field in Xuchang, Henan Province, China, where H. filipjevi had been present for years. Greenhouse experiments were conducted with representative resistant entries from each group of accessions. None of the 78 wheat cultivars and breeding lines from China was resistant in field tests. Wheat cultivar Madsen from Washington State was most resistant among the entries tested both in field and under controlled environment. New sources of resistance to H. filipjevi were identified in some wheat–intermediate wheatgrass [Thinopyrum intermedium (Host) Barkworth & D. R. Dewey] and wheat–tall wheatgrass [Thinopyrum ponticum (Podp.) Barkworth & D. R. Dewey] partial amphiploids, which will diversify resistance resources in enhancing resistance of wheat against CCN.

Molecular mapping of cereal cyst nematode resistance in Triticum monococcum L. and its transfer to the genetic background of cultivated wheat

Triticum monococcum, the diploid A genome species, harbours enormous variability for resistance to biotic stresses. A spring type T. monococcum acc. 14087 was found to be resistant to Heterodera avenae (cereal cyst nematode, CCN). A recombinant inbred line population (RIL) developed by crossing this accession with a CCN susceptible T. boeoticum acc. 5088 was used for studying the inheritance and map location of the CCN resistance. Based on composite interval mapping two QTL, one each on chromosome 1AS and 2AS, were detected. The QTL on 1A, designated as Qcre.pau-1A, appeared to be a major gene with 26% contribution to the overall phenotypic variance whereas the QTL on 2A designated as Qcre.pau-2A contributed 13% to total phenotypic variation. Qcre.pau-1A is novel, being the only CCN resistance gene mapped in any ‘A’ genome species and none of the other known genes have been mapped on chromosome 1A. The QTL Qcre.pau-2A might be allelic to Cre5, a CCN resistance gene transferred from Ae. ventricosa and mapped on 2AS. The Qcre.pau-1A was transferred to cultivated wheat using T. durum cv. PBW114 as the bridging species. Selected CCN resistant F8 lines showed introgression for the molecular markers identified to be linked with CCN resistance locus Qcre.pau-1A. Thus, this gene alone could impart complete resistance against CCN. These introgression lines can be used for marker-assisted transfer of Qcre.pau-1A to elite wheat cultivars.

Molecular characterization of Cereal Cyst Nematode diagnostic markers Cre1 and Cre3 in some winter wheat germplasm and their potential use against Heterodera filipjevi

Two PCR based markers tightly linked to Cre1 and Cre3 resistant genes against the Cereal Cyst Nematode (CCN, causal agent Heterodera avenae) were used to characterize a number of diploid, tetraploid and hexaploid wheat germplasm consisting of obsolete, advanced lines and new cultivars from the Turkish National Breeding Program and the joint TURKEY CIMMYT ICARDA International Winter Wheat Improvement Program. In addition a number of landraces and wild relatives of wheat collected from the Turkish side of the Fertile Crescent, the centre of origin of cereals, were included. None of the genetic material tested positive for either marker except the land race Sardari which originated from neighboring Iran was found positive for Cre1 gene. Combining both of these molecular markers in one assay for fast and cost-effective screening of the germplasm was not effective, so marker genotyping with germplasm was realized using each primer alone. Most work that has been reported is on the CCN species H. avenae, however the predominant species in Turkey is the closely related H. filipjevi. In vitro resistance assaying for this species indicates that Cre1 is a moderate source of resistance against the H. filipjevi population, however Cre3 gene appears ineffective. It is suggested that a larger pool of a range of germplasm should be screened for Cre1 in Turkey. Furthermore, the landrace Sardari could be used as a parent to deploy CCN resistance in to genetic background of Turkish bread wheat cultivars with a targeted Marker Assisted Selection Backcrossing approach.

Characterization and expression profiling of a novel cereal cyst nematode resistance gene analog in wheat

Based on the conserved regions of known resistance genes, a NBS-LRR type CCN resistance gene analog was isolated from the CCN resistant E-10 near isogenic lines (NILs) of wheat, designated as CreZ (GenBank Acc. N: EU327996). It contained a complete ORF that was 2775 bp in length and encoded 924 amino acids. Sequence comparison indicated that it shared 92% nucleotide and 87% amino acid identity with those of the known CCN-resistance gene Cre3 and had similar characteristic conserved motifs as those in other established NBS-LRR disease resistance genes. The expression profiling of CreZ indicated that it was specifically expressed in the roots of resistant plants and real-time PCR analysis demonstrated that expression levels drastically increased when the plants were inoculated with cereal cyst nematodes. It could be inferred, then, that CreZ belongs to the NBS-LRR resistance gene family and is a candidate gene for potential resistance to the cereal cyst nematode.

Cereal cyst nematode resistance conferred by theCre7 gene fromAegilops triuncialisand its relationship withCregenes from Australian wheat cultivars

Cereal cyst nematode (CCN; Heterodera avenae Woll.) is a root pathogen of cereal crops that can cause severe yield losses in wheat (Triticum aestivum). Differential host-nematode interactions occur in wheat cultivars carrying different CCN resistance (Cre) genes. The objective of this study was to determine the CCN resistance conferred by the Cre7 gene from Aegilops triuncialis in a 42-chromosome introgression line and to assess the effects of the Cre1, Cre3, Cre4, and Cre8 genes present in Australian wheat lines on Spanish pathotype Ha71. Inhibition of nematode reproduction was rank-ordered as Cre1 >or = Cre4 > or = Cre7 >> Cre8 > Cre3. Lines carrying Cre1, Cre4, or Cre7 exhibited a significantly higher level of resistance than those carrying Cre8 or Cre3. Allelism tests indicated that Cre7 segregated independently of Cre1 on chromosome 2BL and Cre4 on chromosome 2DL, and these genes could consistently be combined in the same genotype, inducing a more durable resistance. Tests to determine the chromosomal location of Cre7 using addition lines were inconclusive.

Mapping of a novel QTL for resistance to cereal cyst nematode in wheat

Cereal cyst nematode (CCN; Heterodera avenae Woll.) is a root pathogen of cereals that can cause severe yield losses in intolerant wheat cultivars. Loci for resistance to CCN, measured by a seedling bioassay, were identified by creating a genetic map based on a Trident/Molineux doubled haploid population of 182 lines. A novel locus accounting for up to 14% of the resistance to CCN was mapped to chromosome 1B of Molineux by association with microsatellite marker loci Xwmc719 and Xgwm140. This locus acts additively with the previously identified CCN resistance loci identified on chromosomes 6B (Cre8) and 2A (Cre5 on the VPM1 segment) in this population to explain 44% of the genetic variance for this major wheat pathogen.

Impact of Cre1, Cre8 and Cre3 genes on cereal cyst nematode resistance in wheat

The cereal cyst nematode (CCN; Heterodera avenae), a root disease of cereal crops, is a major economic constraint in many wheat (Triticum aestivum)-growing areas of the world. The objective of this study was to assess the impact of the Cre1, Cre8 and Cre3 genes on CCN resistance. A population of 92 doubled-haploid (DH) lines derived from a cross between wheat cvs. Frame and Silverstar as well as 1,851 wheat breeding lines were screened for CCN resistance at the Primary Industries Research Victoria (PIRVic). A second population of 9,470 wheat breeding lines was screened at the South Australian Research and Development Institute (SARDI). Cre3 had the largest impact on reducing the number of female cysts, followed by Cre1 and Cre8. There was no significant difference in number of cysts between DH lines with or without the Cre8 marker, suggesting that the marker is not perfectly linked to Cre8. The estimated heritabilities were 0.32 in the DH population, 0.48 in the PIRVic data set and 0.32 in the SARDI data set, which confirm that this is a trait of low heritability. The repeatability of CCN resistance improved with an increase in the number of plants assessed per line-up to ten. However, 85-88% of the improvement was achieved with the assessments of the first five plants.

Mapping and QTL analysis of the barley population Galleon × Haruna Nijo

A genetic linkage map consisting of 435 molecular markers has been constructed using a doubled-haploid mapping population derived from a cross between the Australian barley feed variety Galleon and Haruna Nijo, a Japanese barley cultivar of high malting quality. This map was used to locate the genes conferring CCN and SFNB resistance from Galleon and to locate malting and brewing quality genes from Haruna Nijo. Closely linked markers to the trait loci have been identified and are now being widely implemented in Australian breeding programs.

Mapping of a QTL contributing to cereal cyst nematode tolerance and resistance in wheat

Cereal cyst nematode (CCN; Heterodera avenae Woll.) is a root pathogen of cereals that can cause severe yield losses in intolerant wheat cultivars. Tolerance to CCN, measured as early vigour in CCN-infested plots, was mapped in a Trident/Molineux doubled-haploid (DH) population. A locus accounting for a significant proportion of the tolerance to CCN was mapped to chromosome 6B of Molineux by association with RFLP loci Xcdo347-6B and Xbcd1 and also by nullisomic/tetrasomic substitution line analysis, and has been designated Cre8. The linkage of CCN tolerance with Xcdo347-6B was validated using a Barunga/Suneca DH population. The Cre8 locus also contributed to CCN resistance in the Trident/Molineux population. The RFLP locus Xbcd175, which is diagnostic for the Aegilops ventricosa segment VPM1 of Trident, explained up to 18% of the variation for early vigour in CCN-infested soils in the Trident/Molineux population. However, the Trident/Molineux population also segregated for early vigour in the absence of CCN, with Xbcd175 explaining up to 7% of the variation for this trait. The VPM1 segment of Trident therefore provides early vigour that may contribute to CCN tolerance in this cultivar.

Molecular-genetic characterisation of a new nematode resistance gene in wheat

Bread wheat lines introgressed with Aegilops ventricosa chromosomes were evaluated for their resistance to the Australian cereal cyst nematode (CCN, Heterodera avenae) pathotype Ha13. Higher levels of resistance relative to the phenotype of the Cre1 CCN resistance gene in wheat were found in the donor Ae. ventricosa parental lines and chromosome-5Nv substitution or addition lines. The newly identified resistance to pathotype Ha13 on chromosome 5Nv, designated, Cre6, was shown to be independent of the Ae. ventricosa-derived Cre2 gene, effective against several European pathotypes. Another Ae. ventricosa derived gene, Cre5, showed partial resistance to pathotype Ha13. Inhibition of Ha13 female nematode reproduction was ranked in the order Cre6 >Cre1 >CreF≥Cre5. Cre6 was inherited as a single dominant locus. Gene sequences encoding nucleotide-binding sites and leucine-rich repeats (NBS-LRR) from the Cre3 CCN-pathotype Ha13 resistance locus were used as probes to isolate related sequences from one of the donor Ae. ventricosa parents. Related sequences from Ae. ventricosa (71–73% similarity at the amino-acid level to the Cre3-derived sequences) of chromosome 5Nv origin were identified and served as diagnostic molecular markers for the presence of 5Nv. CCN-susceptible plants, found as variants in some of the purported chromosome 5Nv lines, were also found to be missing the diagnostic 5Nv RFLP markers assayed by the NBS-LRR probe. An alloplasmic chromosome-5Nv addition line with Ae. ventricosa cytoplasm in the wheat cultivar, Moisson, background was particularly variable, with 43% CCN-susceptible plants and a corresponding loss of the diagnostic chromosome-5 molecular markers.

A directed search for DNA sequences tightly linked to cereal cyst nematode resistance genes in Triticum tauschii

An improved system for identifying DNA sequences linked to a targeted region was developed by fractionating DNA sequences prior to polymerase chain reaction (PCR) analysis. In an attempt to identify DNA markers linked to a strong CCN resistance gene, Ccn-D1, in Triticum tauschii, DNA samples from individuals homozygous for resistance and susceptibility at the Ccn-D1 locus in a segregating progeny were bulked separately to produce "near isogenic" DNA pools. The polymerase chain reaction was employed to generate several DNA amplification products from each of the bulked DNA segregants using 240 random (RAPD) and 4 semirandom (consensus sequences of intron-splice junctions) primers. A DNA polymorphic fragment was apparent between the resistant and susceptible bulks using one of the semirandom primers. Hydroxylapatite chromatography of reannealed DNA (to Cot values > 100) was used to enrich low copy DNA sequences in the bulk DNA segregants (resistant and susceptible DNA pools). PCR analysis on the low copy enriched DNA pool increased the level of polymorphism detected between bulked segregants. One of the RAPD fragments present in only the resistant low copy DNA pool was cloned and mapped to the distal region of the long arm of chromosome 2D. By using the cloned RAPD fragment, csE20-2, to assay an RFLP locus in three independent F2 progenies, complete cosegregation was obtained with the Ccn-D1 locus. Joint segregation analysis from a genome-wide mapping of RFLP markers and a second CCN resistance in T. tauschii, Ccn-D2, showed this locus to be loosely linked to the proximal region of chromosome 2.