Cereal Cyst Nematode Resistance Research Articles

Identification of microsatellite markers associated with the cereal cyst nematode resistance gene Cre3 in wheat

Cereal cyst nematode (CCN) is a root disease caused by the pathogen Heterodera avenae Woll. that significantly reduces wheat (Triticum aestivum L.) grain yields in temperate countries. The Cre3 gene, located on chromosome 2DL, provides high levels of resistance to the Australian pathotype and isolates from Syria and Algeria, and has become available to wheat breeders. Selection for lines carrying the Cre3 gene in Australian wheat breeding programs is currently based on a dominant PCR marker (Cre3spf/r) diagnostic for the Cre3 gene. However, this marker has limitations that increase the cost and reduce selection efficiency in screening early-generation breeding lines. Such limitations would be minimised by the identification of a microsatellite marker linked to the Cre3 gene. We have constructed 2 genetic linkage maps of wheat chromosome 2DL and identified microsatellite markers mapping closely to the diagnostic Cre3spf/r marker. These closely linked markers were validated in a diverse range of germplasm, and one microsatellite marker, Xgwm301, which mapped 4 cM from Cre3spf/r, was shown to be highly associated with the presence of the Cre3 gene. Amplification conditions for the Xgwm301 locus were optimised, and its use in marker-assisted selection to identify Cre3 CCN-resistant wheat in the Australian Grain Technologies breeding program is demonstrated.

The cre1 and cre3 nematode resistance genes are located at homeologous loci in the wheat genome.

Differential responses in host-nematode pathotype interactions occur in wheat lines carrying different cereal cyst nematode resistance (Cre) genes. Cre1, located on chromosome 2B, confers resistance to most European nematodes and the sole Australian pathotype, while Cre3, present on chromosome 2D, is highly resistant to the Australian pathotype and susceptible to a number of European pathotypes. Genes encoding nucleotide binding site-leucine rich repeat (NBS-LRR) proteins that cosegregate with the Cre3 locus cross hybridize to homologues whose restriction fragment length polymorphism (RFLP) patterns distinguish near-isogenic Cre1 nematode-resistant wheat lines. Genetic mapping showed that the NBS-LRR gene members that distinguished the Cre1 near-isogenic lines were located on chromosome 2BL at a locus, designated Xcsl107, that cosegregates with the Cre1 locus. A haplotype of NBS-LRR genes from the Xcsl107 locus provides a diagnostic marker for the presence of Cre1 nematode resistance in a wide collection of wheat lines and segregating families. Genetic analysis of NBS-LRR haplotypes that cosegregate with Cre1 and Cre3 resistance, together with flanking cDNA markers and other markers from homoeologous group 2 chromosomes, revealed a conserved gene order that suggests Cre1 and Cre3 are homeoloci.

Mapping and QTL analysis of the barley population Clipper × Sahara

A genetic linkage map consisting of 211 molecular markers has been generated using a doubled-haploid population derived from a cross between the Australian barley variety Clipper and the Algerian landrace Sahara 3771. The map was used in subsequent trait mapping studies to locate the genes conferring boron tolerance and cereal cyst nematode resistance from Sahara 3371 and to map several plant type and developmental genes. 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.

A comparison of the genetics of seedling and adult plant resistance to the spot form of net blotch (Pyrenophora teres f. maculata)

Spot form of net blotch (SFNB) (Pyrenophora teres f. maculata) is an economically damaging foliar disease of barley in many of the world's cereal-growing areas. The gene Rpt4 that confers seedling resistance to SFNB has been mapped on the long arm of chromosome 7H, but no genes for adult plant resistance (APR) have been identified. A lack of field resistance to SFNB in breeders' lines selected for Rpt4 led us to investigate the genetics of APR to this disease. Five doubled-haploid populations were phenotyped for seedling and adult plant reaction. Markers linked to Rpt4 explained a large part of the seedling variation, but little of the APR. In 2 mapped populations, major quantitative trait loci (QTLs) for APR distal to Rpt4 on chromosome 7H were identified. QTLs contributing to APR on chromosomes 4H or 5H were also identified in each population. Association of the 5H QTL with a gene for cereal cyst nematode resistance and the probable effect of this linkage on the historical development of cultivars with adult plant resistance to SFNB is discussed.

Deletion Mapping of a Nematode Resistance Gene on Rye Chromosome 6R in Wheat

Four deletion mutants of rye chromosome 6R were identified in progeny of wheat (Triticum aestivum L.) lines of ph1bph1b genotype and monosomic for chromosome 6R. The rye chromosome carried a resistance gene against the cereal cyst nematode (CCN) (Heterodera avenae Woll.) and this chromosome originated in triticale line T‐701 (× Triticosecale Witt.). The deletion mutants were selected on the basis of dissociation of three isozyme loci on the long arm of the rye (Secale cereale L.) chromosome. Three plants and their progeny showed expression of the rye genes α‐Amy‐R1 and Got‐R2 but lacked the gene PgdR2 The other plant and its progeny showed expression of the α‐Amy‐R1 gene while the genes Got‐R2 and PgdR2 were absent. The four deletion chromosomes displayed long‐arm terminal deficiencies of different sizes which enabled mapping of the rye isozyme genes and the CCN resistance gene. The distal to proximal order of the 6R isozyme loci was found to be: PgdR2, Got‐R2, and then α‐Amy‐R1 Bioassay tests demonstrated that the CCN resistance gene (CreR) was located on an interstitial section of the long arm of 6R adjacent to Got‐R2

The Aegilops ventricosa segment on chromosome 2AS of the wheat cultivar ‘VPM1’ carries the cereal cyst nematode resistance gene Cre5

AbstractPrevious studies showed that the intermediate level of resistance in bread wheat line ‘VPM1’ to pathotype Ha12 of the cereal cyst nematode could be conferred by an Aegilops ventricosa‐derived gene, CreX, in chromosome arm 2AS, which also carries the rust resistance genes Yrl7, Lr37 and Sr38. Near isogenic lines (NILs) differing for the presence and absence of the Ae. ventricosa‐derived linked genes Yrl7/Lr37/Sr38 were tested with cereal cyst nematode. Lines carrying Yr17 produced significantly fewer nematode cysts than the controls. An infested soil experiment produced better differentiation among resistant and susceptible genotypes. Susceptibility of ‘Trident’ indicated that linkage between CreX and Yr17 is incomplete. Microsatellite markers did not differentiate between ‘Trident’ and CreX‐carrying genotypes. However, Xgwm636 (104) was associated with the presence of Yr17 in all six genetic backgrounds. Since none of the reported cereal cyst nematode resistance genes is located in chromosome 2AS, CreX was designated as Cre5.

Diagnostic DNA markers for cereal cyst nematode resistance in bread wheat

The development of cultivars resistant to cereal cyst nematode (CCN) is a primary objective in wheat breeding in the southern wheatbelt of Australia. Nine CCN resistance genes have been identified in wheat and its relatives, some of which confer resistance to the Australian pathotype of CCN (Ha13). Cultivars released in Australia with CCN resistance carry either the Cre1 or CreF gene, with the Cre3 gene present in advanced breeding lines. The biological assay for CCN resistance screening in wheat is time-consuming, not reliable on a single-plant basis, and prone to inconsistencies, thus reducing the efficiency of selection amongst breeding lines. Using gene sequences initially isolated from the Cre3 locus, a DNA-based marker selection system was developed and applied to unambiguously identify wheat lines carrying resistance alleles at theCre1 and/or Cre3 loci in breeding populations derived from diverse genetic backgrounds. Homologues of sequences from the Cre3 locus, located elsewhere in the wheat genome, can also be used to select wheat lines with a newly identified CCN resistance gene (Cre6) introgressed from Aegilops ventricosa. Application of these markers has become an integral part of the southern Australian breeding programs.

Construction and characterisation of a large DNA insert library from the D genome of wheat

A large DNA fragment library consisting of 144 000 clones with an average insert size of 119 kb was constructed from nuclear DNA isolated from root and leaf tissue from Triticum tauschii (syn. Aegilops tauschii), the D-genome progenitor of wheat. The library was made in a binary vector that had previously been shown to stably maintain large inserts of foreign DNA in Escherichia coli. The use of root nuclei reduced considerably the proportion of the library containing clones derived from chloroplast DNA. Several experimental parameters were investigated and optimised, leading to a high cloning efficiency. Only three ligations were needed to construct the library which was estimated to be equivalent to 3.7 haploid genomes. The accuracy of this estimation was demonstrated by screening this library with three well-defined probes. One probe containing a glutenin gene sequence identified 5 clones covering at least 230 kb of the Glu-D1 locus and contained the two tightly linked high-molecular-weight glutenin genes Glu-D1x and -D1y. Each of the other two single-copy probes derived from the Cre3 cereal cyst nematode resistance gene locus hybridised with 4 clones containing gene sequences encoding nucleotide binding sites and a leucine-rich region. This is the first representative large-insert DNA library for wheat, and the results indicated that large molecules of wheat DNA can be efficiently cloned, stably maintained and manipulated in a bacterial system.

A superfamily of disease resistance gene analogs is located on all homoeologous chromosome groups of wheat (Triticum aestivum)

In this study, resistance gene analogs (RGAs) which were isolated from monocot crop species (wheat, barley, maize and rice) and contained conserved sequence motifs found within the nucleotide binding site - leucine rich repeat (NBS-LRR) class of resistance genes, were used to assess their distribution in the wheat genome. The RGAs showed 30-70% amino acid identity to a previously isolated monocot NBS-LRR sequence from the Cre3 locus for cereal cyst nematode (CCN) resistance in wheat. We used the RGAs as probes to identify and map loci in wheat using recombinant inbred lines of an international Triticeae mapping family. RGA loci mapped across all seven homoeologous chromosome groups of wheat. This study demonstrated that the RGA mapping approach provides potential entry points toward identifying resistance gene candidates in wheat.Key words: wheat, disease resistance genes, nucleotide binding site, leucine rich repeat, resistance gene analogs.

RFLP mapping of a new cereal cyst nematode resistance locus in barley

Cereal cyst nematode (CCN) (Heterodera avenae Woll.) is an economically damaging pest of barley in many of the worlds cereal growing areas. The development of CCN‐resistant cultivars may be accelerated with the application of molecular markers. Three resistance genes against the pest have been mapped previously to chromosome 2 (Ha1, Ha2 and Ha3). In this study, a third gene present in the Australian barley variety ‘Galleon’ derived from the landrace ‘CI3576’ was located. Segregation analysis of CCN resistance data derived from doubled haploid populations of the cross ‘Haruna Nijo’בGalleon’ identified a single major locus controlling CCN resistance in the variety ‘Galleon’. This locus mapped to the long arm of chromosome 5H estimated to be 6.2 cM from the known function restriction fragment length polymorphism marker XYL (xylanase). While five genes for CCN resistance, including Ha2, have been mapped to group 2 chromosomes in the Triticeae, no gene other than Ha4 has been identified on group 5 chromosomes.

RFLP mapping of the Ha 2 cereal cyst nematode resistance gene in barley

The cereal cyst nematode (CCN), Heterodera avenae Woll., is an economically damaging pest of barley in many of the world’s cereal-growing areas. The development of CCN-resistant cultivars may be accelerated through the use of molecular markers. A number of resistance genes against the pest are well known; one of them, the single dominant Ha 2 resistance gene, has been shown to be effective against the Australian pathotype and maps to chromosome 2 of barley. Segregation analysis identified two restriction fragment length polymorphism (RFLP) markers flanking the resistance gene in two doubled-haploid populations of barley. AWBMA 21 and MWG 694 mapped 4.1 and 6.1 cM respectively from the Ha 2 locus in the Chebec×Harrington cross and 4.0 and 9.2 cM respectively in the Clipper×Sahara cross. Analysis of a further seven sources of CCN resistance in the form of near-isogenic lines (NILs) indicates that all available sources of resistance to the Australian pathotype of CCN in barley represent the Ha 2 locus.

Identification of RFLP markers linked to the cereal cyst nematode resistance gene (Cre) in wheat

The cereal cyst nematode (CCN) (Heterodera avenae Woll.) is an economically damaging pest of wheat in many of the worlds cereal growing areas. The development of CCN-resistant cultivars may be accelerated by the use of molecular markers. The Cre gene of the wheat line "AUS 10894" confers resistance to CCN. Using a pair of near-isogenic lines (NILs) that should differ only in a small chromosome segment containing the Cre locus, we screened 58 group-2 probes and found two (Tag605 and CDO588) that detect polymorphism between the NILs. Nulli-tetrasomic and ditelosomic lines confirmed that the restriction fragment length polymorphism (RFLP) markers identified were derived from the long arm of wheat chromosome 2. Crosses between "AUS 10894" and "Spear" and the NIL "AP" and its recurrent parent "Prins" were used to produce F2 populations that gave the expected 3∶1 segregation ratio for the resistance gene. Linkage analysis identified two RFLP markers flanking the resistance gene. Xglk605 and Xcdo588 mapped 7.3 cM (LOD=6.0) and 8.4 cM (LOD=6.7), respectively, from the Cre locus.

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.

From somatic variation to variant plants: mechanisms and applications

Plants from cell cultures show a high incidence of mutation. The causes of somaclonal variation are unknown, but the genetic consequences have been analysed. A range of genetic events are responsible, including single base changes; altered gene copy number; altered expression of multigene families; chromosome fragment interchanges; and mobilisation of transposable elements. Recent applications of these phenomena are discussed in this paper, for example, in vitro selection, somaclonal variation for agronomic traits, and limited gene transfer from cultured sexual and somatic hybrids. Research is outlined attempting to use high frequency chromosomal interchanges in wheat cell cultures to introgress genes from alien chromosomes. Two examples are cereal cyst nematode resistance on a rye chromosome and barley yellow dwarf resistance on a Thinopyrum chromosome. Some presumptive introgressed lines show selfed and backcross segregations, suggesting that the resistance, but not other alien markers, is now on a wheat chromosome.Key words: somaclonal variation, introgression, somatic hybridization, chromosome rearrangement, in vitro selection.

Genetic studies of resistance to the cereal cyst nematode (Heterodera avenae) in oats (Avena spp.)

Genetic analysis of cereal cyst nematode resistance in three genotypes of oats indicates that resistance in Avena sterilis I.376 is controlled by two dominant genes and in A. sativa cv. Mortgage Lifter by two recessive genes. Resistance in A. byzantina P.I. 175021 is dominant and monogenic, and is probably at a locus distinct from the resistance loci in the other two genotypes.

Field experiments with spring barley resistant to cereal cyst nematode, 1965–1968

SUMMARYTwo bulk populations of spring barley lines differing in respect of a single dominant gene for cereal cyst nematode (Heterodera avenae) resistance were used in trials over 4 years to assess the effect of the nematode on grain yield. On an infested site the resistant lines consistently and significantly out‐yielded the susceptible lines by an average of 9 % over the 4 years. On non‐infested sites, there was no difference in yield between the resistant and susceptible lines.To measure changes in the cereal cyst nematode population under continuous barley cultivation, the resistant and susceptible lines were each sown at the infested site on the same plots for 4 consecutive years. The cereal cyst nematode population declined under both susceptible and resistant barley, but more rapidly under the latter. Migratory nematodes, mostly Pratylenchus minyus, were latterly prevalent on all plots.There was no detectable change in the pathogenicity of the cereal cyst nematode population after 3 years of growing resistant barley.