Cereal Cyst Nematode Resistance Research Articles

Identification of genomic regions associated with cereal cyst nematode (Heterodera avenae Woll.) resistance in spring and winter wheat

Cereal cyst nematode (CCN) is a major threat to cereal crop production globally including wheat (Triticum aestivum L.). In the present study, single-locus and multi-locus models of Genome-Wide Association Study (GWAS) were used to find marker trait associations (MTAs) against CCN (Heterodera avenae) in wheat. In total, 180 wheat accessions (100 spring and 80 winter types) were screened against H. avenae in two independent years (2018/2019 “Environment 1” and 2019/2020 “Environment 2”) under controlled conditions. A set of 12,908 SNP markers were used to perform the GWAS. Altogether, 11 significant MTAs, with threshold value of −log10 (p-values) ≥ 3.0, were detected using 180 wheat accessions under combined environment (CE). A novel MTA (wsnp_Ex_c53387_56641291) was detected under all environments (E1, E2 and CE) and considered to be stable MTA. Among the identified 11 MTAs, eight were novel and three were co-localized with previously known genes/QTLs/MTAs. In total, 13 putative candidate genes showing differential expression in roots, and known to be involved in plant defense mechanisms were reported. These MTAs could help us to identify resistance alleles from new sources, which could be used to identify wheat varieties with enhanced CCN resistance.

PAL-mediated SA biosynthesis pathway contributes to nematode resistance in wheat.

The pathogen cereal cyst nematode (CCN) is deleterious to Triticeae crops and is a threat to the global crop yield. Accession no.1 of Aegilops variabilis, a relative of Triticum aestivum (bread wheat), is highly resistant to CCN. Our previous study demonstrated that the expression of the phenylalanine ammonia lyase (PAL) gene AevPAL1 in Ae.variabilis is strongly induced by CCN. PAL, the first enzyme of phenylpropanoid metabolism, is involved in abiotic and biotic stress responses. However, its role in plant-CCN interaction remains unknown. In the present study, we proved that AevPAL1 helps to confer CCN resistance through affecting the synthesis of salicylic acid (SA) and downstream secondary metabolites. The silencing of AevPAL1 increased the incidence of CCN infection in roots and decreased the accumulation of SA and phenylalanine (Phe)-derived specialized metabolites. The exogenous pre-application of SA also improved CCN resistance. Additionally, the functions of PAL in phenylpropanoid metabolism correlated with tryptophan decarboxylase (TDC) functioning in tryptophan metabolism pathways. The silencing of either AevPAL1 or AevTDC1 exhibited a concomitant reduction in the expression of both genes and the contents of metabolites downstream of PAL and TDC. These results suggested that AevPAL1, possibly in coordination with AevTDC1, positively contributes to CCN resistance by altering the downstream secondary metabolites and SA content in Ae.variabilis. Moreover, AevPAL1 overexpression significantly enhanced CCN resistance in bread wheat and did not exhibit significant negative effects on yield-related traits, suggesting that AevPAL1 is valuable for the genetic improvement of CCN resistance in bread wheat.

Molecular mapping of a novel QTL for resistance to cereal cyst nematode in F4 wheat population

A wheat line selected from an off-type plant (Lnm136) was found to be resistant to Heterodera avenae (cereal cyst nematode, CCN). A F4 population developed by crossing this line with a CCN susceptible genotype, Ksu118, was used to study the map location of the CCN resistance. The results showed that the most significant QTLs for the cereal cyst nematode (CCN) score were identified on five chromosomes. One of the QTLs was located on chromosome 1B and linked closely to Xwmc85 based on composite interval mapping. A novel locus accounting for up to 70% of the resistance to CCN was mapped to chromosome 1B of Lnm136, by association with the microsatellite marker Xwmc85-1B. The selected CCN resistant F4 lines showed introgression for molecular markers identified to be linked with the CCN resistance locus Xwmc85-1B. This QTL on chromosome 1BS in the interval of Xwmc85–Xgpw4331 appears to be a major novel resistance locus. Multiple regression analysis showed the importance of the 1B region and marker-assisted selection for the favorable allele at this region should improve the resistance against CCN. The introgression lines can be used for marker-assisted transfer of Xwmc85-1B to elite wheat cultivars.

Resistance to Heterodera filipjevi and H. avenae in Winter Wheat is Conferred by Different QTL.

The coexistence of cereal cyst nematode (CCN) species Heterodera avenae and H. filipjevi, often involving multiple pathotypes, is a limiting factor for wheat production in China. Some of the known genes for resistance to CCN are not effective against both nematode species, hence complicating breeding efforts to develop CCN-resistant wheat cultivars. Here, we demonstrate that the CCN resistance in wheat cultivar Madsen to both Heterodera spp. is controlled by different genetic loci, both of which originated from Aegilops ventricosa. A new quantitative trait locus (QTL), QCre-ma7D, was identified and localized in a 3.77-Mb genomic region on chromosome arm 7DL, which confers resistance to H. filipjevi. QCre-ma2A on chromosome arm 2AS corresponds to CCN resistance gene Cre5 and confers resistance to H. avenae. This QTL is a new locus on chromosome arm 7DL and is designated Cre9. Three Kompetitive allele-specific PCR markers (BS00150072, BS00021745, and BS00154302) were developed for molecular marker-assisted selection of Cre9 and locally adapted wheat lines with resistance to both nematode species were developed. QCre-ma2A on chromosome arm 2AS corresponds to CCN resistance gene Cre5 and confers resistance to H. avenae. The identification of different loci underlying resistance to H. filipjevi and H. avenae and the development of adapted resistant entries will facilitate breeding of wheat cultivars that are resistant to these devastating nematodes in China.

Fine mapping of Rha2 in barley reveals candidate genes for resistance against cereal cyst nematode

Key messageThe cereal cyst nematode resistance locus Rha2 was mapped to a 978 kbp region on the long arm of barley chromosome 2H. Three candidate genes are discussed.The cereal cyst nematode (CCN) Heterodera avenae is a soil-borne obligate parasite that can cause severe damage to cereals. This research involved fine mapping of Rha2, a CCN resistance locus on chromosome 2H of barley. Rha2 was previously mapped relative to restriction fragment length polymorphisms (RFLPs) in two mapping populations. Anchoring of flanking RFLP clone sequences to the barley genome assembly defined an interval of 5077 kbp. Genotyping-by-sequencing of resistant and susceptible materials led to the discovery of potentially useful single nucleotide polymorphisms (SNPs). Assays were designed for these SNPs and applied to mapping populations. This narrowed the region of interest to 3966 kbp. Further fine mapping was pursued by crossing and backcrossing the resistant cultivar Sloop SA to its susceptible ancestor Sloop. Evaluation of F2 progeny confirmed that the resistance segregates as a single dominant gene. Genotyping of 9003 BC2F2 progeny identified recombinants. Evaluation of recombinant BC2F3 progeny narrowed the region of interest to 978 kbp. Two of the SNPs within this region proved to be diagnostic of CCN resistance across a wide range of barley germplasm. Fluorescence-based and gel-based assays were developed for these SNPs for use in marker-assisted selection. Within the candidate region of the reference genome, there are nine high-confidence predicted genes. Three of these, one that encodes RAR1 (a cysteine- and histidine-rich domain-containing protein), one that is predicted to encode an acetylglutamate kinase and one that is predicted to encode a tonoplast intrinsic protein, are discussed as candidate genes for CCN resistance.

Cereal cyst nematode resistance gene CreV effective against Heterodera filipjevi transferred from chromosome 6VL of Dasypyrum villosum to bread wheat

Cereal cyst nematodes (CCN) are a global economic problem for cereal production. Heterodera filipjevi is one of the most commonly identified and widespread CCN species found in many wheat production regions of the world. Transferring novel genes for resistance to H. filipjevi from wild relatives of wheat is a promising strategy for protection of wheat crops. A set of wheat–Dasypyrum villosum chromosome addition lines, T6V#4S·6AL translocation lines and their donor parental lines were tested for their response to the nematode. D. villosum and wheat–D. villosum disomic addition line DA6V#4 were resistant. As T6V#4S·6AL translocation lines were susceptible, resistance was presumed to be located on chromosome 6V#4L. The objective of this study was to produce and characterize wheat–6V#4L translocations and confirm the chromosome location of the resistance. Introgression lines T6V#4L·6AS, T6V#4L-4BL·4BS and DT6V#4L were developed and subjected to molecular cytogenetic analysis. These and four additional wheat–6V#4 introgression lines were tested for response to H. filipjevi in the greenhouse. The results indicated that introgression lines DA6V#4, T6V#4L·6AS, T6V#4L-4BL·4BS, T6V#4L·6V#4S-7BS and DT6VL#4 had higher levels of H. filipjevi resistance than their recurrent parent. However, Del6V#4L-1 and translocation line T6V#4S·6AL were equally susceptible to wheat cv. Chinese Spring. The CCN resistance gene, temporarily named CreV, was therefore physically mapped to chromosome arm 6V#4L FL 0.80–1.00. Translocation chromosomes T6V#4L·6AS transferred to a modern wheat cv. Aikang 58 with its co-dominant molecular markers could be utilized as a novel germplasm for CCN resistance breeding in wheat.

Characterization of Resistance to the Cereal Cyst Nematode in the Soft White Winter Wheat 'Madsen'.

The cereal cyst nematode (CCN) has a significant negative impact on production of wheat in China. The presence of pathotypes of both Heterodera avenae and H. filipjevi makes it necessary to identify genetic resources with a wide spectrum of resistance. Results of this study confirmed that the soft white winter wheat 'Madsen' was resistant to many different populations of both H. filipjevi and H. avenae in both naturally infested fields and artificial inoculation tests in China. Fewer juvenile nematodes penetrated roots of Madsen than susceptible 'Wenmai 19' in the early stages of the interaction between the nematodes and plant. Testing wheat cultivars in the pedigree of Madsen demonstrated that the CCN resistance of Madsen was inherited from 'VPM1' via the line 'VPM1/Moisson 951'. Presence of a 2NS chromosome segment from Aegilops ventricosa was detected in Madsen using a Vrga1D-specific marker. However, it appears that gene Pm4b for resistance to powdery mildew (caused by Blumeria graminis f. sp. tritici) was not transferred from VPM1 into Madsen because these cultivars had different reaction patterns against 20 B. graminis f. sp. tritici isolates from China. Madsen serves as an effective source of host resistance from damage caused by CCN.

Isolation and characterization of a candidate gene for resistance to cereal cyst nematode from Aegilops variabilis in China

Cereal cyst nematode (CCN) ( Heterodera avenae Woll.) is one of the most economically damaging endoparasite pests of wheat worldwide. We isolated and characterized a novel cereal CCN resistance candidate gene, CreV8 , from Aegilops variabilis (2n = 28, UUSvSv). The gene was 3,568 bp long and showed high nucleotide similarity with the known nematode resistance genes Cre3, CreZ, and go35 . CreV8 consisted of two exons, one intron and a complete ORF of 2,763 bp. The deduced peptide sequence consisted of 920 amino acid residues and shared 97.83% amino acid identity with Cre3. The sequence harbored a signal peptide domain, a nucleotide-binding ARC (NB-ARC) domain, and a leucine-rich repeat (LRR) domain, all of which are typical characteristics of resistance genes. We proposed the resistance mechanism of CreV8 based on functional analysis and predictions from its conserved domains and tertiary structure. Real-time polymerase chain reaction (PCR) showed that CreV8 was expressed in CCN-inoculated roots of resistant plants, and its expression levels were 2.80 and 27.45 times higher than that in the untreated control at 3 and 11 days post-inoculation, respectively. These results indicate that CreV8 belongs to the nucleotide binding site (NBS)-LRR resistance gene family and that it is a candidate CCN-resistance gene. Key words : Aegilops variabilis , CreV8, cereal cyst nematode (CCN), Heterodera avenae, tertiary structure.

De novo assembly and characterization of the root transcriptome of Aegilops variabilis during an interaction with the cereal cyst nematode.

BackgroundAegilops variabilis No.1 is highly resistant to cereal cyst nematode (CCN). However, a lack of genomic information has restricted studies on CCN resistance genes in Ae. variabilis and has limited genetic applications in wheat breeding.ResultsUsing RNA-Seq technology, we generated a root transcriptome at a sequencing depth of 4.69 gigabases of Ae. variabilis No. 1 from a pooled RNA sample. The sample contained equal amounts of RNA extracted from CCN-infected and untreated control plants at three time-points. Using the Trinity method, nearly 52,081,238 high-quality trimmed reads were assembled into a non-redundant set of 118,064 unigenes with an average length of 500 bp and an N50 of 599 bp. The total assembly was 59.09 Mb of unique transcriptome sequences with average read-depth coverage of 33.25×. In BLAST searches of our database against public databases, 66.46% (78,467) of the unigenes were annotated with gene descriptions, conserved protein domains, or gene ontology terms. Functional categorization further revealed 7,408 individual unigenes and three pathways related to plant stress resistance.ConclusionsWe conducted high-resolution transcriptome profiling related to root development and the response to CCN infection in Ae. variabilis No.1. This research facilitates further studies on gene discovery and on the molecular mechanisms related to CCN resistance.

Tahıl kist nematodu, Heterodera filipjevi (Madzhidov, 1981) Stelter’nin patotipinin belirlenmesi ve buğday genotiplerinin, H. filipjevi populasyonlarına karşı dayanıklılıklarının araştırılması

*Summary Determination of the pathotype group of Heterodera filipjevi (Madzhidov, 1981) population and resistance of H. populations against wheat genotypes The international pathotype differential lines were used to determine the pathotype group of Heterodera filipjevi (Madzhidov, 1981) Stelter population collected from Yozgat province, Turkey. The reaction of H. filipjevi Yozgat population on the differential lines indicated they were different than the other 5 known H. filipjevi pathotypes. Futherwork was conducted with these H. filipjevi populations to assess the usabilitiy of known published Cereal Cyst Nematode (CCN) resistance genes against a closely related species and pathotypes of Heterodera avenae Wollenweber, 1924. Several of these wheat lines known to possess resistance against H. avenae pathotypes and also the Haymana H. filipjevi populations did not exhibit resistance against H. filipjevi Yozgat populations.

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.

Assignment of Aegilops variabilis Eig chromosomes and translocations carrying resistance to nematodes in wheat

The allotetraploid species Aegilops variabilis Eig (2n = 28, UUSvSv) belongs to the tribe Triticeae and is closely related to wheat. One accession, Ae. variabilis No. 1, was found to be resistant to the cereal cyst nematode (CCN) and the root-knot nematode (RKN). As the genetic variability for resistance to those two pests is limited within wheat, this accession was crossed to bread wheat. Previous work enabled the development of two addition lines and two translocation lines carrying resistance. Here, we demonstrate, using genomic in situ hybridization, that there is no U-Sv interchange in the parental accession of Ae. variabilis. However, there are multiple rearrangements in the Sv chromosomes. The Ae. variabilis chromosome carrying the CreX gene for resistance to CCN combined segments with homoeology to wheat groups 1, 2, 4, and 6. The CreX gene belongs to the group 1 part and it was likely to have been introduced into chromosome 1BL at a similar location as the previously found QTL QCre.srd-1B for CCN resistance. The second Ae. variabilis chromosome carrying CreY and Rkn2 combined segments with homoeology to wheat groups 2, 4, and 7 on its short arm and group 3 on its long arm. It was designated as 3Sv. The two genes for resistance are carried by its long arm and have been transferred to wheat chromosome 3BL through homoeologous and genetically balanced recombination. Different SSR markers present in the introgressed segments could be used in marker-assisted selection.

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.

Mining synthetic hexaploids for multiple disease resistance to improve bread wheat

A collection of 253 synthetic hexaploid wheats (SHWs) produced from 192 Aegilops tauschii accessions and 39 elite durum varieties were studied to identify, characterise, and evaluate potentially untapped diversity of disease resistance in wheat. The diseases for which resistance was sought included cereal cyst nematode (CCN), root lesion nematode (RLN), Stagonospora nodorum blotch (SNB), Septoria tritici blotch (STB), and the 3 rusts, leaf rust, stem rust, and stripe rust, all important diseases of bread wheat worldwide, which can severely reduce wheat yield and quality. The SHWs exhibited a wide spectrum of resistance to the 8 pathogens. The frequency of disease-resistant SHWs ranged from 1% for one species of RLN (Pratylenchus neglectus), 3% and 10% for Septoria nodorum leaf and glume blotch, 10% for seedling resistance to yellow leaf spot, 16% for CCN, 21% for the second species of RLN (Pratylenchus thornei), 73% for Septoria tritici blotch, and 15%, 40%, and 24% for leaf rust, stem rust, and stripe rust, respectively. Five SHWs, Aus26860, Aus30258, Aus30294, Aus30301, and Aus30304, exhibited high levels of resistance to CCN, YLP, STB, LR, and SR, while 56 SHWs showed resistance to either 3 or 4 diseases. The genetics of resistance to CCN in some of the SHWs revealed that some of the accessions carry the same CCN gene(s) against pathotype Ha13, while others may carry different resistance gene(s). Additional studies were carried out to understand the relationship between the resistances identified in SHWs and the ones already present in common wheat, in particular the resistance genes Cre1 and Cre3 against CCN. The use of perfect markers associated with Cre1 and Cre3 suggested that some SHWs may carry a new CCN resistance gene(s), which could be deployed in breeding programs to increase the diversity of available resistance. The identification of SHWs with resistance to a range of diseases provides an opportunity to generate genetic knowledge and resistant germplasm to be used in future variety development.

Designing an optimal marker-based pedigree selection strategy for parent building in barley in the presence of repulsion linkage, using computer simulation

Pyramiding multiple desirable genes is an important method for the development of improved breeding materials and/or new cultivars. When the number of genes to be pyramided is many, or the genes are tightly linked in repulsion, it is practically impossible to recover the desirable recombinants in a single generation using a realistic population size, and repeated selection at several generations is required. The availability of markers tightly linked to the desirable genes makes it possible to conduct effective individual selection at early generations. This reduces the number of lines tested in the later generations and increases the desirable genotype frequency in the selected progeny. Computer simulation was used to develop such a marker-based pedigree selection strategy for the development of a barley line that contains 6 desired genes from 3 parental breeding lines (HS078 (H): 221222; PI366444 (P): 212222; Sloop Vic. (S): 122111; with 1 and 2 representing desirable and undesirable alleles, respectively), using the top cross H/P//S. The 6 genes targetted contribute to photoperiod sensitivity, Russian wheat aphid resistance, leaf rust resistance, boron tolerance, earliness per se, and cereal cyst nematode resistance. Under the assumption that perfect markers were available for all the 6 genes, a TC1 population of 300 plants was required to obtain 3 or more lines of the best genotype ‘211222/122111’, in which 3 loci were fixed for the desirable alleles, while the remaining 3 were kept as heterozygous. When single seed descent was used from the TC2 generation until complete homozygosity, the probability of obtaining lines of the desirable genotype (fixed for the desirable alleles at all 6 loci) was low due to the tight repulsion linkage between some of the genes. About 4000 individuals would be required to ensure with 99% probability the recovery of at least 1 line with the desirable genotype. The total number of lines that would need to be genotyped would be at least 5000. When the pedigree method was used in all test-cross generations, many schemes resulted in more lines of the fixed desirable genotype by genotyping fewer lines. The various options were compared using the genetic simulation software module QuLine, based on the QU-GENE simulation platform. The optimum scheme in terms of high success rate and relatively low genotyping costs consisted of the following steps: (1) in TC1 genotyping of 300 individuals allows for 3 or more individuals with the genotype ‘211222/122111’ to be identified; (2) in the TC2 individuals that are fixed for 3 loci and segregating for the remaining 3, loci can be selected from among 500 TC2 plants; (3) in the TC3, 50 or more individuals per TC3 line are genotyped for the 3 segregating loci, and individuals fixed for 5 loci and segregating for the 6th locus can be detected (genotyping is only needed for the segregating loci); (4) 25 individuals per TC4 line are genotyped for the single remaining segregating locus and several individuals of the desirable genotype (111111/111111) are finally selected. The desirable line is then obtained by collecting selfed seed from the selected TC4 plants. Using this scheme, on average, 320 desired TC5 lines were obtained by genotyping fewer than 2000 lines. When markers were tightly linked to the target genes but not diagnostic (perfect), not only was more genotyping required, but also appropriate phenotyping at the end of the marker selection process was necessary to confirm the presence of all the target genes. Under the assumption that recombination between marker and target gene was 5%, the best selection scheme identified, on average, 30 fixed desirable lines by genotyping 8000 lines and phenotyping 700 TC5 lines. If double haploid lines were produced from the F1 generation between H and P, and marker and phenotypic screening were conducted, followed by crossing of the individual with the target 2 loci in desired homozygous allelic status with parent S, the total amount of genotyping and phenotyping could be halved. This study showed that genetic simulation allows for numerous strategies to be compared using real data, and to develop an optimal crossing and selection strategy to combine desired alleles in the most effective and efficient way. This approach could likewise be used in other marker-assisted breeding programs.

Marker-assisted pyramiding of two cereal cyst nematode resistance genes from Aegilops variabilis in wheat

The cereal cyst nematode (CCN) Heterodera avenae, is a significant pathogen of wheat. The wild grass Aegilops variabilis Accession No.1 has been found to be resistant to pathotypes of CCN; at least two genes transferred to wheat, designated as CreX and CreY, are involved in the resistance response. The CreY gene may be the same as Rkn-mn1, which confers resistance to root knot nematode (RKN) Meloidogyne naasi. The objective of this work was to pyramid the two CCN resistance genes in a wheat background through marker-assisted selection. As a first step, molecular markers flanking CreX were identified. The completely linked RAPD marker of Rkn-mn1 (CreY), OpY16-1065, previously obtained, was converted into a SCAR. All these dominant markers were used to incorporate in the same genotype the two Ae. variabilis chromosome segments carrying the two genes for resistance. CCN bioassays with the Ha12 pathotype showed that the level of resistance of the pyramided line was significantly higher than that of CreX and CreY single introgression lines, but lower than that of Ae. variabilis. This study thus illustrates the utilization of molecular markers in breeding for host resistance.

Cloning and sequence analysis of the leucine-rich repeats region of cereal cyst nematode resistance gene

AbstractAccording to the sequence of Rccn4, which is highly similar to the nucleotide-binding site (NBS) coding region of the cereal cyst nematode resistance gene, Cre3, three 3′ nested primers were designed to amplify its 3′ flanking region through single oligonucleotide nested polymerase chain reaction (SON-PCR). One 1264 bp band, Rccn-L, was amplified from E-10, a wheat–Aegilops variabilis translocation line containing the cereal cyst nematode resistance gene from Ae. variabilis. Sequence analysis showed that Rccn-L possesses the 3′ flanking sequence of Rccn4 and contains a 55 bp-sized consensus sequence with Rccn4. The coding region was 1026 bp, consisting of an incomplete open reading frame, a terminator codon and no initiation codon and intron; it encoded a peptide of 342 amino acid residues and shared 86% nucleotide sequence identity with Cre3. The peptide had a conserved leucine-rich repeat (LRR) domain, containing the imperfect repeats, XXLXXLXXL, comprising 17% leucine residues, and shares, respectively, 89% nucleotide sequence and 78% amino acid sequence identity with the LRR sequence of the Cre3 locus. In the present study, SON-PCR was used successfully, for the first time, in plant genome research and proved to be a valuable method in plant gene cloning. The acquirement of Rccn-L established the foundation for obtaining the complete Rccn gene and further structural and functional investigations.

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.

Breeding for resistance to wheat nematodes (Heterodera avenae)

A new wheat variety resistant to cereal cyst nematode (CCN), CCNRV 1 (Raj Molya Rodhak 1), was developed from two genetically diverse cultivars in a single cross (J 24/AUS 15854). This variety exhibited a higher level of productivity in both CCN-infested and normal soils, with increases in the grain and straw yields of 78.7% and 60.1%, respectively, over Raj 3077 in infested soils. It also gave 19.0% higher yield than local varieties under timely-sown irrigated conditions in normal soils. It possesses superior grain quality along with other desirable agronomic traits. Genetically it carries a dominant gene for CCN resistance. It is a robust and reliable wheat variety that offers a high degree of resistance against nematodes in warmer areas of Rajasthan. It was recommended for timely-sown, irrigated conditions in CCN-infested areas of Rajasthan by the State Seed Sub-Committee on Crop Standards, Notification and Release of Varieties in September 2002. It is envisaged that this variety will help to boost wheat production and alleviate the socio-economic problems of subsistent Indian farmers in CCN-infested areas.