Identification of RFLP markers closely linked to the H1 gene conferring resistance to Globodera rostochiensis in potato

Summary Resistance to the root cyst nematode Globodera rostochiensis is an agronomic trait that is at present incorporated into most new potato varieties. Major dominant genes are available that originate from wild and cultivated Solanum species closely related to the cultivated European potato (Solanum tuberosum ssp. tuberosum). One of those genes, H1, from S. Tuberosum ssp. andigena, was mapped to a distal position on potato chromosome V using restriction fragment length polymorphism (RFLP) markers. The H1 locus segregates independently from Gro1, a second dominant gene presumably from S. Spegazzinii that confers resistance to G. Rostochiensis and which has been mapped to chromosome VII. One marker, CP113, was linked without recombination to the H1 locus.     

A high-resolution map of the H1 locus harbouring resistance to the potato cyst nematode Globodera rostochiensis

The resistance gene H1 confers resistance to the potato cyst nematode Globodera rostochiensis and is located at the distal end of the long arm of chromosome V of potato. For marker enrichment of the H1 locus, a bulked segregant analysis (BSA) was carried out using 704 AFLP primer combinations. A second source of markers tightly linked to H1 is the ultra-high-density (UHD) genetic map of the potato cross SH × RH. This map has been produced with 387 AFLP primer combinations and consists of 10,365 AFLP markers in 1,118 bins (). Comparing these two methods revealed that BSA resulted in one marker/cM and the UHD map in four markers/cM in the H1 interval. Subsequently, a high-resolution genetic map of the H1 locus has been developed using a segregating F₁ SH × RH population consisting of 1,209 genotypes. Two PCR-based markers were designed at either side of the H1 gene to screen the 1,209 genotypes for recombination events. In the high-resolution genetic map, two of the four co-segregating AFLP markers could be separated from the H1 gene. Marker EM1 is located at a distance of 0.2 cM, and marker EM14 is located at a distance of 0.8 cM. The other two co-segregating markers CM1 (in coupling) and EM15 (in repulsion) could not be separated from the H1 gene.Communicated by J.G. Wenzel     

Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis

Summary A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers. The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test. By linkage to nine RFLP markers the resistance locus Gro1 was assigned to the potato linkage group IX which is homologous to the tomato linkage group 7. Deviations from the additivity of recombination frequencies between Gro1 and its neighbouring markers in the pot test led to the detection of a few phenotypic misclassifications of small plants with poor root systems that limited the observation of cysts on susceptible roots. Pooled data from both tests provided better estimates of recombination frequencies in the linkage interval defined by the markers flanking the resistance locus.     

Mapping of resistance to the potato cyst nematode Globodera rostochiensis from the wild potato species Solanum vernei

A population of diploid potato (Solanum tuberosum) was used for the genetic analysis and mapping of a locus for resistance to the potato cyst nematode Globodera rostochiensis, introgressed from the wild potato species Solanum vernei. Resistance tests of 108 genotypes of a F₁ population revealed the presence of a single locus with a dominant allele for resistance to G. rostochiensis pathotype Ro1. This locus, designated GroV1, was located on chromosome 5 with RFLP markers. Fine-mapping was performed with RAPD and SCAR markers. The GroV1 locus was found in the same region of the potato genome as the S. tuberosum ssp. andigena H1 nematode resistance locus. Both resistance loci could not excluded to be allelic. The identification of markers flanking the GroV1 locus offers a valuable strategy for marker-assisted selection for introgression of this nematode resistance.Abbreviations BSA bulked segregant analysis - RAPD random-amplified polymorphic DNA - RFLP restriction fragment length polymorphism - SCAR sequence-characterized amplified region     

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 31 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.     

Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach

The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode-plant interactions and assist in breeding nematode-resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co-localized with a resistance-gene-like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1-4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1-4 encodes a protein of 1136 amino acids that contains Toll-interleukin 1 receptor (TIR), nucleotide-binding (NB), leucine-rich repeat (LRR) homology domains and a C-terminal domain with unknown function. The deduced Gro1-4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon-like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT-PCR products showed that Gro1-4 is expressed, among other members of the family including putative pseudogenes, in non-infected roots of nematode-resistant plants. RT-PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.     

Development of PCR assays diagnostic for RFLP marker alleles closely linked to allelesGro1 andH1, conferring resistance to the root cyst nematodeGlobodera rostochiensis in potato

The use of RFLPs for marker-assisted selection schemes in potato breeding is hampered by the fact that RFLP technology requires good laboratory facilities, technical skills and high financial input. Marker technology based on the polymerase chain reaction (PCR) would facilitate the application of marker-assisted selection. PCR assays have been developed that are diagnostic for RFLP alleles at two marker loci,CP56 andCP113, which are closely linked in coupling to the nematode resistance allelesGro1 on chromosome VII andH1 on chromosome V of potato. By comparing DNA sequences among different marker alleles, point mutations were identified based on which allele-specific oligonucleotides were designed. Using allele-specific oligonucleotides as primers in PCR reactions, single-marker alleles were amplified by which the inheritance ofGro1 andH1 could be followed in crosses of diploid potato genotypes containing the genetically characterizedGro1 orH1 resistance allele. When tested in 136 unrelated tetraploid potato varieties, the marker allele indicative ofGro1 was not correlated with the presence of nematode resistance. The marker allele indicative for theH1 resistance allele was correlated with nematode resistance. It was, however, found in four varieties only of the 136 tested.     

Resolution of natural hatching factors for golden potato cyst nematode, Globodera rostochiensis

The hatching responses of Globodera rostochiensis (golden potato cyst nematode) to purified and partially-purified preparations of natural (including the potato glycoalkaloids solanine and α-chaconine) and artificial hatching factors (HFs) were bimodal. At least 10 HFs, mostly anionic, were resolved from potato root leachate by a combination of gel permeation and ion-exchange chromatography. Whereas potato roots were the principal source of HFs, haulm leachate also contained such chemicals. Root leachate from aseptically-grown potato plants lacked several HFs which were present in conventionally-produced leachate.     

Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi

RNA interference (RNAi) has been used widely as a tool for examining gene function and a method that allows its use with plant-parasitic nematodes recently has been described. Here, we use a modified method to analyze the function of secreted beta-1,4, endoglucanases of the potato cyst nematode Globodera rostochiensis, the first in vivo functional analysis of a pathogenicity protein of a plant-parasitic nematode. Knockout of the beta-1,4, endoglucanases reduced the ability of the nematodes to invade roots. We also use RNAi to show that gr-ams-1, a secreted protein of the main sense organs (the amphids), is essential for host location.     

A technique to assess the efficacy of non-volatile nematicides against the potato cyst nematode Globodera rostochiensis

The assessment of larval emergence from cysts, second stage larval behaviour in soil, invasion of roots, development within roots and the final population provides a technique which can be used for evaluating the efficacy of any non-volatile organophosphate or carbamate nematicide.
Oxamyl delayed emergence from cysts, disorientated second stage larvae in soil, delayed and reduced invasion of roots, reduced the number of eggs in the final population and markedly reduced the final population of cysts.     

RFLP markers linked to the root knot nematode resistance gene Mi in tomato

Summary The Mi gene originating from the wild tomato species Lycopersicon peruvianum confers resistance to all major root knot nematodes (Meloidogyne spp.). This single dominant gene is located on chromosome 6 and is very closely linked to the acid phosphatase-1 (Aps-1) locus. Resistance to nematodes has been introgressed into various cultivars of the cultivated tomato (L. esculentum), in many cultivars along with the linked L. peruvianum Aps-1 ¹ allele. By using a pair of nearly isogenic lines differing in a small chromosomal region containing the Mi and Aps-1 loci, we have identified two RFLP markers, GP79 and H6A2c2, which are located in the introgressed L. peruvianum region. Analysis of a test panel of 51 L. esculentum genotypes of various origins indicated that GP79 is very tightly linked to the Mi gene and allows both homozygous and heterozygous nematode-resistant genotypes to be distinguished from susceptible genotypes, irrespective of their Aps-1 alleles. Marker H6A2c2 is linked to the Aps-1 locus and is capable of discriminating between the L. peruvianum Aps-1 ¹ allele and the L. esculentum Aps-1 ³ and Aps-1 ⁺ alleles. In combination, these RFLP markers may provide a powerful tool in breeding tomatoes for nematode resistance.     

Early responses of resistant and susceptible potato roots during invasion by the potato cyst nematode Globodera rostochiensis

Signals from roots of resistant (cv. Maris Piper) and susceptible (cv. Désirée) potato cultivars during invasion by second stage juveniles (J2s) of the potato cyst nematode, Globodera rostochiensis, were investigated. Novel experimental chambers enabled the recording of electrophysiological responses from roots during nematode invasion. The root cell membrane potentials were maintained throughout the 3 d required to assess invasion and feeding site development. The steady-state resting membrane potentials of Désirée were more negative than those of Maris Piper on day 1, but the reverse on day 3. After 5 d there was no difference between the two cultivars. Intracellular microelectrodes detected marked spike activity in roots after the application of J2s and there were distinct and reproducible differences between the two cultivars, with the response from Désirée being much greater than that from Maris Piper. The responses to mechanical stimulation of roots by blunt micropipettes and sharp electrodes were consistent and similar in both cultivars to the responses in Maris Piper obtained after nematode invasion, but could not account for the marked response found in Désirée. Exogenous application of exoenzymes, used to mimic nematode chemical secretions, resulted in a distinct depolarization pattern that, although similar in both cultivars, was different from patterns obtained during nematode invasion or mechanical stimulation. The pH of homogenates prepared from roots of both cultivars was measured and a Ca2+ channel blocker was used to assess the role of Ca2+ in nematode invasion. The results indicated a role for Ca2+ in the signalling events that occur during nematode invasion.     

Screening for quantitative resistance to the white potato cyst nematode Globodera pallida

The resistance to Globodera pallida of potatoes bred from Solanum uernei was assessed rapidly in pot tests by counting cysts on the exterior of the root ball. Assessments of resistance made in this way in three consecutive years were reproducible on a ranking basis. There was also a significant agreement in the ranked results of pot and field tests. A more accurate assessment of nematode multiplication rates in the field could be provided by laboratory tests in which temperature, moisture and initial nematode numbers were strictly controlled.     

Association of RFLP markers with loci conferring broad-based resistance to the soybean cyst nematode (Heterodera glycines)

Soybean cyst nematode (SCN) is a major soybean yield-limiting pest. The present study was conducted to map broad-based SCN resistance loci from the cultivar Hartwig. Two-hundred F₂₃ lines derived from the cross Williams 82 x Hartwig were screened with a fourth-generation SCN inbred and 56 polymorphic molecular markers. Allele states and phenotypes were analyzed using stepwise regression and the model selection was made at P 0.01. Four unlinked RFLP markers (A006, A567, A487, A112) were associated with SCN resistance and the partial coefficient of determinations (R²) were 91%, 1%, 1%, and 1%. We have mapped a new, major SCN resistance locus (A006) and three minor loci (A567, A487, A112). This complete mapping will accelerate the transfer of broad-based resistance without linkage drag and aid in the determination of relationships among various SCN-resistant germplasm sources.     

Genetic variability and differentiation of three Russian populations of yellow potato cyst nematode Globodera rostochiensis as revealed by nuclear markers

Genetic variability of yellow potato cyst nematode G. rostochiensis from three Russian populations (Karelia, Vladimir oblast, and Moscow oblast) was investigated using two types of nuclear markers. Using RAPD markers identified with the help of six random primers (P-29, OPA-10, OPT-14, OPA-11, OPB-11, and OPH-20), it was possible to distinguish Karelian population from the group consisting of the populations from two adjacent regions (Moscow oblast and Vladimir oblast). Based on the combined matrix, containing 294 RAPD fragments, dendrogram of genetic differences was constructed, and the indices of genetic divergence and partition (P, H, and G(st)), as well as the gene flow indices N(m) between the nematode samples examined, were calculated. The dendrogram structure, genetic diversity indices, and variations of genetic distances between single individuals in each population from Karelia and Central Russia pointed to genetic isolation and higher genetic diversity of the nematodes from Karelia. Based on polymorphism of rDNA first intergenic spacer ITS1, attribution of all populations examined to the species G. rostochiensis was proved. Small variations of the ITS1 sequence in different geographic populations of nematodes from different regions of the species world range did not allow isolation of separate groups within the species. Possible factors (including interregional transportations of seed potato) affecting nematode population structure in Russia are discussed.     

Origin, distribution and 3D-modeling of Gr-EXPB1, an expansin from the potato cyst nematode Globodera rostochiensis

Southern analysis showed that Gr-EXPB1, a functional expansin from the potato cyst nematode Globodera rostochiensis, is member of a multigene family, and EST data suggest expansins to be present in other plant parasitic nematodes as well. Homology modeling predicted that Gr-EXPB1 domain 1 (D1) has a flat beta-barrel structure with surface-exposed aromatic rings, whereas the 3D structure of Gr-EXPB1-D2 was remarkably similar to plant expansins. Gr-EXPB1 shows highest sequence similarity to two extracellular proteins from saprophytic soil-inhabiting Actinobacteria, and includes a bacterial type II carbohydrate-binding module. These results support the hypothesis that a number of pathogenicity factors of cyst nematodes is of procaryotic origin and were acquired by horizontal gene transfer.     

Identification of molecular markers linked to Rdr1, a gene conferring resistance to blackspot in roses

Blackspot resistance in the tetraploid rose genotype 91/100–5 had been characterised previously as a single dominant gene in duplex configuration. In the present study a tetraploid progeny (95/3) segregating for the presence of the blackspot resistance gene Rdr1 were screened with 868 RAPD and 114 AFLP primers/primer combinations. Seven AFLP markers were found to be linked to Rdr1 at distances between 1.1 and 7.6 cM. The most closely linked AFLP marker was cloned and converted into a SCAR marker that could be screened in a larger population than the original AFLP and was linked at a distance of 0.76 cM. The cloned fragment was used as an RFLP probe to locate the marker on a chromosome map of diploid roses. This is the first report of markers linked to a resistance gene in roses, and the possibilities of using them for a marker-assisted selection for blackspot resistance as well as for map-based cloning approaches are discussed. Received: 23 December 1999 / Accepted: 25 March 2000     

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.