R.T. Folkertsma

Use of allele specificity of comigrating AFLP markers to align genetic maps from different potato genotypes

Abstract The allele specificity of AFLP markers was assessed in five relatively unrelated potato genotypes. To this end, two diploid mapping populations of potato, F1SH × RH and F1AM × RH, were analysed using four and six AFLP primer combinations, respectively, recently applied to the analysis of the genetically well characterized backcross population BC_C × E. The AFLP profiles of the five parents revealed 733 AFLP markers and, when identical primer combinations were used, 131 comigrating AFLP markers were identified. After construction of five parental maps, the genomic positions of these comigrating AFLP markers were compared and 117 markers (89%) which targeted the same genomic region were assumed to be homologous. Of these putative homologues, 20 markers, each cloned from at least two genotypes, were sequenced and 19 sets of amplification products were shown to be nearly identical. The number of AFLP markers previously mapped in population BC_C × E ranged from three to eleven per chromosome, which allowed a reliable assessment of chromosome numbers from individual linkage groups obtained in populations F1SH × RH and F1AM × RH. The high incidence of corresponding AFLP alleles was confirmed by using an additional set of five primer combinations. The 733 AFLP markers localized provide a valuable reference collection for future mapping studies in potato. As a consequence AFLP analysis may replace more laborious locus-specific marker techniques.

Gene pool similarities of potato cyst nematode populations assessed by AFLP analysis

AFLP was used to characterize 24 potato cyst nematode populations. This novel DNA fingerprinting technique enabled the identification of 987 marker loci by screening only 12 primer combinations. Data on presence or absence polymorphisms and data on the intensities of corresponding DNA fragments were collected. Separate analysis of both data sets revealed similar dendrograms for the nine G. rostochiensis populations included in this study. Both dendrograms consisted of two groups containing three and five related populations, respectively. One population differed from either of these groups. Each group represented a different pathotype as defined by Kort et al. (J. Kort, H. Ross, H. J. Rumpenhorst, and A. R. Stone, Nematologica 23:333-339, 1977). Previously, a similar arrangement was found after analysis of the genetic variation using random amplified polymorphic DNA (RAPD) (R. T. Folkertsma, J. N. A. M. Rouppe van der Voort, M. P. E. van Gent-Pelzer, K. E. de Groot, W. J. van den Bos, A. Schots, J. Bakker, and F. J. Gommers, Phytopathology 84:807-811, 1994). For the 15 G. pallida populations analyzed, complex AFLP patterns were obtained and therefore only qualitative AFLP data were used. Incongruities were observed between clustering on the basis of AFLP data and classical pathotyping. This strongly confirms earlier findings obtained with RAPDs, because the AFLP markers used in this study outnumbered the population characteristics revealed by RAPDs by a factor of five. To arrive at a reliable pathotype designation of potato cyst nematode populations molecular data and virulence characteristics should be integrated. Possible causes for the difference in distribution of polymorphisms among g. rostochiensis and G. pallida populations are discussed.

Mapping of the cyst nematode resistance locus Gpa2 in potato using a strategy based on comigrating AFLP markers

Abstract The nematode resistance locus Gpa2 was mapped on chromosome 12 of potato using information on the genomic positions of 733 known AFLP markers. The minimum number of AFLP primer combinations required to map Gpa2 was three. This demonstrates that a reference collection of potato AFLP markers may be a valuable tool for mapping studies in potato. By use of RFLP probes, Gpa2 was more precisely mapped at the distal end of chromosome 12. Gpa2 confers resistance to a distinct group of populations of the potato cyst nematode Globodera pallida and originates from the same potato accession as locus H1, conferring resistance to pathotype Ro1 of G. rostochiensis. This study shows that these two nematode resistance loci are unlinked and that Gpa2 is linked to the Rx1 locus conferring resistance to potato virus X. The efficiency of AFLPs for genetic mapping of a highly heterozygous crop like potato is discussed and compared with the RFLP technique.

A QTL for broad-spectrum resistance to cyst nematode species (Globodera spp.) maps to a resistance gene cluster in potato

Abstract Broad-spectrum resistance in potato to the potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida is commonly regarded as a polygenically inherited trait. Yet, by use of QTL analysis and a selected set of PCN populations, resistance to both PCN species could be ascribed to the action of locus Grp1. Grp1 confers major resistance to G. rostochiensis line Ro5-22 and G. pallida population Pa2-D383 and partial resistance to G. pallida population Pa3-Rookmaker. Grp1 was mapped on chromosome 5 using previously characterized AFLP markers. Cleaved amplified polymorphic sequence (CAPS) markers available for RFLP loci GP21 and GP179 revealed that Grp1 maps on a genomic region harboring other resistance factors to viral, fungal and nematodal pathogens. The present data indicate that Grp1 is a compound locus which contains multiple genes involved in PCN resistance.

Single juveniles of the potato cyst nematodes Globodera rostochiensis and G. pallida differentiated by randomly amplified polymorphic DNA

Random amplified polymorphic DNA (RAPD) offers a potential basis for the development of a diagnostic assay to differentiate the potato cyst nematode species Globodera rostochiensis and G. pallida. Nine decamer primers have been tested for their ability to amplify species-specific DNA sequences. Primer OPG-05 produced 2 discrete DNA fragments, which were consistently present in 5 G. rostochiensis populations and absent in 5 G. pallida populations. These fragments were detectable in single females as well as in single 2nd-stage juveniles. Their amplification is extremely efficient, and reproducible over a wide range of template concentrations. One-fifth of a single juvenile is sufficient to generate reproducible RAPD markers. The amplification from single juveniles requires no DNA isolation. The use of a crude homogenate does not impair the polymerase chain reaction.

The effects of founding events and agricultural practices on the genetic structure of three metapopulations of Globodera pallida

To assess the genetic constitution of the Globodera pallida populations in the Netherlands and the effects of agricultural practices, three geographically separated metapopulations, in total consisting of 226 local populations, were analyzed by two-dimensional gel electrophoresis (2-DGE) of total proteins. This technique allows the accurate assessment of allele frequencies in homogenates of mixtures of individuals. Based on the estimated average heterozygosity, the average proportion of polymorphic loci and the average number of alleles per locus, the genetic diversity among 226 local G. pallida populations was small. The small genetic basis of G. pallida in the Netherlands will facilitate the identification of Solanum genotypes with a broad and durable resistance to G. pallida. Instead of clusters of local populations with unique alleles, a continuous range of allele frequencies was observed. Analysis of the three metapopulations by the Shannon-Weaver index and Neis G(ST) revealed that the metapopulation from a region with sandy-loam soils was clearly distinguishable from the remaining two; the local populations within this metapopulation were more similar and the genetic diversity within the individual local populations was significantly higher than the local populations from the two remaining regions. These regions are characterized by wider crop rotation schemes and a very limited use of nematicides. The less intensive cultivation of potatoes in these regions with sandy-clay soils resulted in relatively little variation within and more variation between local nematode populations. To our knowledge, the effects of agricultural practices on the genetic constitution of potato cyst nematode populations have not been pinpointed before.

Cluster analysis of 36 Globodera pallida field populations using two sets of molecular markers.

Thirty-six populations of the potato cyst nematodeGlobodera pallida, all collected in the Netherlands, were analysed twice: by two-dimensional gel electrophoresis of proteins (2-DGE) and by random amplified polymorphic DNA fingerprinting (RAPD). Two-DGE revealed frequencies of 21 alleles at eight putative loci in each population. The same populations were subjected to RAPD analysis. This qualitative technique revealed 38 polymorphic DNA fragments. Both datasets were independently processed to determine the intraspecific variation. UPGMA analysis resulted in a 2-DGE- and a RAPD-based dendrogram with cophenetic correlation coefficients of 0.755 and 0.838 respectively. The correlation between the genetic similarity values for the populations was 0.572. Comparison between the 2-DGE- and the RAPD-based dendrogram revealed that only thirteen of the 36 populations analysed were clustered identically. It is concluded that the gene pool similarity concept is only in some instances applicable to Dutch populations ofG. pallida. For populations that could not be differentiated unequivocally on the basis of molecular markers, markers closely linked to avirulence genes should be identified. Approaches that will lead to the identification of such markers are discussed.

Linkage Mapping in Potato Cyst Nematodes

A Mendelian proof for a gene-for-gene relationship between virulence in Globodera rostochiensis and the H1 resistance gene from Solanum tuberosum spp. andigena CPC 1673 was obtained by Janssen et al., (1991). It was shown that virulence to the H1 gene is recessively inherited at a single locus. As expected from the epigenetic nature of sex determination, this locus is not sex-linked. The resistance conferred by the H1 gene is only effective against avirulent juveniles developing into females. It was shown that avirulent juveniles are still able to develop into males on the resistant cultivar (Janssen et al., 1992).

Identification and management of virulence genes in potato cyst nematodes

Efficient and accurate diagnostic assays are essential for the design and evaluation of control measures of the potato cyst nematodesGlobodera rostochiensis andG. pallida by means of resistance. The hybridoma technology and the polymerase chain reaction (PCR) offer in potential various possibilities to design such diagnostic tests for routine purposes. We set out to devise a refined advisory system based on biochemical assays by using the following stepwise approach.In the early 80s a research program was started to develop an immunoassay to differentiate the two sibling species of potato cyst nematodes. Species specific monoclonal antibodies were raised against nematode proteins which are thermostable, abundant and homologous, and which enable reliable species identification using single eggs.The second step to improve the management of virulence genes is aimed at discriminating groups of populations within a species (‘virulence groups’ or ‘pathotypes’). The concept is that the number of initial populations introduced from South America is limited and that numerous Dutch populations (‘secondary founders’) are closely related by descent. Biochemical characters revealed by two-dimensional gel electrophoresis (2-DGE) of polypeptides, PCR in combination with restriction enzyme digests and RAPD (Random amplified polymorphic DNA) will be used to delineate groups of populations. The final diagnostic assay will be based on PCR. One of the challenges will be to devise a manageable number of primers to recognize all distinct groups.The third research line is aimed at developing a PCR assay based on the virulence genes themselves. Genetic studies showed that virulence inG. rostochiensis towards the H1 resistance gene is inherited at a single locus and is recessive to avirulence. To identify molecular markers linked to the virulence gene, 300 virulent lines were selected via backcrossing the F1 (Aa) with the virulent (aa) parent line. Molecular differences between the parent lines were obtained by 2-DGE, RFLPs (restriction fragment length polymorphisms) and RAPD. Especially RAPD proved to be a valuable technique to construct a linkage map. Screening 80 primers (10-mer) resolved more than 120 markers. RAPD will eventually lead to flanking DNA sequences, which will be used to isolate and characterize the virulence gene. Sequence information of the virulence gene inG. rostochiensis for the H1 resistance gene can be used to devise primers for a PCR assay and may also provide a starting point to isolate other virulence genes.