J.N.A.M. Rouppe van der Voort

Autotetraploids and genetic mapping using common AFLP markers: the R2 allele conferring resistance to Phytophthora infestans mapped on potato chromosome 4

Abstract Due to the complexity of tetrasomic inheritance, mapping studies in potato (Solanum tuberosum L.) are generally conducted at the diploid level. In the present study we tested the feasibility of Bulked Segregant Analysis (BSA) using a tetraploid offspring for the identification of AFLP markers linked to the R2 allele, which confers race-specific resistance to Phytophthora infestans. Eleven bulk-specific AFLP markers, detected in fingerprints of 205 AFLP primer combinations, could be mapped in a linkage group encompassing the R2 locus. The efficiency of BSA at the tetraploid level, determined by the frequency of single-dose restriction fragments (SDRF), was much higher than expected on the basis of overall genetic dissimilarity between the parental clones. The fortuitous detection of AFLPs with linkage to the R2 allele is explained on the basis of specific genetic dissimilarity between cultivated potato and the chromosomal segment introgressed from S. demissum carrying the resistant R2 allele. AFLP markers common to those with linkage to R2 were visually recognized by their electrophoretic mobility in the AFLP fingerprint in a parental clone of a reference mapping population. Using these common AFLP markers we anchored the linkage group comprising the R2 allele to potato chromosome 4.

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.

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.

Tight Physical Linkage of the Nematode Resistance Gene Gpa2 and the Virus Resistance Gene Rx on a Single Segment Introgressed from the Wild Species Solanum tuberosum subsp. andigena CPC 1673 into Cultivated Potato

Comparative mapping studies within several plant genera have shown that genes for resistance to taxonomically diverse pathogens are often co-localized on the maps of these genotypes. Here we describe the genetic analysis of such a gene cluster on chromosome 12 of potato. This cluster harbors the resistance loci Gpa2 and Rx, which confer resistance to the potato cyst nematode Globodera pallida and potato virus X (PVX), respectively. By screening two mapping populations with 10 CAPS (cleaved amplified polymorphic sequence) markers derived from AFLP (amplified fragment length polymorphism) markers and the ends of overlapping bacterial artificial chromosome (BAC) clones, it is demonstrated that Gpa2 and Rx are tightly linked. From a recombination fraction of 1/3386, found after screening two different mapping populations, a genetic distance of 0.02 cM between Gpa2 and Rx was calculated. The physical length of this genetic interval does not exceed 300 kb of DNA as determined by the total size of overlapping BA...

An online catalogue of AFLP markers covering the potato genome

An AFLP marker catalogue is presented for gene mapping within cultivated potato. The catalogue is comprised of AFLP fingerprint images of 733 chromosome-specific AFLP markers which are mapped relative to 220 RFLP loci, isozyme loci, morphological characteristics and disease resistance traits. Use of the catalogue is based on identification of common AFLP markers which are visually recognized on autoradiogram images as co-migrating bands in fingerprints generated from different genotypes. Images of AFLP fingerprints combined with detailed information on the genomic location of all AFLP markers are available at URL: http://www.spg.wau.nl/pv/aflp/catalog.htm. It is demonstrated that the comparison of autoradiogram images and subsequent identification of common AFLP markers solely are efficient means for alignment of linkage groups and mapping target genes.

Identification of Essentially Derived Varieties Obtained from Biparental Crosses of Homozygous Lines. III. AFLP Data from Maize Inbreds and Comparison with SSR Data

Genetic distance (GD) estimates based on molecular markers are a preferred approach to estimate genetic conformity between putative essentially derived varieties (EDVs) and their initial varieties (IVs). The objectives of our study were to (1) estimate the variation in the marker-estimated parental contribution (p) to the genome of the progeny, (2) investigate the power of AFLP-based GD estimates for discriminating between homozygous lines with F2, BC1, and BC2 relationships, and (3) compare AFLP data with SSR data from a companion study, as well as with theoretical and simulated results. In total, 104 European maize inbred lines comprising 66 triplets were analyzed with 20 AFLP primer combinations and 100 SSR primer pairs. Each triplet consisted of one F2-, or BC1-derived progeny line and both parental lines. For lines with both F2 and BC1 relationship, mean estimates of p based on AFLP (pF2 = 0.46; pBC1 = 0.70) or SSR data (pF2 = 0.45; pBC1 = 0.67) were lower than the respective expected values of 0.50 and 0.75. Considerable differences were detected between AFLP- and SSR-based mean GD estimates for unrelated inbred lines due to the lower degree of polymorphism of AFLPs compared with SSRs. With each marker system, the GD between progeny lines and parents was little affected by the variation in GD between the parents, particularly for lines with BC1 relationship. Substantial differences in Type I (α) and Type II (β) errors were detected between flint and dent germplasm pools with different marker systems and when fixed EDV thresholds were considered. To counterbalance advantages and disadvantages of AFLP and SSRs, a complementary application of the two marker systems is suggested for discrimination between EDVs and independently derived varieties (IDVs).

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.

LINKAGE ANALYSIS BY GENOTYPING OF SIBLING POPULATIONS : A GENETIC MAP FOR THE POTATO CYST NEMATODE CONSTRUCTED USING A PSEUDO-F2 MAPPING STRATEGY

Abstract A mapping strategy is described for the construction of a linkage map of a non-inbred species in which individual offspring genotypes are not amenable to marker analysis. After one extra generation of random mating, the segregating progeny was propagated, and bulked populations of offspring were analyzed. Although the resulting population structure is different from that of commonly used mapping populations, we show that the maximum likelihood formula for a normal F2 is applicable for the estimation of recombination. This “pseudo-F2” mapping strategy, in combination with the development of an AFLP assay for single cysts, facilitated the construction of a linkage map for the potato cyst nematode Globodera rostochiensis. Using 12 pre-selected AFLP primer combinations, a total of 66 segregating markers were identified, 62 of which were mapped to nine linkage groups. These 62 AFLP markers are randomly distributed and cover about 65% of the genome. An estimate of the physical size of the Globodera genome was obtained from comparisons of the number of AFLP fragments obtained with the values for Caenorhabditis elegans. The methodology presented here resulted in the first genomic map for a cyst nematode. The low value of the kilobase/centimorgan (kb/cM) ratio for the Globodera genome will facilitate map-based cloning of genes that mediate the interaction between the nematode and its host plant.

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).