Linkage analysis by genotyping of sibling populations: a genetic map for the potato cyst nematode constructed using a "pseudo-F2" mapping strategy.

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.

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

The allele specificity of AFLP markers was assessed in five relatively unrelated potato genotypes. To this end, two diploid mapping populations of potato, F1SH x RH and F1AM x 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 x 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 x E ranged from three to eleven per chromosome, which allowed a reliable assessment of chromosome numbers from individual linkage groups obtained in populations F1SH x RH and F1AM x 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.