Genetic analysis of root-knot nematode resistance in potato

The development of potato varieties with resistance towards the potato cyst nematode, allowed a dramatic decrease of the use of nematicides. Subsequently the population of the free living nematodes and the root-knot nematodes ( Meloidogyne spp.) has increased. Among the root-knot nematodes, three Meloidogyne species are important in the potato cultivation in The Netherlands: M. hapla , M. chitwoodi and M. fallax . The latter two species are the most harmful because they cause malformation in the tubers and are quarantine organisms. Until now no resistant potato cultivars are available. Several wild species of potato show resistance, but several back crossings are required to develop a root-knot nematode resistant potato cultivar. Selection based on molecular markers will speed up this development and will replace the expensive and time consuming bioassays, which have to be carried out under strict quarantine conditions. In this thesis diploid as well as polyploid wild Solanum species are used for the introgression of resistance genes in cultivated potato, and to analyse the inheritance of the various resistances.Chapter 2 describes the genetic analysis of the diploid wild species Solanum chacoense with resistance against M. hapla and M. fallax. An AFLP based linkage map was constructed, resulting in twelve linkage groups of either parent of the mapping population with a total length of 685 and 596 cM each. Due to the large differences in AFLP patterns between S. chacoense and cultivated potato, chromosome numbers could not be assigned to the linkage groups containing the QTLs. Two QTLs for resistance against the M. hapla population Hi were identified in this population. These two QTLs, R Mh-chc A and R Mh-chc B , explained 38% and 13% of the total phenotypic variance of the resistance to M. hapla . As a consequence of the large variation in the results of the bioassay, phenotypic selection may lead to the loss of these resistance QTLs. This loss can be prevented by marker assisted selection with the markers flanking the QTLs. The genetic analysis of the resistance against M. fallax resulted in the detection of one QTL explaining 14% of the phenotypic variance. The high level of resistance to M. fallax as observed in the resistant parent was not observed in the progeny. In view of the modest results that can be expected from S. chacoense , we cannot recommend breeding efforts with this germplasm. Better results in genetic analyses and introgression of resistance against M. fallax were achieved in the mapping studies with the wild Solanum species S. fendleri (Chapter 5), S. hougasii and S. bulbocastanum (Chapter 6).The differential interaction of eight M. hapla populations to the resistance QTLs, R Mh-chc A and R Mh-chc B was studied in Chapter 3. The R Mh-chc A allele allows recognition on virulence factors present in all six tested cytological race A populations. Although the allele R Mh-chc B also showed a reduction in the amount of formed egg masses of population Ham, this allele had only an additive effect with population Hi, the population initially used for the QTL analysis (Chapter 2). No effect of both alleles was noticed for the two race B populations. Besides the distinctive difference in reproduction between race A and B, both races can now also be divided based on their differences in virulence to the R Mh-chc A allele. Furthermore, the molecular fingerprints allow too clearly distinguish between the races A and B. Because the resistant parent of the mapping population is susceptible to the race B populations, it is not worthwhile to check the used mapping population for other resistance QTLs. The breeder should therefore search for new sources of resistance to the race B populations in the wild species. Looking at the higher level of reproduction, the population Han would be the most appropriate population to be used in that survey.In Chapter 4 the diploid wild species S. tarijense was used as the source of resistance to M. hapla. The non absolute level of resistance and the continuous distribution of produced egg masses in the segregating offspring, suggests that the resistance is under polygenic control. Therefore a linkage map of the BC1 mapping population was developed as prerequisite of QTL analysis. The assignment of a chromosome number to the linkage groups was simple, because a detailed genetic map was already constructed of the susceptible parent. The linkage groups of the resistant female parent were subsequently aligned to the genetic map of the susceptible male parent using the AFLP markers that were heterozygous in both parents (so-called ab×ab markers). The QTL mapping approach resulted in only one locus, R Mh-tar , on chromosome 7. In contrast to QTL analysis that leads to a broad QTL interval, qualification of the resistance will lead to a more precise map position of the resistance locus. For that case the progeny of the mapping population was arbitrary classified in two groups: 1) a resistant group with the amount of less than1 egg mass and 2) a susceptible group with more than 10 egg masses. Via this approach the R Mh-tar resistance gene could be located on a distal position of the short arm of chromosome 7, and the low amount of singletons offered an indirect validation of the classification of the resistance phenotypes in discrete groups. The value of the R Mh-tar gene for the potato breeding is depending on the allele frequency of the associated virulence genes in the M. hapla populations and the geographic distribution of such populations.In contrast to the research with M. hapla as described in Chapter 2 and 4, the progeny of the mapping populations with resistance against M. chitwoodi and M. fallax (Chapter 5 and 6) could be easily classified as resistant or susceptible. This suggests that resistance is mediated by a single R-gene. The resistance against both nematode species, M. chitwoodi and M. fallax , was absolutely correlated in all three mapping populations, derived from S. fendleri (Chapter 5), S. hougasii and S. bulbocastanum (Chapter 6). In Chapter 5 the monogenic resistance against M. chitwoodi and M. fallax was mapped in an inter-specific and an intra-specific mapping population of the tetraploid wild species S. fendleri . By using the so-called Bulked Segregant Analysis (BSA) method, a large amount of closely linked markers was identified efficiently. In the intra-specific mapping population a remarkably lower amount of linked AFLP markers was found. This can be due to the fact that S. fendleri is a self-fertilising species, showing a lower level of genetic polymorphisms. On the other hand the genetic dissimilarity between the wild species introgression segments and the recurrent potato parents may have caused an elevated level of polymorphic markers in the BC 2 inter-specific mapping population. Besides the larger amount of polymorphic markers, the utilisation of inter-specific mapping populations is an advantage for the breeder, because back-crossings are already made, necessary to introgress the resistance into potato. Based on the sequence of an R Mc1-fen locus linked AFLP marker, a PCR marker was developed. This marker was used to map its position in a reference map, were it was located on the distal position of the long arm of chromosome 11. The chromosomal position of the R Mc1-fen locus was confirmed by using the chromosome 11 specific CAPS marker M39b. The R Mc1-fen gene is located in a cluster of resistance genes, containing the highly related Rmc1 gene and other fungus, virus and nematode resistance genes. This PCR marker, however, can not yet be used in marker assisted selection because 40% of the progeny with the PCR product will not contain the resistance allele.Chapter six describes the localisation of resistance against M. chitwoodi and M. fallax in a BC2 population of the hexaploid wild species S. hougasii . Also this study used the BSA method, resulting in a linkage group of 14 AFLP markers and the monogenic resistance locus R Mc1-hou . Similar to the linkage group of S. fendleri, the linkage group of S. hougasii has been positioned on the distal position of the long arm of chromosome 11. The differential interaction between the resistance genes R Mc1-blb , R Mc1-fen and R Mc1-hou with six M. chitwoodi populations has been studied. Differences in virulence were not found between race 1 and race 2, but, remarkably, within the two races. Populations Ccl and Cbh seem to have a similar virulence spectrum, while these populations are described as respectively race 1 and race 2. The presence of virulence in the populations will have a negative effect on the durability of the resistance. Because the three resistance genes against M. chitwoodi and M. fallax from the wild species S. bulbocastanum , S. fendleri and S. hougasii are mapped on the same locus and act against the same spectrum of virulence genes, no additional value is expect from the strategy of pyramiding these genes. Consequently, the breeder has to search for new resistance sources against the virulent nematode population Ccl and/or Cbh, in order to develop a potato with broad spectrum resistance.The resistance genes described in this thesis can be introgressed in potato with the help of molecular markers. Moreover the presence of individual resistance genes can be monitored by molecular markers, which is a prerequisite for pyramiding of R-genes. Pyramiding of the resistance genes may lead to a resistance with a broader spectrum, and/or may offer a more durable resistance, providing that it can be performed with loci with a complementary resistance spectrum

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