Characterization and high-resolution mapping of a late blight resistance locus similar to R2 in potato.

Identification of resistance (R) genes to Phytophthora infestans is an essential step in molecular breeding of potato. We identified three specific R genes segregating in a diploid mapping population. One of the R genes is located on chromosome 4 and proved phenotypically indistinguishable from the Solanum demissum-derived R2, although S. demissum is not directly involved in the pedigree of the population. By bulked segregant analysis combined with a resistance assay, a genetic linkage map of the R2-like locus was constructed with 30 coupling and 23 repulsion phase AFLP markers. Two markers flanking the R2-like locus were applied to screen an extended population of 1,586 offspring. About 103 recombinants were selected, and an accurate high-resolution map was constructed. The R2-like resistance was localized in a 0.4 cM interval and was found co-segregating with four AFLP markers, which can be used to isolate the R2-like gene by map-based gene cloning. By analyzing race-specificity and R gene-specific molecular markers, we also found that an R1-like gene and an additional unknown R gene are segregating in the population.

Production of potato monohaploids (2n=x=12) through prickle pollination.

Data are presented on the potential of gynogenesis for the production of monohaploids and on factors affecting their frequency and relative vigour. Diploid Solanum tuberosum L. and S. tuberosumxS. phureja Juz et Buk hybrids were used as maternal parents and selected S. phureja clones as prickle pollinators with embryo-spot and nodal band as dominant seed and plant marker. About 2 million seeds were screened for absence of embryo-spot. After raising plants from phenotypically spotless seeds, further screening for absence of nodal bands and for ploidy level was carried out. Finally more than 500 monohaploid plants from three genetically different groups of maternal parents were obtained. Frequency and vigour of the monohaploids were clearly dependent on their maternal genotypes. The data also indicated an effect of the pollinator genotype, the physiological stage of the maternal plant and the environment on monohaploid frequency. On the basis of these results the possibility of breeding for a higher monohaploid production rate and for more stable and vigorous monohaploids is discussed. Furthermore, gynogenesis and androgenesis are compared. It is suggested that both should be used in order to obtain monohaploids from sufficiently various diploid breeding material.

Identification of alien chromosomes through GISH and RFLP analysis and the potential for establishing potato lines with monosomic additions of tomato chromosomes.

To increase the potential for establishing a complete series of tomato chromosome addition-sbstitution lines in a potato background, six new BC1 progeny were produced. All of them originated from crosses between three different hexaploid potato (+) tomato fusion hybrids. Three different somatic hybrids, viz., C31-17-5, C31-17-24, and C31-17-51, were used as female parents, and four different tetraploids, viz., Katahdin, Frieslander, 6704-1, and AM66.42 were used as male parents. A characterisation of the genomes of the three fusion hybrids and the six BC1 progenies (6739, 2001, 2002, 2003, 2004, and 2005) through genomic in situ hybridization and restriction fragment length polymorphism (RFLP) analysis indicated that there was preferential tomato chromosome elimination in the fusion hybrids. Similar analyses of the six BC1 progeny indicated that a variable number of the alien tomato chromosomes (6-11) were present in individual plants. RFLP analysis using chromosome specific DNA probes indicated that BC1 progenies had retained all 12 tomato chromosomes, albeit in different individual plants. This means that the available BC1 progenies have the potential for establishing a complete series of tomato chromosome addition-substitution lines in a potato background.

Genomic in situ hybridization analysis of a trigenomic hybrid involving Solanum and Lycopersicon species.

A 4x potato (+) tomato fusion hybrid (2n = 4x = 48) was successfully backcrossed with a diploid Lycopersicon pennellii (2n = 2x = 24). Genomic in situ hybridization (GISH) on somatic and meiotic chromosomes confirmed that the progenies were triploids (2n = 3x = 36) and possessed three different genomes: potato, tomato, and L. pennellii. Therefore, they have been called trigenomic hybrids. Total genomic probes of both Lycopersicon species were found to hybridize mutually, whereas the potato genome was clearly differentiated. During metaphase I, bivalents were formed predominantly between tomato and L. pennellii chromosomes and the univalents of potato chromosomes were most common. Trivalents in all cases included homoeologous chromosomes of potato, tomato, and L. pennellii. However, the triploids were totally sterile as determined from extensive crossing. On chromosome doubling of triploids by shoot regeneration from callus, hexaploids (2n = 6x = 72) were obtained. Despite exhibiting clear allohexaploid behaviour by forming 36 bivalents at meiosis, these were also completely sterile like their triploid counterparts. In spite of this drawback, the prospects of chromosome pairing between potato L. pennellii and Solanum genomes does open the possibilities for bringing the two genera close.

The first and second backcross progeny of the intergeneric fusion hybrids of potato and tomato after crossing with potato.

Somatic fusion hybrids between the diploid potato and tomato were backcrossed to several genotypes of potato. Two ploidy levels of fusion hybrids, 4x and 6x, were used as female parents in backcrosses with five clones of 4x-potato. An estimate of the berry set and "seed set" in immature berries harvested 14-21 days after pollination indicated that crosses between certain combinations of 6x-fusion hybrids and male parents were more successful than others. The culture of over 4000 young seeds from berries harvested 2-2.5 weeks after pollination gave rise to a single seedling, 93.6701, from the cross between the 6x-fusion hybrid C 31-17-1 and the 4x-potato AM 66.42. This seedling was found to possess a pentaploid chromosome number, which was expected of a 6x × 4x cross. Isozyme analysis and DNA hybridisation studies confirmed that the seedling 93.6701 was indeed a backcross (BC1) progeny. Morphologically, this BC1 plant resembled potato with respect to plant habit, leaf shape, stolons and tuber characteristics, while some of the characters, such as floral morphology and the fragrance of the crushed leaves (typical of tomato), were intermediate. It was male sterile but could be successfully hybridized with 4x-potato through in vitro culture of yound seeds; thus, BC2 plants were obtained. The possibilities of backcrossing and the potential use of BC1 and BC2 plants in genetics and breeding are discussed.

Isolation and characterization of potato-tomato somatic hybrids using an amylose-free potato mutant as parental genotype.

Using different genotypes of tomato and diploid potato, possessing alien selectable markers as well as endogenous markers, very high frequencies of protoplast fusion hybrids were obtained. One endogenous genetic marker, the amylose-free (amf) mutant of potato, was helpful not only for the confirmation of fusion products but also for the study of genetic complementation and the segregation of amylose-free starch in microspores. Cytological analysis of the fusion hybrids indicated that except for one which was hexaploid, all of them had a perfectly balanced chromosome number of allotetraploid constitution (2n = 4x = 48). Despite normal chromosome pairing and a diploid behaviour, the microspores in some of the fusion hybrids segregated for the recessive amf-locus. This anomalous segregation of a recessive character in these hybrids was shown not to be due to chromosome elimination or to the absence of the wild-type tomato Amf gene. Although all fusion hybrids were totally sterile, the hexaploid produced stainable pollen and berries with badly developed seeds. Embryo rescue has so far failed to produce backcross progeny.

Segregation analysis and RFLP mapping of the R1 and R3 alleles conferring race-specific resistance to Phytophthora infestans in progeny of dihaploid potato parents.

Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato (Solanum tuberosum). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles (R genes) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V. The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F1 populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F1 populations analysed, whereas the R1 allele segregated with a 1:1 ratio as expected in five F1 populations. The mode of inheritance of the R10 allele could not be deduced as only very few F1 hybrids bearing R10 were obtained. Linkage analysis in two F1 populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3, to a distal position on chromosome XI.