An assessment of chromosomal rearrangements in neopolyploids of Lilium hybrids.

Two types of newly induced polyploids (neopolyploids) of Lilium hybrids were monitored for the occurrence of chromosomal rearrangements through genomic in situ hybridization (GISH) technique. One of the populations was obtained through crossing an allotriploid Longiflorum x Oriental hybrid (LLO) with an allotetraploid Longiflorum x Trumpet hybrid (LLTT), both of which were derived from somatic chromosome doubling. The other type of allopolyploid population was derived from meiotic chromosome doubling in which numerically unreduced (2n) gametes from two different interspecific hybrids, namely, Longiflorum x Asiatic (LA) and Oriental x Asiatic (OA), were used to get backcross progeny with the Asiatic parents. GISH clearly discriminated the three constituent genomes (L, T, and O) in the complements of the progeny obtained from mitotic chromosome doubling. A total of 26 individuals were analyzed from this population and there was no evidence of chromosomal rearrangements. However, in the case of meiotically doubled allopolyploid progeny, considerable frequencies of chromosomal rearrangements were observed through GISH. The so-called chromosomal rearrangements in meiotic polyploids are the result of homoeologous recombination rather than translocations. Furthermore, evidence for the occurrence of meiotic recombination in the LA hybrids has been confirmed with GISH on meiotic chromosomes. Thus, there was evidence that neopolyploids of Lilium hybrids did not possess any noticeable chromosome rearrangements.

Construction of chromosomal recombination maps of three genomes of lilies (Lilium) based on GISH analysis.

Chromosomal recombination maps were constructed for three genomes of lily (Lilium) using GISH analyses. For this purpose, the backcross (BC) progenies of two diploid (2n = 2x = 24) interspecific hybrids of lily, viz. Longiflorum x Asiatic (LA) and Oriental x Asiatic (OA), were used. Mostly the BC progenies of LA hybrids consisted of both triploid (2n = 3x = 36) and diploid (2n = 2x = 24) with some aneuploid genotypes and those of OA hybrids consisted of triploid (2n = 3x = 36) and some aneuploid genotypes. In all cases, it was possible to identify the homoeologous recombinant chromosomes as well as accurately count the number of crossover points, which are called "recombination sites". Recombination sites were estimated in the BC progeny of 71 LA and 41 OA genotypes. In the case of BC progenies of LA hybrids, 248 recombination sites were cytologically localized on 12 different chromosomes of each genome (i.e., L and A). Similarly, 116 recombinant sites were localized on the 12 chromosomes each from the BC progenies of OA hybrids (O and A genomes). Cytological maps were constructed on the basis of the percentages of distances (micrometres) of the recombination sites from the centromeres. Since an Asiatic parent was involved in both hybrids, viz. LA and OA, two maps were constructed for the A genome that were indicated as Asiatic (L) and Asiatic (O). The other two maps were Longiflorum (A) and Oriental (A). Remarkably, the recombination sites were highly unevenly distributed among the different chromosomes of all four maps. Because the recombination sites can be unequivocally identified through GISH, they serve as reliable landmarks and pave the way for assigning molecular markers or desirable genes to chromosomes of Lilium and also monitor introgression of alien segments.

Intergenomic recombination in F1 lily hybrids (Lilium) and its significance for genetic variation in the BC1 progenies as revealed by GISH and FISH.

Intergenomic recombination was assessed in a BC1 population of Oriental (O)xAsiatic (A) lilies (Lilium) backcrossed to Asiatic parents. This population consisted of 38 plants generated from the 2n gametes from 2 genotypes (951502-1 and 952400-1) of the diploid F1, Orientalx Asiatic lilies (2n=2x=24) as parents. In the majority of BC1 plants, there was evidence that first division restitution, with and without crossovers, resulted in functional gametes. However, there were 5 BC1 plants in which 2n gametes originated from indeterminate meiotic restitution (IMR). Based on the number of recombinant chromosomes for a particular homoeologous pair, 3 types of plants were identified: (i) those with both the reciprocal product of a crossover (O/A, A/O, where O represents the centromere of the O genome and A the recombinant segment of Asiatic chromosome, and vice versa); (ii) those with 1 normal chromosome of the O genome and a recombinant chromosome (O, A/O); and (iii) those with 1 normal chromosome of the A genome and a recombinant chromosome (A, O/A). An important feature of A x OA backcross progeny is the occurrence of substitutions for the segment distal in the crossover wherever the recombinant chromosome O/A was present. In the case of IMR, the substitution occurred for both proximal and distal recombinant segments. The significance of these substitutions is that they offer the potential for the phenotypic expression of recessive genes in polyploids (i.e., nulliplex genotype).

Use of 2n gametes for the production of sexual polyploids from sterile Oriental x Asiatic hybrids of lilies (Lilium).

Sixteen Oriental and 12 Asiatic cultivars were crossed in 158 different combinations. A total of 708 F1 hybrids were obtained from 86 of the different combinations of 15 Oriental and 11 Asiatic cultivars. Because the Lilium cultivars (2n=2x=24) used for the production of these hybrids belong to two different taxonomic sections-Archelirion (0) and Sinomartagon (A), respectively-the F1 hybrids (OA) could be obtained only through embryo, embryo sac rescue, ovary slice or ovule culture. Most of the F, hybrids were highly sterile (did not produce viable n gametes) due to the failure of chromosome pairing. However, in a few cases F1 plants were found that produced viable 2n pollen at variable frequencies. These 2n pollen grains were successfully used for the production of backcross progenies. Using genomic in situ hybridization we found intergenomic recombinant chromosomes in the sexual polyploid progenies. These results indicate that there are effective prospects for combining important horticultural traits from the two main groups of cultivars of lilies through sexual polyploidization.

Meiotic behaviour of individual chromosomes in allotriploid Alstroemeria hybrids.

Chromosome association and chiasma formation were studied in pollen mother cells at metaphase I of four allotriplod BC1 plants (2n=3x=24) obtained from the backcross of the hybrid Alstroemeria aurea x A. inodora with its parent A. inodora. We distinguished the chromosomes of both parental species by genomic in situ hybridization (GISH), whereas the individual chromosomes were identified on the basis of their multicolour FISH banding patterns obtained after a second hybridization with two species-specific satellite repeats as probes. All the four BC1 plants possessed two genomes of A. inodora and one of A. aurea. Variable numbers of recombinant chromosomes, resulting from meiotic recombination in the interspecific hybrid, were present in these plants. The homologous A. inodora chromosomes generally formed bivalents, leaving the homoeologous A. aurea chromosomes unassociated. High frequencies of trivalents were observed for the chromosome sets that contained recombinant chromosomes, even when the recombinant segments were small. Chromosome associations in the trivalents were restricted to homologous segments. The implications of the absence of homoeologous chromosome pairing on gamete constitution and prospects for introgression in Alstroemeria are discussed.

Evaluation of BC2 progenies derived from 3x-2x and 3x-4x crosses of Lilium hybrids: a GISH analysis.

An allotriploid (ALA, 2n=3 x=36) BC(1) plant was obtained by backcrossing a diploid F(1) interspecific hybrid (LA, 2n=2 x=24), derived from a Lilium longiflorum (L genome) and an Asiatic hybrid (A genome), to the latter parent. This allotriploid was backcrossed to a diploid Asiatic hybrid (2n=2 x=24) and to an allotetraploid (LLAA, 2n=4 x=48) LA hybrid. A total of 25 plants of these crosses were examined for ploidy level, and 12 individuals were analyzed for their genome constitution through genomic in situ hybridization (GISH). In most cases the progenies from the triploid-diploid (3 x-2 x) crosses consisted of aneuploids. Further more, there was evidence for the formation of near-haploid (x=12+2) to triploid (3 x=36) gametes in the allotriploid BC(1) plant. The progenies of triploid-tetraploid (3 x-4 x) cross also consisted of mostly aneuploids but in this case the triploid female parent had contributed predominantly near-triploid (2n) gametes for the origin of BC(2) progenies. The different ploidy levels observed between 3 x-2 x and 3 x-4 x crosses are possibly caused by preferential fertilization or survival resulting in a different ratio of chromosome numbers between the embryo and endosperm. Though Lilium has a tetrasporic, eight-nucleate type of embryo sac formation (Fritillaria type), the observed difference between the progeny types in 3 x-2 x and 3 x-4 x crosses is comparable to that of observed in monosporic eight nucleate types (Polygonum type) that predominate in most genera of Angiosperms. An important feature of the genome constitution of the progenies was that the homoeologous recombinant chromosomes were transmitted intact from BC(1) to BC(2) progenies in variable numbers. In addition, there was evidence for the occurrence of new homoeologous recombinations in the triploid BC(1). Of the two euploid BC(2) plants one had originated through the parthenogenetic development of a 2n egg and the other had originated through indeterminate meiotic restitution (IMR).

Introgression of Lilium rubellum Baker chromosomes into L. longiflorum Thunb.: a genome painting study of the F1 hybrid, BC1 and BC2 progenies.

Interspecific hybrids between Lilium longiflorum (L, 2n = 2x = 24) and Lilium rubellum (R, 2n = 2x = 24) were produced with the aim of transferring desirable horticultural traits from L. rubellum to L. longiflorum. All F1 hybrids (LR, 2n = 2x = 24) and BC1 individuals (LLR, 2n = 3x = 36) were phenotypically uniform for plant height, flowering time, leaf shape and flower colour. The BC1 plants were, in spite of their triploid nature, fertile and could be used as a female parent in backcrossings with autotetraploid L. longiflorum (LLLL, 2n = 4x = 48). Twelve BC2 individuals were obtained and three of them were selected for further chromosome analysis. As L. longiflorum and L. rubellum chromosomes were indistinguishable in the hybrids, genomic in-situ hybridization (GISH) was applied to establish the parentage of the chromosomes of the F1 hybrids and the BC1 and BC2 progenies. GISH confirmed the LLRR constitution of the doubled amphimonoploid (allodiploid), and the LLR constitution of all BC1 plants. The three selected BC2 plants were, as expected, aneuploid, containing three complete sets of L. longiflorum chromosomes and six, seven or eight L. rubellum chromosomes, respectively. However, L/R translocation or recombinant chromosomes could not be demonstrated in the mitotic metaphase complements of the F1, BC1 and BC2 plants. In spite of the high frequencies of homoeologous recombination in the F1 hybrids (LR) pollen was found to be sterile in all cases. At metaphase I of the pollen mother cells of the BC1 plants, genome painting did not reveal any cases of homoeologous pairing and recombination between L and R chromosomes. This lack of exchange between homoeologous chromosome segments indicates complete preferential pairing of the L and R chromosomes in the F1 (amphidiploid) and BC1 plants. It seems that the preferential pairing in the F1 and BC1 hybrids hinder the introgression of the chromosome segments or species-specific genes into the recipient for breeding purposes.

The extent and position of homoeologous recombination in a distant hybrid of Alstroemeria: a molecular cytogenetic assessment of first generation backcross progenies.

To estimate the extent and position of homoeologous recombination during meiosis in an interspecific hybrid between two distantly related Alstroemeria species, the chromosome constitution of six first generation backcross (BC1) plants was analysed using sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) analysis. Four different probes were used for the FISH analysis: two species-specific and two rDNA probes. The six BC1 plants were obtained from crosses between the hybrid A. aurea x A. inodora with its parent A. inodora. GISH clearly identified all chromosomes of both parental genomes as well as recombinant chromosomes. The sequential GISH and FISH analysis enabled the accurate identification of all individual chromosomes in the BC1 plants, resulting in the construction of detailed karyotypes of the plants. The identification of the recombinant chromosomes provided evidence which chromosomes of the two species are homoeologous. Two of the BC1 plants were aneuploid (2n=2x+1=17) and four triploid (2n=3x=24), indicating that both n and 2n gametes were functional in the F1 hybrid. Using GISH, it was possible to estimate homeologous recombination in two different types of gametes in the F1 hyrid. The positions of the crossover points ranged from highly proximal to distal and the maximum number of crossover points per chromosome arm was three. Compared with the aneuploid plants, the triploid plants (which received 2n gametes) clearly possessed fewer crossovers per chromosome, indicating reduced chromosome pairing/recombination prior to the formation of the 2n gametes. Besides homeologous recombination, evidence was found for the presence of structural rearrangements (inversion and translocation) between the chromosomes of the parental species. The presence of the ancient translocation was confirmed through FISH analysis of mitotic and meiotic chromosomes.

Homoeologous chromosome pairing in the distant hybrid Alstroemeria aurea x A. inodora and the genome composition of its backcross derivatives determined by fluorescence in situ hybridization with species-specific probes.

A distant hybrid between two diploid species (2n = 2x = 16), Alstroemeria aurea and A. inodora, was investigated for homoeologous chromosome pairing, crossability with A. inodora and chromosome transmission to its BC1 offspring. Fluorescence in situ hybridization (FISH) with two species-specific probes, A001-I (A. aurea specific) and D32-13 (A. inodora specific), was used to analyse chromosome pairing in the hybrid and the genome constitution of its BC1 progeny plants. High frequencies of associated chromosomes were observed in both genotypes of the F1 hybrid, A1P2-2 and A1P4. In the former, both univalents and bivalents were found at metaphase I, whereas the latter plant also showed tri- and quadrivalents. Based on the hybridization sites of DNA probes on the chromosomes of both parental species, it was established that hybrid A1P4 contains a reciprocal translocation between the short arm of chromosome 1 and the long arm of chromosome 8 of A. inodora. Despite regular homoeologous chromosome pairing in 30% of the pollen mother cells, both hybrids were highly sterile. They were backcrossed reciprocally with one of the parental species, A. inodora. Two days after pollination, embryo rescue was applied and, eventually, six BC1 progeny plants were obtained. Among these, two were aneuploids (2n = 2x + 1 = 17) and four were triploids (2n = 3x = 24). The aneuploid plants had originated when the interspecific hybrid was used as a female parent, indicating that n eggs were functional in the hybrid. In addition, 2n gametes were also functional in the hybrid, resulting in the four triploid BC1 plants. Of these four plants, three had received 2n pollen grains from the hybrid and one a 2n egg. Using FISH, homoeologous crossing over between the chromosomes of the two parental species in the hybrid was clearly detected in all BC1 plants. The relevance of these results for the process of introgression and the origin of n and 2n gametes are discussed.

FISH studies reveal the molecular and chromosomal organization of individual telomere domains in tomato.

The molecular and cytological organization of the telomeric repeat (TR) and the subtelomeric repeat (TGR1) of tomato were investigated by fluorescence in situ hybridization (FISH) techniques. Hybridization signals on extended DNA fibres, visualized as linear fluorescent arrays representing individual telomeres, unequivocally demonstrated the molecular co-linear arrangement of both repeats. The majority of the telomeres consisted of a TR and a TGR1 region separated by a spacer. Microscopic measurements of the TR and TGR1 signals revealed high variation in length of both repeats, with maximum sizes of 223 and 1330 kb, respectively. A total of 27 different combinations of TR and TGR1 was detected, suggesting that all chromosome ends have their own unique telomere organization. The fluorescent tracks on the extended DNA fibres were subdivided into four classes: (i) TR-spacer-TGR1; (ii) TR-TGR1; (iii) only TR; (iv) only TGR1. FISH to pachytene chromosomes enabled some of the TR/TGR1 groups to be assigned to specific chromosome ends and to interstitial regions. These signals also provided evidence for a reversed order of the TR and TGR1 sites at the native chromosome ends, suggesting a backfolding telomere structure with the TGR1 repeats occupying the most terminal position of the chromosomes. The FISH signals on diakinesis chromosomes revealed that distal euchromatin areas and flanking telomeric heterochromatin remained highly decondensed around the chiasmata in the euchromatic chromosome areas. The rationale for the occurrence and distribution of the TR and TGR1 repeats on the tomato chromosomes are discussed.

Physical localisation of repetitive DNA sequences in Alstroemeria: karyotyping of two species with species-specific and ribosomal DNA.

Fluorescence in situ hybridization (FISH) was used to localise two species-specific repetitive DNA sequences, A001-I and D32-13, and two highly conserved 25S and 5S rDNA sequences on the metaphase chromosomes of two species of Alstroemeria. The Chilean species, Alstroemeria aurea (2n = 16), has abundant constitutive heterochromatin, whereas the Brazilian species, Alstroemeria inodora, has hardly any heterochromatin. The A. aurea specific A001-I probe hybridized specifically to the C-band regions on all chromosomes. The FISH patterns on A. inodora chromosomes using species-specific probe D32-13 resembled the C-banding pattern and the A001-I pattern on A. aurea chromosomes. There were notable differences in number and distribution of rDNA sites between the two species. The 25S rDNA probe revealed 16 sites in A. aurea that closely colocalised with A001-I sites and 12 in A. inodora that were predominantly detected in the centromeric regions. FISH karyotypes of the two Alstroemeria species were constructed accordingly, enabling full identification of all individual chromosomes. These FISH karyotypes will be useful for monitoring the chromosomes of both Alstroemeria species in hybrids and backcross derivatives.

Molecular cytogenetics of Alstroemeria: identification of parental genomes in interspecific hybrids and characterization of repetitive DNA families in constitutive heterochromatin.

The genus Alstroemeria consists of diploid (2n = 2x = 16) species originating mainly from Chile and Brazil. Most cultivars are triploid or tetraploid interspecific hybrids. C-banding of eight species revealed obvious differentiation of constitutive heterochromatin within the genus. The present study focused on the molecular (cyto)genetic background of this differentiation. Genomic slot-blot analysis demonstrated strong conservation of major parts of the genomes among six species. The chromosomes of A. aurea and A. ligtu, species with pronounced interstitial C-bands, were found to contain large amounts of highly repetitive and species-specific DNA. The variation in size, number and intensity of strongly probed bands of major repetitive DNA families observed in genomic Southern blots of Sau3A, HaeIII, and MseI digests indicated a strong correlation between variation in genomic DNA composition and different C-banding patterns among Alstroemeria species. Genomic in situ hybridization (GISH) revealed a clear distinction between parental chromosomes in the hybrids between Chilean and Brazilian species and also between Chilean species, as long as at least one of the parental species possessed prominent C-banding. Regarding the latter, discriminative hybridization resulted from highly repetitive species specific DNA in the heterochromatic chromosome regions of A. aurea and A. ligtu, and caused GISH banding patterns that coincided with the C-banding patterns.

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.

Localization of Ds-transposon containing T-DNA inserts in the diploid transgenic potato: linkage to the R1 resistance gene against Phytophthora infestans (Mont.) de Bary.

The Dissociation transposable element (Ds) of maize containing NPTII was introduced into the diploid potato (Solanum tuberosum) clone J91-6400-A16 through Agrobacterium tumefaciens mediated transformation. Genomic DNA sequences flanking the T-DNAs from 312 transformants were obtained with inverse polymerase chain reaction or plasmid rescue techniques and used as probes for RFLP linkage analysis. The RFLP map location of 60 T-DNAs carrying Ds-NPTII was determined. The T-DNA distribution per chromosome and the relative distance between them appeared to be random. All 12 chromosomes have been covered with Ds-containing T-DNAs, potentially enabling tagging of any gene in the potato genome. The T-DNA insertions of two transformants, BET92-Ds-A16-259 and BET92-Ds-A16-416, were linked in repulsion to the position of the resistance gene R1 against Phytophthora infestans. After crossing BET92-Ds-A16-416 with a susceptible parent, 4 desired recombinants (Ds carrying T-DNA linked in coupling phase with the R1 gene) were discovered. These will be used for tagging the R1 gene. The efficiency of the pathway from the introduction to localization of T-DNAs is discussed. Key words : Solanum tuberosum, Phytophthora infestans, Ds element, transposon tagging, R genes, euchromatin.

Pollen markers for gene-centromere mapping in diploid potato.

The utility of two pollen genetic markers for estimating the extent of meiotic recombination between the centromere and a marker gene was tested in 2n pollen of diploid potato clones. One of these markers was the distal locus amylose-free (amf) on chromosome 8 and the other was the isozyme locus alcohol dehydrogenase (Adh-1) on chromosome 4. In the case of the amf locus, the gene-centromere distance was estimated in a normal synaptic and a desynaptic genotype. In both cases the genetic analysis was confined to: (1) a direct estimation of the phenotypic (blue vs red) segregation ratios in FDR (first-division restitution) 2n pollen and (2) a classification of the 4 x progeny from 4x (nulliplex amf) x 2x (Amf/amf) crosses into duplex, simplex and nulliplex classes. The recombination frequency between the centromere and the amf locus in the normal synaptic genotype B92-7015-4 corresponded to a gene-centromere distance of 48.8 cM, whereas this distance amounted to 13.3 cM in the desynaptic genotype RS93-8025-1. Hence desynapsis reduced crossing-over by 73%. The observed genetic distance of 48.8 cM in the normal synaptic clone, B92-7015-4, is the highest gene-centromere distance reported so far in potato and this could be explained on the assumption of absolute chiasma interference. For the Adh-1 locus, it was found that heterozygous 2n pollen grains could be detected in pollen samples of the diploid clones, because of the occurrence of a heterodimeric band of the isozyme. Unlike the amf locus, the genecentromere distance for the Adh-1 locus was estimated only on the basis of the duplex, simplex and nulliplex classes in the progenies from 4x (nulliplex Adh-1 (2) )x B92-7015-4 (Adh-1 (1) /Adh-1 (2) )crosses and was found to be 19.4 cM. Because the accurate positions of centromeres in relation to other loci are not available in the existing genetic maps of potato, which are saturated with molecular markers, halftetrad analysis is a promising additional approach to the basic genetics of this crop.

Synapsis and chiasma formation in four meiotic mutants of tomato (Lycopersicon esculentum).

Synapsis and chiasma formation were studied in pollen mother cells of four meiotic mutants of tomato. The four mutants displayed defects in the assembly of the synaptonemal complex (SC) covering the whole range from almost complete absence of synapsis to complete synapsis at pachytene. In three mutants, we found a good correlation between the number of bivalents connected by at least one tripartite SC segment at pachytene and the number of chiasmatic bivalents at metaphase I. We suggest that in tomato functional chiasmata are only formed in the context of the tripartite SC.

Comparative performance of FDR and SDR progenies from reciprocal 4x-2x crosses in potato.

Numerically unreduced (2n) gametes from first division restitution (FDR) are considered to be superior to 2n-gametes from second division restitution (SDR) because they transfer a larger proportion of the total parental heterozygosity and epistasis intact to the tetraploid progeny. This supposed superiority was investigated by comparing 12 sets of reciprocal 4x-2x crosses. Each diploid parent used in a reciprocal set produced 2n-pollen by FDR and 2n-eggs by SDR. Six agronomic characters were investigated. FDR progenies (from 4x.2x) were found to have higher mean yields due to more and bigger tubers. With respect to underwater weight, the overall progeny mean of FDR progenies was significantly higher than that of SDR progenies (from 2x.4x). However, the absolute difference found between both overall progeny means was too small to be of practical significance. No differences between FDR and SDR progeny means were found for vine maturity and chip colour. In addition to the progeny mean, within-progeny variation is important in potato breeding. For vine maturity a higher within-progeny variation was detected in SDR progenies, whereas within-progeny variations for yield, underwater weight and chip colour were not different in FDR and SDR progenies. With regard to vine maturity, we conclude that SDR 2n-gametes are superior to FDR 2n-gametes because, with the same progeny means of FDR and SDR progenies, the within-progeny variation was higher in SDR progenies. Therefore the assumed superiority of FDR 2n-gametes was confirmed for yield but was not observed for vine maturity, underwater weight and chip colour.

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 of a new paramutagenic allele of thesulfurea locus in the tomato cultivar Moneymaker following in vitro culture.

A new allele, SC148, of thesulfurea locus inLycopersicon esculentum was detected in a line derived after repeated selfing of plants that had been regenerated from tissue culture. Like the originalsulf mutant, SC148 displayed two mutant phenotypes: green-yellow speckled plants in which thesulf (vag) allele is present and pure yellow plants homozygous for thesulf (tpura) allele. Although the mutant alleles are recessive to wild-type, an unpredictable number of variegated and pura plants appeared in F1 progenies that had been derived from crosses between SC148 and wild-type tomato plants. The presence of the wild-typesulf (+) allele in these variegated heterozygotes was demonstrated using a cytological marker that is linked tosulf. It is concluded that the mutantsulf allele of SC148, imposes its variegated expression state on the wild-typesulf (+) allele present insulf (+)/sulf(vag) heterozygotes. This behaviour, known as paramutation, has also been described for the originalsulf allele. The SC148 allele, however, seems to induce changes at an earlier stage in development. The analogy of this paramutagenic system to dominant position effect variegation inDrosophila is 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.