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.

Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species.

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68-127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed.

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.

Characterisation and physical localisation of Ty1-copia-like retrotransposons in four Alstroemeria species.

The genus Alstroemeria contains species with large genomes (2C = 36.5-78.9 pg (17,600-38,000 Mb) in those species with 2n = 2x = 16). We investigated the diversity and genomic and chromosomal organisation of Ty1-copia-like retrotransposons in four Alstroemeria species. Analysis of 33 PCR-amplified sequences corresponding to a conserved domain of the Ty1-copia reverse transcriptase (rt) gene showed high heterogeneity among predicted amino acid sequences; no two sequences were identical, but most fell into one of five subgroups. Levels of inter- and intra-specific heterogeneity of sequences were similar. HaeIII-digested genomic DNA of various Alstroemeria species contained distinct bands upon hybridisation with individual rt gene fragments. Hybridisation with the heterogeneous PCR pool of rt fragments (retrotransposon pool) revealed additional bands; some minor bands were characteristic of either Brazilian or Chilean species. In situ hybridisation of the retrotransposon pool from three species to metaphase chromosomes from the same species showed a dispersed distribution of the retrotransposon pool with exclusion from rDNA and other chromosomal sites. Alstroemeria pelegrina, which is without major heterochromatic sites, showed some clustering and small negative bands. The retrotransposon pool was excluded from major DAPI-staining bands in Alstroemeria aurea, but in contrast, the sites of the major tandemly repeated sequences in Alstroemeria inodora showed a hybridisation signal similar to that in the rest of the chromosomes. The data are discussed in the context of the contribution of Ty1-copia-like retrotransposons to plant genome size, their evolution, and their value for phylogenetic and biodiversity studies.

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.

GBSS T-DNA inserts giving partial complementation of the amylose-free potato mutant can also cause co-suppression of the endogenous GBSS gene in a wild-type background.

The wild-type gene encoding granule-bound starch synthase (GBSS) is capable of both complementing the amylose-free (amf) potato mutant and inhibiting the endogenous GBSS gene expression in wild-type potato. Co-suppression of the endogenous GBSS gene, easily visualised by staining the starch with iodine, occurred when the full-size GBSS sequence (genomic), GBSS cDNA or even the mutant amf allele were introduced into the wild-type potato. Conversely, introduction of the GBSS promoter sequence alone, did not result in co-suppression in the 80 analysed transformants. Neither the orientation of the GBSS gene with respect to kanamycin resistance nor the presence of an enhancer influenced the frequency of plants showing a co-suppression phenotype. After crossing a partially complemented amf mutant with a homozygous wild-type plant, the F1 offspring segregated into plant phenotypes with normal and decreased expression of the GBSS gene. This decreased expression correlated with the presence of a linked block of five T-DNA inserts which was previously shown to be correlated with partial complementation of the amf mutant. This crossing experiment indicates that co-suppression can cause inhibition of gene expression of both inserted and endogenous wild-type GBSS genes. The frequency of partially complemented amf plants was equal to the frequency of co-suppressed wild types when a construct, with an enhancer in front of the GBSS promoter, was used (pWAM 101E). This might suggest that partial complementation of the amf genotype caused by unstable expression of the transgene can be overcome by inserting an enhancer in front of the GBSS promoter.

Generation of mouse chromosome painting probes by DOP-PCR amplification of microdissected meiotic chromosomes.

We describe here the development of four sets of painting probes for mouse chromosomes 1 and 13, 2 and 8, 6 and 15, and X and Y by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification of 10-20 copies of microdissected male meiotic chromosomes. The X,Y probe was obtained from the X-Y bivalent of diakinesis/metaphase I complements of mice with a normal karyotype, whereas the other probes were derived from tri- or quadrivalents in diakinesis/metaphase I of two reciprocal translocations, T(1;13)70H and T(2;8)2Wa, and one inversion heterozygote carrying a small deletion, In(6;15)Rb1Ald del6(15)1Wa. The specificity of these probes was established by fluorescence in situ hybridization (FISH) to meiotic and mitotic metaphase complements. The chromosomes painted by these probes could be identified by single or multicolor FISH.

Factors affecting the inhibition by antisense RNA of granule-bound starch synthase gene expression in potato.

Inhibition of expression of specific genes by means of antisense RNA is widely used, although little information is available regarding conditions that affect the efficacy of inhibition. In this study, inhibition of granule-bound starch synthase (GBSS), a key enzyme in starch biosynthesis, is used as a model system. Eleven antisense constructs derived from the full-length GBSS cDNA, the genomic GBSS coding region (gDNA) or fragments of each of these sequences, were analysed with respect to their inhibitory effect. Introduction of full-length gDNA constructs yielded a lower percentage of transgenic clones showing complete inhibition than did introduction of the full-length cDNA constructs. This may be caused by a lower antisense binding capacity of the former due to the relatively low GC content in intron sequences present in the gDNA constructs. The presence of multiple T-DNA insertions was related to a higher degree of inhibition. Putative polyadenylation signals on the antisense strand of the GBSS gene resulted in a premature stop of transcription of some of the antisense genes, as demonstrated by the expression of smaller antisense RNA transcripts. Introduction of antisense constructs driven by the promoter of the (target) GBSS gene resulted in a higher percentage of clones with complete inhibition than introduction of antisense constructs driven by the 35S CaMV promoter. Complete antisense inhibition was achieved in 25% of the clones carrying the antisense construct pKGBA50, which is based on the GBSS promoter and the full-length GBSS cDNA. Thus, it is concluded that the use of pKGBA50 is very suitable for the modification of the composition of potato tuber starch via antisense RNA.

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth.

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.

Introduction of hygromycin B resistance into Schizophyllum commune: preferential methylation of donor DNA.

Cotransformation of a trp1 strain of Schizophyllum commune with the homologous TRP gene and the Escherichia coli HPT gene was used to study the feasibility of transformation of S. commune to hygromycin B resistance. Southern blot analysis showed that 75% of the TRP transformants contained multiple integrated copies of the HPT gene. However only 7% of the transformants were resistant to 25 micrograms/ml hygromycin B and direct selection for hygromycin B resistance was hampered by the high incidence of spontaneously arising resistant colonies. Rescue of the HPT gene was possible with E. coli JA221 (mcr-) but not with JM83, suggesting methylation of the integrated donor DNA. Isoschizomer analyses confirmed heavy methylation in the HPT gene and flanking vector sequences but not in the homologous donor TRP gene and its flanking vector sequences. Also cotransforming S. commune Sc4 gene and flanking vector sequences were not methylated. A fusion between the S. commune TRP1 and the E. coli HPT genes resulted in only slight or no methylation of both vector and HPT sequences and in a higher hygromycin B resistance level. This suggests that transformation with DNA exclusively containing foreign sequences results in integration into regions where methylation occurs, possibly entailing poor transcription. Methylation of the HPT gene was also indicated by the stimulation of growth by 5-azacytidine of transformants on hygromycin B containing medium.