Identification of the different Brassica nigra chromosomes from both sets of B. oleracea-B. nigra and B. napus-B. nigra addition lines with a special emphasis on chromosome transmission and self-incompatibility

 The F₁ hybrids produced after crosses between B. gra and B. oleracea were backcrossed two or three times to B. oleracea. Among the 14 plants analysed, five were monosomic addition lines (2n=19), six were double monosomic addition lines (2n=20) and three had three or four additional chromosomes. From these lines, 14 isozyme and 80 RAPD loci were localized on the eight chromosomes of B. nigra. The comparison between B. napus-B. nigra, from which five B. nigra chromosomes were already described, and the new set of B. oleracea-B. nigra addition lines was performed using five isozyme and 22 common RAPD loci. The homology of the common RAPD loci was confirmed by hybridization of the two sets of addition lines as well as the presence of duplicated loci on different chromosomes. For the five added chromosomes available on the two genetic backgrounds, i.e. B. napus and B. oleracea, using isozyme markers, the chromosome transmission rate was studied from backcross progeny using the recurrent parent either as male or as female and from the selfing of monosomic addition lines. For each chromosome, no difference was detected between male and female transmission except for chromosome 3. This latter presented a percentage of female transmission of around 20%, close to the ones observed for the other chromosomes, but a very low male transmission (1.3%). The analysis from restriction enzyme digests of PCR products, obtained from primers selected in highly conserved regions of self-incompatible genes, suggested that the chromosome 3 probably carried the SLG-B. nigra locus. Received: 25 September 1996 / Accepted: 18 October 1996     

Analysis of the Brassica oleracea genome by the generation of B. campestris-oleracea chromosome addition lines: characterization by isozymes and rDNA genes

Summary This study aimed at generating chromosome addition lines and disclosing genome specific markers in Brassica. These stocks will be used to study genome evolution in Brassica oleracea L., B. campestris L. and the derived amphidiploid species B. napus L. B. campestris-oleracea monosomic and disomic chromosome addition plants were generated by crossing and backcrossing the natural amphidiploid B. napus to the diploid parental species B. campestris. The pollen viability of the derived sesquidiploid and hyperploid ranged from 63% to 88%, while the monosomic and disomic addition plants had an average pollen fertility of 94% and 91%, respectively. The addition lines were genetically characterized by genome specific markers. The isozymes for 6PGD, LAP, PGI and PGM, and rDNA Eco RI restriction fragments were found to possess the desired genome specificity. Duplicated loci for several of these markers were observed in B. campestris and B. oleracea, supporting the hypothesis that these diploid species are actually secondary polyploids. A total of eight monosomic and eight disomic addition plants were identified and characterized on the basis of these markers. Another 51 plants remained uncharacterized due to the lack of additional markers. rDNA genes were found to be distributed in more than one chromosome, differing in its restriction sites. Intergenomic recombination for some of the markers was detected at frequencies between 6% and 20%, revealing the feasibility of intergenomic gene transfer.     

Inheritance of isozyme and RFLP markers in Brassica campestris and comparison with B. oleracea

Summary Using primarily cDNA restriction fragment length polymorphism markers (RFLPs) previously located to Brassica oleracea (cabbage, 2n=18) chromosomes, we initiated a comparative RFLP map in an F₂ population of B. campestris (turnip x mock pak-choi, 2n=20). As with B. oleracea, the genome of B. campestris showed extensive gene duplication, and the majority of detected duplicated loci were unlinked. Only 6 of the 49 identified loci were represented as a single copy, and 3 of these 6 were clustered on a single linkage group showing a distorted segregation ratio. Comparison with B. Oleracea indicates this synteny is conserved between species. Two other linkage groups also appeared syntenic between B. oleracea and B. campestris. One single copy locus appears to have changed synteny between B. oleracea and B. campestris. These observations suggest that B. oleracea and B. campestris share a common ancestor, but that chromosome repatterning has occurred during or after speciation. Within B. campestris, 5 loci appeared duplicated in one parent or the other, and 2 of these were linked. Differentiation through subspecies-specific duplication or deletion events is suggested as one mechansim for the evolution of numerous morphotypes within each of these species.     

Production and cytogenetics of Brassica campestris-alboglabra chromosome addition lines

 Four different Brassica campestris-alboglabra monosomic addition lines (AA+1 chromosome from C, 2n=21) were obtained after consecutive backcrosses between resynthesized B. napus (AACC, 2n=38) and the parental B. campestris (AA, 2n=20) accession. The alien chromosomes of B. alboglabra (CC, 2n=18) in the addition lines were distinguished by random amplified polymorphic DNA (RAPD) marker analysis and morphology of mitotic chromosomes. Four RAPD marker synteny groups were established, which represented the four different alien chromosomes of B. alboglabra in the four addition lines. Three of the four addition lines were identified to harbour chromosomes 4, 8 or 9 of B. alboglabra. Studies on meiotic pairing in the addition lines revealed intergenomic homoeology relationships among specific chromosome arms between the A- and C-genomes. The long arm of B. campestris chromosome 9 was homoeologous with the long arm of B. alboglabra chromosome 4, while its short arm with the short arms of B. alboglabra chromosomes 8 and 9. Such an intergenomic homoeology relationship supports the hypothesis that B. campestris and B. alboglabra share a common ancestor but that chromosomal rearrangements have occurred during the evolution of the two species. Intergenomic introgression was observed in the progenies of the addition lines. The introgression of an entire B. alboglabra marker synteny group into the B. campestris genome implied the possible occurrence of interspecific chromosomal substitution. Received: 30 May 1996 / Accepted: 18 October 1996     

Construction of synteny groups of Brassica alboglabra by RAPD markers and detection of chromosome aberrations and distorted transmission under the genetic background of B. campestris

Interspecific hybrids were produced by crosses between the inbred lines of B. campestris and B. alboglabra, and were backcrossed twice to B. campestris. Genetical constitutions of the BC₂ plants were analyzed by RAPD (random amplified polymorphic DNA), flow cytometry and cytological observations. By using 140 arbitrary primers, a total of 137 polymorphic bands were obtained and 125 were found to be specific to B. alboglabra. Based on the presence and absence of the specific RAPD markers of B. alboglabra, 13 synteny groups were constructed. The number of markers in each synteny group was found to be different and varied from 2 to 28. This reflects the difference in the degree of genetic variability among the B. alboglabra chromosomes from those of B. campestris. Losses or gains of RAPD markers were observed frequently in most of the synteny groups, which indicated the occurrence of chromosome translocations and/or deletions in the chromosomes of B. alboglabra. In a population of 40 BC₂ plants, chromosome transmission rates were analyzed by using the RAPD markers in each synteny group. Most of the chromosomes of the synteny groups were transmitted with rates of 0.37–0.68. An extremely high transmission rate, 0.98, was however observed in one of the synteny groups. Inheritance data of the synteny groups revealed relationships among themselves. The plants lacking the RAPD markers of two synteny groups tended to lose others belonging to the rest of the synteny groups, indicating the effects of these groups on the transmission of B. alboglabra chromosomes to the B. campestris background. Received: 26 February 1999 / Accepted: 30 December 1999     

Selection of monosomic addition plants in offspring families using repetitive DNA probes in Beta L.

The distribution of two repetitive DNA probes Sat-121 and PB6-4, specific for the section Procumbentes of the genus Beta, was tested in 16 B. patellaris monosomic addition families using a dot-blot hybridization procedure. All monosomic additions were accurately distinguished from diploid sib plants with both DNA probes. The probe PB6-4, with the strongest signal after hybridization, was selected for rapid screening of an extensive number of putative monosomic additions in B. patellaris or B. procumbens addition families using a squash-blot hybridization procedure. The probe PB6-4 detected 118 monosomic additions in 640 plants (18.4%) in eight different B. procumbens addition families. The addition family with chromosome 4 of B. procumbens was semi-lethal and could not be tested. The distribution of PB6-4 in B. patellaris addition families was confirmed in 63 addition families using the squash-blot procedure. In 4580 plants of these addition families, 628 individual monosomic additions (13.7%) were found. The relationship of the morphological characteristics of monosomic addition plants to the results of the squash-blot hybridization (plants with signal) using probe PB6-4 is quite rigorous but not complete. The correlation between plants with a signal and chromosome number (2n=19) is complete. These results indicate that sequences present on PB6-4 are probably present on all chromosomes of B. patellaris and B. procumbens. The possibility of utilizing the sequence information of Sat-121 for a PCR-based assay to screen for putative monosomic addition plants was also investigated as an alternative to chromosome counting. The DNA-amplification profiles using the primers REP and REP.INV clearly distinguished monosomic addition plants from their diploid sibs.     

Assignment of DNA markers to Nicotiana sylvestris chromosomes using monosomic alien addition lines

 A sesquidiploid hybrid (PPS, 2n=32) between Nicotiana plumbaginifolia (PP, 2n=20) and N. sylvestris (SS, 2n=24) was backcrossed to N. plumbaginifolia to produce monosomic alien addition lines. A total of 89 2n=21 plants, each containing two sets of N. plumbaginifolia chromosomes and a single N. sylvestris chromosome, were obtained in the BC₁ and BC₂ generations. These plants were classified into 12 groups based on morphological characteristics. The N. sylvestris chromosomes in these plants were identified by RFLP and karyotype analyses. Among the 84 probes tested, 20 could not detect N. sylvestris-specific DNA bands, and the remaining 64 were assigned to 9 normal and 6 aberrant synteny groups. The 9 normal synteny groups corresponded to chromosomes 2, 4, 5, 6, 7, 8, 9, 10 and 12, respectively. Four aberrant synteny groups were the result of chromosome translocations, and 2 were deletions. Received: 10 April 1996 / Accepted: 5 July 1996     

Generation and characterisation of monosomic chromosome addition lines of Brassica campestris - B. oxyrrhina

 Monosomic chromosome addition lines of Brassica oxyrrhina in the background of alloplasmic B. campestris carrying B. oxyrrhina cytoplasm were generated and characterised through morphology, cytology and molecular (RAPD) analysis. Four successive backcrosses of the synthetic alloploid B. oxycamp with B. campestris yielded 24 monosomic addition plants that were grouped into seven different synteny groups based on morphological similarity and RAPD patterns. Each synteny group exhibited morphological features diagnostic for the presence of individual B. oxyrrhina chromosomes including some novel phenotypes. Meiotic studies of the addition lines revealed the homoeology of four B. oxyrrhina chromosomes (synteny groups 1, 3, 5 and 6 ) with B. campestris chromosomes as indicated by trivalent associations, with the highest homoeology (44.23%) in synteny group 1 and the lowest (6.1%) in synteny group 3. Seed fertility of the addition lines ranged from 94.85% (synteny group 1) to 56.98% (synteny group 5). All of the addition lines were male-sterile except synteny group 6 which had 12–16% stainable pollen. Ovule transmission of the B. oxyrrhina chromosomes added to the progenies of addition lines ranged from 23.52% (synteny group 6) to 14% (synteny group 7). RAPD analysis confirmed the validity of synteny grouping based on morphological observations. Approximately 45% of the primers studied were informative, giving B. oxyrrhina-specific RAPD bands unique for each synteny group, except group 6. Received: 20 October 1997 / Accepted: 31 March 1998     

A Brassica campestris-alboglabra addition line and its use for gene mapping,intergenomic gene transfer and generation of trisomics

Summary Brassica campestris-alboglabra monosomic addition lines were developed from a trigenomic Brassica hybrid (2 n=3 x=29, AAC) obtained by backcrossing a resynthesized B. napus (2 n=4 x=38, AACC) line to its parental B. campestris (2 n=2 x=20, AA) line. One addition line was characterized genetically with three loci specific for the alien chromosome and cytologically by meiotic analysis. The following results were obtained. (1) The same chromosome in the B. alboglabra (2 n= 2 x=18, CC) genome carried the three loci, E _c, W _c and Lap-1 C ^c, which control the biosynthesis of erucic acid, white flower colour and the faster migrating band of leucine aminopeptidase, respectively. The linear order and possible positions of the three loci were inferred. The meiotic behaviour of the alien chromosome was documented and its transmission frequency was assessed. (2) Intergenomic recombination frequently occurred in the monosomic addition line, resulting in the introgression of one or two loci from the alien chromosome into the B. campestris genome. (3) B. campestris trisomics were found in the progeny of the monosomic addition line. (4) The removal of the other eight C-genome chromosomes from the trigenomic Brassica hybrid led to a dramatic increase in the erucic acid content of the monosomic addition line. (5) No offspring of the trigenomic Brassica hybrid showed evidence of intergenomic recombination and introgression of the W _c locus into the B. campestris genome. It is questioned whether such a difference might be due to a possible regulating mechanism for homoeologous chromosome pairing.     

Characterization of disomic addition lines Brassica napus-Brassica nigra by isozyme,fatty acid,and RFLP markers

Summary Six Brassica napus — B. nigra disomic addition lines were characterized by isozyme, fatty acid, and RFLP markers. The markers were arranged in six synteny groups, representing six of the eight chromosomes present in the B. nigra genome. Synteny group 1 displayed high levels of linoleic and linolenic acids in the seeds of the B. nigra parent. Synteny group 3 accumulated higher levels of eicosenoic and erucic acid than B. nigra. Three of the lines transmitted the alien chromosome to 100% of the progeny. The rest had variable transmission rates but all were above 50%. Most of the lines produced disomic addition plants in their progeny, suggesting pollen transmission of the alien chromosome. In addition to the marked lines, six others remained unmarked. These could be grouped into two classes according to their alien chromosome transmission. It is likely that they represent the two other B. nigra chromosomes that remained uncharacterized by the markers. No diploid individuals carrying B. nigra genome-specific markers were detected in the progenies studied.     

Recombination of Solanum brevidens chromosomes in the second backcross generation from a somatic hybrid with S. tuberosum

Solanum brevidens synteny groups were examined with 47 widely-distributed RFLP markers in 17 BC₂ progeny from six fertile BC₁ plants. The BC₁ plants were derived from a single S. brevidens + S. tuberosum somatic hybrid backcrossed with S. tuberosum (potato). Probes which were linked in potato and tomato were also found to be syntenic along each of the 12 S. brevidens chromosomes. More than half of the S. brevidens synteny groups had lost one or more S. brevidens-specific RFLPs in the BC₂, suggesting that recombination had occurred. For 8 of the 12 S. brevidens RFLP synteny groups, the frequency of recombinant chromosomes exceeded that of intact parental chromosomes. Using the RFLP data, 161 RAPD markers were tentatively located throughout the S. brevidens genome. Further analyses with 39 of these 161 RAPD markers generally showed that RAPD and RFLP results were comparable, but some inconsistencies were noted with 14 of the 39 RAPD markers. The extent of marker loss and the high frequency of synteny groups which were marked by a single S. brevidens-specific RFLP marker suggest that the S. brevidens chromosomes have some pairing affinity with potato chromosomes. This interaction should facilitate the transfer of novel disease-resistance traits into potato breeding lines. One plant was recovered with the chromosome number of S. tuberosum (2n=48) that carried a single S. brevidens RFLP marker, suggesting transfer of this S. brevidens marker into the genome of S. tuberosum.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Molecular and cytogenetic characterization of an Oryza officinalis–O. sativa chromosome 4 addition line and its progenies

The wild species Oryza officinalis Wall. ex Watt (2n = 24, CC) is a valuable genetic resource for rice (O. sativa L., 2n = 24, AA) breeding and genomics research. Genomic in situ hybridization (GISH) and molecular approaches were used to determine the nature and composition of the additional chromosome in a monosomic alien addition line (MAAL) of O. officinalis and its backcross progenies. The extra wild species chromosome in the MAAL (2n = 2x = 25) was a mosaic one, comprising of the long arm of chromosome 4 from O. officinalis and the short arm from O. sativa. Comparative analysis showed that O. sativa and O. officinalis shared high synteny of restriction fragment length polymorphism (RFLP) markers and low synteny of simple sequence repeat (SSR) markers. A DNA methylation alteration was revealed at C619 in the MAAL and progenies. Analysis of progenies of the MAAL indicated that introgression segments were small in size and introgression was not evenly distributed along the long arm. One recombination hot spot between C513 and RG177 was identified, which is in a gene-rich region.     

Thinopyrum distichum addition lines: production,morphological and cytological characterisation of 11 disomic addition lines and stable addition-substitution line

Plants of the partial amphiploid Inia 66/Thinopyrum distichum (2n = 70)//Inia 66 (2n = 56) were used as male parents in crosses with the monosomic series in the common wheat cultivar Inia 66. The genome and homoeologous group of the monosomic used in the cross affected the distribution of chromosome number of the progeny plants in the F₂ and F₄. Meiosis in the pollen mother cells of the B₁F₇ partial amphiploids was not stable, and not different from that of the B₁F₁ in which univalents and multivalents were observed. Disomic addition lines were selected on the basis of morphology and meiotic stability in the F₂, F₄ and F₅. Eleven of the fourteen possible wheat-Th. distichum disomic addition lines were identified using chromosome C-band pattern, as well as size and arm ratio, as genetic markers. Addition of T. distichum chromosome J ^dll produced a phenotype indicating homoeology with wheat group-2 chromosomes. Clear indications of homoeology based on morphological characteristics were not obtained in any of the other addition lines, probably due to the mixed homoeology of the Th. distichum chromosomes relative to wheat. The addition lines were all susceptible to leaf rust, unlike the germplasm-line Indis which carries a leaf rust resistance gene on a translocation segment derived from Th. distichum. Instability of meiotic pairing was observed in all addition lines. The stability, or not, of progeny chromosome counts did not reflect the level of chromosome pairing instability in the parental plants. SDS-PAGE for gliadin-type seed proteins revealed two addition lines which expressed seed storage proteins uncommon to Inia 66 but typical of Th. distichum.     

Assignment of RFLP linkage groups to chromosomes using monosomic F1 analysis in hexaploid oat

The availability of molecular genetic maps in oat (Avena spp.) and improved identification of chromosomes by C-banding are two recent developments that have made locating linkage groups to chromosomes possible in cultivated hexaploid oat, 2n=6x=42. Monosomic series derived from Avena byzantina C. Koch cv Kanota and from Avena sativa L. cv Sun II were used as maternal plants in crosses with the parents, Kanota-1 and Ogle-C, of the oat RFLP mapping population. Monosomic F₁ plants were identified by root-tip cell chromosome counts. For marker analysis, DNAs of eight F₂ plants from a monosomic F₁ were combined to provide a larger source of DNA that mimicked that of the monosomic F₁ plant. Absence of maternal alleles in monosomic F₁s served to associate linkage groups with individual chromosomes. Twenty two linkage groups were associated with 16 chromosomes. In seven instances, linkage groups that were independent of each other in recombination analyses were associated with the same chromosome. Five linkage groups were shown to be associated with translocation differences among oat lines. Additionally, the results better-characterized the oat monosomic series through the detection of duplicates and translocation differences among the various monosomic lines. The F₁ monosomic series represents a powerful cytogenetic tool with the potential to greatly improve understanding of the oat genome. Received: 24 April 2000 / Accepted: 10 May 2000     

Cytological and molecular characterization of oat x maize partial hybrids

In cereals, interspecific and intergeneric hybridizations (wide crosses) which yield karyotypically stable hybrid plants have been used as starting points to widen the genetic base of a crop and to construct stocks for genetic analysis. Also, uniparental genome elimination in karyotypically unstable hybrids has been utilized for cereal haploid production. We have crossed hexaploid oat (2n=6x=42, Avena sativa L.) and maize (2n=2x=20, Zea mays L.) and recovered 90 progenies through embryo rescue. Fifty-two plants (58%) produced from oatxmaize hybridization were oat haploids (2n=3x=21) following maize chromosome elimination. Twenty-eight plants (31%) were found to be stable partial hybrids with 1–4 maize chromosomes in addition to a haploid set of 21 oat chromosomes (2n=21+1 to 2n=21+4). Ten of the ninety plants produced were found to be apparent chromosomal chimeras, where some tissues in a given plant contained maize chromosomes while other tissues did not, or else different tissues contained a different number of maize chromosomes. DNA restriction fragment length polymorphisms (RFLPs) were used to identify the maize chromosome(s) present in the various oat-maize progenies. Maize chromosomes 2, 3, 4, 5, 6, 7, 8, and 9 were detected in partial hybrids and chromosomal chimeras. Maize chromosomes 1 and 10 were not detected in the plants analyzed to-date. Furthermore, partial self-fertility, which is common in oat haploids, was also observed in some oat-maize hybrids. Upon selfing, partial hybrids with one or two maize chromosomes showed nearly complete transmission of the maize chromosome to give self-fertile maize-chromosome-addition oat plants. Fertile lines were recovered that contained an added maize chromosome or chromosome pair representing six of the ten maize chromosomes. Four independently derived disomic maize chromosome addition lines contained chromosome 4, one line carried chromosome 7, two lines had chromosome 9, one had chromosome 2, and one had chromosome 3. One maize chromosome-8 monosomic addition line was also identified. We also identified a double disomic addition line containing both maize chromosomes 4 and 7. This constitutes the first report of the production of karyotypically stable partial hybrids involving highly unrelated species from two subfamilies of the Gramineae (Pooideae — oat, and Panicoideae — maize) and the subsequent recovery of fertile oat-maize chromosome addition lines. These represent novel material for gene/ marker mapping, maize chromosome manipulation, the study of maize gene expression in oat, and the transfer of maize DNA, genes, or active transposons to oat.Joint contribution of the Minnesota Agricultural Experiment Station and USDA-ARS. Scientific journal series paper No. 21 859 of the Minnesota Agricultural Experiment Station. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the USDA-ARS or the University of Minnesota and does not imply approval over other products that also may be suitable     

Characterisation of disease resistance gene-like sequences in Brassica oleracea L.

Several cloned disease resistance genes from a wide range of plant species are known to share conserved regions with similar structural motifs. Degenerate primers based on conserved sequences of the nucleotide binding site of the genes RPS2, N and L6 were used for polymerase chain reaction (PCR) amplification from genomic DNA of two doubled haploid lines of Brassica oleracea. Sequences of amplified products were highly variable, but most of them showed similarity to known disease resistance genes, including RPS5, RPS2 and N, and to disease resistance gene-like sequences (RGLs) from different species. Primers based on B. oleracea sequences amplified five groups of RGLs. Products were mapped through cleaved amplified polymorphic sequence assays onto four different linkage groups of B. oleracea. PCR amplification from cDNA and allele analysis indicated that four locus-specific RGL fragments are expressed in cauliflower. Screening of a B. oleracea bacterial artificial chromosome library (BAC) with four B. oleracea RGL probes identified a small number of clones, suggesting that the four RGLs may not be highly copied. Screening of a BAC library of A. thaliana with the same probes identified clones that mapped onto four different chromosomes. These map positions correspond to known disease resistance loci of A. thaliana. Received: 12 November 1999 / Accepted: 19 June 2000     

Resynthesis of Brassica carinata by protoplast fusion and recovery of a novel cytoplasmic hybrid

Brassica carinata (2n=34, BBCC), was synthesized by fusing dark grown etiolated hypocotyl protoplasts of B. nigra (2n=16, BB) with green mesophyll protoplasts of B. oleracea (2n=18,CC) using polyethylene glycol. Heterokaryons could be microscopically distinguished from the parental types by their dark green chloroplasts in the colourless hypocotyl protoplast background. The mean heterokaryotic fusion frequency estimated on the basis of this morphological distinction was about 16%. A total of 626 calli were obtained, of which 92 regenerated shoots after transfer to zeatin (2 mg/l) supplemented MS medium. Of these, 81 calli differentiated into plants morphologically similar to naturally occurring B. carinata and 11 calli yielded plants resembling parental types. Meiosis in seven hybrid plants showed the chromosome number to be 2n=34 the sum of B. nigra and B. oleracea chromosomes. Molecular confirmation of the amphidiploid nature of hybrids was obtained by probing with a B. juncea derived genomic clone. The use of chloroplast and mitochondrial specific gene probes, revealed that one plant was a cytoplasmic hybrid having cp DNA sequences of both B. oleracea and B. nigra and mt DNA sequences of B. nigra.Abbreviations PEG Polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - IBA Indole-3-butyric acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962)     

The morphology, cytology, and C-banded karyotypes of Brassica campestris, B. oleracea, and B. napus plants regenerated from protoplasts

The behaviour of Brassica campestris (2n=20, AA), B. oleracea (2n=18, CC), and B. napus (2n=38, AACC) were studied during a tissue-culturing process. Hypocotyl-protoplasts were cultivated into calli from which new plants were regenerated. The regenerated plants were compared, and mitotic root-tip cells were C-banded and karyotyped. A majority of the plants were tetraploid. The meioses were studied in the PMCs. A number of abberations were observed, mainly due to faulty spindle function. There was a difference between the three species in that B. campestris performed the most poorly with many fewer regenerated plants. These plants were more morphologically disturbed and had more problems during pollen production than B. oleracea and B. napus plants.     

Molecular cytogenetic analysis of Brassica rapa-Brassica oleracea var. alboglabra monosomic addition lines

Interspecific alien chromosome addition lines can be very useful for gene mapping and studying chromosome homoeology between closely related species. In this study we demonstrate a simple but robust manner of identifying individual C-genome chromosomes (C5, C8 and C9) in the A-genome background through the simultaneous use of 5S and 25S ribosomal probes on mitotic and meiotic chromosomes of three different Brassica rapa-B. oleracea var. alboglabra monosomic addition lines. Sequential silver staining and fluorescence in situ hybridisation indicated that 18S-5.8S-25S rRNA genes on the additional chromosome C9 are expressed in the A-genome background. Meiotic behaviour of the additional chromosomes was studied in pollen mother cells at diakinesis and metaphase I. In all of the addition lines the alien chromosome was most frequently observed as a univalent. The alien chromosome C5, which carries an intercalary 5S rDNA locus, occasionally formed trivalents that involved either rDNA- or non rDNA-carrying chromosomes from the A genome. In the case of chromosomes C8 and C9, the most frequently observed intergenomic associations involved the regions occupied by 18S-5.8S-25S ribosomal RNA genes. It is possible that not all such associations represent true pairing but are remnants of nucleolar associations from the preceding interphase. Variations in the numbers and distribution of 5S and 25S rDNA sites between cultivars of B. oleracea, B. oleracea var. alboglabra and B. rapa are discussed.This revised version was published online in April 2005 with corrections to Fig. 2.     

Construction of a binary BAC library for an apomictic monosomic addition line of<Emphasis Type="Italic"> Beta corolliflora</Emphasis> in sugar beet and identification of the clones derived from the alien chromosome

A plant-transformation-competent binary BAC library was constructed from the genomic DNA of the chromosome 9 monosomic addition line of Beta corolliflora Zoss. in sugar beet (B. vulgaris. L). This monosomic addition line (designated M14) is characterized by diplosporic reproduction caused by the alien chromosome carrying the gene(s) responsible for diplospory. The library consists of 49,920 clones with an average insert size of 127 kb, representing approximately 7.5 haploid genome equivalents and providing a greater than 99% probability of isolating a single-copy DNA sequence from the library. To develop the scaffold of a physical map for the alien chromosome, B. corolliflora genome-specific dispersed repetitive DNA sequences were used as probes to isolate BAC clones derived from the alien chromosome in the library. A total of 2,365 positive clones were obtained and arrayed into a sublibrary specific for B. corolliflora chromosome 9 (designated bcBAC-IX). The bcBAC-IX sublibrary was further screened with a subtractive cDNA pool generated from the ovules of M14 and the floral buds of B. vulgaris by the suppression subtractive hybridization method. One hundred and three positive binary BACs were obtained, which potentially contain the genes of the alien chromosome specifically expressed during the ovule and embryo development of M14, and may be associated with apomictic reproduction. Thus, these binary BAC clones will be useful for identification of the genes for apomixis by genetic transformation.Communicated by H. C. Becker