Institut für Pflanzenbau und Pflanzenzüchtung, Georg August Universität Göttingen, von Siebold Str. 8, 37075 Göttingen, GER
tel 0551 - 394381, fax 0551 - 394601, e-mail: UBellin@GWDG.de and firstname.lastname@example.org
Interspecific crosses are possible and common in Brassica to transfer desired characters like disease resistance. The amphidiploid species B. carinata (BBCC) and B. juncea (AABB) are composed of the diploid species B. nigra (BB), B. campestris (AA) and B. oleracea (CC). All species are agronomically important crops. The B-genome of 3 species contains interesting disease resistance genes against for example phoma lingam. By interspecific crossing and backcrossing the B-genome from all three sources was introduced to rape (AACC). To value the introduction of foreign chromatin in rape seed it would be useful to analyse the extent of integration. With banding methods it is not possible to differentiate between the genomes. But clear chromosome painting with GISH (Genomic Fluorescence in situ Hybridisation) proved the ancestral parents in the amphidiploids. Chromosome painting showed species specific sequences mainly around the centromere known as heterochromatic region. Chromosome arms contain more euchromatin and DNA sequences common to all 5 species and were therefore not stained well.
To evaluate the introduction of the foreign chromatin B-genome chromosomes can be proved in the backcrossings. Translocation lines will be checked for B-genome introduction. GISH will be adjusted to meiotic metaphase to value the extend of inter genomic exchange.
KEYWORDS Brassica nigra - B. juncea - B. carinata - interspecific crosses - B-genome
Polyploidization is a common phenomena in the Brassicaceae. Brassica carinata (BBCC) and B. juncea (AABB) are natural allotetraploids derived from the hybridisation of B. oleracea (CC) with B. nigra (BB) and B. campestris (AA) with B. nigra (BB) (MORINAGA, 1931). Both amphidiploids are composed of the complete number of chromosomes of their ancestors. The phylogenetic relation-ship in the genus Brassica is specially interesting for plant breeders because it contains many agronomically important species. Interspecific crosses are possible and common to transfer desired characters like disease resistance (PLIESKE, 1998, JAHIER et al., 1987). It would be very useful to follow more closely the introgression of foreign gene material in the bastards and their progeny. Successful gene transfer and integration of the desired character is most likely to be achieved with recombination between different genomes. Recombination is a result of intergenomic chromosome pairing.
Brassica chromosomes are small and specially in meiosis difficult to distinguish. It is not possible to differentiate between the genomes. Intra- and intergenomic chromosome pairing can`t be differentiated. With GISH (genomic in situ fluorescence hybridisation) it was tried to distinguish between the different genomes in B. juncea and B. carinata. GISH combines cytological and molecular methods. Fixed chromosomes on microscope slides are hybridised with fluorescence colour labelled DNA probes. Differently `painted´ genomes are only possible to be achieved, when the genomes are not too closely related. Enough genome specific sequences have to hybridise with the chromosomes to give a reliable fluorescence signal.
B. carinata no. 3220, B. juncea (Stoke), B. oleracea var. italica (Vitamina), B. nigra no. 2558, B. campestris (Yellow sarson) and B. napus (summer rape Andor) were chosen from the institute collection. Interspecific crosses between all species resulted in B. napus with additional B-genome chromosomes of the 3 species B. nigra, B. carinata and B. juncea.
Cytological preparations and in situ hybridisation were done according to the methods of SNOWDON et al. (1997).
With genomic in situ hybridisation (GISH) is was possible to distinguish between chromosomes of the closely related species B. oleracea (CC) and B. nigra (BB) in B. carinata (BBCC) (Fig 1) and between B. campestris (AA) and B. nigra (BB) in B. juncea (AABB) (Fig.2). Hybridising labelled total B-genome of B. nigra (BB) with Brassica carinata and B. juncea it was possible to count the 16 labelled chromosomes of the B-genome (Fig. 1 and 2). No chromosomes were giving mixed signals. As expected from morphological, isoenzyme (WARWICK and BLACK, 1991) and molecular markers (TRUCO et al., 1996), the B-genome is distantly enough to distinguish from C- and A-genome. It is possible to “paint” the genomes in different fluorescent colours. The results are in accordance with SNOWDON et al.(1997) and FAHLESON et al.( 1997).
Fig. 1: Genomic in situ hybridisation (GISH) of mitotic metaphase chromosomes of B. carinata (BBCC, 2n = 34). Both pictures are showing the same cell seen through different fluorescent filters a) All chromosomes are DAPI stained blue. b)16 red rhodamine signals show the B-genome chromosomes hybridised with total genomic DNA probe of B. nigra (BB, 2n= 16).
It is possible to identify B. napus addition lines, substitution lines and other progenitors of species crosses containing the B-genome.
Fig. 2: Genomic in situ hybridisation (GISH) of mitotic metaphase chromosomes of B. juncea (AABB, 2n = 36). Both pictures are showing the same cell seen through different fluorescent filters: a) All chromosomes are DAPI stained blue. b)16 yellow FITC signals show the B-genome chromosomes hybridised with total genomic DNA probe of B. nigra (BB, 2n = 16).
The chromosome structure of the ancestral parents (A-, B- and C-genome) is still common with the genomes in the amphidiploid species. The complete set of chromosomes of the genomes were showing fluorescent labelling.
`Chromosome painting´ of different genomes in the amphidiploids B. juncea and B. carinata was possible.
Additionlines, substitutionlines can be recognised. To evaluate the introduction of the foreign chromatin B-genome chromosomes can be proved in the backcrossings. Translocation lines will be checked for B-genome introduction. GISH will be adjusted to meiotic metaphase to value the extend of inter genomic exchange.
This work has been supported by a travel grant from the H. Wilhelm Schaumann Stiftung.
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