GENOME INVESTIGATION IN SOUR CHERRY, P. CERASUS L. M. Schuster and H. Schreiber Federal Centre for Breeding Research on Cultivated Plants Institute for Fruit Breeding, D-01326 Dresden, Germany
[email protected] Keywords: P. cerasus, P. avium, P. fruticosa, genomic in situ hybridization, karyotype, C-banding Abstract Mitotic chromosome preparations of the sour cherry ‘Schattenmorelle’ were used to study the genome constitution of Prunus cerasus L. by genomic in situ hybridization (GISH) and karyotype analysis. Total genomic DNA from the presumably donor species P. avium and P. fruticosa were used as the probes. The probes were labelled with Digoxigenin-11dUTP by nick translation. The probe DNA’s of P. avium and of P. fruticosa each hybridized to 16 of the 32 chromosomes of sour cherry. By means of the karyotype analysis of C-banded P. cerasus and P. avium chromosomes, it was possible to detect the P. avium chromosomes in the P. cerasus genome. These results confirmed that P. avium and P. fruticosa are the donor species of the P. cerasus genome. 1.
Introduction
Cherries are one of the main fruit tree species in the northern hemisphere. In cherry production we distinguish between the sweet cherry, Prunus avium L. and the sour cherry, P. cerasus L. P. avium L. is a diploid species (2n=2x=16) and is found from Europe to the Caspian Sea. The sour cherry, P. cerasus L., is a natural allotetraploid hybrid (2n=4x=32). The distribution area of the sour cherry covers an area very similar to that of the sweet cherry, around the Caspian Sea and Minor Asia. The parent species of P. cerasus are, it has been suggested, the sweet cherry, P. avium, and the tetraploid ground cherry P. fruticosa Pall. (2n=4x=32) which has its origins in Western and Central Asia, but is also found in parts of Europe. This hypothesis is a result of morphological, genetical and biochemical studies (Kobel, 1927; Olden and Nybom, 1968, Hancock and Iezzoni, 1988). The present studies were aimed at identification and characterization of the sour cherry genome by genomic in situ hybridization (GISH) technique and by karyotype analysis. 2.
Materials and methods
For cytogenetical investigations actively growing root tips were used from young in vitro propagated plants of P. cerasus ‘Schattenmorelle’. The root tips were pretreated in 8-hydroxychinoline for 4 h and fixed in a 3:1 mixture of absolute ethanol and glacial acetic acid. Chromosome preparations followed the method described by Schuster (1996). GISH was modified according to the procedure described by Fuchs et al. (1995). The total genomic DNA from P. avium ‘Van’ and P. fruticosa clone 26/23 were used as probes. The probes were labelled with Digoxigenin-11-dUTP (Boehringer Mannheim) by nick translation. For blocking, autoclaved genomic DNA of P. fruticosa or P. avium were used. Slides were hybridized with 50 ng labelled genomic probe DNA and 1500-2000 ng (3040x) blocking DNA from the opposite fusion partner. The labelled probe was detected by anti-digoxigenin-FITC and the chromosomes were counterstained with DAPI and 375 Proc. EUCARPIA Symp. on Fruit Breed. and Genetics Eds M. Geibel, M. Fischer & C. Fischer Acta Hort. 538, ISHS 2000
propidium iodide. Slides were examined with a Zeiss Axioskop epifluorescence microscope. Photographs were taken on Fujichrome 400 colour slide film. A modified C-banding technique, reported by Schwarzacher et al. (1980), was used for the karyotype analysis of P. avium ‘Van’ and P. cerasus ‘Schattenmorelle’. The images were taken with a digital CCD camera and processed using Adobe Photoshop 4.0 and Designer 4.1 (Micrografx) software. Arm ratio, relative lengths and standard errors were calculated using Excel 5.0 (Microsoft). 3.
Results and discussion
In these studies, the genome constitution of P. cerasus was analysed by GISH with labelled genomic DNA of the probable parent species and by comparative studies of the C-banded karyotypes of P. cerasus and P. avium. GISH is the method of choice to differentiate between the chromosomes of the parental species. By using genomic DNA as a probe, all species-specific repetitive sequences dispersed over the genome are hybridizied with the probe. With the karyotype analysis by C-banding technique we can identify chromosomes that are difficult to distinguish from one another on the basis of size and centromere position alone. 3.1. GISH 3.1.1. Labelled P. avium DNA blocked with P. fruticosa DNA Digoxigenin-labelled genomic DNA of P. avium blocked with unlabelled P. fruticosa genomic DNA was hybridized in situ to mitotic metaphase chromosomes of P. cerasus. Genomic DNA of P. avium hybridized specifically to 16 of the 32 chromosomes of sour cherry genome, presumably those chromosomes of P. avium origin (Figure 1A). The yellow hybridization signals from the P. avium genome hybridized strongly to the centromeric regions of the P. cerasus chromosomes. Labelling of the whole chromosomes was not visible. Several earlier reports described this phenomenon for species with small chromosomes (Fahleson et al., 1997, Snowdon et al., 1997, Osuji et al., 1997). 3.1.2. Labelled P. fruticosa DNA blocked with P. avium Dann In a second step digoxigenin-labelled genomic DNA of P. fruticosa was also hybridized in situ to the P. cerasus chromosomes, total genomic DNA from P. avium being used as blocking DNA. In this experiment, 16 chromosomes were detected with fluorescence signals for the homologous DNA of P. fruticosa in the sour cherry genome (Figure 1B). The fluorescence marks of the P. cerasus chromosomes also were mainly detected only in centromeric positions and were not as clear when P. avium total genomic DNA was used as a probe. This phenomenon could be caused by a higher intergenomic homology of repetitive sequences on chromosome arms than at the centromeres, with a consequent stronger blocking of these regions, or possibly by a concentration of repetitive sequences at the centromeres (Snowdon et al., 1997). Besides the difficulties caused by the small size of the cherry chromosomes, further experiments will be necessary to improve the GISH methodology for cherry species. 3.2. C-banding The mitotic chromosomes of the sour and sweet cherries were analysed by means of the C-banding technique. The genomes of P. cerasus and P. avium are very small (Schuster and Ahne, 1998). The length of the chromosomes ranges from 2.0 to 4.5 µm. All chromosomes showed C-banding specific bands. Seven metaphase plates were used for the karyotype analysis of P. cerasus and twelve metaphase cells were used for the P. avium karyotype. 376 Proc. EUCARPIA Symp. on Fruit Breed. and Genetics Eds M. Geibel, M. Fischer & C. Fischer Acta Hort. 538, ISHS 2000
The idiotype of P. avium has the following characteristics. Three chromosomes have satellites. Chromosome 1 can simply be recognized by its large size. The other chromosomes show only small differences but can be distinguished by characteristic banding patterns. The P. cerasus karyotype is characterized by two large chromosomes, 1 and 2. Both are distinguished by a heterochromatic pattern near the centromere region. In chromosome 1 this is larger than in chromosome 2. The satellites were only found frequently on three chromosomes. Satellites are very small and often linked with the chromosomes. All chromosomes can be distinguished by characteristic banding patterns. After these karyotype studies in sweet and sour cherries, we compared both idiotypes. As a result of this comparison it was possible to identify the P. avium chromosomes in the P. cerasus genome (Figure 2). The small differences between size and banding patterns can be explained by the difficulties in analysing small chromosomes. 4.
Concluding remarks
Based on these cytogenetical studies, we confirmed the theory that P. cerasus is an allopolyploid hybrid between P. fruticosa and P. avium. The GISH technique and C-banding method are applicable for genome investigations in cherries. In both cytogenetical studies, it was possible to detect one or both donor genomes of the sour cherry. By using the GISH method, it is not possible to distinguish the individual chromosomes. The chromosome morphology changed during the in situ hybridization procedure. GISH, in combination with the C-banding technique, will be able to distinguish individual chromosomes. However, further experiments in the GISH methodology for cherries are necessary before intergenomic translocations can be reliably identified. Acknowledgements The authors thank Dr. Viola Hanke for providing the in vitro plant material used in these studies. References Fahleson J., Lagercrantz U., Mouras A., and Glimmelius K., 1997. Characterization of somatic hybrids between Brassica napus and Eruca sativa using specific repetitive sequences and genomic in situ hybridization. Plant Science 1223: 133-142 Fuchs J., and Schubert I., 1995. Localization of seed protein genes on metaphase chromosomes of Vicia faba via fluorescent in situ hybridization. Chromosome Research 3: 94100. Hancock A.M., and Iezzoni A.F., 1988. Malate dehydrogenase isozyme patterns in seven Prunus species. HortScience 23: 381-383. Kobel F., 1927. Zytologische Untersuchungen an Prunoideen und Pomoideen. Archiv Julius-Klaus-Stiftung 3: 1-84. Olden E.J., and Nybom N., 1968. On the origin of Prunus cerasus L. Hereditas 59, 327345. Osuji J.O., Harrison G., Crouch J., and Heslop-Harrison J.S., 1997. Identification of the genomic constitution of Musa L. lines using molecular cytogenetics. Annals of Botany 80: 787-793 Schuster M., 1996. Cytogenetics in fruit breeding. Preparation methods for mitotic chromosomes. Gartenbauwissenschaft 61: 273-275. Schuster M., and Ahne R., 1998. Karyological studies of Prunus avium L. In: Current Topics in Plant Cytogenetics Related to Plant Improvement. T. Lelley (ed.). WUVUniversitätsverlag, Tulln, p. 129-132 Snowdon R.J., Köhler W., Friedt W., and Köhler A., 1997. Genomic in situ hybridization 377 Proc. EUCARPIA Symp. on Fruit Breed. and Genetics Eds M. Geibel, M. Fischer & C. Fischer Acta Hort. 538, ISHS 2000
in Brassica amphidiploids and interspecific hybrids. Theoretical & Applied Genetics 95: 1320-1324. Schwarzacher T., Ambros P., and Schweizer D., 1980. Application of Giemsa banding to orchid karyotype analysis. Plant Sytematics and Evolution 134: 293-297.
Figures
1. Genomic in situ hybridization to metaphases of P. cerasus. Yellow fluorescence indicates hybridization to the probe. Unlabelled chromatin fluoresces red with propidium iodide. A - probed with P. avium, B - probed with P. fruticosa.
378 Proc. EUCARPIA Symp. on Fruit Breed. and Genetics Eds M. Geibel, M. Fischer & C. Fischer Acta Hort. 538, ISHS 2000
2. C-banded idiograms of P. cerasus and P. avium.
379 Proc. EUCARPIA Symp. on Fruit Breed. and Genetics Eds M. Geibel, M. Fischer & C. Fischer Acta Hort. 538, ISHS 2000
