Fig. 1. Karyotype of MAAL 10 of cultivar Nipponbare having a single O. punctata chromosome. Capital letters, A and B represent O. sativa and O. punctata genomes, respectively.
Faculty of Agriculture, Kyushu University, Fukuoka, 812 Japan
The materials used include Japonica rice cultivar Nipponbare (2n=2X= AA), strain W1514 of O. punctata Kotschy (2n=2X=BB), interspecific hybrids (2n=3X=AAB) obtained from the cross of tetraploid Nipponbare with W1514 and monosomic alien addition lines MAAL 7 (2n=AA+7B) and MAAL 10 (2n=AA+10B) derived from these interspecific hybrids. MAAL7 and MAAL10 were identified by morphological resemblance to related trisomics (Yasui and Iwata 1991). RFLP analysis confirmed the presence of alien chromosomes 7 and 10 in these MAAL lines (Yasui et al. 1992).
Prometaphase cells of interspecific hybrids (2n=3X=AAB), MAAL 7 and MAAL 10 were prepared from freshly emerged root tips using a modification of the procedures described by Kurata (1978). Well spread chromosomes on the slides were stained in 10% Giemsa solution diluted by Sorensen phosphate buffer. Karyotyping of chromosomes in MAAL 7 and MAAL 10 were carried out.
For fluorescence in situ hybridization (FISH), total O. punctata DNA was used as probe to detect O. punctata chromosomes. The DNA in the target cells were denatured by immersion in 70% formamide/0.3 M NaCI/0.03 M sodium citrate (2x SSC) for 3 min at 80 deg C. The slides were rapidly dehydrated in an ethanol series (70%, 100%), and air-dried. The hybridization mixture [270microliters total volume consisting of 10% formamide, 2xSSC, dextran sulfate, 1 mg of sonicated herring DNA per ml, and 700 ng digoxygenin labeled total O. punctata DNA] was then denatured and applied. Hybridization was at 37 deg C overnight (15 hr). Slides were washed in 50% formamide/2x SSC for 15 min at 37 deg C, and rinsed in 2x SSC, 1x SSC, and 4x SSC at room temperature for each 15 min.
For detection of hybridization sites, the slides were counterstained using FITC-PI method. The slides were treated with a solution of a complex of antidigoxygenin-FITC in 1% BSA/4xSSC. After rinsing, the slides were counterstained with 1.25ug/ml propidium iodide for 30 min. Photographs of FISH prometaphase spreads were taken with Zeiss Axioplan photomicroscope using Fuji Fujichrome film.
O. sativa c.v. Nipponbare and O. punctata (a diploid strain) W1514 were confirmed to be well differentiated using FISH technique. Among the prometaphase spreads of interspecific hybrids (2n=3X=AAB), only twelve chromosomes in many nuclei were heavily stained by FITC. In some prometaphase nuclei, however, more than twelve (13-14) chromosomes were heavily stained. These additional stained chromosome(s) probably belong to O. sativa chromosome complement.
MAAL 7 and MAAL 10 were each characterized by a heavily stained chromosome of O. punctate. The application of FISH using total genomic DNA as probe was useful to detect introduced alien chromosomes into cultivated rice. The heavily stained chromosome in MAAL 7 was medium and sub-metacentric type and was completely different from O. sativa chromosome 7 being small and metacentric. The heavily stained chromosome in MAAL 10 (10B) was small in size and sub-metacentric type, and shows heterochromatic region in entire short arm. These characteristics were similar to that of O. sativa chromosome 10 (Fig. 1).
Fig. 1. Karyotype of MAAL 10 of cultivar Nipponbare having a single O.
punctata chromosome. Capital letters, A and B represent O. sativa and O.
punctata genomes, respectively.
References
Kurata, N. and T. Omura, 1978. Karyotype analysis in rice. 1. A. new method for identifying all chromosome pairs. Jpn. J. Genet. 53: 251-255.
Yasui, H. and N. Iwata, 1991. Production of monosomic alien addition lines of Oryza sativa having a single O. punctata chromosome. Rice Genet. 2: 147-155.
Yasui, H. A. Yoshimura and N. Iwata 1992. Characterization of monosomic alien chromosomes of O. punctata transferred to O. sativa using RFLP markers. RGN 9: 138-142.