chromosomes

N. ohmido and K.fukui

Hokuriku National Agricultural Experiment Station 

Joetsu 943-01, Japan

45S ribosomal RNA and rbcS genes have been physically mapped on the rice chromosomes by in situ hybridization (ISH) using RI-labeled probes (Fukui et al. 1987; Wu et al. 1986). Although ISH with RI-labeled probes is very sensitive, the method has several shortcomings (MacGregor 1993). Non-RI ISH method for overcoming these disadvantages was developed using biotin labeling (Langer et al. 1981). Fluorescence in situ hybridization (FISH) makes it possible to attain high sensitivity, short detection time, and safety handling of samples. The FISH method has been applied to detect repeated sequences in wheat (Mukai et al. 1993), barley (Leitch 1993), rye (Albini 1992) , tobacco (Kenton et al. 1993) and tomato (Zhong et al. 1996). The method has also been used to characterize alien chromosomes and chromosome segments (Heslop-Harrison et al. 1990) and to determine the genomic constitution of allopolyploids (Bennett et al. 1992). The FISH method is effective to map physically the single genes (Kamisugi et al. 1996, submitted). Fukui et al. (1994) has reported a reliable FISH method, which is applicable to rice for the first time. In this report, further advanced FISH method is described with detailed experimental procedures to locate genes and DNA sequences more effectively on specific chromosome regions in rice and other plant species.

Preparation method of plant chromosomes

water at 0-4°C for overnight to a day are generally effective to collect metaphase spreads.

3. Fix the root tips in a Fixative fluid (ethanol: acetic acid = 3:1) for 1 hours to several months. Store the root tips at -20°C.

4. Wash the root tips in deionized water for 20 min. or more.

5. Cut the meristematic portion of the root tips on a glass slide and put them into an Eppendurf tube with the enzyme mixture (2% Cellulase Onozuka RS, 0.3% Pectolyase Y-23, 1.5% Macerozyme R200, l0mM EDTA, pH4.2, modified from Fukui and Iijima 1992).

6. Decompress them for 5-10 min. in the enzymatic mixture.

7. Incubate the root tips at 37°C for 30-50 min.

8. Pick up the macerated root tips carefully using a Pasteur pipette and dispense them into distilled water.

9. Wash root tips in distilled water for 15 min.

10. Pick the root tip up by the Pasteur pipette and place the root tips carefully on clean

glass slides and remove water from the root tip using a peace of filter paper. I I. Add two or three drops of the fixative, then tap the macerated root tip by fine forceps until the tissue becomes almost invisible spreads.

12. Check the slides under a phase contrast microscope and select the good ones for in situ hybridization.

13. Slides can be stored at -20°C for several months before use.

Probes labeling for fluorescence in situ hybridization

Probe can be labeled by random primer labeling method PCR method or nick translation method.

1. Dissolve 1 microg probe DNA with distilled water in an Eppendurf tube.

2. Denature the DNA at 100°C for five min. and immediately cool it down on ice.

3. Add hexanucleotide mixture and Klenow enzyme (Boehringer Mannheim) according to the instruction of the supplier and incubate the mixture more than 20 hat 37°C.

4. Add competitor DNA, for example 100 ng salmon sperm DNA or genomic DNA.

5. Remove the unincorporated dNTP by ethanol precipitation.

6. Centrifuge at 13,000 rpm for five min. and wash the pellet with 70% ethanol. Dry it in a vacuum chamber and dissolve the pellet with 50micro1 of dissolving buffer (50% formamide, 2 x SSC, 50mM phosphate buffer pH 7.0).

Posttreatment of chromosome samples

1. Place the 300 micro1 of RNase A (100E g/ml DNase free RNaseA, Sigma) in 2xSSC (0.3M NaCI, 0.3M 0.03M sodium citrate, pH7.0) on each slide, cover with cut parafilm and incubate them at 37°C for 1 h.

2. Wash the slides in 2 x SSC for five min.

3. Dehydrate the slides in a ethanol series of 70%, 90% and 100% for five min. each and air-dry.

4. Check the slides by phase contrast microscopy and select good spreads for FISH.

Research Notes 91 Hybridization

1. Mix the 5 ml dissolving buffer with the labelled probe and the l0ml hybridization buffer.

2. Add the 15 ml aliquot of the hybridization mixture to each glass slide and cover with a 24 x 32mm coverslip.

3. Seal coverslip with liquid Arabian gum (Paper Bond, Kokuyo) and air-dry.

4. Place them on a thermal cycler with flat alminum bed (PHC-3, Techne, Cambridge, UK).

5. The programmed heating sequence is 6 min. at 70°C and 18 h at 37°C.

Detection

1. Wash the slides in 2 x SSC for 5 min. at 40°C, 50% formamide/2 x SSC for 5 min at 40°C, 2 x SSC for 5 min. at 40°C and 4 x SSC for 5 min. at 40°C.

2. Apply 600 ml of 5% BSA (Iwai)/BT buffer (0.01 M Sodium hydrogen carbonate, 0.005% Tween-20) for each glass slide, and incubate them for 5 min. at 37°C.

3. Place 70 ml of Avidin-FITC (20ng/ml. Vector) dissolved with 1%BSA/BT buffer on each glass slide and cover with cut palafilm. Incubate the glass slides for 30min. at 37°C in a humid dark chamber.

4. Wash the slides with BT buffer three times at 37°C for 5min. each.

5. Apply 600 ml of 5% goat serum (Cosmo BIO)/BT buffer for 5min. at 37°C.

6. For signal amplification, apply 70 ml of Bio-Anti-Avidin (5mg/ml. Vector) /BT buffer for 30min. at 37°C in the humid dark chamber.

7. Wash the slides with BT buffer three times at 37°C 5min. each.

8. Apply 600 ml 5% BSA/BT buffer for each glass slides and incubate them at 37°C for 5min.

9. Repeat the immune-reaction if fluorescent signals are week. Apply 70 ml Avidin-FITC (20ng/ml ) in 1% BSA/BT buffer for 30 min. at 37°C in a humid chamber.

10. Wash the slides in BT buffer twice at 37°C for 5 min. each and one time in 2xSSC at 37°C for 5min.

11. Counter-stain the chromosome samples by a 20 ml PI solution (lmg/ml, Sigma) with Vectashield (Vector).

12. Observation by fluorescence microscopy. Digitize the signal and chromosome images by PXL1400 (Photometrics) and image analysis by IP-Lab and Photoshop 3.0.

Figure 1 shows fluorescence signals of rDNA sites in Oryza sativa cv. Nipponbare. One rDNA locus is detected in 0. sativa as consistent with the previous report (Fukui et al. 1987; 1994). In &japonica rice, rDNA is located on chromosome 9. On the other hand, two rDNA loci are observed on chromosome 9 and 10 in an indica variety. Information on the localization of genes and repeated DNA sequences such as rDNA is important for understanding rice genome organization.

Figure 2 shows a result of genomic in situ hybridization in Yuan Zhong (2n=56), a variety of wheat-Agropyron partial amphidiploids (Ma et al. in press). This variety has tolerance to barley yellow dwarf virus by the addition of alien chromosomes. It was, however, difficult to distinguish the chromosomes of Triticum aestivum from these of Ag. intermedium. Application of GISH and C-banding methods makes it possible to identify 14 Ag. intermedium chromosomes (light signals) from 42 wheat chromosomes (dark signals). Arrows point the position where translocation occurred between the two chromosomes.

A map based on molecular markers has now been developed (Kurata et al. 1994), and a number of newly cloned genes and DNA sequences are increasing. The FISH method described here will be useful to localize the genes not only on rice chromosomes but also on the other plant chromosomes. The FISH method is anticipated to elucidate genome organization of the plant cells in the near future.

References 

Albini, S.M. and T. Schwarzacher, 1992. in situ localization of two repetitive DNA sequences to