15　 Forward Genetic Approach: Deletion Mutants

Tsuyoshi Aoyama

Introduction

Forward genetic approach is a useful tool to isolate genes involved in phenomena of interest. In this chapter, new forward genetic approach using deletion mutants and a tiling array is described. Irradiation of ion beam usually induces double strand breaks in the genome. As a result, deletion mutant lines are generated. The deletion regions are detected by hybridization of mutant genomic DNA to a wild type genomic DNA probe on a tiling array.

1. Irradiation of ion beam

Materials

・sporangium

Note: We usually use dried sporangium.

Procedure

1. Irradiate sporangium with 50~300 Gy of Ne ion beam (63 keV/µm)

Note: Irradiation procedure is performed at RIKEN Nishina Center for Accelerator-Based Science.

2. Isolation of mutants

It is necessary to optimize the methods to isolate each mutant for any purpose. In this chapter, the basic method to isolate mutants is introduced.

Materials

・ion beam irradiated sporangium

・Yellow tips and blue tips for pipetman (autoclave)

・Water bath

・15 ml round-bottom tube (Falcon, Iwaki, etc)

・ 1.5 ml microtube

Solution

・BCDAT+ 6 mM Ca (spore germination medium): 1000 ml

H2O	900 ml
Stock B	10 ml
Stock C	10 ml
Stock D	10 ml
500 mM Ammonium tartrate	10 ml (= 5 mM)
Alternative TES	1 ml
CaCl2 2H2O	0.9 g (= 6 mM)
Agar	8 g (= 0.8%)
	Fill up to 1000 ml with H2O

After autoclaving, pour into 9 cm petri dish

・spore sowing medium: 200 ml

H2O	150 ml
Stock B	2 ml
Stock C	2 ml
Stock D	2 ml
500 mM Ammonium tartrate	2 ml (= 5 mM)
Alternative TES	200 µl
CaCl2 2H2O	0.18 g (= 6 mM)
Agar	1.6 g (= 0.8%)
	Fill up to 200 ml with H2O

Autoclave and store at 45°C

・Sterilized water

・10% Sodium Hypochlorite Solution (Antiformin): Do not autoclave

Procedure

1. Put one sporangia in a 1.5 ml microtube, then add 1ml of 10% Antiformin to sterilize.

2. Mix gently for 5 min and discard supernatant using pipetman. Usually the sporangium sinks to the bottom of the tube, but be careful not to discard the sporangium until step 6. Do not touch the sporangium with a tip of pipetman, otherwise the tiny spores are dispersed in the tube and it is not possible to recover them.

3. Add 1 ml of sterilized water into the tube and mix gently for a few minutes.

4. Discard supernatant with a pipetman.

5. Repeat steps 3 and 4 four times.

6. Add 1 ml of sterilized water, crush the sporangium with the tip of a pipetman (yellow tip), and mix gently. When you crush the sporangium, you can see the spores disperse in the tube.

7. Dilute the spore solution 100-fold.

8. Add 3ml of spore sowing medium into the 15 ml round-bottom tube and store at 45°C.

9. Add 1 ml of 100-fold diluted spore solution into the tube.

10. Vortex for 3 seconds and pour onto the spore germination medium.

11. Incubate at 25°C under continuous light for a few weeks.

12. Transfer a small part of independent colonies to a new medium and incubate at 25°C under continuous light for a few weeks.

13. Observe the phenotype and isolate mutants.

3. Detection of deletion regions with tiling array

Materials

・Genomic DNA (mutants and wild type)

・BioPrime DNA labeling system (Invitrogen)

・ Ppa Tiling Array ver.1

Note: We designed a custom Affymetrix tiling array covering the whole P. patens genome sequence of approximately 480 Mb at 67±5 bp resolution excluding repeated sequences. The array contains 6,451,867 perfect match probes along with the controls.

Procedure

1. Label the genomic DNA with BioPrime DNA labeling system according to manufacture’s instruction.

2. Perform tiling array analysis according to manufacture’s instruction.

3. Analyze the data with TAS and IGB to detect candidate deletion regions.

Note: This method is still establishing. It is important to improve the analytic method of tiling array data for detecting deletion regions efficiently.

Note: After you detect deletion regions, it is necessary to examine which deletion regions are responsible for mutant phenotype.