Feb 23, 2026
Protocol for Generating Advanced Intercross Populations Using Ether-Zymolyase for BSA-seq in Yeast
This protocol is a draft, published without a DOI.
- Sonali Lenaduwe1,
- Jeffrey Lewis1
- 1Department of Biological Sciences, University of Arkansas
Protocol Citation: Sonali Lenaduwe, Jeffrey Lewis 2026. Protocol for Generating Advanced Intercross Populations Using Ether-Zymolyase for BSA-seq in Yeast. protocols.io https://protocols.io/view/protocol-for-generating-advanced-intercross-popula-hse6b6bhf
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 23, 2026
Last Modified: February 23, 2026
Protocol Integer ID: 243902
Keywords: Sporulation, Diethyl Ether, Zymolyase Digestion, Ascus Wall Digestion, BSA-seq, Tetrad Dissection Alternative, Advanced Intercross Lines, QTL mapping, advanced intercross populations of saccharomyces cerevisiae, seq in yeast, saccharomyces cerevisiae, generating advanced intercross population, yeast, enzymatic digestion with zymolyase, zymolyase for bsa, dispersed spore, heterozygote, spore, bulk segregant analysis, suitable for bulk segregant analysis, recombination, enzymatic digestion, segregant population, elimination of vegetative cell, meiosi, segregant population with high mapping resolution, vegetative cell
Abstract
This protocol details a method for generating advanced intercross populations of Saccharomyces cerevisiae. The objective is to allow meiosis and recombination to disrupt linkage, thereby creating a segregant population with high mapping resolution suitable for Bulk Segregant Analysis (BSA) experiments. The protocol involves multiple rounds of sporulation, followed by elimination of vegetative cells with diethyl ether. The ascus wall of the remaining tetrads is subjected to enzymatic digestion with Zymolyase. The spores are finally dispersed by sonication, and the dispersed spores are allowed to randomly mate to generate heterozygotes.
Guidelines
All centrifugation steps, unless specified, are carried out at 1500 g x 3 minutes.
We used 1% potassium acetate as our sporulation medium.
Materials
**Reagents**
- Yeast strains in 1% Potassium Acetate (KAc)
- Sterile distilled water
- Diethyl ether
- 1 M Sorbitol
- Zymolyase (5U/µl)
- YPD (liquid)
- 1.5% Triton-X solution
- 70% ethanol
- 50% Glycerol (sterile)
- YPD agar plates
- Triple antibiotic YPD plates:
- Ampicillin (100 µg/mL)
- Tetracycline (10 µg/mL)
- Chloramphenicol (25 µg/mL)
**Equipment Disposables**
- Shaker incubator (30°C, 270 RPM)
- Centrifuge (rotor compatible with 15 mL tubes)
- Fume hood
- Rotoflex rotator
- Vortex mixer
- Sonicator with probe
- Microscope
- Ice bucket
- Scale
- 15 mL sterile centrifuge tubes
- 5 mL Eppendorf tubes
- 1.7 mL Eppendorf tubes
- Pre-autoclaved PCR tubes
- 15 mL sterile glass tubes
- 96-well sterile plate
- Multichannel pipettor tips
- Pipettes sterile tips
- Cell scraper (sterile)
- Kim Wipes
- Ear protection
Troubleshooting
Safety warnings
This protocol uses diethyl ether, which is an extremely flammable liquid and vapor. It is recommended to be trained and to perform all steps involving diethyl ether in the fume hood.
Before start
This protocol requires sporulated cultures. Ensure sporulating cultures have reached a sporulation efficiency of >80%. (Note: If sporulation efficiency is poor, I would typically set up four 5 mL sporulation cultures and combine them to achieve >80% sporulation efficiency. When pooling, I prefer pooling two 5 mL cultures to yield a 10 mL culture. I have found that the resulting pellet is ~1g, which helps in the later steps.)
Preparation
10m
Set up the tube rotator in the fume hood. We used RotoFlex‱ Plus bench-top tube rotator for this purpose. Align the rotisserie paddles perpendicular to the bench surface to allow the suspension to nutate (roll) during the emulsification step.
Weigh a 15 mL sterile centrifuge tube and record the volume.
DAY 1: Ether Treatment to Eliminate Vegetative Cells
1h 15m
Collect the spores by centrifugation. (Note: If the cultures were in glass tubes, move them into sterile 15 mL centrifugation tubes.)
Decant the sporulation media, then resuspend the spore pellet in 1 mL sterile distilled water by vortexing.
Add an equal volume (1 mL) of diethyl ether (Fisher
Scientific, Waltham, MA, USA; Cat. No. 50-180-7006) to the resuspended spores.
Vortex briefly to emulsify.
Place tubes on the Rotoflex and rotate for 1 hour at room temperature to emulsify.
1h
Centrifuge and carefully decant the supernatant (ether + water) into an appropriate waste container
Resuspend the pellet in 1 mL of sterile distilled water.
Repeat steps 7-8 to remove all residual ether 4 more times (for a total of 5 washes).
Zymolyase Digestion of the Ascospore Coat
1h 45m
After the final wash, resuspend the pellet in 1 mL of distilled water and transfer it to a fresh, pre-weighed 15 mL centrifuge tube (See Preparation).
Centrifuge and discard the supernatant.
Calculate the wet pellet weight by weighing the tube and pellet together, then subtracting the total weight from the weight of the centrifuge tube previously recorded. Record the weight.
Resuspend the pellet in 4 mL of 1 M Sorbitol.
Add Zymolyase (Zymo Research, Irvine, CA, USA; Cat. No. E1005) to a final concentration of 500 U per gram of wet pellet.
Example: For a 1g pellet, add 10 µL of 5U/µL Zymolyase
Mix gently by inverting the tube 4-5 times. Do not vortex.
Incubate at 30°C, shaking at 270 RPM, for 1 hour and 30 minutes.
Preparation for Sonication
5m
While samples are incubating, prepare an ice bucket.
Prepare two 15 mL sterile centrifuge tubes: one containing 5-7 mL of 70% ethanol and one containing sterile distilled water, both on ice.
Prepare a 96-well flat-bottom microtiter plate for downstream serial dilutions by adding 180 µL of sterile distilled water into the required wells.
Take out YPD triple antibiotic plates (YPD + ampicillin 100 µg/mL, tetracycline 10 µg/mL, and chloramphenicol 25 µg/mL) and warm them to room temperature.
Tetrad Dispersion (Sonication)
30m
After incubation, pellet the spores by centrifugation and wash once with 5 mL of sterile distilled water to remove Zymolyase.
Resuspend the pellet in 5 mL of 1.5% Triton-X and place the tube on ice to chill.
Clean the sonicator probe:
Wipe all surfaces with 70% ethanol. Set the sonicator to 60% amplitude, 30s ON / 30s OFF for 3 minutes.
Wipe the probe with a Kim Wipe soaked in 70% ethanol.
Immerse the probe in the pre-chilled 15 mL tube of 70% ethanol and run one cycle.
Rinse the probe by immersing it in the 15 mL tube of pre-chilled sterile distilled water.
Sonicate the sample:
Immerse the sterilized probe into the pre-chilled sample tube containing spores in 1.5% Triton-X.
Run one cycle.
Place the sample on ice for 3 minutes to cool.
Repeat the sonication and cooling steps 2 more times for a total of 3 sonication cycles.
Recommended: Check spore dispersion under a microscope to confirm separation.
Tip: If processing multiple strains, repeat the full cleaning procedure (step 24) to prevent cross-contamination.
Cell Plating
After sonication, vortex the sample tube at high speed.
Centrifuge and discard the supernatant.
Resuspend the final spore pellet in 120 µl sterile distilled water.
Plate 100 µl of the suspension onto a triple antibiotic YPD plate. Spread evenly to create a mating lawn.
For cell counts: Use the remaining 20 µl of suspension to perform a 10-fold serial dilution (20 µl into 180 µl water). Plate 100 µl of the 10^-5 and 10^-6 dilutions onto standard YPD plates.
Incubate the YPD triple antibiotic plates overnight (16-24 hours) at 30°C.
Incubate the standard YPD plates for two days at 30 °C
DAY 2: Harvesting Mated Cells and Starting Next Cycle
If advancing the population into the next generation, prepare 15 mL sterile glass tubes, each containing 4 mL of 1% potassium acetate.
For each sample processed, label one sterile 5 mL microfuge tube and four 1.7 mL sterile microfuge tubes.
Optional (for saving): Label an appropriate number of sterile PCR tubes.
Cell Harvesting
1h 30m
Add 2 mL of sterile distilled water onto the mating lawn (from Day 1, step 32).
Gently scrape the cells off the plate using a sterile cell scraper, avoiding lifting the agar.
Pipette the cell suspension into the pre-labeled 5 mL microfuge tube.
Centrifuge and resuspend the pellet in 1 mL of sterile distilled water.
Transfer 200 µl of this suspension (20% of the total) into each of the four labeled 1.7 mL microfuge tubes.
Wash the cells:
Centrifuge the 1.7 mL tubes and discard the supernatant.
Resuspend in 1 mL sterile distilled water.
Repeat the wash step four times total.
After the final wash, resuspend the pellet in each 1.7 mL tube in 1 mL of 1% potassium acetate.
Transfer the entirety of the 1 mL 1% potassium acetate spore suspension from each 1.7 mL tube into four 15 mL glass tubes containing 4 mL of 1% potassium acetate. This yields a total of four 5 mL sporulation cultures.
Incubate the new sporulation cultures at 30°C, shaking at 270 RPM, for 5-7 days.
(Repeat Cycle): After 5-7 days, these cultures are ready for the next intercross cycle, starting from Day 1, Step 1.
Saving the Generation (Optional)
Take the remaining cell suspension from the 5 mL microfuge tube (from step 39).
Centrifuge, discard the supernatant, and resuspend the pellet in 360 µl YPD and 240 µl 50% glycerol (total volume 600 µl).
Vortex well to mix.
Aliquot 100 µL into an appropriate number of labeled PCR tubes for cryopreservation.
Store at -80°C.
DAY 3: Quantifying Spores
Count the colonies on the dilution plates (from Day 1, step 33) to determine the spore concentration (CFU/mL) of the initial sonicated population.
Protocol references
1. Bahalul, M., Kaneti, G. & Kashi, Y. Ether–zymolyase ascospore isolation procedure: an efficient protocol for ascospores isolation in Saccharomyces cerevisiae yeast. Yeast 27, 999-1003 (2010). https://doi.org/https://doi.org/10.1002/yea.1808
2. Treco, D. A. & Winston, F. Growth and Manipulation of Yeast. Current Protocols in Molecular Biology 82, 13.12.11-13.12.12 (2008). https://doi.org/https://doi.org/10.1002/0471142727.mb1302s82