Ecotilling is a high throughput, low cost technique for rapid discovery of polymorphisms in natural populations. Ecotilling is similar to Targeting Induced Local Lesions in Genomes, or TILLING, but Ecotilling differs in that naturally occurring polymorphisms are detected as opposed to experimentally induced mutations. Single nucleotide polymorphisms (SNPs), small insertions and deletions, and variations in microsatellite repeat number can be efficiently detected using the Ecotilling technique on a LI-COR^® 4300 DNA Analysis System.

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Advantages

• Two-Color Imaging Eliminates False Positives

• The LI-COR 4300 DNA Analysis System is uniquely suited for Ecotilling because it uses two-color infrared fluorescence to generate two true gel images during electrophoresis.Two-color imaging makes identification of polymorphisms very accurate and virtually eliminates false positives. A polymorphism (band) on one gel image is confirmed by the presence of a band in the same lane on the second gel image. The band on the second image is the opposite cut strand and it should be in the expected position (the sum of the molecular weights of the two bands should approximately equal the total size of the amplicon). This unique imaging method also localizes polymorphisms to within ±10 base pairs, making them easy to identify by DNA sequencing.

• SNP Discovery Using The Ecotilling Technique
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  [ABOVE] Figure 1.

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  [ABOVE] Figure 2. Polymorphisms in one fluorescence channel can be confirmed by the presence of the opposite cut strand in the same lane in the second fluorescence channel (eliminates false positives). Empty lanes (just top and bottom bands) indicate no genetic variation in any of those eight individuals. The eight individuals in lanes with bands can be rerun in separate lanes or sequenced to find the individual(s) with genetic variations.

• Rapid Haplotyping Using The Ecotilling Technique
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  [ABOVE] Figure 3. For rapid haplotyping, 96 individuals can be loaded in separate gel lanes and compared against a reference DNA in each lane. Bands at the same position in different lanes indicate the same haplotype.

• SNP Discovery At A Fraction Of The Cost

• Ecotilling costs only a fraction of the conventional approach of sequencing a given locus for every sample under investigation. For SNP discovery, Ecotilling can be used to compare alleles for variations at a locus. Samples with no genetic variations are quickly identified, eliminating the need to sequence them.

• Sequencing is further resduced since samples can be scored by haplotype and only one sample from each unique haplotype needs to be sequenced to characterize the haplotype.

• EFFICIENT SCREENING

• Ecotilling can also be used as a high throughput screening technique. A single 4300 DNA Analysis System can achieve the following results*:

• Over 750,000 base pairs screened per run (1 kb per sample).

• Up to 2300 samples, or over 2 million base pairs screened per day.

*Results are dependent on species and other factors
• Infrared Detection for Highest Sensitivity:

• The high sensitivity of infrared detection is criticial because the LI-COR 4300 System maintains a good signal-to-noise ratio even with the signal loss due to exonuclease activity in Ecotilling. The inherent low background of infrared detection compared to visible d etection, combined with LI-COR’s IRDye® Infrared Dyes, results in another key advantage – wide dynamic range. Wide dynamic range is critical for resolving the weaker bands of polymorphisms along with strong wild type bands. Low infrared background from amplification artifacts makes it easy to resolve polymorphisms with the 4300 System.

• Ecotilling Applications

• The Ecotilling technique is applicable to any organism, including those that are heterozygous and polyploid.

• Ecotilling can be used for:

• Mapping: Detection of polymorphisms that can be used as genetic markers.

• Association Analysis: Correlating natural polymorphisms with phenotypic traits.

• Mutational Profiling: Characterizing the amount of genetic variation in a species.

• Biodiversity: Determining the evolutionary history of natural populations.

Technical Resources

Protocols:

• 4300 Applications Manual

• Mung Bean Protocol v1.0

• Using Adobe Photoshop to Postprocess LI-COR Images for TILLING applications v1.0

• Application of TILLING/Ecotilling to Screen for Small Mutations in Mammals v1.0
  Kovar, et. al. (9/2008)

Poster presentation on use of 4300 with Tilling and Ecotilling to screen for small mutations in mammals

PubMed

Publishing with LI-COR data?

References

• Efficient Discovery of DNA Polymorphisms in Natural Populations by Ecotilling.
  Comai, L., Young, K., Till, B.J., Reynolds, S.H., Greene, E.A., Codomo, C.A., Enns, L.C., Johnson, J.E., Burtner, C., Odden, A.R., and Henikoff, S. 2004.
  Plant Journal 2004, 37: 778-786.

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