Tilling and Ecotilling High throughput

discovery of SNP variation

Aladdin Hamwieh

Targeting induced local lesions in genomes (TILLING)

• TILLING is a reverse genetics approach for mutation generation and discovery• Slade et al. (2005) created a TILLING library in both bread and durum

wheat to determine its utility in a complex genome like wheat.• Using locus-specific PCR primers, they were able to identify 246

alleles of the waxy genes by TILLING.• This made available novel genetic diversity at waxy loci and provided

a way for allele mining in important germplasm of wheat.• Using TILLING, Arcadia Biosciences has generated 220 induced

mutations in 19 tomato genes

Targeting induced local lesions in genomes (TILLING)

• for detecting unknown point mutations in mutagenized populations based on CELI cleavage• It has been used in many species including Arabidopsis thaliana

(Colbert et al. 2001), Lotus japonica (Perry et al. 2003), maize (Till et al. 2004a), wheat (Uauy et al. 2009; Dong et al. 2009), tomato (Minoia et al. 2010), rice (Till et al. 2007), zebrafish (Wienholds et al. 2003; Sood et al. 2006) and fruit fly (Winkler et al. 2005).• due to the complexity of the methodology, TILLING has not been used

routinely in most laboratories for the detection of SNPs or InDels

TILLING

DNA pooling

Florescent primers

endonuclease

Discovery of rare polymorphism by pooling

Jiang et al. 2013

They modified the CELI-based technology for genotyping both SNPs and InDels using non-labeled primers in combination with either agarose gel or capillary electrophoresis.

Can the technology used for TILLING

be used to discover natural variation?

Rare haplotype

Identifying polymorphisms

Uses of Ecotilling

1. Screen many individuals at one locus2. Not good for few individuals at many loci3. Discover rare haplotypes4. Good in heterozygous populations

High Resolution Melts (HRM)

• Instrument requires• high-intensity + high sensitivity optics• high-speed data capture• very precise temperature control and resolution

DIVERSITY ARRAYS TECHNOLOGY (DArT)

• Company established in 2001in Canberra, Australia• Global service, performed >1.4 G marker assays

DArT - “Parallel reverse RFLP”

Thank you