Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L)

Rana Elias,Bassam Al-Safadi,Bradley J Till,Chikelu Mba

doi:10.1186/1756-0500-2-141

Abstract

BackgroundThe TILLING and Ecotilling techniques for the discovery of nucleotide polymorphisms were applied to three potato (Solanum tuberosum) cultivars treated with gamma irradiation. The three mutant cultivars tested were previously shown to exhibit salinity tolerance, an important trait in countries like Syria where increasing soil salinity is affecting agricultural production.FindingsThree gene-specific primer pairs were designed from BAC sequence to amplify ~1 to 1.5 kb of gene target. One of the three primer pairs amplified a single gene target. We used this primer pair to optimize enzymatic mismatch cleavage and fluorescence DNA detection for polymorphism discovery. We identified 15 putative nucleotide polymorphisms per kilobase. Nine discovered polymorphisms were unique to one of the three tetraploid cultivars tested.ConclusionThis work shows the utility of enzymatic mismatch cleavage for TILLING and Ecotilling in different varieties of potato. The method allows for rapid germplasm characterization without the cost and high informatics load of DNA sequencing. It is also suitable for mutation discovery in high-throughput reverse genetic screens.

Highlights

The TILLING and Ecotilling techniques for the discovery of nucleotide polymorphisms were applied to three potato (Solanum tuberosum) cultivars treated with gamma irradiation
This work shows the utility of enzymatic mismatch cleavage for TILLING and Ecotilling in different varieties of potato
No new bands were observed when screening pooled samples suggesting that only heterozygous differences exist between the three cultivars in this genomic region. In this pilot study to optimize the TILLING and Ecotilling methods for potato, a range of genomic DNA concentrations between 25 ng and 0.25 ng was identified that can be used for PCR to produce high quality gel images

Summary

Introduction

The TILLING and Ecotilling techniques for the discovery of nucleotide polymorphisms were applied to three potato (Solanum tuberosum) cultivars treated with gamma irradiation. The three mutant cultivars tested were previously shown to exhibit salinity tolerance, an important trait in countries like Syria where increasing soil salinity is affecting agricultural production. Potato is considered the fourth most important food crop in the world due to its high productivity and nutritional value. In Syria, a major stress on agricultural output of potato is increasing soil salinity. Mutagenesis in potato has been used as an alternative approach for generating nucleotide diversity and inducing useful traits. This approach, usually referred to as mutation breeding, has been used in developing several salinity tolerant and disease resistant potato lines in Syria [1,2]

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Conclusion