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A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING

Abstract

We report the use of TILLING (targeting induced local lesions in genomes), a reverse genetic, nontransgenic method, to improve a quality trait in a polyploid crop plant. Waxy starches, composed mostly of amylopectin, have unique physiochemical properties. Wheat with only one or two functional waxy genes (granule-bound starch synthase I, or GBSSI) produces starch with intermediate levels of amylopectin. We have identified 246 alleles of the waxy genes by TILLING each homoeolog in 1,920 allohexaploid and allotetraploid wheat individuals. These alleles encode waxy enzymes ranging in activity from near wild type to null, and they represent more genetic diversity than had been described in the preceding 25 years. A line of bread wheat containing homozygous mutations in two waxy homoeologs created through TILLING and a preexisting deletion of the third waxy homoeolog displays a near-null waxy phenotype. This approach to creating and identifying genetic variation shows potential as a tool for crop improvement.

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Figure 1: PCR assays to test for Wx-B1 locus and to test homoeolog-specific TILLING primers.
Figure 2: EMS-induced mutations identified by TILLING in wheat.
Figure 3: Wheat endosperms stained with iodine solution to detect amylose.

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Acknowledgements

The authors would like to thank Cate McGuire for managing the wheat TILLING libraries, David Pritchard for help with statistical analysis, Leigh-Anne Uribe for DNA preparation and Pauline Sanders for observations. We thank Margaret Miller, Luca Comai, Trent Colbert, Claire McCallum, Charles Moehs and Joseph Koning for helpful discussions. We thank Craig Morris for the waxy null seed.

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Correspondence to Ann J Slade.

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Supplementary Table 1

Additional intronic waxy alleles obtained through TILLING in hexaploid and tetraploid wheat. (PDF 55 kb)

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Slade, A., Fuerstenberg, S., Loeffler, D. et al. A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 23, 75–81 (2005). https://doi.org/10.1038/nbt1043

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