Development of Decreased-Gluten Wheat Enabled by Determination of the Genetic Basis of lys3a Barley.

Celiac disease is the most common food-induced enteropathy in humans, with a prevalence of approximately 1% worldwide. It is induced by digestion-resistant, proline- and glutamine-rich seed storage proteins, collectively referred to as gluten, found in wheat (Triticum aestivum). Related prolamins are present in barley (Hordeum vulgare) and rye (Secale cereale). The incidence of both celiac disease and a related condition called nonceliac gluten sensitivity is increasing. This has prompted efforts to identify methods of lowering gluten in wheat, one of the most important cereal crops. Here, we used bulked segregant RNA sequencing and map-based cloning to identify the genetic lesion underlying a recessive, low-prolamin mutation (lys3a) in diploid barley. We confirmed the mutant identity by complementing the lys3a mutant with a transgenic copy of the wild-type barley gene and then used targeting-induced local lesions in genomes to identify induced single-nucleotide polymorphisms in the three homeologs of the corresponding wheat gene. Combining inactivating mutations in the three subgenomes of hexaploid bread wheat in a single wheat line lowered gliadin and low-molecular-weight glutenin accumulation by 50% to 60% and increased free and protein-bound lysine by 33%.

Development of high amylose wheat through TILLING.

BACKGROUND: Wheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes. RESULTS: Using TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry. CONCLUSIONS: We report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents.

Development of non-transgenic glyphosate tolerant wheat by TILLING.

Glyphosate (N-phosphonomethyl-glycine) is the world's most widely used broad spectrum, post-emergence herbicide. It inhibits the chloroplast-targeted enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), a component of the plant and microorganism-specific shikimate pathway and a key catalyst in the production of aromatic amino acids. Variants of EPSPS that are not inhibited by glyphosate due to particular amino acid alterations in the active site of the enzyme are known. Some of these variants have been identified in weed species that have developed resistance to glyphosate because of the strong selective pressure of continuous, heavy glyphosate use. We have used TILLING (Targeting Induced Local Lesions in Genomes), a non-transgenic, target-selected, reverse genetics, mutation breeding technique, and conventional genetic crosses, to identify and combine, through two rounds of mutagenesis, wheat lines having both T102I and P106S (so-called TIPS enzyme) mutations in both the A and the D sub-genome homoeologous copies of the wheat EPSPS gene. The combined effects of the T102I and P106S mutations are known from previous work in multiple species to minimize the binding of the herbicide while maintaining the affinity of the catalytic site for its native substrates. These novel wheat lines exhibit substantial tolerance to commercially relevant levels of glyphosate.

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

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.