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

Abstract

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 proteinbound lysine by 33%.

Deep sampling and pooled amplicon sequencing reveals hidden genic variation in heterogeneous rye accessions

“…In barley, SNPs located in PBF were associated with crude protein and starch content (Haseneyer et al, 2010), while in wheat, mutating the homeologous PBFs using TILLING resulted in a markedly decreased gluten content and high content of lysine (Moehs et al, 2019).…”

Development of non-transgenic glyphosate tolerant wheat by TILLING

“…This work is another illustration of the practical utility of TILLING and the remarkable ability of wheat to tolerate a high mutation load, which was also demonstrated by the hundreds of new alleles identified in the targeted genes in our earlier work (Moehs et al 2019) (Slade et al 2005) (Slade et al 2012). TILLING in wheat has been applied by others for additional practical breeding efforts, such as generating lines resistant to the powdery mildew pathogen (Acevedo-Garcia et al 2017).…”

“…Then, 5 μl of the pooled DNA samples (or 20 ng wheat DNA) was arrayed on microtiter plates and subjected to gene-specific PCR. Amplification and TILLING was performed exactly as described (Moehs et al 2019).…”

CRISPR/Cas9 Gene Editing of Gluten in Wheat to Reduce Gluten Content and Exposure—Reviewing Methods to Screen for Coeliac Safety

“…When hypoimmunogenic wheat varieties make it to the market, whether made by gene editing, RNAi, alone or in combination with other approaches [such as low prolamin mutations (113)], it will initially require a separate production chain, implying completely separated facilities for hypoimmunogenic wheat on farms, at the processing factory, and all the way to packaging and labeling, to avoid any risks of contamination with other grains containing immunogenic gluten epitopes. If such products are well-accepted and the market grows, consumers may prefer safe wheat over regular wheat, even if they are not coeliac patients themselves, so more products will be made with hypoimmunogenic gluten.…”