Applying a non-GMO breeding approach with an identified natural variation to reduce food allergen Len c3 in Lens culinaris seeds

Lentils (Lens culinaris) are produced in diverse agroecological regions and are consumed as one of the most important food legumes at world-wide. Lentils possess a nutritional profile from a human health perspective that is not only nutrient-dense but also offers a better balance between protein and carbohydrates. However, lentil causes food allergy which has been a significant concern due to increased consumption in parts of the world. Len c3, a none-specific Lipid Transfer Protein (LTP), was identified as one of the allergens in lentil seeds. In this study, we firstly identified the Len c3 encoding gene LcLTP3b via peptide sequence blasting. We then focused on screening natural variations to search for lentil germplasms that harbors natural mutated allergen-encoding genes. A natural variation 11 was identified with mutations at LcLTP3b and low accumulation of vicilin through genomic-assisted approaches. Furthermore, we generated a pool of lentil germplasms with Len c3-free background through crossing the identified natural variation 11 with two lentil cultivars, CDC Redmoon and CDC Gold. These Len c3-free lentil germplasms can be used as a breeding resource targeting at reducing allergen risk in lentil consumption. Total RNA was extracted from dissected embryos and seed coats containing endosperms of CDC Redmoon, CDC Gold, and M11, using a RNeasy plant mini kit (Qiagen, Germany) according to the manufacturer’s instruction. For transcriptome sequencing, cDNA libraries were constructed from the isolated RNA samples by using a TruSeq RNA Sample Preparation kit v2 (Illumina). The cDNA libraries were used for RNAseq. RNAseq were conducted on an Illumina NOVAseq 6000 pair-end sequencing.