FIND-IT: Accelerated trait development for a green evolution

Abstract

Improved agricultural and industrial production organisms are required to meet the future global food demands and minimize the effects of climate change. A new resource for crop and microbe improvement, designated FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal PCR), provides ultrafast identification and isolation of predetermined, targeted genetic variants in a screening cycle of less than 10 days. Using large-scale sample pooling in combination with droplet digital PCR (ddPCR) greatly increases the size of low–mutation density and screenable variant libraries and the probability of identifying the variant of interest. The method is validated by screening variant libraries totaling 500,000 barley ( Hordeum vulgare ) individuals and isolating more than 125 targeted barley gene knockout lines and miRNA or promoter variants enabling functional gene analysis. FIND-IT variants are directly applicable to elite breeding pipelines and minimize time-consuming technical steps to accelerate the evolution of germplasm.

The causal mutation leading to sweetness in modern white lupin cultivars

“…For library construction, we treated around 100,000 seeds (15 kg) with a low dose of mutagen [0.1% ethyl methanesulfonate (EMS)] to minimize the frequency of off-target mutations. We then used FIND-IT technology ( 30 ) to isolate an early stop codon mutant in the AT gene ( AT KO , TGG to TAG at codon 169) (Fig. 4A).…”

“…The narrow-leafed lupin mutant library was constructed essentially as described in Knudsen et al ( 30 ). Briefly, 15 kg of narrow-leafed lupin cultivar Oskar was subjected to mutagenesis using 0.1% EMS for 16 hours at 23°C.…”

“…The narrow-leafed lupin mutant library was screened essentially as described in Knudsen et al ( 30 ). Briefly, a TaqMan assay was designed to identify a specific base change at position 506 in the coding region of AT , which corresponds to the change in amino acid 169 from tryptophan to a premature stop (see DNA Sequence 7 in the Supplementary Materials).…”

“…For the TaqMan assay, we designed a target-specific forward primer (AT_TaqMan_FW), a target-specific reverse primer (AT_TaqMan_RV), a wild-type–specific probe containing both a HEX fluorophore and a BHQ1 quencher (ATWT_TaqMan_HEX), and a mutant-specific probe containing both the FAM fluorophore and a BHQ1 quencher (ATKO_TaqMan_FAM). The TaqMan assay was used to screen the pools of the narrow-leafed lupin mutant DNA library utilizing the FIND-IT workflow ( 30 ). Subsequently, one individual heterozygous mutant seed was extracted from a positive pool of the library by subjecting 768 individual seeds to a nondestructive DNA extraction procedure as described previously for barley ( 30 ).…”

“…The TaqMan assay was used to screen the pools of the narrow-leafed lupin mutant DNA library utilizing the FIND-IT workflow ( 30 ). Subsequently, one individual heterozygous mutant seed was extracted from a positive pool of the library by subjecting 768 individual seeds to a nondestructive DNA extraction procedure as described previously for barley ( 30 ).…”