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.

Fig. 1.. FIND-IT screening procedure.

(A) Barley M3 library generation: Mutagenized barley plants are grown densely to maturity in the field. M3 grains are harvested in pools of approximately 300 plants. (B) Library screening consists of three phases that narrow down the search of a target variant from >500,000 to eventually a single plant within days. See detailed description of the method in Materials and Methods and note S6.1. Screening phase 1: The harvested grain pools are each split into two fractions: One fraction (25%, DNA pool) is used for total DNA extraction, and the other (75%, grain pool) is stored for further analysis. Total DNA from DNA pools is further combined in each well, so one 96-well library plate can contain DNA from up to 100,000 individuals. The following ddPCR screening (fig. S11A), using competing fluorescently labeled TaqMan probes to target the nucleotide variant of interest, identifies one well for further analysis. Screening phase 2: The corresponding 75% grain pool of the identified DNA pool in phase 1 is selected for further analysis. DNA from approximately 1000 grains is sampled nondestructively in subpools of 10 and rescreened using ddPCR (fig. S11B), and several wells are potentially identified to contain the targeted variant. Screening phase 3: The grains from the identified wells in phase 2 are germinated, and total DNA is extracted from the first leaf of each individual plant and rescreened by ddPCR (fig. S11C). From this, a single plant is identified containing the targeted variant, which can be sequence-validated (fig. S11D). Identified variants of interest are immediately available for field trials and agronomic analyses, and incorporation into routine breeding crossing programs.

Fig. 2.. Barley knockout library with selected variants isolated and agronomically evaluated.

(A) Barley physical reference genome map (chromosomes 1H to 7H) with location of more than 150 identified FIND-IT variants (variant type indicated by symbol; table S9). (B) Gene models of selected barley reference targets with known loss-of-function mutations and previously unknown, isolated stop variants (in color). (C) Loss-of-function phenotypes of isolated variants [hull-less grain, nud^W16*; six-rowed spikelets, jmj706^W313*; (1,3;1,4)-β-glucan Calcofluor staining, cslF6^W676*; starch amylose Lugol staining, gbss1^W126*; grain width, gw2^W233*]. (D) TGW and (E) grain yield of field-grown barley variants. (F) Grain (1,3;1,4)-β-glucan content of cslF6^W676* grain. (G) Starch amylose content of gbss1^W126* grain. (H) Grain width distribution of gw2^W233* grain. Scale bars, 5 mm (C). Error bars are SD; two-tailed t test was performed to obtain P values (*P < 0.05, **P < 0.01, and ***P < 0.001; ns, not statistically significant). See table S12 for statistical test details and number of replicates.

Fig. 3.. FIND-IT libraries with distinct nonsynonymous nucleotide variants and variants to modulate transcript abundance for precision breeding.

(A) Identified and isolated variants (in gray and green, respectively) of the SLN1/DELLA protein. (B) Plant height, (C) grain length distribution, and (D) TGW of sln1^A277T. (E) Gene model of barley GRF4 showing the miR396-binding site including a known double substitution in rice (OsGRF4^ngr2, bold) and FIND-IT barley variants (in red). (F) Known cis-acting elements in α-amylase promoters with natural and induced W-box sequence variation: W-boxes with two TGAC core motifs, a natural variation in the Amy1_2 gene in gray and an induced variation in an Amy1_1 gene in red. (G) Expression of α-amylase genes in RGT Planet grain 48 and 72 hours after initiation of germination. (H) Expression of Amy1_1 genes in RGT Planet grain (black) and RGT Planet variant Amy1_1-var (red) 48 and 72 hours after initiation of germination. (I) Protein homology model of CslF6 based on poplar cellulose synthase isoform 8 (Protein Data Bank ID: 6wlb.1.A). Amino acid positions at residue W676, T709, G748, and G847 are shown as spheres. Inset shows uridine diphosphate (UDP)–glucose binding sites as light blue sticks and the TED motif as dark blue sticks. The inset is shown at a slightly tilted angle to visually optimize the position of the residues discussed. (J) Grain (1,3;1,4)-β-glucan content of controls (gray bars) and CslF6 variants. (K) Percentage of broken grain after threshing in controls (gray bars) and CslF6 variants. (L) Field grain yield of controls (gray bars) and CslF6 variants. Error bars are SD; two-tailed t test was performed to obtain P values (*P < 0.05, **P < 0.01, and ***P < 0.001). See table S12 for statistical test details and number of replicates.