Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch

Abstract

Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing. We demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated targeted mutagenesis of two genes involved in amylose biosynthesis, PROTEIN TARGETING TO STARCH (PTST1) or GRANULE BOUND STARCH SYNTHASE (GBSS), can reduce or eliminate amylose content in root starch. Integration of the Arabidopsis FLOWERING LOCUS T gene in the genome-editing cassette allowed us to accelerate flowering-an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes. This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications.

Fig. 1. Genome editing of MeGBSS and MePTST1.

(A) Model for association between GBSS, PTST1, and starch granules as proposed by Seung et al. (35). Image recreated with permission. (B) Binary expression construct containing hptII (hygromycin B resistance) for plant selection; Cas9 codon optimized for cassava usage (pcoCas9) and fused to the eGFP reporter gene and directed to the nucleus via a nucleoplasmin nuclear localization signal (NLS); transcription terminated by sequence from a heat shock protein (tHSP) (56). A synthetic pU6 promoter [(psynU6; 10)] used to drive expression of the protospacer sequence [single guide RNA (sgRNA)] and synthetic scaffold (9) to generate the desired sgRNA. AtFT for early flowering is constitutively expressed by the CaMV35S promoter and terminated by nopaline synthase sequence (tNOS). Binary expression constructs named pCas9-sgGBSS-FT and pCas9-sgPTST-FT. Left and right borders (LB and RB) are shown. Diagram not to scale. (C) Gene maps of MeGBSS and MePTST1. Exons are depicted as blocks, and regions encoding functional domains are shaded. The target sites for the sgRNAs are indicated. Scale bars are shown.

Fig. 2. Sequence analysis of indels and predicted translation of reads in gbss and ptst lines.

(A and B) PacBio SMRT sequencing of amplicons derived from gbss and ptst lines, respectively. The WT sequence is shown at the top of each alignment, with the protospacer adjacent motif (PAM; blue font and underlined) and sgRNA target site (green font). Nucleotide deletions are depicted as red dashes, and nucleotide insertions are in uppercase, red font. The number of reads per line is shown, together with the percentage of reads clustered according to indel sequence in the target site region. The most abundant sequence clusters for each line are shown. Low-abundant clusters (<5% of total reads) are not shown here since they are likely to be artifacts. Sequence data for WT are also shown as a control to determine conservation of sequence, following tissue culture and regeneration from callus. (C and D) Predicted polypeptides from gbss and ptst lines, respectively. The positions of frameshift mutations are indicated by red arrows, and hatched areas represent potentially translated regions until the next predicted termination codon. Predicted amino acid substitutions (white tick) and deletions (white cross) are indicated in gbss-TAB and gbss-TAO.

Fig. 3. Amylose content and immunodetection of GBSS in gbss and ptst storage roots.

(A) Light microscopy images of iodine-stained purified starch granules of selected gbss and ptst lines. Blue-black staining is indicative of amylose-containing starch, while amylose-free starch stains red-brown. A visible intermediate staining was seen in line gbss-TAB (approximately 1.8% amylose), while a higher-percentage amylose (approximately 9.4%) in line gbss-TAO gave a WT-like phenotype. The 20-μm scale bar is representative for all panels. (B) Storage roots from two plants of WT, gbss-TAH, and ptst-TAK were harvested from 6-month-old glasshouse-cultivated material. Root sections were stained with iodine solution and immediately photographed. Coloration described above is evident in amylose-free and amylose-containing lines. (C) Percentage amylose content for gbss (left) and ptst (right) lines using iodine colorimetry as described for potato starch (66). n = 6 to 8 plants; means ± SD are shown. Statistical analysis using Tukey’s multiple comparisons test (****P < 0.0001, ***P < 0.001, and **P < 0.01) is shown. (D) Immunodetection of GBSS in granule-bound proteins extracted from selected plants of gbss (top) and ptst (bottom) lines. Equivalent loading of protein extracted from 0.5 mg of starch and detected using a polyclonal antibody raised against Arabidopsis GBSS. Granule-bound proteins from the Arabidopsis gbss mutant (negative control, lanes −) and from cassava WT (positive control, lanes +).

Fig. 4. Physicochemical properties of gbss and ptst starch.

Viscosity measurements of purified starch from selected gbss and ptst lines. Amylose content for each line is provided in the graph legend. Temperature gradient is depicted as the gray dotted line against the secondary (right) y axis. Mean values (n = 3 to 4 plants) are plotted.

Fig. 5. Early flowering in glasshouse-cultivated gbss lines and molecular characterization of S1 progeny.

(A) Inflorescence on a gbss-TAB plant. (B) Developing fruit following manual pollination (selfing) on a gbss-TAH plant. (C) S1 progeny plantlets following manual pollination (selfing) and seed germination between gbss plants. (D) PCR amplification products of hptII, Cas9-eGFP, AtFT, and endogenous MePP2A. Reactions contained genomic DNA template from S1 progeny samples, a parent control (gbss-TAH), a vector control (binary vector), and WT. (E) Sequence of cloned amplicons derived from the GBSSsgRNA4 genomic target sequence in S1 lines. The WT sequence is shown at the top of the alignment and comprises the PAM (blue font and underlined) and the sgRNA target site (green font). Nucleotide deletions are depicted as red dashes. Number of reads and percentage distribution between indels from the sequenced population are provided.

Fig. 6. Schematic representation of conventional breeding and the designed NPBT for trait improvement.

(A) Pathway for breeding of recessive traits using a wild relative or mutagenized plant as parent material. Multiple crosses are required to introgress a homozygous mutation into a farmer-preferred genotype. Several years are required to generate an improved variety. ^†Information about mutation and availability of genetic marker(s) could allow bypassing of the selfing step. ^‡In the absence of marker(s), self-crosses are performed after every backcross to select offspring with the recessive trait phenotype. ^ΔAvoiding selfing should limit potential inbreeding depression. (B) NPBT described here for accelerated flowering and segregation of genome-edited lines. Agrobacterium-mediated stable transformation provides transgenic lines with different mutant populations. Several lines are screened (phenotype and genotype), and a single segregation from selfing or cross between two Cas9-FT–transformed genotypes can occur ex situ in a glasshouse environment, yielding T-DNA–free progeny with the homozygous recessive mutation.