Plant materials
The non-waxy spring barley (Hordeum vulgare L.) ‘Golden Promise’ was presented by Wendy A. Harwood from Crop Transformation Group, Department of Crop Genetics, John Innes Centre, Norwich, UK. No permission was required in collecting this barley. The barley was grown in a greenhouse under controlled conditions with 18°C during 16 h of light and 15°C during 8 h of darkness. When immature embryos were 1.5 to 2 mm in diameter, immature grains were collected from the center of spikes. Immature embryos were used for Agrobacterium-mediated transformation.
SgRNA design and plasmid construction
Exon 1 of the Waxy gene in ‘Golden Promise’ was cloned and sequenced using the specific primers wxF/R (Table S1). Two target sites located in exon 1 of the Waxy gene were selected using the online software CRISPR-GE (http://skl.scau.edu.cn/home/). The CRISPR/Cas9 system consisting of PMED and pLGYE001 was a gift from the Crop Research Institute, Shandong Academy of Agricultural Sciences. A construct containing two sgRNAs driven by TaU3 was constructed according to previously published protocols [28, 29]. In brief, four primers, T1F, T1F0, T2R and T2R0 (Table S1), containing target sequences were synthesized, and the T1-gRNA-Ter-TaU3-T2 cassette was amplified using plasmid PMED as template in a 50 μL reaction (Sangon Biotech, Shanghai, China). This cassette was then digested using BsaI and ligated to pLGYE001 (NEbiolabs, Beijing, China). The recombinant vector pLGYE001-wx was transferred into Agrobacterium EHA105 competent cells (Huayueyang, Beijing, China).
Agrobacterium-mediated transformation of barley
Agrobacterium-mediated transformation of barley (Hordeum vulgare L.) ‘Golden Promise’ was performed as described [30]. In brief, immature embryos were separated from sterile grains and co-cultivated with Agrobacterium EHA105 harboring the CRISPR/Cas9 vector pLGYE001-wx at 24°C for 3 days in the dark. Embryos were subsequently transferred to selective medium containing 5 mg/L glufosinate ammonium (PPT) (Coolaber, Beijing, China) and cultured at 24°C for 6 to 8 weeks in the dark. Proliferating calli were transferred to transition medium containing 3 mg/L PPT and cultured at 24°C under 16 h low light/8 h dark photoperiod. Small plantlets were transferred to regeneration medium containing 3 mg/L PPT and cultured at 24° under 16 h light/8 h dark photoperiod for 2 weeks. Putative transgenic plants were then transferred to soil and grown to maturity in the greenhouse under controlled conditions of 18°C during 16 h light and 15°C during 8 h darkness.
Detection of transgenic plants and mutations
Transgenic plants were identified using a PAT/bar test strip resistance kit for Bar protein (Youlong, Shanghai, China). Genomic DNA was isolated from leaves of putative transgenic plants using a TaKaRa MineBEAT Plant Genomic DNA Extraction Kit (TaKaRa, Beijing, China). For detection of T-DNA insertions, PCR with primers ubiF/R (Table S1) was performed using 100 ng of DNA in a 20 μL reaction (Sangon Biotech, Shanghai, China). For detection of mutations in transgenic plants, the specific PCR primers wxF/R were used for PCR amplification. Amplified DNA fragments of target genes were ligated into pGEM-Teasy Vector (Promega, Shanghai, China), and 20 positive clones were sequenced. Nucleotide sequencing results were analyzed using the AlignX program (Invitrogen, California, USA).
SDS-PAGE analysis of GBSSI protein
GBSSI protein was extracted according to previous reports with some modifications [13, 31]. Fine powder was scraped from the endosperm of the grain, then homogenized in washing extraction buffer (55 mmol/L Tris-HCl, pH 6.8; 23 g/L SDS; 5% (v/v) β-mercaptoethanol). The mixture was incubated at 4°C overnight and centrifuged at 9000 g for 10 min. The supernatant was discarded, and the sediment was washed twice with distilled water and finally dried overnight. GBSSI protein was released from the starch by boiling (10 mg) in 400 μL of protein extraction buffer (containing 62 mmol/L Tris-HCl, pH 6.8; 23 g/L SDS; 5% (v/v) β-mercaptoethanol; 10% (v/v) glycerol; 0.05 g/L bromophenol blue) for 5 min and centrifuged at 9000 g for 10 min. Protein supernatant (20 μL) was separated using SDS-PAGE gels (Sangon Biotech, Shanghai, China) for 3 h at 50 mA. SDS-PAGE gels were stained using a solution of 0.05% (w/v) Coomassie Blue R-250, 5% (v/v) ethanol and 12% (w/v) acetic acid for 3 h and then immersed in 10% (w/v) acetic acid overnight to remove excess stain.
Iodine staining and scanning electron microscopy of endosperm
Grains of WT and edited lines were randomly selected. Transverse sections of grains were stained using 0.01% (w/v) I2–0.1% (w/v) KI solution. Stained starch granules were examined and images were captured using an Olympus BX53 microscope (Olympus Corp, Tokyo, Japan). The transverse sections of dried grain were attached to metallic stubs using carbon stickers and sputter-coated with gold for 30 s. Images were observed and captured using an SU8100 scanning electron microscope (Hitachi, Tokyo, Japan).
Expression analysis using qRT-PCR
Total RNA was extracted from developing grains of WT and edited lines at 8, 16, 24 and 32 days post-anthesis (dpa) using an RNAprep Pure Plant Kit (Tiangen, Beijing, China). The cDNAs were synthesized using a PrimeScript™M RT reagent Kit with gDNA Eraser (TaKaRa, Tokyo, Japan). The gene-specific qPCR primers wxQF/R (Table S1) were designed according to the barley ‘Golden Promise’ Waxy mRNA sequence. The housekeeping gene α-Tublin was co-amplified as a control for normalizing cDNA templates. qRT-PCR was performed in a 7500 Fast Real-Time PCR System (ABI, Carlsbad, USA) using TB GreenTM Premix Ex TaqTM II (TaKaRa, Tokyo, Japan). Every sample was analyzed with three replicates.
Transcriptome analysis
Total RNA of immature grains of the WT and edited line at 16 dpa was used for transcriptome analysis. Six libraries, with three biological replicates, were prepared and sequenced using an Illumina HiSeq 2000 system (Novogene, China). Raw reads were processed to obtain clean reads by removing low quality reads, and assembly of the clean reads was performed using Trinity. Functional annotation was performed by comparing all unigenes with the following databases: NCBI nonredundant protein sequences (Nr), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Unigene expression levels were calculated using fragments per kb per million reads (FPKM) values. Unigenes with differential expression levels between the WT and edited line were analyzed using a chi-square test with IDEG6 software [32]. The false discovery rate method was employed to determine the threshold P-value at a false discovery rate ≤ 0.001, and the absolute value of |log2ratio| ≥ 2 was used as the threshold to determine the significance of differential expression levels of unigenes. Significantly enriched GO and KEGG terms were obtained from the set of differentially abundant unigenes using the hypergeometric test.
Determination of grain quality and agronomic traits
T1 lines were generated from edited plants grown in the greenhouse under controlled conditions of 18°C during 16 h light and 15°C during 8 h darkness. Analysis of the agricultural traits of WT and edited lines was performed using 20 plants per T1 line. Plant height, number of tillers, spike length and grains per spike were recorded. Six repetitions of thousand-grain weight for the WT and edited lines were also recorded. Amylose/amylopectin, soluble sugar, sucrose, β-glucan and protein contents of grains were determined, respectively, via the dual-wavelength iodine binding method [33], phenol-sulfuric acid colorimetry method, high-performance liquid chromatography [34], streamlined enzymatic method [35] and the Kjeldahl method [36]. Every sample was analyzed three times. The statistical software PASW Statistics 18 (IBM SPSS, Chicago, USA) was used for data analysis.