Peasy blunt3 vector

The total RNA was isolated from the third instar larvae on the second day after molting using the Trizol Plus reagent (Ambion, Austin, TX, USA), following the manufacturer’s recommended protocol. The RNA concentration and quality were assessed using a spectrophotometer (Denovix, Wilmington, DE, USA) and 1% agarose gel electrophoresis. cDNA synthesis was performed by the GoScript™ Reverse Transcription System kit (Promega, Madison, WI, USA) with an oligo (dT)₁₅ primer, and 1 ug of total RNA was used per reaction.
The full-length coding sequence of HcCht5 was amplified using PrimeSTAR^® Max DNA Polymerase (Takara, Shanghai, China). PCR primers were designed based on the reported H. cunea chitinase gene (Accession number: U86877) with the Primer Premier 5 software. The primer sequences are listed in Table S1. The PCR program used was as follows: 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min; and 72 °C for 10 min. The PCR products were purified and cloned into a pEASY-Blunt3 vector (TransGen, Beijing, China) and sequenced at Sangon Biotech (Co., Ltd., Beijing, China).

The sequences of candidate genes were obtained based on the Chinese spring (CS) genome at EnsemblPlants⁴. Primers were designed based on conserved sequences flanking candidate genes utilizing the Oligo 7 software and were further synthesized by Sangon Biotech Co., Ltd. The PCR products were separated by electrophoresis on 1% agarose gels, which were cleaned and cloned into the pEASY-Blunt3 vector (Beijing TransGen Biotech Co., Ltd.⁵). The vector was then transformed into DH5α competent E. coli cells by the heat shock method. Single clones were sequenced by Sangon Biotech Co., Ltd., and sequence analysis (alignment and assembly) was performed using the DNAMAN software⁶ and the GENEDOC software. According to the method used by DuPont et al. (2005) (link), whole wheat flour was used to extract gluten, and RP-HPLC was used to determine gluten and its subunit components (González-Torralba et al., 2011 (link)).
KASP primers were designed based on the differences from the cloned sequences between the RIL parents, which were then used to screen all 196 RILs. The JoinMap software was used to map the gene onto the genetic map based on the genotypes of these marker and neighboring markers. The SNP marker and neighboring markers were evaluated by their physical order on the Chinese Spring reference genome (IWGSC RefSeq v1.1²) to confirm the correct location of cloned genes.

The PCR product of sgRNA and the plasmid of Cr-EF1α>NLS::Cas9::NLS and Cr-EF1α>NLS::Cas9::NLS::mCherry were co-electroporated into the fertilized eggs. After electroporation, embryos were cultured for about 12 h at 18 °C. The embryos with red positive signals were collected and lysed by a direct PCR lysis buffer at 60 °C for 10 min. PCR was performed from the lysate using Phanta^® Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China) as follows: 55 °C annealing temperature for 15 s and 72 °C extension temperature for 30 s, 35 cycles. PCR products were purified by the GeneJET Gel Extraction Kit (Thermo Fisher, Waltham, Massachusetts, USA). Two hundred-nanogram PCR products in the knockout (KO) group and 200-ng control PCR products were diluted to 17 μL by ddH₂O and mixed with 2-μL T7 reaction buffer (Vazyme, Nanjing, China) in a 200-μL PCR tube and incubated with 1-μL T7 endonuclease I (Vazyme, Nanjing, China) at 37 °C for 30 min. The products were detected on a 2% agarose gel at 100 V for 30 min, and the image of the gel was analyzed by ImageJ (National Institutes of Health). Cleavage efficiency was determined according to the previously published protocol [33 (link)]. The PCR product of mutant gDNA was ligated into the pEasy Blunt-3 vector (Transgen, Beijing, China) for Sanger sequencing (GENEWIZ, Suzhou, China).