Plant material and cultivation
Huayu 23, a popular peanut cultivar of runner market type widely accepted by growers and processors, was used in this study. Sanger sequencing of its FAD2A and FAD2B and subsequent sequence alignment revealed that this cultivar had a mutated FAD2A (448 G>A)and a wild type FAD2B. Peanut was sown under film mulching in an isolated region in Laixi Experimental Station on May 5, 2021. Agronomic practices were followed as routine.
Vector construction and transformation of E. coli
Target site of sgRNA, 501-520 position of the coding sequence, was selected based on wild type FAD2B using CRISPR-GE (http://skl.scau.edu.cn/). To facilitate ligation in genome editing vector construction, two oligos which would produce overhangs after mixing, denaturation and annealing were generated with online tool (http://biogle.cn/index/excrispr) and synthesized (Tsingke, Qingdao). CRISPR/Cas9 editing vector with target site incorporated was made with BGK41-Cas9 (Biogle Biotechnology Co. Ltd, Hangzhou) following manufacturer’s instructions. Briefly, BGK41-Cas9, oligo dimers and enzyme mix (Biogle CRISPR/Cas vector construction kit) were mixed on ice bath, and then incubated at 20C for 1 h. The ligation products were transformed into competent cells of E. coli strain DH5α. Positive clones identified using colony-PCR with Cas9-F/Cas9-R primer pair (5’-tcgtgctgaccctgacactgtttga-3’, 5’- cttggcggtagccttgccgatttcc-3’) were sequenced with primer CXYW1 (5’- cccagtcacgacgttgtaaa-3’) (Tsingke, Qingdao) to confirm the inclusion of the target site in the vector. The newly constructed plasmid was named as BGK41-Cas9 recombinant vector FAD2B-1 (Fig. 1)
Transformation of Agrobacterium and node injection transformation of peanut
Genome editing construct was transformed into Agrobacterium tumefaciens strain GV3101 chemically competent cells (Veidi Biotech, Shanghai) according to the attached user’s guide. Preparation of Agrobacterium for injection was based on Pan et al. (2020) with some modifications. Positive single clones were verified by bacterial suspension PCR using Cas9-F/Cas9-R primer pair. 100 μl of freshly prepared bacterial suspension were cultured in 10 ml of YEB (Yeast Extract Beef) liquid medium at 28C with agitation (250 rpm) until OD600 reached 0.6-0.8 (about 12 h). The cultures were centrifuged at 6 000 rpm for 1 min to collect the bacterium. Equal volume of infection solution containing 100 μmol/L acetosyringone (AS), 10 mmol/L MES and 10 mmol/L MgCl2·6H2O was added to the pellets. Resuspended bacterial pellets were used for injection. Node injection procedure was essentially the same as that in our previous report (Wang et al. 2013) (Fig. 2). Injection was done between 6:00-8:00 a.m. on July 17, 2021, and the positions injected were marked with threads (Fig. 2). Inverted U-shaped mental wires were used to facilitate the entry of pegs into the soil from higher nodes (Fig. 2).
Quality analysis of resultant seeds
Pods were harvested when matured (Sept. 17, 2021). These pods were sun-dried and hand shelled. The chemical quality of the individual single seeds was predicted with near infra-red spectroscopy (NIRS) (Wang et al. 2014). Seeds with at least 74% oleic acid along with were further analyzed for fatty acids using gas-chromatography (GC) using cotyledonary slices follow the protocol of Yang et al. (2012).
Comparison of FAD2 sequences between the high-oleic seeds and Huayu 23
FAD2A and FAD2B sequences of the high-oleic seeds and Huayu 23 were amplified by PCR using primers aF19 (5’-gattactgattattgactt-3’)/R1 (5’- ctctgactatgcatcag-3’) and bF19 (5’- cagaaccattagctttg-3’)/R1 respectively (Patel et al. 2014), and templates prepared from cotyledonary slices (Yu et al. 2010). PCR products were directly sequenced by Tsingke, Qingdao. Sequence comparison was done with the DNAStar Lasergene version 7.1.0.