Additional file 1 of Enhancing powdery mildew resistance in soybean by targeted mutation of MLO genes using the CRISPR/Cas9 system
Creators: name: Bui, Thao Phuong
name: Le, Huy
name: Ta, Dong Thi
name: Nguyen, Cuong Xuan
name: Le, Ngoc Thu
name: Tran, Truong Thi
name: Van Nguyen, Phuong
name: Stacey, Gary
name: Stacey, Minviluz G.
name: Pham, Ngoc Bich
name: Chu, Ha Hoang
name: Do, Phat Tien
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: Plant Biology

description: Additional file 1: Fig. S1. Transcriptomics analysis of GmMLO genes in different tissues of soybean plant. Data were obtained from RNA-Seq Atlas of Glycine max. Fig. S2. Induced mutation analysis of hairy roots. A Gel electrophoresis (agarose 1%) of GmMLO20 edited region in wild-type (WT) and hairy root samples (HR1, HR2, HR3) with large deletions. M: 1 kb DNA marker. Shifted bands in lines HR1, HR2 and HR3 indicated the induced mutations of targeted genes. B Sequencing result of the HR1 line for the edited regions of GmMLO20 compared to wild-type allele. Target sequences and PAMs are indicated in red and blue, respectively. Δ indicates targeted sequence changes: - for deletion. Clones indicate number of colonies with the respective alleles out of total of clones sequenced. Fig. S3. Soybean transformation procedure. A Cotyledons at 5 days on the co-cultivation medium. B, C Shoot induction at 14 and 28 days on the selection medium. D Shoot elongation. E Rooted plants on the rooting medium. F, G Leaf painting using 200 mg/L glufosinate. Fig. S4. Gel electrophoresis of PCR-amplicons of GmMLO target expanding regions at T1 generation. WT: Non transgenic wild-type plant; 3.1-2 to 3.1-5: T1 plants from 3.1 line; 15.1-1 to 15.1-6: T1 plants from 15.1 line; M: 1 kb DNA ladder. PCR products amplified by specific primers for extended regions of GmMLO02, GmMLO10, GmMLO18, GmMLO19, GmMLO20 and GmMLO23 genes. Fig. S5. Inheritance of induced mutations in GmMLO02 (A), GmMLO19 (B), GmMLO20 (C) and GmMLO23 (D) genes in T2 plants. Target sequences and PAMs are indicated in red and blue color, respectively. Inserted nucleotides are shown in yellow. Δ indicates targeted sequence changes: 0 for no change, - for deletion, + for insertion. Fig. S6. E. diffusa isolation and characterization. A Conidiophore and conidia of E. diffusa isolated from infected leaves. Scale bar = 10 μm. B Internal transcribed spacer (ITS) sequences of the collected E. diffusa. C Nucleotide BLAST result of the E. diffusa ITS sequence on GenBank, NCBI. Fig. S7. Powdery mildew resistant assessment of T2 offspring Gmmlo mutant lines under the net- house conditions. Infection levels were recorded using a 0 to 5 scale (described by Tran et al., 2015), which according to strong resistance to severe infection. Infection levels were calculated as the average of 20-30 biological replicates and three experiments. Statistical analysis was done using one-way ANOVA followed by a post hoc Turkey’s test. Significant difference was considered at P &lt; 0.05. Fig. S8. Soybean seed production under the net-house conditions. A The total number of pods per plant. B The frequency of pods with 3 seeds. WT: Control line (DT26 cultivar); 3.1-3-41, 3.1-5-87 44, 15.1-2-2, 15.1-6-4: T2 offspring Gmmlo soybean mutant lines. Error bars indicate standard deviations, n = 9-14. Statistical analysis was done using one-way ANOVA followed by a post hoc Turkey’s test. Significant difference was considered at P &lt; 0.05. Fig. S9. Transgene inheritance and segregation at different generations. A Representative results of herbicide leaf painting with glufosinate solution (200 mg/L) on wild-type (WT), herbicide resistant line (3.1) and herbicide susceptible line (3.1-3-41). B Gel electrophoresis of PCR amplicons of transgenes at T0, T1 and T2 generations. M: 1 kb DNA ladder; WT: non transgenic wild-type plant; (+): positive control (CRISPR/Cas9 vector); bar: herbicide resistance gene; 35S:pFGC: transgene region spanning pFGC vector and 35SPPDK promoter of pcoCas9. Table S1. Sequences of oligonucleotides and primer sets used in this study. Table S2. Genotypes of T0 mutant lines. Table S3. Inheritance and segregation of transgenes at different transgenic soybean generations.