CRISPR mutant rapid identification in B. napus: RNA-Seq functional profiling and breeding technology application

Abstract

Introduction: Traditional rapeseed breeding is inefficient and imprecise. CRISPR genome editing offers a precise alternative for trait improvement. Here, we edited the Bnaida gene in elite rapeseed cultivar ZS11 to study its role in floral organ abcission and enable rapid trait transfer to elite lines.

Methods: The BnaIDA gene was CRISPR-edited in ZS11. Phenotypes (petal adhesion time, cracking force of siliques) were statistically analyzed. And analyze the mutants using RNA -Seq. Edited alleles were introgressed into elite line SW1-6 via backcrossing. Locus-specific primers enabled efficient genotyping to distinguish hetero- and homozygous plants during selection.

Results and discussion: In this study, The Bnaida mutant by gene editing in the cv ZS11, which is widely used in rapeseed breeding. The phenotypic analysis showed that the petal was attached to the pod and pods were harder to crack in edited plants, and then we quickly introduced two Bnaida loci into the elite line of SW1-6 by backcrossing with edited ZS11 as the donor plant. Locus-specific primer combinations were designed to differentiate heterozygous and homozygous genotypes in backcrossing generations, enabling efficient and rapid selection. This study highlights the integration of gene editing and genotyping selection, offering insights into the future of gene editing-assisted breeding.

Keywords: Brassica napus L.; CRISPR/Cas9; IDA-HAE/HSL2 signaling pathway; RNA-Seq; rapid identification.

Figure 1

Gene editing strategy and analysis of BnaIDA-A07/C06-ZS11. (A) Schematic illustration of the CRISPR-Cas9 vector targeting the conserved coding sequence region of BnaIDA. (B) Mutation types and frequency at the sgRNA target sites in T0 mutants. The X-axis: I# and D# were the numbers of base pairs inserted and deleted at the sgRNA target sites. (C) showing mutation sites at two homologous copies of T1-BnaIDA (BnaIDA-ZS11-A07/C06). Targets were colored yellow. Red represented the edited base. PAMs were colored green. “-” indicated nucleotide deletions in the target sequence.

Figure 2

The Bnaida exhibited a prolonged flowering period by affecting the isolation of AZ cells. (A) Bnaida mutants are deficient in floral organ abscission. (B) SEMs of floral abscission zones in Bnaida and WT plants. Images were captured at the mid-region (position 1/2) of the primary inflorescence. St, Stamen AZ. Se, sepal AZ. N, Nectaries AZ. P, Petal AZ. The red arrows highlight the residual floral organs. Bars = 500 μm. (C) Quantitative statistics of WT and mutant flowers over 80 days. (n = 10, bars = SD). (D) Schematic diagram of texture analyzer and the force measurements of siliques opening. (n = 22, bars = SD; **** Student′s t-test, P < 0.0001).

Figure 3

RNA-Seq and differential gene expression analysis in mutants and WT. (A) Venn diagram of mutant and WT. (B) The scatter plot. (C) Cluster heat map of differential gene expression. The redder the color, the higher the expression, and the bluer the color, the lower the expression.

Figure 4

GO (A) and KEGG (B) Analysis of DEGs. The vertical axis indicates GO categories and KEGG pathways, while the horizontal axis shows the number of genes and enrichment ratios.

Figure 5

Rapid identification of Bnaida mutants. (A) Design of primers for rapid identification of Bnaida mutants. (B) Rapid identification of Bnaida mutants. M: DL500 Marker(bp); -: Negative control (ddH₂O); WT-1/2: Wild Type (The leaf genome of the ZS11). (C) Bnaida fruits retain floral organs indefinitely.

Figure 6

Gene editing combined with breeding technology. (A) Strategy diagram for cross and backcrossing breeding of Bnaida (T-DNA free) with other rapeseed varieties. (B, C) Primer design diagram for distinguishing dominant and recessive alleles A-A/C-C, as well as distinguishing BnaIDA-A07 and BnaIDA-C06 genes. (D) Sanger sequencing and Hi-TOM of BC₂ generation plants with AaBb genotype. Targets were colored yellow. Red represents the inserted base.