Large-scale screening of genes responsible for silique length and seed size in Brassica Napus via pooled CRISPR library

Abstract

Background: Enhancing rapeseed (Brassica napus, B. napus) yield is critical for ensuring global vegetable oil security. However, yield is heavily influenced by silique development and seed size, the enhancement of which is limited by scarce genetic resources. The CRISPR/Cas9 system has emerged as a powerful tool for constructing genome-wide mutant libraries, even in polyploid crops with complex genomes.

Results: The transcriptome-wide association study (TWAS) data, tissue-specific expression profiles data and reported genes were integrated to identify candidate genes regulating silique development and seed size. We constructed a sgRNA library targeting these genes and generated a CRISPR/Cas9 editing mutant library through genetic transformation. Specifically, 6124 sgRNAs were designed for 1739 candidate genes with ≦ 4 orthologues. 681 T₀ plants were obtained through genetic transformation, which harbor 453 sgRNAs. Of 408 T₀ plants analyzed, 151 (37.00%) exhibited successful gene editing events, targeting 84 candidate genes. Ten homozygous mutant plants were isolated and preliminary phenotypic analysis was performed in mutants targeting the BnaHRDs. The results suggest that mutations in BnaHRD.A03 and BnaHRD.C03 may modulate plant height (PH), main inflorescence length (MIL), silique length (SL), effective silique number per plant (ENS), seed number per silique (SNPS), and thousand-seed weight (TSW).

Conclusions: This study harnessed the CRISPR/Cas9 technology to establish a preliminary library of gene-edited mutants in B. napus, thereby laying a robust foundation for the future screening of candidate genes pertaining to silique development and seed size. Furthermore, this study provides a methodological framework for rapid functional gene discovery in B. napus through CRISPR-based approaches.

Keywords: Brassica Napus; CRISPR; Gene editing; Seed size; Silique length.

Fig. 1

Candidate gene screening from different sources. A Candidate gene from diverse sources; B Integrative comparison of candidate genes derived from different sources. TWAS-20 and TWAS-40 represent differently expressed candidate genes associated with the traits such as oil content at 20 days after flowering and 40 days after flowering. Silique-spe represents candidate genes specifically expressed in silique and seed. Reported represents genes associated with silique or seed size have been reported in plants

Fig. 2

Comparison of Quality Testing Results of the sgRNA plasmid library

Fig. 3

sgRNA typing of T₀ transformed plants. A The status of sgRNA in T₀ transformed plants. B Analysis of T₀ plants containing a single sgRNA

Fig. 4

Analysis of editing efficiency. A The edited rate of T₀ transformed plants. B Analysis of sgRNA in T₀ generation edited plants. C The edited rate of sgRNA in T₀ transformed plants. D The candidate gene sources of T₀ generation edited plants

Fig. 5

Analysis of Edit the type of T₀ generation edited plants. A The edited type of T₀ generation edited plants. SNP: Base substitution; 1I: one base insertion; nI: multiple base insertion; 1D: one base deletion; nD: multiple base deletion. B Frequency of different bases for 1 bp insertion

Fig. 6

The editing status in partial T₁ generation mutants. Dashes (−) represents a missing base; Red font represents base insertion; Blue font represents base substitution; WT represents a wild-type sequence

Fig. 7

Editing and amino acid sequence analysis of BnaHRDs homozygous mutants. A Target editing of some BnaHRDs T₁ homozygous mutants; B Amino acid sequence analysis of some BnaHRDs T₁ homozygous mutants. WT represents the wild-type sequence, a03 represents the BnaHRD.A03 copy on chromosome A03, c03 represents the BnaHRD.C03 copy on chromosome C03, and C03 also represents the wild-type BnaHRD.C03 sequence

Fig. 8

Analysis of Agronomic Traits of BnaHRDs Homozygous Mutant. PH Plant height, MIL Length of main inflorescence, ENS Effective number of siliques, SL Silique length, SNPS Number of seeds per silique, TSW Thousand seed weight. * p < 0.05