Targeted mutation of BnaMS1/ BnaMS2 combined with the RUBY reporter enables an efficient two-line system for hybrid seed production in Brassica napus

Abstract

The recessive genic male sterility (RGMS) method has several benefits in hybrid seed production; however, it is seldom employed in industrial hybrid seed production owing to the difficulty of producing an ample number of pure male-sterile seeds. In this study, we present an efficient methodology for developing a two-line strategy to produce hybrid seed through targeted mutation of BnaMS1 and BnaMS2 in conjunction with the RUBY reporter in Brassica napus. In this method, male-sterile lines were successfully created directly from different elite rapeseed breeding lines through CRISPR/Cas9-mediated mutagenesis and enhanced Agrobacterium-mediated transformation. To establish an efficient transgenic maintainer, three seed production technology (SPT) cassettes carrying a functional BnaMS1 gene linked to different reporters (DsRed, BnaA07.PAP2, and RUBY) were tested and compared in rapeseed. The results indicated that the PMR-based reporter possesses advantages such as phenotypic stability and ease of identification at early stages, making it an ideal tool for rapid and efficient screening. Subsequently, ideal transgenic maintainer lines with a single hemizygous copy of the SPT cassette were successfully developed in the context of Bnams1Bnams2 double mutants. The progeny from crossing the maintainer line with its male-sterile counterpart exhibited a 1:1 segregation pattern of nontransgenic male-sterile and male-fertile maintainer plants, distinguishable by seedling color. This biotechnological approach to male sterility offers promising prospects for improving the propagation of recessive genic male-sterile plants and the development of hybrid seeds in rapeseed. Furthermore, it is simple to adapt this technique to more Brassica crops.

Figure 1

Null mutants of BnaMS1 and BnaMS2 by CRISPR/Cas9 technology. (A) BnaMS1 (BnaA07g0309800ZS) and BnaMS2 (BnaC06g0359600ZS) gene models. There are six exons, denoted by boxes, which are interspersed by five introns, indicated by solid lines. The vertical line within the gene model denotes the specific target site. The target sequences are presented with PAM, the protospacer adjacent motif. (B) The CRISPR/Cas9 system incorporates a hygromycin resistance component, which features the hygromycin phosphotransferase gene under the control of the cauliflower mosaic virus 35S promoter. Additionally, it includes a Cas9 expression module with Cas9 coding sequences regulated by the 35S promoter. Two single-guide RNAs (sgRNAs), labelled S1 and S2, are driven by the AtU3d and AtU6–26 promoters, respectively, which are derived from Arabidopsis. (C) The sequences at the sgRNA target sites of homozygous mutants of BnaMS1 and BnaMS2 in the T₄ or T₅ generation are presented. PAM sequence is underlined, while nucleotide insertions or deletions (indels) are highlighted, with specific details provided on the right side of the sequences. The symbols ‘a’ and ‘c’ denote the mutated alleles of the target gene on BnaMS1 and BnaMS2, respectively. The combinations ‘aaCC,’ ‘AAcc,’ and ‘aacc’ represent homozygous mutations of the target gene in BnaMS1, BnaMS2, and both loci, respectively.

Figure 2

Mutations in the BnaMS1BnaMS2 gene affect plant fertility. (A–F) Phenotypic comparison of flowers and main branches at the flowering stage between the WT and Bnams1Bnams2 double mutant plants; scale bar for A–B, 5 cm; scale bar for C–F, 1 cm. (G, H, K) Morphological analysis of petal-free flowers, anthers, and pollen from WT plants. Bars, 2000 μm. (I, J, L) Phenotypes of petal-removed flowers, anthers, and pollen in the Bnams1Bnams2 double mutant. Bars, 2000 μm. (M–P) Plant height (M), petal area (N), stamen length (O), and pistil length (P) of the WT and Bnams1Bnams2 double mutant plants. The data are displayed as the mean values with standard errors (n ≥ 10). Statistical analysis comparing the WT and double mutant strains was conducted using Student’s t-test (**, P > 0.01).

Figure 3

Schematic diagrams of the visual reporter systems used in this study. (A) MSBD cassette-containing construct based on DsRed. (B) MSPR cassette-containing construct based on BnaA07.PAP2. (C) PMR cassette-featuring construct comprising genes for betalain biosynthesis. LB, left border; RB, right border; HPT, HygR gene; P_35S, cauliflower mosaic virus (CaMV) 35S promoter; Cas9, SpCas9; P_BnaMS1, BnaMS1 native promoter; BnaMS1, BnaMS1 genomic sequence; DsRed, gene-encoding red fluorescent protein; BnaA07. PAP2, a gene involved in anthocyanin biosynthesis; RePAT, a glufosinate ammonium resistance gene. Three genes, namely CYP76AD1, DODA, and glucosyl transferase, play a key role in the biosynthesis of betalains. These genes are associated with the P2A, 2A peptide.

Figure 4

Phenotypic analysis of WT and PMR transgenic rapeseed plants. (A–B) The shoot, (C–D) leaf, (E–F) flower bud, (G–H) flower, (I–J) whole plant at maturity, and (K–L) developing silique morphology of WT (left) and PMR transgenic plants (right). Scale bar for A–J, 1 cm; scale bar for K–L, 2000 μm.

Figure 5

Dosage effect of the homologous/heterozygous insertion of the RUBY gene on seedling color. (A) Localization of T-DNA insertions in the transgenic line PMR-28. (B) Segregation of seedling color in the PMR-28 lines at 2 days after germination. Bar, 1 cm. (C) Segregation of seedling color in Bnams1Bnams2 × PMR-28 plants at 2 days after germination. Bar, 2 cm. (D) Genotypic and phenotypic analysis of Bnams1Bnams2 × PMR-28-1 seedlings at 2 days after germination. The codominant PCR marker was used to distinguish the WT, heterozygous, and homozygous genotypes of the T-DNA containing the SPT cassette. (E) The absorbance of betaines on Bnams1Bnams2 × PMR-28 seedlings at 2 days after germination.

Figure 6

Establishment of a new convenient two-line system for hybrid seed production in B. napus. The non-transgenic RGMS lines from Bnams1Bnams2 double mutants and the ideal PMR reporter lines sharing the same background were pyramided together by crossing. Then, PMR-based maintainer lines harboring Bnams1Bnams2 double mutants (ms1/ms2; PMR/−) were generated in the process of cross-pollination between the male-sterile line and the resulting maintainer line, 50% of the maintainer seedlings propagated, and 50% of the male-sterile seedlings were sorted on the basis of seedling color.