The leucine-rich repeat receptor-like protein kinase (LRR-RLK) family represents the largest group of RLKs in plants and plays vital roles in plant growth, development and the responses to environmental stress. Although LRR-RLK families have been identified in many species, they have not yet been reported in B. napus. In this study, a total of 444 BnLRR-RLK genes were identified in the genome of Brassica napus cultivar “Zhongshuang 11” (ZS11), and classified into 22 subfamilies based on phylogenetic relationships and genome-wide analyses. Conserved motifs and gene structures were shared within but not between subfamilies. The 444 BnLRR-RLK genes were asymmetrically distributed on 19 chromosomes and exhibited specific expression profiles in different tissues and in response to stress. We identified six BnBRI1 homologs and obtained partial knockouts via CRISPR/Cas9 technology, generating semi-dwarf lines without decreased yield compared with controls. This study provides comprehensive insight of the LRR-RLK family in B. napus. Additionally, the semi-dwarf lines expand the “ideotype” germplasm resources and accelerate the breeding process for B. napus.

The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar “Zhongshuang 11” (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in Brassica napus. This study provides a systemic view of BnCYP450s and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out GMS controlling genes.

Cytoplasmic male sterility (CMS) is the main mechanism employed to utilize the heterosis of Brassica napus. CMS three-line rapeseed hybrids have dramatically enhanced yield and brought about the global revolution of hybrid varieties, replacing conventional crop varieties. Over the last half century, China has led the development of hybrid Brassica napus varieties. Two sterile lines, polima (pol) and shaan 2A, were of particular importance for the establishment of three-line hybrid systems in rapeseed, which has opened up a new era of heterosis utilization. However, in current breeding practices, it takes up to three years to identify the restorer or maintainer relationship and the cytoplasmic type of any inbred material. This greatly affects the breeding speed of new varieties and inhibits the rapid development of the rapeseed industry. To address this problem, we developed a set of molecular markers for the identification of fertile cytoplasmic gene N and sterile cytoplasmic gene S, as well as for the fertile nucleus gene R and sterile nucleus gene r, based on differences in the gene sequences between the CMS line, maintainer line and restorer line of Brassica napus. Combining these markers can accurately identify the CMS line, maintainer and restorer of both the pol and shaan systems, as well as their hybrids. These markers can not only be used to identify of the maintainer and restorer relationship of inbred materials; they can also be used as general molecular markers to identify the CMS-type hybrid purity of pol and shaan systems.

MADS-box transcription factors play an important role in regulating floral organ development and participate in environmental responses. To date, the MADS-box gene family has been widely identified in Brassica rapa (B. rapa), Brassica oleracea (B. oleracea), and Brassica napus (B. napus); however, there are no analogous reports in Brassica nigra (B. nigra), Brassica juncea (B. juncea), and Brassica carinata (B. carinata). In this study, a whole-genome survey of the MADS-box gene family was performed for the first time in the triangle of U species, and a total of 1430 MADS-box genes were identified. Based on the phylogenetic relationship and classification of MADS-box genes in Arabidopsis thaliana (A. thaliana), 1430 MADS-box genes were categorized as M-type subfamily (627 genes), further divided into Mα, Mβ, Mγ, and Mδ subclades, and MIKC-type subfamily (803 genes), further classified into 35 subclades. Gene structure and conserved protein motifs of MIKC-type MADS-box exhibit diversity and specificity among different subclades. Comparative analysis of gene duplication events and syngenic gene pairs among different species indicated that polyploidy is beneficial for MIKC-type gene expansion. Analysis of transcriptome data within diverse tissues and stresses in B. napus showed tissue-specific expression of MIKC-type genes and a broad response to various abiotic stresses, particularly dehydration stress. In addition, four representative floral organ mutants (wtl, feml, aglf-2, and aglf-1) in the T0 generation were generated by editing four AGAMOUS (BnaAG) homoeologs in B. napus that enriched the floral organ variant phenotype. In brief, this study provides useful information for investigating the function of MADS-box genes and contributes to revealing the regulatory mechanisms of floral organ development in the genetic improvement of new varieties.