Background: Chinese cabbage (Brassica rapassp.pekinensis) is a member of one of the most important leaf vegetables grown worldwide, which has experienced thousands of years in cultivation and artificial selection. The entire Chinese cabbage genome sequence, and more than forty thousand proteins have been obtained to date. The genome has undergone triplication events since its divergence fromArabidopsis thaliana(13 to 17 Mya), however a high degree of sequence similarity and conserved genome structure remain between the two species.Arabidopsisis therefore a viable reference species for comparative genomics studies. Variation in the number of members in gene families due to genome triplication may contribute to the broad range of phenotypic plasticity, and increased tolerance to environmental extremes observed inBrassicaspecies. Transcription factors are important regulators involved in plant developmental and physiological processes. The AP2/ERF proteins, one of the most important families of transcriptional regulators, play a crucial role in plant growth, and in response to biotic and abiotic stressors. Our analysis will provide resources for understanding the tolerance mechanisms inBrassica rapassp.pekinensis.Results: In the present study, 291 putative AP2/ERF transcription factor proteins were identified from the Chinese cabbage genome database, and compared with proteins from 15 additional species. The Chinese cabbage AP2/ERF superfamily was classified into four families, including AP2, ERF, RAV, and Soloist. The ERF family was further divided into DREB and ERF subfamilies. The AP2/ERF superfamily was subsequently divided into 15 groups. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns, and interaction networks of the AP2/ERF transcription factor superfamily were predicted and analyzed. Distribution mapping results showed AP2/ERF superfamily genes were localized on the 10 Chinese cabbage chromosomes. AP2/ERF transcription factor expression levels exhibited differences among six tissue types based on expressed sequence tags (ESTs). In the AP2/ERF superfamily, 214 orthologous genes were identified between Chinese cabbage andArabidopsis. Orthologous gene interaction networks were constructed, and included seven CBF and four AP2 genes, primarily involved in cold regulatory pathways and ovule development, respectively.Conclusions: The evolution of the AP2/ERF transcription factor superfamily in Chinese cabbage resulted from genome triplication and tandem duplications. A comprehensive analysis of the physiological functions and biological roles of AP2/ERF superfamily genes in Chinese cabbage is required to fully elucidate AP2/ERF, which provides us with rich resources and opportunities to understand crop stress tolerance mechanisms.

Non-heading Chinese cabbage (NHCC) is an important leafy vegetable cultivated worldwide. Here, we report the first high-quality, chromosome-level genome of NHCC001 based on PacBio, Hi-C, and Illumina sequencing data. The assembled NHCC001 genome is 405.33 Mb in size with a contig N50 of 2.83 Mb and a scaffold N50 of 38.13 Mb. Approximately 53% of the assembled genome is composed of repetitive sequences, among which long terminal repeats (LTRs, 20.42% of the genome) are the most abundant. Using Hi-C data, 97.9% (396.83 Mb) of the sequences were assigned to 10 pseudochromosomes. Genome assessment showed that thisB. rapaNHCC001 genome assembly is of better quality than other currently availableB. rapaassemblies and that it contains 48,158 protein-coding genes, 99.56% of which are annotated in at least one functional database. Comparative genomic analysis confirmed thatB. rapaNHCC001 underwent a whole-genome triplication (WGT) event shared with otherBrassicaspecies that occurred after the WGD events shared withArabidopsis. Genes related to ascorbic acid metabolism showed little variation among the threeB. rapasubspecies. The numbers of genes involved in glucosinolate biosynthesis and catabolism were higher in NHCC001 than in Chiifu and Z1, due primarily to tandem duplication. The newly assembled genome will provide an important resource for research onB. rapa, especiallyB. rapassp.chinensis.

Non-heading Chinese cabbage (NHCC), belonging to Brassica, is an important leaf vegetable in Asia. Although genetic analyses have been performed through conventional selection and breeding efforts, the domestication history of NHCC and the genetics underlying its morphological diversity remain unclear. Thus, the reliable molecular markers representative of the whole genome are required for molecular-assisted selection in NHCC. A total of 20,836 simple sequence repeats (SSRs) were detected in NHCC, containing repeat types from mononucleotide to nonanucleotide. The average density was 62.93 SSRs/Mb. In gene regions, 5,435 SSRs were identified in 4,569 genes. A total of 5,008 primer pairs were designed, and 74 were randomly selected for validation. Among these, 60 (81.08%) were polymorphic in 18 Cruciferae. The number of polymorphic bands ranged from two to five, with an average of 2.70 for each primer. The average values of the polymorphism information content, observed heterozygosity, Hardy-Weinberg equilibrium, and Shannon’s information index were 0.2970, 0.4136, 0.5706, and 0.5885, respectively. Four clusters were classified according to the unweighted pair-group method with arithmetic average cluster analysis of 18 genotypes. In addition, a total of 1,228,979 single nucleotide polymorphisms (SNPs) were identified in the NHCC through a comparison with the genome of Chinese cabbage, and the average SNP density in the whole genome was 4.33/Kb. The number of SNPs ranged from 341,939 to 591,586 in the 10 accessions, and the average heterozygous SNPs ratio was ~42.53%. All analyses showed these markers were high quality and reliable. Therefore, they could be used in the construction of a linkage map and for genetic diversity studies for NHCC in future. This is the first systematic and comprehensive analysis and identification of SSRs in NHCC and 17 species. The development of a large number of SNP and SSR markers was successfully achieved for NHCC. These novel markers are valuable for constructing genetic linkage maps, comparative genome analysis, quantitative trait locus (QTL) mapping, genome-wide association studies, and marker-assisted selection in NHCC breeding system research.

Flavonols have been shown to respond to a variety of abiotic stresses in plants, including cold stress. Higher total flavonoid content was found in non-heading Chinese cabbage (NHCC, Brassica campestris (syn. Brassica rapa) ssp. chinensis) after cold stress. A non-targeted metabolome analysis showed a significant increase in flavonol content, including that of quercetin and kaempferol. Here, we found that an R2R3–MYB transcription factor, BcMYB111, may play a role in this process. BcMYB111 was up-regulated in response to cold treatment, with an accompanying accumulation of flavonols. Then, it was found that BcMYB111 could regulate the synthesis of flavonols by directly binding to the promoters of BcF3H and BcFLS1. In the transgenic hairy roots of NHCC or stable transgenic Arabidopsis, overexpression of BcMYB111 increased flavonol synthesis and accumulation, while these were reduced in virus-induced gene silencing lines in NHCC. After cold stress, the higher proline content and lower malondialdehyde (MDA) content showed that there was less damage in transgenic Arabidopsis than in the wild-type (WT). The BcMYB111 transgenic lines performed better in terms of antioxidant capacity because of their lower H₂O₂ content and higher superoxide dismutase (SOD) and peroxidase (POD) enzyme activities. In addition, a key cold signaling gene, BcCBF2, could specifically bind to the DRE element and activate the expression of BcMYB111 in vitro and in vivo. The results suggested that BcMYB111 played a positive role in enhancing the flavonol synthesis and cold tolerance of NHCC. Taken together, these findings reveal that cold stress induces the accumulation of flavonols to increase tolerance via the pathway of BcCBF2–BcMYB111–BcF3H/BcFLS1 in NHCC.