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Male sterility is an ideal character for the female parent in commercial hybrid seed production in Chinese cabbages. We identified three allele male sterile mutants msm2-1/2/3 in progenies of ethyl methane sulfonate mutagenized Chinese cabbage. It was proved that their male sterilities were controlled by a same recessive nuclear gene. Cytological observation showed that the delayed tapetal programmed cell death (PCD) as well as the abnormal pollen exine and intine led to pollen abortion in these mutants. MutMap combined with KASP analyses showed that BraA10g019050.3C, a homologous gene of AtMS1 encoding a PHD-finger transcription factor and regulated pollen development, was the causal gene. A single-nucleotide mutation from G to A occurred at the 2443th base of BrMS1 in msm2-1 which results in premature termination of the PHD-finger protein translation; a single-nucleotide mutation from G to A existed at 1372th base in msm2-2 that makes for frameshift mutation; a single-nucleotide mutation from G to A distributed at 1887th base in msm2-3 which issues in the amino acid changed from Asp to Asn. The three allelic mutations in BrMS1 all led to the male sterile phenotype, which revealed its function in stamen development. Quantitative reverse transcription polymerase chain reaction analysis indicated that BrMS1 specially expressed in the anther at the early stage of pollen development and its expression level was higher in msm2-1/2/3 than that in the wild-type “FT.” BrMS1 was located at the nucleus and a length of 12 amino acid residues at the C-terminus had transcriptional activation activity. RNA-seq indicated that the mutation in BrMS1 affected the transcript level of genes related to the tapetum PCD and pollen wall formation, which brought out the pollen abortion. These male sterile mutants we developed provided a novel gene resource for hybrid breeding in Chinese cabbage.

Cut flower ornamental kale (Brassica oleracea var. acephala) is a biennial cultivar, which completes a sexual reproductive generation in two years. Isolated microspore culture (IMC) can accelerate plant homozygosity instead of self-pollinations. However, the application of IMC in cut flower ornamental kale was rare since its low rate of embryogenesis. It is proved that histone acetylation might affect the gene expression in microspores and led to the transformation of microspores from pollen development pathway to embryogenesis. In this paper, microspores, derived from three varieties of cut flower ornamental kale, Crane Bicolor (CB), Crane Pink (CP) and Crane Feather Queen (CFQ), were treated with histone deacetylation inhibitor (HDACI) trichostatin A (TSA). Results revealed that the appropriate concentration of TSA was 10 nM for CB with obtaining 5.39 embryos per bud, while for CP and CFQ was 5 nM with acquiring 10.89 and 16.99 embryos per bud, respectively. TSA treatment also reduced the embryonic mortality, of which 10 nM TSA treatments CB was the optimal and the embryonic mortality decreased to 25.01%. The double haploid (DH) proportion of regenerated plants reached 37.3%. These results contribute to improving the technology for IMC in cut flower ornamental kale.

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Ornamental kale, as a burgeoning landscaping plant, is gaining popularity for its rich color patterns in leaf and cold tolerance. Leaf variegation endows ornamental kale with unique ornamental characters, and the mutants are ideal materials for exploring the formation mechanisms of variegated phenotype. Herein, we identified a novel variegated leaf kale mutant ‘JC007-2B’ with green margins and white centers. Morphological observations and physiological determinations of the green leaf stage (S1), albino stage (S2) and variegated leaf stage (S3) demonstrated that the chloroplast structure and photosynthetic pigment content in the white sectors (S3_C) of variegated leaves were abnormal. Genetic analysis revealed that a single dominant nuclear gene (BoVl) controlled the variegated leaf trait of ‘JC007-2B’, and three candidate genes for BoVl were fine-mapped to a 6.74 Kb interval on chromosome C03. Multiple sequence alignment among the green-leaf mapping parent ‘BS’, recombinant individuals, mutant parent ‘JC007-2B’ and its same originated DH line population established that the mutation sites in Bo3g002080 exhibited a complete consensus. Bo3g002080, homologous to Arabidopsis MED4, was identified as the candidate gene for BoVl. Expression analysis showed that Bo3g002080 displayed a 2158.85-fold higher expression at albino stage than that in green leaf stage. Transcriptome analysis showed that related pathways of photosynthesis and chloroplast development were significantly enriched in the white sectors, and relevant DEGs involved in these pathways were almost down-regulated. Overall, our study provides a new gene resource for cultivar breeding in ornamental kale and contributes to uncovering the molecular genetic mechanism underlying the variegated leaf formation.