Ornamental kale is a popular decorative plant. We identified a peculiar bicolor leaf double haploid line, with green margins and red centers. The development of bicolor leaves can be divided into three stages: S1, S2, and S3. To probe the reason for bicolor formation, we analyzed the anthocyanin and chlorophyll contents, detected the changes in indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellin 3 (GA3), sugar, and starch contents, and identified the differentially expressed genes (DEGs) using RNA-seq. Results showed that the bicolor leaf phenotype is gradually formed with anthocyanin degrading and chlorophyll accumulation. Anthocyanin content is lower in the green margin (S3_S) than in the red center (S3_C) part at S3. IAA content was positively correlated with anthocyanin content during the bicolor leaf development. During anthocyanin degrading from S1 to S2, cinnamate-4-hydroxylase (C4H) and transport inhibitor response 1 (TIR1) were downregulated, while lateral organ boundaries domain 39 (LBD39) was upregulated. Two peroxidases, two β-glucosidases (BGLU), LBD39, LBD37, detoxifying efflux carrier 35 (DTX35), three no apical meristem (NAC) transcription factors (TFs), and 15 WRKY DNA-binding protein (WRKY) TFs were downregulated in S3_S vs. S3_C. The bicolor phenotype was mainly linked to anthocyanin degrading and chlorophyll accumulation, and that anthocyanin degrading resulted from reduced anthocyanin biosynthesis and increased anthocyanin degradation.

Background: Anther development has been extensively studied at the transcriptional level, but a systematic analysis of full-length transcripts on a genome-wide scale has not yet been published. Here, the Pacific Biosciences (PacBio) Sequel platform and next-generation sequencing (NGS) technology were combined to generate full-length sequences and completed structures of transcripts in anthers of Chinese cabbage. Results: Using single-molecule real-time sequencing (SMRT), a total of 1,098,119 circular consensus sequences (CCSs) were generated with a mean length of 2664 bp. More than 75% of the CCSs were considered full-length non-chimeric (FLNC) reads. After error correction, 725,731 high-quality FLNC reads were estimated to carry 51,501 isoforms from 19,503 loci, consisting of 38,992 novel isoforms from known genes and 3691 novel isoforms from novel genes. Of the novel isoforms, we identified 407 long non-coding RNAs (lncRNAs) and 37,549 open reading frames (ORFs). Furthermore, a total of 453,270 alternative splicing (AS) events were identified and the majority of AS models in anther were determined to be approximate exon skipping (XSKIP) events. Of the key genes regulated during anther development, AS events were mainly identified in the genes SERK1, CALS5, NEF1, and CESA1/3. Additionally, we identified 104 fusion transcripts and 5806 genes that had alternative polyadenylation (APA). Conclusions: Our work demonstrated the transcriptome diversity and complexity of anther development in Chinese cabbage. The findings provide a basis for further genome annotation and transcriptome research in Chinese cabbage.

Purple non-heading Chinese cabbage is one of the most popular vegetables, and is rich in various health-beneficial anthocyanins. Research related to genes associated with anthocyanin biosynthesis in non-heading Chinese cabbage is important. This study performed integrative transcriptome and metabolome analysis in the purple non-heading Chinese cabbage wild type (WT) and its green mutant to elucidate the formation of purple leaves. The anthocyanin level was higher in purple than in green plants, while the contents of chlorophyll and carotenoid were higher in the green mutant than in the purple WT. Twenty-five anthocyanins were identified in purple and green cultivars; eleven anthocyanin metabolites were identified specifically in the purple plants. RNA-seq analysis indicated that 27 anthocyanin biosynthetic genes and 83 transcription factors were significantly differentially expressed between the WT and its mutant, most of them with higher expression in the purple than green non-heading Chinese cabbage. Transcriptome and metabolome analyses showed that UGT75C1 catalyzing the formation of pelargonidin-3,5-O-diglucoside and cyanidin-3,5-O-diglucoside may play a critical role in purple leaf formation in non-heading Chinese cabbage. Therefore, these results provide crucial information for elucidating the formation of purple leaves in non-heading Chinese cabbage.

Mutagenesis is an important tool for breeding and genomic research. In this study, the germinated seeds and isolated microspores of a double haploid line ‘FT’ were treated with EMS, respectively, with the aim of comparing the effects of the two approaches on generating mutants in Chinese cabbage. For microspore EMS mutagenesis, the isolated microspores were treated with 0.12% EMS for 20 min, a total of 1268 plantlets were obtained, and 15 M₁ mutants were screened with a mutation frequency of 1.2%. For seed EMS mutagenesis, 7800 germinated seeds were treated with 0.8% EMS for 12 h, and a total of 701 M₂ mutants were screened, with a mutation frequency of 18.78%. In total, 716 mutants with heritable morphological variation including leaf color, leaf shape, leafy head, bolting, and fertility, were obtained from the EMS mutagenesis experiments. Homozygous mutant plants could be screened from M₁ lines by microspore mutagenesis, and M₂ lines by seed mutagenesis. The mutation frequency was higher in seed mutagenesis than in microspore mutagenesis. Based on these results, we propose that seed EMS mutagenesis is more suitable to generate a large-scale mutant library, and the microspore EMS mutagenesis is conducive to rapidly obtaining homozygous mutants.