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Cabbage (Brassica oleracea) is one of the most important vegetable crops worldwide. qRT-PCR is a sensitive technique for gene expression studies and choosing the appropriate reference gene is essential to obtain reliable results. In the present work, 22 candidate reference genes were evaluated under various experimental conditions, including NaCl, drought stress treatment, temperature treatments (cold and heat) and a set of hormones stress (6-BA, NAA, and ABA) treatments, across a range of tissue types and cultivars. Gene expression data taken from 45 cabbage samples was analyzed using two algorithms, geNorm and NormFinder. Suitable combinations of reference genes for qRT-PCR normalization should be applied according to different experimental conditions. In this study, the genes EF1a, GAPC2 and SAND were verified as the suitable reference genes across all tested samples. Additionally, each experimental condition had a unique set of reference genes best suited to samples within the particular condition. To validate the suitability of the candidate reference genes, the gene expression of WSD1, a gene that may be involved in biosynthesis pathway of wax esters in cabbage, was measured across all 45 samples and normalized using the three best reference gene candidates. WSD1 displayed variation in gene expression across different tissues and cultivars, and exhibited diverse up- or down- regulated expression patterns under various treatments, which indicate that BoWSD1 may play an important role in the response to abiotic stresses in cabbage. Our results provide the foundation for gene expression analysis in Brassica oleracea and other species of Brassica vegetables.

Alternative splicing (AS) events were identified and verified in cabbage by comparative transcriptome analysis. The corresponding markers were developed and the germplasm resources were identified. Alternative splicing (AS) is a central regulatory mechanism that greatly contributes to plant gene expression and transcriptome diversity. A large body of evidence has shown that AS complexity is relevant for plant development, evolution, complexity, and adaptation. Both insertion/deletion (InDel) and single nucleotide polymorphism (SNP) are typically co-dominant inheritance markers and have abundant polymorphisms. These have been widely used for marker-assisted selection, genetic mapping, and germplasm identification in plants. However, little is known about the molecular mechanisms underlying AS events and the development of markers including SNP and InDel from the cabbage transcriptome. In this study, three cabbage transcriptome datasets were collected and aligned to the cabbage reference genome to analyze AS events and marker development. 31,524 AS events were identified from three cabbage genotypes, accounting for 20.8% of the total cabbage genes. Alternative 3' splice site donor (A3SS) was the most frequent type of the four main AS events in cabbage. 70,475 InDels and 706,269 SNPs were identified with average frequencies of 1 InDel/6.9 kb and 1 SNP/0.7 kb, respectively. 71,942 potential SSRs were identified in 53,129 assembled unigenes with a density of 1 SSR/6.8 kb. The ratio of SNPs with synonymous/non-synonymous mutations was 1:0.65. 142 InDels and 36 SNPs were randomly selected and validated via Sanger sequencing and polymorphism was found among 66.2% of the InDels and 78.6% of the SNPs. Furthermore, 35 informative InDel markers were successfully used for genetic diversity analysis on 36 cabbage accessions. These results facilitate understanding of the molecular regulation mechanism underlying AS events in cabbage. They also provide molecular marker resource data for genetic mapping construction and germplasm identification, and facilitate the genetic improvement of cabbage via breeding.

Cabbage (Brassica oleracea L.var. capitata), an important crop of the Brassicaceae family, is a highly valued source of anti-cancer compounds, vitamin C and high-quality protein. Glossiness of the leaf surface is an important character that has an important influence on the quality of commercial products. In this study, two heart-shaped glossy cabbage mutants were examined to determine their characteristics and potential practical applications. Firstly, mutant-type (MT) plants showed a shiny and attractive appearance, and the total wax load in MT leaves was significantly less than that of wild-type (WT) leaves, when assessed by scanning electron micrographs. Then, the results of χ2 tests on the separated proportion proved that the glossy wax-less trait is controlled by a single recessive gene. Unexpectedly, the sulforaphane content, which has been identified as a naturally potent anticarcinogen, showed an increasing trend in MT plants than the corresponding WT plants. Lastly, our work also showed that some genes that are involved in wax biosynthesis and export pathways are down-regulated in MT leaves, which could cause the glossy phenotype of MT plants. These analyses will shed light on the benefits of incorporating the two mutants and other glossy mutants into future breeding programs to develop varieties with morphological markers linked to desirable traits.

Cabbage (Brassica oleracea var. capitata) is one of the most important cruciferous leafy vegetable crops and is widely cultivated all over the world. Its yield and quality are often affected by diseases such as cabbage black rot. 2R is a cabbage line that is newly resistant to black rot, which was created by radiation mutagenesis and backcross transfer. However, the underlying molecular bases and mechanisms of early-phase response of different resistant cabbage lines against black rot infections remain unknown. Here, we completed a comprehensive transcriptome profile analysis between resistant (2R) and susceptible (2T) cabbage lines after black rot inoculations. The results showed that the typical V-shaped lesions were found in inoculated plants after 15 days, and the symptoms in the susceptible cabbage lines (2T) were significant severe than that of the resistant 2R line. A total of 10,030 differentially expressed genes (DEGs) were identified, of which 384 DEGs were found to overlap in resistant and susceptible cabbage lines after black rot infections, suggesting those DEGs may play more important roles in cabbage early responses to black rot infections. We ranked the expression levels of DEGs among the four comparison sets of resistant and susceptible cabbage lines and, interestingly, found the top ten differential expression genes contained NBS-LRR genes, protein kinase genes and expansin genes. These findings provide a comprehensive differential transcriptome profile between resistant and susceptible cabbage lines and indicate some genes play key roles in the regulation of early response to black rot infections, which will help to understand the molecular resistance of cabbage against these infections. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02256-8) contains supplementary material, which is available to authorized users.

As a tool in genetic engineering, isolated microspore cultures have the remarkable quality to produce doubled haploids for plant breeding and gene mapping. Protocols were developed for the induction of microspore-derived embryos and generation of doubled haploid plants from isolated microspores of Brussels sprouts. A cytological analysis showed that two modes of Brussels sprouts microspore embryogenesis exist: a direct route via embryos which was the major developmental pathway, and an indirect route via calli. Cold pretreatment (4 °C) and aminoethoxyvinylglycine addition were tested to determine whether and how they could improve embryogenesis. Cold pretreatment improved the viability of microspores, and stimulated to produce embryos directly. Similarly, aminoethoxyvinylglycine had a positive effect on the number of embryos produced via improving multinucleate structures to further develop. The combination of cold pretreatment for 24 h and 1 μM aminoethoxyvinylglycine addition significantly enhanced microspore embryogenesis efficiency, especially with low responsive genotype ‘1076’ for which it was increased by about 82-fold. Subsequently, the use of solid B5 medium with 1% agarose and 2% sucrose (w/v) achieved an efficient rate of plant regeneration, and more than 67% of regenerated plants were spontaneous diploids.

SUMMARYMicrospore embryogenesis in plants is a process by which microspores can switch their developmental pathway from gametophytic to embryogenic via specific inductive stress treatments, which results in the formation of haploid embryos. However, only a small portion of the cultured cells can undergo the entire process to produce embryos, and most die at different developmental stages during culture. The objective of this study was to improve the induction of embryogenesis in an isolated microspore culture (IMC) of white cabbage (Brassica oleracea L. var. capitata) by focussing on the regulation of dead microspores. The results indicated that microspores died rapidly after 24 h at a heat shock temperature of 32.5°C, and that 80–90% were dead after 3 d in culture. Cell purification performed after 3 d in culture, eliminated most of the dead microspores and had a beneficial effect on embryo yield. Similarly, ascorbic acid (AsA) had a positive effect on the number of embryos produced by decreasing the mor...

Androgenesis is an efficient method of producing doubled haploids (DHs) in plant breeding programs. DH plants derived from the F1 (broccoli × white-headed cabbage) are valuable for accelerating the transfer of excellent genes and increasing biodiversity of species via isolated microspore culture (IMC). Therefore, this study investigated the developmental process of embryogenesis and studied the conditions needed for effective induction of embryogenesis in IMC of broccoli × white-headed cabbage hybrids. The results indicated that the B pathway was the predominant route of androgenic induction in broccoli × white-headed cabbage, which was recognized as a principal route in the formation of haploid embryos. In addition, small amounts of the A–G, A–VG and C pathways were observed. Based on this observation, cold pretreatment (4 °C) and mannitol in a B5 washing medium was tested to determine whether and how they could improve embryogenesis in IMC. Cold pretreatment enhanced microspore embryogenesis by increasing the symmetric division of cells. Similarly, mannitol had a positive effect on the number of embryos produced by affecting the number of microspores that reached the multinucleate stage. The combination of cold pretreatment (4 °C) for 1 day and 0.4 M mannitol had a synergistic effect on microspore development. Moreover, the combined treatment resulted in a higher frequency of plant regeneration and diploid frequency in the regenerated population. This report helps to elucidate in vitro embryo development in seeds plants and IMC systems by providing insight into some similarities and differences between Brassica species.