Comparative transcriptomics analysis uncovers alternative splicing events and molecular markers in cabbage (Brassica oleracea L.).

MAIN CONCLUSION: 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.

Construction and analysis of a high-density genetic linkage map in cabbage (Brassica oleracea L. var. capitata).

BACKGROUND: Brassica oleracea encompass a family of vegetables and cabbage that are among the most widely cultivated crops. In 2009, the B. oleracea Genome Sequencing Project was launched using next generation sequencing technology. None of the available maps were detailed enough to anchor the sequence scaffolds for the Genome Sequencing Project. This report describes the development of a large number of SSR and SNP markers from the whole genome shotgun sequence data of B. oleracea, and the construction of a high-density genetic linkage map using a double haploid mapping population. RESULTS: The B. oleracea high-density genetic linkage map that was constructed includes 1,227 markers in nine linkage groups spanning a total of 1197.9 cM with an average of 0.98 cM between adjacent loci. There were 602 SSR markers and 625 SNP markers on the map. The chromosome with the highest number of markers (186) was C03, and the chromosome with smallest number of markers (99) was C09. CONCLUSIONS: This first high-density map allowed the assembled scaffolds to be anchored to pseudochromosomes. The map also provides useful information for positional cloning, molecular breeding, and integration of information of genes and traits in B. oleracea. All the markers on the map will be transferable and could be used for the construction of other genetic maps.

Genome-Wide Analysis of Simple Sequence Repeats in Cabbage (Brassica oleracea L.).

Cabbage (Brassica oleracea L. var. capitata) accounts for a critical vegetable crop belonging to Brassicaceae family, and it has been extensively planted worldwide. Simple sequence repeats (SSRs), the markers with high polymorphism and co-dominance degrees, offer a crucial genetic research resource. The current work identified totally 64,546 perfect and 93,724 imperfect SSR motifs in the genome of the cabbage 'TO1000.' Then, we divided SSRs based on the respective overall length and repeat number into different linkage groups. Later, we characterized cabbage genomes from the perspectives of motif length, motif-type classified and SSR level, and compared them across cruciferous genomes. Furthermore, a large set of 64,546 primer pairs were successfully identified, which generated altogether 1,113 SSR primers, including 916 (82.3%) exhibiting repeated and stable amplification. In addition, there were 32 informative SSR markers screened, which might decide 32 cabbage genotypes for their genetic diversity, with level of polymorphism information of 0.14-0.88. Cultivars were efficiently identified by the new strategy designating manual diagram for identifying cultivars. Lastly, 32 cabbage accessions were clearly separately by five Bol-SSR markers. Besides, we verified whether such SSRs were available and transferable in 10 Brassicaceae relatives. Based on the above findings, those genomic SSR markers identified in the present work may facilitate cabbage research, which lay a certain foundation for further gene tagging and genetic linkage analyses, like marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Transcriptomic analysis of resistant and susceptible cabbage lines reveals differential expressions and candidate genes involved in cabbage early responses to black rot.

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.

Transcriptome Sequencing and Metabolism Analysis Reveals the role of Cyanidin Metabolism in Dark-red Onion (Allium cepa L.) Bulbs.

Onion (Allium cepa L.) is an important bulbous vegetable crop that possesses important properties related to health as well as extraordinary colors. Naturally white onion bulbs were used in this study to reveal the complex metabolic mechanisms that underlie phenotypic traits, especially bulb pigmentation. Six libraries (three dark-red and three white) were constructed and analyzed to elucidate differences in cyanidin (Cy) metabolism between dark-red and white onion bulbs. Libraries were screened using RNA-sequencing (RNA-seq) to reveal the differentially expressed genes (DEGs) involved in anthocyanin biosynthesis at the transcriptional level. Comparison with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database shows that a total of 27 unigenes participate in onion anthocyanin biosynthesis and 16 DEGs perform critical roles in flavonoid biosynthesis. Expression patterns of color-related flavonoid compounds associated with the onion anthocyanin biosynthesis pathway (ABP) show that flavonoid 3',5'-hydroxylase (F3'5'H) and dihydroflavonol 4-reductase (DFR) genes play crucial roles in the biosynthesis of dark-red bulbs, the expression levels of flavonol synthase (FLS) and DFR genes may act to block blue pigmentation, and the loss of Cy from white onion bulbs might explain multibranching in the synthesis of this compound. Positive variation in the F3'5'H/F3'H ratio also affects onion bulb color diversity. The transcriptome presented here provides a basis for future onion molecular breeding based on variations in the diversity of ornamental plant pigmentation.