Molecular Evaluation of the Effects of FLC Homologs and Coordinating Regulators on the Flowering Responses to Vernalization in Cabbage (Brassica oleracea var. capitata) Genotypes.

The flowering loci of cabbage must be understood to boost their productivity. In this study, to clarify the flowering mechanisms of cabbage, we examined the three flowering repressors BoFLC1, 2 and 3, and the flowering regulators BoGI, BoCOOLAIR, and BoVIN3 of early (CAB1), middle (CAB3), and late (CAB5) flowering cabbage genotypes. Analysis of allele-specifically amplified genomic DNA and various sequence alignments demonstrated that maximal insertions and deletions influenced cabbage flowering behavior, notably in CAB3 and CAB5. Phylogenetic studies showed that BoFLC1, 2, and 3 in the CAB1, 3, and 5 genotypes had the highest homologies to other Brassica species, with CAB3 and 5 the most similar. Although CAB3 and CAB5 have comparable genetic patterns, flowering repressors and flowering regulators were investigated individually with and without vernalization to determine their minor flowering differences. The expression investigation revealed that vernalized CAB5 downregulated all BoFLC genes compared to CAB3 and, in contrast, CAB3 exhibited upregulated BoCOOLAIR. We hypothesized that the CAB3 BoFLC locus' additional insertions may have led to BoCOOLAIR overexpression and BoFLC downregulation. This study sheds light on cabbage genotypes-particularly those of CAB1 and CAB5-and suggests that structural variations in BoFLC2 and 3 bind flowering regulators, such as COOLAIR, which may affect cabbage flowering time.

Genotyping-by-sequencing map permits identification of clubroot resistance QTLs and revision of the reference genome assembly in cabbage (Brassica oleracea L.).

Clubroot is a devastating disease caused by Plasmodiophora brassicae and results in severe losses of yield and quality in Brassica crops. Many clubroot resistance genes and markers are available in Brassica rapa but less is known in Brassica oleracea. Here, we applied the genotyping-by-sequencing (GBS) technique to construct a high-resolution genetic map and identify clubroot resistance (CR) genes. A total of 43,821 SNPs were identified using GBS data for two parental lines, one resistant and one susceptible lines to clubroot, and 18,187 of them showed >5× coverage in the GBS data. Among those, 4,103 were credibly genotyped for all 78 F2 individual plants. These markers were clustered into nine linkage groups spanning 879.9 cM with an average interval of 1.15 cM. Quantitative trait loci (QTLs) survey based on three rounds of clubroot resistance tests using F2 : 3 progenies revealed two and single major QTLs for Race 2 and Race 9 of P. brassicae, respectively. The QTLs show similar locations to the previously reported CR loci for Race 4 in B. oleracea but are in different positions from any of the CR loci found in B. rapa. We utilized two reference genome sequences in this study. The high-resolution genetic map developed herein allowed us to reposition 37 and 2 misanchored scaffolds in the 02-12 and TO1000DH genome sequences, respectively. Our data also support additional positioning of two unanchored 3.3 Mb scaffolds into the 02-12 genome sequence.

Genome-wide SNP identification and QTL mapping for black rot resistance in cabbage.

BACKGROUND: Black rot is a destructive bacterial disease causing large yield and quality losses in Brassica oleracea. To detect quantitative trait loci (QTL) for black rot resistance, we performed whole-genome resequencing of two cabbage parental lines and genome-wide SNP identification using the recently published B. oleracea genome sequences as reference. RESULTS: Approximately 11.5 Gb of sequencing data was produced from each parental line. Reference genome-guided mapping and SNP calling revealed 674,521 SNPs between the two cabbage lines, with an average of one SNP per 662.5 bp. Among 167 dCAPS markers derived from candidate SNPs, 117 (70.1%) were validated as bona fide SNPs showing polymorphism between the parental lines. We then improved the resolution of a previous genetic map by adding 103 markers including 87 SNP-based dCAPS markers. The new map composed of 368 markers and covers 1467.3 cM with an average interval of 3.88 cM between adjacent markers. We evaluated black rot resistance in the mapping population in three independent inoculation tests using F2:3 progenies and identified one major QTL and three minor QTLs. CONCLUSION: We report successful utilization of whole-genome resequencing for large-scale SNP identification and development of molecular markers for genetic map construction. In addition, we identified novel QTLs for black rot resistance. The high-density genetic map will promote QTL analysis for other important agricultural traits and marker-assisted breeding of B. oleracea.

Transcriptome sequencing of two parental lines of cabbage (Brassica oleracea L. var. capitata L.) and construction of an EST-based genetic map.

BACKGROUND: Expressed sequence tag (EST)-based markers are preferred because they reflect transcribed portions of the genome. We report the development of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers derived from transcriptome sequences in cabbage, and their utility for map construction. RESULTS: Transcriptome sequences were obtained from two cabbage parental lines, C1184 and C1234, which are susceptible and resistant to black rot disease, respectively, using the 454 platform. A total of 92,255 and 127,522 reads were generated and clustered into 34,688 and 40,947 unigenes, respectively. We identified 2,405 SSR motifs from the unigenes of the black rot-resistant parent C1234. Trinucleotide motifs were the most abundant (66.15%) among the repeat motifs. In addition, 1,167 SNPs were detected between the two parental lines. A total of 937 EST-based SSR and 97 SNP-based dCAPS markers were designed and used for detection of polymorphism between parents. Using an F2 population, we built a genetic map comprising 265 loci, and consisting of 98 EST-based SSRs, 21 SNP-based dCAPS, 55 IBP markers derived from B. rapa genome sequence and 91 public SSRs, distributed on nine linkage groups spanning a total of 1,331.88 cM with an average distance of 5.03 cM between adjacent loci. The parental lines used in this study are elite breeding lines with little genetic diversity; therefore, the markers that mapped in our genetic map will have broad spectrum utility. CONCLUSIONS: This genetic map provides additional genetic information to the existing B. oleracea map. Moreover, the new set of EST-based SSR and dCAPS markers developed herein is a valuable resource for genetic studies and will facilitate cabbage breeding. Additionally, this study demonstrates the usefulness of NGS transcriptomes for the development of genetic maps even with little genetic diversity in the mapping population.