Genomic analyses reveal the stepwise domestication and genetic mechanism of curd biogenesis in cauliflower.

Cauliflower (Brassica oleracea L. var. botrytis) is a distinctive vegetable that supplies a nutrient-rich edible inflorescence meristem for the human diet. However, the genomic bases of its selective breeding have not been studied extensively. Herein, we present a high-quality reference genome assembly C-8 (V2) and a comprehensive genomic variation map consisting of 971 diverse accessions of cauliflower and its relatives. Genomic selection analysis and deep-mined divergences were used to explore a stepwise domestication process for cauliflower that initially evolved from broccoli (Curd-emergence and Curd-improvement), revealing that three MADS-box genes, CAULIFLOWER1 (CAL1), CAL2 and FRUITFULL (FUL2), could have essential roles during curd formation. Genome-wide association studies identified nine loci significantly associated with morphological and biological characters and demonstrated that a zinc-finger protein (BOB06G135460) positively regulates stem height in cauliflower. This study offers valuable genomic resources for better understanding the genetic bases of curd biogenesis and florescent development in crops.

Map-based cloning and functional analysis of a major quantitative trait locus, BolC.Pb9.1, controlling clubroot resistance in a wild Brassica relative (Brassica macrocarpa).

KEY MESSAGE: A causal gene BoUGT76C2, conferring clubroot resistance in wild Brassica oleracea, was identified and functionally characterized. Clubroot is a devastating soil-borne disease caused by the obligate biotrophic pathogen Plasmodiophora brassica (P. brassicae), which poses a great threat to Brassica oleracea (B. oleracea) production. Although several QTLs associated with clubroot resistance (CR) have been mapped in cultivated B. oleracea, none have been cloned in B. oleracea. Previously, we found that the wild B. oleracea B2013 showed high resistance to clubroot. In this study, we constructed populations using B2013 and broccoli line 90196. CR in B2013 is quantitatively inherited, and a major QTL, BolC.Pb9.1, was identified on C09 using QTL-seq and linkage analysis. The BolC.Pb9.1 was finely mapped to a 56 kb genomic region using F2:3 populations. From the target region, the candidate BoUGT76C2 showed nucleotide variations between the parents, and was inducible in response to P. brassicae infection. We generated BoUGT76C2 overexpression lines in the 90196 background, which showed significantly enhanced resistance to P. brassicae compared to the WT line, suggesting that BoUGT76C2 corresponds to the resistance gene BolC.Pb.9.1. This is the first report on the CR gene map-based cloning and functional analysis from wild relatives, which provides a theoretical basis to the understanding of the molecular mechanism of CR, and lays a foundation to improve the CR of cultivated B. oleracea.

Construction of an SNP fingerprinting database and population genetic analysis of 329 cauliflower cultivars.

Cauliflower is one of the most important vegetable crops grown worldwide. However, the lack of genetic diversity information and efficient molecular markers hinders efforts to improve cauliflower. This study aims to construct DNA fingerprints for 329 cauliflower cultivars based on SNP markers and the KASP system. After rigorous filtering, a total of 1662 candidate SNPs were obtained from nearly 17.9 million SNP loci. The mean values of PIC, MAF, heterozygosity and gene diversity of these SNPs were 0.389, 0.419, 0.075, and 0.506, respectively. We developed a program for in silico simulations on 153 core germplasm samples to generate ideal SNP marker sets from the candidates. Finally, 41 highly polymorphic KASP markers were selected and applied to identify 329 cauliflower cultivars, mainly collected from the public market. Furthermore, based on the KASP genotyping data, we performed phylogenetic analysis and population structure analysis of the 329 cultivars. As a result, these cultivars could be classified into three major clusters, and the classification patterns were significantly related to their curd solidity and geographical origin. Finally, fingerprints of the 329 cultivars and 2D barcodes with the genetic information of each sample were generated. The fingerprinting database developed in this study provides a practical tool for identifying the authenticity and purity of cauliflower seeds and valuable genetic information about the current cauliflower cultivars.

Genome-wide identification and expression analysis of the bHLH gene family in cauliflower (Brassica oleracea L.).

Basic helix-loop-helix (bHLH) transcription factors (TFs) are one of the largest TF families in plant species, and they play important roles in plant growth, development and stress responses. The present study systematically identified members of the cauliflower (Brassica oleracea L.) bHLH gene family based on genomic data. Analysis of bHLH family gene numbers, evolution, collinearity, gene structures and motifs indicated that cauliflower contained 256 bHLH family genes distributed on 10 chromosomes. Most of these genes have been localized in the nucleus, and they were divided into 18 subgroups which have been relatively conserved during evolution. Promoter analysis showed that most cis-acting elements were related to MeJA and ABA. Expression analysis suggested that 14 bHLH genes may be involved in the transformation of cauliflower curd from white to purple. An expression analysis of these 14 genes in FQ136 material was performed using qRT-PCR, and 9 bHLH genes (BobHLH1, 14, 58, 61, 63, 84, 231, 239 and 243) showed significantly increased or decreased expression in cauliflower from white to purple, which suggests that these 9 genes play important roles in the accumulation of anthocyanins in cauliflower. The coexpression network of these 9 genes and anthocyanin synthesis-related key genes was analyzed using weighted gene coexpression network analysis (WGCNA). In conclusion, our observations suggested that the bHLH gene family plays an important role in the accumulation of anthocyanins in cauliflower and provide an important theoretical basis for further research on the functions of the bHLH gene family and the molecular mechanism of cauliflower coloration. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01238-9.

Metabolic and RNA sequencing analysis of cauliflower curds with different types of pigmentation.

Cauliflower (Brassica oleracea var. botrytis) is a popular vegetable worldwide due to its delicious taste, high nutritional value and anti-cancer properties. Cauliflower normally produces white curds, and natural spontaneous mutations lead to the production of orange, purple or green curds. However, some white cauliflowers show uneven purple pigmentation in their curds, which seriously affects the appearance quality and economic value of this crop. The underlying mechanism is still unclear. In this study, we performed comparative transcriptional and metabolic profiling analysis of light orange, white and purplish cauliflower curds. Metabolite analysis revealed that the pigments conferring purple colouration were delphinin and cyanin. Transcriptome analysis showed that the anthocyanin metabolism-related structural genes DFR, ANS and UGT and the transcription factor genes PAP2, TT8, GL3, EGL3 and TTG1 were upregulated in purplish versus white curds. These findings shed light on the formation of purplish curds, which could facilitate the breeding of purely white or red cauliflower.

Draft genome sequence of cauliflower (Brassica oleracea L. var. botrytis) provides new insights into the C genome in Brassica species.

Cauliflower is an important variety of Brassica oleracea and is planted worldwide. Here, the high-quality genome sequence of cauliflower was reported. The assembled cauliflower genome was 584.60 Mb in size, with a contig N50 of 2.11 Mb, and contained 47,772 genes; 56.65% of the genome was composed of repetitive sequences. Among these sequences, long terminal repeats (LTRs) were the most abundant (32.71% of the genome), followed by transposable elements (TEs) (12.62%). Comparative genomic analysis confirmed that after an ancient paleohexaploidy (γ) event, cauliflower underwent two whole-genome duplication (WGD) events shared with Arabidopsis and an additional whole-genome triplication (WGT) event shared with other Brassica species. The present cultivated cauliflower diverged from the ancestral B. oleracea species ~3.0 million years ago (Mya). The speciation of cauliflower (~2.0 Mya) was later than that of B. oleracea L. var. capitata (approximately 2.6 Mya) and other Brassica species (over 2.0 Mya). Chromosome no. 03 of cauliflower shared the most syntenic blocks with the A, B, and C genomes of Brassica species and its eight other chromosomes, implying that chromosome no. 03 might be the most ancient one in the cauliflower genome, which was consistent with the chromosome being inherited from the common ancestor of Brassica species. In addition, 2,718 specific genes, 228 expanded genes, 2 contracted genes, and 1,065 positively selected genes in cauliflower were identified and functionally annotated. These findings provide new insights into the genomic diversity of Brassica species and serve as a valuable reference for molecular breeding of cauliflower.

Transcriptome and DNA methylome reveal insights into yield heterosis in the curds of broccoli (Brassica oleracea L var. italic).

BACKGROUND: Curds are the main edible organs, which exhibit remarkable yield heterosis in F1 hybrid broccoli. However, the molecular basis underlying heterosis in broccoli remains elusive. RESULTS: In the present study, transcriptome profiles revealed that the hybridization made most genes show additive expression patterns in hybrid broccoli. The differentially expressed genes including the non-additively expressed genes detected in the hybrid broccoli and its parents were mainly involved in light, hormone and hydrogen peroxide-mediated signaling pathways, responses to stresses, and regulation of floral development, which suggested that these biological processes should play crucial roles in the yield heterosis of broccoli. Among them, light and hydrogen peroxide-mediated signaling pathways represent two novel classes of regulatory processes that could function in yield or biomass heterosis of plants. Totally, 53 candidate genes closely involved in curd yield heterosis were identified. Methylome data indicated that the DNA methylation ratio of the hybrids was higher than that of their parents. However, the DNA methylation levels of most sites also displayed additive expression patterns. These sites with differential methylation levels were predominant in the intergenic regions. In most cases, the changes of DNA methylation levels in gene regions did not significantly affect their expression levels. CONCLUSIONS: The differentially expressed genes, the regulatory processes and the possible roles of DNA methylation modification in the formation of curd yield heterotic trait were discovered. These findings provided comprehensive insights into the curd yield heterosis in broccoli, and were significant for breeding high-yield broccoli varieties.

[Stable carbon isotopic composition in PM 2.1 in Nanjing Region].

Stable carbon isotopes (delta13C) in aerosol fine particles (PM2.1) collected in Nanjing Normal University representing urban area, and in Nanjing College of Chemical Technology standing for suburban industrial area, were analyzed using EA-IRMS. Besides, sources of carbonaceous contents were studied and the pollution characteristics of total carbon (TC) were evaluated. The annual average concentrations of TC in urban area and suburban industrial area were 15.94 microg.m-3 and 17.17 microg.m-3, respectively. The proportions for TC in PM2.1 were 17.18% and 16. 40% , indicating that carbonaceous pollution was more serious and the pollutants were more complex in suburban industrial area. The average delta13C for winter, spring, summer and autumn were -24. 42 per thousand +/- 1. 12 per thousand, -25. 19 per thousand +/- 1. 92% per thousand, - 25.79 per thousand +/- 0.45 per thousand and - 25.58 per thousand + 0. 65 per thousand, respectively in urban area and - 25.34 per thousand +/- 1. 18 per thousand, -25. 55 per thousand +/- 1. 50 per thousand, -25. 31 per thousand +/- 0. 55%o and -25. 38 per thousand +/- 0. 82 per thousand, respectively in suburban area. Correlation analysis and isotopic signatures of potential sources suggested that carbonaceous contents mainly came from gasoline vehicles exhaust in urban area, and might be attributed to the vehicle exhaust emissions and industrial emissions in suburban area. In addition, coal combustion,biomass burning and geological sources might have important contribution to aerosols in winter and spring. Back trajectory analysis implied that the long-range transport had considerable contribution to the carbonaceous aerosol in winter and spring. However, the major sources might be attributed to local emissions in the other two seasons.

Identification of genes differentially expressed in cauliflower associated with resistance to Xanthomonas campestris pv. campestris.

Black rot, caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc), is one of the most damaging diseases of cauliflower and other crucifers. In order to investigate the molecular resistance mechanisms and to find the genes related to black rot resistance in cauliflower, a suppression subtractive hybridization (SSH) cDNA library was constructed using resistant line C712 and its susceptible near-isogenic line C731 as tester and driver, respectively. A total of 280 clones were obtained from the library by reverse northern blotting. Sequencing analysis and homology searching showed that these clones represent 202 unique sequences. The library included many defense/disease-resistant related genes, such as plant defensin gene PDF1.2, lipid transfer protein, thioredoxin h. Gene expression profiles of 12 genes corresponding to different functional categories were monitored by real-time RT-PCR. The results showed that the expression induction of these genes in the susceptible line C712 in response to Xcc was quicker and more intense, while in C731 the reaction was delayed and limited. Our results imply that these up-regulated genes might be involved in cauliflower responses against Xcc infection. Information obtained from this study could be used to understand the molecular mechanisms of disease response in cauliflower under Xcc stress.

[Construction of genetic linkage map and localization of NBS-LRR like resistance gene analogues in cauliflower (Brassica oleracea var. botrytis)].

Nucleotide binding site (NBS) profiling, a new method was used to map resistance gene analogues (RGAs) in cauliflower (Brassica oleracea var. botrytis). This method allows amplification and the mapping of genetic markers anchored in the conserved NBS encoding domain of plant disease resistance genes. AFLP was also performed to construct the cauliflower intervarietal genetic map. The aim of constructing genetic map was to identify potential molecular markers linked to important agronomic traits that would be particularly useful for development and improving the species. Using 17 AFLP primer combinations and two degeneration primer/enzyme combinations, a total of 234 AFLP markers and 21 NBS markers were mapped in the F2 population derived from self-pollinating a single F1 plant of the cross AD White Flower x C-8. The markers were mapped in 9 of major linkage groups spanning 668.4 cM, with an average distance of 2.9 cM between adjacent mapped markers. The AFLP markers were well distributed throughout the linkage groups. The linkage groups contained from 12 to 47 loci each and the distance between two consecutive loci ranged from 0 to 14.9 cM. NBS markers were mapped on 8 of the 9 linkage groups of the genetic map. Most of these markers were organized in clusters. This result demonstrates the feasibility of the NBS-profiling method for generating NBS markers for resistance loci in cauliflower. The clustering of the markers mapped in this study adds to the evidence that most of them could be real RGAs.