Black spot disease, caused by Alternaria alternata, results in enormous losses in broccoli production. The current measures to prevent black spot disease mainly rely on seed disinfection and chemical control, but excellent disease-resistance resources are relatively scarce. In this study, we screened primers for black spot disease identification and conducted black spot disease resistance identification of 173 lines, including 70 hybrid lines and 103 inbred lines. Based on the phenotype, we have set five grades to present different symptoms of illness: high disease resistance, disease resistance, disease tolerance, susceptibility, and high susceptibility (the disease resistance gradually weakens). According to our phenotypic evaluations, 3, 55, 65, 45, and 5 lines were classified into high disease resistance, disease resistance, disease tolerance, susceptible, and high susceptibility, respectively. By comparing the proportion of resistant lines between hybrid and inbred lines, we noticed that the frequency of hybrid varieties with high disease resistance and disease resistance (28.57%) was lower than that in inbred lines (36.89%), indicating that the resistance resources have not yet been effectively utilized in hybrid broccoli breeding. Therefore, our results identified the resistance resources to black spot disease in broccoli, which lays the foundation for the exploration of disease resistance genes as well as the analysis of disease resistance mechanisms in the future.

Owing to the high anthocyanin content, broccoli varieties with purple curds have become more popular in food inventories, while the genetic mechanisms of anthocyanin biosynthesis pathways remain largely unknown. We bred a pair of near-isogenic lines (NILs), GB767 and PB767, whose curds exhibited green and purple colors, respectively, due to the purple sepals of florets. RNA sequencing and widely targeted metabolic analyses were conducted. Compared with GB767, eighteen anthocyanin biosynthesis-related genes exhibited significantly higher expressions in PB767, and in turn, the expression level of BolMYBL2.1 was attenuated. A comparison of the metabolites in the flavonoid biosynthetic pathways revealed 142 differentially accumulated metabolites, among which higher content of anthocyanins was responsible for the purple color of PB767. Interestingly, the total cyanidin contents were similar between the curds of NILs, whereas total delphinidin contents were increased by more than 170 times in purple curds, presumably due to a non-canonical F3′H/CYP75B gene, BolC02g015480.2J, with elevated expression in PB767. Furthermore, correlation analysis further confirmed that the identified nineteen DEGs were significantly correlated with seven differentially accumulated anthocyanins in PB767. Together, these results identified the metabolic factors and genes that contribute to the purplish curds, which could lay foundations for the breeding programs of purple broccoli.

Broccoli (Brassica oleracea L. var. italica) is a globally popular vegetable because of its nutrient richness, especially its glucosinolates (GSLs). The content of GSLs in different organs of broccoli varies greatly. However, few studies have focused on the differences in the GSLs biosynthesis-related genes in different organs. In this study, we selected extreme individuals from an F₂ population of broccoli and mixed them to form low and high glucoraphanin content pools of leaf, stalk and floret, respectively. Transcriptome and metabolome analyses showed that 539, 755 and 617 genes are significantly differentially expressed, and 44, 66 and 118 metabolites are significantly differentially accumulated in leaf, stalk and floret comparison groups, respectively. The combined analysis revealed that some genes such as Bo5g113720, Bo2g161100 and Bo7g09800, Bo4g018590, Bo5g021810, and Bo2g011730 showed different expression trends between low and high glucoraphanin content pools, which increased the accumulation of glucoraphanin. These genes have different expression levels in the three plant parts. Strikingly, the accumulation of glucoraphanin upregulated the expression of plant hormone signal transduction-related genes TIFY, JAR1, IAA, GH3 and SAU, and also increased the levels of tentatively identified flavonoid metabolites. Our study deepens the understanding of glucosinolate biosynthesis in different organs at the molecular level, and also provides evidence for the crosstalk between glucosinolates and flavonoids biosynthesis pathways.

Green broccoli is a very frequently consumed, nutrient-rich vegetable, whereas purple broccoli, known for its high anthocyanin content, is not so common. To investigate the anthocyanin biosynthesis in broccoli, one green broccoli and four purple broccolis were subjected to integrative analyses with metabolomics and transcriptomics. Our comprehensive examination revealed that in difference to other Brassica vegetables, the main anthocyanins accumulating in purple broccoli are delphinidin compounds, especially delphinidin-3-O-glucoside-5-O-galactoside. Transcriptomic analysis demonstrated significant variations in the expression levels of genes such as BoF3′H, BoDFR, and BoANS among the samples, suggesting the involvement of upstream transcription factors, regulating these genes. Furthermore, the integration of transcriptomic data and transient functional validation identified two MYB transcription factors, BoMYB147 and BoMYB148, as key regulators of anthocyanin biosynthesis. In conclusion, this study identifies the types of anthocyanins present in purple broccoli and enhances the understanding of the regulatory genes involved in anthocyanin synthesis. It provides genetic insights and a conceptual framework for future molecular breeding strategies directed toward enhancing the quality of purple broccoli varieties.