**1. Introduction**

Broccoli (*Brassica oleracea* var. *italic*) is a popular vegetable of *B. oleracea* that differs from most Brassica species, including Chinese cabbage, turnip, cabbage, broccoli, cauliflower, and oilseed rape. Most varieties of broccoli are domesticated from crop wild relatives in the Mediterranean Basin and grow as annuals, producing a large head with florets, buds, leaves, stalks, and stems for consumption. Both of broccoli and cauliflower cultivar groups are members of the CC genome *B. oleracea* (2n = 18) coenospecies. High-quality reference genomes of cauliflower have been reported, and the assembled cauliflower genome was 584.60 Mb in size [1]. As a great food source of essential vitamins and minerals, broccoli contains antioxidant phytochemicals, such as glucoraphanin, which may help prevent cancer [2]. Purple broccoli attracts increasing attention as a functional food, owing to its pleasing appearance and high level of health-promoting effects [3]. The purple coloration has been identified as one of the signs of anthocyanin accumulation [4].

Anthocyanins belong to a class of flavonoid compounds that impart color to plants and play an important role in plant protection against a variety of biotic and abiotic

**Citation:** Liu, C.; Yao, X.; Li, G.; Huang, L.; Wu, X.; Xie, Z. Identification of Major Loci and Candidate Genes for Anthocyanin Biosynthesis in Broccoli Using QTL-Seq. *Horticulturae* **2021**, *7*, 246. https://doi.org/10.3390/ horticulturae7080246

Academic Editor: Yuyang Zhang

Received: 6 June 2021 Accepted: 9 August 2021 Published: 13 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

stresses [5–7]. Anthocyanin biosynthetic pathway genes have been extensively characterized in Arabidopsis (*Arabidopsis thaliana*), maize (*Zea mays*), tomato (*Solanum lycopersicum*), eggplant (*Solanum melongena*), and other plant species [8–11]. The induction of structural genes and transcription factors is considered to be an important mechanism for the regulation of anthocyanin biosynthesis in the Brassica species [12–14]. A cauliflower purple mutation exhibited a tissue-specific pattern of anthocyanin overproduction [15]. Due to the insertion of Harbinger transposon, the upregulation of *BoMYB2* specifically activates BobHLHs and some downstream anthocyanin structural genes to generate the ectopic accumulation of anthocyanin. Similarly, the accumulation of anthocyanin is caused by the activation of *TT8* and *MYB2* genes in red cabbages [14]; of *BrMYB2* and downstream genes, such as *BrTT8*, *BrF3'H*, *BrDFR1*, *BrANS1*, *BrUGTs*, *BrATs*, and *BrGSTs*, under the control of *BrMYB2* in a purple head of Chinese cabbage cultivar 11S91 [16]. In purple cabbages, deleting or replacing nucleotides in the exon of *BoMYBL2-1* is solely responsible for the purple coloration [17]. In addition, temperature and light are the major environmental factors that affect anthocyanin accumulation. In purple head Chinese cabbage, *BrMYB2* and *BrTT8* activated anthocyanin structural genes after low temperature induction [18]. Elevated temperature could suppress anthocyanin accumulation via COP1-HY5 signaling, and *MYBL2* down-regulation partially modulated the high-temperature-associated suppression of anthocyanin production [19]. Some efforts have also led to the identification of candidate genes that regulate the coloration of the Brassica species. In broccoli, three QTLs have been mapped to the purple sepal trait of the flower head on chromosome C01 [20]. In ornamental kale, the genes that individually conferred pink and purple leaf colorations have been mapped to chromosomes C3 [21] and C9 [22], respectively. In Zicaitai, an important locus on chromosome 7 highly controlled the stalk color trait, which was significantly correlated with *bHLH49* expression in the F2 population [23]. In purple-heading Chinese cabbage, the purple inner leaf trait was solely regulated by the dominant gene *BrPur*, which was mapped to A07, between SSR markers A710 and A714, with a genetic distance of 3.1 and 3.5 cM, respectively [24].

The majority of agronomic traits are controlled by QTLs, which are critical for improvement in crop breeding, through marker-assisted selection (MAS). The classical method of QTL mapping is linkage mapping, which is laborious and requires a great amount of time. Nextgeneration sequencing (NGS) has become the new strategy for establishing associations between agronomic traits and biomarkers or genes. It has been demonstrated that the QTLseq method, which combines bulk segregant analysis (BSA) with NGS, is an effective tool for mapping and isolating QTLs [25]. QTL-seq is performed with two groups of individual plants, with a contrasting phenotype on a trait of interest, from segregating population-either F2 recombinant inbred lines, double haploid, or backcross populations. Through high-throughput SNP (Hi-SNP) technology, the genotype analysis of two mixed pools identifies the genomic position of the polymorphic molecular markers, and the major QTL region with significant segregation of genotypes is identified. Hi-SNP is a technique for large-scale SNP genotyping, based on multiplex-PCR, combined with the next generation sequencing and bioinformatics tools. Amplicon sequencing, combined multiplex-PCR and NGS with higher depth and low false discovery rate (and was more accurate than the whole-genome resequencing (WGS)), has been used for known SNPs genotyping in diploid but was not reported in allopolyploid crops. QTL-seq used for SNP genotyping had many advantages, such as simple primer design, single short reads sequences, high-throughput, high-depth sequencing, and easy to automate and process, e.g., recently, the QTL-seq method has been utilized for mapping QTLs related to resistance to rice blast disease and seedling vigor [26], leaf spot resistance in peanuts [27,28], heat tolerance in broccoli [29], resistance to Fusarium oxysporum f. sp. niveum race 1 in watermelon [30], cucumber early flowering [31], and tomato fruit weight and lobule amounts [32].

Mutant analyses, leading to purple or red organs, has been extensively studied in flowers, fruits, and model plants [15]. Although there is much research on the underlying mechanisms of anthocyanin biosynthesis in cauliflower [15], cabbage [17], kohlrabi [33], kale [22], and

Chinese kale [34], only few have focused on broccoli [20]. The purple broccoli mutant represents an interesting mutation, in which the accumulation of anthocyanins in flower buds causes the mutant heads to exhibit purple coloration. The broccoli purple mutation was found to be controlled by QTLs. We designate the symbol *Pur* for the Purple allele.

Through QTL-seq, this work aimed to identify QTLs involved in anthocyanin biosynthesis in the F2 population, which was obtained by crossing the purple broccoli line BT 126 with SN 60 (a cultivar with green heads).
