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. 2022 Apr 1:13:855707.
doi: 10.3389/fpls.2022.855707. eCollection 2022.

Characterisation of the Introgression of Brassica villosa Genome Into Broccoli to Enhance Methionine-Derived Glucosinolates and Associated Health Benefits

Mikhaela Neequaye  1   2 , Burkhard Steuernagel  2 , Shikha Saha  1 , Martin Trick  2 , Perla Troncoso-Rey  1 , Frans van den Bosch  3 , Maria H Traka  1 , Lars Østergaard  2 , Richard Mithen  1   4
Affiliations

Characterisation of the Introgression of Brassica villosa Genome Into Broccoli to Enhance Methionine-Derived Glucosinolates and Associated Health Benefits

Mikhaela Neequaye et al. Front Plant Sci. .

Abstract

Broccoli cultivars that have enhanced accumulation of methionine-derived glucosinolates have been developed through the introgression of a novel allele of the MYB28 transcription factor from the wild species Brassica villosa. Through a novel k-mer approach, we characterised the extent of the introgression of unique B. villosa genome sequences into high glucosinolate broccoli genotypes. RNAseq analyses indicated that the introgression of the B. villosa MYB28 C2 allele resulted in the enhanced expression of the MYB28 transcription factor, and modified expression of genes associated with sulphate absorption and reduction, and methionine and glucosinolate biosynthesis when compared to standard broccoli. A adenine-thymine (AT) short tandem repeat (STR) was identified within the 5' untranslated region (UTR) B. villosa MYB28 allele that was absent from two divergent cultivated forms of Brassica oleracea, which may underpin the enhanced expression of B. villosa MYB28.

Keywords: Brassica oleracea; MYB28; broccoli; glucoraphanin; introgression; sulphur metabolism; transcription factor; transcriptome.

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Conflict of interest statement

The broccoli with elevated glucoraphanin is the subject of patents filed by Plant Bioscience Limited (PBL), the technology transfer company of the John Innes Centre. RM and MHT are inventors named on these patents. FB was employed by Bayer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
(A) Aliphatic (methionine-derived) glucosinolates in the standard broccoli F1 hybrid Ironman and in three high glucoraphanin broccoli genotypes, 1199, 1086, and high-glucoraphanin (HG). Ironman has significantly fewer glucosinolates than each of the three high glucoraphanin genotypes (***p ≤ 0.0001; Tukey’s multiple comparison test details in Supplementary Table 3). (B) Partitioning of sulphur within the major sulphur-containing metabolites in Ironman, 1199, 1086, and HG. There are no significant differences (p < 0.05) in the total sulphur content or any specific metabolite apart from aliphatic glucosinolates as shown in panel (A).
FIGURE 2
FIGURE 2
Signals for potential introgression traces from Brassica villosa Introgression from k-mer analyses. In the 1086 F1 hybrid, each black horizontal line in the plot marks a contig of a draft-assembled high-glucoraphanin genome. The height of each line is according to the fraction of k-mers in a contig that is unique to B. villosa.
FIGURE 3
FIGURE 3
(A) Relative expression (mean and se) of genes from RNAseq analyses in leaves from field-grown Ironman (IM) and high-glucoraphanin (HG) Inbred (HG). (A) MYB transcription factors. There is a significantly greater expression of MYB28 C2 in HG Inbred than IM (p < 0.001). Other MYB28 C7, MYB 28 C9, and MYB29 C3 genes do not differ significantly in expression between HG and IM (p > 0.05). For information, gene expression of MYB28 genes is also included from a single leaf of glasshouse grown Brassica villosa. (B) Sulphate reduction. There was significantly greater expression of sulphate reduction genes in HG Inbred compared to IM (***p < 0.01; **p < 0.05) with the exception of SIR (p > 0.05). (C) Methionine metabolism. Expression of MTO1 was greater in HG Inbred compared to IM (***p < 0.01). METAP2 and HMT3 did not differ significantly between HG Inbred and Ironman. (D) Glucosinolate biosynthesis. There was significantly greater expression of all glucosinolate biosynthesis genes in HG Inbred compared to IM (***p < 0.01).
FIGURE 4
FIGURE 4
Part of the genomic sequence of the MYB28 C2 allele from the standard broccoli Ironman, Brassica villosa, and the high glucoraphanin broccoli genotypes 1086 and high-glucoraphanin (HG). The start ATG codon is highlighted in green. Introns are highlighted in blue. Red boxes are polymorphisms that differentiate the MYB28 allele in B. villosa from that in standard broccoli, including an AT microsatellite upstream of the start codon. The green box is the R2R3 DNA-binding domain. * indicates identical base in the four sequenced genomes.
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