Genome-wide analysis of flavonoid biosynthetic genes in Musaceae ( <i>Ensete</i> , <i>Musella</i> , and <i>Musa</i> species) reveals amplification of flavonoid 3ʹ,5ʹ-hydroxylase

Flavonoids in Musaceae are involved in pigmentation and stress responses, including cold resistance, and are a component of the healthy human diet. Identification and analysis of the sequence and copy number of flavonoid biosynthetic genes are valuable for understanding the nature and diversity of flavonoid evolution in Musaceae species. In this study, we identified 71-80 flavonoid biosynthetic genes in chromosome-scale genome sequence assemblies of Musaceae, including those of <i>Ensete glaucum</i>, <i>Musella lasiocarpa</i>, <i>Musa beccarii</i>, <i>M. acuminata</i>, <i>M. balbisiana</i> and <i>M. schizocarpa,</i> checking annotations with BLAST and determining the presence of conserved domains. The number of genes increased through segmental duplication and tandem duplication. Orthologues of both structural and regulatory genes in the flavonoid biosynthetic pathway are highly conserved across Musaceae. The flavonoid 3',5'-hydroxylase gene <i>F3'5'H</i> was amplified in Musaceae and ginger compared with grasses (rice, Brachypodium, <i>Avena longiglumis,</i> and sorghum). One group of genes from this gene family amplified near the centromere of chromosome 2 in the <i>x</i> = 11 Musaceae species. Flavonoid biosynthetic genes displayed few consistent responses in the yellow and red bracts of <i>Musella lasiocarpa</i> when subjected to low temperatures. The expression levels of <i>MlDFR2/3</i> (dihydroflavonol reductase) increased while <i>MlLAR</i> (leucoanthocyanidin reductase) was reduced by half. Overall, the results establish the range of diversity in both sequence and copy number of flavonoid biosynthetic genes during evolution of Musaceae. The combination of allelic variants of genes, changes in their copy numbers, and variation in transcription factors with the modulation of expression under cold treatments and between genotypes with contrasting bract-colours suggests the variation may be exploited in plant breeding programmes, particularly for improvement of stress-resistance in the banana crop.