Genome-wide analysis of flavonoid biosynthetic genes in Musaceae (Ensete, Musella, and Musa species) reveals amplification of flavonoid 3’,5’-hydroxylase

Flavonoids in Musaceae are involved in pigmentation and stress responses, including pathogen defense and UV protection, 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 to 80 flavonoid biosynthetic genes in chromosome-scale genome sequence assemblies of Musaceae, including those of Ensete glaucum , Musella lasiocarpa , Musa beccarii , M. acuminata , M. balbisiana , and M. schizocarpa, 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 F3’5’Hs was amplified in Musaceae and ginger compared with grasses (rice, Brachypodium, Avena longiglumis, and sorghum). One group of genes from this gene family amplified near the centromere of chromosome 2 in the x = 11 Musaceae species. Flavonoid biosynthetic genes displayed few consistent responses in the yellow and red bracts of Musella lasiocarpa when subjected to low temperatures. The expression levels of MlDFR2/3 increased while MlLAR was reduced by half. These 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 bract-colours suggests the variation may be exploited in plant breeding programmes, particularly for improvement of stress-resistance in the banana crop. ### Competing Interest Statement The authors have declared no competing interest. * 4CL : 4 coumarate CoA : ligase ANR : anthocyanidin reductase ANS/LDOX : anthocyanidin synthase/leucoanthocyanidin dioxygenase C4H : cinnamic acid 4-hydroxylase CHI : chalcone isomerase CHS : chalcone synthase DFR : dihydroflavonol reductase EBG : early biosynthetic gene F3’5’H : flavanone 3’5’-hydroxylase F3’H : flavanone 3’-hydroxylase F3H : flavanone 3-hydroxylase FBG : flavonoid biosynthetic gene FLS : flavonol synthase FNS : flavone synthase GPP : general phenylpropanoid pathway IFS : isoflavone synthase Ka : non-synonymous Ks : synonymous LAR : leucoanthocyanidin reductase LBG : late biosynthetic gene NJ : neighbor joining PAL : phenylalanine ammonia lyase SMART : simple modular architecture research tool TF : transcription factor TMM : trimmed mean of M-values TPM : transcripts per million UGP/UFGT : UDP-flavonoid glucosyl transferase