The genomic and bulked segregant analysis of Curcuma alismatifolia revealed its diverse bract pigmentation

Abstract

Compared with most flowers where the showy part comprises specialized leaves (petals) directly subtending the reproductive structures, most Zingiberaceae species produce showy "flowers" through modifications of leaves (bracts) subtending the true flowers throughout an inflorescence. Curcuma alismatifolia, belonging to the Zingiberaceae family, a plant species originating from Southeast Asia, has become increasingly popular in the flower market worldwide because of its varied and esthetically pleasing bracts produced in different cultivars. Here, we present the chromosome-scale genome assembly of C. alismatifolia "Chiang Mai Pink" and explore the underlying mechanisms of bract pigmentation. Comparative genomic analysis revealed C. alismatifolia contains a residual signal of whole-genome duplication. Duplicated genes, including pigment-related genes, exhibit functional and structural differentiation resulting in diverse bract colors among C. alismatifolia cultivars. In addition, we identified the key genes that produce different colored bracts in C. alismatifolia, such as F3'5'H, DFR, ANS and several transcription factors for anthocyanin synthesis, as well as chlH and CAO in the chlorophyll synthesis pathway by conducting transcriptomic analysis, bulked segregant analysis using both DNA and RNA data, and population genomic analysis. This work provides data for understanding the mechanism of bract pigmentation and will accelerate breeding in developing novel cultivars with richly colored bracts in C. alismatifolia and related species. It is also important to understand the variation in the evolution of the Zingiberaceae family.

Supplementary information: The online version contains supplementary material available at 10.1007/s42994-022-00081-6.

Keywords: Anthocyanin synthesis; Floriculture; Genome evolution; Siam tulip; Zingiberaceae.

Fig. 1

Structure of the C. alismatifolia genome. The innermost circle shows the diversity of bract pigmentation and inflorescence morphology. A GC content. B rRNA distribution. C tRNA distribution. D SSR distribution. E LTR distribution. F 16 chromosomes

Fig. 2

Evolution of the C. alismatifolia genome and gene families. A Phylogenetic tree constructed using maximum likelihood based on the concatenation of single-copy nuclear genes. B The distribution of orthogroups in each species. C Venn diagram of shared and unique gene families in Zingiberales species. D The distribution frequencies of synonymous substitutions (Ks) and substitutions of 4dtv sites. E Synteny patterns between C. alismatifolia and Z. officinale. F The 2:2 syntenic depth pattern between C. alismatifolia and Z. officinale

Fig. 3

Gene duplication and evolution. A Genes derived from different modes of duplication in four different species. The gene types are whole-genome duplication (WGD), tandem duplication (TD), proximal duplication (PD), transposed duplication (TRD), and dispersed duplication (DSD). B Top five enriched pathways for each duplicated type in C. alismatifolia based on the KEGG analysis. C Percentage of different duplicated gene types which contain TEs in an exon, intron, and 1 kb upstream or downstream sequence from each CDS. D Percentage of cytosine methylation in different duplicated gene types in an exon, intron, and 1 kb upstream or downstream sequence from each CDS. E The relationship between methylation and gene expression among different types of duplicated genes

Fig. 4

The anthocyanin and chlorophyll biosynthetic pathway genes identified by RNA-seq in the all-white bract morph C. alismatifolia and C. alismatifolia “Chiang Mai Pink”. A Locations of tissue samples for RNA-seq. Br (outer all-green bract), SeG (inner whorl bract tips), and SeR (inner whorl bract base) of C. alismatifolia “Chiang Mai Pink” (QMF) at different developmental stages and a white morph C. alismatifolia “Country Snow” (XCX) at S4 (bar of S1: 0.2 cm, bar of S2: 0.3 cm, bar of S3: 0.4 cm, bar of S4: 1.5 cm). B KEGG enrichment of DEGs in the QMF SeR vs XCX SeR at S4 stage. C Anthocyanin biosynthetic pathway in C. alismatifolia. Heatmaps show the FPKM with Log2 transformation of genes in SeR of QMF at S1–S4 stages and XCX at the S4 stage. Enzyme abbreviations: PAL phenylalanine ammonium lyase, C4H cinnamate-4-hydroxylase, 4CL 4-coumaroyl-CoA synthase, CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone 3-hydroxylase, F3′H flavonoid-3′-hydroxylase, F3′5′H flavonoid-3′,5′-hydroxylase, DFR dihydroflavonol 4-reductase, ANS anthocyanidin synthase, BZ1 anthocyanidin 3-O-glucosyltransferase. D Chlorophyll biosynthetic and degradation pathway in C. alismatifolia. Enzyme abbreviations: HemA glutamyl-tRNA reductase, HemL glutamate-1-semialdehyde 2,1-aminomutase, HemB porphobilinogen synthase, HemC hydroxymethylbilane synthase, HemD uroporphyrinogen-III synthase, HemE uroporphyrinogen decarboxylase, HemF coproporphyrinogen III oxidase, HemY protoporphyrinogen/coproporphyrinogen III oxidase, chlH magnesium chelatase subunit H, chlM, magnesium-protoporphyrin O-methyltransferase, chlE magnesium-protoporphyrin IX monomethyl ester, por protochlorophyllide reductase, DVR divinyl chlorophyllide a 8-vinyl-reductase, CAO chlorophyllide a oxygenase, chlG chlorophyll/bacteriochlorophyll a synthase, NOL chlorophyll(ide) b reductase, HCAR, 7-hydroxymethyl chlorophyll a reductase, CLH chlorophyllase, SGR magnesium dechelatase PAO pheophorbide a oxygenase, RCCR red chlorophyll catabolite reductase. E Expression heatmaps of genes in the chlorophyll biosynthetic and degradation pathway

Fig. 5

BSA-seq reveals the F3′5'H gene as a candidate gene responsible for red bract pigmentation of C. alismatifolia. A Left: C. alismatifolia “LM”; right: C. alismatifolia “JL”. Bar: 3 cm. B Genome-wide G´ value for allele frequency of SNPs between F1 hybrids S1 (LM) and S2 (JL) pools. C Anthocyanin biosynthesis related genes in BSA signal regions. D Expression heatmap of 31 anthocyanin biosynthesis-related genes from BSA signal regions. E Gene structure of candidate gene according to genome annotation of QMF and sequence depth in parents LM (P1) and JL (P2), and F1 hybrids LM (S1) and JL (S2)