Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility

Abstract

Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines.

Keywords: anther development; cytoplasmic male sterility (CMS); genome assembly and annotation; industrial chicory; pollen development; regulatory pathway; transcriptome profiling (RNA-seq).

Figure 1

Circos plot representing the C. intybus L8001 genome. (A) chromosome-scale assembly (in Mb), (B) heatmap for gene density, (C) heatmap for repeat density, (D) GC%, (E) expression profile of selected genes (outer-inner: Anther, Ligule, Ovule, Petal, Leaf, Seedling, Root, RPKM values ranging from [-10 to 10] with low expression indicated in green and high expression in red), (F) synteny blocks based on genome duplications as reported by i-ADHoRe.

Figure 2

Global transcriptome analysis of flower bud development in fertile line K1337 and male sterile CMS clones CMS36 and CMS30 of industrial chicory (Cichorium intybus var. sativum). (A) Developmental series of flower buds (FB) at increasing size (4-12 mm) sampled for microscopy and RNA-Seq. (B) Principal component analysis on 30971 transcript expression profiles. (C) Ten clusters of differential expression (DE) profiles identified within the > 4-fold-change gene set of the fertile line. Dark shaded areas indicate the periods of high relative expression level. (D) Comparison of expression profiles between fertile line and CMS lines, either having a similar expression profile to the fertile line (like fertile), or DE in fertile line but not DE across the developmental series in the corresponding CMS line (non-DE).

Figure 3

Expression profiles of genes involved in specific pathways important for anther processes of fertile line K1337 and male sterile CMS clones CMS36 and CMS30 of industrial chicory (Cichorium intybus var. sativum). x-axis shows flower bud developmental stages, ranging from 4mm (FB4) to 12mm (FB12). y-axis shows scaled log2 Reads Per Kilobase transcript per Million reads mapped (RPKM) values. Individual genes are shown with different colors, while pathway grouping is shown by different line types. Gene identifiers per subgroup are listed in Supplemental Data Set 2 . For information on ortholog assignment, see Materials and Methods. The following (regulatory) pathways are shown: (A) tapetum development, (B) phenylpropanoid metabolism in early FB stages, (C) hydroxycinnamic acid amides (HCAA) pathway, (D) phenylpropanoid metabolism in mid FB stages, (E) phenylpropanoid metabolism in late FB stages, (F) isoprenoid metabolism in early FB stages, (G) isoprenoid metabolism in mid FB stages, (H) sterol metabolism, (I) genes related to sink strength of energy metabolism, (J) energy metabolism in early FB stages, (K) energy metabolism in mid FB stages, (L) energy metabolism in late FB stages, (M) jasmonate regulated gene cluster linked to anther dehiscence, (N) programmed cell death (PCD) genes linked to anther dehiscence.

Figure 4

Expression profiles of phytohormone biosynthesis pathway genes (red) and response genes (greyscale) of fertile line K1337 and male sterile CMS clones CMS36 and CMS30 of industrial chicory (Cichorium intybus var. sativum). x-axis shows flower bud developmental stages, ranging from 4mm (FB4) to 12mm (FB12). y-axis shows scaled log2 Reads Per Kilobase transcript per Million reads mapped (RPKM) values. Gene identifiers per subgroup are listed in Supplemental Data Set 2 . For information on ortholog assignment, see Materials and Methods. The following phytohormone pathways are shown: (A) abscisic acid, (B) auxin, (C) brassinosteroid, (D) cytokinin, (E) ethylene, (F) gibberellin, (G) jasmonate.

Figure 5

Morphological analysis and comparison of anthers of fertile line L8018 and male sterile CMS clones CMS36 and CMS30 of industrial chicory (Cichorium intybus var. sativum). (A) fertile 4mm flower bud (FB) stage with epidermis (ep), endothecium (en), tapetum (ta) and pollen (po). (B) fertile 5mm FB stage. (C) fertile 6mm FB stage with degraded tapetum cell walls (ta) and developing exine walls of pollen (ex). (D) fertile 7mm FB stage with the start of septum degradation (se). (E) fertile 8mm FB stage. (F) fertile 9mm FB stage with the start of endothecium expansion and lignification (en) and pollen vacuolization (va). (G) fertile 10mm FB stage. (H) fertile 12mm FB stage with stomium opening (st) and pollen starch bodies (po). (I) CMS36 4mm FB stage. (J) CMS36 5mm FB stage. (K) CMS36 6mm FB stage with the start of endothe cium expansion and lignification (en). (L) CMS36 7mm FB stage with degraded tapetum cell walls (ta) and start of pollen exine wall formation (po). (M) CMS36 8mm FB stage with stomium opening (st). (N) CMS36 9mm FB stage with start of pollen degradation (po). (O) CMS36 10mm FB stage. (P) CMS36 12mm FB stage. (Q) CMS30 4mm FB stage. (R) CMS30 5mm FB stage. (S) CMS30 6mm FB stage with start of endothecium expansion and lignification (en). (T) CMS30 7mm FB stage with degraded tapetum cell walls (ta) and start of pollen degradation (po). (U) CMS30 8mm FB stage. (V) CMS30 9mm FB stage with stomium opening (st) and inwards collapse of the anther. (W) CMS30 10mm FB stage. (X) CMS30 12mm FB stage. Scale bar indicates 50 µm.

Figure 6

Timetable of molecular, cellular, metabolic and morphological events during anther development in fertile and CMS clones of industrial chicory (Cichorium intybus var. sativum). (A) Overview of transient activity of processes in the tapetum, developing pollen, epidermis, endothecium, and activation of sink strength and phytohormones. (B) schematic overview of fertile anther development in chicory. (C) schematic overview of anther development in male sterile chicory clone CMS36. (D) schematic overview of anther development in male sterile chicory clone CMS30.