Comparative transcriptomic analyses of normal and malformed flowers in sugar apple (Annona squamosa L.) to identify the differential expressed genes between normal and malformed flowers

Abstract

Background: Sugar apple (Annona squamosa L.), a popular fruit with high medicinal and nutritional properties, is widely cultivated in tropical South Asia and America. The malformed flower is a major cause for a reduction in production of sugar apple. However, little information is available on the differences between normal and malformed flowers of sugar apple.

Results: To gain a comprehensive perspective on the differences between normal and malformed flowers of sugar apple, cDNA libraries from normal and malformation flowers were prepared independently for Illumina sequencing. The data generated a total of 70,189,896 reads that were integrated and assembled into 55,097 unigenes with a mean length of 783 bp. A large number of differentially expressed genes (DEGs) were identified. Among these DEGs, 701 flower development-associated transcript factor encoding genes were included. Furthermore, a large number of flowering- and hormone-related DEGs were also identified, and most of these genes were down-regulated expressed in the malformation flowers. The expression levels of 15 selected genes were validated using quantitative-PCR. The contents of several endogenous hormones were measured. The malformed flowers displayed lower endogenous hormone levels compared to the normal flowers.

Conclusions: The expression data as well as hormone levels in our study will serve as a comprehensive resource for investigating the regulation mechanism involved in floral organ development in sugar apple.

Keywords: A. squamosa; Digital gene expression; Malformed flower; Normal flower; Phytohormone; Transcriptome.

Fig. 1

Illumina sequencing of sugar apple flowers. a The phenotypes of normal and malformation flowers in sugar apple. b Classification of raw reads generated by Illumina sequencing. c The length distribution of assembled transcripts in sugar apple. d The length distribution of assembled unigenes in sugar apple

Fig. 2

Annotation of assembled sugar apple unigenes. (a) The number of unigenes annotated by different databases, including Nr, Swissprot, COG and KEGG, were showed in a Venn diagram. (b) Distribution of E value in various databases. (c) COG classification of all unigenes of sugar apple. Proportion of each second level COG term belonged to “Signal transduction mechanisms” and “Cell cycle control, cell division”

Fig. 3

Transcriptional variation normal and malformed flowers in sugar apple. a Expression profiles of the differential expressed genes between normal and malformed flowers in sugar apple were showed by a heatmap. b The numbers of up-regulated genes and down-regulated genes in malformed flower compared to normal flower. c Significance analysis of all DEGs between normal and malformed flowers by a volcanoplot. d GO enrichment analysis of DEGs between normal and malformed flowers

Fig. 4

Identification and differential analysis of the hormonal network between normal and malformed flowers in sugar apple. a The number of the DEGs encoding the key components involved in various hormonal signaling pathways. b Overview of various hormonal signaling network in sugar apple. Red indicated the normal flower predominantly expressed genes and green indicated the malformed flower predominantly expressed genes

Fig. 5

Transcript abundance changes of hormone-related genes. a The detailed information on genes involved different hormone signaling pathway. The key components in red cycle indicated genes related to hormone biosynthesis and metabolism; the key components in green cycle indicated genes encoding receptor and transporter; and the key components in blue cycle indicated genes related to downstream response. b Expression changes of the genes associated with different hormones, including auxin, ABA, GA and cytokinin. Red indicates up-regulated genes and blue indicates down-regulated genes

Fig. 6

Real-time quantitative PCR validation of several selected hormone-related genes. Total RNA was extracted from normal and malformation flowers. The histogram shows the relative expression level of these genes with respect to the ACTIN in hickory. The specific identities of the genes: Unigene0001697 (auxin-induced protein 5NG4-like), Unigene0004014 (auxin response factor 5-like), Unigene0037190 (IAA29-like), Unigene0033914 (PIN5), Unigene0004847 (gibberellin-regulated protein 9), Unigene0054219 (GRAS family transcription factor), Unigene0010774 (gibberellin 2-oxidase 2), Unigene0048844 (gibberellin 3-beta-hydroxylase), Unigene0011098 (APRR2), Unigene0018960 (PRR95), Unigene0010916 (cytokinin dehydrogenase 3-like), Unigene0051789 (3-ketoacyl-CoA thiolase 2), Unigene0023736 (zeaxanthin epoxidase), Unigene0006250 (ABA 8′-hydroxylase), Unigene0033398 (PYR1-like). The data were analyzed by three independent repeats, and standard deviations were shown with error bars. Significant differences in expression level were indicated by “*”

Fig. 7

Endogenous hormone measurements. The contents of endogenous hormones, including IAA, ABA, GA and ZRs, between normal and malformation flowers were measured. Three independent samples collected from different flowers were used for endogenous hormone measurements. Significant differences in contents were indicated by “*”