Comparative transcriptome sequencing to determine genes related to the nucellar embryony mechanism in citrus

Authors: ÖZHAN ŞİMŞEK, DİCLE DÖNMEZ, SİNAN ETİ, TURGUT YEŞİLOĞLU, YILDIZ KAÇAR

Abstract: Several citrus varieties produce apomictic seeds by the nucellar embryony (NE) mechanism. Nucellar embryos are created from nucellus tissue and have an identical genetic constitution to the mother plant. Nucellar embryony is known to be an unusual feature of seed production in many citrus cultivars. The term "NE" refers to the development of identical embryos from the maternal tissue known as the nucellus surrounding the embryo sac. The authors aimed here to detect differentially expressed genes involved in the NE mechanism. Orlando tangelo (OT), producing apomictic seeds, and a clementine mandarin, Algerian tangerine ranch selection (AT), known as monoembryonic, were used for high-throughput transcriptome sequencing. First of all, histological analysis was used to determine the initial stage of the development of NE cells. Initial NE cells began to develop on the third day after anthesis. Based on the histological analysis, ovules of flower buds for OT were sampled at the balloon stage and 1, 3, and 5 days after anthesis; for AT only ovules of flower buds at the balloon stage and 3 days after anthesis were sampled for comparative transcriptome sequencing. Primary sequencings, known as "raw reads", were produced using Illumina HiSeq 2000. The raw reads were then filtered into clean reads aligned to the reference sequences. The full genome of Citrus clementina was used as the reference genome. Deep analyses based on gene expression and differentially expressed genes (DEGs), including gene ontology (GO) enrichment analysis, were performed. A total of 2359 DEGs (1996 upregulated, 363 downregulated) and 2123 genes (1372 upregulated, 751 downregulated) were identified from the samples at the OT balloon stage, OT third day after anthesis and AT third day after anthesis, and OT third day after anthesis, respectively. These findings provide helpful information regarding citrus transcriptome changes for the NE mechanism and could help with the future identification and functional analysis of genes that are significant for polyembryony.

Keywords: Apomixis, gene, RNA-seq, somatic embryogenesis

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