Turkish Journal of Agriculture and Forestry Turk J Agric For (2019) 43: 58-68 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1806-10

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

1, 2 1 1 1 Özhan ŞİMŞEK *, Dicle DÖNMEZ , Sinan ETİ , Turgut YEŞİLOĞLU , Yıldız AKA KAÇAR  1 Department of Horticulture, Faculty of Agriculture, Çukurova University, Adana, Turkey 2 Biotechnology Research and Application Center, Çukurova University, Adana, Turkey

Received: 04.06.2018 Accepted/Published Online: 14.10.2018 Final Version: 06.02.2019

Abstract: Several citrus varieties produce apomictic 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 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, 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.

Key words: , gene, RNA-seq, somatic embryogenesis

1. Introduction controlling apomictic reproduction and facilitate the Citrus is an important fruit group with immense economical transfer of apomixis into other crops (Zhang et al., 2018). value and significant nutritional resources for human The extra identical embryos from the nucellus tissue give health. There are many major cultivated citrus species, rise to polyembryonic seeds. “Polyembryony” is a term including Citrus sinensis (sweet ), C. reticulata closely related to NE and refers to the development of two (tangerine and mandarin), C. limon (lemon), C. grandis or more embryos in one seed (Zhang et al., 2018). Apomixis (pummelo), and C. paradisi (grapefruit). Many citrus mechanisms are divided into two categories, gametophytic members reproduce apomictically by nucellar embryony and sporophytic, based on whether the embryo develops (NE). In NE, typical in citrus and mango, somatic embryos via a (embryo sac) or directly from diploid initiate directly from nucellus or integument cells in the somatic (sporophytic) cells within the (Hand and ovule. The offspring derived from nucellar embryony in Koltunow, 2014). NE is considered to be a significant trait citrus possess the same genetic constitution as the female in citrus rootstocks because it provides a low-technology, parent. The phenomenon of NE hinders the formation low-cost method for propagation of genetically identical of hybrid offspring and the progress of crossbreeding. citrus rootstock (Kepiro and Roose, 2007). However, the However, it greatly benefits the production of offspring for true power and/or features of citrus species are not passed true-to-type rootstock with good unity and yields virus- to individuals as a result of hybridization due to NE by free seedlings. Therefore, investigating the mechanism of citrus breeders. For researchers conducting citrus breeding NE in citrus will provide deeper insight into the processes studies this situation can lead to quite severe losses of * Correspondence: [email protected] 58

This work is licensed under a Creative Commons Attribution 4.0 International License. ŞİMŞEK et al. / Turk J Agric For time and money. These citrus-unique characteristics have hindered the study of citrus genetics and breeding improvement (Roose and Close, 2008; Gmitter et al., 2012; Xu et al., 2013). Molecular studies of apomixis have been conducted largely with species exhibiting gametophytic apomixis (Bicknell and Koltunow, 2004), while nucellar/ integumental embryony by sporophytic apomixis has received little attention (Kumar et al., 2014). The high- throughput sequencing of cDNA fragment populations is commonly known as RNA sequencing (RNA-seq). RNA sequencing is an effective tool for transcriptome analysis and uses deep sequencing techniques to produce millions of short cDNA reads (Simsek et al., 2017). In the present study, the authors aimed to conduct a deep investigation Figure 1. Three differently sized flower buds of OT. of differentially expressed genes (DEGs) related to the NE mechanism in citrus for understanding the mechanism of NE in citrus, providing deeper insight into the processes were collected before and after anthesis. The samples were controlling apomictic reproduction and facilitating the immediately fixed in an FPA-70 solution. Histological transfer of apomixis into other crops. We selected two analyses were carried out with the paraffin-embedding different citrus cultivars for high-throughput sequencing. method and sections were stained using hematoxylin. Orlando tangelo (OT) produces almost 100% apomictic All sections were observed under fluorescent and light seeds, meaning that it has high-level features of NE, and microscopes. Algerian tangerine ranch selection (AT, clementine), known as monoembryonic, does not produce nucellar 2.4. RNA extraction and RNA-seq preparation embryos. Thus, the differences in their gene expression Ovary samples belonging to OT and AT were immediately levels based on NE could be easily compared. frozen in liquid nitrogen and stored at –80 °C. Total RNA extraction was performed according to Liu et al. 2. Materials and methods (2006). The RNAs were treated with DNAse I (RNase- 2.1. Plant material and controlled hybridization free, Fermentas Life Sciences) to discard contaminating OT, producing almost 100% apomictic seeds, and AT, DNA. The RNA quantity and quality were measured with known as monoembryonic, were used as plant materials the NanoDrop ND1000 instrument (Thermo Scientific). for histological analysis and high-throughput sequencing. Three biological replicates per application were diluted Trees of OT and AT at Çukurova University, Agriculture at the same concentrations and in the same amounts and Faculty, Horticulture Department, Citrus Variety then pooled to obtain a final quantity of 25 μg of RNA, Collection, Adana, Turkey, were used as plant materials. In which was sent to the Beijing Genomics Institute (BGI) the hybridization studies, OT and AT were used as mother (Shenzhen, China) for mRNA purification, cDNA library plants and the Fremont mandarin as pollinizer. First, three construction, and sequencing using Illumina HiSeq2000. differently sized flower buds (small, middle, and large 2.5. Transcriptome sequencing anthesis stage) (Figure 1) were collected before anthesis to Transcriptome sequencing was performed at BGI examine the nucellar embryo upon initial cell formation (Shenzhen, China) using Illumina HiSeq2000. Briefly, 6 during the preanthesis period. After hybridization, daily μg of the total RNA of each sample was used to construct pistil samples were collected over 15 days to examine the cDNA libraries. Sequencing was carried out with the postanthesis nucellar embryo initiation. constructed cDNA libraries. Quality control and filtering 2.2. Determination of polyembryony rate of raw data was performed after sequencing. Clean reads A total of 50 fruits harvested under free pollination were stored in FASTQ format after filtering. Filtering steps conditions were collected to confirm OT’s ratio of were as follows: 1) remove reads with adapters; 2) remove polyembryony. After seeds were removed from each fruit, reads in which unknown bases were more than 10%; 3) the embryo-carrying parts were separated with the help of remove low-quality reads (the percentage of low-quality a pin. In this way it was determined that they contained bases is over 50% in a read; a low-quality base was defined more than one embryo each. to be a base whose sequencing quality was no more than 2.3. Histological analysis 10%). After filtering, the remaining reads were called “clean Histological analysis was used to detect the beginning reads” and were used for downstream bioinformatics stage of initial NE cells developing in OT. OT flowers analysis with the recently released reference genome (Xu

59 ŞİMŞEK et al. / Turk J Agric For et al., 2013). Gene annotation was performed using the 44,975,770. Numbers of expressed genes in the samples Blast2GO program (Götz et al., 2008). Gene ontology ranged between 25,027 and 26,489. The total mapped reads (GO) enrichment analysis provided all the GO terms to reference genes (%) were: AT third day 81.77%, AT that were significantly enriched in the DEGs compared balloon 81.49%, OT first day 80.24%, OT third day 81.02%, with the genome background, and the researchers filtered OT fifth day 80.44%, and OT balloon 80.19% (Table 1). the DEGs that corresponded to biological functions. This 3.4. Identification of differentially expressed genes method first maps all DEGs to GO terms in the database To identify genes associated with NE in OT, the (http://www.geneontology.org/), calculating gene numbers significance of gene expression differences was determined for every term, and then uses a hypergeometric test to find (Figure 4). The number of DEGs between the OT third significantly enriched GO terms in the input list of DEGs, day postanthesis sample in which nucellar embryo cells based on ‘GO::TermFinder’. The biological interpretation of began differentiation and the AT control group third the differential genes was further completed by assigning day postanthesis sample was determined as 2123. It was them to metabolic pathways using Kyoto Encyclopedia of determined that 1372 of these genes were downregulated Genes and Genomes (KEGG) annotation. while the rest were upregulated. Many genes exhibit up- or downregulation in response to their condition and genetic 3. Results background conditions. The number of DEGs between the 3.1. Polyembryony rate of OT balloon stage in the OT genotype that had not yet exhibited The average number of seeds per fruit was found to be 16.22 nucellar differentiation and the OT third day postanthesis in OT. The ratio of polyembryony was calculated as 99.16%. sample where cellular differentiation was detected to be The average number of embryos per seed was calculated as 2359. 4.3 (2–6 nucellar embryos). The seeds obtained from the 3.5. Annotation of differentially expressed genes OT fruits and pieces of the seeds containing embryos are Differentially expressed genes can be involved in different presented in Figures 2 and 3. functions. Gene ontology is an internationally standardized 3.2. Histological analysis gene functional classification system that describes the The histological examination of the ovules from flowers features of genes and their products in any organism. of different sizes and hybridization studies revealed the Based on the cellular component, the most abundant DEGs beginning of nucellar cell differentiation in OT three days were involved in “cell”, “cell part”, and “organelle”. From after anthesis. On the other hand, AT, a monoembryonic the perspective of biological processes, the DEGs were variety, did not exhibit nucellar embryo initiation at any involved in the “metabolic process”, “cellular process”, and stage of ovule development. Therefore, the RNAs from the “single-organism process”. In terms of molecular functions, ovules of flower buds at balloon stage and 1, 3, and 5 days the genes were dominant in “catalytic activity”, “binding”, after anthesis were sampled in OT, but only ovules of flower and “transporter” (Figure 5). To further investigate the buds at balloon stage and 3 days after anthesis in AT were biological functions of these genes, KEGG ontology (http:// sampled for transcriptome sequencing. www.genome.jp/kegg/) was used to classify their functional 3.3. Illumina sequencing and reads assembly annotations. The top 20 statistics of pathway enrichment To characterize all the transcriptome differences among the for the OT third day sample and the AT third day sample six samples, transcriptional analysis was performed using are presented in Figure 6. Among the pathways, zeatin Illumina HiSeq2000. After RNA sequencing, numbers of biosynthesis, metabolic pathways, RNA polymerase, and clean reads in the samples ranged between 42,168,870 and others were highly represented.

Figure 2. A) Fruit and seeds of OT, B) seed and embryos.

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Figure 3. A, B, C) Polyembryonic seeds of OT containing nucellar embryos, D) monoembryonic seeds of AT.

Table 1. Summary of RNA-seq data.

Clean Genome Gene Expressed Expressed Novel Extend Sample name reads map rate map rate genes transcripts transcripts genes Algerian_3rd_day 44,975,770 77.80% 81.77% 19,992 25,900 1134 4144 Algerian_balloon 44,965,352 77.35% 81.49% 20,038 25,885 1172 4074 Orlando_1st_day 43,204,308 76.54% 80.24% 19,976 26,310 1244 3934 Orlando_3rd_day 42,096,154 77.37% 81.02% 20,097 26,361 1277 3789 Orlando_5th_day 42,168,870 77.76% 80.44% 20,081 26,489 1270 3908 Orlando_balloon 44,506,486 74.63% 80.19% 19,612 25,027 1040 3662

4. Discussion from the period when the nucellar embryonic cells began The samples used in this study were selected in accordance to differentiate, based on the histological analysis. Different with the determination of the genes involved in the findings and hypotheses about the NE mechanism were mechanism of nucellar embryo development in citrus. For published. The first genetic analysis of NE using molecular this purpose, OT, producing highly nucellar plants, and AT, markers to analyze a hybrid population was derived from a monoembryonic species, were used. Genes expressing at C. volkameriana and Poncirus trifoliata by García et al. different levels in the samples may be related to the NE (1999). They reported that apomixis is controlled by six mechanism. For this reason, the OT samples were selected quantitative trait loci that either positively or negatively

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Figure 4. Statistics of differentially expressed genes between each two samples. affect apomixis in citrus. From the results it appears that after the formation of the nucellar embryo initiation cells. different genetic mechanisms control the production of Among the common genes, peroxidase and the MP3K nucellar embryos and the proportion of polyembryonic protein kinase (mitogen-activated protein kinase) genes seeds. Polyembryony has also been investigated in P. were expressed at lower levels in AT compared to OT in trifoliata with a population derived from a cross between our study. Additionally, heat-shock protein and alcohol C. maxima and P. trifoliata using amplified fragment dehydrogenase were expressed at a higher level in OT, length polymorphism (AFLP) markers. The researchers while the nuclear and cytoplasmic polyadenylated RNA- detected that five of these AFLP markers were closely binding protein Pub1 gene was expressed at a higher level linked to this trait. These AFLP markers identified a single in AT. Kumar et al. (2014) investigated the NE mechanism genomic region defined by several marker loci that is with microarray technology and used the GeneChip Citrus strongly associated with the percentage of polyembryonic Genome Array (Affymetrix Inc., Santa Clara, CA, USA) seeds produced by trees in a Citrus × Poncirus cross. It is representing over 33,879 citrus transcripts for global gene very likely that this region contains a gene that is essential expression analysis. The results of the microarray remained for the production of polyembryonic seeds by apomixis. limited with transcript numbers on the chip. We tried to However, the proportion of polyembryonic seeds varies identify DEGs involved in the NE mechanism for certain widely among hybrids, probably due to the influence of stages by RNA-seq. RNA-seq technology enables rapid other genes (Kepiro and Roose 2010). In the present study, profiling and deep investigation of the transcriptome for we detected several other genes that could be in control any species. This approach offers a number of advantages of the NE mechanism. Kumar et al. (2014) investigated compared to microarray analysis, a legacy technology often the genes related to NE in citrus fruits using microarray used in gene expression studies. Unlike arrays, RNA-seq analysis. They reported that most frequently genes involved technology does not require species- or transcript-specific in binding activity were in the GO functional classification probes. It can detect novel transcripts, gene fusions, system and, as a result of the studies, a total of 154 single nucleotide variants, indels (small insertions and upregulated and 130 downregulated genes were detected. deletions), and other previously unknown changes that In their studies, C. sinensis cultivars Vaniglia Sanguigno arrays cannot detect. With array hybridization technology, (polyembryonic) and Temple (monoembryonic hybrid gene expression measurement is limited by background at between mandarin and C. sinensis) (three trees of each the low end and signal saturation at the high end. RNA- variety) were used. They identified certain important genes seq technology produces discrete, digital sequencing read that were expressed at different levels in samples before and counts and can quantify expression across a larger dynamic

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1 biological regulation cellular component organization or biogenesis 2 cellular process developmental process 3 establishment of localization growth 4 localization metabolic process 5 multi−organism process multicellular organismal process biological_process regulation of biological process 6 reproduction reproductive process 7 response to stimulus signaling 8 single−organism process cellular_component cell 9 cell part extracellular region 10 macromolecular complex membrane 11 membrane part molecular_function membrane−enclosed lumen 12 organelle organelle part antioxidant activity 13 binding catalytic activity 14 enzyme regulator activity guanyl−nucleotide exchange factor activity 15 molecular transducer activity nucleic acid binding transcription factor activity 16 receptor activity structural molecule activity 17 transporter activity 100 200 300 400 500 600 18 Number of Genes [length = sqrt(num)] Figure 5. GO functional classification between AT third day and OT third day after anthesis. 19 Figure 5. GO functional classification between AT third day and OT third day post-

20 rangeanthesis (Wang et al. 2009; Wilhelm and Landry 2009; both pummelo and sweet orange. Gene ontology analysis Zhao et al., 2014). Compared to microarrays, RNA-seq indicated that the genes associated with microtubule- 21 technology can detect a higher percentage of differentially based movement, DNA packaging, cell cycle, DNA expressed genes, especially genes with low expression (Liu replication, mitosis, and transcription regulator activity et al., 2015; Li et al., 2016). Wang et al. (2017) compared were overrepresented among the genes that were highly 22 the RNA-seq data derived from leaves, ovules, fruits, and expressed in ovules. The present authors detected genes seeds of pummelo (sexual type) and sweet orange (asexual with similar annotations from their results. A list of the 23 type). They found that 1637 and 1840 genes, respectively, overexpressed selected genes related to the NE mechanism were highly expressed in ovules of pummelo and sweet revealed by the data is presented in Table 2. Nakano et al. 24 orange, and 853 of these genes were highly expressed in (2012) identified genomic sequences that were related to

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2 Zeatin biosynthesis Tyrosine metabolism 3 Tropane, piperidine and pyridine alkaloid biosynthesis 4 Stilbenoid, diarylheptanoid and gingerol biosyntesis RNA polymerase 5 Pyrimidine metabolism Qvalue 6 Purine metabolism 0.125 Plant-pathogen interaction 0.100 7 0.075 Phenylpropanoid biosynthesis 0.050 Nitrogen metabolism 8 0.025 Metabolic pathways 9 Gene number Linoleic acid metabolism 100 10 Limonene and pinene degradation 200 Flavonoid biosynthesis 300 11 Flavone and flavonol biosynthesis Cutin, suberine and wax biosynthesis 12 Carotenoid biosynthesis 13 Biosynthesis of secondary metabolites Benzoxazinoid biosynthesis 14 ABC transporters

15 0.10 0.15 0.20 0.25 RichFactor 16 FFigureigure 6. 6 Pathway. Pathw enrichmentay enric forhment AT third for day A Tand th OTird third day day and after O Tanthesis. third day post-anthesis the polyembryony mechanism in citrus. They detected a were expressed at significantly different levels in the total of 70 candidate open reading frames, 40 of which samples. In a somatic embryogenesis study conducted are known to be associated with polyembryony. Among with carrots, SERK genes were reported to be expressed these genes the authors identified 23 genes with the same at different levels during early embryonic development function. (Schmidt et al., 1997). In a different study, the SERK1 The formation of nucellar embryos in citrus is a natural gene was expressed at a very low rate during the forward somatic embryogenesis. This process is a system that can embryonic stage in Arabidopsis thaliana. The reason be applied to all plants in in vitro conditions, and it is for this is that it is already expressed at an early stage known that different genes play a role in this process. A (Hecht et al., 2001; Salaj et al., 2008). It was determined number of genes associated with somatic embryogenesis that the SERK1 gene was expressed at a higher level in were identified. Among these, genes belonging to the the balloon stage of OT flowers and this level decreased family of the somatic embryogenesis receptor kinase in the fifth day postanthesis sample. It is likely that this (SERK) protein are dominant. The somatic embryogenesis level would decrease even further. The SERK genes have receptor kinase is known to be the first identified gene also been associated with somatic embryogenesis in other associated with somatic embryogenesis (Schmidt et plant species: Helianthus annuus (Thomas et al., 2004), al., 1997). In addition, SERK plays an important role Theobroma cacao (de Oliveira Santos et al., 2005), Oryza in many signal transduction pathways (Becraft, 2002). sativa (Hu et al., 2005), and Vitis vinifera (Schellenbaum It was determined that the SERK1 and SERK4 genes et al., 2008).

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Table 2. List of overexpressed selected genes related to nucellar embryony mechanism.

Gene ID (NCBI) Annotation Overexpressed stage 18042205 Flavonol synthase/flavanone 3-hydroxylase AT balloon 18055754 Aminopeptidase AT balloon 18033369 Nucleic acid binding transcription factor activity AT balloon 18055371 Nuclear and cytoplasmic polyadenylated RNA-binding protein Pub1 AT 3rd day 18051641 Methyltransferase AT 3rd day 18035760 Protein phosphatase 2c AT 3rd day 18040803 Cytochrome P450 AT 3rd day 18055916 YGGT family protein AT 3rd day 18032680 Alcohol dehydrogenase OT balloon 18044000 Spermidine synthase OT balloon 18049532 MADS-box protein (CitMADS8) OT balloon 18033849 Ferredoxin-like protein OT balloon 18038079 Myosin XI OT balloon 18040684 Microtubule-based movement OT balloon 18040419 Mitotic cell size control checkpoint OT balloon 18048076 Peroxidase OT 1st day 18033491 Serine threonine kinase OT 1st day 18040365 DNA excision repair protein OT 1st day 18050208 Glutathione S-transferase 2 OT 1st day 18049121 Enhancer of polycomb-like protein OT 1st day 18054563 Polycomb protein EED OT 1st day 18052055 DNA topoisomerase type II (ATP-hydrolyzing) activity OT 1st day 18036310 Somatic embryogenesis receptor kinase 4 OT 3rd day 18049286 Somatic embryogenesis receptor kinase 1 OT 3rd day 18032801 Late embryogenesis abundant protein Lea5 OT 3rd day 18043861 Heat-shock protein OT 3rd day 18036511 Mitogen-activated protein kinase OT 3rd day 18050224 Mitochondrial carrier protein OT 3rd day 18036881 f-box/LRR-repeat protein OT 3rd day 18052018 LRR receptor-like serine/threonine-protein kinase OT 3rd day 18039133 Floral homeotic protein APETALA 2 OT 3rd day 18035085 UDP-glucose iridoid glucosyltransferase OT 3rd day 18041886 General transcription factor IIIA OT 3rd day 18040390 Transcription factor TGA OT 3rd day 18037832 Transcription factor MYC2 OT 3rd day 18037074 WRKY transcription factor 33 OT 3rd day 18044218 Argonaute protein OT 3rd day 18055467 Peptidyl-prolyl cis-trans isomerase OT 5th day 18048134 Chromodomain-helicase-DNA-binding protein 4 OT 5th day

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Another important gene for somatic embryogenesis is (RISC) that are involved in the posttranscriptional glutathione S-transferase (GST). It has many isoforms that silencing of genes via RNA interference (RNAi) (Tolia and are involved in cellular detoxification processes specific to Joshua-Tor, 2007). Based on our results, argonaute protein different substrates. Different GST isoforms were expressed is differently expressed in certain stages. Argonaute among the samples based on the RNA-seq data. Pan et al. proteins associate with small noncoding RNAs (such as (2009) reported that GST genes in oranges were expressed small interfering (si)RNAs and microRNAs (miRNAs)) at different levels during somatic embryo development. and function in RNA-based silencing mechanisms by They reported that GST genes involved in oxidative stress altering protein synthesis and affecting RNA stability reactions with different enzymes regulate important redox (Hutvagner and Simard, 2008). There are several reports changes to trigger somatic embryos. In a study conducted that argonaute proteins are essential for the development with Arabidopsis, the chromodomain-helicase-DNA- of somatic embryos in plants. Tahir et al. (2006) reported binding protein 3 (CHD3) gene was reported to promote that a gene belonging to the argonaute protein (AGO) somatic embryogenesis from root explants (Chanvivattana family is essential for the development of somatic embryos et al., 2004). The CHD4 gene was expressed at different in Picea glauca, which is part of the pine tree family. levels among the samples of the present study. Similarly, in a study conducted with carrot, a gene of the It was also determined that spermidine synthase was AGO homolog was detected at different expression levels another important gene involved in the mechanism of NE. in the formation of somatic embryos, and this gene was Pan et al. (2009) reported that spermidine synthase was involved in RNAi mechanisms and required for somatic expressed during somatic embryo formation in oranges. embryogenesis (Takahata, 2008). According to our results, Nakano et al. (2012) revealed that spermidine synthase is posttranscriptional silencing of some genes affecting NE a gene belonging to the locus associated with NE. It has could be possible via RNAi. This finding can create a new been determined that the spermidine synthase gene is perspective for the NE mechanism in citrus. expressed five times more during the balloon stage of the Different metabolic pathways were identified as a result flower in which differentiation does not begin than in the of standard bioinformatic analysis. In particular, zeatin third day postanthesis sample in which NE cells started to biosynthesis and RNA polymerase are notable between the differentiate. Spermidine is an aliphatic polyamine and a polyembryonic OT and the monoembryonic AT. low-molecular-weight polycation in all living organisms In conclusion, in this study, the authors conducted (Bagni and Tassoni, 2001; Martin-Tanguy, 2001). This RNA-seq analysis to detect genes involved in the NE growth regulator plays a role in various plant growth mechanism. The authors used OT, producing almost and development processes, including cell division, 100% apomictic seeds, and one of the clementine morphogenesis, flowering initiation, pollen tube growth, mandarins, AT, known as monoembryonic, for high- and senescence (Germana, 2006). throughput sequencing. First, the authors performed Another important gene detected in the study was histological analysis and determined the differentiation polycomb group proteins. Polycomb proteins inhibit time of nucellar embryo cells in OT at the third day after the expression of genes that are not essential for cellular anthesis. For transcriptome analysis, ovules of flower buds differentiation by modifying chromatin (Bratzel et al., at the balloon stage and 1, 3, and 5 days after anthesis were 2010). In other words, the presence of polycomb proteins sampled in OT; only the balloon stage and 3 days after in cells indirectly triggers cellular differentiation. Different anthesis were sampled in AT. Differentially expressed polycomb proteins affecting cellular differentiations genes involved in the NE mechanism for certain stages (polycomb protein EED, enhancer of polycomb-like were determined and compared with previous findings. protein) were detected. An important role of transcription This dataset provides information regarding the NE factors has also been identified in the differentiation of mechanism and may help guide future identification NE initiation cells in citrus. The CitMADS8 transcription and functional analysis of genes that are important for factor, giving different expression levels among samples, polyembryony. is reported to play a crucial role in cell differentiation (Nakano et al., 2012). Acknowledgment Proteins associated with the posttranscriptional This research was supported by the Scientific Research silencing of the genes were also identified. Argonaute Projects Coordination Unit of Çukurova University proteins are part of RNA-induced silencing complexes (ZF2012D9).

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