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ISSN : 0250-3360(Print)
ISSN : 2287-5174(Online)
Korean Journal of Breeding Science Vol.44 No.3 pp.229-237

Comparative Analysis among Four Citrus Species by DNA Microarray

Jae Hoon Kim1,2,3*, Jeong Won Park1, Seong Beom Jin2, Kyung Hwan Boo1, Sung Jin Chung1, Su Hyun Yun4, Md. Adnan Al Bachchu3, Jeong Hun Yun3, Song I Han3, Key Zung Riu1,2,3
1Research Institute for Subtropical Horticulture, Jeju National University
2Subtropical/Tropical Pant Gene Bank, Jeju National University, 3Faculty of Biotechnology, College of Applied Life Science, SARI, 4Citrus Research Station, National Institute of Horticultural & Herbal Science, R.D.A.,
(Received on August 17, 2012. Revised on September 17, 2012. Accepted on September 18, 2012)


Citrus is one of the major fruits produced in Korea. There are about 20 species mainly grown in Jeju Island, Korea.Four representative species, which are quite different in the shape of leaf and the taste of fruit, were selected and were usedto profile the transcriptomes. These species are ‘Miyagawa Wase’ (C. unshiu Marcov.) satsuma mandarin, ‘Kiyomi’ (C. unshiuMarcov. × C. sinensis) mandarin hybrid, ‘Dangyuja’ (C. grandis) and ‘Natsudaidai’ (C. natsudaidai). Classification of theup-regulated and down-regulated genes using the Cluster of Orthologous Groups of proteins (COG) database reveals that thenumber of genes included in each group differed significantly among the four species. Several genes that showed significantdifferences in expression on the microarray were selected and their expression patterns were examined by reverse transcription-ploymerasechain reaction. Metabolic genes such as tyrosine decarboxylase and β-glucosidase ligase were found to be highlyexpressed in Miyagawa Wase, relative to other species. On the other hand, the expression level of mannose phosphate isomerasewas lower in Miyagawa Wase. An efflux pump gene was found to be up-regulated in Kiyomi, whereas cinnamyl-alcoholdehydrogenase was down-regulated. β-carotene 15,15’-dioxygenase, which is involved in the vitamin metabolism, wasup-regulated in Natsudaidai. Interspecific differentiations of gene expression are analyzed in terms of the metabolic pathways andtheir possible roles in citrus species.



Citrus is one of the most economically important fruits in the world and is consumed as fresh fruit and is utilized for the production of many other products including juices, soaps, and cosmetics. The taxonomy of citrus species is very complicated and the precise number of natural species is unclear, mainly due to the sexual compatibility, apomixis, the high frequency of bud mutations, and wide dispersion from their places of origin (Grosser and Gmitter 1990). These diversities limit the application of classical molecular biology techniques in the investigation of genetic variability. Microarray analysis is one of the most powerful methods for comparative analysis of gene expression between samples and microarray-based functional genomics offers high throughput means to monitor gene expression patterns (Duggan et al. 1999; Kim et al. 2004).

Functional genomics studies carried out in plants typically focus on the model plant Arabidopsis thaliana (Schaffer et al. 2001). However, more recently there has been increasing interest in the application of functional genomics to investigate various properties of citrus including ethylene-induced differential gene expression (Agusti et al. 2008), spontaneous mutation in sweet orange (Liu et al. 2009), blood orange ripening (Bernardi et al. 2010), tissue-specific transciptome of Clemenucles mandarin (Matas et al. 2010), and stay-green mutation of navel orange (Alos et al. 2008). However, there is no report focusing on the gene expression differences between species. 

 The aim of this study was to investigate gene expression profiles of four citrus species that are grown in Jeju province, Korea: ‘Miyagawa Wase’ (C. unshiu Marcov.) satsuma mandarin, ‘Kiyomi’ (C. unshiu Marcov. × C. sinensis) mandarin hybrid, Dangyuja (C. grandis) and Natsudaidai (C. natsudaidai). ‘Miyagawa Wase’ is one of the most widely grown citrus cultivars in Korea (Yang 1994), and is characterized by its tender peel and seedless tendency. ‘Kiyomi’ is a hybrid of ‘Miyagawa Wase’ and ‘Trovita’ orange (C. unshiu × C. sinensis). ‘Kiyomi’ is relatively sweet and has an aroma that is similar to that of an orange (Akakabe et al. 2010). ‘Dangyuja’ (C. grandis) is cultivated in Jeju Island from over about one thousand years ago. Extract of Dangyuja fruit peel has been reported to have the anticancer, antioxidant, and antidiabetic activities (kim et al. 2009; Lim et al. 2006, 2009). Extract of Dangyuja leaves induced apoptosis in human cervical carcimoma Hela cells (Kim et al. 2010). Natsudaidai was discovered in 1740 in Japan. Natsudaidai’s rough, textured fruit is medium to medium-large (grapefruit size) and can be eaten fresh. From the microarray analysis of four citrus species, we found several hundred genes that are up-regulated or downregulated in each citrus species. When classified with the Cluster of Orthologous Groups of proteins database, we observed variability in the distribution of the genes in each group. The clustering of orthologous genes in each citrus species will provide us with integrated functional information on these genes.


Plant materials and RNA isolation

Healthy leaves from the first flush were collected from sunny side of the canopy in the spring at the Citrus Research Station, National Institute of Horticultural & Herbal Science, Rural Development Administration (Korea). Collected leaves were immediately frozen in liquid nitrogen and stored at -70℃ until RNA isolation. Total RNA was extracted from young leaves using the commercial total RNA extraction kit (easy-BLUETM Total RNA Extraction Kit, iNtRON Biotechnology, Korea). 

Microarray analysis

Expression profiling was conducted with the Citrus 300k microarray (Park et al. 2010). Briefly, the 300k microarray was designed from four major Citrus species (C. clementina, C. reticulata, C. sinensis and C. unshiu) with available Citrus EST sequences from GeneBank (http://www.ncbi.nlm.nih.gov) and Citrus Genetic Resources Bank (http://citrus.cheju.ac.kr). In total, 310,712 probes were designed for 65,636 unigenes. For 53,564 genes of which the transcription direction could be defined, four probes were designed. Eight probes were constructed for the other 12,042 genes that were not clear in direction of their transcription. The average size of probe is 60 nucleotides, which should result in an average Tm value of 75 to 85℃. A Superscript Double-Stranded cDNA Synthesis Kit (Invitrigen, U.S.A.) was used for the synthesis of double strand cDNAs. One μl of oligo dT primer (100 μm) and 10 μl (10 μg) of total RNA were used. First strand cDNA and second strand cDNA were obtained by following a protocol from the manufacturer. After the synthesis of Cy3-labeled target DNA fragments, 13 μg of cDNA was used for microarray hybridization. The microarray was scanned and data acquisition was performed with NimbleScan (NimbleGen, U.S.A) software (http://www.ggbio.com). 

Reverse transcription-polymerase chain reaction (RT-PCR)

Reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the expression pattern of selected genes by using total RNA samples from leaves of four Citrus species. First-strand cDNA was synthesized from 1 μg total RNA using ImProm-IITM Reverse Transcriptase System (Promega) after DNase (RQ1 RNase-Free DNase, Promega) treatment. Amplification of a DNA sequence involved 12 cycles of PCR. Each cycle consisted of 30 s denaturation at 94℃ followed by 1 min of annealing at 55℃ and then 1 min of extension at 72℃. After electrophoresis, the of RT-PCR band was quantified using Image J (http://rsb.info.nih.gov/ij/index.html). 


Microarray analysis was performed on the mRNA isolated from leaves of each species in order to investigate differentially expressed genes among ‘Miyagawa Wase’ (C. unshiu Marcov.) satsuma mandarin, ‘Kiyomi’ (C. unshiu Marcov. × C. sinensis) mandarin hybrid, ‘Dangyuja’ (C. grandis), and ‘Natsudaidai’ (C. natsudaidai). Based on selection criteria of a two-fold difference in signal intensity and a P-value < 0.05, 313 genes of Miyagawa Wase were up-regulated and 404 genes were down-regulated more than two-fold relative to the other three citrus species. In Kiyomi, 129 genes were up-regulated and 281 genes were down-regulated. In the case of Dangyuja, a native species of Jeju Island in Korea, 836 genes in total showed over two-fold differences in gene expression intensities relative to the other three species that were improved in Japan. Natsudaidai was found to have 231 up-regulated genes and 236 down-regulated genes (Table 1). Finally, in the case of Kiyomi, which is a hybrid of Miyagawa Wase and Trovita orange, it shows a relatively small number of up- or down-regulated genes. This is probably due to the characteristic of Kiyomi that has some properties of Miyagawa Wase. Kiyomi has a thin and smooth peel like Miyagawa Wase. The fruit size of Kiyomi is smaller than Trovita orange, but a little larger than Miyagawa Wase (Song et al. 2008). 

Table 1. The number of gene that was over than two-fold up- or down-regulated genes.

The up- or down-regulated genes were classified using the Cluster of Orthologous Groups of proteins (COGs) database as shown in Table 2 and 3, respectively. Although many genes were not clearly annotated into the functional COG catagories, the numbers of genes included in each functional group were significantly different among four citrus species. In ‘Translation, ribosomal structure and biogenesis (COG: J)’ category, up-regulated genes contained five Miyagawa Wase genes, one Kiyomi gene, four Dangyuja genes and three Natsudaidai genes (Table 2). Down-regulated genes included one Miyagawa Wase gene, three Kiyomi genes, nine Dangyuja genes, and nine Natsudaidai genes. The five up-regulated Miyagawa Wase genes included Polyadenylate-binding protein (RRM superfamily), amidases (hydrolase family), H/ACA small nucleolar RNP component GAR1, Ribosome biogenesis protein Nop56p/Sik1p, and mitochondrial translation elongation factor Tu. The down-regulated gene was the Translation initiation factor 5A (eIF-5A) (Table 3).

Table 2. Classification of up-regulated genes from microarray analysis in the COGs functional categories. The relative percentage is shown in bracket.

Table 3. Classification of down-regulated genes from microarray analysis in the COGs functional categories. The relative percentage is shown in bracket.

The ‘transcription (COG: K)’ categor y featured nine up-regulated genes in Natsudaidai, which included six CREB family genes, two GATA-4/5/6 transcription factor genes, and one heat shock gene. The ‘Signal transduction mechanisms(COG: T)’ category contained 14 up-regulated Natsudaidai genes including Serine/threonine protein kinase, Serine/threonine protein phosphatase, MEKK and other related Serine/threonine protein kinases, as well as down-regulated Protein phosphatase 2A regulatory subunit A and - related protein (Tables 2, 3). 

Up-regulated ‘Cell cycle control, cell division, chromosome partitioning (COG: D)’ genes contained Miyagawa Wase genes including cyclin B and related kinase-activated protein, G1/S-specific cyclin D, and G2/Mitotic-specific cyclin A. Nucleosome assembly protein (NAP-1) and Microtubule-binding protein were up-regulated in Dangyuja (Table 2). In this category (COG: D), only one gene (Cdc2-related protein kinase) of Miyagawa Wase was down-regulated (Table 3). 

Many up-regulated genes were grouped as ‘Posttranslational modification, protein turnover, chaperones (COG: O)’ genes. In 25 genes of Dangyuja, 16 genes were molecular chaperone proteins and six genes were ubiquitination-related proteins. Others were glutaredoxin-related proteins and protein disulfide isomerase. Natsudaidai up-regulated seven genes of the glutathine S-transferase family and two genes of AAA+ their expression levels were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR can be used to detect the presence of specific mRNAs and evaluate their expression levels (Choquer et al. 2003). Gene annotation and specific primers for each gene are listed in Table 4. Parallel reactions using primers for Citrus glyceraldehyde-3-phosphate dehydrogenase (CiGAPHD), a house keeping gene, were performed to normalize the amount of template cDNA added to each reaction.

Table 4. Selected genes for semi-quantitative RT-PCR analysis.

For Miyagawa Wase, three genes were selected that showed a reasonable different intensity in the microarray experiment: Up-regulated tyrosine decarboxylase (TYDC) (probe: CITRUS0035864) and down-regulated mannose-6-phosphate isomerase (probe: CITRUS0032047) from the ‘Carbohydrate transport and metabolism (COG: G)’group as well as the up-regulated β-glucosidase (probe: CITRUS0025324) from the ‘Amino acid transport and metabolism (COG: E)’group (Table 4). When comparing microarray data, general expression patterns were similar. 

The TYDC expression level of Miyagawa Wase was 1.5 to 2 times higher than other species in the RT-PCR analysis (Fig. 1a). TYDC is the enzyme that catalyzes the reaction which produces tyramine from tyrosine. TYDC is involved in the biosynthesis of benzylisoquinoline-type alkaliods including octopamine, morphine, codeine, papverine, and synephrine (Facchini et al. 2000). Synephrine is a potential agent for obesity reduction and is found in leaves and fruits of citrus. Our results support the previous report that the gene expression of TYDC was high in mandarin oranges and is correlated to synephine content (Bartley et al. 2010). 

On the other hand, the expression level of mannose-6-phosphate isomerase of Miyagawa Wase was very low (Fig. 1). Mannose phosphate isomerase is an enzyme that utilizes fructose 6-phosphate and mannose-6-phosphate. In Arabidopsis, mannose-6-phosphate isomerase was known to be associated with the L-ascorbic acid biosynthetic process (Maruta et al. 2008). In the case of β-glucosidase, the expression level was elevated about 2 times in Miyagawa Wase (Fig. 1). β-glucosidases, which are a member of the hydrolase family, act upon beta 1-4 linking two glucose or glucose-substituted molecules, and its activity is important for chemical defenses in plants (Morant et al. 2008). Transplastomic tobacco plants expressing β-glucosidase showed early flowering, increases in biomass and protection from aphids or whiteflies (Jin et al. 2011).

Fig. 1. Confirmation of up- or down-regulation of genes listed in Table 4. Total RNAs were prepared from four citrus species (1: Miyagawa Wase, 2: Kiyomi, 3: Dangyuja, 4: Natsudaidai.), and the transcripts were analyzed by semi-quantitative RT-PCR (A). Relative percentage of signal intensities obtained from the microarray data (gray bar) and the band intensities of RT-PCR (white bar). Error bars show the mean ±SD from each probe (B).

 For Kiyomi, we selected three genes: one up-regulated gene for multidrug and toxic compound extrusion (MATE) efflux pump (probe: CITRUS0031247) from the ‘General function prediction only (COG: R)’ group and two down-regulated genes, cinnamyl-alcohol dehydrogenase (probe: CITRUS0024821) and mitochondrial chaperon Cpn60 (probe: CITRUS0024826), from the ‘Posttranslational modification, protein turnover, chaperones (COG: O)’group. DNA band intensities of the efflux pump gene (probe: CITRUS 0031247) were similar to the microarray data, except for Dangyuja. MATE gene expression was shown to confer aluminum tolerance in sorghum (Magalhaes et al. 2007). Additionally, the arabidopsis MATE gene, TT12, is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed (Marinova et al. 2007). Similar to the microarray data, two down-regulated Kiyomi genes (cinnamyl-alcohol dehydrogenase and mitochondrial chaperon Cpn60) showed weaker bands than those of other species in the RT-PCR experiment (Fig. 1a). The expression level of cinnamyl-alcohol dehydrogenase was found to be induced under drought stress in the maize leaf, which suggests that this gene is a candidate gene for improvement for drought tolerance (Hu et al. 2009). In the case of mitochondrial Cpn60, gene expression was strongly increased during flower development of Narcissus pseudonarcissus flowers (Bonk et al. 1996).

For Dangyuja, we could not get reasonable RT-PCR data when comparing microarray data. As microarray probes and PCR-primers are designed from the EST of Miyagawa Wase and the Orange, it is possible that there is some discordance of sequence between probes and genes. In fact, Dangyuja is supposed to originate from Jeju Island, Korea. 

To confirm the up-regulation of genes in Natsudaidai by RT-PCR, two genes were tested: two up-regulated gene products were β-carotene 15,15'-dioxygenase (probe: CITRUS 0011577) from the ‘Secondary metabolites biosynthesis transport and metabolism (COG:Q)’ group and Glutathione-S-transferase (GST) belonging to the tau class (probe: CITRUS 0007947) from the ‘Posttranslational modification, protein turnover, chaperones (COG: O)’ group. These two up-regulated genes (β-carotene 15,15'-dioxygenase and GST) of Natsudaidai showed the most intensive bands in the RT-PCR analysis. β-carotene 15,15'-dioxygenase produces all- trans-retinal from β-carotene, one of the many forms of vitamin A, in the retinol metabolism pathway (Liden et al. 2006). Plant GSTs are a large group of multifunctional proteins and Salicylic-induced GSTs of the tau class were determined in Arabidopsis thaliana (Sappl et al. 2004). 

In this study, a Citrus 300K microarray was used to profile the transcriptomes of four kinds of citrus species (Miyagawa Wase, Kiyomi, Dangyuja and Natsudaidai). Hundreds of genes were up- or down-regulated in each species. These genes are probably related to the characteristic differences of each species and may contribute to molecular breeding of citrus. Future work will focus on the functional roles of select genes from this analysis. 


This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008217), Rural Development Administration, Republic of Korea, and the Priority Research Centers Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2012048080). 


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