95 1. Introduction Citrus is one of the most important fruit crops in Japan and also worldwide. Various accessions of Citrus species are adapted to the southwest of Japan, and although they are cultivated in this region, almost all of them are non- native, that is, they were introduced from abroad, arose as chance seedlings, were selected from bud sports, and were bred by artificial pollination. Only two species, Cit- rus tachibana (Makino) Tanaka (Tachibana) and Citrus depressa Hayata (Shiikuwasha) were present in Japan be- fore recorded history. C. tachibana mainly grows indigenously on the Pacific side of the southwest of Japan’s main islands (Kyushu, Shi- koku, and Honshu). C. tachibana was recorded in “Kojiki”, the oldest chronicle in Japan dating from the early 8th cen- tury. Its indigenous trees were also found on the Ryukyu Islands (islands including the Okinawa Islands, Sakishi- ma Islands, and Amami Islands, which were ruled by Ja- pan from the 17th to19th centuries) and Taiwan (Tanaka, 1931; Lin and Chen, 2006; Inafuku-Teramoto et al., 2010). C. depressa is indigenous to both the Ryukyu Islands and Taiwan (Tanaka, 1936; Lin and Chen, 2006). Compared to C. tachibana, C. depressa is considered to be adapted to a warmer climate; the former is usually used as an ornamental for gardens and its fruit is inedible. On the other hand, fruit of C. depressa is in much demand as an ingredient for food and drinks, to garnish dishes similar to a lemon or lime, to make juice and jam, and as an additive to soy sauce and distilled spirits. Recently, this fruit has attracted attention because it contains high levels of polymethoxyflavonoids, one of the most important health-promoting components of citrus (Inafuku-Teramoto et al., 2010). We have investigated the phylogenetic relationships of Citrus and its relatives through the analysis of genes encoded in chloroplast DNA (cpDNA) (Tshering et al., 2010, 2013). In our recent study (Tshering et al., 2013) in which various Citrus accessions were used as materi- als, we found that C. tachibana and C. depressa possess a characteristic cpDNA genome based on the sequences of the chloroplast matK genes, which encode a maturase involved in splicing type II introns from RNA transcripts (Hilu and Liang, 1997; Hilu et al., 2003; Olmstead and Palmer, 1994). There are many accessions in both species, and intraspecies diversity is found within each species (Hirai et al., 1990; Yamamoto et al., 1998; Kinjo, 2007; Inafuku-Teramoto et al., 2010; Yamamoto et al., 2011). Characterization of chloroplast matK sequences of Citrus tachibana and Citrus depressa, two indigenous species in Japan Y. Nagano1, S. Inafuku-Teramoto2,3, M. Hashimoto4, T. Mimura4, R. Matsumoto4, M. Yamamoto5(*) 1 Analytical Research Center for Experimental Sciences, Saga University, Honjo-machi, Saga 840-8502, Japan. 2 Faculty of Agriculture, University of The Ryukyus, Nishihara, Okinawa 903-0213, Japan. 3 Botswana-JICA Jatropha Project, DAR, Sebele, Gaborone, Botswana. 4 Faculty of Agriculture, Saga University, Honjo-machi, Saga 840-8502, Japan. 5 Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan. Key words: cpDNA, genetic resources, Ryukyu islands, shiikuwasha, tachibana. Abstract: Citrus tachibana, C. nippokoreana, and C. depressa are indigenous mandarin species in Japan. We deduced their phylogenetic relationships from nucleotide sequences of the chloroplast matK gene. The results indicate that C. tachibana, C. nippokoreana, and C. depressa accessions can be classified into two types: type A, all sixteen C. tachibana and six C. depressa; type B, eleven C. depressa and one C. nippokoreana. Both type A and type B accessions of C. depressa were found on the Okinawa Islands, whereas only type B accessions of C. depressa were found on the Sakishima and Amami Islands. This cpDNA divergence seemed to indicate a polyphyletic origin of C. depressa. The matK genes of type A were found only in C. tachibana and some C. depressa. From these results, both species probably possess a characteristic chloroplast genome among various Citrus species. Adv. Hort. Sci., 2014 28(2): 95-99 (*) Corresponding author: yamasa@agri.kagoshima-u.ac.jp Received for publication 31 March 2014 Accepted for publication 1 July 2014 96 However, a limited number of accessions were used in our previous study (Tshering et al., 2013). Therefore, for the present work, we analyzed the matK gene sequences of a number of C. tachibana and C. de- pressa plants grown in various regions in Japan to reveal their characteristic profiles of the cpDNA genome. C. nip- pokoreana (Korai Tachibana), a C. tachibana relative in- digenous to Hagi City, Yamaguchi Prefecture, Japan, and Cheju Island, Korea (Kimura and Taninaka, 1995), was also investigated. 2. Materials and Methods Plant materials Sixteen C. tachibana, one C. nippokoreana, 17 C. depres- sa, and 13 control accessions were used in this study. The sources of the materials are shown in Table 1 and Figure 1. PCR amplification and DNA sequencing Genomic DNA was extracted from leaves using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). By using this genomic DNA as a template, the matK gene was amplified by PCR using proofreading PrimeSTAR GXL DNA Polymerase (TAKARA BIO, Ohtsu, Shiga, Japan). The primers used for PCR amplification of the matK gene were matK1F (5′-ACCGTATCGCACTATGTATC-3′) and matK1R (5′-GAACTAGTCGGATGGAGTAG-3′). The am- plified DNA fragments were purified using the NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL, Düren, Germany). The primers used for sequencing of the matK gene were matK1F, matK2F (5′-ACGGTTCTTTCTCCAC- GAGT-3′), matK3F (5′-GGTCCGATTTCTCTGATTCT-3′), matK1R, matK2R (5′-AGAATCAGAGAAATCG- GACC-3′), and matK3R (5′-ACTCGTGGAGAAAGAAC- CGT-3′). The purified DNA fragments were sequenced in both directions in an Applied Biosystems 3130 Genetic Ana- lyzer (Applied Biosystems) with a BigDye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems) as described previously (Platt et al., 2007). Sequence data were submitted to DDBJ/GenBank/EBI and were assigned accession numbers ranging from AB839905 to AB839932. The sequences of the accessions from No. 29 to No. 34 were deposited in our previous study (Tshering et al., 2013). Phylogenetic analyses The neighbor-joining (NJ) and maximum likelihood (ML) methods from the MEGA (version 5.2.1) program (Tamura et al., 2011) were used to create phylogenetic trees. The reliability of each branch was tested by boot- strap analysis with 1,000 replications. 3. Results and Discussion We constructed multiple sequence alignments of 1,630-bp fragments containing the matK gene from differ- ent Citrus accessions. Each sequence contained a 1,530-bp protein-coding sequence and 100 bp of the 3′ UTR. One exception is the matK gene of trifoliate orange (Poncirus trifoliata), which has a 6 bp insertion at the 3′ UTR. Of these, 23 bases were variable and six bases were phyloge- netically informative. We created phylogenetic trees using the NJ and ML methods. The topologies of the different trees were identical (data not shown). Therefore, we present here only the ML tree (Fig. 2). C. tachibana, C. depressa, and C. nippokore- ana accessions were classified into two types as follows: Type A: all 16 C. tachibana and six C. depressa [Shiiku- washa-Okinawa#1 (No. 17), Shiikuwasha-Okinawa#3 (No. 19), Shiikuwasha-Okinawa#6 (No. 22), and Shiikuwasha- Oku (No. 26), Kabishi (No. 29), and Fusubuta (No. 31)]. Type B: Eleven C. depressa [Shiikuwasha-Taketomi (Nohara) (No. 11), Shiikuwasha-Taketomi (Takana) (No. 12), Shiikuwasha-Iriomote (No. 13), Shiikuwasha-Irio- mote (Katoura) (No. 14), Shiikuwasha-Kohama (Ufu- dake) (No. 15), Shiikuwasha-Kohama (Omori) (No. 16), Ishikunibu (No. 28), Mikanguwa (No. 30), Kaachi (No. 32), Shiikunin (No. 33), and Shiikurubu (No. 34)] and one C. nippokoreana. None of the control accessions belonged to type A, where- as all seven control mandarin accessions belonged to type B. The other control accessions were clearly distinguished from type A and type B. This finding is consistent with the results of our previous study (Tshering et al., 2013). All 16 C. tachibana accessions carried an identical matK sequence. Previous studies (Hirai et al., 1990; Yamamoto and Tominaga, 2003) reported that C. tachibana was geneti- cally differentiated from Citrus species originating from all Fig. 1 - Collection sites of Citrus tachibana, C. nippokoreana, and C. depressa in the present study. 97 Table 1 - Citrus tachibana, C. nipponkoreana, and C. depressa accessions used in the present study No. Accession Latin name Origin Note 1 Tachibana-Dazaifu (uchi) Citrus tachibana (Makino) Tanaka Fukuoka, Kyushu Planted tree 2 Tachibana-Dazaifu (soto) C. tachibana (Makino) Tanaka Fukuoka, Kyushu Planted tree 3 Tachibana-Heian Jingu C. tachibana (Makino) Tanaka Kyoto, Honshu Planted tree 4 Tachibana-Iwashimizu Hachimangu C. tachibana (Makino) Tanaka Kyoto, Honshu Planted tree 5 Tachibana-Kitano Tenmangu C. tachibana (Makino) Tanaka Kyoto, Honshu Planted tree 6 Tachibana-Toshijima (Mie) C. tachibana (Makino) Tanaka Mie, Honshu Native tree 7 Tachibana-Matsuoyama (Kochi) C. tachibana (Makino) Tanaka Kochi, Shikoku Native tree 8 Tachibana-Nangoku (Kochi) C. tachibana (Makino) Tanaka Kochi, Shikoku Planted tree 9 Korai Tachibana C. nippokoreana Tanaka Kochi, Shikoku Planted tree 10 Tachibana-Ishigakijima C. tachibana (Makino) Tanaka Ishigaki-jima, Sakishima Native tree 11 Shiikuwasha-Taketomi (Nohara) C. depressa Hayata Taketomi-jima, Sakishima Native tree 12 Shiikuwasha-Taketomi (Takana) C. depressa Hayata Taketomi-jima, Sakishima Native tree 13 Shiikuwasha-Iriomote C. depressa Hayata Iriomote-jima, Sakishima Native tree 14 Shiikuwasha-Iriomote (Katoura) C. depressa Hayata Iriomote-jima, Sakishima Native tree 15 Shiikuwasha-Kohama (Ufudake) C. depressa Hayata Kohama-jima, Sakishima Native tree 16 Shiikuwasha-Kohama (Omori) C. depressa Hayata Kohama-jima, Sakishima Native tree 17 Shiikuwasha-Okinawa#1 C. depressa Hayata Okinawa-honto Native tree 18 Tanibuta-Okinawa#2 C. tachibana (Makino) Tanaka Okinawa-honto Native tree 19 Shiikuwasha-Okinawa#3 C. depressa Hayata Okinawa-honto Native tree 20 Tanibuta-Okinawa#4 C. tachibana (Makino) Tanaka Okinawa-honto Native tree 21 Tanibuta-Okinawa#5 C. tachibana (Makino) Tanaka Okinawa-honto Native tree 22 Shiikuwasha-Okinawa#6 C. depressa Hayata Okinawa-honto Native tree 23 Tanibuta-Okinawa#7 C. tachibana (Makino) Tanaka Okinawa-honto Native tree 24 Tanibuta-Okinawa#8 C. tachibana (Makino) Tanaka Okinawa-honto Native tree 25 Garagara C. tachibana (Makino) Tanaka Okinawa-honto Native tree 26 Shiikuwasha-Oku C. depressa Hayata Okinawa-honto Native tree 27 Tanibuta C. tachibana (Makino) Tanaka Okinawa-honto Native tree 28 Ishikunibu C. depressa Hayata Okinawa-honto Native tree 29 Kabishi C. depressa Hayata Okinawa-honto Native tree 30 Mikanguwa C. depressa Hayata Okinawa-honto Native tree 31 Fusubuta C. depressa Hayata Okinawa-honto Native tree 32 Kaachi C. depressa Hayata Okinawa-honto Native tree 33 Shiikunin C. depressa Hayata Tokuno-shima, Amami Native tree 34 Shiikuribu C. depressa Hayata Okinoerabu-jima, Amami Native tree Control accessions Satsuma mandarin ‘Aoshima’ C. unshiu Marcow. Ponkan ‘Yoshida Ponkan’ C. reticulata Blanco Mediterranean mandarin C. deliciosa Ten. Dancy C. tangerina hort. ex Tanaka Kinokuni ‘Hirakishu’ C. kinokuni hort. ex Tanaka Sunki C. sunki (Hayata) hort. ex Tanaka Cleopatra C. reshni hort. ex Tanaka Yuzu ‘Yamane’ C. junos Siebold ex Tanaka Sweet orange ‘Fukuhara’ C. sinensis (L.) Osbeck Lemon ‘Eureka’ C. limon (L.) Burm. f. Pummelo ‘Mato Buntan’ C. maxima (Burm.) Merr. Citron ‘Maru Busshukan’ C. medica L.   Trifoliate orange ‘Standard’ Poncirus trifoliata (L.) Raf.     98 other countries except Japan. The present study also con- firmed that the matK sequence of C. tachibana was not identical to those of studied accessions originating from all other countries except Japan. However, we found that the matK sequence of C. tachibana was identical to those of some investigated C. depressa accessions that are indig- enous to the Ryukyu Islands, Japan. This suggests that C. tachibana has been isolated from the mandarins elsewhere, and evolved in Japan in unique ways. We found no diversity within species. However, further study considering more accessions is needed since the materials used here did not cover the entire area where C. tachibana grows. The matK sequence of C. nippokoreana was not identical to that of C. tachibana, indicting genetic differentiation between the two species. Because it is considered that C. nippokoreana is re- lated to C. tachibana (the Japanese name “Korai Tachibana” means “Tachibana from Korea”), this finding is interesting. C. depressa accessions were divided into two types ac- cording to matK sequences. One was the same type as C. tachibana and the other was the same type as several man- darins such as C. reticulata and C. sunki. This result com- pletely agrees with the results of our previous study (Tsher- ing et al., 2013). Differentiation of the cpDNA genome in C. depressa was also reported by Urasaki et al. (2005) and Yamamoto et al. (2013), who analyzed the trnL-trnF and trnF-trnVr regions, respectively. These results strongly sug- gest a polyphyletic origin of C. depressa. This divergence of matK genes was found in C. depressa accessions grown on Okinawa-honto (the main island of Okinawa Islands) but not in those grown on the Sakishima and Amami Islands. C. depressa possessing C. tachibana-type cpDNA (A type) was found only on Okinawa-honto. Similar results were re- ported by Yamamoto et al. (2013) who studied C. depressa on Okinawa-honto and the Amami Islands. However, Ura- saki et al. (2005) found that C. depressa accessions pos- sessed C. tachibana-type cpDNA (trnL-trnF sequence) on the Sakishima Islands. Thus, further study using many C. depressa accessions grown on various islands is necessary to resolve the distribution of each type. There is a possibility that type A C. depressa is genetical- ly closer to C. tachibana than type B. However, this hypoth- esis is not supported since the proportion of common bands from random amplified polymorphic DNA (RAPD) analysis between C. depressa of type A and C. tachibana was not so different from that of type B and C. tachibana (Yamamoto et al., 1998). Since the origin and/or relationship of C. de- pressa to C. tachibana cannot be elucidated only by cpDNA analysis, cpDNA analysis combined with nuclear genome analysis such as simple sequence repeat (SSR), sequence- related amplified polymorphism markers (SRAPs) (Barkley et al., 2006; Uzun et al., 2009), and restriction site-associat- ed DNA sequences (RAD-seq) (Baird et al., 2008) is con- sidered to be necessary. For this purpose, structural analysis (Barkley et al., 2006) seems to be informative. The present work demonstrates the characteristic pro- files of the chloroplast genome of Citrus tachibana and Citrus depressa, two indigenous species in Japan, using a number of accessions grown in various regions based on the results of matK sequencing. Furthermore, the diver- gence of the cpDNA genome of C. depressa seems to in- dicate a polyphyletic origin of this species. These findings are a contribution to progress in the study of the genetic resources in Citrus and related genera. Acknowledgements We would like to express our deep appreciation to Dr. Tetsuo Koyama, Kochi Prefectural Botanical Garden for help in obtaining tachibana samples. We also acknowl- edge the help of staff of the following prestigious shrines, Dazaifu Tenmangu, Kitano Tenmangu, Heian Jingu, and Iwashimizu Hachimangu, for supplying tachibana samples from their sacred trees. 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