JXB Advance Access originally published online on January 10, 2005
Journal of Experimental Botany 2005 56(412):587-596; doi:10.1093/jxb/eri047
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RESEARCH PAPER |
Analysis of mutations induced by carbon ions in Arabidopsis thaliana*
Department of Ion Beam Applied Biology, Japan Atomic Energy Research Institute (JAERI), Watanuki-machi 1233, Takasaki, Gunma 370-1292, Japan
To whom correspondence should be addressed. Fax: +81 27 346 9688. E-mail: naoya{at}taka.jaeri.go.jp
Received 6 July 2004; Accepted 8 October 2004
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Abstract |
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To investigate the nature of mutations induced by accelerated ions in higher plants, the effects of carbon-ion-irradiation were compared with those of electron-irradiation in Arabidopsis thaliana. Point-like mutations and rearrangements were induced at a similar frequency after carbon-ion-irradiation, whereas point-like mutations were more frequently induced after electron-irradiation. Sequence analysis revealed that carbon-ion-induced point-like mutations were mostly short deletions. In the case of rearrangements, deletions, inversions, insertions, and translocations were found. The estimated frequency of deletion induction was comparable to that of fast neutrons. Analysis of chromosome breakpoints revealed that carbon ions frequently deleted small regions around the breakpoints, whereas electron-irradiation often duplicated these regions. Moreover, for both types of radiation, broken ends with microhomologies were frequently rejoined. Results of the breakpoint and broken end analyses suggest that non-homologous end-joining (NHEJ) leads to the rejoining of double strand breaks (dsbs) after cells are exposed to both types of radiation, but the type of NHEJ that occurs as a result of damage is different. The results indicated that carbon-ion-induced mutations are most likely nulls and that the induced rearrangements may arise through a unique mechanism. These findings indicate that accelerated ions are a useful mutagen for both forward and reverse genetics for plants.
Key words: Arabidopsis thaliana, carbon ions, deletion, forward/reverse genetics, non-homologous end-joining, null mutation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Forward genetics has been a powerful means to dissect plant function. However, one mutation often only describes a part of the function of a gene, and only through analysis of numerous alleles can one identify the gene function (Hirschi, 2003
). A novel mutagen may show a mutation spectrum different from those induced by known mutagens, and thus would be valuable for forward genetics. The reverse genetics approach is also essential for gene function analysis. Mutagenesis, along with new technologies such as RNA interference and co-suppression, have been successful at inducing new phenotypes by inactivating a sequenced gene whose function was unknown. However, RNA interference and co-suppression do not entirely ensure that phenotypic traits will be inherited through subsequent generations. In addition, these methods can not always cause a complete null mutation. With these methods, predicting gene function based on a mutated phenotype is not straightforward, because it is not possible to know the extent of inactivation.
The most widely used mutagens in plant genetics are T-DNAs/transposons, chemicals, and ionizing radiation. Insertional mutagenesis has been so far one of the most common ways to produce plant materials for reverse genetics. Projects aiming to create T-DNA insertion lines that contain inserts in each and every gene in Arabidopsis have been set up and Alonso et al. (2003) identified mutations with T-DNA inserts in more than 21 700 of the 
29 454 predicted Arabidopsis genes. In studies with insertional mutagenesis, insertion is assumed to inactivate the gene completely. However, it has been shown in T-DNA-integrated mutants that a part of the gene was transcribed from the region within the T-DNA in some cases (Friesner and Britt, 2003). Thus, caution should be exercised in deducing the gene function from the phenotype of the insertion mutant.
Chemical mutagens and ionizing radiation have long been used as plant mutagens in forward genetic studies. They have a complementary role or even an advantage over insertion mutagenesis in terms of (1) generating varying types of alleles, (2) acquiring mutations with high frequencies, and (3) being easily applicable to various plant species. Chemicals mainly induce point mutations, and are thus considered to be suitable for producing missense mutations, which would provide a series of change-of-function mutations. Ethylmethane sulphonate (EMS), a commonly used chemical mutagen, has been shown mainly to induce base substitutions (G:C to A:T transition) in Arabidopsis (Colbert et al., 2001). A technique called TILLING (targeting induced local lesions in genomes) has been recently developed to identify a point mutation in the genome of a plant, making the reverse genetic approach applicable with this type of mutagen (Colbert et al., 2001). On the other hand, ionizing radiation is reported frequently to induce rearrangements, including deletions, in plant genomes (Shirley et al., 1992; Bruggemann et al., 1996; Cecchini et al., 1998; Shikazono et al., 1998, 2001) and induction of mutants by ionizing radiation has proved to be valuable in the fields of genetics and breeding. To investigate gene function, gene deletions are considered most valuable, because they could be defined as nulls. In Saccharomyces cerevisiae, researchers have created a nearly complete collection of deletion mutants, in which up to 96% of the annotated open reading frames were deleted to determine the consequence of loss of gene function (Giaever et al., 2002). Recently, a fast-neutron deletion mutagenesis-based reverse genetics system was developed to identify and isolate targeted plant genes (Li et al., 2001). Li et al. (2001) claimed that the reverse genetics system using fast neutron-generated deletions is highly efficient and that fast neutrons could develop mutant lines with complete coverage much easier than could T-DNA insertional mutagenesis.
Accelerated ions, a kind of ionizing radiation, are usually more than 10-fold more effective per unit dose in creating somatic and germline mutations in plants than other sources of radiation such as X-rays, 
-rays, or electrons (for a review see Smith, 1972; Shikazono et al., 2003), and are known to cause more localized, dense ionization within cells than X-rays, -rays, or electrons (Blakely and Kronenberg, 1998). Accelerated ions are thought to produce more closely positioned, clustered DNA damage sites, and such damage has been correlated with biological effectiveness (Goodhead, 1994; Ward, 1994; Ottolenghi et al., 1995; Nikjoo et al., 1999). Therefore, ions appear not only to induce mutations more frequently per unit dose than X-rays, -rays, and electrons, but also to generate mutations of a different nature from those produced by X-rays, -rays, and electrons. Using carbon ions, several novel Arabidopsis mutants have recently been isolated (ast, frl1, uvi1, suv1, tt18, and tt19) (Tanaka et al., 1997b, 2002; Hase et al., 2000; Sakamoto et al., 2003; Shikazono et al., 2003; Kitamura et al., 2004). A detailed molecular analysis of the mutations induced by ions is needed to determine whether they differ fundamentally from those of other types of radiation. However, few investigations of mutations have been carried out in plants. Although eight carbon-ion-induced mutations have been analysed in Arabidopsis thaliana (gl1-3, tt4(C1), ttg1-21, suv1-1, tt18-1, tt18-2, tt19-1, and tt19-2) at the nucleotide sequence level, and found that they contain inversions, translocations, and short deletions (Shikazono et al., 1998, 2001, 2003; Sakamoto et al., 2003; Kitamura et al., 2004), the analysed number of mutants is still limited and the nature of accelerated-ion-induced mutations remains to be elucidated in plants.
In the present study, the structural alterations of the mutations were analysed to gain insight into the nature of the mutations and into the applicability of accelerated ions in plant forward and reverse genetics. Carbon-ion- and electron-induced mutations were examined by PCR and by DNA sequencing. It was shown that carbon ions induced rearrangements at a frequency similar to the frequency of point-like mutations, whereas electrons predominantly generated point-like mutations. Sequence analyses of the point-like mutations showed that carbon ions mostly induce short deletions. From the analysis of rearrangements, deletions were found to be generated at a frequency of 6.1x10–5, which was comparable to that induced by fast neutrons (Li et al., 2001). It is further suggested that carbon ion- and electron-induced rearrangements are generated through different mechanisms. These results imply that mutagenesis by accelerated ions could be uniquely used for both forward and reverse genetics in plants.
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Materials and methods |
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Plant material
Plants of Arabidopsis thaliana ecotype Columbia were grown on metro-mix (HYPONEX Co. Ltd., Japan) or on rock wool (Nichiasu Co. Ltd., Japan) at a temperature of 25±3 °C in an air-conditioned greenhouse, and were sub-irrigated at 3 d or 4 d intervals with 0.03% HYPONeX (HYPONEX Co. Ltd., Japan).
Irradiation
Dry seeds were irradiated as previously described (Tanaka et al., 1997a; Shikazono et al., 2002). The carbon ion energy was 220 MeV and the mean LET within the seeds was calculated to be 113 keV µm–1. The energy of the electrons was 2 MeV and the mean LET was calculated to be 0.2 keV µm–1. The dry seeds were irradiated with a dose of 150 Gy for carbon ions, and 750 Gy for electrons. Isolation of tt and gl mutants was carried out as previously described (Shikazono, 2003). Results of gl1-3, tt4(C1), ttg1-21, tt18-1, tt18-2, tt19-1, and tt19-2 (Shikazono et al., 2001, 2003; Kitamura et al., 2004) were included in the present analysis, as the mutants originated from the same irradiation.
DNA extraction and molecular analysis
Genomic DNA was extracted from the M3 mutants with a Plant Mini Kit (Qiagen KK, Japan) following the manufacturer's protocol. The sequences of the PCR primers are given in the supplementary material which is available at JXB on-line. Amplification was carried out at 94 °C for 10 min, followed by 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. At the end of the 40 cycles, the samples were incubated at 72 °C for another 7 min to complete extension. The amplified DNA fragments were analysed by 1.5% agarose gel electrophoresis in TAE buffer and visualized with ethidium bromide. Fragments that showed no apparent change in size were analysed by direct sequencing, whereas thermal asymmetric interlaced-PCR (TAIL-PCR) (Liu et al., 1995) was used to identify the rejoined region in fragments of the rearranged genes. Both specific and arbitrary degenerate primers were designed to amplify the unknown sequences flanking the breaks in the mutants (Liu et al., 1995; McElver et al., 2001). Primer sequences for TAIL-PCR are shown in the supplementary material which is available at JXB on-line. To analyse deletions, the deleted and unaffected regions were roughly identified by examining the products amplified from the flanking regions. Primers were then designed from this outside sequence, and the sequence containing the deletions was amplified by PCR. PCR fragments were sequenced with an automated sequencer (ABI Prism 310 Genetic analyzer, Perkin Elmer, Co., Ltd., USA). The Arabidopsis genome database (http://www.arabidopsis.org/Blast/) was searched for alignments of the sequences of the breakpoints. The sequences were further analysed with the GENETYX MAC program (version 10.0, Software Development Co., Ltd., Japan).
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Results |
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Carbon-ion- and electron-induced mutations were compared in Arabidopsis. Electrons are considered to have the same biological effects as
-rays. gl and tt mutants were screened from a total of 104 088 M2 plants irradiated with carbon ions, and from a total of 80 827 M2 plants irradiated with electrons. In the previous study, approximately half of the latter plants (44 026) were used (Shikazono et al., 2003). The isolated mutants were crossed with known gl and tt mutants to identify the loci responsible for the glabrous and transparent testa phenotypes, respectively. Mutants that failed to complement known mutant lines were mapped further and the genes were identified from the map position (Shikazono et al., 2003). The estimated mutation rate per unit dose for carbon ions (1.9x10–6 gene –1Gy–1) was 20-fold higher than that for electrons (0.097x10–6 gene –1Gy–1). When considering the practical application of a mutagen, the number of mutations in a genome at a given level of sterility would become more of an important issue than the mutation rate per dose. Carbon ions induce a 20-fold increase of mutation rate per dose and a13-fold increase on frequency of sterility per dose over electrons (Shikazono et al., 2002), suggesting that carbon ions induce slightly larger numbers of mutations per genome than electrons do, when the mutagenized plants would show the same level of sterility.
PCR analysis of induced mutations
To study the nature of the mutations generated by carbon ions, the structure of the mutated genes was first looked at by PCR. A total of 29 independent carbon-ion-induced M2 mutant lines and 12 independent electron-induced M2 mutant lines was analysed. Mutants that generated PCR fragments of a size similar to the wild type were classified as having point-like mutations, and mutants that gave rise to no fragments, or fragments of reduced size, were classified as having rearrangements. Thus the term rearrangement includes deletions, inversions, translocations, and insertions. The analysed results are shown in Table 1. Of the 29 carbon-ion-induced alleles, 14 had point-like mutations and 15 had rearrangements. In the case of electrons, nine alleles had point-like mutations and three had rearrangements.
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Sequence analysis of point-like mutations
To clarify the nature of the induced mutations, the fragments amplified from the point-like mutations were directly sequenced. The changes are summarized in Table 2A. Of the 14 point-like mutations induced by carbon ions, 11 were deletions of between 1 bp and 100 bp, one had a 1 bp insertion, and two had base substitutions. Of the nine electron-induced mutations, four were deletions of between 1 bp and 8 bp, four were base substitutions, and one was a 1 bp insertion. No significant differences were observed between carbon ions and electrons with regard to the formation of frameshift mutations (deletions/insertions) or base substitutions (P=0.132 by Fisher's exact probability test).
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Sequence analysis of rearrangements
14 out of 15 carbon-ion-induced rearrangements were analysed successfully. The carbon-ion-induced rearrangements consisted of deletions, inversions, translocations, insertions, and a combination of these (Fig. 1; Table 2B). Six deletions (tt4(C7), gl1-6, gl1-4, gl1-8, gl1-7, tt6-4) were found and their size varied approximately from 5 kbp to 230 kbp. The electron-induced rearrangements consisted of inversions and translocations. Analysis of the inversions, translocations, insertions, and deletions made it possible to determine the nature of the breakpoints and sites of rejoining (Fig. 2; Tables 3, 4). Of the 17 carbon-ion-induced breakpoints, 11 had deletions (ranging in size from 1 to 29 bp) and only four had duplications (1–5 bp). On the other hand, of the eight electron-induced breakpoints, six had duplications (1–7 bp) and one had a deletion (1 bp). These frequencies were clearly different between carbon-ion-induced breakpoints and electron-induced breakpoints (P=0.0154 by Fisher's exact probability test). Twelve of the 19 rejoined sites for carbon ions and six of the seven rejoined sites for electrons used microhomologies for rejoining (Table 4). The length of microhomologies ranged from 1 bp to 5 bp for both types of radiation.
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Discussion |
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PCR and sequence analyses of mutations
Results on the effects of carbon-ion- and electron-irradiation on Arabidopsis mutants revealed that carbon ions induced point-like mutations and rearrangements at a similar frequency, whereas electrons seemed to induce mainly point-like mutations. Carbon-ion-induced mutants may be more likely to contain rearrangements (Table 1). Since the overall mutation rate for carbon ions was 20-fold higher than that for electrons, the frequencies of inducing point-like mutations and rearrangements by carbon ions are estimated to be 13-fold and 41-fold higher, respectively, than those induced by electrons. The probable reason for the higher incidence of alterations by carbon ions is that the DNA damage induced by carbon ions is more clustered.
Since point-like mutations were mainly short deletions and rearrangements are likely to disrupt a gene totally, the analysed carbon-ion-induced mutants were most likely nulls (Table 2). From the analysis of point-like mutations, most (four out of five) of the deletions and insertions with sizes of 1 bp and 2 bp induced by carbon ions and most (three out of four) of those induced by electrons were found within a run of repeated sequences. Kunkel (1990) has suggested that misalignment within a sequence run can easily lead to a deletion or an insertion, especially when the template strand is damaged. Thus, it is considered that at least some of these 1 or 2 bp deletions, together with the 1 bp insertion, probably arose from errors during DNA synthesis.
Few studies on the molecular nature of ionizing radiation-induced mutations have been carried out in higher plants. Bruggemann et al. (1996) found that most (13 out of 18) fast neutron-induced hy4 mutations in Arabidopsis were deletions larger than 5 kbp. In addition, eight out of eight gamma-induced mutations of a negatively selectable suicide marker (tms2), which was integrated in the Arabidopsis genome, were also deletions larger than 5 kbp (Cecchini et al., 1998). These high frequencies of deletion among the induced mutations were in contrast to these results in which deletions larger than several kbp were rather infrequent (occurring in only six out of 28 carbon-ion-induced mutations and in none of 12 electron-induced mutations). The reason for this discrepancy is unclear. The most likely explanation is that the loci flanking the analysed locus strongly affected the outcome. That is, the presence of an essential gene nearby eliminated the possibility of isolating a deletion accompanying the gene. In support of this hypothesis, the frequency of deletions appeared to differ among loci. In the present study, the frequency of deletions at the GL1 locus (four deletions out of six mutations) was higher than the average frequency of deletions for all the loci studied. Some loci had no deletions. The large (>5 kbp) deletions observed in the cases of hy4 and the locus where the tms gene was integrated might be due to these loci being far away from an essential gene. Alternatively, the difference of the frequency of deletions could be due to some physiological and/or irradiation conditions (such as a difference of water content in the seeds or a difference of dose rates), causing a difference in the type of DNA damage generated or a difference in the mode of repair.
The sequences flanking the breakpoints that led to the rearrangements were often deleted in carbon-ion-induced mutations, whereas they were often duplicated in electron-induced mutations (Fig. 2; Table 3). Apart from the present results, hardly any sequences of radiation-induced breakpoints have been analysed in plants. Shirley et al. (1992) demonstrated that two breakpoints in the fast neutron-induced tt5 mutant of Arabidopsis had short deletions, although the number of analysed breakpoints was limited. In the present study, short homologous sequences were often found at the rejoined junctions for both types of radiation (Fig. 2; Table 4). Because rejoining requires only very short homologies (or possibly no homology at all), these results imply that NHEJ occurs after carbon ion- and electron-irradiation. The difference in the types of mutation may arise from (1) the terminal structure of the dsbs, and/or (2) the mode of rejoining. A model of DNA strand breakage and rejoining after carbon ion and electron irradiation is shown in Fig. 3. The quality of dsb is defined by its end structure (i.e. staggered or blunt, with or without additional types of damage at the broken end). Both Monte-Carlo computer simulations on DNA damage generation and experimental evidence regarding low reparability of dsbs and chromatin breaks suggest that the amount of closely spaced single strand breaks and base damage at a broken end increases as the linear energy transfer (LET) of the radiation increases (Ottolenghi et al., 1995; Blakely and Kronenberg, 1998; Nikjoo et al., 1999; Gulston et al., 2002; Sutherland et al., 2000, 2002; Karlsson and Sternlöw, 2004). LET is the amount of energy deposited per unit length of the particle's path. Accelerated ions are considered high LET radiation, whereas X-rays, -rays, and electrons are considered low LET radiation. Rejoining following a break would affect the probability of inducing mutations, as well as the nature of the mutation. The present results indicate that the end-joining process itself may differ after high and low LET radiation. The end-joining pathway used for T-DNA integration has recently been shown to be partly, if not all, Ku independent, which is consistent with the notion that plants have at least two modes of rejoining DNA ends (Friesner and Britt, 2003; Gallego et al., 2003). Analyses of the sequences of rejoined junctions also support the idea that plants have different modes of rejoining. (Gorbunova and Levy, 1999). The indication that carbon ions and electrons have different mutagenic processes supports the notion that ion irradiation can be useful for investigating DNA repair mechanisms in plants, as well as for producing novel mutants/alleles.
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Application of ion mutagenesis to plant genetics
Novel mutants, such as ast, frl1, uvi1, suv1, tt18, and tt19, have been isolated by carbon-ion-irradiation (Tanaka et al., 1997b
, 2002; Hase et al., 2000; Sakamoto et al., 2003; Shikazono et al., 2003; Kitamura et al., 2004). It is speculated that carbon ions produce novel mutants because they induce mainly nulls. The ability to induce novel mutants is expected to be useful in forward genetics.
The number of mutations per plant at a given level of sterility becomes an important factor in terms of the practical application of a mutagen. If the value is high, one could reduce the overall number of M2 plants at the stage of selecting mutants, without facing a difficulty in obtaining viable M2 seeds. The discrepancy between the values for carbon ions and electrons seemed relatively small, suggesting that carbon ions could be practically applied to plant mutagenesis. The ratio between the frequency of chlorophyll mutation and the extent of sterility were found to be similar for X-rays and fast neutrons in Arabidopsis (Timofeev-Resovskii et al., 1971; Dellaert, 1980). It is expected that the number of mutations per genome at a given level of sterility is fairly constant for various kinds of ionizing radiation, as X-rays and electrons could be considered to have the same biological effects. Using Arabidopsis, Mesken and van der Veen (1968) estimated that the frequency of chlorophyll mutants after EMS treatment was approximately 4-fold higher than that after irradiation of X-rays at an equal level of sterility. This result indicates that, with treatments giving the same level of sterility, the number of mutations in the genome is several-fold higher for EMS-mutagenized plants than for plants exposed to carbon ions.
Li et al. (2001) identified deletion mutants for targeted genes in Arabidopsis and rice by screening fast neutron-mutagenized populations. They adjusted PCR conditions preferentially to amplify the deletion alleles and found that the frequency of deletions per M1 line isolated by the deletion-based reverse genetics system was 3.5x10–5. As carbon ions induced large deletions in three loci (tt4, tt6, gl1) out of 12 loci using 26 200 M1 lines (Shikazono et al., 2003), the frequency of deletions induced by carbon ions was estimated to be 1.1x10–5. Although a different method was used to detect deletions, the frequencies that were observed were comparable. The ability to induce deletions at a high frequency implies that accelerated ions can be useful in reverse genetics. One limitation of using carbon ions as a mutagen for creating a deletion library for a reverse genetic approach is that some deletions would affect more than one gene. For instance, in the case of the gl1-7 mutant, which carries a 233 kbp deletion, 54 ORFs are deleted. As a first step, a single gene deletion would be advantageous for reverse genetic analysis. It will be of interest to apply various kinds of ions with a range of energies to find out whether a particular kind of ion could preferentially induce deletions with several kbps in size.
The analysis of many mutants produced by various mutagens would significantly increase current knowledge of gene function in plants. Furthermore, creating deletions efficiently by ions could complement other techniques in reverse genetics (for example, genome-wide insertional mutagenesis and TILLING) for determining the function of gene products in plants.
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Supplementary data |
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Sequences of PCR and TAIL-PCR primers are available at JXB on-line as supplementary material.
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Acknowledgements |
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We thank Drs Y Hase, A Sakamoto, Y Oono, and T Hirose for helpful discussions. We are also grateful to Drs P O'Neill, R Okayasu, and Y Furusawa for critical readings of the manuscript, and to Mr M Taneishi for his technical assistance with thermal asymmetric interlaced-PCR.
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Footnotes |
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* Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos AB117787, AB117788, AB117727, AB117728, AB117729, AB117730, AB117731, AB117732, AB117733, AB117734, AB117735, AB117785, AB117786, AB117780, AB117781, AB117789, AB117790, AB117791, AB084467, AB084468, AB117792, AB111443, AB111444, AB111445, AB117793, AB117738, AB117739, AB117740, AB117741, AB117742, AB117761, AB117762, AB117763, AB117764, AB117765, AB117766, AB117772, AB117773, AB117774, AB117775, AB117776, AB117777, AB117778, AB117779, AB117767, AB117768, AB117769, AB117770, AB117771.
Present address: Namie-machi 242-6, Takasaki, Gunma, 370-0802, Japan.
Present address: Shinmatsudo-minami 1-243, Matsudo, Chiba 270-0035, Japan.
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