Tomoya Miyazaki

Saga University, Сага Япония, Saga, Japan

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Publications (4)2.84 Total impact

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    ABSTRACT: Integration of previously developed Allium cepa linkage maps requires the availability of anchor markers for each of the eight chromosomes of shallot (A. cepa L. common group Aggregatum). To this end, eight RAPD markers originating from our previous research were converted into SCAR markers via cloning and sequencing of RAPD amplicons and designing of 24-mer oligonucleotide primers. Of the eight pairs of SCAR primers, seven resulted in the amplification of single bands of the original RAPDs, and the remaining primer set amplified an additional band. The results of Southern hybridization using RAPD amplicons from genomic DNA of Japanese bunching onion (Allium fistulosum L.)—shallot monosomic addition lines indicated that five SCAR markers were single shallot chromosome-specific markers and were not detected in genomic DNA of A. fistulosum. The eight SCAR primer pairs were applied to other Allium species and exhibited three types of amplification profiles, namely RAPD amplicons observed only in shallot, in shallot and Allium vavilovii, and in several Allium species. A mapping study using 65 F2 plants generated by the selfing of one interspecific cross A. cepa × Allium roylei individual integrated the SCAR marker SAOE17500 into chromosome 5 as expected. The results of the present study show that the eight SCAR primer sets specific to shallot can facilitate the mapping in A. cepa and can also serve as anchor points between maps of different Allium species.
    Scientia Horticulturae 01/2007; 115(4):323-328. DOI:10.1016/j.scienta.2007.10.016 · 1.37 Impact Factor
  • Masayoshi Shigyo · Tomoya Miyazaki · Yosuke Tashiro ·
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    ABSTRACT: Randomly amplified polymorphic DNA (RAPD) analysis was carried out to develop sufficient numbers of PCR-based genetic markers in two cultivated and four wild species of sections Cepa and Phyllodolon in Allium. Sixty random decamer primers were examined, generating large numbers of RAPD bands between the species. A total of 393 RAPDs were detected between the cultivated species, A. fistulosum and shallot (A. cepa var. ascalonicum). The number of RAPDs between A. fistulosum and the four wild species, A. altaicum, A. galanthum, A. oscaninii, A. vavilovii, was respectively 167, 269, 363, 365 and those between shallot and the wild species was respectively 454, 398, 390, 156. These RAPDs will be useful as genetic markers in the two sections. Some of the RAPDs were effective for identifying interspecific hybrids. The RAPD markers successfully confirmed the hybrid nature in ten kinds of F1 plants. Research was also done on the origin of A. wakegi which is well known as a natural hybrid between A. fistulosum and shallot. In 27 clones, species-specific RAPD markers proved the hybrid origin of this plant and that its parental plants are the above mentioned species. Further, the RAPD variations observed in shallot and A. wakegi indicate a polyphyletic generation of initial clones in this species.
    Journal of Horticultural Science and Biotechnology 01/2002; 77(3):373-377. · 0.54 Impact Factor
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    ABSTRACT: Phylogenetic relationships among two cultivated species, Allium fistulosum and A. cepa, and four wild species, A. altaicum, A. galanthum, A. oschaninii, and A. vavilovii, in section Cepa were investigated based on RFLPs of mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA). Using the group average method, cluster analysis was performed in the six species and dendrograms of mt and cpDNAs were constructed. A. cepa formed a subcluster with A. vavilovii at small distance value in the dendrogram of mtDNA. Furthermore, these two species had no distance value each other in dendrograms of cpDNA. A. fistulosum formed subclusters with A. altaicum at small distance value in both dendrograms of mt and cpDNAs. These results represent the close relationships of A. cepa with A. vavilovii and of A. fistulosum with A. altaicum. Because of large distance value of A. oschaninii with other five species in dendrograms of mt and cpDNAs, A. oschaninii is considered to have a long genetic distance from others. This study demonstrates that phylogenetic relationships among the species on RFLPs of mtDNA closely correspond to those of cpDNA in section Cepa.
    Engei Gakkai zasshi 01/2001; 70(2):195-201. DOI:10.2503/jjshs.70.195
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    Masayoshi Shigyo · Tomoya Miyazaki · Shiro Isshiki · Yosuke Tashiro ·
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    ABSTRACT: The chromosomal locations of randomly amplified polymorphic DNA (RAPD) mark-ers were examined in shallot (Allium cepa L. Aggregatum group). A series of alien monosomic addition lines of Japanese bunching onion (A. fistulosum L.) with extra chromosomes (1A–8A) from A. cepa Aggregatum group was used as plant materials. Several decamer (OPERON KITs A, E, and G) and 12-mer (Wako DNA Oligomer sets A-1, C-4, F-4, and F-5) oligonucleotides were applied as random primers. The chro-mosomal locations of the 16 RAPD markers were determined; the RAPD marker OPE09 1600 was located on the chromosome 1A of A. cepa Aggregatum group, RAPD markers were assigned to all the chromosomes of A. cepa Aggregatum group. Shallot, Allium cepa L. Aggregatum group, has the high-est adaptability to tropical and sub-tropical zones in edible Alliums and is an important crop in these zones (Astley et al., 1982). A series of alien monosomic addition lines (AMALs) of Japanese bunching onion (A. fistulosum L.) with extra chromosomes from A. cepa Aggregatum group was produced in our previous study (Shigyo et al., 1996). Further, we assigned several genetic markers (10 isozyme and one 5S rRNA genes) to all the chromosomes of A. cepa Aggregatum group (Shigyo et al., 1994, 1995a, 1995b, 1996). These genetic markers are useful as chromosome markers in A. cepa Aggregatum group. It is necessary to develop many more chromosome markers on each chromo-some for advancing genetics and breeding in Allium. Amplification of genomic DNA by PCR with the single primers of arbitrary sequence (10 to 12 nucleotides) pro-vided polymorphic DNA bands called Randomly Amplified Polymorphic DNA (RAPD). The RAPDs were proposed as useful genetic markers in plant genetics and breeding (Wil-liams et al., 1990). The results of the RAPD analyses us-ing homozygous single chromosome recombinant lines and alien addition lines in wheat demonstrated that RAPD markers were effective in the analysis of genotypes where single chromosomes or chromosome segments were to be manipulated (Devos and Gale, 1992). In Allium, the RAPD analyses have been performed to assist resistance breeding (De Vries et al., 1992), to exam-ine variabilities among species (Wilkie et al., 1993) and cultivars (Roxas and Peffley, 1992), to assess integrities of inbreds (Bardeen and Havey, 1995) and doubled haploid lines (Campion et al., 1995), to confirm hybridities (Dubouzet et al., 1996), and to study phylogenetic relationships (Maaβ and Klaas, 1995; Hong et al., 1996). However, no chromo-somal locations of RAPD markers have been thus far de-termined in Allium. In order to obtain a large number of chromosome mark-ers of A. cepa Aggregatum group, the chromosomal loca-tions of RAPD markers were examined using the series of the AMALs. A series of the AMALs of A. fistulosum with extra chro-mosomes from A. cepa Aggregatum group was used as plant materials. Two plants were analyzed for each AMAL. A. fistulosum and A. cepa Aggregatum groups were also used as controls. Total DNA was isolated from the leaves or flo-rets by the micro extraction method according to Dubouzet et al. (1996). The reaction mixture (25 µl) for PCR was composed of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dCTP, 0.2 mM dTTP, 3.75 pM primer polynucleotide, 25 ng template DNA, and 0.5 unit Taq DNA polymerase, and overlaid with a drop of mineral oil. Amplification was carried out in a Program Temp Control System PC-800 (Astec, Shime, Japan) with
    Genes & Genetic Systems 01/1997; 72(4):249-252. DOI:10.1266/ggs.72.249 · 0.93 Impact Factor