Yosuke Tashiro

Successful interspecific hybridization between Cucumis sativus L. and C. hystrix Chakr.

Interspecific F1 hybrids were obtained from a cross between Cucumis sativus L. (2n = 2x = 14) and C. hystrix Chakr. (2n = 2x = 24). Controlled crossing resulted in fruit containing embryos which were excised and rescued on a Murashige and Skoog solid medium. A total of 59 vigorous plants were obtained from a fruit containing 159 embryos (37.3% regeneration rate). Hybrid plants were morphologically uniform. The multiple branching habit, densely brown hairs (especially on corolla and pistil), orange-yellow collora, and ovate fruit of F1 hybrid plants were similar to that of the C. hystrix paternal parent. While appearance of the first pistillate flower was more similar to that of C. sativus maternal parent than to C. hystrix, staminate flower appearance was mid-parent in occurence. The diameter and internode length of stem, shape and size of leaves and flowers were intermediate when compared to the parents. An elongated green, trilobate style/stigma which was not apparent in either parent was observed in staminate flowers of F1 plants. Similarly, the style/stigma of pistillate flower of F1 plants were longger when compared to their parents. The brown pubescence observed on pistillate flowers of the F1 and C. hystrix was not observed on the C. sativus parent. The somatic chromosome number of F1 plants was 19. Two morphologically distinct groups of chromosomes were observed in the F1 hybrid; 7 relatively large chromosomes characteristic of C. sativus, and 12 smaller chromosomes characteristic of C. hystrix. Analysis of malate dehydrogenase isozyme banding patterns provided additional comfirmation of hybridity. Reciprocal crossing of F1 plants to either parent and self-crossing indicated that the hybrids were male and female sterile.

AFLP linkage group assignment to the chromosomes of Allium cepa L. via monosomic addition lines

Abstract Two complete sets of Allium fistulosum L.– A. cepa monosomic addition lines (2n=2x+1=17) together with an AFLP linkage map based on a cross between A. cepa and A. roylei Stearn were used to re-evaluate the eight A. cepa linkage groups identified in the mapping study. The linkage groups could be assigned to individual, physical chromosomes. The low level of molecular homology between A. cepa and A. fistulosum enabled the identification of 186 amplified fragment length polymorphisms (AFLP™ markers) present in A. cepa and not in A. fistulosum with ten different primer combinations. With the monosomic addition lines the distribution of the markers over the eight chromosomes of A. cepa could be determined. Of these 186 AFLP markers 51 were absent in A. roylei and consequently used as markers in the mapping study (A. cepa ×A. roylei cross). Therefore, these 51 AFLP markers could be used to assign the eight A. cepa linkage groups identified in the mapping study to physical chromosomes. Seven isozyme and three CAPS markers were also included. Two of the linkage groups had to be split because they included two sets of markers corresponding to different chromosomes. A total of 20 (approx. 10%) of the A. cepa-specific AFLP markers were amplified in more than one type of the monosomic addition lines, suggesting unlinked duplications. The co-dominant isozyme and CAPS markers were used to identify the correspondence of linkage groupsoriginating from A. cepa or from A. roylei.

Development of microsatellite markers in cultivated and wild species of sections Cepa and Phyllodolon in Allium

The potential of microsatellite markers for use in genetic studies has been evaluated in Allium cultivated species (Allium cepa, A. fistulosum) and its allied species (A. altaicum, A. galanthum, A. roylei, A. vavilovii). A total of 77 polymerase chain reaction (PCR) primer pairs were employed, 76 of which amplified a single product or several products in either of the species. The 29 AMS primer pairs derived from A. cepa and 46 microsatellites primer pairs from A. fistulosum revealed a lot of polymorphic amplicons between seven Allium species. Some of the microsatellite markers were effective not only for identifying an intraspecific F1 hybrid between shallot and bulb onion but also for applying to segregation analyses in its F2 population. All of the microsatellite markers can be used for interspecific taxonomic analyses among two cultivated and four wild species of sections Cepa and Phyllodolon in Allium. Generally, our data support the results obtained from recently performed analyses using molecular and morphological markers. However, the phylogeny of A. roylei, a threatened species with several favorable genes, was still ambiguous due to its different positions in each dendrogram generated from the two primer sets originated from A. cepa and A. fistulosum.

Chromosome and cytoplasm analyses of somatic hybrids between onion (Allium cepa L.) and garlic (A. sativum L.)

Chromosomes and cytoplasms were analyzed in two lines of a somatic hybrid between onion (Allium cepa L.) and garlic (A. sativum L.). One line of the somatic hybrid had 40 chromosomes and the other 41chromosomes. Genomic in situhybridization successfully revealed the chromosome constitution of the two lines. One line had 20 chromosomes from onion and17 chromosomes from garlic, and the other had 21 chromosomes from onion and 17chromosomes from garlic. Interestingly, both lines had three chimeric chromosomes. PCR-RFLP analyses of chloroplast and mitochondrial DNAs of both lines showed that these were identical to the onion parent.

Comparative analysis of organelle DNAs in acid citrus grown in Japan using PCR-RFLP method

PCR-RFLP analyses of three regions for each of chloroplast DNA (cpDNA; rbcL-ORF106, trnD-trnT, trnH-trnK) and mitochondrial DNA (mtDNA; nad7/exon2-exon3, nad7/exon3-exon4, 18S-5S) were performed in 26 cultivars of acid citrus grown in Japan to identify polymorphisms and classify them. The polymorphisms were compared with those of three true Citrus species, i.e., mandarin, pummelo and citron. Ichang papeda (C. ichangensis) was also included in this study to find its relationship with Yuzu. Inter-species cpDNA variation was recognized and the acid citrus were divided into three groups, namely; I (‘Yuzukichi’ and ‘Kinkoyu’), II [sour oranges (‘Kaiseito’, ‘Daidai’ and ‘China daidai’), ‘Nansho daidai’, ‘Kiku daidai’, C. sudachi (‘Mushi yukaku’, ‘Yushi yukaku’ and ‘Yushi mukaku’), C. sphaerocarpa (‘Kabosu’ and ‘Aka kabosu’), C. kizu (‘Taninaka kizu’, ‘Kinosu’ and ‘Kizu’), ‘Zanbo’, ‘Mochiyu’, ‘Jabara’ and ‘Naoshichi’], and III [Yuzu (‘Tetraploid’, ‘Tochikei yuzu’ and ‘Yamanekei yuzu’), ‘Matsuda sudachi’, ‘Zuishoyu’, ‘Hanayu’ and ‘Yuko’]. CpDNA restriction patterns of the three true Citrus species differed from each other as well as from those of ichang papeda. CpDNA restriction patterns of group I of the acid citrus were identical to those of mandarins. Group II showed the same as pummelos. CpDNA restriction patterns of group III were differed from those of the three true Citrus species in the three regions. This group was differed from ichang papeda after digestion of trnH-trnK PCR products with TaqI, HinfI and AluI, while they showed identical restriction patterns in two regions, rbcL-ORF106 and trnD-trnT. Citrons and ichang papeda were placed in groups IV and V, respectively. Based on mtDNA restriction patterns, the acid citrus were divided into three groups; i, ii and iii. In groups i and ii accessions of groups I and II of cpDNA were placed with mandarins and pummelos, respectively. In group iii accessions of group III of cpDNA were placed with ichang papeda. Citrons were placed in a distinct group, iv.

Chromosomal location of a pollen fertility-restoring gene, Rf , for CMS in Japanese bunching onion ( Allium fistulosum L.) possessing the cytoplasm of A. galanthum Kar. et Kir. revealed by genomic in situ hybridization

In a previous study, we developed cytoplasmic male sterile lines of Allium fistulosum possessing the cytoplasm of A. galanthum, a wild species, by continuous backcrossing. Furthermore, we reported the presence of a pollen fertility-restoring gene (Rf) for cytoplasmic male sterility (CMS) in A. fistulosum from segregation of pollen fertility of backcross progenies. In the present study, genomic in situ hybridization (GISH), using genomic DNA of A. galanthum as the probe DNA and that of A. fistulosum as the blocking DNA, was applied to F1 hybrids between both species and backcross progenies to determine the chromosomal location of the Rf locus. By means of GISH, eight chromosomes from A. galanthum were clearly discriminated from those of A. fistulosum in the F1 hybrids, and chromosome substitution process through continuous backcrossing was visualized. Interestingly, the chromosome region from A. galanthum, specific to male fertile plants, was detected in one chromosome of BC4 to BC7 generations. Based on the karyotype analysis of the male fertile plants, the chromosome was identified as the 5F chromosome. Our results confirm that the Rf locus is located on the 5F chromosome of the male fertile plants. This is the first report that identified the chromosomal location of the pollen fertility-restoring gene in A. fistulosum.

Haploid induction from F1 hybrids between CMS shallot with Allium galanthum cytoplasm and common onion by unpollinated flower culture

The induction of haploid plants from F1 hybrids between CMS shallot with Allium galanthum cytoplasm and common onion was examined. Starting with 535 unpollinated flowers cultured in B5 medium 25 seedlings from part henogenetic embryos were obtained of which 13 seedlings survived. Eleven seedlings were determined as haploid plants (2n = x = 8) and 2 seedlings were doubled haploid plants (2n = 2x = 16). All haploid and doubled haploid plants preserved chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) from A. galanthum. Segregation in different characters was observed among the haploid plants. The haploid and doubled haploid plants exhibited the different combinations of genes from shallot and common onion. Crossing of the doubled haploid plants with other shallot strains, common onion cultivars or related species may produce excellent F1 hybrids for bulb production.

Maternal inheritance of chloroplast DNA in intergeneric sexual hybrids of true citrus fruit trees revealed by PCR-RFLP analysis

Summary PCR-RFLP analysis was employed to clarify the inheritance pattern of chloroplast DNA (cpDNA) in six kinds of intergeneric sexual hybrids among five genera, i.e. Fortunella, Eremocitrus, Poncirus, Microcitrus and Citrus within the “true citrus fruit trees” group of the subtribe Citrinae (Rutaceae). Two cpDNA gene regions were amplified from each material via PCR as following: one region of tRNA (trnD-trnT) and a variable region bounded by the conserved sequences in ribulose-1,5-bisphosphate carboxylase large subunit (rbcL) and open reading frame 106 (ORF106). Twelve restriction endonucleases (MspI, XbaI, RsaI, AluI, HinfI, ScrFI, StyI, HaeIII, Sau3AI, NdeII, HhaI and TaqI) were used for RFLP analysis of the amplified regions of cpDNA. Polymorphisms were detected with five (HinfI, RsaI, NdeII, ScrFI and XbaI) and four (HinfI, RsaI, HaeIII and Sau3AI) enzymes for rbcL-ORFl06 and trnD-trnT regions, respectively. All progeny examined in each kind of the intergeneric sexual hybrid exhibited maternal inheritance of cpDNA. Also, cpDNA analysis for determining the maternal parent of a natural hybrid, Microcitrus virgata (Sydney hybrid) indicated that M. australis (Planch.) Swing. was the female parent of M. virgata.

Biochemical affinities between Cucumis hystrix Chakr. and two cultivated Cucumis species (C. sativus L. and C. melo L.) based on isozyme analysis

Isozymes of eight enzymes in Cucumis hystrix Chakr. and two cultivated Cucumis species (C. sativus L. and C. melo L.) were analysed electrophoretically to investigate the biosystematics of these three species. Cluster analysis using data from six enzymes indicated that considerable genetic distance existed between both C. hystrix and melon and between C. hystrix and cucumber. C. hystrix might be a key species for studying the evolution and taxonomy of genus Cucumis.

Genetic analysis of phosphoglucoisomerase isozymes in cultivated and wild species of section Cepa in Allium

Phosphoglucoisomerase (PGI) was studied to establish the genetic system of the isozymes in section Cepa of Allium. The results of isozyme analyses using 15 cultivars and clones in two cultivated species A. fistulosum and A. cepa, eight strains in four wild species A. altaicum, A. galanthum, A. oschaninii and A. vavilovii and a total of 10 hybrids between A. fistulosum and A. cepa and between the cultivated and wild species revealed that: 1) PGI was a dimeric enzyme; 2) the two cultivated and four wild species had a common gene locus Pgi-1 where six alleles were identified; and 3) A. fistulosum had Pgi-1F-1 and Pgi-1F-2, A. cepa had Pgi-1A-1 and Pgi-1A-2, A. altaicum had Pgi-1F-1, A. galanthum and A. vavilovii had Pgi-1A-1, and A. oschaninii had Pgi-1A-1, Pgi-1O-1 and Pgi-1O-1. The intra- and inter-specific polymorphisms of PGI isozymes are expected to be valuable tools for the genetics and practical utilization of the wild species in section Cepa.