Shiro Isshiki

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.

Genetic variations of isozymes in cultivated sesame (Sesamum indicum L.)

Patterns of variation for seven enzyme systems were studied in 68 accessions of cultivated sesame (Sesamum indicum L.), 12 from Japan, 15 from Korea and 41 from Thailand. Only one enzyme system, isocitrate dehydrogenase (IDH), of these exhibited variation. The IDH isozymes were shown to be controlled by a single locus (Idh) with two alleles. The two alleles were widely distributed in the accessions from the three countries. As few gene markers which have simple genetic control are available in sesame, these IDH isozymes could contribute to a range of studies in the breeding and genetics of sesame.

Molecular characterization and genetic diversity among Japanese acid citrus (Citrus spp.) based on RAPD markers

Summary Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate genetic similarity and inter-relationship among31 acid citrus species and cultivars, including sour oranges (six accessions); ‘Yuzu’ (four accessions) andits relatives (21 accessions). Out of the 60 decamer primers screened, 27 were selected which produced 108 markers; 76 of which were polymorphic. Species or cultivar-specific RAPD markers were also found. A dendrogram based on genetic distance implied that sour oranges were very distinct from ‘Yuzu’ and its relatives. ‘Yuzu’ accessions were very closely linked to each other, however; for the other specimens genetic polymorphism could easily be detected by RAPDs and the genetic variation between accessions was quite high and revealed their different origins. In this study some RAPDs allowed the distinction of very close cultivars, for instance ‘Kabosu’ from ‘Aka kabosu’.

RFLP analysis of mitochondrial DNA in eggplant and related Solanum species

RFLP analysis of mitochondrial DNA (mtDNA) was performed by the Southern hybridization method using total DNA extracted from eggplant, Solanum melongena L., and six related Solanum species, S. surattense Burm. (i.e. S. virginianum L.), S. torvum Swartz, S. gilo Raddi (i.e. S. aethiopicum), S. integrifolium Poir. (i.e. S. aethiopicum), S. indicum auct. non L. (i.e. S. violaceum Ort.) and S. sanitwongsei Craib. Forty-one fragments were detected by the analysis using 12 combinations of four restriction enzymes and three probes of mtDNA clones from rice. Thirty-four out of the 41 fragments were polymorphic among the species, whereas the other seven were monomorphic. This RFLP analysis of mtDNA is demonstrated to be appropriate for assessing phylogenetic relationships in eggplant and related Solanum species at the interspecific level.

Cultivar identification of 'Yuzu' (Citrus junos Sieb. ex Tanaka) and related acid citrus by leaf isozymes

Abstract Leaf extracts of 27 ‘Yuzu’ and related acid citrus cultivars were analyzed using polyacrylamide gel electrophoresis for isozyme variation of glutamate oxaloacetate transaminase (GOT) and shikimate dehydrogenase (SDH). SDH yielded 12 different isozyme phenotypes and six cultivars were discriminated by this enzyme alone. GOT produced 10 different isozyme phenotypes and four cultivars were separated. When both enzyme systems were taken together, 16 cultivars (59%) were uniquely discriminated and the rest could be classified into four groups of 2–4 cultivars each. Mutation originated cultivars could not be discriminated. Differences between cultivars suggested that isozymes may provide useful markers for cultivar identification.

Functional male-sterility expressed in eggplant (Solanum melongena L.) containing the cytoplasm of S. kurzii Brace & Prain

Summary In order to develop a male-sterile eggplant (Solanum melongena L.), cytoplasm substitution lines of eggplant were produced by continuous backcrossing using S. kurzii Brace & Prain as cytoplasm donor and eggplant as nucleus donor. Analyses of chloroplast DNA and mitochondrial DNA confirmed that all backcross progenies had the cytoplasm from S. kurzii. Anthers in some BC1 and BC2 plants, and in most BC3 plants, were of the indehiscent-type (i.e., pollen non-release type). Pollen fertility remained low in all backcross progenies. The number of seeds per fruit in the backcross progenies increased in successive generations. Functional male-sterility of the anther- indehiscent-type was shown to be attributed to incompatibility between the cytoplasm of S. kurzii and nuclear genes of S. melongena. The present male-sterile lines are potentially valuable to reduce the cost of developing hybrid cultivars and to produce seedless eggplant fruit.

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.

Fertility restoration of hybrids between Solanum melongena L. and S. aethiopicum L. Gilo Group by chromosome doubling and cytoplasmic effect on pollen fertility

Reproductive fertility traits were studied in the reciprocal hybrids of the eggplant(Solanum melongena L.) and S. aethiopicum L. Gilo Group, and in synthetic amphidiploids to discover whether fertility in these reciprocal hybrids was restored by chromosome doubling. Isozyme and RAPD analyses confirmed hybridity of the hybrids and amphidiploids. Analyses of chloroplast and mitochondrial DNAs confirmed that the cytoplasm of each of the hybrids and amphidiploids was from the maternal parent. Pollen sterility of S. melongena × S. aethiopicum Gilo Group [F1 (Mel × Aet)] was restored by chromosome doubling, while the reciprocal hybrid S. aethiopicum Gilo Group ×S. melongena [F1 (Aet × Mel)]and its amphidiploid did not produce any pollen grains; their microspores degenerated without being released from tetrads. Hence the cytoplasm of S. aethiopicum Gilo Group seems to beresponsible for their pollen-non-formation type sterility of the hybrid. Both the F1 hybrids did not set any fruits by either selfing or backcrossing, while their amphidiploids set fruits after pollinating with pollen from the amphidiploid of F1 (Mel × Aet). Seeds obtained from both the amphidiploids germinated normally. Chromosome doubling has been effective in restoring fertility of the hybrids.

Genetic control of isozymes in eggplant and its wild relatives

SummaryThis study was conducted to elucidate the inheritance and linkage relationships of isozymes in aspartate aminotransferase (AAT, EC 2.6.1.1), alcohol dehydrogenase (ADH, EC 1.1.1.1), phosphogluconate dehydrogenase (PGD, EC 1.1.1.43), phosphoglucomutase (PGM, EC 2.7.5.1) and shikimate dehydrogenase (SKDH, EC 1.1.1.25) in eggplant and its wild relatives. Segregating populations were generated by backcrossing of hybrids among the species. Evidence of Mendelian inheritance was obtained for seven loci: Aat-1, Adh-1, Adh-2, Pgd-1, Pgm-1, Pgm-2 and Skdh-1. Twenty-one pairs of loci were tested for independent assortment, suggesting three linked pairs, Aat-2 with Pgd-2 (R=0.35±0.07), Adh-2 with Pgm-1 (R=0.33±0.07) and Pgd-2 with Pgm-2 (R=0.32±0.06).

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.