In the present work, the male-associated DNA sequence was analyzed in a dioecious plant, Schisandra nigra. The randomly amplified polymorphic DNA (RAPD) method was used to detect potential male-specific markers. Genomic DNA of Schisandra nigra was isolated from male and female plants and subjected to polymerase chain reaction (PCR) with decamer nucleotide primers of arbitrary sequence. Out of 120 primers tested, only one primer, OPA-17, produced a nucleotide segment of approximately 800 bp that appeared to be male specific. This male-specific segment was cloned and used as a probe for hybridization with genomic DNA. When the male DNA and female DNA were allowed to hybridize, this probe was found to be male specific. Analysis of the nucleotide sequence of the male-specific RAPD marker did not show any significant homology with previously reported sequences. The nucleotide sequence was used to design a sequence characterized amplified region (SCAR) marker of 486 bp in length and male-specific. This SCAR could be useful for a precise, early and rapid identification of male plants during breeding programs of Schisandra nigra.
The seed storage proteins of 434 strains of azuki bean, Vigna angularis (Willd.) Ohwi and Ohashi, from Japan, South Korea, Bhutan, Nepal, China (mainland) and Taiwan, were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Fifteen protein types (types I to XV) were identified on the basis of the combination of the polymorphic bands. The geographical distribution of total protein types differed among regions from where the strains originated. In Bhutan and Nepal, most strains showed region-specific types I and II, whereas a few strains had type III. Genetic variability (H’) of Bhutan and Nepal strains were 0.502 and 0.636, respectively. Four types (III, IV, V and VII) were detected in germplasm from South Korea. Of these, types III and IV were major types. They showed a genetic variability value of 0.854. All strains from China (mainland) and Taiwan belonged to type III and type IV, respectively. In Japan, 311 strains from 38 prefectures were divided into seven geographical groups, i.e., Hokkaido, North, East, Central and West Honshu, Shikoku and Kyushu. In Hokkaido, only types III and IV were detected with equal frequency, whereas type IV was dominant in other six regions. Type V strains were distributed with a low frequency in East, Central and West Honshu, and Shikoku. A small number of strains with the other nine types (VI, VIII-XV) were uniquely observed in Central Honshu, West Honshu, Shikoku and/or Kyushu. Most of these unique types were detected with the strains in the districts along the coast of the Japan Sea. Consequently, the strains of northeastern and central Japan exhibited a lower genetic variability (0.416-0.694) than those of southwestern Japan (0.619-1.286). The highest genetic variability (1.286) was found in West Honshu where 11 kinds of protein types were observed.
To analyze the genetic mechanism regulating root elongation, we characterized five recessive mutants in rice (BRX65, BRX117, BRX430, BRX448 and crl2 mutant). In fifteen-day-old seedlings, the root length of BRX65 was 36% of that of the wild type root, while the length of the roots of BRX117, BRX430 and BRX448 was about 45% of that of the wild type root. These types of mutation were designated as reduced root length with the gene symbol rrl. In contrast, the length of the crl2 mutant root was 114% of that of the wild type root. Allelism test indicated that rrl mutations occurred in two complementation groups and that these mutant genes were not allelic to crl2. The first RRL locus was designated as RRL1 (BRX65) and the other locus as RRL2, and the alleles were designated as rrl2-1 (BRX117), rrl2-2 (BRX430) and rrl2-3 (BRX448). In the rrl1 mutant, only the mature cortical cell length was significantly shorter than that of the wild type, whereas, the values of the cell length, apical meristem size and cell flux of the rrl2-1 mutant root were all clearly lower than those of the wild type root. On the other hand, the values of the mature cortical cell length, apical meristem size and cell flux of the crl2 mutant root were all clearly higher than those of the wild type. Mature cortical cell length of the rrl1crl2 double mutant was intermediate between that of the parental single mutants, while the cell length, meristem size and cell flux of the rrl2-1crl2 double mutant were all intermediate between those of the parental single mutants, respectively. These results suggest that there are genes that inhibit or promote the maintenance of root apical meristem and cell elongation, and the opposite effects between these individual genes determine the extent of root elongation in rice.
Waxy mutant wheat Tanikei A6599-4, which was induced from Tanikei A6099 (low-amylose line), exhibited a unique pasting curve with stable hot paste viscosity in the Rapid ViscoAnalyser (RVA) measurement. Analysis of the amylose content of reciprocal F1 seeds revealed the incomplete dominance and gene dosage effect in hexaploid wheat. In the dominance among multiple Wx-D1 alleles, the Wx-D1 allele of Tanikei A6099 partially dominated that of Tanikei A6599-4, and the allele of Tanikei A6599-4 partially dominated that of Tanikei H1881 (waxy line of amylose-free type). Genetic analysis using DH2 lines suggested that both the waxy character and stable hot paste viscosity of Tanikei A6599-4 are controlled by the same mutated Wx-D1 gene. We studied the pasting properties of Tanikei A6599-4 starch and compared them to those of other starches. At a low suspension concentration (6%), the peak viscosity of Tanikei A6599-4 was closer to that of Tanikei H1881 but the peak viscosity temperature was the same as that of Norin 61 (nonwaxy line). Addition of NaCl did not affect the starch pasting properties of Tanikei A6599-4, while potato starch which shows a more stable hot paste viscosity than cereal starches, was significantly affected by the NaCl treatment.
To better understand the geographic differentiation of the Perilla crops and their weedy types in East Asia, we studied the morphological variation of 60 accessions by examining 22 morphological characters. Var. frutescens (Egoma in Japanese) and var. crispa (Shiso in Japanese) showed significant geographical differences for many characters. None of them, however, individually could discriminate between the two crops as suggested by other studies. Principal component analyses clearly discriminated cultivated var. frutescens from cultivated and weedy types of var. crispa. Seed size, leaf size, plant height, branch number, flower color, leaf and stem color, plant fragrance and degree of pubescence contributed for the classification of var. frutescens and var. crispa. Seed size was the most reliable character for the discrimination between var. frutescens and var. crispa, and between var. frutescens and its weedy type. Most of the characters studied were not different between Korea and Japan, but Chinese accessions were significantly different in many characters from the Korean and Japanese accessions.
Many upland rice cultivars show higher levels of blast field resistance than do lowland cultivars. In this study, we performed a linkage mapping of quantitative trait loci (QTLs) for blast field resistance using F2 plants/F3 progenies derived from a cross between the upland variety Kahei (high level of resistance) and the lowland variety Koshihikari (susceptible). We mapped two putative QTLs on chromosome 4. qBFR4-1 was mapped in the vicinity of restriction fragment length polymorphism (RFLP) marker G264 on chromosome 4. This QTL explained about 62% of the total phenotypic variation in F3 lines. Another QTL, qBFR4-2, was also found near the RFLP marker G271 on chromosome 4. These two QTLs on chromosome 4 explained about 71% of total phenotypic variation based on the analysis of a multiple-QTL model. Alleles of Kahei increased the level of resistance in these two QTLs. The high level of resistance to blast in Kahei is mainly explained by these two QTLs. Applications of newly found QTL for breeding rice with blast field resistance is discussed.
Eleusine africana, which is an important species of the genus Eleusine, grows as an annual wild grass mainly in Africa and in some parts of India. It is an allotetraploid species with the chromosome number of 2n=4x=36 which is considered to be the progenitor of the cultivated species E. coracana. Genomic in situ hybridization (GISH) enabled to identify E. indica and E. floccifolia as ‘A’ and ‘B’ genome donors to E. africana, which are also reported to be genome donors to E. coracana. It was observed that at the genomic level the four diploid species with the chromosome number 2n=18 were closely related to the polyploid species E. africana. The genomic DNA of E. indica, E. floccifolia and E. tristachya hybridized with 15-18 chromosomes and the genomic DNA of E. intermedia labeled about 24-31 chromosomes of E. africana. The same pattern of genomic in situ hybridization of the genomic DNA of these four species with the chromosomes of E. coracana has also been reported. The similar hybridization pattern of the genomic DNA of E. indica, E. floccifolia, E. tristachya and E. intermedia on the chromosomes of the two polyploid species supports the assumption that E. africana is the progenitor of the cultivated species E. coracana.
Thirteen morphological characters including 9 simply inherited and 4 quantitative traits, were analyzed by using a genetic map constructed with F2 progenies from a cross between the peach cultivars ‘Akame’ and ‘Juseitou’. The map consisted of 92 markers (9 morphological and 83 DNA markers) covering approximately 1020cM, in which 9 linkage groups were found. Eleven SSR (simple sequence repeat) markers were mapped. Segregation of 9 qualitative traits in the F2 population fitted to the expected ratio, suggesting that these traits were controlled by single genes. Three characters relating to pigmentation showing a monogenic inheritance, i.e., flesh color around the stone, flower color (pink/pale pink) and fruit skin color, were newly identified. The fruit skin color cosegregated with the leaf color trait located on the linkage group 3. The flower color and the flesh color around the stone were located on the linkage group 4 with a recombination value of 0.13. Furthermore, several DNA markers tightly linked to flesh adhesion and 2 kinds of root-knot nematode resistance were obtained, which could be used as a reliable tool for trait selection. QTL analysis was conducted on 4 morphological traits, flowering time, maturation time, fruit dropping time, and fruit weight. Six, three, three and four QTLs were identified for these 4 traits, respectively. Among them, 12 QTLs tended to gather on the linkage groups 3 and 6, whereas no QTLs were detected on the groups 2, 4, 8 and 9.
Using fluorescence in situ hybridization (FISH), we localized transferred barnase-psl and pHctinG DNA sequences onto chromosomes in a group of rice transgenic plants cotransformed by microprojectile bombardment. In all of the tested rice transgenic lines, the detected cells showed 1-3 signal spots and they were located in the central and terminal regions of chromosome arms. The signals of both barnase-psl and pHctinG genes were mostly detected in close contact on chromosomes, which could not be resolved without using two-color in situ hybridization. In a few cells, the signals were separated from each other for barnase-psl and pHctinG.
For the production of intergeneric hybrid plants with partial alien genome via microprotoplast fusion in the Liliaceous ornamentals, the effects of the DNA synthesis inhibitor, hydroxyurea (HU), and the spindle toxins, colchicine (COL), oryzalin (ORY), amiprophos-methyl (APM), butamiphos (BUT), isopropyl N-(3-chloro-phenyl)carbamate (CIPC) and propyzamide (PRO) on the metaphase index (MI) and the percentage of micronucleated cells (micronucleus index; MNI) were examined in cell suspension cultures of Hemerocallis hybrida cv. Stella d’Oro. Suspension cells were subcultured every three days in MS medium containing 10mgl-1 picloram. Although MI was only 2-3% in the asynchronous control cultures, it increased up to 8.9% and 9.7% by treatment of the cultures with HU and COL, respectively. In addition, MI was further increased by using the sequential treatments of the cultures with HU and each spindle toxin: the highest MI of 30.5% was obtained by treatment with 2mM HU for 24h followed by that with 250μM COL for 20h. COL and ORY were more effective for synchronizing cell division than the other four spindle toxins. The effects of various spindle toxin treatments on the micronucleation of suspension cultures were also examined by combining the HU pre-treatment for 24h. Among the six spindle toxins, COL and ORY induced few micronuclei, whereas APM, BUT, CIPC and PRO induced micronucleation in cells to various extents. The most effective treatment for micronucleation was that with 8μM PRO for 66h, where MNI was 14.7% and the number of micronuclei per cell ranged from 1-7 were obtained.
Quantitative trait loci (QTLs) controlling low temperature germinability (LTG) in rice were identified using 98 backcross inbred lines (BILs) derived from a cross between a japonica variety Nipponbare and an indica variety Kasalath. Seeds of each BIL were stored at 30°C in a drying machine for 7 months to exclude the effects of seed dormancy. Then the germination rate at 15°C for 4 days was scored to represent the LTG. The LTG of Nipponbare and Kasalath was 17 and 61%, respectively, and that of the BILs ranged from 0 to 100%. Five putative QTLs, qLTG-2, qLTG-4-1, qLTG-4-2, qLTG-5 and qLTG-11, were detected on chromosome 2 (G1327: nearest marker locus), 4 (two regions, C946 and C513), 5 (R830) and chromosome 11 (G1465). In the case of qLTG-2, qLTG-4-1 and qLTG-11, Kasalath alleles increased the LTG, while Nipponbare alleles increased it in the case of qLTG-4-2 and qLTG-5. These QTLs explained 40.7% of the total phenotypic variation in the BC1F9 lines. Using chromosome segment substitution lines (SLs), the existence of qLTG-4-1 and qLTG-11 was confirmed and the effect of the Kasalath allele on qLTG-11 was more pronounced than that on qLTG-4-1. The effect of the Kasalath allele on qLTG-4-2, which suppressed the germination, was temporary and disappeared after 7 to 8 month’s storage, suggesting that the disappearance was due to the breaking of seed dormancy and that qLTG-4-2 might control it. Kasalath allele in the case of qLTG-5 decreased the germination rate when the effect of dormancy was completely removed. Therefore, we concluded that qLTG-5 was involved in LTG but not in seed dormancy.