We evaluated the nature of microsatellites in Brassica rapa in order to develop an informative and reliable DNA marker system for Brassica genetic analysis. Microsatellites were isolated by hybridization screening of an unconcentrated small-insert genomic library using tri-and dinucleotide probes. Of 45,000 clones screened, 210 had repeat sequences, in which 228 microsatellites were identified. The most frequent microsatellite motif was (GA)n at a frequency of one every 4.8 × 105 bp, followed by (CAA)n at one every 5.0 × 105 bp. The frequency of the tri- and dinucleotide microsatellites throughout the B. rapa genome was estimated to be one every 120 Kb. The number of repeats and the polymorphism information content of the dinucleotide microsatellites were higher than those of the trinucleotide microsatellites. More than 90 % of the primer pairs successfully amplified the corresponding microsatellite regions in other Brassica species. Furthermore, a considerable portion of them could be used in other Cruciferous species, 78.5 % in Raphanus sativus, 68.6 % in Sinapis alba and 39.8 % in Arabidopsis thaliana. Based on these results, we concluded that B. rapa microsatellites have a high potential for the development of DNA markers that could contribute to the genetic analysis of Brassica and other Cruciferae.
Embryos of Glycine max (L.) Merrill cv. ‘Jack’ were co-transformed with the hygromycin phosphotransferase (hpt) gene and the jellyfish green fluorescent protein (GFP) gene by microprojectile bombardment. The purpose of this study was to determine the optimum conditions which could achieve the highest co-transformation rate using this method. In the whole process of co-transformation, the screening of transformants was effectively monitored by the expression of GFP. Transformation rates were highest when (1) induced embryos were transferred to a solid medium more than 5 times, (2) only the most highly-proliferated embryos forming globular clusters were bombarded, (3) the helium pressure of rupture disc was 1,100 psi, and (4) the embryogenic tissues 5 days after bombardment were cultured for more than 8 weeks in hygromycin-containing liquid medium for selection. In these conditions, the proportion of clones expressing hygromycin-resistance was 7 %, and the proportion of GFP-expressing clones was 18 % of them.
Interspecific crosses between Delphinium elatum and D. nudicaule or D. cardinale were carried out to produce red-flowering Delphinium varieties. An in vitro germination technique was effective to produce interspecific hybrids. From 800 seeds, 379 interspecific hybrid plants were produced. The form of these hybrid plants was intermediate between that of the parents and their flower color was purplish due to the presence of delphinidin derivatives. Most of these hybrid plants were fertile. The F1 generation was self- and sib-crossed. In the second generation, six plants with pink or red flowers were selected from 224 plants.
‘Senno’ (Lychnis senno Siebold et Zucc., Caryophyllaceae), is a traditional and endangered ornamental plant species and presently only eleven strains have been confirmed to be under cultivation in nine localities in Chugoku and Kyushu districts of Japan. Analyses on chromosome number and nuclear DNA contents using flow cytometry revealed that all of the strains are triploid (2n = 36). The average of meiotic chromosome configuration (10.1III + 1.9II + 1.9I) observed in one strain indicated that ‘Senno’ is an autotriploid. Chromosome bridges, lagging chromosomes and micronuclei were observed in microsporogenesis. Although about 70 % of pollen showed stainability with cotton blue solution, ‘Senno’ plants conserved in Japan did not produce any viable seeds by self-pollination. The origin of the ‘Senno’ conserved in Japan is discussed.
Random amplified polymorphic DNA (RAPD) markers linked to the L3 locus were developed by applying the bulked segregant analysis method to two doubled haploid (DH) populations in Capsicum. The codominant RAPD markers, E18272 and E18286, were converted into sequence-characterized amplified region (SCAR) markers by molecular cloning and nucleotide sequencing. A PCR analysis using DH (n = 176) and backcross (n = 190) populations revealed that all the SCAR markers, PMFR11269, PMFR11283 and PMFR21200, co-segregated with the original RAPD markers, and were mapped at a distance of 4.0 cM from the L3 locus. Furthermore, after confirmation of their validity in 18 accessions of Capsicum spp. using the codominant SCAR marker pair, PMFR11269 and PMFR11283, it was suggested that the SCAR markers developed here could become effective in marker-assisted selection programs for the introduction of the L3 gene derived from PI159236 (C. chinense) into sweet pepper for breeding purposes.
Since the changes in the root shape of radish (Raphanus sativus L.) during development cannot be easily observed, it is often difficult to determine the suitable time after sowing for the selection of genotypes with desirable root shapes. To determine the optimum time for selection of radish root in breeding, we conducted diallel analyses of the root shape at different growth stages and investigated the changes in the mode of inheritance of the root shape. A set of diallel crosses of 6 inbred lines was harvested 6 times from 40 to 75 days after sowing. The major shape characteristics, associated with principal components of elliptic Fourier descriptors (EFDs), included the ratio of length to width (AP1), the bluntness of the distal part (AP2), and the curvature of the middle part (BP1). ANOVA indicated that the growth stages, genetic effects, and their interaction were significant in all 3 characteristics. The interaction did not affect appreciably the difference in the mode of inheritance of AP1 among growth stages, suggesting that the time of harvest did not affect the selection of this characteristic. The interaction caused a certain degree of heterogeneity in the ordering of genotypes and in the mode of inheritance of AP2 and BP1 at different growth stages. In AP2, the degree of dominance increased at the later growth stages. In BP1, the genetic parameters varied, but showed no apparent trend with the growth process. The results suggest that AP1 can be efficiently selected at an early stage, i.e., at the beginning of thickening growth. For AP2 and BP1, the selection at a stage around the normal harvest time is recommended. However, the fluctuations in the time of selection within a 2-week period around the normal harvest time did not affect the selection of the characteristics, because the interaction was not significant within this period.
To develop superior rice panicles with a sufficient sink capacity and a high degree of grain filling, we should consider the within-panicle distribution of spikelets showing different degrees of grain filling, as well as the total spikelet number. In the present study, several component traits of the number of spikelets per panicle which are related to the within-panicle distribution, and also the number of panicles per plant, were examined using 68 recombinant inbred lines (RILs) derived from a cross of Asominori/IR24 grown over a period of four years, 1997, 1998, 2000 and 2001. For every trait related to the spikelet number per plant, analyses of variance (ANOVA) were conducted at the 375 position of molecular markers one by one to detect significant main effects of quantitative trait loci (QTLs) and interactions of marker genotype and year of cultivation (QY interactions). Then, genetic associations of these traits were analyzed by examining the positions of the relevant genome regions which showed significant main effects and/or interactions for different traits. Finally, the QTLs causing favorable genetic effects and associations for the development of superior panicles were surveyed. The results showed that most of the genome region identified to increase the spikelet number per panicle also increased the number of spikelets on the secondary branches which are characterized by a low degree of grain filling. One region on chromosome 3, on the other hand, increased the spikelet number per panicle through the increase of the number of spikelets on primary branches which generally show a high degree of grain filling. Significant QY interactions were demonstrated in many genome regions for all traits, whereas the interactions of RIL and year could not be detected in an overall ANOVA in which the marker genotypes of RILs were not being considered. The region on chromosome 3, cited above, did not exhibit a significant QY interaction. This region appears to involve QTL(s) suitable for the development of superior panicle types.
We determined the genomic regions of wild soybean (Glycine max subsp. soja) carrying quantitative trait loci (QTL) for hard seededness, a distinct character between the wild and cultivated soybeans. The segregation of hard seededness, as evaluated using seed coat permeability (SCP), was examined in the progeny of a cross between wild and cultivated soybeans. The high parent-offspring correlation coefficient between F2 and F3 indicated that SCP was a highly heritable character. A marker-assisted analysis of five isozyme and 131 simple sequence repeat (SSR) loci revealed that at least two major and one minor QTL were involved in controlling the trait. The QTL near an SSR marker (Satt459) in molecular linkage group D1b + W had the greatest effect on SCP, and accounted for 23.8 and 38.5 % of the phenotypic variance observed in the F2 and F3 mean, respectively. Seed coat color, determined by a locus inhibiting seed coat color (I) and a pubescence color locus (T), was also closely associated with SCP in F4 to F6, although seed coat color did not appear to be associated with SCP in the F2. The results of this and a previous study suggest that different sets of QTL contribute to the phenotypic difference of seed coat permeability in wild and cultivated soybeans to different degrees.
Ninety-three recombinant inbred lines were produced by single-seed descent from reciprocal crosses of Cucumis melo ‘PMAR No. 5’ and ‘Harukei No. 3’. Using these lines, resistance to race 1 of powdery mildew, cotton aphid resistance and flesh colour were studied. Resistance of ‘PMAR No. 5’ to the powdery mildew fungus appeared to be conferred by two dominant and independent genes, because F1 plants were moderately resistant and the observed segregation ratio of the resistant, moderately resistant and susceptible lines was 42: 31: 19. Genetic analysis revealed that cotton aphid resistance and flesh colour were controlled by single dominant genes. The usefulness of recombinant inbred lines was discussed.
Seed production in rice (Oryza sativa L.) largely depends on the number of flowers, which is in turn regulated by the inflorescence architecture. The developmental process of inflorescence and spikelet in grasses including rice differs considerably from that in other monocotyledonous and dicotyledonous species, and rice, an important crop plant, is often used as a model monocot plant. Nevertheless, the developmental course of wild-type rice has not yet been well characterized. Thus, detailed description of rice inflorescence and spikelet development would be valuable for characterizing mutant phenotypes and also for comparing rice with other grass species. In this study, we showed a number of landmark events in the developmental courses of inflorescence and spikelet, and divided their development into nine and eight stages, respectively. This staging system would be useful as a reference for developmental description.
A novel mutation affecting the lateral symmetry of rice leaves, leaf lateral symmetry 1 (lsy1), was identified. The lsy1 mutant showed two major phenotypes of leaves; a narrow leaf due to the deletion of a part or whole of one lateral half, and a bifurcated leaf in which an additional midrib is formed in one lateral half. Developmental analysis revealed that in the leaf primordia of the latter type, an additional protrusion, which would later develop to a midrib, was formed in the center of one lateral half, suggesting that the lateral half of the primordium was developing as a single leaf. Other phenotypes associated with leaf lateral symmetry were also observed. Thus, in lsy1 leaves, the two lateral halves did not grow synchronously. Lateral asymmetry was also observed in leaf-like spikelet/floral organs such as glumes and carpel. The LSY1 gene is considered to play an important role in establishing lateral symmetry of leaves and leaf-like organs.
Diploid Ipomoea trifida is an ancestral wild species of the cultivated hexaploid sweet potato, and displays a sporophytic self-incompatibility (SI) that is controlled by a single multiallelic S-locus. To characterize the genomic region of the S-locus using a map-based cloning method, a BAC library consisting of approximately 40,000 clones was constructed from genomic DNA of S1-homozygote, and screened using S-linked DNA markers which were mapped in our previous study. We constructed a contig covering the S-locus region with additional screening of fosmid and λ phase libraries. RFLP analysis of recombinant plants using terminal end sequences of the BAC clones as probes indicated that the S-locus region was delimited within a map distance of 0.57 cM, spanning approximately 300 kb in physical distance. Remarkable suppression of genetic recombination was detected in the S-locus region. From sequence analysis of the 313-kp region, 43 ORFs, many repetitive sequences and 5 transposable elements were predicted. None of the ORFs, however, showed a high homology with the SI genes reported to date at the S-locus of other plant families, suggesting that a unique molecular mechanism is involved in the SI system of the Convolvulaceae family.
To promote the implementation of genetic analyses and breeding programs, a set of microsatellite markers in sweetpotato should be developed, which cover the entire sweetpotato genome. To achieve this objective, 102 polymorphic microsatellite markers were developed using three different methods: 1) screening of a small-insert genomic library, 2) construction of a microsatellite-enriched library (51-fold enrichment) and 3) mining of EST databases. Approximately 8,000 clones of the small-insert library were screened with (GA)20 and (CA)20 probes, and 42 positive clones were identified. One hundred and twenty-two clones containing microsatellites were identified from the enriched library, which consisted of 800 clones. Thirty-two and 47 primer pairs were designed for these clones containing microsatellites from the small-insert library and enriched library, respectively. Among the sweetpotato accessions examined, 27 of these microsatellite markers showed polymorphism. Eventually, 4,153 sequences from a published expressed sequence tag (EST) databases were mined as a source for the development of microsatellite markers, and 379 sequences containing microsatellites were identified. A total of 151 primer pairs were designed and 120 scorable microsatellite markers were obtained. Seventy-five EST-SSR loci showed length polymorphisms. Of these polymorphic loci, 71 % were associated with some genes.
In order to gain a better understanding of the genetic basis of the wide differentiations observed in Japanese cultivated radish varieties, structural and sequence variations were studied in the 3′ region of mitochondrial atp6. Three hundred and ninety-four radish plants belonging to eight Chinese, one Korean and 27 Japanese varieties were used as materials, and total DNA was isolated from each plant. Using the DNA as a template, PCRs with three kinds of primer pairs specific to the sequence of normal type cytoplasm were conducted. The radish plants were classified into four types by the combination of amplification patterns based on the three PCRs. Seventy-five percent of the varieties were assigned to one of the four types, and 87 % of the plants were grouped into two types with similar proportions for each (44 and 43 %). Sequence analysis of the plants belonging to the two major types demonstrated that structural rearrangements of the mitochondrial genome had generated the differentiations in the region observed here. The variations in the atp6 3 ′ region corresponded to those of orfB, with a few exceptions. The results indicated the existence of a dual structure of the cytoplasm in Japanese radish cultivars. Additionally, the origin of polymorphic varieties consisting of two or three types was examined.