Two new types of cytoplasmic male sterile (CMS) lines have been developed, one by in vitro chimera formation between Brassica rapa and B. oleracea and another by protoplast fusion of B. oleracea with Raphanus sativus. These CMS lines harbored an Ogura-type mitochondrial gene, orf138. They expressed chlorosis at a young stage and their chloroplast genome changed to the type of Ogura radish based on restriction fragment length polymorphism (RFLP) analysis. Thus, we attempted to study the relationships among cytoplasmic male sterility, chlorosis and the variation of the chloroplast genome using the matK and RuBisCO genes. The two CMS lines, normal and male sterile radishes exhibited similar matK gene sequences, while B. rapa and B. oleracea showed matK sequences of the radish type in very low copy numbers. The CMS lines displayed an identical RFLP pattern with that of the male sterile radish in the RuBisCO gene region. These phenomena indicated that the chloroplast genome organization had changed from Brassica to Raphanus type or Raphanus CMS type by CMS induction, and suggested the existence of heteroplasia in the Brassica chloroplast.
Optimum allocation of locations (sj, subscript j designating stage), replications within locations (uj), fraction of selection (pj) and field area per stage (αj) in three-stage yield selection is discussed on the principle of maximizing the probability of success (S) that an entry with the highest yielding ability is selected from a population of candidate entries. Monte Carlo calculations of S with practically possible conditions of the parameters concerned confirm that the total number and allocation of locations, not of replications, determine S. With the same total number of locations, a uniform allocation s1 = s2 =s3 (or, s1 > s2 > s3 in the presence of a high magnitude of genotype × location interaction) is nearly if not exactly the best. If circumstances permit, the locations should be chosen newly in each stage from the target region. As to selection fraction and field area, allocations directed as p1 < p2 < p3 and α1 > α2 > α3 will be appropriate in most practical conditions. Replications uj should be allocated in accordance with the total available field area and such directed allocations of sj, pj and αj. The previously proposed and widely adopted allocation of locations, i.e., 1(=s1) < s2 < s3, is not advantageous except when multiple locations cannot be allocated to the first stage for some reason, as would be the case when testing many entries with a severely limited capacity of field area. An allocation directed as p1 > p2 > p3 instead of p1 < p2 < p3 is appropriate when genotype × year interaction is the major error component in the yield scores of entries.
Low content of protein and the presence of an anti-nutritional factor, ‘trypsin inhibitor (TI)’ reduce the nutritional value of sweetpotato (Ipomoea batatas (L.) Lam.) when used as animal feed. Attempts have been made to develop sweetpotato varieties with a high protein content and/or low trypsin inhibitor activity (TIA). Using approx. 800 breeding lines of sweetpotato, the crude protein content and TIA were determined during 6 seasons from 1998 to 2003. Fifteen lines with a high protein content (27.8–44.6 mg/g DW) and 7 lines with a low TIA (11–137 U/mg DW) were selected, when compared to the standard variety ‘Shiroyutaka’ (crude protein content: 19.9 mg/g DW, TIA: 186 U/mg DW). Among the lines used, there was a strong positive correlation between TIA and the crude protein content (r = 0.871**). However, lines, ‘Q94128-1’ (crude protein content: 28.9 mg/g DW, TIA: 21 U/mg DW) and ‘KNF94225-13’ (crude protein content: 35.5 mg/g DW, TIA: 63 U/mg DW) showed a high protein content and a low TIA. Electrophoretic analysis and activity staining of TIs extracted from these two lines revealed a considerably lower amount of trypsin inhibitory bands compared to that in ‘Shiroyutaka’. Partially purified TIs from ‘Shiroyutaka’ were estimated to correspond to sporamins, sweetpotato major storage root proteins.
Fusarium head blight (FHB) severity was evaluated in a set of recombinant inbred (RI) lines from a cross between the two-rowed barley varieties, Harbin 2-row (resistant) and Turkey 6 (susceptible). Harbin 2-row was one of the most resistant varieties to FHB among ca. 5,000 barley germplasm accessions stored at Okayama University. The FHB severity of the RI lines and their parents was evaluated by the ‘cut-spike test’ in the 2000–2001 and 2003–2004 seasons. Spikes with disease symptoms were scored based on eleven grades from resistant (0) to susceptible (10). A high-density linkage map, including 328 AFLP, 45 SSR, one RFLP-STS, one AFLP-STS, four resistance gene analog (RGA) and four morphological markers, was constructed to detect QTLs for FHB severity. Composite interval mapping enabled to detect one QTL on chromosome 2H in the 2000–2001 season, and one QTL on chromosome 4H and another QTL on chromosome 6H in the 2003–2004 season. At these three QTLs, the alleles from Harbin 2-row contributed to a lower severity. A QTL on chromosome 2H was located in the vicinity of the cleistogamy locus (cly1 or Cly2) that determines open/closed flowering type. The comparison with previously identified QTLs for FHB severity by the present authors indicated that Harbin 2-row might harbor the same resistance allele in the vicinity of the cly1/Cly2 locus as Russia 6, which was used as a resistant parent in the previous study. The QTL in the vicinity of the cly1/Cly2 locus is one of the promising sources for FHB resistance breeding in barley.
Interspecific hybridization is essential to introgress resistance genes from Capsicum baccatum, a related species of cultivated pepper (C. annuum), since reliable genetic resources resistant to anthracnose have recently been identified within the C. baccatum germplasm. In conventional interspecific hybridization between the two species, hybrids could not be generated due to embryo abortion, which has been known to be a post-fertilization genetic barrier. Some partially compatible cross combinations, determined through observations of embryo development after pollination, were identified using a large number of accessions of C. annuum as pistillate parents. Embryo rescue technique was successfully employed to produce hybrids in these partially compatible crosses. Immature seeds bearing torpedo or early cotyledonary embryos, developed 35–40 days after pollination, were excised and the embryos were cultured on MS medium with sucrose and plant growth regulators. Hybridity was confirmed by observation of corolla yellow spot as a dominant species-specific trait of C. baccatum and using random amplified polymorphic DNA (RAPD) marker analysis. All the hybrid plants displayed vigorous growth but complete pollen sterility. The hybrid sterility was overcome through intensive backcrossing using C. annuum as the pollen parent. Consequently, hundreds of interspecific BC1F1 progenies were raised, and introgression of anthracnose resistance was confirmed in this segregating population.
The overwinter bud of the gentian plant, Gentiana triflora, is an organ that develops from the basal tuber in late spring and becomes dormant and freezing-tolerant for overwintering. Here, proteins specifically enriched in the overwinter buds were identified using two-dimensional polyacrylamide gel electrophoresis followed by amino acid sequencing. Enriched proteins included those so far identified as stress- and cold-inducible in other plants, i.e., two isoforms of ethylene-induced esterase, a dehydration-induced protein, glyoxalase I, thioredoxin peroxidase, two isoforms of ascorbate peroxidase and a membrane pore protein. Interestingly, the stress-related proteins had already accumulated in the overwinter buds under non-stress conditions in early summer. Accumulation of these proteins was also examined in a mutant gentian, which developed overwinter buds but lacked cold tolerance and died under conditions below 3°C. In this mutant, several proteins including one of the putative esterase, enolase and ascorbate peroxidase were absent or greatly decreased. Thus, it appears that these stress-related proteins are expressed under non-stress conditions to aquire cold tolerance before overwintering.
The evaluation of drought tolerance based on the leaf temperature was examined in upland rice breeding. The relationships of the leaf temperature with the transpiration and photosynthetic rates were observed in upland and lowland rice varieties under upland cultivation in 1995, 1996 and 1997. The leaf temperature of the upland rice varieties was lower than that of the lowland rice varieties. Their transpiration and photosynthetic rates were highly correlated with the leaf temperature, measured using an infrared radiation thermometer or portable photosynthesis gas analyzing system in all the three years. The leaf temperature also showed a significant relationship with the grain yield tested in 1995. It is considered that rice varieties with a lower leaf temperature can maintain high transpiration and photosynthetic rates as well as produce a high yield under upland conditions. In addition, there was a significant positive correlation between the leaf temperature and root growth recorded by the trench method. Upland rice varieties with deep rooting showed lower leaf temperatures than those with a shallower root system. On the other hand, in the comparison of leaf temperature in varietal groups of breeding materials, the upland rice lines with medium-late maturity showed the lowest temperature, followed by the early maturing lines and lines for cultivation with sprinkler irrigation. This tendency was in agreement with the general degree of drought tolerance of individual varietal groups. To analyze the mode of inheritance of the leaf temperature, the parent-offspring correlation of leaf temperature measured using an infrared radiation thermometer was examined in breeding materials. Leaf temperature was compared between the progeny lines (F4 generation) of the upland rice variety Kantomochi168 with a high drought tolerance and the upland rice variety Norinmochi4 with a medium drought tolerance. Kantomochi168 progeny showed a lower leaf temperature than Norinmochi4. A similar tendency was confirmed in the F5 generation in the following year. Significant parental-offspring correlation (r = 0.812**) was observed between F4 and F5. Since the leaf temperature of the upland rice progeny may display a relatively higher inheritance, the leaf temperature is, therefore, considered to be a useful indicator to estimate the drought tolerance for line selection in upland rice breeding.
Pungency in the fruit of pepper (Capsicum spp.) is due to the accumulation of capsaicinoids. We isolated two genes characterized as putative Capsicum acyl-transferase genes (Catf-1 and Catf-2) from the placenta of pungent pepper, which displayed consensus motifs of acyl-transferase, H***D and DFG*G. The expression pattern of Catf-1 coincided with the accumulation of capsaicinoids in pungent pepper. The Catf-1 was expressed in placenta of pungent pepper fruit, but not in other organs, such as leaf, flower, seed and pericarp. Accumulation of mRNA of Catf-1 varied with the developmental stages of the pungent pepper fruits. Genomic PCR and Southern blot studies revealed the presence of a sequence deletion at the 5′ end of the Catf-1 coding region in the non-pungent line. In the F2 population obtained from a cross between a non-pungent strain (‘mGTY-1’) and a pungent strain (‘277 long’), the genotype at the Catf-1 locus cosegregated with the phenotype for fruit pungency. These results suggest that Catf-1 is a candidate gene differentiating pungent from non-pungent peppers.
In order to isolate grain-specific genes during kernel development, two differential screening methods—suppression subtractive hybridization (SSH) and differential hybridization (DH)—were applied. Two cDNAs encoding hordoindolines known to be related to grain hardness were isolated and designated as Hordeum vulgare indoline a and b (HvIDa and HvIDb). The cDNAs encoding HvIDa and HvIDb contained a 450 bp and a 444 bp open reading frame (ORF) that encoded the putative hordoindoline-a and -b precursors consisting of 150 and 148 amino acids, respectively. The deduced amino acid sequences of both HvIDa and HvIDb contained one tryptophan-rich domain and ten highly conserved cysteine residues. The expression of the HvIDa gene was high at 5 DAF, reached a peak at 8 DAF and decreased slightly until 20 DAF, while the expression of HvIDb began to be detectable at 8 DAF when it was higher than that of other developmental stages and decreased slightly until 20 DAF. The HvIDa and HvIDb genes were predominantly detected in the aleurone cell layers in the late part of kernel development, e.g. at 20 DAF. These findings may provide clues to the molecular mechanisms of kernel development and contribute to the determination of the grain texture in barley.
In higher plants, alcoholic fermentation is required to supply NAD+ to the glycolytic pathway, which is responsible for ATP synthesis under anaerobic conditions. Matsumura et al. (1995) previously isolated the reduced adh activity (rad) mutant in rice, in which elongation of the coleoptile is repressed under submergence. However, the rad gene had not been characterized. In the present study, we observed that, in the coleoptile, the Adh1 mRNA levels were comparable between the rad mutant and the wild type cultivar Kinmaze, while the amount of ADH1 protein was much lower in the rad mutant than in the wild type. Sequencing showed that G106 of the Adh1 gene of Kinmaze was replaced with A in the rad mutant, resulting in an E36K substitution in the deduced amino acid sequence. A genotyping experiment using F2 plants from a cross of the rad mutant with the indica cultivar Kasalath indicated that the point mutation was involved in the repression of coleoptile elongation. Furthermore, since the reduced ADH activity appeared to cause an ATP deficiency, elongation of the coleoptile was repressed in the submerged rad mutant.
Flowers of Brassica rapa L. produce a nectar guide, which consists of a coloured pattern (the dark, UV-absorbing centre of the flower) invisible to humans but visible to insect pollinators. As a result, the colour of the flowers typically appears as uniform light yellow to human eyes. The objective of the present study was to investigate the mode of inheritance of this character by using two inbred lines and their F1, F2 and F3 progenies with a view to improving this character. After digitizing UV-photographs of each flower, we measured the UV-absorbing area (UVA) and the total flower area (FA), based on image analysis. The ratio of UVA to FA represented the UV colour proportion (UVP). We estimated the broad-sense and narrow-sense heritabilities from within-generation variances in the UVP scores and environmental variance from the average value of the variances in the parental lines. The value of broad-sense heritability of UVP was high (0.75) in the F2 generation (hB2[F2]) and higher (0.84) in the F3 generation (hB2[F3]), indicating that UVP is a heritable character. Moreover, the high value of broad-sense heritability of UVP indicates that breeders have not focused their selection intentionally on this character in B. rapa. In contrast, the value of narrow-sense heritability was much lower: 0.12 (hN2[F2]) and 0.24 (hN2[F3]), respectively, suggesting that the genetic variation in UVP was mainly due to dominance effects. If we attempt to breed new lines with larger or smaller UVP values, we need to select this trait in advanced generations, in which additive effects become larger.