The divergence among hop cultivars, as well as wild hops (Humulus lupulus var. lupulus, neomexicanus and cordifolius) and related species (H.japonicus) was studied using the sequence difference of the partial rbcL fragment, the noncoding regions, the trnL intron and spacer between the trnL3' exon and trnF, of chloroplast DNA and the ITS2, one of the nuclear ribosomal internal transcribed spacer regions. There were no differences within the varieties of H.lupulus and within H.japonicus. A few nucleotide substitutions were observed between the varieties of H.lupulus in the 1260 bp nucleotide sequence of rbcL or the 853 bp total of the noncoding regions of choroplast DNA. There were no nucleotide changes in the 232 bp ITS2, even though this region showed a higher substitution rate when H.lupulus and H.japonicus were compared. H.japonicus showed 13 substitutions in rbcL, 9 in the noncoding regions and 24 in the ITS2 region compared with H.lupulus var.lupulus. Synonymous substitution at tne 3rd codon position of rbcL enabled to estimate the divergence time of the two species at 3.74 million years ago. This study which revealed a low variability within H.lupulus suggests that variation, which could be utilized in hop breeding is limited.
Cowpea (Vigna unguiculata (L.) Walp.) is an important grain legume crop commonly grown and consumed in many parts of the tropics and subtropics. A genetic linkage map of cowpea was construted using randomly amplified polymorphic DNA (RAPD) markers and a recombinant inbred (RI) population derived from the intersubspecific cross between IT84S-2246-4, an improved cowpea line, and TVNu 110-3A (Vigna unguiculata spp. dekindtiana var.pubescens). The map spanned 669.8 centiMorgans (cM) of the genome and comprised 80 mapped loci (77 RAPD and 3 morphological loci) assembled into 12 linkage groups. The 12 linkage groups ranged in size from 14.0 to 175.4cM. The distribution of interval sizes between adjacent markers on the cowpea map ranged from 0.7 to 26.7 cM with an average distance of 9.9 cM. Random amplified polymorphic DNA marker loci exhibiting distorted segregation ratios were detected. The linkage map was used to locate quantitative trait loci (QTLs) for days to flowering, days to maturity, pod length, seed/pod weight percent, leaf length, leaf width, primary leaf length, primary leaf width, and the derived traits: leaf area and primary leaf area in the RI population. Significant (p<0.01) quantitative trait locus (QTL) associations of marker loci were identified for each trait. A total of 88 significant marker locus-trait associations were identified in all the linkage groups. Six markers with effects on these traits are yet unlinked. Both parental types contributed alleles with positive effects on these traits. The percentage of phenotypic variation associated with individual markers ranged from 6.6 to 16.9%. Several regions of the genome affected more than one trait. The coincident map locations of many QTLs for more than one trait corroborate the strong correlations observed between these traits.
In an attempt to use new sources of disease resistance, genomic sequences of two genes Rpr1-1(t) and Rpr1-2(t) of PR-1 proteins, which are one of the major pathogenesis-related (PR) proteins in plants, were obtained by TAIL-PCR on the basis of a registered genomic and from expressed sequence tag (EST) data, respectively. The genetic loci of Rpr1-1(t) and Rpr1-2(t) were identified on chromosome 1 and 5, respectively, using recombinant inbred lines (RILs) from and an indica-japonica cross. Abundant sequence polymorphism between alleles of indica and japonica cultivars were found in the two genes. Polymorphic insertions including the Tnr1 (transposable element in rice #1) sequence in indica cultivars and a minor insertion in japonica cultivars were detected in the ORF flanking regions of Rpr1-1(t). In the ORF flanking regions of Rpr1-2(t), a unique insertion of 827 dp was found in japonica cultivars, while two insertions were found in an indica cultivar, Milyang 23. Despite such diversity in the flanking regions, only a few amino-acid residues were different between the alleles. Based on a test for polymorophism among some cultivars, it was found that variation of inserted/deleted sequences may provide a useful molecular tool for investigating indica-japonica differentiation in rice.
We precisely mapped the Pb1 locus for the panicle blast resistence of a quantitative nature on rice chromosome 11 using RFLP markers. Based on the cosegregation of the resistant genotypes of Pb1 and Stvb-i, a rice stripe virus (RSV) resistance gene derived from an Indica cultivar Modan, we examined the linkage relationships between Pb1 and 13 Stvb-i-linked RFLP markers located on the long arm of chromosome 11. The Pb1 locus was mapped in the Modan-derived chromosomal region in the middle part of the long arm of chromosome 11. Pb1 was located on the telomere side in relation to the Stvb-i locus. The Pb1 gene was closely located at 1.2cM from three RFLP markers: S723, CDO226, C189. Then, we examined the graphical genotypes of 34 Modan-derived RSV-resistant cultivars with or without panicle blast resistance, and 12 susceptible Japonica cultivars, using 21 RFLP markers. Among them, cultivars with panicle blast resistance were classified into four types: A, B, C and D, and those without it into six types: E, F, G, H, I and J. In all of the panicle blast-resistant cultivars, the Modan-type bands were observed in S723, CDO226 and C189. On the other hand, in all the panicle blast-susceptible cultivars, the Japonica-type band was observed in S723, whereas the Modan-type bands were noticed in CDO226 and C189 in Type E cultivars. Consequently, only the genotypes of the S723 locus completely coincided with the genotypes of the Pb1 locus. Although, it was determined, based on linkage analysis, that S723, CDO226 and C189 were located at the same locus, graphical genotyping analysis, using many progeny cultivars derived from Modan, revealed that S723 was the closest marker to Pb1 among the three.
The importance of developing a quantitative shape discriminant method has been widely emphasized to obtain a consistent and objective evaluation. In fact, several methods for shape discrimination have been proposed. However, each of the methods can only be applied to a specific material, requiring specific shape features. Considering the number of shape discrimination problems in agriculture, such as genetic resource management, a single model that can be generally applied to different discrimination problems is highly desirable. A shape discriminant model based on neural networks with image input that did not require any shape features has been recently proposed, and it was shown that this model could be applied to soybean leaflet shape discrimination. This model was expected to be highly suitable for various materials, because it did not require any shape features. In this study, we examined the applicability of this model to the discrimination of several plant materials, by changing the training conditions of the neural networks to find their optimal combination. The plant materials were as follows: maple leaves, Tartary buckwheat kernels, pear fruits, mulberry leaves, and leaf-mustard leaves. The discriminant error rates, which were evaluated by cross-validation examination, differed depending on the materials, the number of training classes, and the training conditions. For example, the cross-validation error rate was 0.202 in the case of maple leaf shape with 10 training classes under the optimal training condition. The optimal training condition was common to all the materials examined in this study. Considering that this model did not require any shape features, its error rate was acceptable. This model can be widely and easily applied even for the evaluation of rather complex shapes such as lobed leaves. We concluded that this model would be highly suitable when various shapes of phytoorgans in many species have to be discriminated, such as in the case of genetic resource management.
The japonica rice cultivar Koshihikari has been used as the crossing parent for breeding of high-eating quality cultivars. An anther culture method has been developed to breed a new rice cultivar with in a short period, and several cultivars have been released in recent years. However, the efficiency of anther culture of Koshihikari was low. We developed a culture medium suitable for Koshihikari callus culture to increase the anther culture efficiency. The medium was a modification of the R2 medium, i.e., the concentrations of potassium nitrate (KNO3) and ammonium sulfate ((NH4)2SO4) in the R2 medium were diluted to 1/5, 5mM aspartic acid and 5mM glutamine were added, and the vitamins were replaced by those of B5 medium. We designated the medium as DKN (dilute KNO3) medium. The anthers of Koshihikari were cultured by using the three-step anther culture method in which DKN medium was used as the basal medium. The green plant regeneration rate peranther on DKN medium was 4.3 times (donor plants cultivated in green house) and 21.4 times (donor plants grown in field) higher than those on 1/2R2 medium which had been used for rice breeding by anther culture. It is concluded that the combination of the three-step anther culture method and DKN medium is efficient for anther culture of Koshihikari.
The efficiency of anther culture of Koshihikari and its related cultivars with high eating quality i.e., Hitomebore, Akitakomachi and Kinuhikari was improved by using DKN medium as basal medium in the three-step anther culture method. The anther culture efficiency of F1 plants derived from crosses between these cultivars was also improved. In this culture method, a positive correlation was recognized between the anther culture efficiency and the coefficient of parentage of the donor plant to Koshihikari. Therefore, this method was an effective means for the anther culture of materials derived from crossings between cultivars related to Koshihikari. On the other hand, the results implied that F1 pollen which has a definite genetic background was regenerated preferentially. To examine the segregation of characteristics both in the anther culture-derived population and F2 population, two populations were produced from the F1 plant derived from a cross between the long-culmed medium-ripening cultivar Koshihikari and short-culmed early-ripening cultivar Niigatawase. No significant differences were observed in the segregation of heading date or culm length between the two populations. It is cocluded that the anther culture method developed in this study is a useful tool for breeding of rice by using Koshihikari and its related cultivars as a crossing parent.
Four single recessive viviparous mutants of rice, riv1-1, riv1-2, riv2 and enl1, were characterized. All riv mutants showed pre-harvest germination (vivipary) during seed development but no defects in the other traits. Vivipary was also observed in the enl1 mutant that lacked endosperm in the seeds. Examination of the viviparous nature of the mutants under three rain conditions, no rain, artificial rain and natural rain, revealed that the induction of vivipary in the riv mutants required a small amount of water. In contrast, precocious germination in the enl1 mutant was caused by the absence of endosperm in the seeds, but not by the external signal of rain. In riv mutants, the precocious germination occurred simultaneously at late stages of seed development. The germination test in the presence of exogenous ABA revealed that sensitivity to ABA was gradually reduced from 15 through 35 days after pollination in the wild-type seeds, and was almost lost at 40 days after pollination. A similar tendency was observed in riv1 and riv2 seeds. However, at 15-35 days after pollination, riv1-1 and riv2 seeds had significantly lower sensitivity to ABA than the wild-type seeds. In additon, the three riv mutants lost the sensitivity to ABA at an earlier stage of seed development than the wild type. These results indicate that riv1 and riv2 have a lower sensitivity and shorter period of sensitivity to ABA.
The Wx gene expression plays a major role in determining the amylose content in the rice endosperm, although Wx alleles and their trans-acting genes like du genes are also known to genetically control the amylose content. Experiments were conducted to determine whether genes inhibiting the Wx expression respond similary to two Wx alleles, Wx-a and Wxb, which are predominantly distributed in cultivated. A recessive mutant with chalky endosperm and showing an independent inheritance of wx was used in this study. Complementation tests showed that the mutant gene was allelic to du2 but the gene was designated as du2-2 as its phenotype was distinct. Nearisogenic lines (NILs) with different combinations of alleles at the Wx and du2-2 loci were then established and evaluated for the Wx gene expression. Waxy or chalky endosperms due to du2-2 were detected only in response to Wxb but not to Wxa, which may account for the fact that no du variants were detected in the Indica type. The potential use of du2-2 for improving the grain quality of the Indica type was discussed.