Ascorbate (vitamin C) is a powerful antioxidant and scavenger of free radicals that protects plants against oxidative damage caused by adverse environmental conditions such as drought, salt stress, and herbicide use. Dehydroascorbate reductase (DHAR; EC 126.96.36.199) and monodehydroascorbate reductase (MDAR; EC 188.8.131.52) are crucial for ascorbate regeneration and the maintenance of a pool of the reduced form. In this study, we report the development of transgenic potato (Solanum tuberosum L.) that overexpresses the Arabidopsis thaliana DHAR gene (AtDHAR1) in the cytosol. The transgenic plants exhibited up to 4.5 times the DHAR activity and up to 2.8 times the level of reduced ascorbate found in the wild-type plants. When subjected to methylviologen treatment, the transgenic plants exhibited enhanced tolerance in terms of less ion leakage, greater chlorophyll contents, less accumulation of hydrogen peroxide, and less severe visual injury symptoms. Moreover, the transgenic plants exhibited faster growth under drought and salt stress. Our results demonstrate that elevating ascorbate contents by overproducing DHAR represents a viable approach for the development of herbicide-tolerant potato.
Glu-B3 alleles on chromosome 1BS of common wheat (Triticum aestivum L.) encoding low-molecular-weight glutenin subunit are associated with dough property. Among them, Glu-B3g is known to increase dough strength. To incorporate efficiently Glu-B3g allele into breeders’ lines, DNA marker-assisted selection (DNA-MAS) is considered to be a powerful tool. In the present study, we examined the applicability of ‘glume color’ (GC) controlled by Rg-B1 (Rg1) locus on 1BS as a ‘field-marker’ to detect Glu-B3 alleles. Three populations of F4 breeding lines were used. In each cross, both genotypes of Glu-B3 and Rg-B1 loci of each parent were different. Glu-B3 allele-specific DNA markers were applied to determine the genotype of Glu-B3 locus of each line. The genotype of Rg-B1 locus of each line was visually determined by GC. Significantly tight cosegregations between Glu-B3 and Rg-B1 loci were found in all the populations of breeding lines. It is considered that GC controlled by Rg-B1 locus is a useful ‘field-marker’ to detect Glu-B3 alleles, as it is less time-consuming and more cost effective than DNA-MAS. By using the ‘field-marker’, breeders can select elite lines carrying Glu-B3g allele in breeding fields only by observing the GC of each line/plant without laboratorial procedures for DNA-MAS.
In cereal breeding, semi-dwarfness and an increased spikelet number are favorable characteristics. We show that the rice DENSE PANICLE 1 (DN1) mutant allele Dn1-1 causes both of these characteristics and that Dn1-1 is a loss-of-function mutation. DN1 is allelic to DENSE AND ERECT PANICLE 1 (DEP1) (=qPE9-1). The expression level of OsCKX2 in the shoot apex of Dn1-1 plants is similar to that in the wild type, indicating that OsCKX2 does not contribute to an increased number of spikelets. A comparison of the Dn1-1 and Dn1-3 alleles suggests that the N-terminal region of DN1 contains a coiled-coil domain and a nuclear localization signal that might be responsible for semi-dwarfness. This comparison also revealed that a single transmembrane alpha-helix, a VWFC module, and a four-disulfide core domain can further increase spikelet number. Subcellular localization analysis of the DN1 protein fused with green fluorescent protein (GFP) implies that DN1 is located in the nucleus and cell membrane and that its N-terminal fragment is cleaved. Dn1-1 plants have normal sensitivity to gibberellin, brassinolide, and kinetin, and we observed no genetic epistasis with brassinolide-related mutants, suggesting that DN1 does not function in the signaling pathways of these phytohormones.
Soybean fasciation is controlled by a recessive gene affecting the shape of the aerial parts of the plant, which is characterized by flattened stem, infrequent branches and clustering of flowers and pods on shoot apices. Morphological feature of the fasciation mutation has been dissected, but molecular information is still limited. We developed three populations derived from the crosses between wild type cultivars and three Japanese fasciation varieties, Shakujodaizu, Taikadaizu and Shakujomame, respectively. The molecular mapping of the three F2 populations revealed that the fasciation locus (F locus) was mapped on the chromosome 2 (LG D1b). Fine mapping experiment with a population consisting of 1536 seeds derived from the F2 lines segregating for fasciation revealed that a DNA marker cosegregated with the fasciation phenotype. This DNA marker was dominant for wild type allele and the flanking region of this marker could not be amplified in Shakujodaizu as well as in the other two fasciation varieties, suggesting that some deletion or major rearrangement probably occurred at the f allele. Genomic information disclosed two predicted genes, Glyma02g36940 and Glyma02g36960, that were annotated in the vicinity of this DNA marker. The relationship between these candidate genes and the fasciation phenotype was discussed.
Most cereal crops have hulless grains (naked caryopses) with a free-threshing trait, whereas the majority of barley cultivars show hulled (covered) caryopses. The naked caryopsis in barley is genetically controlled by a single locus, nud. The Nud gene (the covered caryopsis allele) encodes an ethylene response factor (ERF) family transcription factor that regulates a lipid biosynthetic pathway. For functional analysis of the barley Nud gene, we produced transgenic rice expressing Nud in the developing caryopses. All transgenic lines had caryopses that were easily dehulled at maturity, indicating that the naked caryopsis phenotype remained in spite of expression of the Nud transgene. Histochemical and lipid analyses of the transgenic rice caryopses did not show increased lipid accumulation on the surface of developing caryopses, suggesting that the Nud-mediated lipid pathway may not function in rice caryopses. The predicted rice ortholog of Nud, Os06ERF was expressed specifically in the developing caryopses. However, expression of Os06ERF ceased at an earlier developmental stage than that of the native Nud gene in barley caryopses, which was also the case for expression of the Nud transgene. This raises the alternative hypothesis that the timing of Nud expression may be critical for activating the pathway for hull-caryopsis adhesion.
Phytoextraction by high-cadmium (Cd)–accumulating rice (Oryza sativa L.) cultivars has been proposed as an attractive technique for cleaning up Cd-contaminated paddy field soil. To breed rice cultivars useful for Cd phytoextraction, it is necessary to understand the genetic basis of Cd accumulation in aerial parts. We developed backcross inbred lines (BILs) derived from a cross between a low-Cd–accumulating cultivar, Koshihikari (japonica), and a relatively high-Cd–accumulating cultivar, Jarjan (indica). The BILs were grown in fields containing two low levels of Cd and in pots of soil containing a moderate level of Cd. A quantitative trait locus (QTL) analysis revealed that a major QTL, qCdp7 (QTL potentially useful for Cd-phytoextraction on chromosome 7) contributed to accumulation of Cd in both brown rice and straw of plants grown in any of the three soils, and it accounted for 31% to 54% of the phenotypic variance. To confirm the phytoextraction-promotion ability of qCdp7, we grew BILs carrying the Jarjan allele of qCdp7 in Cd-contaminated soil, and confirmed the reduction of the Cd concentration in the shoots of Koshihikari subsequently grown in the phytoextracted soil. These results demonstrate that the Jarjan qCdp7 allele is effective at phytoextracting Cd from the soil.
To isolate the factors that define variation in domesticated rice (Oryza sativa L.) inflorescence architecture, we performed a principal component analysis (PCA) using 292 accessions (136 indica and 156 japonica). The first component accounted for 40.6% of the total variance, and its main components were interpreted as lengthening of the stage in which secondary branch primordia were formed. The second component accounted for 23.4%, and its main components were explained by formation of more primordia on the rachis and primary branches. Indica tended to have more primary branches and their lateral organs, whereas japonica tended to have a longer rachis with more lateral organs, suggesting that there were heterochronic differences in panicle development. Quantitative trait locus (QTL) analyses for panicle traits and heading date were performed in 5 F2 populations and a set of backcross inbred lines; these were derived from crosses between Koshihikari and six other accessions, including the top three for number of spikelets per panicle. A total of 174 QTLs were detected for 10 panicle traits. Only the QTLs on chromosomes 3, 6, and 7 were shared, and only by two or three populations, suggesting that variation in domesticated rice inflorescence architecture is determined by many genes.
Cold stress at the booting stage in rice induces spikelet sterility because of aberrant microspore development, which often seriously damages seed production. Some breeding lines with high cold tolerance were developed by using tropical japonica variety Silewah as a donor of cold tolerance; however, the genetic factors that confer cold tolerance of this variety have not been comprehensively analyzed. In this study, phenotypic and molecular characterization of novel cold-tolerant strains derived from crosses with Silewah was performed to identify quantitative trait loci (QTLs) responsible for cold tolerance. Molecular marker analysis revealed that 2 cold-tolerant strains carried chromosomal segments of Silewah at the same genomic regions on chromosomes 3, 4 and 11. Single marker analysis in segregating population confirmed that the allele of Silewah on chromosome 3 (qCTB3-Silewah) conferred cold tolerance. The effect of qCTB3-Silewah was supported by the fact that this allele had been a target of selection during developing a breeding line by phenotypic selection from backcrossed progenies with an elite variety as a recurrent parent. qCTB3 is a different QTL from those reported for Silewah previously, suggesting that different QTLs might be exploited in different breeding programs depending on the genetic backgrounds and environmental conditions.
Grain weight (GW) is one of the most important targets for grain yield in rice breeding. In previous studies, two quantitative trait loci (QTLs) for GW, gw8.1 and gw9.1, have been identified using progeny derived from a cross between the japonica cultivar Hwaseong and Oryza rufipogon (IRGC 105491). To test whether these quantitative trait loci (QTLs) have an epistatic interaction, we developed an F2 population by crossing two nearly isogenic lines (NILs) harboring gw8.1 and gw9.1. Simple sequence repeat (SSR) markers tightly linked to the QTLs were used to select F3 QTL-NILs from the F2 population. A two-way ANOVA revealed an epistatic interaction between the two QTLs in the F2 population (P = 0.0084). This interaction was confirmed by an analysis of F3 QTL-NILs indicating that both QTLs are involved in the same genetic mechanism controlling GW. The gw8.1 QTL was further mapped between two SSR markers, RM23204 and RM23211, which are 110.1 kb apart. To our knowledge, this is the first report using QTL-NILs to reveal an epistatic interaction between QTLs for GW.
Backcross populations at advanced generations are useful in evaluating the agronomic traits from unadapted germplasms, such as wild species. In this study, we generated 159 backcross recombinant inbred lines (BRILs) at the BC2F8 generation between Oryza sativa Nipponbare (a recurrent parent) and O. rufipogon W630 (a donor parent). These BRILs were found to have nearly homozygous genome constitutions based on the marker genotypes at 180 SSR loci covering whole chromosomes. Since their selfed progenies have almost identical genotypes in each line, they were further used for QTL analysis of drought tolerance. Out of six QTLs detected, three were found to have wild alleles with favorable effects on drought tolerance. The present BRILs with known marker data can be applied to more QTL analyses of traits specific to wild species.
The short awn 2 (lks2) and dense spike 1 (dsp1) genes are unique to East Asian barley. These two spike-related morphological genes are important because they may be involved in stable production and local adaptation. As a first step of their positional cloning, molecular mapping was conducted in 98 F2 plants derived from a cross between Karafuto Zairai and Aizu Hadaka 3. The dsp1 gene was mapped to the proximal region of the short arm of chromosome 7H. The lks2 gene was located on the long arm of 7H and flanked by EST-based markers k04151 and k06123, with distances of 0.5 cM in the proximal side and 1.0 cM in the distal side. Both k04151 and k06123 shared homology to rice genes on chromosome 6 that were separated with the physical distance of 5.6 Mbp. In this interval, rice–barley microsynteny was exploited for marker enrichment. Of 57 rice genes attempted, 15 (26.3%) yielded polymorphic EST-based markers. Breakdown of collinearity was found in the candidate region of lks2, suggesting occurrence of structural changes in the chromosome region harboring lks2 during divergence of barley and rice from a common ancestor.
‘Tachisuzuka’ is a new rice (Oryza sativa L.) cultivar for whole-crop silage (WCS) use. Agronomic characters were recorded from 2007 to 2009. The culm length of Tachisuzuka was 11 cm longer than that of Kusanohoshi. The short panicle of Tachisuzuka was morphologically similar to that of the short panicle 1 (sp1) mutant. Tachisuzuka exhibited high lodging resistance in the yellow ripening stage, the optimal period for harvesting, and a month after yellow ripening. The straw yield of Tachisuzuka was remarkably higher than that of Kusanohoshi but its grain yield was lower. A portion of grains are undigested and excreted in feces by cattle fed with rice WCS, and this loss of nutrients should not be disregarded. The low grain/straw ratio of Tachisuzuka indicates that the loss of nutrients in cattle fed with Tachisuzuka WCS should be lower than with Kusanohoshi WCS. Low sugar content of rice plants is considered a cause of silage deterioration. The sugar content of Tachisuzuka was markedly higher than that of Kusanohoshi.
Oryzamutaic acids A and D, alkaloids with a novel type of nitrogen-containing heterocyclic ring system, which had previously been isolated only from the brown rice (husked rice) of Hatsuyamabuki, an Oryza sativa artificial mutant cultivar with yellow endosperm, were isolated from the brown rice of Musashino 20, an Oryza sativa spontaneous mutant line with yellow endosperm. The concentrations of oryzamutaic acids A and D in the brown rice of Musashino 20 were similar to those of Hatsuyamabuki, respectively. These results suggest that oryzamutaic acid A may play an important role in the yellow color of the brown rice of Musashino 20.
Microspore culture is an important method for production of haploids and doubled haploids. Although the routine protocol of isolated microspore culture of Brassica species has been established, the protocol for a large number of genotypes is laborious. In this paper, we report an improvement of the microspore culture method for dealing with multiple samples. The improved method showed the same results as the conventional one in the number of isolated microspores per bud and embryo yield per dish. The improved protocol is easier and can deal with two to four times more genotypes than the conventional one during the same period. This method provides some advantages to plant breeders and/or geneticists.