Amyβ is a single gene that encodes β-amylase, a starch-hydrolyzing enzyme that confers sweetness to sweet potato roots upon cooking. To clarify the genetic basis underlying β-amylase null activity, we sequenced a highly conserved region of Amyβ, including the Amyβ active center, from normal and null cultivars. Comparison of the sequences revealed the presence of the In-Del sequences in null cultivars, which was thought to be a loss-of-function mutation causing a change from sweet to non-sweet. The In-Del frequency revealed using a multiple sequencing approach, and its association with β-amylase activity, showed that an inactive allele of Amyβ gene (Amyβ-I) exists. Amyβ expression analysis showed that Amyβ-I is transcribed normally, suggesting that translational control of β-amylase activity occurs in null mutants. The insertion in Amyβ-I caused a sequence frameshift resulting in an amino acid substitution with the incorporation of a stop codon in Amyβ-I. The Amyβ-I amino acid substitution and premature termination affect the substrate binding and catalytic site of the enzyme, which may result in an inactive protein that is responsible for the β-amylase inactivity in non-sweet cultivars. These findings are the basis of selection markers for non-sweetness in sweet potato breeding.
Association or linkage disequilibrium mapping is alternative to identify QTLs in plants. In this study we used SSR based sorghum diversity research set (SDRS) of 107 sorghum accessions. The representative set included a geographically diverse collection of accessions from 27 countries in Asia and Africa. For association analysis 98 sorghum SSR markers were selected from three previously published linkage maps. Phenotypic data was recorded for 26 morphological traits. Different association models were used to identify QTLs controlling major agronomic traits including both single QTL approaches as well as a multiple QTL approach. All models revealed loci having different strength of association with morphological traits. A total of 14 common significant SSR loci were identified by three different models of association analysis namely, single-QTL models with the effects of population structure, single-QTL models with the effects of population structure and familial relatedness, and multiple-QTL model with the effects of population structure. These loci were associated with 12 different morphological traits including days to heading, days to flowering, culm length, number of tillers, number of panicles and panicle length. Comparing results from different models may be an efficient way to detect reliable associations in the genome-wide association studies.
Weedy rice (Oryza sativa L.) plants found in Okayama, Japan, exhibit various morphophysiological characteristics, and are roughly classified into japonica-type and indica-type. We characterized the weedy strains collected from nine sites with molecular markers in flanking regions of two diagnostic isozyme loci, Acp1 and Sdh1. Haplotype analysis for closely-linked SSR markers flanking Acp1 revealed that 1) japonica-type weedy strains shared the same haplotypes with local varieties, and 2) indica-type weedy strains carrying an indica-specific isozyme allele possessed two haplotypes. One of the two haplotypes was common to a forage variety, ‘Moretsu’. PCR analysis revealed that the dwarf weedy strains carried the deletion inside OsGA20ox2 as the semi-dwarf allele (sd1). The deletion was also found in ‘Moretsu’. The other haplotype was shared with indica forage variety, ‘Tetep’, which showed the same sequence haplotype for Sdh1 with the indica-type weedy strains. The results obtained from this study suggested that the weedy strains in Okayama have multiple origins and semi-dwarfness possibly derived from ‘Moretsu’ or indica modern varieties.
Enhancing nitrogen utilization is important in maintaining crop yield under low nitrogen conditions. Therefore, rice plants (Oryza sativa L.) were transformed with pMLH7133-RNRT2, which bears OsNRT2.1, a major gene involved in nitrate uptake under low nitrate concentrations. Two transgenic plants harboring an OsNRT2.1 transgene were identified and used for further study. In transgenic plants, OsNRT2.1 was expressed constitutively in roots, while in wild-type plants, the transcript was detected 30 min after starting to supply nitrate. No co-suppression between transgene and host gene was observed, as was observed on introduction of a nitrate reductase gene from the same species. One of the transformants grew faster than the wild-type cultivars when KNO3 or NH4NO3 was supplied as a nitrogen source. However, no increase in nitrate uptake by young plants was observed. These results suggest that constitutive expression of OsNRT2.1 might enhance vegetative growth and that introduction of genes involved in nitrate uptake from the same species may be useful for genetic improvement of plant growth under low nitrogen conditions. Further studies are necessary to understand the molecular and physiological bases of expression of NRT2.1 in relation to plant growth.
Herbicide resistance genes have been widely used in plant biotechnology both for commercial applications and as selectable markers. Phosphinothricin N-acetyltransferase (PAT) detoxifies the nonselective herbicide phosphinothricin (PPT, the ammonium salt of which is known as glufosinate), which is the active ingredient in bialaphos. Two novel PAT genes, hpat and mat, have been isolated from PPT-resistant soil bacteria, and we have now introduced these genes under the control of the 35S promoter of cauliflower mosaic virus into soybean [Glycine max (L.) Merrill] by particle bombardment and subsequent hygromycin selection. The expression of each enzyme was confirmed in leaves of the transgenic soybean plants by immunoblot analysis with specific antibodies and by measurement of PAT activity. The transgenic plants showed complete resistance to a commercial formulation of PPT at the recommended concentration. Our results suggest that these PAT genes may prove useful as markers for soybean transformation as well as in the production of herbicide-resistant transgenic plants.
We searched for blast resistance genes in an Indica-type rice (Oryza sativa L.) cultivar, Kasalath. The resistance gene was estimated based on the reaction patterns to standard differential blast isolates from Japan and Philippines against the monogenic lines as differential varieties targeting 24 kinds of resistance genes. Kasalath showed complete resistance only to a Japanese isolate, Hoku1, which was avirulent to genotypes containing the Piz allele genes and virulent to genotypes containing Pia and Pish. Kasalath was susceptible, or only moderately resistant, to the Philippine isolates. A QTL analysis for resistance to Hoku1 was performed in backcross inbred lines derived from crosses between Kasalath and a Japonica-type cultivar, Nipponbare (recurrent parent), harboring Pia and Pish. A resistance QTL with the Kasalath genotype was detected in the same region as Pia on chromosome 11. Association analysis in 50 chromosome segment substitution lines detected three associations, on chromosomes 6, 8 and 11; the association located in the same region as the QTL on chromosome 11 had a strong resistance effect. These reaction patterns indicated that Kasalath did not harbor Pia and that the QTL on chromosome 11 was the other allele of Pia or a novel resistance gene.
Fusarium head blight (FHB) is the most serious disease affecting wheat (Triticum aestivum L.) because of its impact on yield and grain quality and its associated mycotoxin accumulation. This study was conducted (1) to develop efficient methods to evaluate varietal resistance against fungal spread after infection, (2) to investigate varietal differences in resistance to FHB infection, FHB spread, and mycotoxin accumulation in 23 Asian varieties, and (3) to analyze relations among FHB-resistant types. Inoculation of Fusarium graminearum isolate, causing virulence to detached spikes, enabled evaluation of FHB spread in a shorter period and smaller space than conventional methods. Among the varieties, the FHB spread differed significantly. ‘Sumai 3’ and its derivatives showed higher resistance to FHB spread than other varieties. The FHB spread showed no significant correlation with FHB infection. It showed significant positive correlation with DON, but some varieties with high resistance to FHB spread showed moderately high DON. Results show that improved methods to evaluate FHB spread can be effective to screen numerous materials. The FHB resistance of western Japanese varieties depends mainly on resistance to FHB spread. For development of FHB resistant varieties, due attention should be devoted to DON.
The objective of this study was to investigate varietal differences in dry matter production and physiological nitrogen use efficiency (PNUE) using a wide range of rice (Oryza sative L.) varieties at an early growth stage. The results provide useful information for breeding suitable varieties for cultivation under conditions of low soil fertility or with reduced fertilizer use for sustainable agriculture. Thirty-one rice varieties were used, and seedlings were grown under 8 nitrogen treatment conditions. The effects of natural variations in embryo and/or endosperm size on initial growth were controlled for by comparing the relative dry weight (RDW) of nitrogen-treated samples and controls. The rice varieties were classified into 5 groups by cluster analysis. Overall, the present study found that there are wide variations in PNUE among varieties with similar levels of RDW under varying nitrogen conditions. Variations in PNUE were greater under lower nitrogen conditions; in contrast, variations in RDW were greater under higher nitrogen conditions. These data, along with those of other studies, indicate differences in nitrogen use efficiency between Indica- and Japonica-types, upland and lowland varieties, and landraces and improved types.
Two tea plants containing little caffeine (caffeine-less) were found in a population derived from natural crossings of an interspecific hybrid ‘Cha Chuukanbohon Nou 6’ (Camellia taliensis × C. sinensis). Two caffeine-less plants showed low caffeine content and rather high theobromine, which is a precursor of caffeine, whereas all tested tea (C. sinensis) cultivars including ‘Cha Chuukanbohon Nou 6’ were observed high caffeine/low theobromine contents. Component analysis by HPLC showed that ‘Taliensis-akame’ (C. taliensis), which is the seed parent of ‘Cha Chuukanbohon Nou 6’, showed low caffeine content and rather high theobromine and may be a caffeine-less character donor. Pedigree analysis using SSR markers suggested that parent-offspring relationship was found between tea plants related to caffeine-less tea breeding lines. Therefore, it is considered that caffeine-less character of caffeine-less plants could be inherited recessively from ‘Taliensis-akame’ via ‘Cha Chuukanbohon Nou 6’. Preliminary genetic analysis using 33 progenies of ‘Cha Chuukanbohon Nou 6’ suggested the possibility that the caffeine-less character might be controlled by one recessive locus. Caffeine-less tea plants found in this study, will be efficiently used for genetic resources to introgress caffeine-less traits to tea cultivars in breeding programs.
To identify quantitative trait loci (QTLs) associated with culm length in Japanese japonica rice varieties, we conducted QTL analyses using a set of reciprocal backcrossed inbred lines (BILs) from crosses between Nipponbare and Koshihikari. We detected five QTLs in the two BILs that are involved in the culm-length difference between Nipponbare and Koshihikari. A QTL located on the short arm of chromosome 1, qCL1, was commonly detected near the simple sequence repeat (SSR) marker RM8068 in both BILs in three growing seasons. The Nipponbare allele of qCL1 shortened from 1.9 to 3.0 cm of culm length. Substitution lines (SLs) of Koshihikari in which the QTL was replaced with the Nipponbare allele exhibited decreased lengths of lower internodes. qCL1 was mapped within about 2.6 Mbp in the region distal from SSR marker RM10132, suggesting qCL1 differed from the dwarf or semi-dwarf genes that have been previously reported. Japanese japonica rice landraces with Nipponbare alleles of DNA markers around qCL1 had significantly shorter culm length than other landraces. The results suggest that the novel allele for short culm length was originally distributed in Japanese rice landraces and has been used for introducing the semi-dwarf phenotype and improving plant architecture during practical breeding.
The distribution and proliferation among Oryza species of miniature Ping (mPing), the only miniature inverted-repeat transposable element (MITE) that is active in all organisms, were investigated through analysis of 78 accessions encompassing nine of the 10 genomic constitutions of the genus Oryza. The mPing family transposons, mPing, Ping, and Pong, were detected in only two of the AA genome species, Oryza sativa and its direct ancestor O. rufipogon, whereas the inactive rice MITE Kiddo was detected in the AA, BB, CC, BBCC and CCDD genome species. Furthermore, the insertion sites and copy numbers of mPing were considerably different among O. rufipogon accessions. A phylogenetic analysis showed that the O. rufipogon accessions used in this study could be grouped into four clusters which were not associated with the copy number of mPing or the presence of Ping. These results suggest that the mPing family was present in the ancestor of O. rufipogon and that mPing proliferated independently in each accession. Based on these results, one possible evolutionary history of the distribution and proliferation of mPing family in O. rufipogon is discussed.
Haruka is a new table-stock potato variety with parti-colored red/white-skinned tubers, white flesh, medium maturing and good culinary quality. It was selected from a cross between T9020-8 as the female parent and Sayaka as the male parent, and was released in 2007. Agronomic characters of the Haruka were checked from 2003 to 2007. Its marketable yield is 103% of the Sayaka and 124% of the Irish Cobbler. The average size of a tuber was 104 g. The starch value of Haruka was 16.2%. Dormancy period of the tuber was 105 days. In comprehensive evaluations of product quality, Haruka tubers shows a little disintegration of the flesh after boiling, the texture was rated as pasty, and the boiled tuber had favorable taste. Haruka was superior or equally good to Irish Cobbler and Sayaka for salad quality and had a high processing ability for chilled potato, whereas it was not suitable for chips and french-fry. Haruka is resistant to the golden nematode, and good resistant to brown rot and tuber rot, while it is susceptible to potato virus Y, common scab and foliage late blight.
In the cultivation of edible mushrooms including Pleurotus pulmonarius, the enormous number of spores produced by fruiting bodies adversely affects the health of mushroom farmers, mushroom cultivation facilities and the genetic diversity of natural populations. A sporeless mutant of P. pulmonarius (TMIC-30058) has been introduced with a high potential for breeding sporeless cultivars to reduce these adverse effects. We report here the conversion of the amplified fragment length polymorphism (AFLP) marker, closely linked to a sporeless locus, to a sequence tagged site (STS) marker for use in marker-assisted selection (MAS). By mainly bulked segregant analysis-based AFLP (BSA-AFLP), a total of 18 markers were identified the linkage to the sporeless locus with genetic distances ranging from 0 (co-segregation) from 3.4 cM. Out of them, 2 AFLP markers could be converted into STS markers, SD192 and SD296, and their implementations in various genetic backgrounds were validated by the amplification test against 15 wild type isolates from different geographical origin. Furthermore, we could amplify these 2 STS markers’ fragments by using multiplex PCR reproducibly. These results indicated that 2 STS markers can be used in MAS targeted at the sporeless trait in P. pulmonarius breeding.
Coenzyme Q (CoQ), also called ubiquinone, is an electron transfer molecule in the respiratory chain; it is also a lipid-soluble antioxidant. Most cereal crops produce mainly CoQ9, which has nine isoprene units, whereas humans produce mainly CoQ10, with 10 isoprene units. CoQ10 is a very popular food supplement. Using the cultivar Nipponbare, we previously produced CoQ10-enriched rice plants (Nipponbare-type CoQ10-enriched rice plants) by introduction of the gene for decaprenyl diphosphate synthase (DdsA). In Nipponbare-type CoQ10-enriched rice plants, seed CoQ10 content per weight was increased to up to 10 times that of wild-type rice. However, seed CoQ10 level of those rice plants is still insufficient for practical use, and further 10-times increase in CoQ10 content have been expected. Here, we confirmed preferential accumulation of CoQ in bran and germ of rice seed, and produced improved CoQ10-enriched rice plants by using 2 kinds of giant embryo rice lines, Haiibuki and Chukei-toku 70 (Toku 70). In giant embryo line-type CoQ10-enriched rice plants, seed CoQ10 content per weight was increased to up to 1.4 times (Haiibuki-type) or 1.8 times (Toku 70-type) that in Nipponbare-type CoQ10-enriched rice plants, demonstrating that use of giant embryo rice lines is effective to increase seed CoQ10 content per weight.