Recently we cloned and characterized the gene for the wheat transcription factor TaWRKY45 and showed that TaWRKY45 was upregulated in response to benzothiadiazole (BTH) and Fusarium head blight (FHB) and that its overexpression conferred enhanced resistance against F. graminearum. To characterize the functional role of TaWRKY45 in the disease resistance of wheat, in the present study we conducted expression analyses of TaWRKY45 with inoculations of powdery mildew and leaf rust and evaluated TaWRKY45-overexpressing wheat plants for resistance to these diseases. TaWRKY45 was upregulated in response to infections with Blumeria graminis, a causal fungus for powdery mildew, and Puccinia triticina, a causal fungus for leaf rust. Constitutive overexpression of the TaWRKY45 transgene conferred enhanced resistance against these two fungi on transgenic wheat plants grown under greenhouse conditions. However, the expression of two resistance-related genes, Pm3 and Lr34, was not induced by the inoculation with powdery mildew in TaWRKY45-overexpressing wheat plants. These results suggest that TaWRKY45 is involved in the defense responses for multiple fungal diseases in wheat but that resistance involving TaWRKY45 differs from at least Pm3 and/or Lr34-related resistance. Our present and previous studies indicate that TaWRKY45 may be potentially utilized to improve a wide range of disease resistance in wheat.

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Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N₂O) gas to obtain 2n male gametes. N₂O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is unknown how N₂O treatment restores fertility in sterile hybrids. To establish optimal N₂O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N₂O gas at different anther developmental stages from fertile and sterile hybrid lilies. N₂O treatment using the optimal 1–4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N₂O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC₁ plants. Thus N₂O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.

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Amylose content (AC) and viscosity profile are primary indices for evaluating eating and cooking qualities of rice grain. Using chromosome segment substitution lines (CSSLs), previous studies identified a QTL cluster of genes for rice eating and cooking quality in the interval R727–G1149 on chromosome 8. In this study we report two QTLs for viscosity parameters, respectively controlling setback viscosity (SBV) and consistency viscosity (CSV), located in the same interval using rapid viscosity analyzer (RVA) profile as an indicator of eating quality. Previously reported QTL for AC was dissected into two components with opposite genetic effects. Of four QTLs, qCSV-8 and qAC-8-2 had stable genetic effects across three and four environments, respectively. qSBV-8, qCSV-8 and qAC-8-1 partly overlapped, but were separated from qAC-8-2. Based on data from an Affymetrix rice GeneChip, two genes related to starch biosynthesis at the qAC-8-2 locus were chosen for further quantitative expression analysis. Both genes showed enhanced expression in sub-CSSLs carrying the target qAC-8-2 allele, but not in sub-CSSLs without the target qAC-8-2 allele, indicating their possible role in rice quality determination. Molecular markers closely linked to the two stable QTL provide the opportunity for marker-assisted selection (MAS) in breeding high quality rice.

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Few genes are available to develop drought-tolerant bread wheat (Triticum aestivum L.) cultivars. One way to enhance bread wheat’s genetic diversity would be to take advantage of the diversity of wild species by creating synthetic hexaploid wheat (SW) with the genomic constitution of bread wheat. In this study, we compared the expression of traits encoded at different ploidy levels and evaluated the applicability of Aegilops tauschii drought-related traits using 33 Ae. tauschii accessions along with their corresponding SW lines under well-watered and drought conditions. We found wide variation in Ae. tauschii, and even wider variation in the SW lines. Some SW lines were more drought-tolerant than the standard cultivar Cham 6. Aegilops tauschii from some regions gave better performing SW lines. The traits of Ae. tauschii were not significantly correlated with their corresponding SW lines, indicating that the traits expressed in wild diploid relatives of wheat may not predict the traits that will be expressed in SW lines derived from them. We suggest that, regardless of the adaptability and performance of the Ae. tauschii under drought, production of SW could probably result in genotypes with enhanced trait expression due to gene interactions, and that the traits of the synthetic should be evaluated in hexaploid level.

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Imported genetically modified (GM) canola (Brassica napus) is approved by Japanese law. Some GM canola varieties have been found around importation sites, and there is public concern that these may have any harmful effects on related species such as reduction of wild relatives. Because B. juncea is distributed throughout Japan and is known to be high crossability with B. napus, it is assumed to be a recipient of B. napus. However, there are few reports for introgression of cross-combination in B. juncea × B. napus. To assess crossability, we artificially pollinated B. juncea with B. napus. After harvesting a large number of progeny seeds, we observed false hybrids and metaxenia of seed coats. Seed coat color was classified into four categories and false hybrids were confirmed by morphological characteristics and random amplified polymorphic DNA (RAPD) markers. Furthermore, the occurrence of false hybrids was affected by varietal differences in B. napus, whereas that of metaxenia was related to hybridity. Therefore, we suggest that metaxenia can be used as a marker for hybrid identification in B. juncea L. cv. Kikarashina × B. napus. Our results suggest that hybrid productivity in B. juncea × B. napus should not be evaluated by only seed productivity, crossability ought to be assessed the detection of true hybrids.

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Two cytologically stable wheat-Dasypyrum breviarisatatum addition lines, Y93-1-6-6 and Y93-1-A6-4, were identified by integrated molecular and cytogenetic techniques. C-banding and genomic in situ hybridization (GISH) showed that Y93-1-6-6 and Y93-1-A6-4 were different wheat-D. breviaristatum additions. A total of 51 markers (primer/enzyme combinations), including 6 PCR-based Landmark Unique Gene (PLUG) markers and 45 Sequence-Tagged-Site (STS) markers, were selected from 3,774 primer/enzyme combinations to further characterize these two additions. Marker haploytpes suggested that both D. breviaristatum chromosomes in Y93-1-6-6 and Y93-1-A6-4 were rearranged. Stem rust resistance screening indicated that both additions were highly resistant to race RKQQC, whereas only Y93-1-6-6 was resistant to race TTKSK (Ug99). Powdery mildew resistance screening showed that only Y93-1-6-6 was resistant. Pedigree analysis suggested that the stem rust and powdery mildew resistance of Y93-1-6-6 was derived from D. breviaristatum, indicating that the D. breviaristatum chromosomes in Y93-1-6-6 possess a new powdery mildew resistance gene(s), and new stem rust resistance gene(s). These two additions could be used as stem rust or powdery mildew resistance sources in wheat breeding programs.

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Intergeneric hybridization was performed between Moricandia arvensis and four inbred lines of Brassica rapa following embryo rescue. Three F₁ hybrid plants were developed from three cross combinations of M. arvensis × B. rapa, and amphidiploids were synthesized by colchicine treatment. Six BC₁ plants were generated from a single cross combination of amphidipolid × B. rapa ‘Ko1-303’ through embryo rescue. One BC₂ and three BC₃ plants were obtained from successive backcrossing with B. rapa ‘Ko1-303’ employing embryo rescue. Alloplasmic and monosomic addition lines of B. rapa (Allo-MALs, 2n = 21) were obtained from backcrossed progeny of three BC₃ plants (2n = 21, 22 and 23) without embryo rescue. An alloplasmic line of B. rapa (2n = 20) degenerated before floliation on 1/2 MS medium due to severe chlorosis. Allo-MALs of B. rapa (2n = 21) showed stable male sterility without any abnormal traits in vegetative growth and female fertility. Molecular analyses revealed that the same chromosome and cytoplasm of M. arvensis had been added to each Allo-MAL of B. rapa. This Allo-MAL of B. rapa may be useful material for producing cytoplasmic male sterile lines of B. rapa.

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The study on the genetic basis of heterosis has received significant attention in recent years. In this study, using a set of introgression lines (ILs) and corresponding testcross F₁ populations, we investigated heterotic loci (HL) associated with six yield-related traits in both Oryza sativa L. subsp. indica and japonica. A total of 41 HL were detected on the basis of mid-parent heterosis values with single-point analysis. The F₁ testcross population showed superiority in most yield-related traits and was characterized by a high frequency of overdominant HL. Thirty-eight of the 41 HL were overdominant, and in the absence of epistasis, three HL were dominant, suggesting that heterotic effects at the single-locus level mainly appeared to be overdominant in rice. Twenty-four HL had a real positive effect, suggesting that they are viable candidates for the improvement of rice yield potential. Compared with the quantitative trait loci (QTLs) detected in the ILs, only six out of the 41 (14.6%) HL were detected in QTL analysis under the same statistical threshold, indicating that heterosis and trait performance may be conditioned by different sets of loci.

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Black root rot (BRR) caused by Thielaviopsis basicola as well as Tomato spotted wilt virus (TSWV) are the most serious problems in tobacco growing regions. We crossed the breeding line WGL 3 carrying BRR resistance derived from N.glauca with the line PW-834 the resistance of which to TSWV was transferred from cultivar Polalta. Anthers obtained from F₁ hybrid plants were cultured to induce haploids combining resistance to Th. basicola and TSWV. Flow cytometry analysis revealed 242 haploids and 2 spontaneous doubled haploids among regenerants. All haploids were cloned and then evaluated for BRR as well as TSWV resistance. The presence of pathogens was detected by microscopic evaluation of roots or using DAS-ELISA test. Microscopic assessment showed that, 132 haploids had no symptoms of Th. basicola which, together with the absence of symptoms in the F₁ hybrids, indicated a dominant monogenic mode of inheritance. At the same time only 30 haploids demonstrated resistance to TSWV. SCAR markers associated with TSWV resistance gene detection was applied. The results indicate that small proportion of TSWV-resistant haploids is probably due to the influence of deleterious genes flanking the resistance factor that reduced vitality of gametophytes. Altogether, 24 haploids showed multiple resistance to Th. basicola and TSWV.

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Photoperiod sensitivity is an important trait related to crop adaptation and ecological breeding in common buckwheat (Fagopyrum esculentum Moench). Although photoperiod sensitivity in this species is thought to be controlled by quantitative trait loci (QTLs), no genes or regions related to photoperiod sensitivity had been identified until now. Here, we identified QTLs controlling photoperiod sensitivity by QTL analysis in a segregating F₄ population (n = 100) derived from a cross of two autogamous lines, 02AL113(Kyukei SC2)LH.self and C0408-0 RP. The F₄ progenies were genotyped with three markers for photoperiod-sensitivity candidate genes, which were identified based on homology to photoperiod-sensitivity genes in Arabidopsis and 76 expressed sequence tag markers. Among the three photoperiod-sensitivity candidate genes (FeCCA1, FeELF3 and FeCOL3) identified in common buckwheat, FeELF3 was associated with photoperiod sensitivity. Two EST regions, Fest_L0606_4 and Fest_L0337_6, were associated with photoperiod sensitivity and explained 20.0% and 14.2% of the phenotypic variation, respectively. For both EST regions, the allele from 02AL113(Kyukei SC2)LH.self led to early flowering. An epistatic interaction was also confirmed between Fest_L0606_4 and Fest_L0337_6. These results demonstrate that photoperiod sensitivity in common buckwheat is controlled by a pathway consisting of photoperiod-sensitivity candidate genes as well as multiple gene action.

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The genotypes of photoperiod response genes Ppd-B1 and Ppd-D1 in Japanese wheat cultivars were determined by a PCR-based method, and heading times were compared among genotypes. Most of the Japanese wheat cultivars, except those from the Hokkaido region, carried the photoperiod-insensitive allele Ppd-D1a, and heading was accelerated 10.3 days compared with the Ppd-D1b genotype. Early cultivars with Ppd-D1a may have been selected to avoid damage from preharvest rain. In the Hokkaido region, Ppd-D1a frequency was lower and heading date was late regardless of Ppd-D1 genotype, suggesting another genetic mechanism for late heading in Hokkaido cultivars. In this study, only 11 cultivars proved to carry Ppd-B1a, and all of them carried another photoperiod-insensitive allele, Ppd-D1a. The Ppd-B1a/Ppd-D1a genotype headed 6.7 days earlier than the Ppd-B1b/Ppd-D1a genotype, indicating a significant effect of Ppd-B1a in the genetic background with Ppd-D1a. Early-maturity breeding in Japan is believed to be accelerated by the introduction of the Ppd-B1a allele into medium-heading cultivars carrying Ppd-D1a. Pedigree analysis showed that Ppd-B1a in three extra-early commercial cultivars was inherited from ‘Shiroboro 21’ by early-heading Chugoku lines bred at the Chugoku Agriculture Experimental Station.

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Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.

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We genotyped strawberry cultivars by two newly selected and two previously reported SSR markers. All four markers produced interpretable electropherograms from 75 accessions consisting of 72 Fragaria × ananassa cultivars or lines and three octoploid Fragaria species accessions. These SSR markers were highly polymorphic; in particular, one of the newly developed markers, FxaHGA02P13, was capable of distinguishing all of the accessions except for a mutant strain that was derived from another accession in the set. When two markers were combined, all 48 full-sib individuals could be distinguished. Fingerprinting patterns were reproducible between multiple samples, including the leaves, sepals, and fruit flesh of the same accession. Principal-coordinate analysis of the 75 accessions detected several groups, which reflect taxon and breeding site. Together with other available markers, these SSR markers will contribute to the management of strawberry genetic resources and the protection of breeders’ rights.

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A Mexican hexaploid wild potato species, Solanum demissum (dms), was only used as a female in previous breeding programs. The resulting clones with dms cytoplasm produced abundant, but non-functional pollen. A 170 bp DNA fragment, named Band 1, was originally detected in the F₁ hybrid between dms and S. tuberosum. In this study, the sequenced region was extended to 1,032 bp; nevertheless, it did not show any homology to known sequences. This extended region harboring Band 1 was, without introns, all transcribed to mRNA and was maternally inherited from dms to S. tuberosum through backcrosses. Three dms accessions, 168 accessions of 38 cultivated and closely related wild species, and 158 varieties and breeding lines were surveyed, which demonstrated that Band 1 was specific to dms and varieties and breeding lines with dms cytoplasm. Thus, Band 1 is a useful marker to distinguish dms cytoplasm, which enables us to design efficient mating combinations in breeding programs.

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