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In this study, DNA markers were developed for discrimination of strawberry (Fragaria × ananassa L.) cultivars based on retrotransposon insertion polymorphisms. We performed a comprehensive genomic search to identify retrotransposon insertion sites and subsequently selected one retrotransposon family, designated CL3, which provided reliable discrimination among strawberry cultivars. Through analyses of 75 strawberry cultivars, we developed eight cultivar-specific markers based on CL3 retrotransposon insertion sites. Used in combination with 10 additional polymorphic markers, we differentiated 35 strawberry cultivars commonly cultivated in Japan. In addition, we demonstrated that the retrotransposon-based markers were effective for PCR detection of DNA extracted from processed food materials, whereas a SSR marker was ineffective. These results indicated that the retrotransposon-based markers are useful for cultivar discrimination for processed food products, such as jams, in which DNA may be fragmented or degraded.

The soil-borne plasmodiophorid Polymyxa graminis is a vector for Barley yellow mosaic virus (BaYMV), which can severely damage barley plants. Although 22 disease resistance genes have been identified, only a few have been used for breeding virus-resistant cultivars. Recently, BaYMV strains capable of overcoming the effects of some of these genes have been detected. In this study, green fluorescent protein (GFP)-expressing BaYMV was constructed and used to examine viral dynamics in inoculated barley plants. Leaf inoculations resulted in higher infection rates than root or crown inoculations. Additionally, inoculations of some resistant cultivars produced infections that were similar to those observed in a field test. The results of this study indicate that the GFP-expressing virus is a useful tool for visualizing virus replication and dynamics, and for understanding resistance mechanisms.

Owing to its high ornamental value, the double flower phenotype of hydrangea (Hydrangea macrophylla) is one of its most important traits. In this study, genome sequence information was obtained to explore effective DNA markers and the causative genes for double flower production in hydrangea. Single molecule real-time sequencing data followed by a HiC analysis was employed. The resultant haplotype-phased sequences consisted of 3,779 sequences (2.256 Gb in length and N50 of 1.5 Mb), and 18 pseudomolecules comprising 1.08 Gb scaffold sequences along with a high-density SNP genetic linkage map. Using the genome sequence data obtained from two breeding populations, the SNPs linked to double flower loci (Djo and Dsu), were discovered for each breeding population. DNA markers J01 linked to Djo and S01 linked to Dsu were developed, and these could be used successfully to distinguish the recessive double flower allele for each locus respectively. The LEAFY gene was suggested as the causative gene for Dsu, since frameshift was specifically observed in double flower accession with dsu. The genome information obtained in this study will facilitate a wide range of genomic studies on hydrangea in the future.

To investigate the mode of inheritance of apomixis in Chinese chive, the degrees of diplospory and parthenogenesis were evaluated in F(1) and BC(1) progenies derived from crosses between amphimictic and apomictic diploids (2n = 16, 2x). The F(1) population was generated by crossing three amphimictic diploids 94Mo13, 94Mo49 and 94Mo50 with an apomictic diploid KaD2 and comprised 110 diploids and 773 triploids. All the diploid F(1) plants examined were completely or highly eusporous and completely syngamic. All the triploid F(1) plants examined were highly diplosporous and highly parthenogenetic. KaD2 could not transmit its high level of apomixis via monoploid pollen grains. The BC(1) population, generated by crossing 94Mo49 with apomictic triploids found in the F(1) offspring, exhibited heteroploidy; it comprised haploid, diploid, triploid, tetraploid and various aneuploid individuals. In this generation, clear segregation was observed between diplospory and parthenogenesis. Analysis of the BC(1) population suggests that diplospory and parthenogenesis are each controlled by single dominant genes, D and P, respectively. However, all the BC(1) plants characterized as parthenogenetic were diplosporous. The absence of phenotypically eusporous parthenogenetic plants can be explained by assuming that the presence of diplospory gene is a prerequisite for the parthenogenesis gene expression in Chinese chive.

ニラのアポミクシス（複相大胞子形成と単為発生から構成される）の遺伝様式を解明する手がかりを得るため，アポミクシス四倍体ニラ（Allium ramosum, syn. A. tuberosum 2n=32）の交雑集団において，胚珠観察法を用いて単為発生を調査した結果，10個体の非単為発生個体を選抜することができた．フローサイトメーターによる倍数性調査を行ったところ，全個体が四倍体であった．9個体の非単為発生個体については，交雑を確認するためにアポミクシス品種と交雑検定を行った．この結果，1個体（00-03-14）を除いて100%の交雑率を示した．「テンダーポール」を検定親（花粉親）とした組合せでは，単為発生と非単為発生個体がほぼ1:1に分離したが，他の組合せでは単為発生個体の頻度が高く，非単為発生因子を優性と仮定した場合に矛盾が生じた．このことから，単為発生を支配する主働遺伝子は単一の優性因子であると推察され，カイ二乗検定を行った結果，「テンダーポール」はその遺伝子座について単式遺伝子型であることが示唆された．5個体の非単為発生個体（97-11-7，97-12-43，97-12-85，00-02-3，00-02-187）を種子親とした検定交雑では，F1世代に六倍体が高頻度で出現した．このうち（97-12-43×「テンダーポール」）F1の六倍体80個体について花粉親特異的なRAPDマーカー4個の遺伝子型を調べたところ，76個体（95%）でマーカーの分離が認められた．このことから，種子親である非単為発生個体において，複相大胞子形成に起因する非減数性卵細胞が受精して六倍体を生じたものと推察された．以上の結果，本研究において四倍体ニラの両性生殖個体が得られ，F1で六倍体を生じた両性生殖個体は複相大胞子を形成していると推察された．

We previously demonstrated that cowpea [Vigna unguiculata (L.) Walp.] cultivar Kurodane-Sanjaku contains the Cry gene, which confers resistance against Cucumber mosaic cucumovirus infection. In this paper, randomly amplified polymorphic DNA (RAPD) analysis was carried out to tag the Cry locus. Bulked segregant analysis for RAPD resulted in many polymorphisms in amplified DNA patterns. Candidates were further screened using parental and/or F2 cowpea DNAs. As a result, we obtained three RAPD markers, D13/E14-350, WA3-850 and OPE3-500, flanking the Cry locus. In addition, we amplified cowpea sequences coding for the putative nucleotide-binding site (NBS). Degenerate primers based on NBS sequences of tobacco N and Arabidopsis RPS2 disease resistance genes were used for polymerase chain reaction, and resultant products were cloned and sequenced. Among eight independent clones, cowpea resistance gene analog (CRGA) 5 showed a distinct polymorphism when used as a probe for restriction fragment length polymorphism analysis against the susceptible cowpea cultivar PI 189375 and a near-isogenic line for the Cry. Linkage analyses of these molecular markers showed that genetic distances of CRGA5, D13/E14-350, WA3-850 and OPE3-500 to the Cry locus were 0.7, 5.2, 11.5 and 24.5 cM, respectively.

The elicitation of the hypersensitive response (HR) is known to depend on the interaction between a resistance gene of a host plant and a corresponding avirulence gene of a pathogen. The cv. Kurodane-Sanjaku of cowpea (Vigna unguiculata) has the Cry locus that confers resistance against cucumber mosaic virus strain Y (CMV-Y). The resistance is overcome by infection with a legume strain of CMV (CMV-L). RNA 2, which codes for the 2a protein, a subunit of the viral replicase components, has been known to control virulent/avirulent phenotypes. We generated chimeric constructs of full-length cDNA clones of RNA 2 of both strains and inoculated infectious transcripts to delimit the domain controlling symptoms. A 243-base pair fragment containing a coding region for the GDD RNA-dependent RNA polymerase core sequence was shown to be responsible for the phenotypic differences. From sequence alignment analysis, two amino acids (Phe631 and Ala641) of the HR-type 2a protein encoded in this fragment were specifically exchanged to Tyr and Ser, respectively, in the 2a proteins of resistance-breaking strains. Point mutations introduced into RNA 2 backgrounds of both strains that were designed to change the amino acid at position 631 resulted in a change of symptoms, indicating that a single nucleotide change determines the reactions elicited by both strains. Analysis for one additional mutant RNA 2 showed that symptom determination may be correlated with the nature of the lateral chain of amino acid 631.

Transposon Tn4431 was used to introduce a luciferase (lux) operon into Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174. Two transconjugants which were highly bioluminescent and indistinguishable in pathogenicity with the parental strain were selected. The transconjugants produced more bioluminescence in the logarithmic phase in liquid cultures. Bioluminescence was also detected from rice leaves infected with the transconjugants. In the compatible rice cultivar IR24 bacteria multiplied and moved prior to the expression of disease symptoms. In contrast, in the incompatible rice cultivar IR-BB2, which has resistance genes Xa1 and Xa2, bioluminescence was restricted near the inoculation sites, but light produced by the transconjugants was detected even 3 weeks after inoculation, suggesting that bacteria could multiply, although restrictedly, and remain viable. No bacterium was reisolated from the leaf sections beyond the light-producing regions. The detection of bioluminescence makes it possible to examine the actual location or infection process of X. o. pv. oryzae continuously without destroying the infected leaves.

Ralstonia solanacearum OE1-1 was transformed with pNP126 carrying a luxCDABE operon of Vibrio fisheri and a promoter region derived from the genomic DNA of Burkholderia glumae. Intensity of bioluminescence from YN5 thus obtained positively correlated with growth of the bacteria from the lag phase to the stationary phase in vitro. In this study, we used the VIM camera equipped with the ARGUS 50 to successively observe bioluminescence and the development of bacterial wilt in singly and doubly grafted tomato plants in various combinations of the resistant LS-89 and the susceptible Oogata-Fukuju. The degree of bacterial growth in the roots and collars was one of the determinants for the induction of bacterial wilt in tomato plants. Resistance of LS-89 resulted from suppression of bacterial proliferation in the roots and stems below the first leaf. In grafted tomato plants using LS-89 as the rootstock, all rootstocks were latently infected with the bacteria. The bacteria were also recovered from susceptible scions even though the plants did not wilt. In some plants, the bacteria proliferated in the susceptible scions. These plants wilted heavily. Taken together, suppression of bacterial proliferation in the roots and the stems below the first leaf of resistant rootstocks affected the bacterial growth in the susceptible scions and quantative control of resistance against development of the disease.