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Self-incompatibility in the Brassicaceae family is governed by SRK and SCR, which are two highly polymorphic genes located at the S-locus. Previously, the Arabidopsis lyrata SRK and SCR genes were introduced into Arabidopsis thaliana to generate self-incompatible lines. However, there are no reports showing that Brassica SRK and SCR genes confer self-incompatibility in A. thaliana. Doing so would further advance the mechanistic understanding of self-incompatibility in Brassicaceae. Therefore, we attempted to generate A. thaliana transformants showing the self-recognition activity of Brassica rapa by introducing BrSCR along with a chimeric BrSRK (BrSRK chimera, in which the kinase domain of BrSRK was replaced with that of AlSKR-b). We found that the BrSRK chimera and BrSCR of B. rapa S-9 and S-46 haplotypes, but not those of S-29, S-44, and S-60 haplotypes, conferred self-recognition activity in A. thaliana. Analyses of A. thaliana transformants expressing mutant variants of the BrSRK-9 chimera and BrSCR-9 revealed that mutations at the amino acid residues involved in BrSRK9-BrSCR9 interaction caused defects in the self-incompatibility response. The method developed in this study for generating self-incompatible A. thaliana transformants showing B. rapa self-recognition activity will be useful for analysis of self-recognition mechanisms in Brassicaceae.

Gamma-rays are the most widely used mutagenic radiation in plant mutation breeding, but detailed characteristics of mutated DNA sequences have not been clarified sufficiently. In contrast, newly introduced physical mutagens, e.g., heavy-ion beams, have attracted geneticists’ and breeders’ interest and many studies on their mutation efficiency and mutated DNA characteristics have been conducted. In this study, we characterized mutations induced by gamma rays and carbon(C)-ion beams in rice (Oryza sativa L.) mutant lines at M5 generation using whole-genome resequencing. On average, 57.0 single base substitutions (SBS), 17.7 deletions, and 5.9 insertions were detected in each gamma-ray-irradiated mutant, whereas 43.7 single SBS, 13.6 deletions, and 5.3 insertions were detected in each C-ion-irradiated mutant. The structural variation (SV) analysis detected 2.0 SVs (including large deletions or insertions, inversions, duplications, and reciprocal translocations) on average in each C-ion-irradiated mutant, while 0.6 SVs were detected on average in each gamma-ray-irradiated mutant. Furthermore, complex SVs presumably having at least two double-strand breaks (DSBs) were detected only in C-ion-irradiated mutants. In summary, gamma-ray irradiation tended to induce larger numbers of small mutations than C-ion irradiation, whereas complex SVs were considered to be the specific characteristics of the mutations induced by C-ion irradiation, which may be due to their different radiation properties. These results could contribute to the application of radiation mutagenesis to plant mutation breeding.

Common buckwheat (Fagopyrum esculentum) is a heteromorphic self-incompatible (SI) species with two types of floral architecture: thrum (short style) and pin (long style). The floral morphology and intra-morph incompatibility are controlled by a single genetic locus, S. However, the molecular mechanisms underlying the heteromorphic self-incompatibility of common buckwheat remain unclear. To identify these mechanisms, we performed proteomic, quantitative reverse-transcription PCR, and linkage analyses. Comparison of protein profiles between the long and short styles revealed a protein unique to the short style. Amino-acid sequencing revealed that it was a truncated form of polygalacturonase (PG); we designated the gene encoding this protein FePG1. Phylogenetic analysis classified FePG1 into the same clade as PGs that function in pollen development and floral morphology. FePG1 expression was significantly higher in short styles than in long styles. It was expressed in flowers of a short-homostyle line but not in flowers of a long-homostyle line. Linkage analysis indicated that FePG1 was not linked to the S locus; it could be a factor downstream of this locus. Our finding of a gene putatively working under the regulation of the S locus provides useful information for elucidation of the mechanism of heteromorphic self-incompatibility.

High temperature disrupts the targeting of SRK to the plasma membrane, resulting in breakdown of the stigmatic self-incompatibility response in self-incompatible Arabidopsis transformants., Commercial seeds of Brassicaceae vegetable crops are mostly F1 hybrids, the production of which depends on self-incompatibility during pollination. Self-incompatibility is known to be weakened by exposure to elevated temperatures, which may compromise future breeding and seed production. In the Brassicaceae, self-incompatibility is controlled by two genes, SRK and SCR, which function as female and male determinants of recognition specificity, respectively. However, the molecular mechanisms underlying the breakdown of self-incompatibility under high temperature are poorly understood. In this study, we examined the self-incompatibility phenotypes of self-incompatible Arabidopsis thaliana SRK-SCR transformants under normal (23 °C) and elevated (29 °C) temperatures. Exposure to elevated temperature caused defects in the stigmatic, but not the pollen, self-incompatibility response. In addition, differences in the response to elevated temperature were observed among different S haplotypes. Subcellular localization revealed that high temperature disrupted the targeting of SRK to the plasma membrane. SRK localization in plants transformed with different S haplotypes corresponded to their self-incompatibility phenotypes, further indicating that defects in SRK localization were responsible for the breakdown in the self-incompatibility response at high temperature. Our results provide new insights into the causes of instability in self-incompatibility phenotypes.

SCR/SP11 encodes the male determinant of recognition specificity of self-incompatibility (SI) in Brassica species and is sporophytically expressed in the anther tapetum. Based on dominance relationships in pollen and nucleotide sequence similarity, the S haplotypes in Brassica have been classified as class I or class II, with class-I S haplotypes being dominant over class-II S haplotypes. Here, we revealed that S-22 in B. rapa belonging to class I is recessive to class-II S-44 and class-I S-36 in pollen, whereas it is dominant over S-60, S-40, and S-29 based on pollination tests. SCR/SP11 of S-22 (SCR-22) was sequenced, revealing that the deduced amino-acid sequence of SCR-22 has the longest C-terminal domain among the SCR/SP11 sequences. The expression of SCR-22 was found to be suppressed in S-22/S-44 and S-22/S-36 heterozygotes. Normal transcription of SCR-44 was considered to be due to the transcription suppression of Smi sRNA of the S-22 haplotype and a very low methylation state of the SCR-44 promoter region in the tapetum of S-22/S-44 heterozygotes. In SCR-22, only the cytosine residue located at the –37 bp position of the promoter region was hypermethylated in the tapetum of S-22/S-44 heterozygotes, and few methylated cytosines were detected in the promoter and coding regions of SCR-22 in S-22/S-36 heterozygotes. SCR-22 was also expressed in microspores in S-22 homozygotes but not in S-22/S-44 and S-22/S-36 heterozygotes. These results suggest that a mechanism different from class-II SCR/SP11 suppression may operate for the suppression of recessive class-I SCR-22 in S heterozygotes., Self-incompatibility: Rethinking hierarchy Studies in cabbage plants shed new light on self-incompatibility mechanisms to avoid self-fertilization. In Brassica, self-incompatibility is controlled by the expression of dominant and recessive genetic variants in the S-locus region. These variants encode proteins that mediate the rejection of self-pollen, but little is known about how the dominance relationship is established between them. Takeshi Nishio at Tohoku University in Japan and colleagues have examined the sequence, expression pattern and dominance relationships of the gene encoding the pollen-coat protein SCR-22. Despite sharing features with dominant SCR variants, SCR-22 can act in a recessive manner. Interestingly, unlike other recessive SCR variants, the suppression of SCR-22 expression does not depend on the addition of methyl groups that prevent transcription factor binding. This finding suggests the dominance hierarchy is governed by different mechanisms.

Self-incompatibility in the Brassicaceae family is governed by two-linked highly polymorphic genes located at theSlocus,SRKandSCR. Previously, theSRKandSCRgenes ofArabidopsis lyratawere introduced intoArabidopsis thalianatransformants to generate self-incompatible lines. However, it has not been reported that theSRKandSCRgenes ofBrassicaspecies confer self-incompatibility inA. thaliana. In this study, we attempted to construct self-incompatibleA. thalianatransformants expressing the self-recognition activity ofBrassica rapaby introducing theBrSCRgene along with a chimericBrSRKgene (BrSRK chimera, in which the kinase domain ofBrSRKwas replaced with that ofAlSKRb). We found thatBrSRK chimeraandBrSCRofB. rapa S-9andS-46haplotypes, but not those ofS-29,S-44, andS-60haplotypes, conferred self-recognition activity inA. thaliana. We also investigated the importance of amino acid residues involved in the BrSRK9–BrSCR9 interaction usingA. thalianatransformants expressing mutant variants ofBrSRK-9 chimeraandBrSCR-9. The results showed that some of the amino acid residues are essential for self-recognition. The method developed in this study for the construction of self-incompatibleA. thalianatransformants showingB. rapaself-recognition activity will be useful for analysis of self-recognition mechanisms in Brassicaceae.

The timing of flower formation and length of the seed-filling period of indeterminate growth soybean varieties vary more than those of determinate varieties (Glycine max (L.) Merrill). These variations have been hypothesized to affect single seed weight and its uniformity which determine the processing quality of soybean used in foods. We derived near isogenic lines (NILs) with different growth characteristics from an indeterminate line (donor parent) and three determinate lines with heavy seeds (recurrent parents), and evaluated the effects of growth habit on seed weight and its uniformity. Each NIL population consisting of five indeterminate and five determinate BC4F4 lines tested at two locations in two different years with two replications. Split-plot analysis of variance, with main-plot and sub-plot being cross combination and growth habit, respectively, showed that indeterminate varieties had slightly heavier seeds than determinate varieties and that there was no significant difference in uniformity of single seed weights. The effects of growth habit on seed uniformity was related to genetic background, but differences between the two growth characteristics were less than the differences among genetic background. This indicates that indeterminate growth habit did not much influence seed weight or its uniformity.

In general, axonal regeneration is very limited after transection of adult rat spinal cord. We previously demonstrated that regenerative axons reached the lesion site within 6 h of sharp transection with a thin scalpel. However, they failed to grow across the lesion site, where injured axon fragments (axon-glial complex, AGC) were accumulated. Considering a possible role of these axon fragments as physicochemical barriers, we examined the effects of prompt elimination of the barriers on axonal growth beyond the lesion site. In this study, we made additional oblique section immediately after the primary transection and surgically eliminated the AGC (debridement). Under this treatment, regenerative axons successfully traversed the lesion site within 4 h of surgery. To exclude axonal sparing, we further inserted a pored sheet into the debrided lesion and observed the presence of fascicles of unmyelinated axons traversing the sheet through the pores by electron microscopy, indicating bona fide regeneration. These results suggest that the sequential trial of reduction and early elimination of the physicochemical barriers is one of the effective approaches to induce spontaneous and rapid regeneration beyond the lesion site.

Internalization of plasma membrane (PM)-localized ligand-activated receptor kinases and their trafficking to sorting endosomes have traditionally been viewed as functioning primarily in the down-regulation of receptor signaling, but are now considered to be also essential for signaling by some receptors. A major mechanism for internalization of PM proteins is clathrin-mediated endocytosis (CME). CME is mediated by the Adaptor Protein Complex 2 (AP2), which is involved in interaction of the AP2 μ-adaptin subunit with a tyrosine-based Yxxϕ motif located in the cytoplasmic domain of the cargo protein. In this study, we investigated the role of AP2-mediated CME for signaling by the S-locus receptor kinase (SRK), a protein localized in the PM of stigma epidermal cells, which, together with its pollen coat-localized S-locus cysteine-rich (SCR) ligand, functions in the self-incompatibility (SI) response of the Brassicaceae. Using Arabidopsis thaliana plants that were made self-incompatible by transformation with an A. lyrata-derived SRK/SCR gene pair, we tested the effect on SI of site-directed mutations in each of the two Yxxϕ motifs in SRK and of a CRISPR/Cas9-induced null mutation in the AP2 μ-adaptin gene AP2M. Both in vitro SRK kinase activity and the in planta SI response were abolished by substitution of tyrosine in one of the two Yxxϕ motifs, but were unaffected by elimination of either the second Yxxϕ motif or AP2M function. Thus, AP2-mediated CME is considered to be unnecessary for SRK signaling in the SI response.

Plum pox virus (PPV) is transmitted by infected buds and aphids. It is important to analyze the outbreak trends and viruliferous rate of aphids in areas where the occurrence of PPV is reported, so as to develop strategies for disease control. Between April 2011 and December 2012, yellow insect-trapping adhesive plates were placed for 2 days at a time each week in an area where PPV is occurring in Japan. Outbreak trends were analyzed based on the trapped alate aphid samples, and up to 50 of them were tested per week to identify species and determine the rate of viruliferous specimens. Although the number of aphids varied according to survey year, three peaks were noticeable in each year. Based on the sequence data for the mitochondrial cytochrome c oxidase I region, approximately 40 different species of aphid were trapped in both years. Of the five dominant species of aphids identified during the 2 years, Aphis spiraecola was trapped in large numbers. PPV-positive aphids were higher in fall onward, when the total number of trapped aphids decreased, than in spring and summer, when a larger number of aphids was caught. PPV transmission tests using the most abundant species revealed that A. spiraecola, A. craccivora, A. gossypii, and Rhopalosiphum maidis were transmitters, although A. spiraecola is likely of epidemiological significance.

In our previous study, we identified a QTL for cold tolerance at the booting stage of ‘Ukei840’, the cold tolerance of which is derived from one of the most tolerant cultivars, ‘Lijiangxintuanheigu’, in Yunnan province of China, in a 1.2-Mb region between RM7000 and RM3719 markers on chromosome 3. In the present study, we delimited the previously identified QTL to a region of about 35 kb by SSR and dot-blot-SNP marker analysis and nucleotide sequencing using a newly produced near-isogenic line having the ‘Lijiangxintuanheigu’ allele in the QTL region. Comparison of nucleotide sequences of the published ‘Nipponbare’ and ‘Hitomebore’ genome sequences and our previously determined sequence of ‘Ukei840’ revealed nine SNPs in the delimited 35-kb region harboring six genes. LOC-Os03g59190 containing two SNPs in its coding region was a retrotransposon-like sequence having long terminal repeats, which has at least seven other copies in the rice genome. Expression levels of the other five genes in spikelets at the booting stage were not different between ‘Ukei840’ and ‘Hitomebore’. There was one SNP in the coding region of Os03g0806700 which causes amino acid substitution in ‘Ukei840’. However, in Os03g0806700, the genotype of ‘Ukei840’ is the same as that of cold sensitive Indica cultivars. Among the other six SNPs in intergenic regions, one SNP was present in a locus for small RNAs. Possible candidates for the cold-tolerance gene derived from ‘Lijiangxintuanheigu’ are discussed.

The medullary vagal motor nuclei, the nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMV), innervate the respiratory and gastrointestinal tracts. We conducted immunohistochemical analysis of expression of the androgen receptor (AR) and estrogen receptor α (ERα), in relation to innervation of the trachea and esophagus via vagal motor nuclei in mice. AR and ERα were expressed in the rostral NA and in part of the DMV. Tracing experiments using cholera toxin B subunit demonstrated that neurons of vagal motor nuclei that innervate the trachea and esophagus express AR and ERα. There was no difference in expression of sex steroid hormone receptors between trachea- and esophagus-innervating neurons. These results suggest that sex steroid hormones may act on vagal motor nuclei via their receptors, thereby regulating functions of the trachea and esophagus.

The mammalian inner ear mediates hearing and balance and during development generates both cochleo-vestibular ganglion neurons and sensory epithelial receptor cells, that is, hair cells and support cells. Cell marking experiments have shown that both hair cells and support cells can originate from a common progenitor. Here, we demonstrate the lineage potential of individual otic epithelial cell clones using three cell lines established by a combination of limiting dilution and gene-marking techniques from an embryonic day 12 (E12) rat otocyst. Cell-type specific marker analyses of these clonal lines under proliferation and differentiation culture conditions demonstrate that during differentiation immature cell markers (Nanog and Nestin) were downregulated and hair cell (Myosin VIIa and Math1), support cell (p27Kip1 and cytokeratin) and neuronal cell (NF-H and NeuroD) markers were upregulated. Our results suggest that the otic epithelium of the E12 mammalian inner ear possess multipotent progenitor cells able to generate cell types of both sensory epithelial and neural cell lineages when cultured under a differentiation culture condition. Understanding the molecular mechanisms of proliferation and differentiation of multipotent otic progenitor cells may provide insights that could contribute to the development of a novel cell therapy with a potential to initiate or stimulate the sensorineural repair of damaged inner ear sensory receptors. Anat Rec, 303:451-460, 2020. © 2019 American Association for Anatomy.

The present study was conducted to evaluate the benefits of indeterminate growth habit in breeding to improve yield potential of Japanese soybean varieties, which exclusively have determinate growth habit. Two populations of recombinant inbred lines (RILs) derived from crosses between determinate Japanese cultivars and indeterminate US cultivars were grown in Akita and Kyoto, and seed weight per plant (SW) and its components were compared between indeterminate and determinate RILs. The difference of SW between the two growth habits in RILs varied depending on maturation time. The SW of early indeterminate lines was significantly higher than that of early determinate ones in Akita, but not in Kyoto. Among yield components, the number of seeds per pod was constantly larger in indeterminate lines than that in determinate ones irrespective of maturation time. The number of seeds per plant and the number of pods per plant of the indeterminate lines were greater than those of the determinate lines in early maturation in Akita. These results suggest that the indeterminate growth habit is an advantageous characteristic in breeding for high yield of early maturing soybean varieties in the Tohoku region.

The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allele-specific interaction with its pollen coat-localized ligand, the S-locus cysteine-rich protein SCR, SRK is responsible for recognition and inhibition of self pollen in the self-incompatibility response of the Brassicaceae. The SRK extracellular ligand binding domain contains several potential N-glycosylation sites that exhibit varying degrees of conservation among SRK variants. However, the glycosylation status and functional importance of these sites are currently unclear. We investigated this issue in transgenic Arabidopsis thaliana stigmas that express the Arabidopsis lyrata SRKb variant and exhibit an incompatible response toward SCRb-expressing pollen. Analysis of single- and multiple-glycosylation site mutations of SRKb demonstrated that, although five of six potential N-glycosylation sites in SRKb are glycosylated in stigmas, N-glycosylation is not important for SCRb-dependent activation of SRKb. Rather, N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking of SRK to the plasma membrane. The study provides insight into the function of a receptor that regulates a critical phase of the plant life cycle and represents a valuable addition to the limited information available on the contribution of N-glycosylation to the subcellular trafficking and function of plant receptor kinases.

Monosomic addition lines (MALs) are useful materials not only for cytogenetic and molecular genetic studies but also for plant breeding as gene sources. In our previous study, two MALs in the tribe Brassiceae were developed, one being Raphanus sativus lines with alien chromosomes of Brassica rapa (B. rapa-monosomic addition lines; BrMALs) and the second being those with alien chromosomes of Brassica oleracea (B. oleracea-monosomic addition lines; BoMALs). We developed species-specific DNA markers from the genomic sequences of B. rapa and B. oleracea comparing them with those of R. sativus, and identified chromosomes added in BrMALs and BoMALs using these markers. It was revealed that eight types of BrMALs have seven chromosomes of B. rapa and seven types of BoMALs have six chromosomes of B. oleracea. Furthermore, chromosome breakage and homoeologous recombination were suggested to have occurred in some MALs. The developed species-specific DNA markers are considered to be useful for producing MALs and also for assessing chromosome abnormality in MALs.

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥ 300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.

Fruit size is one of the most important traits in fruit trees. We found a bud sport mutant bearing giant fruits of pear in an orchard in Yamagata prefecture, Japan. The fruit weight of this mutant was more than twice that of an original cultivar ‘La France’. The size of the receptacle was already larger in the mutant than in the original cultivar at full bloom, and size differences between the mutant and the original cultivar were observed throughout fruit development. Microscopic observation of fruit cortex cells revealed that the size of cells was larger in the mutant than in the original cultivar, and that the number of cells was comparable between the mutant and the original cultivar. Flow cytometric analysis revealed an increase in the number of cells having doubled amounts of DNA (4C cells) in the fruit cortex, but such an increase was not found in leaves, suggesting chimeric polyploidization or endoreduplication in fruits of the mutant. Mutant fruits exhibited significantly higher titratable acidity and lower firmness than those of the original cultivar. These differences were observed from 110 days after full bloom to 165 days (the commercial harvesting time). However, the concentration of soluble solids was not different between the mutant and the original cultivar. There were no significant differences in polyuronide contents between the mutant and the original cultivar fruits, but hemicellulose content in fruits of the mutant was significantly lower than in those of the original cultivar.

Water deficit imposed by either drought or salinity brings about severe growth retardation and yield loss of crops. Since Brassica crops are important contributors to total oilseed production, it is urgently needed to develop tolerant cultivars to ensure yields under such adverse conditions. There are various physiochemical mechanisms for dealing with drought and salinity in plants at different developmental stages. Accordingly, different indicators of tolerance to drought or salinity at the germination, seedling, flowering and mature stages have been developed and used for germplasm screening and selection in breeding practices. Classical genetic and modern genomic approaches coupled with precise phenotyping have boosted the unravelling of genes and metabolic pathways conferring drought or salt tolerance in crops. QTL mapping of drought and salt tolerance has provided several dozen target QTLs in Brassica and the closely related Arabidopsis. Many drought- or salt-tolerant genes have also been isolated, some of which have been confirmed to have great potential for genetic improvement of plant tolerance. It has been suggested that molecular breeding approaches, such as marker-assisted selection and gene transformation, that will enhance oil product security under a changing climate be integrated in the development of drought- and salt-tolerant Brassica crops.

The whiteness of cooked rice and rice cakes was evaluated using a portable spectrophotometer with a whiteness index (WI). Also, by using boiled rice for measurement of Mido values by Mido Meter, it was possible to infer the whiteness of cooked rice without rice cooking. In the analysis of varietal differences of cooked rice, 'Tsuyahime', 'Koshihikari' and 'Koshinokaori' showed high whiteness, while 'Satonoyuki' had inferior whiteness. The whiteness of rice cakes made from 'Koyukimochi' and 'Dewanomochi' was higher than the whiteness of those made from 'Himenomochi' and 'Koganemochi'. While there was a significant correlation (r = 0.84) between WI values and whiteness scores of cooked rice by the sensory test, no correlation was detected between the whiteness scores and Mido values, indicating that the values obtained by a spectrophotometer differ from those obtained by a Mido Meter. Thus, a spectrophotometer may be a novel device for measurement of rice eating quality.

An unusually high temperature during the grain-filling period, such as that caused by global warming, impairs the quality of rice (Oryza sativa L.) grains. This sensitivity to high-temperature stress is different among cultivars, suggesting the possibility of developing a high-temperature-tolerant cultivar. Since marker-assisted selection would reduce time and labor in breeding for such a quantitative trait, we determined the chromosomal region responsible for high-temperature tolerance during the grain-filling period. A high-temperature-sensitive japonica cultivar Tohoku 168 and a tolerant japonica cultivar Kokoromachi were selected as the parental lines of recombinant inbred lines (RILs) by high-temperature stress treatment from 5 to 10 days after anthesis, which was found to be the period most critical for grain quality. Using the RILs, whose genotypes were determined by analysis with 131 DNA markers which were selected as polymorphic markers between these two cultivars from 2,648 DNA markers tested, the quantitative trait locus (QTL) for the percentage of white-back grains was mapped on chromosome 6. The Kokoromachi allele of the QTL, which had a positive additive effect on the high-temperature tolerance, was introduced into the Tohoku 168 genome by repeated backcrossings with marker-assisted selection. Using high-temperature stress treatment of the near isogenic lines developed, the QTL on chromosome 6 was localized within a 1.9-Mb region between two DNA markers, ktIndel001 and RFT1. These DNA markers would be useful not only for breeding high-temperature-tolerant cultivars but also for map-based cloning of the QTL.

One hundred sixty-one EST-SNP markers were newly developed for analysis of QTLs for resistance to black rot caused by Xanthomonas campestris pv. campestris by determining EST sequences of a resistant line obtained from cabbage ‘Early Fuji’ and a susceptible broccoli line. A linkage map consisting of nine linkage groups was constructed with a total of 209 markers, including these new SNP markers and previously reported DNA markers. F2 plants grown in a field for 1 month were inoculated by spraying bacteria of race 1, and disease severity of each plant was recorded. Three QTLs, i.e., QTL-1, QTL-2, and QTL-3, were detected on linkage group C2, C4 and C5, respectively. QTL-1, which showed the highest LOD score and additive effect, was again detected in another F2 population used the next year, suggesting QTL-1 to be a major QTL. QTL-2 and QTL-3 could be minor QTLs influenced by environmental factors. The genomic region harboring QTL-1 showed synteny with a region from 5.3 to 7.4 Mb from the short arm end of chromosome 5 of Arabidopsis thaliana, which is rich in TIR-NBS-LRR family genes. The identified SNP markers in QTL-1 are considered to be useful in marker-assisted selection for black rot resistance in Brassica oleracea lines.

A hybridization barrier leads to the inability of seed formation after intergeneric crossings between Brassica rapa and Raphanus sativus. Most B. rapa lines cannot set intergeneric hybrid seeds because of embryo breakdown, but a B. rapa line obtained from turnip cultivar 'Shogoin-kabu' is able to produce a large number of hybrid seeds as a maternal parent by crossings with R. sativus. In 'Shogoin-kabu' crossed with R. sativus, developments of embryos and endosperms were slower than those in intraspecific crossings, but some of them grew to mature seeds without embryo breakdown. Intergeneric hybrid seeds were obtained in a 'Shogoin-kabu' line at a rate of 0.13 per pollinated flower, while no hybrid seeds were obtained in a line developed from Chinese cabbage cultivar 'Chiifu'. F(1) hybrid plants between the lines of 'Shogoin-kabu' and 'Chiifu' set a larger number of hybrid seeds per flower, 0.68, than both the parental lines. Quantitative trait loci (QTLs) for hybrid seed formation were analyzed after intergeneric crossings using two different F(2) populations derived from the F(1) hybrids, and three QTLs with significant logarithm of odds scores were detected. Among them, two QTLs, i.e., one in linkage group A10 and the other in linkage group A01, were detected in both the F(2) populations. These two QTLs had contrary effects on the number of hybrid seeds. Epistatic interaction between these two QTLs was revealed. Possible candidate genes controlling hybrid seed formation ability in QTL regions were inferred using the published B. rapa genome sequences.

Stable genic male sterility (GMS), which is not influenced by environmental factors, has not been used for F1 hybrid seed production because male-sterile inbred lines cannot be developed and male-sterile plants must be selected from segregating populations every time. However, the stability of male sterility may provide a reliable system for F1 seed production without contamination of selfed seeds. A genic male-sterile mutant in rice (Oryza sativa L.), C204, which was selected from progeny of the cultivar ‘Koshihikari’ irradiated by gamma rays, has shorter and whiter anthers than those of ‘Koshihikari’ and has no pollen grains. Segregation analysis of C204 suggested the male sterility of this mutant to be controlled by a recessive allele of a single gene. Linkage analysis of a mutated gene responsible for the male sterility revealed the gene to be in a region of ca. 75 kb on the long arm of chromosome 9. The nine genes predicted in the 75-kb region were sequenced, and compared with the published Nipponbare genome sequences. A single-base deletion was found in the first exon of a C204 allele of Os09g0493500, which encodes an NAD-dependent epimerase/dehydratase family protein, resulting in a frameshift causing a premature stop codon. A dot-blot single nucleotide polymorphism marker for detection of the single-base deletion in Os09g0493500 was developed. We herein propose an F1 hybrid seed production system using stable GMS with a simple selection method of GMS plants.

Glucosinolates (GSLs) are secondary metabolites whose degradation products confer intrinsic flavors and aromas to Brassicaceae vegetables. Several structures of GSLs are known in the Brassicaceae, and the biosynthetic pathway and regulatory networks have been elucidated in Arabidopsis (Arabidopsis thaliana). GSLs are precursors of chemical defense substances against herbivorous pests. Specific GSLs can act as feeding blockers or stimulants, depending on the pest species. Natural selection has led to diversity in the GSL composition even within individual species. However, in radish (Raphanus sativus), glucoraphasatin (4-methylthio-3-butenyl glucosinolate) accounts for more than 90% of the total GSLs, and little compositional variation is observed. Because glucoraphasatin is not contained in other members of the Brassicaceae, like Arabidopsis and cabbage (Brassica oleracea), the biosynthetic pathways for glucoraphasatin remain unclear. In this report, we identified and characterized a gene encoding GLUCORAPHASATIN SYNTHASE 1 (GRS1) by genetic mapping using a mutant that genetically lacks glucoraphasatin. Transgenic Arabidopsis, which overexpressed GRS1 cDNA, accumulated glucoraphasatin in the leaves. GRS1 encodes a 2-oxoglutarate-dependent dioxygenase, and it is abundantly expressed in the leaf. To further investigate the biosynthesis and transportation of GSLs in radish, we grafted a grs1 plant onto a wild-type plant. The grafting experiment revealed a leaf-to-root long-distance glucoraphasatin transport system in radish and showed that the composition of GSLs differed among the organs. Based on these observations, we propose a characteristic biosynthesis pathway for glucoraphasatin in radish. Our results should be useful in metabolite engineering for breeding of high-value vegetables.

Pear, the third most important temperate fruit species after grape and apple, belongs to the subfamily Pomoideae in the family Rosaceae. The majority of cultivated pears are functional diploids (2n = 34). As a popular fruit in the world market, pear has widespread cultivation on six continents, with major production in China, the United States, Italy, Argentina, and Spain (Supplemental Fig. 1). Pears are among the oldest of the world's fruit crops, with >3000 yr of cultivation history (Lombard and Westwood 1987), likely originating during the Tertiary period (65–55 million years ago [MYA]) in the mountainous regions of southwestern China and, from there, spreading on to both the East and West (Rubtsov 1944; Zeven and Zhukovsky 1975). Central Asia and eastern China are identified as two subcenters of genetic diversity for pear (Vavilov 1951). The Pyrus genus is genetically diverse with thousands of cultivars, but it can be divided into two major groups, Occidental pears (European pears) and Oriental pears (Asiatic pears). At least 22 primary species are well-recognized in Pyrus; however, only a few species, including Pyrus bretschneideri, Pyrus pyrifolia, Pyrus ussuriensis, Pyrus sinkiangensis, and Pyrus communis, have been utilized for fruit production. Herein, we report on a high-quality draft genome sequence of the diploid P. bretschneideri Rehd. cv. ‘Dangshansuli’ (also known as ‘Suli’), the most important commercial Asiatic pear cultivar grown in the world (>4 million tons per year), having >500 yr of cultivated history in China. Pear is highly heterozygous due to self-incompatibility and interspecies compatibility. The genome is known to have an abundance of repetitive DNA sequences. In this study, a novel combination of BAC-by-BAC (bacterial artificial chromosome) strategy, with Illumina sequencing technology, is used for the first time for de novo assembly of a highly heterozygous genome of this size with highly repetitive DNA sequences. This has demonstrated that a complex plant genome sequence can be assembled and characterized using these technologies without the availability of a physical reference. Additionally, we also report on primary factors contributing to genome size differences between pear and apple, both belonging to the subfamily Pomoideae; chromosomal evolution of Rosaceae; and genes controlling valuable traits of pear, including self-incompatibility, lignified stone cells in flesh of fruit (unique to pear), sugar, and aroma.

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase (SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers.

Mutations in the KCNA1 gene, which encodes for the α subunit of the voltage-gated potassium channel Kv1.1, cause episodic ataxia type 1 (EA1). EA1 is a dominant human neurological disorder characterized by variable phenotypes of brief episodes of ataxia, myokymia, neuromyotonia, and associated epilepsy. Animal models for EA1 include Kcna1-deficient mice, which recessively display severe seizures and die prematurely, and V408A-knock-in mice, which dominantly exhibit stress-induced loss of motor coordination. In the present study, we have identified an N-ethyl-N-nitrosourea-mutagenized rat, named autosomal dominant myokymia and seizures (ADMS), with a missense mutation (S309T) in the voltage-sensor domain, S4, of the Kcna1 gene. ADMS rats dominantly exhibited myokymia, neuromyotonia and generalized tonic-clonic seizures. They also showed cold stress-induced tremor, neuromyotonia, and motor incoordination. Expression studies of homomeric and heteromeric Kv1.1 channels in HEK cells and Xenopus oocytes, showed that, although S309T channels are transferred to the cell membrane surface, they remained non-functional in terms of their biophysical properties, suggesting a dominant-negative effect of the S309T mutation on potassium channel function. ADMS rats provide a new model, distinct from previously reported mouse models, for studying the diverse functions of Kv1.1 in vivo, as well as for understanding the pathology of EA1.

To reveal varietally differing glucosinolate (GSL) contents in radish (Raphanus sativus L.) cultivated in Japan, the total and individual GSLs of 28 cultivars were analyzed using high-performance liquid chromatography. In these cultivars, GSL types including three aliphatic GSLs (glucoraphenin, glucoerucin, and 4-methylthio-3-butenyl GSL (4MTB-GSL)) and three indolyl GSLs (4-hydroxyglucobrassicin, glucobrassicin, and 4-methoxy-glucobrassicin) were detected. No cultivar-specific type of GSL was identified. The dominant GSL was 4MTB-GSL, but its contents differed remarkably: 8.6 μmol/g in 'Koushin' to 135.7 μmol/g in 'Karami 199'. Over about 90% of all GSLs in Japanese radish type are 4MTB-GSL, a higher percentage than in Chinese or European garden radish cultivars. A simple, rapid method for estimating total GSL contents in crude extracts was established because of the small variation of glucosinolate composition in Japanese cultivars. The total GSL content can be estimated using an equation for prediction with absorbance at 425 nm in a mixture of GSL crude extract and palladium (II) chloride solution: Total GSL (μmol/g) = 305.47 × A(425) - 29.66. Its coefficient of determination (R(2)) and standard error of prediction (SEP) are 0.968 and 8.052. This method enables total GSL content estimation from more than 200 samples per person per day.

It has long been known that a bacterial leaf blight-resistant line in rice obtained from a crossing using ‘Asominori’ as a resistant parent also has resistance to blast, but a blast resistance gene in ‘Asominori’ has not been investigated in detail. In the present study, a blast resistance gene in ‘Asominori’, tentatively named Pias(t), was revealed to be located within 162-kb region between DNA markers YX4-3 and NX4-1 on chromosome 4 and to be linked with an ‘Asominori’ allele of the bacterial leaf blight resistance gene Xa1, tentatively named Xa1-as(t). An ‘Asominori’ allele of Pias(t) was found to be dominant and difference of disease severity between lines having the ‘Asominori’ allele of Pias(t) and those without it was 1.2 in disease index from 0 to 10. Pias(t) was also closely linked with the Ph gene controlling phenol reaction, suggesting the possibility of successful selection of blast resistance using the phenol reaction. Since blast-resistant commercial cultivars have been developed using ‘Asominori’ as a parent, Pias(t) is considered to be a useful gene in rice breeding for blast resistance.

Low temperature at the booting stage of rice causes male sterility resulting in severe yield loss. Cold tolerance has long been an important objective in rice breeding. We identified a quantitative trait locus (QTL) for cold tolerance on the long arm of chromosome 3 from the cold-tolerant breeding line 'Ukei 840' by using F(2) and BC(1)F(2) populations from crosses between 'Ukei 840' and 'Hitomebore'. The cold tolerance of 'Ukei 840' is derived from the Chinese cultivar 'Lijiangxintuanheigu'. The effect of this QTL on cold tolerance was confirmed by developing 'Hitomebore' chromosome segment substitution lines having 'Lijiangxintuanheigu' alleles on chromosome 3. By producing recombinants in chromosome 3, the QTL region for cold tolerance was delimited to the region of about 1.2-Mb region between RM3719 and RM7000. All lines heterozygous for the QTL showed seed fertilities as low as that of 'Hitomebore', suggesting that the 'Lijiangxintuanheigu' allele for cold tolerance in the QTL region is recessive. Determination of a 1.2-Mb nucleotide sequence of 'Ukei 840' and comparison with the published genomic sequence of 'Nipponbare' showed 254 SNPs, of which 11 were in coding regions of genes, seven in five genes being non-synonymous. SNPs were detected in the 5-kb upstream regions of 89 genes, but no differences of gene expression levels were detected between alleles of these genes. Although further delimitation is required to identify the gene responsible for cold tolerance of 'Lijiangxintuanheigu', SNP markers developed here will be useful for marker-assisted selection in a breeding program using 'Lijiangxintuanheigu' as a donor of cold tolerance.

The self-incompatibility (SI) system of the Brassicaceae is based on allele-specific interactions among haplotypes of the S locus. In all tested self-incompatible Brassicaceae, the S haplotype encompasses two linked genes, one encoding the S -locus receptor kinase (SRK), a transmembrane kinase displayed at the surface of stigma epidermal cells, and the other encoding its ligand, the S -locus cysteine-rich (SCR) protein, which is localized in the pollen coat. Transfer of the two genes to self-fertile Arabidopsis thaliana allowed the establishment of robust SI in several accessions, indicating that the signaling cascade triggered by this receptor–ligand interaction and the resulting inhibition of “self” pollen by the stigma have been maintained in extant A. thaliana . Based on studies in Brassica species, the membrane-tethered kinase MLPK, the ARM repeat-containing U-box protein ARC1, and the exocyst subunit Exo70A1 have been proposed to function as components of an SI signaling cascade. Here we tested the role of these molecules in the SI response of A. thaliana SRK-SCR plants. We show that the A. thaliana ARC1 ortholog is a highly decayed pseudogene. We also show that, unlike reports in Brassica , inactivation of the MLPK ortholog AtAPK1b and overexpression of Exo70A1 neither abolish nor weaken SI in A. thaliana SRK-SCR plants. These results do not support a role for these molecules in the SI response of A. thaliana .

A linkage map of expressed sequence tag (EST)-based markers in radish (Raphanus sativus L.) was constructed using a low-cost and high-efficiency single-nucleotide polymorphism (SNP) genotyping method named multiplex polymerase chain reaction–mixed probe dot-blot analysis developed in this study. Seven hundred and forty-six SNP markers derived from EST sequences of R. sativus were assigned to nine linkage groups with a total length of 806.7 cM. By BLASTN, 726 markers were found to have homologous genes in Arabidopsis thaliana, and 72 syntenic regions, which have great potential for utilizing genomic information of the model species A. thaliana in basic and applied genetics of R. sativus, were identified. By construction and analysis of the genome structures of R. sativus based on the 24 genomic blocks within the Brassicaceae ancestral karyotype, 23 of the 24 genomic blocks were detected in the genome of R. sativus, and half of them were found to be triplicated. Comparison of the genome structure of R. sativus with those of the A, B, and C genomes of Brassica species and that of Sinapis alba L. revealed extensive chromosome homoeology among Brassiceae species, which would facilitate transfer of the genomic information from one Brassiceae species to another.

Quantitative trait loci (QTLs) for low cadmium concentration in brown rice were analyzed in 2008 and 2009 using 126 RIL (F6 and F7 plants) grown in a paddy field with high Cd concentration, which were derived from a cross between a high yielding Japanese rice cultivar ‘Fukuhibiki’ and an African upland rice cultivar ‘LAC23’ with the lowest cadmium concentration in rice grains. Totally two QTLs controlling low cadmium concentration in grains were identified in both 2008 and 2009, i.e., one on chromosome 3 (qLCdG3) and one on chromosome 11 (qLCdG11), which had opposite additive effects. In qLCdG11, ‘LAC23’ alleles had the effect of reducing cadmium concentration, but qLCdG3 had the opposite effect. The explained phenotypic variances of qLCdG11 were 9.4% in 2008 and 12.9% in 2009, and those of qLCdG3 were 13.9% in 2008 and 8.3% in 2009. Cadmium concentrations in brown rice showed positive correlations with grain length and grain weight. QTLs for grain length and grain weight were detected on chromosomes 3 and 11, but their positions differed from those for cadmium concentrations. These results suggest a possibility of successful marker-assisted selection of low cadmium trait derived from ‘LAC23’.

A 5-bp deletion in exon 3 of a Brassica rapa ortholog of GLABRA1 (BrGL1) was previously thought to be responsible for the hairless trait of a doubled haploid line. In this study, the sequence variation of Brassica GL1 was characterized. The DNA-binding domain of GL1 in Arabidopsis thaliana was found to be highly conserved in Brassica species, while in hairless B. oleracea lines, frameshifts and nonsynonymous substitutions were detected in the coding sequences of BoGL1. Phylogenetic analysis showed that BnGL1 in hairless B. napus lines is closely related to BiGL1 in a hairy B. incana line. In 11 lines of B. rapa germplasm, nucleotide polymorphisms were only detected in exon 3 of BrGL1. Regarding the nucleotide polymorphisms in the DNA-binding domain of BrGL1, four alleles were identified, and corresponding dot-blot single nucleotide polymorphism (SNP) markers were developed and used for genotyping of 63 lines of B. rapa. The genotyping results showed that nucleotide polymorphisms in the DNA-binding domain of BrGL1 are associated with leaf hairiness. Reverse-transcription PCR analysis indicated that different BrGL1 alleles are all expressed normally. These findings reveal the key nucleotide polymorphisms of BrGL1 affecting leaf hairiness in the natural B. rapa population.

Simple, reliable methods for identification of species are required for management of many species and lines in a plant gene bank. Species-specific probes were designed from published sequences of the ITS1 region in rDNA of 16 species in Brassica and its related genera, and used as probes for dot-blot hybridization with plant genomic DNA. All the probes detected species-specific signals at dot-blots of genomic DNAs of the 16 species in Brassica, Diplotaxis, Eruca, and Raphanus. Signals of the Brassica digenomic species in the U's triangle, i.e., B. napus, B. juncea, and B. carinata, were detected by the probes of their parental monogenomic species, i.e., B. rapa, B. nigra, and B. oleracea. The probe for B. oleracea showed signals of B. balearica, B. cretica, B. incana, B. insularis, and B. macrocarpa, which have the C genome as B. oleracea. Eruca vesicaria DNA was detected by the probe for E. sativa, which has been classified as a subspecies of E. vescaria. DNA of leaf tissue extracted by an alkaline solution and seed DNA prepared by the NaI method can be used directly for dot-blotting. Misidentification of species was revealed in 20 accessions in the Tohoku University Brassica Seed Bank. These results indicate dot-blot hybridization to be a simple and efficient technique for identification of plant species in a gene bank.

Although the dot-blot-SNP technique is a simple cost-saving technique suitable for genotyping of many plant individuals, optimization of hybridization and washing conditions for each SNP marker requires much time and labor. For prediction of the optimum hybridization conditions for each probe, we compared T (m) values estimated from nucleotide sequences using the DINAMelt web server, measured T (m) values, and hybridization conditions yielding allele-specific signals. The estimated T (m) values were comparable to the measured T (m) values with small differences of less than 3 degrees C for most of the probes. There were differences of approximately 14 degrees C between the specific signal detection conditions and estimated T (m) values. Change of one level of SSC concentrations of 0.1, 0.2, 0.5, and 1.0x SSC corresponded to a difference of approximately 5 degrees C in optimum signal detection temperature. Increasing the sensitivity of signal detection by shortening the exposure time to X-ray film changed the optimum hybridization condition for specific signal detection. Addition of competitive oligonucleotides to the hybridization mixture increased the suitable hybridization conditions by 1.8. Based on these results, optimum hybridization conditions for newly produced dot-blot-SNP markers will become predictable.

In interspecific pollination of Brassica rapa stigmas with Brassica oleracea pollen grains, pollen tubes cannot penetrate stigma tissues. This trait, called interspecific incompatibility, is similar to self-incompatibility in pollen tube behaviors of rejected pollen grains. Since some B. rapa lines have no interspecific incompatibility, genetic analysis of interspecific incompatibility was performed using two F(2) populations. Analysis with an F(2) population between an interspecific-incompatible line and a self-compatible cultivar 'Yellow sarson' having non-functional alleles of S-locus genes and MLPK, the stigmas of which are compatible with B. oleracea pollen grains, revealed no involvement of the S locus and MLPK in the difference of their interspecific incompatibility phenotypes. In QTL analysis of the strength of interspecific incompatibility, three peaks of LOD scores were found, but their LOD scores were as high as the threshold value, and the variance explained by each QTL was small. QTL analysis using another F(2) population derived from selected parents having the highest and lowest levels of interspecific incompatibility revealed five QTLs with high LOD scores, which did not correspond to those found in the former population. The QTL having the highest LOD score was found in linkage group A02. The effect of this QTL on interspecific incompatibility was confirmed by analyzing backcrossed progeny. Based on synteny of this QTL region with Arabidopsis thaliana chromosome 5, a possible candidate gene, which might be involved in interspecific incompatibility, is discussed.

Plant genetic resources are important sources of genetic variation for improving crop varieties as breeding materials. Conservation of such resources of allogamous species requires maintenance of the genetic diversity within each accession to avoid inbreeding depression and loss of rare alleles. For assessment of genetic diversity in the self-incompatibility locus (S locus), which is critically involved in the chance of mating, we developed a dot-blot genotyping method for self-incompatibility (S) haplotypes and applied it to indigenous, miscellaneous landraces of Brassica rapa, provided by the IPK Gene Bank (Gatersleben, Germany) and the Tohoku University Brassica Seed Bank (Sendai, Japan), in which landraces are maintained using different population sizes. This method effectively determined S genotypes of more than 500 individuals from the focal landraces. Although our results suggest that these landraces might possess sufficient numbers of S haplotypes, the strong reduction of frequencies of recessive S haplotypes occurred, probably owing to genetic drift. Based on these results, we herein discuss an appropriate way to conserve genetic diversity of allogamous plant resources in a gene bank.

Using 77 single-nucleotide polymorphic (SNP) markers and dot-blot analysis, we examined 218 rice cultivars, respectively occupying 99% and 92% of the planted areas of non-glutinous and glutinous rice for three consecutive years from 2003 to 2005 in Japan. Among them, 205 cultivars were identified at one time by the genotypes of 18 markers, but 13 cultivars belonged to six groups in which cultivars were indistinguishable from each other. The 205 cultivars were individually distinguished from the others using combinations of up to six markers. This result was considered to be useful for the identification of Japanese commercial rice cultivars, monitoring the contamination of rice with other cultivars, and rice breeding using these cultivars.

Although the dot-blot-SNP technique is a laborsaving, cost-effective method for SNP genotyping of a large number of plants, the synthesis of 5′-digoxigenin (DIG)-labeled oligonucleotides for use as probes is still costly. We developed two probe-labeling methods for this technique, one being digoxigenin labeling of oligonucleotides by PCR (PCR-DIG labeling) and the other being hybridization using a bridge probe and a 5′-DIG-labeled oligonucleotide (bridge hybridization). Bridge hybridization detected allele-specific signals under hybridization conditions similar to those for the 5′-DIG-labeled oligonucleotides and biotin-labeled oligonucleotides, while signals were detected only under a lower stringency condition by PCR-DIG labeling. As a method for genotyping using many markers at one time, two methods, i.e., PCR using mixed primer pairs and hybridization using mixed probes, were examined with successful results. Eighty-five SNP markers designed for genotyping of rice cultivars detected allele-specific signals, the genotyping results corresponding to the previously reported ones.

For identification of genes responsible for varietal differences in flowering time and leaf morphological traits, we constructed a linkage map of Brassica rapa DNA markers including 170 EST-based markers, 12 SSR markers, and 59 BAC sequence-based markers, of which 151 are single nucleotide polymorphism (SNP) markers. By BLASTN, 223 markers were shown to have homologous regions in Arabidopsis thaliana, and these homologous loci covered nearly the whole genome of A. thaliana. Synteny analysis between B. rapa and A. thaliana revealed 33 large syntenic regions. Three quantitative trait loci (QTLs) for flowering time were detected. BrFLC1 and BrFLC2 were linked to the QTLs for bolting time, budding time, and flowering time. Three SNPs in the promoter, which may be the cause of low expression of BrFLC2 in the early-flowering parental line, were identified. For leaf lobe depth and leaf hairiness, one major QTL corresponding to a syntenic region containing GIBBERELLIN 20 OXIDASE 3 and one major QTL containing BrGL1, respectively, were detected. Analysis of nucleotide sequences and expression of these genes suggested possible involvement of these genes in leaf morphological traits.

In order to analyze mutations induced by gamma irradiation in higher plants, we irradiated rice with gamma rays and screened for mutations expressing phenotypes of glutinous endosperm (wx), chlorophyll b deficiency, endosperm protein deficiency, gibberellin-related dwarfism, and shortened plastochron-in order to clarify types of mutations. Nucleotide sequence analysis showed that the most frequent mutation induced by gamma rays was deletion, particularly small deletion. Of the 24 mutations, 15 were small deletions (1-16 bp), four were large deletions (9.4-129.7 kbp), three were single-base substitutions, and two were inversions. Deletions 100 bp-8 kbp in length were not found, suggesting that gamma irradiation is unlikely to induce deletions of 100 bp to 8 kbp but is more likely to induce deletions between 1 and several ten bp or those of around 10 kbp or more. Based on the results, reverse genetics applications may be possible for gamma irradiation-induced deletions in rice by mismatch cleavage analysis used in Targeting Induced Local Lesions IN Genomes (TILLING) to detect small deletions and base substitutions or by using array comparative genomic hybridization (aCGH) to detect large deletions.

One of the causes of genome size expansion is considered to be amplification of retrotransposons. We determined nucleotide sequences of 24 PCR products for each of six retrotransposons in Brassica rapa and Brassica oleracea. Phylogenetic trees of these sequences showed species-specific clades. We also sequenced STF7a homologs and Tto1 homologs, 24 PCR products each, in nine diploids and three allopolyploids, and constructed phylogenetic trees. In these phylogenetic trees, species-specific clades of diploid species were also formed, but retrotransposons of allopolyploids were clustered into the clades of their original genomes, indicating that these two retrotransposons amplified after speciation of the nine diploids. Genetic variation in these retrotransposons may have arisen before emergence of allopolyploid species. There was a positive correlation between the genome size and the average number of substitutions of STF7a and Tto1 homologs in at least seven diploids. The implications of these results in the genome evolution of Brassicaceae are herein discussed.