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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.

Rose yellow mosaic virus, which belongs to the Roymovirus genus in the Potyviridae family, was isolated here in Japan from a rose cultivar, Irish Mist. The full-length genomic sequence was similar to a strain in Minnesota, United States, which was the first ever isolated, with 85% nucleotide and 94% amino acid sequence similarities. Unlike the Minnesota strain, which lacks 220 nt including the 6K1 protein region, the Japanese isolate contains this coding region, which is commonly found in those of the Potyviridae.

Two potyviruses, uraria mosaic virus (UMV) and passiflora foetida virus Y (PfVY), have been detected in Japan for the first time, from newly bred passionfruit cultivars. Host range analyses and phylogenetic analyses based on full and partial polyprotein coding regions of UMV and PfVY, respectively, suggested they were distinct from viruses previously isolated from passionfruit. Both species belong to the BCMV subgroup within the genus Potyvirus, which contains other pathogenic passionfruit viruses.

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.

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.

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.

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.

We identified three physical positions associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis. We also confirmed their genetic effects on the embryo yield. Isolated microspore culture is well utilized for the production of haploid or doubled-haploid plants in Brassica crops. Brassica rapa cv. ‘Ho Mei’ is one of the most excellent cultivars in embryo yield of microspore culture. To identify the loci associated with microspore embryogenesis, segregation analysis of 154 DNA markers anchored to B. rapa chromosomes (A01–A10) was performed using a population of microspore-derived embryos obtained from an F1 hybrid between ‘CR-Seiga’, a low yield cultivar in microspore-derived embryos, and ‘Ho Mei’. Three regions showing significant segregation distortion with increasing ‘Ho Mei’ alleles were detected on A05, A08 and A09, although these regions showed the expected Mendelian segregation ratio in an F2 population. The additive effect of alleles in these regions on embryo yield was confirmed in a BC3F1 population. One region on A08 containing Br071-5c had a higher effect than the other regions. Polymorphism of nucleotide sequences around the Br071-5c locus was investigated to find the gene possibly responsible for efficient embryogenesis from microspores.

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.

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.

Radish, belonging to the family Brassicaceae, has a self-incompatibility which is controlled by multiple alleles on the S locus. To employ the self-incompatibility in an F1 breeding system, identification of S haplotypes is necessary. Since collection of S haplotypes and determination of nucleotide sequences of SLG, SRK, and SCR alleles in cultivated radish have been conducted by different groups independently, the same or similar sequences with different S haplotype names and different sequences with the same S haplotype names have been registered in public databases, resulting in confusion of S haplotype names for researchers and breeders. In the present study, we developed S homozygous lines from radish F1 hybrid cultivars in Japan and determined the nucleotide sequences of SCR, the S domain and the kinase domain of SRK, and the SLG of a large number of S haplotypes. Comparing these sequences with our previously published sequences, the haplotypes were ordered into 23 different S haplotypes. The sequences of the 23 S haplotypes were compared with S haplotype sequences registered by different groups, and we suggested a unification of these S haplotypes. Furthermore, dot-blot hybridization using SRK allele-specific probes was examined for developing a standard method for S haplotype identification.

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 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.

A QTL for cold tolerance at the booting stage of rice cultivar ‘Kuchum’ was detected and delimited into a 1.36 Mb region, and a cold-tolerant line was developed by QTL pyramiding. Low temperature in summer causes pollen sterility in rice, resulting in a serious loss of yield. The second most widely grown rice cultivar in Japan, ‘Hitomebore’, has been developed as a cultivar highly tolerant to low temperature at the booting stage. However, even ‘Hitomebore’ exhibits sterility at a temperature lower than 18.5 °C. Further improvement of cold tolerance of rice is required. In the present study, QTLs for cold tolerance in a Bhutanese rice variety, ‘Kuchum’, were analyzed using backcrossed progenies and a major QTL, named qCT-4, was detected on chromosome 4. Evaluating cold tolerance of seven types of near isogenic lines having ‘Kuchum’ alleles around qCT-4 with a ‘Hitomebore’ genetic background, qCT-4 was delimited to a region of ca. 1.36 Mb between DNA markers 9_1 and 10_13. Homozygous ‘Kuchum’ alleles at qCT-4 showed an effect of increasing seed fertility by ca. 10 % under cold-water treatment. Near isogenic lines of ‘Hitomebore’ having ‘Silewah’ alleles of Ctb1 and Ctb2 and a ‘Hokkai PL9’ allele of qCTB8 did not exhibit higher cold tolerance than that of ‘Hitomebore’. On the other hand, a qLTB3 allele derived from a Chinese cultivar ‘Lijiangxintuanheigu’ increased cold tolerance of ‘Hitomebore’, and pyramiding of the qCT-4 allele and the qLTB3 allele further increased seed fertility under cold-water treatment. Since NILs of ‘Hitomebore’ with the ‘Kuchum’ allele of qCT-4 were highly similar to ‘Hitomebore’ in other agronomic traits, the qCT-4 allele is considered to be useful for developing a cold-tolerant cultivar.

Crop plant architecture plays a highly important role in its agronomic performance. Plant height (PH) and primary branch number (PB) are two major factors that affect the plant architecture of rapeseed (Brassica napus). Previous studies have shown that these two traits are controlled by multiple quantitative trait loci (QTL); however, QTLs have not been delimited to regions less than 10cM. Genome-wide association study (GWAS) is a highly efficient approach for identifying genetic loci controlling traits at relatively high resolution. In this study, variations in PH and PB of a panel of 472 rapeseed accessions that had previously been analyzed by a 60k SNP array were investigated for three consecutive years and studied by GWAS. Eight QTLs on chromosome A03, A05, A07 and C07 were identified for PH, and five QTLs on A01, A03, A07 and C07 were identified for PB. Although most QTLs have been detected in previous studies based on linkage analyses, the two QTLs of PH on A05 and the QTL of PB on C07 were novel. In the genomic regions close to the GWAS peaks, orthologs of the genes involved in flower development, phytohormone biosynthesis, metabolism and signaling in Arabidopsis were identified.

By genome-wide association study, QTLs for salt tolerance in rapeseed were detected, and a TSN1 ortholog was identified as a candidate gene responsible for genetic variation in cultivars. Dissecting the genomic regions governing abiotic stress tolerance is necessary for marker-assisted breeding to produce elite breeding lines. In this study, a world-wide collection of rapeseed was evaluated for salt tolerance. These rapeseed accessions showed a large variation for salt tolerance index ranging from 0.311 to 0.999. Although no significant correlation between salt tolerance and Na+ content was observed, there was a significant negative correlation between shoot biomass production under a control condition and salt tolerance. These rapeseed accessions were genotyped by DArTseq for a total of 51,109 genetic markers, which were aligned with ‘pseudomolecules’ representative of the genome of rapeseed to locate their hypothetical order for association mapping. A total of 62 QTLs for salt tolerance, shoot biomass, and ion-homeostasis-related traits were identified by association mapping using both the P and Q+K models. Candidate genes located within the QTL regions were also shortlisted. Sequence analysis showed many polymorphisms for BnaaTSN1. Three of them in the coding region resulting in a premature stop codon or frameshift were found in most of the sensitive lines. Loss-of-function mutations showed a significant association with salt tolerance in B. napus.

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.

We developed a method for detection of mutants in a large number of plants, and found this method to be applicable to detection of a mutant allele at a concentration of 1/1000. Many techniques for SNP analysis have been developed, but most of these techniques are not so sensitive to be used for detection of mutants in a large number of plants. Although some highly sensitive methods of SNP analysis have been reported, they are costly. In the present study, a method for concentrating mutant DNA was examined for sensitive detection of an SNP allele in a bulked DNA sample. PCR products of mutant alleles were captured by biotin-labeled oligonucleotide conjugated with streptavidin-coated magnetic beads. By repeated captures of each strand and combining both strands, mutant alleles with a concentration of 1/1000 in wild-type alleles were detectable by CAPS or dCAPS analysis. Indirect capture of a mutant allele was possible, but efficiency was slightly lower than that of the direct capture. The developed method was applied to detection of contamination of rice grains by grains of a different cultivar. Possible applications of this method are discussed.

Five years have passed since the triple disaster of the March 2011 earthquake and the ensuing tsunami and radioactive pollution from the Fukushima Daiichi Nuclear Power Station. Along the coastline of Tohoku region, one of Japan’s most important agricultural areas, activities to revive farmland that suffered salt damage due to the tsunami are still continuing to this day. This chapter documents the current state of recovery of the farmland and agriculture, the work being carried out by the Tohoku University Rapeseed Project for Restoring Tsunami-Salt-Damaged Farmland that was initiated in the immediate aftermath of the earthquake disaster, and the future vision for the project.

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.

The first agri-environmental programme in Japan, which started in 2011, is aimed at the conservation of biodiversity on farmland in Japan. For its smooth enforcement, a framework is required in which biodiversity conservation is visible and clear to farmers and the public. Such programmes will be effective when options can easily be adopted and the concept is appropriate and regionally specific for the farmers. While the result-oriented payment approach provides biodiversity’s concrete contribution to the improvement of farm settings, entry-level incentives are also necessary to encourage farmers’ initial participation, as biodiversity has an environmental value as public goods.

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.

Radish (Raphanus sativus L.) has a self-incompatibility system similar to those of Brassica rapa and Brassica oleracea. Self-recognition specificities of pollen and stigmas are determined by multiple alleles of SP11 and SRK, respectively, both of which are located on the S locus. As in B. rapa and B. oleracea, stigmas in radish also have S glycoprotein encoded by SLG closely linked to SP11 and SRK. The set of alleles of SP11, SRK, and SLG is called S haplotype, and S haplotypes are numbered as S-1, S-2, S-3, etc. Although radish belongs to a genus different from Brassica, nucleotide sequences of SP11, SRK, and SLG alleles of radish and Brassica are intermingled in phylogenetic trees of SP11, SRK, and SLG, respectively, indicating that diversification of these alleles predates speciation of these genera. SP11, SRK, and SLG alleles of some S haplotypes in radish are highly similar to those of some S haplotypes in Brassica, and one S haplotype in radish has been revealed to have the same recognition specificity as that of one S haplotype in B. rapa. Comparison of nucleotide sequences of SP11 and SRK alleles and recognition specificities between similar S haplotypes of radish and Brassica may provide information useful for understanding the molecular structures of SP11 and SRK proteins. The self-incompatibility system is used for seed production in F1 hybrid cultivars of radish, in which characterization of each S haplotype, especially the strength of the self-incompatibility phenotype, is required. However, the numbering of S haplotypes in radish varies by researchers, and nucleotide sequence information on S haplotypes is thus confusing. The importance of unification of S haplotype nomenclature is herein discussed.

Radish is important as a root vegetable, a leafy vegetable, a fruit vegetable, an oil crop, and also as a cover plant. The economic importance and characteristics of radish differ between the East and the West of the world. In the East, there are radish cultivars having large roots with various shapes called “Asian big radish” and those grown for production of immature pods or oil seeds, whereas radish is a small vegetable grown within one month in the West. Asian big radish is expected to eventually become popular in the West. Radish belongs to the genus Raphanus, but is similar to the Brassica species except for the shape of pods and seeds. Despite their similarities, the order of genes in chromosomes is quite different between Raphanus and Brassica. Radish genome sequences have been published from three groups using similar cultivars, and therefore, collaboration for combining sequence data is considered to be effective for determination of more reliable genome sequences. Some radish lines have high salt tolerance and disease resistance different from Brassica crops. Radish also has a characteristic glucosinolate composition. Since radish can be crossed with Brassica species, it is also important as a genetic resource for Brassica crop breeding.

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.

An F2 population was developed from a cross between a mur-cytoplasmic male sterile broccoli line and a restorer Chinese kale line. Phenotypic analysis of F2 plants indicated that the pollen fertility is controlled by two genes and segregated in a duplicate gene interaction mode with a ratio of 15:1. A total of 236 single nucleotide polymorphism (SNP) markers were developed utilizing 1,448 primers designed for production of expressed sequence tag (EST)-SNP markers of Raphanus sativus and analyzed by the dot-blot technique in 205 F2 individuals. A linkage map was constructed with a total of 142 markers and these markers were assigned to nine linkage groups together with simple sequence repeat markers mapped previously on the published linkage maps of Brassica oleracea. The linkage map spanned 909 cM with an average marker distance of 6.4 cM. A fertility restorer locus (Rfm1) was mapped on LG1, corresponding to chromosome 3, along with a flower color locus at a distance of 25 cM. SNP markers flanking the Rfm1 locus were BoCL2642s at a distance of 2.5 cM on one side and BoCL2901s at a distance of 7.5 cM on the other side. All the SNP markers showed homology with Arabidopsis thaliana and Brassica rapa genome sequences. Three pentatricopeptide repeat genes of the P-subfamily, particularly expressed in buds of the restorer line, were identified and these genes could be potential candidate fertility restorer genes.

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.