Allelic variation of a clubroot resistance gene (Crr1a) in Japanese cultivars of Chinese cabbage (Brassica rapa L.).

Clubroot resistance (CR) is an important trait in Chinese cabbage breeding worldwide. Although Crr1a, the gene responsible for clubroot-resistance, has been cloned and shown to encode the NLR protein, its allelic variation and molecular function remain unknown. Here, we investigated the sequence variation and function of three Crr1a alleles cloned from six CR F1 cultivars of Chinese cabbage. Gain-of-function analysis revealed that Crr1aKinami90_a isolated from the cv. 'Kinami 90' conferred clubroot resistance as observed for Crr1aG004 . Because two susceptible alleles commonly lacked 172 amino acids in the C-terminal region, we investigated clubroot resistance in transgenic Arabidopsis harboring the chimeric Crr1a, in which 172 amino acids of the functional alleles were fused to the susceptible alleles. The fusion of the C-terminal region to the susceptible alleles restored resistance, indicating that their susceptibility was caused by the lack of the C-terminus. We developed DNA markers to detect the two functional Crr1a alleles, and demonstrated that the functional Crr1a alleles were frequently found in European fodder turnips, whereas they were rarely introduced into Japanese CR cultivars of Chinese cabbage. These results would contribute to CR breeding via marker-assisted selection and help our understanding of the molecular mechanisms underlying clubroot resistance.

Development of novel clubroot resistant rapeseed lines (Brassica napus L.) effective against Japanese field isolates by marker assisted selection.

Clubroot is an important disease infectible to cruciferous plants and a major threat to rapeseed production in Japan. However, no clubroot resistant rapeseed cultivars have been released. We surveyed pathotype variation of six isolates collected from rapeseed fields and found they were classified as pathotype groups 2 and 4 using Japanese F1 Chinese cabbage cultivars. We produced the resynthesized clubroot resistant Brassica napus harboring two resistant loci, Crr1 and Crr2, by interspecific crossing and developed resistant rapeseed lines for southern and northern regions by marker-assisted selection and backcrossing. We improved the DNA marker for erucic acid content to remove linkage drag between Crr1 and high erucic acid content and successfully selected lines with clubroot resistance and zero erucic acid for northern regions. A novel line, 'Tohoku No. 106', suitable for southern regions showed stable resistance against all six isolates and high performance in infested fields. We conclude that Crr1 and Crr2 are important genes for CR rapeseed breeding and marker-assisted selection is effective in improving clubroot resistance.

Production of interspecific hybrids between Japanese gentians and wild species of Gentiana.

Wide hybridization, which is a powerful tool to broaden genetic variation, has been used in breeding of many crops. However, in ornamental gentian few wide hybridizations have been reported. Interspecific hybridizations between two gentian cultivated species (Gentiana scabra and G. triflora) and 11 wild species, which were classified in five sections, were carried out using ovule culture. When G. scabra was used as a female parent, normal seedlings and hybrid plants were obtained from eight and five interspecific combinations, respectively. The yield of seedling produced from ovule culture depended on interspecific combinations, ranging from 0.3 to 427.7 normal seedling per flower. In the hybridization of G. triflora with five wild species, normal seedlings and plants were produced in five and four interspecific combinations, respectively. The yield of normal seedling ranging from 0.4 to 228.3 was different between not only interspecific combinations but also reciprocal crosses. Two cultivated species are classified in sect. Pneumonanthe, and successful production of hybrids was obtained from the hybridization with species classified in sections Pneumonanthe or Cruciata. The hybrid nature of the produced plants was confirmed by molecular marker and morphology. The production of interspecific hybrids opens a novel prospect in ornamental gentian breeding.

QTL-seq analysis identifies two genomic regions determining the heading date of foxtail millet, Setaria italica (L.) P.Beauv.

Heading date is an important event to ensure successful seed production. Although foxtail millet (Setaria italica (L.) P.Beauv.) is an important foodstuff in semiarid regions around the world, the genetic basis determining heading date is unclear. To identify genomic regions regulating days to heading (DTH), we conducted a QTL-seq analysis based on combining whole-genome re-sequencing and bulked-segregant analysis of an F2 population derived from crosses between the middle-heading cultivar Shinanotsubuhime and the early-heading cultivar Yuikogane. Under field conditions, transgressive segregation of DTH toward late heading was observed in the F2 population. We made three types of bulk samples: Y-bulk (early-heading), S-bulk (late-heading) and L-bulk (extremely late-heading). By genome-wide comparison of SNPs in the Y-bulk vs. the S-bulk and the Y-bulk vs. the L-bulk, we identified two QTLs associated with DTH. The first QTL, qDTH2, was detected on chromosome 2 from the Y-bulk and S-bulk comparison. The second QTL, qDTH7, was detected on chromosome 7 from the Y-bulk and L-bulk comparison. The Shinanotsubuhime allele for qDTH2 caused late heading in the F2 population, whereas the Yuikogane allele for qDTH7 led to extremely late heading. These results suggest that allelic differences in both qDTH2 and qDTH7 determine regional adaptability in S. italica.

Identification of loci associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis.

KEY MESSAGE: 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.

Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (Gentiana spp.).

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.

W14/15 esterase gene haplotype can be a genetic landmark of cultivars and species of the genus Gentiana L.

We have identified multiple alleles for a single gene termed W14/15. This gene encodes closely related but not identical proteins W14 and W15 that accumulate in overwinter buds of Gentiana triflora (Takahashi et al. in Breed Sci 56:39-46, 2006; Hikage et al. in Mol Genet Genomics 278:95-104, 2007). In this study, structural analysis of the W14/15 gene was carried out for 21 different gentian lines/cultivars consisting of 5 different species, to survey species- or line/cultivar-specific haplotypes. Within the samples examined, multiple variant forms were found. Those were categorized into seven major types (type I-VII) and ten subtypes based on the presence of three short insertion/deletion sites, three RFLP sites, and several SNP sites. Each line/cultivar had a distinct set of W14/15 gene variants for an allelic pair. Phylogenetic analysis showed that the W14/15 alleles cluster into groups that are characteristic of gentian species, i.e., G. triflora, G. scabra, G. pneumonanthe, G. septemfida and an unknown species other than the former four. In addition, within the same gentian species, different sets of haplotypes were found. Thus, the W14/15 alleles provide useful landmarks to resolve phylogenies of the genus or section Gentiana, as well as to analyze pedigree and breeding history of the cultivars derived from those Gentiana sp.

Gynogenesis in gentians (Gentiana triflora, G. scabra): production of haploids and doubled haploids.

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0.93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding.

Arabidopsis EMBRYOMAKER encoding an AP2 domain transcription factor plays a key role in developmental change from vegetative to embryonic phase.

Although several types of plant cells retain the competence to enter into embryonic development without fertilization, the molecular mechanism(s) underlying ectopic embryogenesis is largely unknown. To gain insight into this mechanism, in a previous study we identified 136 ESTs specifically expressed in microspore embryogenesis of Brassica napus. Here, we describe the characterization of the Arabidopsis EMBRYOMAKER (EMK) gene, which is homologous to one of the identified Brassica ESTs (BnGemb-58) and encodes an AP2 domain transcription factor. The AtEMK was expressed in developing and mature embryos, but its rapid disappearance occurred during germination. After germination, the expression of AtEMK was found in the root apical meristem and the distal parts of cotyledons. Although a mutant lacking AtEMK exhibited no distinctive defects in the embryo, ectopic expression of AtEMK induced embryo-like structures from cotyledons. The embryo-like structures contained high concentration of lipids, expressed several embryo-specific genes, and could convert into independent plants, indicating that the structures are somatic embryos. In vitro culture, AtEMK enhanced the efficiency of somatic embryogenesis. Furthermore, ectopic expression of AtEMK caused the formation of trichomes on cotyledons, dedifferentiated several tissues into calli, and retarded root development, demonstrating that AtEMK is harmful for the normal development of plants after germination. From these results, we conclude that the AtEMK is a key player to maintain embryonic identity, and the rapid disappearance of AtEMK expression during germination is essential for the developmental transition between the embryonic and vegetative phases in plants.

GASSHO1 and GASSHO2 encoding a putative leucine-rich repeat transmembrane-type receptor kinase are essential for the normal development of the epidermal surface in Arabidopsis embryos.

Receptor-like kinases (RLKs) containing leucine-rich repeats (LRRs) act as both signal receptor and signal transducer in ligand-mediated communication between cells. It is believed that many LRR-RLKs are present in the Arabidopsis genome, but the functions of most are unknown. We recently identified Bnms4D-82, an expressed sequence tag (EST) in Brassica napus that encodes an LRR-RLK and is expressed at an early stage of its microspore embryogenesis. To elucidate the function of this gene we used GASSHO1 (GSO1) and GSO2, two Arabidopsis genes with a high degree of homology with Bnms4D-82. The products of transcripts of GSO1 and GSO2 accumulate in parts of the embryo and in seedlings, but not in true leaves. Plants that lacked both GSO1 and GSO2 exhibited pleiotropy, including abnormal bending of embryos, ectopic adhesion between cotyledons, a highly permeable epidermal structure, and an abnormal pattern of distribution of stomata on cotyledons in embryos and seedlings. However, plants homozygous for either gso1-1 or gso2-1 had no visible abnormality. These results suggest that GASSHO genes are essential for the formation of a normal epidermal surface during embryogenesis.

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans.

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T(1) soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.

Acylated cyanidin 3-sophoroside-5-glucoside in purple-violet flowers of Moricandia arvensis (Brassicaceae).

A new acylated anthocyanin was isolated as a major pigment, along with a known anthocyanin (Moricandia arvensis anthocyanin 1: MAA-1), from a strain of Moricandia arvensis (Code No. MOR-ARV-3) with purple-violet flowers, and identified as cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(ß-glucopyranosyl)-trans-caffeoyl)-ß-glucopyranosyl)-trans-sinapoyl)-ß-glucopyranoside]-5-O-[6-O-(malonyl)-ß-glucopyranoside].

The advantages of cDNA microarray as an effective tool for identification of reproductive organ-specific genes in a model legume, Lotus japonicus.

To understand the molecular mechanisms intrinsic to reproductive organ development a cDNA microarray, fabricated from flower bud cDNA clones, was used to isolate genes, which are specifically expressed during the development of the anther and pistil in Lotus japonicus. Cluster analysis of the microarray data revealed 21 and 111 independent cDNA groups, which were specifically expressed in immature and mature anthers, respectively. RT-PCR was performed to provide a direct assessment of the accuracy and reproducibility of our approach. Confirmation of our results suggests that cDNA microarray technology is an effective tool for identification of novel reproductive organ-specific genes.

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus.

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.

Isolation and expression of Lea gene in desiccation-tolerant microspore-derived embryos in Brassica spp.

Desiccation tolerance can be induced in microspore-derived embryos of Brassica spp. by application of abscisic acid (ABA). As part of our effort to elucidate the molecular mechanism in induction of desiccation tolerance of embryos, we have isolated Lea genes and examined their expression in microspore-derived embryos of B. napus and B. campestris. The cDNA clones of Lea genes were isolated from desiccation-tolerant microspore-derived embryos of both species. Two cDNA clones of B. napus and B. campestris shared a similar homology of 92.9% identity between the two deduced proteins. These proteins also had high homology with Lea 76 which was isolated from zygotic embryos of B. napus, but were 54 amino acids shorter than Lea 76. Acquisition of desiccation tolerance of the microspore-derived embryos and accumulation of mRNA and its transcripts of the Lea genes began within 12 h of ABA treatment. The embryos treated with ABA for 48-168 h showed a high desiccation tolerance and accumulation of Lea proteins at high levels. The close relationships between the induction of desiccation tolerance of embryos and the expression level of Lea genes suggest an important function of Lea proteins for desiccation tolerance in embryos.

Draft sequences of the radish (Raphanus sativus L.) genome.

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.

Triacylated peonidin 3-sophoroside-5-glucosides from the purple flowers of Moricandia ramburii Webb.

Triacylated peonidin 3-sophoroside-5-glucosides were isolated from the purple flowers of Moricandia ramburii Webb. (Family: Brassicaceae), and determined to be peonidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (1), peonidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)-6-O-(cis-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (2) and peonidin 3-O-[2-O-(2-O-(trans-sinapoyl)-glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (3), respectively, by chemical and spectroscopic methods. In addition, one known acylated cyanidin glycoside, cyanidin 3-O-[2-O-(2-O-(trans-feruloyl)-glucosyl)-6-O-(trans-p-coumaroyl)-glucoside]-5-O-[6-O-(malonyl)-glucoside] (4), was also identified in the flowers. Peonidin glycosides have not been reported hitherto in floral tissues in to Brassicaceae.

Identification and characterization of genes expressed in early embryogenesis from microspores of Brassica napus.

To understand the mechanism in induction of embryogenesis from microspores of Brassica napus, we isolated exhaustively the genes expressed differentially during the early stage of microspore culture. A subtracted cDNA library composed of up-regulated genes during androgenic initiation was produced by suppression subtractive hybridization followed by differential screening by dot blot hybridization, and a total of 136 non-redundant expressed sequence tags were identified. Analysis of the potential functions of the genes showed that 64% of these genes were homologous to known genes, and the remaining ones have not been previously reported to participate in embryogenesis. Many embryo-specific genes were contained in the isolated genes, for example, genes cording lipid transfer protein, napin, cruciferin, oleosin, and phytosulfokine. Real-time RT-PCR analysis for 15 selected genes, which are understood to not be related with embryogenesis, demonstrated that all genes were expressed highly in the early stage of microspore embryogenesis. A few genes also showed higher expression in microspores cultured in non-embryogenic condition or in later stages of embryos. A principal component analysis based on expression profiles of the 15 genes demonstrated that these genes were classified into 2 groups, one characterized by their high expression in initiation of embryogenesis, and the other characterized by their expression in the early to middle stage of embryogenesis. The expressions of these genes were confirmed in zygotic embryos. The identification and characterization of the genes isolated in the present study provide novel information on microspore embryogenesis in Brassica.

Soybean dwarf virus-resistant transgenic soybeans with the sense coat protein gene.

We transformed a construct containing the sense coat protein (CP) gene of Soybean dwarf virus (SbDV) into soybean somatic embryos via microprojectile bombardment to acquire SbDV-resistant soybean plants. Six independent T(0) plants were obtained. One of these transgenic lines was subjected to further extensive analysis. Three different insertion patterns of Southern blot hybridization analysis in T(1) plants suggested that these insertions introduced in T(0) plants were segregated from each other or co-inherited in T(1) progenies. These insertions were classified into two types, which overexpressed SbDV-CP mRNA and accumulated SbDV-CP-specific short interfering RNA (siRNA), or repressed accumulation of SbDV-CP mRNA and siRNA by RNA analysis prior to SbDV inoculation. After inoculation of SbDV by the aphids, most T(2) plants of this transgenic line remained symptomless, contained little SbDV-specific RNA by RNA dot-blot hybridization analysis and exhibited SbDV-CP-specific siRNA. We discuss here the possible mechanisms of the achieved resistance, including the RNA silencing.

Structure and allele-specific expression variation of novel alpha/beta hydrolase fold proteins in gentian plants.

Previously, we identified two closely related proteins termed W14 and W15 that were enriched in the overwinter buds of the gentian plant Gentiana triflora. Expression of the latter protein W15 has been implicated in its association with cold hardiness, because of its absence in a cold-sensitive mutant. Here, we characterized these two proteins and the genes encoding them. Amino acid sequences of the W14 and W15 proteins showed difference at only three amino acid positions, and both of them showed homologies to alpha/beta hydrolase fold superfamily. Consistently, GST-fused W14 and W15 proteins expressed in bacteria showed hydrolase activity toward 1-naphtyl acetate. Structural analysis of these two genes in seven different gentian strains/cultivars including an anther culture-derived homozygous diploid revealed that W14 and W15 genes are allelic. Three genotypes were found; two strains carried both alleles (W14/W15), one carried the W15 genes in both alleles (W15/W15), and others were homozygous of W14 (W14/W14). Interestingly, expression of the two proteins exhibited allele-specificity. In one W14/W15 strain, expression of the W15 allele was almost repressed. In addition, organ specific expression of the alleles was observed in different cultivars. These observations were discussed in relation to winter hardiness of the gentian plants.