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.

Mutation of the imprinted gene OsEMF2a induces autonomous endosperm development and delayed cellularization in rice.

In angiosperms, endosperm development comprises a series of developmental transitions controlled by genetic and epigenetic mechanisms that are initiated after double fertilization. Polycomb repressive complex 2 (PRC2) is a key component of these mechanisms that mediate histone H3 lysine 27 trimethylation (H3K27me3); the action of PRC2 is well described in Arabidopsis thaliana but remains uncertain in cereals. In this study, we demonstrate that mutation of the rice (Oryza sativa) gene EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component of PRC2, causes an autonomous endosperm phenotype involving proliferation of the central cell nuclei with separate cytoplasmic domains, even in the absence of fertilization. Detailed cytological and transcriptomic analyses revealed that the autonomous endosperm can produce storage compounds, starch granules, and protein bodies specific to the endosperm. These events have not been reported in Arabidopsis. After fertilization, we observed an abnormally delayed developmental transition in the endosperm. Transcriptome and H3K27me3 ChIP-seq analyses using endosperm from the emf2a mutant identified downstream targets of PRC2. These included >100 transcription factor genes such as type-I MADS-box genes, which are likely required for endosperm development. Our results demonstrate that OsEMF2a-containing PRC2 controls endosperm developmental programs before and after fertilization.

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.

The tandem repeated organization of NB-LRR genes in the clubroot-resistant CRb locus in Brassica rapa L.

To facilitate prevention of clubroot disease, a major threat to the successful cultivation of Chinese cabbage (Brassica rapa L.), we bred clubroot-resistant (CR) cultivars by introducing resistance genes from CR turnips via conventional breeding. Among 11 CR loci found in B. rapa, we identified CRb in Chinese cabbage cultivar 'CR Shinki' as a single dominant gene for resistance against Plasmodiophora brassicae pathotype group 3, against which the stacking of Crr1 and Crr2 loci was not effective. However, the precise location and pathotype specificity of CRb have been controversial, because CRa and Rcr1 also map near this locus. Previously, our fine-mapping study revealed that CRb is located in a 140-kb genomic region on chromosome A03. Here, we determined the nucleotide sequence of an approximately 64-kb candidate region in the resistant line; this region contains six open reading frames (ORFs) similar to NB-LRR encoding genes that are predicted to occur in tandem with the same orientation. Among the six ORFs present, only four on the genome of the resistant line showed a strong DNA sequence identity with each other, and only one of those four could confer resistance to P. brassicae isolate No. 14 of the pathotype group 3. These results suggest that these genes evolved through recent gene duplication and uneven crossover events that could lead to the acquisition of clubroot resistance. The DNA sequence of the functional ORF was identical to that of the previously cloned CRa gene; thus, we showed that the independently identified CRb and CRa are one and the same clubroot-resistance gene.

Novel glucosinolate composition lacking 4-methylthio-3-butenyl glucosinolate in Japanese white radish (Raphanus sativus L.).

KEY MESSAGE: Genetic analysis and gene mapping of the 4-methylthio-3-butenyl glucosinolate-less trait of white radish were performed and a white radish cultivar with new glucosinolate composition was developed. A spontaneous mutant having significantly low 4-methylthio-3-butenyl glucosinolate (4MTB-GSL) content was identified from a landrace of Japanese white radish (Raphanus sativus L.) through intensive evaluation of glucosinolate profiles of 632 lines including genetic resources and commercial cultivars using high-performance liquid chromatography (HPLC) analysis. A line lacking 4MTB-GSL was developed using the selected mutant as a gene source. Genetic analyses of F1, F2, and BC1F1 populations of this line suggested that the 4MTB-GSL-less trait is controlled by a single recessive allele. Using SNP and SCAR markers, 96 F2 plants were genotyped, and a linkage map having nine linkage groups with a total map distance of 808.3 cM was constructed. A gene responsible for the 4MTB-GSL-less trait was mapped between CL1753 and CL5895 at the end of linkage group 1. The genetic distance between these markers was 4.2 cM. By selfing and selection of plants lacking 4MTB-GSL, a new cultivar, 'Daikon parental line No. 5', was successfully developed. This cultivar was characterized by glucoerucin, which accounted for more than 90% of the total glucosinolates (GSLs). The total GSL content in roots was ca. 12 µmol/g DW, significantly lower than those in common white radish cultivars. Significance of this line in radish breeding is discussed.

Arabidopsis dual resistance proteins, both RPS4 and RRS1, are required for resistance to bacterial wilt in transgenic Brassica crops.

Bacterial wilt phytopathogen Ralstonia solanacearum is a serious soil-borne disease that attacks several economically important plants worldwide, including Brassicaceae. Previous studies indicate that recognition of avirulence (Avr)-effector PopP2 by resistance (R) protein, RRS1-R, and physical interaction between RRS1-R and PopP2 in the nucleus are required for resistance. Of late, we showed that a pair of Arabidopsis thaliana TIR-NLR proteins, RRS1 and RPS4, function together in disease resistance against multiple pathogen isolates. Here, we report that dual R proteins, RRS1 and RPS4, from A. thaliana ecotype Wassilewskija confer resistance to bacterial wilt in transgenic Brassica crops. For practical applications, this finding may provide a new strategy for developing disease resistant plants that express R genes from other plants.

Fine mapping of the clubroot resistance gene CRb and development of a useful selectable marker in Brassica rapa.

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) gene CRb is effective against Plasmodiophora brassicae isolate No. 14, which is classified as pathotype group 3. Although markers linked to CRb have been reported, an accurate position in the genome and the gene structure are unknown. To determine the genomic location and estimate the structure of CRb, we developed 28 markers (average distance, 20.4 kb) around CRb and constructed a high-density partial map. The precise position of CRb was determined by using a population of 2,032 F2 plants generated by selfing B. rapa 'CR Shinki.' We determined that CRb is located in the 140-kb genomic region between markers KB59N07 and B1005 and found candidate resistance genes. Among other CR genes on chromosome R3, a genotype of CRa closest marker clearly matched those of CRb and Crr3 did not confer resistance to isolate No. 14. Based on the genotypes of 11 markers developed near CRb and resistance to isolate No. 14, 82 of 108 cultivars showed a strong correlation between genotypes and phenotypes. The results of this study will be useful for isolating CRb and breeding cultivars with resistance to pathotype group 3 by introducing CRb into susceptible cultivars through marker-assisted selection.

Breaking restricted taxonomic functionality by dual resistance genes.

NB-LRR-type disease resistance (R) genes have been used in traditional breeding programs for crop protection. However, functional transfer of NB-LRR-type R genes to plants in taxonomically distinct families to establish pathogen resistance has not been successful. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and B. napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Thus the successful transfer of two R genes at the family level overcomes restricted taxonomic functionality. This implies that the downstream components of R genes must be highly conserved and interfamily utilization of R genes can be a powerful strategy to combat pathogens.

Identification and characterization of Crr1a, a gene for resistance to clubroot disease (Plasmodiophora brassicae Woronin) in Brassica rapa L.

Clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae Woronin, is one of the most economically important diseases of Brassica crops in the world. Although many clubroot resistance (CR) loci have been identified through genetic analysis and QTL mapping, the molecular mechanisms of defense responses against P. brassicae remain unknown. Fine mapping of the Crr1 locus, which was originally identified as a single locus, revealed that it comprises two gene loci, Crr1a and Crr1b. Here we report the map-based cloning and characterization of Crr1a, which confers resistance to clubroot in Brassica rapa. Crr1a(G004), cloned from the resistant line G004, encodes a Toll-Interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) protein expressed in the stele and cortex of hypocotyl and roots, where secondary infection of the pathogen occurs, but not in root hairs, where primary infection occurs. Gain-of-function analysis proved that Crr1a(G004) alone conferred resistance to isolate Ano-01 in susceptible Arabidopsis and B. rapa. In comparison, the susceptible allele Crr1a(A9709) encodes a truncated NB-LRR protein, which lacked more than half of the TIR domain on account of the insertion of a solo-long terminal repeat (LTR) in exon 1 and included several substitutions and insertion-deletions in the LRR domain. This study provides a basis for further molecular analysis of defense mechanisms against P. brassicae and will contribute to the breeding of resistant cultivars of Brassica vegetables by marker-assisted selection.Data deposition The sequence reported in this paper has been deposited in the GenBank database (accession no. AB605024).

Interfamily transfer of dual NB-LRR genes confers resistance to multiple pathogens.

A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens.

Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.).

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) genes Crr1 and Crr2 are effective against the mild Plasmodiophora brassicae isolate Ano-01 and the more virulent isolate Wakayama-01, but not against isolate No. 14, classified into pathotype group 3. 'Akiriso', a clubroot-resistant F(1) cultivar, showed resistance to isolate No. 14. To increase the durability of resistance, we attempted to identify the CR locus in 'Akiriso'. CR in 'Akiriso' segregated as a single dominant gene and was linked to several molecular markers that were also linked to CRb, a CR locus from cultivar 'CR Shinki'. We developed additional markers around CRb and constructed partial genetic maps of this region in 'Akiriso' and 'CR Shinki'. The positions and order of markers in the genetic maps of the two cultivars were very similar. The segregation ratios for resistance to isolate No. 14 in F(2) populations derived from each of the two cultivars were also very similar. These results suggest that the CR locus in 'Akiriso' is CRb or a tightly linked locus. The newly developed markers in this study were more closely linked to CRb than previously reported markers and will be useful for marker-assisted selection of CRb in Chinese cabbage breeding.

Microstructure of a Brassica rapa genome segment homoeologous to the resistance gene cluster on Arabidopsis chromosome 4.

Genome evolution is a continuous process and genomic rearrangement occurs both within and between species. With the sequencing of the Arabidopsis thaliana genome, comparative genetics and genomics offer new insights into plant biology. The genus Brassica offers excellent opportunities with which to compare genomic synteny so as to reveal genome evolution. During a previous genetic analysis of clubroot resistance in Brassica rapa, we identified a genetic region that is highly collinear with Arabidopsis chromosome 4. This region corresponds to a disease resistance gene cluster in the A. thaliana genome. Relying on synteny with Arabidopsis, we fine-mapped the region and found that the location and order of the markers showed good correspondence with those in Arabidopsis. Microsynteny on a physical map indicated an almost parallel correspondence, with a few rearrangements such as inversions and insertions. The results show that this genomic region of Brassica is conserved extensively with that of Arabidopsis and has potential as a disease resistance gene cluster, although the genera diverged 20 million years ago.

Development of full-length cDNAs from Chinese cabbage (Brassica rapa Subsp. pekinensis) and identification of marker genes for defence response.

Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.

Mapping of quantitative trait loci for high level of self-incompatibility in Brassica rapa L.

The level of self-incompatibility (SI) is important to the purity of F1 seeds produced using the SI system of Brassica vegetables. To analyze the genetic basis of the level of SI, we generated an F2 population derived from a cross between a turnip inbred line showing a high level of SI and a Chinese cabbage inbred line showing a low level, and evaluated the level of SI under insect pollination in two years. We constructed a detailed linkage map of Brassica rapa from the F2 progeny, consisting of SSR, SNP, indel, and CAPS loci segregating into 10 linkage groups covering approximately 700 cM. Five quantitative trait loci (QTL) for high-level SI were identified. The phenotypic variation explained by the QTL ranged between 7.2% and 23.8%. Two QTL were detected in both years. Mapping of SI-related genes revealed that these QTL were co-localized with SLG on R07 and MLPK on R03. This is the first report of QTL for high-level SI evaluated under insect pollination in a Brassica vegetable. Our results could be useful for the marker-assisted selection of parental lines with a stable SI.

Ultrastructural characterization of exine development of the transient defective exine 1 mutant suggests the existence of a factor involved in constructing reticulate exine architecture from sporopollenin aggregates.

A male-sterile mutant of Arabidopsis thaliana, in which filament elongation was defective although pollen fertility was normal, was isolated by means of T-DNA tagging. Transmission electron microscopy (TEM) analysis revealed that primexine synthesis and probacula formation, which are thought to be the initial steps of exine formation, were defective, and that globular sporopollenin aggregation was randomly deposited onto the microspore at the early uninucleate microspore stage. Sporopollenin aggregation, which failed to anchor to the microspore plasma membrane, was deposited on the locule wall and in the locule at the uninucleate microspore stage. However, visually normal exine with a basic reticulate structure was observed at the middle uninucleate microspore stage, indicating that the exine formation was restored in the mutant. Thus, the mutant was designated transient defective exine 1 (tde1). These results indicated that tde1 mutation affects the initial process of the exine formation, but does not impair any critical processes. Our results also suggest the existence of a certain factor responsible for exine patterning in A. thaliana. The TDE1 gene was found to be identical to the DE-ETIOLATED 2 gene known to be involved in brassinosteroid (BR) biosynthesis, and the tde1 probacula-defective phenotypes were recovered in the presence of BR application. These results suggest that BRs control the rate or efficiency of initial process of exine pattern formation.

Simple sequence repeat-based comparative genomics between Brassica rapa and Arabidopsis thaliana: the genetic origin of clubroot resistance.

An SSR-based linkage map was constructed in Brassica rapa. It includes 113 SSR, 87 RFLP, and 62 RAPD markers. It consists of 10 linkage groups with a total distance of 1005.5 cM and an average distance of 3.7 cM. SSRs are distributed throughout the linkage groups at an average of 8.7 cM. Synteny between B. rapa and a model plant, Arabidopsis thaliana, was analyzed. A number of small genomic segments of A. thaliana were scattered throughout an entire B. rapa linkage map. This points out the complex genomic rearrangements during the course of evolution in Cruciferae. A 282.5-cM region in the B. rapa map was in synteny with A. thaliana. Of the three QTL (Crr1, Crr2, and Crr4) for clubroot resistance identified, synteny analysis revealed that two major QTL regions, Crr1 and Crr2, overlapped in a small region of Arabidopsis chromosome 4. This region belongs to one of the disease-resistance gene clusters (MRCs) in the A. thaliana genome. These results suggest that the resistance genes for clubroot originated from a member of the MRCs in a common ancestral genome and subsequently were distributed to the different regions they now inhabit in the process of evolution.

Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen, resulting in male sterility in Arabidopsis thaliana.

A novel male-sterile mutant of Arabidopsis thaliana was isolated by means of T-DNA tagging. Pollen abortion of the mutant was evident after microspore release, and pollen grains were completely absent at anthesis. Transmission electron microscope analysis revealed that primexine was coarsely developed, and that although sporopollenin was produced, it was not deposited onto the microspore plasma membrane. The sporopollenin that failed to be deposited aggregated and accumulated within the locule and on the locule wall. Finally, as no exine formation was observed, the mutant was named nef1. The plastoglobuli within the plastids of the tapetum were reduced, and lipid accumulation was considerably decreased. The mutant had a significantly altered leaf chloroplast ultrastructure and showed various growth defects. Lipid analysis revealed that the total lipid content in nef1 was lower than that in the wild type, which indicated that Nef1 was involved in lipid metabolism. Cloning of the full-length Nef1 indicated that the gene encodes a novel plant protein of 1123 amino acids with limited sequence similarities to membrane proteins or transporter-like proteins, and the NEF1 is predicted to be a plastid integral membrane protein. Motif analysis revealed that NEF1 contains prokaryotic membrane lipoprotein lipid attachment sites that are involved in maintaining cell envelope integrity. It is predicted that the Nef1 encodes a membrane protein that maintains the envelope integrity in the plastids.

A novel male-sterile mutant of Arabidopsis thaliana, faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine.

A mutant exhibiting conditional male sterility, in which fertility was restored under conditions of high humidity, was identified in T-DNA tagged lines of Arabidopsis thaliana. Scanning electron microscopy (SEM) demonstrated that the pollen surface was almost smooth and the reticulate pattern not prominent. Thus, the mutant was named faceless pollen-1 (flp1). Transmission electron microscopy (TEM) revealed that the smooth appearance was due to tryphine filling in the exine cavities and covering the pollen surface. The lipid droplets in the tryphine of mutant pollen were smaller and more numerous than those of the wild type. SEM analysis also demonstrated that pollen exine was easily damaged by acetolysis, suggesting that a component of exine, sporopollenin, was defective in the mutant. In addition, the stems and siliques had reduced amounts of wax crystals. A predicted amino acid sequence of the cDNA that corresponded to the tagged gene, fip1, showed sequence similarity to proteins involved in wax biosynthesis. The FLP1 protein is likely to play a role in the synthesis of the components of tryphine, sporopollenin of exine and the wax of stems and siliques.