Hiroyuki Fukuoka

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.

SNP Discovery and Linkage Map Construction in Cultivated Tomato

Few intraspecific genetic linkage maps have been reported for cultivated tomato, mainly because genetic diversity within Solanum lycopersicum is much less than that between tomato species. Single nucleotide polymorphisms (SNPs), the most abundant source of genomic variation, are the most promising source of polymorphisms for the construction of linkage maps for closely related intraspecific lines. In this study, we developed SNP markers based on expressed sequence tags for the construction of intraspecific linkage maps in tomato. Out of the 5607 SNP positions detected through in silico analysis, 1536 were selected for high-throughput genotyping of two mapping populations derived from crosses between ‘Micro-Tom’ and either ‘Ailsa Craig’ or ‘M82’. A total of 1137 markers, including 793 out of the 1338 successfully genotyped SNPs, along with 344 simple sequence repeat and intronic polymorphism markers, were mapped onto two linkage maps, which covered 1467.8 and 1422.7 cM, respectively. The SNP markers developed were then screened against cultivated tomato lines in order to estimate the transferability of these SNPs to other breeding materials. The molecular markers and linkage maps represent a milestone in the genomics and genetics, and are the first step toward molecular breeding of cultivated tomato. Information on the DNA markers, linkage maps, and SNP genotypes for these tomato lines is available at http://www.kazusa.or.jp/tomato/.

Draft genome sequence of eggplant (Solanum melongena L.): the representative solanum species indigenous to the old world.

Unlike other important Solanaceae crops such as tomato, potato, chili pepper, and tobacco, all of which originated in South America and are cultivated worldwide, eggplant (Solanum melongena L.) is indigenous to the Old World and in this respect it is phylogenetically unique. To broaden our knowledge of the genomic nature of solanaceous plants further, we dissected the eggplant genome and built a draft genome dataset with 33,873 scaffolds termed SME_r2.5.1 that covers 833.1 Mb, ca. 74% of the eggplant genome. Approximately 90% of the gene space was estimated to be covered by SME_r2.5.1 and 85,446 genes were predicted in the genome. Clustering analysis of the predicted genes of eggplant along with the genes of three other solanaceous plants as well as Arabidopsis thaliana revealed that, of the 35,000 clusters generated, 4,018 were exclusively composed of eggplant genes that would perhaps confer eggplant-specific traits. Between eggplant and tomato, 16,573 pairs of genes were deduced to be orthologous, and 9,489 eggplant scaffolds could be mapped onto the tomato genome. Furthermore, 56 conserved synteny blocks were identified between the two species. The detailed comparative analysis of the eggplant and tomato genomes will facilitate our understanding of the genomic architecture of solanaceous plants, which will contribute to cultivation and further utilization of these crops.

Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum

Solanum torvum Sw. cv. Torubamubiga (TB) is a low cadmium (Cd)-accumulating plant. To elucidate the molecular mechanisms of the Cd acclimation process in TB roots, transcriptional regulation was analysed in response to mild Cd treatment: 0.1 μM CdCl2 in hydroponic solution. A unigene set consisting of 6296 unigene sequences was constructed from 18 816 TB cDNAs. The distribution of functional categories was similar to tomato, while 330 unigenes were suggested to be TB specific. For expression profiling, the SuperSAGE method was adapted for use with Illumina sequencing technology. Expression tag libraries were constructed from Cd-treated (for 3 h, 1 d, and 3 d) and untreated roots, and 34 269 species of independent tags were collected. Moreover, 6237 tags were ascribed to the TB or eggplant (aubergine) unigene sequences. Time-course changes were examined, and 2049 up- and 2022 down-regulated tags were identified. Although no tags annotated to metal transporter genes were significantly regulated, a tag annotated to AtFRD3, a xylem-loading citrate transporter, was down-regulated. In addition to induction of heavy metal chaperone proteins, antioxidative and sulphur-assimilating enzymes were induced, confirming that oxidative stress developed even using a mild Cd concentration. Rapid repression of dehydration-related transcription factors and aquaporin isoforms suggests that dehydration stress is a potential constituent of Cd-induced biochemical impediments. These transcriptional changes were also confirmed by real-time reverse transcription-PCR. Further additions of TB unigene sequences and functional analysis of the regulated tags will reveal the molecular basis of the Cd acclimation process, including the low Cd-accumulating characteristics of TB.

Structures and antioxidant activity of anthocyanins in many accessions of eggplant and its related species.

Anthocyanins were detected in extracts from the peels of 123 accessions of eggplant (Solanum melongena) and its related species. Their anthocyanin profiles were classified into four types, including known Japanese eggplant type (type 1) and non-Japanese eggplant type (type 2). Although most of the eggplant accessions had one of the two known profiles, one accession had a novel profile (type 3). Two accessions of related species showed another novel profile (type 4). The major anthocyanins were identified as delphinidin 3-(p-coumaroylrutinoside)-5-glucoside (nasunin) (type 1), delphinidin 3-rutinoside (type 2), delphinidin 3-glucoside (type 3), and petunidin 3-(p-coumaroylrutinoside)-5-glucoside (petunidin 3RGc5G) (type 4). Delphinidin 3-caffeoylrutinoside-5-glucoside (delphinidin 3RGcaf5G) was isolated from the hybrid (F1) plants of a type 1 cultivar and a type 3 germplasm. Among the five purified anthocyanins, delphinidin 3RGcaf5G showed the highest radical-scavenging activities toward both 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and linoleic acid radical, followed in order by nasunin and petunidin 3RGc5G.

Read2Marker: a data processing tool for microsatellite marker development from a large data set.

Microsatellites, or simple sequence repeats (SSRs), are widely considered to be one of the best sources of DNA polymorphism that can be utilized for developing genetic markers (1). Recently, several methods for making microsatellite-enriched genomic libraries were reported (2,3) and, as a consequence, the random sequencing of a microsatellite-enriched genomic library becomes a possible strategy for the mass development of microsatellite markers. Despite the progress in microsatellite isolation, it remains timeconsuming and laborious to handle the large amounts of sequence data. A set of script programs, read2Marker, was developed as a solution to this problem (Figure 1). In cooperation with phred/ phrap (4), BLAST (5), Primer3 (6), and T-Coffee (7), read2Marker can process an unlimited number of raw output data files from an automatic sequencer. It can run on a MacOS X or Linux platform and generates a report on many aspects of the nonredundant microsatellite-containing clones such as whole sequence, microsatellite core sequences, and PCR primers. A set of trace data obtained by sequencing from both ends of a clone is base-called by phred and assembled by phrap. Based on quality values (QVs), the high QV part of the assembled sequence is used for further processing. When data in plain text format without QVs are to be processed, all of the nucleotides are automatically assigned a QV of 20 to disable the quality threshold aspect of the read2Marker program. Subsequently, a microsatellite core is screened in the clone sequence by a newly developed tool, srchssr2. Initially, srchssr2 searches for repeated sequences of four dinucleotide motifs (GA, GT, AT, GC) and 10 trinucleotide motifs (AAC, AAG, ACC, ACG, ACT, AGC, AGG, ATC, TAT, CGC) independently in the clone sequence, and the core with the highest repeat number is selected. Next, a core structure neighboring the selected longest core is rescreened. If the gap between these cores is shorter than a maximum gap length set by the user in advance, then the two cores and gap sequence are connected to make a new core sequence. These procedures are repeated, and finally the composite microsatellite core in the sequence is identified. Finally, a PCR primer set for amplifying the microsatellite core is designed using the Primer3 program. The algorithm for finding microsatellites with srchssr2 is quite simple, but it is efficient enough to find cores. It can find composite repeats composed of different motifs and extracts 5′and 3′-flanking sequences that will be used for a redundancy check in the next step. Several SSR-finding programs with more sophisticated algorithms such as Msatfinder (M.I. Thurston and D. Field, www.bioinf.ceh.ac.uk/msatfinder), TROLL (8), Sputnik (C. Abajian, espressosoftware.com/pages/sputnik. jsp), and Tandem repeats finder (9) are available, and some of them could be substituted for srchssr2 with some modification, especially in cases where screening for motifs of more than three nucleotides is required. In order to remove redundant sequences, a homology check is performed using the BLAST algorithm. Two outer sequences of the clone flanking the microsatellite core are independently set as query sequences and BLASTed to a database comprising unique clones. If at least one of the two core-flanking sequences does not provide a hit from the database, the clone is judged as unique and added to

A protocol for the construction of microsatellite enriched genomic library

An improved protocol for constructing microsatellite-enriched libraries was developed. The procedure depends on digesting genomic DNA with a restriction enzyme that generates blunt-ends, and on ligating linkers that, when dimerized, create a restriction site for a different blunt-end producing restriction enzyme. Efficient ligation of linkers to the genomic DNA fragments is achieved by including restriction enzymes in the ligation reaction that eliminate unwanted ligation products. After ligation, the reaction mixture is subjected to subtractive hybridization without purification. DNA fragments containing microsatellites are captured by biotin-labeled oligonucleotide repeats and recovered using streptavidin-coated beads. The recovered fragments are amplified by PCR using the linker sequence as primer, and cloned directly into a plasmid vector. The linker has the sequence GTTT on the 5′ end, which promotes efficient adenylation of the 3′ ends of the PCR products. Consequently, the amplified fragments could be cloned into vectors without purification. This procedure enables efficient enrichment and cloning of microsatellite sequences, resulting in a library with a low level of redundancy.

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. Crr1aG004, 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 Crr1aG004 alone conferred resistance to isolate Ano-01 in susceptible Arabidopsis and B. rapa. In comparison, the susceptible allele Crr1aA9709 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).

Genome-Wide Association Studies Using Single Nucleotide Polymorphism Markers Developed by Re-Sequencing of the Genomes of Cultivated Tomato

With the aim of understanding relationship between genetic and phenotypic variations in cultivated tomato, single nucleotide polymorphism (SNP) markers covering the whole genome of cultivated tomato were developed and genome-wide association studies (GWAS) were performed. The whole genomes of six tomato lines were sequenced with the ABI-5500xl SOLiD sequencer. Sequence reads covering ∼13.7× of the genome for each line were obtained, and mapped onto tomato reference genomes (SL2.40) to detect ∼1.5 million SNP candidates. Of the identified SNPs, 1.5% were considered to confer gene functions. In the subsequent Illumina GoldenGate assay for 1536 SNPs, 1293 SNPs were successfully genotyped, and 1248 showed polymorphisms among 663 tomato accessions. The whole-genome linkage disequilibrium (LD) analysis detected highly biased LD decays between euchromatic (58 kb) and heterochromatic regions (13.8 Mb). Subsequent GWAS identified SNPs that were significantly associated with agronomical traits, with SNP loci located near genes that were previously reported as candidates for these traits. This study demonstrates that attractive loci can be identified by performing GWAS with a large number of SNPs obtained from re-sequencing analysis.

Construction of a high-density reference linkage map of tea (Camellia sinensis)

A few linkage maps of tea have been constructed using pseudo-testcross theory based on dominant marker systems. However, dominant markers are not suitable as landmark markers across a wide range of materials. Therefore, we developed co-dominant SSR markers from genomic DNA and ESTs and constructed a reference map using these co-dominant markers as landmarks. A population of 54 F1 clones derived from reciprocal crosses between ‘Sayamakaori’ and ‘Kana-Ck17’ was used for the linkage analysis. Maps of both parents were constructed from the F1 population that was taken for BC1 population. The order of most of the dominant markers in the parental maps was consistent. We constructed a core map by merging the linkage data for markers that detected polymorphisms in both parents. The core map contains 15 linkage groups, which corresponds to the basic chromosome number of tea. The total length of the core map is 1218 cM. Here, we present the reference map as a central core map sandwiched between the parental maps for each linkage group; the combined maps contain 441 SSRs, 7 CAPS, 2 STS and 674 RAPDs. This newly constructed linkage map can be used as a basic reference linkage map of tea.