Genomic variation across distribution of Micro-Tom, a model cultivar of tomato (Solanum lycopersicum).

Micro-Tom is a cultivar of tomato (Solanum lycopersicum), which is known as a major crop and model plant in Solanaceae. Micro-Tom has phenotypic traits such as dwarfism, and substantial EMS-mutagenized lines have been reported. After Micro-Tom was generated in Florida, USA, it was distributed to research institutes worldwide and used as a genetic resource. In Japan, the Micro-Tom lines have been genetically fixed; currently three lines have been re-distributed from three institutes, but many phenotypes among the lines have been observed. We have determined the genome sequence de novo of the Micro-Tom KDRI line, one of the Micro-Tom lines distributed from Kazusa DNA Research Institute (KDRI) in Japan, and have built chromosome-scale pseudomolecules. Genotypes among six Micro-Tom lines, including three in Japan, one in the United States, one in France, and one in Brazil showed phenotypic alternation. Here, we unveiled the swift emergence of genetic diversity in both phenotypes and genotypes within the Micro-Tom genome sequence during its propagation. These findings offer valuable insights crucial for the management of bioresources.

Development of 12 sets of chromosome segment substitution lines that enhance allele mining in Asian cultivated rice.

Many agronomic traits that are important in rice breeding are controlled by multiple genes. The extensive time and effort devoted so far to identifying and selecting such genes are still not enough to target multiple agronomic traits in practical breeding in Japan because of a lack of suitable plant materials in which to efficiently detect and validate beneficial alleles from diverse genetic resources. To facilitate the comprehensive analysis of genetic variation in agronomic traits among Asian cultivated rice, we developed 12 sets of chromosome segment substitution lines (CSSLs) with the japonica background, 11 of them in the same genetic background, using donors representing the genetic diversity of Asian cultivated rice. Using these materials, we overviewed the chromosomal locations of 1079 putative QTLs for seven agronomic traits and their allelic distribution in Asian cultivated rice through multiple linear regression analysis. The CSSLs will allow the effects of putative QTLs in the highly homogeneous japonica background to be validated.

An improved reference genome for Trifolium subterraneum L. provides insight into molecular diversity and intra-specific phylogeny.

Subterranean clover (Trifolium subterraneum L., Ts) is a geocarpic, self-fertile annual forage legume with a compact diploid genome (n = x = 8, 544 Mb/1C). Its resilience and climate adaptivity have made it an economically important species in Mediterranean and temperate zones. Using the cultivar Daliak, we generated higher resolution sequence data, created a new genome assembly TSUd_3.0, and conducted molecular diversity analysis for copy number variant (CNV) and single-nucleotide polymorphism (SNP) among 36 cultivars. TSUd_3.0 substantively improves prior genome assemblies with new Hi-C and long-read sequence data, covering 531 Mb, containing 41,979 annotated genes and generating a 94.4% BUSCO score. Comparative genomic analysis among select members of the tribe Trifolieae indicated TSUd 3.0 corrects six assembly-error inversion/duplications and confirmed phylogenetic relationships. Its synteny with T. pratense, T. repens, Medicago truncatula and Lotus japonicus genomes were assessed, with the more distantly related T. repens and M. truncatula showing higher levels of co-linearity with Ts than between Ts and its close relative T. pratense. Resequencing of 36 cultivars discovered 7,789,537 SNPs subsequently used for genomic diversity assessment and sequence-based clustering. Heterozygosity estimates ranged from 1% to 21% within the 36 cultivars and may be influenced by admixture. Phylogenetic analysis supported subspecific genetic structure, although it indicates four or five groups, rather than the three recognized subspecies. Furthermore, there were incidences where cultivars characterized as belonging to a particular subspecies clustered with another subspecies when using genomic data. These outcomes suggest that further investigation of Ts sub-specific classification using molecular and morpho-physiological data is needed to clarify these relationships. This upgraded reference genome, complemented with comprehensive sequence diversity analysis of 36 cultivars, provides a platform for future gene functional analysis of key traits, and genome-based breeding strategies for climate adaptation and agronomic performance. Pangenome analysis, more in-depth intra-specific phylogenomic analysis using the Ts core collection, and functional genetic and genomic studies are needed to further augment knowledge of Trifolium genomes.

MORE PANICLES 3, a natural allele of OsTB1/FC1, impacts rice yield in paddy fields at elevated CO2 levels.

Improving crop yield potential through an enhanced response to rising atmospheric CO2 levels is an effective strategy for sustainable crop production in the face of climate change. Large-sized panicles (containing many spikelets per panicle) have been a recent ideal plant architecture (IPA) for high-yield rice breeding. However, few breeding programs have proposed an IPA under the projected climate change. Here, we demonstrate through the cloning of the rice (Oryza sativa) quantitative trait locus for MORE PANICLES 3 (MP3) that the improvement in panicle number increases grain yield at elevated atmospheric CO2 levels. MP3 is a natural allele of OsTB1/FC1, previously reported as a negative regulator of tiller bud outgrowth. The temperate japonica allele advanced the developmental process in axillary buds, moderately promoted tillering, and increased the panicle number without negative effects on the panicle size or culm thickness in a high-yielding indica cultivar with large-sized panicles. The MP3 allele, containing three exonic polymorphisms, was observed in most accessions in the temperate japonica subgroups but was rarely observed in the indica subgroup. No selective sweep at MP3 in either the temperate japonica or indica subgroups suggested that MP3 has not been involved and utilized in artificial selection during domestication or breeding. A free-air CO2 enrichment experiment revealed a clear increase of grain yield associated with the temperate japonica allele at elevated atmospheric CO2 levels. Our findings show that the moderately increased panicle number combined with large-sized panicles using MP3 could be a novel IPA and contribute to an increase in rice production under climate change with rising atmospheric CO2 levels.

Development of RIKEN Plant Metabolome MetaDatabase.

The advancement of metabolomics in terms of techniques for measuring small molecules has enabled the rapid detection and quantification of numerous cellular metabolites. Metabolomic data provide new opportunities to gain a deeper understanding of plant metabolism that can improve the health of both plants and humans that consume them. Although major public repositories for general metabolomic data have been established, the community still has shortcomings related to data sharing, especially in terms of data reanalysis, reusability and reproducibility. To address these issues, we developed the RIKEN Plant Metabolome MetaDatabase (RIKEN PMM, http://metabobank.riken.jp/pmm/db/plantMetabolomics), which stores mass spectrometry-based (e.g. gas chromatography-MS-based) metabolite profiling data of plants together with their detailed, structured experimental metadata, including sampling and experimental procedures. Our metadata are described as Linked Open Data based on the Resource Description Framework using standardized and controlled vocabularies, such as the Metabolomics Standards Initiative Ontology, which are to be integrated with various life and biomedical science data using the World Wide Web. RIKEN PMM implements intuitive and interactive operations for plant metabolome data, including raw data (netCDF format), mass spectra (NIST MSP format) and metabolite annotations. The feature is suitable not only for biologists who are interested in metabolomic phenotypes, but also for researchers who would like to investigate life science in general through plant metabolomic approaches.

Chromosome-level genome assembly of Ophiorrhiza pumila reveals the evolution of camptothecin biosynthesis.

Plant genomes remain highly fragmented and are often characterized by hundreds to thousands of assembly gaps. Here, we report chromosome-level reference and phased genome assembly of Ophiorrhiza pumila, a camptothecin-producing medicinal plant, through an ordered multi-scaffolding and experimental validation approach. With 21 assembly gaps and a contig N50 of 18.49 Mb, Ophiorrhiza genome is one of the most complete plant genomes assembled to date. We also report 273 nitrogen-containing metabolites, including diverse monoterpene indole alkaloids (MIAs). A comparative genomics approach identifies strictosidine biogenesis as the origin of MIA evolution. The emergence of strictosidine biosynthesis-catalyzing enzymes precede downstream enzymes' evolution post γ whole-genome triplication, which occurred approximately 110 Mya in O. pumila, and before the whole-genome duplication in Camptotheca acuminata identified here. Combining comparative genome analysis, multi-omics analysis, and metabolic gene-cluster analysis, we propose a working model for MIA evolution, and a pangenome for MIA biosynthesis, which will help in establishing a sustainable supply of camptothecin.

Whole-genome sequence diversity and association analysis of 198 soybean accessions in mini-core collections.

We performed whole-genome Illumina resequencing of 198 accessions to examine the genetic diversity and facilitate the use of soybean genetic resources and identified 10 million single nucleotide polymorphisms and 2.8 million small indels. Furthermore, PacBio resequencing of 10 accessions was performed, and a total of 2,033 structure variants were identified. Genetic diversity and structure analysis congregated the 198 accessions into three subgroups (Primitive, World, and Japan) and showed the possibility of a long and relatively isolated history of cultivated soybean in Japan. Additionally, the skewed regional distribution of variants in the genome, such as higher structural variations on the R gene clusters in the Japan group, suggested the possibility of selective sweeps during domestication or breeding. A genome-wide association study identified both known and novel causal variants on the genes controlling the flowering period. Novel candidate causal variants were also found on genes related to the seed coat colour by aligning together with Illumina and PacBio reads. The genomic sequences and variants obtained in this study have immense potential to provide information for soybean breeding and genetic studies that may uncover novel alleles or genes involved in agronomically important traits.

A chromosome-scale draft genome sequence of horsegram (Macrotyloma uniflorum).

Horsegram (Macrotyloma uniflorum [Lam.] Verdc.) is an underutilized warm-season diploid legume (2n = 20, 22). Because of its ability to grow under water-deficient and marginal soil conditions, horsegram is a preferred choice in the era of global climate change. In recognition of its potential as a crop species, we generated and analyzed a draft genome sequence for a horsegram variety, HPK-4. Ten chromosome-scale pseudomolecules were created by aligning Illumina scaffold sequences onto a linkage map. The total length of the ten pseudomolecules was 259.2 Mbp, covering 89% of the total length of the assembled sequences. A total of 36,105 genes were predicted on the assembled sequences. Diversity analysis of 89 horsegram accessions by dd-RAD-Seq identified 277 single nucleotide polymorphisms (SNPs), suggesting narrow genetic diversity among the horsegram accessions. This is the first attempt to generate a draft genome sequence of horsegram and will provide a reference for sequence-based analysis of horsegram germplasm.

Correction: The persimmon genome reveals clues to the evolution of a lineage-specific sex determination system in plants.

[This corrects the article DOI: 10.1371/journal.pgen.1008566.].

The persimmon genome reveals clues to the evolution of a lineage-specific sex determination system in plants.

Most angiosperms bear hermaphroditic flowers, but a few species have evolved outcrossing strategies, such as dioecy, the presence of separate male and female individuals. We previously investigated the mechanisms underlying dioecy in diploid persimmon (D. lotus) and found that male flowers are specified by repression of the autosomal gene MeGI by its paralog, the Y-encoded pseudo-gene OGI. This mechanism is thought to be lineage-specific, but its evolutionary path remains unknown. Here, we developed a full draft of the diploid persimmon genome (D. lotus), which revealed a lineage-specific whole-genome duplication event and provided information on the architecture of the Y chromosome. We also identified three paralogs, MeGI, OGI and newly identified Sister of MeGI (SiMeGI). Evolutionary analysis suggested that MeGI underwent adaptive evolution after the whole-genome duplication event. Transformation of tobacco plants with MeGI and SiMeGI revealed that MeGI specifically acquired a new function as a repressor of male organ development, while SiMeGI presumably maintained the original function. Later, a segmental duplication event spawned MeGI's regulator OGI on the Y-chromosome, completing the path leading to dioecy, and probably initiating the formation of the Y-chromosome. These findings exemplify how duplication events can provide flexible genetic material available to help respond to varying environments and provide interesting parallels for our understanding of the mechanisms underlying the transition into dieocy in plants.

Phased genome sequence of an interspecific hybrid flowering cherry, 'Somei-Yoshino' (Cerasus × yedoensis).

We report the phased genome sequence of an interspecific hybrid, the flowering cherry 'Somei-Yoshino' (Cerasus × yedoensis). The sequence data were obtained by single-molecule real-time sequencing technology, split into two subsets based on genome information of the two probable ancestors, and assembled to obtain two haplotype phased genome sequences of the interspecific hybrid. The resultant genome assembly consisting of the two haplotype sequences spanned 690.1 Mb with 4,552 contigs and an N50 length of 1.0 Mb. We predicted 95,076 high-confidence genes, including 94.9% of the core eukaryotic genes. Based on a high-density genetic map, we established a pair of eight pseudomolecule sequences, with highly conserved structures between the two haplotype sequences with 2.4 million sequence variants. A whole genome resequencing analysis of flowering cherries suggested that 'Somei-Yoshino' might be derived from a cross between C. spachiana and either C. speciosa or its relatives. A time-course transcriptome analysis of floral buds and flowers suggested comprehensive changes in gene expression in floral bud development towards flowering. These genome and transcriptome data are expected to provide insights into the evolution and cultivation of flowering cherry and the molecular mechanism underlying flowering.

Draft Sequencing of the Heterozygous Diploid Genome of Satsuma (Citrus unshiu Marc.) Using a Hybrid Assembly Approach.

Satsuma (Citrus unshiu Marc.) is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma ("Miyagawa Wase") was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome.

DNApod: DNA polymorphism annotation database from next-generation sequence read archives.

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information.

Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle Genomes.

Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy-Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies.

DNA data bank of Japan (DDBJ) progress report.

The DNA Data Bank of Japan Center (DDBJ Center; http://www.ddbj.nig.ac.jp) maintains and provides public archival, retrieval and analytical services for biological information. The contents of the DDBJ databases are shared with the US National Center for Biotechnology Information (NCBI) and the European Bioinformatics Institute (EBI) within the framework of the International Nucleotide Sequence Database Collaboration (INSDC). Since 2013, the DDBJ Center has been operating the Japanese Genotype-phenotype Archive (JGA) in collaboration with the National Bioscience Database Center (NBDC) in Japan. In addition, the DDBJ Center develops semantic web technologies for data integration and sharing in collaboration with the Database Center for Life Science (DBCLS) in Japan. This paper briefly reports on the activities of the DDBJ Center over the past year including submissions to databases and improvements in our services for data retrieval, analysis, and integration.

The Naming of Names: Guidelines for Gene Nomenclature in Marchantia.

While Marchantia polymorpha has been utilized as a model system to investigate fundamental biological questions for over almost two centuries, there is renewed interest in M. polymorpha as a model genetic organism in the genomics era. Here we outline community guidelines for M. polymorpha gene and transgene nomenclature, and we anticipate that these guidelines will promote consistency and reduce both redundancy and confusion in the scientific literature.

Genetic architecture of variation in heading date among Asian rice accessions.

BACKGROUND: Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions. RESULTS: To genetically dissect naturally occurring variation in rice heading date, we collected QTLs in advanced-backcross populations derived from multiple crosses of the japonica rice accession Koshihikari (as a common parental line) with 11 diverse rice accessions (5 indica, 3 aus, and 3 japonica) that originate from various regions of Asia. QTL analyses of over 14,000 backcrossed individuals revealed 255 QTLs distributed widely across the rice genome. Among the detected QTLs, 128 QTLs corresponded to genomic positions of heading date genes identified by previous studies, such as Hd1, Hd6, Hd3a, Ghd7, DTH8, and RFT1. The other 127 QTLs were detected in different chromosomal regions than heading date genes. CONCLUSIONS: Our results indicate that advanced-backcross progeny allowed us to detect and confirm QTLs with relatively small additive effects, and the natural variation in rice heading date could result from combinations of large- and small-effect QTLs. We also found differences in the genetic architecture of heading date (flowering time) among maize, Arabidopsis, and rice.

A Bead-based Normalization for Uniform Sequencing depth (BeNUS) protocol for multi-samples sequencing exemplified by HLA-B.

BACKGROUND: Human leukocyte antigen (HLA) is a group of genes that are extremely polymorphic among individuals and populations and have been associated with more than 100 different diseases and adverse drug effects. HLA typing is accordingly an important tool in clinical application, medical research, and population genetics. We have previously developed a phase-defined HLA gene sequencing method using MiSeq sequencing. RESULTS: Here we report a simple, high-throughput, and cost-effective sequencing method that includes normalized library preparation and adjustment of DNA molar concentration. We applied long-range PCR to amplify HLA-B for 96 samples followed by transposase-based library construction and multiplex sequencing with the MiSeq sequencer. After sequencing, we observed low variation in read percentages (0.2% to 1.55%) among the 96 demultiplexed samples. On this basis, all the samples were amenable to haplotype phasing using our phase-defined sequencing method. In our study, a sequencing depth of 800x was necessary and sufficient to achieve full phasing of HLA-B alleles with reliable assignment of the allelic sequence to the 8 digit level. CONCLUSIONS: Our HLA sequencing method optimized for 96 multiplexing samples is highly time effective and cost effective and is especially suitable for automated multi-sample library preparation and sequencing.

Genome-wide analysis of intraspecific DNA polymorphism in 'Micro-Tom', a model cultivar of tomato (Solanum lycopersicum).

Tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. The genome sequencing of the tomato cultivar 'Heinz 1706' was recently completed. To accelerate the progress of tomato genomics studies, systematic bioresources, such as mutagenized lines and full-length cDNA libraries, have been established for the cultivar 'Micro-Tom'. However, these resources cannot be utilized to their full potential without the completion of the genome sequencing of 'Micro-Tom'. We undertook the genome sequencing of 'Micro-Tom' and here report the identification of single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) between 'Micro-Tom' and 'Heinz 1706'. The analysis demonstrated the presence of 1.23 million SNPs and 0.19 million indels between the two cultivars. The density of SNPs and indels was high in chromosomes 2, 5 and 11, but was low in chromosomes 6, 8 and 10. Three known mutations of 'Micro-Tom' were localized on chromosomal regions where the density of SNPs and indels was low, which was consistent with the fact that these mutations were relatively new and introgressed into 'Micro-Tom' during the breeding of this cultivar. We also report SNP analysis for two 'Micro-Tom' varieties that have been maintained independently in Japan and France, both of which have served as standard lines for 'Micro-Tom' mutant collections. Approximately 28,000 SNPs were identified between these two 'Micro-Tom' lines. These results provide high-resolution DNA polymorphic information on 'Micro-Tom' and represent a valuable contribution to the 'Micro-Tom'-based genomics resources.

DDBJ read annotation pipeline: a cloud computing-based pipeline for high-throughput analysis of next-generation sequencing data.

High-performance next-generation sequencing (NGS) technologies are advancing genomics and molecular biological research. However, the immense amount of sequence data requires computational skills and suitable hardware resources that are a challenge to molecular biologists. The DNA Data Bank of Japan (DDBJ) of the National Institute of Genetics (NIG) has initiated a cloud computing-based analytical pipeline, the DDBJ Read Annotation Pipeline (DDBJ Pipeline), for a high-throughput annotation of NGS reads. The DDBJ Pipeline offers a user-friendly graphical web interface and processes massive NGS datasets using decentralized processing by NIG supercomputers currently free of charge. The proposed pipeline consists of two analysis components: basic analysis for reference genome mapping and de novo assembly and subsequent high-level analysis of structural and functional annotations. Users may smoothly switch between the two components in the pipeline, facilitating web-based operations on a supercomputer for high-throughput data analysis. Moreover, public NGS reads of the DDBJ Sequence Read Archive located on the same supercomputer can be imported into the pipeline through the input of only an accession number. This proposed pipeline will facilitate research by utilizing unified analytical workflows applied to the NGS data. The DDBJ Pipeline is accessible at http://p.ddbj.nig.ac.jp/.