Development of an SSR marker-based genetic linkage map and identification of a QTL associated with flowering time in Eustoma.

Lisianthus (Eustoma grandiflorum) is an important floricultural crop cultivated worldwide. Despite its commercial importance, few DNA markers are available for molecular genetic research. In this study, we constructed a genetic linkage map and to detect quantitative trait loci (QTLs) for important agronomic traits of lisianthus. To develop simple sequence repeat (SSR) markers, we used 454-pyrosequencing technology to obtain genomic shotgun sequences and subsequently identified 8263 putative SSRs. A total of 3990 primer pairs were designed in silico and 1189 unique primer pairs were extracted through a BLAST search. Amplification was successful for more than 1000 primer pairs, and ultimately 278 SSR markers exhibited polymorphism between the two lisianthus accessions evaluated. Based on these markers, a genetic linkage map was constructed using a breeding population derived from crosses between the two accessions, for which flowering time differed (>140 days when grown under 20°C). We detected one QTL associated with flowering time (phenotypic variance, 27%; LOD value, 3.7). The SSR marker located at this QTL may account for variation in flowering time among accessions (i.e., three accessions whose nodes of the first flower were over 30 had late-flowering alleles of this QTL).

An efficient approach for the development of genome-specific markers in allohexaploid wheat (Triticum aestivum L.) and its application in the construction of high-density linkage maps of the D genome.

In common wheat, the development of genotyping platforms has been hampered by the large size of the genome, its highly repetitive elements and its allohexaploid nature. However, recent advances in sequencing technology provide opportunities to resolve these difficulties. Using next-generation sequencing and gene-targeting sequence capture, 12,551 nucleotide polymorphisms were detected in the common wheat varieties 'Hatsumochi' and 'Kitahonami' and were assigned to chromosome arms using International Wheat Genome Sequencing Consortium survey sequences. Because the number of markers for D genome chromosomes in commercially available wheat single nucleotide polymorphism arrays is insufficient, we developed markers using a genome-specific amplicon sequencing strategy. Approximately 80% of the designed primers successfully amplified D genome-specific products, suggesting that by concentrating on a specific subgenome, we were able to design successful markers as efficiently as could be done in a diploid species. The newly developed markers were uniformly distributed across the D genome and greatly extended the total coverage. Polymorphisms were surveyed in six varieties, and 31,542 polymorphic sites and 5,986 potential marker sites were detected in the D genome. The marker development and genotyping strategies are cost effective, robust and flexible and may enhance multi-sample studies in the post-genomic era in wheat.

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (Malus × domestica).

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant 'McIntosh Wijcik', which was discovered as a bud mutation from 'McIntosh', exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in 'McIntosh Wijcik' but not in 'McIntosh'. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in 'McIntosh Wijcik' is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B.

BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

The Glycine max cv. Enrei Genome for Improvement of Japanese Soybean Cultivars.

We elucidated the genome sequence of Glycine max cv. Enrei to provide a reference for characterization of Japanese domestic soybean cultivars. The whole genome sequence obtained using a next-generation sequencer was used for reference mapping into the current genome assembly of G. max cv. Williams 82 obtained by the Soybean Genome Sequencing Consortium in the USA. After sequencing and assembling the whole genome shotgun reads, we obtained a data set with about 928 Mbs total bases and 60,838 gene models. Phylogenetic analysis provided glimpses into the ancestral relationships of both cultivars and their divergence from the complex that include the wild relatives of soybean. The gene models were analyzed in relation to traits associated with anthocyanin and flavonoid biosynthesis and an overall profile of the proteome. The sequence data are made available in DAIZUbase in order to provide a comprehensive informatics resource for comparative genomics of a wide range of soybean cultivars in Japan and a reference tool for improvement of soybean cultivars worldwide.

The radish genome and comprehensive gene expression profile of tuberous root formation and development.

Understanding the processes that regulate plant sink formation and development at the molecular level will contribute to the areas of crop breeding, food production and plant evolutionary studies. We report the annotation and analysis of the draft genome sequence of the radish Raphanus sativus var. hortensis (long and thick root radish) and transcriptome analysis during root development. Based on the hybrid assembly approach of next-generation sequencing, a total of 383 Mb (N50 scaffold: 138.17 kb) of sequences of the radish genome was constructed containing 54,357 genes. Syntenic and phylogenetic analyses indicated that divergence between Raphanus and Brassica coincide with the time of whole genome triplication (WGT), suggesting that WGT triggered diversification of Brassiceae crop plants. Further transcriptome analysis showed that the gene functions and pathways related to carbohydrate metabolism were prominently activated in thickening roots, particularly in cell proliferating tissues. Notably, the expression levels of sucrose synthase 1 (SUS1) were correlated with root thickening rates. We also identified the genes involved in pungency synthesis and their transcription factors.

Construction of pseudomolecule sequences of the aus rice cultivar Kasalath for comparative genomics of Asian cultivated rice.

Having a deep genetic structure evolved during its domestication and adaptation, the Asian cultivated rice (Oryza sativa) displays considerable physiological and morphological variations. Here, we describe deep whole-genome sequencing of the aus rice cultivar Kasalath by using the advanced next-generation sequencing (NGS) technologies to gain a better understanding of the sequence and structural changes among highly differentiated cultivars. The de novo assembled Kasalath sequences represented 91.1% (330.55 Mb) of the genome and contained 35 139 expressed loci annotated by RNA-Seq analysis. We detected 2 787 250 single-nucleotide polymorphisms (SNPs) and 7393 large insertion/deletion (indel) sites (>100 bp) between Kasalath and Nipponbare, and 2 216 251 SNPs and 3780 large indels between Kasalath and 93-11. Extensive comparison of the gene contents among these cultivars revealed similar rates of gene gain and loss. We detected at least 7.39 Mb of inserted sequences and 40.75 Mb of unmapped sequences in the Kasalath genome in comparison with the Nipponbare reference genome. Mapping of the publicly available NGS short reads from 50 rice accessions proved the necessity and the value of using the Kasalath whole-genome sequence as an additional reference to capture the sequence polymorphisms that cannot be discovered by using the Nipponbare sequence alone.

A BAC physical map of aus rice cultivar 'Kasalath', and the map-based genomic sequence of 'Kasalath' chromosome 1.

Comparative analysis using available genomic resources within closely related species is an effective way to investigate genomic sequence and structural diversity. Rice (Oryza sativa L.) has undergone significant physiological and morphological changes during its domestication and local adaptation. We present a complete bacterial artificial chromosome (BAC) physical map for the aus rice cultivar 'Kasalath', which covers 90% of the sequence of temperate japonica rice cultivar 'Nipponbare'. Examination of physical distances between computational and experimental measurements of 'Kasalath' BAC insert size revealed the presence of more than 500 genomic regions that appear to have significant chromosome structural changes between the two cultivars. In particular, a genomic region on the long arm of 'Kasalath' chromosome 11 carrying a disease-resistance gene cluster was greatly expanded relative to the 'Nipponbare' genome. We also decoded 41.37 Mb of high-quality genomic sequence from 'Kasalath' chromosome 1. Extensive comparisons of chromosome 1 between 'Kasalath' and 'Nipponbare' led to the discovery of 317,843 single-nucleotide polymorphisms (SNPs) and 66,331 insertion/deletion (indel) sites. Nearly two-thirds of the expressed genes on rice chromosome 1 carried natural variations involving SNPs and/or indels that resulted in substitutions, insertions or deletions of amino acids in one cultivar relative to the other. We also observed gain and loss of genes caused by large indels. This study provides an important framework and an invaluable dataset for further understanding of the molecular mechanisms underlying the evolution and functions of the rice genome.

KONAGAbase: a genomic and transcriptomic database for the diamondback moth, Plutella xylostella.

BACKGROUND: The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests for crucifer crops worldwide. DBM has rapidly evolved high resistance to most conventional insecticides such as pyrethroids, organophosphates, fipronil, spinosad, Bacillus thuringiensis, and diamides. Therefore, it is important to develop genomic and transcriptomic DBM resources for analysis of genes related to insecticide resistance, both to clarify the mechanism of resistance of DBM and to facilitate the development of insecticides with a novel mode of action for more effective and environmentally less harmful insecticide rotation. To contribute to this goal, we developed KONAGAbase, a genomic and transcriptomic database for DBM (KONAGA is the Japanese word for DBM). DESCRIPTION: KONAGAbase provides (1) transcriptomic sequences of 37,340 ESTs/mRNAs and 147,370 RNA-seq contigs which were clustered and assembled into 84,570 unigenes (30,695 contigs, 50,548 pseudo singletons, and 3,327 singletons); and (2) genomic sequences of 88,530 WGS contigs with 246,244 degenerate contigs and 106,455 singletons from which 6,310 de novo identified repeat sequences and 34,890 predicted gene-coding sequences were extracted. The unigenes and predicted gene-coding sequences were clustered and 32,800 representative sequences were extracted as a comprehensive putative gene set. These sequences were annotated with BLAST descriptions, Gene Ontology (GO) terms, and Pfam descriptions, respectively. KONAGAbase contains rich graphical user interface (GUI)-based web interfaces for easy and efficient searching, browsing, and downloading sequences and annotation data. Five useful search interfaces consisting of BLAST search, keyword search, BLAST result-based search, GO tree-based search, and genome browser are provided. KONAGAbase is publicly available from our website (http://dbm.dna.affrc.go.jp/px/) through standard web browsers. CONCLUSIONS: KONAGAbase provides DBM comprehensive transcriptomic and draft genomic sequences with useful annotation information with easy-to-use web interfaces, which helps researchers to efficiently search for target sequences such as insect resistance-related genes. KONAGAbase will be continuously updated and additional genomic/transcriptomic resources and analysis tools will be provided for further efficient analysis of the mechanism of insecticide resistance and the development of effective insecticides with a novel mode of action for DBM.

DaizuBase, an integrated soybean genome database including BAC-based physical maps.

Soybean [Glycine max (L) Merrill] is one of the most important leguminous crops and ranks fourth after to rice, wheat and maize in terms of world crop production. Soybean contains abundant protein and oil, which makes it a major source of nutritious food, livestock feed and industrial products. In Japan, soybean is also an important source of traditional staples such as tofu, natto, miso and soy sauce. The soybean genome was determined in 2010. With its enormous size, physical mapping and genome sequencing are the most effective approaches towards understanding the structure and function of the soybean genome. We constructed bacterial artificial chromosome (BAC) libraries from the Japanese soybean cultivar, Enrei. The end-sequences of approximately 100,000 BAC clones were analyzed and used for construction of a BAC-based physical map of the genome. BLAST analysis between Enrei BAC-end sequences and the Williams82 genome was carried out to increase the saturation of the map. This physical map will be used to characterize the genome structure of Japanese soybean cultivars, to develop methods for the isolation of agronomically important genes and to facilitate comparative soybean genome research. The current status of physical mapping of the soybean genome and construction of database are presented.

Development of cultivar-specific DNA markers based on retrotransposon-based insertional polymorphism in Japanese pear.

We developed retrotransposon-based insertional polymorphism (RBIP) markers based on the long terminal repeat (LTR) sequences of copia-like retrotransposon Ppcrt4 and flanking genome sequences, which were derived from 454 sequencing data from Japanese pear (Pyrus pyrifolia) 'Hosui'. Out of 40 sequences including both LTR and flanking genome regions, we developed 22 RBIP markers and used them for DNA profiling of 80 pear cultivars: 64 Japanese, 10 Chinese (Pyrus ussuriensis) and 6 European (Pyrus communis). Three RBIP markers were enough to differentiate 'Hosui' from the other Japanese pear cultivars. The 22 RBIP markers could also distinguish 61 of the 64 Japanese pear cultivars. European pears showed almost no amplification of the 22 RBIP markers, which might suggest that retrotransposons had transposed during Asian pear evolution or reflect the genetic relationship between Asian and European pears. Sixteen of the RBIP markers could be positioned on a genetic linkage map of 'Hosui'. The RBIP loci were distributed in 10 linkage groups, and some loci were very closely located within the same linkage group. The information obtained will be applicable to developing cultivar-specific RBIP marker sets in plants.

Comprehensive sequence analysis of 24,783 barley full-length cDNAs derived from 12 clone libraries.

Full-length cDNA (FLcDNA) libraries consisting of 172,000 clones were constructed from a two-row malting barley cultivar (Hordeum vulgare 'Haruna Nijo') under normal and stressed conditions. After sequencing the clones from both ends and clustering the sequences, a total of 24,783 complete sequences were produced. By removing duplicates between these and publicly available sequences, 22,651 representative sequences were obtained: 17,773 were novel barley FLcDNAs, and 1,699 were barley specific. Highly conserved genes were found in the barley FLcDNA sequences for 721 of 881 rice (Oryza sativa) trait genes with 50% or greater identity. These FLcDNA resources from our Haruna Nijo cDNA libraries and the full-length sequences of representative clones will improve our understanding of the biological functions of genes in barley, which is the cereal crop with the fourth highest production in the world, and will provide a powerful tool for annotating the barley genome sequences that will become available in the near future.

Distinct evolutionary patterns of Oryza glaberrima deciphered by genome sequencing and comparative analysis.

Here we present the genomic sequence of the African cultivated rice, Oryza glaberrima, and compare these data with the genome sequence of Asian cultivated rice, Oryza sativa. We obtained gene-enriched sequences of O. glaberrima that correspond to about 25% of the gene regions of the O. sativa (japonica) genome by methylation filtration and subtractive hybridization of repetitive sequences. While patterns of amino acid changes did not differ between the two species in terms of the biochemical properties, genes of O. glaberrima generally showed a larger synonymous-nonsynonymous substitution ratio, suggesting that O. glaberrima has undergone a genome-wide relaxation of purifying selection. We further investigated nucleotide substitutions around splice sites and found that eight genes of O. sativa experienced changes at splice sites after the divergence from O. glaberrima. These changes produced novel introns that partially truncated functional domains, suggesting that these newly emerged introns affect gene function. We also identified 2451 simple sequence repeats (SSRs) from the genomes of O. glaberrima and O. sativa. Although tri-nucleotide repeats were most common among the SSRs and were overrepresented in the protein-coding sequences, we found that selection against indels of tri-nucleotide repeats was relatively weak in both African and Asian rice. Our genome-wide sequencing of O. glaberrima and in-depth analyses provide rice researchers not only with useful genomic resources for future breeding but also with new insights into the genomic evolution of the African and Asian rice species.

Comparative analysis of complete orthologous centromeres from two subspecies of rice reveals rapid variation of centromere organization and structure.

Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. This function is conserved across species, but the DNA components that are involved in kinetochore formation differ greatly, even between closely related species. To shed light on the nature, evolutionary timing and evolutionary dynamics of rice centromeres, we decoded a 2.25-Mb DNA sequence covering the centromeric region of chromosome 8 of an indica rice variety, 'Kasalath' (Kas-Cen8). Analysis of repetitive sequences in Kas-Cen8 led to the identification of 222 long terminal repeat (LTR)-retrotransposon elements and 584 CentO satellite monomers, which account for 59.2% of the region. A comparison of the Kas-Cen8 sequence with that of japonica rice 'Nipponbare' (Nip-Cen8) revealed that about 66.8% of the Kas-Cen8 sequence was collinear with that of Nip-Cen8. Although the 27 putative genes are conserved between the two subspecies, only 55.4% of the total LTR-retrotransposon elements in 'Kasalath' had orthologs in 'Nipponbare', thus reflecting recent proliferation of a considerable number of LTR-retrotransposons since the divergence of two rice subspecies of indica and japonica within Oryza sativa. Comparative analysis of the subfamilies, time of insertion, and organization patterns of inserted LTR-retrotransposons between the two Cen8 regions revealed variations between 'Kasalath' and 'Nipponbare' in the preferential accumulation of CRR elements, and the expansion of CentO satellite repeats within the core domain of Cen8. Together, the results provide insights into the recent proliferation of LTR-retrotransposons, and the rapid expansion of CentO satellite repeats, underlying the dynamic variation and plasticity of plant centromeres.

Gene organization in rice revealed by full-length cDNA mapping and gene expression analysis through microarray.

Rice (Oryza sativa L.) is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA) sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE) genes, 33K annotated non-expressed (ANE) genes, and 5.5K non-annotated expressed (NAE) genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria.