Identification of QTLs for root color and carotenoid contents in Japanese orange carrot F2 populations.

Carrot is a major source of provitamin A in a human diet. Two of the most important traits for carrot breeding are carotenoid contents and root color. To examine genomic regions related to these traits and develop DNA markers for carrot breeding, we performed an association analysis based on a general liner model using genome-wide single nucleotide polymorphism (SNPs) in two F2 populations, both derived from crosses of orange root carrots bred in Japan. The analysis revealed 21 significant quantitative trait loci (QTLs). To validate the detection of the QTLs, we also performed a QTL analysis based on a composite interval mapping of these populations and detected 32 QTLs. Eleven of the QTLs were detected by both the association and QTL analyses. The physical position of some QTLs suggested two possible candidate genes, an Orange (Or) gene for visual color evaluation, and the α- and ß-carotene contents and a chromoplast-specific lycopene ß-cyclase (CYC-B) gene for the ß/α carotene ratio. A KASP marker developed on the Or distinguished a quantitative color difference in a different, related breeding line. The detected QTLs and the DNA marker will contribute to carrot breeding and the understanding of carotenoid biosynthesis and accumulation in orange carrots.

Development of PCR-based DNA marker for detection of white carrot contamination caused by Y2 locus.

In carrot (Daucus carota L.), the taproot colors orange, yellow and white are determined mostly by the Y, Y2, and Or loci. One of the most severe issues in carrot seed production is contamination by wild white carrot. To evaluate the contamination ratio, easily detectable DNA markers for white carrot are desired. To develop PCR-based DNA markers for the Y2 locus, we have re-sequenced two orange-colored carrot cultivars at our company (Fujii Seed, Japan), as well as six white- and one light-orange-colored carrots that contaminated our seed products. Within the candidate region previously reported for the Y2 locus, only one DNA marker, Y2_7, clearly distinguished white carrots from orange ones in the re-sequenced samples. The Y2_7 marker was further examined in 12 of the most popular hybrid orange cultivars in Japan, as well as 'Nantes' and 'Chantenay Red Cored 2'. The Y2_7 marker showed that all of the orange cultivars examined had the orange allele except for 'Beta-441'. False white was detected in the orange-colored 'Beta-441'. The Y2_7 marker detected white root carrot contamination in an old open-pollinated Japanese cultivar, 'Nakamura Senkou Futo'. This marker would be a useful tool in a carrot seed quality control for some cultivars.

Regenerative treatment for male stress urinary incontinence by periurethral injection of adipose-derived regenerative cells: Outcome of the ADRESU study.

OBJECTIVES: To report the outcome of the ADRESU study, a multicenter, single-arm, investigator-initiated clinical trial to confirm the efficacy and safety of regenerative treatment for male patients with stress urinary incontinence. METHODS: The participants were male patients with mild-to-moderate stress urinary incontinence persisting for >1 year after prostatectomy. Autologous adipose-derived regenerative cells were isolated using the Celution system from adipose tissue obtained by liposuction. Adipose-derived regenerative cells and mixture of adipose-derived regenerative cells with adipose tissue were transurethrally injected into the rhabdosphincter and submucosal space of the urethra, respectively. The primary end-point was the proportion of patients with improvement of the urine leakage volume at 52 weeks (or last visit within 52 weeks). Improvement of leakage volume was defined as a decrease from baseline >50% by the 24-h pad test. A total of 10 secondary end-points were set. RESULTS: A total of 45 patients satisfying the eligibility criteria were enrolled. The primary end-point was met; the proportion of patients with improvement in leakage volume at 52 weeks was 37.2% (95% confidence interval 23.0-53.3%). No serious adverse events with causal relationships to the adipose-derived regenerative cells were encountered. There was a progressive improvement in secondary end-points. In the King's Health Questionnaire, improvement of quality of life scores showed greater improvement in responders, as compared with non-responders. CONCLUSIONS: Findings from the ADRESU study suggest the efficacy and safety of regenerative treatment for male patients with mild-to-moderate stress urinary incontinence.

Design of a single-arm clinical trial of regenerative therapy by periurethral injection of adipose-derived regenerative cells for male stress urinary incontinence in Japan: the ADRESU study protocol.

BACKGROUND: Male stress urinary incontinence is a prevalent condition after radical prostatectomy. While the standard recommendation for the management of urine leakage is pelvic floor muscle training, its efficacy is still unsatisfactory. Therefore, we have focused on regenerative therapy, which consists of administering a periurethral injection of autologous regenerative cells from adipose tissue, separated using the Celution® system. Based on an interim data analysis of our exploratory study, we confirmed the efficacy and acceptable safety profile of this treatment. Accordingly, we began discussions with Japanese regulatory authorities regarding the development of this therapy in Japan. The Ministry of Health, Labour and Welfare suggested that we implement a clinical trial of a new medical device based on the Pharmaceutical Affaires Act in Japan. Next, we discussed the design of this investigator-initiated clinical trial (the ADRESU study) aimed at evaluating the efficacy and safety of this therapy, in a consultation meeting with the Pharmaceuticals and Medical Device Agency. METHODS: The ADRESU study is an open-label, multi-center, single-arm study involving a total of 45 male stress urinary incontinence patients with mild-to-moderate urine leakage persisting more than 1 year after prostatectomy, in spite of behavioral and pharmacological therapies. The primary endpoint is the rate of patients at 52 weeks with improvement of urine leakage volume defined as a reduction from baseline greater than 50% by 24-h pad test. Our specific hypothesis is that the primary endpoint result will be higher than a pre-specified threshold of 10%. DISCUSSION: The ADRESU study is the first clinical trial of regenerative treatment for stress urinary incontinence by adipose-derived regenerative cells using the Celution® system based on the Japanese Pharmaceutical Affaires Act. We will evaluate the efficacy and safety in this trial to provide an adequate basis for marketing approval with the final objective of making this novel therapy widely available for Japanese patients. TRIAL REGISTRATION: This trial was registered at the University Hospital Medical information Network Clinical Trial Registry (UMIN-CTR Unique ID: UMIN000017901 ; Registered July 1, 2015) and at ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT02529865 ; Registered August 18, 2015).

Sequencing and comparative analyses of the genomes of zoysiagrasses.

Zoysiais a warm-season turfgrass, which comprises 11 allotetraploid species (2n= 4x= 40), each possessing different morphological and physiological traits. To characterize the genetic systems of Zoysia plants and to analyse their structural and functional differences in individual species and accessions, we sequenced the genomes of Zoysia species using HiSeq and MiSeq platforms. As a reference sequence of Zoysia species, we generated a high-quality draft sequence of the genome of Z. japonica accession 'Nagirizaki' (334 Mb) in which 59,271 protein-coding genes were predicted. In parallel, draft genome sequences of Z. matrella 'Wakaba' and Z. pacifica 'Zanpa' were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions, Z. japonica'Kyoto', Z. japonica'Miyagi' and Z. matrella'Chiba Fair Green', were accumulated, and aligned against the reference genome of 'Nagirizaki' along with those from 'Wakaba' and 'Zanpa'. As a result, we detected 7,424,163 single-nucleotide polymorphisms and 852,488 short indels among these species. The information obtained in this study will be valuable for basic studies on zoysiagrass evolution and genetics as well as for the breeding of zoysiagrasses, and is made available in the 'Zoysia Genome Database' at http://zoysia.kazusa.or.jp.

Survey of genome sequences in a wild sweet potato, Ipomoea trifida (H. B. K.) G. Don.

Ipomoea trifida (H. B. K.) G. Don. is the most likely diploid ancestor of the hexaploid sweet potato, I. batatas (L.) Lam. To assist in analysis of the sweet potato genome, de novo whole-genome sequencing was performed with two lines of I. trifida, namely the selfed line Mx23Hm and the highly heterozygous line 0431-1, using the Illumina HiSeq platform. We classified the sequences thus obtained as either 'core candidates' (common to the two lines) or 'line specific'. The total lengths of the assembled sequences of Mx23Hm (ITR_r1.0) was 513 Mb, while that of 0431-1 (ITRk_r1.0) was 712 Mb. Of the assembled sequences, 240 Mb (Mx23Hm) and 353 Mb (0431-1) were classified into core candidate sequences. A total of 62,407 (62.4 Mb) and 109,449 (87.2 Mb) putative genes were identified, respectively, in the genomes of Mx23Hm and 0431-1, of which 11,823 were derived from core sequences of Mx23Hm, while 28,831 were from the core candidate sequence of 0431-1. There were a total of 1,464,173 single-nucleotide polymorphisms and 16,682 copy number variations (CNVs) in the two assembled genomic sequences (under the condition of log2 ratio of >1 and CNV size >1,000 bases). The results presented here are expected to contribute to the progress of genomic and genetic studies of I. trifida, as well as studies of the sweet potato and the genus Ipomoea in general.

Development of EST-SSR markers and construction of a linkage map in faba bean (Vicia faba).

To develop a high density linkage map in faba bean, a total of 1,363 FBES (Faba bean expressed sequence tag [EST]-derived simple sequence repeat [SSR]) markers were designed based on 5,090 non-redundant ESTs developed in this study. A total of 109 plants of a 'Nubaria 2' × 'Misr 3' F2 mapping population were used for map construction. Because the parents were not pure homozygous lines, the 109 F2 plants were divided into three subpopulations according to the original F1 plants. Linkage groups (LGs) generated in each subpopulation were integrated by commonly mapped markers. The integrated 'Nubaria 2' × 'Misr 3' map consisted of six LGs, representing a total length of 684.7 cM, with 552 loci. Of the mapped loci, 47% were generated from multi-loci diagnostic (MLD) markers. Alignment of homologous sequence pairs along each linkage group revealed obvious syntenic relationships between LGs in faba bean and the genomes of two model legumes, Lotus japonicus and Medicago truncatula. In a polymorphic analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES markers showed polymorphisms. Along with the EST-SSR markers, the dense map developed in this study is expected to accelerate marker assisted breeding in faba bean.

Dissection of the octoploid strawberry genome by deep sequencing of the genomes of Fragaria species.

Cultivated strawberry (Fragaria x ananassa) is octoploid and shows allogamous behaviour. The present study aims at dissecting this octoploid genome through comparison with its wild relatives, F. iinumae, F. nipponica, F. nubicola, and F. orientalis by de novo whole-genome sequencing on an Illumina and Roche 454 platforms. The total length of the assembled Illumina genome sequences obtained was 698 Mb for F. x ananassa, and ∼200 Mb each for the four wild species. Subsequently, a virtual reference genome termed FANhybrid_r1.2 was constructed by integrating the sequences of the four homoeologous subgenomes of F. x ananassa, from which heterozygous regions in the Roche 454 and Illumina genome sequences were eliminated. The total length of FANhybrid_r1.2 thus created was 173.2 Mb with the N50 length of 5137 bp. The Illumina-assembled genome sequences of F. x ananassa and the four wild species were then mapped onto the reference genome, along with the previously published F. vesca genome sequence to establish the subgenomic structure of F. x ananassa. The strategy adopted in this study has turned out to be successful in dissecting the genome of octoploid F. x ananassa and appears promising when applied to the analysis of other polyploid plant species.

Sequence analysis of the genome of carnation (Dianthus caryophyllus L.).

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. 'Francesco' was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568,887,315 bp, consisting of 45,088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16,644 bp and 60,737 bp, respectively, and the longest scaffold was 1,287,144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼ 98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp.

Construction of an integrated high density simple sequence repeat linkage map in cultivated strawberry (Fragaria × ananassa) and its applicability.

The cultivated strawberry (Fragaria × ananassa) is an octoploid (2n = 8x = 56) of the Rosaceae family whose genomic architecture is still controversial. Several recent studies support the AAA'A'BBB'B' model, but its complexity has hindered genetic and genomic analysis of this important crop. To overcome this difficulty and to assist genome-wide analysis of F. × ananassa, we constructed an integrated linkage map by organizing a total of 4474 of simple sequence repeat (SSR) markers collected from published Fragaria sequences, including 3746 SSR markers [Fragaria vesca expressed sequence tag (EST)-derived SSR markers] derived from F. vesca ESTs, 603 markers (F. × ananassa EST-derived SSR markers) from F. × ananassa ESTs, and 125 markers (F. × ananassa transcriptome-derived SSR markers) from F. × ananassa transcripts. Along with the previously published SSR markers, these markers were mapped onto five parent-specific linkage maps derived from three mapping populations, which were then assembled into an integrated linkage map. The constructed map consists of 1856 loci in 28 linkage groups (LGs) that total 2364.1 cM in length. Macrosynteny at the chromosome level was observed between the LGs of F. × ananassa and the genome of F. vesca. Variety distinction on 129 F. × ananassa lines was demonstrated using 45 selected SSR markers.

Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1.

We established a gene tagging population of the model legume Lotus japonicus using an endogenous long terminal repeat (LTR) retrotransposon Lotus Retrotransposon 1 (LORE1). The population was composed of 2450 plant lines, from which a total of 4532 flanking sequence tags of LORE1 were recovered by pyrosequencing. The two-dimensional arrangement of the plant population, together with the use of multiple identifier sequences in the primers used to amplify the flanking regions, made it possible to trace insertions back to the original plant lines. The large-scale detection of new LORE1 insertion sites revealed a preference for genic regions, especially in exons of protein-coding genes, which is an interesting feature to consider in the interaction between host genomes and chromoviruses, to which LORE1 belongs, a class of retrotransposon widely distributed among plants. Forward screening of the symbiotic mutants from the population succeeded to identify five symbiotic mutants of known genes. These data suggest that LORE1 is robust as a genetic tool.

Sequence analysis of the genome of an oil-bearing tree, Jatropha curcas L.

The whole genome of Jatropha curcas was sequenced, using a combination of the conventional Sanger method and new-generation multiplex sequencing methods. Total length of the non-redundant sequences thus obtained was 285 858 490 bp consisting of 120 586 contigs and 29 831 singlets. They accounted for ~95% of the gene-containing regions with the average G + C content was 34.3%. A total of 40 929 complete and partial structures of protein encoding genes have been deduced. Comparison with genes of other plant species indicated that 1529 (4%) of the putative protein-encoding genes are specific to the Euphorbiaceae family. A high degree of microsynteny was observed with the genome of castor bean and, to a lesser extent, with those of soybean and Arabidopsis thaliana. In parallel with genome sequencing, cDNAs derived from leaf and callus tissues were subjected to pyrosequencing, and a total of 21 225 unigene data have been generated. Polymorphism analysis using microsatellite markers developed from the genomic sequence data obtained was performed with 12 J. curcas lines collected from various parts of the world to estimate their genetic diversity. The genomic sequence and accompanying information presented here are expected to serve as valuable resources for the acceleration of fundamental and applied research with J. curcas, especially in the fields of environment-related research such as biofuel production. Further information on the genomic sequences and DNA markers is available at http://www.kazusa.or.jp/jatropha/.

Complete genomic structure of the cultivated rice endophyte Azospirillum sp. B510.

We determined the nucleotide sequence of the entire genome of a diazotrophic endophyte, Azospirillum sp. B510. Strain B510 is an endophytic bacterium isolated from stems of rice plants (Oryza sativa cv. Nipponbare). The genome of B510 consisted of a single chromosome (3,311,395 bp) and six plasmids, designated as pAB510a (1,455,109 bp), pAB510b (723,779 bp), pAB510c (681,723 bp), pAB510d (628,837 bp), pAB510e (537,299 bp), and pAB510f (261,596 bp). The chromosome bears 2893 potential protein-encoding genes, two sets of rRNA gene clusters (rrns), and 45 tRNA genes representing 37 tRNA species. The genomes of the six plasmids contained a total of 3416 protein-encoding genes, seven sets of rrns, and 34 tRNAs representing 19 tRNA species. Eight genes for plasmid-specific tRNA species are located on either pAB510a or pAB510d. Two out of eight genomic islands are inserted in the plasmids, pAB510b and pAB510e, and one of the islands is inserted into trnfM-CAU in the rrn located on pAB510e. Genes other than the nif gene cluster that are involved in N(2) fixation and are homologues of Bradyrhizobium japonicum USDA110 include fixABCX, fixNOQP, fixHIS, fixG, and fixLJK. Three putative plant hormone-related genes encoding tryptophan 2-monooxytenase (iaaM) and indole-3-acetaldehyde hydrolase (iaaH), which are involved in IAA biosynthesis, and ACC deaminase (acdS), which reduces ethylene levels, were identified. Multiple gene-clusters for tripartite ATP-independent periplasmic-transport systems and a diverse set of malic enzymes were identified, suggesting that B510 utilizes C(4)-dicarboxylate during its symbiotic relationship with the host plant.

Genome structure of the legume, Lotus japonicus.

The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L. japonicus genome. The 315.1-Mb sequences determined in this and previous studies correspond to 67% of the genome (472 Mb), and are likely to cover 91.3% of the gene space. Linkage mapping anchored 130-Mb sequences onto the six linkage groups. A total of 10,951 complete and 19,848 partial structures of protein-encoding genes were assigned to the genome. Comparative analysis of these genes revealed the expansion of several functional domains and gene families that are characteristic of L. japonicus. Synteny analysis detected traces of whole-genome duplication and the presence of synteny blocks with other plant genomes to various degrees. This study provides the first opportunity to look into the complex and unique genetic system of legumes.

Complete genomic structure of the bloom-forming toxic cyanobacterium Microcystis aeruginosa NIES-843.

The nucleotide sequence of the complete genome of a cyanobacterium, Microcystis aeruginosa NIES-843, was determined. The genome of M. aeruginosa is a single, circular chromosome of 5,842,795 base pairs (bp) in length, with an average GC content of 42.3%. The chromosome comprises 6312 putative protein-encoding genes, two sets of rRNA genes, 42 tRNA genes representing 41 tRNA species, and genes for tmRNA, the B subunit of RNase P, SRP RNA, and 6Sa RNA. Forty-five percent of the putative protein-encoding sequences showed sequence similarity to genes of known function, 32% were similar to hypothetical genes, and the remaining 23% had no apparent similarity to reported genes. A total of 688 kb of the genome, equivalent to 11.8% of the entire genome, were composed of both insertion sequences and miniature inverted-repeat transposable elements. This is indicative of a plasticity of the M. aeruginosa genome, through a mechanism that involves homologous recombination mediated by repetitive DNA elements. In addition to known gene clusters related to the synthesis of microcystin and cyanopeptolin, novel gene clusters that may be involved in the synthesis and modification of toxic small polypeptides were identified. Compared with other cyanobacteria, a relatively small number of genes for two component systems and a large number of genes for restriction-modification systems were notable characteristics of the M. aeruginosa genome.

Characterization of the soybean genome using EST-derived microsatellite markers.

We generated a high-density genetic linkage map of soybean using expressed sequence tag (EST)-derived microsatellite markers. A total of 6920 primer pairs (10.9%) were designed to amplify simple sequence repeats (SSRs) from 63,676 publicly available non-redundant soybean ESTs. The polymorphism of two parent plants, the Japanese cultivar 'Misuzudaizu' and the Chinese line 'Moshidou Gong 503', were examined using 10% polyacrylamide gel electrophoresis. Primer pairs showing polymorphism were then used for genotyping 94 recombinant inbred lines (RILs) derived from a cross between the parents. In addition to previously reported markers, 680 EST-derived microsatellite markers were selected and subjected to linkage analysis. As a result, 935 marker loci were mapped successfully onto 20 linkage groups, which totaled 2700.3 cM in length; 693 loci were detected using the 668 EST-derived microsatellite markers developed in this study, the other 242 loci were detected with 105 RFLP markers, 136 genome-derived microsatellite markers, and one phenotypic marker. We examined allelic variation among 23 soybean cultivars/lines and a wild soybean line using 668 mapped EST-derived microsatellite markers (corresponding to 686 marker loci), in order to determine the transferability of the markers among soybean germplasms. A limited degree of macrosynteny was observed at the segmental level between the genomes of soybean and the model legume Lotus japonicus, which suggests that considerable genome shuffling occurred after separation of the species and during establishment of the paleopolyploid soybean genome.