Targeted deletion of grape retrotransposon associated with fruit skin color via CRISPR/Cas9 in Vitis labrascana 'Shine Muscat'.

Transposition of transposable elements affect expression levels, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus. For example, in grape, presence of the Gret1 retrotransposon in the promoter region of the VvMYBA1a allele at the VvMYBA1 locus suppress the expression of the VvMYBA1 transcription factor gene for the anthocyanin biosynthesis and this transposon insertion is responsible for the green berry skin color of Vitis labrascana, 'Shine Muscat', a major grape cultivar in Japan. To prove that transposons in grape genome can be removed by genome editing, we focused on Gret1 in the VvMYBA1a allele as a target of CRISPR/Cas9 mediated transposon removal.

Gene prediction for leaf margin phenotype and fruit flesh color in pineapple (Ananas comosus) using haplotype-resolved genome sequencing.

Pineapple (Ananas comosus (L.) Merr.) is one of the most economically important tropical fruit species.

Tracing founder haplotypes of Japanese apple varieties: application in genomic prediction and genome-wide association study.

Haplotypes provide useful information for genomics-based approaches, genomic prediction, and genome-wide association study. As a small number of superior founders have contributed largely to the breeding history of fruit trees, the information of founder haplotypes may be relevant for performing the genomics-based approaches in these plants. In this study, we proposed a method to estimate 14 haplotypes from 7 founders and automatically trace the haplotypes forward to apple parental (185 varieties) and breeding (659 F individuals from 16 full-sib families) populations based on 11,786 single-nucleotide polymorphisms, by combining multiple algorithms.

Stable and efficient transformation of apple.

Apple is one of precious fruit crop grown in temperate zone. In the post genomic era, the analysis of gene functions in horticultural crops such as apple is required for agricultural utilization. For analysis of such crops, the protocol establishment of tissue culture and transformation is essential.

SSR markers developed using next-generation sequencing technology in pineapple, (L.) Merr.

Simple sequence repeat (SSR) markers provide a reliable tool for the identification of accessions and the construction of genetic linkage maps because of their co-dominant inheritance. In the present study, we developed new SSR markers with next-generation sequencing using the Roche 454 GS FLX+ platform. Five hundred SSR primer sets were tested for the genetic identification of pineapple, including 100 each for the di-, tri-, tetra-, penta-, and hexa-nucleotide motif SSRs.

TRANSNAP: a web database providing comprehensive information on Japanese pear transcriptome.

Japanese pear (Pyrus pyrifolia) is a major fruit tree in the family Rosaceae and is bred for fruit production. To promote the development of breeding strategies and molecular research for Japanese pear, we sequenced the transcripts of Japanese pear variety 'Hosui'. To exhaustively collect information of total gene expression, RNA samples from various organs and stages of Japanese pear were sequenced by three technologies, single-molecule real-time (SMRT) sequencing, 454 pyrosequencing, and Sanger sequencing.

The use of a fertile doubled haploid apple line for QTL analysis of fruit traits.

Apple is an economically important crop, and various approaches to genetic analysis in breeding programs have been attempted, including the production of doubled haploid (DH) lines, which are genetically homozygous. In this study, we used a DH line for QTL analyses, for the first time in a fruit tree, expecting it to simplify the analysis of the inheritance of quantitative traits and thus to enhance QTL detection power. Using an F population from 'Prima' × 'Apple Chukanbohon 95P6' (DH), we constructed a genetic map of 'Prima', and identified 19 QTLs for 13 traits.

CRISPR-Cas9-mediated genome editing in apple and grapevine.

The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR-Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components.

Comparison of the gut microbiota of captive common bottlenose dolphins Tursiops truncatus in three aquaria.

Aims: This study was conducted to assess the presence and extent of differences in the gut microbiota of common bottlenose dolphins depending on rearing facilities.

Methods And Results: Faecal samples were collected from 16 common bottlenose dolphins at three aquaria in Japan. After extracting DNA from the faeces, the V3-V4 region of bacterial 16S rRNA was amplified and sequenced using Illumina MiSeq platform.

Surfactant Protein A Inhibits Growth and Adherence of Uropathogenic To Protect the Bladder from Infection.

Surfactant protein A (SP-A) is a multifunctional host defense collectin that was first identified as a component of pulmonary surfactant. Although SP-A is also expressed in various tissues, including the urinary tract, its innate immune functions in nonpulmonary tissues are poorly understood. In this study, we demonstrated that adherence of uropathogenic (UPEC) to the bladder was enhanced in SP-A-deficient mice, which suggests that SP-A plays an important role in innate immunity against UPEC.

Genomic dissection of a 'Fuji' apple cultivar: re-sequencing, SNP marker development, definition of haplotypes, and QTL detection.

'Fuji' is one of the most popular and highly-produced apple cultivars worldwide, and has been frequently used in breeding programs. The development of genotypic markers for the preferable phenotypes of 'Fuji' is required. Here, we aimed to define the haplotypes of 'Fuji' and find associations between haplotypes and phenotypes of five traits (harvest day, fruit weight, acidity, degree of watercore, and flesh mealiness) by using 115 accessions related to 'Fuji'.

Efficient Genome Editing in Apple Using a CRISPR/Cas9 system.

Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system.

Fine mapping of the gene for susceptibility to black spot disease in Japanese pear (Pyrus pyrifolia Nakai).

Black spot disease, which is caused by the Japanese pear pathotype of the filamentous fungus Alternaria alternata (Fries) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We mapped a gene for susceptibility to black spot disease in the Japanese pear (Pyrus pyrifolia Nakai) cultivar 'Kinchaku' (Aki gene) at the top of linkage group 11, similar to the positions of the susceptibility genes Ani in 'Osa Nijisseiki' and Ana in 'Nansui'. Using synteny-based marker enrichment, we developed novel apple SSR markers in the target region.

Direct introduction of neomycin phosphotransferase II protein into apple leaves to confer kanamycin resistance.

The recent developments of transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) have expanded plant breeding technology. One technical issue related to the current genome editing process is residual transgenes for TALEN and CRISPR/Cas9 left in plant genomes after the editing process. Here, we aim to add transient kanamycin resistance into apple leaf cells by introducing neomycin phosphotransferase II (NPTII) into apple leaf cells using the fusion peptide system.

S-genotype identification based on allele-specific PCR in Japanese pear.

Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear.

Identification of QTLs controlling harvest time and fruit skin color in Japanese pear (Pyrus pyrifolia Nakai).

Using an F1 population from a cross between Japanese pear (Pyrus pyrifolia Nakai) cultivars 'Akiakari' and 'Taihaku', we performed quantitative trait locus (QTL) analysis of seven fruit traits (harvest time, fruit skin color, flesh firmness, fruit weight, acid content, total soluble solids content, and preharvest fruit drop). The constructed simple sequence repeat-based genetic linkage map of 'Akiakari' consisted of 208 loci and spanned 799 cM; that of 'Taihaku' consisted of 275 loci and spanned 1039 cM. Out of significant QTLs, two QTLs for harvest time, one for fruit skin color, and one for flesh firmness were stably detected in two successive years.

Identification of QTLs for fruit quality traits in Japanese apples: QTLs for early ripening are tightly related to preharvest fruit drop.

Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars 'Orin' and 'Akane' using F1 seedlings derived from a cross between these cultivars.

Surfactant protein D suppresses lung cancer progression by downregulation of epidermal growth factor signaling.

Surfactant protein D (SP-D) is a member of the collectin family that has an important role in maintaining pulmonary homeostasis. In this study, we demonstrated that SP-D inhibited the proliferation, migration and invasion of A549 human lung adenocarcinoma cells. We found that SP-D suppressed epidermal growth factor (EGF) signaling in A549 cells, H441 human lung adenocarcinoma cells and human EGF receptor (EGFR) stable expression CHO-K1 cells.

Remarkably delayed occurrence of normal surfactant composition in an extremely preterm infant.

Reported herein is the first case of a remarkably delayed occurrence of normal surfactant composition in an extremely preterm infant who required a total of 15 doses of artificial pulmonary surfactant (Surfacten®). A male infant, born at 26 weeks gestation, developed respiratory distress at birth. Chest radiography was consistent with respiratory distress syndrome.

Characterization of 10 MADS-box genes from Pyrus pyrifolia and their differential expression during fruit development and ripening.

We cloned 10 Japanese pear (Pyrus pyrifolia) MIKC-type II MADS-box genes, and analyzed their expression during fruit development and ripening. PpMADS2-1 was APETALA (AP)1-like; PpMADS3-1 was FRUITFULL (FUL)/SQUAMOSA (SQUA)-like; PpMADS4-1 was AGAMOUS-like (AGL)6; PpMADS5-1 and PpMADS8-1 were SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC)-like; PpMADS9-1, PpMADS12-1, PpMADS14-1 and PpMADS16-1 were SEPALLATA (SEP)-like; while PpMADS15-1 was AGL/SHATTERPROOF (SHP)-like. Phylogenetic analysis showed their grouping into five major clades (and 10 sub-clades) that was consistent with their diverse functional types.

TLR4-MD-2 complex is negatively regulated by an endogenous ligand, globotetraosylceramide.

Although endogenous ligands for Toll-like receptor (TLR)4-myeloid differentiation factor 2 (MD2) have not been well-understood, we here report that a globo-series glycosphingolipid, globotetraosylceramide (Gb4), attenuates the toxicity of lipopolysaccharides (LPSs) by binding to TLR4-MD-2. Because α1,4-galactosyltransferase (A4galt)-deficient mice lacking globo-series glycosphingolipids showed higher sensitivity to LPS than wild-type mice, we examined mechanisms by which globo-series glycosphingolipids attenuate LPS toxicity. Cultured endothelial cells lacking A4galt showed higher expression of LPS-inducible genes upon LPS treatment.

DNA profiling of pineapple cultivars in Japan discriminated by SSR markers.

We developed 18 polymorphic simple sequence repeat (SSR) markers in pineapple (Ananas comosus) by using genomic libraries enriched for GA and CA motifs. The markers were used to genotype 31 pineapple accessions, including seven cultivars and 11 breeding lines from Okinawa Prefecture, 12 foreign accessions and one from a related species. These SSR loci were highly polymorphic: the 31 accessions contained three to seven alleles per locus, with an average of 4.