Assessment of genetic relationships among cultivated and wild Rubus accessions using AFLP markers

The genus Rubus comprises more than 740 species divided into 12 subgenera and contains the raspberries, blackberries, arctic fruits, and flowering raspberries, all of which have been utilized in breeding programs. The objective of this study was to evaluate the phylogenetic relationships among wild and cultivated Rubus species mainly collected in Japan. To evaluate genetic resources in Rubus, 81 accessions were analyzed with three amplified fragment length polymorphism (AFLP) primer pairs and data were analyzed with the neighbor-joining and unweighted pair group methods with arithmetic mean. Two of the generated phylogenetic trees grouped subgenera Anoplobatus, Eubatus, Idaeobatus, and Malachobatus into different clusters. Accessions of Rubus idaeus L. var. aculeatissimus collected from four regions in Hokkaido formed distinct clusters reflecting sampling sites. Four hybrid accessions between raspberry cultivars (R. idaeus L.) and R. idaeus L. var. aculeatissimus, and one hybrid accession between a raspberry cultivar and Rubus spectabilis Pursh were clearly distinguished from parental accessions. These results indicated that AFLP markers are a reliable technique for assessing genetic diversity and studying phylogenetic relationships in Rubus. Data from diversity and phylogenetic studies revealed valuable information on the availability of unique fragments in different accessions that would be useful for the development of improved genotypes through conventional breeding and marker-assisted selection

Abnormal asexual reproduction of thalli of Ecklonia stolonifera (Laminariales, Phaeophyceae) off the coast of Nakanoshima in the Oki Islands, Japan

Multiple abnormal individuals of Ecklonia stolonifera with thalli that developed numerous shoot-like structures on the lateral blades were found in two littoral regions of Nakanoshima in the Oki Islands, Shimane Prefecture, Japan. Except for the presence of these diagnostic structures, no other morphological differences were observed between the abnormal and normal individuals. The development of the shoot-like structures isolated from the lateral blade was the same as that of typical vegetative reproductive sporophytes that formed on stolons. In the abnormal individuals, when cultivation was performed using zoospores, the shoot-like structures were observed on the progeny after 18 months of culture and 20.0% of individuals exhibited these structures

Planned seedling production in the distribution area of Saccharina japonica: Sorus formation through the induction of sporophyte maturation and the culture of the seedlings produced

Inconsistency in maturation of saccharinan kelp due to changes in the marine environment is a problem in terms of securing seedlings for aquaculture. In Hokkaido, the main producing area of Saccharina japonica, it has become a challenge to stably secure the parental algae of the species for aquaculture. When maturation was induced by short-day conditions in naturally collected S. japonica sporophytes as well as in the sporophytes collected from them and cultivated, sorus formation began in approximately 3 weeks and spread to 0.4%-16.8% on the upper side area and 23.2%-41.1% on the underside area after about 5 weeks, with the highest rates, 23.2%-59.5%, observed around the base part of the underside. Sorus formation began from the base and middle parts in the top-intact thalli and from the marginal part in the top-cut thalli. The released zoospore germlings grew steadily as aquaculture seedlings, and the blade width and wet weight of the seedlings collected in August and subjected to mariculture in November were significantly larger after approximately five and a half months compared to those of thalli cultivated using a conventional method (i.e. thalli collected just prior to the mariculture). Seedling production through maturation induction in sporophytes would be a feasible option for seedling production facilities and would be helpful for stably producing seedlings without the effect of the natural environment

Effective DNA extraction method for fragment analysis using capillary sequencer of the kelp, Saccharina

The DNA fragment analysis can become an effective tool to study genetic differences between not only species but also individuals on saccharinan kelp from which the little genetic diversity was reported. This time, extraction methods of suitable DNA for use in the analysis with a capillary sequencer was examined on Saccharina japonica var. diabolica that contains polysaccharide abundantly. When AFLP was performed using genomic DNA extracted by seven different methods: (1) commercial kit, (2) original CTAB method, (3)-(5) three types of modified CTAB method, (6) modified SDS method, (7) combination of CTAB method and SDS method, high reproducible peak that was worth for the analysis was noticeable in the electropherogram in the experiment with the last combination method (7). It is considered that the pretreatment washing of polysaccharide and the subsequent purification for protein and RNA in SDS method and for polysaccharide in CTAB method are effective to obtain the high purity DNA

Investigation of Proteomic Profiles of Lamina of Ecklonia kurome (Laminariales): Homology-Based Cross-Species Protein Identification and Analysis of the Post-translational Processing of Vanadium-Dependent Bromoperoxidases Using MALDI-TOF/TOF

Proteomic profiles of the lamina of Ecklonia kurome Okamura, one of the Japanese dominant laminarialean kelps, were investigated by two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF. Due to the absence of E. kurome DNA or protein databases, homology-based cross-species protein identification was performed using a combination of three database-searching algorithms, Mascot peptide mass fingerprinting, Mascot MS/MS ion search, and mass spectrometry-based BLAST. Proteins were extracted from the lamina by an ethanol/phenol method and subjected to 2-DE (pI 4-7, 10 % polyacrylamide gel). More than 700 spots were detected in the 2-DE gel with CBB, and 93 spots (24 proteins) were successfully identified by MALDI-TOF/TOF and the cross-species database searching. The identified proteins mainly consisted of cytoplasmic carbohydrate metabolic enzymes, chloroplast proteins involved in photosynthesis, and haloperoxidases. Interestingly, vanadium-dependent bromoperoxidases (vBPO), which is thought to be involved in halogen uptake, synthesis of halogenated products, and detoxification of reactive oxygen species, were separated into at least 23 different spots. By comparing mass spectra, amino acid sequences predicted from tandem mass spectra and haloperoxidase activities of the vBPOs, we found that (1) at least two types of vBPOs were expressed in the lamina of E. kurome and (2) two pro-vBPOs might be activated by specific cleavage at N- and C-terminal regions

Evaluation of the morphological characteristics and culture performance of Cladosiphon okamuranus strains

This study aimed to determine whether morphological differences of Cladosiphon okamuranus strains at different aquaculture sites were inherent characteristics and to select useful forms for aquaculture production and processing. Three candidate strains with potentially excellent morphological characteristics, that is ON, SY and KT, were selected from six local strains collected from Okinawa Islands. Then, these three candidate strains were transplanted to four aquaculture sites to evaluate their characteristics. The thallus length was significantly larger (e.g. 1.6-2.1 times) in the SY strain than in the ON strain in each area and the density of the primary lateral branches of the latter was significantly (e.g. 2.4-5.9 times) higher than that of the other two strains. The SY and ON strain characteristics were also distinguished by a comprehensive evaluation of eight traits with principal component analysis. Conversely, the KT strain tended to have an intermediate length between ON and SY strains, the biomass yield of the SY and KT strains was higher than that of ON strain. These results, indicating that some morphological differences are intrinsic strain characteristics, will provide information to aquaculture producers using appropriate strains to maximize the unit yield and thalli quality

Genetic differences among varieties of Saccharina japonica in northern Japan as determined by AFLP and SSR analyses

The genetic diversity of Saccharina japonica inhabiting the Hokkaido coastline and the surrounding area was investigated by AFLP and SSR analyses. A STRUCTURE analysis based on the AFLP data identified two clusters in the species, although a few local populations with genetic structures different from that of the neighboring populations were detected. The two clusters could be identified even within each current variety. In the SSR analysis, genetic variation in nucleotide sequences in all samples was located in four DNA regions (MS10, MS11, MS16, and MS29), and it is thought that these regions can be used as markers to detect individual-level variation in S. japonica. The STRUCTURE analysis of the SSR data identified four clusters in the species. Since the geographical distribution of these four different clusters does not correspond exactly to the currently recognized varieties in this species, it will be necessary to re-evaluate the distribution of the varieties of S. japonica