Electrophoretic evidence for the origins of tetraploids and hybrids ofSphenomeris chinensis sensu lato (Lindsaeaceae) in Japan

Hypotheses for the origins of tetraploids and hybrids ofSphenomeris chinensis sensu lato in Japan were tested using electrophoretic analysis of isozymes. Comparison of banding patterns supported the hypothesis that plants having abortive spores had resulted following hybridization between diploidS. chinensis var.chinensis andS. biflora and between diploid var.chinensis andS. gracilis. Results from isozymic analyses also revealed thatS. intermedia probably originated through allotetraploid speciation following hybridization between var.chinensis andS. biflora. There were two electrophoretic variants among tetraploids ofS. chinensis. One variant, restricted to the southern Ryukyu Islands, is probably an allotetraploid of hybrid origin between diploidS. chinensis andS. gracilis. It has been described as a new species,S. yaeyamensis Lin, Kato & K. Iwatsuki. The other variant, morphologically corresponding to var.chinensis, is distributed at least throughout southern Japan and also in northern Thailand. Its origin is uncertain, but one of the parents is probably diploidS. chinensis. Sphenomeris intermedia andS. yaeyamensis occur inland, whereas the parentalS. biflora andS. gracilis are halophilous and rheophllous, respectively.     

Chemical Components,Biological Activities,and Toxicological Evaluation of the Fruit (Aril) of Two Precious Plant Species from Genus Taxus

The fruit (aril) of the endangered genus Taxus plants is an abandoned herbal resource. Traditionally, people enthusiastically focus on its bark, its renewable, tremendous arils fall into the soil with seeds after they are mature. The present research investigated the fruit of two species from the genus Taxus, Taxus chinensis var. mairei, and Taxus media, with regards to their antioxidant and antihyperglycaemic activities, safety, and bioactive constituents. Results showed that T. chinensis var. mairei and T. media both had certain biological activities with T. chinensis var. mairei better in antioxidant activity and T. media better in antihyperglycaemic activity. Correlation analysis revealed that the differences in bioactivities depended on content of their mainly chemical components. The mice acute oral toxicity test indicated that the methanol extracts of the two biotypes of Taxus were safe. And nineteen compounds were tentatively assigned from the two varieties, via tandem mass spectrometry using a LC‐ESI‐Q‐TOF‐MS instrument, which included phenols, flavonoids, and terpenes. These results indicate a possible application of Taxus fruit extracts in various fields like in food industry, however, this still needs further investigations.     

Resistance to Phytophthora fragariae var. fragariae in strawberry: the Rpf2 gene

  Phytophthora fragariae var. fragariae is the causal agent of red stele (red core) root rot in strawberry (Fragaria spp.). The inheritance of resistance to one isolate of this fungus was studied in 12 segregating populations of F.×ananassa derived from crosses between four resistant cultivars (‘Climax’, ‘Redgauntlet’, ‘Siletz’, and ‘Sparkle’) and three susceptible cultivars (‘Blakemore’, ‘Glasa’, and ‘Senga’ Sengana’). The analysis clearly supports the hypothesis of a single segregating dominant resistance gene. It is proposed that this gene be designated Rpf2. Received 12 November 1996 / Accepted: 22 November 1996     

Callus induction and plant regeneration from broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. italica) for transformation

We evaluated the efficiency of callus induction and plantlet regeneration from hypocotyl explants of broccoli (Brassica oleracea var. italica). The cultivars were ‘Marathon’, ‘Greenbelt’, and ‘Shogun’. Transformation success was not affected by the presence of tobacco feeder-cell layers on the culture media. The frequency of shoot regeneration was greater from 10-d-old hypocotyls than from 14-d-old hypocotyls. Both ‘Marathon’ and ‘Greenbelt’ had higher potentials for tissue regeneration than did ‘Shogun’. We found that for transformation selection, the optimum concentration was either 50 mg/L kanamycin or 100 mg/L genetkin.     

Systematics of Juniperus from eastern Asia based on Random Amplified Polymorphic DNAs (RAPDs)

DNA was examined by RAPD banding for Junipers chinensis, J.c. var. sargentii, J.c. var. tsukusiensis, J. communis, J.c. var. nipponica, J.c. var. saxatilis, J. conferta, J. formosana, J. procumbens, J. rigida, J. taxifolia and J.t. var. lutchuensis. The DNA data readily separated junipers of section Sabina from section Juniperus. J.c. var. tsukusiensis from Taiwan was found to be sufficiently different from J.c. var. tsukusiensis (Yakushima) to warrant the recognition of a new variety: J. chinensis var. taiwanensis R.P. Adams and C-F. Hsieh nov. var. Juniperus formosana from mainland China was found to be different from J. formosana from Taiwan and a new variety is recognized: J. formosana var. mairei (Lemee and Lev.) R.P. Adams and C-F. Hsieh comb. nov. Juniperus communis var. nipponica was found to be very distinct from J. communis and this supports its recognition as a variety. The recognition of J. conferta as a variety of J. rigida [J. rigida var. conferta (Parl.) Patschke] is supported by the data. The data also supports the recognition of J. lutchuensis Koidz. [=J. taxifolia var. lutchuensis (Koldz.) Satake] and J. morrisonicola Hayata [=J. squamata var. morrisonicola (Hayata) H.L. Li and H. Keng] at the specific levels.     

Development of sex-linked PCR markers for gender identification in Actinidia

 Two sex-linked random amplified polymorphic DNA (RAPD) markers identified from Actinidia chinensis were converted into sequence-characterised amplified regions (SCARs) for the large-scale screening of Actinidia breeding populations. Initial SCAR primers converted one RAPD (SmX) into a dominant marker, but the other (SmY), which was potentially more useful because of its linkage to the male determining ‘Y’ locus, failed to retain polymorphism. This difficulty was overcome by cloning and sequencing the alternate ‘allele’ from female plants, and then designing ‘allele’-specific primers that utilised nucleotide differences between the sexes. Using a quick squash-blot method of DNA extraction, the SCAR primers were tested in 120 A. chinensis plants to determine their gender. The system is now in use for large-scale screening of seedling populations in the Actinidia breeding programme. The sex-linked SCAR primers also functioned with plants from some other geographically separate accessions of A. chinensis and with plants in the closely related polyploid species A. deliciosa, but did not amplify a sex-linked band in more distantly related species of Actinidia. Received: 27 December 1997 / Accepted: 5 March 1998     

Multiple lines of evidence for an Australasian geographic boundary in the Indo-Pacific humpback dolphin (<Emphasis Type="Italic">Sousa chinensis</Emphasis>): population or species divergence?

The taxonomic status of humpback dolphins (genus Sousa, sub-family Delphininae) is unresolved. While the classification of this genus ranges from a single to three nominal species, the International Union for Conservation of Nature and the International Whaling Commission only recognise a ‘two-species’ taxonomy (S. teuszii in west Africa, and S. chinensis in the Indo-Pacific). Under the IUCN (2008), S. chinensis is listed as ‘near threatened’, but is only considered as a ‘migratory’ species in Australia. Taxonomic resolution of the genus Sousa is needed to define particular conservation status and develop appropriate management actions. Using phylogenetic analyses of 1,082 bp of mitochondrial and 1,916 bp of nuclear DNA, we provide multiple lines of genetic evidence for the genetic distinction of S. chinensis in China and Indonesia from S. chinensis in Australia. The separation of Australian Sousa from Sousa of Southeast Asia requires a review of their current conservation status and respective management actions.     

Response of antioxidant activity to excess copper in two cultivars of <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis> Makino

Changes in ascorbic acid content and antioxidant enzyme activities were investigated in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) leaves of ‘Wutacai’ and ‘Erqing’ exposed to excess copper (Cu). Cu treatment reduced the fresh weight of shoot and root by 57% and 46% in ‘Wutacai’, and 60 and 54% in ‘Erqing’, respectively. The accumulation of copper in leaves was higher in ‘Wutacai’ than that in ‘Erqing’. Compared to the control, ascorbic acid (AsA) contents were significantly decreased after copper treatment in both cultivars, while they were higher in ‘Wutacai’ than in ‘Erqing’, which may explain the higher copper-tolerance of ‘Wutacai’ with higher copper accumulation. The higher AsA contents of ‘Wutacai’ resulted from their lower activities of degrading enzymes, such as ascorbate oxydase (AAO) and ascorbate peroxidase (APX), as well as the increasing activity of dehydroascorbate reductase (DHAR) after copper treatment compared with ‘Erqing’. Copper stimulated superoxide dismutase (SOD) activity in both cultivars, but for catalase (CAT), there was little difference between both cultivars. Peroxidases (POD) activity was decreased after copper treatment in ‘Erqing’, while in ‘Wutacai’, it was significantly increased at 14 days, and POD activity was higher in ‘Wutacai’ than that in ‘Erqing’ at 21 and 28 days. Therefore, the induced increasing activity of POD in ‘Wutacai’ also played an important role in its copper tolerance.     

Evolutionary relationships of membrers of the generaTaphrina, Protomyces, Schizosaccharomyces, and related taxa within the archiascomycetes: Integrated analysis of genotypic and phenotypic characters

To study the phylogeny and evolution of archiascomycetes, we determined the full sequence of the nuclear 18S rRNA gene from 14Taphrina species and 2Protomyces species, and the partial sequence ofSchizosaccharomyces japonicus var.japonicus. The sequences were phylogenetically analyzed by the neighbor-joining, maximum parsimony, and maximum-likelihood methods. We also looked at their principal phenotypic characters and genotypic character. Relationships within the Ascomycota are concordant with the previously published phylogenies inferred from 18S rDNA sequence divergence and divide the archi-, hemi-and euascomycetes into distinct major lineages. All the trees show that, within the archiascomycete lineage, 11 of the 14Taphrina species and the 2Protomyces species are monophyletic. A core groups ofTaphrina andProtomyces is always monophyletic. The evidence from molecular and phenotypic characters such as cell wall sugar composition, ubiquinone, cell wall ultrastructure, and mode of conidium ontogeny, strongly suggests that ‘T’. californica CBS 374.39, ‘T’. maculans CBS 427.69 and ‘T’. farlowii CBS 376.39 should be excluded from the archiascomycete lineage. ‘Taphrina’ farlowii CBS 376.39 groups withCandida albicans in the Saccharomycetales, whereas ‘T’. californica CBS 374.39 and ‘T’. maculans CBS 427.69 have a basidiomycete affinity and group with Tremellalean members in the hymenomycete lineage.Schizosaccharomyces is monophyletic. The strictly anamorphic yeastSaitoella complicata groups with the apothecial ascomyceteNeolecta vitellina rather than theTaphrina/Protomyces branch.     

Hybrid origin of some ornamentals of Allium subgenus Melanocrommyum verified with GISH and RAPD

 Random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) methods have been used to verify the hybridogenic origin and to identify the parental species of some ornamental cultivars in the subgenus Melanocrommyum of the genus Allium. The cultivars had been selected from seed obtained after uncontrolled pollination in breeders’ fields. The combination of GISH analysis with RAPD markers is very suitable for testing the hybridogenic origin of plants and to ascertain the parental species of the hybrids in such cases. As suspected, A. macleanii and A. cristophii are the parental species of ‘Globemaster’. The parental species of cultivar ‘Globus’ are A. karataviense and A. stipitatum, and not A. cristophii and A. giganteum as has been assumed on morphological grounds. Cultivars ‘Lucy Ball’ and ‘Gladiator’ are of hybrid origin, though only one of the parental species, A. hollandicum, could be confirmed. The cultivars ‘Purple Sensation’, ‘Mount Everest’, ‘White Giant’, ‘Michael H. Hoog’ and ‘Mars’ are not hybrids since neither GISH nor RAPD suggest the presence of a second genome. ‘Purple Sensation’ belongs to A. hollandicum, ‘Mount Everest’, ‘White Giant’ and ‘Mars’ to A. stipitatum,‘Michael H. Hoog’ to A. rosenorum. Received: 3 July 1997 / Accepted: 9 October 1997     

Macrozoobenthic assemblages in littoral sediments in the enclosed Rhine-Meuse Delta

Littoral macrozoobenthos in the enclosed Rhine-Meuse Delta was investigated by taking 95 sediment samples from 17 sites between 1984 and 1990. In addition, a set of environmental parameters was determined. The aim was to identify the main assemblages and the environmental conditions under which they occur. By the use of TWINSPAN, three main littoral zoobenthic assemblages were distinguished, which were related to geographical zones and differences in sediment grain size distribution. The ‘litoral river sand’ assemblage was found in the most upstream part; it mainly consisted of ‘interstitial’ invertebrates, including the indicator speciesVejdovskiella comata, Propappus sp. andKloosia pusilla. The ‘littoral sedimentation area silt’ assemblage was dominated byGammarus tigrinus, Einfeldia dissidens andPisidium sp. It was found in several river sections and contained the indicator speciesEinfeldia dissidens, Potamopyrgus antipodarum andValvata piscinalis. The ‘littoral sandy basin’ assemblage was concentrated in the littoral fine sands of the Haringvliet and contained the indicator speciesPisidium henslowanum, P. moitessierianum, Cladotanytarsus sp. andLipiniella arenicola. These three assemblages are the reflection of an interaction between habitat, food and disturbance. Palaeoecological analysis of insect remains revealed that 14 out of the 24 insect taxa, that were formerly common in the river sand habitat, are now extinct from the Rhine. The river silt habitat seems less impoverished: two out of the 19 insect taxa found in palaeoecological analysis are now extinct from the Rhine and seven are rate. Exotic species (Corbicula fluminea, C. fluminalis andCorophium curvispinum) have recently colonized the Rhine-Meuse Delta, but their impact on the macrozoobenthos seems limited.Corbicula spp. have become abundant in the ‘littoral river sand’ assemblage only.     

Towards a legal framework for systematic conservation: identification and development of allele-specific PCR markers for conspecific varieties of an endangered perennial herb <Emphasis Type="Italic">Primula kisoana</Emphasis> Miquel based on sequence variation of chloroplast DNA

Primula kisoana var. kisoana (Primulaceae) is a narrow endemic found only in extremely restricted areas of central Honshu Island, Japan. Although the species is included as ‘Critically Endangered (CR)’ in the latest Japanese Red List, it is not covered by legislation such as the ‘Law for the Conservation of Endangered Species of Wild Fauna and Flora, Japan’. This poor conservation status is due, largely, to the presence of another conspecific variety, var. shikokiana, which is regarded as less threatened than var. kisoana. In this study, we investigated the genetic dissimilarity between the two varieties by examining sequence variation in three noncoding regions of chloroplast DNA. Ten distinct haplotypes were detected, none of which were distributed across populations and varieties. The genetic differentiation between the two varieties revealed by an AMOVA and a minimum spanning network suggests that var. kisoana and var. shikokiana should be conserved and managed as separate units. Using the allele-specific PCR method, four primer pairs were developed at the point mutations and insertion/deletion loci that exhibited only inter-variety variation. These markers will facilitate reliable identification of var. kisoana, assisting in the implementation of restoration programs including the introduction of seedlings and construction of a legal framework for conservation.     

Adventitious shoot regeneration of pear (<Emphasis Type="Italic">Pyrus</Emphasis> spp.) genotypes

Adventitious shoot regeneration of twenty-four pear genotypes was compared in a common in vitro shoot induction and development protocol. This study also compared cultures newly established from scionwood with cultures that been in long-term cold storage. In vitro cultures of 13 Pyrus genotypes and budwood from 23 Pyrus genotypes were obtained from the National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon. With the exception of one genotype of P. elaeagrifolia Pall., and ‘Ya Li’ (P. pyrifolia var. sinensis Teng & Tanabe), all were P. communis L. cultivars. The basal shoot induction media consisted of Chevreau and Leblay (CL) basal nutrients, vitamins, and organics (Chevreau and Leblay in Acta Hortic 336: 263–268, 1993). The analysis of variance indicated that differences among genotypes were highly significant and the main effect of culture origin was non-significant. However, there was a significant interaction between genotype and culture origin, with percentage regeneration of ‘Abate Fetel’ from new budwood significantly greater than that from long-term in vitro cultures, while ‘Jesinji Vodenac’ cultures derived from the old NCGR cultures regenerated significantly more adventitious shoots. The ranges of mean regeneration frequency were similar for both in vitro (0–87.7%) and scionwood-derived cultures (0–70.7%). Maximum regeneration was observed for ‘Conference’, followed by ‘Magness’, ‘Dr. Jules Guyot’, and Packham’s Triumph’. The range of number of adventitious shoots was relatively narrow, with the minimum of 1.0 for seven genotypes to 2.2 for ‘Conference’.     

Microsatellite diversity in tetraploid <Emphasis Type="Italic">Gossypium</Emphasis> germplasm: assembling a highly informative genotyping set of cotton SSRs

A series of 320 mapped simple sequence repeats (SSRs) have been used to screen the allelic diversity of tetraploid Gossypium species. Fourty-seven genotypes were analyzed representing (i) the wide spectrum of diversity of the cultivated pool and of the primitive landraces of species G. hirsutum (‘marie-galante’, ‘punctatum’, ‘richmondi’, ‘morrilli’, ‘palmeri’, and ‘latifolium’, and ‘yucatanense’), and (ii) species G. barbadense, G. darwinii and G. tomentosum. The polymorphism of 201 SSR loci revealed 1128 allelic variants ranging from 3 to 17 per locus. Neighbor-joining (NJ) method based on genetic dissimilarities produced groupings consistent with the assignments of accessions both at species and at race level. Our data confirmed the proximity of the Galapagos endemic species G. darwinii to species G. barbadense. Within species G. hirsutum, and as compared to the other 6 races, race yucatanense appeared as the most distant from cultivated genotypes. Race yucatanense also exhibited the highest number of unique alleles. The important informative heterogeneity of the 201 SSR loci was exploited to select the most polymorphic ones that were assembled into three series of genome-wide (i.e. each homoeologous AD chromosome pair being equally represented) and mutliplexable (× 3) SSRs. Using one of these ‘genotyping set’, consisting of 39 SSRs (one 3-plex for each of the 13 AD chromosomes pairs) or 45 loci, we were able to assess the relationships between accessions and the topology in the genetic diversity sampled. Such genotyping set of highly informative SSR markers assembled in PCR-multiplex, while increasing genotyping throughput, will be applicable for molecular genetic diversity studies of large germplasm collections. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Taxonomic status of the hominid mandible KNM-ER TI 13150 from the middle pliocene of tabarin,in Kenya

A partial mandible with two molars intact was recovered between 1981 and 1984 from deposits of the Middle Pliocene at Tabarin, in Kenya. It has been described and assigned toAustralopithecus cf.afarensis Johanson, White, andCoppens, 1978, with the condition that if ‘A. afarensis’ is revised, then the attribution may change. The taxon ‘A. afarensis’ was found to be invalid and was revised. The smaller specimens of ‘A. afarensis,’ to which the Tabarin mandible was said to be similar, were redescribed asHomo antiquus Ferguson, 1984. Since the Tabarin mandible andH. antiquus are successive transients of the same gens and are allopatric, the Tabarin hominid population is described as an earlier chronosubspecies,Homo antiquus praegens ssp. n.     

Exogenous ethylene influences flower opening of cut roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis>) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1-aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh-ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-induced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhibited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.     

Transformation of a muskmelon ‘Galia’ hybrid parental line (Cucumis melo L. var. reticulatus Ser.) with an antisense ACC oxidase gene

‘Galia’ muskmelon (Cucumis melo L. var. reticulatus Ser.) has been recalcitrant to transformation by Agrobacterium tumefaciens. Transformation of the ‘Galia’ male parental line, ‘Krymka’, with an ACC oxidase (CMACO-1) gene in antisense orientation is described herein. Explants were transformed using A. tumefaciens strain ABI, which contained a vector pCmACO1-AS plasmid, bearing an antisense gene of CMACO-1 and the CP4 syn gene (glyphosate-tolerance). Both CMACO-1 and CP4 syn genes were assessed by a polymerase chain reaction method. Flow cytometry analysis was performed to determine plant ploidy level of primary transformants. Two completely diploid independent transgenic plants were obtained. Southern blot and segregation analysis in the T₁ generation determined that each independent transgenic line had one single insertion of the transgene. These transgenic muskmelon male parental lines have potential for use in the production of ‘Galia’ F₁ hybrids with improved shelf life.     

Parasitism ofLeptinotarsa decemlineata [Coleoptera: Chrysomelidae] byEdovum puttleri [Hymenoptera: Eulophidae] in different cultivars of eggplant

Experiments were conducted to quantify parasitism of Colorado potato beetle,Leptinotarsa decemlineata (Say), by the egg parasitoid,Edovum puttleri Grissell, on 3 different cultivars of eggplant,Solanum melongena L. Levels of parasitism were higher (P<0.05) on ‘Black Pride’ than on other cultivars. The percentage of egg masses that were parasitized was 1.2-fold higher (P<0.05) on ‘Black Pride’ than on ‘Harris Special’ and ‘White’. The number of eggs per mass that were parasitized was 1.3- and 1.4- fold greater (P<0.05) on ‘Black Pride’ than on ‘Harris Special’ and ‘White’, respectively. The percentage of eggs that were parasitized per mass and percentage of emerged adult parasitoids did not differ (P>0.05) among cultivars; between 2.1- to 2.6- fold more females than males emerged from eggs on all cultivars during the growing season.Edovum puttleri suppressed the 2nd generation ofL. decemlineata on ‘Black Pride’ and ‘Harris Special’, but did not suppress populations on ‘White’.      

Critique of ‘Australopithecus afarensis’ as a single species based on dental metrics and morphology

Leonard andHegmon (1987) compare a series of dental metrics of ‘Australopithecus afarensis Johanson, White, andCoppens, 1978’ with criteria for modern apes, to test the hypothesis that ‘A. afarensis’ represents a single species. They also compare the morphology of the lower third premolar. The dental breadth of ‘A. afarensis’ shows a wide range of variation, particularly in the lower third premolar morphology which displays greater variation than in modern apes—yet the study concludes that the single species hypothesis cannot be rejected. The study is flawed by applying criteria for pongids inappropriate for a hominid. When ‘A. afarensis’ is compared with criteria for hominids, the range of variation in dental size, breadth, and third premolar morphology is greater than that in any hominid species. The single species hypothesis is, therefore, once again rejected. Moreover, the name ‘A. afarensis’ is preoccupied byPraeanthropus africanus (Weinert) and must be dropped.