Development of SSR primers using ISSR–PCR in Diospyros kaki Thunb.

Nine polymorphic single sequence repeat (SSR) primers were developed in Japanese persimmon using inter‐SSR (ISSR) suppression polymerase chain reaction (PCR). These primers were tested on 30 individuals from Japan and China. The number of alleles per locus ranged from five to 20. Expected (H_E) and observed (H_O) heterozygosities at each locus ranged from 0.42 to 0.77 and 0.27 to 0.59, respectively. The SSR primers developed herein could be applied to cultivar identification, estimation of genetic diversity and divergence in Diospyros spp.     

柿SCoT-PCR体系优化及品种遗传多样性分析

选用国家柿种质资源圃中24个牛心柿和22个野柿为实验材料,采用L16(45)正交试验设计,建立了最优SCoT-PCR反应体系(20μL反应体系中,含Mg2+2.0mmol/L,dNTPs 0.2mmol/L,Taq聚合酶1.0U,引物0.5μmol/L,DNA模板80ng),并对不同引物的退火温度进行了优化,以探讨SCoT标记在柿亲缘关系鉴定和品种分类方面的应用。结果显示:(1)10条SCoT引物对46份材料共扩增出176个位点,其中多态性位点170个,多态率达96.6%;46份材料的遗传相似系数在0.619~0.852之间。(2)UPGMA聚类分析显示,SCoT标记能将46份材料完全区分开,其中:红花雄野毛柿、红花雌野毛柿单独被聚为一组,独立于牛心柿和野柿之外,与形态特征观察结果一致;璧山牛心柿与野柿聚为一组,在形态特征上璧山牛心柿也更趋于野柿,且璧山牛心柿为四倍体,与其他牛心柿(六倍体)不同,璧山牛心柿应属于野生柿。研究表明,SCoT标记是研究柿种质资源遗传多样性的有效手段;46份材料存在着较高的遗传多样性,在一定程度上反映了品种的地理分布情况,也说明地理因素对柿的亲缘关系有很大影响。     

Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species

To elucidate the relationships among Diospyros kaki and species closely related in previous studies, the nuclear ribosomal internal transcribed spacer (ITS) DNA sequence and the chloroplast matK gene were sequenced and compared with those of nine Diospyros species from Thailand, four species from temperate regions, and one species of southern Africa, D. lycioides. Maximum parsimony, maximum likelihood, and neighbor joining analyses of the matK and ITS data sets revealed that D. kaki is closely related to two diploid species, D. oleifera and D. glandulosa. D. kaki, D. glandulosa, and D. oleifera were placed differently in the trees obtained from ITS and matK data sets, suggesting that hybridization and/or introgression may have occurred during the development of these species. D. kaki was not found to be closely related to D. ehretioides, a diploid species from Thailand. These results differed from a prior analysis of this genus performed with chloroplast DNA (cpDNA) restriction site mutations in 3.2- and 2.1-kb amplified sequences. The results supported Ng’s hypothesis that D. glandulosa and D. kaki may share a common ancestor. D. oleifera was also closely associated with D. kaki.     

Hyperparasitism by Mesochorus spp. (Hymenoptera: Ichneumonidae) in Peristenus sp. (Hymenoptera: Braconidae) and development of PCR primers for hyperparasitoid detection

Peristenus sp. pupae collected from Lygus spp. nymphs in 2001 and 2002 were over-wintered in the laboratory. In both years, more than 30% of adults emerging from over-wintering pupae were identified as ichneumonid hyperparasitoids, Mesochorus curvulus Thomson and Meschorus sp. (Hymenoptera: Ichneumonidae). At the end of the over-wintering period, Peristenus sp. males emerged first followed by Peristenus sp. females and finally Mesochorus spp. The male:female ratio in emerging Peristenus sp. adults was skewed towards males. The Internal Transcribed Spacer (ITS) region and the cytochrome oxidase I (COI) gene from Mesochorus spp. were sequenced. ITS sequences were used to develop PCR primers to detect Mesochorus spp. hyperparasitism in the primary host, Lygus spp. PCR analysis of field-collected Lygus spp. nymphs gave similar estimates of Mesochorus spp. hyperparasitism to the rearing protocols (25–28%). Sequence analysis of COI and ITS regions and subsequent restriction endonuclease analysis of ITS PCR products from Mesochorus spp. indicate the presence of two genotypes in the population. The possibility that these two genotypes represent separate or cyrptic species is discussed.     

广西柿种质资源果实性状多样性分析与模糊综合评价

为进一步揭示广西柿种质资源信息,运用多样性分析和模糊综合评价法对不同生态气候区的柿种质资源果实性状进行研究和评价。结果表明:不同柿种质的果实性状存在丰富的多样性。86份柿种质的模糊综合评价指数FCI值在1.849～3.947之间,平均2.835。根据FCI取值范围将所调查的柿种质分成5个等级,FCI值最高的第5级种质数量最少,占所调查种质的3.49%;FCI值最低的第1级种质数量最多,占所调查种质的48.84%。月柿、牛心柿、大方柿、京柿、鸡心柿、小方柿和火柿等柿品种及一些实生单株的模糊综合评价指数较高,它们的果实综合性状表现较优,建议作为优良品种和单株加以开发和利用。部分单个性状比较优良的实生单株虽不适合于直接推广种植,但可作为柿品种改良的资源加以利用。     

基于叶绿体DNA ndhA和nrDNA ITS序列变异分析栽培柿及其近缘种亲缘关系

以柿属植物（Diospyros spp.）中与柿近缘的8种共30个基因型为试材,进行核糖体DNA(nrDNA)内转录间隔区（ITS）和叶绿体DNA ndhA序列变异分析,并通过软件计算两个序列及合并后的进化模型,依据进化模型采用ML法(maximum likelihood method)分析进化关系。为进一步弄清柿属植物种间亲缘关系和供试柿（Diospyros kaki Thunb.）种内分子差异提供了理论依据。结果表明：（1）ndhA序列长度变异范围在1 492~1 511,14个信息位点;ITS序列长度变异范围在660~761,56个信息位点。ITS、ndhA和ndhA+ITS（ndhA和ITS合并）最适碱基进化模型分别为（TrN+I+G）、（F81+I）和（GTR+I+G）。综合ITS和ndhA序列分析表明：柿与油柿和云南野毛柿亲缘关系最近,与美洲柿和乌柿最远。（2）21份柿品种材料的ITS长度均为730,包括4个变异位点,据此4个变异位点对供试柿种内21个品种进行聚类分析。研究认为,ndhA和ITS能较清楚解释了柿与其近缘种间的亲缘关系,并通过柿品种ITS的差异位点分析鉴别出栽培柿种内的差异。     

Terminal repeat retrotransposon in miniature (TRIM) as DNA markers in Brassica relatives

We have developed a display system using a unique sequence of terminal repeat retrotransposon in miniature (TRIM) elements, which were recently identified from gene-rich regions of Brassica rapa. The technique, named TRIM display, is based on modification of the AFLP technique using an adapter primer for the restriction fragments of BfaI and a primer derived from conserved terminal repeat sequences of TRIM elements, Br1 and Br2. TRIM display using genomic DNA produced 50–70 bands ranging from 100 to 700 bp in all the species of the family Brassicaceae. TRIM display using B. rapa cDNA produced about 20 bands. Sequences of 11 randomly selected bands, 7 from genomic DNA and 4 from cDNA, begin with about 104 bp of the terminal repeat sequences of TRIM elements Br1 or Br2 and end with unique sequences indicating that all bands are derived from unique insertion sites of TRIM elements. Furthermore, 7 of the 11 unique sequences showed significant similarity with expressed gene. Most of the TRIM display bands were polymorphic between genera and about 55% (132 of 239 bands) are polymorphic among 19 commercial F1 hybrid cultivars. Analysis of phylogenetic relationships shows clear-cut lineage among the 19 cultivars. Furthermore, a combination of 11 polymorphic bands derived from only one primer combination can clearly distinguish one cultivar from the others. TRIM display bands were reproducible and inheritable through successive generations that is revealed by genetic mapping of 6 out of 27 polymorphic TRIM markers on the genetic map of Brassica napus. Collective data provide evidence that TRIM display can provide useful DNA markers in Brassica relatives because these markers are distributed in gene-rich regions, and are sometimes involved in the restructuring of genes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank accession nos.: AC190326 (TRIM display fragment gCP01); AC190327 (TRIM display fragment gCP02); AC190328 (TRIM display fragment gCP02) AC190329 (TRIM display fragment gCP04) AC190330 (TRIM display fragment gDP01); AC190331 (TRIM display fragment gDP02); AC190332 (TRIM display fragment gDP03); AC190333 (TRIM display fragment gDP04); AC190334 (TRIM display fragment gDP05); AC190335 (TRIM display fragment gDP06); AC190336 (TRIM display fragment gDP07).     

Quantitative real-time PCR to determine allele number for the astringency locus by analysis of a linked marker in Diospyros kaki Thunb

Persimmon (Diospyros kaki Thunb.) is a polyploidy fruit tree species of economic importance to East Asia. Natural astringency loss is an important trait in persimmon breeding programs. Quantitative real-time PCR was used to determine the number of AST/ast alleles for fruit astringency in persimmon (D. kaki Thunb.). To this end, the cultivar Jiro was transformed with one or two copies of a gene encoding NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH), which was used as a standard for measuring the allele number of a sequenced marker tightly linked to the recessive ast locus for nonastringency. Primers for markers linked to the AST or ast allele were then used to measure the AST to ast ratio directly in the progeny of a full-sib cross. From determination of the AST to ast ratio and the results of the S6PDH copy number, the number of AST and ast alleles at the AST/ast locus was estimated. This research supported the hypothesis that D. kaki is a hexaploid with six AST and/or ast alleles. In addition to the determination of the allelic status of the AST locus, the application of real-time PCR for confirmation of the ploidy level and allelic composition of target genes in autopolyploids or allopolyploids was demonstrated.     

丹霞柿，广东柿属(柿科)一新组合及其一新异名

通过查阅模式标本后发现丹霞南烛(Lyonia danxiaensis R. H. MiaoW. Q. Liu)具有聚伞花序,花部为4数,雄蕊16枚且成对着生,花丝非膝曲状等特征与珍珠花属(Lyonia)不符,而与柿科(Ebenaceae)柿属(Diospyros)一致,因此提出一新组合:丹霞柿[Diospyros danxiaensis (R. H. MiaoW. Q. Liu) Y. H. TongN. H. Xia]。且经标本比对后发现,新近发表的彭华柿(Diospyros penghuae W. B. Liao, Q. FanW. Y. Zhao)与丹霞柿实为同种,在此也予以合并。     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Development of 10 polymorphic microsatellite loci primers for Codonopsis pilosula Nannf. (Campanulaceae)

Codonopsis pilosula Nannf., as an important traditional Chinese medicinal plant species, has been used to treat fatigue, thirst and loss of appetite. In this study, we developed 10 new microsatellite loci primers from the genome of this species using the combined biotin capture method. The polymorphism of each locus was further assessed through 27 individuals from four geographically distant populations. The number of alleles per locus ranged from 6 to 11. The observed and expected heterozygosity ranged from 0.15 to 0.24 and 0.27 to 0.40, respectively. We further performed cross-priming tests of these loci in the other three congeneric species. These microsatellite markers will be useful for investigating genetic diversity within and between populations of these species.     

Molecular identification of symbiotic dinoflagellates (Symbiodinium spp.) from Palythoa spp. (Anthozoa: Hexacorallia) in Japan

In Japan, zooxanthellate Palythoa tuberculosa Klunzinger and Palythoa mutuki Verrill (Anthozoa: Hexacorallia: Zoantharia) are found over a 1,000 + km latitudinal range, often in environments where most other zooxanthellate anthozoans are not found (i.e. tidal lagoon pools, around shallow water hydrothermal vents, subtropical rocky shorelines). Sequences of internal transcribed spacer of ribosomal DNA (ITS-rDNA) of the symbiotic dinoflagellate genus Symbiodinium (zooxanthellae) Freudenthal (Order Suessiales) from P. tuberculosa and P. mutuki from several locations in Japan (34°11′N–24°16′N) were analysed. Unexpectedly, despite the ability of the genus Palythoa to be flexible in association with different Symbiodinium subclades, most (35 of 36) Palythoa investigated here specifically associate with subclade C1 and closely related types. Symbiodinium subclade C1 has been characterized as a “generalist” in terms of the ability to associate with a range of hosts, but present results suggest that subclade C1 may also be a “generalist” in terms of being able to live in a variety of environments over a latitudinal range. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

rDNA cloning and rapid hybrid identification inPopulus spp. (Salicaceae)

Ribosomal DNA genes fromP. deltoides have been cloned and specific sequences of the 25 S and 18 S rDNA region, labelled by digoxigenin, have been used to determine the rDNA structure ofPopulus tremula, P. fremontii, P. maximowiczii, P. yunnanensis, P. nigra, P. wislizenii, P. alba. The restriction maps of the coding region appeared to be similar among the examined species and with those ofP. deltoides andP. trichocarpa, reported in a previous paper. Inter- and intraspecific variation in rDNA repeat unit length have been revealed after EcoRI digestions. SstI and XbaI restriction sites have been found at different positions in the IGS of some species. The polymorphic fragments generated by SstI digestion allowed the identification of the hybrid origin of some genotypes. The number of rDNA genes in the genome ofP. deltoides has been estimated to be about 2 000 copies. Finally, the usefulness of these studies inPopulus spp. taxonomy and forestry genetics is discussed.Ribosomal RNA gene structure in somePopulus spp. (Salicaceae) and their hybrids 2.     

Biology of Lilioceris spp. (Coleoptera: Chrysomelidae) and their parasitoids in Europe

The biology and parasitoid complex of the lily leaf beetle (LLB), Lilioceris lilii Scopoli, and two congeneric species were investigated in Europe, as part of a biological control program against the LLB in North America. Eggs, larvae, and adults of L. lilii were collected in several countries in Europe, on both cultivated and wild Lilium spp., and reared in the laboratory and under natural conditions. Parasitoids were obtained and their biologies were studied. Similar investigations were made in Switzerland on two closely related species Lilioceris tibialis (Villa) found on wild Lilium spp., and Lilioceris merdigera (L.) on several other Liliaceae. The three species are strictly univoltine. Adults overwinter and lay eggs on leaves in early spring. The three beetle species have four instars, which were characterized by their head capsule width. Pupation occurs in a cocoon in the soil. Adults emerge in late summer and start feeding before reaching overwintering sites. Egg and larval parasitoids were obtained. Eggs of L. lilii and L. merdigera were parasitized by the mymarid Anaphes sp., a multivoltine species that needs alternate hosts for overwintering. Larvae were heavily attacked by several parasitoids, among which the most abundant were three ichneumonids, Lemophagus pulcher (Szepligeti), L. errabundus (Gravenhorst), and Diaparsis jucunda (Holmgren), and the eulophid Tetrastichus setifer Thomson. All four parasitoid species were found in the three beetles and in most European regions, but strong variations were observed in their relative abundance among hosts and geographic regions. Three of the four main larval parasitoids are strictly univoltine, whereas L. pulcher has a partial second generation. Lemophagus spp. are frequently parasitized by the ichneumonid hyperparasitoid Mesochorus lilioceriphilus Schwenke. Further details of the biology of the parasitoids are described, and their potential as biological control agents is discussed.     

The infestation and dispersion patterns of Carcinonemertes spp. (Nemertea) on their crab hosts

Seasonal changes in the infestation and dispersion patterns of egg predatory nemerteans on their crab hosts were analyzed. Marked differences in the seasonal patterns of infestation were noted between worm species, yet, common patterns in the aggregation of the worms were found. Worm aggregation increased at the onset of the reproductive seasons of the hosts, and at the nadirs of the reproductive seasons for those hosts with year round breeding. The aggregation patterns of two worm species fluctuated with the physical environment of their estuarine hosts. Salinity changes as a result of seasonal rains may have caused changes in the underlying dispersion patterns of Carcinonemertes epialti on Hemiarapsus oregonensis, and C. mitsukurii on Portunus pelagicus. Lastly, the embryogenic cycle of the host species was significant in shaping the infestation and aggregation patterns of C. epialti on Cancer anthonyi and C. regicides on Paralithodes camtschaticus. Worm immigration and emigration were linked to crab embryogenesis and directly influenced the dispersion patterns of the worms.     

Molecular identification of Taenia spp. in wolves (Canis lupus), brown bears (Ursus arctos) and cervids from North Europe and Alaska

Taenia tapeworms of Finnish and Swedish wolves (Canis lupus) and Finnish brown bears (Ursus arctos), and muscle cysticerci of Svalbard reindeer (Rangifer tarandus platyrhynchus), Alaskan Grant's caribou (Rangifer tarandus granti) and Alaskan moose (Alces americanus) were identified on the basis of the nucleotide sequence of a 396 bp region of the mitochondrial cytochrome c oxidase subunit 1 gene. Two species were found from wolves: Taenia hydatigena and Taenia krabbei. The cysticerci of reindeer, caribou and one moose also represented T. krabbei. Most of the cysticercal specimens from Alaskan moose, however, belonged to an unknown T. krabbei-like species, which had been reported previously from Eurasian elks (Alces alces) from Finland. Strobilate stages from two bears belonged to this species as well. The present results suggest that this novel Taenia sp. has a Holarctic distribution and uses Alces spp. as intermediate and ursids as final hosts.     

RAPD fingerprints for identification and for taxonomic studies of elite poplar (Populus spp.) clones

RAPD (Random Amplified Polymorphic DNA) fingerprints have recently been used to estimate genetic and taxonomic relationships in plants. In this study RAPD analysis was performed on 32 clones belonging to different species of the genus Populus. Of these, 25 clones are registered in several countries for commercial use and, altogether, cover almost 50% of the worlds cultivated poplars. DNA was prepared from leaves and amplified by PCR using random oligonucleotide primers. Amplification products were separated by agarose-gel electrophoresis to reveal band polymorphisms. Four primers out of the 18 tested, were selected on the basis of the number and frequency of the polymorphisms produced. With these a total of 120 different DNA bands were reproducibly obtained, 92% of which were polymorphic. The polymorphisms were scored and used in band-sharing analyses to identify genetic relationships. With a few but interesting exceptions, these are consistent with the present taxonomy of the genus Populus and with the known predigrees of cultivated poplars. Moreover, the results show that RAPD analysis allows one to discriminate among all tested clones and can, therefore, be recommended as a convenient tool to defend plant breeders rights.