Species identification of Radix Astragali (Huangqi) by DNA sequence of its 5S-rRNA spacer domain

About 300 species and varieties of Astragalus are identified in China, making the identification of the origin of a particular Astragalus species on the consumer market difficult. A molecular genetic approach was developed to identify various species of Astragalus. Although the 5S-rRNA coding sequence is conserved in higher eukaryotes, the spacer domain of the 5S-rRNA gene has great diversity among different species. The 5S-rRNA spacer domain was amplified by polymerase chain reaction (PCR) from the isolated genomic DNA, and the PCR products (approximately 300 bp) covering the 5S-rRNA spacer domain were sequenced. The nucleotide sequences of Astragalus membranaceus, A. membranaceus var. mongholicus, A. lehmannianus, A. hoantchy, and of one closely related species Hedysarum polybotrys (Hongqi), were determined. Diversity in DNA sequence and restriction enzyme mapping among various species was found in their 5S-rRNA spacer domains. This is the first report on the detection of 5S-rRNA spacer region sequence of Astragalus, and the results could be used for genetic identification of Huangqi.     

PCR and PCR-RFLP of the 5S-rRNA-NTS region and salvinorin A analyses for the rapid and unequivocal determination of Salvia divinorum

Salvia divinorum Epling & Játiva-M. is a perennial herb belonging to the Lamiaceae family; its active ingredient, the neoclerodane diterpene salvinorin A, is a psychotropic molecule that produces hallucinations. A comparative evaluation of S. divinorum fresh and dried leaves, S. officinalis fresh leaves, and dried powdered leaves claimed to be S. divinorum was done. HPLC-MS data confirmed the presence of salvinorin A in both S. divinorun leaf extracts and the powdered leaves, whereas no salvinorin A was found in S. officinalis. The non-transcribed spacer (NTS) in the 5S-rRNA gene of all leaf samples and the dried powdered leaves was amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 500bp for S. divinorum and 300bp for S. officinalis) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences, great diversities were found in the spacer region of the two species. Specific S. divinorum primers were designed on the sequence of the 5S-rRNA gene spacer region. In addition, a PCR-restriction fragment length polymorphism (PCR-RFLP) method was applied using NdeI and TaqI restriction enzymes. An NdeI site, absent in S. officinalis, was found in S. divinorum NTS region at 428-433bp. For TaqI, multiple sites (161-164, 170-173, and 217-220bp) were found in S. officinalis, whereas a unique site was found in S. divinorum (235-238bp). The results of this work show that the combined use of analytical chemical (HPLC-MS) and molecular (DNA fingerprinting) methods lead to the precise and unequivocal identification of S. divinorum.     

Phylogenetic relationship of ribosomal ITS2 and mitochondrial COI among diploid and triploid Paragonimus westermani isolates

We compared patterns of intraspecific polymorphism of two markers with contrasting modes of evolution, nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA), in the lung fluke, diploid and triploid Paragonimus westermani from three geographical regions of Korea. The genetic distances between three populations of Korean diploid and triploid P. westermani showed no significant difference in the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (mtCOI) and ribosomaal second internal transcribed spacer (ITS2) genes. A highly resolved strict-consensus tree was obtained that illustrated phylogenetically useful information of the ITS2 and mtCOI sequences from diploid and triploid P. westermani.     

Volatiles from leaves and rhizomes of Fragrant Acorus spp. (Acoraceae)

Three horticultural selections of Acorus gramineus Soland were investigated to determine the chemical composition of their leaves and rhizomes. The variety 'liquorice' was found to contain methylchavicol (49%) which accounts for the unusual anisic odor of this variety, while beta-asarone was the main component of A. christophii (43%) and 'yodo-no-yuki' (20%). The results are compared with calamus oils, and the possible biosynthetic precursors of the main components methylchavicol and beta-asarone are considered.     

Commercial-scale sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas

Cryopreservation of sperm from tetraploid organisms (the possession of four chromosome sets) is essentially unexplored. This is the first cryopreservation study to address sperm from tetraploid Pacific oysters, Crassostrea gigas, and addresses the commercial production of triploid oysters (three chromosome sets). Initial motility, refrigerated storage of undiluted sperm, osmolality of extender solutions, sperm concentrations, equilibration time, and cryoprotectants of propylene glycol and dimethyl sulfoxide were evaluated with sperm from diploid and tetraploid oysters. Unlike most teleost fishes, in which the duration of active motility is typically brief, the motility of sperm from oysters lasts for hours. The present study showed that responses to treatment effects by sperm from tetraploids were different from diploids. The majority of tetraploid experiments resulted in less than 10% motility after thawing and less than 5% fertilization. The highest fertilization obtained for thawed sperm was 96% for sperm from diploid oysters and 28% for sperm from tetraploid oysters. Differential responses to treatments by sperm from tetraploid and diploid oysters may be due to differences in gonadal development. However, the use of cryopreserved sperm from tetraploid Pacific oysters produced 100% triploid offspring by fertilization of eggs from diploid females as determined by flow cytometry of larvae. This study demonstrates that sperm from tetraploid oysters can be collected, frozen, and stored for production of triploid offspring.     

Comparative characterization of Santolina insularis chemotypes by essential oil composition, 5S-rRNA-NTS sequencing and EcoRV RFLP-PCR

Santolina insularis (Genn ex Fiori) Arrig. is a medicinal plant whose essential oil shows antiviral and antibacterial activities and potent and selective cytotoxic activity against the human colon carcinoma cell line. The occurrence of several chemotypes makes the taxonomic identification of S. insularis hard to achieve.GC–MS essential oil analyses of four chemotypes (SI1, SI2, SI3 and SI4) revealed the presence of different percentages of santolina triene, β-pinene, myrcene, β-phellandrene, artemisia ketone and cis-chrysanthemol, allowing a chemical discrimination. Single fragments of the 5S-rRNA-NTS region of approximately 150, 170, 260 and 280 bp were produced by SI1, SI2, SI3 and SI4, respectively, and the sequence alignment of the 5S-rRNA spacer region flanked by the 3′-and 5′-ends of the coding region confirmed a consistent difference between chemotypes. Furthermore, a PCR-RFLP method was applied. From the identified sequences, an EcoRV site could be found in chemotypes SI1, SI2 and SI3 in the 5S-rRNA spacer regions at 81 bp position; however, this site was absent in the chemotype SI4.This study, by showing remarkable chemical variation in the terpenoid profile and consistent genomic difference in the 5S-rRNA spacer regions, identified four chemotypes of S. insularis which could be grouped into two ecotypes, based on chemical and genomic analyses. The identification of specific gene sequences of the 5S-rRNA-NTS region and of a EcoRV site identified in this work can be used for a rapid and precise identification of the plant chemo-/ecotypes, complementing the essential oil chemical analysis.     

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm.     

Flesh qualities and muscle fiber characteristics in triploid and diploid rainbow trout

Analyzed with regard to their slaughter weights and flesh quality 78‐month‐old diploid and triploid rainbow trout (full sibs) were reared together in a pond after tagging at an age of 12 months. Triploids had higher body weights and carcass percentages than diploids (6 kg vs 4 kg and 66% vs 52%). Triploid fish also displayed lower electrical conductivity values and darker (L* value) and redder (a* value) flesh color. The fillets of the triploid trout contained more crude fat and less moisture than the diploids (6% vs 3% and 68% vs 74%, respectively). No effect of ploidy was found with regard to the protein contents. Triploid rainbow trout had larger mean white and intermediate muscle fiber areas than diploid fish in the dorsal and pelvic fin regions. In the pelvic fin part, the white muscle fiber areas were larger than in the dorsal fin part. In conclusion, adult triploid rainbow trout grow faster especially by fiber hypertrophy and have better flesh quality parameters than diploid fish.     

Comparison of gene representation between diploid and triploid Paragonimus westermani by expressed sequence tag analyses

Expressed sequence tag (EST) analysis of the diploid and triploid Paragonimus westermani genes was done to have a rapid and informative outlook of the gene-expression profiles of the parasites. Totals of 506 and 505 ESTs were generated from the diploid and triploid P. westermani cDNA libraries. Based on the BLASTx search results of the diploid P. westermani ESTs, 308 (60.9%) matched significantly with formerly identified genes and 198 (39.1%) showed no significant homology in the GenBank database. A similar homology pattern was shown from the triploid EST BLASTx search results with 346 (68.5%) sharing homology with previously identified genes and 159 (31.5%) showing no significant homology. The EST data from both libraries were analyzed and grouped into 9 categories. Comparison of the 2 EST pools revealed high similarities among the categories of the significantly matched genes. Single genes matched repeatedly were also observed in the 2 EST data. Some genes were found that are not yet characterized in P. westermani; these genes were matched by both the diploid and triploid ESTs. Further study of these genes may provide us with more understanding on the parasite's biology and their specific functions in the 2 strains.     

Evaluation of anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and synergy of some bioactive plant extracts

Anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of ethanolic extracts of four medicinal plants namely Acorus calamus (rhizome) Hemidesmus indicus (stem), Holarrhena antidysenterica (bark), and Plumbago zeylanica (root), were detected with inhibition zone size ranged from 11 to 44 mm and minimum inhibitory concentration (MIC) varied from 0.32 to 3.25 mg/mL. Further, ethyl acetate, acetone and methanol fractions of above plants demonstrated antibacterial activity. The potency of these fractions based on zone of inhibition and MIC value was relatively higher in P. zeylanica (ethylacetate fraction), followed by acetone fractions of H. indicus, A. calamus, and H. antidysenterica. Time kill assay with most promising fractions of these plant extracts, demonstrated concentration-dependent killing of MRSA within 9-12 h of incubation. Interestingly, synergistic interaction among alcoholic extracts and some fractions of above four plants was evident against MRSA. Further, synergistic interaction of these extracts was detected with one or more antibiotics tested (tetracycline, chloramphenicol, ciprofloxacin, cefuroxime and ceftidizime). The findings also validate the traditional uses of above plants against infectious diseases. Phytochemical studies demonstrated flavonoids and phenols as major active constituents. Further investigations are needed to characterize the active principle and its interaction mechanism with antibiotics.     

Antimutagenic activity of methanolic extracts of four ayurvedic medicinal plants

Methanolic extracts of Acorus calamus (Rhizome), Hemidesmus indicus (Stem), Holarrhena antidysenterica (Bark) and Plumbago zeylanica (Root), were tested for their antimutagenic potential. These extracts, at tested concentrations, showed no sign of mutagenicity to Salmonella typhimurium tester strains. The extracts of the plants exhibited varying level of antimutagenicity. At a dose of 100 microg/plate, the extracts exhibited the inhibition of His+ revertants from 18.51% to 82.66% against direct acting mutagens, methyl methanesulphonate (MMS) and sodium azide (NaN3) induced mutagenicity in Salmonella tester strains TA 97a, TA 100, TA 102 and TA 104. However, at lower concentrations (25 and 50 mcirog/plate) of the plant extracts, a decrease in antimutagenic activity was recorded. Dose dependent antimutagenic activity of the extracts is also evident from linear regression analysis of the data. The over all antimutagenic potential of above four extracts was found to be in order of A. calamus > H. indicus > H. antidysenterica > P. zeylanica. Further, total phenolic content of these extracts did not correlate with its antimutagenic activity in A. calamus and P. zeylanica.     

基于三个DNA片段讨论菖蒲科的分子系统学

菖蒲属长期以来被置于天南星科,但近年来分子系统学的研究表明菖蒲属与天南星科的关系较远,应成立一个单型科,即菖蒲科（Acoraceae)。本文选择菖蒲科3个种及1个变种即金钱蒲（Acorus gramineus),石菖蒲（A.ttrinowii)。金边菖蒲（A.tatarinowii),金边菖蒲（A.tatarinowii var.flawo-marginatus)和菖蒲（A.calamus)不同染色体倍性的4个居群进行了核糖体RNA内源转录间隔段（ITS）进行测序。并据此讨论了菖蒲属的属下系统关系,结果表明,菖蒲科为一个自然的单系类群,不同倍性的菖蒲构成一个自展数据支持率为100%的分支,无中肋的金钱蒲,石菖蒲和金边石菖蒲构成另一个自展数据支持率为100%的分支,通过比较分析菖蒲科与其相关类群之间18S和rbcL两段DNA序列,对菖蒲科的系统位置进行了讨论,在基于18S和rbcL的系统树中,菖蒲均处于单子叶植物中较为孤立的位置。     

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

The origin of the triploid in Paragonimus westermani on the basis of variable regions in the mitochondrial DNA

Triploid, parthenogenetic forms of the lungfluke, Paragonimus westermani, occur in Japan, Korea and China. The origin(s) of triploidy has been debated over the years. Sequences of two regions in the mitochondrial DNA, i.e. partial lrRNA (16S), and a portion of the non-coding region, were obtained from natural populations of P. westermani. All triploid individuals (Japan, Korea, China) and a single tetraploid individual (China) had identical sequences in the 16S region studied. Some sequence variation was observed among diploids, with those from Taiwan being distinct from the remainder. Both neighbour joining and parsimony trees using the 16S region placed diploid individuals from southwestern Japan close to the triploids and the tetraploid. The fragment amplified from the mitochondrial non-coding region showed dimorphism. One form (type A) consisted of 239 bp comprising two identical tracts of 70 bp separated by a tract of 93 bp. The second form (Type B) consisted of only a single 70 bp tract. All diploid individuals from Taiwan, China and Korea possessed type A, while those from Japan were polymorphic; individuals from Oita and Hyogo had type B, those from Chiba had type A, but both types were found in Mie. On the other hand, all of the triploid individuals and two tetraploid individuals possessed type B. Both the form present in the non-coding region and the 16S sequence suggest an affinity between a south-eastern group of diploid populations in Japan and the triploid form. A possible mechanism responsible for the origin of the triploid is discussed.     

STABLE DIPLOIDS FROM INTRAGROUP ZYGOSPORES OF CLOSTERIUM EHRENBERGII MENEGH. (CONJUGATOPHYCEAE)1

Two pairs of stable diploid clones were obtained as aberrant forms among F₁ progeny of an intragroup (intraspecific) cross between R-11-4 (mating type +) and M-16-4b (mating type -) of Group A of Closterium ehrenbergii Menegh. Each pair was derived from the two germination products of a single zygospore, and both clones were mating type minus. The cell size range of these four diploid minus clones was considerably above that of normal (haploid) Group A clones. Chromosome counts at the second meiotic metaphase indicated that these clones were diploid with approximately 200 chromosomes, which was double the number for normal Group A clones. Diploid minus clones conjugated normally with any haploid Group A plus clones, and yielded many triploid zygospores. Triploid zygospores germinated normally as did intragroup diploid zygospores. In metaphase I preparations, only bivalents were observed except on a few occasions where some uni- and multivalents were also detected. Viability of F₁ progeny from triploid zygospores (55–74%) was somewhat lower than from diploid zygospores of Japanese Group A populations (65–90%), but higher than intergroup (interspecific) hybrid zygospores from Groups A, B and H (0–12%). In addition to lower viability, some F₁ progeny from triploid zygospores exhibited slow vegetative growth. Almost all pairs of F₁ clones from single triploid zygospores were of opposite mating type, similar to normal diploid zygospores of the intragroup cross. Morphological variability of F₁ progeny of triploid zygospores was great. The apparently normal meiosis of triploid zygospores and the high viability of F₁ progeny suggested that the genome of Group A contains several sets of chromosome complements with mechanisms by which bivalents are regularly formed in the first meiotic division.     

Cytological investigations on two species of allocreadiid trematodes with special reference to the occurrence of triploidy and parthenogenesis in Allocreadium fasciatusi

Karyotypes and cytological details of gametogenesis were analysed for Allocreadium handiai and A. fasciatusi. Mitotic plates studied in squashes of testes, ovary, eggs and intramolluscan stages showed that A. handiai is a diploid (2n = 14) with six pairs of submetacentrics and one pair of subtelocentrics. Total chromosome length of the diploid complement was 86.57 μm, the largest chromosome measured 8.87 μm (10.24% total chromosome length [TCL]) and the smallest was 2.83 μm (3.28% TCL). Squashes of testes revealed the presence of all stages of spermatogenesis with spermatocytes in various stages of meiotic activity and spermatids containing bundles of spermatozoa. Stages of development of the ovum conclusively proved that reproduction takes place by amphimixis. Mitotic figures of A. fasciatusi, on the other hand, revealed that it is a triploid (3n = 21) with three metacentrics, 12 submetacentrics and six subtelocentrics. The mean total chromosome length of the triploid complement was 137.54 μm. The largest chromosome measured 10.37 μm (7.54% TCL) and the smallest measured 2.67 μm (1.94% TCL). Spermatogenesis was abortive with no evidence of synaptic pairing and spermatozoa were not produced. Eggs remained unfertilized and reproduction was achieved by parthenogenesis which is of mitotic type. The karyotypes of the two species differed not only in the ploidy level but also in the centromeric indexes of certain chromosomes.     

The feeding response of diploid and triploid Atlantic salmon and brook trout

Pair and group experiments were conducted to determine whether differences exist in feeding success between juvenile diploid and triploid salmonids in a competitive situation. In the pair experiments, 22 pairs (one diploid and one triploid) of size-matched Quebec-strain brook trout Salvelinus fontinalis (7·2–46·3 g) were fed an unlimited number of pellets three times a day for 5 days. Dominance was assigned to the fish which ate the most pellets within each pair. In the group experiments, groups of three diploid and three triploid size-matched fish were fed a restricted ration three times a day for 5 days. Hierarchical rank within the group was assigned based on the number of pellets consumed by each fish. The group experiment was repeated 10 times with Atlantic salmon Salmo salar (5·1–62·7 g), Quebec-strain brook trout (11·8-110·8 g), and large UNB-strain brook trout (18·2–33·0 g), and 12 times with smaller UNB-strain brook trout (0·6–2·0 g). A statistically significant difference in rank between ploidies was found only for the smaller UNB-strain brook trout in the group experiments, with diploids dominant over triploids. This suggests that there may be a difference in competitive feeding success between diploid and triploid brook trout early in development, but that this difference diminishes as the fish grow.