八角莲组织培养的器官发生途径研究

为探究小檗科植物八角莲组织培养的器官发生方式,该研究以八角莲离体叶片、叶柄在MS培养基上诱导产生的愈伤组织、不定芽、不定根为对象,用连续石蜡切片技术分析八角莲组织培养的器官发生途径。结果表明:八角莲愈伤组织形成的解剖学特征是靠近表皮的薄壁细胞经激素刺激恢复分裂能力,继续培养形成拟分生组织。拟分生组织可形成许多分化中心。通过对八角莲组织培养产生的不定芽细胞组织学观察发现芽原基起源于愈伤组织外侧的几层薄壁细胞,芽原基背离愈伤组织中央生长形成不定芽,故八角莲脱分化形成的芽起源方式为外起源。而八角莲的根原基起源于组织深处髓部薄壁细胞和部分维管形成层细胞,进而形成类似球形或楔形并朝韧皮部突起的根原基轮廓,根原基继续发育会突破表皮生成不定根,起源方式为内起源。八角莲离体再生途径为器官发生型,在组培苗生长过程中先诱导形成不定芽,再诱导形成不定根,在愈伤组织上形成维管组织将不定芽和不定根连接成完整植株。     

Development and characterization of microsatellite markers for a mangrove tree species Sonneratia caseolaris (L.) Engler (Lythraceae sensu lato)

Sonneratia caseolaris is a typical non-viviparous mangrove species and a key component of mangrove community in the Indo-West Pacific region. Here we isolated nine microsatellite simple sequence repeat (SSR) loci from the genome of S. caseolaris. Our isolated loci provided SSR markers with polymorphism of 2–6 alleles per locus. The expected and observed heterozygosities ranged from 0.242 to 0.745 and from 0.083 to 0.417, respectively. Cross-species amplification in S. alba and S. ovata showed that a subset of these markers holds promise for these congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on S. caseolaris as well as other congeneric species.     

Isolation and characterization of microsatellites in Drosophila montana and their cross‐species amplification in D. virilis

We report the isolation of 20 microsatellite loci from Drosophila montana and their cross amplification in the relative D. virilis. All microsatellite loci were polymorphic in the focal species D. montana, with gene diversities ranging from 0.23 to 0.93. In D. virilis only eight loci (40%) amplified and two loci were polymorphic (10%). These markers represent the first report of microsatellites isolated in D. montana. They could be applied for studying population structure and phylogeography. The largest benefit, however, will be their use in studies of quantitative trait loci, such as the mapping of behavioural quantitative trait loci.     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

Isolation of eight microsatellites loci from the saddled bream, <Emphasis Type="Italic">Oblada melanura</Emphasis> and cross-species amplification in two sea bream species of the genus <Emphasis Type="Italic">Diplodus</Emphasis>

We have developed eight new microsatellite markers for the saddled bream (Oblada melanura) from an enriched genome library protocol. All these loci are polymorphic, with mean allelic diversity of 14.75 (range 3–22), and expected and observed heterozygosities from 0.233 to 0.918 and 0.212 to 0.913, respectively. Cross-species tests in two close relatives of the genus Diplodus (D. sargus and D. vulgaris) revealed successful amplifications at 6 out of 8 loci, with means allele number of 6.67 (range 4–10) and 6.50 (range 4–10), respectively. These results are consistent with the close phylogenetic relationships between the three species, indicating this set of primers might proved useful for studying the levels of genetic diversity and population differentiation in these three species and in other phylogenetically close species of the genus Diplodus and Sparus.     

Isolation and characterization of microsatellite loci in the Japanese pipistrelle (Pipistrellus abramus)

We describe the first set of ten microsatellite markers isolated in Pipistrellus abramus. The number of alleles per locus ranged from 7 to 13. The observed and expected heterozygosities values ranged from 0.486 to 0.971 and from 0.752 to 0.876, respectively. Three loci revealed significant departure from Hardy–Weinberg equilibrium and no linkage disequilibrium was found between loci pairs. These informative microsatellite markers will be a powerful molecular tool for studying the population genetic structure of P. abramus, as well as other species of this genus.     

Isolation and characterization of eleven microsatellite loci in small abalone, Haliotis diversicolor Reeve

Eleven novel microsatellite markers were isolated from small abalone, Haliotis diversicolor Reeve. These loci were tested on 22 individuals from two different geographic populations. We identified a total of 162 alleles from the 11 microsatellite loci. All of the loci were highly polymorphic. Polymorphism information content (PIC) is ranging from 0.7276 to 0.9163. Observed and expected heterozygosities ranged from 0.2727 to 1.0000 and from 0.7738 to 0.9429, respectively. Three loci deviated significantly from Hardy–Weinberg equilibrium. No pairs of loci displayed linkage disequilibrium. These polymorphic markers will be used to analyze population structure, genetic diversity and construct a genetic linkage map. Xin Zhan and Hai-Yan Hu contribute equally to this study.     

Characterisation of 14 blue crane <Emphasis Type="Italic">Grus paradisea</Emphasis> (Gruidae,AVES) microsatellite loci for use in detecting illegal trade

Forty-two unique microsatellite loci were isolated from an unenriched and a tetranucleotide-enriched blue crane (Grus paradisea) genomic library. Fourteen polymorphic loci were characterised in 20 unrelated wild blue crane individuals from the Karoo region, South Africa, and displayed 4–27 alleles with observed heterozygosities ranging between 0.50 and 0.95. All 14 loci were also polymorphic in the grey-crowned crane (Balearica regulorum) and the wattled crane (Grus carunculatus). These markers are aimed at assisting the identification of illegal trade in the blue crane but have wider population and conservation applications for most, if not all, crane species.     

Development and characterization of novel tetra-, tri- and di-nucleotide microsatellite markers in cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.)

The cacao plant, Theobroma cacao L., produces white seeds (beans) that form the major ingredient of processed chocolate. A great deal of research effort has been expended to the development of new genetically modified cacao plants with improved productivity and resistance and beans of good industrial quality. The availability of suitable genetic markers is an important aspect of the efficient selection and breeding of this perennial species. We describe the development of 123 microsatellite loci of cacao. An optimized protocol was used to construct and screen a microsatellite-enriched genomic library from which we isolated 64 di-nucleotide, 45 tri-nucleotide and 14 tetra-nucleotide microsatellite loci. The primers were tested on samples from five different T. cacao accessions, one accession from T. grandiflorum and one accession from Herranea sp. Among the 123 loci, 54 were polymorphic, 61 were monomorphic and eight did not present an amplification product. These new markers will be useful in future studies by increasing the accuracy of genotypic assessments in diverse cocoa tree populations as well as in other species of the Theobroma genus.     

Development and characterization of polymorphic microsatellite DNA markers for Sasa senanensis (Poaceae: Bambuseae)

Sasa senanensis is a dwarf bamboo species distributed on the floors of cool temperate forests in Japan and adjacent regions. We isolated eight polymorphic microsatellite loci from this species. The number of alleles ranged from two to eight and the observed heterozygosity per locus from 0.13 to 0.74. Seven of the eight loci were also polymorphic in Sasa nipponica. Most of these markers were successfully amplified in other dwarf bamboo species. These markers will be useful for investigating clonal structure and population genetics in some dwarf bamboo species.     

Low temperature enhanced the podophyllotoxin accumulation vis-a-vis its biosynthetic pathway gene(s) expression in Dysosma versipellis (Hance) M. Cheng – A pharmaceutically important medicinal plant

Dysosma versipellis (Hance) M. Chang is an endemic and endangered Chinese medicinal herb. Podophyllotoxin, a lead compound for the semi-synthesis of clinically useful anticancer compounds, is the main active aryltetralin lignan accumulated in D. versipellis. In this study, morphology and physiology traits in response to podophyllotoxin accumulation and its biosynthetic pathway genes expression were investigated in D. versipellis plants grown at low temperature (4∼6 °C) and greenhouse (25∼30 °C) temperature. Our study found that low temperature significantly increased plant biomass (2.7-fold), chlorophyll, and carotenoid content compared to greenhouse temperature. The mRNA expression analysis of podophyllotoxin pathway genes secoisolariciresinol dehydrogenase and pinoresionol lariciresinol reductase expression levels were also up-regulated by low temperature. In addition, plants grown at low temperature markedly increased podophyllotoxin accumulation to 3.49-fold in rhizome of D. versipellis than the greenhouse temperature. These results suggest that plants at low temperature enhance the accumulation of podophyllotoxin in rhizome of D. versipellis by over-expressing podophyllotoxin pathway genes. This study finding could be useful in the producing pharmaceutically important podophyllotoxin without overexploiting this endangered species.     

Nine new microsatellite loci for the orange roughy (Hoplostethus atlanticus)

We isolated nine new dinucleotide microsatellites for the deep-sea fish Hoplostethus atlanticus. These loci are generally highly polymorphic, with allele number ranging from 3 to 31 and observed heterozygosity ranging from 0.067 to 0.933. In conjuction with previously published loci, these markers will be useful for fine-scale analysis of population structure in this commercially important and threatened species.     

dbEST-derived SSR markers in sea urchin (Hemicentrotus pulcherrimus)

Sea urchin is an important model organism for evolutionary biology, embryology, and gene regulation study. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Strongylocentrotus purpuratus and Hemicentrotus pulcherrimus. Characteristics of nine EST-SSR loci were investigated using 41 Hemicentrotus pulcherrimus individuals. The number of alleles per locus ranged from two to five. The observed heterozygosity ranged from 0.122 to 0.7805, while the expected heterozygosity ranged from 0.4472 to 0.7696. These loci and markers will be useful for population genetics and systemic evolution among species of sea urchin.     

Development of microsatellite markers for <Emphasis Type="Italic">Metrosideros boninensis</Emphasis> (Myrtaceae), an endangered endemic plant species from the Bonin Islands,Japan

Metrosideros boninensis is an endangered endemic plant species from the Bonin Islands. We isolated and characterized nine microsatellite loci in this species. The expected heterozygosities of these nine markers ranged from 0.127 to 0.768. These markers described here will be useful for investigating the genetic diversity, genetic structure and gene flow, and planning for conservation of M. boninensis.     

Linkages between restriction fragment length,isozyme, and morphological markers in lentil

Summary A genetic linkage map of lentil comprising 333 centimorgans (cM) was constructed from 20 restriction fragment length, 8 isozyme, and 6 morphological markers segregating in a single interspecific cross (Lens culinaris × L. orientalis). Because the genotypes at marker loci were determined for about 66 F₂ plants, linkages are only reported for estimates of recombination less than 30 cM. Probes for identification of restriction fragment length polymorphisms (RFLPs) were isolated from a cDNA and EcoRI and PstI partial genomic libraries of lentil. The cDNA library gave the highest frequency of relatively low-copy-number probes. The cDNAs were about twice as efficient, relative to random genomic fragments, in RFLP detection per probe. Nine markers showed significant deviations from the expected F₂ ratios and tended to show a predominance of alleles from the cultigen. Assuming a genome size of 10 Morgans, 50% of the lentil genome could be linked within 10 cM of the 34 markers and the map is of sufficient size to attempt mapping of quantitative trait loci.     

Antiproliferation and cell apoptosis inducing bioactivities of constituents from Dysosma versipellis in PC3 and Bcap-37 cell lines

Background

Recently, interest in phytochemicals from traditional Chinese medicinal herbs with the capability to inhibit cancer cells growth and proliferation has been growing rapidly due to their nontoxic nature. Dysosma versipellis as Bereridaceae plants is an endemic species in China, which has been proved to be an important Chinese herbal medicine because of its biological activity. However, systematic and comprehensive studies on the phytochemicals from Dysosma versipellis and their bioactivity are limited.

Results

Fifteen compounds were isolated and characterized from the roots of Dysosma versipellis, among which six compounds were isolated from this plant for the first time. The inhibitory activities of these compounds were investigated on tumor cells PC3, Bcap-37 and BGC-823 in vitro by MTT method, and the results showed that podophyllotoxone (PTO) and 4'-demethyldeoxypodophyllotoxin (DDPT) had potent inhibitory activities against the growth of human carcinoma cell lines. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in PC3 and Bcap-37 cells, and the apoptosis ratios reached the peak (12.0% and 14.1%) after 72 h of treatment at 20 μM, respectively.

Conclusions

This study suggests that most of the compounds from the roots of D. versipellis could inhibit the growth of human carcinoma cells. In addition, PTO and DDPT could induce apoptosis of tumor cells.     

Development of single sequence repeat markers for the ant Aphaenogaster senilis and cross-species amplification in A. iberica, A . gibbosa, A. subterranea and Messor maroccanus

Eight polymorphic microsatellite loci were isolated and characterized for Aphaenogaster senilis, a common ant species distributed in the Western Mediterranean. Characterization of 15 individuals form southern Spain showed moderate to high allelic diversity ranging from 2 to 9 alleles per locus. Cross-species tests on 10 individuals of A. iberica, A. gibbosa, A. subterranea and Messor maroccanus revealed successful amplification for most loci. This set of markers can be useful for population genetic studies and might even prove useful in other phylogenetically close species of the subfamily Myrmicinae.     

Wheat genome structure: translocations during the course of polyploidization

The genomic organization of Triticum timopheevii (2n=28, A^tA^tGG) was compared with hexaploid wheat T. aestivum (2n=42, AABBDD) by comparative mapping using microsatellites derived from bread wheat. Genetic maps for the two crosses T. timopheevii var. timopheevii × T. timopheevii var. typica and T. timopheevii K-38555×T. militinae were constructed. On the first population, 121 loci were mapped, and on the second population 103 loci. The transferability of the wheat markers to T. timopheevii was generally better for the A genome-specific markers (76–78% produced amplification products; 26 and 29% were polymorphic) than for B genome-specific markers (54% produced amplification products; 14 and 16% were polymorphic). Of the D genome-specific markers, one third produced amplification products in T. timopheevii, but only 5 and 2% were polymorphic in the corresponding mapping populations. The maps constructed confirmed the previously described translocation between chromosome arms 6A^tS and 1GS and revealed at least two yet unknown rearrangements on chromosomes 4A^t and 6A^t. The presence of other translocations and rearrangements between T. timopheevii and T. aestivum was demonstrated by a variety of markers mapping to nonhomoeologous positions.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Characterization of novel microsatellite loci in the great leaf-nosed bat, Hipposideros armiger and cross-amplification in other related species

Eight microsatellite loci were isolated from an enriched genomic library of the great leaf-nosed bat, Hipposideros armiger. The polymorphism of these loci was tested on a population of 48 individuals from Anhui Province, China. All loci revealed the polymorphism ranging from three to 12 alleles. The observed heterozygosity values were from 0.213 to 0.875 and expected heterozygosity from 0.232 to 0.820. No significant linkage disequilibrium was detected. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. In addition, successful cross-amplification also suggested that these microsatellite markers will facilitate research on the population genetics and gene flow of H. armiger and other related species.