Development of SCAR marker for simultaneous identification of Miscanthus sacchariflorus, M. sinensis and M. x giganteus

The sequence-characterized amplified region (SCAR) marker for simultaneous identification of Miscanthus sacchariflorus, Miscanthus sinensis, and Miscanthus x giganteus was developed. In this study, it was attempted for the first time to develop the SCAR marker for detecting the molecular phenotypes among Miscanthus species. Randomly amplified polymorphic DNA technique was applied for this study and one fragment which is unique to M. sacchariflorus was identified and then sequenced. Based on the specific fragment, one SCAR primer pair designated as MS62-5F and MS62-5R was designed to amplify an approximately 1,000 bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer pair. Using this SCAR marker, approximately 1,000 bp and 1,200 bp DNA fragments were obtained in M. sacchariflorus and M. sinensis, respectively. Moreover, M. x giganteus was obtained both bands at the same time. The result showed that this SCAR marker can clearly distinguish the M. sacchariflorus, M. sinensis, and M. x giganteus, respectively.     

SSR-based genetic maps of Miscanthus sinensis and M. sacchariflorus, and their comparison to sorghum

We present SSR-based genetic maps from a cross between Miscanthus sacchariflorus Robustus and M. sinensis, the progenitors of the promising cellulosic biofuel feedstock Miscanthus × giganteus. cDNA-derived SSR markers were mapped by the two-way pseudo-testcross model due to the high heterozygosity of each parental species. A total of 261 loci were mapped in M. sacchariflorus, spanning 40 linkage groups and 1,998.8 cM, covering an estimated 72.7% of the genome. For M. sinensis, a total of 303 loci were mapped, forming 23 linkage groups and 2,238.3 cM, covering 84.9% of the genome. The use of cDNA-derived SSR loci permitted alignment of the Miscanthus linkage groups to the sorghum chromosomes, revealing a whole genome duplication affecting the Miscanthus lineage after the divergence of subtribes Sorghinae and Saccharinae, as well as traces of the pan-cereal whole genome duplication. While the present maps provide for many early research needs in this emerging crop, additional markers are also needed to improve map density and to further characterize the structural changes of the Miscanthus genome since its divergence from sorghum and Saccharum.     

芒属植物荻愈伤组织的诱导与植株再生

利用资源植物荻(Miscanthus sacchariflorus)的幼穗作为外植体,通过愈伤组织途径建立了植株再生体系.结果表明:在附加600 mg·L-1脯氨酸、500 mg·L-1水解酪蛋白、2.0mg ·L-12,4-D和0.1mg·L-16-BA的MS培养基上能高效诱导出生长良好的愈伤组织;将其转移到MS+1.5 mg·L-1 6-BA培养基上诱导产生芽,再转移到1/2 MS+0.25 mg·L-1 NAA+0.25 mg·L-1 IAA培养基上生根后,可发育为生长健壮的植株.     

Genetic Differentiation between Sympatric Populations of Bacillus cereus and Bacillus thuringiensis

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species—B. thuringiensis or B. cereus—were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.     

Silicon Distribution and Anatomy of the Grass Rhizome, with Special Reference to Miscanthus sacchariflorus (Maxim.) Hackel.

Rhizome anatomy is described for Miscanthus sacchariflorus (Maxim.)Hackel. Solid silica deposits, detected as elemental siliconby electron-probe microanalysis and energy-dispersive X-rayanalysis, are confined to cell walls of three concentric zonesconsisting of the uniseriate epidermis, and parenchyma layersaround the cortical air lacunae, and the central cavity, respectively.Si is localized in outer tangential walls of the epidermis,while occurring in all walls of nucleated, parenchyma cellsforming the two internal zones. In comparison, the root exhibitsonly one Si zone. Rhizome Si distribution more closely resemblesthat for Phragmites australis, than for related members of theAndropogoneae. P. australis similarly exhibits aerenchyma anda central cavity. Thus, internal anatomy may strongly influencesilicon distribution. A comparison of taxa of four tribes indicatesthat epidermal wall deposition is common, followed by specificinternal localization in up to three zones of perivascular tissues. Silicon accumulation occurs early in the epidermis of the youngapex of M. sacchariflorus, decreasing sharply across an internodetransection. In comparison, the oldest, basal internodes exhibitvery high Si X-ray counts in each of the three zones, the highestoccurring in the most internal zone around the central cavity.Early Si mobilization in the rhizome apex may resist shearingand abrasion during horizontal growth extension, while depositsbordering aerenchyma of older internodes may resist compression. Miscanthus sacchariflorus (Maxim.) Hackel, plume grass, rhizome, silicification, anatomy, aerenchyma     

Genetic Analysis of Natural Populations of DROSOPHILA MELANOGASTER in Japan. III. Genetic Variability of Inducing Factors of Amylase and Fitness

"Inducibility" of amylase in Drosophila melanogaster was defined and investigated in a natural population from Japan. Inducibility represents the effects of factors remote from the structural gene that control the amount of enzyme produced. Inducibility of an isogenic line is measured as the ratio of the enzyme's specific activity in two different inducing environments. There was considerable genetic variability with respect to inducibility of amylase in 44 isogenic lines derived from a natural population of D. melanogaster . Net fitness and its components in these isogenic lines were also measured. The results indicated that, although the inducibility of the enzyme was positively correlated with the net fitness (r_g = 0.63 ± 0.2), the enzyme activities in the normal medium were not (r_g = 0.12 ± 0.37). The analysis of the data shows that the differences in inducing factors are mainly responsible for the differences in the fitness of lines and are the genetic materials for the adaptive evolution of organisms.     

A new genotype of Miscanthus sacchariflorus Geodae-Uksae 1, identified by growth characteristics and a specific SCAR marker

Miscanthus is referred to as an ideal lignocellulosic bioenergy crop, which can be used to generate heat, power, and fuel, as well as to reduce carbon dioxide emissions. The new Miscanthus sacchariflorus genotype named Geodae-Uksae 1 was recently collected from damp land in southern Korea. This study investigated the growth characteristics of Miscanthus genotypes, and developed a specific, sensitive, and reproducible sequence characterized amplified region (SCAR) marker to distinguish new M. sacchariflorus genotype Geodae-Uksae 1 from other native Miscanthus species in Korea. Growth characteristics such as stem length, stem diameter, and dry weight of Geodae-Uksae 1 were greater than those of normal M. sacchariflorus. The genotypes within Geodae-Uksae 1 were had the highest genetic similarity. A putative 1,800-bp polymorphic sequence specific to Geodae-Uksae 1 was identified with the random amplified polymorphic DNA (RAPD) N8018 primer. The sequence-characterized amplified region (SCAR) primers Geodae 1-F and Geodae 1-R were designed based on the unique RAPD amplicon. The SCAR primers produced a specific 1,799-bp amplicon in authentic Geodae-Uksae 1, whereas no amplification was observed in other Miscanthus species. The SCAR marker could contribute to identify Geodae-Uksae 1 among native Miscanthus species. The new Miscanthus genotype Geodae-Uksae 1 has great potential as an alternative lignocellulosic biomass feedstock for bioenergy productions.     

长萼堇菜多倍体复合体种群的分布格局研究

采用常规压片方法观察了长萼堇菜Viola inconspicua Blume多倍体复合体18个种群的染色体数目,发现其中4个种群的染色体为2n=4x=24,14个种群的染色体为2n=8x=48.讨论了长萼堇菜四倍体和八倍体种群的分布,并推测其可能的起源和造成目前分布格局的原因.     

Analysis of lignocelluloses and lignins from Arundo donax L. and Miscanthus sinensis Anderss., and hydroliquefaction of Miscanthus

In a comparative investigation of the chemical composition of Arundo donax L. and Miscanthus sinensis Anderss. the following experiments were performed: ash determination and ash characterization by energy dispersive X-ray analysis; determination of solubility in cyclohexane/ ethanol, hot water, 1% hydrochloric acid and sodium hydroxide; C, H and N determination; determination of Klason lignin and acid soluble lignin content; sulfuric acid hydrolysis followed by borate complex ion exchange chromatography of the monomeric sugars; isolation of milled wood lignins (MWL) and dioxane lignins (DIL) and their analysis by C, H, and OMe determination; quantitative FTIR spectroscopy of MWL; recording the molecular weight distribution curves using high performance size exclusion chromatography (HPSEC); calculation of average molecular weights, such as M_w and M_n; calculation of the heating values of the lignocellulosics and their components. The quantitative composition of the lignin from the three basic phenylpropane units is presented.M. sinensis was submitted to hydroliquefaction. The conversion process yielded 35% of a net product oil (NPO) with low oxygen content (11%), low viscosity (10⁻² NS m⁻²), low asphaltene content (3·5%) low molecular weight (M_w 200) and with a specific gravity of c. 0·93 g cm⁻³. The NPO has a heating value of 39·4 MJ kg⁻¹ and contains 55% of the carbon of the starting material and 59% of the combined heating value from the biomass and the hydrogen used for hydroliquefaction. The process yields 28% water which contains 58% of the original oxygen of the biomass. The process gives rise to 9 g solids and 32–35 g gases, whose energy content can easily be recovered.     

The effects of breaking or bending the stems of two rhizomatous plants, Phragmites australis and Miscanthus sacchariflorus, on their communities

To understand the effects of disturbance on vegetation, bending and cutting experiments were conducted on two rhizomatous plant species, Phragmites australis and Miscanthus sacchariflorus, in a floodplain area of the Arakawa River, Japan. The plants were damaged in the late development growth stage on 3 August 2004 (August disturbance) and in the middle development growth stage on 29 June 2005 (June disturbance). The severity of the damage was evaluated based on shoot morphology and belowground biomass. The recoveries of the two plants from the flood-like artificial disturbance were compared with undisturbed stands. The morphological response of the shoot was higher after the June disturbance than after the August disturbance in both plants. In contrast, the recovery of belowground biomass in P. australis at the end of the growth season was higher after the August disturbance (87 and 72% for bending and cutting, respectively) compared with the June disturbance (84 and 60% for the same). The recoveries in M. sacchariflorus for the two disturbances showed the opposite trend (73 and 59% for bending and cutting, respectively, after the August disturbance, and 90 and 73% after the same disturbance in June). The study demonstrated that an event like flooding, whether it is breaking or bending, will cause greater damage if it occurs at the late development growth stage in M. sacchariflorus compared with the middle development growth stage. In contrast, P. australis tolerated disturbances up to a certain magnitude; after that, the effect was more severe in the middle development growth stage compared with late development growth stage.     

Structure and functioning of Miscanthus sinensis grassland in Sugadaira,Central Japan

To be able to deseribe the structure and functioning of Miscanthus sinensis grassland, its floristie composition, life-form spectra, stand productivity and dry matter allocation were studied. 43 species were to be found during the growing season of 1976. 29 species, and 345 individuals per 1 m², were observed in the sample areas in September. The values for standing crop of constituent species have been arranged in ascending order. The dominant Raunkiaer life-forms were the hemicryptophyte and geophyte. The stand was characterized by erect and tufted forms in Numata's system of growth form classification. The standing crop of the whole stand decreased from April to June, then increased from mid-July. The maximum standing crop of 3346 g m^-2was observed in September. The net production of the stand was 959.4 g m^-2year^-1. Fifty one percent of this net production was allocated to the underground organs which consume it for respiration during winter and for initial growth in the next growing season.     

Genetic Variation Segregating in Natural Populations of Tribolium Castaneum Affecting Traits Observed in Hybrids with T. Freemani

We investigated patterns of within-species genetic variation for traits observed in hybrids (hybrid numbers, hybrid sex ratios, and hybrid male deformities) between two species of flour beetles, Tribolium castaneum and T. freemani. We found genetic variation segregating among four natural populations of T. castaneum as well as within these populations. For some hybrid traits, we observed as much variation among populations 750 km apart as between populations on different continents, suggesting genetic differentiation at a local scale. Within natural populations, the variation segregating among sires is greater than that found in an earlier study for an outbred laboratory population and comparable to that observed between inbred lines derived from the outbred stock by eight generations of brother-sister mating. When sires from T. castaneum are mated to conspecific and heterospecific females, we do not observe a significant correlation at the level of the family mean between the intraspecific and interspecific phenotypes, suggesting the independence of the hybrid traits from comparable traits within species. We discuss our findings in relation to the evolutionary genetics of speciation and the expression of epistatic genetic variance in interspecific crosses.     

Genetic Analysis of Natural Populations of DROSOPHILA MELANOGASTER in Japan. II. the Measurement of Fitness and Fitness Components in Homozygous Lines

Fifty lethal-free, sterility-free isogenic lines of Drosophila melanogaster that were randomly sampled from a natural population were tested for net fitness and other components of fitness by competition with D. hydei. Larval viability and developmental time were also measured using the balanced marker method. Distribution patterns of these fitness components were similar, but correlation between the fitness components varied depending on the combinations used. The highest correlations were obtained between net fitness and productivity (r_p = 0.6987, r_g = 0.9269). The correlation between net fitness and total larval viability was much lower (r_p = 0.1473 and r_g = 0.2171). These results indicate that measuring net fitness, not just a component of fitness, is necessary for the good understanding of the genetic structures of natural populations.     

红泡刺藤居群的遗传多样性研究

利用ISSR分子标记对红泡刺藤的12个居群共242个个体进行了遗传多样性分析.结果表明:(1)16个ISSR引物共扩增到257个位点,其中236个是多态性位点,占91.83％.(2)红泡刺藤居群具有较高水平的遗传多样性,在物种水平上,平均每个位点的多态位点百分率为97.50％,有效等位基因数为1.267,Nei's遗传多样性为0.177,Shannon's多态信息指数为0.296;居群水平上多态位点百分率为51.43％,有效等位基因数为1.205,Nei's 遗传多样性为0.127,Shannon's多态信息指数为0.202.(3)居群间基因分化系数为0.2815,AMOVA分析居群间遗传变异占总量的34.47％,二者结果相近,说明红泡刺藤居群间存在一定程度的遗传分化.居群内的遗传变异为65.53％,基因流为1.2762.(4) Mantel检测显示居群间的遗传距离与地理距离不存在相关性.UPGMA聚类分析和二维主成分分析结果一致,红泡刺藤居群可分为2个居群组,即金沙江居群和维西居群为一个类群,其他居群为另外一大类,表明生态地理条件相似的居群优先集中.