Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

Estimation of nuclear DNA content in Nasturtium R. Br. by flow cytometry

The most common and widespread species of Nasturtium in central Europe are the tetraploid Nasturtium officinale (2n = 4x = 32), the octoploid Nasturtium microphyllum (2n = 8x = 64), and their hexaploid hybrid Nasturtium × sterile (2n = 6x = 48). For the first time, flow cytometry was used to measure the genome size (2C DNA content) of these taxa. The highest nuclear DNA content was found in the octoploid N. microphyllum (2C = 1.43 pg) and the lowest in the tetraploid N. officinale (2C = 0.76 pg). Some differences in the amount of nuclear DNA were observed for the hexaploid N. × sterile (2C = 1.09-1.12 pg). Genome size analysis was thus proposed as a very useful tool for the identification of species of Nasturtium in their vegetative stage.     

Little population structuring and recent evolution of the Pacific saury (Cololabis saira) as indicated by mitochondrial and nuclear DNA sequence data

Genetic population structure of the Pacific saury (Cololabis saira) was investigated using nucleotide sequence analysis on the mitochondrial DNA control region (355-361 bp). Although the left domain of the control region is known to be highly variable in many species, extremely low nucleotide and haplotype diversities (π = 0.17% and h = 0.418, respectively) were observed in a total of 141 individuals collected from five distant locales (East China Sea, Sea of Okhotsk, northwest Pacific, central North Pacific and northeast Pacific). No significant haplotype frequency differences were detected among widely separated samples, therefore we were unable to reject the null hypothesis of no genetic structuring in the Pacific saury population. Moderate levels of nucleotide substitution (p-distance) were observed between the Pacific saury and its Atlantic counterpart (Scomberesox saurus) in the control region (7.44%), cytochrome b gene (4.64%), and internal transcribed spacer (ITS1) (11.49%), indicating that the low sequence diversity of the control region in the Pacific saury is not due to the slow mutation rate. The molecular data suggest the Pacific saury may be a relatively recent offshoot among the extant members in the family Scomberesocidae.     

基于流式细胞术的乌饭树核DNA含量(2C-值)测定

核DNA含量(2C-值)是描述植物生物多样性的一个重要特征参数。该研究利用流式细胞仪检测越橘属植物乌饭树核DNA含量,建立了适合乌饭树的流式细胞术测定方法:以野生乌饭树的嫩叶为材料,以已知核DNA含量的水稻品种‘日本晴’为内标,采用GPB解离液,细胞核悬液加入50μL獉mL-1碘化丙啶染色5 min即可上机检测。结果表明:(1) 9个乌饭树单株的核DNA含量平均值为(1.22±0.03) pg,最小值为1.18 pg,最大值为1.27 pg。(2)检测结果与已知的越橘属二倍体植株的2C-值含量相似,且不同地理来源的单株DNA含量没有显著差异(P>0.05),推测这9个单株为二倍体植株。(3)测定的乌饭树核DNA含量(2C-值)可丰富越橘属植物的C-值库;基于流式细胞术建立的乌饭树核DNA含量测定方法可为该属其他植物的相关研究提供借鉴。     

Phylogenetic relationships of the Aurantioideae (Rutaceae) based on the nuclear ribosomal DNA ITS region and three noncoding chloroplast DNA regions, atpB-rbcL spacer, rps16, and trnL-trnF

The tribes and subtribes of Aurantioideae, an economically important subfamily of the Rutaceae, have a controversial taxonomic history because a phylogenetic framework has been lacking. In order to construct an evolutionary history and evaluate the most recent classification system [Swingle and Reece 1967. The botany of Citrus and its wild relatives, in: The Citrus Industry, vol. 1, History, World Distribution, Botany, and Varieties. University of California, Berkeley, pp. 190-430], one nuclear and three noncoding chloroplast genes were sequenced and analyzed phylogenetically along with selected non-molecular characters. Taxa representing tribes Citreae and Clauseneae and their six subtribes were sampled. In all analyses Aurantioideae is monophyletic. The majority-rule consensus tree from the combined analysis indicates that the two tribes are not monophyletic. The combined topology is not congruent with the widely used classification of Aurantioideae by Swingle and Reece (1967). The tribes and subtribes are in need of revision.     

Patterns of endopolyploidy and 2C nuclear DNA content (Feulgen) inScilla (Liliaceae)

The Feulgen-DNA content of 3558 nuclei from 21 different tissues and organs ofScilla decidua (Liliaceae) was measured by a scanning cytophotometer interfaced to a computer. The basic nuclear DNA content (2 C value) was 13.62 pg, and 71 per cent of the nuclei were polyploid. The highest DNA values were found in the antipodal cells of the ovule, and the elaiosomes of the seeds (512 C). In addition to polyploidy, the 2 C values exhibited tissue-specific variation which was statistically significant (0.05% level of probability), It is suggested that differential DNA replication and endopolyploidization may be basic factors in the complex mechanism of cell and tissue differentiation.Dedicated to Professor Dr.Lothar Geitler in honour of his 80th birthday.     

The effects of nuclear DNA content (C-value) on the quality and utility of AFLP fingerprints

BACKGROUND AND AIMS: Nuclear DNA content (C-value) varies approximately 1000-fold across the angiosperms, and this variation has been reported to have an effect on the quality of AFLP fingerprints. Various methods have been proposed for circumventing the problems associated with small and large genomes. Here we investigate the range of nuclear DNA contents across which the standard AFLP protocol can be used. METHODS: AFLP fingerprinting was conducted on an automated platform using the standard protocol (with 3 + 3 selective bases) in which DNA fragments are visualized as bands. Species with nuclear DNA contents ranging from 1C = 0.2 to 32.35 pg were included, and the total number of bands and the number of polymorphic bands were counted. For the species with the smallest C-value (Bixa orellana) and for one of the species with a large C-value (Damasonium alisma), alternative protocols using 2 + 3 and 3 + 4 selective bases, respectively, were also used. KEY RESULTS: Acceptable AFLP traces were obtained using the standard protocol with 1C-values of 0.30-8.43 pg. Below this range, the quality was improved by using 2 + 3 selective bases. Above this range, the traces were generally characterized by a few strongly amplifying bands and noisy baselines. Damasonium alisma, however, gave more even traces, probably due to it being a tetraploid. CONCLUSIONS: We propose that for known polyploids, genome size is a more useful indicator than the 1C-value in deciding which AFLP protocol to use. Thus, knowledge of ploidy (allowing estimation of genome size) and C-value are both important. For small genomes, the number of interpretable bands can be increased by decreasing the number of selective bases. For larger genomes, increasing the number of bases does not necessarily decrease the number of bands as predicted. The presence of a small number of strongly amplifying bands is likely to be linked to the presence of repetitive DNA sequences in high copy number in taxa with large genomes.     

Population-genetic structure of Chuvashia (from data on eight DNA loci in the nuclear genome)

Population-genetic study of indigenous populations representing three ethnic Chuvash group: highland (Cheboksarsk and Morgaush district), lowland (Kanash district) and mid-lowland (Marposad district). Eight polymorphic DNA loci of the nuclear genome (VNTR/PAH, STR/PAH, VNTR/ApoB, VNTR/DAT1, APF, VNTR/eNOS, IVS6aGATT, and KM.19/PstI) were examined in the population of each district. For each of the four population, we estimated the allele and genotype frequency distributions at each polymorphic system, heterozygosities HS and between-population differences FST. In the combined Chuvash sample, HS = 0.464 and FST = 0.006. Loci VNTR(DAT) and VNTR(ApoB) showed highest between-population differentiation (0.009 < or = FST < or = 0.012), and loci IVS6aGATT, APF, VNTR/eNOS, and D7S23 (KM.19), lowest differentiation (0.001 < or = FST < or = 0.003). Analysis of genetic distances revealed somewhat higher genetic similarity between the Cheboksarsk and Morgaush populations belonging to the highland Chuvash group, whereas the highland Chuvash population from the Marposad district, which belong to the mid-lowland group, was more distant from the former populations.     

Inter-familial relationships of the shorebirds (Aves: Charadriiformes) based on nuclear DNA sequence data

Background  

Phylogenetic hypotheses of higher-level relationships in the order Charadriiformes based on morphological data, partly disagree with those based on DNA-DNA hybridisation data. So far, these relationships have not been tested by analysis of DNA sequence data. Herein we utilize 1692 bp of aligned, nuclear DNA sequences obtained from 23 charadriiform species, representing 15 families. We also test earlier suggestions that bustards and sandgrouses may be nested with the charadriiforms. The data is analysed with methods based on the parsimony and maximum-likelihood criteria.     

Variability in nuclear DNA content within pigeonpea,Cajanus cajan (Fabaceae)

4C DNA values have been estimated in 16 cultivars ofCajanus cajan by cytophotometry. The values range between 6.19 pg to 7.97 pg, a 23.6% variation. The cultivars form four groups which differ significantly from each other but have insignificant difference within them. The implications of this variability with respect to the heterogeneity and origin of this legume crop are discussed.     

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.     

Discordance of chloroplast and nuclear ribosomal DNA data in Osmorhiza (Apiaceae)

Phylogenetic studies were conducted to evaluate interspecific relationships in Osmorhiza (Apiaceae: Apioideae) using sequences of the ITS regions of nuclear ribosomal DNA, the chloroplast ndhF gene, and two noncoding regions (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer). All data sets suggest the monophyly of the New World taxa and showed that Osmorhiza aristata from Asia is relatively divergent from other members of the genus, even though it is morphologically similar to the eastern North American O. claytonii and O. longistylis. The ITS and chloroplast DNA trees differ in the relationships among the New World taxa, especially the phylogenetic position of O. occidentalis, O. glabrata, and O. depauperata. The lack of congruence between the two data sets may be a result of hybridization or introgression. Although there is high discordance between nrITS and two chloroplast DNA data sets, the latter two show similar topologies.     

Genetic analyses of two spawning stocks of the short-finned squid (Illex argentinus) using nuclear and mitochondrial data

Short-finned squid Illex argentinus (Omastrephidae) is an economically important species which supports a long-term and intensive commercial fishery in the South-West Atlantic Ocean. In this study, the genetic variability of allozymes (40 loci) and mitochondrial regions, COI (556 bp) and 16S rDNA (439 bp), were addressed in the two most important species’ spawning stocks, the Summer Spawning Stock (SSS) and the South Patagonic Stock (SPS). Five out of 20 polymorphic allozyme loci were polymorphic at 95% criterion and heterozygosity levels were low. The concatenated analysis of mitochondrial molecular markers revealed high to low values of haplotype and nucleotide diversity, respectively. Nuclear and mitochondrial data revealed no significant genetic differences between these two spawning stocks. Our results and the biological characteristics of I. argentinus deal with a no simple genetic structure of populations described as spatial and temporal chaotic patchiness. SSS and SPS spawning stocks of I. argentinus could not be elevated to the status of genetic stocks.     

Phylogenetic relationships in Bupleurum (apiaceae) based on nuclear ribosomal DNA its sequence data

BACKGROUND AND AIMS: The genus Bupleurum has long been recognized as a natural group, but its infrageneric classification is controversial and has not yet been studied in the light of sequence data. METHODS: Phylogenetic relationships among 32 species (35 taxa) of the genus Bupleurum were investigated by comparative sequencing of the ITS region of the 18-26S nuclear ribosomal DNA repeat. Exemplar taxa from all currently accepted sections and subsections of the genus were included, along with outgroups from four other early branching Apioideae genera (Anginon, Heteromorpha, Physospermum and Pleurospermum). RESULTS: Phylogenies generated by maximum parsimony, maximum likelihood, and neighbour-joining methods show similar topologies, demonstrating monophyly of Bupleurum and the division of the genus into two major clades. This division is also supported by analysis of the 5.8S coding sequence alone. The first branching clade is formed by all the species of the genus with pinnate-reticulate veined leaves and B. rigidum with a unique type of leaf venation. The other major clade includes the remaining species studied, all of which have more or less parallel-veined leaves. CONCLUSIONS: These phylogenetic results do not agree with any previous classifications of the genus. Molecular data also suggest that the endemic Macaronesian species B. salicifolium is a neoendemic, as the sequence divergence between the populations in Madeira and Canary Islands, and closer mainland relatives in north-west Africa is small. All endemic north-west African taxa are included in a single unresolved but well-supported clade, and the low nucleotide variation of ITS suggests a recent radiation within this group. The only southern hemisphere species, B. mundii (southern Africa), is shown to be a neoendemic, apparently closely related to B. falcatum, a Eurasian species.     

Environmentally induced nuclear 2C DNA content instability in Helianthus annuus (Asteraceae)

Experiments were conducted to detect developmental and environmental factors that influence nuclear DNA content in H. annuus inbred lines RHA 271 and RHA 299. DNA content (2C) was determined by laser flow cytometry of nuclei isolated from the first leaf pair of seedlings grown in a greenhouse and in growth chambers. DNA content of greenhouse grown seedlings was highly variable, ranging from 3.2 to 8.0 pg for RHA 299 and 5.2 to 8.2 pg for RHA 271. DNA content only weakly correlated to the position of the achene in the head from which the seedlings derived, and not at all to the position of the head on the plant. Experimentally varied environmental parameters of heat stress and water deficit, phosphate fertilizer levels in the substrate, and pH had little or no effect on the DNA content of seedlings. Seedlings grown with increased levels of substrate nitrogen in the form of NH₄NO₃ showed a significant increase in the mean DNA content. Plants grown in one of two types of growth chambers possessed less variability in DNA content, 6.2–8.4 pg for RHA 299 and 6.8–7.4 pg for RHA 271. Plants grown in a second growth chamber were highly variable with DNA content ranging from 3.0 to 8.6 pg for RHA 299 and 3.0 to 7.8 pg for RHA 271. Measurable physical differences between the growth chambers were irradiance level and the ratio of red to far red light. The hypothesis is presented that DNA stability of sunflowers is influenced by light quantity and/or quality.