Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean α‐amylase inhibitor gene

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for α‐amylase inhibitor‐1 (αAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean αAI1 protein and the corresponding αAI1‐free segregating lines and non‐GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for αAI1. Proteomic analysis showed that in addition to the presence of αAI1, 33 other proteins were differentially accumulated in the αAI1‐expressing GM lines compared with their non‐GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of αAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium‐mediated transformation events. Sixteen proteins were identified after MALDI‐TOF‐TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin‐containing protein. Two proteins were uniquely expressed in the αAI1‐expressing GM lines and one new protein was present in both the αAI1‐expressing GM lines and their αAI1‐free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.     

Microsatellite analysis of the maternal origin of <Emphasis Type="Italic">Myrica rubra</Emphasis> seeds in the feces of Japanese macaques

Identifying the maternal origin of dispersed seeds is a challenging task because it is impossible to directly track seed movement once an animal has ingested them. However, recent development of molecular techniques allows us to identify the maternal origin of dispersed seeds in natural plant populations. Here we analyzed the maternal origin of Myrica rubra seeds found in the feces of Yakushima macaques. We detected a high level of diversity among the dispersed seeds, with an average of 3.0 maternal origins (range 1–9) in each feces. The average dispersal distance was 270.0 m (range 20.4–634.0 m) and the average of distance between maternal trees of dispersed seeds in the same feces was 161.5 m (range 0–573.9 m). These results suggest that seed dispersal by macaques plays an important role in maintaining the genetic diversity of Myrica rubra populations.     

Genetic analysis of the dwarfing gene Rht8 in wheat. Part II. The distribution and adaptive significance of allelic variants at the Rht8 locus of wheat as revealed by microsatellite screening

 Wheat microsatellite WMS 261 whose 192-bp allele has been shown to be diagnostic for the commercially important dwarfing gene Rht8 was used to screen over 100 wheat varieties to determine the worldwide spread of Rht8. The results showed Rht8 to be widespread in southern European wheats and to be present in many central European wheats including the Russian varieties ‘Avrora’, ‘Bezostaya’ and ‘Kavkaz’. Rht8 appears to be of importance to South European wheats as alternative giberellic acid (GA)-insensitive dwarfing genes do not appear to be adapted to this environment. The very successful semi-dwarf varieties bred by CIMMYT, Mexico, for distribution worldwide have been thought to carry Rht8 combined with GA-insensitive dwarfing genes. Additional height reduction would have been obtained from pleiotropic effects of the photoperiod-response gene Ppd1 that is essential to the adaptability of varieties bred for growing under short-winter days in tropical and sub-tropical areas. The microsatellite analysis showed that CIMMYT wheats lack Rht8 and carry a WMS 261 allelic variant of 165 bp that has been associated with promoting height. This presumably has adaptive significance in partly counteracting the effects of other dwarfing genes and preventing the plants being too short. Most UK, German and French wheats carry an allelic variant at the WMS 261 locus with 174 bp. This could be selected because of linkage with the recessive photoperiod-sensitive ppd1 allele that is thought to offer adaptive significance northern European wheats. Received: 17 October 1997 / Accepted: 12 November 1997     

Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis

Summary Restriction fragment analysis of chloroplast (cp) DNAs from 35 wheat (Triticum) and Aegilops species, including their 42 accessions, was carried out with the use of 13 restriction enzymes to clarify variation in their cpDNAs. Fourteen fragment size mutations (deletions/insertions) and 33 recognition site changes were detected among 209 restriction sites sampled. Based on these results, the 42 accessions of wheat-Aegilops could be classified into 16 chloroplast genome types. Most polyploids and their related diploids showed identical restriction fragment patterns, indicating the conservatism of the chloroplast genome during speciation, and maternal lineages of most polyploids were disclosed. This classification of cpDNAs was principally in agreement with that of the plasma types assigned according to phenotypes arising from nucleus-cytoplasm interactions. These mutations detected by restriction fragment analysis were mapped on the physical map of common wheat cpDNA, which was constructed with 13 restriction endonucleases. Length mutations were more frequently observed in some regions than in others: in a 16.0 kilo base pairs (kbp) of DNA region, including rbcL and petA genes, 6 of 14 length mutations were concentrated. This indicates that hot spot regions exist for deletions/insertions in chloroplast genome. On the other hand, 33 recognition site mutations seemed to be distributed equally throughout the genome, except in the inverted repeat region where only one recognition site change was observed. Base substitution rate (p) of cpDNA was similar to that of other plants, such as Brassica, pea and Lycopersicon, showing constant base substitution rates among related taxa and slow evolution of cpDNA compared with animal mitochondrial DNA. Phylogenetic relationships among Triticum and Aegilops species were discussed, based on the present data.Contributions no. 45 and no. 490 from the Kihara Institute for Biological Research, Yokohama City University and the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, respectively.     

Genetic diversity and population structure of the medicinal orchid Gastrodia elata revealed by microsatellite analysis

The wild resources of Gastrodia elata are currently threatened with extinction due to over-harvesting because of their high medicinal value. Genetic diversity plays a key role in the survival of endangered orchid species. In this study we investigated the genetic pattern in eight microsatellite loci within eight G. elata populations from central China. Compared with the other orchids, G. elata showed a low level of genetic variation within populations (H_E = 0.356–0.622). The main factors responsible for the genetic pattern were the plant's inbreeding system due to mating within clone patches, and the genetic bottlenecks and genetic drift caused by a long-history over-collecting. The significant heterozygote deficit was detected in all the populations. The F statistics calculated by different approaches consistently revealed a clear genetic differentiation among populations, contributing about 20% of the total gene diversity. The results are discussed in relation to both in situ and ex situ conservation efforts of the species. The populations with a high level of genetic diversity or with great genetic distinction were identified, which should be a high priority for conservation managers.     

High outcrossing and random pollen dispersal in a planted stand of Acacia saligna subsp. saligna revealed by paternity analysis using microsatellites

The mating system, patterns of pollen mediated gene flow and levels of genetic contamination were investigated in a planted stand of Acacia saligna subsp. saligna via paternity analysis using microsatellite markers. High levels of outcrossing were detected within the stand (t _m = 0.98), and the average pollen dispersal distance was 37 m with the majority of progeny sired by paternal trees within a 50-m neighbourhood of the maternal tree. Genetic contamination from the natural background population of A. saligna subsp. lindleyi was detected in 14% of the progeny of A. saligna subsp. saligna and varied among maternal trees. Long distance inter-subspecific pollen dispersal was detected for distances of over 1,500 m. The results provide information for use in the breeding and domestication programme aimed at developing A. saligna as an agroforestry crop for the low rainfall areas of southern Australia.     

Speciation analysis of nickel in soil solutions and availability to oat plants

Soil solutions were collected for speciation analysis of nickel from a pot experiment with oats. Oat plants (Avena sativa L.) were grown on 3 soils differing in total amount and origin of nickel (Ni) (Luvisol, LS with 28 mg kg^-1; sludge amended Luvisol, LS+SS with 32 mg kg^-1; Cambisol, CS with 95 mg kg^-1). Results were compared with those for soil solutions obtained from corresponding unplanted pots. Separation methods were used for characterization of size and charge distribution and stability of the Ni species. In addition, short-term experiments were performed on the uptake rates of Ni by oat plants from the different soil solutions as well as from nutrient solutions with increasing concentrations of a synthetic chelator.The Ni concentrations in the soil solutions of unplanted soils increased in the order: LS5000 g mol^-1) was the predominant form, whereas in the other soils the low-molecular-size cationic and chelated Ni species (500–1000 g mol^-1) dominated in the soil solution. In the short-term uptake studies, the uptake rates of Ni from the solutions decreased in the order: nutrient solution > soil solutions, and in the latter in the order: LS>LS+SS>CS, which was inversely related to the concentrations of dissolved organic carbon in the soil solutions.The results demonstrate that Ni availability to plants is not only affected by total concentration of Ni in the soil solution and the rate of replenishment from the solid phase, but also by Ni species, which can differ considerably between soil types.     

Genetic diversity in a collection of old and new bread wheat cultivars from Iran as revealed by simple sequence repeat-based analysis

Genetic diversity in a collection of 70 bread wheat ( Triticum aestivum ) genotypes was studied using 73 microsatellite [simple sequence repeat (SSR)] loci evenly spaced on wheat chromosomes. A total of 592 alleles with an average of 8.53 allele/locus were detected, of which 185 (31.25%) occurred only in a specific group of genotypes. A set of SSR markers consisted of 22 loci with polymorphic information content values of 0.80 or higher were selected for rapid fingerprinting of many genotypes. Average of gene diversity was 0.74 ± 0.017, and significant difference between observed and maximum theoretical values of gene diversity in the analysed SSR loci was obtained using a paired t -test. Genetic distance-based clustering methods including unweighted pair group method with arithmetic average and neighbour joining (NJ) were used for grouping of genotypes. The resulted dendrogram based on NJ and number of differences coefficient hinted of the existence of three groups. This grouping was in agreement with the pedigree information and confirmed by high within-group bootstrap value. A comparatively higher genetic diversity in the studied wheat collection as revealed by presence of high allelic diversity and large number of specific alleles could be utilised in development of new cultivars with desired characteristics.     

Origins of weedy rice revealed by polymorphisms of chloroplast DNA sequences and nuclear microsatellites

Conspecific weeds that permanently infest worldwide agroecosystems are evolved from their crop species. These weeds cause substantial problems for crop production by competing for resources in agricultural fields. Weedy rice represents such a conspecific weed infesting rice ecosystems, and causing tremendous rice yield losses owing to its strong competitiveness and abundant genetic diversity, likely resulted from its complex origins. Here, we report the use of chloroplast DNA (cpDNA) fingerprints to determine whether weedy rice is evolved from its wild (exo‐feral) or cultivated (endo‐feral) rice progenitor as the maternal donor in recent hybridization events. In addition, we also applied nuclear simple sequence repeat (SSR) markers to confirm the exo‐feral or endo‐feral origins of weedy rice accessions determined by the cpDNA fingerprints. We found that the studied weedy rice accessions evolved either from their wild or cultivated rice progenitor, as the maternal donor, based on the cpDNA network and structure analyses. Combined analyses of cpDNA and nuclear SSR markers indicated that a much greater proportion of weedy rice accessions had the endo‐feral origin. In addition, results from the genetic structure of nuclear SSR markers indicated that weedy rice accessions from the endo‐feral pathway are distinctly associated with either indica or japonica rice cultivars, suggesting their complex origins through crop–weed introgression. The complex pathways of origin and evolution could greatly promote genetic diversity of weedy rice. Therefore, innovative methods should be developed for effective weedy rice control.     

Identification of cross-contaminated animal cells by PCR and isoenzyme analysis

Animal cell lines have become very popular substrates for the production of vaccines and biopharmaceuticals. Characterization of candidate production cell lines is central to ensure product safety and maintenance of consistency in the manufacture of biologicals. Nested PCR and isoenzyme analysis have been used widely to prove the identity and purity of various cell lines and primary cells individually and also after deliberate cross-contamination. The nested PCR based on the Cytochrome b (Cyt b) gene of mitochondrial DNA (Mt DNA) was found to be more sensitive than isoenzyme analysis in detecting low levels of contaminants (as low as 1%). Interestingly, competition between different co-cultured cell lines has shown in one case that cross-contamination need not always results in a mixed cell population. The nested PCR technique for the Cyt b gene described in this study appears to be a potential replacement for isoenzyme analysis and here we demonstrate the PCR method used is sensitive and reliable for cell line authentication in a simple, rapid and reliable format to help assure the authenticity of cell substrates for the production of safe vaccines and biopharmaceuticals.     

Gene flow and spatio-temporal genetic variation among sympatric populations of Tetranychus kanzawai (Acari: Tetranychidae) occurring on different host plants, as estimated by microsatellite gene diversity

We investigated spatio-temporal genetic variation in allele frequency and estimated gene flow among sympatric populations of Tetranychus kanzawai on different host plants by the use of microsatellite markers. In the analysis of spatial genetic variation, no isolation by distance was detected among the populations. Gene flow between populations on Hydrangea macrophylla and those on other host plants was relatively restricted, whereas the populations on Akebia quinata and Clerodendrum trichotomum were almost panmictic. Our study on temporal genetic variation showed (1) that population differentiation was slightly reduced during the period from April to May owing to frequent gene flow among populations; and (2) that population differentiation was greatly enhanced from May to October because of bottleneck effects. Genetic differentiation among T. kanzawai populations was caused by the effect of host plants rather than by the effect of geographic distance among populations, suggesting possibility of sympatric host race formation in this species.This revised version was published online in May 2005 with a corrected cover date.     

Comparative analysis of genetic relationship and diagnostic markers of several taxa of Guizotia Cass. (Asteraceae) as revealed by AFLPs and RAPDs

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) were employed to examine the genetic relationship between Guizotia taxa, to suggest the taxonomic status of some of these taxa, and to identify their diagnostic markers. Results from AFLPs and RAPDs share some features in common, both revealing G. scabra ssp. schimperi as the most closely related taxon to G. abyssinica, and indicating that G. arborescens and G. zavattarii are the most divergent taxa. Most of the diagnostic markers revealed in this study were specific to G. arborescens and G. zavattarii. Our analysis suggests that G. scabra ssp. scabra, G. scabra ssp. schimperi, Chelelu and Ketcha are separate species. In this study, AFLP was found to be superior to RAPD in detecting genetic variation, in internal consistency of the data and in the fitness of its clusters to genetic similarity data. AFLPs revealed genetic relationship between Guizotia taxa that is more inline with the cytogenetic and hybridization studies than that revealed by RAPDs.     

Spatio-temporal changes in germination and radical elongation of barley seeds tracked by proteome analysis of dissected embryo, aleurone layer, and endosperm tissues

Germination of barley is accompanied by changes in water-soluble seed proteins. 2-DE was used to describe spatio-temporal proteome differences in dissected seed tissues associated with germination and the subsequent radicle elongation. Protein identification by MS enabled assignment of proteins and functions to the seed embryo, aleurone, and endosperm. Abundance in 2-DE patterns was monitored for 48 different proteins appearing in 79 gel spots at 8 time-points up to 72 h post imbibition (PI). In embryo, a beta-type proteasome subunit and a heat shock protein 70 fragment were among the earliest proteins to appear (at 4 h PI). Other early changes were observed that affected spots containing desiccation stress-associated late embryogenesis abundant and abscisic acid (ABA)-induced proteins. From 12 h PI proteins characteristic for desiccation stress disappeared rapidly, as did a putative embryonic protein and an ABA-induced protein, suggesting that these proteins are also involved in desiccation stress. Several redox-related proteins differed in spatio-temporal patterns at the end of germination and onset of radicle elongation. Notably, ascorbate peroxidase that was observed only in the embryo, increased in abundance at 36 h PI. The surprisingly early changes seen in the protein profiles already 4 h after imbibition indicate that germination is programmed during seed maturation.     

Gene flow and mating system in five <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don seed orchards as revealed by analysis of microsatellite markers

We investigated gene flow in five Cryptomeria japonica D. Don seed orchards of two different types (common and miniature) at widely spaced locations using microsatellite markers. The quality of a seed crop is determined by many factors, including pollen contamination from outside sources, self-fertilization, and the proportion of contributions from constituent clones. Contamination rates were found to vary among ramets both within seed orchards (10.0–76.7% in the most variable seed orchard) and among seed orchards (35.0–65.8% on average). Among ramets, there were significant negative correlations between pollen contamination rate and their distance from the orchard edge; among seed orchards, there were significant positive correlations between the pollen contamination rate and the C. japonica forest area nearby. Some proportion of the pollen (10.7% of total contamination) also migrated from parts of the orchards that had not been treated with gibberellin to induce flowering. Self-fertilization rates varied among seed orchards (1.4–4.4% on average), and there were significant positive correlations between self-fertilization rate and the number of ramets per clone in both types of seed orchard. Contributions as pollen donors differed significantly among clones in all seed orchards. The distance between planted ramets, flowering phenology, and relative pollen fecundity may also have contributed to observed differences in paternal contribution. The influence of these factors on genetic potential did not differ greatly between the two types of orchards. This work was supported by grants from the Pioneer Special Studies Program of the Japanese Ministry of Agriculture, Fishery, and Forestry; the Program for Promotion of Basic Research Activities for Innovative Biosciences; and research fellowships from the Japan Society for the Promotion of Science for Young Scientists.     

Genetic structure and reproduction dynamics of Salix reinii during primary succession on Mount Fuji,as revealed by nuclear and chloroplast microsatellite analysis

The early stage of volcanic desert succession is underway on the southeastern slope of Mount Fuji. We used markers of nuclear microsatellites (simple sequence repeats; SSR) and chloroplast microsatellites (cpSSR) to investigate the population genetic structure and reproduction dynamics of Salix reinii, one of the dominant pioneer shrubs in this area. The number of S. reinii genets in a patch and the area of the largest genet within the patch increased with patch area, suggesting that both clonal growth and seedling recruitment are involved in the reproduction dynamics of S. reinii. Five polymorphic cpSSR markers were developed for S. reinii by sequencing the noncoding regions between universal sequences in the chloroplast genome. Nineteen different cpSSR haplotypes were identified, indicating that S. reinii pioneer genets were created by the long-distance dispersal of seeds originating from different mother genets around the study site, where all vegetation was destroyed during the last eruption. Furthermore, the clustered distributions of different haplotypes within each patch or plot suggested that newly colonized genets tended to be generated from seeds dispersed near the initially established mother genets. These results revealed that the establishment of the S. reinii population on the southeastern slope of Mount Fuji involved two sequential modes of seed dispersal: long-distance dispersal followed by short-distance dispersal.     

Pattern and timing of diversification of Cetartiodactyla (Mammalia, Laurasiatheria), as revealed by a comprehensive analysis of mitochondrial genomes

The order Cetartiodactyla includes cetaceans (whales, dolphins and porpoises) that are found in all oceans and seas, as well as in some rivers, and artiodactyls (ruminants, pigs, peccaries, hippos, camels and llamas) that are present on all continents, except Antarctica and until recent invasions, Australia. There are currently 332 recognized cetartiodactyl species, which are classified into 132 genera and 22 families. Most phylogenetic studies have focused on deep relationships, and no comprehensive time-calibrated tree for the group has been published yet. In this study, 128 new complete mitochondrial genomes of Cetartiodactyla were sequenced and aligned with those extracted from nucleotide databases. Our alignment includes 14,902 unambiguously aligned nucleotide characters for 210 taxa, representing 183 species, 107 genera, and all cetartiodactyl families. Our mtDNA data produced a statistically robust tree, which is largely consistent with previous classifications. However, a few taxa were found to be para- or polyphyletic, including the family Balaenopteridae, as well as several genera and species. Accordingly, we propose several taxonomic changes in order to render the classification compatible with our molecular phylogeny. In some cases, the results can be interpreted as possible taxonomic misidentification or evidence for mtDNA introgression. The existence of three new cryptic species of Ruminantia should therefore be confirmed by further analyses using nuclear data. We estimate divergence times using Bayesian relaxed molecular clock models. The deepest nodes appeared very sensitive to prior assumptions leading to unreliable estimates, primarily because of the misleading effects of rate heterogeneity, saturation and divergent outgroups. In addition, we detected that Whippomorpha contains slow-evolving taxa, such as large whales and hippos, as well as fast-evolving taxa, such as river dolphins. Our results nevertheless indicate that the evolutionary history of cetartiodactyls was punctuated by four main phases of rapid radiation during the Cenozoic era: the sudden occurrence of the three extant lineages within Cetartiodactyla (Cetruminantia, Suina and Tylopoda); the basal diversification of Cetacea during the Early Oligocene; and two radiations that involve Cetacea and Pecora, one at the Oligocene/Miocene boundary and the other in the Middle Miocene. In addition, we show that the high species diversity now observed in the families Bovidae and Cervidae accumulated mainly during the Late Miocene and Plio-Pleistocene.     

Patterns and levels of gene flow in Rhododendron metternichii var. hondoense revealed by microsatellite analysis

Parentage analysis was conducted to elucidate the patterns and levels of gene flow in Rhododendron metternichii Sieb. et Zucc. var. hondoense Nakai in a 150 x 70 m quadrant in Hiroshima Prefecture, western Japan. The population of R. metternichii occurred as three subpopulations at the study site. Seventy seedlings were randomly collected from each of three 10 x 10 m plots (S1, S2, and S3) on the forest floor of each subpopulation (A1, A2, and A3). Almost all parents (93.8%) of the 70 seedlings were unambiguously identified by using 12 pairs of microsatellite markers. Within the quadrant, adult trees less than 5 m from the centre of the seedling bank (plots S1, S2, and S3) produced large numbers of seedlings. The effects of tree height and distance from the seedling bank on the relative fertilities of adult trees were highly variable among subpopulations because of the differences in population structure near the seedling bank: neither distance nor tree height had any significant effect in subpopulation A1; distance from the seedling bank had a significant effect in subpopulation A2; and tree height had a significant effect in subpopulation A3. Although gene flow within each subpopulation was highly restricted to less than 25 m and gene flow among the three subpopulations was extremely small (0-2%), long-distance gene flow from outside the quadrant reached 50%. This long-distance gene flow may be caused by a combination of topographical and vegetational heterogeneity, differences in flowering phenology, and genetic substructuring within subpopulations.     

Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis

The FliF ring is the base for self-assembly of the bacterial flagellum and the FliF/FliG ring complex is the core of the rotor of the flagellar motor. We report the structures of these two ring complexes obtained by electron cryomicroscopy and single-particle image analysis at 22A and 25A resolution, respectively. Direct comparison of these structures with the flagellar basal body made by superimposing the density maps on the central section reveals many interesting features, such as how the mechanically stable connection between the ring and the rod is formed, how directly FliF domains are involved in the near axial density of the basal body forming the proximal end of the central channel for a potential gating mechanism, some indication of flexibility in the connection of FliF and FliG, and structural and functional similarities to the head-to-tail connectors of bacteriophages.     

Identification and mapping on chromosome 9 of RAPD markers linked to Sw-5 in tomato by bulked segregant analysis

Bulked segregant analysis was used to identify random amplified polymorphic DNA (RAPD) markers linked to the Sw-5 gene for resistance to tomato spotted wilt virus (TSWV) in tomato. Using two pools of phenotyped individuals from one segregating population, we identified four RAPD markers linked to the gene of interest. Two of these appeared tightly linked to Sw-5, whereas another, linked in repulsion phase, enabled the identification of heterozygous and susceptible plants. After linkage analysis of an F₂ population, the RAPD markers were shown to be linked to Sw-5 within a distance of 10.5 cM. One of the RAPD markers close to Sw-5 was used to develop a SCAR (sequence characterized amplified region) marker. Another RAPD marker was stabilized into a pseudo-SCAR marker by enhancing the specificity of its primer sequence without cloning and sequencing. RAPD markers were mapped to chromosome 9 on the RFLP tomato map developed by Tanksley et al. (1992). The analysis of 13 F₃ families and eight BC₂ populations segregating for resistance to TSWV confirmed the linkage of the RAPD markers found. These markers are presently being used in marker-assisted plant breeding.