Genetic and physical maps and a clone bank of mitochondrial DNA from rice

Summary Mitochondrial DNA (mtDNA) was isolated from young green leaves of rice plants. DNA fragments were cloned into lambda DNA, and clones that hybridized to mitochondrial genes from other plants were selected. Distal restriction fragments of these clones were used as probes for the selection of overlapping clones. A genetic map was finally created from the library by walking along the genome. The mitochondrial genome consists of five basic circles, with each circle sharing homologous sequences with one or two other circles. A master circle was constructed from the results of recombination across repeated sequences, and its size was estimated to be 492 kb. A physical map and a bank of overlapping clones were also constructed.     

Importance of a pilot study for non-invasive genetic sampling: genotyping errors and population size estimation in red deer

Population size information is critical for managing endangered or harvested populations. Population size can now be estimated from non-invasive genetic sampling. However, pitfalls remain such as genotyping errors (allele dropout and false alleles at microsatellite loci). To evaluate the feasibility of non-invasive sampling (e.g., for population size estimation), a pilot study is required. Here, we present a pilot study consisting of (i) a genetic step to test loci amplification and to estimate allele frequencies and genotyping error rates when using faecal DNA, and (ii) a simulation step to quantify and minimise the effects of errors on estimates of population size. The pilot study was conducted on a population of red deer in a fenced natural area of 5440 ha, in France. Twelve microsatellite loci were tested for amplification and genotyping errors. The genotyping error rates for microsatellite loci were 0–0.83 (mean=0.2) for allele dropout rates and 0–0.14 (mean=0.02) for false allele rates, comparable to rates encountered in other non-invasive studies. Simulation results suggest we must conduct 6 PCR amplifications per sample (per locus) to achieve approximately 97% correct genotypes. The 3% error rate appears to have little influence on the accuracy and precision of population size estimation. This paper illustrates the importance of conducting a pilot study (including genotyping and simulations) when using non-invasive sampling to study threatened or managed populations.     

Regeneration of whole plants from callus culture of diverse genetic lines of Pisum sativum L.

Sixteen genetic lines of peas were screened for their ability to regenerate whole plants from callus cultures. Epicotyl sections from germinating seeds were placed on callus-inducing medium; the resulting callus was subcultured monthly and was tested every other month for its regeneration ability. Six lines were found that would regenerate after 2 months' growth as callus. Four of these continued to regenerate after 4 months and, of these, two after 6 months. The cultivars Frosty and Alaska were among the lines that would not regenerate at all.Michigan Agricultural Experiment Station Journal Article No. 8932     

Tissue-specific expression of the chalcone synthase multigene family in Phaseolus vulgaris: development of a RT-PCR method for the expression profiling of the chs isogenes

Chalcone synthase is a key metabolic control point in the biosynthesis of a large number of flavonoids and isoflavonoid metabolites. Chs genes in bean comprise a multigene family, of which certain individual members can be differentially induced with respect to kinetics and extent of accumulation. A RT-PCR technique, based on primers designed complementary to a common conserved region and divergent 3′ sequences of the bean chs family, was developed to detect the expression of individual members of the chs family. The semi-quantitative technique is based on the amplification of short, overlapping sequences differing in size. The method was found to be sensitive, rapid, and capable of distinguishing among the individual chs members (chs 1, 4, 14, and 17). The tissue-specific expression of chs isogenes in bean seedlings, flowers and callus, as well as the effect of light on chs expression in etiolated tissue was documented.     

Genetic structure of brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> L.) from the Hardangervidda mountain plateau (Norway) analyzed by microsatellite DNA: a basis for conservation guidelines

The Hardangervidda in southern Norway, the largest mountain plateau in Europe, has thousands of lakes and streams, mainly between 1000 and 1300 m above sea level, where brown trout is the only fish species. To describe the current genetic diversity of brown trout in this area, a total of 863 fish from 20 lakes were genotyped with eleven microsatellites. Most diversity is within lake populations, but diversity among geographical groups and populations within groups was significant, too. Neighbor-joining, principle coordinate analysis and Bayesian clustering show three major geographic groups in accordance with the river systems. Bias was caused by recent stocking in two lakes. Low/no genetic differentiation among some populations indicates that intermixing is common when lakes are well-connected, as was also shown by assignment test. We recommend preserving the genetic diversity of brown trout in this unique area by managing stocking in lake systems according to genetic structure.     

Genetic variability in mitochondrial DNA in a regional population of the great tit (Parus major)

Genetic variation of mitochondrial DNA (mtDNA) in 18 great tits (Parus major) from three neighboring localities in Sweden was investigated with eight tetranucleotide restriction endonucleases. The 18 individuals could be separated into 13 different maternal lineages. The high number of female lineages present in this regional population contrasts with a low level of sequence divergence between the different mtDNA clones, with a mean of 0.19% sequence divergence between all individuals. There was no obvious spatial structuring of mtDNA clones among the three localities. The presence of a high number of different clones with a low degree of sequence divergence could be explained by the effects of a large long-term effective population size, with the mtDNA clones having diverged about 25,000–200,000 years ago.This study was supported by the Swedish Natural Science Research Council, the Erik Philip-Sörensen Foundation, and the Nilsson-Ehle Foundation.     

The use of RAPD markers for detecting genetic diversity,relationship and molecular identification of Chinese elite tea genetic resources [<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze] preserved in a tea germplasm repository

The genetic diversity, relationship and molecular identification of 15 well known, widely planted traditional Chinese elite tea genetic resources [Camellia sinensis (L.) O. Kuntze] preserved in the China National Germplasm Hangzhou Tea Repository in the Tea Research Institute of the Chinese Academy of Agricultural Sciences located in Zhejiang province, China, were investigated using RAPD markers. A total of 1050 bands with an average of 52.5 bands per primer, 70 bands per genetic resource were generated by the 20 selected primers from the 15 tea genetic resources. In the total of 137 amplified products, 129 were polymorphic, corresponding to 94.2% genetic diversity. The relative frequency of polymorphic products was from 0.24 to 0.83, with an average of 0.47. In general, this average frequency was relatively high. The genetic distances among the genetic resources were from 0.16 to 0.62, with an average of 0.37. The 15 tea genetic resources were grouped into three groups by UPGMA cluster analysis based on RAPD data. By using the presence of 20 unique RAPD markers and the absence of 11 unique markers, all the 15 investigated tea genetic resources could be easily identified. RAPD markers provided a practical method not only to evaluate the genetic diversity and relationship, but also to identify tea genetic resources.     

Innate versus adaptive immunity in sticklebacks: evidence for trade-offs from a selection experiment

In vertebrates, the immune system consists of two arms of different characteristics: the innate and the acquired immune response. Parasites that are only shortly exposed to the immune system are most efficiently attacked by fast, constitutive innate immune mechanisms. Here, we experimentally selected within four fish families for high innate resistance versus susceptibility of three-spined sticklebacks (Gasterosteus aculeatus) against infection with the eye-fluke (Diplostomum pseudospathacaeum), a parasite whose metacercariae are protected from the immune system within the eye lens. We predicted that in families with high susceptibility, the adaptive immune system would be upregulated when challenged with infection. In accordance, we found that MHC class IIB expression is increased by approximately 50% in those lines selected for higher parasite load (i.e. low innate response). This suggests extensive genetic correlations between innate and adaptive immune system and/or crosstalk between both lines of defense. An efficient, specific innate immune response might reduce overall activation of the immune system and potentially alleviate associated effects of immunopathology.     

A simple and rapid procedure to establish embryogenic cell suspensions as a source of protoplasts for efficient plant regeneration from two Chinese commercial rice cultivars

An efficient and rapid procedure has been developed to establish embryogenic cell suspension cultures of two Japonica Chinese commercial rice cultivars, Zhonghua 8 and Eryi 105. Embryogenic cell suspensions of both varieties were established from 0.5–1.0 g fresh weight of embryogenic callus in AA medium within 2.5 months of the initiation of callus from sterilised seeds. The previously reported subculture of callus on semi-solid medium for 4–8 weeks prior to transfer into liquid medium was unnecessary and caused delay in the establishment of embryogenic cell suspensions. Protoplasts were isolated reproducibly from cell suspensions up to 18 months after their initiation, with protoplast plating efficiencies attaining 0.15–0.37%. Reproducible plant regeneration from 14–26% of the protoplast-derived tissues was achieved without the requirement for nurse cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Consequences of harvesting for genetic diversity in American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.): a simulation study

American ginseng, Panax quinquefolius L., is one of the most heavily traded medicinal plants in North America. The effect of harvest on genetic diversity in ginseng was measured with a single generation culling simulation program. Culling scenarios included random harvest at varying levels, legal limit random harvest and legal limit mature plant harvest. The legal limit was determined by the proportion of legally harvestable plants per population (% mature plants per population). Random harvest at varying levels resulted in significant loss of genetic diversity, especially allelic richness. Relative to initial levels, average within-population genetic diversity (H _e) was significantly lower when plants were culled randomly at the legal limit (Mann–Whitney U=430, p<0.001) or when only mature plants were culled (Mann–Whitney U=394, p<0.01). Within-population genetic diversity was significantly higher with legal limit mature plant harvest (H _e=0.068) than when plants were culled randomly at the legal limit (H _e=0.064; U=202, p<0.01). Based on these simulations of harvest over one generation, we recommend that harvesting fewer than the proportion of mature plants could reduce the negative genetic effects of harvest on ginseng populations.     

Consensual predictions of potential distributional areas for invasive species: a case study of Argentine ants in the Iberian Peninsula

Invasive species are known to influence the structure and function of invaded ecological communities, and preventive measures appear to be the most efficient means of controlling these effects. However, management of biological invasions requires use of adequate tools to understand and predict invasion patterns in recently introduced areas. The present study: (1) estimates the potential geographic distribution and ecological requirements of the Argentine ant (Linepithema humile Mayr), one of the most conspicuous invasive species throughout the world, in the Iberian Peninsula using ecological niche modeling, and (2) provides new insights into the process of selection of consensual areas among predictions from several modeling methodologies. Ecological niche models were developed using 5 modeling techniques: generalized linear models (GLM), generalized additive models (GAM), generalized boosted models (GBM), Genetic Algorithm for Rule-Set Prediction (GARP), and Maximum Entropy (Maxent). Models for the eastern and western portions of the Iberian Peninsula were built using subsets of occurrence and environmental data to investigate the potential for ecological niche differences between the invading populations. Our results indicate geographic differences between predictions of different approaches, and the utility of ensemble predictions in identifying areas of uncertainty regarding the species’ invasive potential. More generally, our models predict coastal areas and major river corridors as highly suitable for Argentine ants, and indicate that western and eastern Iberian Peninsula populations occupy similar environmental conditions.

Identification and purification of a nuclease from Zinnia elegans L.: a potential molecular marker for xylogenesis

A single-strand specific nuclease was identified during a particular stage of a defined cellular differentiation pathway characteristic of xylem development. Using a hormone-inducible system in which cultured mesophyll cells of Zinnia elegans differentiated to xylem cells in synchrony, the enzymatic activity on single-stranded (ss) DNA was highest during the maturation phase of differentiation. Nondifferentiating cells contained little of this activity throughout a similar course of culture. After electrophoresis of extracts from differentiating cells, a 43-kilodalton (kDa) polypeptide was detected by its activity in the gels containing either ssDNA or RNA. Lectins specific for mannose residues on glycoproteins bound to the 43-kDa nuclease and were used to distinguish it from several ribonucleases. The nuclease was purified by a two-step chromatographic procedure: a lectin-affinity column followed by a phosphocellulose column. The purified protein was determined to be a single polypeptide with a relative molecular mass of 43000 by the analysis of its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme. A sensitive detection system using biotinylated-concanavalin A and avidin was demonstrated to be specific as a probe for the nuclease protein. An N-terminal amino-acid sequence was derived from 5 pmol of the enzyme. The nuclease was most active on ssDNA at pH 5.5 in the presence of Zn²⁺ and dithiothreitol. The purified preparation hydrolyzed RNA and to a lesser extent, native DNA. It digested closed circular duplex DNA by introducing a single endonucleolytic cleavage followed by random hydrolysis. During the induced pathway of synchronous differentiation in Zinnia the 43-kDa nuclease rapidly increased just prior to the onset of visibly differentiated features, and developed to a maximum level during xylem cell maturation. At this time a similar but slightly smaller nuclease appeared and became dominant as differentiation continued, and subsequently both enzymes decayed. After autolysis, a nuclease of about 37 kDa was found together with the 43-kDa enzyme in the culture medium. Complementing these analyses was the examination of the tissue distribution of the 43-kDa enzyme in Zinnia and other dicotyledonous plants, which also indicated an invivo role of the nuclease in autolysis, the terminal stage of vascular differentiation in plants. The Zinnia nuclease is therefore a potential marker for xylogenesis.Abbreviations Con A Canavalia ensiformis (concanavalin) agglutinin - DNase deoxyribonuclease - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - kDa kilodalton - M_r relative molecular mass - RNase ribonuclease - ss single-stranded - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis