Genetic diversity of Ustilago maydis strains

Thirty wild isolates belonging to five different locations in Mexico plus two laboratory strains of Ustilago maydis were characterized by restriction fragment length polymorphism (RFLP) analysis using 23 different clones as probes derived from a PstI library and two restriction enzymes. All loci analysed presented a high level of polymorphism, including one locus with thirty one different alleles. Geographical grouping of the populations was based on Nei's genetic distance and there was no correlation between genetic and geographic distances among these isolates. Our results suggest that DNA fingerprinting is a useful method for detecting genetic variation in populations of U. maydis. This work demonstrated that considerable genetic variation may be present within field populations of U. maydis.     

Effect of plant genotype on the transformation of cultivated alfalfa (Medicago sativa) by Agrobacterium tumefaciens

The trait for somatic embryogenesis is being introduced sexually into alfalfa (Medicago sativa) breeding populations to facilitate genetic transformation of this crop. Cocultivation experiments were conducted with an agronomically-improved embryogenic clone from one such population as well as with two other embryogenic clones, one of which was the source of the embryogenic trait in the breeding populations. Transgenic plants were produced from the agronomically-improved clone whereas none were produced from the other two clones. Among the 16 transgenic plants analyzed there was a range in both copy number and number of integration sites for the NPT-II gene; those plants regenerated after a prolonged selection phase in vitro generally had the highest numbers in both respects. There was no evidence of sectoral chimerism of the transgene in a subsample of transgenic plants analyzed by PCR.     

不同年龄巴山木竹种群克隆结构

很多竹类植物是典型的克隆植物,也是大熊猫的食物。研究典型竹子种群克隆结构的形成和发展对竹林的生产和抚育具有理论和实践意义,可为预测该竹林群落的演替趋势和大熊猫保护提供科学依据。利用SSR标记研究不同年龄A(7龄)、B(30龄)和C(60龄)巴山木竹种群的克隆结构和多样性,探讨小尺度范围内不同年龄巴山木竹种群的克隆结构及斑块的建立和发展。8对SSR引物共扩增出了118个位点,3个种群样地的256个样本共检测到了49个克隆(基因型),A、B和C种群分别检测出31、10个和8个克隆。随着种群年龄的增长,巴山木竹克隆面积增加,克隆数量减少;A和B样地各克隆分布格局为团块状,而C样地克隆既有团块状又有离散状。这一结果显示出在幼苗定居的初期,基株可能以短距离的克隆延伸为主从而呈现出团块状;而随着年龄的增长,克隆面积不断扩大,当复轴混生型的巴山木竹克隆受到强大的压迫时,基株可能会进行较多的单轴和长距离克隆延伸,呈现出离散状。Mantel检测和空间自相关分析都支持3个样地在小尺度范围内存在明显的克隆空间遗传结构。3个样地在10 m等级下显著的正相关空间遗传结构距离为3.1、28、48 m,X-轴截距为9.051、30.698和50.536,空间自相关系数的范围分别为0.1—0.167、0.008—0.703和0.006—0.735。由此可推断,随着年龄的增长,巴山木竹克隆斑块的规模在不断地扩大,同一克隆的分株数量增加,在均匀取样情况下,正相关空间遗传结构距离范围内取到具有相同基因型的可能性越大。A、B和C 3样地的基因型比率(G/N)为1、0.14和0.055,Simpson多样性指数(D)分别为1、0.876和0.744。这说明巴山木竹幼苗期基因型比例远远高于成年的竹林,随着年龄的增长巴山木竹克隆多样性虽有所降低,但由于有性繁殖的作用仍然保持了较高的多样性。聚类和主坐标分析均表明总体上各样地的克隆被聚为一类,但不同样地少数克隆的基因型有重叠和聚集,可推断出不同巴山木竹种群之间可能存在着基因流动和近似的克隆起源。     

Genetic variation in two sympatric European populations of Bosmina spp. (Cladocera) tested with RAPD markers

We used RAPDs (Random Amplified Polymorphic DNA) to test genetic divergence between two populations of Bosmina spp. in Lake Östersjön, Sweden. Previous taxonomic studies on European species within the genus Bosmina have been based on morphological characters alone. RAPD markers distinguished the two populations and supported the specific status of B. coregoni and B. longispina based on morphological characters. Furthermore, juveniles with a long antennule and a mucro were classified as B. coregoni. RAPDs also revealed genetic differences among the tested individuals, suggesting several clones within each species.     

Genetic variation in the effect of a facultative symbiont on host-plant use by pea aphids

Ecological specialisation on different host plants occurs frequently among phytophagous insects and is normally assumed to have a genetic basis. However, insects often carry microbial symbionts, which may play a role in the evolution of specialisation. The bacterium Regiella insecticola is a facultative symbiont of pea aphids (Acyrthosiphon pisum) where it is found most frequently in aphid clones feeding on Trifolium giving rise to the hypothesis that it may improve aphid performance on this plant. A study in which R. insecticola was eliminated from a single naturally infected aphid clone supported the hypothesis, but a second involving two aphid clones did not find the same effect. We created a series of new pea aphid–R. insecticola associations by injecting different strains of bacteria into five aphid clones uninfected by symbionts. For all aphid clones, the bacteria decreased the rate at which aphids accepted Vicia faba as a food plant and reduced performance on this plant. Their effect on aphids given Trifolium pratense was more complex: R. insecticola negatively affected acceptance by all aphid clones, had no effect on the performance of four aphid clones, but increased performance of a fifth, thus demonstrating genetic variation in the effect of R. insecticola on pea aphid host use. We discuss how these results may explain the distribution and frequency of this symbiont across different aphid populations. Julia Ferrari and Claire L. Scarborough contributed equally to the work.     

Genetic variation in Myzus persicae populations associated with host-plant and life cycle category

Random amplified polymorphic DNA (RAPD) analysis was applied on 96 clones of Myzus persicae (Sulzer) (Homoptera: Aphididae) representing seven populations collected from different host-plants and regions of Greece. Ten decamer random primers were used to evaluate genetic variation among the examined samples. Despite the variability found between clones, no specific RAPD marker was detected to discriminate the different populations. A significant finding was that aphids from peach and pepper, which were collected far away from tobacco-growing regions, especially those from peach, showed genetic divergence from the tobacco-feeding clones. Moreover, data analysis revealed a significant genetic divergence between holocyclic and anholocyclic populations from tobacco. Lastly, holocyclic clones showed higher level of estimated heterozygosity than the nonholocyclic (anholocyclic, androcyclic and intermediate) ones.     

Variation in performance on two grape cultivars within and among populations of grape Phylloxera from wild and cultivated habitats

When an indigenous insect becomes a pest, comparisons of performance of pest and non-pest populations on crop plants and of genetic variation in that performance may provide insight into the evolution of pest populations. To measure such genetic variation, 8–15 clones of the grape phylloxera (Daktulosphaira vitifoliae Fitch) were collected from wild grapevines in each of 3 geographically isolated sites (populations) and from commercial vineyards in northern California. A complete life table was made for clonal replicates from populations collected from wild grapevines on each of two commercial grape cultivars, the susceptibleVitis vinifera (L.) cultivar Cabernet Sauvignon, and the phylloxera-resistant rootstock ‘AxR # 1’. Variation in mean performance on these two hosts was partitioned among clones within collection sites and among sites. Performance measures included an individual analog to the intrinsic rate of increase (r), age at first oviposition, fecundity in the first ten days of reproduction, total fecundity, and longevity. The overall performance of phylloxera from the wild grapevines on the resistant cultivar AxR # 1 was greater than or equal to that on the susceptible cultivar Cabernet Sauvignon. There was significant variation among clones within populations from wild grapes in the rate of increase on ‘AxR # 1’ and marginally significant clonal variation in some of the component paramters. There was no significant variation among clones within populations on ‘Cabernet Sauvignon’ and no significant differences between populations on either crop in any trait. In a second experiment we compared the relative performance of 15–17 clones from wild grapevines and from commercial vineyards when reared on ‘Cabernet Sauvignon’ and ‘AxR # 1’. Phylloxera from commercial vineyards had much higher overall performance on ‘Cabernet Sauvignon’ than did phylloxera from the wild grapevines. Phylloxera from the commercial vineyard also had higher performance on ‘Cabernet Sauvignon’ than on ‘AxR′ 1’ but the performance of the phylloxera from wild and commercial grapes did not differ on ‘AxR # 1’. Our results show that there is genetic variation in traits related to performance on a resistant rootstock within these indigenous non-pest populations of phylloxera, but not among them. The pattern of performance of pest and non-pest populations on two commercial cultivars suggests that current levels of phylloxera performance on crop cultivars are the result of adaptation to those cultivars which has occurred while phylloxera has been associated with viticulture. Implications of these results for understanding the recent adaptation of phylloxera to ‘AxR # 1’ in California are also discussed.     

Isolation and characterization of first microsatellite markers for the noble crayfish, <Emphasis Type="Italic">Astacus astacus</Emphasis>

Noble crayfish (Astacus astacus) is listed as vulnerable by the IUCN Red List of Threatened Species in Europe. However, very little is known about genetic diversity and structuring of noble crayfish populations, mainly because of the lack of informative genetic markers. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4,000 recombinant clones. Eight loci revealed polymorphisms in a panel of 172 individuals from seven populations in Northern Europe. Number of alleles per locus ranged from two to 10 (average 4.4) and heterozygosity levels among populations varied from 0 to 0.80 for H _o and from 0 to 0.72 for H _e.     

Genetic processes in intergeneric cell hybrids Atropa + Nicotiana

Summary The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120–150 chromosomes including 50–70 of Atropa and 50–90 of Nicotiana. Other clones are polyploid and possess 200–250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.Dedicated to Professor G. Melchers with gratitude     

Behavioural Reproductive Isolation in a Rotifer Hybrid Zone

A hybrid zone between two Brachionus plicatilis rotifer mitochondrial DNA (mtDNA) lineages was recently described in the Iberian Peninsula between a pond (Santed 2) and a lake (Gallocanta). The patterns of mitochondrial and nuclear genetic variation observed suggested that gene flow is mainly male-mediated from the lake to the pond. Here we test two hypotheses: (a) that male-mediated gene flow occurs through assortative mating between individuals from these ponds, (b) that behavioural isolation occurs between the two mtDNA lineages. We isolated, reared and genotyped rotifer clones from resting eggs collected in the sediments of these and two other distant ponds. We devised a quick, inexpensive RFLP method to discriminate between B. plicatilis and its sibling species B. ‘Manjavaeas’ and between both mtDNA B. plicatilis lineages. Behavioural no-choice tests using new-born, virgin males and females were performed between five clones. B. ‘Manjavacas’ and B. plicatilis were reproductively isolated. B. plicatilis clones did not show evidence of reproductive isolation, regardless of their mtDNA lineage, except Santed 2 males, which discriminated strongly against Gallocanta females. These results could help to explain the discrepancies between mitochondrial and nuclear genetic variation reported in the two populations.     

Genetic Diversity and Geographic Differentiation in the Threatened Species Dysosma pleiantha in China as Revealed by ISSR Analysis

Dysosma pleiantha, an important threatened medicinal plant species, is restricted in distribution to southeastern China. The species is capable of reproducing both sexually and asexually. In this study, inter-simple sequence repeat marker data were obtained and analyzed with respect to genetic variation and genetic structure. The extent of clonality, together with the clonal and sexual reproductive strategies, varied among sites, and the populations under harsh ecological conditions tended to have large clones with relatively low clonal diversity caused by vegetative reproduction. The ramets sharing the same genotype show a clumped distribution. Across all populations surveyed, average within-population diversity was remarkably low (e.g., 0.111 for Nei’s gene diversity), with populations from the nature reserves maintaining relatively high amounts of genetic diversity. Among all populations, high genetic differentiation (AMOVA: Φ_ST = 0.500; Nei’s genetic diversity: G _ST = 0.465, Bayesian analysis: Φ_B = 0.436) was detected, together with an isolation-by-distance pattern. Low seedling recruitment due to inbreeding, restricted gene flow, and genetic drift are proposed as determinant factors responsible for the low genetic diversity and high genetic differentiation observed.     

An analysis of the phototactic behaviour of Daphnia magna clones and their sexual descendants

Daphnia magna clones, isolated from different natural populations, were inbred and crossbred. Some aspects of the heredity of the phototactic behaviour, quantified by an index, were analysed. Heritability in the broad sense was determined for several assemblages of clones through calculation of the clonal repeatability. Heritability sensu strictu was determined by offspring-parent regressions. Frequency distributions of a phototactic index of several inbred and crossbred families are given.The results indicate a significant contribution of additive genetic variance to the total phenotypic variance. Comparison of heritability estimates suggests an important genetic interaction component.Some implications of these results on the variability of vertical migration patterns in natural populations are discussed.     

Genetic variation in the diatomFragilaria capucina (Fragilariaceae) along a latitudinal gradient across North America

Genetic variation in 126 clones of the planktonic diatom,Fragilaria capucina, collected from seven lakes from Manitoba to Texas, was determined by RAPD analysis. Five primers yielded 48 scorable RAPD fragments in 123 unique combinations. Patterns of genetic variation were analysed by cluster analysis, which showed that most clones grouped together according to the site from which they were collected. This pattern in genetic variation may be a result of the geographically disjunct nature of these populations and/or the different environmental conditions, especially temperature, in the different lakes.     

Genetic comparison of wild populations ofLepilemur septentrionalis andLepilemur dorsalis using RAPD markers

The genetic polymorphism of Malagasy prosimian populations is studied by using RAPD markers. The analysis includes twoLepilemur septentrionalis populations of the area of Analamera separated by a deforested hill crest and one population ofL. dorsalis from the island of Nosy-Be. The genetic diversity is higher in the two populations of Analamera than in that of Nosy-Be and the level of genetic differentiation is higher between the population ofL. dorsalis and the two populations ofL. septentrionalis than between the two populations ofL. septentrionalis themselves. Despite the hill crest separating the two populations ofL. septentrionalis, our results demonstrate that they belong to one population. The respective roles of the geographical barriers and the reproductive barrier between the two species, are discussed.     

Effect of population spatial structure on pollination and seed set of a clonal distylous plant,Persicaria japonica (Polygonaceae)

In order to assess the mechanisms through which the spatial structure of the population influences female reproductive success, spatial distribution of clones, degrees of limitation of legitimate (inter-morph) pollination, type and abundance of pollen loaded on the stigmas, and seed set were measured for many clones of two natural populations of the distylous clonal plant,Persicaria japonica. Within the populations, according to the spatial relation to the nearest opposite morph clone, individual clones were assorted into two spatial types,i.e., clones that congregated with clones of the opposite morph (congregating clones), and clones that occurred singly at a considerable distance from the nearest opposite-morph clone (single clones). The pollination success,i.e., the proportion of legitimately pollinated flowers, and seed set were severely limited in the single clones compared to the congregating clones. Since artificial legitimate pollination improved the seed set in single clones, at least to some degree pollination failure was responsible for the reduced seed set in the single clones.     

Clonality in the Endangered <Emphasis Type="Italic">Ambrosia pumila</Emphasis> (Asteraceae) Inferred from RAPD Markers; Implications for Conservation and Management

Clonal plants have the ability to spread and survive over long periods of time by vegetative growth. For endangered species, the occurrence of clonality can have significant impacts on levels of genetic diversity, population structure, recruitment, and the implementation of appropriate conservation strategies. Here we␣examine clone structure in three populations of Ambrosia pumila (Nutt.) Gray (Asteraceae), a federally endangered clonal species from southern California. Ambrosia pumila is a perennial herbaceous species spreading from a rhizome, and is frequently found in dense patches of several hundred stems in a few square meters. The primary habitat for this species is upper terraces of rivers and drainages in areas that have been heavily impacted by anthropogenic disturbances and changing flood regimes. RAPD markers were employed to document the number and distribution of clones within multiple 0.25 m² plots from each of three populations. Thirty-one multi-locus genotypes were identified from the 201 stems sampled. The spatial distribution of clones was limited with no genotypes shared between plots or populations. Mean clone size was estimated at 9.10 ramets per genet. Genets in most plots were intermingled, conforming to a guerrilla growth form. The maximum genet spread was 0.59 m suggesting that genets can be larger than the sampled 0.25 m² plots. Spatial autocorrelation analysis found a lack of spatial genetic structure at short distances and significant structure at large distances within populations. Due to the occurrence of multiple genets within each population, the limited spread of genets, and a localized genetic structure, conservation activities should focus on the maintenance of multiple populations throughout the species range.     

Similarities and relationships among populations of the bulb onion as estimated by nuclear RFLPs

Random nuclear restriction fragment length polymorphisms (RFLPs) were used to assess similarities and relationships among open-pollinated (OP) populations of the cultivated bulb onion (Allium cepa). Seventeen OP populations and 2 inbreds of contrasting daylength response [termed by convention as long (LD) and short (SD) day], 1 shallot (A. cepa var. ascalonicum), and one cultivar of bunching onion (Allium fistulosum) were examined with 104 cDNA clones and two to four restriction enzymes. Sixty (58%) clones detected at least 1 polymorphic fragment scorable among the OP populations and were used for analyses. The average number of polymorphic fragments per polymorphic probe-enzyme combination was 1.9, reflecting that numerous monomorphic fragments were usually present. Similarities were estimated as the proportion of polymorphic fragments shared by 2 populations. Average similarity values among LD, among SD, and between LD and SD OP populations were 0.79, 0.67, and 0.68, respectively. Relationships among the OP populations were estimated by parsimony, cluster analysis of similarities using the unweighted-pair-group method (UPGMA), and multivariate analysis using principle components. Parsimony analysis generated a strict consensus tree that grouped all but 1 LD onion with unresolved relationships to the SD OP populations. The UPGMA analysis placed together the LD storage OP populations. Principal component analysis grouped all but 2 LD onions; the other OP populations were dispersed. The results suggest that LD and SD onions do not represent distinct germ plasm, but that LD storage onions represent a derived group selected for production at higher latitudes. If it is assumed that the sampled populations are representative of all onion OP populations, the lower similarities among SD OP populations indicate that their collection and maintenance in germ plasm collections is important for the preservation of genetic diversity.     

Methods used to reveal genetic diversity in the colony-forming prymnesiophytes Phaeocystis antarctica, P. globosa and P. pouchetii—preliminary results

Previous work on the genetic diversity of Phaeocystis used ribosomal DNA and internal transcribed spacer (ITS) sequence analyses to show that there is substantial inter- and intraspecific variation within the genus. First attempts to trace the biogeographical history of strains in Antarctic coastal waters were based on a comparison of ITS sequences. To gain deeper insights into the population structure and bloom dynamics of this microalga it is necessary to quantify the genetic diversity within populations of P. antarctica from different locations (i.e., each of the three major gyres in the Antarctic continental waters) and to calculate the gene flow between them. Here we describe methods to quantify genetic diversity and our preliminary results for P. antarctica in comparison to two other colonial species: P. globosa and P. pouchetii. For this study of genetic diversity, two fingerprinting techniques were used. First, amplified fragment-length polymorphisms (AFLPs) were established as a pre-screening tool to assess clone diversity and to select divergent clones prior to physiological investigations. Second, the more-powerful microsatellite markers were established to assess population structure and biogeography more accurately. Results show differences in the AFLP patterns between isolates of P. antarctica from different regions, and that a wide variety of microsatellite motifs could be obtained from the three Phaeocystis species.     

Genetic differentiation among Greek lake populations of Carassius gibelio and Cyprinus carpio carpio

The genetic structure of the Western Greece lake populations of Carassius gibelio and Cyprinus carpio carpio populations was characterized by using a PCR-based RFLP and sequencing analysis of mitochondrial rDNA genes and regions (16S rDNA, cytochrome b and D-loop). Our analysis was able to detect: (a) two haplotypes in C. c. carpio populations and two haplotypes in C. gibelio populations (b) a high nucleotide divergence between the two species and (c) two genetically distinct C. gibelio populations, one existing in the Amvrakia habitat (AMV1) with a second in Ozeros and Trichonida (OZE1 and TRI1) habitat. The present analysis indicates that genetic diversity observed was limited with a haplotype index between 0.0 and 55.6%, and a nucleotide diversity within and among populations between 0.0 and 1.27%. It also underlines a restricted mtDNA-based evaluation of the phylogenetic relationships among C. gibelio and C. c. carpio populations. In addition, the present study contributed knowledge on the genetic variation and structure of these populations which is absolutely necessary for any efficient fish management and/or conservation programme.     

New Resources for Marine Genomics: Bacterial Artificial Chromosome Libraries for the Eastern and Pacific Oysters (Crassostrea virginica and C. gigas)

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at