Comparative Analysis of MGE 412 Patterns in 18 Isogenic Lines of Drosophila melanogaster

Comparative analysis of patterns of mobile genetic element 412 was conducted in 18 isogenic lines of Drosophila melanogaster isolated in three isogenic experiments in 1987 through 1999. Twelve extra-hot isogenization sites (in 15–18 lines) and 23 hot isogenization sites (10 lines) were found; of these, 19 occurred in the original heterogeneous line. These sites virtually do not overlap with hot induction sites of transposition. Sites of the latter group generally retain their positions during isogenization. It was shown that no more than 20% of the new sites were brought from the balancer by double recombination, while inbreeding and outbreeding caused 80% of them. Different factors were shown to have different hot isogenization sites. A similarity tree was constructed for the patterns of 18 isogenic lines. The maximum peak of the tree was very low (< 0.25), i.e., the isogenic lines are more similar to than different from one another. The tree was subdivided into subtrees. The division was in good agreement with the isogenization groups corresponding to individual isogenization experiments. Significant correlation was found between the total fragment length and the number of new sites per lines.     

Comparative contribution of different genetic factors in induction of MGE transpositions under isogenization

Localization patterns of mobile genetic element 412 in polytene chromosomes of larvae from the control (riC) line, the balancer line, the F1 and F2 generations of the isogenization scheme, and 10 final isogenic lines were obtained and compared. The contributions of the recombination transfer of mobile genetic element copies from the balancer line, the outbreeding of control and balancer lines, and the inbreeding of isogenized lines to the rate of transposition were determined and estimated. These constituted < 0.187, < 0.30, and > 0.207 events per initial mobile genetic element copy per isogenized haploid genome per isogenization, respectively. During consecutive steps of isogenization (F1-F2-isogenic lines), the total transposition rate decreased: 2.09, 1.78, and 0.69. This was explained in terms of the existence of large selective and random losses in the variability of mobile genetic elements within the sites of their patterns during isogenization. The existence of a recombination transfer does not change the main conclusions and estimates regarding isogenization-induced transpositions.     

Molecular genetic analysis of hobo mobile genetic element polymorphism in the genome of Drosophila melanogaster line subjected to long-term selection

The distribution of mobile genetic element hobo was examined in Drosophila melanogaster lines HA (high male mating activity) and LA (low male mating activity) before and after their isogenization using Southern blot hybridization. The probe containing a full-size hobo copy was shown to produce polymorphic multilocus hybridization with chromosomal DNA. The polymorphism was line-specific. A comparison of hybridization patterns in isogenic and original lines showed that isogenization in dysgenic crosses resulted in the appearance of additional hobo localization sites in LA but not in HA. The hobo destabilization in the LA genome correlated with genetic instability and the ability to induce H-E hybrid dysgenesis. The results obtained are discussed in relation to the possible role of hobo in inducing genetic variability in lines with low male mating activity, which may counteract deleterious consequences of inbreeding and selection in the negative direction.     

Change in distribution of mobile genetic elements in genome of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: Cause or consequence of selection for quantitative traits?

The distribution pattern of the hobo transposon and Dm412 retrotransposon hybridization sites on the salivary gland polytene chromosomes from larvae of the Drosophila melanogaster isogenic strain 51 used to analyze the effect of the transposition of transposable elements (TEs) on selection for quantitative traits was studied. It was shown that no more than half of the Dm412 hybridization sites were retained 15 years after isogenization; the frequency of the Dm412 transposition varied from 2.0 × 10⁻⁴ to 8.8 × 10⁻⁵ sites per genome for generation depending on whether the appearance of the same hybridization sites in a part of individuals was considered as independent events or as the manifestation of the appearing sample heterogeneity. The distribution patterns of hobo hybridization sites in two isofemale strains derived from the isogenic strain 51 differed much more markedly; the number of the hobo sites in one of the derivative strains was threefold smaller than in the other one and only some of the sites were common. Within each derivative strain, the TE distribution was uniform, which suggests that inbreeding had no effect on Dm412 activity in this strain. The rates of change in the distribution patterns of various TE in the strain 51 corresponded to their spontaneous transposition rates. Since the isogenic strain accumulates polymorphism in the TE distribution without selection, the TEs are more likely to be the markers of selection events rather than their inducers. Thus, when studying the effects of various environmental factors on TE transposition even in isogenic strains, it is necessary to perform additional close inbreeding to reduce the potential polymorphism.     

Transposable elements and the penetrance of quantitative characters in Drosophila melanogaster

We wanted to determine whether there is a correlation between the quantitative character, the penetrance of the loss of humeral bristles in scute lines, and the distribution of transposable genetic elements in their genomes. We derived 18 isogenic lines with penetrance ranging between 2.8% and 92.0% from six mutant lines. The localization of the transposable elements (TEs) P, mdg1, Dm412, copia, gypsy and B104 was determined in all isogenic derivatives by in situ hybridization. The total number of the TE sites over all lines was 180. A comparison of the distribution of the TEs in the isogenic lines revealed the location of sites typical of lines with similar penetrance, no matter which parental line was involved. The results obtained suggest that such typical sites appear to tag the genome regions where the polygenes affecting the character in question are most likely to be found.     

Associations among inbred lines of maize using electrophoretic,chromatographic, and pedigree data

Summary Associations among 18 Lancaster Sure Crop derived inbred lines of maize (Zea mays L.) were determined using multivariate and cluster analysis. Objectives were to assess the degree of unique characterization among lines afforded by reversed-phase high-performance liquid chromatography (RP-HPLC) and starch gel electrophoresis of allozymes and to compare associations among lines revealed by biochemical and pedigree data. RP-HPLC revealed 11 different chromatograms that uniquely identified 79% of lines that differed by more than isogenic or near isogenic segments. Allozymic data for 21 loci provided unique discrimination among 93% of non-isogenic lines. Chromatographic and allozymic data together provided unique characterization of all non-isogenic lines. Cluster and multivariate analyses of biochemical data associated lines into three groups that would have been expected on the basis of pedigree breeding records. More detailed associations were dependent upon the data set employed. Multivariate and cluster analysis of chromatographic, electrophoretic, and pedigree data could be useful in revealing more detailed associations among elite germplasm than hitherto available, thus providing data pertinent to line and hybrid development, plant variety protection, and germplasm security.     

Analysis of MGE Dm412 localization pattern in Drosophila melanogaster lines undergoing long-term selection for a quantitative character]

The subpopulation composed of the mixture of Drosophila isogenic lines with interrupted wing radial vein (mutation radius incompletus, ri) was subjected to long-term selection in different directions for increase or decrease in expression of the ri gene. As a result, the lines with contrasting different values of mean character phenotype were developed. The isogenic lines of mean character phenotype were developed. The isogenic lines and F2 from their crosses with selected lines were analysed by the pattern of copia-like MGE DM412 localization. The isogenic lines were shown to have individual pattern, the selected lines differing strongly from them. Selection led to the loss of Dm412 localization sites during negative selection, while positive selection results both in loss and acquisition of sites. Correlation between the phenotype of the quantitative character and the pattern of MGE Dm412 was revealed.     

A novel approach to the analysis of the initiation of embryo development in gramineae

An in vivo model system to study the initiation of embryo development is presented. From the so-called Salmon system of wheat (alloplasmic lines with a 1BL-1RS chromosome translocation), three completely isogenic and homozygous lines were produced by selection for uniformity in about 20 selfing/backcross generations as well as between sublines of doubled haploids. The line (aestivum)-Salmon is male fertile and sexual. The lines (caudata)-Salmon and (kotschyi)-Salmon are male sterile and have a parthenogenetic capacity of about 90%. The expression of nuclear-cytoplasmic male sterility is different for the two parthenogenetic lines. The initiation of autonomous embryo development at defined developmental stages of the ovaries and the maximum degree of parthenogenesis are identical in both parthenogenetic lines as proved by the auxin test and progeny analyses. The protein patterns from ovary extracts of the three isogenic lines were identical for more than 200 spots of 2-D polyacrylamide gels, confirming their homogeneity. However, one protein (P 115.1) was found 3 days before and during anthesis only in ovaries of the parthenogenetic lines. It seems to be involved in the initiation of parthenogenesis.     

Analysis of a dispersed repetitive DNA sequence in isogenic lines of Drosophila

The location of sequences homologous to a cloned D. melanogaster DNA segment, Dm 25, has been examined in polytene chromosomes by hybridization in situ. Dm 25 localizes to multiple sites and shows variation in patterns between different strains and among individuals within wild-type laboratory strains. Analysis of numerous geographically distinct isogenic lines suggests that Dm 25 patterns are determined by germ-line factors and are not the product of strictly somatic events. In general there is wide variation in Dm 25 patterns among different lines, but a significant number of sites are common to two or more distinct lines. Hybridization to restriction digests of genomic DNA suggests that Dm 25 is a moderately repetitive, conserved sequence whose copies are dispersed throughout the genome. Analysis of species other than melanogaster indicates a significant divergence in structure of sequences homologous to Dm 25 as well as a drastic reduction in amount of homology to the melanogaster sequence.     

Current regeneration patterns at the tree line in the Pyrenees indicate similar recruitment processes irrespective of the past disturbance regime

Aim Impacts of global change, such as land‐use and climate changes, could produce significant alterations in the elevational patterns of alpine tree line ecotones and their adjacent vegetation zones. Because the responses of the tree line to environmental variations are directly related to successful tree regeneration, understanding recruitment dynamics is an indispensable step in tree line research. We aimed to compare potential ecological limitations on recent tree line regeneration in undisturbed and disturbed sites by analysing the demographic structure and spatiotemporal patterns of recruits and large trees. Location Alpine tree line ecotones comprising Pinus uncinata in the Catalan Pyrenees (north‐east Spain) and Andorra. Methods We assessed the demographic structure and spatial pattern of recent recruitment using techniques of point‐pattern and autocorrelation analyses. A total of 3639 P. uncinata individuals were mapped, measured and aged at 12 sites. To evaluate the effects of past disturbances on recent tree line response we compared tree lines that had either been recently affected by human‐induced disturbances or had remained undisturbed for many years. Results The age structure of the tree lines, together with the lack of an age gap between seedlings and saplings, did not indicate recent episodes of high seedling mortality and suggest that recruitment has been frequent under current climate conditions. Seedlings appeared highly aggregated at short distances (up to 3 m), irrespective of disturbance history, and were spatially segregated with respect to large trees. However, we found no evidence of patches of even‐aged seedlings, and our results suggest that dispersal events at intermediate distances (10–17 m) may be frequent. Autocorrelation analyses revealed different patterns of density and age of recruits between disturbed and undisturbed tree lines, but the strength and small‐scale clustering of seedlings and saplings were very similar between sites. Main conclusions We found no recruitment limitation on recent tree line dynamics in the Pyrenees. Furthermore, processes affecting tree recruitment seem to be similar among populations regardless of their past disturbance regime. Our results suggest that constraints on tree line dynamics causing differential responses between sites may operate on older life stages and not upon recruits, and that such constraints may be more contingent on local site conditions than on disturbance history.     

A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).     

How can we distinguish between mutational "hot spots" and "old sites" in human mtDNA samples?

New research into variation in mutation rates across nucleotide positions in human mitochondrial DNA (mtDNA) calls into question population genetics models that assume a constant mutation rate for all sites in a sequence, particularly for hypervariable control region segments I and II. Related to this research is discovering the extent to which highly polymorphic sites are really mutational "hot spots" rather than "old" sites rooted early in the phylogenetic tree. This issue is addressed through the analysis of linkage disequilibrium patterns in the mtDNAs of 10 human populations. Hot spots can be expected to show little or no disequilibrium since they can be interpreted as having randomly expressed patterns. In fact, the results suggest that many highly polymorphic sites are not old sites, but instead are hot spots. Suspected hot spots are listed and compared with hypervariable sites given by Wakeley (1993) and Hasegawa et al. (1993).     

Induction of MGE 412 Transpositions in Spermatogenesis of Drosophila Males Separately by Heat and Cold Shock

Effects of temperature treatment (heavy heat shock, HHS; heat shock, HS; and cold shock, CS) on the daily productivity of treated males in different spermatogenesis stages have been studied in isogenic line 51 of Drosophila melanogaster. The average productivity was shown to substantially decrease in all cases. The sum of the HS and CS contributions to this decrease was nearly equal to the HHS (the combined HS and CS) contribution, i.e., these contributions were almost additive. The temperature treatments did not kill mature sperm. In the control, mating productivity of day 1 exceeded that of the next day at least by 10–20%. Each day, most sperm in matings was new, i.e., matured during that day. Transposition induction of MGE 412was studied at four spermatogenesis stages after HS and CS. Both temperature treatments were effective but CS had a more pronounced inducing effect. Most temperature-induced transpositions occurred at stage III (meiosis) and IV (spermiogenesis). The day rates of transpositions at different stages were estimated. After HS at the meiosis stage, = 0.11 events per initial MGE copy per sperm per day of mating, which is approximately equal to the previous estimates after HHS. After CS at the meiosis stage, = 0.51. The transposition hot sites (including the previously known 43B and 97DE as well as a number of new sites) were detected. The lists of transpositions after CS completely included the corresponding lists after HS, which suggests similarity of induction mechanisms underlying CS and HS.     

ES Cell Cycle Rates Affect Gene Targeting Frequencies

We have investigated the gene targeting frequency at thehprtlocus in a range of embryonic stem cell lines selected for variations in cell cycle parameters. Our results show that targeting frequency varies with cell line by as much as 12-fold between nonisogenic lines and 3-fold between isogenic lines and that a nonisogenic line can support homologous recombination events by up to 21-fold more frequently than an isogenic line. This variation is consistent with both insertion and replacement vectors. These results can be explained by an inverse linear correlation of targeting frequencies with cell doubling times. Additionally, by reducing serum concentration in the culture medium the mean cell doubling time for R1 ES cells can be increased from 11.4 to 15.7 h, with a subsequent 15-fold decrease in gene targeting frequency. This change fits the correlation found for the different nonisogenic cell lines. Our observations have important implications when performing gene targeting experiments and explain some of the variation noted between experiments.     

Proteomic approach for investigation of cytoplasmic male sterility (CMS) in Brassica

The alloplasmic ‘Tournefortii’ CMS system fromB. napus was chosen for a comprehensive investigation of the protein content of the mitochondrial compartment in male sterile and fertile near isogenic lines. As proteins involved in the male sterile phenotype may be expressed in a tissue-specific manner, mitochondrial preparations were made from different tissues and checked for the purity of the organelles with a new and highly sensitive method (BN-PAGE). InBrassica, only mitochondrial preparations from 4-5-day-old seedlings and from flower buds were found to be sufficiently pure. High resolution, two-dimensional gel electrophoresis of total protein from anthers and from mitochondrial protein of male sterile and fertile seedlings of near isogenic lines revealed distinct differences in the protein patterns. Several explanations for the occurrence of these differences, which may be the starting point for a detailed molecular analysis and a better understanding of the CMS syndrome, are discussed.     

Transposition induction of the mobile genetic element Dm412 in the Drosophila genome using heat shock]

Males of Drosophila melanogaster isogenic line with oligogene mutation radius incompletus (ri) were exposed to standard heat-shock (SHS: t = 37 degrees C, 90 min) and heavy heat-shock (SHS: three-fold transfer of males from t = 37 degrees C, 2h, t0t = 4 degrees C 1 h, and back). At F1 of the treated males with untreated females of the same isogenic line mass transpositions of MGE Dm412 were found. The new positions of MGE seem to be not random, and 5 "hot sites" of transpositions were detected. The probabilities of transpositions were estimated after SHS and HHS and in control sample. They were, correspondingly, 3.4 x 10(-2), 8.7 x 10(-2) and less than 4.1 x 10(-4) transpositions per genome, per site occupied, per generation. Therefore, as a result of HS treatment, the probabilities of transpositions were two orders of magnitude increased as compared to control, directly at next generation after induction. Comparison of these results with those obtained after step-wise temperature treatment shows that induction is dependent rather of "stressor effect" of temperature treatment than of treatment way used.     

Virulence variation of Puccinia striiformis Westend. f. sp. tritici in Pakistan

Yellow rust populations of Pakistan were characterised for their virulence pathotypes/races and pathogenetic variation using seedling evaluation of differential genotypes under glasshouse conditions in Murree (6000 feet above sea level). Differential genotypes comprised a world set, an European set, near isogenic lines and the universally susceptible bread wheat cultivar “Morocco”. Over the two-year study a total of 18 race groups were identified. Out of these 18 race groups, several (68E0, 64E0, 66E0, 70E0, 6E0, 71E0, 6E0, 2E0, 67E0, and 68E16) were found previously. The new race group 70E32 found probably evolved because of mutation from the previously existing 70E16. Virulence frequencies of yellow rust (Yr) resistance genes were also determined on near isogenic lines. The highest virulence frequencies (%) were found for Yr7 (88%), Yr9 (57%), Yr18 (70%), and Yr24 (69%). Virulence frequencies were low for Yr 1 (4%), Yr5 (7%), Yr10 (10%) and Yr15 (4%). Our studies indicated that virulence existed for almost all yr genes, necessitating regular monitoring of the yellow rust populations and intensifying efforts to identify new sources of resistance to this pathogen.     

Within-tree and among-tree variation in growth of Epirrita autumnata on mountain birch leaves

Abstract.

• 1 We studied among-tree and within-tree variation in the growth of larvae of a geometrid, Epirrita autumnata, on mountain birch (Betula pubescens ssp. tortuosa) leaves at different hierarchical levels: among trees (genets), among ramets within trees, among branches within ramets, among shoots within branches and among leaves within shoots. We used only short shoot leaves, which burst simultaneously in spring.
• 2 Trees accounted for most of the variation in larval growth rate, but there was substantial variation also among ramets within trees, among branches within ramets, and among short shoots within branches. Variation among leaves within short shoots was negligible. When the probabilities from different experiments were combined, the differences were statistically significant at the tree, ramet and branch levels, and approached significance at the shoot level.
• 3 In different experiments, larval growth was from 9% to 54% lower on the worst tree than on the best tree of the experiment. On average, larval growth rate was in different experiments from 11% to 32% lower on the worst ramet than on the best ramet within tree, from 8% to 18% lower on the worst than on the best branch within ramet, and from 12% to 30% lower on the worst than on the best shoot within branch.
• 4 The amount of among-and within-tree variation shown by our results may have ecological and evolutionary implications: among-tree variation should select for discrimination by ovipositing females and dispersing larvae, within-tree variation should select also for optimal foraging behaviour of larvae.

     

The Effect of Level of Heterozygosity on the Performance of Hybrids between Isogenic Lines of Barley

Sixteen "isogenic" lines of Atlas 46 barley differing in one to four short chromosome segments, and 16 heterozygotes obtained by crossing these lines to male-sterile Atlas, were used to study the effect of level of heterozygosity on performance. In field tests conducted in four environments (two planting dates in two years) significant differences were found among the homozygous isogenic lines for the traits seed yield, kernel weight, tiller number, plant height, and heading time; thus each of the marked chromosome segments carries genes which, when homozygous, affect these quantitative characters. It was also found that heterozygotes produced more and heavier kernels and were taller and earlier than homozygotes but there was no clear indication that the degree of heterosis increased as the number of heterozygous segments increased from one to five. Degree of heterosis was, however, strongly affected by the environment, by allelic state at each segment (especially the segment marked by the two-row, six-row spike locus), and also by genotype for other marked segments. These results indicate that heterosis in barley has a more complex structure than can be adequately represented by simple models, such as the multiplicative model in which fitnesses are the product of fitnesses at individual loci, or threshold models in which optimum fitness is approached asymptotically as the number of heterozygous loci increases.     

Uncorrelated mistletoe infection patterns and mating success with local host specialization in <Emphasis Type="Italic">Psittacanthus calyculatus</Emphasis> (Loranthaceae)

Mistletoe infection between conspecific and interspecific hosts can be restricted by seed dispersal, host-mistletoe compatibility and abiotic factors, yet no studies have linked mistletoe infection patterns and pollination together for understanding mistletoe distribution at a local scale. Psittacanthus calyculatus (Loranthaceae) is a hemiparasitic plant with a broad host range across its geographic distribution. The potential for local host adaptation has been shown using cross-inoculation experiments, in which plants of mistletoe seeds collected from a given host are more likely to survive when they are inoculated on conspecific host trees compared with those inoculated on other host provenances. Here we evaluate host adaptation by describing the local patterns of infection (prevalence and intensity) of P. calyculatus mistletoes on three native host tree species (Alnus acuminata, Quercus crassipes, Salix bonplandiana) and one introduced species (Populus alba) and carried out cross-pollination experiments to examine how pollination affects infection patterns of different host species. Mistletoe infection prevalence (proportion of infection) and infection intensity (mean number of mistletoes per tree) were in general disproportional with respect to the availability of native host tree species but higher to that of non-native host tree species. Cross-pollination experiments showed higher mating success on the native host tree species, suggesting higher local adaptation to specially Q. crassipes. The observed spatial distribution of host tree species and mistletoe infection along with the non-random mating could contribute to local genetic structuring of mistletoe populations.