A DNA Bank of People Having Experienced Occupational Irradiation: Aims and Prospects

A DNA bank was created to preserve the genetic material of staff of the first Russian atomic plant, Production Association (PO) Mayak, who were exposed to chronic external -irradiation at various doses in the early years of plant operation. Some of the workers experienced combined irradiation from external and internal (incorporated plutonium-239) sources. The unique genetic material of the subjects and their children can be used to study the remote consequences of irradiation by means of modern molecular-genetic techniques. At present, the bank contains the genetic material of 500 people. A database was constructed to include medical, demographic, professional, and dose characteristics of each individual and quantitative and qualitative parameters of each DNA preparation. The bank is continuously augmented with new DNA samples of the irradiated people and members of their families.     

Genetic comparisons between seed bank and Stipa krylovii plant populations

The soil seed bank represents the potential plant population since it is the source for population replacement. The genetic structure of a Stipa krylovii (Roshev.) plant population and its soil seed bank was investigated in the Xilinguole Steppe of Inner Mongolia using random amplified polymorphic DNA (RAPD) analyses. The population was sampled at two sites that were in close proximity to each other (0.5 km apart). Thirty plants and 18 seed bank samples were taken from each site to determine the genetic diversity between sites and between sources (plant or seed). The material was analyzed using 13 primers to produce 92 loci. Eighty-six were multi-loci, of which 23 loci (26.74%) of allele frequencies showed significant differences (P < or = 0.05). The genetic similarity between two seed bank sites was 0.9843 while the genetic similarity between two plant sites was 0.9619. Their similarities were all greater than that between the seed bank and plant populations. An analysis of their genetic structure showed that 87.86% of total variation was derived by two-loci. Genetic structures between plant and soil seed bank populations in S. krylovii were different due to the variance of mean gametic disequilibria and mean gene diversity. AMOVA results showed that the majority of variance (88.62%) occurred within sites, 12.75% was from between-groups. Further research is needed to investigate the selective function in maintaining the genetic diversity of Stipa krylovii plant populations.     

Genetic comparisons between seed bank and <Emphasis Type="Italic">Stipa krylovii</Emphasis> plant populations

The soil seed bank represents the potential plant population since it is the source for population replacement. The genetic structure of a Stipa kryiovii (Roshev.) plant population and its soil seed bank was investigated in the Xilinguole Steppe of Inner Mongolia using random amplified polymorphic DNA (RAPD) analyses. The population was sampled at two sites that were in close proximity to each other (0.5 km apart). Thirty plants and 18 seed bank samples were taken from each site to determine the genetic diversity between sites and between sources (plant or seed). The material was analyzed using 13 primers to produce 92 loci. Eighty-six were multi-loci, of which 23 loci (26.74%) of allele frequencies showed significant differences (P ≤ 0.05). The genetic similarity between two seed bank sites was 0.9843 while the genetic similarity between two plant sites was 0.9619. Their similarities were all greater than that between the seed bank and plant populations. An analysis of their genetic structure showed that 87.86% of total variation was derived by two-loci. Genetic structures between plant and soil seed bank populations in S. krylovii were different due to the variance of mean gametic disequilibria and mean gene diversity. AMOVA results showed that the majority of variance (88.62%) occurred within sites, 12.75% was from between-groups. Further research is needed to investigate the selective function in maintaining the genetic diversity of Stipa krylovii plant populations.     

Genetic efficacy of low doses of ionizing radiation in chronically-irradiated small mammals

Earlier we have established the genetic effects of low dose chronic irradiation in bank vole (somatic and germ cells, embryos), in pond carp (fertilized eggs, embryos, fry) and in laboratory mice (somatic and germ cells) in the range of doses from near-background to 10 cGy. These low dose effects observed in mammals and fish are not expected from extrapolation of high dose experiments. For understanding reasons this discrepancy the comparative analysis of genetic efficiency of low dose chronic irradiation and the higher doses of acute irradiation was carried out with natural populations of bank vole which inhabited the two sites differing in ground of radionuclide deposition. For comparing efficiency the linear regression model of dose-effect curve was used. Dose-effect equations were obtained for animals from two chronically irradiated bank vole populations. The mean population absorbed doses were in the range 0.04-0.68 cGy, the main part of absorbed doses consisted of external radiation of animals exposed to 137Cs gamma-rays. Dose-effect equations for acute irradiation to 137Cs gamma-rays (10-100 cGy) were determined for the same populations. Comparison of genetic efficiency was made by extrapolation, using regression coefficient beta and doubling dose estimation. For chronic exposure the doubling doses calculated from low-dose experiments are 0.1-2 cGy and the doubling doses determined from high-dose experiments are in the range of 5-20 cGy. Our hypothesis that the doubling dose estimate is calculated in higher-dose ionizing radiation experiments should be much higher than the deduced from the low dose line regression equation was verified. The doubling dose estimates for somatic cells of bank vole and those for germ cells of laboratory mice are in close agreement. The radiosensitivity of bank vole chromosomes were shown is practically the same as that for human lymphocytes since doubling dose estimates for acute irradiation close to each other. For low LET radiation a higher genetic efficiency of chronic low doses in comparison with the higher doses of acute gamma-irradiation (137Cs source) was proved by three methods.     

The role of genetic factors in human radioresistance

The role of genetic factors in the development of chronic radiation disease (CRD), mostly caused by occupational external gamma-exposure, was evaluated. The data of molecular genetic survey of a cohort of 985 workers at the nuclear power plant, the Mayak PA, were analyzed. Among the genetic markers tested, an association between the haptoglobin (Hp) genetic system and the development of CRD was established. It was demonstrated that the contribution of genetic factors to the CRD onset was realized not within the whole, but in a relatively narrow dose interval (70 to 400 cGy), i.e., was relative. Furthermore, at equal irradiation doses, relatively higher risk of CRD was observed among the Hp 2-2 phenotype carriers (1.96) compared to lower risk among the Hp 1-1 and Hp 2-1 phenotype carriers (0.64). It was shown that with the increase of the irradiation dose, genotypic differences in the CRD frequency decreased to the point of their complete disappearance. Comparison of the roles of the genetic factors in the onset of such deterministic irradiation effect as CRD, with their roles in the onset of lung cancer in tobacco smokers revealed similar patterns. A scheme of the relationships between the effector intensity and the differences in the genetically determined radioresistance is presented. The data obtained do not support the idea that the survivals of the atomic bombing of Hiroshima and Nagasaki were the most radioresistant individuals, who are not representative for evaluating the radiation risk.     

Effect of dehydration duration of apices on characteristics of in vitro plants of <Emphasis Type="Italic">Fragaria vesca</Emphasis> after cryopreservation

Effect of different duration of dehydration of the apices isolated from in vitro plants on genetic stability was investigated in regenerated plants of wild strawberry (Fragaria vesca L., var. alpine) recovered after cryopreservation according to a precultivation-dehydration protocol. Plant material belongs to a clone (cv. Reine des Vallees) that has been maintained in vitro for more than 25 years in Timiryazev Institute of Plant Physiology. It was shown that duration of desiccation the apices before freezing appreciably affected the rate of postcryogenic recovery of plant growth and coefficient of their subsequent propagation. After 5-h-long desiccation, apices were notable for the highest growth rate. The plants restored from such apices also had the highest coefficient of propagation. For DNA analysis, the samples of leaves were taken separately from each plant after hardening and after cryopreservation. According to the results of RAPD, ISSR, and REMAP analyses, the plants from the chosen clone of strawberry showed some genetic variation prior to cryopreservation (percentage of polymorphic fragments was 9.0%). Plant adaptation to cold did not change the level of genetic variation. Among postcryogenic regenerants, morphologically modified plant forms were not observed, with the level of DNA marker variation decreasing almost two times irrespective of the duration of dehydration. However, in one plant restored after 5-h-long dehydration and cryogenic freezing, a 1200 bp fragment of DNA was lacking, which was detected in all other examined samples (frequency of deviation was 0.9%). Earlier, we did not reveal plant polymorphism of investigated strawberry clone associated with this fragment. Probably, this modification of DNA resulted from the exposure of plant material to dehydration and freezing in liquid nitrogen.     

Historic DNA reveals genetic consequences of fragmentation in an endangered,endemic mustard

Understanding how anthropogenic disturbance affects genetic diversity is essential to appropriately incorporating genetic considerations into conservation plans. Unfortunately, we rarely have information about a population’s genetic diversity before it becomes imperiled. Here we reconstruct the historic range of the naturally rare annual mustard Streptanthus glandulosus subsp. niger (Sgn) and use herbarium specimens to quantify pre-disturbance genetic diversity. We compare this to the genetic diversity in the contemporary plant populations and to plants in the seed bank. We conclude that Sgn was recently a single, panmictic population composed of orders of magnitude more plants than exist today but experienced recent and abrupt declines following housing development. Today Sgn persists as two disjunct populations, the larger of which has retained historic levels of diversity although there is a downward trend in all measures. The smaller population has lost 21–28% of the diversity that was present only 50 years ago with an N_e?~?5–16. The contemporary populations have differentiated from each other due to drift. The seed bank contained no novel alleles and had high levels of homozygosity, indicating that it is incapable of providing genetic rescue. This novel combination of hDNA, the aboveground plant population and the seed bank can be used to design high impact conservation plans that appropriately incorporate genetic diversity for this and other imperiled species.

     

Population genetic structure of Vitex negundo (Verbenaceae) in Three-Gorge Area of the Yangtze River: The riverine barrier to seed dispersal in plants

Previous studies have demonstrated that large rivers can influence inter- and intra-specific gene flow for many animals. The effects of large rivers on the genetics of plant populations have focused on either hydrochoric impacts of water current on gene flow or genetic differentiation among populations from different watersheds. Few studies have explicitly tested the barrier effects on plant gene flow across banks of large rivers, especially their relative effects on pollen and seed dispersals. The Yangtze River (Changjiang River), one of the major rivers of the world, provides an excellent model to evaluate the impacts of rivers on gene flow in plants. Using RAPD (random amplified polymorphic DNA) and cpDNA (chloroplast DNA) markers, we investigated the genetic structure of 10 populations of Vitex negundo in two regions of Three-Gorge Area along the Yangtze River. Each region contained two populations on the north bank, two on the south bank and one island population along the river. The analyses indicated low RAPD between banks, and similar or a little higher differentiation between populations within the same bank. In contrast, a large proportion of chloroplast polymorphism was ascribed to among-bank variation but much lower cpDNA differentiation was among populations within the same bank. These results indicate that the Yangtze River represents a general barrier to the dispersal of seeds but not to the movement of pollen in V. negundo. The cpDNA genetic distances or differentiations between the island populations and those on either bank of the river are intermediate to those between the banks across the river, implying that the islands in the Yangtze River may serve as a stepping-stone for seed dispersal. Our results suggest that large rivers may serve as a general barrier, not only for the movement of animals, but also for the dispersal of plants, which should be of great significance for the conservation of biodiversity around the rivers.     

Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419.

Four Tn5-induced mutants of Rhizobium meliloti WSM419 were unable to grow or maintain intracellular pH at an external pH of 5.6. Restriction analysis of DNA fragments carrying Tn5 and flanking sequences cloned from these mutants indicated that all four cloned mutations are unique and that the two strains (TG1-6 and TG1-11) carry Tn5 insertions which are within 4.4 kilobases of each other on a single EcoRI fragment. Southern analysis of total mutant DNA indicated a single copy of Tn5 in each mutant. A limited cosmid gene bank of wild-type WSM419 DNA was probed for homology to mutant DNA cloned from the acid-sensitive mutants. Dot hybridization experiments identified one cosmid element within this bank carrying wild-type DNA sequences corresponding to DNA implicated in acid tolerance. This cosmid was able to complement defects in growth and intracellular pH maintenance in TG1-11 but not TG1-6.     

Easy strategy used to detect the genetic variability in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

A priority in the management and use of elite plant materials for breeding has been based on molecular markers or DNA sequencing of entire genomes, in order to perform genetic differentiation which is still quite costly. Chickpea (Cicer arietinum) is one of the species with genomic monotony and very low polymorphism, and its detection even with DNA markers has not been easy. In germplasm banks, the genetic distinction is a priority in order to use properly selected lines. In this study, 57 chickpea accessions from a germplasm bank were analyzed by using nrRAMP (non-radioactive Random Amplified Microsatellite Polymorphism) markers, and their genetic variability was determined. Our results showed DNA polymorphisms, which are enough to differentiate between the accessions and between C. arietinum and Cicer reticulatum (out-group); this last wild species is closely related to chickpea. We concluded that the nrRAMP technique was an effective and a highly useful method to assess the genetic diversity and variability among closely related plants, such as chickpea; in addition, this technique can be easily implemented in laboratories.     

Novel perspectives in wood certification and forensics: dry wood as a source of DNA

The importance of wood for human societies can hardly be understated. If dry wood were amenable to molecular genetic investigations, this could lead to major applications in wood forensics, certification, archaeology and palaeobotany. To evaluate the potential of wood for molecular genetic investigations, we have attempted to isolate and amplify, by PCR, DNA fragments of increasing size corresponding to all three plant genomes from different regions of 10 oak logs. Stringent procedures to avoid contamination with external DNA were used in order to demonstrate the authenticity of the fragments amplified. This authenticity was further confirmed by demonstrating genetic uniformity within each log using both nuclear and chloroplast microsatellites. For most wood samples DNA was degraded, and the sequences that gave the best results were those of small size and present in high copy number (chloroplast, mitochondrial, or repeated nuclear sequences). Both storage conditions and storage duration play a role in DNA conservation. Overall, this work demonstrates that molecular markers from all three plant genomes can be used for genetic analysis on dry oak wood, but outlines some limitations and the need for further evaluation of the potential of wood for DNA analysis.     

Ex situ conservation of underutilised fruit tree species: establishment of a core collection for Ficus carica L. using microsatellite markers (SSRs)

Ex situ germ plasm collections of woody crops are necessary to ensure the optimal use of plant genetic resources. The fig tree (Ficus carica L.) germ plasm bank, consisting of 229 accessions, is located in Centro de Investigación ‘La Orden’. Despite great progress in conservation, ex situ collections face size and organization problems. Core collections obtained from structured samples of bigger collections are a useful tool to improve germ plasm management. In this work, we used simple sequence repeat (SSR) markers to establish a core collection in this underutilised Mediterranean fruit tree species. Four approaches have been carried out (random sampling, maximization, simulated annealing and stepwise clustering) to determine the best method to develop a core collection in this woody plant. The genetic diversity obtained with each subset was compared with that of the complete collection. It was found that the most efficient way to achieve the maximum diversity was the maximization strategy, which, with 30 accessions, recovers all the SSR alleles and does not show significant differences in allele frequency distribution in any of the loci or in the variability parameters (H _O, H _E) between the whole and core collections. Thus, this core collection, a representative of most fig diversity conserved in the germ plasm bank, could be used as a basis for plant material exchange among researchers and breeders.     

The role of genetic factors in human radioresistance

The role of genetic factors in the development of chronic radiation disease (CRD), mostly caused by occupational external -exposure, was evaluated. The data of molecular genetic survey of a cohort of 985 workers at the nuclear power plant, the Mayak PA, were analyzed. Among the genetic markers tested, an association between the haptoglobin (Hp) genetic system and the development of CRD was established. It was demonstrated that the contribution of genetic factors to the CRD onset was realized not within the whole, but in a relatively narrow dose interval (70 to 400 cGy), i.e., was relative. Furthermore, at equal irradiation doses, relatively higher risk of CRD was observed among the Hp 2-2 phenotype carriers (1.96) compared to lower risk among the Hp 1-1 and Hp 2-1 phenotype carriers (0.64). It was shown that with the increase of the irradiation dose, genotypic differences in the CRD frequency decreased to the point of their complete disappearance. Comparison of the roles of the genetic factors in the onset of such deterministic irradiation effect as CRD, with their roles in the onset of lung cancer in tobacco smokers revealed similar patterns. A scheme of the relationships between the effector intensity and the differences in the genetically determined radioresistance is presented. The data obtained do not support the idea that the survivals of the atomic bombing of Hiroshima and Nagasaki were the most radioresistant individuals, who are not representative for evaluating the radiation risk.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 85–92.Original Russian Text Copyright © 2005 by Telnov.     

Frequency of dominant lethal mutations induced by combined exposure to incorporated 137Cs and external gamma-irradiation in mice

The genetic effect of combined exposure to incorporated 137Cs and external gamma-irradiation was studied in germ cells of male mice. The activity of incorporated 137Cs was 3.7 x 10(4) Bq/g. The dose of external gamma-irradiation was 1.5 Gy. In the case of combined exposure, mice were treated with a cesium solution immediately after irradiation. The genetic effect was estimated by the frequency of dominant lethal mutations (DLM) induced at different stages of spermatogenesis. Upon combined exposure to external and internal irradiation, the frequency of DLM in premeiotic cells significantly exceeded the total frequency of DLM induced by separate exposure to external and internal irradiation; i.e., a marked synergistic interaction of external and internal irradiation was observed.     

Traditional ecological knowledge and use of vegetation in southeastern Mexico: a case study from Solferino, Quintana Roo

In order to assess traditional ecological knowledge of the Maya people in southeastern Mexico, we interviewed local people in Quintana Roo and estimated a number of vegetation variables in two different types of forest which are currently locally exploited, namely Monte alto (medium statured forest) and Sakal che' (low forest). We employed the Use Value index for each plant species (UV_s) to quantify the importance of each plant for each inhabitant. The results showed that this Maya community classify the different forest types by species associations and size, and according to soil appearance. A total of nine categories of use were defined for three plant forms (tree, palm and vine). Manilkara zapota (zapote), Thrinax radiata (chiit) and Macfadyena uncata (bilin kok) showed the highest use values for each plant form. The most common uses were construction (35.5%), medicine (19.0%), craft (17.9%) and edibility (10.3%). There was a weak relationship between the cultural importance of plant species, expressed by the UV_s, and their availability in the medium statured forest and the medium statured–low forest transition expressed by the Importance Value index (IVI). The medium statured forest was the most used forest type, as it provides many species for construction due to external demands rather than to local needs.     

Can a seed bank maintain the genetic variation in the above ground plant population?

There are indications that a persistent seed bank can protect small and isolated plant populations from local extinction. Genetic mechanisms contributing to this phenomenon are the increase of local effective population size – and hence the decrease of genetic drift – through a reservoir of persistent seeds, and the accumulation of intergenerational genetic diversity in the seed bank. To find evidence for these mechanisms, we conducted two formal meta-analyses. First, we analyzed 42 published habitat fragmentation studies and investigated whether the degree of genetic differentiation between fragmented plant populations was mediated by seed longevity. Second, we reviewed 13 published studies reporting the genetic diversity of both the seed bank and the above ground plants, aiming at comparing genetic diversity contained in the seed bank with the above ground vegetation. We conclude that a persistent seed bank may indeed mitigate the consequences of habitat fragmentation and protect a species from genetic drift and population genetic differentiation. We found no evidence, however, of high levels of genetic diversity accumulating in the soil seed bank. If genetic differences are present between the standing crop and the seed bank, they are very likely the result of local selection acting either directly or indirectly as a filter on the alleles present in the seed bank. We finally suggest that 1) the role of the seed bank should not be neglected in habitat fragmentation studies and 2) it is not very fruitful to continue comparing seed bank genetic diversity with above ground plant genetic diversity, unless this is performed under different selection regimes.     

Comparison of genetic and cytogenetic maps of hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) using SSR and DArT markers

A number of technologies are available to increase the abundance of DNA markers and contribute to developing high resolution genetic maps suitable for genetic analysis. The aim of this study was to expand the number of Diversity Array Technology (DArT) markers on the wheat array that can be mapped in the wheat genome, and to determine their chromosomal location with respect to simple sequence repeat (SSR) markers and their position on the cytogenetic map. A total of 749 and 512 individual DArT and SSR markers, respectively, were identified on at least one of four genetic maps derived from recombinant inbred line (RIL) or doubled haploid (DH) populations. A number of clustered DArT markers were observed in each genetic map, in which 20–34% of markers were redundant. Segregation distortion of DArT and SSR markers was also observed in each mapping population. Only 14% of markers on the Version 2.0 wheat array were assigned to chromosomal bins by deletion mapping using aneuploid lines. In this regard, methylation effects need to be considered when applying DArT marker in genetic mapping. However, deletion mapping of DArT markers provides a reference to align genetic and cytogenetic maps and estimate the coverage of DNA markers across the wheat genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A predatory mechanism dramatically increases the efficiency of lateral gene transfer in Streptococcus pneumoniae and related commensal species

Bacteria that are competent for natural genetic transformation, such as pneumococci and their commensal relatives Streptococcus mitis and Streptococcus oralis , take up exogenous DNA and incorporate it into their genomes by homologous recombination. Traditionally, it has been assumed that genetic material leaking from dead bacteria constitutes the sole source of external DNA for competent streptococci. Here we describe a mechanism for active acquisition of homologous DNA that dramatically increases the efficiency of gene exchange between and within the streptococcal species mentioned above. This mechanism gives competent streptococci access to a common gene pool that is significantly larger than their own genomes, a property representing a considerable advantage when these bacteria are subjected to external selection pressures, such as vaccination and treatment with antibiotics.     

Computer simulations to determine the efficacy of different genome resource banking strategies for maintaining genetic diversity

Genome resource banks (GRBs) and assisted reproductive techniques are increasingly recognized as useful tools for the management and conservation of biodiversity, including endangered species. Cryotechnology permits long-term storage of valuable genetic material. Although, the actual application to endangered species management requires technical knowledge about sperm freezing and thawing, a systematic understanding of the quantitative impacts of various germ plasm storage and use scenarios is also mandatory. In this study, various GRB strategies were analyzed using the historical data from three managed populations of endangered species with varied pedigrees (Eld's deer, Przewalski's horse, and Sumatran tiger). The following types of sperm banks were assessed: (1) a "Wild Bank" consisting of sperm (i.e., genes) from 5 to 10 males unrelated to the managed population and to each other; and (2) a "Best Male" bank containing sperm from only the most genetically valuable males alive in the ex situ population at the time the bank was established. These different bank types were then used to evaluate the effectiveness of different bank usage frequencies. The efficiency of each scenario was assessed by examining the level of inbreeding and gene diversity in the population. Overall, a sperm usage frequency of five times per year was determined to be the most efficient and "wild banks" were highly successful at enhancing genetic diversity. The value of a GRB established from the ex situ population depends on how closely related the banked males are to future generations. A GRB will have significantly less benefit when banked males also produce many successful offspring, or when donors are already genetically over-represented in the population at the time of establishing the GRB.