The diversity of chloroplast microsatellite loci in Siberian fir (Abies sibirica Ledeb.) and two Far East fir species A. nephrolepis (Trautv.) Maxim. and A. sachalinensis Fr. Schmidt

The genetic variability in 29 populations of Abies sibirica, three of A. nephrolepis, and seven of A. sachalinensis was studied using SSR markers of chloroplast DNA. Among ten primer pairs examined, pairs Pt71936 and Pt30204 gave stable amplification and polymorphic products (with nine and fourteen alleles, respectively). Totally, 70 haplotypes were found, 43 in A. sibirica, 49 in A. sachalinensis, and 31 in A. nephrolepis. The highest values of genetic diversity parameters were observed in A. sachalinensis, and the lowest in A. sibirica. The Siberian fir differs from Far East species by the uneven multimodal frequency distributions of allele length in both cpSSR loci, which is explained by the presence of few separated from each other dominating haplotypes. This fact indicates that A. sibirica and the Far East species have different demographic histories. In A. sibirica, the proportion of diversity between populations in the total genetic diversity, calculated taking into account the differences between haplotypes (R _ST) was 8.34 and 4.42% without accounting for haplotypes differences (R _ST > G _ST, P = 0.01). The pairwise G _ST correlate significantly with geographic distances between the populations A. sibirica and with genetic distances D calculated from allozyme data. No such correlations were found with the R _ST parameter. The results of cpSSR variability analysis strongly support the conclusions inferred from allozyme data: several geographic groups of comparatively genetically close populations are identified, which may be explained by the history of colonization of the present-day Siberian fir range. Original Russian Text ? S.A. Semerikova, V.L. Semerikov, 2007, published in Genetika, 2007, Vol. 43, No. 12, pp. 1637–1646.     

Genetic variability of Siberian fir (Abies sibirica Ledeb.) inferred from AFLP markers

Genetic variability of AFLP markers was studied in 20 populations of Siberian fir (Abies sibirica (Pinaceae) and in two populations of Far-Eastern Manchurian fir A. nephrolepis and Sakhalin fir A. sachalinensis each. Four pairs of selective primers were used. In total, 168 samples from three fir species were genotyped for 117 polymorphic loci. According to the AMOVA results, the variability proportion characterizing the differences between three Abies species was several times higher (F(CT) = 0.53) than that accounting for among-population differences within the species (F(SC) = 0.125). Differentiation of the A. sibirica populations based on AFLP markers exceeded 14% (F(ST) = 0.141). Significant correlation between the genetic distances calculated from the AFLP data and the geographic distances between populations was found. The results of AFLP variability analysis supported and supplemented the conclusions inferred previously from allozyme and cpSSR data: several genetically similar geographic groups of Siberian fir were identified. These groups differ both in allele frequencies and in the levels of genetic variation.     

Lichens on branches of Siberian fir (Abies sibirica Ledeb.) as indicators of atmospheric pollution in forests

The abundance distribution of different ecological groups of lichens depending on bark pH has been studied on 1- to 24-year shoots of Siberian fir in the mountains of southern Siberia. Along with acidophytic lichens commonly found on the Siberian fir (Usnea sp., Bryoria sp., etc.), its young shoots are also colonized by nitrophytic species (Physcia tenella, Melanelia exasperatula, etc.), which is evidence for the increasing pH of shoot bark. The proportion of thalli of nitrophytic lichen species shows a significant positive correlation with the pH of the upper (dusted) bark layer and is greater in the Eastern Sayan (at bark pH averaging 5.4) than in the Western Sayan (pH 4.7). The trends revealed in this study may be used for indication of pollution and ecological monitoring of forest ecosystems.     

The effect of individual genetic heterozygosity on general homeostasis,heterosis and resilience in Siberian larch (Larix sibirica Ledeb.) using dendrochronology and microsatellite loci genotyping

The genetic mechanisms underlying the relationship of individual heterozygosity (IndHet) with heterosis and homeostasis are not fully understood. Such an understanding, however, would have enormous value as it could be used to identify trees better adapted to environmental stress. Dendrochronology data, in particular the individual average radial increment growth of wood measured as the average tree ring width (AvTRW) and the variance of tree ring width (VarTRW) were used as proxies for heterosis (growth rate measured as AvTRW) and homeostasis (stability of the radial growth of individual trees measured as VarTRW), respectively. These traits were then used to test the hypothesis that IndHet can be used to predict heterosis and homeostasis of individual trees. Wood core and needle samples were collected from 100 trees of Siberian larch (Larix sibirica Ledeb.) across two populations located in Eastern Siberia. DNA samples were obtained from the needles of each individual tree and genotyped for eight highly polymorphic microsatellite loci. Then mean IndHet calculated based on the genotypes of eight loci for each tree was correlated with the statistical characteristics of the measured radial growth (AvTRW and VarTRW) and the individual standardized chronologies. The analysis did not reveal significant relationships between the studied parameters. In order to account for the strong dependence of the radial growth on tree age the age curves were examined. An original approach was employed to sort trees into groups based on the distance between these age curves. No relationship was found between these groups and the groups formed based on heterozygosity. However, further work with more genetic markers and increased sample sizes is needed to test this novel approach for estimating heterosis and homeostasis.     

Structural and chemical characterization of abienan, a pectin from the greenery of Siberian fir (Abies sibirica L.)

A pectic polysaccharide, earlier called abienan, has been extracted from the fresh greenery of Siberian fir (Abies sibirica Ledeb.) and described. Like many pectic polysaccharides, the abienan macromolecule consists of linear and branched regions. Results of the ion-exchange chromatography and partial acidolysis demonstrate that linear regions of the abienan macromolecule consist of 1,4-??-D-galacturonan. The side carbohydrate chains of branched regions consist mainly of arabinofuranose and galactopyranose residues.     

A karyological study of swamp and dry valley populations of Siberian fir (<Emphasis Type="Italic">Abies sibirica</Emphasis> Ledeb.)

The karyological data on populations of Siberian fir (Abies sibirica Ledeb.) growing in lowland swamp and dry valleys are given. The diploid set of both populations included 24 chromosomes (2n = 24). Polykaryogram analysis revealed seven pairs of metacentric and five pairs of submetacentric chromosomes. The revealed differences between the populations included the absolute length of chromosomes, number of nucleolar organizer regions, and number of nucleoli. Changed chromosome numbers (mixoploidy and aneuploidy) as well as chromosomal aberrations were recorded. For the first time, mitosis was studied in this species and anaphase/telophase aberrations were revealed. The population of Siberian fir growing under extreme conditions of lowland swamp featured the widest range of mutations.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 23–29.Original Russian Text Copyright © 2005 by Sedelnikova, Pimenov.     

Genetic diversity and differentiation of two Mediterranean pines (Pinus halepensis Mill. and Pinus pinaster Ait.) along a latitudinal cline using chloroplast microsatellite markers

Several studies have reported glacial refugia and migration pathways for different pine species in the Iberian Peninsula, all of them based on a single‐species approach. In this paper, chloroplast microsatellites (cpSSRs) are used to compare population genetic structure and diversity estimates for interspecific pairs of populations located along a cline from southwestern (latitude 36°32′ N, longitude 5°17′ W) to northeastern Spain (latitude 42°14′ N, longitude 2°47′ E) in two widely distributed Mediterranean pines, Pinus halepensis Mill. and Pinus pinaster Ait. Some cpSSRs were shared between species, facilitating comparison of levels of gene diversity at the species level and inferences about within and among species differentiation. P. pinaster showed a much higher number of variants (29) and haplotypes (69) than P. halepensis (20 and 21, respectively). Moreover, genetic diversity estimates for interspecific pairs of populations along the cline were negatively correlated. Three main causes may explain the differences between species in the present‐day distribution of genetic diversity: (1) the distribution of genetic variability before the Quaternary glaciations, with an earlier presence of P. pinaster in the Iberian Peninsula and a late spread of P. halepensis from eastern and central Europe, (2) the location of the Holocene glacial refugia and the migration pathways from these refugia to the present‐day range (from northeast to southwest in P. halepensis and from southwest to northeast and northwest in P. pinaster) and (3) the interactions between species during the postglacial spread.     

Isolation and characterization of eight dinucleotide microsatellite loci from two closely related clupeid species (Alosa alosa and A. fallax)

Eight dinucleotide microsatellite loci were developed through an enrichment protocol for allis shad (Alosa alosa) and twaite shad (A. fallax). Cross‐species amplification was successful for all loci isolated. The number of alleles per locus ranged from three to nine for A. alosa and from two to seven for A. fallax, while the observed heterozygosity ranged from 0.267 to 0.926 and from 0.240 to 0.727, respectively. These markers will constitute useful tools for studies of population structure and gene flow between two closely related hybridizing species.     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

Genetic structure based on nuclear and chloroplast microsatellite loci of Solanum lycocarpum A. St. Hil. (Solanaceae) in Central Brazil

Solanum lycocarpum (Solanaceae) is a woody species found in the Brazilian Cerrado. The flowers are pollinated by Xylocopa spp bees, and seeds are dispersed by mammals with distinct home range sizes. As a consequence, relative contributions of pollen and seeds to overall gene flow can vary according to different spatial scales. We studied the genetic structure of four natural populations of S. lycocarpum separated by 19 to 128 km, including individuals located along dirt roads that interlink three of the populations. A total of 294 individuals were genotyped with five nuclear and six chloroplast microsatellite markers. Significant spatial genetic structure was found in the total set of individuals; the Sp statistic was 0.0086. Population differentiation based on the six chloroplast microsatellite markers (θ(pC) = 0.042) was small and similar to that based on the five nuclear microsatellite markers (θ(p) = 0.054). For this set of populations, pollen and seed flow did not differ significantly from one another (pollen-to-seed flow ratio = 1.22). Capability for long distance seed dispersion and colonization of anthropogenic sites contributes to the ability of S. lycocarpum to maintain genetic diversity. Seed dispersion along dirt roads may be critical in preserving S. lycocarpum genetic diversity in fragmented landscapes.     

A panel of microsatellite loci from two species of octopus, Pareledone turqueti (Joubin, 1905) and Pareledone charcoti (Joubin, 1905)

Eighteen dinucleotide microsatellite loci were isolated from two octopus species, Pareledone turqueti and Pareledone charcoti, which are endemic to the Southern Ocean. Genetic diversity was assessed in samples of P. charcoti and P. turqueti from Elephant Island and Shag Rocks respectively. All except one locus (which has proved to be polymorphic in other species) were variable in the focal species and amplified between six and 30 and between four and 28 alleles for P. charcoti and P. turqueti respectively; mean expected heterozygosities varied between 0.38 and 0.95 (P. charcoti) and between 0.34 and 0.97 (P. turqueti), with significant (P < 0.05) departures from Hardy–Weinberg equilibrium at seven loci; three of these loci provided significant (P < 0.05) evidence for null alleles. Two pairs of loci isolated from P. turqueti demonstrated significant (P < 0.05) linkage disequilibrium. We are presently using these genetic markers to quantify spatial genetic structure in the genus Pareledone.     

Characterization of 13 polymorphic microsatellite loci for two land snail species, Trochulus villosus and T. sericeus (Gastropoda: Pulmonata: Hygromiidae)

Thirteen new microsatellite loci were isolated and tested on two land snail species, Trochulus villosus and T. sericeus (Pulmonata: Hygromiidae), resulting in a set of eight polymorphic markers for each species. The expected heterozygosity was high for all loci and species (between 0.616 and 0.944). Such levels of variability will allow detailed insights into the population genetic structure of some Trochulus species.     

Isolation and characterization of microsatellite loci for alligator gar (Atractosteus spatula) and their variability in two other species (Lepisosteus oculatus and L. osseus) of Lepisosteidae

We report on the isolation of 17 polymorphic microsatellite loci from alligator gar (Atractosteus spatula), a large-bodied species that has experienced population declines across much of its range. These loci possessed 2-19 alleles and observed heterozygosities of 0-0.974. All loci conformed to Hardy-Weinberg equilibrium expectations, and none exhibited linkage disequilibrium. Nine and eight of these loci were found to be polymorphic in the related species Lepisosteus oculatus and L. osseus, respectively. These microsatellite loci should prove useful in conservation efforts of A. spatula through the study of population structure and hatchery broodstock management.     

A microsatellite marker for studying the ecology and diversity of fungal endophytes (Epichloë spp.) in grasses.

Randomly amplified polymorphic DNA fingerprinting, which is based on PCR with arbitrary 10-nucleotide primers, were used to analyze genetic diversity among isolates of the endophytic ascomycete Epichloë typhina, which were collected at a single field site from a population of one of its hosts, the grass Bromus erectus. One of the polymorphic randomly amplified polymorphic DNA PCR products occurred in all isolates as single bands with different but closely related sizes. Two of the size variants of this product were cloned and sequenced, and they were found to represent the same DNA sequence, except for a stretch of tandem repeats of the trinucleotide AAG.TTC, which differed in size, consisting of 8 and 18 repeats, respectively. Tandem repeats of this type are called microsatellites. Oligonucleotides were synthesized corresponding to portions of the sequence flanking the microsatellite and were used for PCR amplification of the loci from the genomic DNAs of different Epichloë isolates. A single PCR product was found for most isolates, indicating that the sequence represented a single genetic locus. Five alleles that could clearly be distinguished in size were found in a population of 91 field isolates. PCR with (AAC)8 and (AAG)8 as primers yielded a number of amplified bands from genomic DNA of Epichloë isolates, indicating that these types of microsatellites occur frequently in the genome of this fungus. A survey of all fungal DNA sequences currently deposited in the DNA sequence databases of EMBL and GenBank revealed that microsatellites of different repeating units are widespread in fungi.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new genus and two new nematode species (Drilonematoidea: Ungellidae: Synoecneminae) parasitic in two morphs of Drawida ghilarovi Gates,endemic earthworm from the Russian Far East

Drasico n. g. is erected to accommodate two new species of nematode, Drasico nemoralis n. sp. and D. paludigenus n. sp., recovered from coelomic cavities of Drawida ghilarovi Gates, endemic earthworms of the Russian Far East. The new genus is characterised by the following unique for the Synoecneminae characters: apical portion of the head attenuated, cephalic hooks displaced to the base of attenuated portion, amphids displaced posterior to cephalic hooks, excretory duct short and weak, males possessing several genital papilliform sensilla. The new species are differentiated by the size, number and disposition of the male genital sensilla (larger and more numerous in D. nemoralis n. sp.); the body shape of females (with thinner neck and wider mid-body in D. paludigenus n. sp.) and the ovarian tube arranged in transversal folds in D. paludigenus (vs longitudinal folds in D. nemoralis n. sp.). Nucleotide sequences of D2–D3 expansion segment of 28S rDNA for the two new species differed at 13 positions. Phylogenetic analysis revealed close relationships of Drasico n. g. with species of Siconema Timm, 1966. The host species was represented by two morphs (blue-grey forest and tar-black meadow-swamp morph) with intraspecific divergence of 16–17% for cytochrome c oxidase subunit 1 (COI) gene, and each host morph was found infected by a different nematode species. A co-infection with the plectid nematode Creagrocercus drawidae Ivanova & Spiridonov, 2011 was recorded together with D. nemoralis n. sp. in the blue-grey forest morph.     

Establishing genomic tools and resources for Guizotia abyssinica (L.f.) Cass.-the development of a library of expressed sequence tags, microsatellite loci, and the sequencing of its chloroplast genome

We present an EST library, chloroplast genome sequence, and nuclear microsatellite markers that were developed for the semi-domesticated oilseed crop noug (Guizotia abyssinica) from Ethiopia. The EST library consists of 25 711 Sanger reads, assembled into 17 538 contigs and singletons, of which 4781 were functionally annotated using the Arabidopsis Information Resource (TAIR). The age distribution of duplicated genes in the EST library shows evidence of two paleopolyploidizations—a pattern that noug shares with several other species in the Heliantheae tribe (Compositae family). From the EST library, we selected 43 microsatellites and then designed and tested primers for their amplification. The number of microsatellite alleles varied between 2 and 10 (average 4.67), and the average observed and expected heterozygosities were 0.49 and 0.54, respectively. The chloroplast genome was sequenced de novo using Illumina’s sequencing technology and completed with traditional Sanger sequencing. No large re-arrangements were found between the noug and sunflower chloroplast genomes, but 1.4% of sites have indels and 1.8% show sequence divergence between the two species. We identified 34 tRNAs, 4 rRNA sequences, and 80 coding sequences, including one region (trnH-psbA) with 15% sequence divergence between noug and sunflower that may be particularly useful for phylogeographic studies in noug and its wild relatives.     

Strength, diversity and plasticity of postmating reproductive barriers between two hybridizing oak species (Quercus robur L. and Quercus petraea (Matt) Liebl.)

Very little is known about the nature and strength of reproductive isolation (RI) in Quercus species, despite extensive research on the estimation and evolutionary significance of hybridization rates. We characterized postmating pre- and postzygotic RI between two hybridizing oak species, Quercus robur and Quercus petraea, using a large set of controlled crosses between different genotypes. Various traits potentially associated with reproductive barriers were quantified at several life history stages, from pollen-pistil interactions to seed set and progeny fitness-related traits. Results indicate strong intrinsic postmating prezygotic barriers, with significant barriers also at the postzygotic level, but relatively weaker extrinsic barriers on early hybrid fitness measures assessed in controlled conditions. Using general linear modelling of common garden data with clonal replicates, we showed that most traits exhibited important genotypic differences, as well as different levels of sensitivity to micro-environmental heterogeneity. These new findings suggest a large potential genetic diversity and plasticity of reproductive barriers and are confronted with hybridization evidence in these oak species.     

Genetic diversity and structure of populations in Pilosocereus gounellei (F.A.C.Weber ex K.Schum.) (Cactaceae) in the Caatinga biome as revealed by heterologous microsatellite primers

Microsatellite transferability was used as a method to examine the genetic diversity and structure of populations in Pilosocereus gounellei seedling samples that have potential to implement effective restoration strategies for degraded and disturbed areas of the Caatinga biome. Genomic DNA was extracted from 85 seedlings obtained from fruit collected from plants growing in native areas in the Brazilian states of Piaui (PI), Rio Grande do Norte (RN), and Bahia (BA). Six microsatellite primers were polymorphic. AMOVA showed higher genetic variation within (72%) than among (28%) the samples from the three states. The high level of similarity between the seedlings from PI, BA, and RN indicated that samples collected at any of the three sites can be used to represent the genetic diversity of the species. Seeds of plants from the three States are recommended as samples for germplasm banks and/or the production of plantlets to i) plant in areas of strategic reserves for forage, ii) deploy new cultivation areas, iii) restore degraded areas in the semi-arid Northeast, and iv) maintain ecological reserve banks and fodder with genetically divergent plants.