Expression analysis and functional characterization of a novel cold-responsive gene <Emphasis Type="Italic">CbCOR15a</Emphasis> from <Emphasis Type="Italic">Capsella bursa</Emphasis>-<Emphasis Type="Italic">pastoris</Emphasis>

The cold-responsive (COR) genes involved in C-repeat binding factor signaling pathway function essentially in cold acclimation of higher plants. A novel COR gene CbCOR15a from shepherd’s purse (Capsella bursa-pastoris) was predicted to be a homolog of COR15 in Arabidopsis. The analysis of tissue specific expression pattern as well as characterization of the CbCOR15a promoter revealed that the expression of CbCOR15a was induced by coldness not only in leaves and stem but also in roots. Sequence analysis showed that a 909 bp promoter region of CbCOR15a contained two CRT/DRE elements, two ABRE elements, one auxin-responsive TGA-element and one MeJA-responsive CGTCA-motif. In young seedlings the expression of CbCOR15a could be apparently increased by SA, ABA, MeJA and IAA, and transiently increased by GA₃ accompanied by obvious feedback suppression. According to the altered physiological index values in tobacco under cold treatments, the overexpression of CbCOR15a significantly increased the cold tolerance of transgenic tobacco plants. It can be suggested that CbCOR15a was involved in cold response of Capsella bursa-pastoris associated with SA, ABA, MeJA, IAA and GA₃ regulation and confers enhanced cold acclimation in transgenic plants.     

The “<Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>” complex of cryptic species

Cryptic species are common among protists and have long been known in ciliates. The ciliate genus Tetrahymena contains a large group of morphologically indistinguishable species referred to as the ‘T. pyriformis’ complex. These species include those reproductively isolated by mating type as well as asexual species characterized by the absence of the germinal micronucleus. This paper examines the molecular diversity of the species and describes the biogeography of ‘T. pyriformis’ species. Most species are globally distributed, though the best studied species, T. thermophila, is confined to North America and gives evidence of population structure in local populations. Selfers and asexual species are common and arise from sexual species, a possible exploitation of nuclear dimorphism. It is argued that the cryptic species likely have different ecological roles and that the biodiversity of Tetrahymena in particular, and ciliates in general, is underestimated. Special Issue: Protist diversity and geographic distribution. Guest editor: W. Foissner.     

Novel molecular markers of <Emphasis Type="Italic">Chlamydia pecorum</Emphasis> genetic diversity in the koala (<Emphasis Type="Italic">Phascolarctos cinereus</Emphasis>)

Background  

Chlamydia pecorum is an obligate intracellular bacterium and the causative agent of reproductive and ocular disease in several animal hosts including koalas, sheep, cattle and goats. C. pecorum strains detected in koalas are genetically diverse, raising interesting questions about the origin and transmission of this species within koala hosts. While the ompA gene remains the most widely-used target in C. pecorum typing studies, it is generally recognised that surface protein encoding genes are not suited for phylogenetic analysis and it is becoming increasingly apparent that the ompA gene locus is not congruent with the phylogeny of the C. pecorum genome. Using the recently sequenced C. pecorum genome sequence (E58), we analysed 10 genes, including ompA, to evaluate the use of ompA as a molecular marker in the study of koala C. pecorum genetic diversity.     

Identification of a major QTL for time of initial vegetative budbreak in apple (<Emphasis Type="Italic">Malus</Emphasis> x <Emphasis Type="Italic">domestica</Emphasis> Borkh.)

In the Western Cape region of South Africa, dormancy release and the onset of growth does not occur normally in apple (Malus x domestica Borkh.) trees during spring due to the mild winter conditions experienced and fluctuations in temperatures experienced during and between winters. In this region, the application of chemicals to induce the release of dormancy forms part of standard orchard management. Increasing awareness of the environmental impact of chemical sprays and global warming has led to the demand for new apple cultivars better adapted to local climatic conditions. We report the construction of framework genetic maps in two F1 crosses using the low chilling cultivar ‘Anna’ as common male parent and the higher chill requiring cultivars ‘Golden Delicious’ and ‘Sharpe’s Early’ as female parents. The maps were constructed using 320 simple sequence repeats, including 116 new markers developed from expressed sequence tags. These maps were used to identify quantitative trait loci (QTL) for time of initial vegetative budbreak (IVB), a dormancy related characteristic. Time of IVB was assessed four times over a 6-year period in ‘Golden Delicious’ x ‘Anna’ seedlings kept in seedling bags under shade in the nursery. The trait was assessed for 3 years on adult full-sib trees derived from a cross between ‘Sharpe’s Early’ and ‘Anna’ as well as for 3 years on replicates of these seedlings obtained by clonal propagation onto rootstocks. A single major QTL for time of IVB was identified on linkage group (LG) 9. This QTL remained consistent in different genetic backgrounds and at different developmental stages. The QTL may co-localize with a QTL for leaf break identified on LG 3 by Conner et al. (1998), a LG that was, after the implementation of transferable microsatellite markers, shown to be homologous to the LG now known to be LG 9 (Kenis and Keulemans 2004). These results contribute towards a better understanding regarding the genetic control of IVB in apple and will also be used to elucidate the genetic basis of other dormancy related traits such as time of initial reproductive budbreak and number of vegetative and reproductive budbreak.     

Spatial genetic structure of <Emphasis Type="Italic">Coccoloba cereifera</Emphasis> (Polygonaceae), a critically endangered microendemic species of Brazilian rupestrian fields

Coccoloba cereifera (Polygonaceae) is an extremely rare endemic shrub found exclusively in the rupestrian fields of Serra do Cipó, southeastern, Brazil. We assessed the genetic diversity and structure across the single occurrence area of C. cereifera. The genetic variation at 13 microsatellite loci was estimated from 139 individuals sampled in nine patches. The number of alleles per locus varied from two to ten; the expected and observed heterozygosity ranged from 0.324 to 0.566 and 0.337 to 0.529, respectively. Microsatellites detected low but statistically significant levels of differentiation among patches (F _ST = 0.123, R _ST = 0.105), whereas Mantel test results showed a weak but significant pattern of isolation by distance (r ² = 0.31, P < 0.002). Bayesian clustering indicated two subdivisions connected via admixture. Habitat heterogeneity across the drainage basin of the Rio Indequicé is likely limiting gene flow within patches of the geographically restricted population. While there is currently no evidence for a direct genetic risk to species survival, the apparent natural segregation occurring within the species could be exacerbated by future land use changes and the influx of alien species which could lead to demographic reductions in population size leading to a reduction in genetic diversity and an increase in population subdivision. We suggest that maintaining the integrity of the habitat within the small range of the species and continued monitoring of the effects of alien species would be the wisest use of management resources.     

Biochemical changes induced in seeds of <Emphasis Type="Italic">Brassicaceae</Emphasis> wild species during ageing

The aim of the present study was to determine whether the loss of seed germination capacity and vigour in seeds of four wild Brassicaceae species (Brassica repanda, Moricandia arvensis, Rorippa nasturtium-aquaticum and Sinapis alba) during ageing at 45°C and 90% relative humidity was related to changes in lipid peroxidation and membrane integrity. For all of the species, ageing reduced the final germination percentage and increased the length of time required to reach 50% of final germination (T ₅₀). Large differences in longevity were observed among the species. The times required for viability to be reduced to 80 and 50% of maximum germination (P80 and P50) were the lowest for B. repanda, and these values were two times longer for M. arvensis and R. nasturtium-aquaticum and five times longer for S. alba compared with B. repanda. A loss of seed viability was not associated with malondialdehyde accumulation, suggesting that lipid peroxidation did not cause seed deterioration under these conditions. However, the conductivity test effectively detected seed deterioration in these wild Brassicaceae species, and membrane permeability correlated with both germination and vigour loss. This correlation may provide a valuable mean for early detection of seed viability in wild Brassicaceae species.     

The order of the <Emphasis Type="Italic">bs,Skdh</Emphasis>, and <Emphasis Type="Italic">Aadh1</Emphasis> genes in chromosome 5R of rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

Segregation analysis was performed in the progenies obtained in analyzing crosses of hybrids of spring and winter accessions of rye Secale cereale L. and wild S. montanum subsp. anatolicum (Grossh.) Tzvel. (syn. S. strictum (J. Presl) J. Presl). The test genes controlled the brittle stem (bs), the allelic variants of aromatic alcohol dehydrogenase (Aadh1) and shikimate dehydrogenase (Skdh), and the growth habit (Vrn1). A linkage was observed in the inheritance of the brittle stem and the aromatic alcohol dehydrogenase and shikimate dehydrogenase alloenzymes. The order of genes was established to be bs-Skdh-Aadh1, and the genetic distances were estimated to be bs-(9.0%)-Skdh, bs-(10.8%)-Aadh1, and Skdh-(5.3%)-Aadh1. The recombination coefficient between the Skdh and Aadh1 genes varied from 2.2 to 18.2%, averaging 5.3%. The growth habit was inherited independently of the bs-Skdh-Aadh1 linkage group.     

Development of 37 microsatellite loci for the great gray owl (<Emphasis Type="Italic">Strix nebulosa</Emphasis>) and other <Emphasis Type="Italic">Strix</Emphasis> spp. owls

We developed 37 great gray owl (Strix nebulosa) microsatellite primers from CA and TAGA enriched genomic libraries. Primers were tested in 15 great gray owls from California, USA and Alberta, Canada as well as two other Strix species, spotted owl (S. occidentalis) and barred owl (S. varia). These markers will have broad application in investigations of Strix population structure and genetic diversity.     

Biochemical and agro-biological diversity of <Emphasis Type="Italic">Viburnum opulus</Emphasis> genotypes

Interest in the biochemical composition of Viburnum opulus fruit has intensified due to the food industry’s demand for natural vitamins, pigments and other substances that enhance the value of different foods. The present study was conducted to determine the agro-biological and biochemical variability of V. opulus and to select the genotypes that could best serve as sources of health promoting substances. Twelve selected genotypes were evaluated. ‘Leningradskaya Otbornaya’, V. opulus var. americanum, ‘Zarnitsa’, and local clone P2 were determined to be the best genotypes for growth in commercial plantations. Fruits of the local clone P3 were characterised by large amounts of total phenolics, ascorbic acid, and reducing sugars. V. opulus var. sargentii and V. opulus var. americanum contained exceptionally large amounts of total phenolics, 1460.0 and 1400.0 mg/100 g, respectively. The amount of ascorbic acid varied from 12.4 to 41.4 mg/100 g, the amount of carotenoids varied from 1.4 to 2.8 mg/100 g, the amount of anthocyanins varied from 23.2 to 44.6 mg/100 g, and the amount of total phenolics varied from 753.0 to 1460.0 mg/100 g. The presence of these large amounts of biologically active compounds enables their use as potent antioxidants. The data describing agro-biological characteristics, biochemical components, and health promoting activities of V. opulus fruits will increase the understanding of this plant and facilitate its use in the food and pharmaceuticals industry.     

Comparison of anonymous and targeted molecular markers for the estimation of genetic diversity in <Emphasis Type="Italic">ex situ</Emphasis> conserved <Emphasis Type="Italic">Lactuca</Emphasis>

The anonymous marker systems microsatellites (simple sequence repeats), amplified fragment length polymorphisms and sequence-specific amplified polymorphisms were compared with the targeted marker systems sequence-related amplified polymorphisms, target region amplification polymorphisms and nucleotide binding site profiling for their ability to describe the genetic diversity in a selected set of 80 Lactuca accessions. The accessions were also described morphologically, and all characterisation methods were evaluated against the genetic diversity assessed by a panel of three crop experts. The morphological data showed a low level of association with the molecular data, and did not display a consistently better relationship with the experts’ assessments in comparison with the molecular data. In general, the diversity described by the targeted molecular markers did not differ markedly from that of the anonymous markers, resulting in only slight differences in performance when related to the expert-based assessments. It was argued that markers targeted to specific gene sequences may still behave as anonymous markers and that the type of marker system used is irrelevant when at low taxonomic levels a clear genetic structure is absent due to intensive breeding activities.     

Regional genetic variability among South African vervet monkey<Emphasis Type="Italic">Chlorocebus aethiops</Emphasis> populations

A South African rehabilitation centre for illegally kept vervet monkeysChlorocebus aethiops Linnaeus, 1758 required an evaluation of the genetic status of vervet monkeys, to determine whether animals from different geographical areas may be mixed. We studied animals originating from three geographical regions and used biochemical genetic and morphological approaches to address this question. The most prominent trend from allozyme data was derived from the locusPrt-2 (an unspecified serum protein), where each of the three populations could be characterised by the absence or presence of unique alleles. Nevertheless, statistical coefficients indicated little genetic divergence, with genetic distance values of 0.001–0.003 and an overall fixation index value of 0.046. Average heterozygosity did not differ appreciably among populations (2.5–3.3%). The morphological study identified suitable traits, free from the influence of growth allometry, that can be used for inter-population comparisons. No significant morphological differences between con-specific populations were however found. It is concluded that vervet monkeys from the species’ wider distribution range is relatively monotypic, but that monkeys from different geographical areas should not be unduly mixed, pending the results of finer grained molecular studies.     

Molecular characterization and mating type analysis of oyster mushroom (<Emphasis Type="Italic">Pleurotus</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>) using single basidiospores for strain improvement

Existence of variability in morphological traits and growth rate of mycelium of homokaryotic single basidiospores can be exploited for the development of inter-strainal hybrids. We isolated 182 single basidiospores from mushroom bodies of P. sajor-caju, P. florida, P. eous and one wild relative of Pleurotus called Hypsizygus ulmaris. The single spores were isolated using a new technique that is less prone to contamination and more efficient than the common techniques used by earlier workers. All the isolates showed a varied range of cultural morphology. Mating types of all the isolates within the species were identified on the basis of hyphal fusion via anastomosis with the tester strains. Four compatible pairs of isolates with well prominent tuft in the contact zone were selected for dikaryon isolation. Dikaryons were used for spawn preparation and mushroom cultivation. The dikaryotic isolates with their replicates were evaluated for spawn run period, yield and biological efficiency. 42 isolates (10 di- and 32 mono-karyotic isolates) were analyzed with RAPD genetic markers. Phenotypic characters and mating types of all the 42 isolates analyzed genetically were correlated with their genetic polymorphism data. The isolates showed very large diversity both at the phenotypic and the genotypic level. Available phenotypic and genotypic data can further help in the selection of monosporous isolates for developing inter-strainal hybrids which can lead to better prospects for genetic improvement in different species of Pleurotus.     

Conservation genetics of the black rhinoceros, <Emphasis Type="Italic">Diceros bicornis bicornis</Emphasis>, in Namibia

Poaching and habitat destruction across sub-Saharan Africa brought the black rhinoceros (Diceros bicornis) close to extinction. Over the past few decades, however, one of four subspecies, D. b. bicornis, has experienced a significant population increase as a consequence of its protection within Etosha National Park (ENP), Namibia. We report here on the level and spatial distribution of black rhinoceros genetic diversity within ENP. Using nine microsatellite loci, genetic variation was assessed from 144 individuals. Our results are consistent with the observation of lower levels of genetic diversity in D. b. bicornis, when compared to D. b. michaeli, but greater diversity when compared to D. b. minor. We also showed that ENP’s black rhino genetic diversity is well represented in Waterberg National Park, originally founded with ENP individuals. We found no genetic signature of a recent bottleneck in ENP, however, suggesting that the genetic diversity within ENP has not been adversely affected by the recent severe population decline. Using Bayesian clustering methods, we observed no significant population structure within ENP, but positive spatial genetic correlation is observed at distances up to 25 km. This relationship exists in females but not males, suggesting reduced dispersal among females, the first evidence of limited female dispersal or philopatry in any species of rhinoceros.     

Development and characterisation of microsatellite markers for <Emphasis Type="Italic">Cordia verbenacea</Emphasis> (Boraginaceae), an important medicinal species from the Brazilian coast

Cordia verbenacea DC (Boraginaceae) is a Brazilian coastal shrub species extensively utilized in the folk medicine and also by the pharmaceutical industry. Despite its enormous economic and pharmacological potential, little is known about its reproductive biology, genetic structure and genetic diversity of natural populations. Due to the rapid degradation of its natural habitat, this knowledge became imperative. We developed a set of eight highly polymorphic microsatellite markers for the study of genetic structure and diversity of C. verbenacea natural populations and germplasm collections. Six of them revealed a multibanded pattern typical of polyploid species. The total number of bands per locus ranged from 2 to 12 and the number of bands per locus per individual ranged from 1 to 8. The primer pairs presented here will be used to generate information to guide conservation programs and rational exploitation of C. verbenacea.     

Conservation genetics of xerothermic beetles in Europe: the case of <Emphasis Type="Italic">Centricnemus leucogrammus</Emphasis>

Xerothermic habitats are closely related to continental steppes of Eurasia and contain communities rich in rare and endemic species, especially insects. Considering the dramatic loss of xerothermic habitats due to climatic and anthropogenic changes the evaluation of genetic variation and phylogeographical patterns in xerothermic species is a matter of utmost importance if appropriate conservation measures are to be undertaken. In this paper 3 mitochondrial genes and 3 nuclear markers were used for evaluation of genetic diversity of populations of the weevil Centricnemus leucogrammus from central-east Europe. These data were used for the recognition of conservation units as well as centers of genetic diversity which could be considered as present “warm-stage” refugia for xerothermic beetle assemblages. The most diverse are eastern and central populations from Ukraine and central Poland, however, also isolated groups of populations from the Pannonian Basin and northern Poland possessed a unique genetic signature. This lead to the conclusion that all three regional groups of populations are significantly isolated from each other and represent distinct evolutionary lineages so they should be considered as separate Management Units and Evolutionary Significant Units. C. leucogrammus can be regarded as an excellent representative for typically xerothermophilous, flightless beetles and knowledge about its genetic diversity may be used for the conservation and management of entire xerothermic communities, especially for those with low mobility invertebrate taxa. Further research on the genetic diversity of various organisms with varying levels of mobility should be undertaken to broaden our knowledge on the conservations needs of xerothermic assemblages.     

Morphological and molecular characterization of two ITS groups of <Emphasis Type="Italic">Erysiphe</Emphasis> (Erysiphales) occurring on <Emphasis Type="Italic">Syringa</Emphasis> and <Emphasis Type="Italic">Ligustrum</Emphasis> (Oleaceae)

ITS sequences determined for 53 Erysiphe specimens on Syringa and Ligustrum collected in Europe, East Asia, and North and South America were divided into two ITS groups, S and K types. Phylogenetic analysis showed that these two ITS types do not share a common ancestor and form separate clades. The K type on Ligustrum was identified as Erysiphe ligustri based on the three-dimensional branching pattern of appendages. Morphological observations showed that there are some morphological differences—pigmentation of appendages and number of ascospores per ascus—between the S and K types on Syringa. Based on these morphological observations, the S and K types on Syringa were identified as E. syringae and E. syringae-japonicae, respectively. The recent abundant production of chasmothecia by lilac powdery mildew in Europe was caused by E. syringae-japonicae introduced from East Asia. DNA sequence analyses of the rDNA ITS region and the 28S rDNA, tub2, CYP51, and Chs1 genes did not support an interspecific hybrid origin for E. syringae-japonicae. Haplotype analysis suggested that E. syringae originated in North America and independently migrated to East Asia and Europe/South America.     

Identification and assessment of genetic relationships in three <Emphasis Type="Italic">Chlorophytum</Emphasis> species and two high yielding genotypes of <Emphasis Type="Italic">C. borivilianum</Emphasis> through RAPD markers

Conservation of identified germplasm is an important component for efficient and effective management of plant genetic resources. Since Chlorophytum species are important medicinal plants, studies were carried out for identification and establish genetic relationships in three species of Chlorophytum and two high yielding genotypes of Chlorophtum borivilianum using RAPD markers. Out of one hundred primers tested, 47 decamers amplified a total of 454 distinct bands ranging from 0.25–3.0 kbp to identify and to evaluate genetic relationships between and among three species of Chlorophytum and two genotypes of Chlorophtum borivilianum. The cluster analysis indicated that three species of Chlorophytum and two genotypes (NRCCB-1 and NRCCB-2) of C. borivilianum formed two major clusters. The first major cluster constituted C. arundinaceum and C. tuberosum, and the second major cluster composed of two subclusters; the first subcluster represented NRCB-1 and NRCB-2 where as the second subcluster represented C. borivilianum. Thus, the RAPD markers have the potential for identification and characterization of genetic relatedness among the species and genotypes. C. borivilianum along with two genotypes also showed similar banding patterns which could be chosen as candidate markers for differentiating the other two species such as C. arundinaceum and C. tuberosum. This would helpful for breeding programmes and provides an important input in conservation biology.     

Characterization and distribution of <Emphasis Type="Italic">Daucus</Emphasis> species in Syria

Syria is considered as one of the important centres for Daucus diversity including Daucus carota. Therefore, it is essential to study the distribution and characterization of these species in Syria. An exploration of plants belonging to the Apiaceae family was conducted on road and field sides in several areas of Syria. Seeds (fruits) from these plants have been also collected. The seeds were sown in pots containing peatmoss in a glasshouse and emerging plants were grown until flowering and seed formation. The plants were classified based on leaf, umbel, and seed shape. Proteins were extracted from the leaves and analysed using electrophoresis to establish genetic relationships among species. Seven Daucus species have been identified to grow in Syria. These are D. aureus, D. bicolor, D. carota, D. durieua, D. guttatus, D. littoralis, and D. muricatus. Isozyme and total protein analysis, cluster analysis, and correlation matrix have revealed considerable genetic variation among studied Daucus species. Wild carrot (D. carota) came in one group with its cultivated form (D. carota ssp. sativus) and the closest species to them was D. guttatus. Species D. bicolor and D. durieua were in the same group and D. aureus and D. littoralis were in another group farther from the previous groups. The farthest species on the genetic tree was D. muricatus.