Mitochondrial DNA evolution in the genus Equus

Employing mitochondrial DNA (mtDNA) restriction-endonuclease maps as the basis of comparison, we have investigated the evolutionary affinities of the seven species generally recognized as the genus Equus. Individual species' cleavage maps contained an average of 60 cleavage sites for 16 enzymes, of which 29 were invariant for all species. Based on an average divergence rate of 2%/Myr, the variation between species supports a divergence of extant lineages from a common ancestor approximately 3.9 Myr before the present. Comparisons of cleavage maps between Equus przewalskii (Mongolian wild horse) and E. caballus (domestic horse) yielded estimates of nucleotide sequence divergence ranging from 0.27% to 0.41%. This range was due to intraspecific variation, which was noted only for E. caballus. For pairwise comparisons within this family, estimates of sequence divergence ranged from 0% (E. hemionus onager vs. E. h. kulan) to 7.8% (E. przewalskii vs. E. h. onager). Trees constructed according to the parsimony principle, on the basis of 31 phylogenetically informative restriction sites, indicate that the three extant zebra species represent a monophyletic group with E. grevyi and E. burchelli antiquorum diverging most recently. The phylogenetic relationships of E. africanus and E. hemionus remain enigmatic on the basis of the mtDNA analysis, although a recent divergence is unsupported.      

Cassava leaf harvesting as vegetables; a cause of vulnerability of cassava plant to cassava mosaic disease and eventual yield reduction: Ernte von cassavablättern als gemüse; eine ursache für die anfälligkeit der cassavapflanze für die cassava-mosaikkrankheit und für spätere ertragseinbußen

The consequence of harvesting young leaves of cassava as vegetable on the vulnerability of the crop to cassava mosaic disease (CMD) and on storage root yield was investigated using 30 cassava genotypes planted in IITA fields located in the humid forest (Port Harcourt?:?Onne), forest-savannah transition (Ibadan), southern guinea savannah (Mokwa) and northern guinea savannah (Zaria) agroecologies in Nigeria. Tender apical leaves and shoots of the cassava genotypes were removed from forty plants per cassava genotype with the same number of plants considered as control. Whitefly infestation, disease incidence (DI) and symptom severity (ISS) of the disease were assessed at monthly interval for six months and also at the ninth month after planting (MAP). Yield reduction due to this treatment was calculated as percentage harvest index (HI). Whitefly population fluctuated throughout the period of observation at all locations with higher population obtained generally for treated plants compared to control plants. Sprouting leaves of some treated genotypes were observed with severe mosaic symptoms, while corresponding control showed no mosaic symptoms. Contrarily, no remarkable difference was observed in Zaria between the mean ISS of treated and control cassava genotypes. There was a highly significant difference (P?<?0.01) in DI and ISS among cassava genotypes across all locations. Also, there was a highly significant interaction (P?<?0.01) in symptom severity between location (loc) and genotype, genotype and treatment (trt), loc and trt. Interaction between loc, genotypes and trt with regard to DI was highly significant at 2, 3 and 4 MAP, while with ISS, the interaction was highly significant all through the counting period. There was a positive relationship between DI and ISS on plants of genotypes 96/1039 and ISU. The percentage HI (27.4) of treated plants of genotype 95/0166 in Ibadan was remarkably lower than the value obtained for corresponding control (41.9) plants. Also, sharp distinction in% HI of treated (39.5) and control (43.8) ISU was observed in Onne with their respective ISS values as 3.7 and 3.2. Therefore, harvesting tender apical leaves and shoots of cassava as vegetables should be discouraged as it increases the severity of CMD infection in the regenerating shoots of cassava with attendant storage root yield reduction.     

Genetic diversity in a germplasm collection of roseroot (Rhodiola rosea) in Norway studied by AFLP

Rhodiola rosea is widely distributed in Norway, but so far limited knowledge exists on the level of genetic diversity. To initiate a selective breeding program, Amplified Fragment Length Polymorphism (AFLP) analysis was used to estimate genetic diversity within the Norwegian R. rosea germplasm collection. AFLP analysis of 97 R. rosea clones using five primer combinations gave a total of 109 polymorphic bands. We detected high percentage of polymorphic bands (PPB) with a mean of 82.3% among the clones of R. rosea. Each of the 97 R. rosea clones could be unambiguously identified based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the Unweighted Pair Group Method with Arithmetic mean (UPGMA). Genetic similarity based on the AFLP data ranged from 0.440 to 0.950 with a mean of 0.631. This genetic analysis showed that there was no close genetic similarity among clones related to their original growing county. No gender-specific markers were found in the R. rosea clones. Analysis of molecular variance (AMOVA) revealed a significantly greater variation within regions (92.03%) than among regions (7.97%). A low level of genetic differentiation (F_ST = 0.043) was observed, indicating a high level of gene flow, which had a strong influence on the genetic structure at different counties. Our results indicate high gene flow among R. rosea clones that might be a result of seed dispersal rather than cross-pollination. Further world-wide studies are required to compare the level of genetic diversity and more studies in R. rosea detailing the consequences of different patterns of gene flow (pollen spread and dispersal of seeds and clonal plants) will be useful for characterization of roseroot.     

Biomarkers of toxicity in Clarias gariepinus exposed to sublethal concentrations of polycyclic aromatic hydrocarbons

Physiological, biochemical and histological indices in Clarias gariepinus broodstock, and teratogenic indices in embryos exposed to sublethal concentrations of naphthalene, phenanthrene and pyrene were investigated in 2014 using a static-renewal bioassay protocol. Phenanthrene (1.41 mg l^?1) was the most toxic, followed by pyrene (1.53 mg l^?1) and naphthalene (7.21 mg l^?1), based on 96 h LC50 values. Hepatosomatic indices were significantly higher in naphthalene- and pyrene-treated males compared with solvent controls, whereas fecundity in females was significantly lower by factors of 2.4 (naphthalene), 2.8 (phenanthrene) and 2.4 (pyrene), compared with controls. Catalase levels were lower in female phenanthrene-treated fish compared with controls. Histological alterations observed in PAH-treated fish include oedema, inflammatory cells, epithelial lifting and hyperplasia in the gills, vacuolation, haemosiderin pigments and sinusoidal congestion in the liver, and degenerated zona radiata in the ovary. Teratogenic effects were not observed, as evidenced by the lack of histological alterations in embryos spawned from pre-exposed broodstock. Sex-specific responses and the utility of biomarkers at cellular and individual levels of organisation are therefore demonstrated for holistic evaluations of polycyclic aromatic hydrocarbons in ecotoxicological studies.     

Genetic mapping of DArT markers in the Festuca�CLolium complex and their use in freezing tolerance association analysis

Species belonging to the Festuca-Lolium complex are important forage and turf species and as such, have been studied intensively. However, their out-crossing nature and limited availability of molecular markers make genetic studies difficult. Here, we report on saturation of F. pratensis and L. multiflorum genetic maps using Diversity Array Technology (DArT) markers and the DArTFest array.The 530 and 149 DArT markers were placed on genetic maps of L. multiflorum and F. pratensis, respectively, with overlap of 20 markers, which mapped in both species. The markers were sequenced and comparative sequence analysis was performed between L. multiflorum, rice and Brachypodium. The utility of the DArTFest array was then tested on a Festulolium population FuRs0357 in an integrated analysis using the DArT marker map positions to study associations between markers and freezing tolerance. Ninety six markers were significantly associated with freezing tolerance and five of these markers were genetically mapped to chromosomes 2, 4 and 7. Three genomic loci associated with freezing tolerance in the FuRs0357 population co-localized with chromosome segments and QTLs previously identified to be associated with freezing tolerance. The present work clearly confirms the potential of the DArTFest array in genetic studies of the Festuca-Lolium complex. The annotated DArTFest array resources could accelerate further studies and improvement of desired traits in Festuca-Lolium species.     

High-throughput genotyping of unknown genomic terrain in complex plant genomes: lessons from a case study

Novel high-throughput genotyping technologies have facilitated rapid genotyping of single nucleotide polymorphisms in non-model organisms. Most plant species have complex genomes with a large proportion of their genes having one or more paralogous copies due to single gene duplications and ancient or recent polyploidization events. These paralogous gene copies are potential sources of genotyping errors, and hence genotyping of plant genomes is inherently difficult. Here we present a case study that exemplifies paralog-related problems in high-throughput genotyping of plant genomes. We used the MassARRAY genotyping platform to genotype the LpIRI locus in L. perenne populations; this gene is thought to be involved in low-temperature stress tolerance. The dissection of the molecular genetics underlying the genotyping results provides a good example of how unknown paralogs can mask the true genotype of the locus, instructive to the non-specialist plant researcher and breeder.     

Direct haplotype-specific DNA sequencing

Determining haplotype-specific DNA sequence information is very important in a wide range of research fields. However, no simple and robust approaches are currently available for determining haplotype-specific sequence information. We have addressed this problem by developing a very simple and robust haplotype-specific sequencing approach. We utilise the fact that DNA sequencing polymerases are sensitive to 3'end mismatches in the sequencing primer. By using two sequencing primers with 3'end corresponding to the two alleles in a given SNP locus, we are able to obtain allele-specific DNA sequences from both alleles. We evaluated this direct haplotype-specific approach by determining haplotypes within the intron 2 sequence of the fructan-6-fructosyltransferase (6-ft) gene in Lolium perenne L. We obtained reliable haplotype-specific sequences for all primers and genotypes evaluated. We conclude that the haplotype-specific sequencing is robust, and that the approach has a potentially very wide application range for any diploid organism.     

Spatial models of pollen dispersal in the forage grass meadow fescue

Several bivariate probability distributions, generated by different underlying dispersal mechanisms, are fitted to the observed frequencies of an isozyme marker gene using a maximum likelihood approach. The pollen dispersal data were generated using two experimental populations of meadow fescue (Festuca pratensis Huds.), homozygous for different allozymes at the (Pgi-2) locus, arranged in a circular donor–acceptor field design. The contribution of a plant depends on plant position, fecundity and flowering time, factors which are taken into account when fitting the different models. Several approximate likelihood-ratio tests are done between alternative nested models, and a wind threshold model with bimodality in the wind direction is selected. The evolutionarily important variances and expectations of gene displacement under the selected model are calculated. It is also shown that the underlying probability distribution is significantly more than exponentially leptokurtic. By fitting a distribution of deposition in all three dimensions to the data, taking into account differences in plant height, separate estimates of additional physical parameters are obtained, showing that gravity and vertical random movements are more important than intervening vegetation in limiting pollen dispersal in meadow fescue. According to the model, plants with a high seed yield contribute pollen over-proportionally to neighbouring plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogeographical history of the widespread meadow fescue (Festuca pratensis Huds.) inferred from chloroplast DNA sequences

Aim Here we explore the variation in chloroplast DNA (cpDNA) in a widespread Eurasian diploid forage grass, meadow fescue (Festuca pratensis Huds.), to address its phylogeographical history. In particular, we aim to answer whether the post‐glacial migration routes of meadow fescue are associated with the spread of agriculture or concurrent with well‐documented natural migration pathways from glacial refugia. Location A total of 56 Eurasian accessions of F. pratensis were analysed, representing the entire native distribution area as well as non‐native areas in northernmost Europe. Methods Based on initial sequencing of 10 non‐coding cpDNA regions, three regions were sequenced for all F. pratensis accessions. For reference, three closely related species [the diploid Lolium perenne L. and the polyploids Festuca arundinacea Schreb. and Festuca gigantea (L.) Vill.] were also sequenced, as well as the more distantly related Festuca ovina L. Divergence times were estimated assuming a simple molecular clock, calibrated using a previously published estimate of 9 Myr for the divergence between fine‐leaved (F. ovina) and broad‐leaved fescues (F. pratensis, F. arundinacea and F. gigantea). Results Limited, but geographically structured, cpDNA variation was observed in F. pratensis. Three haplotypes, estimated to have diverged 0.16 Ma, were identified: one western European (A), one with a wide eastern distribution from central‐eastern Europe into Asia (B) and one Caucasian (C). The haplotypes of the polyploids and L. perenne were estimated to have diverged from haplotype A in F. pratensis 0.8–1.3 Ma. Main conclusions We found no definite evidence for migration of the diploid F. pratensis associated with the spread of agriculture from the Fertile Crescent after the last glaciation. The distinct geographical structuring of the present‐day variation in cpDNA can rather be explained by northwards expansion of the western haplotype from an Iberian refugium, expansion of the eastern haplotype from an unlocated (south‐)eastern refugium and glacial survival without subsequent expansion from a Caucasian refugium. The high level of cpDNA divergence observed between this diploid and the polyploids which have probably been derived from it may suggest that the very low level of cpDNA variation in the diploid is caused by a recent bottleneck. Today, F. pratensis is widespread in the open agricultural landscape but appears otherwise confined to naturally open habitats such as river banks, and its populations may have been decimated when dense forests dominated in the previous interglacial.     

Changes in plasmalemma K+Mg2+ -ATPase dephosphorylating activity and H+ transport in relation to seasonal growth and freezing tolerance of Festuca pratensis Huds

Changes in plasmalemma K⁺Mg²⁺-ATPase dephosphorylating activity and H⁺ transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and ΔμH⁺ were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K⁺Mg²⁺-ATPase activity was found throughout the seasonal growth of the plants (i). The K⁺Mg²⁺-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K⁺Mg²⁺-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K⁺Mg²⁺-ATPase activity than in that of H⁺ transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4–2 °C) and at freezing (−8 °C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed.