Ultrastructural and morphometric study of the lung of the European salamander,Salamandra salamandra L.

Ultrastructural and morphometric investigations were performed on the lung of the European salamander, Salamandra salamandra L. Folds of first and second order are covered with a ciliated epithelium containing goblet cells. The respiratory surface of the lung is lined by a single type of cell which, in amphibians, combines features of type I and type II alveolar cells of the mammalian lung. In the salamander the respiratory and ciliated epithelial cells as well as goblet cells possess electron dense and lucent vesicles in their cytoplasm as well as lamellar bodies. A small amount of surfactant, composed most probably of phospholipids and mucopolysaccharides, was observed covering the entire inner surface of the lung. Morphometric methods were used to determine the dimensions of the perinuclear region of pneumocytes, the thickness of the air-blood barrier and lung wall, and also the diameter of capillaries. The thickness of the respiratory air-blood barrier was found to be considerably higher than that of the corresponding barrier in mammals.     

A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L

European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) sustains a regional fishery and is commonly farmed in the Mediterranean basin, but has not undergone much long-term genetic improvement. An updated genetic linkage map of the European sea bass was constructed using 190 microsatellites, 176 amplified fragment length polymorphisms and two single nucleotide polymorphisms. From the 45 new microsatellite markers (including 31 type I markers) reported in this study, 28 were mapped. A total of 368 markers were assembled into 35 linkage groups. Among these markers, 28 represented type I (coding) markers, including those located within the peptide Y, SOX10, PXN1, ERA and TCRB genes (linkage groups 1, 7, 16, 17 and 27 respectively). The sex-averaged map spanned 1373.1 centimorgans (cM) of the genome. The female map measured 1380.0 cM, whereas the male map measured 1046.9 cM, leading to a female-to-male (F:M) recombination rate ratio of 1.32:1. The intermarker spacing of the second-generation linkage map of the European sea bass was 3.67 cM, which is smaller than that of the first-generation linkage map (5.03 cM). Comparative mapping of microsatellite flanking regions was performed with five model teleosts and this revealed a high percentage (33.6%) of evolutionarily conserved regions with the three-spined stickleback.     

Chloroplast DNA diversity within and among populations of the allotetraploid Prunus spinosa L.

High chloroplast DNA (cpDNA) diversity was found within and among populations of Prunus spinosa sampled from seven European deciduous forests. A study of 12% of the total chloroplast genome detected 44 mutations, which were distributed over 24 haplotypes; four were common to two or more populations and the rest were unique haplotypes. The most-abundant and widely distributed haplotype was H2 (frequency = 41% approximately). Six of the seven populations were polymorphic. All of the six polymorphic populations had ”private” haplotypes (frequency < 5%) in addition to common haplotypes. The UPGMA dendrogram demonstrated a correlation between populations and their geographical locations. The total diversity was high (h_T= 0.824) and a major portion of it was within populations (h_s= 0.663). The level of population subdivision for unordered alleles was low (G_ST= 19.5%) and for ordered alleles was lower (N_ST= 13.6%). No phylogeographic structure could be demonstrated in the present geographical scale. High polymorphism in the cpDNA of P. spinosa has to be considered carefully when planning phylogenetic studies involving this species. Received: 20 September 1999 / Accepted: 10 November 1999     

A first-generation genetic linkage map of the European flat oyster Ostrea edulis (L.) based on AFLP and microsatellite markers

This study presents the first genetic linkage map for the European flat oyster Ostrea edulis . Two hundred and forty-six AFLP and 20 microsatellite markers were genotyped in a three-generation pedigree comprising two grandparents, two parents and 92 progeny. Chi-square goodness-of-fit tests revealed high segregation distortion, which was significant for 32.8% of markers. Sixteen microsatellites and 235 AFLPs (170 type 1:1 AFLPs and 65 type 3:1 AFLPs) were used to build sex-specific linkage maps using crimap software. The first parental map (P₁) consisted of 104 markers grouped in nine linkage groups, and spanned 471.2 cM with an average spacing of 4.86 cM. The second parental map (P₂) consisted of 117 markers grouped in 10 linkage groups (which equals the haploid chromosome number), and covered 450.0 cM with an average spacing of 4.21 cM. The estimated coverage of the genome was 82.4% for the P₁ map and 84.2% for the P₂ map. Eight linkage groups that were probably homologous between the two parents contained the same microsatellites and 3:1 AFLPs (segregating through both parents). Distorted markers were not randomly distributed across the genome and tended to cluster in a few linkage groups. Sex-specific differences in recombination rates were evident. This first-generation genetic linkage map for O. edulis represents a major step towards the mapping of QTL such as resistance to bonamiasis, a parasitosis that has drastically decreased populations of flat oysters since the 1960s.     

Analysis of genetic diversity in Aconitum kongboense L. revealed by AFLP markers

The systematic evaluation of the molecular diversity encompassed in Aconitum kongboense L. inbreds or parental lines offers an efficient means of exploiting the heterosis in A. kongboense as well as for management of biodiversity. An excellent and novel DNA-based molecular, amplified fragment length polymorphisms (AFLPs) markers, was firstly used to analysis the genetic diversity in A. kongboense genotypes. Out of 256 primers screened, a total of ten combinations successfully produced scorable, clear, reproducible and relatively high polymorphism bands, 64.12% of which were polymorphic. The values of number of polymorphic loci (NPL), percentage of polymorphic loci (PPB), observed number of alleles (Na) and effective number of alleles (Ne) were highest in population 3 (NPL = 77, PPB = 68.75%, Na = 1.688 ± 0.466, Ne = 1.412 ± 0.397) and lowest in population 2 (NPL = 57, PPB = 50.89%, Na = 1.509 ± 0.502, Ne = 1.273 ± 0.340). In addition, Jaccard's similarity coefficients among different populations varied from 0.45 to 1.00 with an average of 0.55. The data collected will contribute to identification, rational exploitation and conservation of germplasms of A. kongboense, and potentially useful to aid its breeding.     

A morphometric and comparative study of the malleus.

This study has attempted to research, in detail, the dimensions and form of the malleus, and to indicate possible differences with regard to race, sex and side of origin (left or right). The ossicles were obtained from 75 adult cadavers and the dimensions were determined with the aid of a reflection microscope. Clear statistically significant differences were found between the Negroid and Caucasoid races, as well as between the right and left ossicles. No meaningful differences were determined between male and female ossicles.     

Phylogenetic relationships and genetic diversity among members of theFestuca-Lolium complex (Poaceae) based on ITS sequence data

There have been few DNA sequencebased studies of phylogenetic relationships within theFestuca-Lolium complex. Here we infer the phylogeny of 31Festuca-Lolium taxa with a dataset of 116 ITS sequences. The results are consistent with previous studies that resolved two majorFestuca clades: one clade of fine fescues and another clade that containsLolium and associatedFestuca species. This study is unique in suggesting a third, basalFestuca clade, but support for the basal position of this group is low. Extensive sampling permitted investigation of the effects of lineage sorting and reticulate events on the evolution of the complex. Roughly half of the taxa show evidence of lineage sorting or reticulation, and the monophyly ofLolium has likely been obscured by reticulate events. Overall, polyploid species harbor higher levels of ITS sequence diversity than diploids; ITS sequence variants may provide clues to the identity of allopolyploid parents.     

SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.     

Hepatic autophagy and intracellular ATP. A morphometric study

In order to estimate the sensitivity of macroautophagy in liver toward changes in ATP we have analyzed the volume density of the autophagic/lysosomal system in isolated rat hepatocytes, incubated under conditions where intracellular ATP was partially depleted. (a) It appeared that reduction of the intracellular ATP concentration by 30-50% decreased the volume density of autophagic vacuoles by 70%. (b) Partial ATP depletion did not involve significant changes in the volume density of dense bodies. Together with studies showing that the rate of overall proteolysis via macroautophagy decreases with decreasing ATP concentration (P.J.A.M. Plomp, E.J. Wolvetang, A.K. Groen, A.J. Meijer, P.B. Gordon, and P.O. Seglen (1987) Eur. J. Biochem. 164, 197-203) our data indicate that changes in intracellular ATP primarily affect early steps in the autophagic/proteolytic pathway.     

Genetic diversity and differentiation in Eryngium alpinum L. (Apiaceae): comparison of AFLP and microsatellite markers

Genetic diversity and structure of 12 populations of Eryngium alpinum L. were investigated using 63 dominant amplified fragment length polymorphism (AFLP) and seven codominant microsatellite (48 alleles) markers. Within-population diversity estimates obtained with both markers were not correlated, but the microsatellite-based fixation index Fis was correlated with both AFLP diversity indices (number of polymorphic bands and Nei's expected heterozygosity). Only AFLP diversity indices increased with the size of populations, although they did not significantly differ among them (Kruskall-Wallis test). The discrepancy between AFLPs and microsatellites may be explained by a better coverage of the genome with numerous AFLPs, the higher mutation rates of microsatellites or the absence of significant difference among within-population diversity estimates. Genetic differentiation was higher with AFLPs (theta=0.40) than with microsatellites (theta=0.23), probably due to the higher polymorphism of microsatellites. Thus, we considered global qualitative patterns rather than absolute estimates to compare the performance of both types of markers. On a large geographic scale, the Mantel test and multivariate analysis showed that genetic patterns were more congruent with the spatial arrangement of populations when inferred from microsatellites than from AFLPs, suggesting higher homoplasy of AFLP markers. On a small spatial scale, AFLPs managed to discriminate individuals from neighboring populations whereas microsatellites did not (multivariate analysis), and the percentage of individuals correctly assigned to their population of origin was higher with AFLPs than with microsatellites. However, dominant AFLPs cannot be used to study heterozygosity-related topics. Thus, distinct molecular markers should be used depending on the biological question and the geographical scale investigated.     

Chloroplast DNA study in wild populations and some cultivars of Prunus avium L.

The PCR-RFLP technique was used to detect chloroplast DNA diversity in wild populations of Prunus avium from five European deciduous forests and some cultivars. A study of 10.8% of the total chloroplast genome detected eight insertion-deletion (indel) mutations, distributed over 12 haplotypes. Six haplotypes (H1, H2, H3, H4, H5 and H6) were found in wild populations and eight (H2, H6, H7, H8, H9, H10, H11 and H12) in the cultivars. Only two haplotypes (H2 and H6) are shared by the wild populations and the cultivars. The most-abundant and frequent haplotype in wild populations is H2 (frequency=78%). The wider geographical distribution along with the high frequency reflects its ancient origin. Of the five populations, three are polymorphic. Populations GA (Scotland) and KE (Germany) have unique haplotypes. The total cpDNA diversity in wild populations is h_T=0.40, and a major portion of it is within populations (h_S=0.37). The genetic differentiation among populations was low (G_STC=0.08) and no genetic structure among wild populations was observed. A minimum-length spanning tree, demonstrating relationships among the haplotypes in wild populations, indicated two possible chloroplast lineages. The ten identified cultivars were represented by seven haplotypes; this result proposes the possible utilisation of the PCR-RFLP technique for the characterisation of sweet cherry cultivars. The cpDNA diversity in P. avium should be considered carefully for phylogenetic studies involving this species. Received: 10 July 2000 / Accepted: 19 October 2000     

Genetic diversity in colonial bentgrass (Agrostis capillaris L.) revealed by EcoRI-MseI and PstI-MseI AFLP markers.

Colonial bentgrass (Agrostis capillaris L.) is a potential source for genetic improvement of resistance to environmental stress and disease for other bentgrass species (Agrostis spp.). To conserve and study the existing genetic resources of colonial bentgrass for use in breeding, genetic diversity was investigated using amplified fragment length polymorphism (AFLP) markers. Included in this study were 22 accessions from US Department of Agriculture germplasm collected from 11 countries, in conjunction with 14 accessions from northern Spain and 3 commercial cultivars. Ten EcoRI-MseI and 6 PstI-MseI AFLP primer combinations produced 181 and 128 informative polymorphic bands, respectively. Cluster analysis of genetic similarity estimates revealed a high level of diversity in colonial bentgrass species with averages of 0.51 (EcoRI-MseI) and 0.63 (PstI-MseI). Greater genetic diversity was detected by the EcoRI-MseI AFLP primer combinations. A low but significant positive correlation (r = 0.44, p = 0.0099) between the 2 Jaccard similarity matrices was obtained by the Mantel test. Commercial cultivars of bentgrass showed a narrow genetic background. The assessment of genetic diversity among colonial bentgrass accessions suggested the potential value of the colonial bentgrass germplasm in turfgrass cultivar improvement.     

A study of genetic diversity in Populus nigra subsp. betulifolia in the Upper Severn area of the UK using AFLP markers

A survey of amplified fragment length polymorphism (AFLP) was conducted to examine genetic diversity in 146 individuals of British, native black poplar ( Populus nigra subsp. betulifolia ) and three individuals considered by collectors to be non- betulifolia poplars. Using two primer pairs, a total of 147 bands were detected of which 82 (56%) were polymorphic in at least one individual. Cluster analysis and principal coordinates analysis of the calculated similarity matrix revealed a low level of genetic diversity, although a loose clustering into five groups could be identified, one of which contained the non- betulifolia individuals. Examination of the spatial distribution of the other four groups (all betulifolia ) revealed a general correlation between geographic proximity and genetic similarity. On the basis of the polymorphism observed, it was possible to identify a small number of individual plants which exhibit maximum diversity and might therefore be suitable for inclusion in a replanting programme designed to maintain at least the existing low level of polymorphism observed within British black poplar.     

Phylogeography and molecular diversity analysis of Jatropha curcas L. and the dispersal route revealed by RAPD,AFLP and nrDNA-ITS analysis

Jatropha curcas L. (Euphorbiaceae) has acquired a great importance as a renewable source of energy with a number of environmental benefits. Very few attempts were made to understand the extent of genetic diversity and its distribution. This study was aimed to study the diversity and deduce the phylogeography of Jatropha curcas L. which is said to be the most primitive species of the genus Jatropha. Here we studied the intraspecific genetic diversity of the species distributed in different parts of the globe. The study also focused to understand the molecular diversity at reported probable center of origin (Mexico), and to reveal the dispersal route to other regions based on random amplified polymorphic DNA, amplified fragment length polymorphism and nrDNA-ITS sequences data. The overall genetic diversity of J. curcas found in the present study was narrow. The highest genetic diversity was observed in the germplasm collected from Mexico and supports the earlier hypothesis based on morphological data and natural distribution, it is the center for origin of the species. Least genetic diversity found in the Indian germplasm and clustering results revealed that the species was introduced simultaneously by two distinct germplasm and subsequently distributed in different parts of India. The present molecular data further revealed that J. curcas might have spread from the center of the origin to Cape Verde, than to Spain, Portuguese to other neighboring countries and simultaneously to Africa. The molecular evidence supports the Burkill et al. (A dictionary of the economic products of the Malay Peninsula, Governments of Malaysia and Singapore by the Ministry of Agriculture and Co-operatives. Kuala Lumpur, Malaysia, 1966) view of Portuguese might have introduced the species to India. The clustering pattern suggests that the distribution was interfered by human activity.