Molecular tools in marine ecology

Molecular tools have diverse applications in marine ecology. In microbial systems, DNA sequences of rRNA and other genes have identified a variety of novel lineages of bacteria inhabiting marine environments that have resisted traditional culture methods. However, relatively few natural populations have been characterized due to the rather labor-intensive methodologies employed. Recent technological developments such as in situ PCR and flow cytometry promise to greatly enhance the speed at which microbial taxa can be identified and enumerated in field collected water and substrate samples; such advances will allow future work to employ the spatial and temporal field sampling required to monitor the impact of natural and anthropogenic changes in the environment. This approach also holds promise for examining physiological status of field collected cells, garnering information on such elusive parameters as growth rates and the extent of nutrient limitation under natural conditions. Studies of macrobiota have similarly benefited from the use of molecular approaches to species identification. This has been particularly true with regard to distinguishing among larval forms of closely related taxa which are nearly identical morphologically. Genetic variation within species assayed by molecular tools has been useful in examining the stability of populations through time and in assessing patterns of recruitment to geographically separated populations. Enhanced understanding of these ecological problems will also require intensive spatial and temporal monitoring of both larval and adult populations. Often, the newer techniques based on DNA sequence variation have practical advantages over allozyme techniques: e.g., PCR allows assay of minute quantities of DNA that may come from ethanol preserved samples. However, when ample allozyme variation exists to address a given issue, these older techniques may be favored on a variety of criteria, including speed and cost. Hence, choice of methodology should be based on the expected efficiency of a given approach to a specific problem rather than the apparent sophistication of the method itself.     

Patterns of nucleotide change in mitochondrial ribosomal RNA genes and the phylogeny of piranhas

The patterns and rates of nucleotide substitution in mitochondrial ribosomal RNA genes are described and applied in a phylogenetic analysis of fishes of the subfamily Serrasalminae (Teleostei, Characiformes, Characidae). Fragments of 345 bp of the 12S and 535 bp of the 16S genes were sequenced for 37 taxa representing all but three genera in the subfamily. Secondary-structure models based on comparative sequence analysis were derived to characterize the pattern of change among paired and unpaired nucleotides, forming stem and loop regions, respectively. Base compositional biases were in the direction of A-rich loops and G-rich stems. Ninety-five percent of substitutions in stem regions were compensatory mutations, suggesting that selection for maintenance of base pairing is strong and that independence among characters cannot be assumed in phylogenetic analyses of stem characters. The relative rate of nucleotide substitution was similar in both fragments sequenced but higher in loop than in stem regions. In both genes, C-T transitions were the most common type of change, and overall transitions outnumbered transversions by a factor of two in 16S and four in 12S. Phylogenetic analysis of the mitochondrial DNA sequences suggests that a clade formed by the generaPiaractus, Colossoma, andMylossoma is the sister group to all other serrasalmins and that the generaMyleus, Serrasalmus, andPristobrycon are paraphyletic. A previous hypothesis concerning relationships for the serrasalmins, based on morphological evidence, is not supported by the molecular data. However, phylogenetic analysis of host-specific helminth parasites and cytogenetic data support the phylogeny of the Serrasalminae obtained in this study and provide evidence for coevolution between helminth parasites and their fish hosts.     

The complete mitochondrial DNA (mtDNA) of the donkey and mtDNA comparisons among four closely related mammalian species-pairs

The nucleotide sequence of the complete mitochondrial genome of the donkey, Equus asinus, was determined. The length of the molecule is 16,670 bp. The length, however, is not absolute due to pronounced heteroplasmy caused by variable numbers of two types of repetitive motifs in the control region. The sequence of the repeats is (a) 5′-CACACCCA and (b) 5′-TGCGCGCA, respectively. The order of (a) and (b) can be expressed as {n[2(a)+(b)]+m(a)}. In 32 different clones analyzed the number of n and m ranged from 0 to 9 and 1 to 7. The two rRNA genes, the 13 peptide-coding genes, and the 22 tRNA genes of the donkey and the horse, Equus caballus, were compared in detail. Total nucleotide difference outside the control region was 6.9%. Nucleotide difference between peptide-coding genes ranged from 6.4% to 9.4% with a mean of 8.0%. In the inferred protein sequences of the 13 peptide-coding genes the amino acid difference was 0.2–8.8%, and the mean for the 13 concatenated amino acid sequences was 1.9%. In the 22 tRNA genes, the mean difference was 3.5%, and that in the two rRNA genes was 4.1%. The mtDNA differences between the donkey and the horse suggest that the evolutionary separation of the two species occurred ≈9 million years ago. Analyses of differences among the mtDNAs of three other species-pairs, harbor seal/grey seal, fin whale/blue whale, and Homo/common chimpanzee, showed that the relative evolutionary rate of individual peptide-coding genes varies among different species-pairs and modes of comparison. The findings show that the superimposition of sequence data of one lineage for resolving and dating evolutionary divergences of other lineages should be performed with caution unless based on comprehensive data. Received: 15 October 1995 / Accepted: 15 April 1996     

comparison between the complete mitochondrial DNA sequences ofHomo and the common chimpanzee based on nonchimeric sequences

The complete mitochondrial DNA (mtDNA) molecules ofHomo and of the common chimpanzee were sequenced. Each sequence was established from tissue of one individual and thus nonchimeric. Both sequences were assembled in their entirety from natural (not PCR amplified) clones. Comparison with sequences in the literature identified the chimpanzee specimen asPan troglodytes verus, the West African variety of the species. The nucleotide difference between the complete human and chimpanzee sequences is 8.9%. The difference between the control regions of the two sequences is 13.9% and that between the remaining portions of the sequences 8.5%. The mean amino acid difference between the inferred products of the 13 peptide-coding genes is 4.4%. Sequences of the complete control regions, the complete 12S rRNA genes, the complete cytochromeb genes, and portions of the NADH4 and NADH5 genes of two other chimpanzee specimens showed that they were similar but strikingly different from the same regions of the completely sequenced molecule fromPan troglodytes verus. The two specimens were identified asPan troglodytes troglodytes, the Central African variety of the common chimpanzee.     

Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.)

Microsatellite markers containing simple sequence repeats (SSR) are a valuable tool for genetic analysis. Our objective is to augment the existing RFLP map of rice with simple sequence length polymorphisms (SSLP). In this study, we describe 20 new microsatellite markers that have been assigned to positions along the rice chromosomes, characterized for their allelic diversity in cultivated and wild rice, and tested for amplification in distantly related species. Our results indicate that the genomic distribution of microsatellites in rice appears to be random, with no obvious bias for, or clustering in particular regions, that mapping results are identical in intersubspecific and interspecific populations, and that amplification in wild relatives ofOryza sativa is reliable in species most closely related to cultivated rice but becomes less successful as the genetic distance increases. Sequence analysis of SSLP alleles in three relatedindica varieties demonstrated the clustering of complex arrays of SSR motifs in a single 300-bp region with independent variation in each. Two microsatellite markers amplified multiple loci that were mapped onto independent rice chromosomes, suggesting the presence of duplicated regions within the rice genome. The availability of increasing numbers of mapped SSLP markers can be expected to increase the power and resolution of genome analysis in rice.     

Identification of genetic factors for alachlor tolerance in maize by molecular markers analysis

Genetic factors controlling tolerance to the herbicide Alachlor in maize were localised by means of two different strategies. In the first approach, backcross (BC) plants, derived from pollen which had been subjected to selective pressure for resistance to the herbicide, were analysed for segregation distortion at 47 RFLP loci and compared to BC plants obtained from non-selected pollen. Preferential transmission of five chromosomal regions where putative QTLs (Quantitative Trait Loci) are localised was revealed in the BC plants from selected pollen. A second approach was based on a classical linkage analysis for segregation of the same set of RFLPs and factors controlling the trait, in a BC population of 210 individuals, by means of regression analysis. This study detected seven significant loci in four genomic regions. Overall, two loci revealed both segregation distortion and association with the expression of the trait, indicating linkage to genes expressed in both gametophytic and sporophytic phase. Three chromosomal regions appeared to carry factors involved in plant tolerance to Alachlor which are not expressed in pollen. Conversely, three loci were linked to factors selectable in pollen, but did not reveal significant association with tolerance in the plant in the segregating populations.     

铃兰族rbcL基因的PCR—RFLP分析

本文用7属9种铃兰族Tribe convallarieae植物的叶绿体DNA中rbcL基因片段的PCR产物的RFLP结果进行聚类分析。结果表明:开口箭属与蜘蛛抱蛋属关系密切,夏须草属与族内其余各属亲缘关系稍远,与外部器官形态、核型和孢粉学资料所得出的结论基本一致。此外,本文对铃兰属的系统位置也进行了讨论。     

Molecular dynamics study of interaction of dimyristoyl phosphotidyl choline with water

We present here results of molecular dynamics (MD) simulations on hydrated bilayers of 40 molecules of 1-2-dimyristoyl-sn-glycero-3-phosphatidyl choline (DMPC) in liquid crystalline (Lα) phase using two different models (i) with same (A) conformation for all DMPC molecules, (ii) with alternate rows having different (A and B reported in crystallographic studies on DMPC) conformations. The bilayers were hydrated using 776 and 1064 water molecules. Simulations have been carried out at 310K with AMBER 4.0 program, using united atom force field for 200 pico seconds (ps) after equilibration. During heating and equilibration constant pressure temperature (PT) conditions were maintained while in simulation of equillibrated bilayers constant volume temperature (VT) conditions were used. Subaveraged atomic coordinates were used to calculate geometric parameters of lipid molecules and lipid water interaction. Our results show larger flexibility of polar head group and glycerol region in Lα phase compared to gel or non-hydrated bilayers. Chain disorder was more towards end. Sn-2 chains were more disordered. Use of two types of starting conformations increased disorder. Trans fraction of chain torsional angle was higher in non-hydrated bilayer. However it was more disordered due to ‘swing’ movement of chains because of distortion in torsional angles α2 and 03 due to absence of water molecules. Trans fraction of the chains, order parameter and water penetration showed general agreement with the available experimental results. On the whole MD technique was found to be quite useful for depicting microscopic behaviour of liquid crystalline system and correlating the same with macroscopic changes observed experimentally.     

Estimation of the position and effect of a lethal factor locus on a molecular marker linkage map

In the mapping of DNA markers the distortion of segregation of marker genotypes is often observed, which may be caused by a lethal factor acting in filial generations derived from distant crosses. A method is presented for estimating the recombination values between a lethal factor locus and neighboring molecular markers, and the relative viability or fertilization ability of gametes or zygotes affected by the lethal factor in an F₂ population using the maximum likelihood method and the expectation conditional maximization (ECM) algorithm. Three selection models of gamete or zygote were considered, and the most likely one was determined by goodness of fit of the observed frequency of the phenotypes to the expected ones under the models. The method was applied to segregation data of molecular markers of an F₂ population consisting of 144 individuals derived from a cross between an Indica and a Japonica rice variety. The presence of a lethal factor locus (L) located on chromosome III that caused partial gametic selection in both the male and female sides was suggested. The locus L was tightly linked to RFLP marker number 23 of the RFLP linkage map of Saito et al. (1991a), and the fertilization chance of a male or female gamete possessing the lethal factor was, on average, 41.5% of that of the normal gamete.