Mitochondrial DNA as effective molecular markers for the genetic variation and phylogeny of the family Osteoglossidae

The present study examined the genetic variation of the family Osteoglossidae from different geographical locations based on the mitochondrial NADH dehydrogenase subunit 2 (ND2) and ATPase subunit 6 (ATPase6) genes; we then re-constructed the phylogenetic relationships using the two sequences in combination. The results showed that the partial sequences of mitochondrial ND2 and ATPase6 of the family Osteoglossidae were 813 bp and 669 bp, respectively. A total of 42 species-specific nucleotide positions of the family Osteoglossidae were found to be useful for molecular identification. The sequence variation showed greater differences (8.3% ~ 28.1% for the combined sequences, 8.3% ~ 26.7% for the ND2 gene, and 9.3% ~ 28.7% for the ATPase6 gene) among the different species of Osteoglossidae, and there was a significant association between the genetic difference and geographical location. Phylogenetic analyses using neighbor-joining, Bayesian inference, and maximum parsimony (MP) methods based on the combined sequences of the two genes were able to distinguish the different species and were in agreement with the existing taxonomy based on morphological characters and in association with the geographical distribution among seven species of the family Osteoglossidae.     

Molecular markers: a potential resource for ginger genetic diversity studies

Ginger is an economically important and valuable plant around the world. Ginger is used as a food, spice, condiment, medicine and ornament. There is available information on biochemical aspects of ginger, but few studies have been reported on its molecular aspects. The main objective of this review is to accumulate the available molecular marker information and its application in diverse ginger studies. This review article was prepared by combing material from published articles and our own research. Molecular markers allow the identification and characterization of plant genotypes through direct access to hereditary material. In crop species, molecular markers are applied in different aspects and are useful in breeding programs. In ginger, molecular markers are commonly used to identify genetic variation and classify the relatedness among varieties, accessions, and species. Consequently, it provides important input in determining resourceful management strategies for ginger improvement programs. Alternatively, a molecular marker could function as a harmonizing tool for documenting species. This review highlights the application of molecular markers (isozyme, RAPD, AFLP, SSR, ISSR and others such as RFLP, SCAR, NBS and SNP) in genetic diversity studies of ginger species. Some insights on the advantages of the markers are discussed. The detection of genetic variation among promising cultivars of ginger has significance for ginger improvement programs. This update of recent literature will help researchers and students select the appropriate molecular markers for ginger-related research.     

Microsatellite DNA markers for population genetic studies in the dinoflagellate Karenia brevis

Nine nuclear‐encoded microsatellites from an enriched genomic DNA library of the HAB (harmful algal bloom) dinoflagellate Karenia brevis were isolated and characterized. The microsatellites include five perfect (three dinucleotide and two trinucleotide) and four imperfect (two dinucleotide and two trinucleotide) repeat motifs. Gene (haplotype) diversity ranged from 0.153 to 0.750 among a sample of 13 isolates; the number of alleles among the isolates ranged from two to six and pairwise tests of genotypic disequilibria were nonsignificant. The microsatellites developed in this study will provide insight into the genetic diversity of this HAB species and tools that may prove useful in predicting source populations and physiological parameters of individual K. brevis blooms.     

Plant protoplasts as genetic tool: selectable markers for developmental studies.

Protoplasts have usually been presented as a methodological tool. Even as such, they make possible an impressive array of applications in plant biology. Here we report on the use of protoplast-derived selectable markers in the study of several disturbed genetic systems with obvious effects on plant development: (1) auxotrophic mutants and the control of amino acid biosynthesis and transport in vegetative and reproductive tissues; (2) introgression of alien genetic information across phylogenetic boundaries by protoplast fusion, a consequence of controlled dedifferentiation-redifferentiation processes and attenuated incompatibility reactions in cultured cells; (3) expression (in)stability of foreign genes in transgenic plants during successive meiotic generations and in crosses between independent transformants.     

Detection of universal variable fragments as markers for genetic studies. A novel technology for DNA fingerprinting

A novel DNA technology enables the detection of universal variable fragments (UVF), thus revealing genetic variation without a priori sequence information. The detection of UVF markers is based on two amplifications of genomic DNA with the polymerase chain reaction. In the first amplification, two short oligonucleotide primers produce a large number of fragments. One primer is based on a microsatellite sequence, whereas the second primer can have any sequence. In the second amplification, the length of the primers is increased in order to decrease the number of amplicons. This enables the selection of polymorphic fragments. Restriction digestion can be used to further increase the number of polymorphisms. Until now, we have demonstrated UVF in several different species. In addition, with the present study we have contributed to the linkage map of the rabbit by localizing 11 UVF markers on different linkage groups. Mendelian inheritance was shown in this linkage study through a backcross of two inbred rabbit strains. The power of the UVF technique is based on the selection for microsatellite variation in combination with the detection of single-nucleotide polymorphisms. UVF thus offers the possibility of increasing the clustering of markers and localizing genes in species for which sequence information is either not present or only scarcely present.     

Mitochondrial DNA control region polymorphisms: genetic markers for ecological studies of marine turtles

We describe a rapid and sensitive method for the detection of population-specific genetic markers in mitochondrial DNA (mtDNA) and the use of such markers to analyse population structure of marine turtles. A series of oligonucleotide primers specific for the amplification of the mtDNA control region in Cheloniid turtles were designed from preliminary sequence data. Using two of these primers, a 384–385-bp sequence was amplified from the 5′ portion of the mtDNA control region of 15 green turtles Chelonia mydas from 12 different Indo-Pacific rookeries. Fourteen of the 15 individuals, including some with identical whole-genome restriction fragment patterns, had sequences that differed by one or more base substitutions. Analysis of sequence variation among individuals identified a total of 41 nucleotide substitutions and a 1-bp insertion/deletion. Comparison with evidence from whole-genome restriction enzyme analysis of the same individuals indicated that this portion of the control region is evolving approximately eight times faster than the average rate and that the sequence analysis detected approximately one fifth of the total variation present in the genome. Restriction enzyme analysis of amplified products from an additional 256 individuals revealed significant geographic structuring in the distribution of mtDNA genotypes among five of the 10 rookeries surveyed extensively. Additional geographic structuring of genotypes was identified through denaturing gradient gel electrophoresis (DGGE) of amplified products. Only two of the 10 rookeries surveyed could not be differentiated, indicating that the Indo-Pacific C. mydas include a number of genetically differentiated populations, with minimal female-mediated gene flow among them. Important applications for genetic markers in the conservation and management of marine turtles include the identification of appropriate demographic units for research and management (i.e. genetically discrete populations) and assessment of the composition of feeding and harvested populations.     

SSR mining in coffee tree EST databases: potential use of EST-SSRs as markers for the Coffea genus

Expressed sequence tags (ESTs) from Coffea canephora leaves and fruits were used to search for types and frequencies of simple sequence repeats (EST–SSRs) with a motif length of 1–6 bp. From a non-redundant (NR) EST set of 5,534 potential unigenes, 6.8% SSR-containing sequences were identified, with an average density of one SSR every 7.73 kb of EST sequences. Trinucleotide repeats were found to be the most abundant (34.34%), followed by di- (25.75%) and hexa-nucleotide (22.04%) motifs. The development of unique genic SSR markers was optimized by a computational approach which allowed us to eliminate redundancy in the original EST set and also to test the specificity of each pair of designed primers. Twenty-five EST–SSRs were developed and used to evaluate cross-species transferability in the Coffea genus. The orthology was supported by the amplicon sequence similarity and the amplification patterns. The >94% identity of flanking sequences revealed high sequence conservation across the Coffea genus. A high level of polymorphic loci was obtained regardless of the species considered (from 75% for C. liberica to 86% for C. canephora). Moreover, the polymorphism revealed by EST–SSR was similar to that exposed by genomic SSR. It is concluded that Coffea ESTs are a valuable resource for microsatellite mining. EST-SSR markers developed from C. canephora sequences can be easily transferred to other Coffea species for which very little molecular information is available. They constitute a set of conserved orthologous markers, which would be ideal for assessing genetic diversity in coffee trees as well as for cross-referencing transcribed sequences in comparative genomics studies.     

Microsatellite DNA markers for population‐genetic studies of Atlantic bluefin tuna (Thunnus thynnus thynnus) and other species of genus Thunnus

Twenty‐five microsatellites from Atlantic bluefin tuna (Thunnus thynnus thynnus) were characterized. All 25 microsatellites were polymorphic; the number of alleles among up to 56 individuals surveyed ranged from two to 23. Atlantic bluefin tuna are highly exploited and major questions remain as to stock structure and abundance in the eastern and western North Atlantic. The microsatellites will be useful in testing stock‐structure hypotheses and in generating estimates of effective population size. The polymerase chain reaction primer sets developed also amplified identifiable alleles in three other species of genus Thunnus: T. albacares (yellowfin tuna), T. alalunga (albacore tuna) and T. obesus (bigeye tuna).     

Monoclonal antibody to single-stranded DNA: a potential tool for DNA repair studies

Growing evidence suggests that DNA repair capacity is an important factor in cancer risk and is therefore essential to assess. Immunochemical assays are amenable to the detection of repair products in complex matrices, such as urine, facilitating noninvasive measurements, although diet and extra-DNA sources of lesion can confound interpretation. The production of single-stranded, lesion-containing DNA oligomers characterises nucleotide excision repair (NER) and hence defines the repair pathway from which a lesion may be derived. Herein we describe the characterisation of a monoclonal antibody which recognises guanine moieties in single-stranded DNA. Application of this antibody in ELISA, demonstrated such oligomers in supernatants from repair-proficient cells post-insult. Testing of urine samples from volunteers demonstrated a relationship between oligomer levels and two urinary DNA damage products, thymine dimers and 8-oxo-2'-deoxyguanosine, supporting our hypothesis that NER gives rise to lesion-containing oligomers which are specific targets for the investigation of DNA repair.     

Microsatellite DNA markers for population genetic studies and parentage assignment in cobia,Rachycentron canadum

Twenty nuclear‐encoded microsatellites from a genomic DNA library of cobia, Rachycentron canadum, were isolated and characterized. The microsatellites include two tetranucleotide, one trinucleotide, three combination tetranucleotide/dinucleotide, nine dinucleotide, and five imperfect (dinucleotide) repeat motifs. Gene diversity ranged between zero to 0.910; the number of alleles among a sample of 24 fish ranged from one to 15. Cobia support an important recreational fishery in the southeastern United States and recently have become of interest to aquaculture. The microsatellites developed will be useful tools for studying both population genetics (e.g. stock structure, effective population size) and inheritance of traits important to aquaculture.     

The evolutionary change of the genetic code as restricted by the anticodon and identity of transfer RNA

The discovery of non-universal genetic codes in several mitochondria and nuclear systems during the past ten years has necessitated a reconsideration of the concept that the genetic code is universal and frozen, as was once believed. Here, the flexibility of the relationship between codons and amino acids is discussed on the basis of the distribution of non-universal genetic codes in various organisms insofar as has been observed to date. Judging from the result of recent investigations into tRNA identity, it would appear that the non-participation of the anticodon in recognition by aminoacyl-tRNA synthetase has significantly influenced the variability of codons.     

QTL analysis and genomic selection using RADseq derived markers in Sitka spruce: the potential utility of within family data

Sitka spruce (Picea sitchensis (Bong.) Carr) is the most common commercial plantation species in Britain and a breeding programme based on traditional lines has been in operation since the early 1960s. Rotation lengths of 40-years have led breeders to adopt a process of indirect selection at younger ages based on traits well correlated with final selection, but still the generation interval is unlikely to reduce much below twenty years. Recent successful developments with genomic selection in animal breeding have led tree breeders to consider the application of this technology. In this study a RAD sequence assay was developed as a means of investigating the potential of molecular breeding in a non-model species. DNA was extracted from nearly 500 clonally replicated trees growing in a single full-sibling family at one site in Britain. The technique proved successful in identifying 132 QTLs for 5-year bud-burst and 2 QTLs for 6-year height. In addition, the accuracy of predicting phenotypes by genomic selection was strikingly high at 0.62 and 0.59 respectively. Sensitivity analysis with 200 offspring found only a slight fall in correlation values (0.54 and 0.38) although when the training population reduced to 50 offspring predictive values fell further (0.33 and 0.25). This proved an encouraging first investigation into the potential use of genomic selection in the breeding of Sitka spruce. The authors investigate how problems associated with effective population size and linkage disequilibrium can be avoided and suggest a practical way of incorporating genomic selection into a dynamic breeding programme.     

Influence of salt concentration on the susceptibility of moderately halophilic bacteria to antimicrobials and its potential use for genetic transfer studies

The influence of salinity on the susceptibility of 13 moderately halophilic collection strains belonging to the genera Chromohalobacter, Deleya, Halomonas, Vibrio, and Volcaniella to 10 common antimicrobials has been studied. Three different patterns of tolerance were found when salinity was varied from 10 to 1% (wt/vol) total salts in the testing media. The first one included the responses to ampicillin and rifampicin, where only minimal effects on the susceptibility were found. All moderate halophiles showed a high sensitivity to rifampicin regardless of the salt concentration. In the second group, including the responses to the aminoglycosides gentamycin, kanamycin, neomycin, and streptomycin, a remarkable and gradual increase of the toxicity was detected at lower salinities. Thirdly, the highest heterogeneity was found for the rest of antimicrobials assayed (trimethoprim, nalidixic acid, spectinomycin, and tetracycline), where the effect of salinity was moderate and dependent on both the individual strain and the antimicrobial tested. The data presented here should facilitate genetic studies on moderate halophiles. Thus, they simplify the design of selection media for genetic exchange experiments. Besides, by using low-salinity media, genes encoding resistance to a number of antimicrobials, especially to aminoglycosides, can be used as genetic markers for plasmids or transposons to be transferred to these extremophiles.     

Immunohistological markers for proliferative events,gliogenesis, and neurogenesis within the adult hippocampus

Biologists long believed that, once development is completed, no new neurons are produced in the forebrain. However, as is now firmly established, new neurons can be produced at least in two specific forebrain areas: the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampal formation. Neurogenesis within the adult DG occurs constitutively throughout postnatal life, and the rate of neurogenesis within the DG can be altered under various physiological and pathophysiological conditions. The process of adult neurogenesis within the DG is a multi-step process (proliferation, differentiation, migration, targeting, and synaptic integration) that ends with the formation of a post-mitotic functionally integrated new neuron. Various markers are expressed during specific stages of adult neurogenesis. The availability of such markers allows the time-course and fate of newly born cells to be followed within the DG in a detailed and precise fashion. Several of the available markers (e.g., PCNA, Ki-67, PH3, MCM2) are markers for proliferative events, whereas others are more specific for early phases of neurogenesis and gliogenesis within the adult DG (e.g., nestin, GFAP, Sox2, Pax6). In addition, markers are available allowing events to be distinguished that are related to later steps of gliogenesis (e.g., vimentin, BLBP, S100beta) or neurogenesis (e.g., NeuroD, PSA-NCAM, DCX).     

Comparative study of the L1 family in the genus Mus. Possible role of retroposition and conversion events in its concerted evolution

The long interspersed repetitive family L1 was analysed in different species belonging to the genus Mus. It is shown to be highly conserved even in M.n. setulosus, which diverged from the other species around ten million years ago. The study of the linkage between diagnostic restriction sites in the various species and the sequence variations of different regions of the L1Md repeat shows that the L1 family undergoes concerted changes involving subsets of repeats. The rate at which this homogenization process occurs does not appear to be the same for all the subfamilies detected. The L1Md repeat in the twelfth intron of the serum albumin gene of Balb/c mice is shown to be a recent insertion. The role retroposon- and gene conversion-like events may play in the concerted evolution of the L1 family is discussed.     

Microsatellite DNA markers for American shad (Alosa sapidissima) and cross‐species amplification within the family Clupeidae

Sixteen microsatellite loci were identified and characterized for American shad (Alosa sapidissima). The number of alleles per locus observed ranged from eight to 32 and averaged 15.4 alleles. Average observed heterozygosity was 81.1%. The markers were screened using four other species from the family Clupeidae. Amplification success among Alosa species was 79.2% with 81.6% polymorphism among those markers that amplified successfully. Amplification success was poor in Dorosoma (31.3%). Due to allelic diversity and estimates of heterozygosity, these markers can be useful in A. sapidissima for population level analyses, parentage assignment and broodstock management.