A simple and sensitive high-throughput GFP screening in woody and herbaceous plants

Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T₀ seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.     

The Evolutionary History of the Transposable Element Penelope in the Drosophila virilis Group of Species

We have used phylogenetic techniques to study the evolutionary history of the Penelope transposable element in the Drosophila virilis species group. Two divergent types of Penelope have been detected, one previously described, clade I, and a new one which we have termed clade III. The phylogeny of some copies of the Penelope clade I element was partially consistent with the species phylogeny of the D. montana subphylad, suggesting cospeciation and allowing the estimation of the evolutionary rate of Penelope. Divergence times of elements found in different species are younger than the age of the species, suggesting horizontal transfer events. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov]     

Rapid isolation, purification and characterisation of type II restriction enzymes from Lactococcus spp.

An inexpensive procedure that uses small volumes (5–10 ml) of cell culture for the rapid isolation of restriction enzymes, sufficiently pure to allow preliminary characterisation, is presented. The method was designed initially to screen for Type II restriction enzymes, but different assays can be devised to screen for other types of restriction enzymes. Although initially optimised in Lacotococcus lactis subsp. cremoris LC17-1, this method potentially holds wider applications in other lactococcal species as was shown by its successful application to Lactococcus lactis subp. lactis. Without the necessity for chromatographic techniques that are often expensive and time consuming, the convenience of the technique makes it suitable for rapid, routine screening of a large number of lactic acid bacterial strains, or restriction and modification systems cloned into them, for restriction enzyme activity.     

Isolation and characterization of microsatellite loci in Ostryopsis davidiana (Betulaceae)

Ostryopsis (Betulaceae) is a samll genus endemic to China with only two species. Both of them play an important role in restoring the local ecosystems. The distribution of genetic diversity between and within populations in each species are important to further utilize the wild genetic resources and explore the interspecific divergence. In this study, we developed 10 microsatellite loci from O. davidiana by the combining biotin capture method for the first time. A total of 27 microsatellite sequences were recovered through screening the library and 10 of them are polymorphic. The number of alleles per locus in 18 sampled individuals ranged from 3 to 6, expected heterozygosity and observed heterozygosity ranged from 0.2958 to 0.4767 and from 0.1591 to 0.2997, respectively. In addition, all markers have been crossly checked in the other congeneric species. These microsatellite markers would together provide a useful tool for investigating the genetic diversity and structure of both species and speciation mechanism between them.     

Improving salinity tolerance in crop plants: a biotechnological view

Salinity limits the production capabilities of agricultural soils in large areas of the world. Both breeding and screening germplasm for salt tolerance encounter the following limitations: (a) different phenotypic responses of plants at different growth stages, (b) different physiological mechanisms, (c) complicated genotype × environment interactions, and (d) variability of the salt-affected field in its chemical and physical soil composition. Plant molecular and physiological traits provide the bases for efficient germplasm screening procedures through traditional breeding, molecular breeding, and transgenic approaches. However, the quantitative nature of salinity stress tolerance and the problems associated with developing appropriate and replicable testing environments make it difficult to distinguish salt-tolerant lines from sensitive lines. In order to develop more efficient screening procedures for germplasm evaluation and improvement of salt tolerance, implementation of a rapid and reliable screening procedure is essential. Field selection for salinity tolerance is a laborious task; therefore, plant breeders are seeking reliable ways to assess the salt tolerance of plant germplasm. Salt tolerance in several plant species may operate at the cellular level, and glycophytes are believed to have special cellular mechanisms for salt tolerance. Ion exclusion, ion sequestration, osmotic adjustment, macromolecule protection, and membrane transport system adaptation to saline environments are important strategies that may confer salt tolerance to plants. Cell and tissue culture techniques have been used to obtain salt tolerant plants employing two in vitro culture approaches. The first approach is selection of mutant cell lines from cultured cells and plant regeneration from such cells (somaclones). In vitro screening of plant germplasm for salt tolerance is the second approach, and a successful employment of this method in durum wheat is presented here. Doubled haploid lines derived from pollen culture of F₁ hybrids of salt-tolerant parents are promising tools to further improve salt tolerance of plant cultivars. Enhancement of resistance against both hyper-osmotic stress and ion toxicity may also be achieved via molecular breeding of salt-tolerant plants using either molecular markers or genetic engineering.     

Development and Evaluation of a PCR Based Assay for Detection of the Toxic Dinoflagellate, <Emphasis Type="Italic">Gymnodinium catenatum</Emphasis>(Graham) in Ballast Water and Environmental Samples

Gymnodinium catenatum is a bloom forming dinoflagellate that has been known to cause paralytic shellfish poisoning (PSP) in humans. It is being reported with increased frequency around the world, with ballast water transport implicated as a primary vector that may have contributed to its global spread. Major limitations to monitoring and management of its spread are the inability for early, rapid, and accurate detection of G. catenatum in plankton samples. This study explored the feasibility of developing a PCR-based method for specific detection of G. catenatumin cultures and heterogeneous ballast water and environmental samples. Sequence comparison of the large sub unit (LSU) ribosomal DNA locus of several strains and species of dinoflagellates allowed the design of G. catenatum specific PCR primers that are flanked by conserved regions. Assay specificity was validated through screening a range of dinoflagellate cultures, including the morphologically similar and taxonomically closely related species G. nolleri. Amplification of the diagnostic PCR product from all the strains of G. catenatum but not from other species of dinoflagellates tested imply the species specificity of the assay. Sensitivity of the assay to detect cysts in ballast water samples was established by simulated spiked experiments. The assay could detect G. catenatum in all ‘blank’ plankton samples that were spiked with five or more cysts. The assay was used to test environmental samples collected from the Derwent river estuary, Tasmania. Based on the results we conclude that the assay may be utilized in large scale screening of environmental and ballast water samples.     

Embryo sac isolation inArabidopsis thaliana: A simple and efficient technique for structure analysis and mutant selection

Arabidopsis thaliana has been widely used as a model plant in gene function analysis. However, its tiny flower and curved embryo sac make it difficult to study gene expression during megagametogenesis, fertilization, and early embryogenesis, especially in the screening of mutants from those developmental processes. The techniques currently available are sectioning and whole-mount clearing of ovules; however, sectioning is time consuming and laborious for quantitative analysis, and whole-mount clearing, makes clear cytological observation impossible. Reported here is a simple and efficient method based on enzymatic isolation of embryo sacs that enables both quantitative analysis and elaborate cytological observation for gene expression investigation and mutant screening.     

Screening methods for salinity tolerance: a case study with tetraploid wheat

Fast and effective glasshouse screening techniques that could identify genetic variation in salinity tolerance were tested. The objective was to produce screening techniques for selecting salt-tolerant progeny in breeding programs in which genes for salinity tolerance have been introduced by either conventional breeding or genetic engineering. A set of previously unexplored tetraploid wheat genotypes, from five subspecies of Triticum turgidum, were used in a case study for developing and validating glasshouse screening techniques for selecting for physiologically based traits that confer salinity tolerance. Salinity tolerance was defined as genotypic differences in biomass production in saline versus non-saline conditions over prolonged periods, of 3–4 weeks. Short-term experiments (1 week) measuring either biomass or leaf elongation rates revealed large decreases in growth rate due to the osmotic effect of the salt, but little genotypic differences, although there were genotypic differences in long-term experiments. Specific traits were assessed. Na⁺ exclusion correlated well with salinity tolerance in the durum subspecies, and K⁺/Na⁺ discrimination correlated to a lesser degree. Both traits were environmentally robust, being independent of root temperature and factors that might influence transpiration rates such as light level. In the other four T. turgidum subspecies there was no correlation between salinity tolerance and Na⁺ accumulation or K⁺/Na⁺ discrimination, so other traits were examined. The trait of tolerance of high internal Na⁺ was assessed indirectly, by measuring chlorophyll retention. Five landraces were selected as maintaining green healthy leaves despite high levels of Na⁺ accumulation. Factors affecting field performance of genotypes selected by trait-based techniques are discussed.     

Molecular Discrimination of Perna (Mollusca: Bivalvia) Species Using the Polymerase Chain Reaction and Species-Specific Mitochondrial Primers

This work was prompted by the need to be able to identify the invasive mussel species, Perna viridis, in tropical Australian seas using techniques that do not rely solely on morphology. DNA-based molecular methods utilizing a polymerase chain reaction (PCR) approach were developed to distinguish unambiguously between the three species in the genus Perna. Target regions were portions of two mitochondrial genes, cox1 and nad4, and the intergenic spacer between these that occurs in at least two Perna species. Based on interspecific sequence comparisons of the nad4 gene, a conserved primer has been designed that can act as a forward primer in PCRs for any Perna species. Four reverse primers have also been designed, based on nad4 and intergenic spacer sequences, which yield species-specific products of different lengths when paired with the conserved forward primer. A further pair of primers has been designed that will amplify part of the cox1 gene of any Perna species, and possibly other molluscs, as a positive control to demonstrate that the PCR is working.     

Molecular identification of crocodile species using novel primers for forensic analysis

All crocodilians are under varying degrees of threat due to over exploitation and these species have been listed in Appendix I or II of CITES. The lack of molecular techniques for the identification of confiscated samples makes it difficult to enforce the law. Conclusive forensic identification of species requires a complete gene sequence which is difficult in case of degraded samples. We have developed two novel sets of primers to amplify two partial cytochrome b gene sequences of six crocodile species i.e. Crocodylus palustris, Crocodylus porosus, Crocodylus siamensis, Crocodylus niloticus, Gavialis gangeticus and Caiman crocodilus. These partial sequences were edited to give a complete cyt b gene sequence, which can be used as an effective tool for forensic authentication of crocodile species. A phylogeny of crocodile species was reconstructed using these sequences. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these ancient species.     

The utility and limitations of genetic data for stock identification and management of North Pacific rockfish (Sebastes spp.)

Rockfishes (Sebastes spp.) represent a speciose and ecologically important group of marine fishes found in both the Pacific and Atlantic oceans, with approximately 105 species found world-wide (Hyde and Vetter 2007). They also comprise the majority of species found in the Pacific groundfish fishery. Thorough species assessments in terms of harvest management have been done for only 11 species, and of the 11 species, seven have been declared overfished. Having accurate genetic information is critical to the continuing effort at stock assessments, but sampling is often difficult in marine fishes. Genetic techniques are a powerful tool in the effort to better characterize the ecology of these species. These techniques can be used to investigate multiple biological traits, including species identity, intra- and interspecific genetic variation, migration patterns, and effective population size. There are important caveats and limitations when applying specific genetic methods, especially in marine species that lack discrete spawning aggregates. Nevertheless, it is clear from a review of recent literature that genetic tools have already provided very specific insight regarding rockfish population dynamics. The results are diverse and difficult to synthesize; however, existing studies show five primary patterns to population groupings in rockfishes: no obvious pattern of structure, structure consistent with isolation by distance, structure evident but inconsistent with isolation by distance, structure that correlates to oceanographic features, and potential genetic introgression. Clearly the study of rockfish population genetics is poised for rapid expansion that will unquestionably aid management of the rockfish fisheries and general understanding of rockfish evolutionary systematics. A principle challenge at this point is to derive generalized inferences from such a diverse array of study results across the vast North Pacific range of Sebastes. This review summarizes existing genetic studies in Sebastes spp. in the North Pacific to assist in identifying knowledge gaps for this ecologically important and diverse group.     

Biochemical variation to determine phylogenetic relationships betweenHordeum chilense and other American species of the genusHordeum (Poaceae)

Slab gel electrophoresis techniques have been applied to the study of isozyme and kernel protein patterns in 20 accessions ofHordeum chilense and related species in order to elucidate their phylogenetic relationships. On the basis of our results we can conclude that: (1) Conventional classification based on morphological characters does not totally agree with biochemical data. (2) Sectt.Anisolepis andCritesion seem to be clearly differentiated. (3) The accessions classified asH. compressum present biochemical phenotypes quite different from the rest of the species. (4)H. stenostachys, H. muticum andH. chilense constitute a group of variable species with many biochemical similarities and close phylogenetic relationships. (5) The evolutionary pattern of these American species seems to follow a model of reticulate evolution.     

Use of restriction fragment length polymorphism to fingerprint beets at the genotype and species levels

Summary cDNA probes have been used to assess genetic variation in beet using hybridisation techniques that detect restriction fragment length polymorphism. Probes have been identified which differ in the levels of variation that they can detect (i) within closely related genetic material of sugar beet, and (ii) between sugar beet and a taxonomically distant Beta species.     

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene‐edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high‐throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high‐throughput quantitative real‐time (qPCR)‐based method. The qPCR‐based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild‐type and a gene‐edited mutant. We showed that the qPCR‐based method can accurately distinguish CRISPR/Cas9‐induced mutants from the wild‐type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR‐based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T₀ transgenic plants. In a 384‐well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post‐polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T₀ transgenic plants, which will be widely used in the area of plant gene editing.     

PARALYTIC SHELLFISH TOXINS ARE RESTRICTED TO FEW SPECIES AMONG AUSTRALIA'S TAXONOMIC DIVERSITY OF CULTURED MICROALGAE1

There are at least 40,000 species of microalgae in the aquatic environment. Fifteen species of marine dinoflagellates and freshwater cyanobacteria are known to produce paralytic shellfish toxins (PSTs) and represent a threat to human and/or livestock health. Although known toxic species are regularly monitored, the wider cross‐section of microalgae has not been systematically tested for PSTs. Advances in rapid screening techniques have resulted in the development of highly sensitive and specific methods to detect PSTs, including the sodium channel and saxiphilin binding assays. These assays were used in this study in 96‐well formats to screen 234 highly diverse isolates of Australian freshwater and marine microalgae for PSTs. The screening assays detected five toxic species, representing one freshwater cyanobacterium (Anabaena circinalis Rabenhorst) and four species of marine dinoflagellates (Alexandrium minutum Halim, A. catenella Balech, A. tamarense Balech, and Gymnodinium catenatum Graham). Liquid chromatography‐fluorescence detection was used to identify 14 saxitoxin analogues across the five species, and each species exhibited distinct toxin profiles. These results indicate that PST production is restricted to a narrow range of microalgal species found in Australian waters.     

Caiman latirostris (broad-snouted caiman) as a sentinel organism for genotoxic monitoring: Basal values determination of micronucleus and comet assay

Caiman latirostris is one of the two crocodilian species that inhabit Argentina. In this country, as a consequence of agricultural frontiers expansion during the last years, many areas of the geographic distribution of the broad snouted caiman overlap with regions of intensive agricultural activity. Contaminants released to the environment may induce genetic alterations in wildlife, which could lead to mutations and/or carcinogenesis. Up to the moment, no studies had been made concerning the possibbility to apply biomarkers of genotoxic evaluation in C. latirostris.The aim of this study was to adapt two widely used genotoxic techniques, the comet assay and the micronucleus test, for their application in C. latirostris and to determine the baseline values in this species, in order to establish its suitability as a sentinel organism for future genotoxic monitoring of environmental pollutants.A total of 41 juvenile caimans of 4 months old (FMO) and 10 months old (TMO) were used. Genotoxic techniques were applied on peripheral blood erythrocytes introducing the necessary modifications required by the material, which are presented here.Our results show that baseline values of DNA damage are quite stable among juvenile caimans (MN: FMO animals 0.87 ± 0.74 and TMO animals 1.04 ± 0.92; DI: FMO animals 103.40 ± 3.36 and TMO animals 120.08 ± 11.33), being independent of the nest of origin, sex and size of the animals and confirm the potential value of both short term tests as accurate screening tools for the evaluation of genotoxic agents in C. latirostris. This is the first reference to the application of genotoxic techniques on C. latirostris and the second in crocodilians.Data provided here will be useful for future studies involving the biomonitoring of natural regions where C. latirostris occurs, employing this species as a sentinel organism for genotoxic assessment of environmental pollutants.     

Real-time PCR reveals a high incidence of <Emphasis Type="Italic">Symbiodinium </Emphasis>clade D at low levels in four scleractinian corals across the Great Barrier Reef: implications for symbiont shuffling

Reef corals form associations with an array of genetically and physiologically distinct endosymbionts from the genus Symbiodinium. Some corals harbor different clades of symbionts simultaneously, and over time the relative abundances of these clades may change through a process called symbiont shuffling. It is hypothesized that this process provides a mechanism for corals to respond to environmental threats such as global warming. However, only a minority of coral species have been found to harbor more than one symbiont clade simultaneously and the current view is that the potential for symbiont shuffling is limited. Using a newly developed real-time PCR assay, this paper demonstrates that previous studies have underestimated the presence of background symbionts because of the low sensitivity of the techniques used. The assay used here targets the multi-copy rDNA ITS1 region and is able to detect Symbiodinium clades C and D with >100-fold higher sensitivity compared to conventional techniques. Technical considerations relating to intragenomic variation, estimating copy number and non-symbiotic contamination are discussed. Eighty-two colonies from four common scleractinian species (Acropora millepora, Acropora tenuis, Stylophora pistillata and Turbinaria reniformis) and 11 locations on the Great Barrier Reef were tested for background Symbiodinium clades. Although these colonies had been previously identified as harboring only a single clade based on SSCP analyses, background clades were detected in 78% of the samples, indicating that the potential for symbiont shuffling may be much larger than currently thought.     

Biotechnology and the conservation of forest genetic resources: in vitro strategies and cryopreservation

In vitro techniques have a clear role within ex situ conservation strategies for trees and crop genetic resources, particularly where it is important to conserve specific genotypes or where normal propagules such as recalcitrant seed may not be suitable for long-term storage. These involve the use of conventional micropropagation, restricted growth techniques and cryopreservation. Although these techniques have been used primarily with herbaceous species, increasing attention is being given to woody species. Cryopreservation techniques for both woody and herbaceous species and new approaches which do not require freeze-induced cell dehydration, referred to as the encapsulation-dehydration and the vitrification techniques are described. Illustrative data are presented for the cryopreservation of willow using the encapsulation-dehydration technique.     

Recent developments inRhizobium taxonomy

Recent developments inRhizobium taxonomy are presented from a molecular and evolutionary point of view. Analyses of ribosomal RNA gene sequences provide a solid basis to infer phylogenies in the Rhizobiaceae family. These studies confirmed thatRhizobium andBradyrhizobium are only distantly related and showed thatRhizobium andBradyrhizobium are related to other groups of bacteria that are not plant symbionts.Rhizobium andAgrobacterium species are intermixed. Differences in plasmid content may explain to a good extent the different behavior ofRhizobium andAgrobacterium as symbionts or pathogens. Other approaches to identify and classify bacteria such as DNA-DNA hybridization, fatty acid analysis, RFLP and RPD-PCR techniques and phylogenies derived from other genes are in general agreement to the groupings derived by ribosomal sequences. Only a small proportion of nodulated legumes have been sampled for their symbionts and more knowledge is required on the systematics and taxonomy ofRhizobium andBradyrhizobium species.