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Abstract Background Despite a low rate of infant mortality, Aotearoa New Zealand has a high rate of Sudden Unexpected Death in Infants (SUDI), with disproportionate impact for Pacific infants. This study explored the infant care practices, factors and relationships associated with increased risk of SUDI amongst Tongan, Samoan, Cook Islands Māori, and Niuean mothers in New Zealand, to inform evidence-based interventions for reducing the incidence of SUDI for Pacific families and their children. Methods Analysis comprised of data collected in 2009–2010 from 1089 Samoan, Tongan, Cook Islands Māori and Niuean mothers enrolled in the Growing Up in New Zealand longitudinal cohort study. The sleeping environment (bed-sharing and sleep position) of the infants was assessed at 6 weeks. Multivariable logistic regression analysis were conducted, controlling for sociodemographic factors to explore the association between selected maternal and pregnancy support and environment factors and the sleeping environment for infants. Results Mothers who converse in languages other than English at home, and mothers who consulted alternative practitioners were less likely to follow guidelines for infant sleeping position. Similarly language, smoking, alcohol, household dwelling, crowding and access to a family doctor or GP were associated with mothers following guidelines for bed-sharing. Conclusion The impact of SUDI on Pacific infants may be lessened or prevented if communication about risk factors is more inclusive of diverse ethnic, cultural worldviews, and languages. Societal structural issues such as access to affordable housing is also important. This research suggests a need for more targeted or tailored interventions which promote safe sleeping and reduce rates of SUDI in a culturally respectful and meaningful way for Pasifika communities in Aotearoa, New Zealand.

Abstract Background The current biodiversity crisis underscores the urgent need for sustainable management of the human uses of nature. In the context of sustainability management, adopting the ecosystem service (ES) concept, i.e., the benefits humans obtain from nature, can support decisions aimed at benefiting both nature and people. However, marine ecosystems in particular endure numerous direct drivers of change (i.e., habitat loss and degradation, overexploitation, pollution, climate change, and introduction of non-indigenous species) all of which threaten ecosystem structure, functioning, and the provision of ES. Marine ecosystems have received less attention than terrestrial ecosystems in ES literature, and knowledge on marine ES is hindered by the highly heterogeneous scientific literature with regard to the different types of marine ecosystem, ES, and their correlates. Here, we constructed a systematic map of the existing literature to highlight knowledge clusters and knowledge gaps on how changes in marine ecosystems influence the provision of marine ES. Method We searched for all evidence documenting how changes in structure and functioning of marine ecosystems affect the delivery of ES in academic and grey literature sources. In addition to Scopus, Web of Science, and Google Scholar, we searched 6 online databases from intergovernmental agencies, supranational or national organizations, and NGOs. We screened English-language documents using predefined inclusion criteria on titles, abstracts, and then full texts, without any geographic or temporal limitations. All qualifying literature was coded and metadata were extracted. No formal validity appraisal was undertaken. We identified knowledge clusters and gaps in terms of which ecosystem types, biodiversity components, or ES types have been studied and how these categories are linked. Review findings Our searches identified 41 884 articles published since 1968 of which 12 140 were duplicates; 25 747 articles were excluded at the title-screening stage, then 2774 at the abstract stage. After full-text screening, a total of 653 articles—having met the eligibility criteria—were included in the final database, spanning from 1977 to July 2021. The number of studies was unevenly distributed across geographic boundaries, ecosystem types, ES, and types of pressure. The most studied ecosystems were pelagic ecosystems on continental shelves and intertidal ecosystems, and deep-sea habitats and ice-associated ecosystems were the least studied. Food provision was the major focus of ES articles across all types of marine ecosystem (67%), followed by climate regulation (28%), and recreation (14%). Biophysical values were assessed in 91% of the analysed articles, 30% assessed economic values, but only 3% assessed socio-cultural values. Regarding the type of impact on ecosystems, management effects were the most studied, followed by overexploitation and climate change (with increase in seawater temperature being the most commonly assessed climate change pressure). Lastly, the introduction of non-indigenous species and deoxygenation were the least studied. Conclusions This systematic map provides, in addition to a database, knowledge gaps and clusters on how marine ecosystem changes impact ES provision. The current lack of knowledge is a threat to the sustainability of human actions and knowledge-based nature conservation. The knowledge gaps and clusters highlighted here could guide future research and impact the beneficial development of policy and management practices.

Abstract Background Bread wheat is one of the world’s most important food crops and considerable efforts have been made to develop genomic resources for this species. This includes an on-going project by the International Wheat Genome Sequencing Consortium to assemble its large and complex genome, which is hexaploid and contains three closely related ‘homoeologous’ copies for each chromosome. This multi-national effort avoids the complications polyploidy entails for correct assembly of the genome by sequencing flow-sorted chromosome arms one at a time. Here we report on an alternate approach, a direct homoeolog-specific assembly of the expressed portion of the genome, the transcriptome. Results After assessment of the ability of various assemblers to generate homoeolog-specific assemblies, we employed a two-stage assembly process to produce a high-quality assembly of the transcriptome of hexaploid wheat from Roche-454 and Illumina GAIIx paired-end sequence reads. The assembly process made use of a rapid partitioning of expressed sequences into homoeologous clusters, followed by a parallel high-fidelity assembly of each cluster on a 1150-processor compute cloud. We assessed assembly quality through comparison to known wheat gene sequences and found that in ca. 98.5% of cases the assembly was sufficiently accurate for homoeologous triplets to be cleanly separated into either two or three separate contigs. Comparison to publicly available transcript collections suggests that the assembly covers ~75-80% of the complete transcriptome. Conclusions This work therefore describes the first homoeolog-specific sequence assembly of the wheat transcriptome and provides a reference transcriptome for future wheat research. Furthermore, our assembly methodology is transferable to other polyploid organisms.

Background Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. Result Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with 250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. Conclusion BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.

Abstract Background MicroRNAs are important components of the regulatory network of biological systems and thousands have been discovered in both animals and plants. Systematic investigations performed in species with sequenced genomes such as Arabidopsis, rice, poplar and Brachypodium have provided insights into the evolutionary relationships of this class of small RNAs among plants. However, miRNAs from barley, one of the most important cereal crops, remain unknown. Results We performed a large scale study of barley miRNAs through deep sequencing of small RNAs extracted from leaves of two barley cultivars. By using the presence of miRNA precursor sequences in related genomes as one of a number of supporting criteria, we identified up to 100 miRNAs in barley. Of these only 56 have orthologs in wheat, rice or Brachypodium that are known to be expressed, while up to 44 appear to be specifically expressed in barley. Conclusions Our study, the first large scale investigation of small RNAs in barley, has identified up to 100 miRNAs. We demonstrate that reliable identification of miRNAs via deep sequencing in a species whose genome has not been sequenced requires a more careful analysis of sequencing errors than is commonly performed. We devised a read filtering procedure for dealing with errors. In addition, we found that the use of a large dataset of almost 35 million reads permits the use of read abundance distributions along putative precursor sequences as a practical tool for isolating miRNAs in a large background of reads originating from other non-coding and coding RNAs. This study therefore provides a generic approach for discovering novel miRNAs where no genome sequence is available.

Abstract Background Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum), Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat. Results We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs. Conclusions The physical map reported here is the first physical map using fingerprinting of a complete Triticeae genome. This study demonstrates that global fingerprinting of the large plant genomes is a viable strategy for generating physical maps. Physical maps allow the description of the co-linearity between wheat and grass genomes and provide a powerful tool for positional cloning of new genes.

Abstract Background Barley and particularly wheat are two grass species of immense agricultural importance. In spite of polyploidization events within the latter, studies have shown that genotypically and phenotypically these species are very closely related and, indeed, fertile hybrids can be created by interbreeding. The advent of two genome-scale Affymetrix GeneChips now allows studies of the comparison of their transcriptomes. Results We have used the Wheat GeneChip to create a "gene expression atlas" for the wheat transcriptome (cv. Chinese Spring). For this, we chose mRNA from a range of tissues and developmental stages closely mirroring a comparable study carried out for barley (cv. Morex) using the Barley1 GeneChip. This, together with large-scale clustering of the probesets from the two GeneChips into "homologous groups", has allowed us to perform a genomic-scale comparative study of expression patterns in these two species. We explore the influence of the polyploidy of wheat on the results obtained with the Wheat GeneChip and quantify the correlation between conservation in gene sequence and gene expression in wheat and barley. In addition, we show how the conservation of expression patterns can be used to elucidate, probeset by probeset, the reliability of the Wheat GeneChip. Conclusion While there are many differences in expression on the level of individual genes and tissues, we demonstrate that the wheat and barley transcriptomes appear highly correlated. This finding is significant not only because given small evolutionary distance between the two species it is widely expected, but also because it demonstrates that it is possible to use the two GeneChips for comparative studies. This is the case even though their probeset composition reflects rather different design principles as well as, of course, the present incomplete knowledge of the gene content of the two species. We also show that, in general, the Wheat GeneChip is not able to distinguish contributions from individual homoeologs. Furthermore, the comparison between the two species leads us to conclude that the conservation of both gene sequence as well as gene expression is positively correlated with absolute expression levels, presumably reflecting increased selection pressure on genes coding for proteins present at high levels. In addition, the results indicate the presence of a correlation between sequence and expression conservation within the Triticeae.

Abstract Background Chromosome pairing, recombination and DNA repair are essential processes during meiosis in sexually reproducing organisms. Investigating the bread wheat (Triticum aestivum L.) Ph2 (Pairing homoeologous) locus has identified numerous candidate genes that may have a role in controlling such processes, including TaMSH7, a plant specific member of the DNA mismatch repair family. Results Sequencing of the three MSH7 genes, located on the short arms of wheat chromosomes 3A, 3B and 3D, has revealed no significant sequence divergence at the amino acid level suggesting conservation of function across the homoeogroups. Functional analysis of MSH7 through the use of RNAi loss-of-function transgenics was undertaken in diploid barley (Hordeum vulgare L.). Quantitative real-time PCR revealed several T0 lines with reduced MSH7 expression. Positive segregants from two T1 lines studied in detail showed reduced MSH7 expression when compared to transformed controls and null segregants. Expression of MSH6, another member of the mismatch repair family which is most closely related to the MSH7 gene, was not significantly reduced in these lines. In both T1 lines, reduced seed set in positive segregants was observed. Conclusion Results presented here indicate, for the first time, a distinct functional role for MSH7 in vivo and show that expression of this gene is necessary for wild-type levels of fertility. These observations suggest that MSH7 has an important function during meiosis and as such remains a candidate for Ph2.

Abstract Background Our understanding of the mechanisms that govern the cellular process of meiosis is limited in higher plants with polyploid genomes. Bread wheat is an allohexaploid that behaves as a diploid during meiosis. Chromosome pairing is restricted to homologous chromosomes despite the presence of homoeologues in the nucleus. The importance of wheat as a crop and the extensive use of wild wheat relatives in breeding programs has prompted many years of cytogenetic and genetic research to develop an understanding of the control of chromosome pairing and recombination. The rapid advance of biochemical and molecular information on meiosis in model organisms such as yeast provides new opportunities to investigate the molecular basis of chromosome pairing control in wheat. However, building the link between the model and wheat requires points of data contact. Results We report here a large-scale transcriptomics study using the Affymetrix wheat GeneChip® aimed at providing this link between wheat and model systems and at identifying early meiotic genes. Analysis of the microarray data identified 1,350 transcripts temporally-regulated during the early stages of meiosis. Expression profiles with annotated transcript functions including chromatin condensation, synaptonemal complex formation, recombination and fertility were identified. From the 1,350 transcripts, 30 displayed at least an eight-fold expression change between and including pre-meiosis and telophase II, with more than 50% of these having no similarities to known sequences in NCBI and TIGR databases. Conclusion This resource is now available to support research into the molecular basis of pairing and recombination control in the complex polyploid, wheat.

Abstract Background The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination. Unfortunately, such cDNA libraries prepared directly in yeast can not be used for the efficient recovery of purified plasmids and thus are incompatible with existing yeast one-hybrid systems, which use yeast transformation for the library screen. Results Here we propose an adaptation of the yeast one-hybrid system for identification and cloning of transcription factors using a MATCHMAKER cDNA library. The procedure is demonstrated using a cDNA library prepared from the liquid part of the multinucleate coenocyte of wheat endosperm. The method is a modification of a standard one-hybrid screening protocol, utilising a mating step to introduce the library construct and reporter construct into the same cell. Several novel full length transcription factors from the homeodomain, AP2 domain and E2F families of transcription factors were identified and isolated. Conclusion In this paper we propose a method to extend the compatibility of MATCHMAKER cDNA libraries from yeast two-hybrid screens to one-hybrid screens. The utility of the new yeast one-hybrid technology is demonstrated by the successful cloning from wheat of full-length cDNAs encoding several transcription factors from three different families.

Abstract Background Climate change and the current phase-out of fossil fuel-fired power generation are currently expanding the market of renewable energy and more especially photovoltaic (PV) panels. Contrary to other types of renewable energies, such as wind and hydroelectricity, evidence on the effects of PV panels on biodiversity has been building up only fairly recently. PV panels have been linked to substantial impacts on species and ecosystems, the first and most obvious one being the degradation of natural habitats but they may also lead to mortality of individuals and displacements of populations. Hence, we propose a systematic map aiming to draw a comprehensive panorama of the available knowledge on the effects of photovoltaic and solar thermal (PVST) installations, whatever their scales (i.e. cells, panels, arrays, utility-scale facilities), on terrestrial and semi-aquatic species and natural/semi-natural habitats and ecosystems. This work aims at providing decision-makers with a better understanding of the effects of PVST installations and, therefore, help them further protect biodiversity while also mitigating anthropogenic climate change. Methods We will follow the collaboration for environmental evidence guidelines and search for relevant peer-reviewed and grey literature in English or French. The search string will combine population (all wild terrestrial and semi-aquatic species—e.g. animals, plants, fungi, microorganisms—as well as natural/semi-natural terrestrial habitats and ecosystems) and exposure/intervention (all technologies of PVST panels at all scales of installations and therefore excluding concentrated solar power) terms. A pre-built test list of relevant articles will be used to assess the comprehensiveness of the search string. Extracted citations will be screened at title and full-text stages thanks to pre-defined inclusion/exclusion criteria. Accepted citations will then be split into studies and observations, from which relevant metadata (e.g. taxon, exposure/intervention, outcome) will be extracted and their internal validity assessed through a critical appraisal. The database will be accessible alongside a map report which will draw a landscape of eligible studies. By describing studied populations, exposures/interventions, outcomes and internal study validity results, the report will identify potential knowledge clusters and gaps regarding the effects of PVST installations on biodiversity and ecosystems.

Abstract Background The current biodiversity crisis calls for an urgent need to sustainably manage human uses of nature. The Ecosystem Services (ES) concept defined as « the benefits humans obtain from nature » support decisions aimed at promoting nature conservation. However, marine ecosystems, in particular, endure numerous direct pressures (e.g., habitat loss and degradation, overexploitation, pollution, climate change, and the introduction of non-indigenous species) all of which threaten ecosystem structure, functioning, and the very provision of ES. While marine ecosystems often receive less attention than terrestrial ecosystems in ES literature, it would also appear that there is a heterogeneity of knowledge within marine ecosystems and within the different ES provided. Hence, a systematic map on the existing literature will aim to highlight knowledge clusters and knowledge gaps on how changes in marine ecosystems influence the provision of marine ecosystem services. This will provide an evidence base for possible future reviews, and may help to inform eventual management and policy decision-making. Methods We will search for all evidence documenting how changes in structure and functioning of marine ecosystems affect the delivery of ES, across scientific and grey literature sources. Two bibliographic databases, Scopus and Web of Science Core Collection, will be used with a supplementary search undertaken in Google scholar. Multiple organisational websites related to intergovernmental agencies, supra-national or national structures, and NGOs will also be searched. Searches will be performed with English terms only without any geographic or temporal limitations. Literature screening, against predefined inclusion criteria, will be undertaken on title, abstract, and then full texts. All qualifying literature will be subjected to coding and meta-data extraction. No formal validity appraisal will be undertaken. Indeed, the map will highlight how marine ecosystem changes impact the ES provided. Knowledge gaps will be identified in terms of which ecosystem types, biodiversity components, or ES types are most or least studied and how these categories are correlated. Finally, a database will be provided, we will narratively describe this evidence base with summary figures and tables of pertinent study characteristics.

Abstract Background Ecosystem degradation, mainly through overexploitation and destruction of natural habitats, is a well-known threat to the viability and persistence of many species’ populations worldwide. The use of translocations as a viable conservation tool in conjunction with protected areas has been rapidly increasing over the last few decades. Protected areas such as strict nature reserves, national parks, and species management areas continue to be central tools for biodiversity conservation as they provide vital habitats set aside from various human pressures. Because action consistently runs ahead of policy, the need for a clearer evidence base on the outcomes of wildlife translocations undertaken at a global scale is becoming increasingly urgent for scientific and decision-making communities, in order to build clear strategy frameworks around conservation translocations. We therefore conducted a systematic mapping exercise to provide an overview of the existing evidence on the outcomes of wildlife translocations in protected areas. Methods We searched two bibliographic databases, four web-based search engines with search-by-key-words capacity, 5 specialist websites, and conducted a grey literature call through two project stakeholders. We screened articles by title, abstract, and full text using pre-defined inclusion criteria all the while assessing the consistency of the reviewers. All relevant translocations were coded from retained publications. Key variables of interest were extracted and coded for each translocation event. The quantity and characteristics of the available evidence and knowledge gaps/clusters are summarised. The distribution and frequency of translocations are presented in heat- and geographical maps. Review findings A total of 613 articles were considered eligible for coding bibliometric data. Metapopulation management and review articles were not coded for quantitative and qualitative variables. Linked data (duplicated translocations) were also excluded. Finally, 841 studies of different translocation events were fully coded from 498 articles. Most of these translocations were carried out in North America and Oceania. The most commonly undertaken intervention types were one-off supplementations and “supplemented reintroductions”. Mammals were by far the most transferred group among animals. Magnoliopsida was the most translocated plant group. Survival, space use, and demography metrics were the most studied outcomes on translocated species. Conclusions This systematic map provides an up-to-date global catalogue of the available evidence on wildlife translocations to, from, or within protected areas. It should enable protected area managers to better understand their role in the global network of protected areas, regarding translocation practice, both as suppliers or recipients of translocated species. It may help managers and practitioners make their own choices by comparing previous experiences, regarding both the species concerned and the precise translocation modalities (number of individuals, etc.). Finally, it constitutes a decision-making tool for managers as well as for policy makers for future translocations.

Abstract Background Chickpea is an important legume and is moderately tolerant to salinity stress during the growing season. However, the level and mechanisms for salinity tolerance can vary among accessions and cultivars. A large family of CaRab-GTP genes, previously identified in chickpea, is homologous to intracellular vesicle trafficking superfamily genes that play essential roles in response to salinity stress in plants. Results To determine which of the gene family members are involved in the chickpea salt response, plants from six selected chickpea accessions (Genesis 836, Hattrick, ICC12726, Rupali, Slasher and Yubileiny) were exposed to salinity stress and expression profiles resolved for the major CaRab-GTP gene clades after 5, 9 and 15 days of salt exposure. Gene clade expression profiles (using degenerate primers targeting all members of each clade) were tested for their relationship to salinity tolerance measures, namely plant biomass and Na+ accumulation. Transcripts representing 11 out of the 13 CaRab clades could be detected by RT-PCR, but only six (CaRabA2, −B, −C, −D, −E and −H) could be quantified using qRT-PCR due to low expression levels or poor amplification efficiency of the degenerate primers for clades containing several gene members. Expression profiles of three gene clades, CaRabB, −D and −E, were very similar across all six chickpea accessions, showing a strongly coordinated network. Salt-induced enhancement of CaRabA2 expression at 15 days showed a very strong positive correlation (R2 = 0.905) with Na+ accumulation in leaves. However, salinity tolerance estimated as relative plant biomass production compared to controls, did not correlate with Na+ accumulation in leaves, nor with expression profiles of any of the investigated CaRab-GTP genes. Conclusion A coordinated network of CaRab-GTP genes, which are likely involved in intracellular trafficking, are important for the salinity stress response of chickpea plants.

Abstract Background A family of genes designated as the Zinc finger A20/AN1 Transcription factors encoding stress-associated proteins (SAP) are well described in Arabidopsis and rice, and include 14 AtSAP and 18 OsSAP genes that are associated with variable tolerances to multiple abiotic stresses. The SAP gene family displays a great diversity in its structure and across different plant species. The aim of this study was to identify all HvSAP genes in barley (Hordeum vulgare L.), to analyse the expression of selected genes in response to salinity in barley leaves and develop SNP marker for HvSAP12 to evaluate the association between genotypes of barley plants and their grain yield in field trials. Results In our study, 17 HvSAP genes were identified in barley, which were strongly homologous to rice genes. Five genes, HvSAP5, HvSAP6, HvSAP11, HvSAP12 and HvSAP15, were found to be highly expressed in leaves of barley plants in response to salt stress in hydroponics compared to controls, using both semi-quantitative RT-PCR and qPCR analyses. The Amplifluor-like SNP marker KATU-B30 was developed and used for HvSAP12 genotyping. A strong association (R2 = 0.85) was found between KATU-B30 and grain yield production per plant of 50 F3 breeding lines originating from the cross Granal × Baisheshek in field trials with drought and low to moderate salinity in Northern and Central Kazakhstan. Conclusions A group of HvSAP genes, and HvSAP12 in particular, play an important role in the tolerance of barley plants to salinity and drought, and is associated with higher grain yield in field trials. Marker-assisted selection with SNP marker KATU-B30 can be applied in barley breeding to improve grain yield production under conditions of abiotic stress.

Abstract Background Conversion, fragmentation, and loss of natural habitats are among the main causes of declining species’ populations worldwide. Protected areas are therefore crucial for biodiversity as they provide refuge and ensure key ecological processes. Wildlife translocations, defined as “the deliberate movement of organisms from one site for release in another”, have been used in conjunction as a conservation tool for a number of decades as wild populations become increasingly fragmented and endangered. Not only are translocations used to bolster the viability of imperiled species but are also recommended for improving population resilience and adapting species’ ranges in response to climate change. Despite translocation being a recognised conservation tool, it remains complex with variable results due to the different factors that can determine its success. Accordingly, the Map will investigate the existing evidence on the links between different types of wildlife translocation interventions and factors that may be important to consider for planning. This will provide an overview of relevant studies for possible future syntheses, and may help to inform management decisions. Method We will perform a thorough search of peer-reviewed journal articles and grey literature sources documenting the occurrence of translocations in the context of protected areas. Two databases will be used: Web of science core collection and Scopus, with a supplementary search in Google Scholar. Multiple key specialized websites will also be used. All bibliographic data will be extracted, managed, and screened in Microsoft excel. Three screening stages will be undertaken (title, then abstract, then full texts) against predefined inclusion criteria. The retained relevant literature will be subjected to coding and meta-data extraction. No formal validity appraisal will be undertaken. The Map will particularly highlight translocation operations in terms of origin and destination (i.e. translocating from one protected area to another, within the same area, and from and to non-protected areas) by taxonomic group, among other important factors (e.g. number of individuals, age class, release strategy, distance between capture and release sites etc.). Finally, a database will be provided along with a Map narratively describing the evidence with summary figures and tables of pertinent study characteristics.

Abstract Background The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate. Results The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat. Conclusions We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.

Abstract Background The relatively low efficiency of biolistic transformation and subsequent integration of multiple copies of the introduced gene/s significantly complicate the genetic modification of wheat (Triticum aestivum) and other plant species. One of the key factors contributing to the reproducibility of this method is the uniformity of the DNA/gold suspension, which is dependent on the coating procedure employed. It was also shown recently that the relative frequency of single copy transgene inserts could be increased through the use of nanogram quantities of the DNA during coating. Results A simplified DNA/gold coating method was developed to produce fertile transgenic plants, via microprojectile bombardment of callus cultures induced from immature embryos. In this method, polyethyleneglycol (PEG) and magnesium salt solutions were utilized in place of the spermidine and calcium chloride of the standard coating method, to precipitate the DNA onto gold microparticles. The prepared microparticles were used to generate transgenics from callus cultures of commercial bread wheat cv. Gladius resulting in an average transformation frequency of 9.9%. To increase the occurrence of low transgene copy number events, nanogram amounts of the minimal expression cassettes containing the gene of interest and the hpt gene were used for co-transformation. A total of 1538 transgenic wheat events were generated from 15,496 embryos across 19 independent experiments. The variation of single copy insert frequencies ranged from 16.1 to 73.5% in the transgenic wheat plants, which compares favourably to published results. Conclusions The DNA/gold coating procedure presented here allows efficient, large scale transformation of wheat. The use of nanogram amounts of vector DNA improves the frequency of single copy transgene inserts in transgenic wheat plants.

Abstract Background KASP (KBioscience Competitive Allele Specific PCR) and Amplifluor (Amplification with fluorescence) SNP markers are two prominent technologies based upon a shared identical Allele-specific PCR platform. Methods Amplifluor-like SNP and KASP analysis was carried out using published and own design of Universal probes (UPs) and Gene-specific primers (GSPs). Results Advantages of the Amplifluor-like system over KASP include the significantly lower costs and much greater flexibility in the adjustment and development of ‘self-designed’ dual fluorescently-labelled UPs and regular GSPs. The presented results include optimisation of ‘tail’ length in UPs and GSPs, protocol adjustment, and the use of various fluorophores in different qPCR instruments. The compatibility of the KASP Master-mix in both original and Amplifluor-like systems has been demonstrated in the presented results, proving their similar principles. Results of SNP scoring with rare alleles in addition to more frequently occurring alleles are shown. Conclusions The Amplifluor-like system produces SNP genotyping results with a level of sensitivity and accuracy comparable to KASP but at a significantly cheaper cost and with much greater flexibility for UPs with self-designed GSPs.

Abstract Background Selecting for low concentration of Na+ in the shoot provides one approach for tackling salinity stress that adversely affects crop production. Novel alleles for Na+ exclusion can be identified and then introduced into elite crop cultivars. Results We have identified loci associated with lower Na+ concentration in leaves of durum wheat landraces originating from Afghanistan. Seedlings of two F2 populations derived from crossings between Australian durum wheat (Jandaroi) and two Afghani landraces (AUS-14740 and AUS-14752) were grown hydroponically and evaluated for Na+ and K+ concentration in the third leaf. High heritability was found for both third leaf Na+ concentration and the K+/Na+ ratio in both populations. Further work focussed on line AUS-14740. Bulk segregant analysis using 9 K SNP markers identified two loci significantly associated with third leaf Na+ concentration. Marker regression analysis showed a strong association between all traits studied and a favourable allele originating from AUS-14740 located on the long arm of chromosome 4B. Conclusions The candidate gene in the relevant region of chromosome 4B is likely to be the high affinity K+ transporter B1 (HKT1;5-B1). A second locus associated with third leaf Na+ concentration was located on chromosome 3BL, with the favourable allele originating from Jandaroi; however, no candidate gene can be identified.

Abstract Background The plant cuticle is the outermost layer covering aerial tissues and is composed of cutin and waxes. The cuticle plays an important role in protection from environmental stresses and glaucousness, the bluish-white colouration of plant surfaces associated with cuticular waxes, has been suggested as a contributing factor in crop drought tolerance. However, the cuticle structure and composition is complex and it is not clear which aspects are important in determining a role in drought tolerance. Therefore, we analysed residual transpiration rates, cuticle structure and epicuticular wax composition under well-watered conditions and drought in five Australian bread wheat genotypes, Kukri, Excalibur, Drysdale, RAC875 and Gladius, with contrasting glaucousness and drought tolerance. Results Significant differences were detected in residual transpiration rates between non-glaucous and drought-sensitive Kukri and four glaucous and drought-tolerant lines. No simple correlation was found between residual transpiration rates and the level of glaucousness among glaucous lines. Modest differences in the thickness of cuticle existed between the examined genotypes, while drought significantly increased thickness in Drysdale and RAC875. Wax composition analyses showed various amounts of C31 β-diketone among genotypes and increases in the content of alkanes under drought in all examined wheat lines. Conclusions The results provide new insights into the relationship between drought stress and the properties and structure of the wheat leaf cuticle. In particular, the data highlight the importance of the cuticle’s biochemical makeup, rather than a simple correlation with glaucousness or stomatal density, for water loss under limited water conditions.

Abstract Background Publicly insured women usually have a different demographic background to privately insured women, which is related to poor neonatal outcomes after birth. Given the difference in nature and risk of preterm versus term births, it would be important to compare adverse neonatal outcomes after preterm birth between these groups of women after eliminating the demographic differences between the groups. Methods The study population included 3085 publicly insured and 3380 privately insured, singleton, preterm deliveries (32–36 weeks gestation) from Western Australia during 1998–2008. From the study population, 1016 publicly insured women were matched with 1016 privately insured women according to the propensity score of maternal demographic characteristics and pre-existing medical conditions. Neonatal outcomes were compared in the propensity score matched cohorts using conditional log-binomial regression, adjusted for antenatal risk factors. Outcomes included Apgar scores less than 7 at five minutes after birth, time until establishment of unassisted breathing (>1 minute), neonatal resuscitation (endotracheal intubation or external cardiac massage) and admission to a neonatal special care unit. Results Compared with infants of privately insured women, infants of publicly insured women were more likely to receive a low Apgar score (ARR = 2.63, 95% CI = 1.06-6.52) and take longer to establish unassisted breathing (ARR = 1.61, 95% CI = 1.25-2.07), yet, they were less likely to be admitted to a special care unit (ARR = 0.84, 95% CI = 0.80-0.87). No significant differences were evident in neonatal resuscitation between the groups (ARR = 1.20, 95% CI = 0.54-2.67). Conclusions The underlying reasons for the lower rate of special care admissions in infants of publicly insured women compared with privately insured women despite the higher rate of low Apgar scores is yet to be determined. Future research is warranted in order to clarify the meaning of our findings for future obstetric care and whether more equitable use of paediatric services should be recommended.

Abstract Background Transgenic chloroplasts are potential bioreactors for recombinant protein production, especially for achievement of high levels of protein expression and proper folding. Production of therapeutic proteins in leaves provides transgene containment by elimination of reproductive structures. Therefore, in this study, human Insulin like Growth Factor-1 is expressed in transgenic chloroplasts for evaluation of structural identity and function. Results Expression of the synthetic Insulin like Growth Factor 1 gene (IGF-1s, 60% AT) was observed in transformed E. coli. However, no native IGF-1 gene (IGF-1n, 41% AT) product was detected in the western blots in E. coli. Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR. Southern blot analysis confirmed that the transgenic lines were homoplasmic. The transgenic plant lines had IGF-1s expression levels of 11.3% of total soluble protein (TSP). The IGF-1n plants contained 9.5% TSP as IGF-1n, suggesting that the chloroplast translation machinery is more flexible than E. coli in codon preference and usage. The expression of IGF-1 was increased up to 32% TSP under continuous illumination by the chloroplast light regulatory elements. IgG-Sepharose affinity column chromatographic separation of Z domain containing chloroplast derived IGF-1 protein, single and two dimensional electrophoresis methods and mass spectrometer analysis confirmed the identity of human IGF-1 in transgenic chloroplasts. Two spots analyzed from 2-D focusing/phoresis acrylamide gel showed the correct amino acid sequence of human IGF-1 and the S. aureus Z-tag. Cell proliferation assays in human HU-3 cells demonstrated the biological activity of chloroplast derived IGF-1 even in the presence of the S. aureus Z tag. Conclusion This study demonstrates that the human Insulin like Growth Factor-1 expressed in transgenic chloroplasts is identical to the native protein and is fully functional. The ability to use plant chloroplasts as bioreactors to generate proteins of great economic value that retain their biological activity is an exciting and achievable goal that appears to be within our grasp.

Abstract Background Of the > 2000 serovars of Salmonella enterica subspecies I, most cause self-limiting gastrointestinal disease in a wide range of mammalian hosts. However, S. enterica serovars Typhi and Paratyphi A are restricted to the human host and cause the similar systemic diseases typhoid and paratyphoid fever. Genome sequence similarity between Paratyphi A and Typhi has been attributed to convergent evolution via relatively recent recombination of a quarter of their genomes. The accumulation of pseudogenes is a key feature of these and other host-adapted pathogens, and overlapping pseudogene complements are evident in Paratyphi A and Typhi. Results We report the 4.5 Mbp genome of a clinical isolate of Paratyphi A, strain AKU_12601, completely sequenced using capillary techniques and subsequently checked using Illumina/Solexa resequencing. Comparison with the published genome of Paratyphi A ATCC9150 revealed the two are collinear and highly similar, with 188 single nucleotide polymorphisms and 39 insertions/deletions. A comparative analysis of pseudogene complements of these and two finished Typhi genomes (CT18, Ty2) identified several pseudogenes that had been overlooked in prior genome annotations of one or both serovars, and identified 66 pseudogenes shared between serovars. By determining whether each shared and serovar-specific pseudogene had been recombined between Paratyphi A and Typhi, we found evidence that most pseudogenes have accumulated after the recombination between serovars. We also divided pseudogenes into relative-time groups: ancestral pseudogenes inherited from a common ancestor, pseudogenes recombined between serovars which likely arose between initial divergence and later recombination, serovar-specific pseudogenes arising after recombination but prior to the last evolutionary bottlenecks in each population, and more recent strain-specific pseudogenes. Conclusion Recombination and pseudogene-formation have been important mechanisms of genetic convergence between Paratyphi A and Typhi, with most pseudogenes arising independently after extensive recombination between the serovars. The recombination events, along with divergence of and within each serovar, provide a relative time scale for pseudogene-forming mutations, affording rare insights into the progression of functional gene loss associated with host adaptation in Salmonella.