Southern marsh deer (Blastocerus dichotomus) populations assessed using Amplicon Sequencing on fecal samples.

Populations in isolated and small fragments lose genetic variability very fast and are usually of conservation concern because they are at greater risk of local extinction. The largest native deer in South America, Blastocerus dichotomus (Illiger, 1815), is a Vulnerable species according to the IUCN categorization, which inhabits tropical and subtropical swampy areas. In Argentina, its presence has been restricted to four isolated fragments. Here we examine the genetic diversity and differentiation among three of them, including the three different patches that form the southernmost population, using 18 microsatellite markers genotyped by Amplicon Sequencing of DNA extracted from fecal samples. Genetic diversity was low (HE < 0.45) in all three populations studied. We found three genetic clusters compatible with the geographic location of the samples. We also found a metapopulation dynamics that involves the patches that make up the southernmost population, with evidence of a barrier to gene flow between two of them. Our results point to the creation of a corridor as a necessary and urgent management action. This is the first study, at the population level, employing microsatellite genotyping by Amplicon Sequencing with non-invasive samples in an endangered species.

Optical Genome Mapping for Applications in Repeat Expansion Disorders.

Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System Basic Protocol 3: Manual De Novo Assembly workflow Basic Protocol 4: Local guided assembly workflow Basic Protocol 5: EnFocus Fragile X workflow Basic Protocol 6: Molecule distance script workflow.

Development of microsatellite markers for the soft tick Ornithodoros phacochoerus.

BACKGROUND: Soft ticks of the genus Ornithodoros are responsible for the maintenance and transmission of the African swine fever (ASF) virus in the sylvatic and domestic viral cycles in Southern Africa. They are also the main vectors of the Borrelia species causing relapsing fevers. Currently, no genetic markers are available for Afrotropical Ornithodoros ticks. As ASF spreads globally, such markers are needed to assess the role of ticks in the emergence of new outbreaks. The aim of this study is to design microsatellite markers that could be used for ticks of the Ornithodoros moubata complex, particularly Ornithodoros phacochoerus, to assess population structure and tick movements in ASF endemic areas. METHODS: A total of 151 markers were designed using the O. moubata and O. porcinus genomes after elimination of repeated sequences in the genomes. All designed markers were tested on O. phacochoerus and O. porcinus DNA to select the best markers. RESULTS: A total of 24 microsatellite markers were genotyped on two populations of O. phacochoerus and on individuals from four other Ornithodoros species. Nineteen markers were selected to be as robust as possible for population genetic studies on O. phacochoerus. CONCLUSIONS: The microsatellite markers developed here represent the first genetic tool to study nidicolous populations of O. phacochoerus.

Genetic and phenotypic diversification in a widespread fish, the Sailfin Molly (Poecilia latipinna).

Widespread species often experience significant environmental clines over the area they naturally occupy. We investigated a widespread livebearing fish, the Sailfin molly (Poecilia latipinna) combining genetic, life-history, and environmental data, asking how structured populations are. Sailfin mollies can be found in coastal freshwater and brackish habitats from roughly Tampico, Veracruz in Mexico to Wilmington, North Carolina, in the USA. In addition, they are found inland on the Florida peninsula. Using microsatellite DNA, we genotyped 168 individuals from 18 populations covering most of the natural range of the Sailfin molly. We further determined standard life-history parameters for both males and females for these populations. Finally, we measured biotic and abiotic parameters in the field. We found six distinct genetic clusters based on microsatellite data, with very strong indication of isolation by distance. However, we also found significant numbers of migrants between adjacent populations. Despite genetic structuring we did not find evidence of cryptic speciation. The genetic clusters and the migration patterns do not match paleodrainages. Life histories vary between populations but not in a way that is easy to interpret. We suggest a role of humans in migration in the sailfin molly, for example in the form of a ship channel that connects southern Texas with Louisiana which might be a conduit for fish migration.

Clonal growth characteristics and diversity patterns of different Clintonia udensis (Liliaceae) diploid and tetraploid cytotypes in the Hualongshan Mountains.

Polyploidization plays an important role in plant evolution and biodiversity. However, intraspecific polyploidy compared to interspecific polyploidy received less attention. Clintonia udensis (Liliaceae) possess diploid (2n = 2x = 14) and autotetraploid (2n = 4x = 28) cytotypes. In the Hualongshan Mountains, the autotetraploids grew on the northern slope, while the diploids grew on the southern slopes. The clonal growth characteristics and clonal architecture were measured and analyzed by field observations and morphological methods. The diversity level and differentiation patterns for two different cytotypes were investigated using SSR markers. The results showed that the clonal growth parameters, such as the bud numbers of each rhizome node and the ratio of rhizome branches in the autotetraploids were higher than those in the diploids. Both the diploids and autotetraploids appeared phalanx clonal architectures with short internodes between ramets. However, the ramets or genets of the diploids had a relatively scattered distribution, while those of the autotetraploids were relatively clumping. The diploids and autotetraploids all allocated more biomass to their vegetative growth. The diploids had a higher allocation to reproductive organs than that of autotetraploids, which indicated that the tetraploids invested more resources in clonal reproduction than diploids. The clone diversity and genetic diversity of the autotetraploids were higher than that of the diploids. Significant genetic differentiation between two different cytotypes was observed (P < 0.01). During establishment and evolution, C. udensis autotetraploids employed more clumping phalanx clonal architecture and exhibited more genetic variation than the diploids.

Genetic analyses and dispersal patterns unveil the Amazonian origin of guava domestication.

Guava (Psidium guajava L.) is a semi-domesticated fruit tree of moderate importance in the Neotropics, utilized for millennia due to its nutritional and medicinal benefits, but its origin of domestication remains unknown. In this study, we examine genetic diversity and population structure in 215 plants from 11 countries in Mesoamerica, the Andes, and Amazonia using 25 nuclear microsatellite loci to propose an origin of domestication. Genetic analyses reveal one gene pool in Mesoamerica (Mexico) and four in South America (Brazilian Amazonia, Peruvian Amazonia and Andes, and Colombia), indicating greater differentiation among localities, possibly due to isolation between guava populations, particularly in the Amazonian and Andean regions. Moreover, Mesoamerican populations show high genetic diversity, with moderate genetic structure due to gene flow from northern South American populations. Dispersal scenarios suggest that Brazilian Amazonia is the probable origin of guava domestication, spreading from there to the Peruvian Andes, northern South America, Central America, and Mexico. These findings present the first evidence of guava domestication in the Americas, contributing to a deeper understanding of its evolutionary history.

The origin and insecticide resistance of Aedes albopictus mosquitoes established in southern Mozambique.

BACKGROUND: The Aedes albopictus mosquito is of medical concern due to its ability to transmit viral diseases, such as dengue and chikungunya. Aedes albopictus originated in Asia and is now present on all continents, with the exception of Antarctica. In Mozambique, Ae. albopictus was first reported in 2015 within the capital city of Maputo, and by 2019, it had become established in the surrounding area. It was suspected that the mosquito population originated in Madagascar or islands of the Western Indian Ocean (IWIO). The aim of this study was to determine its origin. Given the risk of spreading insecticide resistance, we also examined relevant mutations in the voltage-sensitive sodium channel (VSSC). METHODS: Eggs of Ae. albopictus were collected in Matola-Rio, a municipality adjacent to Maputo, and reared to adults in the laboratory. Cytochrome c oxidase subunit I (COI) sequences and microsatellite loci were analyzed to estimate origins. The presence of knockdown resistance (kdr) mutations within domain II and III of the VSSC were examined using Sanger sequencing. RESULTS: The COI network analysis denied the hypothesis that the Ae. albopictus population originated in Madagascar or IWIO; rather both the COI network and microsatellites analyses showed that the population was genetically similar to those in continental Southeast Asia and Hangzhou, China. Sanger sequencing determined the presence of the F1534C knockdown mutation, which is widely distributed among Asian populations, with a high allele frequency (46%). CONCLUSIONS: These results do not support the hypothesis that the Mozambique Ae. albopictus population originated in Madagascar or IWIO. Instead, they suggest that the origin is continental Southeast Asia or a coastal town in China.

Population dynamics and genetic isolation in the Central Himalayan region: insights from Sikkim population, India.

India's rich diversity encompasses individuals from varied geographical, cultural, and ethnic backgrounds. In the field of population genetics, comprehending the genetic diversity across distinct populations plays a crucial role. This study presents significant findings from genetic data obtained from the Sikkimese population of India. Autosomal markers were crucial for evaluating forensic parameters, with a combined paternity index of 1 × 109. Notably, Penta E emerged as a distinguishing marker for individual identification in the Sikkim population. Fst genetic distance values revealed insights into genetic isolation among different groups, enhancing our understanding of population dynamics in the central Himalayan region. The NJ-based phylogenetic tree highlighted close genetic relationships, of the Sikkim population with the Nepalese population surrounding neighbouring Himalayan populations providing glimpses into common ancestry. In summary, this study contributes valuable data to population genetics and underscores the importance of genetic variation in comprehending population dynamics and forensic applications.

Validation of the Utility of the Genetically Shared Regions of Chromosomes (GD-ICS) Measuring Method in Identifying Complicated Genetic Relatedness.

BACKGROUND: Relatives share more genomic regions than unrelated individuals, with closer relatives sharing more regions. This concept, paired with the increased availability of high-throughput single nucleotide polymorphism (SNP) genotyping technologies, has made it feasible to measure the shared chromosomal regions between individuals to assess their level of relation to each other. However, such techniques have remained in the conceptual rather than practical stages in terms of applying measures or indices. Recently, we developed an index called "genetic distance-based index of chromosomal sharing (GD-ICS)" utilizing large-scale SNP data from Korean family samples and demonstrated its potential for practical applications in kinship determination. In the current study, we present validation results from various real cases demonstrating the utility of this method in resolving complex familial relationships where information obtained from traditional short tandem repeats (STRs) or lineage markers is inconclusive. METHODS: We obtained large-scale SNP data through microarray analysis from Korean individuals involving 13 kinship cases and calculated GD-ICS values using the method described in our previous study. Based on the GD-ICS reference constructed for Korean families, each disputed kinship was evaluated and validated using a combination of traditional STRs and lineage markers. RESULTS: The cases comprised those A) that were found to be inconclusive using the traditional approach, B) for which it was difficult to apply traditional testing methods, and C) that were more conclusively resolved using the GD-ICS method. This method has overcome the limitations faced by traditional STRs in kinship testing, particularly in a paternity case with STR mutational events and in confirming distant kinship where the individual of interest is unavailable for testing. It has also been demonstrated to be effective in identifying various relationships without specific presumptions and in confirming a lack of genetic relatedness between individuals. CONCLUSION: This method has been proven effective in identifying familial relationships across diverse complex and practical scenarios. It is not only useful when traditional testing methods fail to provide conclusive results, but it also enhances the resolution of challenging kinship cases, which suggests its applicability in various types of practical casework.

Spontaneous single-nucleotide substitutions and microsatellite mutations have distinct distributions of fitness effects.

The fitness effects of new mutations determine key properties of evolutionary processes. Beneficial mutations drive evolution, yet selection is also shaped by the frequency of small-effect deleterious mutations, whose combined effect can burden otherwise adaptive lineages and alter evolutionary trajectories and outcomes in clonally evolving organisms such as viruses, microbes, and tumors. The small effect sizes of these important mutations have made accurate measurements of their rates difficult. In microbes, assessing the effect of mutations on growth can be especially instructive, as this complex phenotype is closely linked to fitness in clonally evolving organisms. Here, we perform high-throughput time-lapse microscopy on cells from mutation-accumulation strains to precisely infer the distribution of mutational effects on growth rate in the budding yeast, Saccharomyces cerevisiae. We show that mutational effects on growth rate are overwhelmingly negative, highly skewed towards very small effect sizes, and frequent enough to suggest that deleterious hitchhikers may impose a significant burden on evolving lineages. By using lines that accumulated mutations in either wild-type or slippage repair-defective backgrounds, we further disentangle the effects of 2 common types of mutations, single-nucleotide substitutions and simple sequence repeat indels, and show that they have distinct effects on yeast growth rate. Although the average effect of a simple sequence repeat mutation is very small (approximately 0.3%), many do alter growth rate, implying that this class of frequent mutations has an important evolutionary impact.

Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

Genetic Identification of Medicinal Citrus Cultivar 'Local Juhong' Using Molecular Markers and Genomics.

The citrus cultivar 'Local Juhong', which has historically been used as a traditional Chinese medicinal material, originated in Yuanjiang County, Hunan Province.Its parental type and genetic background are indistinct as of yet. Morphological observation shows that 'Local Juhong' has a slight oblateness in fruit shape, a relatively smooth pericarp, a fine and slightly raised oil vacuole, and an inward concave at the blossom end. The tree form and fruit and leaf morphology of 'Local Juhong' are similar to those of 'Huangpi' sour orange. To reveal the genetic background of 'Local Juhong', 21 citrus accessions were evaluated using nuclear and chloroplast SSR markers and whole-genome SNP information. 'Local Juhong' was grouped with mandarins and sub-grouped with 'Miyagawa Wase' and 'Yanxi Wanlu' in a nuclear SSR analysis, which indicated that its pollen parent might be mandarins. It was closely clustered with orange and pummelo in the chloroplast SSR analysis. The genomic sequence similarity rate of 'Local Juhong' with mandarin and pummelo heterozygosity was 70.88%; the main part was the heterozygosity, except for the unknown (19.66%), mandarin (8.73%), and pummelo (3.9%) parts. Thus, 'Local Juhong' may be an F1 hybrid with pummelo as the female parent and mandarin as the male parent, sharing sisterhood with 'Huangpi' sour orange.

Genetic Differentiation of Reed Canarygrass (Phalaris arundinacea L.) within Eastern Europe and Eurasia.

Worldwide molecular research of economically important Phalaris arundinacea (Poaceae) is mainly focused on the invasions of this species from Europe to North America. Until the present study, the genetic diversity of the P. arundinacea had not been studied across the Baltic countries. The objective of this research is to evaluate the diversity of Lithuanian populations of P. arundinacea at simple sequence repeat (SSR) loci comparatively among populations of the Baltic countries, Luxembourg, and the Russian Far East (Eurasian), evaluating differentiation between Lithuanian populations and ornamental accessions, and relating these with environmental features. For six selected Lithuanian river basin populations, GBS low density SNPs were used to determine genetic diversity. Bayesian analysis showed that Eurasian populations of Phalaris arundinacea consist of two gene clusters. Statistically significant genetic differentiation among European and Eurasian populations was documented. Lithuanian genotypes growing naturally along rivers are genetically distinct from cultivated ornamentals. GBS-SNPs divided the six selected Nemunas river basins into three distinct groups with one, two, or three rivers in separate groupings for genetic diversity. Genetic diversity is primarily within, rather than among, Lithuanian, eastern European, and Eurasian populations of P. arundinacea across the continent. Thus, restoration efforts would benefit from local population seed origination.

The Usefulness of qPCR Data for Sample Pre-Assessment and Interpretation of Genetic Typing Results.

DNA quantification is a crucial step in the STR typing workflow for human identification purposes. Given the reaction's nature, qPCR assays may be subjected to the same stochastic effects of traditional PCR for low-input concentrations. The study aims to evaluate the precision of the PowerQuant® (Promega) kit assay measurements and the degree of variability for DNA templates falling below the optimal threshold of the PowerPlex® ESX-17 Fast STR typing kit (Promega). Five three-fold dilutions of the 2800 M control DNA (Promega) were set up. Each dilution (concentrations: 0.05, 0.0167, 0.0055, 0.00185, and 0.000617 ng/µL) was quantified and amplified in four replicates. Variability for qPCR results, STR profile completeness, and EPGs' peak height were evaluated. The qPCR-estimated concentration of casework samples was correlated with profile completeness and peak intensity, to assess the predictive value of qPCR results for the successful STR typing of scarce samples. qPCR was subjected to stochastic effects, of which the degree was inversely proportional to the initial input template. Quantitation results and the STR profile's characteristics were strongly correlated. Due to the intrinsic nature of real casework samples, a qPCR-derived DNA concentration threshold for correctly identifying probative STR profiles may be difficult to establish. Quantitation data may be useful in interpreting and corroborating STR typing results and for clearly illustrating them to the stakeholders.

An Assessment of the Performance Limitations of the Integrated QuantifilerTM Trio-HRM Assay: A Forensic Tool Designed to Identify Mixtures at the Quantification Stage.

Although guidelines exist for identifying mixtures, these measures often occur at the end-point of analysis and are protracted. To facilitate early mixture detection, we integrated a high-resolution melt (HRM) mixture screening assay into the qPCR step of the forensic workflow, producing the integrated QuantifilerTM Trio-HRM assay. The assay, when coupled with a prediction tool, allowed for 75.0% accurate identification of the contributor status of a sample (single source vs. mixture). To elucidate the limitations of the developed qPCR-HRM assay, developmental validation studies were conducted assessing the reproducibility and samples with varying DNA ratios, contributors, and quality. From this work, it was determined that the integrated QuantifilerTM Trio-HRM assay is capable of accurately identifying mixtures with up to five contributors and mixtures at ratios up to 1:100. Further, the optimal performance concentration range was found to be between 0.025 and 0.5 ng/µL. With these results, evidentiary-like DNA samples were then analyzed, resulting in 100.0% of the mixture samples being accurately identified; furthermore, every time a sample was predicted as a single source, it was true, giving confidence to any single-source calls. Overall, the integrated QuantifilerTM Trio-HRM assay has exhibited an enhanced ability to discern mixture samples from single-source samples at the qPCR stage under commonly observed conditions regardless of the contributor's sex.

Characterization and Comparative Analysis of Complete Chloroplast Genomes of Four Bromus (Poaceae, Bromeae) Species.

Bromus (Poaceae Bromeae) is a forage grass with high adaptability and ecological and economic value. Here, we sequenced Bromus ciliatus, Bromus benekenii, Bromus riparius, and Bromus rubens chloroplast genomes and compared them with four previously described species. The genome sizes of Bromus species ranged from 136,934 bp (Bromus vulgaris) to 137,189 bp (Bromus ciliates, Bromus biebersteinii), with a typical quadripartite structure. The studied species had 129 genes, consisting of 83 protein-coding, 38 tRNA-coding, and 8 rRNA-coding genes. The highest GC content was found in the inverted repeat (IR) region (43.85-44.15%), followed by the large single-copy (LSC) region (36.25-36.65%) and the small single-copy (SSC) region (32.21-32.46%). There were 33 high-frequency codons, with those ending in A/U accounting for 90.91%. A total of 350 simple sequence repeats (SSRs) were identified, with single-nucleotide repeats being the most common (61.43%). A total of 228 forward and 141 palindromic repeats were identified. No reverse or complementary repeats were detected. The sequence identities of all sequences were very similar, especially with respect to the protein-coding and inverted repeat regions. Seven highly variable regions were detected, which could be used for molecular marker development. The constructed phylogenetic tree indicates that Bromus is a monophyletic taxon closely related to Triticum. This comparative analysis of the chloroplast genome of Bromus provides a scientific basis for species identification and phylogenetic studies.

Genetic diversity, chemical constituents and anatomical analysis of eight popular Olive (Olea europaea L.) cultivars in Al-Jouf region, Saudi Arabia.

In light of the multitude of olive trees cultivated and the lack of the genetic diversity of available genotypes to select varieties and lines that are characterized by high diversity and better performance under the corresponding conditions, A comparison analysis of the genotyping and morphological characteristics of eight olive cultivars growing in Saudi Arabia's Al-Jouf region was conducted and analyzed. Morpho-anatomical and chemical characteristics along with both inter-simple-sequence repeats (ISSRs) and start-codon-targeted (SCoT) markers were used to evaluate the genetic diversity among eight olive varieties in Al-Jouf, Saudi Arabia. Analyses of 27 morphological, chemical, and anatomical characteristics concluded the existence of genetic differences among the studied varieties. Moreover, six ISSR and eight SCoT primer combinations produced a total of 48 loci, of which 18 (10 ISSR and 8 SCoT) were polymorphic. The average polymorphism information content (PIC values of 0.48 and 0.44, respectively) and marker index (MI of 0.79 and 0.48, respectively) detected for ISSR and SCoT markers revealed the prevalence of high genetic diversity among the studied olive varieties. Based on chemical and anatomical characteristics and the selected molecular markers, the eight olive cultivars were grouped into two distinct clusters. Clusters in the adjacent joint dendrogram produced using ISSR, SCoT and combined data were similar, and grouped all individuals into two groups. However, the dendrogram generated on the basis of SCoT separated individuals into subgroups containing at least two varieties. The findings showed that both methods were effective in assessing diversity, and that SCoT markers can be used as a reliable and informative method for assessing genetic diversity and relationships among olive varieties and can serve as a complementary tool to provide a more complete understanding of the genetic diversity available in Olea europaea populations in Saudi Arabia.

Genetic variability of the endangered fish lake minnow (Eupallasella percnurus) in populations newly established by translocation and those existing long term in Poland.

The lake minnow Eupallasella percnurus is a small leuciscid fish. In Poland, this species has been in a continuous decline since the mid-20th century and is presently considered as a extremely endangered. According to Polish law, E. percnurus is a strictly protected species that requires active conservation measures. In Poland, one the most common and effective measure of active protection E. percnurus is initiation of new populations. For this purpose, in 2004-2012, juvenile individuals originating from aquaculture conditions were translocated to group of isolated water bodies not inhabited by this species. The juveniles were offspring of parental fish belonging to the same local population, which is extinct at present. Five of those attempts were successful. The aim of the present study was to assess the genetic variation in a group new populations and compare genetic variation indicators with 13 old populations that had existed for decades. The polymorphism of 13 microsatellite markers was investigated, significance of differences in the genetic variation indicators between the groups were tested using a one-way analysis of variance (ANOVA). The mean values of all summary statistics under study, i.e. observed heterozygosity, expected heterozygosity and the total number of alleles, were higher in the group of new populations compared to almost all old ones. A similar dependence was found for Garza-Williamson M values, where the mean for the group of new populations was higher than in almost all old populations. Our results indicate that all recently established E. percnurus populations have not yet experienced any extensive founder effects or bottlenecks. They have preserved a large part of the genetic variability typical of their maternal population, which might also have been relatively high. This feature of new populations, may give them a relatively high ability to adapt to changing environments in the future.

Genetic tracing of the illegal trade of the white-bellied pangolin (Phataginus tricuspis) in western Central Africa.

The white-bellied pangolin is subject to intense trafficking, feeding both local and international trade networks. In order to assess its population genetics and trace its domestic trade, we genotyped 562 pangolins from local to large bushmeat markets in western central Africa. We show that the two lineages described from the study region (WCA and Gab) were overlapping in ranges, with limited introgression in southern Cameroon. There was a lack of genetic differentiation across WCA and a significant signature of isolation-by-distance possibly due to unsuspected dispersal capacities involving a Wahlund effect. We detected a c. 74.1-82.5% decline in the effective population size of WCA during the Middle Holocene. Private allele frequency tracing approach indicated up to 600 km sourcing distance by large urban markets from Cameroon, including Equatorial Guinea. The 20 species-specific microsatellite loci provided individual-level genotyping resolution and should be considered as valuable resources for future forensic applications. Because admixture was detected between lineages, we recommend a multi-locus approach for tracing the pangolin trade. The Yaoundé market was the main hub of the trade in the region, and thus should receive specific monitoring to mitigate pangolins' domestic trafficking. Our study also highlighted the weak implementation of CITES regulations at European borders.

High-yield hybrid breeding of Camellia oleifolia based on ISSR molecular markers.

BACKGROUND: C. Oleifera is among the world's largest four woody plants known for their edible oil production, yet the contribution rate of improved varieties is less than 20%. The species traditional breeding is lengthy cycle (20-30 years), occupation of land resources, high labor cost, and low accuracy and efficiency, which can be enhanced by molecular marker-assisted selection. However, the lack of high-quality molecular markers hinders the species genetic analysis and molecular breeding. RESULTS: Through quantitative traits characterization, genetic diversity assessment, and association studies, we generated a selection population with wide genetic diversity, and identified five excellent high-yield parental combinations associated with four reliable high-yield ISSR markers. Early selection criteria were determined based on kernel fresh weight and cultivated 1-year seedling height, aided by the identification of these 4 ISSR markers. Specific assignment of selected individuals as paternal and maternal parents was made to capitalize on their unique attributes. CONCLUSIONS: Our results indicated that molecular markers-assisted breeding can effectively shorten, enhance selection accuracy and efficiency and facilitate the development of a new breeding system for C. oleifera.