Species-specific eDNA assay development for enhanced box jellyfish risk management in coastal environments.

Human interaction with marine creatures holds both positive and negative dimensions. Coastal communities benefit from marine environments, relying on them for sustenance and livelihoods. Fishing activities support economies, and marine biodiversity contributes to overall ecosystem health. However, challenges like overfishing, habitat destruction, and pollution pose threats to both marine life and human communities. Recently, there has been widespread concern regarding the potential increase in jellyfish populations across global marine ecosystems, attributed mainly to environmental factors such as climate drivers and anthropogenic forces, or their complex interactions. Encounters with hazardous marine species, such as box jellyfish, exemplify the dangers associated with coastal activities. Unintended interactions may lead to stings, injuries, and even fatalities, necessitating proactive measures and advanced technologies. This study addresses the inadequacies of existing measures in preventing box jellyfish incidents by introducing environmental DNA (eDNA) assays for detecting the deadly Chiropsoides buitendijki and focuses on developing qPCR and dPCR-based eDNA assays. Emphasising prevention over treatment, the study establishes a proactive system to assess C. buitendijki distribution across 63 tourist beaches in the Gulf of Thailand. Comparative analysis highlights the superior performance of dPCR over qPCR and traditional surveys. The dPCR experiment yielded positive results for all eDNA samples collected at sites where C. buitendijki had previously been identified. Remarkably, the eDNA testing also detected positive results in 16 additional sample locations where no physical specimens were collected, despite reported jellyfish stings at some of these sites. These findings underscore the precision and efficacy of the proposed eDNA detection technology in the early detection and assessment of box jellyfish distribution. This advancement therefore not only aids ecological research but also serves as a valuable tool for safeguarding public health, providing an early warning system for potential jellyfish encounters. Balancing positive human-marine interactions with effective risk mitigation strategies is crucial for sustainable coexistence, the preservation of marine ecosystems, and human well-being.

A comparative study on eDNA-based detection of Siamese bat catfish (Oreoglanis siamensis) in wet and dry conditions.

The use of environmental DNA (eDNA) analysis has demonstrated notable efficacy in detecting the existence of freshwater species, including those that are endangered or uncommon. This application holds significant potential for enhancing environmental monitoring and management efforts. However, the efficacy of eDNA-based detection relies on several factors. In this study, we assessed the impact of rainfall on the detection of eDNA for the Siamese bat catfish (Oreoglanis siamensis). Quantitative polymerase chain reaction (qPCR) analysis indicated that samples from days with average rainfall exceeding 35 mm (classified as heavy and very heavy rain) yielded negative results. While eDNA detection remains feasible on light or moderate rainy days, a noteworthy reduction in eDNA concentration and qPCR-positive likelihood was observed. Analysis across 12 sampling sites established a statistically significant negative relationship (p < 0.001) between eDNA detection and rainfall. Specifically, for each 1 mm increase in rainfall, there was an observed drop in eDNA concentration of 0.19 copies/mL (±0.14). The findings of this study provide definitive evidence that precipitation has a significant impact on the detection of eDNA in Siamese bat catfish. However, in the case of adverse weather conditions occurring on the day of sampling, our research indicates that it is acceptable to continue with the task, as long as the rainfall is not heavy or very heavy. To enhance the effectiveness of an eDNA survey, it is crucial to consider many factors related to climatic conditions. The aforementioned factor holds significant importance not only for the specific species under scrutiny but also for the broader dynamics of the climate.

An eDNA-based assessment of Garra cambodgiensis (stonelapping minnow) distribution on a megadiverse river, the Mekong.

Garra cambodgiensis (stonelapping minnow) has experienced significant population declines, prompting intensive research and management, although its distribution in river systems such as the Mekong remains obscure. Effective conservation and management necessitate accurate monitoring and survey data on the distribution of freshwater species. Traditional surveying techniques for fish may be challenging and generate insufficient data on species distribution. This study developed an eDNA-based method for detecting G. cambodgiensis to address this void. Twenty-one locations were surveyed. Water samples were collected in triplicate from the river's surface at each site and processed within 48 h in a dedicated laboratory. Primers and probes for G. cambodgiensis were meticulously designed and species-specificity tested to ensure accurate detection without interference from co-occurring species in the same geographic range. Each water sample was analysed by qPCR using six technical replicates. The results of qPCR were reported as positive with quantifiable eDNA concentration (copies/mL), below the limit of quantification, or non-detectable. G. cambodgiensis eDNA was detected in water samples collected from 10 out of 21 sampling sites, with concentrations ranging from 8.5 to 2990.0 copies/mL. Importantly, G. cambodgiensis eDNA was consistently detected in all three replicate water samples at each site where the qPCR experiment yielded positive results. The findings of this study demonstrate the feasibility and effectiveness of incorporating eDNA-based monitoring or surveys for G. cambodgiensis in the ecologically diverse Mekong River. Monitoring based on eDNA can aid in targeting and informing conservation and management of G. cambodgiensis in its natural habitat. Comprehensive and robust information on species distribution can be obtained via an eDNA-based survey, which could contribute to more efficient and informed decision-making processes in fisheries management and conservation efforts.

In-vitro antimalarial activity of methanolic leaf- and stem-derived extracts from four Annonaceae plants.

OBJECTIVE: Plants in the Annonaceae family are known for having abundant biologically active secondary metabolites. They have been used in alternative drugs for various diseases in several countries, for instance, the bark of Cananga odorata (Lam.) Hook and Thomson is used for Ophthalmic inflammation and wound healing in Malaysia. Extracts from the leaves and stems of four Annonaceae plants, namely Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Dasymaschalon sp., Artabotrys burmanicus A.DC, and Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders were investigated for growth inhibitory activity against blood-stage Plasmodium falciparum growth in vitro and for non-specific cytotoxicity against normal peripheral blood mononuclear cells (PBMCs). Antimalarial activity was assessed by invasion inhibition assay and the percentage of infected red blood cells on blood smears were determined. Cytotoxicity was tested by culturing PBMCs with the extracts, and viabilities were determined by Annexin V/propidium iodide staining. RESULTS: A. burmanicus stem extract and M. modestum leaf extract were capable of inhibiting growth of P. falciparum when used at 200 µg/mL compared to chloroquine. The extracts at effective concentrations, did not affect the viability of PBMCs. These results support further need for characterization of active compounds from specific Annonaceae plants in order to exploit their components for potential malaria treatment.

Distinguishing fanged frogs (Limnonectes) species (Amphibia: Anura: Dicroglossidae), from Thailand using high resolution melting analysis.

Morphologically, species of fanged frogs (Limnonectes) are exceedingly similar, making it difficult to distinguish them within the complex. In Thailand, it has been difficult to distinguish between the sympatric species L. bannaensis and L. taylori, particularly among tadpoles, adolescents, and adult females. A precise identification contributes to a greater understanding of biodiversity, particularly for assessing distributions and population dynamics. Therefore, a novel approach is required. The objective of this study was to develop a high resolution melting analysis (HRM) for the rapid and accurate identification of six species of Limnonectes of the L. kuhlii complex found in Thailand, particularly the two sympatric fanged frogs. Here, HRM assays using 16S rRNA mitochondrial primers were designed and developed. There was as much as a 25.3% variation in the nucleotide sequence of the fragment amplified by HRM16S primers among the six species of Limnonectes. Prior to conducting an in vitro HRM, the DNA sequences were used in a simulation HRM, uMELT Quartz, to predict the melting curve for each species of Limnonectes. There were discrepancies between the predicted melting curves of each species generated by the programme. Consequently, in vitro HRM tests were conducted. The obtained melting curve and Tm values were consistent with those predicted, albeit with a slightly different Tm value and a more distinct melting curve. All evaluated species of Limnonectes could be easily distinguished from one another by comparing the melting curve shapes. The HRM assay was then used to confirm the species of 18 Limnonectes samples in comparison to the reference samples (confidence interval > 90%). In addition, the results of HRM were consistent with those of experts who used morphological analysis to identify species. The HRM was found to be useful, and therefore the method would also contribute to future ecological and systematic studies on the target species.

Sustainable fisheries management through reliable restocking and stock enhancement evaluation with environmental DNA.

The practise of restocking and stock improvement as a means of managing fisheries and aquaculture has been widely used. However, it is difficult to claim that fish stocking is effective due to a number of challenges. One of those is the lack of suitable monitoring and assessment methods, although all assessment approaches have their strengths and weaknesses. If the full benefits of fisheries and their long-term sustainability are to be realised, it is necessary to examine the effectiveness of restocking and stock enhancement. Therefore, effective, rapid, and dependable monitoring techniques are necessary. In this study, we used an eDNA-based method to identify G. cambodgiensis at 14 sites throughout Thailand's restocking and stock enhancement programme. eDNA from this species was identified in water samples using quantitative polymerase chain reaction (qPCR) tests with primers and a probe specific to G. cambodgiensis. A successful stocking would show positive eDNA results in water samples collected from the studied sites. Only five of the studied sites returned positive eDNA readings, which could be considered a successful stocking. The locations that contained G. cambodgiensis eDNA were either confirmed to be natural habitats or were regularly stocked with a large number of hatchery fish. In this study, we demonstrated that eDNA is a reliable, fast and accurate alternative method for measuring stock improvement.

eDNA testing reveals surprising findings on fish population dynamics in Thailand.

COVID-19, a global health concern, has an effect on all aspects of the economy. The aquaculture and fishing industries were severely harmed as a result of the closures in multiple nations. Regular systems for inventory monitoring, production, and supply were disrupted. Cancellation of programmes for research, fieldwork, sampling, and tagging influences management-required data. For effective species management, fish dispersion assessments are indispensable. However, due to the difficulty of accessing sampling sites and the associated costs, there is frequently a lack of comprehensive information regarding the distribution and abundance of organisms. The COVID-19 prohibition made fish monitoring more problematic. Due to constant pressure, populations of the stone lapping minnow (Garra cambodgiensis), one of Thailand's overfished fish, are rapidly declining. Therefore, eDNA-based monitoring was devised and implemented to reveal the likely dispersal of the species in Thailand prior to and following the lockdown. At 28 locations within the Chao Phraya River Basin, water samples were collected. qPCR was used to determine the presence or absence of G. cambodgiensis in water samples. In 78 of 252 water samples, a wide range of computed copy numbers for G. cambodgiensis eDNA was observed. It was discovered that samples collected in 2021 (after the lockdown) contain a higher concentration of G. cambodgiensis eDNA than samples collected in 2018 or 2019 (prior to the lockdown). The closure appears to be a boon and may result in a substantial restocking of the fish we have studied. Overall, eDNA-based analysis is an extremely promising new survey instrument.

Detection of the Endangered Siamese Bat Catfish (Oreoglanis siamensis Smith, 1933) in Doi Inthanon National Park Using Environmental DNA.

Siamese bat catfish (Oreoglanis siamensis Smith, 1993) has been listed as an endangered species, and its abundance has been severely declining due to habitat degradation and overfishing. To establish an appropriate management strategy, it is crucial to gain information about the distribution of this endangered species. As O. siamensis live under rocks in streams, detecting their presence is difficult. Recently, environmental DNA (eDNA)-based detection has been demonstrated to be a valid tool for monitoring rare species, such as O. siamensis. Therefore, this study developed an eDNA assay targeting a 160 bp fragment of the COI region to detect the presence of this species in its natural habitat. An amount of 300 mL of water samples (0.7 µm filtered) were collected from 15 sites in the Mae Klang sub-basin, where this fish species was visually detected at two locations. O. siamensis eDNA was detected at 12 of the 15 sites sampled with varying concentrations (0.71-20.27 copies/mL), including at the sites where this species was visually detected previously. The developed O. siamensis eDNA assay was shown to be effective for detecting the presence of this endangered species in the Klang Phat and Klang Rivers within the Doi Inthanon National Park.

Seasonal Shifts in Soil Microbiome Structure Are Associated with the Cultivation of the Local Runner Bean Variety around the Lake Mikri Prespa.

Leguminous crops play a key role in food production and agroecosystem sustainability. However, climate change and agricultural intensification have a significant impact on the available arable land, soil microbiome functions, and ultimately, crop productivity. The "Prespa bean" (Phaseolous coccineous L.) is an important leguminous crop for the agricultural economy of the rural areas surrounding the lake, Mikri Prespa, which is of significant ecological importance. The seasonal effects on soil microbiome structure, diversity and functions associated with the runner bean cultivation were investigated using 16S rRNA amplicon sequencing. The results indicated that the presence of the runner bean differentially shaped the soil microbial community structure. The runner bean was implicated in the recruitment of specific bacteria, by favouring or excluding specific classes or even phyla. Soil functions involved in nutrient availability and carbon metabolism, among other pathways, were associated with microbiome-plant interactions. The temporal relative abundance shifts could be explained by the impact of soil organic matter, the fertilization regime, and the equilibrium in carbon metabolic processes. This research has shown the effect of runner bean cultivation on the soil microbiome which, in future, may potentially contribute to research into sustainable agricultural productivity and the protection of soil ecosystem services.

Environmental DNA detection of giant snakehead in Thailand's major rivers for wild stock assessment.

Capture-based aquaculture is now gaining much attention in Southeast Asia. This system was used to produce several fish species with social and economic implications, including the giant snakehead (Channa micropeltes). As wild harvesting of organisms for seed stock is one of main practices in capture-based aquaculture, abundance and distribution of the wild stock are essential for both environmental impact evaluation and stock management. Mark and recapture, visual observation and physical capture of target species are costly, ineffective, and labour intensive for fish surveys in several cases. Detection of target organisms using eDNA (environmental DNA) could be a good alternative as it has proved to be a non-invasive, rapid, and sensitive method for aquatic species monitoring and surveying. Here, we developed a TaqMan assay that targets the 16S region of giant snakehead DNA to amplify eDNA captured in water samples. 300 µl of water samples were collected from 15 sites located in the Chao Phraya River Basin (Ping, Wang, Yom, Nan, and Chao Phraya River) and filtered with 0.7 µm glass fibre membrane filter. Giant snakehead eDNA was detected in most tributaries (60%) with concentrations ranging from 74.0 copies/ml in Wang River sites to 7.4 copies/ml in Nan River sites. As intensification of capture-based aquaculture could lead to depleting of wild fish stocks, urgent management is needed. However, the existing conventional approaches for assessment of fish overexploitation, survey and monitoring have several limitations.

DNA-Based Identification of Eurasian Vicia Species Using Chloroplast and Nuclear DNA Barcodes.

Many legume species of the Vicia L. genus (Fabaceae Lindl.) are key components of the Mediterranean diet and have an integral role in sustainable agriculture. Given the importance of the Vicia species for Eurasian culture, it is necessary to implement methodologies, such as DNA barcoding, that can enable the effective authentication and identification of species in the genus. In this study, we analysed the chloroplast trnL and rpoC1, as well as the nuclear ITS2 DNA barcoding regions, to identify 71 Vicia specimens of Eurasian descent. Both the trnL and ITS2 regions were highly effective in discriminating the analysed taxa, while the more conserved rpoC1 region could not identify all of the selected species due to high sequence conservation or non-annotated or absent rpoC1 species sequences in GenBank. A dendrographic representation of the generated trnL data showed sufficient clustering for most of the analysed taxa, although some topological discrepancies were observed. ITS2 and rpoC1 reconstructions were also used for resolving the topological discrepancies observed in the trnL tree. Our analysis suggests that a combination of DNA barcoding regions is essential for accurate species discrimination within the Vicia genus, while single-locus analyses do not provide the necessary resolution.

ITS Metabarcoding Reveals the Effects of Oregano Essential Oil on Fusarium oxysporum and Other Fungal Species in Soil Samples.

The destructive effects of Fusarium wilts are known to affect the production of many crops. The control of Fusarium oxysporum and other soilborne pathogens was mainly based on soil fumigation (methyl bromide), which has long been prohibited and, nowadays, is based on a limited number of available fungicides due to legislation restrictions on residue tolerances and environmental impacts. Alternatively, natural and environmentally safe compounds, such as essential oils, are being investigated for their efficacy in the control of soilborne diseases. The great fungicidal ability of the oregano essential oil components (carvacrol and thymol) has been reported to inhibit the germination and the mycelial development of several fungal species, including F. oxysporum. The aim of our study was to demonstrate how the metabarcoding approach can provide valuable information about the positive or negative impacts of two different doses of oregano essential oil on Fusarium oxysporum and other fungal species which were present in the studied soil samples through the amplification of the ITS1 and ITS2 regions, which were analyzed on a MiSeq platform. A higher dose of oregano essential oil decreased the abundance of F. oxysporum, along with other fungal species, but also had negative effects on Trichoderma evansii and Mortierella chlamydospora, species with possible fungicidal properties. Soil properties, essential oil properties, the fungal composition, and interactions between fungal species should be considered as factors influencing the effectiveness of essential oils as biological control agents for soilborne pathogens.

Exploring plant diversity through soil DNA in Thai national parks for influencing land reform and agriculture planning.

BACKGROUND: The severe deforestation, as indicated in national forest data, is a recurring problem in many areas of Northern Thailand, including Doi Suthep-Pui National Park. Agricultural expansion in these areas, is one of the major drivers of deforestation, having adverse consequences on local plant biodiversity. Conserving biodiversity is mainly dependent on the biological monitoring of species distribution and population sizes. However, the existing conventional approaches for monitoring biodiversity are rather limited. METHODS: Here, we explored soil DNA at four forest types in Doi Suthep-Pui National Park in Northern Thailand. Three soil samples, composed of different soil cores mixed together, per sampling location were collected. Soil biodiversity was investigated through eDNA metabarcoding analysis using primers targeting the P6 loop of the plastid DNA trnL (UAA) intron. RESULTS: The distribution of taxa for each sample was found to be similar between replicates. A strong congruence between the conventional morphology- and eDNA-based data of plant diversity in the studied areas was observed. All species recorded by conventional survey with DNA data deposited in the GenBank were detected through the eDNA analysis. Moreover, traces of crops, such as lettuce, maize, wheat and soybean, which were not expected and were not visually detected in the forest area, were identified. It is noteworthy that neighboring land and areas in the studied National Park were once used for crop cultivation, and even to date there is still agricultural land within a 5-10 km radius from the forest sites where the soil samples were collected. The presence of cultivated area near the forest may suggest that we are now facing agricultural intensification leading to deforestation. Land reform for agriculture usage necessitates coordinated planning in order to preserve the forest area. In that context, the eDNA-based data would be useful for influencing policies and management towards this goal.

Molecular detection of giant snakeheads, Channa micropeltes (Cuvier, 1831), one of the most troublesome fish species.

A lack of reliable tools for determining the presence and distribution of fish species can impede understanding of predator-prey interactions and fishery management. Conventional fish survey methods are invasive, and can be size or species selective. Combining netting and electrofishing is a current method used to monitor fish species in Phayao Lake (Kwan Phayao), Thailand. However, the methods are inefficient and time-consuming. Recently, locals who rely on inland fisheries in Kwan Phayao expressed their deep concerns about the giant snakehead, Channa micropeltes (Cuvier, 1831) destroying other fish there. The giant snakehead prey on many commercially important fish species, as the prey species is reduced, negative effects on both biodiversity and the fishery sector could follow. Here, an eDNA-based survey was developed to detect the presence of the giant snakehead. Water samples were collected from six sites within Kwan Phayao and 17 sites in Ing River where water flowed into and out of Kwan Payao. The eDNA of the giant snakehead was detected in water samples from all collection sites using the developed qPCR assay with various concentrations. The eDNA was shown here to be a sensitive and reliable tool for fish surveillance so there will be a better chance for developing an effective management strategy.

Mapping biodiversity hotspots of fish communities in subtropical streams through environmental DNA.

Large tropical and subtropical rivers are among the most biodiverse ecosystems worldwide, but also suffer from high anthropogenic pressures. These rivers are hitherto subject to little or no routine biomonitoring, which would be essential for identification of conservation areas of high importance. Here, we use a single environmental DNA multi-site sampling campaign across the 200,000 km2 Chao Phraya river basin, Thailand, to provide key information on fish diversity. We found a total of 108 fish taxa and identified key biodiversity patterns within the river network. By using hierarchical clustering, we grouped the fish communities of all sites across the catchment into distinct clusters. The clusters not only accurately matched the topology of the river network, but also revealed distinct groups of sites enabling informed conservation measures. Our study reveals novel opportunities of large-scale monitoring via eDNA to identify relevant areas within whole river catchments for conservation and habitat protection.

A molecular survey based on eDNA to assess the presence of a clown featherback (Chitala ornata) in a confined environment.

BACKGROUND: The importance of the inland fisheries sector in food security as a provider of much-needed protein and income supplier has been highlighted. This is especially the case in poor rural communities in developing countries. Inland capture fisheries in Thailand are in place nationwide in rivers, lakes, swamps and reservoirs. The clown featherback (Chitala ornata) is popularly consumed and is an economically important fish in Thailand which is often used in food products such as fish balls and fish cakes. Along with other fish species, the clown featherback is one of fish of inland fisheries at Phayao Lake. Recent fish surveys from 2016-2018 at Phayao Lake using netting and electrofishing found that the number of clown featherback have been reducing since 2016 and could not be detected at all by 2018. This is despite the fact that there are still reports of their presence in the lake from locals. METHODS: We developed an eDNA-based method for detection of the clown featherback in Phayao Lake as an alternative tool. Water samples were collected in three different sampling months (February, June and September) at six sites located in the lake. Species-specific primers and the probe were designed to amplify a 183 bp fragment of the cytB region of the clown featherback. RESULTS: eDNA of the clown featherback can be detected in all different sampling months and sites. Concentration of the clown featherback found in Prayao Lake showed no difference over sampling month but between collecting sites. This proves that eDNA based survey is a sensitive and useful tool for monitoring and surveying the clown featherback at any time of the year.

Projection range of eDNA analysis in marshes: a suggestion from the Siberian salamander (Salamandrella keyserlingii) inhabiting the Kushiro marsh, Japan.

BACKGROUND: Freshwater ecosystems are rapidly declining. The Siberian salamander (Salamandrella keyserlingii) which inhabits the Kushiro marsh in Hokkaido, Japan has lost some habitat due to human activity. There are many challenges associated with conventional monitoring methods, including cost, the need for specialist personnel, environmental impact, and ability to detect the presence of this species; thus, we investigated the feasibility of using environmental DNA (eDNA) analysis to detect its presence and identify its breeding grounds. METHODS: We performed tank experiments to confirm eDNA emission from egg sacs, larvae, and adult Siberian salamanders in the water. We also performed water sampling and visual observation of egg sacs in the Kushiro marsh during the end of the breeding season and the larval season. RESULTS: The tank experiments found eDNA emission from all growth stages. It also implied concentrated emissions just after spawning and after hatching, and limited emissions during the incubation phase in egg sacs. We also detected eDNA in the field, likely reflecting the distribution of egg sacs or larvae. Combining this data with visual observations, it was determined that the eDNA results from the field were best explained by the number of egg sacs within 7-10 m of the sampling point. CONCLUSIONS: The results of this investigation show that the breeding sites and habitats of marshland species can successfully be monitored using eDNA analysis. They also suggest that the eDNA results from the marshes may reflect the biomass that is in close range to the sampling point. These results support the increased use of eDNA analysis in marshes and provide knowledge that could improve the interpretation of future results.

Detection and quantification of cashew in commercial tea products using High Resolution Melting (HRM) analysis.

Tea, a popular aromatic infusion and food supplement, prepared from Camellia sinensis (L.) Kuntze leaves, is often subjected to adulteration with various undeclared inorganic and plant-derived materials. Cashew (Anacardium occidentale L.) nut husk is one of the most common plant tea adulterants. To date, there are limited DNA-based technologies for tea authentication and quantitative detection of adulterants. Herein, we used a universal plant DNA barcoding marker coupled with High Resolution Melting (Bar-HRM) analysis to authenticate tea products from cashew ground nut. Additionally, cashew-specific markers coupled with HRM technology were used to detect and quantify adulteration of tea with cashew DNA. This methodology can reliably detect admixtures as low as 1% v/v cashew in commercial tea products. Overall, our results demonstrate that the HRM technology is a strong molecular approach in tea authentication, capable of detecting very low adulterations in DNA admixtures. PRACTICAL APPLICATION: In this study, we established the use of high-resolution DNA-based technologies for the detection of cashew adulteration in tea, even in very low quantities. The technology could be applied to a greater range of plant-based tea adulterants. This work is expected to facilitate the traceability and authenticity of tea products and form the basis for the development of strategies against fraudulent practices.

DNA Fingerprinting and Species Identification Uncovers the Genetic Diversity of Katsouni Pea in the Greek Islands Amorgos and Schinoussa.

Pea (P. sativum L.), one of the most important legume crops worldwide, has been traditionally cultivated in Lesser Cyclades since ancient times. The commonly known traditional pea cultivar, 'Katsouni', is endemic to the islands of Amorgos and Schinoussa and is of great local economic importance. Despite the widespread cultivation of 'Katsouni' in both islands, it is still unknown whether the current Schinoussa and Amorgos pea populations are distinct landraces, and if they have common evolutionary origin. To assist conservation and breeding of the pea crop, the genetic diversity and phylogenetic relationships of 39 pea samples from Amorgos and 86 from Schinoussa were studied using DNA barcoding and ISSR marker analyses. The results indicate that both populations are different landraces with distinct geographical distribution and are more closely related to P. sativum subsp. elatius than the P. abyssinicum and P. fulvum species. Further characterization of the 'Katsouni' landraces for functional polymorphisms regarding pathogen resistance, revealed susceptibility to the powdery mildew (Erysiphe pisi DC.). This work represents the first investigation on the genetic diversity and population structure of the 'Katsouni' cultivar. Exploiting the local genetic diversity of traditional landraces is fundamental for conservation practices and crop improvement through breeding strategies.

Associations among thermal biology, preovulatory follicle diameter, follicular and luteal vascularities, and sex steroid hormone concentrations during preovulatory and postovulatory periods in tropical beef cows.

The objectives were to evaluate effects of tropical seasons on thermal biology, preovulatory follicle (POF) diameter, POF and luteal vascularities, and estradiol (E2) and progesterone (P4) concentrations; and to determine the associations among the values for these variables during preovulatory and postovulatory periods in Thai native cows in tropical climates: cold, hot, and rainy seasons. Development and vascularity of the POF and corpora lutea (CL) were evaluated using color Doppler ultrasonography. The temperature-humidity index (THI) was greater when the preovulatory period occurred during the rainy season when compared with the occurrence during the hot and cold seasons of the year. Furthermore, POF diameter was less when the THI was greater. The THI was greater when the postovulatory period occurred during the rainy season when compared to the occurrence of the postovulatory period during the hot and cold seasons of the year. Furthermore, the CL vascularity and P4 concentration were less when the THI was greater. The THI was inversely correlated with CL vascularity and P4 concentrations. When the THI was greatest during the hot and rainy seasons of the year, there were the greatest negative effects on POF size, POF and CL blood flow, and concentrations of E2 and P4 during the preovulatory and postovulatory periods. While native Bos indicus are capable of adapting to tropical conditions, there are still negative effects, such as impaired POF and CL functions, when the THI induces heat stress.