Fungal biodiversity in Arctic paleoecosystems assessed by metabarcoding of lake sedimentary ancient DNA

Fungi are crucial organisms in most ecosystems as they exert ecological key functions and are closely associated with land plants. Fungal community changes may therefore help reveal biodiversity changes in past ecosystems. Lake sediments contain DNA of organisms in the catchment area, which allows reconstructing past biodiversity by using metabarcoding of ancient sedimentary DNA. We developed a novel PCR primer combination for fungal metabarcoding targeting a short amplicon to account for length bias of amplification due to ancient DNA degradation. In-silico PCRs showed higher diversity using this primer combination than using previously established fungal metabarcoding primers. We analyzed existing data from sediment cores from four artic and one boreal lake in Siberia. These cores had been stored for 2-22 years and examined degradation effects of ancient DNA and storage time-related bias in fungal communities. Amplicon size differed between fungal divisions, however, we observed no significant effect of sample age on amplicon length and GC content, suggesting robust results. We also found no indication of post-coring fungal growth during storage distorting ancient fungal communities. Terrestrial soil fungi, including mycorrhizal fungi and saprotrophs, were predominant in all lakes, which supports the use of lake sedimentary ancient DNA for reconstructing terrestrial communities.

A Model and Simulation of the Influence of Temperature and Amplicon Length on Environmental DNA Degradation Rates: A Meta-Analysis Approach

Environmental DNA (eDNA) analysis can detect aquatic organisms, including rare and endangered species, in a variety of habitats. Degradation can influence eDNA persistence, impacting eDNA-based species distribution and occurrence results. Previous studies have investigated degradation rates and associated contributing factors. It is important to integrate data from across these studies to better understand and synthesize eDNA degradation in various environments. We complied the eDNA degradation rates and related factors, especially water temperature and amplicon lengths of the measured DNA from 28 studies, and subjected the data to a meta-analysis. In agreement with previous studies, our results suggest that water temperature and amplicon length are significantly related to the eDNA degradation rate. From the 95% quantile model simulation, we predicted the maximum eDNA degradation rate in various combinations of water temperature and amplicon length. Predicting eDNA degradation could be important for evaluating species distribution and inducing innovation (e.g., sampling, extraction, and analysis) of eDNA methods, especially for rare and endangered species with small population size.

Degradation factors of environmental DNA evaluated by experiments and meta-analysis

Environmental DNA (eDNA) methods have been developed to detect organisms' distributions and abundance/biomass in various environments. eDNA degradation is critical for eDNA evaluation, but, the dynamics and mechanisms of eDNA degradation are largely unknown, especially when considering different eDNA sources, e.g., cell-derived and fragmental DNA. In this study, we conducted the degradation experiments (Saito and Doi 2020a) and a meta-analysis (Saito and Doi 2020b). Firstly, we experimentally evaluated the degradation rates of eDNA derived from multiple sources, including fragmental DNA (the DNA of internal positive control, IPC), free cells from Oncorhynchus kisutch, and the resident species (Saito and Doi 2020a). We conducted the experiments with pond and seawater to evaluate the differences between freshwater and marine habitats. Our results showed that eDNA derived from the both cells and fragmental DNA decreased exponentially in the both sea and pond samples. The degradation of eDNA from the resident species showed similar behavior to the cell-derived eDNA. As a meta-analysis, we complied the degradation rates of eDNA in laboratory experiment and field studies from 28 studies (Saito and Doi 2020b). We also collected the related factors, including water sources, water temperature, DNA regions, and PCR amplicon lengths of the measured DNA. Our results suggested that water temperature and amplicon length were significantly related to the degradation rate of eDNA. From the simulation based on the 95% quantile model, we predicted the maximum degradation rate of eDNA in various combinations of water temperature and PCR amplicon length.

DETEKSI DAN IDENTIFIKASI Austropuccinia psidii PADA MYRTACEAE DI YOGYAKARTAINDONESIA

Austropuccinia psidii is a pathogenic fungus that causes rust in the Myrtaceae plant. The extensive plantation of the host of this fungus has increased the attack of fungal pathogen, therefore, will increase the threat tothe presence of Myrtaceae species around the globe including in Indonesia. This present study was aiming to detect and identify the presence of this pathogen by morphologicaland molecular observation. Morphological observationrevealed the presence of A. psidii urediniospores onsalam (Syzygium polyanthum) andkayuputih (Melaleuca cajuputi)leaves collected from the arboretum of the Indonesian Center for Forest Biotechnology and Tree Improvement (CFBTI), and the presence of teliospores on young Syzygium leaves. PCR amplification using specific primers of Ppsi1 / Ppsi6 succeeded in detecting the presence of A. psidii fungi Melaleuca and Syzygium showed by DNA amplicon length around 500bp. Efforts to obtain ITS DNA sequences to compare the molecular characteristics of fungi from two different hosts have been carried out, however, the sequencing electropherogram was unreadable, so the comparison can not be performed. This study reported that A. psidii is currently present in Myrtaceae species in Yogyakarta, therefore precaution efforts should be conducted to avoid economic and ecological impact from this pathogen.

A viability qPCR dilemma: Are longer amplicons better?

The development of viability qPCR (v-qPCR) has allowed for a more accurate assessment of the viability of a microbial sample by limiting the amplification of DNA from dead cells. Although valuable, v-qPCR is not infallible. One of the most limiting factors for accurate live/dead distinction is the length of the qPCR amplicon used. However, no consensus or guidelines exist for selecting and designing amplicon lengths for optimal results. In this study, a wide range of incrementally increasing amplicon lengths (68-906 bp) was used on live and killed cells of nine bacterial species treated with viability dye (PMA). Increasing amplicon lengths up to approximately 200 bp resulted in increasing quantification cycle (Cq) differences between live and killed cells, while maintaining a good qPCR efficiency. Longer amplicon lengths, up to approximately 400 bp, further increased Cq difference, but at the cost of qPCR efficiency. Above 400 bp, no valuable increase in Cq differences was observed. Importance Viability qPCR (v-qPCR) has evolved to a valuable, mainstream technique for determining the number of viable micro-organisms in samples by qPCR. Amplicon length is known to be positively correlated with the ability to distinguish between live and dead bacteria but is negatively correlated with qPCR efficiency. This trade-off is often not taken into account and might have an impact on the accuracy of v-qPCR data. Currently there is no consensus on the optimal amplicon length. This paper provides methods to determine the optimal amplicon length and suggests an amplicon length range for optimal v-qPCR, taking into consideration the trade-off between qPCR efficiency and live-dead distinction.

Antha-guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries

Protein engineering through directed evolution facilitates the screening and characterization of protein libraries. Efficient and effective methods for multiple site-saturation mutagenesis, such as Darwin Assembly, can accelerate the sampling of relevant sequence space and the identification of variants with desired functionalities. Here, we present the automation of the Darwin Assembly method, using a Gilson PIPETMAX™ liquid handling platform under the control of the Antha software platform, which resulted in the accelerated construction of complex, multiplexed gene libraries with minimal hands-on time and error-free, while maintaining flexibility over experimental parameters through a graphical user interface rather than requiring user-driven library-dependent programming of the liquid handling platform. We also present an approach for barcoding libraries that overcomes amplicon length limitations in next generation sequencing and enables fast reconstruction of library reads.

Diagnosis and molecular characterization of Anaplasma platys in dog patients in Yogyakarta area, Indonesia

Anaplasma platys is a tick-borne, Gram-negative bacterium that causes anaplasmosis, a companion vector-borne disease impacting dogs. Information on this disease remains limited in Indonesia. Its symptoms are not specific, so molecular analysis is required for a rapid and accurate diagnosis. GroEL is an essential gene commonly used for classification and species identification of many groups of bacteria, including Anaplasma spp. In this study, a molecular diagnosis of anaplasmosis based on the groEL gene sequence was conducted using PCR. In addition, the genetic diversity of Anaplasma platys in infected dogs was determined. Blood samples were collected from 51 dogs suspected of anaplasmosis from Prof. Dr. Soeparwi Animal Hospital, animal clinics, and pet shops in the Yogyakarta area, Indonesia, based on anamnesis, histories of tick infestations, and clinical symptom examinations. DNA extraction and PCR targeting the groEL gene were performed, followed by sequencing. Phylogenetic tree analysis and construction were carried out using the BLAST and MEGA programs. Positive PCR sample results (amplicon length of 624 bp) were found in 6 of 51 dogs. Samples A1 (KHJ/C2), A2 (KHJ/A2), A3 (KSK/L), A4 (KHJ/L), and A5 (KNP/M2) had close ties to Anaplasma platys (AF478129.1) from GenBank. Phylogenetic analysis showed a very high homology value (100%) and bootstrap value of 100%. It can be concluded that there was no genetic diversity in the Anaplasma platys found in infected dogs in the Yogyakarta area.