DNA Analysis Research Articles

Novel Insights into Hb Shaare Zedek Associated with β0-Thalassemia: Molecular Characteristics, Genetic Origin and Diagnostic Approaches.

Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype-phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and β-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a β-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with β0-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.

When less is more: The association between the expression of polymorphic CYPs and AFB1-induced HCC.

An individual's genetic fingerprint is emerging as a pivotal predictor of numerous disease- and treatment-related factors. Single nucleotide polymorphisms (SNPs) in drug-metabolizing enzymes play key roles in an individual's exposure to a malignancy-associated risk, such as Aflatoxin B1 (AFB1)-induced hepatocellular carcinoma (HCC). This study aimed at reviewing literature on the polymorphisms that exist in CYP enzymes and their possible link with susceptibility to AFB1-induced HCC. A set of keywords associated with the study subject of interest was used to search the Google Scholar and the PubMed database. The last ten years' worth of research projects were included in the results filter. The research involved HCC patients and any connection between polymorphic forms of CYP enzymes and their susceptibility to AFB1-induced HCC, including older but significant data. Variations in CYP1A2 and CYP3A4 were reported to impact the rate and magnitude of AFB1 bio-activation, thus influencing an individual's vulnerability to develop HCC. In HCC patients, the activity of CYP isoforms varies, where increased activity has been reported with CYP2C9, CYP2D6, and CYP2E1, while CYP1A2, CYP2C8, and CYP2C19 exhibit decreased activity. CYP2D6*10 frequency has been discovered to differ considerably in HCC patients. Rs2740574 (an upstream polymorphism in CYP3A4 as detected in CYP3A4*1B) and rs776746 (which affects CYP3A5 RNA splicing), both of which influence CYP3A expression, thus impacting the variability of AFB1-epoxide adducts in HCC patients. CYP1A2 is the primary enzyme accountable for the formation of harmful AFBO globally. CYP3A4, CYP3A5, CYP3A7, CYP2B7, and CYP3A3 are also implicated in the bio-activation of AFB1 to mutagenic metabolites. It is thought that CYP3A4 is the protein that interacts with AFB1 metabolism the most. Polymorphic variants of CYP enzymes have a functional impact on the susceptibility to AFB1-induced HCC. Outlining such variation and their implications may provide deeper insights into approaching HCC in a more personalized manner for guiding future risk-assessment, diagnosis, and treatment.

Assessment of DNA quality for whole genome library preparation

In recent years, more sophisticated DNA technologies for genotyping have enabled considerable progress in various fields such as clinical genetics, archaeogenetics and forensic genetics. DNA samples previously rejected as too challenging to analyze due to low amounts of degraded DNA can now provide useful information. To increase the chances of success with the new methodologies, it is crucial to know the fragment size of the template DNA molecules, and whether the DNA in a sample is mostly single or double stranded. With this knowledge, an appropriate library preparation method can be chosen, and the DNA shearing parameters of the protocol can be adjusted to the DNA fragment size in the sample. In this study, we first developed and evaluated a user-friendly fluorometry-based protocol for estimation of DNA strandedness. We also evaluated different capillary electrophoresis methods for estimation of DNA fragmentation levels. Next, we applied the developed methodologies to a broad variety of DNA samples processed with different DNA extraction protocols. Our findings show that both the applied DNA extraction method and the sample type affect the DNA strandedness and fragmentation. The established protocols and the gained knowledge will be applicable for future sequencing-based high-density SNP genotyping in various fields.

Detection and Quantification of Programmed Cell Death in Chlamydomonas reinhardtii: The Example of S-Nitrosoglutathione.

Chlamydomonas (Chlamydomonas reinhardtii) is a unicellular model alga that has been shown to undergo programmed cell death (PCD) that can be triggered in response to different stresses. We have recently shown that Chlamydomonas is particularly well suited to the study and quantification of PCD. We have shown for the first time that S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor, is able to induce PCD and can be used as a study system in Chlamydomonas. In this article, we provide a simple and robust protocol for quantifying GSNO-induced PCD, which can be adapted to any other treatment. We explain how to detect NO production in the cell following GSNO treatment. We show how PCD can be identified simply by analyzing the degradation profile of genomic DNA. We also provide an easy and reproducible cell death quantification protocol, which makes it possible to follow the course of PCD over time and highlight very fine differences in the number of affected cells between different samples. Key features • Use of S-nitrosoglutathione (GSNO) as a means to study programmed cell death (PCD) in Chlamydomonas. • Discrimination of PCD vs. necrosis. • In vivo determination of NO production in the cell. • A simple, robust protocol for PCD quantification.

Is it worthy to use environmental DNA instead of scientific trawling or video survey to monitor taxa in soft-bottom habitats?

Non-extractive techniques such as video analysis are increasingly used by scientists to study marine communities instead of extractive methods such as trawling. Currently, environmental DNA (eDNA) analysis is seen as a revolutionary tool to study taxonomic diversity. We aimed to determine which method is the most appropriate to describe fish and commercial invertebrate diversity comparing bottom trawl hauls, video transects and seawater eDNA. Our results reveal that video detected the lowest number of taxa and trawling the highest. eDNA analysis is powerful to describe marine bony fish communities, but some taxa of importance for the ecosystem such as elasmobranchs, crustaceans or molluscs are poorly detected. This may be due to several factors such as marker specificity, incomplete reference gene databases or low DNA release in the environment. For now, the various methods provide different information and none is exhaustive enough to be used alone for biodiversity characterisation.

Microbial Forensics: Comparison of MLVA Results According to NGS Methods, and Forensic DNA Analysis Using MLVA

Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying the origin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiological analysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, and advances in next generation sequencing(NGS) technology have enabled <i>in silico</i> anlysis of full-length whole genome sequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The United Nations Secretary-General’s Mechanism(UNSGM)’s external quality assessment exercise(EQAE) project, which we officially participated in 2023. We confirmed that the 3 unknown samples were <i>B. anthracis</i> through nucleic acid isolation and genetic sequence analysis studies. MLVA results on 32 loci of <i>B. anthracis</i> were analysed by using genome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing using short-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of the tandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy than NextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel error caused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 published MLVA databases of <i>B. anthracis</i>, and MLVA results of 10 strains were identical with 3 unkonwn samples. As a result of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as <i>B. anthracis</i> strain A4564 which is associated with injectional anthrax isolates in heroin users.

Growth characteristics and molecular identification of indigenous Limnospira strains from Ethiopian soda lakes as a protein source

Malnutrition poses a significant global challenge in regions such as Ethiopia, particularly in children with limited dietary diversity. Arthrospira, a protein-rich cyanobacterium known for its rapid growth even under extreme conditions, is a promising solution for biomass production. This study addresses knowledge gaps in the morphological characterization and taxonomic classification of Arthrospira by focusing on precise species identification and strategic strain selection for optimal biomass production. Cyanobacterial strains were cultivated in a modified Spirulina Ogawa Terui medium at 35 °C under the photosynthetically active radiation of 160 μmol photons m−2 s−1 with a 12/12 h light/dark period in laboratory conditions. One hundred Arthrospira-like strains were isolated and characterized based on their morphological attributes according to accepted criteria, from Ethiopian soda lakes. Systematic evaluation of the growth rate comparison test identified four exceptionally fast-growing strains, with LC-30 exhibiting the highest dry weight (3.27 ± 0.51 g L˗1) and specific growth rate (1.63 ± 0.05 d˗1) among them. DNA analysis confirmed that all four examined strains belong to the Limnospira genus, specifically identified as Limnospira fusiformis. LC-30 and LA-08 demonstrated protein and phycocyanin levels (582.80 ± 0.07 and 557.90 ± 0.09 mg g˗1, respectively) comparable to those reported for Arthrospira platensis, and surpassing conventional protein sources, thus suggesting their potential for nutritional supplementation. The study highlights the importance of bioprospecting indigenous strains from soda lakes, revealing promising candidates for large-scale biomass production.

Transfer and recovery of DNA and metal particles: A proof-of-concept application of a parallel strategy by DNA and environmental scanning electron microscopy analysis

According to the principle of Locard “Every contact leaves a trace", when touching a surface, a bi-directional transfer of self and non-self-DNA residing on the hands and touched objects can occur. Metals are commonly encountered in forensic evidence and, during hand contact with these surfaces, a transfer of metal particles could occur together with the transfer of human DNA. This study proposes a proof-concept approach for the original detection of metal particles and touch DNA to track the activity performed by a donor and particularly to assess the metallic substrate touched before the contact with a subsequent surface. To this scope, a scenario of contact events was simulated by three volunteers, who participated in fingerprint deposition firstly on copper and then on plastic and glass surfaces. Twenty-four stubs were collected on the hands of volunteers and the secondary surfaces and then analyzed by environmental scanning electron microscopy (ESEM). DNA was quantified only from copper and plastic surfaces. Ten additional volunteers followed the same protocol of deposition on copper and then on plastic surfaces to evaluate DNA transfer only. On 20 touch DNA samples, the copper surface yielded significantly lower DNA amounts, ranging from 0.001 to 0.129 ng/μl, compared to the secondary touched plastic surface, ranging from 0.007 to 0.362 ng/μl. ESEM-EDS analysis showed that copper particles could be abundantly detected on the hands of the volunteers after contact with the copper surface. Particles containing silicates with copper were shown on plastic, while they were only found in 1/3 of samples on glass. Our proof-of-concept study has shown that ESEM-EDS analysis has the potential to detect copper particles transferred to the hands of volunteers during contact with a copper metallic surface and deposited on secondarily touched items. The results suggest that this original ESEM-DNA parallel approach could potentially allow the tracking of DNA transfer and metal particles at a crime scene, although this represents only a first step and further research on a wider casuistry could help to address the interpretation of results given activity level propositions.

Development of single nucleotide polymorphism (SNP) markers and construction of DNA fingerprinting of Alcea rosea L. based on specific-locus amplified fragment sequencing (SLAF-seq) technology

Development of single nucleotide polymorphism (SNP) markers and construction of DNA fingerprinting of Alcea rosea L. based on specific-locus amplified fragment sequencing (SLAF-seq) technology

Assessing actionability and incidental findings of germline variants in two precision oncology trials

IntroductionHigh-throughput sequencing techniques have revolutionized oncology. Paired germline-tumor DNA analysis has emerged as a comprehensive strategy to uncover actionable alterations in advanced cancer patients (ACP) enrolled in precision oncology trials. However, challenges persist in variant interpretation and managing incidental germline findings. MethodsWe conducted a study involving 288 ACP from MOSCATO (NCT01566019) and MATCHR (NCT02517892) trials to assess germline variants impacting cancer-related genes. Germline DNA sequencing was performed using a panel of 250 cancer-related genes, and the results were discussed during tumor molecular board sessions. ResultsGermline pathogenic variants (PV) were classified according to the ESCAT classification. Lung cancer (36.8 %), followed by prostate (18.4 %) and breast cancer (17.7 %), comprised the most prevalent tumor types. PVs were found in 12.5 % of patients. Most PVs were classified as ESCAT X (63.9 %), highlighting limited therapeutic actionability. Notably,2 % of patients had actionable variants (ESCAT I-A/II-A). Incidental findings included 7.3 % of patients with PVs in cancer-predisposition genes, with 2.4 % having very high-risk potential, necessitating mandatory oncogenetic counseling. Nearly one in five patients (21.9 %) had at least one VUS. DiscussionOur study underscores the significance of germline sequencing in identifying actionable alterations and the need for improved variant interpretation as well as pretest counseling plans in precision oncology trials.

Where did it go? A study of DNA transfer in a social setting

The sensitivity of DNA analysis has progressed to the point that trace levels of DNA, originating from only a few cells, can generate informative profiles. This means that virtually any item or surface can be sampled with a reasonable chance of obtaining a DNA profile. As the presence of DNA does not suggest how it was deposited, questions are often raised as to how the DNA came to be at a particular location and the activity that led to its deposition. Therefore, understanding different modes of DNA deposition, reflective of realistic forensic casework situations, is critical for proper evaluation of DNA results in court. This study aimed to follow the movements of DNA to and from individuals and common household surfaces in a residential premises, while socially interacting. This took place over an hour and involved four participants, with known shedder status, designated as visitors (a male and a female) and hosts (a male and a female), who engaged in the activity of playing a board game while being served food. During the study, the participants were instructed to use the toilet on a single occasion to assess the transfer of DNA to new and unused underwear that was provided. All contacts made by the participants in the dining room and kitchen were video recorded to follow the movements of DNA. Samples were collected based on the history of contact, which included hands, fingernails and penile swabs. Direct contacts resulted in detectable transfer (LR > 1) in 87 % (87/100) of the non-intimate samples and clothing. For surfaces touched by multiple participants, DNA from the person who made the last contact was not always detectable. The duration and number of contacts did not significantly affect the detection of the person contacting the item. On the other hand, presence of background DNA and participant’s shedder status appear to play an important role. Further, unknown contributors were detected in the majority of samples. Finally, indirect transfer was observed on a number of occasions including co-habiting partners of guests who were not present at the study location. The results of this study may assist with decision making for exhibit selection or targeting areas for sampling within the home environment. Our findings can also be used in conjunction with previous literature to develop activity-level evaluations in such situations where the source of the DNA is conceded, but the mode of deposition is disputed.

Assessing benthic macroinvertebrate communities’ spatial heterogeneity in Mediterranean transitional waters through eDNA metabarcoding

Transitional waters are important habitats both for biodiversity and ecological functions, providing valuable natural resources and relevant ecosystem services. However, they are highly susceptible to climate changes and anthropogenic pressures responsible for biodiversity losses and require specific biomonitoring programs. Benthic macroinvertebrates are suitable as ecological indicators of transitional waters, being affected by biological, chemical, and physical conditions of the ecosystems about their life cycles and space-use behaviour. The advent of high-throughput sequencing technologies has allowed biodiversity investigations, at the molecular level, in multiple ecosystems and for different ecological guilds. Benthic macroinvertebrate communities’ composition has been investigated, at the molecular level, mainly through DNA extracted from sediments in marine and riverine ecosystems. In this work, benthic macroinvertebrate communities are explored through eDNA metabarcoding from water samples in a Mediterranean transitional water ecosystem. This research highlighted the validity of eDNA metabarcoding as an efficient tool for the assessment of benthic macroinvertebrate community structure in transitional waters, unveiling the spatial heterogeneity of benthic macroinvertebrate communities correlated to the measured environmental gradients. The results suggest that peculiar features of transitional water ecosystems, such as shallow waters and limited currents, facilitate the assessment of benthic macroinvertebrate communities through environmental DNA analysis from surface water samples, opening for more rapid and accurate monitoring programs for these valuable ecosystems.

Searching for alternative high DNA-yielding bone types for DNA analysis of aged skeletal remains

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Črnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Črnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations.

Report: Establishment of a DNA Fingerprinting Database in the United Arab Emirates

DNA fingerprinting represents a pivotal advancement in forensic science since the 1980s. Recognizing its significance, the United Arab Emirates (UAE) implemented the first law pertaining to this technology through Federal Decree by Law No. (39) of 2023, titled ‘Concerning the Regulation of the Federal DNA Fingerprinting Database’. This law came into effect on 1 November 2023. The report will delve into the concept of DNA fingerprinting and its database, focusing on the administration of the Federal DNA Fingerprinting Database and its practical applications. The discussion will cover various aspects, including the origins of biological specimens, managing biological samples preservation and execution, the retention duration of DNA fingerprinting, and the measures in place to ensure legal safeguards for genetic fingerprint databases. This brief overview endeavours to offer a concise insight into the legal framework and operational factors concerning DNA fingerprinting in the context of recent legislative changes.

Circulating tumour DNA analysis for early detection of lung cancer: a systematic review.

Circulating tumor DNA (ctDNA) analysis has been applied in cancer diagnostics including lung cancer. Specifically for the early detection purpose, various modalities of ctDNA analysis have demonstrated their potentials. Such analyses have showed diverse performance across different studies. We performed a systematic review of original studies published before 1 January 2023. Studies that evaluated ctDNA alone and in combination with other biomarkers for early detection of lung cancer were included. The systematic review analysis included 56 original studies that were aimed for early detection of lung cancer. There were 39 studies for lung cancer only and 17 for pan-cancer early detection. Cancer and control cases included were heterogenous across studies. Different molecular features of ctDNA have been evaluated, including 7 studies on cell-free DNA concentration, 17 on mutation, 29 on methylation, 5 on hydroxymethylation and 8 on fragmentation patterns. Among these 56 studies, 17 have utilised different combinations of the above-mentioned ctDNA features and/or circulation protein markers. For all the modalities, lower sensitivities were reported for the detection of early-stage cancer. The systematic review suggested the clinical utility of ctDNA analysis for early detection of lung cancer, alone or in combination with other biomarkers. Future validation with standardised testing protocols would help integration into clinical care.

High levels of consanguinity in a child from Paquimé, Chihuahua, Mexico

Paquimé (also known as Casas Grandes), situated in northern Chihuahua between Mesoamerican and Ancestral Puebloan groups, was a vibrant multicultural centre during the thirteenth and fourteenth centuries AD. Substantial debate surrounds the social organisation of Paquimé's inhabitants. Here, the authors report on the analysis of ancient DNA from a unique child burial beneath a central support post of a room in the House of the Well. They argue that the close genetic relationship of the child's parents, revealed through this analysis, and the special depositional context of the burial reflect one family's attempts to consolidate and legitimise their social standing in this ancient community.

Unlocking rivers' hidden diversity and ecological status using DNA metabarcoding in Northwest Spain.

Rivers are crucial ecosystems supporting biodiversity and human well-being, yet they face increasing degradation globally. Traditional river biomonitoring methods based on morphological identification of macroinvertebrates present challenges in terms of taxonomic resolution and scalability. This study explores the application of DNA metabarcoding analysis in both bulk and environmental DNA (eDNA) samples for comprehensive assessment of macrozoobenthic biodiversity, detection of invasive and endangered species, and evaluation of river ecological status in northwestern Spain. DNA metabarcoding of homogenized bulk samples and water eDNA revealed a mean of 100 and 87 macrozoobenthos species per sample respectively. However, the specific composition was significantly different with only 27.3% of the total species being shared. It was not possible to identify all the OTUs to species level; only 17.43% and 49.4% of the OTUs generated could be identified to species level in the bulk and eDNA samples, respectively. Additionally, a total of 11 exotic species (two first records for the Iberian Peninsula and another three first records for Asturias region) and one endangered species were detected by molecular tools. Molecular methods showed significant correlations with morphological identification for EQR values (Ecological Quality Ratio) of IBMWP index, yet differences in inferred river ecological status were noted, with bulk samples tending to indicate higher status. Overall, DNA metabarcoding offers a promising approach for river biomonitoring, providing insights into biodiversity, invasive species, and ecological status within a single analysis. Further optimization and intercalibration are required for its implementation in routine biomonitoring programmes, but its scalability and multi-tasking capabilities position it as a valuable tool for integrated monitoring of river ecosystems.

Next generation triplex-forming PNAs for site-specific genome editing of the F508del CFTR mutation

BackgroundCystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein for which there is no cure. One approach to cure CF is to correct the underlying mutations in the CFTR gene. We have used triplex-forming peptide nucleic acids (PNAs) loaded into biodegradable nanoparticles (NPs) in combination with donor DNAs as reagents for correcting mutations associated with genetic diseases including CF. Previously, we demonstrated that PNAs induce recombination between a donor DNA and the CFTR gene, correcting the F508del CFTR mutation in human cystic fibrosis bronchial epithelial cells (CFBE cells) and in a CF murine model leading to improved CFTR function with low off-target effects, however the level of correction was still below the threshold for therapeutic cure. MethodsHere, we report the use of next generation, chemically modified gamma PNAs (γPNAs) containing a diethylene glycol substitution at the gamma position for enhanced DNA binding. These modified γPNAs yield enhanced gene correction of F508del mutation in human bronchial epithelial cells (CFBE cells) and in primary nasal epithelial cells from CF mice (NECF cells). ResultsTreatment of CFBE cells and NECF cells grown at air-liquid interface (ALI) by NPs containing γtcPNAs and donor DNA resulted in increased CFTR function measured by short circuit current and improved gene editing (up to 32 %) on analysis of genomic DNA. ConclusionsThese findings provide the basis for further development of PNA and NP technology for editing of the CFTR gene.

Identification and Characterization Colletotrichum spp. Causing Mango Dieback in Indonesia.

Dieback disease in mango trees has been observed in Indonesia, particularly in Java Island, with the causal agent remaining unidentified. One of the important pathogens that are responsible for causing mango dieback is Colletotrichum. Field surveys were conducted in various mango cultivating areas in Java Island, Indonesia to assess prevalence of Colletotrichum as dieback disease pathogen. Eleven Colletotrichum isolates were recovered from symptomatic dieback twigs and morphologically characterized. Genetic diversity fingerprint analysis was carried out using rep-PCR. Phylogenetic analysis identified isolates as belonging to Colletotrichum asianum and Colletotrichum cairnsense using partial sequences of four gene regions, including ITS, ACT, GAPDH, and TUB2. Pathogenicity tests on mango seedlings cv. Arumanis showed that all fungal isolates were responsible for causing dieback symptoms. Subsequently, symptomatic tissue was reisolated to fulfill Koch's Postulate. This study represented new funding for two species of Colletotrichum causing mango dieback in Indonesia.

Applications and challenges of DNA barcoding in rapid radiation groups: Rhodiola (Crassulaceae) as a case study

ObjectiveRhodiolae Crenulatae Radix et Rhizoma (Hongjingtian in Chinese, RCRR), the roots and rhizomes of Rhodiola crenulata and its application in the medicinal market is very chaotic. In this study, DNA barcoding database and identification engine of Rhodiola species were established, decoction pieces from the medicinal market were identified, and the application and challenges of DNA barcoding in the rapid radiation of Rhodiola species were analyzed. This study provides reference for the protection, rational development, and utilization of endangered resources within Rhodiola species. MethodsA total of 50 original plant samples from 20 species of the genus Rhodiola from Hebei, Xinjiang, Tibet, Jilin, and other major production areas were collected. Theses samples cover the typical distribution area (Qinghai-Tibetan Platea) of Rhodiola species and other scattered alpine regions (Changbai Mountain, Taibai Mountain, Lushan Mountain, etc.), it encompasses all Rhodiola species with thick rhizomes in China. ITS2 and psbA-trnH barcode of Rhodiola database (BORD) were established and an identification engine named Rhodiola-IDE was developed. The stability and accuracy of the standard DNA barcoding database were evaluated using two datasets. Rhodiola-IDE identified 31 decoction pieces of RCRR from the medicinal material market. ResultsThe BORD containing 1 532 sequences of 88 Rhodiola species has been established, and the identification efficiency results showed good accuracy and stability. According to the Chinese Pharmacopoeia (2020 edition), 23 samples (74.2%) were identified as authentic R. crenulata, while the rest of the marketed varieties were R. kirilowii, R. dumulosa, and R. fastigiata. The product label “Larger flower, Hongjingtian” was identified as R. crenulata. Samples labeled as “Smaller flower, Hongjingtian” were identified as R. crenulata, R. kirilowii, and R. fastigiata. ConclusionITS2 and psbA-trnH barcodes can identify monophyletic groups represented by R. crenulata. However, for non-monophyletic species, it is necessary to collect as many samples as possible and combine them with multiple markers for joint identification. This study discussed the application and challenges of DNA barcodes in Rhodiola under rapid radiation conditions, providing a scientific basis for the rational development and utilization of Rhodiola varieties.