基于环境DNA宏条形码技术的辽东湾典型围海养殖池塘内水母多样性研究

研究使用环境DNA宏条形码技术(eDNA metabarcoding)检测辽东湾东北部河口区围海养殖池塘水母种类多样性,探索适用于水母种类物种鉴定和监测的新方法。利用环境DNA宏条形码技术,分别基于18S rDNA和COI宏条形码检测了辽东湾东北部河口区围海养殖池塘水母种类多样性,通过水样采集、过滤、eDNA提取、遗传标记扩增、测序与生物信息分析的环境DNA宏条形码标准化分析流程,从围海养殖池塘7个采样点中获得可检测的采样点数据。结果显示,基于18S rDNA宏条形码检测出8种水母种类,其中钵水母纲大型水母2种、水螅水母总纲小型水母6种;基于COI宏条形码技术共检测出19种水母种类,其中钵水母纲大型水母5种、水螅水母总纲小型水母14种;两种DNA条形码标记都显示养殖种类海蜇(Rhopilema esculentum)为优势种。研究结果表明,环境DNA宏条形码技术作为一种新兴的生物多样性监测手段可用于快速检测水母种类多样性,在水母类物种鉴定、监测及早期预警中有较大的应用潜能。     

Environmental DNA (eDNA) metabarcoding reveals strong discrimination among diverse marine habitats connected by water movement

While in recent years environmental DNA (eDNA) metabarcoding surveys have shown great promise as an alternative monitoring method, the integration into existing marine monitoring programs may be confounded by the dispersal of the eDNA signal. Currents and tidal influences could transport eDNA over great distances, inducing false‐positive species detection, leading to inaccurate biodiversity assessments and, ultimately, mismanagement of marine environments. In this study, we determined the ability of eDNA metabarcoding surveys to distinguish localized signals obtained from four marine habitats within a small spatial scale (<5 km) subject to significant tidal and along‐shore water flow. Our eDNA metabarcoding survey detected 86 genera, within 77 families and across 11 phyla using three established metabarcoding assays targeting fish (16S rRNA gene), crustacean (16S rRNA gene) and eukaryotic (cytochrome oxidase subunit 1) diversity. Ordination and cluster analyses for both taxonomic and OTU data sets show distinct eDNA signals between the sampled habitats, suggesting dispersal of eDNA among habitats was limited. Individual taxa with strong habitat preferences displayed localized eDNA signals in accordance with their respective habitat, whereas taxa known to be less habitat‐specific generated more ubiquitous signals. Our data add to evidence that eDNA metabarcoding surveys in marine environments detect a broad range of taxa that are spatially discrete. Our work also highlights that refinement of assay choice is essential to realize the full potential of eDNA metabarcoding surveys in marine biodiversity monitoring programs.     

Comparative study of the validity of three regions of the 18S‐rRNA gene for massively parallel sequencing‐based monitoring of the planktonic eukaryote community

The nuclear 18S‐rRNA gene has been used as a metabarcoding marker in massively parallel sequencing (MPS)‐based environmental surveys for plankton biodiversity research. However, different hypervariable regions have been used in different studies, and their utility has been debated among researchers. In this study, detailed investigations into 18S‐rRNA were carried out; we investigated the effective number of sequences deposited in international nucleotide sequence databases (INSDs), the amplification bias, and the amplicon sequence variability among the three variable regions, V1–3, V4–5 and V7–9, using in silico polymerase chain reaction (PCR) amplification based on INSDs. We also examined the primer universality and the taxonomic identification power, using MPS‐based environmental surveys in the Sea of Okhotsk, to determine which region is more useful for MPS‐based monitoring. The primer universality was not significantly different among the three regions, but the number of sequences deposited in INSDs was markedly larger for the V4–5 region than for the other two regions. The sequence variability was significantly different, with the highest variability in the V1–3 region, followed by the V7–9 region, and the lowest variability in the V4–5 region. The results of the MPS‐based environmental surveys showed significantly higher identification power in the V1–3 and V7–9 regions than in the V4–5 region, but no significant difference was detected between the V1–3 and V7–9 regions. We therefore conclude that the V1–3 region will be the most suitable for future MPS‐based monitoring of natural eukaryote communities, as the number of sequences deposited in INSDs increases.     

Responses of foraminifera communities to aquaculture‐derived organic enrichment as revealed by environmental DNA metabarcoding

Current monitoring methods to assess benthic impacts of marine finfish aquaculture are based on complex biological indices and/or geochemistry data. The former requires benthic macrofauna morpho‐taxonomic characterization that is time‐ and cost‐intensive, while the latter provides rapid assessment of the organic enrichment status of sediments but does not directly measure biotic impacts. In this study, sediment samples were collected from seven stations at six salmon farms in British Columbia, Canada, and analyzed for geochemical parameters and by eDNA metabarcoding to investigate linkages between geochemistry and foraminifera. Sediment texture across farm sites ranged from sand to silty loam, while the maximum sediment pore‐water sulphide concentration at each site ranged from 1,000 to 13,000 μM. Foraminifera alpha diversity generally increased with distance from cage edge. Adonis analyses revealed that farm site explained the most variation in foraminifera community, followed by sediment type, enrichment status, and distance from cage edge. Farm‐specific responses were observed in diversity analyses, taxonomic difference analyses, and correlation analyses. Results demonstrated that species diversity and composition of foraminifera characterized by eDNA metabarcoding generated signals consistent with benthic biodiversity being impacted by finfish farming activities. This substantiates the validity of eDNA metabarcoding for augmenting current approaches to benthic impact assessments by providing more cost‐effective and practicable biotic measures than traditional morpho‐taxonomy. To capitalize on this potential, further work is needed to design a new nomogram that combines eDNA metabarcoding data and geochemistry data to enable accurate monitoring of benthic impacts of fish farming in a time‐ and cost‐efficient way.     

基于环境DNA宏条形码的洱海鱼类多样性研究

研究使用环境DNA宏条形码(eDNA metabarcoding)检测洱海鱼类多样性,探索适用于洱海鱼类多样性监测和保护的新方法。通过水样采集、过滤、eDNA提取、遗传标记扩增、测序与生物信息分析的环境DNA宏条形码标准化分析流程,从洱海16个采样点中获得可检测的9个采样点数据,共检测出17种鱼类,其中土著种5种、外来种12种;鲫(Carassius auratus)、鳙(Hypophthalmichthys nobilis)、麦穗鱼(Pseudorasbora parva)、泥鳅(Misgurnus anguillicaudatus)和食蚊鱼(Gambusia affinis)为优势种。研究结果表明虽然环境DNA宏条形码无法完全替代传统的鱼类监测方法,但作为一种新兴的生物多样性监测手段,其可用于快速检测洱海鱼类多样性及其空间分布。     

Simultaneous analysis of seven 16S rRNA hypervariable gene regions increases efficiency in marine bacterial diversity detection

Environmental DNA sequencing is the gold standard to reveal microbial community structures. In most applications, a one-fragment PCR approach is applied to amplify a taxonomic marker gene, usually a hypervariable region of the 16S rRNA gene. We used a new reverse complement (RC)-PCR-based assay that amplifies seven out of the nine hypervariable regions of the 16S rRNA gene, to interrogate bacterial communities in sediment samples collected from different coastal marine sites with an impact gradient. In parallel, we employed a traditional one-fragment analysis of the hypervariable V3–V4 region to investigate whether the RC-PCR reveals more of the ‘unseen’ diversity obtained by the one-fragment approach. As a benchmark for the full deck of diversity, we subjected the samples to PCR-free metagenomic sequencing. None of the two PCR-based approaches recorded the full taxonomic repertoire obtained from the metagenomics datasets. However, the RC-PCR approach detected 2.8 times more bacterial genera compared to the near-saturation sequenced V3–V4 samples. RC-PCR is an ideal compromise between the standard one-fragment approach and metagenomics sequencing and may guide future environmental sequencing studies, in which bacterial diversity is a central subject.     

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

Preserving biodiversity is a global challenge requiring data on species’ distribution and abundance over large geographic and temporal scales. However, traditional methods to survey mobile species’ distribution and abundance in marine environments are often inefficient, environmentally destructive, or resource‐intensive. Metabarcoding of environmental DNA (eDNA) offers a new means to assess biodiversity and on much larger scales, but adoption of this approach for surveying whole animal communities in large, dynamic aquatic systems has been slowed by significant unknowns surrounding error rates of detection and relevant spatial resolution of eDNA surveys. Here, we report the results of a 2.5 km eDNA transect surveying the vertebrate fauna present along a gradation of diverse marine habitats associated with a kelp forest ecosystem. Using PCR primers that target the mitochondrial 12S rRNA gene of marine fishes and mammals, we generated eDNA sequence data and compared it to simultaneous visual dive surveys. We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 m. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.     

Hypervariable regions (V1–V9) of the dinoflagellate 18S rRNA using a large dataset for marker considerations

DNA sequencing methods have been used for the molecular taxonomic discrimination of dinoflagellate protists, particularly using partial 18S rRNA sequences. This study evaluated the taxonomic discrimination power of rRNA gene hypervariable regions (V1 to V9) in dinoflagellates from a large dataset. These included 77 dinoflagellate species (9 orders, 17 families, 40 genera). The complete 18S rRNA sequences of the dinoflagellates ranged from 1,787 to 1,813?bp in length, and consisted of eight V regions with a total combined length of 678 to 699?bp. Regions longer than 100?bp were recoded for V2, V4, and V8 regions; high nucleotide divergences were detected in V1, V2, and V4 regions. Statistic tests showed that the divergences of individual V regions were significantly different (t-test, P?<?0.05) compared with the complete 18S rRNA. The V2 region showed the highest score (83.5%) for PI sites. Moreover, intra-genus DNA similarities of the V2 were considerably low (<93%). Neighbor-joining analyses showed that phylogenetic resolution in the V2–V4 region was 1.32-fold higher than that of the complete 18S rRNA. These results demonstrate that V2 has the highest taxonomic resolving power within the 18S rRNA gene of dinoflagellates, suggesting the V2 and adjacent regions (e.g., V1 to V4) may be the best for marker considerations.     

Detection of a diverse marine fish fauna using environmental DNA from seawater samples

Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days.Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.     

Investigating the Potential Use of Environmental DNA (eDNA) for Genetic Monitoring of Marine Mammals

The exploitation of non-invasive samples has been widely used in genetic monitoring of terrestrial species. In aquatic ecosystems, non-invasive samples such as feces, shed hair or skin, are less accessible. However, the use of environmental DNA (eDNA) has recently been shown to be an effective tool for genetic monitoring of species presence in freshwater ecosystems. Detecting species in the marine environment using eDNA potentially offers a greater challenge due to the greater dilution, amount of mixing and salinity compared with most freshwater ecosystems. To determine the potential use of eDNA for genetic monitoring we used specific primers that amplify short mitochondrial DNA sequences to detect the presence of a marine mammal, the harbor porpoise, Phocoena phocoena, in a controlled environment and in natural marine locations. The reliability of the genetic detections was investigated by comparing with detections of harbor porpoise echolocation clicks by static acoustic monitoring devices. While we were able to consistently genetically detect the target species under controlled conditions, the results from natural locations were less consistent and detection by eDNA was less successful than acoustic detections. However, at one site we detected long-finned pilot whale, Globicephala melas, a species rarely sighted in the Baltic. Therefore, with optimization aimed towards processing larger volumes of seawater this method has the potential to compliment current visual and acoustic methods of species detection of marine mammals.     

Assessing eukaryotic biodiversity in the Florida Keys National Marine Sanctuary through environmental DNA metabarcoding

Environmental DNA (eDNA) is the DNA suspended in the environment (e.g., water column), which includes cells, gametes, and other material derived from but not limited to shedding of tissue, scales, mucus, and fecal matter. Amplifying and sequencing marker genes (i.e., metabarcoding) from eDNA can reveal the wide range of taxa present in an ecosystem through analysis of a single water sample. Metabarcoding of eDNA provides higher resolution data than visual surveys, aiding in assessments of ecosystem health. This study conducted eDNA metabarcoding of two molecular markers (cytochrome c oxidase I (COI) and 18S ribosomal RNA (rRNA) genes) to survey eukaryotic diversity across multiple trophic levels in surface water samples collected at three sites along the coral reef tract within the Florida Keys National Marine Sanctuary (FKNMS) during four research cruises in 2015. The 18S rRNA gene sequences recovered 785 genera while the COI gene sequences recovered 115 genera, with only 33 genera shared between the two datasets, emphasizing the complementarity of these marker genes. Community composition for both genetic markers clustered by month of sample collection, suggesting that temporal variation has a larger effect on biodiversity than spatial variability in the FKNMS surface waters. Sequences from both marker genes were dominated by copepods, but each marker recovered distinct phytoplankton groups, with 18S rRNA gene sequences dominated by dinoflagellates and COI sequences dominated by coccolithophores. Although eDNA samples were collected from surface waters, many benthic species such as sponges, crustaceans, and corals were identified. These results show the utility of eDNA metabarcoding for cataloging biodiversity to establish an ecosystem baseline against which future samples can be compared in order to monitor community changes.     

Individual haplotyping of whale sharks from seawater environmental DNA

Population genetic data can provide valuable information on the demography of a species. For rare and elusive marine megafauna, samples for generating the data are traditionally obtained from tissue biopsies, which can be logistically difficult and expensive to collect and require invasive sampling techniques. Analysis of environmental DNA (eDNA) offers an alternative, minimally invasive approach to provide important genetic information. Although eDNA approaches have been studied extensively for species detection and biodiversity monitoring in metabarcoding studies, the potential for the technique to address population-level questions remains largely unexplored. Here, we applied “eDNA haplotyping” to obtain estimates of the intraspecific genetic diversity of a whale shark (Rhincodon typus) aggregation at Ningaloo reef, Australia. Over 2 weeks, we collected seawater samples directly behind individual sharks prior to taking a tissue biopsy sample from the same animal. Our data showed a 100% match between mtDNA sequences recovered in the eDNA and tissue sample for all 28 individuals sampled. In the seawater samples, >97% of all reads were assigned to six dominant haplotypes, and a clear dominant signal (~99% of sample reads) was recovered in each sample. Our study demonstrates accurate individual-level haplotyping from seawater eDNA. When DNA from one individual clearly dominates each eDNA sample, it provides many of the same opportunities for population genetic analyses as a tissue sample, potentially removing the need for tissue sampling. Our results show that eDNA approaches for population-level analyses have the potential to supply critical demographic data for the conservation and management of marine megafauna.     

Marine water environmental DNA metabarcoding provides a comprehensive fish diversity assessment and reveals spatial patterns in a large oceanic area

Current methods for monitoring marine fish (including bony fishes and elasmobranchs) diversity mostly rely on trawling surveys, which are invasive, costly, and time‐consuming. Moreover, these methods are selective, targeting a subset of species at the time, and can be inaccessible to certain areas. Here, we used environmental DNA (eDNA), the DNA present in the water column as part of shed cells, tissues, or mucus, to provide comprehensive information about fish diversity in a large marine area. Further, eDNA results were compared to the fish diversity obtained in pelagic trawls. A total of 44 5 L‐water samples were collected onboard a wide‐scale oceanographic survey covering about 120,000 square kilometers in Northeast Atlantic Ocean. A short region of the 12S rRNA gene was amplified and sequenced through metabarcoding generating almost 3.5 million quality‐filtered reads. Trawl and eDNA samples resulted in the same most abundant species (European anchovy, European pilchard, Atlantic mackerel, and blue whiting), but eDNA metabarcoding resulted in more detected bony fish and elasmobranch species (116) than trawling (16). Although an overall correlation between fishes biomass and number of reads was observed, some species deviated from the common trend, which could be explained by inherent biases of each of the methods. Species distribution patterns inferred from eDNA metabarcoding data coincided with current ecological knowledge of the species, suggesting that eDNA has the potential to draw sound ecological conclusions that can contribute to fish surveillance programs. Our results support eDNA metabarcoding for broad‐scale marine fish diversity monitoring in the context of Directives such as the Common Fisheries Policy or the Marine Strategy Framework Directive.     

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.     

Mitochondrial Genome Sequencing and Development of Genetic Markers for the Detection of DNA of Invasive Bighead and Silver Carp (Hypophthalmichthys nobilis and H. molitrix) in Environmental Water Samples from the United States

Invasive Asian bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix) pose a substantial threat to North American aquatic ecosystems. Recently, environmental DNA (eDNA), genetic material shed by organisms into their environment that can be detected by non-invasive sampling strategies and genetic assays, has gained recognition as a tool for tracking the invasion front of these species toward the Great Lakes. The goal of this study was to develop new species-specific conventional PCR (cPCR) and quantitative (qPCR) markers for detection of these species in North American surface waters. We first generated complete mitochondrial genome sequences from 33 bighead and 29 silver carp individuals collected throughout their introduced range. These sequences were aligned with those from other common and closely related fish species from the Illinois River watershed to identify and design new species-specific markers for the detection of bighead and silver carp DNA in environmental water samples. We then tested these genetic markers in the laboratory for species-specificity and sensitivity. Newly developed markers performed well in field trials, did not have any false positive detections, and many markers had much higher detection rates and sensitivity compared to the markers currently used in eDNA surveillance programs. We also explored the use of multiple genetic markers to determine whether it would improve detection rates, results of which showed that using multiple highly sensitive markers should maximize detection rates in environmental samples. The new markers developed in this study greatly expand the number of species-specific genetic markers available to track the invasion front of bighead and silver carp and will improve the resolution of these assays. Additionally, the use of the qPCR markers developed in this study may reduce sample processing time and cost of eDNA monitoring for these species.     

Haptophyte Diversity and Vertical Distribution Explored by 18S and 28S Ribosomal RNA Gene Metabarcoding and Scanning Electron Microscopy

Haptophyta encompasses more than 300 species of mostly marine pico‐ and nanoplanktonic flagellates. Our aims were to investigate the Oslofjorden haptophyte diversity and vertical distribution by metabarcoding, and to improve the approach to study haptophyte community composition, richness and proportional abundance by comparing two rRNA markers and scanning electron microscopy (SEM). Samples were collected in August 2013 at the Outer Oslofjorden, Norway. Total RNA/cDNA was amplified by haptophyte‐specific primers targeting the V4 region of the 18S, and the D1‐D2 region of the 28S rRNA. Taxonomy was assigned using curated haptophyte reference databases and phylogenetic analyses. Both marker genes showed Chrysochromulinaceae and Prymnesiaceae to be the families with highest number of Operational Taxonomic Units (OTUs), as well as proportional abundance. The 18S rRNA data set also contained OTUs assigned to eight supported and defined clades consisting of environmental sequences only, possibly representing novel lineages from family to class. We also recorded new species for the area. Comparing coccolithophores by SEM with metabarcoding shows a good correspondence with the 18S rRNA gene proportional abundances. Our results contribute to link morphological and molecular data and 28S to 18S rRNA gene sequences of haptophytes without cultured representatives, and to improve metabarcoding methodology.     

eDNA metabarcoding survey reveals fine‐scale coral reef community variation across a remote,tropical island ecosystem

Environmental DNA (eDNA) metabarcoding, a technique for retrieving multispecies DNA from environmental samples, can detect a diverse array of marine species from filtered seawater samples. There is a growing potential to integrate eDNA alongside existing monitoring methods in order to establish or improve the assessment of species diversity. Remote island reefs are increasingly vulnerable to climate‐related threats and as such there is a pressing need for cost‐effective whole‐ecosystem surveying to baseline biodiversity, study assemblage changes and ultimately develop sustainable management plans. We investigated the utility of eDNA metabarcoding as a high‐resolution, multitrophic biomonitoring tool at the Cocos (Keeling) Islands, Australia (CKI)—a remote tropical coral reef atoll situated within the eastern Indian Ocean. Metabarcoding assays targeting the mitochondrial 16S rRNA and CO1 genes, as well as the 18S rRNA nuclear gene, were applied to 252 surface seawater samples collected from 42 sites within a 140 km² area. Our assays successfully detected a wide range of bony fish and elasmobranchs (244 taxa), crustaceans (88), molluscs (37) and echinoderms (7). Assemblage composition varied significantly between sites, reflecting habitat partitioning across the island ecosystem and demonstrating the localisation of eDNA signals, despite extensive tidal and oceanic movements. In addition, we document putative new occurrence records for 46 taxa and compare the efficiency of our eDNA approach to visual survey techniques at CKI. Our study demonstrates the utility of a multimarker metabarcoding approach in capturing multitrophic biodiversity across an entire coral reef atoll and sets an important baseline for ongoing monitoring and management.     

分子标记及其在海洋动物遗传研究中的应用

分子遗传标记在农业动植物育种和生产上得到了广泛的应用,且取得了可喜的成果,但在水生生物上的应用还处于初始阶段。本文简要介绍了限制性片段长度多态性（RFLP）、随机扩增多态性DNA（RAPD）、扩增片段长度多态性（AFLP）、小卫星DNA和微卫星DNA（或称简单序列重复,SSR）等分子标记的概念、基本原理及其特点,重点介绍了第三代分子标记单核苷酸多态性（SNP）技术。综述了这些分子标记在海洋动物遗传结构分析、亲缘关系鉴定、遗传图谱的构建和标记辅助育种等方面的应用。     

Improved Methods for Capture,Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.