The speciation of coliform genera from above and below a sewer outfall and their susceptibilities to antimicrobial agents

The occurrence of coliforms in a small water course was shown to increase by a factor of thirty six below the outfall of a sewage treatment plant. Speciation of the bacteria from above and below the sewer outfall showed thatEscherichia coli andEnterobacter species predominated. Drug resistance levels were significant in microorganisms from both sampling sites and the occurrence of a significant number of multiple-resistant microorganisms, particularlyE. coli, is reported. BothE. coli andEnterobacter species from below the sewer outfall show a statistically significant increase in resistance to ampicillin andE. coli from below the outfall also shows a statistically significant increase in resistance to sulphamethoxazole as compared with isolates from above the outfall.     

Glacial and postglacial sedimentation in the Fryxell basin, Taylor Valley, southern Victoria Land, Antarctica

A 9.14 m long sediment sequence was recovered from Lake Fryxell, Taylor Valley, southern Victoria Land, Antarctica, and investigated for its chronology and sedimentological, mineralogical, and biogeochemical changes. The basal part of the sequence is dominated by coarse clastic matter, i.e., mainly sand. The sediment composition suggests that a lake existed in Fryxell basin during the Middle Weichselian by ca. 48,000 cal. year BP. After a short period of lake-level lowstand ca. 43,000 cal. year BP, lower Taylor Valley became occupied by the proglacial Lake Washburn, which was at least partly supplied by meltwater and sediments from the Ross Ice Sheet that was advanced to the mouth of Taylor Valley. Evaporation of Lake Washburn to lower levels started during the Last Glacial Maximum at ca. 22,000 cal. year BP, long before the Ross Ice Sheet retreated significantly. Lake-level lowering was discontinuous with a series of high and low stands. From ca. 4000 cal. year BP environmental conditions were similar to those of today and lower Fryxell basin was occupied by a small lake. This lake evaporated to a saline or hypersaline pond between ca. 2500 and 1000 cal. year BP and refilled subsequently.     

Analyses of three native aquatic plant species to assess spatial gradients of lake trace element contamination

A bioaccumulation study was performed on Lake Averno, a volcanic lake close to Naples (southern Italy), to ascertain the degree of trace element contamination. Good information to assess spatial gradients of Cd, Cr, Cu, Fe, Ni, Pb, V and Zn contamination was provided by analysing Phragmites communis roots, Najas marina shoots, Potamogeton pectinatus leaves and surface sediments of Lake Averno collected from July 1999 to July 2000 at three sites in the littoral zone. The highest concentrations of the trace elements were measured in sediments and plants from the site where a nearby sewage outfall channel reaches the lake, the lowest at the site below woodland. Significant correlations were found between sediment and plant element concentrations. For almost all the elements, values 2–4-fold higher were found for the site affected by the sewage effluent. Among the plant species, Ph. communis is the most useful species in biomonitoring studies due to its ability to accumulate trace elements in the roots and its collectability throughout the year. Given the absence of systematic water quality monitoring, trace elements in plants, rather than sediments, provide a cost-effective means for assessing trace element accumulation in aquatic systems during plant organ lifespan.     

Increased death of new leaves of coastal banksia (Banksia integrifolia L.f) around ocean sewage outfall sites

Abstract Salt‐spray damage to coastal vegetation is a worldwide phenomenon. Worsening of such damage by surfactants present in sewage disposed of at sea has been observed in Europe and on trees planted around metropolitan beaches in Australia. Whether adverse impacts of sewage occur on the more widespread native vegetation around non‐metropolitan outfalls in Australia is unknown. The present study investigated the survivorship of existing leaves, and of new shoots and leaves, on Banksia integrifolia around three outfall and three non‐outfall sites in spring/summer of 1997‐1998. Survivorship of existing leaves was almost identical at outfall and non‐outfall sites. When plants initiated new shoots over the spring/summer, plants at outfall sites initiated more than plants at non‐outfall sites; but the proportion of these new shoots lost subsequently differed little between outfall and non‐outfall sites. Survivorship of newly initiated leaves on the new shoots differed between outfall and non‐outfall sites, with 65% of the new leaves on average being lost at outfall sites, and 47% being lost at non‐outfall sites by the end of summer. The probability of observing a difference of this magnitude ranged between P= 0.057 and 0.11 (February‐April 1998). This is the first report of a possible adverse impact on an Australian native plant from sewage discharged to the ocean.     

Pattern of natural 15N abundance in lakeside forest ecosystem affected by cormorant-derived nitrogen

Waterbirds are one of the most important groups of organisms inhabiting the land–water interface, especially with regard to mediating the transport of materials from the aquatic to the terrestrial environment. The great cormorant (Phalacrocorax carbo) is a colonial piscivorous bird that transports nutrients from fresh water to forest. We measured cormorant-derived nitrogen at two nesting colonies on the Isaki Peninsula and Chikubu Island at Lake Biwa, Japan, and analyzed the long-term effects of cormorant colonization on the forest nitrogen cycle, and the mechanisms of nitrogen retention. Three sites were examined in each colony: a currently occupied area, a previously occupied but now abandoned area, and a control area never colonized by cormorants. High nitrogen stable isotope ratios of cormorant excreta, the forest floor, mineral soil, and living plants showed cormorant-derived nitrogen in both occupied and abandoned areas. The relationship between δ¹⁵N and N content showed that the high δ¹⁵N of the excreta and N turnover in the soil were important at the occupied sites, whereas high δ¹⁵N of litter was important at the abandoned sites. Physiological changes of various organisms are also important for the N decomposition process. In conclusion, cormorant-derived nitrogen remains in the forest ecosystem as a result of two cormorant activities: heavy deposition of excreta and collection of nitrogen-rich nest material. Colony stage (occupied, abandoned, or never inhabited) and historical change of N decomposition process of an area can be identified from the relationship between δ¹⁵N and N content.     

Use of environmental DNA to detect Eastern Sand Darter (Ammocrypta pellucida Putnam, 1863) in large Laurentian Great Lakes embayments

A single-species environmental DNA (eDNA) method was developed to sample for a small, benthic rare species, Eastern Sand Darter (Ammocrypta pellucida Putnam, 1863) in two large Lake Ontario embayments. Summer water sampling allowed for: (a) surveys of habitats (Wellers Bay) where traditional fish sampling gear could not be used; and, (b) a comparison between eDNA and seining-based detection probabilities at known occupied habitats (West Lake). In 2018, replicate (n = 3) 1 L water samples were collected from 90 Wellers Bay sites and 71 West Lake sites. A site-occupancy model, a hierarchical logistic regression model, was fitted to determine site occupancy, sample occupancy (presence of Eastern Sand Darter DNA in a water sample) and probability of detection (p) based on replicate quantitative polymerase chain reaction (qPCR) results for each water sample. Eastern Sand Darter was detected at 10 West Lake sites, but not from Wellers Bay. Mean site occupancy was 0.31 (0.12–0.70; 95% CLs), mean sample occupancy was 0.28 (0.09–0.58; 95% CLs), and mean detection probability in a subsample (i.e., successful qPCR amplification) given it was present was 0.40 (0.25–0.55; 95% CLs). While the eDNA method successfully detected Eastern Sand Darter from known occupied areas in West Lake, it was not more effective for assessing local site occupancy than traditional sampling methods, such as the seine.     

Genetic Diversity of Escherichia coli Isolated from Urban Rivers and Beach Water

Repetitive element anchored PCR was used to evaluate the genetic profiles of Escherichia coli isolated from surface water contaminated with urban stormwater, sanitary sewage, and gull feces to determine if strains found in environmental samples reflect the strain composition of E. coli obtained from host sources. Overall, there was less diversity in isolates collected from river and beach sites than with isolates obtained from human and nonhuman sources. Unique strain types comprised 28.8, 29.2, and 15.0% of the isolate data sets recovered from stormwater, river water, and beach water, respectively. In contrast, 50.4% of gull isolates and 41.2% of sewage isolates were unique strain types. River water, which is expected to contain E. coli strains from many diffuse sources of nonpoint source pollution, contained strains most closely associated with other river water isolates that were collected at different sites or on different days. However, river sites impacted by sewage discharge had approximately 20% more strains similar to sewage isolates than did sites impacted by stormwater alone. Beach sites with known gull fecal contamination contained E. coli most similar to other beach isolates rather than gull isolates collected at these same sites, indicating underrepresentation of possible gull strains. These results suggest large numbers of strains are needed to represent contributing host sources within a geographical location. Additionally, environmental survival may influence the composition of strains that can be recovered from contaminated waters. Understanding the ecology of indicator bacteria is important when interpreting fecal pollution assessments and developing source detection methodology.     

Some factors influencing the density of invertebrates near a sewage outfall

The factors influencing the density of benthic and planktonic invertebrates near a sewage outfall on Great Slave Lake in the Canadian subarctic were determined between June 1975 and May 1977. Densities were measured along 3 transects extending 500 m from the outfall and at 3 stations located 0.5–6.0 km from the outfall. Almost all benthic (Chironomus decorus, Procladius denticulatus, Polypedilum nebeculosum, Tanytarsus sp., Limnodrilus hoffmeisteri, Pontoporeia afftnis, Pisidium casternatum, Valvata sincera helicoidea) and planktonic (Keratella earlinae, Kellicottia longispina, Synchaeta stylata) species increased in abundance moving towards the sewage discharge point. Multiple regression analyses indicated that the elevated levels of algae and the total amount of food available in the environment had little if any influence on all species. Detritus and associated bacteria were the most important factors affecting densities and were probably the major food source for all species. Low winter oxygen levels (10–60% staturation), water depth and temperature had negligible effect on most species.     

The balance between photosynthesis and grazing in Antarctic mixotrophic cryptophytes during summer

SUMMARY 1. Grazing and photosynthetic contributions to the carbon balance of planktonic, mixotrophic cryptophytes in Lakes Fryxell and Hoare in the Taylor Valley, Antarctica were measured during November and December 2000.
2. The cryptophytes never became entirely photosynthetic, although carbon derived from grazing decreased in December. Individual grazing rates ranged between 5.28 and 10.08 bacteria cell⁻¹ day⁻¹ in Lake Fryxell and 0.36–11.76 bacteria cell⁻¹ day⁻¹ in Lake Hoare. Grazing rates varied temporally and with depth in the water column. In Lake Fryxell, which is a meromictic lake, highest grazing occurred just above the chemocline. Individual photosynthetic rates ranged from 0.23 to 1.35 pg C cell⁻¹ h⁻¹ in Lake Fryxell and 0.074 to 1.08 pg C cell⁻¹ h⁻¹ in Lake Hoare.
3. Carbon acquisition by the cryptophyte community gained through grazing ranged between 8 and 31% during November in Lake Fryxell, dropping to between 2 and 24% in December. In Lake Hoare grazing contributed 12–21% of the community carbon budget in November and 1–28% in December. Around 4% of the carbon acquired from grazing and photosynthesis was remineralised through respiration.
4. Mixotrophy is probably a major survival strategy for cryptophytes in the extreme lakes of the Dry Valleys, because perennial ice-cover severely limits light penetration to the water column, whereas these phytoflagellates are not normally mixotrophic in lower latitude lakes. The evidence suggests that mixotrophy may be a mechanism for supplementing the carbon budget, as well as a means of acquiring nutrients for growth.     

Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real‐time PCR for the detection of viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan^® real‐time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan^® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false‐negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 μg ml^?1, was demonstrated to effectively bind DNA from 10⁸ CFU ml^?1 dead cells, and the optimized method could detect as low as 10⁴ CFU g^?1 of viable E. coli O157:H7 cells in ground beef without interference from 10⁸ CFU g^?1 of dead cells. Conclusions: EMA real‐time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real‐time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.     

The presence of atrazine and atrazine-degrading bacteria in the residential, cattle farming, forested and golf course regions of Lake Oconee

Aims: To assess the concentration of atrazine in Lake Oconee and develop a qPCR assay as a potential marker for the presence of atrazine‐degrading bacteria indicating atrazine contamination. Methods and Results: Water and sediment samples were collected from the Oconee Lake at four golf course sites, two residential sites, one cattle farming site and a forested site. Atrazine concentration at the study sites was determined using an ELISA kit and indicated the presence of atrazine from 0·72 ppb at the forested sites to 1·84 ppb at the golf course sites. QPCR results indicate the presence of atzA gene (atrazine chlorohydrolase) from 1·51 × 10² gene copies at the residential sites to 3·31 × 10⁵ gene copies per 100 ml of water at the golf course regions of the lake and correlated (r = 0·64) with atrazine concentration. Sediment samples had higher atzA gene copies compared with the water samples (P < 0·05). Conclusions: Atrazine concentration and the highest quantity of atzA gene were detected in the golf course regions of the lake. Overall, atrazine concentration monitored in Lake Oconee was below the Environment Protection Agency (EPA) regulatory standards. Significance and Impact of the Study: Quantitative PCR is an efficient technique for assessing the presence of atrazine catabolism gene as a functional marker for atrazine‐degrading bacteria and the presence of atrazine contamination.     

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans

Aims: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR‐based diagnostic tool for P. penetrans. Methods and Results: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria‐specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Conclusions: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. Significance and Impact of the Study: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro‐organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.     

Detection of Genetic Markers of Fecal Indicator Bacteria in Lake Michigan and Determination of Their Relationship to Escherichia coli Densities Using Standard Microbiological Methods

Lake Michigan surface waters impacted by fecal pollution were assessed to determine the occurrence of genetic markers for Bacteroides and Escherichia coli. Initial experiments with sewage treatment plant influent demonstrated that total Bacteroides spp. could be detected by PCR in a 25- to 125-fold-higher dilution series than E. coli and human-specific Bacteroides spp., which were both found in similar dilution ranges. The limit of detection for the human-specific genetic marker ranged from 0.2 CFU/100 ml to 82 CFU/100 ml culturable E. coli for four wastewater treatment plants in urban and rural areas. The spatial and temporal distributions of these markers were assessed following major rain events that introduced urban storm water, agricultural runoff, and sewage overflows into Lake Michigan. Bacteroides spp. were detected in all of these samples by PCR, including those with <1 CFU/100 ml E. coli. Human-specific Bacteroides spp. were detected as far as 2 km into Lake Michigan during sewage overflow events, with variable detection 1 to 9 days postoverflow, whereas the cow-specific Bacteroides spp. were detected in only highly contaminated samples near the river outflow. Lake Michigan beaches were also assessed throughout the summer season for the same markers. Bacteroides spp. were detected in all beach samples, including 28 of the 74 samples that did not exceed 235 CFU/100 ml of E. coli. Human-specific Bacteroides spp. were detected at three of the seven beaches; one of the sites demonstrating positive results was sampled during a reported sewage overflow, but E. coli levels were below 235 CFU/100 ml. This study demonstrates the usefulness of non-culture-based microbial-source tracking approaches and the prevalence of these genetic markers in the Great Lakes, including freshwater coastal beaches.     

The microfauna of algal mats and artificial substrates in Southern Victoria Land lakes of Antarctica

The first microfaunal colonization study of continental lakes in Antarctica is reported. In addition to the general taxonomic composition of six Antarctic lakes, detailed community composition analyses of two adjacent lakes, Lake Fryxell and Lake Hoare, using polyurethane foam artificial substrates suspended in the open water and placed directly over benthic algal mats are also presented. Protozoans, rotifers, tardigrades and nematodes constituted the microfaunal community in all lakes. Higher metazoans do not occur in lakes that we studied. Protozoans were the most diverse of the four groups, and the first dinoflagellates reported from southern Victoria Land, Gymnodinium and Glenodinium, were collected from Lake Fryxell. The greatest number of protozoan taxa (55) were associated with the algal mats in the more productive Lake Fryxell. Both the physical and chemical properties and taxonomic diversity showed marked variability between all lakes that were studied, especially the adjacent lakes, Fryxell and Hoare. Ecological variables which regulate taxonomic diversity in other lakes of the world also appear to exert similar influences in remote Antarctic lakes.     

Incidence of Mentum Deformities in Midge Larvae (Diptera:Chironomidae) from Northern Nova Scotia, Canada

Deformities in the mouthparts of larval Chironomidae, particularly of the teeth on the mentum, have been proposed as a bioindicator of sediment quality and environmental stress. Most work to date has concentrated on relatively few abundant, responsive genera common in soft-bottom lakes. We examined mentum deformities in 25 genera of Chironominae, Orthocladiinae and Diamesinae (one genus) from streams and a lake in rural Nova Scotia where farming and forestry are the principal land uses. Incidence of deformity at similar stream sites varied across genera from zero to >10%. Average frequencies of deformity across all three subfamilies at sites with no known sources of contamination ranged from <4% to 8%, and increased to nearly 15% at a site receiving treated municipal sewage effluent. Differences in chironomid community structure and rates of leaf litter decomposition above and below the sewage effluent outfall were congruent with the difference in mentum deformities. Frequencies of deformity observed here are an order of magnitude greater than in similar studies of rural areas. Low-level stress from agriculture or forest harvesting may be widespread in rural regions even in aquatic ecosystems that are seemingly free of industrial discharges or sediment contamination.     

Satellite tracking of gulls and genomic characterization of faecal bacteria reveals environmentally mediated acquisition and dispersal of antimicrobial‐resistant Escherichia coli on the Kenai Peninsula,Alaska

Gulls (Larus spp.) have frequently been reported to carry Escherichia coli exhibiting antimicrobial resistance (AMR E. coli); however, the pathways governing the acquisition and dispersal of such bacteria are not well described. We equipped 17 landfill‐foraging gulls with satellite transmitters and collected gull faecal samples longitudinally from four locations on the Kenai Peninsula, Alaska to assess: (a) gull attendance and transitions between sites, (b) spatiotemporal prevalence of faecally shed AMR E. coli, and (c) genomic relatedness of AMR E. coli isolates among sites. We also sampled Pacific salmon (Oncorhynchus spp.) harvested as part of personal‐use dipnet fisheries at two sites to assess potential contamination with AMR E. coli. Among our study sites, marked gulls most commonly occupied the lower Kenai River (61% of site locations) followed by the Soldotna landfill (11%), lower Kasilof River (5%) and upper Kenai River (<1%). Gulls primarily moved between the Soldotna landfill and the lower Kenai River (94% of transitions among sites), which were also the two locations with the highest prevalence of AMR E. coli. There was relatively high spatial and temporal variability in AMR E. coli prevalence in gull faeces and there was no evidence of contamination on salmon harvested in personal‐use fisheries. We identified E. coli sequence types and AMR genes of clinical importance, with some isolates possessing genes associated with resistance to as many as eight antibiotic classes. Our findings suggest that gulls acquire AMR E. coli at habitats with anthropogenic inputs and subsequent movements may represent pathways through which AMR is dispersed.     

Long-term Changes in Littoral Habitats and Communities in Lake Mikolajskie (Poland)

Lake Mikolajskie is seriously affected by Mikolajki town and tourism. The amount of sewage entering the littoral zone is increasing as also is the area of the artificially transformed shore. During the past 20 years the area occupied by macrophytes has decreased markedly. The decline of Phragmites australis, changes in species composition, and reductions in biomass and in the depth-range colonized by submerged plants have been observed. Simultaneously the biomass of algae has increased markedly. Animals characteristic of polluted sites have become more abundant. Changes in animal numbers result to a great extent from changes in the availability of natural substrates. Zonation of the occurrence of organisms has been observed for years on sites directly affected by sewage—quantitatively and qualitatively poor communities are found at sewage discharge points and the richer ones at a greater distance in the zone fertilized by diluted sewage.     

Particulate organic and dissolved inorganic carbon stable isotopic compositions in Taylor Valley lakes,Antarctica: the effect of legacy

In perennially ice-covered lakes of Taylor Valley, Antarctica, “legacy”, a carryover of past ecosystem events, has primarily been discussed in terms of nutrient and salinity concentrations and its effect on the current ecology of the lakes. In this study, we determine how residual pools of ancient carbon affect the modern carbon abundance and character in the water columns of Lakes Fryxell, Hoare, and Bonney. We measure the stable carbon isotopic compositions and concentrations of particulate organic carbon (POC) and dissolved inorganic carbon (DIC) in the water column of these lakes over four seasons (1999–2002). These data are presented and compared with all the previously published Taylor Valley lacustrine carbon stable isotopic data. Our results show that the carbon concentrations and isotopic compositions of the upper water columns of those lakes are controlled by modern processes, while the lower water columns are controlled to varying degrees by inherited carbon pools. The water column of the west lobe of Lake Bonney is dominated by exceptionally high concentrations of DIC (55,000–75,000 μmol l⁻¹) reflecting the long period of ice-cover on this lake. The east lobe of Lake Bonney has highly enriched δ¹³C_DIC values resulting from paleo-brine evaporation effects in its bottom waters, while its high DIC concentrations provide geochemical evidence that its middle depth waters are derived from West Lake Bonney during a hydrologically connected past. Although ancient carbon is present in both Lake Hoare and Lake Fryxell, the δ¹³C_DIC values in bottom waters suggest dominance by modern primary productivity-related processes. Anaerobic methanogenesis and methanotrophy are also taking place in the lower water column of Lake Fryxell with enough methane, oxidized anaerobically, to contribute to the DIC pool. We also show how stream proximity and high flood years are only a minor influence on the carbon isotopic values of both POC and DIC. The Taylor Valley lake system is remarkably stable in both inter-lake and intra-lake carbon dynamics. Handling editor: K. Martens     

Isolation and functional expression of the bop gene from Halobiforma lacisalsi

A novel bop gene was described from Halobiforma lacisalsi strain AJ5^T, an extremely halophilic archaeon isolated from Ayakekum Lake, China. Following six rounds of PCR amplification based on the conserved fragment of the bop gene, the complete sequence of the bop gene, including the 5′ and 3′ flanking regions of the conserved fragment, was obtained by the ligation-mediated PCR amplification (LPA) approach. The data presented provide us with further insight into the distribution of bop-like genes in the family Halobacteriaceae. This is the first example of a bop-like gene in halophiles found in the high-pH environment. Alignment and hydropathy analysis of the deduced amino acid sequence identified the conserved functional sites as well as some variations compared with other bacterio-opsins. Molecular phylogenetic analysis revealed the position of the bacterio-opsin of strain AJ5, which is closest to that of Haloterrigena sp. arg-4 with 85% identity. In the presence of all-trans retinal, recombinant Escherichia coli cells expressing the gene turned dark purple. The purple membrane from the recombinant E. coli showed maximal absorption at 540 nm.     

Multiplex restriction amplicon sequencing: a novel next‐generation sequencing‐based marker platform for high‐throughput genotyping

To enable rapid selection of traits in marker‐assisted breeding, markers must be technically simple, low‐cost, high‐throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’‐ends, preceded by 6‐10 bases of specific or degenerate nucleotide sequences and then by a unique M13‐tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next‐generation sequencing‐based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding and many other genetics and breeding applications of various species.