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Using Next-Generation Sequencing to detect mutations endowing resistance to pesticides: application to acetolactate-synthase (ALS) based resistance in barnyard-grass, a polyploid grass weed.
Pest Management Science
Abstract
Background
The Next Generation Sequencing (NGS) technologies offer tremendous possibilities for accurate detection of mutations endowing pesticide resistance. Yet, their use for this purpose has not emerged in crop protection. This study aims at promoting NGS use for pesticide resistance diagnosis. It describes a simple procedure accessible to virtually any scientist and implementing freely accessible programs for the analysis of NGS data.ResultsThree PCR amplicons encompassing seven codons of the acetolactate-synthase gene crucial for herbicide resistance were sequenced using non-quantified pools of crude DNA extracts from 40 plants in each of 28 field populations of barnyard-grass, a polyploid weed. A total of 63,959 quality NGS sequence runs were obtained using the 454 technology. Three herbicide resistance-endowing mutations (Pro-197-Ser, Pro-197-Leu and/or Trp-574-Leu) were identified in seven populations. The NGS results were confirmed by individual plant Sanger sequencing.Conclusion
This work demonstrated the feasibility of NGS-based detection of pesticide resistance, and the interest of NGS compared to other molecular biology techniques for analysing large numbers of individuals. NGS-based resistance diagnosis has the potential to play a substantial role in monitoring resistance, maintaining pesticide efficacy and optimising pesticide applications.
... target-site resistance, TSR), that change specific amino acids of the target protein (ALS), thus reducing its affinity for this class of herbicides [7]. So far, TSR to ALS inhibitors in Echinochloa involve three amino acid positions : Ala-122, with three allelic variants (Val [8], Thr [8] and Asn [9], in E. crusgalli), Pro-197, with two allelic variants (Ser and Leu, in E. cru-galli [10]), and Trp-574, with one allelic variant (Leu, in E. crus-galli [10,11] and E. oryzicola [12]). ...
... target-site resistance, TSR), that change specific amino acids of the target protein (ALS), thus reducing its affinity for this class of herbicides [7]. So far, TSR to ALS inhibitors in Echinochloa involve three amino acid positions : Ala-122, with three allelic variants (Val [8], Thr [8] and Asn [9], in E. crusgalli), Pro-197, with two allelic variants (Ser and Leu, in E. cru-galli [10]), and Trp-574, with one allelic variant (Leu, in E. crus-galli [10,11] and E. oryzicola [12]). ...
... Although NTSR mechanisms cannot be ruled out [10,41], TS ALS1 mutations seem to be the most evolutionarily favored mechanism endowing ALS inhibitor resistance in Echinochloa spp. It would be interesting to conduct a broader survey of Echinochloa spp. to determine whether resistance-conferring mutations in the other ALS copies are occasionally present and, if so, to quantify the level of whole-plant resistance that they confer. ...
The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target-site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance.
... Ten cases of ALS inhibitor resistance and 12 cases of ACCase inhibitor resistance have been recorded in this species [3] and include cases of multiple-herbicide resistance to ALS and ACCase inhibitors [10][11][12]. Less frequent but significant cases of herbicide resistance in E. phyllopogon have been found in the USA, Korea, Greece and France [3,[13][14][15]. The USA and Korean populations exhibit multipleherbicide resistance to ALS and ACCase inhibitors. ...
... Recently, several groups have reported on the molecular mechanisms of ALS inhibitor resistance in Echinochloa spp. Substitutions within ALS amino acid residues have been found, although no studies thus far have included analyses of ALS gene copy numbers or have identified which copies of the genes carry mutations [10,13,[15][16][17]. In addition, we have partly elucidated the mechanism for NTSR in a case of ALS inhibitor resistance in the USA biotype of E. phyllopogon [18]. ...
... On the other hand, the presence of only three copies of the ALS genes in E. crus-galli might not be sufficient to constrict the evolution of target-site-based ALS inhibitor resistance. Resistance-conferring mutations in at least a single ALS gene have been reported in some populations of E. crus-galli in Italy, the USA, Brazil and France, although the involvement of other additive mechanisms cannot be ruled out as suggested in the USA populations [10,[15][16][17]. ...
Biotypes of Echinochloa crus-galli var. formosensis with resistance to cyhalofop-butyl, an acetyl-CoA carboxylase (ACCase) inhibitor, have been found in dry-seeded rice fields in Okayama, Japan. We collected two lines with suspected resistance (Ecf27 and Ecf108) from dry-seeded rice fields and investigated their sensitivity to cyhalofop-butyl and other herbicides. Both lines exhibited approximately 7-fold higher resistance to cyhalofop-butyl than a susceptible line. Ecf108 was susceptible to penoxsulam, an acetolactate synthase (ALS) inhibitor. On the other hand, Ecf27 showed resistance to penoxsulam and two other ALS inhibitors: propyrisulfuron and pyriminobac-methyl. The alternative herbicides butachlor, thiobencarb, and bispyribac-sodium effectively controlled both lines. To examine the molecular mechanisms of resistance, we amplified and sequenced the target-site encoding genes in Ecf27, Ecf108, and susceptible lines. Partial sequences of six ACCase genes and full-length sequences of three ALS genes were examined. One of the ACCase gene sequences encodes a truncated aberrant protein due to a frameshift mutation in both lines. Comparisons of the genes among Ecf27, Ecf108, and the susceptible lines revealed that none of the ACCases and ALSs in Ecf27 and Ecf108 have amino acid substitutions that are known to confer herbicide resistance, although a single amino acid substitution was found in each of three ACCases in Ecf108. Our study reveals the existence of a multiple-herbicide resistant biotype of E. crus-galli var. formosensis at Okayama, Japan that shows resistance to cyhalofop-butyl and several ALS inhibitors. We also found a biotype that is resistant only to cyhalofop-butyl among the tested herbicides. The resistance mechanisms are likely to be non-target-site based, at least in the multiple-herbicide resistant biotype.
Copyright © 2015 Elsevier Inc. All rights reserved.
... The first one described the detection of mutations at seven codons of one gene involved in herbicide resistance in two polyploid Echinochloa species using the now obsolete 454 sequencing technology. 8 A total of 1120 individual plants were screened as 28 bulks, each corresponding to one field population. 454 reads corresponding to each population were www.soci.org ...
... Herein, we targeted 15 codons in one ALS gene in 96 batches of 50 plants (ragweed) and in two ALS homeologs in 96 other batches of 50 plants (groundsel), which surpasses all preceding studies having applied NGS to pesticide resistance detection. [8][9][10] This merely necessitated 192 DNA extractions followed by the production of three amplicons, each in triplicate PCR reactions per extraction, i.e., 1728 PCRs, and two Illumina MiSeq runs. (It should be mentioned that, given the sequencing depth achieved, one single such run would have been sufficient to analyze all 192 pools). ...
BACKGROUND
Next Generation Sequencing (NGS) technologies offer tremendous possibilities for high‐throughput pesticide resistance diagnosis via massive genotyping‐by‐sequencing. Herein, we used Illumina sequencing combined with a simple, non‐commercial bioinformatics pipe‐line to seek mutations involved in herbicide resistance in two weeds.
RESULTS
DNA was extracted from 96 pools of 50 plants for each species. Three amplicons encompassing 15 ALS (acetolactate‐synthase) codons crucial for herbicide resistance were amplified from each DNA extract. Above 18 and 20 million quality 250‐nucleotide sequence reads were obtained for groundsel (Senecio vulgaris, tetraploid) and ragweed (Ambrosia artemisiifolia, diploid), respectively. Herbicide resistance‐endowing mutations were identified in 45 groundsel and in eight ragweed field populations. The mutations detected and their frequencies assessed by NGS were checked by individual plant genotyping or Sanger sequencing. NGS results were fully confirmed, except in three instances out of 12 where mutations present at a frequency of 1% were detected below the threshold set for reliable mutation detection.
CONCLUSION
Analyzing 9600 plants requested 192 DNA extractions followed by 1728 PCRs and two Illumina runs. Equivalent results obtained by individual analysis would have necessitated 9600 individual DNA extractions followed by 216 000 genotyping PCRs, or by 121 500 PCRs and 40 500 Sanger sequence runs. This clearly demonstrates the interest and power of NGS‐based detection of pesticide resistance from pools of individuals for diagnosing resistance in massive numbers of individuals. © 2019 Society of Chemical Industry
... 32 The 454-technology has been used as an efficient method in detecting mutations of the ALS gene in ALS-inhibitor-resistant weeds. 33 However, the Illumina MiSeq platform provides greater read depth, fewer sequencing errors, and lower per-base cost than 454 technology. 32,34 Illumina MiSeq is performed on a bench-top device and is designed for speed and simplicity for targeted resequencing, for example PCR amplicons and small-genome sequencing such as microorganisms, mitochondrial, and chloroplast genomes. ...
... As the average pool size was 24 samples, the expected frequency was 1 of 48 alleles. 33 In comparison with the waterhemp PPX2 allele, the Palmer amaranth-type has two repeats and the tumble pigweed-type has only one repeat in the R128 region. The frequency of ATGTA and TTGTAT repeats from MiSeq results of each population were observed in IGV software (Table S1). ...
BACKGROUND
Waterhemp (Amaranthus tuberculatus (Moq.) J. D. Sauer) is one of the most pernicious weeds in cropping systems of the USA due to its evolved resistance against several herbicide sites‐of‐action, including protoporphyrinogen oxidase inhibitors (PPO‐R). Currently, the only source of PPO‐R documented in waterhemp is ΔG210 of PPX2. Gene flow may not only lead to a transfer of herbicide‐resistant alleles, but also produce a hybrid genotype more competitively fit than one or both parents. However, investigating gene flow of Amaranthus species has been of interest in the past two decades with limited evidence.
RESULTS
Here, a high‐throughput MiSeq amplicon sequencing method was used to investigate alterations of the PPX2 gene in 146 PPO‐R waterhemp populations across five Midwest states of the USA. Five R128 codons of PPX2, novel to waterhemp, were found including AGG (R), GGA (G), GGG (G), AAA (K) and ATA (I). R128G, R128I, and R128K were found in 11, 3, and 2 populations, respectively. R128G and R128I, but not R128K, conferred fomesafen resistance in a bacterial system. Sequence alignment of the R128 region of PPX2 identified a tumble pigweed (Amaranthus albus)‐type and Palmer amaranth (Amaranthus palmeri)‐type PPX2 allele to be present and widespread in the surveyed waterhemp populations, thus providing strong evidence of gene flow between Amaranthus species.
CONCLUSION
Using a next‐generation sequencing method, we identified two PPO target‐site mutations R128G/I novel to waterhemp and provided evidence of gene flow of Amaranthus species in a large group of screened waterhemp populations from five Midwest states of the USA. © 2019 Society of Chemical Industry
... To date, 12 different ALS gene mutations have been reported to endow resistance to ALS inhibitors in Echinochloa spp. (Table 1) for TSR mechanism (Amaro-Blanco et al., 2021;Deĺye et al., 2015;Fang et al., 2019a, Fang et al., 2019bFeng et al., 2022;Liu et al., 2019;Panozzo et al., 2017;Tranel et al., 2024). Among these, mutations at positions Pro-197 and Trp-574 have commonly been documented in resistant weeds (Tranel et al., 2024). ...
Recently, due to the widespread use of the acetolactate synthase (ALS)-inhibiting herbicide penoxsulam in paddy fields in China, Echinochloa crus-galli (L.) P. Beauv. has become a problematic grass weed that is frequently not controlled, posing a threat to weed management and rice yield. There are many reports on target-site mutations of ALS inhibiting herbicides; however, the detailed penoxsulam resistance mechanism in E. crus-galli remains to be determined. Greenhouse and laboratory studies were conducted to characterize target-site resistance mechanisms in JL-R, AH-R, and HLJ-R suspected resistant populations of E. crus-galli survived the field-recommended dose of penoxsulam. The whole-plant dose–response testing of E. crus-galli to penoxsulam confirmed the evolution of moderate-level resistance in two populations, JL-R (9.88-fold) and HLJ-R (8.66-fold), and a high-level resistance in AH-R (59.71-fold) population. ALS gene sequencing identified specific mutations in resistant populations, including Pro-197-His in ALS1 for JL-R, Trp-574-Leu in ALS1 for AH-R, and Pro-197-Leu in ALS2 for HLJ-R. In vitro ALS activity assays demonstrated a significantly higher activity in AH-R compared to the susceptible population (YN-S). Molecular docking studies revealed that Trp-574-Leu mutation primarily reduced the enzyme’s ability to bind to the triazole-pyrimidine ring of penoxsulam due to decreased π–π stacking interactions, while Pro-197-His/Leu mutations impaired binding to the benzene ring by altering hydrogen bonds and hydrophobic interactions. Additionally, the Pro-197-His/Leu amino acid residue changes resulted in alterations in the shape of the active channel, impeding the efficient entry of penoxsulam into the binding site in the ALS protein. The three mutant ALS proteins expressed via the Bac-to-Bac baculovirus system exhibited notably lower activity inhibition rates than the non-mutant ALS proteins to penoxsulam, indicating all three ALS mutations reduce sensitivity to penoxsulam. This study elucidated the distinct impacts of the Pro-197-His/Leu and Trp-574-Leu mutations in E. crus-galli to penoxsulam resistance. Notably, the Trp-574-Leu mutation conferred stronger resistance to penoxsulam compared to the Pro-197-His/Leu mutations in E. crus-galli. The Pro-197-His/Leu mutations were first detected in E. crus-galli conferring penoxsulam resistance. These findings provide deeper insights into the molecular mechanisms underlying target-site resistance to penoxsulam in E. crus-galli.
... 8 They include restriction fragment length polymorphism, PCR amplification of specific alleles, cleaved amplified polymorphic sequences (CAPS), derived CAPS, real-time PCR and next-generation sequencing. 9 Very recently, also droplet digital PCR 10 and third generation long-read sequencing technologies (e.g. Pacific Biosciences Menlo Park, CA, USA; and Oxford Nanopore Technologies, Oxford, UK) have been applied to detect herbicide resistance. ...
BACKGROUND
The early detection of herbicide resistance in weeds is a key factor to avoid herbicide waste and improve agriculture sustainability. The present study aimed to develop and validate an allele‐specific loop‐mediated isothermal amplification (AS‐LAMP) assay for the quick on‐site detection of the resistance‐endowing point mutation Trp‐574‐Leu in the acetolactate synthase (ALS) gene in three widely diffused Amaranthus weed species: Amaranthus retroflexus, Amaranthus hybridus and Amaranthus tuberculatus.
RESULTS
The AS‐LAMP protocol was developed on wild‐type and ALS‐mutant plants of the three species and revealed that the amplification approach with only the primer set specific for the mutant allele (574‐Leu) was the most promising. The validation and estimation of the AS‐LAMP performance evaluated by comparing the results with those of the molecular marker (cleaved amplified polymorphic sequences) indicated that, although the sensitivity and specificity were relatively high in all species (overall 100 and > 65%, respectively), precision was high for A. hybridus L. and A. retroflexus L. (75 and 79%, respectively), but quite low for A. tuberculatus (Moq.) J. D. Sauer (59%). The LAMP assay was also effective on crude genomic DNA extraction, allowing the quick detection of mutant plants in field situation (on site resistance detection).
CONCLUSION
The proposed AS‐LAMP method has proven to be a promising technique for rapid detection of resistance as a result of Trp‐574‐Leu on the two monoecious weedy Amaranthus species but resulted less effective in the genetically variable dioecious species A. tuberculatus. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
... [50]. Genome-wide signatures had been used to detect 2,4-D resistance in Trifolium pratense [51], and the next-generation sequencing had been reported to detect ALS inhibitor resistance in E. crusgalli [52]. However, these methods are mutation-specific and limited to known mutations. ...
In this study, rapid resistance in-season quick (RISQ) tests were developed for detecting the resistance in Echinochloa spp. to penoxsulam, metamifop, and quinclorac, which are widely used in rice fields to control E. spp. biotypes. Seedlings in 1–2 leaf stages from nine biotypes of E. crusgalli, E. crusgalli var. zelayensis, and E. glabrescens, with different susceptibility to the three herbicides tested, were transplanted to plates containing nutrient agar and different rates of herbicides. The survival rates were recorded at 8 days after treatment when no more new roots emerged for all the treatments. By comparing the results from RISQ tests and whole-plant pot bioassays statistically, discrimination rates could be determined to distinguish resistant plants from susceptible plants. For penoxsulam, metamifop, and quinclorac, the discrimination rates were 0.3, 0.6, and 2.4 µmol/L, respectively. Two additional biotypes of E. crusgalli collected in rice fields were used to confirm the validation of the RISQ test and the obtained results by the RISQ test were consistent with that of the whole-plant pot bioassay. Therefore, the developed RISQ test would be a possible alternative method to determine the susceptibility of E. spp. to certain herbicides.
... Pool sequencing with Illumina short reads offers a cost-and time-saving option for the analysis of many individuals by combining barcoded DNA from multiple samples before sequencing (Ferretti et al., 2013;Schlötterer et al., 2014). This has included pooled amplicon sequencing approaches for resistance diagnosis assays in multiple species (Délye et al., 2020(Délye et al., , 2015Schlipalius et al., 2019). Unfortunately, due to the limited read lengths (from 50 to 300 bases in paired-end mode), to preserve haplotype information of an entire gene, variant calls have to be phased based on known patterns of linkage disequilibrium, with phasing accuracy depending on the co-occurrence of variants within paired-end reads. ...
Rapid adaptation of weeds to herbicide applications in agriculture through resistance development is a widespread phenomenon. In particular, the grass Alopecurus myosuroides is an extremely problematic weed in cereal crops with the potential to manifest resistance in only a few generations. Target‐site resistances (TSRs), with their strong phenotypic response, play an important role in this rapid adaptive response. Recently, using PacBio's long‐read amplicon sequencing technology in hundreds of individuals, we were able to decipher the genomic context in which TSR mutations occur. However, sequencing individual amplicons is costly and time consuming, thus impractical to implement for other resistance loci or applications. Alternatively, pool‐based approaches overcome these limitations and provide reliable allele frequencies, albeit at the expense of not preserving haplotype information. In this proof‐of‐concept study, we sequenced with PacBio High Fidelity (HiFi) reads long‐range amplicons (13.2 kb) encompassing the entire ACCase gene in pools of over hundred individuals, and resolved them into haplotypes using the clustering algorithm PacBio amplicon analysis (pbaa), a new application for pools in plants and other organisms. From these amplicon pools, we were able to recover most haplotypes from previously sequenced individuals of the same population. In addition, we analyzed new pools from a Germany‐wide collection of A. myosuroides populations and found that TSR mutations originating from soft sweeps of independent origin were common. Forward‐in‐time simulations indicate that TSR haplotypes will persist for decades even at relatively low frequencies and without selection, highlighting the importance of accurate measurement of TSR haplotype prevalence for weed management.
... The trait of resistance is one that has developed over the course of evolutionary history. Herbicides such as ALS inhibitors and ACCase inhibitors have the ability to select resistant biotypes within one to five election cycles, which renders the continued use of herbicides worthless and leads to a rise in resistance [ 7 ] .Target site resistance refers to a mutation that occurs in genes that encode target enzymes and can cause weeds to be resistant to herbicides such as ALS and ACCase-inhibiting herbicides (TSR). In most cases, resistance to particular herbicide compounds is conferred on the weed L. rigidum by mutations at specific locations in the ALS or ACCase genes L. rigidum [ 8 ]. ...
During the winter agricultural season 2021-2022, a field experiment was conducted in one of the fields located in the Al-Hindiyah District of the Holy Karbala Governorate. The purpose of the experiment was to study potential mutations that could make plants resistant to the action of certain herbicides belonging to the genus Lolium. The experiment was designed according to the randomized complete block design (R.C.B.D) with split plots arrangement and three replications, as the main plots included four different types of herbicides, namely Chevalier, Pallas, Tobik, and Axial, along with a comparison treatment (spraying with water only), while the sub plts included planting Lolium bush seeds. The holy province of Karbala, the province of Babylon, and the province of Wasit are the three provinces that make up this grouping in the Iraqi country of Iraq. The results revealed mutations in the ALS gene in the mRNA sequences of plants from Karbala, Wasit, and Babylon that are resistant to the pesticides Chevalier and Pallas, by substituting nitrogenous bases with other bases in codons 195 and 200 of AGC and AGT, which encode for the amino acid Ser to ACC, and GGT, which encodes for Thr and Gly. Iraqi weed mutations matched global mutations. Two missense mutations were found in codons 175 and 266 of the succession of Wasit governorate plants, by substituting codons GTC and CGC, which encode for Val and Arg, by codons GGC and TGC, which encode for Gly and Cys. The results of this gene in the weed (Lolium temulentum) were identical to the weed Lolium rigidum, except for one silent mutation at codon number 150 GCT that encodes for the amino acid Ala. This codon was changed to GCC in the weed of Wasit province. The results revealed a number of mutations resistant to the action of the two pesticides, Tobic and Axial, in the ACCase gene in the mRNA sequences of the plants of the Holy Karbala, Wasit and Babel governorates. These mutations are similar to the mutation in the resistant plants recorded globally, by substitution of nitrogenous bases with others in codons 25, 29, 33, 167, and 209 from CGA, GAA, AAT, TTG, and ACA, These mutations in the Iraqi weed coincided with mutations recorded globally, and the results showed that there is a missense mutation in codon 161 of the sequence of Wasit and Karbala pesticide-resistant plants, by substituting two nitrogenous bases from AAG, which encodes Lys, to AGA, which encodes Arg. In Karbala and Wasit, pesticide-resistant plants had two frame shift mutations.
... a) Next Generation Sequencing (NGS): NGS has been recognised for studies associated with resistance, pesticide use, and associated problems. Furthermore, NGS-based studies for analysing increased numbers of mutation have better prospects compared with molecular techniques (Délye et al. 2015). b) Targeting Induced Local Lesions in Genomes (TILLING): ...
... Targeted sequencing can detect SDN1 as well as SDN2 and SDN3 edits. The primary consideration for using targeted sequencing for detection of rare edits in tissue culture is the depth of sequencing to cover the necessary statistical power of detection of low frequency edits (Délye et al. 2015). Increasing the sequencing depth will increase the statistical power of detection of rare mutations, but also increases the cost of sequencing (Liu et al. 2014). ...
Genome editing (also known as gene editing) employs a range of tools such as Meganucleases, Zinc Finger Nucleases, TALENs, and more recently CRISPR to make defined changes in genes, regulatory sequences, untranslated regions, or intergenic regions. It is increasingly being applied in plant science research and to improve plant varieties. The benefits of having effective detection tools begin with optimization of the genome editing process itself and continue with selection and characterization of tissue cultures and/or regenerated plants. Detection tools are also used throughout the breeding process, and for preparation of regulatory dossiers when required, as well as for seed production, and may be necessary for monitoring products in the marketplace. Detection and identification of genome edits employs a wide range of analytical approaches including PCR, digital PCR, and sequencing methods. This article examines the applicability of each category of detection or identification approach, from the optimization of genome editing processes, through creation of edits, selection and characterization, and breeding. The challenges surrounding the detection of genome edits present at low levels in large seed, plant, or grain populations and of differentiating directed genome edits from conventional mutations are also explained.
... Depending on the derived amino acid, mutation at the position Ala-122 confers a broad spectrum of (cross-) resistance to ALS inhibitors (Riar et al. 2013;Panozzo et al. 2017). Mutations at Pro-197 provide high resistance to sulfonylureas (Délye et al. 2015), and mutations at Trp-574 confer resistance to sulfonylureas and imidazolinones (Panozzo et al. 2013;Yu and Powles 2014) as well as the triazolopyrimidine and pyrimidinylthiobenzoate herbicides (Patzold and Tranel 2002). ...
Echinochloa crus-galli (L.) Pal. Beauv. is one of the most noxious weeds in maize cultivation and has evolved target-site resistance to ALS-inhibiting herbicides. Due to the hexaploid nature of E. crus-galli, resistance inducing mutations can be harbored by multiple gene copies, but up to now, studies did not include an analysis of the ALS gene copies conferring resistance. Investigations on E. crus-galli populations from different countries in Central Europe revealed the presence of several point mutations conferring resistance and occurring independently at the positions Ala-122, Pro-197 and Trp-574. Moreover, an Asp-376-Glu mutation in the ALS gene in E. crus-galli and a Ser-653-Tyr amino acid change in the ALS protein of a weed were detected for the first time. Additionally, the study revealed the first case of an ALS-resistant population (Trp-574-Leu) in the Czech Republic. The detection of mutations is relevant to the evolution and management of herbicide resistance. Dose–response experiments showed different effects depending on the population and the individual active ingredients and mixtures. Due to the genetic variability of E. crus-galli, results were highly variable. Overall, the populations showed a high level of resistance. For the prevailing mutated positions, i.e., Ala-122, Pro-197 and Trp-574, gene copies were examined separately using molecular genetic methods. A single mutation in at least one out of three ALS gene copies was sufficient to confer resistance at the positions Pro-197 and Trp-574. At Ala-122, point mutations co-occurring in the ALS 1, ALS 2 and ALS 3 gene copy were identified. The study provides a starting point for differentiation of the resistance mechanisms in polyploid E. crus-galli.
... A resistência foi quantificada pelo Factor de Resistência (FR=ED50 R/ED50 S) -proporção entre as doses que provocam o mesmo efeito em populações resistentes (R) e suscetíveis (S) ao herbicida ( Oliveira et al., 2016 c O FR ao penoxsulame variou entre 18,6 e 27,8, confirmando a resistência ao penoxsulame destas três populações de milhãs. Estes valores estão em concordância com os referidos na bibliografia (Yasuor et al., 2009;Panozzo et al., 2013;Delye et al., 2014). Nenhuma das populações apresentou resistência cruzada ao profoxidime, herbicida com diferente modo de ação. ...
INTRODUÇÃO A produtividade do arroz é significativamente afectada pelo poten-cial da cultivar e pelo grau de competição das infestantes. Quarenta a 45 espécies infestam, em maior ou menor grau, os arrozais portu-gueses durante todo o seu ciclo (Catarino et al.,2005). Nos arrozais do Ribatejo as espécies infestantes mais importantes são o arroz-bravo (Oryza sativa L.) e as milhãs-milhã-peluda (Echinochloa phyllopo-gon (Stapf) Stapf ex Kossenko) e milhã-branca ou milhã-do-arroz (E. crus-galli (L.) P. Beauv. subsp. hispidula (Retz.) Honda). Novas es-pécies, como leptocloa (Leptochloa fusca (L.) Kunthsubsp. fascicularis (Lam.) N. Snow) começam a tornar-se problemáticas pela sua impor-tância crescente e dificuldade no controlo (Oliveira, 2016)-Figura 1. De todas as espécies, o grupo das milhãs representa a componente mais importante da flora infestante, pelo seu potencial competitivo, pela dificuldade no combate, visto pertencerem à mesma família botânica da cultura com exigências térmicas e características morfológicas simila-res (Vasconcelos et al., 1999). Um levantamento detalhado por todo o País revelou a existência de um complexo de espécies, designadamente Vasinger)) (Medina,2005). Outras espécies foram também identificadas mas com relevância ape-nas a nível regional: E. oryzoides (Ard.) Fritsch (milhã-do-arroz) e E. co-lonum (L.) Link (Vasconcelos e Forte, 1999; Vasconcelos et al., 1999). As perdas de produção provocadas pela competição com as milhãs podem atingir 38%, enquanto que para a negrinha (Cyperus difformis L.) as perdassão de 23% (Catonet al., 2001). As infestantes de baixo porte como a falsa-alisma (Heteranthera limosa (Sw.) Willd.)) e orelha-de-mu-la (Alisma plantago-aquatica) são pouco competitivas (Smith, 1988). (Quadro 1) Em Portugal estima-se que as perdas de produção provocadas pela competição com as infestantes sejam da ordem dos 20% (Silva, 1983) e, diferentemente de outras culturas, 100% da área cultivada de arroz é tratada com herbicidas (Nunes & Silva, 1996). Atualmen-te, o penoxsulame e o profoxidime estão entre as principais subs-tâncias ativas herbicidas utilizadas no controlo de milhãs em arroz. Figura 1-Principais infestantes dos arrozais do Tejo e Sorraia: ar-roz-bravo (Oryza sativa); milhã-peluda (Echinochloa phyllopogon); leptocloa (Leptochloa fusca subsp. fascicularis)
... Herbicide resistance in E. phyllopogon has been reported in populations in France, Greece, Brazil, South Korea, and the United States ( Délye et al. 2015;Kaloumenos et al. 2012;Matzenbacher et al. 2014;Song et al. 2017). The populations in France, Greece, and Brazil are resistant to ALS inhibitors due mainly to TSR mechanisms, although additional NTSR mechanisms were not excluded. ...
Several grass and broadleaf weed species around the world have evolved multiple-herbicide resistance at alarmingly increasing rates. Research on the biochemical and molecular resistance mechanisms of multiple-resistant weed populations indicate a prevalence of herbicide metabolism catalyzed by enzyme systems such as cytochrome P450 monooxygenases and glutathione S-transferases and, to a lesser extent, by glucosyl transferases. A symposium was conducted to gain an understanding of the current state of research on metabolic resistance mechanisms in weed species that pose major management problems around the world. These topics, as well as future directions of investigations that were identified in the symposium, are summarized herein. In addition, the latest information on selected topics such as the role of safeners in inducing crop tolerance to herbicides, selectivity to clomazone, glyphosate metabolism in crops and weeds, and bioactivation of natural molecules is reviewed.
... Next-generation sequencing-based assays have proven to be of great interest for this purpose. 47 Such assays can easily be developed for S. vulgaris, especially because of the absolute sequence conservation of ALS1 and ALS2 around this codon in S. vulgaris. ...
Following control failure by herbicides inhibiting acetolactate-synthase (ALS) in French wheat fields and vineyards, we aimed at confirming resistance evolution and investigating the evolutionary origin and spread of resistance in the tetraploid species Senecio vulgaris (common groundsel), a widespread, highly mobile weed.
Sequencing two ALS homeologs in S. vulgaris enabled the first identification and characterisation of ALS-based resistance in this species. Cross-resistance patterns associated with Leu-197 and Ser-197 ALS1 were established using eight herbicides. Sequencing and genotyping showed that ALS-based resistance evolved by multiple, independent appearances of mutant ALS1 and ALS2 alleles followed by spread. Spread of a mutant ALS1 allele issued from one particular appearance event was observed over 60 km. Independent resistance appearance events and easy seed dispersion are the most likely reasons for populations of S. vulgaris containing different mutant ALS alleles. Accumulation of different alleles likely due to sexual reproduction was observed in the same plant.
Mutant ALS alleles and possibly other mechanisms cause resistance to ALS inhibitors in S. vulgaris. Management strategies should aim at limiting S. vulgaris establishment and seed set. Considering the mobility of this species, control coordination at a regional level is clearly necessary if resistance spread is to be contained.
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... 5 To date, no complete genome has been assembled from any weed species, and only a small number of publications have reported the use of NGS for assembly and analysis of transcriptome or chloroplast genome, 6 -9 for marker development 7,8,10,11 and for addressing herbicide/pesticide resistance problems in weed species. 12 -14 Poa annua is one of the most widespread grass species worldwide. It is adapted to diverse environments, including extreme areas such as remote subarctic meadows, 15 the Antarctic Peninsula 16 and Mount Kilimanjaro in Africa close to the equator. ...
Background:
Detection of single nucleotide polymorphisms (SNPs) related to herbicide resistance in non-model polyploid weed species is fraught with difficulty owing to the gene duplication and lack of reference sequences. Our research seeks to overcome these obstacles by Illumina HiSeq read mapping, SNP calling and allele frequency determinations. Our focus is on the acetolactate synthase (ALS) gene, the target site of ALS-inhibiting herbicides, in Poa annua, an allotetraploid weed species originating from two diploid parents, P. supina and P. infirma.
Results:
ALS contigs with complete coding regions of P. supina, P. infirma and P. annua were assembled and compared with ALS genes from other plant species. The ALS infirma-homeolog of P. annua showed higher levels of nucleotide sequence variability than the supina-homeolog. Comparisons of read mappings of P. annua and a simulated P. supina × P. infirma hybrid showed high resemblance. Two homeolog-specific primer pairs were designed and used to amplify a 1860 bp region covering all resistance-conferring codons in the ALS gene. Four P. annua populations, GN, RB, GW and LG, showed high resistance to two ALS inhibitors, bispyribac-sodium and foramsulfuron, and two populations, HD and RS, showed lower resistance in the rate-response trial. Mutations conferring Trp-574-Leu substitution were observed in the infirma-homeolog of GN and RB and in the supina-homeolog of GW and LG, but no resistance-conferring mutation was observed in the two populations of lower resistance, HD and RS.
Conclusion:
In this study we have demonstrated the use of NGS data to study homeologous polymorphisms, parentage and herbicide resistance in an allotetraploid weed species, P. annua. Complete coding sequences of the ALS gene were assembled for P. infirma, P. supina, infirma-homeolog and supina-homeolog in P. annua. A pipeline consisting of read mapping, SNP calling and allele frequency calculation was developed to study the parentage of P. annua, which provided a new perspective on this topic besides the views of morphology, karyotype and phylogeny. Our two homeolog-specific primer pairs can be utilized in future research to separate the homeologs of the ALS gene in P. annua and cover all the codons that have been reported to confer herbicide resistance.
While genetic manipulation for improvement of agricultural crops is not new, technologies such as gene editing that allow precise and targeted gene modifications are becoming more common. Highly specific, sensitive, and practical methods to detect such edits are desirable for a number of reasons. In this chapter, we provide readers with an overview of their application and what different technology platforms that can used to develop detection methods for edits. The platforms include PCR, digital PCR and sequencing and are discussed in the context of their strengths and weakness. This chapter also includes a discussion on challenges to differentiate a genetic change created through gene-editing technologies from same changes that might arise through other means such as conventional breeding or mutagenesis.
American sloughgrass (Beckmannia syzigachne Steud.) has become a dominant weed in fields with rice-wheat rotation. Moreover, herbicide resistance has rendered weed control difficult. We identified a biotype showing resistance to ALS inhibitor mesosulfuron-methyl with a resistant index 3.3, but without any ALS mutation. This study aims to identify and confirm the factors associated with non-target site resistance of this biotype to mesosulfuron-methyl using RNA-Seq. 118,111 unigenes were assembled, and 50.9% of these were annotated across seven databases. Eleven contigs related to metabolic resistance were identified based on differential expression via RNA-Seq which include a novel resistance-related transcription factor (MYC3) and two disease resistance proteins were also identified (At1g58602 and At1g15890). Fold changes in expression of these genes in comparison M-R vs. M-S ranged from 3.9 to 11.6, as confirmed by qPCR. The expression of a contig annotated as cytochrome P450 (CYP86B1) in resistant individuals was over 3 times higher than that in sensitive individuals at 0–72 h after mesosulfuron-methyl treatment. A similar trend was noted for three other genes annotated as glutathione S-transferase (GST), namely GST-T3, GST-U6, and GST-U14; the expression of GST-U6 in resistant individuals was up to 142.3 times higher than that in sensitive individuals at 24 h after mesosulfuron-methyl treatment. In addition, GST activity in resistant individuals was 2.1 to 5.3 times higher than that in sensitive individuals. The GR50 of resistant biotype decreased from 24.4 to 11.3 g a.i. ha⁻¹ after P450 inhibitor malathion treatment. This study identified a cytochrome P450 gene CYP86B1 and three GST genes GST-T3, GST-U6, and GST-U14 that have higher expression in mesosulfuron-methyl resistant B. syzigachne, suggesting that both P450- and GST-based activities could be involved in resistance.
Resistance to pyrethroid insecticides is widespread among populations of Aedes aegypti, the main vector for the dengue virus. Several different point mutations within the voltage-gated sodium channel (VGSC) gene contribute to such resistance. A mutation at position 1016 in domain II, segment 6 of the VGSC gene in Ae. aegypti leads to a valine to glycine substitution (V1016G) that confers resistance to deltamethrin.
This study developed and utilized an allele-specific PCR (AS-PCR) assay that could be used to detect the V1016G mutation. The assay was validated against a number of sequenced DNA samples of known genotype and was determined to be in complete agreement. Larvae and pupae were collected from various localities throughout Thailand. Samples were reared to adulthood and their resistance status against deltamethrin was determined by standard WHO susceptibility bioassays. Deltamethrin-resistant and susceptible insects were then genotyped for the V1016G mutation. Additionally, some samples were genotyped for a second mutation at position 1534 in domain III (F1534C) which is also known to confer pyrethroid resistance.
The bioassay results revealed an overall mortality of 77.6%. Homozygous 1016G individuals survived at higher rates than either heterozygous or wild-type (1016 V) mosquitoes. The 1016G mutation was significantly and positively associated with deltamethrin resistance and was widely distributed throughout Thailand. Interestingly, wild-type 1016 V mosquitoes tested were homozygous for the 1534C mutation, and all heterozygous mosquitoes were also heterozygous for 1534C. Mutant homozygous (G/G) mosquitoes expressed the wild-type (F/F) at position 1534. However, the presence of the 1534C mutation was not associated with deltamethrin resistance.
Our bioassay results indicate that all populations sampled display some degree of resistance to deltamethrin. Homozygous 1016G mosquitoes were far likelier to survive such exposure. However, resistance in some populations cannot be explained due to kdr mutations and indicates that other resistance mechanisms are operating. The presence of this mutation alone does not fully explain the resistance phenotype we see among Thai Ae. aegypti populations.
Antiviral therapy for cytomegalovirus (CMV) plays an important role in the management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a bench-top next-generation sequencing instrument. Plasmid clones of the UL97 and UL54 genes were used to demonstrate a low overall empiric error rate of the assay (0.189%), and to develop a statistical algorithm for identifying authentic low-abundance variants. The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples known to contain mutations that confer resistance. Finally, 48 clinical plasma specimens with a range of viral loads (394 - 2,191,011 copies/mL plasma) were sequenced, using multiplexing of up to 24 specimens per run. This led to the identification of seven resistance mutations, three of which were present at < 20% of the sequenced population. Thus this assay offers more sensitive minor variant detection and increased multiplexing relative to current methods for genotypic detection of CMV drug resistance mutations.
The past 60 years have seen a revolution in our understanding of the molecular genetics of insecticide resistance. While at first the field was split by arguments about the relative importance of mono- vs. polygenic resistance and field- vs. laboratory-based selection, the application of molecular cloning to insecticide targets and to the metabolic enzymes that degrade insecticides before they reach those targets has brought out an exponential growth in our understanding of the mutations involved. Molecular analysis has confirmed the relative importance of single major genes in target-site resistance and has also revealed some interesting surprises about the multi-gene families, such as cytochrome P450s, involved in metabolic resistance. Identification of the mutations involved in resistance has also led to parallel advances in our understanding of the enzymes and receptors involved, often with implications for the role of these receptors in humans. This Review seeks to provide an historical perspective on the impact of molecular biology on our understanding of resistance and to begin to look forward to the likely impact of rapid advances in both sequencing and genome-wide association analysis.
Papaver rhoeas, an annual plant species in the Papaveraceae family, is part of the biodiversity of agricultural ecosystems and also a noxious agronomic weed. We developed microsatellite markers to study the genetic diversity of P. rhoeas, using an enriched microsatellite library coupled with 454 next-generation sequencing. A total of 13,825 sequences were obtained that yielded 1795 microsatellite loci. After discarding loci with less than six repeats of the microsatellite motif, automated primer design was successful for 598 loci. We tested 74 of these loci for amplification with a total of 97 primer pairs. Thirty loci passed our tests and were subsequently tested for polymorphism using 384 P. rhoeas plants originating from 12 populations from France. Of the 30 loci, 11 showed reliable polymorphism not affected by the presence of null alleles. The number of alleles and the expected heterozygosity ranged from 3 to 7.4 and from 0.27 to 0.73, respectively. A low but significant genetic differentiation among populations was observed (FST = 0.04; p < 0.001). The 11 validated polymorphic microsatellite markers developed in this work will be useful in studies of genetic diversity and population structure of P. rhoeas, assisting in designing management strategies for the control or the conservation of this species.
Background:
Studies were carried out to elucidate the mechanism of resistance to ALS-inhibiting herbicides in 29 Echinochloa accessions from water-seeded rice fields of northern Greece and to discriminate the Echinochloa species.
Results:
Two E. oryzicola accessions were found to be cross-resistant to penoxsulam, bispyribac-sodium, imazamox, foramsulfuron, nicosulfuron and rimsulfuron, whereas all accessions were susceptible (S) to profoxydim. Sequencing of the ALS gene revealed that resistant (R) accessions had a Trp574Leu mutation, which was also confirmed by TspRI endonuclease digestion. Use of cpDNA sequence comparison analysis of Echinochloa species discriminated successfully E. crus-galli and E. oryzicola accessions.
Conclusion:
This is the first report of Echinochloa oryzicola cross-resistance to ALS-inhibiting herbicides as a result of Trp574Leu mutation. The cpDNA sequence comparison analysis is a reliable tool for discrimination of conventionally classified E. crus-galli and E. oryzicola accessions.
Background:
Target-site resistance to ALS- and ACCase-inhibiting herbicides in the grass weed Alopecurus myosuroides is associated with well-characterised allelic variants encoding ALS- and ACCase-based resistance. The potential for combined ALS and ACCase resistance presents a threat to future control, given the extent to which these herbicides are used. The authors present a primer extension method for rapid detection of known resistance-conferring substitutions.
Results:
Individuals showing combined resistance to field-rate mesosulfuron + iodosulfuron and cycloxydim were identified in four field-collected populations, with proportions ranging from 30 to 100%. Genotyping with the SNaPshot primer extension kit showed the T197 and L574 ALS and L1781 ACCase isoforms to be associated with ALS and ACCase resistance whenever they occurred.
Conclusion:
Combined ALS and ACCase target-site resistance threatens future control of A. myosuroides. The SNaPshot extension assay provides a reliable new multiplexable method for characterising known allelic variants of the ALS and ACCase genes of A. myosuroides. The method offers significant advantages over both CAPS/dCAPS and PASA in that full genotyping can be accomplished at any nucleotide position using a single extension primer.
Many herbicide-resistant weed species are polyploids, but far too little about the evolution of resistance mutations in polyploids is understood. Hexaploid wild oat (Avena fatua) is a global crop weed and many populations have evolved herbicide resistance. We studied plastidic acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicide resistance in hexaploid wild oat and revealed that resistant individuals can express one, two or three different plastidic ACCase gene resistance mutations (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg). Using ACCase resistance mutations as molecular markers, combined with genetic, molecular and biochemical approaches, we found in individual resistant wild-oat plants that (1) up to three unlinked ACCase gene loci assort independently following Mendelian laws for disomic inheritance, (2) all three of these homoeologous ACCase genes were transcribed, with each able to carry its own mutation and (3) in a hexaploid background, each individual ACCase resistance mutation confers relatively low-level herbicide resistance, in contrast to high-level resistance conferred by the same mutations in unrelated diploid weed species of the Poaceae (grass) family. Low resistance conferred by individual ACCase resistance mutations is likely due to a dilution effect by susceptible ACCase expressed by homoeologs in hexaploid wild oat and/or differential expression of homoeologous ACCase gene copies. Thus, polyploidy in hexaploid wild oat may slow resistance evolution. Evidence of coexisting non-target-site resistance mechanisms among wild-oat populations was also revealed. In all, these results demonstrate that herbicide resistance and its evolution can be more complex in hexaploid wild oat than in unrelated diploid grass weeds. Our data provide a starting point for the daunting task of understanding resistance evolution in polyploids.Heredity advance online publication, 10 October 2012; doi:10.1038/hdy.2012.69.
The weed Senecio vulgaris acquired high levels of resistance to triazine herbicides soon after the latter's introduction. As in most weeds, triazine resistance is conferred by a point mutation in the chloroplast psbA gene that negatively affects the fitness of its carrier. To assess levels of triazine resistance in S. vulgaris field populations, we adopted a PCR-RFLP-based molecular diagnostic test recently developed for the triazine resistance-conferring region of the psbA gene of other weeds, including Brassica napus, Chenopodium spp. and Amaranthus spp., and compared these molecular results to the phenotypic response after triazine application. A highly significant linear correlation was found between phytotoxic symptoms and biomass reduction. Variability in phenotypic response was not only found between populations or inbred lines of S. vulgaris but also within replicates of the same inbred line. No clear relationship, however, was found between the DNA restriction pattern and the phenotypic response to triazine application, thereby throwing doubt on the use of such molecular diagnostic tests to track triazine resistance in S. vulgaris. Our results indicate that the chloroplast genome of S. vulgaris is polymorphic and that the level of polymorphism may be variable within single leaves of individual plants. We discuss the possible genetic basis of this polymorphism and its consequence for the acquisition and inheritance of chloroplast-based traits.
Even with recent reductions in sequencing costs, most plants lack the genomic resources required for successful short-read transcriptome analyses as performed routinely in model species. Several approaches for the analysis of short-read transcriptome data are reviewed for nonmodel species for which the genome of a close relative is used as the reference genome. Two approaches using a data set from Phytophthora-challenged Rubus idaeus (red raspberry) are compared. Over 70000000 86-nt Illumina reads derived from R. idaeus roots were aligned to the Fragaria vesca genome using publicly available informatics tools (Bowtie/TopHat and Cufflinks). Alignment identified 16956 putatively expressed genes. De novo assembly was performed with the same data set and a publicly available transcriptome assembler (Trinity). A BLAST search with a maximum e-value threshold of 1.0 × 10(-3) revealed that over 36000 transcripts had matches to plants and over 500 to Phytophthora. Gene expression estimates from alignment to F. vesca and de novo assembly were compared for raspberry (Pearson's correlation = 0.730). Together, alignment to the genome of a close relative and de novo assembly constitute a powerful method of transcriptome analysis in nonmodel organisms. Alignment to the genome of a close relative provides a framework for differential expression testing if alignments are made to the predefined gene-space of a close relative and de novo assembly provides a more robust method of identifying unique sequences and sequences from other organisms in a system. These methods are considered experimental in nonmodel systems, but can be used to generate resources and specific testable hypotheses.
The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.
Multiple sequence alignments are fundamental to many sequence analysis methods. Most alignments are computed using the progressive alignment heuristic. These methods are starting to become a bottleneck in some analysis pipelines when faced with data sets of the size of many thousands of sequences. Some methods allow computation of larger data sets while sacrificing quality, and others produce high-quality alignments, but scale badly with the number of sequences. In this paper, we describe a new program called Clustal Omega, which can align virtually any number of protein sequences quickly and that delivers accurate alignments. The accuracy of the package on smaller test cases is similar to that of the high-quality aligners. On larger data sets, Clustal Omega outperforms other packages in terms of execution time and quality. Clustal Omega also has powerful features for adding sequences to and exploiting information in existing alignments, making use of the vast amount of precomputed information in public databases like Pfam.
Here, we present PRINSEQ for easy and rapid quality control and data preprocessing of genomic and metagenomic datasets. Summary statistics of FASTA (and QUAL) or FASTQ files are generated in tabular and graphical form and sequences can be filtered, reformatted and trimmed by a variety of options to improve downstream analysis.
Availability and Implementation: This open-source application was implemented in Perl and can be used as a stand alone version or accessed online through a user-friendly web interface. The source code, user help and additional information are available at http://prinseq.sourceforge.net/.
Contact: rschmied@sciences.sdsu.edu; redwards@cs.sdsu.edu
Knockdown resistance (kdr) mutation (L1014F) is a well-defined mechanism of resistance to pyrethroids and DDT in many insect species. Sensitive detection of the mutations associated with resistance is a prerequisite for resistance management strategies. The authors have developed a new real-time molecular diagnostic assay based on SimpleProbe(®)/melting curve analysis for large-scale kdr genotyping in the wild population of Culex quinquefasciatus Say, the principal vector of bancroftian filariasis. Melting curve analysis is based on the thermal stability difference between matched and mismatched DNA duplexes. The application of SimpleProbe(®) chemistry in insects described here is novel in entomology research.
The mosquitoes homozygous for knockdown-resistant and knockdown-susceptible allele showed melting peaks at 60.45 °C (±0.25) and 64.09 °C (±0.24) respectively. The heterozygous mosquitoes yielded both peaks at approximately 60.5 °C (±0.2) and 64.20 °C (±0.23). Among the 92 samples genotyped, 16 were found to be homozygous resistant, 44 homozygous susceptible and 32 heterozygous. Comparative assessments were made of all the reported methods for kdr genotyping.
The present method is cheaper, faster, more reliable and versatile than other alternatives proposed in detecting correct kdr genotypes in mosquitoes. This is the first report using a single-labelled hybridisation probe to detect point mutations in insect populations.
We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.
Cultivars of several cereal crops have been developed with acetohydroxyacid synthase (AHAS) insensitivity to imidazolinone herbicides and are now an important tool for weed management. Options for screening for imidazolinone resistant lines include direct herbicide application, biochemical assays for AHAS activity, and DNA-based methods. Herbicide and biochemical assays for AHAS activity provide limited information as to mutation copy number and provide no information as to the genome on which the mutation is located without extensive test crossing. Wheat (Triticum aestivum L.) can have between one and six copies of the resistant acetohydroxyacid synthase on any of three genomes. A novel DNA-based screening protocol is described here in which pyrosequencing is used to screen for the S653N imidazolinone tolerant mutation in wheat. One assay is shown to successfully detect zero to four copies of the S653N mutation, while additional assays can detect the presence of S653N in individual wheat genomes. All of these assays are based on a single 298-bp polymerase chain reaction (PCR) fragment and can be easily scaled up or down depending on the number of lines that need to be screened. Potential applications include detection of mutant copy number in segregating populations, and the selection of parental lines with genome specific mutant composition.
Thiabendazole (TBZ) is commonly used as a postharvest treatment for control of blue mold in apples caused by Penicillium expansum. Different point mutations in the β-tubulin gene conferring benzimidazole resistance have been reported in plant pathogens, but molecular mechanisms of TBZ resistance in P. expansum from apple in Washington State are unknown. Determination of TBZ resistance level showed that all 102 TBZ-resistant (TBZ-R) isolates were highly resistant. Sequencing of the majority of the β-tubulin gene showed that 76 TBZ-R isolates harboured the E198V mutation and 26 harboured the F167Y mutation, and all the sensitive isolates did not possess any of the mutations, indicating that these two point mutations in the β-tubulin gene were correlated with TBZ resistance in P. expansum from apple in Washington State. There was no association between levels of TBZ resistance and types of point mutations (E198V or F167Y) in the β-tubulin gene. A multiplex allele-specific PCR assay was developed to detect these two mutations simultaneously. Microsatellite-primed PCR derived presence-absence matrix used to assess the genetic relationship among 56 isolates suggested that the resistance mutations originated several times independently and that there was no correlation between the types of point mutation and the genetic background of the isolates.
In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.
With no major new site-of-action herbicide introduced into the marketplace in the last 20 years, the stagnation or decline in available herbicides in the past decade in a number of jurisdictions, and ever-increasing incidence of herbicide-resistant (HR) weeds, more efficient use of our existing herbicide tools will be required to proactively or reactively manage HR weed populations. Herbicide-resistant weed management can be aided by crop cultivars with alternative single or stacked herbicide-resistance traits, such as synthetic auxins, which will become increasingly available to growers in the future. An examination of cross-resistance patterns in HR weed populations may inform proactive or reactive HR weed management through better insights into the potential for HR trait-stacked crops to manage HR weed biotypes as well as identify possible effective alternative herbicide options for growers. Clethodim is the lowest resistance risk acetyl-CoA carboxylase (ACC) inhibiting herbicide, with only two of eleven target-site mutations (amino acid substitutions) in weed populations that confer resistance. However, there are no reduced-risk acetolactate synthase/acetohydroxyacid synthase (ALS/AHAS) herbicides or herbicide classes. Growers will be increasingly reliant on reduced-risk herbicide sites of action (groups), such as microtubule assembly inhibitors (e.g., trifluralin, pendimethalin), synthetic auxins (e.g., 2,4-D, dicamba), some photosystem-II inhibitors (nitriles such as bromoxynil), protoporphyrinogen oxidase (PPO) or hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, glyphosate, or glutamine synthetase inhibitor (glufosinate), used in sequences, mixtures, or rotations, to manage HR weed populations.
Echinochloa crus-galli (L.) Beauv., barnyard grass (pied-de-coq), is an annual grass and a cosmopolitan weed with a range from 50°N to 40°S latitude. In Canada, it extends up to 53°N latitude in the prairies and is widely distributed along roadsides and in cultivated fields of all provinces. The success of this weed may be attributed to the production of large numbers of small, easily dispersed seeds per plant, possession of seed dormancy, rapid development and ability to flower under a wide range of photoperiods, and relative resistance of mature plants to herbicide sprays. In this review a summary of biological information on E. crus-galli is presented.Key words: Barnyard grass, weed biology, Echinochloa crus-galli
Weed resistance monitoring has been routinely conducted in the Northern Great Plains of Canada (Prairies) since the mid-1990s. Most recently, random surveys were conducted in Alberta in 2001, Manitoba in 2002, and Saskatchewan in 2003 totaling nearly 800 fields. In addition, nearly 1,300 weed seed samples were submitted by growers across the Prairies between 1996 and 2006 for resistance testing. Collected or submitted samples were screened for group 1 [acetyl-CoA carboxylase (ACCase) inhibitor] and/or group 2 [acetolactate synthase (ALS) inhibitor] resistance. Twenty percent of 565 sampled fields had an herbicide-resistant (HR) wild oat biotype. Most populations exhibited broad cross-resistance across various classes of group 1 or group 2 herbicides. In Manitoba, 22% of 59 fields had group 1-HR green foxtail. Group 2-HR biotypes of kochia were documented in Saskatchewan, common chickweed and spiny sowthistle in Alberta, and green foxtail and redroot pigweed in Manitoba. Across the Prairies, HR weeds are estimated to occur in fields covering an area of nearly 5 million ha. Of 1,067 wild oat seed samples submitted by growers and industry for testing between 1996 and 2006, 725 were group 1 HR, 34 group 2 HR, and 55 groups 1 and 2 HR. Of 80 submitted green foxtail samples, 26 were confirmed group 1 HR; most populations originated from southern Manitoba where the weed is most abundant. Similar to the field surveys, various group 2-HR biotypes were confirmed among submitted samples: kochia, wild mustard, field pennycress, Galium spp., common chickweed, and common hempnettle. Information from grower questionnaires indicates patterns of herbicide usage are related to location, changing with cropping system. Two herbicide modes of action most prone to select resistance, groups l and 2, continue to be widely and repeatedly used. There is little evidence that growers are aware of the level of resistance within their fields, but a majority have adopted herbicide rotations to proactively or reactively manage HR weeds.
Resistance to herbicides in arable weeds is increasing rapidly worldwide and threatening global food security. Resistance has now been reported to all major herbicide modes of action despite the development of resistance management strategies in the 1990s. We review here recent advances in understanding the genetic bases and evolutionary drivers of herbicide resistance that highlight the complex nature of selection for this adaptive trait. Whereas early studied cases of resistance were highly herbicide-specific and largely under monogenic control, cases of greatest concern today generally involve resistance to multiple modes of action, are under polygenic control, and are derived from pre-existing stress response pathways. Although 'omics' approaches should enable unraveling the genetic bases of complex resistances, the appearance, selection, and spread of herbicide resistance in weed populations can only be fully elucidated by focusing on evolutionary dynamics and implementing integrative modeling efforts.
The chromosome numbers determined from material of 55 Canadian weeds collected in the wild are reported. The chromosome numbers for 45 of the 55 weeds studied were previously obtained by other workers, mostly on non-Canadian material. The chromosome numbers of the following 10 taxa are reported for the first time: Chenopodium berlandieri Moq. var. zschackei (Murr.) Murr. 2n = 36; Chenopodium botrys L. 2n = 18; Corispermum villosum Rydb. 2n = 18; Iva axillaris Pursh 2n = 36 and 54; Lactuca pulchella (Pursh) DC. 2n = 18; Descurainia pinnata (Walt.) Britt. ssp. brachycarpa (Richards.) Detling 2n = 28; Descurainia richardsonii (Sweet) O. E. Schulz ssp. richardsonii 2n = 14; Descurainia sophioides (Fisch.) O. E. Schulz 2n = 14; Dracocephalum thymiflorum L. 2n = 20; Sium suave Walt. 2n = 22.
The chromosome numbers of 58 Canadian weeds are reported. Counts obtained on material of Ambrosia psilostachya DC. and Epilobium angustifolium L. differ from those obtained by other workers. The chromosome numbers of the following species had not previously been reported: Silene cserei Baumg. 2n = 24; Axyris amaranthoides L. 2n = 18; Erigeron philadelphicus L. 2n = 18; Lepidium ramosissimum Nels. 2n = 64; Dracocephalum parviflorum Nutt. 2n = 14; Rumex fennicus Murb. 2n = 40; R. occidentalis Wats. 2n = ca. 140; R. stenophyllus Ledeb. 2n = 60. The significance of some of these chromosome numbers is discussed.
a b s t r a c t Sensitivity to benzimidazoles in isolates of the brown rot pathogen (Monilinia laxa) collected from stone fruit in central and northern Greece was evaluated and the molecular basis for resistance was investigated. M. laxa isolates were classified as benzimidazole e sensitive (S) or highly resistant (HR) based on their sensitivity profiles to carbendazim. Thirty seven percent of the isolates belonged to the HR phenotype, carried no apparent fitness penalties and exhibited resistance factor values (based on EC 50 values) greater than 500. Highly resistant isolates were also less sensitive to the benzimidazoles benomyl and thiophanate-methyl but more sensitive to the N-phenylcarbamate diethofencarb and the benzamide zoxamide compared to isolates belonging to the S phenotype. Fungitoxicity tests with fungicides belonging to other chemical classes revealed no cross resistance relationships between benzimidazoles and the dicarboximide iprodione, the phenylpyrrole fludioxonil, the hydroxyanilide fenhexamid, the carboxamide boscalid, the triazole tebuconazole and the strobilurin-type fungicide pyraclostrobin, indicating that a target site modification is probably responsible for the resistant phenotypes observed. Comparison of the b-tubulin gene DNA sequences between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in all HR isolates tested. An Eco31I restriction site in the b-tubulin gene, which was destroyed in HR M. laxa isolates, allowed the development of a PCR-RFLP diagnostic for the detection of the E198A resistance mutation.
Pathogenic Alu element insertions are rarely reported while their occurrence is expected to be much higher. Alu containing alleles are usually out-competed during the PCR process and consequently undetectable with the classical screening methods. However, with the introduction of the Next Generation Sequencing technology in the diagnostic field new opportunities are emerging. NGS sequencing data for a particular genomic region can be seen as the summation of all the individual sequences (reads) obtained for that region and no longer as the mean of this sum as it is the case for traditional Sanger sequencing. Since each single read covering that region is expected to be generated from a different template molecule, the presence of one single mutant read must theoretically be sufficient to identify the mutation. However, generation and identification of mutant reads bearing Alu insertions remains challenging and several wet/dry bench parameters need to be optimized. Hereby we present the proof of principle of a NGS based mutation screening procedure allowing the detection of inherited Alu insertions within any predefined sequence by investigating 2 cases: c.1739_1740insAlu in BRCA1 and c.156_157insAlu in BRCA2.
Background
Given that there is limited available information on the insensitive acetylcholinesterase in insect species in Malaysia, the present study aims to detect the presence of G119S mutation in the acetylcholinesterase gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia. ResultsThe ace-1 sequence and PCR-RFLP test revealed the presence of glycine-serine ace-1 mutation in the wild populations of Cx. quinquefasciatus. Both direct sequencing and PCR-RFLP methods demonstrated similar results and revealed the presence of a heterozygous genotype at a very low frequency (18 out of 140 individuals), while a homozygous resistant genotype was not detected across any study site in Malaysia. In addition, statistical analysis also revealed that malathion resistance is associated with the frequency of ace-1(R) in Cx. quinquefasciatus populations. Conclusion
This study has demonstrated the first field-evolved instance of G119S mutation in Malaysian populations. Molecular identification of insensitive acetylcholinesterase provides significant insights into the evolution and adaptation of the Malaysian Cx. quinquefasciatus populations. (c) 2013 Society of Chemical Industry
Acetolactate synthase (ALS) inhibitors are widely used herbicides in rice and their recurrent use has resulted in several resistant weed populations. Recent reports from Italian rice growers indicated that resistance to ALS inhibitors evolved in the polyploid species Echinochloa crus-galli (L.) Beauv. (barnyardgrass), which is the most noxious weed infesting Italian rice fields. Fourteen E. crus-galli populations were confirmed to be resistant to at least one ALS-inhibiting herbicide. Three patterns of herbicide resistance were identified: seven populations were highly cross-resistant to ALS inhibitors, two were resistant to a sulfonylurea but not to an imidazolinone and five were multiple resistant to ALS and the ACCase inhibitor profoxydim. The level of resistance to the latter herbicide was low. Molecular analyses yielded the first reported consensus sequence for E. crus-galli ALS gene, encompassing all known mutation sites conferring herbicide resistance. The nucleotide substitution of a G with a T, giving a Trp to Leu change at amino acid 574 was detected in plants of five resistant populations analyzed, confirming an ALS target-site-mediated resistance mechanism. The W574L is a common ALS mutation endowing cross-resistance to all ALS inhibitor chemical families, as confirmed by the high levels of resistance observed for ALS inhibitors at both whole-plant and enzyme activity levels. ALS-resistant, and especially ALS- and ACCase multiple resistant barnyardgrass are threatening the sustainability of Italian rice crops due to the lack of alternative post-emergence herbicides.
Barnyardgrass biotypes from Arkansas (AR1 and AR2) and Mississippi (MS1) have evolved cross-resistance to imazamox, imazethapyr, and penoxsulam. Additionally, AR1 and MS1 have evolved cross-resistance to bispyribac-sodium. Studies were conducted to determine if resistance to acetolactate synthase (ALS)-inhibiting herbicides in these biotypes is target site or non-target site based. Sequencing and analysis of a 1701 base pair ALS coding sequence revealed an Ala122 to Val and Ala122 to Thr substitution in AR1 and AR2, respectively. The imazamox concentrations required for 50% inhibition of ALS enzyme activity in vitro of AR1 and AR2 were 2.0 and 5.8 times, respectively, greater than the susceptible biotype. Absorption of 14C-bispyribac-sodium, -imazamox, and -penoxsulam was similar in all biotypes. 14C-penoxsulam translocation out of the treated leaf (<2%) was similar among all biotypes. 14C-bispyribac-treated AR1 and MS1 translocated 31 to 43% less radioactivity to aboveground tissue below the treated leaf compared to the susceptible biotype. 14C-imazamox-treated AR1 plants translocated 39% less radioactivity above the treated leaf and aboveground tissue below the treated leaf, and MS1 translocated 54 and 18% less radioactivity to aboveground tissue above and below the treated leaf, respectively, compared to the susceptible biotype. Phosphorimaging results further corroborated the above results. This study shows that altered target site is a mechanism of resistance to imazamox in AR2 and probably in AR1. Additionally, reduced translocation, which may be a result of metabolism, could contribute to imazamox and bispyribac-sodium resistance in AR1 and MS1.
A 7 year experiment was set up in 2002 to evaluate the long-term effects of weed management strategies based on graminicidal sulfonylureas (SUs) on the evolution of a Lolium rigidum population resistant to ACCase inhibitors in a continuous wheat cropping system. The strategies included the continued use of ALS inhibitors, the continued application of ACCase inhibitors and a simple resistance management strategy based on a biennial rotation of herbicide mode of action (MoA).
The efficacy of the tested SUs in the field decreased significantly, starting from the fourth treatment in all control strategies. Regardless of control strategy, the few survivors of the ALS treatment in the third season produced a significant number of ACCase- and ALS-resistant (multiple-resistant) progeny. Continuous ALS and biennial rotation of herbicides reduced weed densities, but L. rigidum conserved its ACCase resistance trait. Enhanced metabolism was detected in ALS-resistant plants, whereas target site was primarily involved in the ACCase-resistant individuals.
At the end of the experiment, multiple-resistant individuals were found in all samples coming from the control strategies investigated. The biennial rotation between ALS and other MoA appeared to delay the development of resistance to SUs over continuous treatments, but additional measures will likely need to be taken in order to make this sustainable in the long term, whereas the field efficacy of SUs remained relatively high until the end of the experiment. Integrated weed management with more diversity should be introduced in oversimplified cropping systems in order to sustainably manage resistant L. rigidum populations. Copyright
Research in molecular ecology is now often based on large numbers of DNA sequence reads. Given a time and financial budget for DNA sequencing, the question arises as to how to allocate the finite number of sequence reads among three dimensions: (i) sequencing individual nucleotide positions repeatedly and achieving high confidence in the true genotype of individuals, (ii) sampling larger numbers of individuals from a population, and (iii) sampling a larger fraction of the genome. Leaving aside the question of what fraction of the genome to sample, we analyze the trade-off between repeatedly sequencing the same nucleotide position (coverage depth) and the number of individuals in the sample. We review simple Bayesian models for allele frequencies and utilize these in the analysis of how to obtain maximal information about population genetic parameters. The models indicate that sampling larger numbers of individuals, at the expense of coverage depth per nucleotide position, provides more information about population parameters. Dividing the sequencing effort maximally among individuals and obtaining approximately one read per locus and individual (1 × coverage) yields the most information about a population. Some analyses require genetic parameters for individuals, in which case Bayesian population models also support inference from lower coverage sequence data than are required for simple likelihood models. Low coverage sequencing is not only sufficient to support inference, but it is optimal to design studies to utilize low coverage because they will yield highly accurate and precise parameter estimates based on more individuals or sites in the genome.
Productivity of crops grown for human consumption is at risk due to the incidence of pests, especially weeds, pathogens and animal pests. Crop losses due to these harmful organisms can be substantial and may be prevented, or reduced, by crop protection measures. An overview is given on different types of crop losses as well as on various methods of pest control developed during the last century.
Estimates on potential and actual losses despite the current crop protection practices are given for wheat, rice, maize, potatoes, soybeans, and cotton for the period 2001–03 on a regional basis (19 regions) as well as for the global total. Among crops, the total global potential loss due to pests varied from about 50% in wheat to more than 80% in cotton production. The responses are estimated as losses of 26–29% for soybean, wheat and cotton, and 31, 37 and 40% for maize, rice and potatoes, respectively. Overall, weeds produced the highest potential loss (34%), with animal pests and pathogens being less important (losses of 18 and 16%). The efficacy of crop protection was higher in cash crops than in food crops. Weed control can be managed mechanically or chemically, therefore worldwide efficacy was considerably higher than for the control of animal pests or diseases, which rely heavily on synthetic chemicals. Regional differences in efficacy are outlined. Despite a clear increase in pesticide use, crop losses have not significantly decreased during the last 40 years. However, pesticide use has enabled farmers to modify production systems and to increase crop productivity without sustaining the higher losses likely to occur from an increased susceptibility to the damaging effect of pests.
The concept of integrated pest/crop management includes a threshold concept for the application of pest control measures and reduction in the amount/frequency of pesticides applied to an economically and ecologically acceptable level. Often minor crop losses are economically acceptable; however, an increase in crop productivity without adequate crop protection does not make sense, because an increase in attainable yields is often associated with an increased vulnerability to damage inflicted by pests.
Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence ∼1/3500). There is considerable phenotype and genotype heterogeneity, making a specific diagnosis very difficult without molecular testing. We investigated targeted capture combined with next-generation sequencing using Nimblegen 12plex arrays and the Roche 454 sequencing platform to explore its potential for clinical diagnostics in two common types of IRD, retinitis pigmentosa and cone-rod dystrophy. 50 patients (36 unknowns and 14 positive controls) were screened, and pathogenic mutations were identified in 25% of patients in the unknown, with 53% in the early-onset cases. All patients with new mutations detected had an age of onset <21 years and 44% had a family history. Thirty-one percent of mutations detected were novel. A de novo mutation in rhodopsin was identified in one early-onset case without a family history. Bioinformatic pipelines were developed to identify likely pathogenic mutations and stringent criteria were used for assignment of pathogenicity. Analysis of sequencing metrics revealed significant variability in capture efficiency and depth of coverage. We conclude that targeted capture and next-generation sequencing are likely to be very useful in a diagnostic setting, but patients with earlier onset of disease are more likely to benefit from using this strategy. The mutation-detection rate suggests that many patients are likely to have mutations in novel genes.European Journal of Human Genetics advance online publication, 12 September 2012; doi:10.1038/ejhg.2012.172.
Délye C, Pernin F & Michel S (2011). ‘Universal’ PCR assays detecting mutations in acetyl-coenzyme A carboxylase or acetolactate synthase that endow herbicide resistance in grass weeds. Weed Research51, 353–362.
Herbicides inhibiting acetyl-coenzyme A carboxylase (ACCase) or acetolactate synthase (ALS) are key for grass weed control. Yet, numerous cases of resistance have evolved. Using the derived cleaved amplified polymorphic sequence method, we developed molecular assays to detect amino acid replacements at the seven ACCase codons (1781, 1999, 2027, 2041, 2078, 2088 and 2096) and at two ALS codons (197 and 574) known to play a role in herbicide resistance in grass weeds. For each codon, one assay detecting all known amino acid replacements endowing herbicide resistance was developed. The nine assays were successfully used to genotype ACCase and ALS in 39 grass species. Their flexible design enables easy detection of new mutations at the targeted codons. Because they can be implemented with basic molecular biology facilities and no previous knowledge of the ACCase or ALS sequence of the grass weed of interest, these assays are tools of choice to easily detect resistance caused by alteration(s) of ACCase or ALS in such species.
Polymerase chain reaction (PCR) and polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) techniques were applied for establishing the reliable practice in identification of Echinochloa oryzicola Vasing. and E. crus-galli (L.) Beauv. (barnyardgrass). Total DNA was extracted from 18 accessions and 86 individuals of E. oryzicola, 33 accessions and 140 individuals of E. crus-galli var. crus-galli, 23 individuals of E. crus-galli var. praticola, and six individuals of E. crus-galli var. formosensis that were collected from Japan. A partial region of intergenetic spacer between trnT and trnL, and an intron of trnL were amplified separately using a trn-a and trn-b1 primer set, and a trn-c and trn-d primer set, respectively. All individuals of E. oryzicola showed the same fragment amplified by the trn-a and trn-b1 primer set. The fragment was 481 bp in length, and was undigested by EcoR I, whereas all individuals of E. crus-galli, including three botanical varieties, showed the same fragment with a 449-bp length. The fragment was digested by EcoR I into two fragments (178 and 271 bp). The fragment amplified by the trn-c and trn-d primer set in all individuals of E. oryzicola was digested by Alu I into two fragments (174 and 452 bp), but undigested by Dra I. In contrast, the fragment amplified by the trn-c and trn-d primer set in all individuals of E. crus-galli was digested by Dra I into two fragments (134 and 487 bp), but undigested by Alu I. There was no intraspecific variation in these regions; thus, these two species are easily identifiable by using our method.
Lolium species (ryegrasses) are genetically highly variable plants that are both forage crops and major weeds across the globe. As weeds, they rapidly evolve resistance under the selective pressure of acetolactate-synthase (ALS) inhibitors, the most resistance-prone herbicide group. Quick and accurate diagnosis is therefore of importance to prevent resistance spread in ryegrass. To develop proactive molecular tools for the detection of mutant, resistant ALS alleles, we assessed variation in the ryegrass ALS gene. Sequencing the full 1929-bp ALS coding sequence in 59 plants from six distant locations revealed a total of 208 polymorphic nucleotide positions (one every 9.3 nucleotides). The heterogeneous distribution of synonymous and non-synonymous substitutions along the ALS coding sequence suggested that nucleotide variation of ALS is shaped by purifying and background selection. Using regions of the ALS coding sequence with a low number of polymorphic nucleotide sites, five derived cleaved amplified polymorphic sequence (dCAPS) assays were developed targeting codons crucial for herbicide sensitivity. These enabled the first detection in ryegrass of a Pro-197-Thr substitution that confers herbicide resistance. Most assays could also be used to genotype Festuca and Vulpia plants. These dCAPS assays should prove powerful tools for both resistance diagnosis and population genetics studies.
Although fungicide treatments are a key component in the integrated management of many plant diseases, the appearance of resistance has become an important factor in limiting the efficacy and useful lifetime of fungicides developed at increasingly higher costs. Extensive molecular studies have led to advances in our understanding of mechanisms of fungicide resistance and in developing effective, rapid methods for detection of resistant genotypes of pathogens. This paper reviews recent advances in our understanding of resistance mechanisms of phytopathogenic fungi to some major classes of fungicides (benzimidazoles, demethylation inhibitors [DMIs], Qo respiration inhibitors [QoIs], and dicarboximides [DCFs]) at a molecular level and developments in molecular detection of fungicide-resistant fungi.
Substitution of isoleucine by leucine at the equivalent of residue 1781 of acetyl CoA carboxylase (ACCase) in Alopecurus myosuroides (I1781L) has been shown to be a key point mutation conferring resistance to most aryloxypropionate and cyclohexanedione herbicides in Lolium spp., A. myosuroides, Avena fatua and Setaria viridis. This substitution results from changing an adenine residue to either thymine or cytosine at position 5341 in the ACCase coding sequence of A. myosuroides and at the homologous position in the other species. The I1781L mutation can be detected by allele-specific amplification assays. These are, however, very dependent on the conservation of the nucleotide sequences flanking the causative single nucleotide polymorphism. Moreover, such assays cannot distinguish between homozygous and heterozygous individuals in a single polymerase chain reaction reaction. Here we present an alternative derived Cleaved Amplified Polymorphic Sequence (dCAPS) method to define I1781L status in the ACCase enzyme of four grass weeds. This dCAPS approach is simple, economical, highly transferable between species and can readily distinguish homozygous Leu/Leu 1781 and heterozygous Ile/Leu 1781 resistant individuals, providing the basis for accurate measures of the frequency of the dominant Leu allele in a given population.
Agricultural weeds evolve in response to crop cultivation. Nevertheless, the central importance of evolutionary ecology for understanding weed invasion, persistence and management in agroecosystems is not widely acknowledged. This paper calls for more evolutionarily-enlightened weed management, in which management principles are informed by evolutionary biology to prevent or minimize weed adaptation and spread. As a first step, a greater knowledge of the extent, structure and significance of genetic variation within and between weed populations is required to fully assess the potential for weed adaptation. The evolution of resistance to herbicides is a classic example of weed adaptation. Even here, most research focuses on describing the physiological and molecular basis of resistance, rather than conducting studies to better understand the evolutionary dynamics of selection for resistance. We suggest approaches to increase the application of evolutionary-thinking to herbicide resistance research. Weed population dynamics models are increasingly important tools in weed management, yet these models often ignore intrapopulation and interpopulation variability, neglecting the potential for weed adaptation in response to management. Future agricultural weed management can benefit from greater integration of ecological and evolutionary principles to predict the long-term responses of weed populations to changing weed management, agricultural environments and global climate.
A new PCR/RFLP method is presented to identify all described alleles involved in resistance at the Ester locus in the mosquito Culex pipiens. The efficiency of this method as well as its advantages compared with the traditional identification technique (starch gel electrophoresis) have been tested in four natural samples from France, Tunisia and California. This simple and fast molecular test is a very convenient tool for studies in field populations and laboratory strains.© 2000 Society of Chemical Industry
Citrus exocortis viroid (CEV) is widespread in citrus production areas where trifoliate orange [Poncirus trifoliata (L.) Raf.] is used as rootstock. Citrus reticulata Blanco cv. Red tangerine, a different rootstock, is tolerant to CEV. Embryogenic protoplasts of C. reticulata cv. Red tangerine were electrically fused with mesophyll protoplasts from P. trifoliata, and five embryoids were regenerated after 40 days of culture. The embryoids were cut into several pieces and subcultured on shoot induction medium. After 5 months and several subcultures, shoots initially regenerated. The plants grew vigorously with well-developed root systems and exhibited the trifoliate leaf character of P. trifoliata. Chromosome counts on four randomly selected root tips revealed them to be tetraploids (2n=4x=36). RAPD analysis of four randomly selected plants verified their hybridity. This hybridity was further confirmed by AFLP analysis using four primer pairs, from which a total of 65 specific bands were detected. Cytoplasmic genome analysis using universal primers revealed that their chloroplast DNA banding pattern was identical to that of trifoliate orange, while the banding pattern of mitochondrial DNA was identical to that of Red tangerine. The potential of this somatic hybrid as a means to control tree size and provide multi-resistance is discussed.
A number of next-generation sequencing (NGS) technologies such as Roche/454, Illumina and AB SOLiD have recently become available. These technologies are capable of generating hundreds of thousands or tens of millions of short DNA sequence reads at a relatively low cost. These NGS technologies, now referred as second-generation sequencing (SGS) technologies, are being utilized for de novo sequencing, genome re-sequencing, and whole genome and transcriptome analysis. Now, new generation of sequencers, based on the 'next-next' or third-generation sequencing (TGS) technologies like the Single-Molecule Real-Time (SMRT™) Sequencer, Heliscope™ Single Molecule Sequencer, and the Ion Personal Genome Machine™ are becoming available that are capable of generating longer sequence reads in a shorter time and at even lower costs per instrument run. Ever declining sequencing costs and increased data output and sample throughput for NGS and TGS sequencing technologies enable the plant genomics and breeding community to undertake genotyping-by-sequencing (GBS). Data analysis, storage and management of large-scale second or TGS projects, however, are essential. This article provides an overview of different sequencing technologies with an emphasis on forthcoming TGS technologies and bioinformatics tools required for the latest evolution of DNA sequencing platforms.
Target-site resistance is the major cause of herbicide resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in arable weeds, whereas non-target-site resistance is rarely reported. In the Echinochloa phyllopogon biotypes resistant to these herbicides, target-site resistance has not been reported, and non-target-site resistance is assumed to be the basis for resistance. To explore why target-site resistance had not occurred, the target-site genes for these herbicides were isolated from E. phyllopogon, and their expression levels in a resistant biotype were determined.
Two complete ALS genes and the carboxyltransferase domain of four ACCase genes were isolated. The expression levels of ALS and ACCase genes were higher in organs containing metabolically active meristems, except for ACC4, which was not expressed in any organ. The differential expression among examined organs was more prominent for ALS2 and ACC2 and less evident for ALS1, ACC1 and ACC3.
E. phyllopogon has multiple copies of the ALS and ACCase genes, and different expression patterns were observed among the copies. The existence of three active ACCase genes and the difference in their relative expression levels could influence the occurrence of target-site resistance to ACCase inhibitors in E. phyllopogon.
DNA sequencing is undergoing a revolution. Sanger sequencing has reached its limits and is being replaced by a new generation of technologies that promise faster and cheaper delivery of sequencing information. The application of these technologies is already having a huge impact in fields as diverse wholeâgenome sequencing, disease genetics and epigenomics.
Rapid and accurate detection of mutations related to insecticide resistance is essential for development of resistance management strategies to support sustainable agriculture. The M918V, L925I and T929V mutations of the voltage-gated sodium channel gene (vgsc) and the F392W mutation of the acetylcholinesterase I gene (ace1) are reportedly associated with resistance to pyrethroids and organophosphates, respectively, in Bemisia tabaci. In order to detect known base substitutions in the ace1 and vgsc genes, a low-density microarray with an allele-specific probe was developed.
Specific regions of the ace1 and vgsc gene mutations were amplified by multiplex asymmetrical PCR using Cy3-labelled primers, and then the PCR products were hybridised on the microarray. After analysing the probe signal data, the microarray containing 12 allele-specific probes produced a unique pattern of probe signals for field DNA samples of B. tabaci. To determine the optimal cut-off value of each probe, receiver operating characteristic (ROC) curve analysis was conducted using SPSS. Among 60 individual samples, microarray data for 57 samples were consistent with direct sequencing data.
Although many molecular detection methods have been employed to monitor insecticide resistance, the present microarray provides rapid and accurate identification of target mutations in B. tabaci for resistance management.
Pollen beetle (Meligethes spp.) is a major pest of European oilseed rape crops. Its resistance to pyrethroid insecticides has been recorded in samples of beetles collected in Europe since at least 1999, and problems with the control of the beetle in the field have been widely reported. In 2007, a Pollen Beetle Working Group was formed through the Insecticide Resistance Action Committee (IRAC) in order to coordinate efforts for surveying pyrethroid resistance development.
The results of the first 3 years of the pollen beetle pyrethroid susceptibility survey using a laboratory test are presented in this paper. Resistant beetle samples were collected from 20 of the 21 countries surveyed, with a general trend of increasing frequency and spread of resistant samples in European oilseed-rape-growing regions.
Pyrethroid-resistant beetles dominate in Western and Central Europe and are becoming established in the North and East, the main oilseed-rape-growing areas of Europe. The development and spread of pyrethroid-resistant pollen beetles highlights the need for effective management strategies for oilseed rape insect pests.
The increasing occurrence of QoI fungicide resistance in Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew. In Japan, the existence of QoI-fungicide-resistant P. viticola was reported in 2009.
The QoI fungicide resistance in P. viticola samples collected from vineyards in Japan in 2008 and 2009 was monitored. Resistant P. viticola were detected in the regions where QoI fungicides have been introduced in accordance with the pest management programme, whereas in Hokkaido vineyards, where QoI fungicides have not yet been introduced, QoI-fungicide-resistant P. viticola were not found.
Japan comprises thousands of islands and is physically isolated from other countries by the sea. Monitoring the emergence, incidence and distribution of QoI fungicide resistance in P. viticola populations in Japan is necessary to improve pest management strategies for downy mildew disease in Japanese vineyards.
*The geographical structure of resistance to herbicides inhibiting acetyl-coenzyme A carboxylase (ACCase) was investigated in the weed Alopecurus myosuroides (black-grass) across its geographical range to gain insight into the process of plant adaptation in response to anthropogenic selective pressures occurring in agricultural ecosystems. *We analysed 297 populations distributed across six countries in A. myosuroides' main area of occupancy. The frequencies of plants resistant to two broadly used ACCase inhibitors and of seven mutant, resistant ACCase alleles were assessed using bioassays and genotyping, respectively. *Most of the resistance was not endowed by mutant ACCase alleles. Resistance and ACCase allele distribution patterns were characterized by mosaicism. The prevalence of resistance and of ACCase alleles differed among countries. *Resistance clearly evolved by redundant evolution of a set of resistance alleles or genes, most of which remain unidentified. Resistance in A. myosuroides was shaped by variation in the herbicide selective pressure at both the individual field level and the national level.
Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.
A point mutation often confers resistance of organisms against medical drugs and agricultural pesticides. Allele-specific nucleotide polymerase chain reaction (ASPCR) and allele-specific quantitative real-time PCR using SYBR Green (ASQPCR) are widely and effectively applied to detect and monitor this type of resistance. However, the former is unsuitable for high-throughput detection, and the latter often reduces the accuracy of detection.
In order to decrease background amplification, a rapid and high-throughput genotyping method with mismatch primers was developed (ASQPCR-MP) and applied specifically to survey the frequency of the highly benzimidazole-resistant MBC(HR) mutation (E198A) in the beta-tubulin gene of Sclerotinia sclerotiorum (Lib.) de Bary populations. Genomic DNA from 223 sclerotia was analysed. Similar genotype results were also obtained using ASPCR with mismatch primers and a mycelial growth inhibition assay. It was found that ASQPCR-MP clearly differentiated MBC(HR) and benzimidazole-sensitive MBC(S) phenotypes. Moreover, ASQPCR-MP took less than 6 h to complete.
ASQPCR-MP appears suitable for large epidemiological studies involving resistant genotypes and requiring high-throughout formats.