A survey of natural and ethyl methane sulfonate-induced variations of eIF4E using high-resolution melting analysis in Capsicum

Allele mining is a method used to find undiscovered natural variations or induced mutations in a plant, and has become increasingly important as more genomic information is available in plants. A high-throughput method is required to facilitate the identification of novel alleles in a large number of samples. In this paper we describe the application of a high-resolution melting (HRM) method to detect natural variations and ethyl methane sulfonate (EMS)-induced mutations in Capsicum. We have scanned single polymorphic mutations in the first exon of the eIF4E gene, wherein the mutations confer resistance to potyviruses. Sixteen allelic variations out of 248 germplasm collections were identified using HRM analysis, and one accession carrying an allelic variation (pvrHRM1 ¹³ ) was confirmed to be resistant to the TEV-HAT strain. In addition, five single polymorphic mutations in the eIF4E gene were identified in an EMS-induced mutant population. These results demonstrate that HRM allows for the rapid identification of new allelic variants in both natural and artificial mutant populations.     

Mimicking natural polymorphism in eIF4E by CRISPR‐Cas9 base editing is associated with resistance to potyviruses

In many crop species, natural variation in eIF4E proteins confers resistance to potyviruses. Gene editing offers new opportunities to transfer genetic resistance to crops that seem to lack natural eIF4E alleles. However, because eIF4E are physiologically important proteins, any introduced modification for virus resistance must not bring adverse phenotype effects. In this study, we assessed the role of amino acid substitutions encoded by a Pisum sativum eIF4E virus‐resistance allele (W69L, T80D S81D, S84A, G114R and N176K) by introducing them independently into the Arabidopsis thaliana eIF4E1 gene, a susceptibility factor to the Clover yellow vein virus (ClYVV). Results show that most mutations were sufficient to prevent ClYVV accumulation in plants without affecting plant growth. In addition, two of these engineered resistance alleles can be combined with a loss‐of‐function eIFiso4E to expand the resistance spectrum to other potyviruses. Finally, we use CRISPR‐nCas9‐cytidine deaminase technology to convert the Arabidopsis eIF4E1 susceptibility allele into a resistance allele by introducing the N176K mutation with a single‐point mutation through C‐to‐G base editing to generate resistant plants. This study shows how combining knowledge on pathogen susceptibility factors with precise genome‐editing technologies offers a feasible solution for engineering transgene‐free genetic resistance in plants, even across species barriers.     

Marker assisted pea breeding: <Emphasis Type="Italic">eIF4E</Emphasis> allele specific markers to <Emphasis Type="Italic">pea seed-borne mosaic virus</Emphasis> (PSbMV) resistance

Traditional plant breeding relies upon crosses and subsequent selection of genotypes to meet desirable traits. The incorporation of marker-assisted selection into breeding strategies would result in a reduction in the number of offspring to be propagated, selected and tested. In the case of pea (Pisum sativum L.), the testing of resistance to viral pathogens such as pea seed-borne mosaic virus (PSbMV) is included in the breeding process. Resistance to the common strains of PSbMV is conferred by a single recessive gene (eIF4E), localized on LG VI (sbm-1 locus). We have analyzed for variation in the eIF4E genomic sequences from 43 pea varieties and breeding lines, reported as donors of resistance. This enabled a comprehensive investigation of the eIF4E gene structure and mutations responsible for PSbMV resistance were identified. Subsequently, PCR-based and gene-specific single nucleotide polymorphism and co-dominant amplicon length polymorphism markers were developed. All together 60 accessions were analyzed using sequence data and/or allele specific DNA markers. Developed allele specific markers were reproducibly amplified across a broad spectra of pea varieties and breeding lines. These were found to be 100% accurate in detecting the presence of the respective alleles when compared to symptomology and ELISA, testing (74% reliable). Hence, these molecular markers will substantially speed-up PSbMV diagnosis and resistance breeding processes in pea.     

A TILLING approach to generate broad‐spectrum resistance to potyviruses in tomato is hampered by eIF4E gene redundancy

Genetic resistance to pathogens is important for sustainable maintenance of crop yields. Recent biotechnologies offer alternative approaches to generate resistant plants by compensating for the lack of natural resistance. Tomato (Solanum lycopersicum) and related species offer a model in which natural and TILLING‐induced potyvirus resistance alleles may be compared. For resistance based on translation initiation factor eIF4E1, we confirm that the natural allele Sh–eIF4E1^PI24–pot1, isolated from the wild tomato species Solanum habrochaites, is associated with a wide spectrum of resistance to both potato virus Y and tobacco etch virus isolates. In contrast, a null allele of the same gene, isolated through a TILLING strategy in cultivated tomato S. lycopersicum, is associated with a much narrower resistance spectrum. Introgressing the null allele into S. habrochaites did not extend its resistance spectrum, indicating that the genetic background is not responsible for the broad resistance. Instead, the different types of eIF4E1 mutations affect the levels of eIF4E2 differently, suggesting that eIF4E2 is also involved in potyvirus resistance. Indeed, combining two null mutations affecting eIF4E1 and eIF4E2 re‐establishes a wide resistance spectrum in cultivated tomato, but to the detriment of plant development. These results highlight redundancy effects within the eIF4E gene family, where regulation of expression alters susceptibility or resistance to potyviruses. For crop improvement, using loss‐of‐function alleles to generate resistance may be counter‐productive if they narrow the resistance spectrum and limit growth. It may be more effective to use alleles encoding functional variants similar to those found in natural diversity.     

Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum

Background

Translation initiation factors of the 4E and 4G protein families mediate resistance to several RNA plant viruses in the natural diversity of crops. Particularly, a single point mutation in melon eukaryotic translation initiation factor 4E (eIF4E) controls resistance to Melon necrotic spot virus (MNSV) in melon. Identification of allelic variants within natural populations by EcoTILLING has become a rapid genotype discovery method.

Results

A collection of Cucumis spp. was characterised for susceptibility to MNSV and Cucumber vein yellowing virus (CVYV) and used for the implementation of EcoTILLING to identify new allelic variants of eIF4E. A high conservation of eIF4E exonic regions was found, with six polymorphic sites identified out of EcoTILLING 113 accessions. Sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with MNSV resistance. Except for the MNSV case, no correlation was found between variation of eIF4E and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. We have also characterized a new allele of eIF4E from Cucumis zeyheri, a wild relative of melon. Functional analyses suggested that this new eIF4E allele might be responsible for resistance to MNSV.

Conclusion

This study shows the applicability of EcoTILLING in Cucumis spp., but given the conservation of eIF4E, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. Part of the methodology described here could alternatively be used in TILLING experiments that serve to generate new eIF4E alleles.     

Plastid genome characterisation in Brassica and Brassicaceae using a new set of nine SSRs

The objective of the present study was to identify favourable exotic Quantitative Trait Locus (QTL) alleles for the improvement of agronomic traits in the BC₂DH population S42 derived from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). QTLs were detected as a marker main effect and/or a marker × environment interaction effect (M × E) in a three-factorial ANOVA. Using field data of up to eight environments and genotype data of 98 SSR loci, we detected 86 QTLs for nine agronomic traits. At 60 QTLs the marker main effect, at five QTLs the M × E interaction effect, and at 21 QTLs both the effects were significant. The majority of the M × E interaction effects were due to changes in magnitude and are, therefore, still valuable for marker assisted selection across environments. The exotic alleles improved performance in 31 (36.0%) of 86 QTLs detected for agronomic traits. The exotic alleles had favourable effects on all analysed quantitative traits. These favourable exotic alleles were detected, in particular on the short arm of chromosome 2H and the long arm of chromosome 4H. The exotic allele on 4HL, for example, improved yield by 7.1%. Furthermore, the presence of the exotic allele on 2HS increased the yield component traits ears per m² and thousand grain weight by 16.4% and 3.2%, respectively. The present study, hence, demonstrated that wild barley does harbour valuable alleles, which can enrich the genetic basis of cultivated barley and improve quantitative agronomic traits.     

An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations

In barley, the eukaryotic translation initiation factor 4E (eIF4E) gene situated on chromosome 3H is recognized as an important source of resistance to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In modern barley cultivars, two recessive eIF4E alleles, rym4 and rym5, confer different isolate-specific resistances. In this study, the sequence of eIF4E was analysed in 1090 barley landraces and noncurrent cultivars originating from 84 countries. An exceptionally high nucleotide diversity was evident in the coding sequence of eIF4E but not in either the adjacent MCT-1 gene or the sequence-related eIF(iso)4E gene situated on chromosome 1H. Surprisingly, all nucleotide polymorphisms detected in the coding sequence of eIF4E resulted in amino acid changes. A total of 47 eIF4E haplotypes were identified, and phylogenetic analysis using maximum likelihood provided evidence of strong positive selection acting on this barley gene. The majority of eIF4E haplotypes were found to be specific to distinct geographic regions. Furthermore, the eI4FE haplotype diversity (uh) was found to be considerably higher in East Asia, whereas SNP genotyping identified a comparatively low degree of genome-wide genetic diversity in 16 of 17 tested accessions (each carrying a different eIF4E haplotype) from this same region. In addition, selection statistic calculations using coalescent simulations showed evidence of non-neutral variation for eIF4E in several geographic regions, including East Asia, the region with a long history of the bymovirus-induced yellow mosaic disease. Together these findings suggest that eIF4E may play a role in barley adaptation to local habitats.     

When a knockout is an Achilles’ heel: Resistance to one potyvirus species triggers hypersusceptibility to another one in Arabidopsis thaliana

The translation initiation factors 4E are a small family of major susceptibility factors to potyviruses. It has been suggested that knocking out these genes could provide genetic resistance in crops when natural resistance alleles, which encode functional eIF4E proteins, are not available. Here, using the well-characterized Arabidopsis thaliana–potyvirus pathosystem, we evaluate the resistance spectrum of plants knocked out for eIF4E1, the susceptibility factor to clover yellow vein virus (ClYVV). We show that besides resistance to ClYVV, the eIF4E1 loss of function is associated with hypersusceptibility to turnip mosaic virus (TuMV), a potyvirus known to rely on the paralog host factor eIFiso4E. On TuMV infection, plants knocked out for eIF4E1 display striking developmental defects such as early senescence and primordia development stoppage. This phenotype is coupled with a strong TuMV overaccumulation throughout the plant, while remarkably the levels of the viral target eIFiso4E remain uninfluenced. Our data suggest that this hypersusceptibility cannot be explained by virus evolution leading to a gain of TuMV aggressiveness. Furthermore, we report that a functional eIF4E1 resistance allele engineered by CRISPR/Cas9 base-editing technology successfully circumvents the increase of TuMV susceptibility conditioned by eIF4E1 disruption. These findings in Arabidopsis add to several previous findings in crops suggesting that resistance based on knocking out eIF4E factors should be avoided in plant breeding, as it could also expose the plant to the severe threat of potyviruses able to recruit alternative eIF4E copies. At the same time, it provides a simple model that can help understanding of the homeostasis among eIF4E proteins in the plant cell and what makes them available to potyviruses.     

High resolution melting analysis of almond SNPs derived from ESTs

High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.     

Engineering of CRISPR/Cas9‐mediated potyvirus resistance in transgene‐free Arabidopsis plants

Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as recessive resistance alleles against various potyviruses in a range of different hosts. However, the identification and introgression of these alleles into important crop species is often limited. In this study, we utilise CRISPR/Cas9 technology to introduce sequence‐specific deleterious point mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field‐grown vegetable crops. By segregating the induced mutation from the CRISPR/Cas9 transgene, we outline a framework for the production of heritable, homozygous mutations in the transgene‐free T₂ generation in self‐pollinating species. Analysis of dry weights and flowering times for four independent T₃ lines revealed no differences from wild‐type plants under standard growth conditions, suggesting that homozygous mutations in eIF(iso)4E do not affect plant vigour. Thus, the established CRISPR/Cas9 technology provides a new approach for the generation of Potyvirus resistance alleles in important crops without the use of persistent transgenes.     

Evaluation of genes from <Emphasis Type="Italic">eIF4E</Emphasis> and <Emphasis Type="Italic">eIF4G</Emphasis> multigenic families as potential candidates for partial resistance QTLs to <Emphasis Type="Italic">Rice yellow mottle virus</Emphasis> in rice

QTLs for partial resistance to Rice yellow mottle virus (RYMV) in rice were mapped in two populations of doubled-haploid lines (DHLs) and recombinant inbred lines (RILs) derived from the same cross but evaluated for different resistance criteria (virus content and symptom severity). An integrative map was used to compare the two genetic maps and a global analysis of both populations was performed. Most of the QTLs previously identified in DHL population were confirmed with increased significance and precision. As many recent studies evidenced the role of eukaryotic translation initiation factors (eIF) of 4E and 4G families in plant susceptibility to RNA viruses, we checked if these genes co-locate with QTLs of resistance to RYMV. Their systematic in silico identification was carried out on the rice genome and their physical locations were compared to QTL positions on the integrative map. In order to confirm or not the co-locations observed, the analysis was completed by evaluation of near-isogenic lines, QTL fine mapping and sequencing of candidate genes. Three members from eIF4G family could be retained as reliable candidates whereas eIF4E genes, commonly found to govern resistances in other plant/virus interactions, were discarded. Together with the recent identification of an eIF(iso)4G as a major resistance gene, data suggests an important role of genes from eIF4G family in rice resistance to RYMV but does not exclude the contribution of factors different from the translation initiation complex. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence that the recessive bymovirus resistance locus rym4 in barley corresponds to the eukaryotic translation initiation factor 4E gene

Recent studies have shown that resistance in several dicotyledonous plants to viruses in the genus Potyvirus is controlled by recessive alleles of the plant translation initiation factor eIF4E or eIF ( iso ) 4E genes. Here we provide evidence that the barley rym 4 gene locus, controlling immunity to viruses in the genus Bymovirus , corresponds to eIF4E . A molecular marker based on the sequence of eIF4E was developed and used to demonstrate that eIF4E and rym 4 map to the same genetic interval on chromosome 3HL in barley . Another genetic marker was developed that detects a polymorphism in the coding sequence of eIF4E and consistently distinguishes between rym 4 and susceptible barley cultivars of diverse parentage. The eIF4E gene product from barley genotypes carrying rym 4 and allelic rym 5 and rym 6 genes, originating from separate exotic germplasm, and a novel resistant allele that we identified through a reverse genetics approach all contained unique amino acid substitutions compared with the wild-type protein. Three-dimensional models of the barley eIF4E protein revealed that the polymorphic residues identified are all located at or near the mRNA cap-binding pocket, similarly to recent findings from studies on recessive potyvirus resistance in dicotyledonous plants. These new data complement our earlier observations that specific mutations in bymovirus VPg are responsible for overcoming rym 4/5-controlled resistance. Because the potyviral VPg is known to interact with eIF4E in dicotyledonous plants, it appears that monocotyledonous and dicotyledonous plants have evolved a similar strategy to combat VPg-encoding viruses in the family Potyviridae .     

Structure-based mutational analysis of eIF4E in relation to sbm1 resistance to pea seed-borne mosaic virus in pea

Background

Pea encodes eukaryotic translation initiation factor eIF4E (eIF4E^S), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4E^R) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection.

Methodology/Principal Findings

To study structure-function relationships between pea eIF4E and PSbMV VPg, we obtained an X-ray structure for eIF4E^S bound to m⁷GTP. The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E. To assess indirectly the importance of key residues in binding to VPg and/or m⁷GTP, an extensive range of point mutants in eIF4E was tested for their ability to complement PSbMV multiplication in resistant pea tissues and for complementation of protein translation, and hence growth, in an eIF4E-defective yeast strain conditionally dependent upon ectopic expression of eIF4E. The mutants also dissected individual contributions from polymorphisms present in eIF4E^R and compared the impact of individual residues altered in orthologous resistance alleles from other crop species. The data showed that essential resistance determinants in eIF4E differed for different viruses although the critical region involved (possibly in VPg-binding) was conserved and partially overlapped with the m⁷GTP-binding region. This overlap resulted in coupled inhibition of virus multiplication and translation in the majority of cases, although the existence of a few mutants that uncoupled the two processes supported the view that the specific role of eIF4E in potyvirus infection may not be restricted to translation.

Conclusions/Significance

The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance. In addition to defining functional domains within the eIF4E structure, we identified eIF4E alleles with the potential to convey novel virus resistance phenotypes.     

Calreticulin Mutations in Myeloproliferative Neoplasms: Comparison of Three Diagnostic Methods

Calreticulin (CALR) mutations have recently been reported in 70–84% of JAK2V617F-negative myeloproliferative neoplasms (MPN), and this detection has become necessary to improve the diagnosis of MPN. In a large single-centre cohort of 298 patients suffering from Essential Thrombocythemia (ET), the JAK2V617F, CALR and MPL mutations were noted in 179 (60%), 56 (18.5%) and 13 (4.5%) respectively. For the detection of the CALR mutations, three methods were compared in parallel: high-resolution melting-curve analysis (HRM), product-sizing analysis and Sanger sequencing. The sensitivity for the HRM, product-sizing analysis and Sanger sequencing was 96.4%, 98.2% and 89.3% respectively, whereas the specificity was 96.3%, 100% and 100%. In our cohort, the product-sizing analysis was the most sensitive method and was the easiest to interpret, while the HRM was sometimes difficult to interpret. In contrast, when large series of samples were tested, HRM provided results more quickly than did the other methods, which required more time. Finally, the sequencing method, which is the reference method, had the lowest sensitivity but can be used to describe the type of mutation precisely. Altogether, our results suggest that in routine laboratory practice, product-sizing analysis is globally similar to HRM for the detection of CALR mutations, and that both may be used as first-line screening tests. If the results are positive, Sanger sequencing can be used to confirm the mutation and to determine its type. Product-sizing analysis provides sensitive and specific results, moreover, with the quantitative measurement of CALR, which might be useful to monitor specific treatments.     

Trans‐species synthetic gene design allows resistance pyramiding and broad‐spectrum engineering of virus resistance in plants

To infect plants, viruses rely heavily on their host's machinery. Plant genetic resistances based on host factor modifications can be found among existing natural variability and are widely used for some but not all crops. While biotechnology can supply for the lack of natural resistance alleles, new strategies need to be developed to increase resistance spectra and durability without impairing plant development. Here, we assess how the targeted allele modification of the Arabidopsis thaliana translation initiation factor eIF4E1 can lead to broad and efficient resistance to the major group of potyviruses. A synthetic Arabidopsis thaliana eIF4E1 allele was designed by introducing multiple amino acid changes associated with resistance to potyvirus in naturally occurring Pisum sativum alleles. This new allele encodes a functional protein while maintaining plant resistance to a potyvirus isolate that usually hijacks eIF4E1. Due to its biological functionality, this synthetic allele allows, at no developmental cost, the pyramiding of resistances to potyviruses that selectively use the two major translation initiation factors, eIF4E1 or its isoform eIFiso4E. Moreover, this combination extends the resistance spectrum to potyvirus isolates for which no efficient resistance has so far been found, including resistance‐breaking isolates and an unrelated virus belonging to the Luteoviridae family. This study is a proof‐of‐concept for the efficiency of gene engineering combined with knowledge of natural variation to generate trans‐species virus resistance at no developmental cost to the plant. This has implications for breeding of crops with broad‐spectrum and high durability resistance using recent genome editing techniques.     

Discovery and assay of single-nucleotide polymorphisms in barley (Hordeum vulgare)

The least ambiguous genetic markers are those based on completely characterized DNA sequence polymorphisms. Unfortunately, assaying allele states by allele sequencing is slow and cumbersome. The most desirable type of genetic marker would be unambiguous, inexpensive to assay and would be assayable singly or in parallel with hundreds of other markers (multiplexable). In this report we sequenced alleles at 54 barley (Hordeum vulgare ssp. vulgare) loci, 38 of which contained single-nucleotide polymorphisms (SNPs). Many of these 38 loci contained multiple polymorphisms, and a total of 112 polymorphisms were scored in five barley genotypes. The polymorphism data set was analyzed both by using the individual mutations as cladistic characters and by reducing data for each locus to haplotypes. We compared the informativeness of these two approaches by consensus tree construction and bootstrap analysis. Both approaches provided similar results. Since some of the loci sequenced contained insertion/deletion events and multiple point mutations, we thought that these multiple-mutated loci might represent old alleles that predated the divergence of barley from H. spontaneum. We evaluated sequences from a sample of H. spontaneum accessions from the Eastern Mediterranean, and observed similar alleles present in both cultivated barley and H. spontaneum, suggesting either multiple domestication events or multiple transfers of genes between barley and its wild ancestor.     

Improving the limit of detection for Sanger sequencing: A comparison of methodologies for KRAS variant detection

Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in which different alleles were expected as heterozygous mixtures of 50%. Increasingly, the requirements for variant detection are an analytic sensitivity for minor alleles of <20%, in particular, when assessing the mutational status of heterogeneous tumor samples. Here, we describe a simple modification to the FTSS workflow that improves the limit of detection of cell-line gDNA mixtures from 50%-20% to 5% for G>A transitions and from 50%-5% to 5% for G>C and G>T transversions. In addition, we use two different sample types to compare the limit of detection of sequence variants in codons 12 and 13 of the KRAS gene between Sanger sequencing and other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), and real-time PCR (qPCR).     

Multiple copies of eukaryotic translation initiation factors in Brassica rapa facilitate redundancy,enabling diversification through variation in splicing and broad‐spectrum virus resistance

Recessive strain‐specific resistance to a number of plant viruses in the Potyvirus genus has been found to be based on mutations in the eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E. We identified three copies of eIF(iso)4E in a number of Brassica rapa lines. Here we report broad‐spectrum resistance to the potyvirus Turnip mosaic virus (TuMV) due to a natural mechanism based on the mis‐splicing of the eIF(iso)4E allele in some TuMV‐resistant B. rapa var. pekinensis lines. Of the splice variants, the most common results in a stop codon in intron 1 and a much truncated, non‐functional protein. The existence of multiple copies has enabled redundancy in the host plant's translational machinery, resulting in diversification and emergence of the resistance. Deployment of the resistance is complicated by the presence of multiple copies of the gene. Our data suggest that in the B. rapa subspecies trilocularis, TuMV appears to be able to use copies of eIF(iso)4E at two loci. Transformation of different copies of eIF(iso)4E from a resistant B. rapa line into an eIF(iso)4E knockout line of Arabidopsis thaliana proved misleading because it showed that, when expressed ectopically, TuMV could use multiple copies which was not the case in the resistant B. rapa line. The inability of TuMV to access multiple copies of eIF(iso)4E in B. rapa and the broad spectrum of the resistance suggest it may be durable.     

应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型

氯吡格雷是一种广泛用于预防静脉血栓形成的抗血小板药物。研究表明, 携带有CYP2C19基因功能缺失型等位基因CYP2C19*2、CYP2C19*3的病人, 其体内代谢氯吡格雷成为其活性形式的能力降低, 导致氯吡格雷抑制血小板聚集功能减弱。文章旨在建立一种利用高分辨率熔解曲线分析(High-resolution melting curve analysis,HRM)技术在闭合单管中同时对CYP2C19*2、CYP2C19*3两个多态性位点进行简便、准确分型的方法。本实验针对两个SNP位点分别设计特异性的HRM引物, 并在两个位点引物的5′端分别加上富含AT和GC的序列, 保证两个位点的扩增产物熔解峰无重叠。利用HRM技术, 快速、灵敏地对64例随机DNA样本的CYP2C19*2 、CYP2C19*3两个多态性位点进行了基因分型, 且HRM方法的分型结果与测序验证结果完全一致。因此, 利用HRM技术可以实现在闭合单管中简便、准确地对CYP2C19*2 、CYP2C19*3两个多态性位点同时进行基因分型。该方法有望应用于临床, 指导氯吡格雷的个体化用药。     

Rapid identification of chloroplast haplotypes using High Resolution Melting analysis

We have evaluated High Resolution Melting (HRM) analysis as a method for one‐step haplotype identification in phylogeographic analysis. Using two adjoined internal amplicons (c. 360 and 390 bp) at the chloroplast rps16 intron (c. 750 bp) we applied HRM to identify haplotypes in 21 populations of two European arctic‐alpine herb species Arenaria ciliata and Arenaria norvegica (Caryophyllaceae). From 446 accessions studied, 20 composite rps16 haplotypes were identified by the melting‐curve protocol, 18 of which could be identified uniquely. In a comparative sensitivity analysis with in silico PCR‐RFLP, only seven of these 20 haplotypes could be identified uniquely. Observed in vitro experimental HRM profiles were corroborated by in silico HRM analysis generated on uMelt^SM. In silico mutation analysis carried out on a 360 bp wild‐type rps16I amplicon determined that the expected rate of missed single‐nucleotide polymorphisms (SNP) detection in vitro was similar to existing evaluations of HRM sensitivity, with transversion SNPs being more likely to go undetected compared to transition SNPs. In vitro HRM successfully discriminated between all amplicon templates differing by two or more base changes (352 cases) and between 11 pairs of amplicons where the only difference was a single transition or transversion SNP. Only one pairwise comparison yielded no discernable HRM curve difference between haplotypes, and these samples differed by one transversion (C/G) SNP. HRM analysis represents an untapped resource in phylogeographic analysis, and with appropriate primer design any polymorphic locus is potentially amenable to this single‐reaction method for haplotype identification.