RESUMO
Anthracnose leaf blight (ALB) is an economically important disease of sorghum [Sorghum bicolor (L.) Moench] caused by the fungal pathogen Colletotrichum sublineola Henn. ex Sacc. & Trotter. Although qualitative and quantitative resistance have been identified for ALB, the usefulness of resistance loci differs depending on the pathogen pathotype. Identifying resistance effective against unique pathogen pathotypes is critical to managing ALB, as the disease is managed primarily through the deployment of host resistance. We isolated C. sublineola from ALB-infected leaves collected in Illinois and found that the strain was a novel pathotype, as it produced a unique combination of virulence against a set of differential lines. Using this isolate, we inoculated 579 temperate-adapted sorghum conversion lines in 2019 and 2020. We then conducted a genome-wide association study (GWAS) and a metabolic pathway analysis using the Pathway Associated Study Tool (PAST). We identified 47 significant markers distributed across all chromosomes except chromosome 8. We identified 32 candidate genes based on physical proximity with significant markers, some of which have a known role in host defense. We identified 47 pathways associated with ALB resistance, indicating a role for secondary metabolism in defense to ALB. Our results are important to improve the understanding of the genetic basis of ALB resistance in sorghum and highlight the importance of developing durable resistance to ALB.
Assuntos
Colletotrichum , Sorghum , Grão Comestível/genética , Estudo de Associação Genômica Ampla , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Sorghum/genética , Sorghum/microbiologiaRESUMO
Northern corn leaf blight (NCLB) and sorghum leaf blight (SLB) are significant diseases of maize and sorghum, respectively, caused by the filamentous fungus Setosphaeria turcica. Strains of S. turcica are typically host-specific and infect either maize or sorghum. Host specificity in this pathogen is attributed to a single locus for maize and a second distinct locus for sorghum. To identify the genetic basis of host specificity in S. turcica, we generated a biparental population of S. turcica by crossing strains specific to maize and sorghum, phenotyped the population for leaf blight on sorghum and maize, genotyped the population to create a linkage map of S. turcica, and located candidate virulence regions. A total of 190 ascospores from 35 pseudothecia were isolated from the cross of maize and sorghum-specific strains. Greenhouse phenotyping of the biparental population (n = 144) showed independent inheritance of virulence, as indicated by a 1:1:1:1 segregation for virulence to maize, sorghum, both maize and sorghum, and avirulence to both crops. The population and host-specific parent strains were genotyped using genome skim sequencing on an Illumina NovaSeq 6000 platform resulting in over 780 million reads. A total of 32,635 variants including single nucleotide polymorphisms and indels were scored. There was evidence for a large deletion in the sorghum-specific strain of S. turcica. A genetic map consisting of 17 linkage groups spanning 3,069 centimorgans was constructed. Virulence to sorghum and maize mapped on distinct linkage groups with a significant QTL detected for virulence to maize. Furthermore, a single locus each for the in vitro traits hyphal growth rate and conidiation were identified and mapped onto two other linkage groups. In vitro traits did not correlate with in planta virulence complexity, suggesting that virulence on both hosts does not incur a fitness cost. Hyphal growth rate and conidiation were negatively correlated, indicating differences in hyphal growth versus dispersal ability for this pathogen. Identification of genetic regions underlying virulence specificity and saprotrophic growth traits in S. turcica provides a better understanding of the S. turcica- Andropogoneae pathosystem.
Assuntos
Doenças das Plantas , Zea mays , Ascomicetos , Mapeamento Cromossômico , Genômica , Doenças das Plantas/microbiologia , Virulência/genética , Zea mays/microbiologiaRESUMO
Pathogens that infect more than one host offer an opportunity to study how resistance mechanisms have evolved across different species. Exserohilum turcicum infects both maize and sorghum and the isolates are host-specific, offering a unique system to examine both compatible and incompatible interactions. We conducted transcriptional analysis of maize and sorghum in response to maize-specific and sorghum-specific E. turcicum isolates and identified functionally related co-expressed modules. Maize had a more robust transcriptional response than sorghum. E. turcicum responsive genes were enriched in core orthologs in both crops, but only up to 16% of core orthologs showed conserved expression patterns. Most changes in gene expression for the core orthologs, including hub genes, were lineage specific, suggesting a role for regulatory divergent evolution. We identified several defense-related shared differentially expressed (DE) orthologs with conserved expression patterns between the two crops, suggesting a role for parallel evolution of those genes in both crops. Many of the differentially expressed genes (DEGs) during the incompatible interaction were related to quantitative disease resistance (QDR). This work offers insights into how different hosts with relatively recent divergence interact with a common pathogen. Our results are important for developing resistance to this critical pathogen and understanding the evolution of host-pathogen interactions.
RESUMO
Clavibacter nebraskensis causes Goss's bacterial wilt and leaf blight, a major disease of maize. Infected crop residue is the primary inoculum source and infection can occur via wounds or natural openings, such as stomata or hydathodes. The use of resistant hybrids is the primary control method for Goss's wilt. In this study, colonization and movement patterns of C. nebraskensis during infection were examined using green fluorescent protein (GFP)-labeled bacterial strains. We successfully introduced a plasmid to C. nebraskensis via electroporation, which resulted in GFP accumulation. Fluorescence microscopy revealed that in the absence of wounding, bacteria colonize leaf tissue via entry through the hydathodes when guttation droplets are present. Stomatal penetration was not observed under natural conditions. Bacteria initially colonize the xylem and subsequently the mesophyll, which creates the freckles that are characteristic of the disease. Bacteria infiltrated into the mesophyll did not cause disease symptoms, could not enter the vasculature, and did not spread from the initial inoculation point. Bacteria were observed exuding through stomata onto the leaf surface, resulting in the characteristic sheen of diseased leaves. Resistant maize lines exhibited decreased bacterial spread in the vasculature and the mesophyll. These tools to examine C. nebraskensis movement offer opportunities and new insights into the pathogenesis process and can form the basis for improved Goss's wilt management through host resistance.
Assuntos
Clavibacter , Zea mays , Proteínas de Fluorescência Verde/genética , Doenças das PlantasRESUMO
Plants have the capacity to respond to conserved molecular features known as microbe-associated molecular patterns (MAMPs). The goal of this work was to assess variation in the MAMP response in sorghum, to map loci associated with this variation, and to investigate possible connections with variation in quantitative disease resistance. Using an assay that measures the production of reactive oxygen species, we assessed variation in the MAMP response in a sorghum association mapping population known as the sorghum conversion population (SCP). We identified consistent variation for the response to chitin and flg22-an epitope of flagellin. We identified two SNP loci associated with variation in the flg22 response and one with the chitin response. We also assessed resistance to Target Leaf Spot (TLS) disease caused by the necrotrophic fungus Bipolaris cookei in the SCP. We identified one strong association on chromosome 5 near a previously characterized disease resistance gene. A moderately significant correlation was observed between stronger flg22 response and lower TLS resistance. Possible reasons for this are discussed.
Assuntos
Moléculas com Motivos Associados a Patógenos , Doenças das Plantas/imunologia , Sorghum/genética , Sorghum/imunologia , Bipolaris , Quitina/imunologia , Resistência à Doença/genética , Flagelina/imunologia , Estudo de Associação Genômica Ampla , Doenças das Plantas/microbiologia , Pseudomonas syringae , Sorghum/microbiologiaRESUMO
Herbicide application is crucial for weed management in most crop production systems, but for sorghum herbicide options are limited. Sorghum is sensitive to residual protoporphyrinogen oxidase (PPO)-inhibiting herbicides, such as fomesafen, and a long re-entry period is required before sorghum can be planted after its application. Improving sorghum for tolerance to such residual herbicides would allow for increased sorghum production and the expansion of herbicide options for growers. In this study, we observed sorghum tolerance to residual fomesafen. To investigate the underlying tolerance mechanism a genome-wide association mapping study was conducted using field-collected sorghum biomass panel (SBP) data, and a greenhouse assay was developed to confirm the field phenotypes. A total of 26 significant SNPs (FDR<0.05), spanning a 215.3 kb region on chromosome 3, were detected. The ten most significant SNPs included two in genic regions (Sobic.003G136800, and Sobic.003G136900) and eight SNPs in the intergenic region encompassing the genes Sobic.003G136700, Sobic.003G136800, Sobic.003G137000, Sobic.003G136900, and Sobic.003G137100. The gene Sobic.003G137100 (PPXI), which encodes the PPO1 enzyme, one of the targets of PPO-inhibiting herbicides, was located 12kb downstream of the significant SNP S03_13152838. We found that PPXI is highly conserved in sorghum and expression does not significantly differ between tolerant and sensitive sorghum lines. Our results suggest that PPXI most likely does not underlie the observed herbicide tolerance. Instead, the mechanism underlying herbicide tolerance in the SBP is likely metabolism-based resistance, possibly regulated by the action of multiple genes. Further research is necessary to confirm candidate genes and their functions.
Assuntos
Benzamidas/farmacologia , Resistência a Herbicidas , Polimorfismo de Nucleotídeo Único , Protoporfirinogênio Oxidase/genética , Sorghum/crescimento & desenvolvimento , Biomassa , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Protoporfirinogênio Oxidase/antagonistas & inibidores , Sorghum/efeitos dos fármacos , Sorghum/genéticaRESUMO
Crops are hosts to numerous plant pathogenic microorganisms. Maize has several major disease issues; thus, breeding multiple disease resistant (MDR) varieties is critical. While the genetic basis of resistance to multiple fungal pathogens has been studied in maize, less is known about the relationship between fungal and bacterial resistance. In this study, we evaluated a disease resistance introgression line (DRIL) population for the foliar disease Goss's bacterial wilt and blight (GW) and conducted quantitative trait locus (QTL) mapping. We identified a total of ten QTL across multiple environments. We then combined our GW data with data on four additional foliar diseases (northern corn leaf blight, southern corn leaf blight, gray leaf spot, and bacterial leaf streak) and conducted multivariate analysis to identify regions conferring resistance to multiple diseases. We identified 20 chromosomal bins with putative multiple disease effects. We examined the five chromosomal regions (bins 1.05, 3.04, 4.06, 8.03, and 9.02) with the strongest statistical support. By examining how each haplotype effected each disease, we identified several regions associated with increased resistance to multiple diseases and three regions associated with opposite effects for bacterial and fungal diseases. In summary, we identified several promising candidate regions for multiple disease resistance in maize and specific DRILs to expedite interrogation.
Assuntos
Micoses , Zea mays , Ascomicetos , Resistência à Doença/genética , Melhoramento Vegetal , Doenças das Plantas/genética , Locos de Características Quantitativas , Zea mays/genéticaRESUMO
BACKGROUND: Exserohilum turcicum is an important pathogen of both sorghum and maize, causing sorghum leaf blight and northern corn leaf blight. Because the same pathogen can infect and cause major losses for two of the most important grain crops, it is an ideal pathosystem to study plant-pathogen evolution and investigate shared resistance mechanisms between the two plant species. To identify sorghum genes involved in the E. turcicum response, we conducted a genome-wide association study (GWAS). RESULTS: Using the sorghum conversion panel evaluated across three environments, we identified a total of 216 significant markers. Based on physical linkage with the significant markers, we detected a total of 113 unique candidate genes, some with known roles in plant defense. Also, we compared maize genes known to play a role in resistance to E. turcicum with the association mapping results and found evidence of genes conferring resistance in both crops, providing evidence of shared resistance between maize and sorghum. CONCLUSIONS: Using a genetics approach, we identified shared genetic regions conferring resistance to E. turcicum in both maize and sorghum. We identified several promising candidate genes for resistance to leaf blight in sorghum, including genes related to R-gene mediated resistance. We present significant advancements in the understanding of host resistance to E. turcicum, which is crucial to reduce losses due to this important pathogen.
Assuntos
Ascomicetos/fisiologia , Genes de Plantas , Ligação Genética , Doenças das Plantas/genética , Sorghum/genética , Zea mays/genética , Meio Ambiente , Estudo de Associação Genômica Ampla , Doenças das Plantas/microbiologiaRESUMO
Quantification of the simultaneous contributions of loci to multiple traits, a phenomenon called pleiotropy, is facilitated by the increased availability of high-throughput genotypic and phenotypic data. To understand the prevalence and nature of pleiotropy, the ability of multivariate and univariate genome-wide association study (GWAS) models to distinguish between pleiotropic and non-pleiotropic loci in linkage disequilibrium (LD) first needs to be evaluated. Therefore, we used publicly available maize and soybean genotypic data to simulate multiple pairs of traits that were either (i) controlled by quantitative trait nucleotides (QTNs) on separate chromosomes, (ii) controlled by QTNs in various degrees of LD with each other, or (iii) controlled by a single pleiotropic QTN. We showed that multivariate GWAS could not distinguish between QTNs in LD and a single pleiotropic QTN. In contrast, a unique QTN detection rate pattern was observed for univariate GWAS whenever the simulated QTNs were in high LD or pleiotropic. Collectively, these results suggest that multivariate and univariate GWAS should both be used to infer whether or not causal mutations underlying peak GWAS associations are pleiotropic. Therefore, we recommend that future studies use a combination of multivariate and univariate GWAS models, as both models could be useful for identifying and narrowing down candidate loci with potential pleiotropic effects for downstream biological experiments.
RESUMO
Vascular wilt bacteria such as Pantoea stewartii, the causal agent of Stewart's bacterial wilt of maize (SW), are destructive pathogens that are difficult to control. These bacteria colonize the xylem, where they form biofilms that block sap flow leading to characteristic wilting symptoms. Heritable forms of SW resistance exist and are used in maize breeding programs but the underlying genes and mechanisms are mostly unknown. Here, we show that seedlings of maize inbred lines with pan1 mutations are highly resistant to SW. However, current evidence suggests that other genes introgressed along with pan1 are responsible for resistance. Genomic analyses of pan1 lines were used to identify candidate resistance genes. In-depth comparison of P. stewartii interaction with susceptible and resistant maize lines revealed an enhanced vascular defense response in pan1 lines characterized by accumulation of electron-dense materials in xylem conduits visible by electron microscopy. We propose that this vascular defense response restricts P. stewartii spread through the vasculature, reducing both systemic bacterial colonization of the xylem network and consequent wilting. Though apparently unrelated to the resistance phenotype of pan1 lines, we also demonstrate that the effector WtsE is essential for P. stewartii xylem dissemination, show evidence for a nutritional immunity response to P. stewartii that alters xylem sap composition, and present the first analysis of maize transcriptional responses to P. stewartii infection.
Assuntos
Resistência à Doença , Pantoea , Zea mays , Resistência à Doença/genética , Genoma de Planta/genética , Pantoea/fisiologia , Plântula/microbiologia , Xilema/microbiologia , Zea mays/genética , Zea mays/microbiologiaRESUMO
Goss's bacterial wilt and leaf blight is one of the most important foliar diseases of maize ( L.). To date, neither large-effect resistance genes, nor practical chemical controls exist to manage the disease. Thus, the importance of discovering durable host resistance necessitates additional genetic mapping for this disease. Unfortunately, because of the biology of the pathogen and the highly significant genotype-by-environment interaction effect observed with Goss's wilt, consistent phenotyping across multiple years poses a hurdle for genetic studies and conventional breeding methods. The objective of this study was to perform a genome-wide association study (GWAS) to identify regions of the genome associated with Goss's wilt resistance as well as to use genomic prediction models to evaluate the utility of genomic selection (GS) in predicting Goss's wilt phenotypes in a panel of diverse maize lines. Using genome-wide association mapping, we were unable to identify any variants significantly associated with Goss's wilt. However, using genomic prediction we were able to train a model with an accuracy of 0.69. Taken together, this suggests that resistance to Goss's wilt is highly polygenic. In addition, when evaluating the accuracy of our prediction model under reduced marker density, it was shown that only 10,000 single nucleotide polymorphisms (SNPs), or â¼20% of our total marker set, was necessary to achieve prediction accuracies similar to the full marker set. This is the first report of genomic prediction for a bacterial disease of maize, and these results highlight the potential of GS for disease resistance in maize.
Assuntos
Actinobacteria , Resistência à Doença/genética , Doenças das Plantas/genética , Zea mays/genética , Clavibacter , Estudo de Associação Genômica Ampla , Doenças das Plantas/microbiologia , Zea mays/microbiologiaRESUMO
The logistic mixed model (LMM) is well-suited for the genome-wide association study (GWAS) of binary agronomic traits because it can include fixed and random effects that account for spurious associations. The recent implementation of a computationally efficient model fitting and testing approach now makes it practical to use the LMM to search for markers associated with such binary traits on a genome-wide scale. Therefore, the purpose of this work was to assess the applicability of the LMM for GWAS in crop diversity panels. We dichotomized three publicly available quantitative traits in a maize diversity panel and two quantitative traits in a sorghum diversity panel, and them performed a GWAS using both the LMM and the unified mixed linear model (MLM) on these dichotomized traits. Our results suggest that the LMM is capable of identifying statistically significant marker-trait associations in the same genomic regions highlighted in previous studies, and this ability is consistent across both diversity panels. We also show how subpopulation structure in the maize diversity panel can underscore the LMM's superior control for spurious associations compared to the unified MLM. These results suggest that the LMM is a viable model to use for the GWAS of binary traits in crop diversity panels and we therefore encourage its broader implementation in the agronomic research community.
Assuntos
Variação Genética , Estudo de Associação Genômica Ampla , Modelos Estatísticos , Sorghum/genética , Zea mays/genética , Modelos LinearesRESUMO
The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc.
Assuntos
Fungos/patogenicidade , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Zea mays/microbiologia , Automação , Imagem Óptica/métodos , Doenças das Plantas/microbiologia , Manejo de Espécimes/métodos , Coloração e Rotulagem/métodosRESUMO
The recent rapid emergence of maize lethal necrosis (MLN), caused by coinfection of maize with Maize chlorotic mottle virus (MCMV) and a second virus usually from the family Potyviridae, is causing extensive losses for farmers in East Africa, Southeast Asia, and South America. Although the genetic basis of resistance to potyviruses is well understood in maize, little was known about resistance to MCMV. The responses of five maize inbred lines (KS23-5, KS23-6, N211, DR, and Oh1VI) to inoculation with MCMV, Sugarcane mosaic virus, and MLN were characterized. All five lines developed fewer symptoms than susceptible controls after inoculation with MCMV; however, the virus was detected in systemic leaf tissue from each of the lines similarly to susceptible controls, indicating that the lines were tolerant of MCMV rather than resistant to it. Except for KS23-5, the inbred lines also developed fewer symptoms after inoculation with MLN than susceptible controls. To identify genetic loci associated with MCMV tolerance, large F2 or recombinant inbred populations were evaluated for their phenotypic responses to MCMV, and the most resistant and susceptible plants were genotyped by sequencing. One to four quantitative trait loci (QTL) were identified in each tolerant population using recombination frequency and positional mapping strategies. In contrast to previous studies of virus resistance in maize, the chromosomal positions and genetic character of the QTL were unique to each population. The results suggest that different, genotype-specific mechanisms are associated with MCMV tolerance in maize. These results will allow for the development of markers for marker-assisted selection of MCMV- and MLN-tolerant maize hybrids for disease control.
Assuntos
Cromossomos de Plantas/genética , Gammaherpesvirinae , Doenças das Plantas/genética , Doenças das Plantas/virologia , Locos de Características Quantitativas/genética , Zea mays/genética , Mapeamento Cromossômico , Predisposição Genética para Doença , GenótipoRESUMO
Despite the reduction in the price of sequencing, it remains expensive to sequence and assemble whole, complex genomes of multiple samples for population studies, particularly for large genomes like those of many crop species. Enrichment of target genome regions coupled with next generation sequencing is a cost-effective strategy to obtain sequence information for loci of interest across many individuals, providing a less expensive approach to evaluating sequence variation at the population scale. Here we evaluate amplicon-based enrichment coupled with semiconductor sequencing on a validation set consisting of three maize inbred lines, two hybrids and 19 landrace accessions. We report the use of a multiplexed panel of 319 PCR assays that target 20 candidate loci associated with photoperiod sensitivity in maize while requiring 25 ng or less of starting DNA per sample. Enriched regions had an average on-target sequence read depth of 105 with 98% of the sequence data mapping to the maize 'B73' reference and 80% of the reads mapping to the target interval. Sequence reads were aligned to B73 and 1,486 and 1,244 variants were called using SAMtools and GATK, respectively. Of the variants called by both SAMtools and GATK, 30% were not previously reported in maize. Due to the high sequence read depth, heterozygote genotypes could be called with at least 92.5% accuracy in hybrid materials using GATK. The genetic data are congruent with previous reports of high total genetic diversity and substantial population differentiation among maize landraces. In conclusion, semiconductor sequencing of highly multiplexed PCR reactions is a cost-effective strategy for resequencing targeted genomic loci in diverse maize materials.
Assuntos
Flores/fisiologia , Genes de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Zea mays/genética , Algoritmos , Mapeamento Cromossômico , Biologia Computacional , Genômica , Genótipo , Heterozigoto , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Zea mays/fisiologiaRESUMO
KEY MESSAGE: Quantitative disease resistance is used by plant breeders to improve host resistance. We demonstrate a role for a maize remorin ( ZmREM6.3 ) in quantitative resistance against northern leaf blight using high-resolution fine mapping, expression analysis, and mutants. This is the first evidence of a role for remorins in plant-fungal interactions. Quantitative disease resistance (QDR) is important for the development of crop cultivars and is particularly useful when loci also confer multiple disease resistance. Despite its widespread use, the underlying mechanisms of QDR remain largely unknown. In this study, we fine-mapped a known quantitative trait locus (QTL) conditioning disease resistance on chromosome 1 of maize. This locus confers resistance to three foliar diseases: northern leaf blight (NLB), caused by the fungus Setosphaeria turcica; Stewart's wilt, caused by the bacterium Pantoea stewartii; and common rust, caused by the fungus Puccinia sorghi. The Stewart's wilt QTL was confined to a 5.26-Mb interval, while the rust QTL was reduced to an overlapping 2.56-Mb region. We show tight linkage between the NLB QTL locus and the loci conferring resistance to Stewart's wilt and common rust. Pleiotropy cannot be excluded for the Stewart's wilt and the common rust QTL, as they were fine-mapped to overlapping regions. Four positional candidate genes within the 243-kb NLB interval were examined with expression and mutant analysis: a gene with homology to an F-box gene, a remorin gene (ZmREM6.3), a chaperonin gene, and an uncharacterized gene. The F-box gene and ZmREM6.3 were more highly expressed in the resistant line. Transposon tagging mutants were tested for the chaperonin and ZmREM6.3, and the remorin mutant was found to be more susceptible to NLB. The putative F-box is a strong candidate, but mutants were not available to test this gene. Multiple lines of evidence strongly suggest a role for ZmREM6.3 in quantitative disease resistance.
Assuntos
Proteínas de Transporte/genética , Resistência à Doença/genética , Fosfoproteínas/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Locos de Características Quantitativas , Zea mays/genética , Ascomicetos/patogenicidade , Basidiomycota/patogenicidade , Mapeamento Cromossômico , Ligação Genética , Genótipo , Filogenia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Polimorfismo de Nucleotídeo Único , Zea mays/microbiologiaRESUMO
Quantitative trait locus (QTL) mapping in plants dates to the 1980s (Stuber et al. Crop Sci 27: 639-648, 1987; Paterson et al. Nature 335: 721-726, 1988), but earlier studies were often hindered by the expense and time required to identify large numbers of polymorphic genetic markers that differentiated the parental genotypes and then to genotype them on large segregating mapping populations. High-throughput sequencing has provided an efficient means to discover single nucleotide polymorphisms (SNPs) that can then be assayed rapidly on large populations with array-based techniques (Gupta et al. Heredity 101: 5-18, 2008). Alternatively, high-throughput sequencing methods such as restriction site-associated DNA sequencing (RAD-Seq) (Davey et al. Nat Rev Genet 12: 499-510, 2011; Baird et al. PloS ONE 3: e3376, 2008) and genotyping-by-sequencing (GBS) (Elshire et al. PLoS One 6: 2011; Glaubitz et al. PLoS One 9: e90346, 2014) can be used to identify and genotype polymorphic markers directly. Linkage disequilibrium (LD) between markers and causal variants is needed to detect QTL. The earliest QTL mapping methods used backcross and F2 generations of crosses between inbred lines, which have high levels of linkage disequilibrium (dependent entirely on the recombination frequency between chromosomal positions), to ensure that QTL would have sufficiently high linkage disequilibrium with one or more markers on sparse genetic linkage maps. The downside of this approach is that resolution of QTL positions is poor. The sequencing technology revolution, by facilitating genotyping of vastly more markers than was previously feasible, has allowed researchers to map QTL in situations of lower linkage disequilibrium, and consequently, at higher resolution. We provide a review of methods to identify QTL with higher precision than was previously possible. We discuss modifications of the traditional biparental mapping population that provide higher resolution of QTL positions, QTL fine-mapping procedures, and genome-wide association studies, all of which are greatly facilitated by high-throughput sequencing methods. Each of these procedures has many variants, and consequently many details to consider; we focus our chapter on the consequences of practical decisions that researchers make when designing QTL mapping studies and when analyzing the resulting data. The ultimate goal of many of these studies is to resolve a QTL to its causal sequence variation.
Assuntos
Mapeamento Cromossômico/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Locos de Características Quantitativas , Clonagem Molecular , Cruzamentos Genéticos , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Desequilíbrio de Ligação , Fenótipo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Multiple disease resistance has important implications for plant fitness, given the selection pressure that many pathogens exert directly on natural plant populations and indirectly via crop improvement programs. Evidence of a locus conditioning resistance to multiple pathogens was found in bin 1.06 of the maize genome with the allele from inbred line "Tx303" conditioning quantitative resistance to northern leaf blight (NLB) and qualitative resistance to Stewart's wilt. To dissect the genetic basis of resistance in this region and to refine candidate gene hypotheses, we mapped resistance to the two diseases. Both resistance phenotypes were localized to overlapping regions, with the Stewart's wilt interval refined to a 95.9-kb segment containing three genes and the NLB interval to a 3.60-Mb segment containing 117 genes. Regions of the introgression showed little to no recombination, suggesting structural differences between the inbred lines Tx303 and "B73," the parents of the fine-mapping population. We examined copy number variation across the region using next-generation sequencing data, and found large variation in read depth in Tx303 across the region relative to the reference genome of B73. In the fine-mapping region, association mapping for NLB implicated candidate genes, including a putative zinc finger and pan1. We tested mutant alleles and found that pan1 is a susceptibility gene for NLB and Stewart's wilt. Our data strongly suggest that structural variation plays an important role in resistance conditioned by this region, and pan1, a gene conditioning susceptibility for NLB, may underlie the QTL.