Fine mapping a QTL for BYDV-PAV resistance in maize

Fine mapping a QTL for BYDV-PAV resistance in maize | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fine mapping a QTL for BYDV-PAV resistance in maize Maria Schmidt, Ricardo Guerreiro, Nadia Baig, Antje Habekuß, Torsten Will, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3863035/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Jun, 2024 Read the published version in Theoretical and Applied Genetics → Version 1 posted 5 You are reading this latest preprint version Abstract Barley yellow dwarf (BYD) is one of the economically most important virus diseases of cereals worldwide, causing yield losses of up to 80 %. BYD is caused by at least ten different phloem-limited viruses called BYD viruses (BYDVs) and Cereal yellow dwarf viruses (CYDVs). Means to control BYD are limited and the use of genetically resistant cultivars is the most economic and environmentally friendly approach. Maize plays a central role in the BYD infection cycle, serving as a reservoir for BYD-causing viruses and their vectors in summer. Growing BYD resistant maize varieties would reduce BYD pressure on maize and cereals. Using two biparental mapping populations, we were able to reduce a previously published QTL for BYDV-PAV resistance in maize to ~0.3 Mbp, comprising nine genes. Association mapping and gene expression analysis further reduced the number of candidate genes for BYDV-PAV resistance in maize to two: Zm00001eb428010 and Zm00001eb428020. Predicted functions of these genes suggest that they confer BYDV-PAV resistance either via interfering with virus replication or induction of ROS signaling. The sequence of one of these genes, Zm00001eb428010, is affected by a 54 bp deletion in the 5`-UTR and a protein altering variant in BYDV-PAV resistant maize inbreds but not BYDV-PAV susceptible and BYDV-PAV tolerant inbreds. This suggests that altered abundance and/or properties of the proteins that are encoded by Zm00001eb428010 may lead to BYDV-PAV resistance. Figures Figure 1 Figure 2 Figure 3 Introduction World food consumption heavily relies on cereals. The three most important food crops in the world are rice, wheat, and maize (corn), accounting for about 42% of the world’s human food calorie intake and 37% of protein intake (Erenstein et al. 2022 ). With an expected increase of the world population (United Nations 2022 ), an increasing demand for cereals is expected (FAO 2023). However, global cereal production is threatened by climate change effects, herbivore pests and several diseases caused by fungi, bacteria, and viruses (Rivero et al. 2022 ; Savary et al. 2019 ). Barley Yellow Dwarf (BYD) is one of the economically most important diseases in small grain cereals (Choudhury et al. 2017 ; van den Eynde et al. 2020 ). This disease can infect all members of the grass family ( Poaceae ), causing yield losses in cereals of up to 80% but also negatively affects grain quality (Choudhury et al. 2019 ; Nancarrow et al. 2021 ; Peiris et al. 2019 ). BYD is caused by at least ten different phloem-limited single stranded, positive sense RNA viruses called Barley yellow dwarf viruses (BYDV) and Cereal yellow dwarf viruses (CYDV) (Walls et al. 2019 ; Miller and Lozier 2022 ). BYDVs and CYDVs are transmitted by more than 25 aphid species worldwide (Halbert and Voegtlin 1995 ). Worldwide, BYDV-PAV is the most prevalent virus species that causes BYD and is mainly transmitted by Rophalosiphum padi (Aradottir and Crespo-Herrera 2021 ). Several studies suggest that climate change will promote spread of R. padi and therefore spread of BYDV-PAV (for review see Irwin and Thresh 1990 ; Moreno-Delafuente et al. 2020 ). Viruses possess no own metabolism which makes it difficult to control them directly. The use of insecticides to limit the spread of vectors is not desirable because the application of insecticides is costly and not environmentally friendly (Chagnon et al. 2015 ; Serrão et al. 2022 ). Due to their harmful effects on beneficial insects, neonicotinoids - a very efficient and previously widely used class of insecticides (Simon-Delso et al. 2015 ) - are banned from application in the field in the European Union (Regulation (EU) No. 485/2013). Moreover, there is evidence for resistance against pyrethroid insecticides in some R. padi populations (Walsh et al. 2020 ). Thus, employment of genetically resistant cereal cultivars is the most promising approach to limit spread of BYD. However, breeding for BYD resistance is hampered by the unavailability of reliable high-throughput phenotyping methods. On one hand, BYD symptoms are influenced by the environment and might be confounded with symptoms of other diseases, nutrient deficiency, waterlogging or mechanical injury (Grüntzig et al. 1997 ). On the other hand, BYD-tolerant genotypes show no symptoms but the virus is able to replicate and move systemically in these plants. Thus, BYD-tolerant plants act as a virus reservoir and are a source for infection of other cereals. In contrast, resistant plants inhibit virus replication and/or systemic movement. A more reliable alternative to visual symptom scoring is the quantification of the virus titer in plants by quantitative real time PCR (qRT-PCR), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) or tissue blot immunoassay (TBIA) (Canning et al. 1996 ; Chéour et al. 1993 ; Choudhury et al. 2018 ). However, these methods are from a breeder’s perspective laborious and time-consuming. Thus, the use of molecular markers that are at least closely linked to the resistance gene will help to accelerate the breeding process for BYD resistant varieties. To date, four loci - r yd1, Ryd2, Ryd3 , and Ryd4 Hb - are known to convey tolerance or resistance to BYD in barley, but only Ryd2 was introduced into commercial cultivars so far (Choudhury et al. 2017 ; Jarošová et al. 2016 ; Walls et al. 2019 ). Ryd1 was less efficient for breeding purpose (Suneson, 1955 ) compared to the other loci. Ryd2 reduces the virus titer of BYDV-PAV and BYDV-MAV in young plants but does not convey resistance against all BYD-causing virus species and is not effective in adult plants (Baltenberger et al 1987 ; Riedel et al 2011 ; Šip et al 2004 ). Ryd3 is a major gene for BYDV-PAV resistance (Niks et al 2004 ). Despite the presence of Ryd3 seems to prevent BYDV-PAV replication, 20% of the plants carrying the Ryd3 gene developed symptoms and had virus concentrations similar to those of susceptible accessions (Niks et al 2004 ). Ryd4 Hb was introgressed into cultivated barley from the wild relative H. bulbosum and was not used in breeding programs because of linkage drag with a recessive sublethality factor (Scholz et al. 2009 ). Recently, Ryd4 Hb was fine-mapped to a 66.5 kbp physical interval in the barley ‘Morex’ reference genome that comprises one complete and one partial gene from the nucleotide-binding and leucine-rich repeat immune receptor family (Pidon et al. 2023 ). Additional QTL for tolerance to BYD-PAV and BYDV-RPV were mapped to chromosomes 1H, 2H, 4H, and 7H of barley (del Blanco et al. 2014; Kraakman et al. 2006 ; Scheurer et al. 2001 ; Toojinda et al. 2000 ). In wheat, no naturally occurring resistance gene or QTL against BYD is known from the primary gene pool. Only one locus – bdv1 – confers tolerance to BYDV-MAV (Singh et al. 1993 ) and is used in commercial breeding programs. However, BYD resistance was found in the tertiary gene pool of wheat. Three BYD resistance loci – Bdv2 , Bdv3 , and Bdv4 – were introgressed from Thinopyrum intermedium into wheat via translocation lines (for review see Zhang et al. 2009 ). However, the only locus used in wheat breeding is Bdv2 , which conveys a broad-spectrum resistance to BYD viruses (Brettell et al. 1988 ; Zhang et al. 1999 ). A reduction of plant height, ear height, biomass, and grain yield as well as an earlier flowering was observed in BYD infected maize compared to non-inoculated maize plants (Beuve et al. 1999 ; Loi et al. 2004 : Panayotou 1977 ). In addition, maize plays an important role in the BYD transmission cycle serving as a ‘green bridge‘ between harvest of small-grain cereals in early summer and sowing of winter cereals in autumn (Brown et al. 1984 ; Haack et al. 1999 ; Rashidi et al. 2021 ). This effect is intensified by increasing viruliferous aphid populations under global warming conditions (Moreno-Delafuente et al. 2020 ). Cultivation of BYD-resistant maize is expected to reduce BYD pressure on maize and small-grain cereals like wheat and barley. BYDV-PAV resistance in maize shows high genotypic variance and high heritability (Horn et al. 2013 , 2014 , and 2015 ), making it a promising target for breeding efforts. Recently, a QTL for BYDV-PAV resistance was discovered in maize on the distal end of chromosome 10 (Horn et al. 2014 and 2015 ). In a genome wide association study (GWAS), Horn et al. ( 2014 ) identified three single nucleotide polymorphims (SNPs) in gene GRMZM2G018027 (Zm00001eb428020) that were associated with BYDV-PAV resistance. These SNPs explained between 16 and 21% of the phenotypic variance of the trait virus titer (EX) as well as between 11 and 18% of the phenotypic variance of the trait infection rate (IR). Similarly, in another study employing five connected linkage mapping populations, Horn et al. ( 2015 ) identified a QTL on the distal end of chromosome 10 which overlapped with the above described gene GRMZM2G018027 and that explained 45% of the phenotypic variance for the traits EX and IR. However, the causative gene underlying this QTL was not identified. The objectives of this study were i) to identify the causative gene for BYDV-PAV resistance in maize, ii) to identify SNPs and/or structural variations in the gene sequences that may cause differences in susceptibility to BYDV-PAV of maize inbreds, and iii) to characterize the effect of BYDV-PAV infection on gene expression of susceptible, tolerant, and resistant maize inbreds. The findings may be used to develop markers for marker assisted breeding of BYDV-PAV resistant maize and as well as a starting point for the investigation of the resistance mechanism. However, the cloning of this BYDV-PAV resistance QTL will be also informative for the breeding of BYD resistant barley and wheat genotypes by providing targets for mutagenesis experiments. Methods Plant cultivation and aphid rearing Maize plants ( Zea mays L.) were grown in a greenhouse (16h light, 20°C / 8h darkness, 16°C) for phenotyping of segregating material or in a climate chamber (16h light, 24°C / 8h darkness, 22°C) for all other experiments. BYDV-PAV carrying and virus-free aphids of species Rhopalosiphum padi were reared on BYDV-susceptible barley cv. ‘Rubina’ at room temperature under artificial light conditions. Viruferous and virus-free aphids were kept at physical distance and checked regularly for presence of BYDV-PAV using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) method with in-house polyclonal antisera for BYDV-PAV from the Julius Kühn-Institute as described by Horn et al. ( 2013 ). Fine mapping of the BYDV-PAV resistance gene Plant material Our study was based on heterogenous inbred family (HIF) populations developed from recombinant inbred lines (RILs) which were derived from crosses of BYDV-PAV tolerant inbred line P092 with BYDV-PAV resistant inbred lines FAP1360A and Ky226, designated as population A and B, respectively (Horn et al. 2015 ). The progenies derived from each RIL were designated in the following as sub-populations. The RILs that were selfed were heterozygous for the QTL interval but homozygous for the rest of the genome. HIFs were genotyped using total of 39 SNP-based Kompetitive allele-specific polymerase chain reaction (KASP™) marker (see below). Furthermore, these individuals were selfed to generate seeds for replicated infection experiments as described below. Heterozygous HIF offspring were subjected to another round of selfing. DNA extraction, KASP™ marker design and application DNA extraction was conducted using an in-house protocol. In brief, about 25 to 50 mg frozen plant material was homogenized using Tissue Lyzer II (Qiagen, Hilden, Germany) and 150 µl extraction buffer was added. After centrifugation (10 min, 4°C, 4.000 RCF), 75 µl supernatant was transferred to a new plate containing 60 µl isopropanol, gently mixed, and centrifuged (10 min, 4°C, 4.000 RCF). The supernatant was discarded, the pellet was washed with 150 µl ethanol (70%) and eluted in 100 µl TE buffer. DNA concentration of random samples was checked with a nanophotometer. KASP™ marker were designed in several rounds based on different sources of SNP information. No matter of the source, SNP information was filtered for identical alleles for inbreds Ky226 and FAP1360A but different to P092. In the first round, molecular marker information from Horn et al. ( 2015 ) was employed. Sequences flanking the SNPs at least 50 bp upstream and downstream were retrieved from the maize genetics and genomics database ( https://www.maizegdb.org/ ) using reference version 4 of the B73 genome. Later, information from targeted sequencing of parental inbred lines was used for marker design. Such SNPs were preferred that have identical sequences of P092, Ky226, and FAP1360A in the 50 bp flanking regions. We aimed to spread markers evenly across the QTL confidence interval. Sequences were send to the manufacturer LGC Genomics Ltd. (Hoddesdon, Herts, UK) for the design of the markers. Genotyping was conducted as recommended by the manufacturer. An ABI Quantstudio 5 (Applied Biosystems) was used for analysis. Inoculation and quantification of virus titer The above described HIF populations were evaluated for IR and EX in four replications per genotype, where an experimental unit comprised eight to ten plants of one genotype. In all experiments, founder inbred lines FAP1360A, Ky226, and P092 but also two additional inbreds which are resistant and susceptible to BYDV-PAV, D408 and W64A, respectively, were used as controls. BYDV-PAV carrying R. padi were removed from barley plants used for rearing when the maize plants reached the two-leaf stage. Aphids were distributed evenly across the maize plants in a way that approximately ten aphids per plant were applied. This time point was in the following designated as start of inoculation. After one week, plants were treated with insecticide “Careo” (Substral Celaflor) or “Confidor” (Bayer CropScience). Leaf material from the sixth leaf of each plant was harvested separately six weeks after the start of the inoculation and virus titer was determined using the DAS-ELISA method with in-house polyclonal antisera for BYDV-PAV as described by Horn et al. ( 2013 ). The infection rate (IR) was calculated as the percentage of plants of one experimental unit with virus titer ≥ 0.5. EX was calculated as the mean virus titer per experimental unit. Phenotypic data analyses and genetic mapping Estimated marginal means of EX and IR across all replications of an experiment were calculated using the following mixed linear model: Y ij = µ + g i + r j + e ij where Y ij was the phenotypic observation for the ith genotype for the jth replicate, µ the general mean, g i the effect of the ith genotype, r j the effect of the jth replication, and e ij the residual. With the same model but with genotype as random effect, genotypic σ g 2 and error variance σ e 2 were calculated. Broad-sense heritability on an entry mean basis (H 2 ) was calculated. In order to associate the above described marginal means of each genotype of the HIF populations with the molecular marker profile, the following linear model was used for each of the 39 SNP-based KASP™ markers: Y ik = µ + s k + m i + e ik were s k was the effect of the HIF population and m i the effect of the marker which significance was tested with a F-test. In a second mapping approach, we used the phenotypic data for BYDV-PAV resistance of an association panel from Horn et al. ( 2014 ). HapMap3.2.1 genotypic data (Bokowski et al. 2018) corresponding to the 300 Kbp QTL confidence interval were retrieved from the PANZEA website. Ambiguous data points were removed and sequence variants were filtered for minor allele frequency > 0.025 and missing values < 20%. Association analysis was conducted as described by Horn et al. ( 2014 ) using the Q matrix from Flint-Garcia et al. ( 2005 ) and the K matrix from Horn et al. ( 2014 ). Analyses were conducted using R version 3.6.3 (R Core Team 2020 , https://www.R-project.org/ ) with packages “lme4” version 1.1–23 (Bates et al 2015 ), “emmeans” version 1.5.1 (Lenth 2020 ), “car” version 3.0–10 (Fox and Weisberg 2019), and RStudio version 1.3.1073 (RStudio Team 2020 , http://www.rstudio.com/ ). Protein sequences and information on gene annotation were retrieved from the maize genetics and genomics database ( https://www.maizegdb.org/ ). Protein sequences were loaded into InterPro ( https://www.ebi.ac.uk/interpro/ ; Paysan-Lafosse et al. 2022 ) to predict functional protein domains. Degree of dominance of the resistance gene Six sub-populations of HIFs were created to estimate the degree of dominance. Each of these sub-populations consisted of one genotype that was homozygous at marker SYN4811 for the allele of P092 and a sibling that was homozygous for the allele of Ky226 or FAP1360A. Additionally, one or two heterozygous genotypes per sub-population were included that were siblings or offspring of crosses between the homozygous genotypes. Plants were inoculated with BYDV-PAV carrying R. padi in two replications. Virus titer was measured and EX and IR per genotype were calculated (see previous section). Mean EX and IR per group (homozygous resistant, homozygous susceptible or heterozygous) were calculated. Genomic characterization of founder maize inbreds Probe design Probes for target enrichment sequencing of founder inbred lines were designed for the QTL confidence interval identified by Horn et al. ( 2014 ) plus 1 Mbp to the distal end of the chromosome. At the time of probe design, reference sequences of seven maize inbred lines were available. These were B73 (Zm00001d.2), CML247 (Zm00024a.1), EP1 (Zm00010a.1), F7 (Zm00011a.1), Mo17 (Zm00014a.1), PH207 (Zm00008a.1), and W22 (Zm00004a.1). The sequences were used by the probe design team of the manufacturer (Roche/Nimblegen) to design after masking of repetitive sequences, 2 million probes optimized for PacBio sequencing where up to three matches to the reference genome of B73 version 4 (Zm00001d.2) were allowed. DNA extraction and sequencing DNA was extracted using NucleoMag Plant Kit (Macherey & Nagel GmbH & Co. KG Düren, Germany) following manufacturer's instructions. DNA concentration and quality were assessed with a nanophotometer, a Qubit fluorometer (Invitrogen) with a Qubit dsDNA HS Assay Kit, and a Fragment Analyzer (Advanced Analytical Technologies). Sample preparation was conducted following PacBio protocol “Multiplex Genomic DNA Target Capture Using SeqCap EZ Libraries” (PN 100-893-500 version 03). In brief, genomic DNA was fragmented using gTUBES (Covaris), end-repaired and A-tailed using a KAPA HyperPlus Kit (Roche Sequencing Solutions, Pleasanton, CA, USA), barcoded and adapters were ligated. DNA fragments were then amplified using a universal primer (Sigma-Aldrich) and Takara LA Taq DNA polymerase hot-start version (Takara). PCR fragments were size selected for fragment length greater than 4.5 kbp with a BluePippin™ automated DNA size selection device (Sage Science), pooled, hybridized with SeqCap EZ Prime Developer Probes (Roche Diagnostics GmbH, Mannheim, Germany) and captured using HyperCap Target Enrichment Kit (Roche Diagnostics GmbH, Mannheim, Germany) and Dynabeads M-270 Streptavidin (Invitrogen by Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania). Captured DNA fragments were amplified using a universal primer and Takara LA Taq DNA polymerase hot-start version (Takara). SMRTbell™ library preparation was performed following the manufacturer's instructions. Sequencing was conducted on a Sequel II plattform (PacBio) to deliver highly accurate long reads appropriate for the identification of structural variants. Data processing, SNP calling, and prediction of SVs Obtained reads were demultiplexed with python package demultiplex and trimmed with bbmap (sourceforge.net/projects/bbmap/). Trimmed reads were used for a reference guided assembly of the QTL confidence interval with RaGOO (Alonge et al. 2019 ). Trimmed reads were mapped to B73 reference sequence version 5 (Zm00001eb) using minimap2 (Li et al. 2018 ) with parameter -ax asm20 and coverage was calculated with samtools as well as custom awk and python scripts. From reads that mapped to the QTL interval, SNPs and insertions/deletions of less than 50 bp length (InDels) were called using freebayes (Garrison et al. 2012). SNPs and InDels were subjected to variant effect prediction using Variant Effect Predictor tool from Gramene ( https://ensembl.gramene.org/Oryza_sativa/Tools/VEP# ) that employs the SIFT algorithm (Ng and Henikoff 2003 ). Insertions and deletions ≥ 50 bp were defined as structural variations (SVs) and were called by re-mapping reads with restrictive parameters and exploiting cuteSV (Jiang et al. 2020 ). Genome-wide gene expression analysis RNA extraction and sequencing Two independent experiments were conducted to assess gene expression differences between the maize founder inbreds as well as upon infection with BYDV. In both experiments, plants of inbreds FAP1360A, P092, and W64A were treated with BYDV-PAV carrying R. padi , virus-free R. padi , or without aphids as control. Approximately ten BYDV-PAV carrying aphids per plant were applied when plants reached two leaf stage. After one week, all plants including controls were sprayed with insecticide “Careo” (Substral Celaflor). Samples were taken 24 hpi and 96 hpi in experiment 1 from 4–8 pooled plants per inbred, treatment and timepoint in four replications and two weeks after inoculation in experiment 2, where 2–4 single plants per inbred and treatment were tested. Leaves were harvested, frozen immediately in liquid nitrogen, and stored at -80°C until further analysis. RNA was extracted with TRIzol (Ambion by Life Technologies, Carlsbad, USA) and Direct-zol RNA MiniPrep Kit (Zymo Research; experiment 1) or RNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany; experiment 2) following the manufacturer’s recommendations. All samples were treated with RNase-free DnaseI (ThermoFisher Scientific). RNA concentration was quantified using a Qubit fluorometer (Invitrogen) and a Qubit RNA HS Assay kit (Life Technologies, Eugene, USA) and quality was assessed with a nanophotometer. The RNA was paired end sequenced with 150 bp reads on an Illumina (experiment 1) or DNBseq™ (experiment 2) plattform, respectively. BYDV infection status was confirmed via DAS-ELISA six weeks after inoculation from the sixth leaf (experiment 1) or two weeks after inoculation from the youngest fully developed leaf (experiment 2). RNAseq data processing RNAseq reads were filtered, including removing adaptor sequences, contamination and low-quality reads from raw reads. Unpaired reads were discarded. Exon and splice site information was retrieved from B73 reference genome. Reads were aligned to this reference genome using HISAT2 version 2.1.0 (Kim et al. 2019 ). SamTools version1.6 (Li et al. 2009 , Danecek et al. 2021 ) was used to index, sort, and filter mapped reads. Duplicates were removed. Reads per gene were counted with HTSeq version 0.11.1 (Anders et al. 2015 , Putri et al. 2022 ). Analysis of DEGs Analysis of differently expressed genes was conducted with edgeR version 3.28.1 (Robinson, McCarthy and Smyth 2010 ) where the following contrasts were considered: Aphid infested plants versus Control (Aphid_vs_Ctrl), BYDV infected plants versus Control (BYDV_vs_Ctrl), and BYDV infected plants versus aphid infested plants (BYDV_vs_Aphid). The lists of DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using ShinyGO 0.76.3 (Ge et al. 2020 ). Pathway databases “KEGG”, “GO Biological Process”, “GO Cellular Component”, and “GO Molecular Function” were used and parameters were set to FDR = 0.05, Pathway size: min = 2 and max = 2000, and redundancy was removed. No background gene list was provided because ShinyGO 0.76.3 employs protein coding genes as default. Results Mapping of the BYDV-PAV resistance gene For fine mapping of the BYDV-PAV resistance in maize, homozygous genotypes were selected that were recombinant in the QTL confidence interval. This selection procedure resulted in 83 genotypes originating from selfings of two RILs derived from P092 x FAP1360A (population A) and 102 individuals from selfings of RILs derived from Ky226 x P092 (population B). These 185 homozygous recombinants were subjected to phenotyping for BYDV-PAV resistance. Broad sense heritability ( H 2 ) was estimated as 0.89 for EX and 0.82 for IR across the homozygous recombinants of both populations. Analyzing both populations separately, H 2 of population A was slightly lower with 0.79 for EX and 0.70 for IR, compared to 0.92 for EX and 0.85 for IR in population B. Estimated marginal means ranged from 0.11 to 1.65 for the trait EX and − 0.05 to 1.14 for the trait IR. For both traits, estimated marginal means followed a continuous distribution (Fig. 1 ). For that subset of genotypes, for which heterozygote siblings or progenies were available, the degree of dominance was estimated. Across all sub-populations and replications, the degree of dominance was − 0.44 for EX and − 0.18 for IR. The statistical test associating the genotyping profiles of the 185 homozygous recombinants with their marginal means for EX and IR resulted in the fine mapping of the resistance factor to the genome region between marker PZE-110080306 and the newly developed marker BYDV-M20 as flanking marker of the QTL interval as for them the slope of the trendline changed the direction (Fig. 2 ). The physical position of these markers delimits the resistance factor to the region between 137,131,915 and 137,409,058 bp on chromosome 10, which comprises nine genes. These genes are Zm00001eb428020 (GRMZM2G018027), a candidate gene for BYDV-PAV resistance identified by Horn et al. ( 2014 ), two transcription factors (Zm00001eb427970 and Zm00001eb427980), a putative WAK-related receptor-like protein kinase family protein (Zm00001eb427960), a putative RING zinc finger domain superfamily protein (Zm00001eb427950), a P-loop containing nucleoside triphosphate hydrolases superfamily protein (Zm00001eb428010), and three genes of unknown function (Zm00001eb427940, Zm00001eb427990, and Zm00001eb428000) (Table 1 ). Table 1 Genes in the ~ 0.3 Mbp long QTL confidence interval for BYDV-PAV resistance in maize on chromosome 10. Start and end positions are given based on the reference sequence of B73, version 5. Gene ID Start End Description / suggested function Zm00001eb427940 137133463 137134445 unknown Zm00001eb427950 137197560 137198870 RING zinc finger domain superfamily protein Zm00001eb427960 137214761 137217464 WAK-related receptor-like protein kinase family protein Zm00001eb427970 137229874 137233357 ABI3-VP1-transcription factor 2 Zm00001eb427980 137263651 137266456 Transcription factor bHLH28 like Zm00001eb427990 137278582 137280136 unknown Zm00001eb428000 137280991 137284148 unknown Zm00001eb428010 137285187 137290824 DNA2/NAM7 helicase-like protein Zm00001eb428020 137348959 137349907 response to oxidative stress, response to cadmium ion To further reduce the number of candidate genes, we used an association mapping approach based on the BYDV-PAV phenotyping data from an association mapping panel described by Horn et al. ( 2014 ) and the genotypic data from HapMap3.2.1 (Bokowski et al. 2018). The strongest association for BYDV-PAV resistance was found for sequence variants located in genes GRMZM2G322506 (Zm00001eb428010) and GRMZM2G018027 (Zm00001eb428020) and the intergenic space in between these two genes (Fig. 3 ). Analysis of sequence variation in the QTL interval Targeted long read sequencing of the five founder inbred lines FAP1360A, Ky226, P092, D408, and W64A resulted in 1,579,826 raw reads and 8,421,284,727 bases sequenced. Reads were filtered, mapped against B73 v5 reference genome (Zm00001eb) and assembled to contigs. The total length of contigs was between 9,747,441 and 14,948,168 bp per inbred. The three BYDV-PAV resistant genotypes had similar numbers of variants when compared to reference B73. We counted 1972, 1911, and 1869 SNPs and InDels for D408, FAP1360A, and Ky226, respectively. BYDV-PAV susceptible genotype W64A had slightly less variants (1797) and for BYDV-PAV tolerant genotype P092 the lowest number of variants (1139) compared to B73 was detected. SNPs and InDels were subjected to variant effect prediction. More than 94% of SNPs and InDels were predicted to be modifiers such as upstream and downstream gene variants, intron variants, intergenic variants, and 5’- and 3’-UTR variants (Table 2 ). The SIFT algorithm predicted a high impact for 19 SNPs and InDels. However, of those only one SNP, which leads to a frameshift in gene Zm00001eb428000, was shared by the three BYDV-PAV resistant founder inbreds but not by P092 and W64A (Supplementary table 1). Additionally, six protein altering variants were detected. One protein altering variant in gene Zm00001eb428010 was shared by the three BYDV-PAV resistant founder inbreds but not by P092 and W64A. The other five variants were located in gene Zm00001eb427970, of which three were shared between D408 and FAP1360A, and two were unique to Ky226. Table 2 Variant effect prediction of single nucleotide polymorphisms (SNPs) and InDels (< 50 bp) in the ~ 0.3 Mbp QTL confidence interval on chromosome 10 between 137,131,915 and 137,409,058 bp (B73 ref v5) of five founder inbred lines. Impact Consequence Count High stop gained 2 start lost 1 stop gained, frameshift variant 1 frameshift variant 12 splice acceptor variant, coding sequence variant 1 splice acceptor variant, intron variant 2 Moderate protein altering variant 6 inframe deletion 6 inframe insertion 4 missense variant, splice region variant 1 missense variant 159 Low splice region variant, intron variant 11 splice region variant, synonymous variant 1 stop retained variant 1 synonymous variant 85 Modifier 5 prime UTR variant 63 3 prime UTR variant 148 intron variant 244 upstream gene variant 1688 downstream gene variant 1258 intergenic variant 1699 In addition, 34 SVs were detected in the 0.3 Mbp QTL confidence interval, where the majority of them (24) were unique to one inbred (Supplementary table 2). Only nine SVs were located in a gene. Remarkably, the three BYDV-PAV resistant inbreds shared a 54 bp deletion located in the 5’ UTR of gene Zm00001eb428010, a 91 bp insertion in intron 6 and a 362 bp deletion in intron 7 of the same gene. These were not present in susceptible and tolerant genotypes, respectively. Only SNPs and InDels with low (synonymous variants) or modifier effect (intron or upstream/down-stream gene variants) but no SNPs with predicted high or protein altering effect or SVs were detected for the BYDV-PAV resistance candidate gene Zm00001eb428020. Gene expression Two independent experiments were conducted to analyze the effect of BYDV-PAV infection on genome-wide gene expression in maize. Samples were taken 24 hours past infection (hpi) and 96 hpi in experiment 1 as well as two weeks after inoculation in experiment 2. Only a small number genes was significantly differently expressed in experiment 1 (Table 3 ) among treatments. P092 had the most differently expressed genes (DEGs) with a total of 111 DEGs for all time points and comparisons. A total of 88 DEGs were found for FAP1360A but no DEGs were found for W64A. Also, we did not find any DEGs in FAP1360A for the comparison BYDV_vs_Aphid. Most DEGs were found among treatments in the upregulated group at 96 hpi in both FAP1360A and P092. Remarkably, the 25 downregulated genes in BYDV vs Control of FAP1360A at 24 hpi were enriched for nucloetide and nucleoside biosynthesis and metabolism processes. Table 3 Counts of differently expressed genes (DEGs) in experiment 1 at 24 and 96 hours past infection (hpi) and in experiment 2 2 weeks past infection (wpi). Genotype Comparison 24 hpi 96 hpi 2 wpi up down up down up down FAP1360A BYDV vs Control 4 25 26 0 347 290 BYDV vs Aphid 0 0 0 0 0 0 Aphid vs Control 0 1 31 1 18 36 ‍ P092 BYDV vs Control 0 1 61 5 2904 3509 BYDV vs Aphid 0 1 43 0 3058 3546 Aphid vs Control 0 0 0 0 350 383 ‍ W64A BYDV vs Control 0 0 0 0 4577 4880 BYDV vs Aphid 0 0 0 0 5137 5010 Aphid vs Control 0 0 0 0 3671 4001 Supplementary table 1: Single Nucleotide Polymorphisms (SNPs) and InDels (< 50 bp) with high impact and selected moderate impact consequences in the ~ 0.3 Mbp long QTL confidence interval on chromosome 10 of five maize inbreds. In the second experiment, a considerably higher number of DEGs was detected (Table 3 ). R. padi infestation and BYDV-PAV infection had a low effect on gene expression in FAP1360A in comparison to P092 and W64A. We found eight to 19 times more DEGs in P092 and 13 to 204 times more DEGs in W64A than in FAP1360A, respectively. Interestingly, there were no DEGs in BYDV_vs_Aphid in FAP1360A. In contrast, BYDV_vs_Aphid was the comparison with most DEGs in P092 and W64A for up-regulated and down-regulated genes, respectively. Among the downregulated genes in P092 in BYDV_vs_Aphid, KEGG pathways „Phagosome“ (zma04145) and „Spliceosome“ (zma03040) were enriched 2.8-fold and 1.9-fold . Only two genes of the 0.3 Mbp QTL confidence interval – Zm00001eb428010 and Zm00001eb428020 – were expressed in both experiments. Additionally, Zm00001eb428000 was expressed in experiment 2 but with lower abundance than Zm00001eb428010 and Zm00001eb428020. None of these three genes was differently expressed in any genotype in any treatment combination. Discussion Barley Yellow Dwarf (BYD) is one of the economically most important diseases in small grain cereals (Choudhury et al. 2017 ; van den Eynde et al. 2020 ). Due to increasing autumn and winter temperatures, it is expected that BYD will become an increasing problem (Pidon et al. 2023 ). For maize, BYD infection has a direct negative effect on different phenotypic characters (Beuve et al. 1999 ; Loi et al. 2004 ; Panayotou 1977 ). In addition, maize plays an important role in the BYD transmission cycle serving as a ‘green bridge‘ between harvest of small-grain cereals in early summer and sowing of winter cereals in autumn (Brown et al. 1984 ; Haack et al. 1999 ; Rashidi et al. 2021 ). Cultivation of BYD-resistant maize is expected to reduce BYD pressure on maize and small-grain cereals like wheat and barley. BYD is caused by different viruses of which BYDV-PAV is the most prevalent virus worldwide. The breeding of BYDV-PAV resistant maize is strongly facilitated by the availability of markers that are closely linked to the resistance gene. Furthermore, the cloning of this BYDV-PAV resistance QTL will be also informative for the breeding of BYD-resistant barley and wheat genotypes by providing targets for mutagenesis experiments. Therefore, the BYDV-PAV QTL identified by Horn et al. ( 2015 ) was fine mapped in our study. Fine mapping of the BYDV-PAV resistance in maize In order to avoid the potential problem related to marker-trait associations that are due to population structure (Stich et al. 2008 ), in our study HIF populations were exploited. Despite the observed high heritabilities around 0.8, the marginal means of the homozygous recombinants in the HIF populations showed no distinct categories for the virus titer phenotypes EX and IR but a continuous distribution with a trend towards a bimodal distribution (Fig. 1 ). The reasons for this observation are the heritabilities lower than one together with a limited difference in the virus titer phenotypes EX and IR between resistant and susceptible/tolerant genotypes. Therefore, an ANOVA approach was used in our study to fine map the resistance factor. Furthermore, as we observed a difference in virus titer phenotypes EX and IR between both HIF populations (Fig. 2 ), we fitted a population effect in our linkage analyses of BYDV-PAV titer. These analyses allowed to reduce the QTL confidence interval from 8 Mbp (Horn et al. 2015 ) to ~ 0.3 Mbp (Fig. 2 ). The interval comprised nine annotated genes in the fifth version of the B73 reference genome (Zm00001eb) (Table 1 ). The putative function of these nine genes suggest that some of them might be involved in virus defense-related processes and, thus, convey resistance against BYDV-PAV in maize. However, as these links were rather weak, we performed an association study using BYDV-PAV resistance data from an association mapping population (Horn et al. 2014 ) and HapMap3.2.1 genotypic data (Bokowski et al. 2018) for the 0.3 Mbp QTL confidence interval to further reduce the number of candidate genes. This analysis showed strong associations of BYDV-PAV resistance with sequence variants located in genes Zm00001eb428010 and Zm00001eb428020 but not with sequence variants located in other genes of the 0.3 Mbp QTL confidence interval. This confirms that either Zm00001eb428010 or Zm00001eb428020 confers BYDV-PAV resistance in maize. Two candidate genes in the QTL for BYDV-PAV resistance in maize The protein encoded by Zm00001eb428010 contains two AAA domains. GO-terms for this gene are RNA binding (GO:0003723) and helicase activity (GO:0004386). AAA domain containing proteins possess diverse functions, including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication (Khan et al. 2022). The protein encoded by Zm00001eb428010 is predicted to belong to the DNA2/NAM7-like helicase family. Nam7, also known as Upstream frameshift 1 (Upf1), targets plant and animal viruses for nonsense-mediated mRNA decay (NMD) (for review see May and Simon 2021 ). However, many viruses escape Upf1-mediated decay through cis -acting RNA sequences and trans -acting viral proteins (May and Simon 2021 ). Horn et al. ( 2014 ) identified three SNPs in Zm00001eb428020 (GRMZM2G018027) that were significantly associated with EX and IR and proposed this gene as a candidate gene for BYDV-PAV resistance in maize. Zm00001eb428020 is associated with GO terms „response to oxidative stress“ (GO:0006979) and „response to cadmium ion“ (GO:0046686) in the molecular function category and „nuclear speck“ (GO:0016607) in the cellular component category. Nuclear speckles (NS) are nuclear membranless bodies enriched in splicing factors (Hasenson and Shav-Tal 2020 ). Fungal effectors are able to induce susceptibility of host plants by inducing alternative splicing of host transcripts at NS (Tang et al. 2022 ). The same process is suspected for oomycete effectors (Wang et al. 2015 ). The best BLAST hit for Zm00001eb428020 in Arabidopsis thaliana is the gene OXS3 (Horn et al. 2014 ). OXS3 is expressed during response reactions to oxidative stress (Blanvillain et al. 2009 ) and likely improves resistance to Tobacco mosaic virus in A. thaliana by the production of hydrogen-peroxide (Wang and Culver 2012 ). In both RNAseq experiments, Zm00001eb428010 and Zm00001eb428020 were the only two genes in the 0.3 Mbp QTL confidence interval that were expressed, indicating that either one of them is the causative agent for BYDV-PAV resistance in maize. However, neither Zm00001eb428010 nor Zm00001eb428020 were differently expressed among the different treatments, suggesting that BYDV-PAV resistance in maize might act at time points that were not covered by our experiments. The more likely explanation is that the difference among resistant and susceptible/tolerant genotypes appears on the protein level and not at gene expression level. Protein abundance might be shaped by post-transcriptional gene regulation (for review see Prall et al .2019). Protein substrate specificity and kinetics might be influenced by changes in amino acid sequence evoked through SNPs or alternative splicing. Indeed, alternative splicing has been shown for maize upon viral infection (Du et al. 2020 , Zhou et al. 2022 ). In addition, three SV were observed for the candidate genes Zm00001eb428010. In contrast to most other detected SV in the QTL confidence interval were these three SVs shared between all three BYDV-PAV resistant inbreds but not present in susceptible and tolerant inbreds. The relatively small size of the SVs in Zm00001eb428010 (54 bp, 91 bp, and 362 bp) is in accordance with findings by Hufford et al. ( 2021 ). Two SVs were located in intronic regions of Zm00001eb428010 and one 54 bp deletion was located in the 5`-UTR (untranslated region). Some 5`-UTR are known to influence translation efficiency (Yamasaki et al 2018 ). Generally, 5`- and 3`-UTR possess cis -acting elements for post-transcriptional control that regulate mRNA stability, transport, and translation efficiency as well as the functioning and subcellular localization of the translated proteins (Mignone et al. 2002 ). Thus, the deletion in the 5`-UTR may influence protein abundance and/or properties. Thus, we speculate that variants in Zm00001eb428010 may influence the encoded protein. However, further work on the protein altering effect is necessary to identify isoforms of Zm00001eb428010 that are expressed in different inbreds or under different conditions and analyze differences in protein substrate specifity and kinetics. Zm00001eb428010 and Zm00001eb428020 are located at the distal end of maize chromosome 10, a genomic region that contains multiple overlapping QTL for resistance to diverse viruses (Redinbaugh et al. 2018 ). This suggests that the BYDV-PAV resistance gene may be efficient to other viruses of maize as well. Changes in genome-wide gene expression following BYDV-PAV infection In contrast to other gene expression studies on BYD infection in cereals or virus infection in maize (Cao et al. 2019 ; Li et al. 2018 ; Rong et al. 2018 ; Shen et al. 2020 ; Wang et al. 2013 ; Zhou et al. 2016 ), only a low number of DEGs was detected (Table 3 ). We suspect that early reactions to BYDV-PAV infection are limited to the phloem cells that are penetrated by aphids during feeding and maybe a few adjacent cells. Using whole leaves might have led to “dilution effects“ that prevent detection of DEGs because unaffected cells outnumber infected cells. Thus, single cell sequencing might be a more feasible approach. Experiment 2 represents processes in the plant at a later infection stage in systemic leaves compared to experiment 1. Virus titer corresponded with number of DEGs. Very low virus titer and numbers of DEGs were detected in the BYDV-PAV resistant inbred FAP1360A when compared to BYDV-PAV tolerant P092 and BYDV-PAV susceptible W64A. Together with the fact that no DEGs were found in BYDV_vs_Aphid (Table 4), this leads to the conclusion that the BYDV-PAV resistance gene may act at early stages after infection, hampering virus replication and/or movement, enabling the plant to grow relatively unaffected. A lower number of genes of BYDV-PAV tolerant inbred P092 were differently expressed compared to the BYDV-PAV susceptible inbred W64A (Table 4), which potentially reflects the lack of symptom formation (Horn et al. 2013 ; this study). Consistently, DEGs of the BYDV-PAV tolerant genotype P092 were not enriched for genes related to chloroplasts or photosynthesis. This might be a starting point to answer the question why BYDV-PAV is able to replicate and spread in P092 but does not cause visible symptoms. Conclusion Combining biparental mapping, association mapping, gene expression profiling, and targeted sequencing, we identified two candidate genes for BYDV-PAV resistance in maize: Zm00001eb428010 and Zm00001eb428020. The predicted functions of these genes suggest a rather unspecific resistance mechanism, potentially by interfering with virus replication or induction of ROS signaling. Expression of Zm00001eb428010 and Zm00001eb428020 was not influenced by BYDV-PAV infection in any inbred. However, sequence variants of Zm00001eb428010 that are present in BYDV-PAV resistant inbreds but absent in BYDV-PAV susceptible or BYDV-PAV tolerant inbreds suggest that abundance and/or properties of the proteins that are encoded by Zm00001eb428010 may lead to BYDV-PAV resistance. Providing closely linked markers to this gene strongly facilitates the selection of resistant material. Finally, orthologs of these two genes in barley, wheat and other cereals are promising targets for mutagenesis experiments to generate BYDV resistant genotypes. Declarations Funding This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project number 403095468. The funders did not influence the study design, the collection, analysis and interpretation of data, the writing of the manuscript, and the decision to submit the manuscript for publication. Competing Interests Benjamin Stich is a member of the editorial board of Theoretical and Applied Genetics. The authors have no other relevant financial or non-financial interests to disclose. Author contributions Maria Schmidt: Experimental work, Data analysis, Manuscript drafting Ricardo Guerreiro: Data analysis targeted sequencing Nadia Baig: Data analysis RNA sequencing Antje Habekuß: Conceptualization, Funding acquisition, Project coordination Torsten Will: Revision of the manuscript Britta Ruckwied: Revision of the manuscript Benjamin Stich: Conceptualization, Funding acquisition, Project coordination, Manuscript drafting All authors approved the submitted version of the manuscript Data availability The original sequencing datasets will be uploaded upon the acceptance of the manuscript. Acknowledgements We would like to thank Kerstin Becker, Karl Köhrer (both Genomics & Transcriptomics Laboratory, Biological and Medical Research Centre, Heinrich Heine University, Düsseldorf, Germany), and Bruno Huettel (Max Planck Institute for Plant Breeding Research, Cologne) for advice and support with PacBio sample preparation and sequencing. Furthermore, we like to thank Stephanie Krey, Florian Esser, Vesna Lamesic and Konstantin Shek for technical support. References Alonge M, Soyk S, Ramakrishnan S, Wang X, Goodwin S, Sedlazeck FJ, Lippman ZB, Schatz MC (2019) RaGOO: fast and accurate reference-guided scaffolding of draft genomes. Genome Biol 20:224. https://doi.org/10.1186/s13059-019-1829-6 Anders S, Pyl PT, Huber W (2015) HTSeq - a Python framework to work with high-throughput sequencing data. 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Agron J 47. https://doi.org/10.2134/agronj1955.00021962004700060014x Tang C, Xu Q, Zhao J, Yue M, Wang J, Wang X, Kang Z, Wang X (2022) A rust fungus effector directly binds plant pre-mRNA splice site to reprogram alternative splicing and suppress host immunity. Plant Biotechnol J 20:1167–1181. https://doi.org/10.1111/pbi.13800 Toojinda T, Broers LH, Chen XM, Hayes PM, Kleinhofs A, Korte J, Kudrna D, Leung H, Line RF, Powell W, Ramsey L, Vivar H, Waugh R (2000) Mapping quantitative and qualitative disease resistance genes in a doubled haploid population of barley (Hordeum vulgare). Theor Appl Genet 101:580–589. https://doi.org/10.1007/s001220051519 United Nations (2022) World Population Prospects 2022. Summary of Results van den Eynde R, van Leeuwen T, Haesaert G (2020) Identifying drivers of spatio-temporal dynamics in Barley Yellow Dwarf Virus epidemiology as a critical factor in disease control. Pest Manag Sci 76:2548–2556. https://doi.org/10.1002/ps.5851 Walls J, Rajotte E, Rosa C (2019) The past, present, and future of barley yellow dwarf management. Agriculture 9:23. https://doi.org/10.3390/agriculture9010023 Walsh LE, Ferrari E, Foster SP, Gaffney MT (2020) Evidence of pyrethroid tolerance in the bird cherry-oat aphid Rhopalosiphum padi in ireland. Outlooks on Pest Management 31:5–9 Wang X, Culver JN (2012) DNA binding specificity of ATAF2, a NAC domain transcription factor targeted for degradation by Tobacco mosaic virus. BMC Plant Biol 12. https://doi.org/10.1186/1471-2229-12-157 Wang X, Boevink P, McLellan H, Armstrong M, Bukharova T, Qin Z, Birch PRJ (2015) A host KH RNA-binding protein is a susceptibility factor targeted by an RXLR effector to promote late blight disease. Mol Plant 8:1385–1395. https://doi.org/10.1016/j.molp.2015.04.012 Wang X, Liu Y, Chen L, Zhao D, Wang X, Zhang Z (2013) Wheat resistome in response to Barley yellow dwarf virus infection. Funct Integr Genomics 13:155–165. https://doi.org/10.1007/s10142-013-0309-4 Yamasaki S, Suzuki A, Yamano Y, Kawabe H, Ueno D, Demura T, Kato K (2018) Identification of 5′-untranslated regions that function as effective translational enhancers in monocotyledonous plant cells using a novel method of genome-wide analysis. Plant Biotechnol (Tokyo) 35:365–373. https://doi.org/10.5511/plantbiotechnology.18.0903a Zhang Z, Lin Z, Xin Z (2009) Research progress in BYDV resistance genes derived from wheat and its wild relatives. J Genet Genomics 36:567–573. https://doi.org/10.1016/S1673-8527(08)60148-4 Zhang Z, Xin Z, Ma Y, Chen X, Xu Q, Lin Z (1999) Mapping of a BYDV resistance gene from Thinopyrum intermedium in wheat background by molecular markers. Sci China C Life Sci 42:663–668. https://doi.org/10.1007/BF02881585 Zhou Y, Lu Q, Zhang J, Zhang S, Weng J, Di H, Zhang L, Li X, Liang Y, Dong L, Zeng X, Liu X, Guo P, Zhang H, Li X, Wang Z (2022) Genome-wide profiling of alternative splicing and gene fusion during Rice black-streaked dwarf virus stress in maize (Zea mays L). Genes 13. https://doi.org/10.3390/genes13030456 Zhou Y, Xu Z, Duan C, Chen Y, Meng Q, Wu J, Hao Z, Wang Z, Li M, Yong H, Zhang D, Zhang S, Weng J, Li X (2016) Dual transcriptome analysis reveals insights into the response to Rice black-streaked dwarf virus in maize. J Exp Bot 67:4593–4609. https://doi.org/10.1093/jxb/erw244 Cite Share Download PDF Status: Published Journal Publication published 18 Jun, 2024 Read the published version in Theoretical and Applied Genetics → Version 1 posted Editorial decision: Major revisions 18 Mar, 2024 Reviewers agreed at journal 25 Jan, 2024 Reviewers invited by journal 17 Jan, 2024 Editor assigned by journal 16 Jan, 2024 First submitted to journal 13 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3863035","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267775628,"identity":"ca39cf35-7248-4232-9f36-2aeddd69fa9e","order_by":0,"name":"Maria Schmidt","email":"","orcid":"","institution":"HHU: Heinrich-Heine-Universitat Dusseldorf","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Schmidt","suffix":""},{"id":267775629,"identity":"784d77ba-c211-4c11-ba0e-20cc789a089f","order_by":1,"name":"Ricardo Guerreiro","email":"","orcid":"","institution":"Heinrich Heine University Düsseldorf: Heinrich-Heine-Universitat Dusseldorf","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"","lastName":"Guerreiro","suffix":""},{"id":267775630,"identity":"27197e03-d0de-48e3-8598-923e529ac69b","order_by":2,"name":"Nadia Baig","email":"","orcid":"","institution":"Heinrich-Heine-Universitat Dusseldorf","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Baig","suffix":""},{"id":267775631,"identity":"1f45d13e-798b-4f97-bc30-204ed77cdddf","order_by":3,"name":"Antje Habekuß","email":"","orcid":"","institution":"Julius Kühn-Institut Bundesforschungsinstitut für Kulturpflanzen: Julius Kuhn-Institut","correspondingAuthor":false,"prefix":"","firstName":"Antje","middleName":"","lastName":"Habekuß","suffix":""},{"id":267775632,"identity":"4d349c77-afb9-4ce2-87cc-e117a35151a3","order_by":4,"name":"Torsten Will","email":"","orcid":"","institution":"Julius Kühn-Institut Bundesforschungsinstitut für Kulturpflanzen: Julius Kuhn-Institut","correspondingAuthor":false,"prefix":"","firstName":"Torsten","middleName":"","lastName":"Will","suffix":""},{"id":267775633,"identity":"064dc8e2-28f5-4ce7-b29a-430bb8cf6a92","order_by":5,"name":"Britta Ruckwied","email":"","orcid":"","institution":"Julius Kühn-Institut Bundesforschungsinstitut für Kulturpflanzen: Julius Kuhn-Institut","correspondingAuthor":false,"prefix":"","firstName":"Britta","middleName":"","lastName":"Ruckwied","suffix":""},{"id":267775634,"identity":"f4d04ab6-c68c-4907-9e7a-640b42583944","order_by":6,"name":"Benjamin Stich","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-6791-8068","institution":"Julius Kühn-Institut Bundesforschungsinstitut für Kulturpflanzen: Julius Kuhn-Institut","correspondingAuthor":true,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"Stich","suffix":""}],"badges":[],"createdAt":"2024-01-14 11:02:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3863035/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3863035/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00122-024-04668-z","type":"published","date":"2024-06-19T00:25:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49866760,"identity":"5650fd43-b668-41dd-842a-adf644f6b682","added_by":"auto","created_at":"2024-01-19 10:41:09","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206443,"visible":true,"origin":"","legend":"\u003cp\u003eDensity curves of estimated marginal means of virus titer (EX, left) and infection rate (IR, right), separated by population. One-hundred and eighty-five homozygous offspring from two heterozygous inbred families and the parental lines FAP1360A (F), Ky226 (K), P092 (P) were infected with BYDV-PAV and virus titer was analyzed six weeks after infection.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3863035/v1/c42e899a6a503577ec3d171f.jpeg"},{"id":49866761,"identity":"433541e6-06a8-42ad-aff8-5a8b3c1ff1ac","added_by":"auto","created_at":"2024-01-19 10:41:09","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189292,"visible":true,"origin":"","legend":"\u003cp\u003eManhattan plots for the association between BYDV-PAV resistance in 185 maize inbreds and genotypic marker. Left: trait virus titer (EX), left: trait infection rate (IR). Marker positions are given based on the reference sequence of B73, version 5. Up-facing triangle: marker SYN4811. Down-facing triangles: flanking marker PZE-110080306 (left) and BYDV-M20 (right).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3863035/v1/ea83a401b7896511885736e0.jpeg"},{"id":49866762,"identity":"2fc4c23e-e799-415d-902d-aa004a9f451c","added_by":"auto","created_at":"2024-01-19 10:41:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":169153,"visible":true,"origin":"","legend":"\u003cp\u003eManhattan plots for the association analysis of BYDV-PAV resistance and HapMap3.2.1 marker. The association of genetic marker with IR (left) and EX (right) is shown. Variant positions and gene names are given based on B73 reference genome version 3.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3863035/v1/866de729588198200eb496c9.png"},{"id":58691485,"identity":"5af2dc55-3f3d-462d-8c9d-71886c60e342","added_by":"auto","created_at":"2024-06-20 00:25:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1391119,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3863035/v1/9d7a7db4-58a0-4112-b502-b94db33ba18c.pdf"}],"financialInterests":"","formattedTitle":"Fine mapping a QTL for BYDV-PAV resistance in maize","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWorld food consumption heavily relies on cereals. The three most important food crops in the world are rice, wheat, and maize (corn), accounting for about 42% of the world\u0026rsquo;s human food calorie intake and 37% of protein intake (Erenstein et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). With an expected increase of the world population (United Nations \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), an increasing demand for cereals is expected (FAO 2023). However, global cereal production is threatened by climate change effects, herbivore pests and several diseases caused by fungi, bacteria, and viruses (Rivero et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Savary et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBarley Yellow Dwarf (BYD) is one of the economically most important diseases in small grain cereals (Choudhury et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; van den Eynde et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This disease can infect all members of the grass family (\u003cem\u003ePoaceae\u003c/em\u003e), causing yield losses in cereals of up to 80% but also negatively affects grain quality (Choudhury et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nancarrow et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Peiris et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). BYD is caused by at least ten different phloem-limited single stranded, positive sense RNA viruses called Barley yellow dwarf viruses (BYDV) and Cereal yellow dwarf viruses (CYDV) (Walls et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Miller and Lozier \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBYDVs and CYDVs are transmitted by more than 25 aphid species worldwide (Halbert and Voegtlin \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Worldwide, BYDV-PAV is the most prevalent virus species that causes BYD and is mainly transmitted by \u003cem\u003eRophalosiphum padi\u003c/em\u003e (Aradottir and Crespo-Herrera \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Several studies suggest that climate change will promote spread of \u003cem\u003eR. padi\u003c/em\u003e and therefore spread of BYDV-PAV (for review see Irwin and Thresh \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Moreno-Delafuente et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eViruses possess no own metabolism which makes it difficult to control them directly. The use of insecticides to limit the spread of vectors is not desirable because the application of insecticides is costly and not environmentally friendly (Chagnon et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Serr\u0026atilde;o et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Due to their harmful effects on beneficial insects, neonicotinoids - a very efficient and previously widely used class of insecticides (Simon-Delso et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) - are banned from application in the field in the European Union (Regulation (EU) No. 485/2013). Moreover, there is evidence for resistance against pyrethroid insecticides in some \u003cem\u003eR. padi\u003c/em\u003e populations (Walsh et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, employment of genetically resistant cereal cultivars is the most promising approach to limit spread of BYD.\u003c/p\u003e \u003cp\u003eHowever, breeding for BYD resistance is hampered by the unavailability of reliable high-throughput phenotyping methods. On one hand, BYD symptoms are influenced by the environment and might be confounded with symptoms of other diseases, nutrient deficiency, waterlogging or mechanical injury (Gr\u0026uuml;ntzig et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). On the other hand, BYD-tolerant genotypes show no symptoms but the virus is able to replicate and move systemically in these plants. Thus, BYD-tolerant plants act as a virus reservoir and are a source for infection of other cereals. In contrast, resistant plants inhibit virus replication and/or systemic movement.\u003c/p\u003e \u003cp\u003eA more reliable alternative to visual symptom scoring is the quantification of the virus titer in plants by quantitative real time PCR (qRT-PCR), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) or tissue blot immunoassay (TBIA) (Canning et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Ch\u0026eacute;our et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Choudhury et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, these methods are from a breeder\u0026rsquo;s perspective laborious and time-consuming. Thus, the use of molecular markers that are at least closely linked to the resistance gene will help to accelerate the breeding process for BYD resistant varieties.\u003c/p\u003e \u003cp\u003eTo date, four loci - r\u003cem\u003eyd1, Ryd2, Ryd3\u003c/em\u003e, and \u003cem\u003eRyd4\u003c/em\u003e\u003csup\u003e\u003cem\u003eHb\u003c/em\u003e\u003c/sup\u003e- are known to convey tolerance or resistance to BYD in barley, but only \u003cem\u003eRyd2\u003c/em\u003e was introduced into commercial cultivars so far (Choudhury et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Jarošov\u0026aacute; et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Walls et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eRyd1\u003c/em\u003e was less efficient for breeding purpose (Suneson, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1955\u003c/span\u003e) compared to the other loci. \u003cem\u003eRyd2\u003c/em\u003e reduces the virus titer of BYDV-PAV and BYDV-MAV in young plants but does not convey resistance against all BYD-causing virus species and is not effective in adult plants (Baltenberger et al \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Riedel et al \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Šip et al \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). \u003cem\u003eRyd3\u003c/em\u003e is a major gene for BYDV-PAV resistance (Niks et al \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Despite the presence of \u003cem\u003eRyd3\u003c/em\u003e seems to prevent BYDV-PAV replication, 20% of the plants carrying the \u003cem\u003eRyd3\u003c/em\u003e gene developed symptoms and had virus concentrations similar to those of susceptible accessions (Niks et al \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). \u003cem\u003eRyd4\u003c/em\u003e\u003csup\u003e\u003cem\u003eHb\u003c/em\u003e\u003c/sup\u003e was introgressed into cultivated barley from the wild relative \u003cem\u003eH. bulbosum\u003c/em\u003e and was not used in breeding programs because of linkage drag with a recessive sublethality factor (Scholz et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Recently, \u003cem\u003eRyd4\u003c/em\u003e\u003csup\u003e\u003cem\u003eHb\u003c/em\u003e\u003c/sup\u003e was fine-mapped to a 66.5 kbp physical interval in the barley \u0026lsquo;Morex\u0026rsquo; reference genome that comprises one complete and one partial gene from the nucleotide-binding and leucine-rich repeat immune receptor family (Pidon et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additional QTL for tolerance to BYD-PAV and BYDV-RPV were mapped to chromosomes 1H, 2H, 4H, and 7H of barley (del Blanco et al. 2014; Kraakman et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Scheurer et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Toojinda et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn wheat, no naturally occurring resistance gene or QTL against BYD is known from the primary gene pool. Only one locus \u0026ndash; \u003cem\u003ebdv1 \u0026ndash;\u003c/em\u003e confers tolerance to BYDV-MAV (Singh et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) and is used in commercial breeding programs. However, BYD resistance was found in the tertiary gene pool of wheat. Three BYD resistance loci \u0026ndash; \u003cem\u003eBdv2\u003c/em\u003e, \u003cem\u003eBdv3\u003c/em\u003e, and \u003cem\u003eBdv4\u003c/em\u003e \u0026ndash; were introgressed from \u003cem\u003eThinopyrum intermedium\u003c/em\u003e into wheat via translocation lines (for review see Zhang et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, the only locus used in wheat breeding is \u003cem\u003eBdv2\u003c/em\u003e, which conveys a broad-spectrum resistance to BYD viruses (Brettell et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA reduction of plant height, ear height, biomass, and grain yield as well as an earlier flowering was observed in BYD infected maize compared to non-inoculated maize plants (Beuve et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Loi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2004\u003c/span\u003e: Panayotou \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). In addition, maize plays an important role in the BYD transmission cycle serving as a \u0026lsquo;green bridge\u0026lsquo; between harvest of small-grain cereals in early summer and sowing of winter cereals in autumn (Brown et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Haack et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Rashidi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This effect is intensified by increasing viruliferous aphid populations under global warming conditions (Moreno-Delafuente et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Cultivation of BYD-resistant maize is expected to reduce BYD pressure on maize and small-grain cereals like wheat and barley. BYDV-PAV resistance in maize shows high genotypic variance and high heritability (Horn et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, and \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), making it a promising target for breeding efforts.\u003c/p\u003e \u003cp\u003eRecently, a QTL for BYDV-PAV resistance was discovered in maize on the distal end of chromosome 10 (Horn et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e and \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In a genome wide association study (GWAS), Horn et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) identified three single nucleotide polymorphims (SNPs) in gene GRMZM2G018027 (Zm00001eb428020) that were associated with BYDV-PAV resistance. These SNPs explained between 16 and 21% of the phenotypic variance of the trait virus titer (EX) as well as between 11 and 18% of the phenotypic variance of the trait infection rate (IR). Similarly, in another study employing five connected linkage mapping populations, Horn et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) identified a QTL on the distal end of chromosome 10 which overlapped with the above described gene GRMZM2G018027 and that explained 45% of the phenotypic variance for the traits EX and IR. However, the causative gene underlying this QTL was not identified.\u003c/p\u003e \u003cp\u003eThe objectives of this study were i) to identify the causative gene for BYDV-PAV resistance in maize, ii) to identify SNPs and/or structural variations in the gene sequences that may cause differences in susceptibility to BYDV-PAV of maize inbreds, and iii) to characterize the effect of BYDV-PAV infection on gene expression of susceptible, tolerant, and resistant maize inbreds. The findings may be used to develop markers for marker assisted breeding of BYDV-PAV resistant maize and as well as a starting point for the investigation of the resistance mechanism. However, the cloning of this BYDV-PAV resistance QTL will be also informative for the breeding of BYD resistant barley and wheat genotypes by providing targets for mutagenesis experiments.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant cultivation and aphid rearing\u003c/h2\u003e \u003cp\u003eMaize plants (\u003cem\u003eZea mays\u003c/em\u003e L.) were grown in a greenhouse (16h light, 20\u0026deg;C / 8h darkness, 16\u0026deg;C) for phenotyping of segregating material or in a climate chamber (16h light, 24\u0026deg;C / 8h darkness, 22\u0026deg;C) for all other experiments.\u003c/p\u003e \u003cp\u003eBYDV-PAV carrying and virus-free aphids of species \u003cem\u003eRhopalosiphum padi\u003c/em\u003e were reared on BYDV-susceptible barley cv. \u0026lsquo;Rubina\u0026rsquo; at room temperature under artificial light conditions. Viruferous and virus-free aphids were kept at physical distance and checked regularly for presence of BYDV-PAV using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) method with in-house polyclonal antisera for BYDV-PAV from the Julius K\u0026uuml;hn-Institute as described by Horn et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFine mapping of the BYDV-PAV resistance gene\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eOur study was based on heterogenous inbred family (HIF) populations developed from recombinant inbred lines (RILs) which were derived from crosses of BYDV-PAV tolerant inbred line P092 with BYDV-PAV resistant inbred lines FAP1360A and Ky226, designated as population A and B, respectively (Horn et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The progenies derived from each RIL were designated in the following as sub-populations. The RILs that were selfed were heterozygous for the QTL interval but homozygous for the rest of the genome. HIFs were genotyped using total of 39 SNP-based Kompetitive allele-specific polymerase chain reaction (KASP\u0026trade;) marker (see below). Furthermore, these individuals were selfed to generate seeds for replicated infection experiments as described below. Heterozygous HIF offspring were subjected to another round of selfing.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction, KASP\u0026trade; marker design and application\u003c/h2\u003e \u003cp\u003eDNA extraction was conducted using an in-house protocol. In brief, about 25 to 50 mg frozen plant material was homogenized using Tissue Lyzer II (Qiagen, Hilden, Germany) and 150 \u0026micro;l extraction buffer was added. After centrifugation (10 min, 4\u0026deg;C, 4.000 RCF), 75 \u0026micro;l supernatant was transferred to a new plate containing 60 \u0026micro;l isopropanol, gently mixed, and centrifuged (10 min, 4\u0026deg;C, 4.000 RCF). The supernatant was discarded, the pellet was washed with 150 \u0026micro;l ethanol (70%) and eluted in 100 \u0026micro;l TE buffer. DNA concentration of random samples was checked with a nanophotometer.\u003c/p\u003e \u003cp\u003eKASP\u0026trade; marker were designed in several rounds based on different sources of SNP information. No matter of the source, SNP information was filtered for identical alleles for inbreds Ky226 and FAP1360A but different to P092. In the first round, molecular marker information from Horn et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) was employed. Sequences flanking the SNPs at least 50 bp upstream and downstream were retrieved from the maize genetics and genomics database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.maizegdb.org/\u003c/span\u003e\u003cspan address=\"https://www.maizegdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using reference version 4 of the B73 genome. Later, information from targeted sequencing of parental inbred lines was used for marker design. Such SNPs were preferred that have identical sequences of P092, Ky226, and FAP1360A in the 50 bp flanking regions. We aimed to spread markers evenly across the QTL confidence interval. Sequences were send to the manufacturer LGC Genomics Ltd. (Hoddesdon, Herts, UK) for the design of the markers.\u003c/p\u003e \u003cp\u003eGenotyping was conducted as recommended by the manufacturer. An ABI Quantstudio 5 (Applied Biosystems) was used for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eInoculation and quantification of virus titer\u003c/h2\u003e \u003cp\u003eThe above described HIF populations were evaluated for IR and EX in four replications per genotype, where an experimental unit comprised eight to ten plants of one genotype. In all experiments, founder inbred lines FAP1360A, Ky226, and P092 but also two additional inbreds which are resistant and susceptible to BYDV-PAV, D408 and W64A, respectively, were used as controls. BYDV-PAV carrying \u003cem\u003eR. padi\u003c/em\u003e were removed from barley plants used for rearing when the maize plants reached the two-leaf stage. Aphids were distributed evenly across the maize plants in a way that approximately ten aphids per plant were applied. This time point was in the following designated as start of inoculation. After one week, plants were treated with insecticide \u0026ldquo;Careo\u0026rdquo; (Substral Celaflor) or \u0026ldquo;Confidor\u0026rdquo; (Bayer CropScience). Leaf material from the sixth leaf of each plant was harvested separately six weeks after the start of the inoculation and virus titer was determined using the DAS-ELISA method with in-house polyclonal antisera for BYDV-PAV as described by Horn et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe infection rate (IR) was calculated as the percentage of plants of one experimental unit with virus titer\u0026thinsp;\u0026ge;\u0026thinsp;0.5. EX was calculated as the mean virus titer per experimental unit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic data analyses and genetic mapping\u003c/h2\u003e \u003cp\u003eEstimated marginal means of EX and IR across all replications of an experiment were calculated using the following mixed linear model:\u003c/p\u003e \u003cp\u003eY\u003csub\u003eij\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026micro;\u0026thinsp;+\u0026thinsp;g\u003csub\u003ei\u003c/sub\u003e + r\u003csub\u003ej\u003c/sub\u003e + e\u003csub\u003eij\u003c/sub\u003e\u003c/p\u003e \u003cp\u003ewhere Y\u003csub\u003eij\u003c/sub\u003e was the phenotypic observation for the ith genotype for the jth replicate, \u0026micro; the general mean, g\u003csub\u003ei\u003c/sub\u003e the effect of the ith genotype, r\u003csub\u003ej\u003c/sub\u003e the effect of the jth replication, and e\u003csub\u003eij\u003c/sub\u003e the residual.\u003c/p\u003e \u003cp\u003eWith the same model but with genotype as random effect, genotypic σ\u003csub\u003eg\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e and error variance σ\u003csub\u003ee\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e were calculated. Broad-sense heritability on an entry mean basis (H\u003csup\u003e2\u003c/sup\u003e) was calculated.\u003c/p\u003e \u003cp\u003eIn order to associate the above described marginal means of each genotype of the HIF populations with the molecular marker profile, the following linear model was used for each of the 39 SNP-based KASP\u0026trade; markers:\u003c/p\u003e \u003cp\u003eY\u003csub\u003eik\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026micro;\u0026thinsp;+\u0026thinsp;s\u003csub\u003ek\u003c/sub\u003e + m\u003csub\u003ei\u003c/sub\u003e + e\u003csub\u003eik\u003c/sub\u003e\u003c/p\u003e \u003cp\u003ewere s\u003csub\u003ek\u003c/sub\u003e was the effect of the HIF population and m\u003csub\u003ei\u003c/sub\u003e the effect of the marker which significance was tested with a F-test. In a second mapping approach, we used the phenotypic data for BYDV-PAV resistance of an association panel from Horn et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). HapMap3.2.1 genotypic data (Bokowski et al. 2018) corresponding to the 300 Kbp QTL confidence interval were retrieved from the PANZEA website. Ambiguous data points were removed and sequence variants were filtered for minor allele frequency\u0026thinsp;\u0026gt;\u0026thinsp;0.025 and missing values\u0026thinsp;\u0026lt;\u0026thinsp;20%. Association analysis was conducted as described by Horn et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) using the Q matrix from Flint-Garcia et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and the K matrix from Horn et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnalyses were conducted using R version 3.6.3 (R Core Team \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with packages \u0026ldquo;lme4\u0026rdquo; version 1.1\u0026ndash;23 (Bates et al \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u0026ldquo;emmeans\u0026rdquo; version 1.5.1 (Lenth \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u0026ldquo;car\u0026rdquo; version 3.0\u0026ndash;10 (Fox and Weisberg 2019), and RStudio version 1.3.1073 (RStudio Team \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rstudio.com/\u003c/span\u003e\u003cspan address=\"http://www.rstudio.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProtein sequences and information on gene annotation were retrieved from the maize genetics and genomics database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.maizegdb.org/\u003c/span\u003e\u003cspan address=\"https://www.maizegdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein sequences were loaded into InterPro (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; Paysan-Lafosse et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) to predict functional protein domains.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDegree of dominance of the resistance gene\u003c/h2\u003e \u003cp\u003eSix sub-populations of HIFs were created to estimate the degree of dominance. Each of these sub-populations consisted of one genotype that was homozygous at marker SYN4811 for the allele of P092 and a sibling that was homozygous for the allele of Ky226 or FAP1360A. Additionally, one or two heterozygous genotypes per sub-population were included that were siblings or offspring of crosses between the homozygous genotypes. Plants were inoculated with BYDV-PAV carrying \u003cem\u003eR. padi\u003c/em\u003e in two replications. Virus titer was measured and EX and IR per genotype were calculated (see previous section). Mean EX and IR per group (homozygous resistant, homozygous susceptible or heterozygous) were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGenomic characterization of founder maize inbreds\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eProbe design\u003c/h2\u003e \u003cp\u003eProbes for target enrichment sequencing of founder inbred lines were designed for the QTL confidence interval identified by Horn et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) plus 1 Mbp to the distal end of the chromosome. At the time of probe design, reference sequences of seven maize inbred lines were available. These were B73 (Zm00001d.2), CML247 (Zm00024a.1), EP1 (Zm00010a.1), F7 (Zm00011a.1), Mo17 (Zm00014a.1), PH207 (Zm00008a.1), and W22 (Zm00004a.1). The sequences were used by the probe design team of the manufacturer (Roche/Nimblegen) to design after masking of repetitive sequences, 2\u0026nbsp;million probes optimized for PacBio sequencing where up to three matches to the reference genome of B73 version 4 (Zm00001d.2) were allowed.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and sequencing\u003c/h2\u003e \u003cp\u003eDNA was extracted using NucleoMag Plant Kit (Macherey \u0026amp; Nagel GmbH \u0026amp; Co. KG D\u0026uuml;ren, Germany) following manufacturer's instructions. DNA concentration and quality were assessed with a nanophotometer, a Qubit fluorometer (Invitrogen) with a Qubit dsDNA HS Assay Kit, and a Fragment Analyzer (Advanced Analytical Technologies).\u003c/p\u003e \u003cp\u003eSample preparation was conducted following PacBio protocol \u0026ldquo;Multiplex Genomic DNA Target Capture Using SeqCap EZ Libraries\u0026rdquo; (PN 100-893-500 version 03). In brief, genomic DNA was fragmented using gTUBES (Covaris), end-repaired and A-tailed using a KAPA HyperPlus Kit (Roche Sequencing Solutions, Pleasanton, CA, USA), barcoded and adapters were ligated. DNA fragments were then amplified using a universal primer (Sigma-Aldrich) and Takara LA Taq DNA polymerase hot-start version (Takara). PCR fragments were size selected for fragment length greater than 4.5 kbp with a BluePippin\u0026trade; automated DNA size selection device (Sage Science), pooled, hybridized with SeqCap EZ Prime Developer Probes (Roche Diagnostics GmbH, Mannheim, Germany) and captured using HyperCap Target Enrichment Kit (Roche Diagnostics GmbH, Mannheim, Germany) and Dynabeads M-270 Streptavidin (Invitrogen by Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania). Captured DNA fragments were amplified using a universal primer and Takara LA Taq DNA polymerase hot-start version (Takara). SMRTbell\u0026trade; library preparation was performed following the manufacturer's instructions. Sequencing was conducted on a Sequel II plattform (PacBio) to deliver highly accurate long reads appropriate for the identification of structural variants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eData processing, SNP calling, and prediction of SVs\u003c/h2\u003e \u003cp\u003eObtained reads were demultiplexed with python package demultiplex and trimmed with bbmap (sourceforge.net/projects/bbmap/). Trimmed reads were used for a reference guided assembly of the QTL confidence interval with RaGOO (Alonge et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Trimmed reads were mapped to B73 reference sequence version 5 (Zm00001eb) using minimap2 (Li et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with parameter -ax asm20 and coverage was calculated with samtools as well as custom awk and python scripts. From reads that mapped to the QTL interval, SNPs and insertions/deletions of less than 50 bp length (InDels) were called using freebayes (Garrison et al. 2012). SNPs and InDels were subjected to variant effect prediction using Variant Effect Predictor tool from Gramene (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ensembl.gramene.org/Oryza_sativa/Tools/VEP#\u003c/span\u003e\u003cspan address=\"https://ensembl.gramene.org/Oryza_sativa/Tools/VEP#\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) that employs the SIFT algorithm (Ng and Henikoff \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsertions and deletions\u0026thinsp;\u0026ge;\u0026thinsp;50 bp were defined as structural variations (SVs) and were called by re-mapping reads with restrictive parameters and exploiting cuteSV (Jiang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGenome-wide gene expression analysis\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eRNA extraction and sequencing\u003c/h2\u003e \u003cp\u003eTwo independent experiments were conducted to assess gene expression differences between the maize founder inbreds as well as upon infection with BYDV. In both experiments, plants of inbreds FAP1360A, P092, and W64A were treated with BYDV-PAV carrying \u003cem\u003eR. padi\u003c/em\u003e, virus-free \u003cem\u003eR. padi\u003c/em\u003e, or without aphids as control. Approximately ten BYDV-PAV carrying aphids per plant were applied when plants reached two leaf stage. After one week, all plants including controls were sprayed with insecticide \u0026ldquo;Careo\u0026rdquo; (Substral Celaflor).\u003c/p\u003e \u003cp\u003eSamples were taken 24 hpi and 96 hpi in experiment 1 from 4\u0026ndash;8 pooled plants per inbred, treatment and timepoint in four replications and two weeks after inoculation in experiment 2, where 2\u0026ndash;4 single plants per inbred and treatment were tested. Leaves were harvested, frozen immediately in liquid nitrogen, and stored at -80\u0026deg;C until further analysis. RNA was extracted with TRIzol (Ambion by Life Technologies, Carlsbad, USA) and Direct-zol RNA MiniPrep Kit (Zymo Research; experiment 1) or RNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany; experiment 2) following the manufacturer\u0026rsquo;s recommendations. All samples were treated with RNase-free DnaseI (ThermoFisher Scientific).\u003c/p\u003e \u003cp\u003eRNA concentration was quantified using a Qubit fluorometer (Invitrogen) and a Qubit RNA HS Assay kit (Life Technologies, Eugene, USA) and quality was assessed with a nanophotometer.\u003c/p\u003e \u003cp\u003eThe RNA was paired end sequenced with 150 bp reads on an Illumina (experiment 1) or DNBseq\u0026trade; (experiment 2) plattform, respectively.\u003c/p\u003e \u003cp\u003eBYDV infection status was confirmed via DAS-ELISA six weeks after inoculation from the sixth leaf (experiment 1) or two weeks after inoculation from the youngest fully developed leaf (experiment 2).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRNAseq data processing\u003c/h2\u003e \u003cp\u003eRNAseq reads were filtered, including removing adaptor sequences, contamination and low-quality reads from raw reads. Unpaired reads were discarded. Exon and splice site information was retrieved from B73 reference genome. Reads were aligned to this reference genome using HISAT2 version 2.1.0 (Kim et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). SamTools version1.6 (Li et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Danecek et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was used to index, sort, and filter mapped reads. Duplicates were removed. Reads per gene were counted with HTSeq version 0.11.1 (Anders et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Putri et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of DEGs\u003c/h2\u003e \u003cp\u003eAnalysis of differently expressed genes was conducted with edgeR version 3.28.1 (Robinson, McCarthy and Smyth \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) where the following contrasts were considered: Aphid infested plants versus Control (Aphid_vs_Ctrl), BYDV infected plants versus Control (BYDV_vs_Ctrl), and BYDV infected plants versus aphid infested plants (BYDV_vs_Aphid).\u003c/p\u003e \u003cp\u003eThe lists of DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using ShinyGO 0.76.3 (Ge et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Pathway databases \u0026ldquo;KEGG\u0026rdquo;, \u0026ldquo;GO Biological Process\u0026rdquo;, \u0026ldquo;GO Cellular Component\u0026rdquo;, and \u0026ldquo;GO Molecular Function\u0026rdquo; were used and parameters were set to FDR\u0026thinsp;=\u0026thinsp;0.05, Pathway size: min\u0026thinsp;=\u0026thinsp;2 and max\u0026thinsp;=\u0026thinsp;2000, and redundancy was removed. No background gene list was provided because ShinyGO 0.76.3 employs protein coding genes as default.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMapping of the BYDV-PAV resistance gene\u003c/h2\u003e \u003cp\u003eFor fine mapping of the BYDV-PAV resistance in maize, homozygous genotypes were selected that were recombinant in the QTL confidence interval. This selection procedure resulted in 83 genotypes originating from selfings of two RILs derived from P092 x FAP1360A (population A) and 102 individuals from selfings of RILs derived from Ky226 x P092 (population B). These 185 homozygous recombinants were subjected to phenotyping for BYDV-PAV resistance.\u003c/p\u003e \u003cp\u003eBroad sense heritability (\u003cem\u003eH\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e) was estimated as 0.89 for EX and 0.82 for IR across the homozygous recombinants of both populations. Analyzing both populations separately, H\u003csup\u003e2\u003c/sup\u003e of population A was slightly lower with 0.79 for EX and 0.70 for IR, compared to 0.92 for EX and 0.85 for IR in population B.\u003c/p\u003e \u003cp\u003eEstimated marginal means ranged from 0.11 to 1.65 for the trait EX and \u0026minus;\u0026thinsp;0.05 to 1.14 for the trait IR. For both traits, estimated marginal means followed a continuous distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For that subset of genotypes, for which heterozygote siblings or progenies were available, the degree of dominance was estimated. Across all sub-populations and replications, the degree of dominance was \u0026minus;\u0026thinsp;0.44 for EX and \u0026minus;\u0026thinsp;0.18 for IR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe statistical test associating the genotyping profiles of the 185 homozygous recombinants with their marginal means for EX and IR resulted in the fine mapping of the resistance factor to the genome region between marker PZE-110080306 and the newly developed marker BYDV-M20 as flanking marker of the QTL interval as for them the slope of the trendline changed the direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The physical position of these markers delimits the resistance factor to the region between 137,131,915 and 137,409,058 bp on chromosome 10, which comprises nine genes. These genes are Zm00001eb428020 (GRMZM2G018027), a candidate gene for BYDV-PAV resistance identified by Horn et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), two transcription factors (Zm00001eb427970 and Zm00001eb427980), a putative WAK-related receptor-like protein kinase family protein (Zm00001eb427960), a putative RING zinc finger domain superfamily protein (Zm00001eb427950), a P-loop containing nucleoside triphosphate hydrolases superfamily protein (Zm00001eb428010), and three genes of unknown function (Zm00001eb427940, Zm00001eb427990, and Zm00001eb428000) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenes in the ~\u0026thinsp;0.3 Mbp long QTL confidence interval for BYDV-PAV resistance in maize on chromosome 10. Start and end positions are given based on the reference sequence of B73, version 5.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStart\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDescription / suggested function\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZm00001eb427940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e137133463\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137134445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eunknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZm00001eb427950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e137197560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137198870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRING zinc finger domain superfamily protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZm00001eb427960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e137214761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137217464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWAK-related receptor-like protein kinase family protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZm00001eb427970\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e137229874\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137233357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eABI3-VP1-transcription factor 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZm00001eb427980\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e137263651\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137266456\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTranscription factor bHLH28 like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZm00001eb427990\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e137278582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137280136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eunknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZm00001eb428000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e137280991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137284148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eunknown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZm00001eb428010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e137285187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137290824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDNA2/NAM7 helicase-like protein\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZm00001eb428020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e137348959\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e137349907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eresponse to oxidative stress, response to cadmium ion\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo further reduce the number of candidate genes, we used an association mapping approach based on the BYDV-PAV phenotyping data from an association mapping panel described by Horn et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and the genotypic data from HapMap3.2.1 (Bokowski et al. 2018). The strongest association for BYDV-PAV resistance was found for sequence variants located in genes GRMZM2G322506 (Zm00001eb428010) and GRMZM2G018027 (Zm00001eb428020) and the intergenic space in between these two genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of sequence variation in the QTL interval\u003c/h2\u003e \u003cp\u003eTargeted long read sequencing of the five founder inbred lines FAP1360A, Ky226, P092, D408, and W64A resulted in 1,579,826 raw reads and 8,421,284,727 bases sequenced. Reads were filtered, mapped against B73 v5 reference genome (Zm00001eb) and assembled to contigs. The total length of contigs was between 9,747,441 and 14,948,168 bp per inbred.\u003c/p\u003e \u003cp\u003eThe three BYDV-PAV resistant genotypes had similar numbers of variants when compared to reference B73. We counted 1972, 1911, and 1869 SNPs and InDels for D408, FAP1360A, and Ky226, respectively. BYDV-PAV susceptible genotype W64A had slightly less variants (1797) and for BYDV-PAV tolerant genotype P092 the lowest number of variants (1139) compared to B73 was detected.\u003c/p\u003e \u003cp\u003eSNPs and InDels were subjected to variant effect prediction. More than 94% of SNPs and InDels were predicted to be modifiers such as upstream and downstream gene variants, intron variants, intergenic variants, and 5\u0026rsquo;- and 3\u0026rsquo;-UTR variants (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The SIFT algorithm predicted a high impact for 19 SNPs and InDels. However, of those only one SNP, which leads to a frameshift in gene Zm00001eb428000, was shared by the three BYDV-PAV resistant founder inbreds but not by P092 and W64A (Supplementary table 1). Additionally, six protein altering variants were detected. One protein altering variant in gene Zm00001eb428010 was shared by the three BYDV-PAV resistant founder inbreds but not by P092 and W64A. The other five variants were located in gene Zm00001eb427970, of which three were shared between D408 and FAP1360A, and two were unique to Ky226.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVariant effect prediction of single nucleotide polymorphisms (SNPs) and InDels (\u0026lt;\u0026thinsp;50 bp) in the ~\u0026thinsp;0.3 Mbp QTL confidence interval on chromosome 10 between 137,131,915 and 137,409,058 bp (B73 ref v5) of five founder inbred lines.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImpact\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConsequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCount\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003estop gained\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003estart lost\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003estop gained, frameshift variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eframeshift variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esplice acceptor variant, coding sequence variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esplice acceptor variant, intron variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eprotein altering variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einframe deletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003einframe insertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emissense variant, splice region variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emissense variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e159\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esplice region variant, intron variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esplice region variant, synonymous variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003estop retained variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esynonymous variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eModifier\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 prime UTR variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 prime UTR variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eintron variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e244\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eupstream gene variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1688\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edownstream gene variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1258\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eintergenic variant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1699\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn addition, 34 SVs were detected in the 0.3 Mbp QTL confidence interval, where the majority of them (24) were unique to one inbred (Supplementary table 2). Only nine SVs were located in a gene. Remarkably, the three BYDV-PAV resistant inbreds shared a 54 bp deletion located in the 5\u0026rsquo; UTR of gene Zm00001eb428010, a 91 bp insertion in intron 6 and a 362 bp deletion in intron 7 of the same gene. These were not present in susceptible and tolerant genotypes, respectively.\u003c/p\u003e \u003cp\u003eOnly SNPs and InDels with low (synonymous variants) or modifier effect (intron or upstream/down-stream gene variants) but no SNPs with predicted high or protein altering effect or SVs were detected for the BYDV-PAV resistance candidate gene Zm00001eb428020.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGene expression\u003c/h2\u003e \u003cp\u003eTwo independent experiments were conducted to analyze the effect of BYDV-PAV infection on genome-wide gene expression in maize. Samples were taken 24 hours past infection (hpi) and 96 hpi in experiment 1 as well as two weeks after inoculation in experiment 2. Only a small number genes was significantly differently expressed in experiment 1 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) among treatments. P092 had the most differently expressed genes (DEGs) with a total of 111 DEGs for all time points and comparisons. A total of 88 DEGs were found for FAP1360A but no DEGs were found for W64A. Also, we did not find any DEGs in FAP1360A for the comparison BYDV_vs_Aphid. Most DEGs were found among treatments in the upregulated group at 96 hpi in both FAP1360A and P092. Remarkably, the 25 downregulated genes in BYDV vs Control of FAP1360A at 24 hpi were enriched for nucloetide and nucleoside biosynthesis and metabolism processes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCounts of differently expressed genes (DEGs) in experiment 1 at 24 and 96 hours past infection (hpi) and in experiment 2 2 weeks past infection (wpi).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eComparison\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e24 hpi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e96 hpi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e2 wpi\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eFAP1360A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBYDV vs Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e290\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBYDV vs Aphid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAphid vs Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026zwj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eP092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBYDV vs Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2904\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e3509\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBYDV vs Aphid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e3546\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAphid vs Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e383\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026zwj;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eW64A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBYDV vs Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4880\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBYDV vs Aphid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAphid vs Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3671\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003eSupplementary table 1: Single Nucleotide Polymorphisms (SNPs) and InDels (\u0026lt;\u0026thinsp;50 bp) with high impact and selected moderate impact consequences in the ~\u0026thinsp;0.3 Mbp long QTL confidence interval on chromosome 10 of five maize inbreds.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the second experiment, a considerably higher number of DEGs was detected (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eR. padi\u003c/em\u003e infestation and BYDV-PAV infection had a low effect on gene expression in FAP1360A in comparison to P092 and W64A. We found eight to 19 times more DEGs in P092 and 13 to 204 times more DEGs in W64A than in FAP1360A, respectively. Interestingly, there were no DEGs in BYDV_vs_Aphid in FAP1360A. In contrast, BYDV_vs_Aphid was the comparison with most DEGs in P092 and W64A for up-regulated and down-regulated genes, respectively. Among the downregulated genes in P092 in BYDV_vs_Aphid, KEGG pathways \u0026bdquo;Phagosome\u0026ldquo; (zma04145) and \u0026bdquo;Spliceosome\u0026ldquo; (zma03040) were enriched 2.8-fold and 1.9-fold .\u003c/p\u003e \u003cp\u003eOnly two genes of the 0.3 Mbp QTL confidence interval \u0026ndash; Zm00001eb428010 and Zm00001eb428020 \u0026ndash; were expressed in both experiments. Additionally, Zm00001eb428000 was expressed in experiment 2 but with lower abundance than Zm00001eb428010 and Zm00001eb428020. None of these three genes was differently expressed in any genotype in any treatment combination.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBarley Yellow Dwarf (BYD) is one of the economically most important diseases in small grain cereals (Choudhury et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; van den Eynde et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Due to increasing autumn and winter temperatures, it is expected that BYD will become an increasing problem (Pidon et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For maize, BYD infection has a direct negative effect on different phenotypic characters (Beuve et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Loi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Panayotou \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). In addition, maize plays an important role in the BYD transmission cycle serving as a \u0026lsquo;green bridge\u0026lsquo; between harvest of small-grain cereals in early summer and sowing of winter cereals in autumn (Brown et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Haack et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Rashidi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cultivation of BYD-resistant maize is expected to reduce BYD pressure on maize and small-grain cereals like wheat and barley. BYD is caused by different viruses of which BYDV-PAV is the most prevalent virus worldwide. The breeding of BYDV-PAV resistant maize is strongly facilitated by the availability of markers that are closely linked to the resistance gene. Furthermore, the cloning of this BYDV-PAV resistance QTL will be also informative for the breeding of BYD-resistant barley and wheat genotypes by providing targets for mutagenesis experiments. Therefore, the BYDV-PAV QTL identified by Horn et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) was fine mapped in our study.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eFine mapping of the BYDV-PAV resistance in maize\u003c/h2\u003e \u003cp\u003eIn order to avoid the potential problem related to marker-trait associations that are due to population structure (Stich et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), in our study HIF populations were exploited. Despite the observed high heritabilities around 0.8, the marginal means of the homozygous recombinants in the HIF populations showed no distinct categories for the virus titer phenotypes EX and IR but a continuous distribution with a trend towards a bimodal distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The reasons for this observation are the heritabilities lower than one together with a limited difference in the virus titer phenotypes EX and IR between resistant and susceptible/tolerant genotypes. Therefore, an ANOVA approach was used in our study to fine map the resistance factor. Furthermore, as we observed a difference in virus titer phenotypes EX and IR between both HIF populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we fitted a population effect in our linkage analyses of BYDV-PAV titer.\u003c/p\u003e \u003cp\u003eThese analyses allowed to reduce the QTL confidence interval from 8 Mbp (Horn et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) to ~\u0026thinsp;0.3 Mbp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The interval comprised nine annotated genes in the fifth version of the B73 reference genome (Zm00001eb) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The putative function of these nine genes suggest that some of them might be involved in virus defense-related processes and, thus, convey resistance against BYDV-PAV in maize. However, as these links were rather weak, we performed an association study using BYDV-PAV resistance data from an association mapping population (Horn et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and HapMap3.2.1 genotypic data (Bokowski et al. 2018) for the 0.3 Mbp QTL confidence interval to further reduce the number of candidate genes. This analysis showed strong associations of BYDV-PAV resistance with sequence variants located in genes Zm00001eb428010 and Zm00001eb428020 but not with sequence variants located in other genes of the 0.3 Mbp QTL confidence interval. This confirms that either Zm00001eb428010 or Zm00001eb428020 confers BYDV-PAV resistance in maize.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eTwo candidate genes in the QTL for BYDV-PAV resistance in maize\u003c/h2\u003e \u003cp\u003eThe protein encoded by Zm00001eb428010 contains two AAA domains. GO-terms for this gene are RNA binding (GO:0003723) and helicase activity (GO:0004386). AAA domain containing proteins possess diverse functions, including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication (Khan et al. 2022). The protein encoded by Zm00001eb428010 is predicted to belong to the DNA2/NAM7-like helicase family. Nam7, also known as Upstream frameshift 1 (Upf1), targets plant and animal viruses for nonsense-mediated mRNA decay (NMD) (for review see May and Simon \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, many viruses escape Upf1-mediated decay through \u003cem\u003ecis\u003c/em\u003e-acting RNA sequences and \u003cem\u003etrans\u003c/em\u003e-acting viral proteins (May and Simon \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHorn et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) identified three SNPs in Zm00001eb428020 (GRMZM2G018027) that were significantly associated with EX and IR and proposed this gene as a candidate gene for BYDV-PAV resistance in maize. Zm00001eb428020 is associated with GO terms \u0026bdquo;response to oxidative stress\u0026ldquo; (GO:0006979) and \u0026bdquo;response to cadmium ion\u0026ldquo; (GO:0046686) in the molecular function category and \u0026bdquo;nuclear speck\u0026ldquo; (GO:0016607) in the cellular component category.\u003c/p\u003e \u003cp\u003eNuclear speckles (NS) are nuclear membranless bodies enriched in splicing factors (Hasenson and Shav-Tal \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Fungal effectors are able to induce susceptibility of host plants by inducing alternative splicing of host transcripts at NS (Tang et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The same process is suspected for oomycete effectors (Wang et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe best BLAST hit for Zm00001eb428020 in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e is the gene \u003cem\u003eOXS3\u003c/em\u003e (Horn et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eOXS3\u003c/em\u003e is expressed during response reactions to oxidative stress (Blanvillain et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and likely improves resistance to Tobacco mosaic virus in \u003cem\u003eA. thaliana\u003c/em\u003e by the production of hydrogen-peroxide (Wang and Culver \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn both RNAseq experiments, Zm00001eb428010 and Zm00001eb428020 were the only two genes in the 0.3 Mbp QTL confidence interval that were expressed, indicating that either one of them is the causative agent for BYDV-PAV resistance in maize. However, neither Zm00001eb428010 nor Zm00001eb428020 were differently expressed among the different treatments, suggesting that BYDV-PAV resistance in maize might act at time points that were not covered by our experiments. The more likely explanation is that the difference among resistant and susceptible/tolerant genotypes appears on the protein level and not at gene expression level. Protein abundance might be shaped by post-transcriptional gene regulation (for review see Prall et al .2019). Protein substrate specificity and kinetics might be influenced by changes in amino acid sequence evoked through SNPs or alternative splicing. Indeed, alternative splicing has been shown for maize upon viral infection (Du et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhou et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, three SV were observed for the candidate genes Zm00001eb428010. In contrast to most other detected SV in the QTL confidence interval were these three SVs shared between all three BYDV-PAV resistant inbreds but not present in susceptible and tolerant inbreds. The relatively small size of the SVs in Zm00001eb428010 (54 bp, 91 bp, and 362 bp) is in accordance with findings by Hufford et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Two SVs were located in intronic regions of Zm00001eb428010 and one 54 bp deletion was located in the 5`-UTR (untranslated region). Some 5`-UTR are known to influence translation efficiency (Yamasaki et al \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Generally, 5`- and 3`-UTR possess \u003cem\u003ecis\u003c/em\u003e-acting elements for post-transcriptional control that regulate mRNA stability, transport, and translation efficiency as well as the functioning and subcellular localization of the translated proteins (Mignone et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Thus, the deletion in the 5`-UTR may influence protein abundance and/or properties. Thus, we speculate that variants in Zm00001eb428010 may influence the encoded protein. However, further work on the protein altering effect is necessary to identify isoforms of Zm00001eb428010 that are expressed in different inbreds or under different conditions and analyze differences in protein substrate specifity and kinetics.\u003c/p\u003e \u003cp\u003eZm00001eb428010 and Zm00001eb428020 are located at the distal end of maize chromosome 10, a genomic region that contains multiple overlapping QTL for resistance to diverse viruses (Redinbaugh et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This suggests that the BYDV-PAV resistance gene may be efficient to other viruses of maize as well.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eChanges in genome-wide gene expression following BYDV-PAV infection\u003c/h2\u003e \u003cp\u003eIn contrast to other gene expression studies on BYD infection in cereals or virus infection in maize (Cao et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rong et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), only a low number of DEGs was detected (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We suspect that early reactions to BYDV-PAV infection are limited to the phloem cells that are penetrated by aphids during feeding and maybe a few adjacent cells. Using whole leaves might have led to \u0026ldquo;dilution effects\u0026ldquo; that prevent detection of DEGs because unaffected cells outnumber infected cells. Thus, single cell sequencing might be a more feasible approach.\u003c/p\u003e \u003cp\u003eExperiment 2 represents processes in the plant at a later infection stage in systemic leaves compared to experiment 1. Virus titer corresponded with number of DEGs. Very low virus titer and numbers of DEGs were detected in the BYDV-PAV resistant inbred FAP1360A when compared to BYDV-PAV tolerant P092 and BYDV-PAV susceptible W64A. Together with the fact that no DEGs were found in BYDV_vs_Aphid (Table\u0026nbsp;4), this leads to the conclusion that the BYDV-PAV resistance gene may act at early stages after infection, hampering virus replication and/or movement, enabling the plant to grow relatively unaffected.\u003c/p\u003e \u003cp\u003eA lower number of genes of BYDV-PAV tolerant inbred P092 were differently expressed compared to the BYDV-PAV susceptible inbred W64A (Table\u0026nbsp;4), which potentially reflects the lack of symptom formation (Horn et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; this study). Consistently, DEGs of the BYDV-PAV tolerant genotype P092 were not enriched for genes related to chloroplasts or photosynthesis. This might be a starting point to answer the question why BYDV-PAV is able to replicate and spread in P092 but does not cause visible symptoms.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCombining biparental mapping, association mapping, gene expression profiling, and targeted sequencing, we identified two candidate genes for BYDV-PAV resistance in maize: Zm00001eb428010 and Zm00001eb428020. The predicted functions of these genes suggest a rather unspecific resistance mechanism, potentially by interfering with virus replication or induction of ROS signaling. Expression of Zm00001eb428010 and Zm00001eb428020 was not influenced by BYDV-PAV infection in any inbred. However, sequence variants of Zm00001eb428010 that are present in BYDV-PAV resistant inbreds but absent in BYDV-PAV susceptible or BYDV-PAV tolerant inbreds suggest that abundance and/or properties of the proteins that are encoded by Zm00001eb428010 may lead to BYDV-PAV resistance. Providing closely linked markers to this gene strongly facilitates the selection of resistant material. Finally, orthologs of these two genes in barley, wheat and other cereals are promising targets for mutagenesis experiments to generate BYDV resistant genotypes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) \u0026ndash; Project number 403095468. The funders\u003c/p\u003e\n\u003cp\u003edid not influence the study design, the collection, analysis and interpretation of\u003c/p\u003e\n\u003cp\u003edata, the writing of the manuscript, and the decision to submit the manuscript for\u003c/p\u003e\n\u003cp\u003epublication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBenjamin Stich is a member of the editorial board of Theoretical and Applied Genetics.\u003c/p\u003e\n\u003cp\u003eThe authors have no other relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaria Schmidt: Experimental work, Data analysis, Manuscript drafting\u003c/p\u003e\n\u003cp\u003eRicardo Guerreiro: Data analysis targeted sequencing\u003c/p\u003e\n\u003cp\u003eNadia Baig: Data analysis RNA sequencing\u003c/p\u003e\n\u003cp\u003eAntje Habeku\u0026szlig;: Conceptualization, Funding acquisition, Project coordination\u003c/p\u003e\n\u003cp\u003eTorsten Will: Revision of the manuscript\u003c/p\u003e\n\u003cp\u003eBritta Ruckwied: Revision of the manuscript\u003c/p\u003e\n\u003cp\u003eBenjamin Stich: Conceptualization, Funding acquisition, Project coordination, Manuscript drafting\u003c/p\u003e\n\u003cp\u003eAll authors approved the submitted version of the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original sequencing datasets will be uploaded upon the acceptance of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Kerstin Becker, Karl K\u0026ouml;hrer (both Genomics \u0026amp; Transcriptomics Laboratory, Biological and Medical Research Centre, Heinrich Heine University, D\u0026uuml;sseldorf, Germany), and Bruno Huettel (Max Planck Institute for Plant Breeding Research, Cologne) for advice and support with PacBio sample preparation and sequencing. 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J Exp Bot 67:4593\u0026ndash;4609. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/erw244\u003c/span\u003e\u003cspan address=\"10.1093/jxb/erw244\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3863035/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3863035/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBarley yellow dwarf (BYD) is one of the economically most important virus diseases of cereals worldwide, causing yield losses of up to 80 %. BYD is caused by at least ten different phloem-limited viruses called BYD viruses (BYDVs) and Cereal yellow dwarf viruses (CYDVs). Means to control BYD are limited and the use of genetically resistant cultivars is the most economic and environmentally friendly approach. Maize plays a central role in the BYD infection cycle, serving as a reservoir for BYD-causing viruses and their vectors in summer. Growing BYD resistant maize varieties would reduce BYD pressure on maize and cereals.\u003c/p\u003e\n\u003cp\u003eUsing two biparental mapping populations, we were able to reduce a previously published QTL for BYDV-PAV resistance in maize to ~0.3 Mbp, comprising nine genes. Association mapping and gene expression analysis further reduced the number of candidate genes for BYDV-PAV resistance in maize to two: Zm00001eb428010 and Zm00001eb428020. Predicted functions of these genes suggest that they confer BYDV-PAV resistance either via interfering with virus replication or induction of ROS signaling. The sequence of one of these genes, Zm00001eb428010, is affected by a 54 bp deletion in the 5`-UTR and a protein altering variant in BYDV-PAV resistant maize inbreds but not BYDV-PAV susceptible and BYDV-PAV tolerant inbreds. This suggests that altered abundance and/or properties of the proteins that are encoded by Zm00001eb428010 may lead to BYDV-PAV resistance.\u003c/p\u003e","manuscriptTitle":"Fine mapping a QTL for BYDV-PAV resistance in maize","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-19 10:41:04","doi":"10.21203/rs.3.rs-3863035/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-03-18T07:26:04+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-01-26T01:11:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-17T13:12:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-16T12:37:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Theoretical and Applied Genetics","date":"2024-01-13T09:29:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bdeaaef3-4fa9-4f9a-b1a7-2a7c4c67b5eb","owner":[],"postedDate":"January 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-20T00:25:17+00:00","versionOfRecord":{"articleIdentity":"rs-3863035","link":"https://doi.org/10.1007/s00122-024-04668-z","journal":{"identity":"theoretical-and-applied-genetics","isVorOnly":false,"title":"Theoretical and Applied Genetics"},"publishedOn":"2024-06-19 00:25:17","publishedOnDateReadable":"June 19th, 2024"},"versionCreatedAt":"2024-01-19 10:41:04","video":"","vorDoi":"10.1007/s00122-024-04668-z","vorDoiUrl":"https://doi.org/10.1007/s00122-024-04668-z","workflowStages":[]},"version":"v1","identity":"rs-3863035","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3863035","identity":"rs-3863035","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}