A Genome-Wide Association Study of the Maize Hypersensitive Defense Response Identifies Genes That Cluster in Related Pathways

<div><p>Much remains unknown of molecular events controlling the plant hypersensitive defense response (HR), a rapid localized cell death that limits pathogen spread and is mediated by resistance (R-) genes. Genetic control of the HR is hard to quantify due to its microscopic and rapid nature. Natural modifiers of the ectopic HR phenotype induced by an aberrant auto-active R-gene (<i>Rp1-D21</i>), were mapped in a population of 3,381 recombinant inbred lines from the maize nested association mapping population. Joint linkage analysis was conducted to identify 32 additive but no epistatic quantitative trait loci (QTL) using a linkage map based on more than 7000 single nucleotide polymorphisms (SNPs). Genome-wide association (GWA) analysis of 26.5 million SNPs was conducted after adjusting for background QTL. GWA identified associated SNPs that colocalized with 44 candidate genes. Thirty-six of these genes colocalized within 23 of the 32 QTL identified by joint linkage analysis. The candidate genes included genes predicted to be in involved programmed cell death, defense response, ubiquitination, redox homeostasis, autophagy, calcium signalling, lignin biosynthesis and cell wall modification. Twelve of the candidate genes showed significant differential expression between isogenic lines differing for the presence of <i>Rp1-D21</i>. Low but significant correlations between HR-related traits and several previously-measured disease resistance traits suggested that the genetic control of these traits was substantially, though not entirely, independent. This study provides the first system-wide analysis of natural variation that modulates the HR response in plants.</p></div

Correlation coefficients between mean values of NAM RILs for lesion mimic traits and disease resistance score values obtained from previous studies (SLB, Kump et al. [34]; NLB, Poland et al. [35]; GLS, Benson [33]).

<p>LES, lesion score; HTR, height ratio; SWR, stalk width ratio; DTAR, days to anthesis ratio; SLB, southern leaf blight resistance score ; GLS, gray leaf spot resistance score; NLB, northern leaf blight resistance score; N, sample size and <i>_inv</i> indicates that the original lesion/disease rating scale was inverted so that the correlation sign was consistent between comparisons so that in every case, a positive correlation implies increased HR was correlated with increased HR or with increased disease resistance. Significance of correlation coefficients (<i>r</i>);</p><p>****<i>P</i><0.0001,</p><p>***P<i><</i>0.001,</p><p>**P<i><</i>0.01,</p><p>*P<i><</i>0.05.</p><p>ns- not significant.</p><p>Correlation coefficients between mean values of NAM RILs for lesion mimic traits and disease resistance score values obtained from previous studies (SLB, Kump et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004562#pgen.1004562-Kump1" target="_blank">[34]</a>; NLB, Poland et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004562#pgen.1004562-Poland1" target="_blank">[35]</a>; GLS, Benson <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004562#pgen.1004562-Benson1" target="_blank">[33]</a>).</p

Heat map showing additive allelic effects for the HTR across 24 NAM founder lines relative to the common B73 parent.

<p>Chromosome and genetic map positions (centiMorgans; cM) of QTL peaks are shown on the left vertical axis, the contribution to variance among RIL mean values across all 24 NAM populations are shown on right vertical axis and the NAM founder lines are shown on the horizontal axis. Scale below heat map indicates range of allelic effect values and corresponding color intensity. Boxes with asterisks indicate significant (<i>p</i><0.05) allelic effects.</p

SNPs significantly associated with HR-related traits (LES, HTR, SWR and DTAR), and closest predicted maize genes (more details can be found in File S5).

1<p>chromosome;</p>2<p>physical map position based on B73 maize reference genome v2;</p>3<p>−log10 of average <i>p</i> value from GWA analysis based on 100 resampling method;</p>4<p>resampling model inclusion probability (RMIP);</p>5<p>positive and negative allelic effects relative to B73 imply enhancing and suppressing effect on the HR phenotype, respectively;</p>6<p>Number of HR-related trait QTL colocalizing with associated SNP hits (maximum is 4);</p>7<p>mutations of these candidate genes induce lesion phenotypes;</p>8<p>differentially expressed genes (DEG), expression level ratios of candidate genes between mutant and wild type F1 plants from the cross B73×<i>Rp1-D21</i>-H95 and Mo17: <i>Rp1-D21</i>-H95.</p>9<p>Associated SNPs/candidate genes colocalized within QTL that were detected in more than 12 of the 24 NAM populations.</p><p>Key: Lignification: LIG; Programed cell death: PCD,; Autophagy, AUT; monoubiquitination, mUBQ; Calcium signaling, Ca²⁺Sig; defense response, DFR; redox homeostasis, RxH; ubiquitination, UBQ; CWM: cell wall modification; TF, transcription factor; STR: stress response; CHP: molecular chaperone; RNAp, RNA processing.</p><p>SNPs significantly associated with HR-related traits (LES, HTR, SWR and DTAR), and closest predicted maize genes (more details can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004562#pgen.1004562.s014" target="_blank">File S5</a>).</p

NAM joint-linkage QTL analysis for HR-related traits (LES, HTR, SWR and DTAR) across all the 10 maize chromosomes/linkage groups.

<p>Units on vertical bars are in centi-Morgan (cM) genetic distance. Colored bars on right side of chromosome bar indicate the HR-related trait QTL support intervals: purple- DTAR; red-HTR; green-LES pink-SWR, while green and red segments inside chromosome bars correspond to SLB and NLB resistance QTL support intervals, respectively (Derived from KUMP et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004562#pgen.1004562-Kump1" target="_blank">[34]</a>; POLAND et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004562#pgen.1004562-Poland1" target="_blank">[35]</a>). Horizontal lines on the right side of chromosomes 5, 7,9,10 indicate significant SNP hits for HR-related traits from a previous study (OLUKOLU et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004562#pgen.1004562-Olukolu1" target="_blank">[27]</a>).</p

LES QTL obtained from single and joint-linkage QTL analysis across all the 10 maize chromosomes/linkage groups.

<p>Parental inbred lines crossed with the common B73 inbred line to derive each bi-parental sub-population are shown on the vertical axis. The results of joint linakage analysis across the NAM population comprising 24 populations is indicated as JL. The genetic distance for each chromosome is represented in cM (centi-Morgan) unit on the horizontal axis. Different colours are used to indicate alternating chromosomes.</p