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RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation and expanded function including anti-viral immunity. Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we discovered that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. We determined that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins or long double-stranded RNA. These results suggest that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement. Our results demonstrate that GIGS is active across a range of plant species, evidence similar to recent findings in an insect system, which suggests that GIGS is potentially active across many eukaryotes.

Local landrace and breeding germplasm is a useful source of genetic diversity for regional and global crop improvement initiatives. Sorghum (Sorghum bicolorL. Moench) in West Africa has diversified across a mosaic of cultures and end-uses, and along steep precipitation and photoperiod gradients. To facilitate germplasm utilization, a West African sorghum association panel (WASAP) of 756 accessions from national breeding programs of Niger, Mali, Senegal, and Togo was assembled and characterized. Genotyping-by-sequencing was used to generate 159,101 high-quality biallelic SNPs, with 43% in intergenic regions and 13% in genic regions. High genetic diversity was observed within the WASAP (π = 0.00045), only slightly less than in a global diversity panel (π = 0.00055). Linkage disequilibrium decayed to background level (r2< 0.1) by ~50 kb in the WASAP. Genome-wide diversity was structured both by botanical type, and by populations within botanical type, with eight ancestral populations identified. Most populations were distributed across multiple countries, suggesting several potential common gene pools across the national programs. Genome-wide association studies of days to flowering and plant height revealed eight and three significant quantitative trait loci (QTL), respectively, with major height QTL at canonical height lociDw3andSbHT7.1. Colocalization of two of eight major flowering time QTL with flowering genes previously described in US germplasm (Ma6andSbCN8) suggests that photoperiodic flowering in WA sorghum is conditioned by both known and novel genes. This genomic resource provides a foundation for genomics-enabled breeding of climate-resilient varieties in West Africa.

Forward genetics determines the function of genes underlying trait variation by identifying the change in DNA responsible for changes in phenotype. Detecting phenotypically-relevant variation outside protein coding sequences and distinguishing this from neutral variants is not trivial; partly because the mechanisms by which DNA polymorphisms in the intergenic regions affect gene regulation are poorly understood. Here we utilized a dominant genetic marker with a convenient phenotype to investigate the effect of cis and trans-acting regulatory variation. We performed a forward genetic screen for natural variation that suppress or enhance the semi-dominant mutant alleleOy1-N1989,encoding the magnesium chelatase subunit I of maize. This mutant permits rapid phenotyping of leaf color as a reporter for chlorophyll accumulation, and mapping of natural variation in maize affecting chlorophyll metabolism. We identified a single modifier locus segregating between B73 and Mo17 that was linked to the reporter gene itself, which we callvery oil yellow1. Based on the variation in OY1 transcript abundance and genome-wide association data,vey1is predicted to consist of multiple cis-acting regulatory sequence polymorphisms encoded at the wild-typeoy1alleles. Thevey1allele appears to be a common polymorphism in the maize germplasm that alters the expression level of a key gene in chlorophyll biosynthesis. Thesevey1alleles have no discernable impact on leaf chlorophyll in the absence of theOy1-N1989reporter. Thus, use of a mutant as a simple and efficient reporter for magnesium chelatase activity resulted in the detection of expression-level polymorphisms not readily visible in the laboratory.

We previously demonstrated that maize (Zea mays) locus very oil yellow1 (vey1) encodes a putative cis-regulatory expression polymorphism at the magnesium chelatase subunit I gene (aka oil yellow1) that strongly modifies the chlorophyll content of the semi-dominant Oy1-N1989 mutants. The vey1 allele of Mo17 inbred line reduces chlorophyll content in the mutants leading to reduced photosynthetic output. Oy1-N1989 mutants in B73 reached reproductive maturity four days later than wild-type siblings. Enhancement of Oy1-N1989 by the Mo17 allele at the vey1 QTL delayed maturity further, resulting in detection of a flowering time QTL in two bi-parental mapping populations crossed to Oy1-N1989. The near isogenic lines of B73 harboring the vey1 allele from Mo17 delayed flowering of Oy1-N1989 mutants by twelve days. Just as previously observed for chlorophyll content, vey1 had no effect on reproductive maturity in the absence of the Oy1-N1989 allele. Loss of chlorophyll biosynthesis in Oy1-N1989 mutants and enhancement by vey1 reduced CO2 assimilation. We attempted to separate the effects of photosynthesis on the induction of flowering from a possible impact of chlorophyll metabolites and retrograde signaling by manually reducing leaf area. Removal of leaves, independent of the Oy1-N1989 mutant, delayed flowering but surprisingly reduced chlorophyll contents of emerging leaves. Thus, defoliation did not completely separate the identity of the signal(s) that regulates flowering time from changes in chlorophyll content in the foliage. These findings illustrate the necessity to explore the linkage between metabolism and the mechanisms that connect it to flowering time regulation.

In the cereal crop sorghum (Sorghum bicolor) inflorescence morphology variation underlies yield variation and confers adaptation across precipitation gradients, but its genetic basis is poorly understood. We characterized the genetic architecture of sorghum inflorescence morphology using a global nested association mapping (NAM) population (2200 recombinant inbred lines) and 198,000 phenotypic observations from multi-environment trials for four inflorescence morphology traits (upper branch length, lower branch length, rachis length, and rachis diameter). Trait correlations suggest that lower and upper branch length are under somewhat independent control, while lower branch length and rachis diameter are highly pleiotropic. Joint linkage and genome-wide association mapping revealed an oligogenic architecture with 1–22 QTL per trait, each explaining 0.1–5.0% of variation across the entire NAM population. There is a significant enrichment (2.twofold) of QTL colocalizing with grass inflorescence gene homologs, notably with orthologs of maize Ramosa2 and rice Aberrant Panicle Organization1 and TAWAWA1. Still, many QTL do not colocalize with inflorescence gene homologs. In global georeferenced germplasm, allelic variation at the major inflorescence QTL is geographically patterned but only weakly associated with the gradient of annual precipitation. Comparison of NAM with diversity panel association suggests that naive association models may capture some true associations not identified by mixed linear models. Overall, the findings suggest that global inflorescence diversity in sorghum is largely controlled by oligogenic, epistatic, and pleiotropic variation in ancestral regulatory networks. The findings also provide a basis for genomics-enabled breeding of locally-adapted inflorescence morphology.

We previously demonstrated that maize (Zea mays) locusvery oil yellow1 (vey1)encodes a putative cis-regulatory expression polymorphism at the magnesium chelatase subunit I gene (akaoil yellow1) that strongly modifies the chlorophyll content of the semi-dominantOy1-N1989mutants. Thevey1allele of Mo17 inbred line reduces chlorophyll content in the mutants leading to reduced photosynthetic output.Oy1-N1989mutants in B73 reached reproductive maturity four days later than wild-type siblings. Enhancement ofOy1-N1989by the Mo17 allele at thevey1QTL delayed maturity further, resulting in detection of a flowering time QTL in two bi-parental mapping populations crossed toOy1-N1989. The near isogenic lines of B73 harboring thevey1allele from Mo17 delayed flowering ofOy1-N1989mutants by twelve days. Just as previously observed for chlorophyll content,vey1had no effect on reproductive maturity in the absence of theOy1-N1989allele. Loss of chlorophyll biosynthesis inOy1-N1989mutants and enhancement byvey1reduced CO2assimilation. We attempted to separate the effects of photosynthesis on the induction of flowering from a possible impact of chlorophyll metabolites and retrograde signaling by manually reducing leaf area. Removal of leaves, independent of theOy1-N1989mutant, delayed flowering but surprisingly reduced chlorophyll contents of emerging leaves. Thus, defoliation did not completely separate the identity of the signal(s) that regulates flowering time from changes in chlorophyll content in the foliage. These findings illustrate the necessity to explore the linkage between metabolism and the mechanisms that connect it to flowering time regulation.

Dissecting the genetic architecture of stress tolerance in crops is critical to understand and improve adaptation. In temperate climates, early planting of chilling-tolerant varieties could provide longer growing seasons and drought escape, but chilling tolerance ((Sorghum bicolor[L.] Moench). The NAM resource, developed from reference line BTx623 and three chilling-tolerant Chinese lines, is comprised of 771 recombinant inbred lines genotyped by sequencing at 43,320 single nucleotide polymorphisms. We phenotyped the NAM population for emergence, seedling vigor, and agronomic traits (>75,000 data points from ∼16,000 plots) in multi-environment field trials in Kansas under natural chilling stress (sown 30–45 days early) and normal growing conditions. Joint linkage mapping with early-planted field phenotypes revealed an oligogenic architecture, with 5–10 chilling tolerance loci explaining 20–41% of variation. Surprisingly, several of the major chilling tolerance loci co-localize precisely with the classical grain tannin (Tan1andTan2) and dwarfing genes (Dw1andDw3) that were under strong directional selection in the US during the 20th century. These findings suggest that chilling sensitivity was inadvertently selected due to coinheritance with desired nontannin and dwarfing alleles. The characterization of genetic architecture with NAM reveals why past chilling tolerance breeding was stymied and provides a path for genomics-enabled breeding of chilling tolerance.Article SummaryChilling sensitivity limits productivity of tropical-origin crops in temperate climates, and remains poorly understood at a genetic level. We developed a nested association mapping resource in sorghum, a tropical-origin cereal, to understand the genetic architecture of chilling tolerance. Linkage mapping of growth traits from early-planted field trials revealed several major chilling tolerance loci, including some colocalized with genes that were selected in the origin of US grain sorghum. These findings suggest chilling sensitivity was inadvertently selected during 20th century breeding, but can be bypassed using a better understanding of the underlying genetic architecture.DisclaimerMention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the USDA and does not imply approval or recommendation of the product to the exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.

Adult plant resistance (APR) is an enigmatic phenomenon in which resistance genes are ineffective in protecting seedlings from disease but confer robust resistance at maturity. Maize has multiple cases in which genes confer APR to northern leaf spot, a lethal disease caused by Cochliobolus carbonum race 1 (CCR1). The first identified case of APR in maize is encoded by a hypomorphic allele, Hm1A, at the hm1 locus. In contrast, wild-type alleles of hm1 provide complete protection at all developmental stages and in every part of the maize plant. Hm1 encodes an NADPH-dependent reductase, which inactivates HC-toxin, a key virulence effector of CCR1. Cloning and characterization of Hm1A ruled out differential transcription or translation for its APR phenotype and identified an amino acid substitution that reduced HC-toxin reductase (HCTR) activity. The possibility of a causal relationship between the weak nature of Hm1A and its APR phenotype was confirmed by the generation of two new APR alleles of Hm1 by mutagenesis. The HCTRs encoded by these new APR alleles had undergone relatively conservative missense changes that partially reduced their enzymatic activity similar to HM1A. No difference in accumulation of HCTR was observed between adult and juvenile plants, suggesting that the susceptibility of seedlings derives from a greater need for HCTR activity, not reduced accumulation of the gene product. Conditions and treatments that altered the photosynthetic output of the host had a dramatic effect on resistance imparted by the APR alleles, demonstrating a link between the energetic or metabolic status of the host and disease resistance affected by HC-toxin catabolism by the APR alleles of HCTR., Author summary Adult plant resistance (APR) is a phenomenon in which disease resistance genes are able to confer resistance at the adult stages of the plant but somehow fail to do so at the seedling stages. Despite the widespread occurrence of APR in various plant diseases, the mechanism underlying this trait remains obscure. It is not due to the differential transcription of these genes, and here we show that it is also not due to the differential translation or activity of the APR alleles of the maize hm1 gene at different stages of development. Using a combination of molecular genetics, biochemistry and physiology, we present multiple lines of evidence that demonstrate that APR is a feature or symptom of weak forms of resistance. While the mature parts of the plant are metabolically robust enough to manifest resistance, seedling tissues are not, leaving them vulnerable to disease. Growth conditions that compromise the photosynthetic output of the plant further deteriorate the ability of the seedlings to protect themselves from pathogens.

Background Plant height is an important morphological and developmental phenotype that directly indicates overall plant growth and is widely predictive of final grain yield and biomass. Currently, manually measuring plant height is laborious and has become a bottleneck for genetics and breeding programs. The goal of this research was to evaluate the performance of five different sensing technologies for field-based high throughput plant phenotyping (HTPP) of sorghum [Sorghum bicolor (L.) Moench] height. With this purpose, (1) an ultrasonic sensor, (2) a LIDAR-Lite v2 sensor, (3) a Kinect v2 camera, (4) an imaging array of four high-resolution cameras were evaluated on a ground vehicle platform, and (5) a digital camera was evaluated on an unmanned aerial vehicle platform to obtain the performance baselines to measure the plant height in the field. Plot-level height was extracted by averaging different percentiles of elevation observations within each plot. Measurements were taken on 80 single-row plots of a US × Chinese sorghum recombinant inbred line population. The performance of each sensing technology was also qualitatively evaluated through comparison of device cost, measurement resolution, and ease and efficiency of data analysis. Results We found the heights measured by the ultrasonic sensor, the LIDAR-Lite v2 sensor, the Kinect v2 camera, and the imaging array had high correlation with the manual measurements (r ≥ 0.90), while the heights measured by remote imaging had good, but relatively lower correlation to the manual measurements (r = 0.73). Conclusion These results confirmed the ability of the proposed methodologies for accurate and efficient HTPP of plant height and can be extended to a range of crops. The evaluation approach discussed here can guide the field-based HTPP research in general.

International audience; Improving adaptation of staple crops in developing countries is important to ensure food security. In the West African country of Niger, the staple crop sorghum (Sorghum bicolor) is cultivated across diverse agroclimatic zones, but the genetic basis of local adaptation has not been described. The objectives of this study were to characterize the genomic diversity of sorghum from Niger and to identify genomic regions conferring local adaptation to agroclimatic zones and farmer preferences. We analyzed 516 Nigerien accessions for which local variety name, botanical race, and geographic origin were known. We discovered 144 299 single nucleotide polymorphisms (SNPs) using genotyping-by-sequencing (GBS). We performed discriminant analysis of principal components (DAPC), which identified six genetic groups, and performed a genome scan for loci with high discriminant loadings. The highest discriminant coefficients were on chromosome 9, near the putative ortholog of maize flowering time adaptation gene Vgt1. Next, we characterized differentiation among local varieties and used a genome scan of pairwise F sr values to identify SNPs associated with specific local varieties. Comparison of varieties named for light- versus dark-grain identified differentiation near Tannin1, the major gene responsible for grain tannins. These findings could facilitate genomics-assisted breeding of locally adapted and farmer-preferred sorghum varieties for Niger.; L’amélioration de cultures dans les pays en développement est importante afin d’assurer sécurité alimentaire. En Afrique de l’Ouest, particulièrement au Niger, le sorgho (Sorghum bicolor) est cultivé dans différentes zones agroclimatiques. Cependant les bases génétiques de leur adaptation locale sont peu décrites. Cette étude a pour objectifs de caractériser la structure génétique du sorgho au Niger et d’identifier les régions génomiques associées à l’adaptation locale aux zones agroclimatiques et aux préférences des agriculteurs. L’analyse par le génotypage par séquençage (GBS) de 516 accessions du Niger, dont le nom local, la race botanique et l’origine sont connus, nous a permis d’identifier 144 299 polymorphismes nucléotidiques (SNPs). La méthode d’analyse discriminante des composantes principales a identifié six clusters génétiques. Le balayage génomique des coefficients de discrimination a montré des locus aux coefficients élevés au niveau du chromosome 9, colocalisés avec le gene Vgt1 responsable de la variation de la date de floraison. Aussi, nous avons caractérisé la différentiation des variétés locales. Le balayage génomique de FST entre les variétés locales Mota (grains blancs) et Jenjari (grains sombres) a identifié des locus près du Tannin1, le gène responsable des tanins. Ces résultats permettront de faciliter la sélection assistée par la génomique de variétés de sorgho localement adaptées et préférées en Niger.

Chilling temperatures (0 to 15°C) are a major constraint for temperate cultivation of tropical-origin crops, including the cereal crop sorghum ( [L.] Moench). Northern Chinese sorghums have adapted to early-season chilling, but molecular mechanisms of chilling tolerance are unknown. We used RNA sequencing of seedlings to compare the chilling-responsive transcriptomes of a chilling-tolerant Chinese accession with a chilling-sensitive US reference line, and mass spectrometry to compare chilling-responsive lipidomes of four chilling-tolerant Chinese accessions with two US reference lines. Comparative transcriptomics revealed chilling-induced up-regulation of cold-response regulator C-repeat binding factor (CBF) transcription factor and genes involved in reactive oxygen detoxification, jasmonic acid (JA) biosynthesis, and lipid remodeling phospholipase Dα1 (α) gene in the chilling-tolerant Chinese line. Lipidomics revealed conserved chilling-induced increases in lipid unsaturation, as well as lipid remodeling of photosynthetic membranes that is specific to chilling-tolerant Chinese accessions. Our results point to CBF-mediated transcriptional regulation, galactolipid and phospholipid remodeling, and JA as potential molecular mechanisms underlying chilling adaptation in Chinese sorghums. These molecular systems underlying chilling response could be targeted in molecular breeding for chilling tolerance.

Heat stress reduces grain yield and quality worldwide. Enhancing heat tolerance of crops at all developmental stages is one of the essential strategies required for sustaining agricultural production especially as frequency of temperature extremes escalates in response to climate change. Although heat tolerance mechanisms have been studied extensively in model plant species, little is known about the genetic control underlying heat stress responses of crop plants at the vegetative stage under field conditions. To dissect the genetic basis of heat tolerance in sorghum [ (L.) Moench], we performed a genome-wide association study (GWAS) for traits responsive to heat stress at the vegetative stage in an association panel. Natural variation in leaf firing (LF) and leaf blotching (LB) were evaluated separately for 3 yr in experimental fields at three locations where sporadic heat waves occurred throughout the sorghum growing season. We identified nine single-nucleotide polymorphisms (SNPs) that were significantly associated with LF and five SNPs that were associated with LB. Candidate genes near the SNPs were investigated and 14 were directly linked to biological pathways involved in plant stress responses including heat stress response. The findings of this study provide new knowledge on the genetic control of leaf traits responsive to heat stress in sorghum, which could aid in elucidating the genetic and molecular mechanisms of vegetative stage heat tolerance in crops. The results also provide candidate markers for molecular breeding of enhanced heat tolerance in cereal and bioenergy crops.

Mining crop genomic variation can facilitate the genetic research of complex traits and molecular breeding. In sorghum [ L. (Moench)], several large-scale single nucleotide polymorphism (SNP) datasets have been generated using genotyping-by-sequencing of KI reduced representation libraries. However, data reuse has been impeded by differences in reference genome coordinates among datasets. To facilitate reuse of these data, we constructed and characterized an integrated 459,304-SNP dataset for 10,323 sorghum genotypes on the version 3.1 reference genome. The SNP distribution showed high enrichment in subtelomeric chromosome arms and in genic regions (48% of SNPs) and was highly correlated ( = 0.82) to the distribution of KI restriction sites. The genetic structure reflected population differences by botanical race, as well as familial structure among recombinant inbred lines (RILs). Faster linkage disequilibrium decay was observed in the diversity panel than in the RILs, as expected, given the greater opportunity for recombination in diverse populations. To validate the quality and utility of the integrated SNP dataset, we used genome-wide association studies (GWAS) of genebank phenotype data, precisely mapping several known genes (e.g and ) and identifying novel associations for other traits. We further validated the dataset with GWAS of new and published plant height and flowering time data in a nested association mapping population, precisely mapping known genes and identifying epistatic interactions underlying both traits. These findings validate this integrated SNP dataset as a useful genomics resource for sorghum genetics and breeding.

In crop species, adaptation to different agroclimatic regions creates useful variation but also leads to unwanted genetic correlations. Bouchet......., Adaptation of domesticated species to diverse agroclimatic regions has led to abundant trait diversity. However, the resulting population structure and genetic heterogeneity confounds association mapping of adaptive traits. To address this challenge in sorghum [Sorghum bicolor (L.) Moench]—a widely adapted cereal crop—we developed a nested association mapping (NAM) population using 10 diverse global lines crossed with an elite reference line RTx430. We characterized the population of 2214 recombinant inbred lines at 90,000 SNPs using genotyping-by-sequencing. The population captures ∼70% of known global SNP variation in sorghum, and 57,411 recombination events. Notably, recombination events were four- to fivefold enriched in coding sequences and 5′ untranslated regions of genes. To test the power of the NAM population for trait dissection, we conducted joint linkage mapping for two major adaptive traits, flowering time and plant height. We precisely mapped several known genes for these two traits, and identified several additional QTL. Considering all SNPs simultaneously, genetic variation accounted for 65% of flowering time variance and 75% of plant height variance. Further, we directly compared NAM to genome-wide association mapping (using panels of the same size) and found that flowering time and plant height QTL were more consistently identified with the NAM population. Finally, for simulated QTL under strong selection in diversity panels, the power of QTL detection was up to three times greater for NAM vs. association mapping with a diverse panel. These findings validate the NAM resource for trait mapping in sorghum, and demonstrate the value of NAM for dissection of adaptive traits.

Mutations, occurring either naturally or induced, are permanent alterations of the nucleotide sequence in organisms. These alterations occurring throughout the evolution of a species are responsible for creating immense genetic diversity. Such natural variation generated by mutations has been selected and extensively utilized in crop improvement in several agronomically important crops. In sorghum, mutations producing key agronomic traits such as nonshattering, dwarfing, photoperiod insensitivity, improved protein digestibility, and brown midrib phenotype resulted in establishment of sorghum as the fifth major cereal crop of the world. In addition to successful utilization of these mutations in breeding, understanding the mechanistic basis underlying these traits is equally important to assist in trait advancement. Hence, mutations underlying important agronomic traits were identified using approaches such as positional or map-based cloning, candidate-gene approach, and whole genome sequencing. This chapter provides an overview of key sorghum mutations that resulted in evolution of sorghum as a major source of food, forage, and bioenergy. Additionally, cloning strategies used to identify the underlying mutations and mechanistic basis of the phenotype of interest are discussed. Identification of mutations underlying agriculturally important traits can assist in developing molecular markers to enable precise introgression of selected traits into elite inbreds used in sorghum improvement programs.

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 (Rp1-D21), 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 Rp1-D21. 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., Author Summary The hypersensitive pathogen defense response (HR) in plants typically consists of a rapid, localized cell death around the point of attempted pathogen penetration. It is found in all plant species and is associated with high levels of resistance to a wide range of pathogens and pests including bacteria, fungi, viruses, nematodes, parasitic plants and insects. Little is known about the control of HR after initiation, largely because it is so rapid and localized and therefore difficult to quantify. Here we use a mutant maize gene conferring an exaggerated HR to quantify HR levels in a set of 3,381 mapping lines characterised at 26.5 million loci to identify genes associated with naturally-occurring variation in HR. Many of these genes seem to be involved in a set of connected regulatory pathways including protein degradation, control of programmed cell death, recycling of cellular components and regulation of oxidative stress. We have also shown that several of these genes show high levels of expression induction during HR. The study provides the first comprehensive list of natural variants in maize genes that modulate HR and cluster within reported pathways underlying molecular events during HR.

Rp1-D21 is a maize auto-active resistance gene conferring a spontaneous hypersensitive response (HR) of variable severity depending on genetic background. We report an association mapping strategy based on the Mutant Assisted Gene Identification and Characterization approach to identify naturally occurring allelic variants associated with phenotypic variation in HR. Each member of a collection of 231 diverse inbred lines of maize constituting a high-resolution association mapping panel were crossed to a parental stock heterozygous for Rp1-D21, and the segregating F1 generation testcrosses were evaluated for phenotypes associated with lesion severity for 2 years at two locations. A genome-wide scan for associations with HR was conducted with 47,445 SNPs using a linear mixed model that controlled for spurious associations due to population structure. Since the ability to identify candidate genes and the resolution of association mapping are highly influenced by linkage disequilibrium (LD), we examined the extent of genome-wide LD. On average, marker pairs separated by >10 kbp had an r2 value of