Precise colocalization of sorghum's major chilling tolerance locus with Tannin1 due to tight linkage drag rather than antagonistic pleiotropy.

Chilling tolerance in crops can increase resilience through longer growing seasons, drought escape, and nitrogen use efficiency. In sorghum (Sorghum bicolor [L.] Moench), breeding for chilling tolerance has been stymied by coinheritance of the largest-effect chilling tolerance locus, qSbCT04.62, with the major gene underlying undesirable grain proanthocyanidins, WD40 transcriptional regulator Tannin1. To test if this coinheritance is due to antagonistic pleiotropy of Tannin1, we developed and studied near-isogenic lines (NILs) carrying chilling tolerant haplotypes at qCT04.62. Whole-genome sequencing of the NILs revealed introgressions spanning part of the qCT04.62 confidence interval, including the Tannin1 gene and an ortholog of Arabidopsis cold regulator CBF/DREB1G. Segregation pattern of grain tannin in NILs confirmed the presence of wildtype Tannin1 and the reconstitution of a functional MYB-bHLH-WD40 regulatory complex. Low-temperature germination did not differ between NILs, suggesting that Tannin1 does not modulate this component of chilling tolerance. Similarly, NILs did not differ in seedling growth rate under either of two contrasting controlled environment chilling scenarios. Finally, while the chilling tolerant parent line had notably different photosynthetic responses from the susceptible parent line - including greater non-photochemical quenching before, during, and after chilling - the NIL responses match the susceptible parent. Thus, our findings suggest that tight linkage drag, not pleiotropy, underlies the precise colocalization of Tan1 with qCT04.62 and the qCT04.62 quantitative trait nucleotide lies outside the NIL introgressions. Breaking linkage at this locus should advance chilling tolerance breeding in sorghum and the identification of a novel chilling tolerance regulator.

Pilot-scale genome-wide association mapping in diverse sorghum germplasms identified novel genetic loci linked to major agronomic, root and stomatal traits.

This genome-wide association studies (GWAS) used a subset of 96 diverse sorghum accessions, constructed from a large collection of 219 accessions for mining novel genetic loci linked to major agronomic, root morphological and physiological traits. The subset yielded 43,452 high quality single nucleotide polymorphic (SNP) markers exhibiting high allelic diversity. Population stratification showed distinct separation between caudatum and durra races. Linkage disequilibrium (LD) decay was rapidly declining with increasing physical distance across all chromosomes. The initial 50% LD decay was ~ 5 Kb and background level was within ~ 80 Kb. This study detected 42 significant quantitative trait nucleotide (QTNs) for different traits evaluated using FarmCPU, SUPER and 3VmrMLM which were in proximity with candidate genes related and were co-localized in already reported quantitative trait loci (QTL) and phenotypic variance (R2) of these QTNs ranged from 3 to 20%. Haplotype validation of the candidate genes from this study resulted nine genes showing significant phenotypic difference between different haplotypes. Three novel candidate genes associated with agronomic traits were validated including Sobic.001G499000, a potassium channel tetramerization domain protein for plant height, Sobic.010G186600, a nucleoporin-related gene for dry biomass, and Sobic.002G022600 encoding AP2-like ethylene-responsive transcription factor for plant yield. Several other candidate genes were validated and associated with different root and physiological traits including Sobic.005G104100, peroxidase 13-related gene with root length, Sobic.010G043300, homologous to Traes_5BL_8D494D60C, encoding inhibitor of apoptosis with iWUE, and Sobic.010G125500, encoding zinc finger, C3HC4 type domain with Abaxial stomatal density. In this study, 3VmrMLM was more powerful than FarmCPU and SUPER for detecting QTNs and having more breeding value indicating its reliable output for validation. This study justified that the constructed subset of diverse sorghums can be used as a panel for mapping other key traits to accelerate molecular breeding in sorghum.

Parallel tuning of semi-dwarfism via differential splicing of Brachytic1 in commercial maize and smallholder sorghum.

In the current genomic era, the search and deployment of new semi-dwarf alleles have continued to develop better plant types in all cereals. We characterized an agronomically optimal semi-dwarf mutation in Zea mays L. and a parallel polymorphism in Sorghum bicolor L. We cloned the maize brachytic1 (br1-Mu) allele by a modified PCR-based Sequence Amplified Insertion Flanking Fragment (SAIFF) approach. Histology and RNA-Seq elucidated the mechanism of semi-dwarfism. GWAS linked a sorghum plant height QTL with the Br1 homolog by resequencing a West African sorghum landraces panel. The semi-dwarf br1-Mu allele encodes an MYB transcription factor78 that positively regulates stalk cell elongation by interacting with the polar auxin pathway. Semi-dwarfism is due to differential splicing and low functional Br1 wild-type transcript expression. The sorghum ortholog, SbBr1, co-segregates with the major plant height QTL qHT7.1 and is alternatively spliced. The high frequency of the Sbbr1 allele in African landraces suggests that African smallholder farmers used the semi-dwarf allele to improve plant height in sorghum long before efforts to introduce Green Revolution-style varieties in the 1960s. Surprisingly, variants for differential splicing of Brachytic1 were found in both commercial maize and smallholder sorghum, suggesting parallel tuning of plant architecture across these systems.

Genomics and phenomics enabled prebreeding improved early-season chilling tolerance in Sorghum.

In temperate climates, earlier planting of tropical-origin crops can provide longer growing seasons, reduce water loss, suppress weeds, and escape post-flowering drought stress. However, chilling sensitivity of sorghum, a tropical-origin cereal crop, limits early planting, and over 50 years of conventional breeding has been stymied by coinheritance of chilling tolerance (CT) loci with undesirable tannin and dwarfing alleles. In this study, phenomics and genomics-enabled approaches were used for prebreeding of sorghum early-season CT. Uncrewed aircraft systems (UAS) high-throughput phenotyping platform tested for improving scalability showed moderate correlation between manual and UAS phenotyping. UAS normalized difference vegetation index values from the chilling nested association mapping population detected CT quantitative trait locus (QTL) that colocalized with manual phenotyping CT QTL. Two of the 4 first-generation Kompetitive Allele Specific PCR (KASP) molecular markers, generated using the peak QTL single nucleotide polymorphisms (SNPs), failed to function in an independent breeding program as the CT allele was common in diverse breeding lines. Population genomic fixation index analysis identified SNP CT alleles that were globally rare but common to the CT donors. Second-generation markers, generated using population genomics, were successful in tracking the donor CT allele in diverse breeding lines from 2 independent sorghum breeding programs. Marker-assisted breeding, effective in introgressing CT allele from Chinese sorghums into chilling-sensitive US elite sorghums, improved early-planted seedling performance ratings in lines with CT alleles by up to 13-24% compared to the negative control under natural chilling stress. These findings directly demonstrate the effectiveness of high-throughput phenotyping and population genomics in molecular breeding of complex adaptive traits.

CRISPR guides induce gene silencing in plants in the absence of Cas.

BACKGROUND: 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 antiviral immunity. RESULTS: Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe 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. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design. CONCLUSIONS: Collectively, our results suggest 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. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics.

A genomics resource for genetics, physiology, and breeding of West African sorghum.

Local landrace and breeding germplasm is a useful source of genetic diversity for regional and global crop improvement initiatives. Sorghum (Sorghum bicolor L. Moench) in western Africa (WA) 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 (GBS) was used to generate 159,101 high-quality biallelic single nucleotide polymorphisms (SNPs), with 43% in intergenic regions and 13% in genic regions. High genetic diversity was observed within the WASAP (π = .00045), only slightly less than in a global diversity panel (GDP) (π = .00055). Linkage disequilibrium (LD) decayed to background level (r2 < .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 (GWAS) of days to flowering (DFLo) and plant height (PH) revealed eight and three significant quantitative trait loci (QTL), respectively, with major height QTL at canonical height loci Dw3 and SbHT7.1. Colocalization of two of eight major flowering time QTL with flowering genes previously described in U.S. germplasm (Ma6 and SbCN8) suggests that photoperiodic flowering in West African sorghum is conditioned by both known and novel genes. This genomic resource provides a foundation for genomics-enabled breeding of climate-resilient varieties in WA.

Dissecting Adaptive Traits with Nested Association Mapping: Genetic Architecture of Inflorescence Morphology in Sorghum.

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.

Interaction Between Induced and Natural Variation at oil yellow1 Delays Reproductive Maturity in Maize.

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.

Genetic Architecture of Chilling Tolerance in Sorghum Dissected with a Nested Association Mapping Population.

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 (<15°) is generally lacking in tropical-origin crops. Here we developed a nested association mapping (NAM) population to dissect the genetic architecture of early-season chilling tolerance in the tropical-origin cereal sorghum (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 (Tan1 and Tan2) and dwarfing genes (Dw1 and Dw3) 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.

An Integrated Genotyping-by-Sequencing Polymorphism Map for Over 10,000 Sorghum Genotypes.

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.

A Very Oil Yellow1 Modifier of the Oil Yellow1-N1989 Allele Uncovers a Cryptic Phenotypic Impact of Cis-regulatory Variation in Maize.

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 reporter to investigate the effect of cis and trans-acting regulatory variation. We performed a forward genetic screen for natural variation that suppressed or enhanced the semi-dominant mutant allele Oy1-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 call very oil yellow1 (vey1). Based on the variation in OY1 transcript abundance and genome-wide association data, vey1 is predicted to consist of multiple cis-acting regulatory sequence polymorphisms encoded at the wild-type oy1 alleles. The vey1 locus appears to be a common polymorphism in the maize germplasm that alters the expression level of a key gene in chlorophyll biosynthesis. These vey1 alleles have no discernable impact on leaf chlorophyll in the absence of the Oy1-N1989 reporter. Thus, the use of a mutant as a reporter for magnesium chelatase activity resulted in the detection of expression-level polymorphisms not readily visible in the laboratory.

Adult plant resistance in maize to northern leaf spot is a feature of partial loss-of-function alleles of Hm1.

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.

Field-based high-throughput phenotyping of plant height in sorghum using different sensing technologies.

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.

Population genomics of sorghum (Sorghum bicolor) across diverse agroclimatic zones of Niger.

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 FST 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.

Genome-Wide Association Study of Developing Leaves' Heat Tolerance during Vegetative Growth Stages in a Sorghum Association Panel.

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.

Increased Power To Dissect Adaptive Traits in Global Sorghum Diversity Using a Nested Association Mapping Population.

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.

Comparative Transcriptome and Lipidome Analyses Reveal Molecular Chilling Responses in Chilling-Tolerant Sorghums.

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.

A genome-wide association study of the maize hypersensitive defense response identifies genes that cluster in related pathways.

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.

A connected set of genes associated with programmed cell death implicated in controlling the hypersensitive response in maize.

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 F(1) 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 r(2) value of <0.1. Genomic regions surrounding SNPs significantly associated with HR traits were locally saturated with additional SNP markers to establish local LD structure and precisely identify candidate genes. Six significantly associated SNPs at five loci were detected. At each locus, the associated SNP was located within or immediately adjacent to candidate causative genes predicted to play significant roles in the control of programmed cell death and especially in ubiquitin pathway-related processes.