Genome-wide association identifies a BAHD acyltransferase activity that assembles an ester of glucuronosylglycerol and phenylacetic acid.

Genome-wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD-family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl-CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP-sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate-starvation stress. GGES is a member of a well-supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant-specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism.

Differential environmental and genomic architectures shape the natural diversity for trichome patterning and morphology in different Arabidopsis organs.

Despite the adaptive and taxonomic relevance of the natural diversity for trichome patterning and morphology, the molecular and evolutionary mechanisms underlying these traits remain mostly unknown, particularly in organs other than leaves. In this study, we address the ecological, genetic and molecular bases of the natural variation for trichome patterning and branching in multiple organs of Arabidopsis (Arabidopsis thaliana). To this end, we characterized a collection of 191 accessions and carried out environmental and genome-wide association (GWA) analyses. Trichome amount in different organs correlated negatively with precipitation in distinct seasons, thus suggesting a precise fit between trichome patterning and climate throughout the Arabidopsis life cycle. In addition, GWA analyses showed small overlapping between the genes associated with different organs, indicating partly independent genetic bases for vegetative and reproductive phases. These analyses identified a complex locus on chromosome 2, where two adjacent MYB genes (ETC2 and TCL1) displayed differential effects on trichome patterning in several organs. Furthermore, analyses of transgenic lines carrying different natural alleles demonstrated that TCL1 accounts for the variation for trichome patterning in all organs, and for stem trichome branching. By contrast, two other MYB genes (TRY and GL1), mainly showed effects on trichome patterning or branching, respectively.

Genetic architecture of root and shoot ionomes in rice (Oryza sativa L.).

KEY MESSAGE: Association analysis for ionomic concentrations of 20 elements identified independent genetic factors underlying the root and shoot ionomes of rice, providing a platform for selecting and dissecting causal genetic variants. Understanding the genetic basis of mineral nutrient acquisition is key to fully describing how terrestrial organisms interact with the non-living environment. Rice (Oryza sativa L.) serves both as a model organism for genetic studies and as an important component of the global food system. Studies in rice ionomics have primarily focused on above ground tissues evaluated from field-grown plants. Here, we describe a comprehensive study of the genetic basis of the rice ionome in both roots and shoots of 6-week-old rice plants for 20 elements using a controlled hydroponics growth system. Building on the wealth of publicly available rice genomic resources, including a panel of 373 diverse rice lines, 4.8 M genome-wide single-nucleotide polymorphisms, single- and multi-marker analysis pipelines, an extensive tome of 321 candidate genes and legacy QTLs from across 15 years of rice genetics literature, we used genome-wide association analysis and biparental QTL analysis to identify 114 genomic regions associated with ionomic variation. The genetic basis for root and shoot ionomes was highly distinct; 78 loci were associated with roots and 36 loci with shoots, with no overlapping genomic regions for the same element across tissues. We further describe the distribution of phenotypic variation across haplotypes and identify candidate genes within highly significant regions associated with sulfur, manganese, cadmium, and molybdenum. Our analysis provides critical insight into the genetic basis of natural phenotypic variation for both root and shoot ionomes in rice and provides a comprehensive resource for dissecting and testing causal genetic variants.

Mining the natural genetic variation in Arabidopsis thaliana for adaptation to sequential abiotic and biotic stresses.

MAIN CONCLUSION: In this genome-wide association study, we obtained novel insights into the genetic basis of the effect of herbivory or drought stress on the level of resistance against the fungus Botrytis cinerea. In nature, plants function in complex environments where they encounter different biotic and abiotic stresses individually, sequentially or simultaneously. The adaptive response to a single stress does not always reflect how plants respond to such a stress in combination with other stresses. To identify genetic factors that contribute to the plant's ability to swiftly adapt to different stresses, we investigated the response of Arabidopsis thaliana to infection by the necrotrophic fungus B. cinerea when preceded by Pieris rapae herbivory or drought stress. Using 346 natural A. thaliana accessions, we found natural genetic variation in the level of resistance against single B. cinerea infection. When preceded by herbivory or drought stress, the level of B. cinerea resistance was differentially influenced in the 346 accessions. To study the genetic factors contributing to the differential adaptation of A. thaliana to B. cinerea infection under multi-stress conditions, we performed a genome-wide association study supported by quantitative trait loci mapping and fine mapping with full genome sequences of 164 accessions. This yielded several genes previously associated with defense to B. cinerea and additional candidate genes with putative roles in the plant's adaptive response to a combination of herbivory, drought and B. cinerea infection.

Natural allelic variation of GVS1 confers diversity in the regulation of leaf senescence in Arabidopsis.

Leaf senescence affects plant fitness. Plants that evolve in different environments are expected to acquire distinct regulations of leaf senescence. However, the adaptive and evolutionary roles of leaf senescence are largely unknown. We investigated leaf senescence in 259 natural accessions of Arabidopsis by quantitatively assaying dark-induced senescence responses using a high-throughput chlorophyll fluorescence imaging system. A meta-analysis of our data with phenotypic and climatic information demonstrated biological and environmental links with leaf senescence. We further performed genome-wide association mapping to identify the genetic loci underlying the diversity of leaf senescence responses. We uncovered a new locus, Genetic Variants in leaf Senescence (GVS1), with high similarity to reductase, where a single nonsynonymous nucleotide substitution at GVS1 mediates the diversity of the senescence trait. Loss-of-function mutations of GVS1 in Columbia-0 delayed leaf senescence and increased sensitivity to oxidative stress, suggesting that this GVS1 variant promotes optimal responses to developmental and environmental signals. Intriguingly, gvs1 loss-of-function mutants display allele- and accession-dependent phenotypes, revealing the functional diversity of GVS1 alleles not only in leaf senescence, but also oxidative stress. Our discovery of GVS1 as the genetic basis of natural variation in senescence programs reinforces its adaptive potential in modulating life histories across diverse environments.

Response of Tibetan Wild Barley Genotypes to Drought Stress and Identification of Quantitative Trait Loci by Genome-Wide Association Analysis.

Tibetan wild barley has been identified to show large genetic variation and stress tolerance. A genome-wide association (GWA) analysis was performed to detect quantitative trait loci (QTLs) for drought tolerance using 777 Diversity Array Technology (DArT) markers and morphological and physiological traits of 166 Tibetan wild barley accessions in both hydroponic and pot experiments. Large genotypic variation for these traits was found; and population structure and kinship analysis identified three subpopulations among these barley genotypes. The average LD (linkage disequilibrium) decay distance was 5.16 cM, with the minimum on 6H (0.03 cM) and the maximum on 4H (23.48 cM). A total of 91 DArT markers were identified to be associated with drought tolerance-related traits, with 33, 26, 16, 1, 3, and 12 associations for morphological traits, H⁺K⁺-ATPase activity, antioxidant enzyme activities, malondialdehyde (MDA) content, soluble protein content, and potassium concentration, respectively. Furthermore, 7 and 24 putative candidate genes were identified based on the reference Meta-QTL map and by searching the Barleymap. The present study implicated that Tibetan annual wild barley from Qinghai⁻Tibet Plateau is rich in genetic variation for drought stress. The QTLs detected by genome-wide association analysis could be used in marker-assisting breeding for drought-tolerant barley genotypes and provide useful information for discovery and functional analysis of key genes in the future.

Genome-wide association study reveals novel players in defense hormone crosstalk in Arabidopsis.

Jasmonic acid (JA) regulates plant defenses against necrotrophic pathogens and insect herbivores. Salicylic acid (SA) and abscisic acid (ABA) can antagonize JA-regulated defenses, thereby modulating pathogen or insect resistance. We performed a genome-wide association (GWA) study on natural genetic variation in Arabidopsis thaliana for the effect of SA and ABA on the JA pathway. We treated 349 Arabidopsis accessions with methyl JA (MeJA), or a combination of MeJA and either SA or ABA, after which expression of the JA-responsive marker gene PLANT DEFENSIN1.2 (PDF1.2) was quantified as a readout for GWA analysis. Both hormones antagonized MeJA-induced PDF1.2 in the majority of the accessions but with a large variation in magnitude. GWA mapping of the SA- and ABA-affected PDF1.2 expression data revealed loci associated with crosstalk. GLYI4 (encoding a glyoxalase) and ARR11 (encoding an Arabidopsis response regulator involved in cytokinin signalling) were confirmed by T-DNA insertion mutant analysis to affect SA-JA crosstalk and resistance against the necrotroph Botrytis cinerea. In addition, At1g16310 (encoding a cation efflux family protein) was confirmed to affect ABA-JA crosstalk and susceptibility to Mamestra brassicae herbivory. Collectively, this GWA study identified novel players in JA hormone crosstalk with potential roles in the regulation of pathogen or insect resistance.

Genome-wide signatures of flowering adaptation to climate temperature: Regional analyses in a highly diverse native range of Arabidopsis thaliana.

Current global change is fueling an interest to understand the genetic and molecular mechanisms of plant adaptation to climate. In particular, altered flowering time is a common strategy for escape from unfavourable climate temperature. In order to determine the genomic bases underlying flowering time adaptation to this climatic factor, we have systematically analysed a collection of 174 highly diverse Arabidopsis thaliana accessions from the Iberian Peninsula. Analyses of 1.88 million single nucleotide polymorphisms provide evidence for a spatially heterogeneous contribution of demographic and adaptive processes to geographic patterns of genetic variation. Mountains appear to be allele dispersal barriers, whereas the relationship between flowering time and temperature depended on the precise temperature range. Environmental genome-wide associations supported an overall genome adaptation to temperature, with 9.4% of the genes showing significant associations. Furthermore, phenotypic genome-wide associations provided a catalogue of candidate genes underlying flowering time variation. Finally, comparison of environmental and phenotypic genome-wide associations identified known (Twin Sister of FT, FRIGIDA-like 1, and Casein Kinase II Beta chain 1) and new (Epithiospecifer Modifier 1 and Voltage-Dependent Anion Channel 5) genes as candidates for adaptation to climate temperature by altered flowering time. Thus, this regional collection provides an excellent resource to address the spatial complexity of climate adaptation in annual plants.

Phenotypic and genome-wide association analysis of spike ethylene in diverse wheat genotypes under heat stress.

The gaseous phytohormone ethylene plays an important role in spike development in wheat (Triticum aestivum). However, the genotypic variation and the genomic regions governing spike ethylene (SET) production in wheat under long-term heat stress remain unexplored. We investigated genotypic variation in the production of SET and its relationship with spike dry weight (SDW) in 130 diverse wheat elite lines and landraces under heat-stressed field conditions. We employed an Illumina iSelect 90K single nucleotide polymorphism (SNP) genotyping array to identify the genetic loci for SET and SDW through a genome-wide association study (GWAS) in a subset of the Wheat Association Mapping Initiative (WAMI) panel. The SET and SDW exhibited appreciable genotypic variation among wheat genotypes at the anthesis stage. There was a strong negative correlation between SET and SDW. The GWAS uncovered five and 32 significant SNPs for SET, and 22 and 142 significant SNPs for SDW, in glasshouse and field conditions, respectively. Some of these SNPs closely localized to the SNPs for plant height, suggesting close associations between plant height and spike-related traits. The phenotypic and genetic elucidation of SET and its relationship with SDW supports future efforts toward gene discovery and breeding wheat cultivars with reduced ethylene effects on yield under heat stress.

Identification of manganese efficiency candidate genes in winter barley (Hordeum vulgare) using genome wide association mapping.

BACKGROUND: Manganese (Mn) has several essential functions in plants, including a role as cofactor in the oxygen evolving complex (OEC) of photosystem II (PSII). Manganese deficiency is a major plant nutritional disorder in winter cereals resulting in significant yield reductions and winter kill in more severe cases. Among the winter cereals, genotypes of winter barley are known to differ considerably in tolerance to Mn deficiency, but the genes controlling the Mn deficiency trait remains elusive. RESULTS: Experiments were conducted using 248 barley varieties, cultivated in six distinct environments prone to induce Mn deficiency. High-throughput phenotyping for Mn deficiency was performed by chlorophyll a (Chl a) fluorescence analysis to quantify the quantum yield efficiency of PSII. High-throughput phenotyping in combination with ICP-OES based multi-element analyses allowed detection of marker-trait associations by genome wide association (GWA) mapping. Several key candidate genes were identified, including PSII subunit proteins, germin like proteins and Mn superoxide dismutase. The putative roles of the encoded proteins in Mn dependent metabolic processes are discussed. CONCLUSIONS: Fifty-four candidate genes were identified by Chl a fluorescence phenotyping and association genetics. Tolerance of plants to Mn deficiency, which is referred to as Mn efficiency, appeared to be a complex trait involving many genes. Moreover, the trait appeared to be highly dependent on the environmental conditions in field. This study provides the basis for an improved understanding of the parameters influencing Mn efficiency and is valuable in future plant breeding aiming at producing new varieties with improved tolerance to cultivation in soil prone to induce Mn deficiency.

Natural genetic variation in Arabidopsis for responsiveness to plant growth-promoting rhizobacteria.

The plant growth-promoting rhizobacterium (PGPR) Pseudomonas simiae WCS417r stimulates lateral root formation and increases shoot growth in Arabidopsis thaliana (Arabidopsis). These plant growth-stimulating effects are partly caused by volatile organic compounds (VOCs) produced by the bacterium. Here, we performed a genome-wide association (GWA) study on natural genetic variation in Arabidopsis for the ability to profit from rhizobacteria-mediated plant growth-promotion. To this end, 302 Arabidopsis accessions were tested for root architecture characteristics and shoot fresh weight in response to exposure to WCS417r. Although virtually all Arabidopsis accessions tested responded positively to WCS417r, there was a large variation between accessions in the increase in shoot fresh weight, the extra number of lateral roots formed, and the effect on primary root length. Correlation analyses revealed that the bacterially-mediated increase in shoot fresh weight is related to alterations in root architecture. GWA mapping for WCS417r-stimulated changes in root and shoot growth characteristics revealed 10 genetic loci highly associated with the responsiveness of Arabidopsis to the plant growth-promoting activity of WCS417r. Several of the underlying candidate genes have been implicated in important plant growth-related processes. These results demonstrate that plants possess natural genetic variation for the capacity to profit from the plant growth-promoting function of a beneficial rhizobacterium in their rhizosphere. This knowledge is a promising starting point for sustainable breeding strategies for future crops that are better able to maximize profitable functions from their root microbiome.

Genome-wide association mapping of cadmium accumulation in different organs of barley.

The threshold value of cadmium (Cd) concentration in grains of barley (Hordeum vulgare) is the lowest among cereal crops; however, it is poorly understood how Cd accumulation in barley grain is genetically controlled. We investigated genotypic variation in Cd accumulation of different organs in 100 accessions from a subset of the barley core collection using both hydroponic and Cd-contaminated soil culture. We also performed a genome-wide association (GWA) mapping for Cd accumulation in different organs. A large genotypic variation in the Cd concentration was found in all organs. There was a good correlation between shoot Cd of solution and soil culture, the shoot Cd and grain Cd, but no correlation between the root Cd and grain Cd. GWA mapping detected 9 quantitative trait loci (QTL) for root Cd, 21 for shoot Cd, 14 for root-to-shoot translocation and 15 for grain Cd. A common QTL for the shoot Cd and root-to-shoot translocation was found at 132.6 cM on chromosome 5H. Two major QTL for grain Cd were identified on chromosome 2H and chromosome 5H. The genetic variation in Cd accumulation and major QTL detected provide useful information helpful for cloning candidate genes for Cd accumulation and breeding low-Cd barley cultivars in future.

A guide to genome-wide association analysis and post-analytic interrogation.

This tutorial is a learning resource that outlines the basic process and provides specific software tools for implementing a complete genome-wide association analysis. Approaches to post-analytic visualization and interrogation of potentially novel findings are also presented. Applications are illustrated using the free and open-source R statistical computing and graphics software environment, Bioconductor software for bioinformatics and the UCSC Genome Browser. Complete genome-wide association data on 1401 individuals across 861,473 typed single nucleotide polymorphisms from the PennCATH study of coronary artery disease are used for illustration. All data and code, as well as additional instructional resources, are publicly available through the Open Resources in Statistical Genomics project: http://www.stat-gen.org.

Position of Hungarian Merino among other Merinos, within-breed genetic similarity network and markers associated with daily weight gain.

OBJECTIVE: In this study, we aimed to position the Hungarian Merino among other Merino-derived sheep breeds, explore the characteristics of our sampled animals' genetic similarity network within the breed, and highlight single nucleotide polymorphisms (SNPs) associated with daily weight-gain. METHODS: Hungarian Merino (n = 138) was genotyped on Ovine SNP50 Bead Chip (Illumina, San Diego, CA, USA) and positioned among 30 Merino and Merino-derived breeds (n = 555). Population characteristics were obtained via PLINK, SVS, Admixture, and Treemix software, within-breed network was analysed with python networkx 2.3 library. Daily weight gain of Hungarian Merino was standardised to 60 days and was collected from the database of the Association of Hungarian Sheep and Goat Breeders. For the identification of loci associated with daily weight gain, a multi-locus mixed-model was used. RESULTS: Supporting the breed's written history, the closest breeds to Hungarian Merino were Estremadura and Rambouillet (pairwise FST values are 0.035 and 0.036, respectively). Among Hungarian Merino, a highly centralised connectedness has been revealed by network analysis of pairwise values of identity-by-state, where the animal in the central node had a betweenness centrality value equal to 0.936. Probing of daily weight gain against the SNP data of Hungarian Merinos revealed five associated loci. Two of them, OAR8_17854216.1 and s42441.1 on chromosome 8 and 9 (-log10P>22, false discovery rate<5.5e-20) and one locus on chromosome 20, s28948.1 (-log10P = 13.46, false discovery rate = 4.1e-11), were close to the markers reported in other breeds concerning daily weight gain, six-month weight, and post-weaning gain. CONCLUSION: The position of Hungarian Merino among other Merino breeds has been determined. We have described the similarity network of the individuals to be applied in breeding practices and highlighted several markers useful for elevating the daily weight gain of Hungarian Merino.

Effect of dietary calcium on the gender-specific association between polymorphisms in the PTPRD locus and osteoporosis.

BACKGROUND & AIMS: Osteoporosis is the most common bone disease and is characterized by low bone mineral density (BMD) and a high risk of fracture. Despite advances in our understanding of the pathogenesis of osteoporosis, complex gene-environment interactions that influence osteoporosis development remain largely unexplored. In this study, we aimed to identify genetic loci associated with low BMD and to evaluate these genetic variants under individual and environmental factors. METHODS: A genome-wide association analysis was conducted using 500,568 single-nucleotide polymorphisms (SNPs) in 8842 individuals aged 40-69 years using clinical, demographic, and dietary data (>260 traits) established by the Korean Genome and Epidemiology Study. The gPLINK program was used to detect SNPs associated with osteoporosis at a genome-wide significance level (P < 1.0 × 10-05) and conduct a haplotype analysis. Statistical differences between the osteoporosis and control groups in categorical variables (sex and dietary profiles) were assessed based on frequency distributions using the chi-squared test. RESULTS: Of the seven SNPs that were associated with osteoporosis, both rs10977574 and rs4390000 lay in the PTPRD locus encoding a protein tyrosine phosphatase-receptor type D, which has been implicated in bone metabolism. Haplotype analysis identified two minor alleles, C and G, at the rs10977574 and rs4390000 loci, respectively, forming a linkage disequilibrium block. The subsequent gender-stratified analysis using dietary calcium intake revealed an increased correlation between the CG haplotype and osteoporosis (OR = 2.069) in the low-calcium-intake-female group but not in the high-calcium-intake-female or any male group. CONCLUSIONS: This study revealed novel evidence of the sex-specific association of the CG haplotype in the PTPRD locus with osteoporosis and indicated that the association can be influenced by dietary calcium intake.

Analyses of Natural Variation: Field Experiments and Nucleotide Diversity for Your Favorite Gene.

Arabidopsis has become a model plant for ecological and population genomics, owing to the substantial phenotypic and genotypic variation that exists among and within natural populations. Specially, the recent availability of large worldwide collections of accessions, together with their full genome sequences, has triggered the study of Arabidopsis natural variation. In this chapter, we describe two protocols that exploit these new resources to understand the natural variation for any trait and gene: (1) the phenotypic analysis of Arabidopsis plants grown in field experiments; (2) the analysis of nucleotide diversity and environmental associations for specific genes.

PDE4B Proposed as a High Myopia Susceptibility Gene in Chinese Population.

Myopia is the most common cause of refractive error worldwide. High myopia is a severe type of myopia, which usually accompanies pathological changes in the fundus. To identify high myopia susceptibility genes, DNA-pooling based genome-wide association analysis was used to search for a correlation between single nucleotide polymorphisms and high myopia in a Han Chinese cohort (cases vs. controls in discovery stage: 507 vs. 294; replication stage 1: 991 vs. 1,025; replication stage 2: 1,021 vs. 52,708). Three variants (rs10889602T/G, rs2193015T/C, rs9676191A/C) were identified as being significantly associated with high myopia in the discovery, and replication stage. rs10889602T/G is located at the third intron of phosphodiesterase 4B (PDE4B), whose functional assays were performed by comparing the effects of rs10889602T/T deletion of this risk allele on PDE4B and COL1A1 gene and protein expression levels in the rs10889602T/Tdel/del, rs10889602T/Tdel/wt, and normal control A549 cell lines. The declines in the PDE4B and COL1A1 gene expression levels were larger in the rs10889602T/T deleted A549 cells than in the normal control A549 cells (one-way ANOVA, p < 0.001). The knockdown of PDE4B by siRNA in human scleral fibroblasts led to downregulation of COL1A1. This correspondence between the declines in rs10889602 of the PDE4B gene, PDE4B knockdown, and COL1A1 protein expression levels suggest that PDE4B may be a novel high myopia susceptibility gene, which regulates myopia progression through controlling scleral collagen I expression levels. More studies are needed to determine if there is a correlation between PDE4B and high myopia in other larger sample sized cohorts.

Genetic Drivers of Pancreatic Islet Function.

The majority of gene loci that have been associated with type 2 diabetes play a role in pancreatic islet function. To evaluate the role of islet gene expression in the etiology of diabetes, we sensitized a genetically diverse mouse population with a Western diet high in fat (45% kcal) and sucrose (34%) and carried out genome-wide association mapping of diabetes-related phenotypes. We quantified mRNA abundance in the islets and identified 18,820 expression QTL. We applied mediation analysis to identify candidate causal driver genes at loci that affect the abundance of numerous transcripts. These include two genes previously associated with monogenic diabetes (PDX1 and HNF4A), as well as three genes with nominal association with diabetes-related traits in humans (FAM83E, IL6ST, and SAT2). We grouped transcripts into gene modules and mapped regulatory loci for modules enriched with transcripts specific for α-cells, and another specific for δ-cells. However, no single module enriched for ß-cell-specific transcripts, suggesting heterogeneity of gene expression patterns within the ß-cell population. A module enriched in transcripts associated with branched-chain amino acid metabolism was the most strongly correlated with physiological traits that reflect insulin resistance. Although the mice in this study were not overtly diabetic, the analysis of pancreatic islet gene expression under dietary-induced stress enabled us to identify correlated variation in groups of genes that are functionally linked to diabetes-associated physiological traits. Our analysis suggests an expected degree of concordance between diabetes-associated loci in the mouse and those found in human populations, and demonstrates how the mouse can provide evidence to support nominal associations found in human genome-wide association mapping.