Combined GWAS and eGWAS reveals the genetic basis underlying drought tolerance in emmer wheat (Triticum turgidum L.).

Drought is one of the major environmental constraints for wheat production world-wide. As the progenitor and genetic reservoir of common wheat, emmer wheat is considered as an invaluable gene pool for breeding drought-tolerant wheat. Combining GWAS and eGWAS analysis of 107 accessions, we identified 86 QTLs, 105 462 eQTLs as well as 68 eQTL hotspots associating with drought tolerance (DT) in emmer wheat. A complex regulatory network composed of 185 upstream regulator and 2432 downstream drought-responsive candidates was developed, of which TtOTS1 was found to play a negative effect in determining DT through affecting root development. This study sheds light on revealing the genetic basis underlying DT, which will provide the indispensable genes and germplasm resources for elite drought tolerance wheat improvement and breeding.

New whole-genome alignment tools are needed for tapping into plant diversity.

Genome alignment is one of the most foundational methods for genome sequence studies. With rapid advances in sequencing and assembly technologies, these newly assembled genomes present challenges for alignment tools to meet the increased complexity and scale. Plant genome alignment is technologically challenging because of frequent whole-genome duplications (WGDs) as well as chromosome rearrangements and fractionation, high nucleotide diversity, widespread structural variation, and high transposable element (TE) activity causing large proportions of repeat elements. We summarize classical pairwise and multiple genome alignment (MGA) methods, and highlight techniques that are widely used or are being developed by the plant research community. We also outline the remaining challenges for precise genome alignment and the interpretation of alignment results in plants.

Combined analysis of transposable elements and structural variation in maize genomes reveals genome contraction outpaces expansion.

Structural differences between genomes are a major source of genetic variation that contributes to phenotypic differences. Transposable elements, mobile genetic sequences capable of increasing their copy number and propagating themselves within genomes, can generate structural variation. However, their repetitive nature makes it difficult to characterize fine-scale differences in their presence at specific positions, limiting our understanding of their impact on genome variation. Domesticated maize is a particularly good system for exploring the impact of transposable element proliferation as over 70% of the genome is annotated as transposable elements. High-quality transposable element annotations were recently generated for de novo genome assemblies of 26 diverse inbred maize lines. We generated base-pair resolved pairwise alignments between the B73 maize reference genome and the remaining 25 inbred maize line assemblies. From this data, we classified transposable elements as either shared or polymorphic in a given pairwise comparison. Our analysis uncovered substantial structural variation between lines, representing both simple and complex connections between TEs and structural variants. Putative insertions in SNP depleted regions, which represent recently diverged identity by state blocks, suggest some TE families may still be active. However, our analysis reveals that within these recently diverged genomic regions, deletions of transposable elements likely account for more structural variation events and base pairs than insertions. These deletions are often large structural variants containing multiple transposable elements. Combined, our results highlight how transposable elements contribute to structural variation and demonstrate that deletion events are a major contributor to genomic differences.

Cloning of the wheat leaf rust resistance gene Lr47 introgressed from Aegilops speltoides.

Leaf rust, caused by Puccinia triticina Eriksson (Pt), is one of the most severe foliar diseases of wheat. Breeding for leaf rust resistance is a practical and sustainable method to control this devastating disease. Here, we report the identification of Lr47, a broadly effective leaf rust resistance gene introgressed into wheat from Aegilops speltoides. Lr47 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that is both necessary and sufficient to confer Pt resistance, as demonstrated by loss-of-function mutations and transgenic complementation. Lr47 introgression lines with no or reduced linkage drag are generated using the Pairing homoeologous1 mutation, and a diagnostic molecular marker for Lr47 is developed. The coiled-coil domain of the Lr47 protein is unable to induce cell death, nor does it have self-protein interaction. The cloning of Lr47 expands the number of leaf rust resistance genes that can be incorporated into multigene transgenic cassettes to control this devastating disease.

Pan-European study of genotypes and phenotypes in the Arabidopsis relative Cardamine hirsuta reveals how adaptation, demography, and development shape diversity patterns.

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species.

Phylogenomic discovery of deleterious mutations facilitates hybrid potato breeding.

Hybrid potato breeding will transform the crop from a clonally propagated tetraploid to a seed-reproducing diploid. Historical accumulation of deleterious mutations in potato genomes has hindered the development of elite inbred lines and hybrids. Utilizing a whole-genome phylogeny of 92 Solanaceae and its sister clade species, we employ an evolutionary strategy to identify deleterious mutations. The deep phylogeny reveals the genome-wide landscape of highly constrained sites, comprising ∼2.4% of the genome. Based on a diploid potato diversity panel, we infer 367,499 deleterious variants, of which 50% occur at non-coding and 15% at synonymous sites. Counterintuitively, diploid lines with relatively high homozygous deleterious burden can be better starting material for inbred-line development, despite showing less vigorous growth. Inclusion of inferred deleterious mutations increases genomic-prediction accuracy for yield by 24.7%. Our study generates insights into the genome-wide incidence and properties of deleterious mutations and their far-reaching consequences for breeding.

A role for heritable transcriptomic variation in maize adaptation to temperate environments.

BACKGROUND: Transcription bridges genetic information and phenotypes. Here, we evaluated how changes in transcriptional regulation enable maize (Zea mays), a crop originally domesticated in the tropics, to adapt to temperate environments. RESULT: We generated 572 unique RNA-seq datasets from the roots of 340 maize genotypes. Genes involved in core processes such as cell division, chromosome organization and cytoskeleton organization showed lower heritability of gene expression, while genes involved in anti-oxidation activity exhibited higher expression heritability. An expression genome-wide association study (eGWAS) identified 19,602 expression quantitative trait loci (eQTLs) associated with the expression of 11,444 genes. A GWAS for alternative splicing identified 49,897 splicing QTLs (sQTLs) for 7614 genes. Genes harboring both cis-eQTLs and cis-sQTLs in linkage disequilibrium were disproportionately likely to encode transcription factors or were annotated as responding to one or more stresses. Independent component analysis of gene expression data identified loci regulating co-expression modules involved in oxidation reduction, response to water deprivation, plastid biogenesis, protein biogenesis, and plant-pathogen interaction. Several genes involved in cell proliferation, flower development, DNA replication, and gene silencing showed lower gene expression variation explained by genetic factors between temperate and tropical maize lines. A GWAS of 27 previously published phenotypes identified several candidate genes overlapping with genomic intervals showing signatures of selection during adaptation to temperate environments. CONCLUSION: Our results illustrate how maize transcriptional regulatory networks enable changes in transcriptional regulation to adapt to temperate regions.

Chromosome-scale genome assembly of Camellia sinensis combined with multi-omics provides insights into its responses to infestation with green leafhoppers.

The tea plant (Camellia sinensis) is an important economic crop, which is becoming increasingly popular worldwide, and is now planted in more than 50 countries. Tea green leafhopper is one of the major pests in tea plantations, which can significantly reduce the yield and quality of tea during the growth of plant. In this study, we report a genome assembly for DuyunMaojian tea plants using a combination of Oxford Nanopore Technology PromethION™ with high-throughput chromosome conformation capture technology and used multi-omics to study how the tea plant responds to infestation with tea green leafhoppers. The final genome was 3.08 Gb. A total of 2.97 Gb of the genome was mapped to 15 pseudo-chromosomes, and 2.79 Gb of them could confirm the order and direction. The contig N50, scaffold N50 and GC content were 723.7 kb, 207.72 Mb and 38.54%, respectively. There were 2.67 Gb (86.77%) repetitive sequences, 34,896 protein-coding genes, 104 miRNAs, 261 rRNA, 669 tRNA, and 6,502 pseudogenes. A comparative genomics analysis showed that DuyunMaojian was the most closely related to Shuchazao and Yunkang 10, followed by DASZ and tea-oil tree. The multi-omics results indicated that phenylpropanoid biosynthesis, α-linolenic acid metabolism, flavonoid biosynthesis and 50 differentially expressed genes, particularly peroxidase, played important roles in response to infestation with tea green leafhoppers (Empoasca vitis Göthe). This study on the tea tree is highly significant for its role in illustrating the evolution of its genome and discovering how the tea plant responds to infestation with tea green leafhoppers will contribute to a theoretical foundation to breed tea plants resistant to insects that will ultimately result in an increase in the yield and quality of tea.

A multiple alignment workflow shows the effect of repeat masking and parameter tuning on alignment in plants.

Alignments of multiple genomes are a cornerstone of comparative genomics, but generating these alignments remains technically challenging and often impractical. We developed the msa_pipeline workflow (https://bitbucket.org/bucklerlab/msa_pipeline) to allow practical and sensitive multiple alignment of diverged plant genomes and calculation of conservation scores with minimal user inputs. As high repeat content and genomic divergence are substantial challenges in plant genome alignment, we also explored the effect of different masking approaches and parameters of the LAST aligner using genome assemblies of 33 grass species. Compared with conventional masking with RepeatMasker, a masking approach based on k-mers (nucleotide sequences of k length) increased the alignment rate of coding sequence and noncoding functional regions by 25 and 14%, respectively. We further found that default alignment parameters generally perform well, but parameter tuning can increase the alignment rate for noncoding functional regions by over 52% compared with default LAST settings. Finally, by increasing alignment sensitivity from the default baseline, parameter tuning can increase the number of noncoding sites that can be scored for conservation by over 76%. Overall, tuning of masking and alignment parameters can generate optimized multiple alignments to drive biological discovery in plants.

QTL mapping and genomic analyses of earliness and fruit ripening traits in a melon Recombinant Inbred Lines population supported by de novo assembly of their parental genomes.

Earliness and ripening behavior are important attributes of fruits on and off the vine, and affect quality and preference of both growers and consumers. Fruit ripening is a complex physiological process that involves metabolic shifts affecting fruit color, firmness, and aroma production. Melon is a promising model crop for the study of fruit ripening, as the full spectrum of climacteric behavior is represented across the natural variation. Using Recombinant Inbred Lines (RILs) population derived from the parental lines "Dulce" (reticulatus, climacteric) and "Tam Dew" (inodorus, non-climacteric) that vary in earliness and ripening traits, we mapped QTLs for ethylene emission, fruit firmness and days to flowering and maturity. To further annotate the main QTL intervals and identify candidate genes, we used Oxford Nanopore long-read sequencing in combination with Illumina short-read resequencing, to assemble the parental genomes de-novo. In addition to 2.5 million genome-wide SNPs and short InDels detected between the parents, we also highlight here the structural variation between these lines and the reference melon genome. Through systematic multi-layered prioritization process, we identified 18 potential polymorphisms in candidate genes within multi-trait QTLs. The associations of selected SNPs with earliness and ripening traits were further validated across a panel of 177 diverse melon accessions and across a diallel population of 190 F1 hybrids derived from a core subset of 20 diverse parents. The combination of advanced genomic tools with diverse germplasm and targeted mapping populations is demonstrated as a way to leverage forward genetics strategies to dissect complex horticulturally important traits.

AnchorWave: Sensitive alignment of genomes with high sequence diversity, extensive structural polymorphism, and whole-genome duplication.

Millions of species are currently being sequenced, and their genomes are being compared. Many of them have more complex genomes than model systems and raise novel challenges for genome alignment. Widely used local alignment strategies often produce limited or incongruous results when applied to genomes with dispersed repeats, long indels, and highly diverse sequences. Moreover, alignment using many-to-many or reciprocal best hit approaches conflicts with well-studied patterns between species with different rounds of whole-genome duplication. Here, we introduce Anchored Wavefront alignment (AnchorWave), which performs whole-genome duplication-informed collinear anchor identification between genomes and performs base pair-resolved global alignment for collinear blocks using a two-piece affine gap cost strategy. This strategy enables AnchorWave to precisely identify multikilobase indels generated by transposable element (TE) presence/absence variants (PAVs). When aligning two maize genomes, AnchorWave successfully recalled 87% of previously reported TE PAVs. By contrast, other genome alignment tools showed low power for TE PAV recall. AnchorWave precisely aligns up to three times more of the genome as position matches or indels than the closest competitive approach when comparing diverse genomes. Moreover, AnchorWave recalls transcription factor-binding sites at a rate of 1.05- to 74.85-fold higher than other tools with significantly lower false-positive alignments. AnchorWave complements available genome alignment tools by showing obvious improvement when applied to genomes with dispersed repeats, active TEs, high sequence diversity, and whole-genome duplication variation.

Using high-throughput multiple optical phenotyping to decipher the genetic architecture of maize drought tolerance.

BACKGROUND: Drought threatens the food supply of the world population. Dissecting the dynamic responses of plants to drought will be beneficial for breeding drought-tolerant crops, as the genetic controls of these responses remain largely unknown. RESULTS: Here we develop a high-throughput multiple optical phenotyping system to noninvasively phenotype 368 maize genotypes with or without drought stress over a course of 98 days, and collected multiple optical images, including color camera scanning, hyperspectral imaging, and X-ray computed tomography images. We develop high-throughput analysis pipelines to extract image-based traits (i-traits). Of these i-traits, 10,080 were effective and heritable indicators of maize external and internal drought responses. An i-trait-based genome-wide association study reveals 4322 significant locus-trait associations, representing 1529 quantitative trait loci (QTLs) and 2318 candidate genes, many that co-localize with previously reported maize drought responsive QTLs. Expression QTL (eQTL) analysis uncovers many local and distant regulatory variants that control the expression of the candidate genes. We use genetic mutation analysis to validate two new genes, ZmcPGM2 and ZmFAB1A, which regulate i-traits and drought tolerance. Moreover, the value of the candidate genes as drought-tolerant genetic markers is revealed by genome selection analysis, and 15 i-traits are identified as potential markers for maize drought tolerance breeding. CONCLUSION: Our study demonstrates that combining high-throughput multiple optical phenotyping and GWAS is a novel and effective approach to dissect the genetic architecture of complex traits and clone drought-tolerance associated genes.

Conserved noncoding sequences provide insights into regulatory sequence and loss of gene expression in maize.

Thousands of species will be sequenced in the next few years; however, understanding how their genomes work, without an unlimited budget, requires both molecular and novel evolutionary approaches. We developed a sensitive sequence alignment pipeline to identify conserved noncoding sequences (CNSs) in the Andropogoneae tribe (multiple crop species descended from a common ancestor ∼18 million years ago). The Andropogoneae share similar physiology while being tremendously genomically diverse, harboring a broad range of ploidy levels, structural variation, and transposons. These contribute to the potential of Andropogoneae as a powerful system for studying CNSs and are factors we leverage to understand the function of maize CNSs. We found that 86% of CNSs were comprised of annotated features, including introns, UTRs, putative cis-regulatory elements, chromatin loop anchors, noncoding RNA (ncRNA) genes, and several transposable element superfamilies. CNSs were enriched in active regions of DNA replication in the early S phase of the mitotic cell cycle and showed different DNA methylation ratios compared to the genome-wide background. More than half of putative cis-regulatory sequences (identified via other methods) overlapped with CNSs detected in this study. Variants in CNSs were associated with gene expression levels, and CNS absence contributed to loss of gene expression. Furthermore, the evolution of CNSs was associated with the functional diversification of duplicated genes in the context of maize subgenomes. Our results provide a quantitative understanding of the molecular processes governing the evolution of CNSs in maize.

Gene expression evolution in pattern-triggered immunity within Arabidopsis thaliana and across Brassicaceae species.

Plants recognize surrounding microbes by sensing microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). Despite their significance for microbial control, the evolution of PTI responses remains largely uncharacterized. Here, by employing comparative transcriptomics of six Arabidopsis thaliana accessions and three additional Brassicaceae species to investigate PTI responses, we identified a set of genes that commonly respond to the MAMP flg22 and genes that exhibit species-specific expression signatures. Variation in flg22-triggered transcriptome responses across Brassicaceae species was incongruent with their phylogeny, while expression changes were strongly conserved within A. thaliana. We found the enrichment of WRKY transcription factor binding sites in the 5'-regulatory regions of conserved and species-specific responsive genes, linking the emergence of WRKY-binding sites with the evolution of gene expression patterns during PTI. Our findings advance our understanding of the evolution of the transcriptome during biotic stress.

Transcriptome profiling analysis of tea plant (Camellia sinensis) using Oxford Nanopore long-read RNA-Seq technology.

Transcriptome profiles have been widely captured using short-read sequencing technology, but there are still limitations partially due to the read length. Here, we generated long reads using Oxford Nanopore PromethION™ technology and short reads using the Illumina sequencing platform to study the transcriptome of root, stem, and leaf of Camellia sinensis cv. Fudingdabai. We mapped the Nanopore reads to the Shuchazao of C. sinensis genome sequence, and the mapping rates ranged from 82.63% to 90.59% (average 86.44%); this is lower than that of the Illumina reads which was 87.83% to 91.14% (average 90.12%). Gene expression level was quantified using the Nanopore and Illumina data and we observed a good agreement. The same tea leaf flavor synthesis pathways were highlighted using both sequencing technologies when analyzing the differentially expressed genes between leaf and root. Alternative splicing was then analyzed, and the intron-retention was observed as the most common alternative splicing. Moreover Nanopore long reads could correct transcript isoform annotation for differential expression investigation purposes. Nanopore sequencing techniques can provide a novel reference basis for molecular analysis of tea plants.

Mutagenesis of a Quintuple Mutant Impaired in Environmental Responses Reveals Roles for CHROMATIN REMODELING4 in the Arabidopsis Floral Transition.

Several pathways conferring environmental flowering responses in Arabidopsis (Arabidopsis thaliana) converge on developmental processes that mediate the floral transition in the shoot apical meristem. Many characterized mutations disrupt these environmental responses, but downstream developmental processes have been more refractory to mutagenesis. Here, we constructed a quintuple mutant impaired in several environmental pathways and showed that it possesses severely reduced flowering responses to changes in photoperiod and ambient temperature. RNA-sequencing (RNA-seq) analysis of the quintuple mutant showed that the expression of genes encoding gibberellin biosynthesis enzymes and transcription factors involved in the age pathway correlates with flowering. Mutagenesis of the quintuple mutant generated two late-flowering mutants, quintuple ems1 (qem1) and qem2 The mutated genes were identified by isogenic mapping and transgenic complementation. The qem1 mutant is an allele of the gibberellin 20-oxidase gene ga20ox2, confirming the importance of gibberellin for flowering in the absence of environmental responses. By contrast, qem2 is impaired in CHROMATIN REMODELING4 (CHR4), which has not been genetically implicated in floral induction. Using co-immunoprecipitation, RNA-seq, and chromatin immunoprecipitation sequencing, we show that CHR4 interacts with transcription factors involved in floral meristem identity and affects the expression of key floral regulators. Therefore, CHR4 mediates the response to endogenous flowering pathways in the inflorescence meristem to promote floral identity.

Complement Genome Annotation Lift Over Using a Weighted Sequence Alignment Strategy.

With the broad application of high-throughput sequencing, more whole-genome resequencing data and de novo assemblies of natural populations are becoming available. For a particular species, in general, only the reference genome is well established and annotated. Computational tools based on sequence alignment have been developed to investigate the gene models of individuals belonging to the same or closely related species. During this process, inconsistent alignment often obscures genome annotation lift over and leads to improper functional impact prediction for a genomic variant, especially in plant species. Here, we proposed the zebraic striped dynamic programming algorithm, which provides different weights to genetic features to refine genome annotation lift over. Testing of our zebraic striped dynamic programming algorithm on both plant and animal genomic data showed complementation to standard sequence approach for highly diverse individuals. Using the lift over genome annotation as anchors, a base-pair resolution genome-wide sequence alignment and variant calling pipeline for de novo assembly has been implemented in the GEAN software. GEAN could be used to compare haplotype diversity, refine the genetic variant functional annotation, annotate de novo assembly genome sequence, detect homologous syntenic blocks, improve the quantification of gene expression levels using RNA-seq data, and unify genomic variants for population genetic analysis. We expect that GEAN will be a standard tool for the coming of age of de novo assembly population genetics.

Recovery of novel association loci in Arabidopsis thaliana and Drosophila melanogaster through leveraging INDELs association and integrated burden test.

Short insertions, deletions (INDELs) and larger structural variants have been increasingly employed in genetic association studies, but few improvements over SNP-based association have been reported. In order to understand why this might be the case, we analysed two publicly available datasets and observed that 63% of INDELs called in A. thaliana and 64% in D. melanogaster populations are misrepresented as multiple alleles with different functional annotations, i.e. where the same underlying variant is represented by inconsistent alignments leading to different variant calls. To address this issue, we have developed the software Irisas to reclassify and re-annotate these variants, which we then used for single-locus tests of association. We also integrated them to predict the functional impact of SNPs, INDELs, and structural variants for burden testing. Using both approaches, we re-analysed the genetic architecture of complex traits in A. thaliana and D. melanogaster. Heritability analysis using SNPs alone explained on average 27% and 19% of phenotypic variance for A. thaliana and D. melanogaster respectively. Our method explained an additional 11% and 3%, respectively. We also identified novel trait loci that previous SNP-based association studies failed to map, and which contain established candidate genes. Our study shows the value of the association test with INDELs and integrating multiple types of variants in association studies in plants and animals.

The Cardamine hirsuta genome offers insight into the evolution of morphological diversity.

Finding causal relationships between genotypic and phenotypic variation is a key focus of evolutionary biology, human genetics and plant breeding. To identify genome-wide patterns underlying trait diversity, we assembled a high-quality reference genome of Cardamine hirsuta, a close relative of the model plant Arabidopsis thaliana. We combined comparative genome and transcriptome analyses with the experimental tools available in C. hirsuta to investigate gene function and phenotypic diversification. Our findings highlight the prevalent role of transcription factors and tandem gene duplications in morphological evolution. We identified a specific role for the transcriptional regulators PLETHORA5/7 in shaping leaf diversity and link tandem gene duplication with differential gene expression in the explosive seed pod of C. hirsuta. Our work highlights the value of comparative approaches in genetically tractable species to understand the genetic basis for evolutionary change.