afila, the origin and nature of a major innovation in the history of pea breeding.

The afila (af) mutation causes the replacement of leaflets by a branched mass of tendrils in the compound leaves of pea - Pisum sativum L. This mutation was first described in 1953, and several reports of spontaneous af mutations and induced mutants with a similar phenotype exist. Despite widespread introgression into breeding material, the nature of af and the origin of the alleles used remain unknown. Here, we combine comparative genomics with reverse genetic approaches to elucidate the genetic determinants of af. We also investigate haplotype diversity using a set of AfAf and afaf cultivars and breeding lines and molecular markers linked to seven consecutive genes. Our results show that deletion of two tandemly arranged genes encoding Q-type Cys(2)His(2) zinc finger transcription factors, PsPALM1a and PsPALM1b, is responsible for the af phenotype in pea. Eight haplotypes were identified in the af-harbouring genomic region on chromosome 2. These haplotypes differ in the size of the deletion, covering more or less genes. Diversity at the af locus is valuable for crop improvement and sheds light on the history of pea breeding for improved standing ability. The results will be used to understand the function of PsPALM1a/b and to transfer the knowledge for innovation in related crops.

Genome-wide circadian gating of a cold temperature response in bread wheat.

Circadian rhythms coordinate the responses of organisms with their daily fluctuating environments, by establishing a temporal program of gene expression. This schedules aspects of metabolism, physiology, development and behaviour according to the time of day. Circadian regulation in plants is extremely pervasive, and is important because it underpins both productivity and seasonal reproduction. Circadian regulation extends to the control of environmental responses through a regulatory process known as circadian gating. Circadian gating is the process whereby the circadian clock regulates the response to an environmental cue, such that the magnitude of response to an identical cue varies according to the time of day of the cue. Here, we show that there is genome-wide circadian gating of responses to cold temperatures in plants. By using bread wheat as an experimental model, we establish that circadian gating is crucial to the programs of gene expression that underlie the environmental responses of a crop of major socioeconomic importance. Furthermore, we identify that circadian gating of cold temperature responses are distributed unevenly across the three wheat subgenomes, which might reflect the geographical origins of the ancestors of modern wheat.

Low-temperature and circadian signals are integrated by the sigma factor SIG5.

Chloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis, a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions.

Genomics and biochemical analyses reveal a metabolon key to ß-L-ODAP biosynthesis in Lathyrus sativus.

Grass pea (Lathyrus sativus L.) is a rich source of protein cultivated as an insurance crop in Ethiopia, Eritrea, India, Bangladesh, and Nepal. Its resilience to both drought and flooding makes it a promising crop for ensuring food security in a changing climate. The lack of genetic resources and the crop's association with the disease neurolathyrism have limited the cultivation of grass pea. Here, we present an annotated, long read-based assembly of the 6.5 Gbp L. sativus genome. Using this genome sequence, we have elucidated the biosynthetic pathway leading to the formation of the neurotoxin, ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP). The final reaction of the pathway depends on an interaction between L. sativus acyl-activating enzyme 3 (LsAAE3) and a BAHD-acyltransferase (LsBOS) that form a metabolon activated by CoA to produce ß-L-ODAP. This provides valuable insight into the best approaches for developing varieties which produce substantially less toxin.

Layers of crosstalk between circadian regulation and environmental signalling in plants.

Circadian regulation has a pervasive influence upon plant development, physiology and metabolism, impacting upon components of fitness and traits of agricultural importance. Circadian regulation is inextricably connected to the responses of plants to their abiotic environments, from the cellular to whole plant scales. Here, we review the crosstalk that occurs between circadian regulation and responses to the abiotic environment from the intracellular scale through to naturally fluctuating environments. We examine the spatial crosstalk that forms part of plant circadian regulation, at the subcellular, tissue, organ and whole-plant scales. This includes a focus on chloroplast and mitochondrial signalling, alternative splicing, long-distance circadian signalling and circadian regulation within natural environments. We also consider mathematical models for plant circadian regulation, to suggest future areas for advancing understanding of roles for circadian regulation in plant responses to environmental cues.

Novelty and Convergence in Adaptation to Whole Genome Duplication.

Whole genome duplication (WGD) can promote adaptation but is disruptive to conserved processes, especially meiosis. Studies in Arabidopsis arenosa revealed a coordinated evolutionary response to WGD involving interacting proteins controlling meiotic crossovers, which are minimized in an autotetraploid (within-species polyploid) to avoid missegregation. Here, we test whether this surprising flexibility of a conserved essential process, meiosis, is recapitulated in an independent WGD system, Cardamine amara, 17 My diverged from A. arenosa. We assess meiotic stability and perform population-based scans for positive selection, contrasting the genomic response to WGD in C. amara with that of A. arenosa. We found in C. amara the strongest selection signals at genes with predicted functions thought important to adaptation to WGD: meiosis, chromosome remodeling, cell cycle, and ion transport. However, genomic responses to WGD in the two species differ: minimal ortholog-level convergence emerged, with none of the meiosis genes found in A. arenosa exhibiting strong signal in C. amara. This is consistent with our observations of lower meiotic stability and occasional clonal spreading in diploid C. amara, suggesting that nascent C. amara autotetraploid lineages were preadapted by their diploid lifestyle to survive while enduring reduced meiotic fidelity. However, in contrast to a lack of ortholog convergence, we see process-level and network convergence in DNA management, chromosome organization, stress signaling, and ion homeostasis processes. This gives the first insight into the salient adaptations required to meet the challenges of a WGD state and shows that autopolyploids can utilize multiple evolutionary trajectories to adapt to WGD.

De Novo Mutation and Rapid Protein (Co-)evolution during Meiotic Adaptation in Arabidopsis arenosa.

A sudden shift in environment or cellular context necessitates rapid adaptation. A dramatic example is genome duplication, which leads to polyploidy. In such situations, the waiting time for new mutations might be prohibitive; theoretical and empirical studies suggest that rapid adaptation will largely rely on standing variation already present in source populations. Here, we investigate the evolution of meiosis proteins in Arabidopsis arenosa, some of which were previously implicated in adaptation to polyploidy, and in a diploid, habitat. A striking and unexplained feature of prior results was the large number of amino acid changes in multiple interacting proteins, especially in the relatively young tetraploid. Here, we investigate whether selection on meiosis genes is found in other lineages, how the polyploid may have accumulated so many differences, and whether derived variants were selected from standing variation. We use a range-wide sample of 145 resequenced genomes of diploid and tetraploid A. arenosa, with new genome assemblies. We confirmed signals of positive selection in the polyploid and diploid lineages they were previously reported in and find additional meiosis genes with evidence of selection. We show that the polyploid lineage stands out both qualitatively and quantitatively. Compared with diploids, meiosis proteins in the polyploid have more amino acid changes and a higher proportion affecting more strongly conserved sites. We find evidence that in tetraploids, positive selection may have commonly acted on de novo mutations. Several tests provide hints that coevolution, and in some cases, multinucleotide mutations, might contribute to rapid accumulation of changes in meiotic proteins.

Formation and diversification of a paradigm biosynthetic gene cluster in plants.

Numerous examples of biosynthetic gene clusters (BGCs), including for compounds of agricultural and medicinal importance, have now been discovered in plant genomes. However, little is known about how these complex traits are assembled and diversified. Here, we examine a large number of variants within and between species for a paradigm BGC (the thalianol cluster), which has evolved recently in a common ancestor of the Arabidopsis genus. Comparisons at the species level reveal differences in BGC organization and involvement of auxiliary genes, resulting in production of species-specific triterpenes. Within species, the thalianol cluster is primarily fixed, showing a low frequency of deleterious haplotypes. We further identify chromosomal inversion as a molecular mechanism that may shuffle more distant genes into the cluster, so enabling cluster compaction. Antagonistic natural selection pressures are likely involved in shaping the occurrence and maintenance of this BGC. Our work sheds light on the birth, life and death of complex genetic and metabolic traits in plants.

Interspecific introgression mediates adaptation to whole genome duplication.

Adaptive gene flow is a consequential phenomenon across all kingdoms. Although recognition is increasing, there is no study showing that bidirectional gene flow mediates adaptation at loci that manage core processes. We previously discovered concerted molecular changes among interacting members of the meiotic machinery controlling crossover number upon adaptation to whole-genome duplication (WGD) in Arabidopsis arenosa. Here we conduct a population genomic study to test the hypothesis that adaptation to WGD has been mediated by adaptive gene flow between A. arenosa and A. lyrata. We find that A. lyrata underwent WGD more recently than A. arenosa, suggesting that pre-adapted alleles have rescued nascent A. lyrata, but we also detect gene flow in the opposite direction at functionally interacting loci under the most extreme levels of selection. These data indicate that bidirectional gene flow allowed for survival after WGD, and that the merger of these species is greater than the sum of their parts.

Pervasive population genomic consequences of genome duplication in Arabidopsis arenosa.

Ploidy-variable species allow direct inference of the effects of chromosome copy number on fundamental evolutionary processes. While an abundance of theoretical work suggests polyploidy should leave distinct population genomic signatures, empirical data remains sparse. We sequenced ~300 individuals from 39 populations of Arabidopsis arenosa, a naturally diploid-autotetraploid species. We find that the impacts of polyploidy on population genomic processes are subtle yet pervasive, such as reduced efficiency of purifying selection, differences in linked selection and rampant gene flow from diploids. Initial masking of deleterious mutations, faster rates of nucleotide substitution and interploidy introgression likely conspire to shape the evolutionary potential of polyploids.

A critical comparison of technologies for a plant genome sequencing project.

BACKGROUND: A high-quality genome sequence of any model organism is an essential starting point for genetic and other studies. Older clone-based methods are slow and expensive, whereas faster, cheaper short-read-only assemblies can be incomplete and highly fragmented, which minimizes their usefulness. The last few years have seen the introduction of many new technologies for genome assembly. These new technologies and associated new algorithms are typically benchmarked on microbial genomes or, if they scale appropriately, on larger (e.g., human) genomes. However, plant genomes can be much more repetitive and larger than the human genome, and plant biochemistry often makes obtaining high-quality DNA that is free from contaminants difficult. Reflecting their challenging nature, we observe that plant genome assembly statistics are typically poorer than for vertebrates. RESULTS: Here, we compare Illumina short read, Pacific Biosciences long read, 10x Genomics linked reads, Dovetail Hi-C, and BioNano Genomics optical maps, singly and combined, in producing high-quality long-range genome assemblies of the potato species Solanum verrucosum. We benchmark the assemblies for completeness and accuracy, as well as DNA compute requirements and sequencing costs. CONCLUSIONS: The field of genome sequencing and assembly is reaching maturity, and the differences we observe between assemblies are surprisingly small. We expect that our results will be helpful to other genome projects, and that these datasets will be used in benchmarking by assembly algorithm developers.

Fluctuating selection on migrant adaptive sodium transporter alleles in coastal Arabidopsis thaliana.

Stressors such as soil salinity and dehydration are major constraints on plant growth, causing worldwide crop losses. Compounding these insults, increasing climate volatility requires adaptation to fluctuating conditions. Salinity stress responses are relatively well understood in Arabidopsis thaliana, making this system suited for the rapid molecular dissection of evolutionary mechanisms. In a large-scale genomic analysis of Catalonian A. thaliana, we resequenced 77 individuals from multiple salinity gradients along the coast and integrated these data with 1,135 worldwide A. thaliana genomes for a detailed understanding of the demographic and evolutionary dynamics of naturally evolved salinity tolerance. This revealed that Catalonian varieties adapted to highly fluctuating soil salinity are not Iberian relicts but instead have immigrated to this region more recently. De novo genome assembly of three allelic variants of the high-affinity K+ transporter (HKT1;1) locus resolved structural variation between functionally distinct alleles undergoing fluctuating selection in response to seasonal changes in soil salinity. Plants harboring alleles responsible for low root expression of HKT1;1 and consequently high leaf sodium (HKT1;1HLS ) were migrants that have moved specifically into areas where soil sodium levels fluctuate widely due to geography and rainfall variation. We demonstrate that the proportion of plants harboring HKT1;1HLS alleles correlates with soil sodium level over time, HKT1;1HLS -harboring plants are better adapted to intermediate levels of salinity, and the HKT1;1HLS allele clusters with high-sodium accumulator accessions worldwide. Together, our evidence suggests that HKT1;1 is under fluctuating selection in response to climate volatility and is a worldwide determinant in adaptation to saline conditions.

Building a locally diploid genome and transcriptome of the diatom Fragilariopsis cylindrus.

The genome of the cold-adapted diatom Fragilariopsis cylindrus is characterized by highly diverged haplotypes that intersperse its homozygous genome. Here, we describe how a combination of PacBio DNA and Illumina RNA sequencing can be used to resolve this complex genomic landscape locally into the highly diverged haplotypes, and how to map various environmentally controlled transcripts onto individual haplotypes. We assembled PacBio sequence data with the FALCON assembler and created a haplotype resolved annotation of the assembly using annotations of a Sanger sequenced F. cylindrus genome. RNA-seq datasets from six different growth conditions were used to resolve allele-specifc gene expression in F. cylindrus. This approach enables to study differential expression of alleles in a complex genomic landscape and provides a useful tool to study how diverged haplotypes in diploid organisms are used for adaptation and evolution to highly variable environments.

Comparative analysis of targeted long read sequencing approaches for characterization of a plant's immune receptor repertoire.

BACKGROUND: The Oxford Nanopore Technologies MinION™ sequencer is a small, portable, low cost device that is accessible to labs of all sizes and attractive for in-the-field sequencing experiments. Selective breeding of crops has led to a reduction in genetic diversity, and wild relatives are a key source of new genetic resistance to pathogens, usually via NLR immune receptor-encoding genes. Recent studies have demonstrated how crop NLR repertoires can be targeted for sequencing on Illumina or PacBio (RenSeq) and the specific gene conveying pathogen resistance identified. RESULTS: Sequence yields per MinION run are lower than Illumina, making targeted resequencing an efficient approach. While MinION generates long reads similar to PacBio it doesn't generate the highly accurate multipass consensus reads, which presents downstream bioinformatics challenges. Here we demonstrate how MinION data can be used for RenSeq achieving similar results to the PacBio and how novel NLR gene fusions can be identified via a Nanopore RenSeq pipeline. CONCLUSION: The described library preparation and bioinformatics methods should be applicable to other gene families or any targeted long DNA fragment nanopore sequencing project.

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Targeted capture and sequencing of gene-sized DNA molecules.

Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family-key genes in plant immune systems.

Iron Age and Anglo-Saxon genomes from East England reveal British migration history.

British population history has been shaped by a series of immigrations, including the early Anglo-Saxon migrations after 400 CE. It remains an open question how these events affected the genetic composition of the current British population. Here, we present whole-genome sequences from 10 individuals excavated close to Cambridge in the East of England, ranging from the late Iron Age to the middle Anglo-Saxon period. By analysing shared rare variants with hundreds of modern samples from Britain and Europe, we estimate that on average the contemporary East English population derives 38% of its ancestry from Anglo-Saxon migrations. We gain further insight with a new method, rarecoal, which infers population history and identifies fine-scale genetic ancestry from rare variants. Using rarecoal we find that the Anglo-Saxon samples are closely related to modern Dutch and Danish populations, while the Iron Age samples share ancestors with multiple Northern European populations including Britain.