The reference genome of an endangered Asteraceae, Deinandra increscens subsp. villosa, endemic to the Central Coast of California.

We present a reference genome for the federally endangered Gaviota tarplant, Deinandra increscens subsp. villosa (Madiinae, Asteraceae), an annual herb endemic to the Central California coast. Generating PacBio Hifi, Oxford Nanopore Technologies, and Dovetail Omni-C data, we assembled a haploid consensus genome of 1.67 Gbp as 28.7 K scaffolds with a scaffold N50 of 74.9 Mb. We annotated repeat content in 74.8% of the genome. Long terminal repeats (LTR) covered 44.0% of the genome with Copia families predominant at 22.9% followed by Gypsy at 14.2%. Both Gypsy and Copia elements were common in ancestral peaks of LTR, and the most abundant element was a Gypsy element containing nested Copia/Angela sequence similarity, reflecting a complex evolutionary history of repeat activity. Gene annotation produced 33,257 genes and 68,942 transcripts, of which 99% were functionally annotated. BUSCO scores for the annotated proteins were 96.0% complete of which 77.6% was single copy and 18.4% duplicates. Whole genome duplication (WGD) synonymous mutation rates of Gaviota tarplant and sunflower (Helianthus annuus) shared peaks that correspond to the last Asteraceae polyploidization event and subsequent divergence from a common ancestor at ∼27 mya. Regions of high-density tandem genes were identified, pointing to potentially important loci of environmental adaptation in this species.

Critical considerations for communicating environmental DNA science.

The economic and methodological efficiencies of environmental DNA (eDNA) based survey approaches provide an unprecedented opportunity to assess and monitor aquatic environments. However, instances of inadequate communication from the scientific community about confidence levels, knowledge gaps, reliability, and appropriate parameters of eDNA-based methods have hindered their uptake in environmental monitoring programs and, in some cases, has created misperceptions or doubts in the management community. To help remedy this situation, scientists convened a session at the Second National Marine eDNA Workshop to discuss strategies for improving communications with managers. These include articulating the readiness of different eDNA applications, highlighting the strengths and limitations of eDNA tools for various applications or use cases, communicating uncertainties associated with specified uses transparently, and avoiding the exaggeration of exploratory and preliminary findings. Several key messages regarding implementation, limitations, and relationship to existing methods were prioritized. To be inclusive of the diverse managers, practitioners, and researchers, we and the other workshop participants propose the development of communication workflow plans, using RACI (Responsible, Accountable, Consulted, Informed) charts to clarify the roles of all pertinent individuals and parties and to minimize the chance for miscommunications. We also propose developing decision support tools such as Structured Decision-Making (SDM) to help balance the benefits of eDNA sampling with the inherent uncertainty, and developing an eDNA readiness scale to articulate the technological readiness of eDNA approaches for specific applications. These strategies will increase clarity and consistency regarding our understanding of the utility of eDNA-based methods, improve transparency, foster a common vision for confidently applying eDNA approaches, and enhance their benefit to the monitoring and assessment community.

Chromosome-level reference genome of stinkwort, Dittrichia graveolens (L.) Greuter: A resource for studies on invasion, range expansion, and evolutionary adaptation under global change.

Dittrichia graveolens (L.) Greuter, or stinkwort, is a weedy annual plant within the family Asteraceae. The species is recognized for the rapid expansion of both its native and introduced ranges: in Europe, it has expanded its native distribution northward from the Mediterranean basin by nearly 7 °C latitude since the mid-20th century, while in California and Australia the plant is an invasive weed of concern. Here, we present the first de novo D. graveolens genome assembly (1N = 9 chromosomes), including complete chloroplast (151,013 bp) and partial mitochondrial genomes (22,084 bp), created using Pacific Biosciences HiFi reads and Dovetail Omni-C data. The final primary assembly is 835 Mbp in length, of which 98.1% are represented by 9 scaffolds ranging from 66 to 119 Mbp. The contig N50 is 74.9 Mbp and the scaffold N50 is 96.9 Mbp, which, together with a 98.8% completeness based on the BUSCO embryophyta10 database containing 1,614 orthologs, underscores the high quality of this assembly. This pseudo-molecule-scale genome assembly is a valuable resource for our fundamental understanding of the genomic consequences of range expansion under global change, as well as comparative genomic studies in the Asteraceae.

Environmental DNA as an emerging tool in botanical research.

Over the past quarter century, environmental DNA (eDNA) has been ascendant as a tool to detect, measure, and monitor biodiversity (species and communities), as a means of elucidating biological interaction networks, and as a window into understanding past patterns of biodiversity. However, only recently has the potential of eDNA been realized in the botanical world. Here we synthesize the state of eDNA applications in botanical systems with emphases on aquatic, ancient, contemporary sediment, and airborne systems, and focusing on both single-species approaches and multispecies community metabarcoding. Further, we describe how abiotic and biotic factors, taxonomic resolution, primer choice, spatiotemporal scales, and relative abundance influence the utilization and interpretation of airborne eDNA results. Lastly, we explore several areas and opportunities for further development of eDNA tools for plants, advancing our knowledge and understanding of the efficacy, utility, and cost-effectiveness, and ultimately facilitating increased adoption of eDNA analyses in botanical systems.

A manager's guide to using eDNA metabarcoding in marine ecosystems.

Environmental DNA (eDNA) metabarcoding is a powerful tool that can enhance marine ecosystem/biodiversity monitoring programs. Here we outline five important steps managers and researchers should consider when developing eDNA monitoring program: (1) select genes and primers to target taxa; (2) assemble or develop comprehensive barcode reference databases; (3) apply rigorous site occupancy based decontamination pipelines; (4) conduct pilot studies to define spatial and temporal variance of eDNA; and (5) archive samples, extracts, and raw sequence data. We demonstrate the importance of each of these considerations using a case study of eDNA metabarcoding in the Ports of Los Angeles and Long Beach. eDNA metabarcoding approaches detected 94.1% (16/17) of species observed in paired trawl surveys while identifying an additional 55 native fishes, providing more comprehensive biodiversity inventories. Rigorous benchmarking of eDNA metabarcoding results improved ecological interpretation and confidence in species detections while providing archived genetic resources for future analyses. Well designed and validated eDNA metabarcoding approaches are ideally suited for biomonitoring applications that rely on the detection of species, including mapping invasive species fronts and endangered species habitats as well as tracking range shifts in response to climate change. Incorporating these considerations will enhance the utility and efficacy of eDNA metabarcoding for routine biomonitoring applications.

Revisiting the effect of PCR replication and sequencing depth on biodiversity metrics in environmental DNA metabarcoding.

Environmental DNA (eDNA) metabarcoding is an increasingly popular tool for measuring and cataloguing biodiversity. Because the environments and substrates in which DNA is preserved differ considerably, eDNA research often requires bespoke approaches to generating eDNA data. Here, we explore how two experimental choices in eDNA study design-the number of PCR replicates and the depth of sequencing of PCR replicates-influence the composition and consistency of taxa recovered from eDNA extracts. We perform 24 PCR replicates from each of six soil samples using two of the most common metabarcodes for Fungi and Viridiplantae (ITS1 and ITS2), and sequence each replicate to an average depth of ~84,000 reads. We find that PCR replicates are broadly consistent in composition and relative abundance of dominant taxa, but that low abundance taxa are often unique to one or a few PCR replicates. Taxa observed in one out of 24 PCR replicates make up 21-29% of the total taxa detected. We also observe that sequencing depth or rarefaction influences alpha diversity and beta diversity estimates. Read sampling depth influences local contribution to beta diversity, placement in ordinations, and beta dispersion in ordinations. Our results suggest that, because common taxa drive some alpha diversity estimates, few PCR replicates and low read sampling depths may be sufficient for many biological applications of eDNA metabarcoding. However, because rare taxa are recovered stochastically, eDNA metabarcoding may never fully recover the true amplifiable alpha diversity in an eDNA extract. Rare taxa drive PCR replicate outliers of alpha and beta diversity and lead to dispersion differences at different read sampling depths. We conclude that researchers should consider the complexity and unevenness of a community when choosing analytical approaches, read sampling depths, and filtering thresholds to arrive at stable estimates.

Landscape analyses using eDNA metabarcoding and Earth observation predict community biodiversity in California.

Ecosystems globally are under threat from ongoing anthropogenic environmental change. Effective conservation management requires more thorough biodiversity surveys that can reveal system-level patterns and that can be applied rapidly across space and time. Using modern ecological models and community science, we integrate environmental DNA and Earth observations to produce a time snapshot of regional biodiversity patterns and provide multi-scalar community-level characterization. We collected 278 samples in spring 2017 from coastal, shrub, and lowland forest sites in California, a complex ecosystem and biodiversity hotspot. We recovered 16,118 taxonomic entries from eDNA analyses and compiled associated traditional observations and environmental data to assess how well they predicted alpha, beta, and zeta diversity. We found that local habitat classification was diagnostic of community composition and distinct communities and organisms in different kingdoms are predicted by different environmental variables. Nonetheless, gradient forest models of 915 families recovered by eDNA analysis and using BIOCLIM variables, Sentinel-2 satellite data, human impact, and topographical features as predictors, explained 35% of the variance in community turnover. Elevation, sand percentage, and photosynthetic activities (NDVI32) were the top predictors. In addition to this signal of environmental filtering, we found a positive relationship between environmentally predicted families and their numbers of biotic interactions, suggesting environmental change could have a disproportionate effect on community networks. Together, these analyses show that coupling eDNA with environmental predictors including remote sensing data has capacity to test proposed Essential Biodiversity Variables and create new landscape biodiversity baselines that span the tree of life.

Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing.

Long-read sequencing facilitates assembly of complex genomic regions. In plants, loci containing nucleotide-binding, leucine-rich repeat (NLR) disease resistance genes are an important example of such regions. NLR genes constitute one of the largest gene families in plants and are often clustered, evolving via duplication, contraction, and transposition. We recently mapped the Xo1 locus for resistance to bacterial blight and bacterial leaf streak, found in the American heirloom rice variety Carolina Gold Select, to a region that in the Nipponbare reference genome is NLR gene-rich. Here, toward identification of the Xo1 gene, we combined Nanopore and Illumina reads and generated a high-quality Carolina Gold Select genome assembly. We identified 529 complete or partial NLR genes and discovered, relative to Nipponbare, an expansion of NLR genes at the Xo1 locus. One of these has high sequence similarity to the cloned, functionally similar Xa1 gene. Both harbor an integrated zfBED domain, and the repeats within each protein are nearly perfect. Across diverse Oryzeae, we identified two sub-clades of NLR genes with these features, varying in the presence of the zfBED domain and the number of repeats. The Carolina Gold Select genome assembly also uncovered at the Xo1 locus a rice blast resistance gene and a gene encoding a polyphenol oxidase (PPO). PPO activity has been used as a marker for blast resistance at the locus in some varieties; however, the Carolina Gold Select sequence revealed a loss-of-function mutation in the PPO gene that breaks this association. Our results demonstrate that whole genome sequencing combining Nanopore and Illumina reads effectively resolves NLR gene loci. Our identification of an Xo1 candidate is an important step toward mechanistic characterization, including the role(s) of the zfBED domain. Finally, the Carolina Gold Select genome assembly will facilitate identification of other useful traits in this historically important variety.

A novel hydroxycinnamoyl transferase for synthesis of hydroxycinnamoyl spermine conjugates in plants.

BACKGROUND: Hydroxycinnamoyl-spermine conjugates (HCSpm) are a class of hydroxycinnamic acid amides (HCAAs), which not only are instrumental in plant development and stress response, but also benefit human health. However, HCSpm are not commonly produced in plants, and the mechanism of their biosynthesis remains unclear. In previous investigations of phenolics in Solanum fruits related to eggplant (Solanum melongena L.), we discovered that Solanum richardii, an African wild relative of eggplant, was rich in HCSpms in fruits. RESULTS: The putative spermine hydroxycinnamoyl transferase (HT) SpmHT was isolated from S. richardii and eggplant. SrSpmHT expression was high in flowers and fruit, and was associated with HCSpm accumulation in S. richardii; however, SpmHT was hardly detected in eggplant cultivars and other wild relatives. Recombinant SpmHT exclusively selected spermine as the acyl acceptor substrate, while showing donor substrate preference in the following order: caffeoyl-CoA, feruloyl-CoA, and p-coumaroyl-CoA. Molecular docking revealed that substrate binding pockets of SpmHT could properly accommodate spermine but not the shorter, more common spermidine. CONCLUSION: SrSpmHT is a novel spermine hydroxycinnamoyl transferase that uses Spm exclusively as the acyl acceptor substrate to produce HCSpms. Our findings shed light on the HCSpm biosynthetic pathway that may allow an increase of health beneficial metabolites in Solanum crops via methods such as introgression or engineering HCAA metabolism.

Eggplant Domestication: Pervasive Gene Flow, Feralization, and Transcriptomic Divergence.

In the context of food security, examining the genomics of domestication will help identify genes underlying adaptive and economically important phenotypes, for example, larger fruit, improved taste, and loss of agronomically inferior phenotypes. Examination of genome-scale single nucleotide polymorphisms demonstrates the relationships between wild ancestors of eggplant (Solanum melongena L.), confirming that Solanum insanum L. is the wild progenitor. This species is split roughly into an Eastern (Malaysian, Thai, and Vietnamese) and Western (Indian, Madagascan, and Sri Lankan) group, with domesticates derived from the former. Additional "wild" accessions from India appear to be feral escapes, derived multiple times from domesticated varieties through admixture. Accessions with small egg-shaped fruit are generally found intermixed with East Asian Solanum insanum confirming they are primitive relative to the large-fruited domesticates. Comparative transcriptomics was used to track the loci under selection. Sequence analysis revealed a genetic bottleneck reducing variation by almost 50% in the primitive accessions relative to the wild species and a further 10% in the landraces. We also show evidence for selection on genes with a role in response to wounding and apoptosis. Genes showing a significant difference in expression between wild and primitive or between primitive and landrace genepools were mostly (>75%) downregulated in the derived populations and enriched for gene ontologies related to defense, flowering, signaling, and response to biotic and abiotic stimuli. This work reveals genomic changes involved in crop domestication and improvement, and the population genetics work explains why defining the eggplant domestication trajectory has been so challenging.

Genetic control of seed shattering during African rice domestication.

Domestication represents a unique opportunity to study the evolutionary process. The elimination of seed dispersal traits was a key step in the evolution of cereal crops under domestication. Here, we show that ObSH3, a YABBY transcription factor, is required for the development of the seed abscission layer. Moreover, selecting a genomic segment deletion containing SH3 resulted in the loss of seed dispersal in populations of African cultivated rice (Oryza glaberrima Steud.). Functional characterization of SH3 and SH4 (another gene controlling seed shattering on chromosome 4) revealed that multiple genes can lead to a spectrum of non-shattering phenotypes, affecting other traits such as ease of threshing that may be important to tune across different agroecologies and postharvest practices. The molecular evolution analyses of SH3 and SH4 in a panel of 93 landraces provided unprecedented geographical detail of the domestication history of African rice, tracing multiple dispersals from a core heartland and introgression from local wild rice. The cloning of ObSH3 not only provides new insights into a critical crop domestication process but also adds to the body of knowledge on the molecular mechanism of seed dispersal.

ranacapa: An R package and Shiny web app to explore environmental DNA data with exploratory statistics and interactive visualizations.

Environmental DNA (eDNA) metabarcoding is becoming a core tool in ecology and conservation biology, and is being used in a growing number of education, biodiversity monitoring, and public outreach programs in which professional research scientists engage community partners in primary research. Results from eDNA analyses can engage and educate natural resource managers, students, community scientists, and naturalists, but without significant training in bioinformatics, it can be difficult for this diverse audience to interact with eDNA results. Here we present the R package ranacapa, at the core of which is a Shiny web app that helps perform exploratory biodiversity analyses and visualizations of eDNA results. The app requires a taxonomy-by-sample matrix and a simple metadata file with descriptive information about each sample. The app enables users to explore the data with interactive figures and presents results from simple community ecology analyses. We demonstrate the value of ranacapa to two groups of community partners engaging with eDNA metabarcoding results.

A single-nucleotide polymorphism causes smaller grain size and loss of seed shattering during African rice domestication.

Grain size is one of the most important components of grain yield and selecting large seeds has been a main target during plant domestication. Surprisingly, the grain of African cultivated rice (Oryza glaberrima Steud.) typically is smaller than that of its progenitor, Oryza barthii. Here we report the cloning and characterization of a quantitative trait locus, GL4, controlling the grain length on chromosome 4 in African rice, which regulates longitudinal cell elongation of the outer and inner glumes. Interestingly, GL4 also controls the seed shattering phenotype like its orthologue SH4 gene in Asian rice. Our data show that a single-nucleotide polymorphism (SNP) mutation in the GL4 gene resulted in a premature stop codon and led to small seeds and loss of seed shattering during African rice domestication. These results provide new insights into diverse domestication practices in African rice, and also pave the way for enhancing crop yield to meeting the challenge of cereal demand in West Africa.

Tracing ancestor rice of Suriname Maroons back to its African origin.

African rice (Oryza glaberrima) and African cultivation practices are said to have influenced emerging colonial plantation economies in the Americas1,2. However, the level of impact of African rice practices is difficult to establish because of limited written or botanical records2,3. Recent findings of O. glaberrima in rice fields of Suriname Maroons bear evidence of the high level of knowledge about rice among African slaves and their descendants, who consecrate it in ancestor rituals4,5. Here we establish the strong similarity, and hence likely origin, of the first extant New World landrace of O. glaberrima to landraces from the Upper Guinean forests in West Africa. We collected African rice from a Maroon market in Paramaribo, Suriname, propagated it, sequenced its genome6 and compared it with genomes of 109 accessions representing O. glaberrima diversity across West Africa. By analysing 1,649,769 single nucleotide polymorphisms (SNPs) in clustering analyses, the Suriname sample appears sister to an Ivory Coast landrace, and shows no evidence of introgression from Asian rice. Whereas the Dutch took most slaves from Ghana, Benin and Central Africa7, the diaries of slave ship captains record the purchase of food for provisions when sailing along the West African Coast8, offering one possible explanation for the patterns of genetic similarity. This study demonstrates the utility of genomics in understanding the largely unwritten histories of crop cultures of diaspora communities.

Domestication history and geographical adaptation inferred from a SNP map of African rice.

African rice (Oryza glaberrima Steud.) is a cereal crop species closely related to Asian rice (Oryza sativa L.) but was independently domesticated in West Africa ∼3,000 years ago. African rice is rarely grown outside sub-Saharan Africa but is of global interest because of its tolerance to abiotic stresses. Here we describe a map of 2.32 million SNPs of African rice from whole-genome resequencing of 93 landraces. Population genomic analysis shows a population bottleneck in this species that began ∼13,000-15,000 years ago with effective population size reaching its minimum value ∼3,500 years ago, suggesting a protracted period of population size reduction likely commencing with predomestication management and/or cultivation. Genome-wide association studies (GWAS) for six salt tolerance traits identify 11 significant loci, 4 of which are within ∼300 kb of genomic regions that possess signatures of positive selection, suggesting adaptive geographical divergence for salt tolerance in this species.

Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop.

Date palms (Phoenix dactylifera) are the most significant perennial crop in arid regions of the Middle East and North Africa. Here, we present a comprehensive catalogue of approximately seven million single nucleotide polymorphisms in date palms based on whole genome re-sequencing of a collection of 62 cultivars. Population structure analysis indicates a major genetic divide between North Africa and the Middle East/South Asian date palms, with evidence of admixture in cultivars from Egypt and Sudan. Genome-wide scans for selection suggest at least 56 genomic regions associated with selective sweeps that may underlie geographic adaptation. We report candidate mutations for trait variation, including nonsense polymorphisms and presence/absence variation in gene content in pathways for key agronomic traits. We also identify a copia-like retrotransposon insertion polymorphism in the R2R3 myb-like orthologue of the oil palm virescens gene associated with fruit colour variation. This analysis documents patterns of post-domestication diversification and provides a genomic resource for this economically important perennial tree crop.

Parallel reductions in phenolic constituents resulting from the domestication of eggplant.

Crop domestication is often accompanied by changes in metabolite compositions that alter traits such as flavor, color, or other beneficial properties. Fruits of eggplants (Solanum melongena L.) and related species are abundant and diverse in pharmacologically interesting phenolic compounds, particularly hydroxycinnamic acid (HCA) conjugates such as the antioxidant caffeoylquinic acids (CQA) and HCA-polyamine amides (HCAA). To understand metabolite variability through the lens of natural and artificial selection, HPLC-DAD was used to generate phenolic profiles for 32 compounds in fruits from 93 accessions representing 9 Solanum species. Profiles were used for identification of species-level and infraspecific chemical patterns across both genetic distance and landscape. Sampling of plant lines included the undomesticated progenitor of eggplant and Asian landraces with a genetic background associated with three Asian regions near proposed separate centers of domestication to test whether chemical changes were convergent despite different origins. Results showed ten compounds were unique to species, and ten other compounds varied significantly in abundance among species. Five CQAs and three HCA-polyamine conjugates were more abundant in wild (undomesticated) versus domesticated eggplant, indicating that artificial selection may have led to reduced phenolic levels. No chemical abundance patterns were associated with site-origin. However, one genetically distinct lineage of geographically-restricted SE Asian eggplants (S. melongena subsp. ovigerum) had a higher HCAA content and diversity than other lineages, which is suggested to be related to artificial selection for small, firm fruit. Overall, patterns show that fruit size, palatability and texture were preferentially selected over health-beneficial phytochemical content during domestication of several nightshade crops.

Location of chlorogenic acid biosynthesis pathway and polyphenol oxidase genes in a new interspecific anchored linkage map of eggplant.

BACKGROUND: Eggplant is a powerful source of polyphenols which seems to play a key role in the prevention of several human diseases, such as cancer and diabetes. Chlorogenic acid is the polyphenol most present in eggplant, comprising between the 70% and 90% of the total polyphenol content. Introduction of the high chlorogenic acid content of wild relatives, such as S. incanum, into eggplant varieties will be of great interest. A potential side effect of the increased level polyphenols could be a decrease on apparent quality due to browning caused by the polyphenol oxidase enzymes mediated oxidation of polyphenols. We report the development of a new interspecific S. melongena × S. incanum linkage map based on a first backcross generation (BC1) towards the cultivated S. melongena as a tool for introgressing S. incanum alleles involved in the biosynthesis of chlorogenic acid in the genetic background of S. melongena. RESULTS: The interspecific genetic linkage map of eggplant developed in this work anchor the most informative previously published genetic maps of eggplant using common markers. The 91 BC1 plants of the mapping population were genotyped with 42 COSII, 99 SSRs, 88 AFLPs, 9 CAPS, 4 SNPs and one morphological polymorphic markers. Segregation marker data resulted in a map encompassing 1085 cM distributed in 12 linkage groups. Based on the syntheny with tomato, the candidate genes involved in the core chlorogenic acid synthesis pathway in eggplant (PAL, C4H, 4CL, HCT, C3'H, HQT) as well as five polyphenol oxidase (PPO1, PPO2, PPO3, PPO4, PPO5) were mapped. Except for 4CL and HCT chlorogenic acid genes were not linked. On the contrary, all PPO genes clustered together. Candidate genes important in domestication such as fruit shape (OVATE, SISUN1) and prickliness were also located. CONCLUSIONS: The achievements in location of candidate genes will allow the search of favorable alleles employing marker-assisted selection in order to develop new varieties with higher chlorogenic content alongside a lower polyphenol oxidase activity. This will result into an enhanced product showing a lower fruit flesh browning with improved human health properties.