Genomic mechanisms of climate adaptation in polyploid bioenergy switchgrass.

Long-term climate change and periodic environmental extremes threaten food and fuel security1 and global crop productivity2-4. Although molecular and adaptive breeding strategies can buffer the effects of climatic stress and improve crop resilience5, these approaches require sufficient knowledge of the genes that underlie productivity and adaptation6-knowledge that has been limited to a small number of well-studied model systems. Here we present the assembly and annotation of the large and complex genome of the polyploid bioenergy crop switchgrass (Panicum virgatum). Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive genomic evidence of climate adaptation. Climate-gene-biomass associations were abundant but varied considerably among deeply diverged gene pools. Furthermore, we found that gene flow accelerated climate adaptation during the postglacial colonization of northern habitats through introgression of alleles from a pre-adapted northern gene pool. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome. In addition to investigating patterns of climate adaptation, the genome resources and gene-trait associations developed here provide breeders with the necessary tools to increase switchgrass yield for the sustainable production of bioenergy.

Genomic variation in 3,010 diverse accessions of Asian cultivated rice.

Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.

Full-Length Transcriptome Sequencing Reveals Alternative Splicing and lncRNA Regulation during Nodule Development in Glycine max.

Alternative splicing (AS) is a ubiquitous phenomenon among eukaryotic intron-containing genes, which greatly contributes to transcriptome and proteome diversity. Here we performed the isoform sequencing (Iso-Seq) of soybean underground tissues inoculated and uninoculated with Rhizobium and obtained 200,681 full-length transcripts covering 26,183 gene loci. It was found that 80.78% of the multi-exon loci produced more than one splicing variant. Comprehensive analysis of these identified 7874 differentially splicing events with highly diverse splicing patterns during nodule development, especially in defense and transport-related processes. We further profiled genes with differential isoform usage and revealed that 2008 multi-isoform loci underwent stage-specific or simultaneous major isoform switches after Rhizobium inoculation, indicating that AS is a vital way to regulate nodule development. Moreover, we took the lead in identifying 1563 high-confidence long non-coding RNAs (lncRNAs) in soybean, and 157 of them are differentially expressed during nodule development. Therefore, our study uncovers the landscape of AS during the soybean-Rhizobium interaction and provides systematic transcriptomic data for future study of multiple novel directions in soybean.

The chromosome-scale reference genome of safflower (Carthamus tinctorius) provides insights into linoleic acid and flavonoid biosynthesis.

Safflower (Carthamus tinctorius L.), a member of the Asteraceae, is a popular crop due to its high linoleic acid (LA) and flavonoid (such as hydroxysafflor yellow A) contents. Here, we report the first high-quality genome assembly (contig N50 of 21.23 Mb) for the 12 pseudochromosomes of safflower using single-molecule real-time sequencing, Hi-C mapping technologies and a genetic linkage map. Phyloge nomic analysis showed that safflower diverged from artichoke (Cynara cardunculus) and sunflower (Helianthus annuus) approximately 30.7 and 60.5 million years ago, respectively. Comparative genomic analyses revealed that uniquely expanded gene families in safflower were enriched for those predicted to be involved in lipid metabolism and transport and abscisic acid signalling. Notably, the fatty acid desaturase 2 (FAD2) and chalcone synthase (CHS) families, which function in the LA and flavonoid biosynthesis pathways, respectively, were expanded via tandem duplications in safflower. CarFAD2-12 was specifically expressed in seeds and was vital for high-LA content in seeds, while tandemly duplicated CarFAD2 genes were up-regulated in ovaries compared to CarFAD2-12, which indicates regulatory divergence of FAD2 in seeds and ovaries. CarCHS1, CarCHS4 and tandem-duplicated CarCHS5˜CarCHS6, which were up-regulated compared to other CarCHS members at early stages, contribute to the accumulation of major flavonoids in flowers. In addition, our data reveal multiple alternative splicing events in gene families related to fatty acid and flavonoid biosynthesis. Together, these results provide a high-quality reference genome and evolutionary insights into the molecular basis of fatty acid and flavonoid biosynthesis in safflower.

Genome puzzle master (GPM): an integrated pipeline for building and editing pseudomolecules from fragmented sequences.

MOTIVATION: Next generation sequencing technologies have revolutionized our ability to rapidly and affordably generate vast quantities of sequence data. Once generated, raw sequences are assembled into contigs or scaffolds. However, these assemblies are mostly fragmented and inaccurate at the whole genome scale, largely due to the inability to integrate additional informative datasets (e.g. physical, optical and genetic maps). To address this problem, we developed a semi-automated software tool-Genome Puzzle Master (GPM)-that enables the integration of additional genomic signposts to edit and build 'new-gen-assemblies' that result in high-quality 'annotation-ready' pseudomolecules. RESULTS: With GPM, loaded datasets can be connected to each other via their logical relationships which accomplishes tasks to 'group,' 'merge,' 'order and orient' sequences in a draft assembly. Manual editing can also be performed with a user-friendly graphical interface. Final pseudomolecules reflect a user's total data package and are available for long-term project management. GPM is a web-based pipeline and an important part of a Laboratory Information Management System (LIMS) which can be easily deployed on local servers for any genome research laboratory. AVAILABILITY AND IMPLEMENTATION: The GPM (with LIMS) package is available at https://github.com/Jianwei-Zhang/LIMS CONTACTS: jzhang@mail.hzau.edu.cn or rwing@mail.arizona.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome.

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.

BAC-end sequences analysis provides first insights into coffee (Coffea canephora P.) genome composition and evolution.

Coffee is one of the world's most important agricultural commodities. Coffee belongs to the Rubiaceae family in the euasterid I clade of dicotyledonous plants, to which the Solanaceae family also belongs. Two bacterial artificial chromosome (BAC) libraries of a homozygous doubled haploid plant of Coffea canephora were constructed using two enzymes, HindIII and BstYI. A total of 134,827 high quality BAC-end sequences (BESs) were generated from the 73,728 clones of the two libraries, and 131,412 BESs were conserved for further analysis after elimination of chloroplast and mitochondrial sequences. This corresponded to almost 13 % of the estimated size of the C. canephora genome. 6.7 % of BESs contained simple sequence repeats, the most abundant (47.8 %) being mononucleotide motifs. These sequences allow the development of numerous useful marker sites. Potential transposable elements (TEs) represented 11.9 % of the full length BESs. A difference was observed between the BstYI and HindIII libraries (14.9 vs. 8.8 %). Analysis of BESs against known coding sequences of TEs indicated that 11.9 % of the genome corresponded to known repeat sequences, like for other flowering plants. The number of genes in the coffee genome was estimated at 41,973 which is probably overestimated. Comparative genome mapping revealed that microsynteny was higher between coffee and grapevine than between coffee and tomato or Arabidopsis. BESs constitute valuable resources for the first genome wide survey of coffee and provide new insights into the composition and evolution of the coffee genome.

Human gut microbiota in obesity and after gastric bypass.

Recent evidence suggests that the microbial community in the human intestine may play an important role in the pathogenesis of obesity. We examined 184,094 sequences of microbial 16S rRNA genes from PCR amplicons by using the 454 pyrosequencing technology to compare the microbial community structures of 9 individuals, 3 in each of the categories of normal weight, morbidly obese, and post-gastric-bypass surgery. Phylogenetic analysis demonstrated that although the Bacteria in the human intestinal community were highly diverse, they fell mainly into 6 bacterial divisions that had distinct differences in the 3 study groups. Specifically, Firmicutes were dominant in normal-weight and obese individuals but significantly decreased in post-gastric-bypass individuals, who had a proportional increase of Gammaproteobacteria. Numbers of the H(2)-producing Prevotellaceae were highly enriched in the obese individuals. Unlike the highly diverse Bacteria, the Archaea comprised mainly members of the order Methanobacteriales, which are H(2)-oxidizing methanogens. Using real-time PCR, we detected significantly higher numbers of H(2)-utilizing methanogenic Archaea in obese individuals than in normal-weight or post-gastric-bypass individuals. The coexistence of H(2)-producing bacteria with relatively high numbers of H(2)-utilizing methanogenic Archaea in the gastrointestinal tract of obese individuals leads to the hypothesis that interspecies H(2) transfer between bacterial and archaeal species is an important mechanism for increasing energy uptake by the human large intestine in obese persons. The large bacterial population shift seen in the post-gastric-bypass individuals may reflect the double impact of the gut alteration caused by the surgical procedure and the consequent changes in food ingestion and digestion.

Sequencing, mapping, and analysis of 27,455 maize full-length cDNAs.

Full-length cDNA (FLcDNA) sequencing establishes the precise primary structure of individual gene transcripts. From two libraries representing 27 B73 tissues and abiotic stress treatments, 27,455 high-quality FLcDNAs were sequenced. The average transcript length was 1.44 kb including 218 bases and 321 bases of 5' and 3' UTR, respectively, with 8.6% of the FLcDNAs encoding predicted proteins of fewer than 100 amino acids. Approximately 94% of the FLcDNAs were stringently mapped to the maize genome. Although nearly two-thirds of this genome is composed of transposable elements (TEs), only 5.6% of the FLcDNAs contained TE sequences in coding or UTR regions. Approximately 7.2% of the FLcDNAs are putative transcription factors, suggesting that rare transcripts are well-enriched in our FLcDNA set. Protein similarity searching identified 1,737 maize transcripts not present in rice, sorghum, Arabidopsis, or poplar annotated genes. A strict FLcDNA assembly generated 24,467 non-redundant sequences, of which 88% have non-maize protein matches. The FLcDNAs were also assembled with 41,759 FLcDNAs in GenBank from other projects, where semi-strict parameters were used to identify 13,368 potentially unique non-redundant sequences from this project. The libraries, ESTs, and FLcDNA sequences produced from this project are publicly available. The annotated EST and FLcDNA assemblies are available through the maize FLcDNA web resource (www.maizecdna.org).

Genome assembly of the JD17 soybean provides a new reference genome for comparative genomics.

Cultivated soybean (Glycine max) is an important source for protein and oil. Many elite cultivars with different traits have been developed for different conditions. Each soybean strain has its own genetic diversity, and the availability of more high-quality soybean genomes can enhance comparative genomic analysis for identifying genetic underpinnings for its unique traits. In this study, we constructed a high-quality de novo assembly of an elite soybean cultivar Jidou 17 (JD17) with chromosome contiguity and high accuracy. We annotated 52,840 gene models and reconstructed 74,054 high-quality full-length transcripts. We performed a genome-wide comparative analysis based on the reference genome of JD17 with 3 published soybeans (WM82, ZH13, and W05), which identified 5 large inversions and 2 large translocations specific to JD17, 20,984-46,912 presence-absence variations spanning 13.1-46.9 Mb in size. A total of 1,695,741-3,664,629 SNPs and 446,689-800,489 Indels were identified and annotated between JD17 and them. Symbiotic nitrogen fixation genes were identified and the effects from these variants were further evaluated. It was found that the coding sequences of 9 nitrogen fixation-related genes were greatly affected. The high-quality genome assembly of JD17 can serve as a valuable reference for soybean functional genomics research.

Rice structural variation: a comparative analysis of structural variation between rice and three of its closest relatives in the genus Oryza.

Rapid progress in comparative genomics among the grasses has revealed similar gene content and order despite exceptional differences in chromosome size and number. Large- and small-scale genomic variations are of particular interest, especially among cultivated and wild species, as they encode rapidly evolving features that may be important in adaptation to particular environments. We present a genome-wide study of intermediate-sized structural variation (SV) among rice (Oryza sativa) and three of its closest relatives in the genus Oryza (Oryza nivara, Oryza rufipogon and Oryza glaberrima). We computationally identified regional expansions, contractions and inversions in the Oryza species genomes relative to O. sativa by combining data from paired-end clone alignments to the O. sativa reference genome and physical maps. A subset of the computational predictions was validated using a new approach for BAC size determination. The result was a confirmed catalog of 674 expansions (25-38 Mb) and 611 (4-19 Mb) contractions, and 140 putative inversions (14-19 Mb) between the three Oryza species and O. sativa. In the expanded regions unique to O. sativa we found enrichment in transposable elements (TEs): long terminal repeats (LTRs) were randomly located across the chromosomes, and their insertion times corresponded to the date of the A genome radiation. Also, rice-expanded regions contained an over-representation of single-copy genes related to defense factors in the environment. This catalog of confirmed SV in reference to O. sativa provides an entry point for future research in genome evolution, speciation, domestication and novel gene discovery.

Construction, characterization, and preliminary BAC-end sequence analysis of a bacterial artificial chromosome library of the tea plant (Camellia sinensis).

We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%-87.24%), followed by DNA retrotransposons (11.16%-11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers.

Improved chromosome-level genome assembly and annotation of the seagrass, Zostera marina (eelgrass).

Background: Seagrasses (Alismatales) are the only fully marine angiosperms.  Zostera marina (eelgrass) plays a crucial role in the functioning of coastal marine ecosystems and global carbon sequestration. It is the most widely studied seagrass and has become a marine model system for exploring adaptation under rapid climate change. The original draft genome (v.1.0) of the seagrass  Z. marina (L.) was based on a combination of Illumina mate-pair libraries and fosmid-ends. A total of 25.55 Gb of Illumina and 0.14 Gb of Sanger sequence was obtained representing 47.7× genomic coverage. The assembly resulted in ~2000 unordered scaffolds (L50 of 486 Kb), a final genome assembly size of 203MB, 20,450 protein coding genes and 63% TE content. Here, we present an upgraded chromosome-scale genome assembly and compare v.1.0 and the new v.3.1, reconfirming previous results from Olsen et al. (2016), as well as pointing out new findings.   Methods: The same high molecular weight DNA used in the original sequencing of the Finnish clone was used. A high-quality reference genome was assembled with the MECAT assembly pipeline combining PacBio long-read sequencing and Hi-C scaffolding.  Results: In total, 75.97 Gb PacBio data was produced. The final assembly comprises six pseudo-chromosomes and 304 unanchored scaffolds with a total length of 260.5Mb and an N50 of 34.6 MB, showing high contiguity and few gaps (~0.5%). 21,483 protein-encoding genes are annotated in this assembly, of which 20,665 (96.2%) obtained at least one functional assignment based on similarity to known proteins.  Conclusions: As an important marine angiosperm, the improved  Z. marina genome assembly will further assist evolutionary, ecological, and comparative genomics at the chromosome level. The new genome assembly will further our understanding into the structural and physiological adaptations from land to marine life.

Two gap-free reference genomes and a global view of the centromere architecture in rice.

Rice (Oryza sativa), a major staple throughout the world and a model system for plant genomics and breeding, was the first crop genome sequenced almost two decades ago. However, reference genomes for all higher organisms to date contain gaps and missing sequences. Here, we report the assembly and analysis of gap-free reference genome sequences for two elite O. sativa xian/indica rice varieties, Zhenshan 97 and Minghui 63, which are being used as a model system for studying heterosis and yield. Gap-free reference genomes provide the opportunity for a global view of the structure and function of centromeres. We show that all rice centromeric regions share conserved centromere-specific satellite motifs with different copy numbers and structures. In addition, the similarity of CentO repeats in the same chromosome is higher than across chromosomes, supporting a model of local expansion and homogenization. Both genomes have over 395 non-TE genes located in centromere regions, of which ∼41% are actively transcribed. Two large structural variants at the end of chromosome 11 affect the copy number of resistance genes between the two genomes. The availability of the two gap-free genomes lays a solid foundation for further understanding genome structure and function in plants and breeding climate-resilient varieties.

A draft physical map of a D-genome cotton species (Gossypium raimondii).

BACKGROUND: Genetically anchored physical maps of large eukaryotic genomes have proven useful both for their intrinsic merit and as an adjunct to genome sequencing. Cultivated tetraploid cottons, Gossypium hirsutum and G. barbadense, share a common ancestor formed by a merger of the A and D genomes about 1-2 million years ago. Toward the long-term goal of characterizing the spectrum of diversity among cotton genomes, the worldwide cotton community has prioritized the D genome progenitor Gossypium raimondii for complete sequencing. RESULTS: A whole genome physical map of G. raimondii, the putative D genome ancestral species of tetraploid cottons was assembled, integrating genetically-anchored overgo hybridization probes, agarose based fingerprints and 'high information content fingerprinting' (HICF). A total of 13,662 BAC-end sequences and 2,828 DNA probes were used in genetically anchoring 1585 contigs to a cotton consensus genetic map, and 370 and 438 contigs, respectively to Arabidopsis thaliana (AT) and Vitis vinifera (VV) whole genome sequences. CONCLUSION: Several lines of evidence suggest that the G. raimondii genome is comprised of two qualitatively different components. Much of the gene rich component is aligned to the Arabidopsis and Vitis vinifera genomes and shows promise for utilizing translational genomic approaches in understanding this important genome and its resident genes. The integrated genetic-physical map is of value both in assembling and validating a planned reference sequence.

Genomic structure and evolution of the Pi2/9 locus in wild rice species.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a devastating disease of rice worldwide. Among the 85 mapped resistance (R) genes against blast, 13 have been cloned and characterized. However, how these genes originated and how they evolved in the Oryza genus remains unclear. We previously cloned the rice blast R-genes Pi2, Pi9, and Piz-t, and analyzed their genomic structure and evolution in cultivated rice. In this study, we determined the genomic sequences of the Pi2/9 locus in four wild Oryza species representing three genomes (AA, BB and CC). The number of Pi2/9 family members in the four wild species ranges from two copies to 12 copies. Although these genes are conserved in structure and categorized into the same subfamily, sequence duplications and subsequent inversions or uneven crossing overs were observed, suggesting that the locus in different wild species has undergone dynamic changes. Positive selection was found in the leucine-rich repeat region of most members, especially in the largest clade where Pi9 is included. We also provide evidence that the Pi9 gene is more related to its homologues in the recurrent line and other rice cultivars than to those in its alleged donor species O. minuta, indicating a possible origin of the Pi9 gene from O. sativa. Comparative sequence analysis between the four wild Oryza species and the previously established reference sequences in cultivated rice species at the Pi2/9 locus has provided extensive and unique information on the genomic structure and evolution of a complex R-gene cluster in the Oryza genus.

A genome assembly and the somatic genetic and epigenetic mutation rate in a wild long-lived perennial Populus trichocarpa.

BACKGROUND: Plants can transmit somatic mutations and epimutations to offspring, which in turn can affect fitness. Knowledge of the rate at which these variations arise is necessary to understand how plant development contributes to local adaption in an ecoevolutionary context, particularly in long-lived perennials. RESULTS: Here, we generate a new high-quality reference genome from the oldest branch of a wild Populus trichocarpa tree with two dominant stems which have been evolving independently for 330 years. By sampling multiple, age-estimated branches of this tree, we use a multi-omics approach to quantify age-related somatic changes at the genetic, epigenetic, and transcriptional level. We show that the per-year somatic mutation and epimutation rates are lower than in annuals and that transcriptional variation is mainly independent of age divergence and cytosine methylation. Furthermore, a detailed analysis of the somatic epimutation spectrum indicates that transgenerationally heritable epimutations originate mainly from DNA methylation maintenance errors during mitotic rather than during meiotic cell divisions. CONCLUSION: Taken together, our study provides unprecedented insights into the origin of nucleotide and functional variation in a long-lived perennial plant.

A platinum standard pan-genome resource that represents the population structure of Asian rice.

As the human population grows from 7.8 billion to 10 billion over the next 30 years, breeders must do everything possible to create crops that are highly productive and nutritious, while simultaneously having less of an environmental footprint. Rice will play a critical role in meeting this demand and thus, knowledge of the full repertoire of genetic diversity that exists in germplasm banks across the globe is required. To meet this demand, we describe the generation, validation and preliminary analyses of transposable element and long-range structural variation content of 12 near-gap-free reference genome sequences (RefSeqs) from representatives of 12 of 15 subpopulations of cultivated Asian rice. When combined with 4 existing RefSeqs, that represent the 3 remaining rice subpopulations and the largest admixed population, this collection of 16 Platinum Standard RefSeqs (PSRefSeq) can be used as a template to map resequencing data to detect virtually all standing natural variation that exists in the pan-genome of cultivated Asian rice.

The genomic landscape of molecular responses to natural drought stress in Panicum hallii.

Environmental stress is a major driver of ecological community dynamics and agricultural productivity. This is especially true for soil water availability, because drought is the greatest abiotic inhibitor of worldwide crop yields. Here, we test the genetic basis of drought responses in the genetic model for C4 perennial grasses, Panicum hallii, through population genomics, field-scale gene-expression (eQTL) analysis, and comparison of two complete genomes. While gene expression networks are dominated by local cis-regulatory elements, we observe three genomic hotspots of unlinked trans-regulatory loci. These regulatory hubs are four times more drought responsive than the genome-wide average. Additionally, cis- and trans-regulatory networks are more likely to have opposing effects than expected under neutral evolution, supporting a strong influence of compensatory evolution and stabilizing selection. These results implicate trans-regulatory evolution as a driver of drought responses and demonstrate the potential for crop improvement in drought-prone regions through modification of gene regulatory networks.

Publisher Correction: Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

This article was not made open access when initially published online, which was corrected before print publication. In addition, ORCID links were missing for 12 authors and have been added to the HTML and PDF versions of the article.