Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations.

Structural variations (SVs) and gene copy number variations (gCNVs) have contributed to crop evolution, domestication, and improvement. Here, we assembled 31 high-quality genomes of genetically diverse rice accessions. Coupling with two existing assemblies, we developed pan-genome-scale genomic resources including a graph-based genome, providing access to rice genomic variations. Specifically, we discovered 171,072 SVs and 25,549 gCNVs and used an Oryza glaberrima assembly to infer the derived states of SVs in the Oryza sativa population. Our analyses of SV formation mechanisms, impacts on gene expression, and distributions among subpopulations illustrate the utility of these resources for understanding how SVs and gCNVs shaped rice environmental adaptation and domestication. Our graph-based genome enabled genome-wide association study (GWAS)-based identification of phenotype-associated genetic variations undetectable when using only SNPs and a single reference assembly. Our work provides rich population-scale resources paired with easy-to-access tools to facilitate rice breeding as well as plant functional genomics and evolutionary biology research.

Genomic basis of geographical adaptation to soil nitrogen in rice.

The intensive application of inorganic nitrogen underlies marked increases in crop production, but imposes detrimental effects on ecosystems1,2: it is therefore crucial for future sustainable agriculture to improve the nitrogen-use efficiency of crop plants. Here we report the genetic basis of nitrogen-use efficiency associated with adaptation to local soils in rice (Oryza sativa L.). Using a panel of diverse rice germplasm collected from different ecogeographical regions, we performed a genome-wide association study on the tillering response to nitrogen-the trait that is most closely correlated with nitrogen-use efficiency in rice-and identified OsTCP19 as a modulator of this tillering response through its transcriptional response to nitrogen and its targeting to the tiller-promoting gene DWARF AND LOW-TILLERING (DLT)3,4. A 29-bp insertion and/or deletion in the OsTCP19 promoter confers a differential transcriptional response and variation in the tillering response to nitrogen among rice varieties. The allele of OsTCP19 associated with a high tillering response to nitrogen is prevalent in wild rice populations, but has largely been lost in modern cultivars: this loss correlates with increased local soil nitrogen content, which suggests that it might have contributed to geographical adaptation in rice. Introgression of the allele associated with a high tillering response into modern rice cultivars boosts grain yield and nitrogen-use efficiency under low or moderate levels of nitrogen, which demonstrates substantial potential for rice breeding and the amelioration of negative environment effects by reducing the application of nitrogen to crops.

Evolutionary Dynamics of Chromatin Structure and Duplicate Gene Expression in Diploid and Allopolyploid Cotton.

Polyploidy is a prominent mechanism of plant speciation and adaptation, yet the mechanistic understandings of duplicated gene regulation remain elusive. Chromatin structure dynamics are suggested to govern gene regulatory control. Here, we characterized genome-wide nucleosome organization and chromatin accessibility in allotetraploid cotton, Gossypium hirsutum (AADD, 2n = 4X = 52), relative to its two diploid parents (AA or DD genome) and their synthetic diploid hybrid (AD), using DNS-seq. The larger A-genome exhibited wider average nucleosome spacing in diploids, and this intergenomic difference diminished in the allopolyploid but not hybrid. Allopolyploidization also exhibited increased accessibility at promoters genome-wide and synchronized cis-regulatory motifs between subgenomes. A prominent cis-acting control was inferred for chromatin dynamics and demonstrated by transposable element removal from promoters. Linking accessibility to gene expression patterns, we found distinct regulatory effects for hybridization and later allopolyploid stages, including nuanced establishment of homoeolog expression bias and expression level dominance. Histone gene expression and nucleosome organization are coordinated through chromatin accessibility. Our study demonstrates the capability to track high-resolution chromatin structure dynamics and reveals their role in the evolution of cis-regulatory landscapes and duplicate gene expression in polyploids, illuminating regulatory ties to subgenomic asymmetry and dominance.

Distinct composition and amplification dynamics of transposable elements in sacred lotus (Nelumbo nucifera Gaertn.).

Sacred lotus (Nelumbo nucifera Gaertn.) is a basal eudicot plant with a unique lifestyle, physiological features, and evolutionary characteristics. Here we report the unique profile of transposable elements (TEs) in the genome, using a manually curated repeat library. TEs account for 59% of the genome, and hAT (Ac/Ds) elements alone represent 8%, more than in any other known plant genome. About 18% of the lotus genome is comprised of Copia LTR retrotransposons, and over 25% of them are associated with non-canonical termini (non-TGCA). Such high abundance of non-canonical LTR retrotransposons has not been reported for any other organism. TEs are very abundant in genic regions, with retrotransposons enriched in introns and DNA transposons primarily in flanking regions of genes. The recent insertion of TEs in introns has led to significant intron size expansion, with a total of 200 Mb in the 28 455 genes. This is accompanied by declining TE activity in intergenic regions, suggesting distinct control efficacy of TE amplification in different genomic compartments. Despite the prevalence of TEs in genic regions, some genes are associated with fewer TEs, such as those involved in fruit ripening and stress responses. Other genes are enriched with TEs, and genes in epigenetic pathways are the most associated with TEs in introns, indicating a dynamic interaction between TEs and the host surveillance machinery. The dramatic differential abundance of TEs with genes involved in different biological processes as well as the variation of target preference of different TEs suggests the composition and activity of TEs influence the path of evolution.

Large structural variations in the haplotype-resolved African cassava genome.

Cassava (Manihot esculenta Crantz, 2n = 36) is a global food security crop. It has a highly heterozygous genome, high genetic load, and genotype-dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi-C to resolve each haplotype of the genome of a farmer-preferred cassava line, TME7 (Oko-iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON-Phase and Hi-C read data. The ultralong-range information from Hi-C sequencing was also used for scaffolding. Comparison of the two phases revealed >5000 large haplotype-specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele-specific expression was further explored. RNA-sequencing data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy-to-use web-based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype-specific structural variation, gene hemizygosity, and allele-specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.

Replaying the evolutionary tape to investigate subgenome dominance in allopolyploid Brassica napus.

Allopolyploidisation merges evolutionarily distinct parental genomes (subgenomes) into a single nucleus. A frequent observation is that one subgenome is 'dominant' over the other subgenome, often being more highly expressed. Here, we 'replayed the evolutionary tape' with six isogenic resynthesised Brassica napus allopolyploid lines and investigated subgenome dominance patterns over the first 10 generations postpolyploidisation. We found that the same subgenome was consistently more dominantly expressed in all lines and generations and that >70% of biased gene pairs showed the same dominance patterns across all lines and an in silico hybrid of the parents. Gene network analyses indicated an enrichment for network interactions and several biological functions for the Brassica oleracea subgenome biased pairs, but no enrichment was identified for Brassica rapa subgenome biased pairs. Furthermore, DNA methylation differences between subgenomes mirrored the observed gene expression bias towards the dominant subgenome in all lines and generations. Many of these differences in gene expression and methylation were also found when comparing the progenitor genomes, suggesting that subgenome dominance is partly related to parental genome differences rather than just a byproduct of allopolyploidisation. These findings demonstrate that 'replaying the evolutionary tape' in an allopolyploid results in largely repeatable and predictable subgenome expression dominance patterns.

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice.

Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security.

Assessing genome assembly quality using the LTR Assembly Index (LAI).

Assembling a plant genome is challenging due to the abundance of repetitive sequences, yet no standard is available to evaluate the assembly of repeat space. LTR retrotransposons (LTR-RTs) are the predominant interspersed repeat that is poorly assembled in draft genomes. Here, we propose a reference-free genome metric called LTR Assembly Index (LAI) that evaluates assembly continuity using LTR-RTs. After correcting for LTR-RT amplification dynamics, we show that LAI is independent of genome size, genomic LTR-RT content, and gene space evaluation metrics (i.e., BUSCO and CEGMA). By comparing genomic sequences produced by various sequencing techniques, we reveal the significant gain of assembly continuity by using long-read-based techniques over short-read-based methods. Moreover, LAI can facilitate iterative assembly improvement with assembler selection and identify low-quality genomic regions. To apply LAI, intact LTR-RTs and total LTR-RTs should contribute at least 0.1% and 5% to the genome size, respectively. The LAI program is freely available on GitHub: https://github.com/oushujun/LTR_retriever.

LTR_retriever: A Highly Accurate and Sensitive Program for Identification of Long Terminal Repeat Retrotransposons.

Long terminal repeat retrotransposons (LTR-RTs) are prevalent in plant genomes. The identification of LTR-RTs is critical for achieving high-quality gene annotation. Based on the well-conserved structure, multiple programs were developed for the de novo identification of LTR-RTs; however, these programs are associated with low specificity and high false discovery rates. Here, we report LTR_retriever, a multithreading-empowered Perl program that identifies LTR-RTs and generates high-quality LTR libraries from genomic sequences. LTR_retriever demonstrated significant improvements by achieving high levels of sensitivity (91%), specificity (97%), accuracy (96%), and precision (90%) in rice (Oryza sativa). LTR_retriever is also compatible with long sequencing reads. With 40k self-corrected PacBio reads equivalent to 4.5× genome coverage in Arabidopsis (Arabidopsis thaliana), the constructed LTR library showed excellent sensitivity and specificity. In addition to canonical LTR-RTs with 5'-TG…CA-3' termini, LTR_retriever also identifies noncanonical LTR-RTs (non-TGCA), which have been largely ignored in genome-wide studies. We identified seven types of noncanonical LTRs from 42 out of 50 plant genomes. The majority of noncanonical LTRs are Copia elements, with which the LTR is four times shorter than that of other Copia elements, which may be a result of their target specificity. Strikingly, non-TGCA Copia elements are often located in genic regions and preferentially insert nearby or within genes, indicating their impact on the evolution of genes and their potential as mutagenesis tools.

Correction to: OsbZIP71, a bZIP transcription factor, confers salinity and drought tolerance in rice.

Due to an error in combining the figure, an incorrect version of Fig. 9e was presented in the original publication.

Integrating GWAS and gene expression data for functional characterization of resistance to white mould in soya bean.

White mould of soya bean, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is a necrotrophic fungus capable of infecting a wide range of plants. To dissect the genetic architecture of resistance to white mould, a high-density customized single nucleotide polymorphism (SNP) array (52 041 SNPs) was used to genotype two soya bean diversity panels. Combined with resistance variation data observed in the field and greenhouse environments, genome-wide association studies (GWASs) were conducted to identify quantitative trait loci (QTL) controlling resistance against white mould. Results showed that 16 and 11 loci were found significantly associated with resistance in field and greenhouse, respectively. Of these, eight loci localized to previously mapped QTL intervals and one locus had significant associations with resistance across both environments. The expression level changes in genes located in GWAS-identified loci were assessed between partially resistant and susceptible genotypes through a RNA-seq analysis of the stem tissue collected at various time points after inoculation. A set of genes with diverse biological functionalities were identified as strong candidates underlying white mould resistance. Moreover, we found that genomic prediction models outperformed predictions based on significant SNPs. Prediction accuracies ranged from 0.48 to 0.64 for disease index measured in field experiments. The integrative methods, including GWAS, RNA-seq and genomic selection (GS), applied in this study facilitated the identification of causal variants, enhanced our understanding of mechanisms of white mould resistance and provided valuable information regarding breeding for disease resistance through genomic selection in soya bean.

OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice.

It has long been established that premature leaf senescence negatively impacts the yield stability of rice, but the underlying molecular mechanism driving this relationship remains largely unknown. Here, we identified a dominant premature leaf senescence mutant, prematurely senile 1 (ps1-D). PS1 encodes a plant-specific NAC (no apical meristem, Arabidopsis ATAF1/2, and cup-shaped cotyledon2) transcriptional activator, Oryza sativa NAC-like, activated by apetala3/pistillata (OsNAP). Overexpression of OsNAP significantly promoted senescence, whereas knockdown of OsNAP produced a marked delay of senescence, confirming the role of this gene in the development of rice senescence. OsNAP expression was tightly linked with the onset of leaf senescence in an age-dependent manner. Similarly, ChIP-PCR and yeast one-hybrid assays demonstrated that OsNAP positively regulates leaf senescence by directly targeting genes related to chlorophyll degradation and nutrient transport and other genes associated with senescence, suggesting that OsNAP is an ideal marker of senescence onset in rice. Further analysis determined that OsNAP is induced specifically by abscisic acid (ABA), whereas its expression is repressed in both aba1 and aba2, two ABA biosynthetic mutants. Moreover, ABA content is reduced significantly in ps1-D mutants, indicating a feedback repression of OsNAP on ABA biosynthesis. Our data suggest that OsNAP serves as an important link between ABA and leaf senescence. Additionally, reduced OsNAP expression leads to delayed leaf senescence and an extended grain-filling period, resulting in a 6.3% and 10.3% increase in the grain yield of two independent representative RNAi lines, respectively. Thus, fine-tuning OsNAP expression should be a useful strategy for improving rice yield in the future.

Variations in CYP78A13 coding region influence grain size and yield in rice.

Grain size is one of the most important determinants of crop yield in cereals. Here, we identified a dominant mutant, big grain2 (bg2-D) from our enhancer-trapping population. Genetic analysis and SiteFinding PCR (polymerase chain reaction) revealed that BG2 encodes a cytochrome P450, OsCYP78A13. Sequence search revealed that CYP78A13 has a paralogue Grain Length 3.2 (GL3.2, LOC_Os03g30420) in rice with distinct expression patterns, analysis of transgenic plants harbouring either CYP78A13 or GL3.2 showed that both can promote grain growth. Sequence polymorphism analysis with 1529 rice varieties showed that the nucleotide diversity at CYP78A13 gene body and the 20 kb flanking region in the indica varieties were markedly higher than those in japonica varieties. Further, comparison of the genomic sequence of CYP78A13 in the japonica cultivar Nipponbare and the indica cultivar 9311 showed that there were three InDels in the promoter region and eight SNPs (single nucleotide polymorphism) in its coding sequence. Detailed examination of the transgenic plants with chimaeric constructs suggested that variation in CYP78A13 coding region is responsible for the variation of grain yield. Taken together, our results suggest that the variations in CYP78A13 in the indica varieties hold potential in rice breeding for application of grain yield improvement.

OsbZIP71, a bZIP transcription factor, confers salinity and drought tolerance in rice.

The bZIP transcription factor (TF) family plays an important role in the abscisic acid (ABA) signaling pathway of abiotic stress in plants. We here report the cloning and characterization of OsbZIP71, which encodes a rice bZIP TF. Functional analysis showed that OsbZIP71 is a nuclear-localized protein that specifically binds to the G-box motif, but has no transcriptional activity both in yeast and rice protoplasts. In yeast two-hybrid assays, OsbZIP71 can form both homodimers and heterodimers with Group C members of the bZIP gene family. Expression of OsbZIP71 was strongly induced by drought, polyethylene glycol (PEG), and ABA treatments, but repressed by salt treatment. OsbZIP71 overexpressing (p35S::OsbZIP71) rice significantly improved tolerance to drought, salt and PEG osmotic stresses. In contrast, RNAi knockdown transgenic lines were much more sensitive to salt, PEG osmotic stresses, and also ABA treatment. Inducible expression (RD29A::OsbZIP71) lines were significantly improved their tolerance to PEG osmotic stresses, but hypersensitivity to salt, and insensitivity to ABA. Real-time PCR analysis revealed that the abiotic stress-related genes, OsVHA-B, OsNHX1, COR413-TM1, and OsMyb4, were up-regulated in overexpressing lines, while these same genes were down-regulated in RNAi lines. Chromatin immunoprecipitation analysis confirmed that OsbZIP71 directly binds the promoters of OsNHX1 and COR413-TM1 in vivo. These results suggest that OsbZIP71 may play an important role in ABA-mediated drought and salt tolerance in rice.

Gapless assembly of complete human and plant chromosomes using only nanopore sequencing.

The combination of ultra-long Oxford Nanopore (ONT) sequencing reads with long, accurate PacBio HiFi reads has enabled the completion of a human genome and spurred similar efforts to complete the genomes of many other species. However, this approach for complete, "telomere-to-telomere" genome assembly relies on multiple sequencing platforms, limiting its accessibility. ONT "Duplex" sequencing reads, where both strands of the DNA are read to improve quality, promise high per-base accuracy. To evaluate this new data type, we generated ONT Duplex data for three widely-studied genomes: human HG002, Solanum lycopersicum Heinz 1706 (tomato), and Zea mays B73 (maize). For the diploid, heterozygous HG002 genome, we also used "Pore-C" chromatin contact mapping to completely phase the haplotypes. We found the accuracy of Duplex data to be similar to HiFi sequencing, but with read lengths tens of kilobases longer, and the Pore-C data to be compatible with existing diploid assembly algorithms. This combination of read length and accuracy enables the construction of a high-quality initial assembly, which can then be further resolved using the ultra-long reads, and finally phased into chromosome-scale haplotypes with Pore-C. The resulting assemblies have a base accuracy exceeding 99.999% (Q50) and near-perfect continuity, with most chromosomes assembled as single contigs. We conclude that ONT sequencing is a viable alternative to HiFi sequencing for de novo genome assembly, and has the potential to provide a single-instrument solution for the reconstruction of complete genomes.

A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.).

A contiguous assembly of the inbred 'EL10' sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long-read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short-read error correction. The EL10.1 assembly was 540 Mb, of which 96.2% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNA-seq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals producing estimates from 633 to 875 Mb/1C. Read-depth mapping with short-read whole-genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

The genome of the Wollemi pine, a critically endangered "living fossil" unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.