Reference Genome for the Highly Transformable Setaria viridis ME034V

Setaria viridis (green foxtail) is an important model system for improving cereal crops due to its diploid genome, ease of cultivation, and use of C4 photosynthesis. The S. viridis accession ME034V is exceptionally transformable, but the lack of a sequenced genome for this accession has limited its utility. We present a 397 Mb highly contiguous de novo assembly of ME034V using ultra-long nanopore sequencing technology (read N50 = 41kb). We estimate that this genome is largely complete based on our updated k-mer based genome size estimate of 401 Mb for S. viridis. Genome annotation identified 37,908 protein-coding genes and >300k repetitive elements comprising 46% of the genome. We compared the ME034V assembly with two other previously sequenced Setaria genomes as well as to a diversity panel of 235 S. viridis accessions. We found the genome assemblies to be largely syntenic, but numerous unique polymorphic structural variants were discovered. Several ME034V deletions may be associated with recent retrotransposition of copia and gypsy LTR repeat families, as evidenced by their low genotype frequencies in the sampled population. Lastly, we performed a phylogenomic analysis to identify gene families that have expanded in Setaria, including those involved in specialized metabolism and plant defense response. The high continuity of the ME034V genome assembly validates the utility of ultra-long DNA sequencing to improve genetic resources for emerging model organisms. Structural variation present in Setaria illustrates the importance of obtaining the proper genome reference for genetic experiments. Thus, we anticipate that the ME034V genome will be of significant utility for the Setaria research community.

However, transformation efficiency for A10 is low, demonstrated at 6.3% infected calli giving rise to at least one independent transgenic line (Nguyen et al. 2020). Transformation efficiency for S. italica appears similarly low, ranging from 5.5-19.2% depending on the accession and protocol (Santos et al. 2020). In contrast, transformation efficiency of S. viridis ME034V (hereafter referred to as ME034V) has been informally described as greater than 80% (Acharya et al. 2017;Zhu et al. 2017), with a recent demonstration of 89-98% efficiency (Weiss et al. 2020). Given its high transformability and phenotypic similarity to A10, transitioning to ME034V as the accession of choice for research involving genetic modification offers significant advantages by reducing the resources required for time consuming and highly technical transformation protocols.
In this study, we report a new de novo genome assembly for ME034V. This genome was generated using a multi-step assembly approach, with overlap and layout performed using ultra-long Oxford Nanopore Technologies (ONT) reads (read N50 = 41 kb) and consensus polishing with Illumina sequencing. This assembly captures 397.03 Mb of total sequence, represented in just 44 contigs (contig N50 = 19.5 Mb). Additionally, we provide an updated average S. viridis genome size estimate of 401 Mb based on k-mer representation of multiple S. viridis accessions. If accurate, this estimate suggests that our new assembly, as well as previous genome assemblies, capture approximately 99% of the total genetic content of green foxtail. Included with this genome release is the de novo annotation of 37,908 genes, of which greater than 96% were assigned to orthologous gene families with other grasses and 60% were assigned a functional annotation. The ME034V genome assembly is highly syntenic with the two other Setaria genomes that are publicly available (Zhang et al. 2012;Mamidi et al. 2020). Our long-read sequencing provides increased resolution in regions of high repeat content, allowing for the discovery of novel insertions and other structural variants. We extended this analysis to short read alignments from over 200 additional Setaria accessions to identify a dataset of 421 polymorphic structural variants, many of which contained transposable elements (TEs). Our analysis indicates that particular repeat families (e.g., copia, gypsy, and MULE families) show recent retrotransposition potential in Setaria. Taken together, the results of our study highlight genome variation between closely related accessions of the same species and will be a valuable genetic resource for the research community that takes advantage of the uniquely high transformation efficiency of ME034V.

MATERIALS AND METHODS
Genome size estimation Unprocessed Illumina sequencing data were acquired from the SRA for S. italica Zhang gu and 10 S. viridis accessions (Table S1). K-mer distributions were evaluated using Jellyfish v2.3.0 (Marçais and Kingsford 2011) using a k-mer size of 21, and the output was evaluated in GenomeScope v1.0.0 (Vurture et al. 2017) with no max k-mer coverage cutoff. Data were evaluated without processing (raw), after read quality trimming to maintain . Q20 and minimum read lengths of 35 bp with cutadapt (trimmed), and after removing any reads that align to S. viridis chloroplast (NC_028075.1) or barley mitochondrial (AP017301.1) sequences (trimmed and filtered).
Plant growth S. viridis ME034V-1 (ME034V) seed was provided by Dr. George Jander at the Boyce Thompson Institute, and grown at Johns Hopkins University Applied Physics Laboratory in growth chambers (400 PAR, 31°/22°day/night temperatures, and 12-hour light:dark cycles). Leaf tissue was harvested two weeks post-germination, following a 48-hour dark cycle to reduce starch content. Upon harvest, tissue was stored at -80°until DNA extraction.
DNA sequencing DNA isolation was performed with frozen leaf tissue that was disrupted using a liquid nitrogen cooled mortar and pestle, and incubated in CTAB buffer with 20ug/mL proteinase K at 55°for 30 min prior to purification with one round of chloroform and two subsequent rounds of phenol:chloroform:isoamyl alcohol.
ONT sequencing on the MinION platform utilized a ligationbased motor protein attachment approach (SQK-LSK108) and R9.4.1 flow cells (FLO-MIN106) using the protocol "1D gDNA long reads without BluePippin" (version GLRE_9052_v108_revD_19Dec2017). This protocol incorporates treatment of input DNA with NEBNext FFPE DNA Repair Mix (M6630) prior to end repair, A-tailing, and subsequent ligation of the Oxford Nanopore motor protein complex. Base calling was performed using Oxford Nanopore Guppy v2.3.5. Reads with quality scores less than 7 were discarded.
In parallel, samples were sequenced on the Illumina NextSeq platform using Nextera XT library preparation reagents and v2 2x150bp paired-end sequencing reagents. Both ONT and Illumina sequencing libraries were generated from the same source material, and reads are available for download at the NCBI Sequence Read Archive (NCBI: PRJNA560942). Additional Illumina sequencing data for ME034V was downloaded from the NCBI SRA (NCBI: SRR1587768).

Genome assembly
An overlapping read file from ONT data was generated using minimap2 v2.15-r911-dirty (Li 2016). These super-contiguous sequences and the original input read file were then assembled using miniasm v0.3-r179 (Li 2016). In order to find the best initial assembly for polishing and scaffolding, a range of miniasm parameter combinations were executed with each resulting assembly and evaluated for total contig count and length. Specifically, a four-feature parameter space for miniasm was explored, yielding 54 unique parameter set tests ( Figure 1). 1) Minimum match lengths [-m] 25, 50, and 100 were evaluated with 100 selected as the optimal parameter. 2) Minimum overlap identities [-i] 0.01, 0.03 and 0.05 were evaluated with 0.05 selected as the optimal parameter. 3) Minimum spans [-s] 100, 500, and 2000 were evaluated with 100 selected as the optimal parameter. 4) Maximum overhang lengths [-h] 1e4 and 1e5 were evaluated with 1e4 selected as the optimal parameter. The resulting miniasm assembly was error corrected via three rounds of polishing with ONT reads followed by 2 rounds of polishing with Illumina reads using Racon v1.4.3 (Vaser et al. 2017). Prior to contig polishing, the Illumina data were processed with Trimmomatic v0.35, which clipped adapters, removed bases with quality scores below Phred Q20, and removed reads less than 50 bp in length (Bolger et al. 2014). For SRAacquired data, a base call quality threshold of Phred Q20 and a minimum length of 50 bp were applied using cutadapt v2.5 (Martin 2011). To annotate the four chloroplast-derived contigs, the contigs were automatically annotated using Prokka v1.14.0 (Seemann 2014), and OGDRAW v1.3.1 (Greiner et al. 2019) was used to generate a physical map of each annotated contig.
To orient the contigs into chromosome-level scaffolds, the fast and accurate reference-guided scaffolding tool RaGOO v1.1 (Alonge et al. 2019) was used to scaffold the polished contigs onto the Joint Genome Institute's A10 genome assembly (NCBI: GCA_ 005286985.1; JGI Sviridis_500_v2.0) and the S. viridis chloroplast DNA sequence (NCBI: NC028075.1). Assembly statistics were calculated using QUAST-LG v5.0.2 (Gurevich et al. 2013). Lastly, assembly gaps in the ME034V genome were manually inserted as 100 Ns between contigs already anchored to the A10 assembly in a reference-guided manner.

RNA sequencing and processing
Total RNA was extracted from leaf, root, and sheath tissue from four ME034V plants using the Promega SV total RNA isolation kit. RNAseq libraries were constructed and sequenced at the Purdue Genomics Core Facility at Purdue University using Illumina's ribominus TruSeq Stranded Total RNA library prep kit. The libraries were sequenced on the Illumina NovaSeq S4 platform (2x150), resulting in approximately 700 Gb of raw read sequence. Initial quality filtration of FASTQ with Trimmomatic v0.36 (Bolger et al. 2014) removed reads less than 100 bp in length (MINLEN:100), Illumina TruSeq adapters (ILLUMINACLIP:TruSeq_PEall.fa:2:30:10), performed sliding window quality filtrations (SLIDINGWINDOW:4:20), and required average total read quality scores to be at least 20 (AVGQUAL:20). Reads were merged across all samples and error corrected using tadpole (BBTools v37.93) (Bushnell et al. 2017) using a k-mer size of 50. Error-corrected RNA-Seq reads were normalized using bbnorm (BBTools v37.93) (Bushnell et al. 2017) to a read-depth of 25 and a minimum k-mer depth of 10 using a unique k-mer size of 31. The normalized reads were aligned to the ME034V assembly with STAR v2.5.4b (Dobin et al. 2013) allowing alignments with an acceptable intron size range of 10-100,000 bp and a maximum multi-mapping allowance of 10. Quantification of expression was completed using Kallisto v0.45.0 (Bray et al. 2016). First, an index was built using the final GTF of the annotation gene set with k-mer size of 31. Raw RNA-Seq FASTQ files (non-normalized) for twelve ME034V and eleven A10 samples (Table S2) were used as inputs for expression quantification and quality assessment of the gene annotation set. Following calculations of expression with Kallisto, gene expression values were calculated as the sum of transcripts per million mapped (TPM) across isoforms for each of the final primary genes (N = 37,908 gene models).

Genome annotation
De novo annotation of repetitive elements was conducted with RepeatModeler v1.09 (http://www.repeatmasker.org), which also implements the de novo repeat finder RECON v1.08 (Bao and Eddy 2002). Nucleotide sequences of the resulting consensus repeat families were propagated using RepeatClassifier from the RepeatModeler package and used as the repeat library for RepeatMasker v4.0.7 annotation and soft masking (http://www.repeatmasker.org). This pipeline was implemented for the ME034V assembly as well as for the S. italica v2 (Bennetzen et al. 2012), A10 v2 (Mamidi et al. 2020), Sorghum bicolor (Ensembl 45 release), and Zea mays B73 (Ensembl 45 release) genome assemblies to facilitate comparisons.
Utilizing the RNA-Seq alignment file, a genome-guided de novo transcriptome assembly was generated by Trinity v2.5.1 (Haas et al. 2013) using the following parameters: no_normalize_ reads, genome_guided_max_intron 100000, SS_lib_type RF. Gene model and protein prediction was conducted with MAKER v2.31.10 Figure 1 Multistage assembly pipeline. ONT long-reads were assembled de novo to generate assembly v0.1. The resulting assembly was first polished with ONT reads and then with Illumina short-reads to create assemblies v0.2 and v0.3, respectively. The assembly contigs were scaffolded to chromosome-level pseudo chromosomes to generate the final assembly v1.0. (Holt and Yandell 2011). Evidence for the first round of MAKER included the de novo transcript FASTA sequences from Trinity as direct EST support, the de novo consensus repeat calls from RepeatModeler, the FASTA sequences of the primary transcripts of A10 v2.1 (Mamidi et al. 2020) as alternate EST support, and alternate protein support came from over 400,000 protein sequences from closely related grass species from the Ensembl release 43 (Kersey et al. 2018) of S. italica (v2), Panicum hallii Fil (v3.1), P. hallii HAL (v2.1), Oryza sativa (IRGSP v1.0), Z. mays (B73 v4.0) and S. bicolor (NCBI v3.0) and the JGI annotation of A10 v2.1 (Mamidi et al. 2020). Following the first MAKER round, Hidden Markov Model (hmm) files were generated by SNAP v2013-02-16 (Korf 2004) to inform subsequent MAKER annotations as well as training by BRAKER2 (Stanke et al. 2008;Hoff et al. 2019). The final MAKER iteration utilized SNAP, BRAKER2, all previously mentioned resources, as well as the GFF from the first MAKER round as evidence. We removed all final MAKER predicted gene models with Annotation Edit Distance (AED) scores greater than 0.5. A primary gene set was determined to represent the best gene model per locus, therefore the model with the lowest AED score (best supported model per Maker) was selected. The recovery of conserved protein-coding genes was assessed using BUSCO v3 (Waterhouse et al. 2018) with the Eukaryota_odb9 dataset. Functional annotations were performed on the gene models using Interproscan v5.29-68.0 (Jones et al. 2014). Annotations of plant secondary metabolite genes was performed by hmmsearch (e-value , 1e-10; HMMER v3.1) (Eddy 2011), using hmms for 62 metabolite domains from the plantiSMASH database (Kautsar et al. 2017).
Orthogroups specific to ME034V were used to perform a final filtration of the gene set. We observed that many genes belonging to these orthogroups were single copy, short, and encompassed by other primary gene models. Therefore, we removed primary gene models shorter than 50 bp, as well as single-exon genes from ME034Vspecific, single-copy orthogroups either without RNA-Seq expression support (TPM , 1) or mostly encompassed by another gene model (. 90% coverage). This resulted in the final gene set of 37,908 gene models.
OrthoFinder inferred duplications with clade support $ 0.90 were parsed from the OrthoFinder duplications.csv output. Tests for functional enrichment were performed using the plantiSMASH hmms and the goslim_generic gene ontology. Hypergeometric tests were performed in python using the SciPy library hypergeom, and p-values were adjusted for multiple comparisons using the StatsModels library multitest with the Benjamini & Hochberg (BH) method (Benjamini and Hochberg 1995).

Structural variation analysis
Genome-level synteny was identified using nucmer from the MUMmer package v4 (Marçais et al. 2018) between ME034V, A10 and S. italica assemblies with minimum cluster size of 65 (default, C = 65) and minimum max lengths of 250 bp (L = 250). Dot plots were visualized using mummerplot. Smaller variations in genome alignments were identified using MUMmer's show-diff using default parameters. Synteny plots of these small regions were generated using minimap2 v2.13 with -cx asm5 flag enabled (Li 2018) and xmatchview v1.1 (Warren 2018). Finally, read mapping support was visualized using the python-powered script package genomeview (Spies et al. 2018).
Enrichment of genome content at assembly gaps in the ME034V genome was first performed by using bedtools genomecov v2.29.1 (Quinlan 2014) to calculate the repetitive element content in nonoverlapping windows across the genome. Permutation tests were completed by randomly shuffling the gap coordinates on the nuclear chromosomes 1,000 times, extracting the nucleotide sequence either 50, 100, 500, 1,000, or 1,500 base-pairs from the gap boundaries, and calculating the repeat content in these windows using bedtools. P-values were calculated as the proportion of permuted gaps with higher average repeat content than the observed gap content. The same process was completed using percent GC in windows surrounding known and permuted gaps.
Whole-genome sequencing data from 220 S. viridis and 15 S. italica libraries were obtained from the NCBI Sequence Read Archive (Table S3). For each library, the reads were aligned to the ME034V, A10 (v2.1), and S. italica (v2) genome assemblies using BWA mem v0.7.17 (Li 2013) and PCR duplicate reads were marked using Picard MarkDuplicates v2.9.0 (http://broadinstitute.github.io/ picard/). The resulting BAM files were sorted using SAMtools v1.8 (Li and Durbin 2009) and passed to Delly v0.8.1 (Rausch et al. 2012) to predict inversions, tandem duplications, and deletions relative to each of the reference genomes. Delly was run with a minimum paired-end read mapping score of 20 (q = 20) and a MAD insert size cutoff of 7 (s = 7) for deletions. Final structural variant call sets were identified as calls with precise breakpoint support (i.e., split-read support), less than 5 Mb in length, passing quality scores (per Delly), mapping quality scores greater than zero, and at least five pairedreads spanning the breakpoint.

Data availability
Seed of the sequenced ME034V accession is available via USDA-NPGS-GRIN (https://npgsweb.ars-grin.gov/gringlobal/accessiondetail.aspx? id=1918592). Raw sequencing reads used for de novo whole-genome assembly and the final genome have been deposited in the Sequence Read Archive database under BioProject PRJNA560942. The genome assembly has been submitted to NCBI under GenBank accession CP050795. The gene, repeat, and structural variant annotation set described in this manuscript is available for upload via a custom track hub for the University of California Santa Cruz (UCSC) Genome Browser (https://github.rcac.purdue.edu/JenniferWisecaverGroup/ ME034V_Trackhub). Nucleotide sequences of the chloroplastderived contigs are available in Supplementary File S1. Functional annotations per Interproscan and PlantiSMASH of ME034V gene models are in Supplementary File S2. Orthologous gene families from OrthoFinder analyses are in Supplementary File S3.

Genome size evaluation
To assess the genome size of S. viridis, unprocessed Illumina sequencing data were downloaded from ten S. viridis accessions and one S. italica accession in the NCBI Sequence Read Archive (SRA). We performed a k-mer frequency analysis via GenomeScope using three levels of filtration: no-filtration (i.e., raw data), quality-trimmed, and quality-trimmed with removal of organellar sequences (Table  S1). For ME034V, we observed a maximum haploid genome size of 432.1 Mb from raw unprocessed sequence data and a minimum haploid genome size of 391.9 Mb from quality-trimmed, organellarfiltered sequence data ( Figure S1). Across all accessions, S. viridis ME043V produced the largest haploid genome size estimates, with a maximum of 465.6 Mb from raw unprocessed sequence data, and 421.0 Mb from quality trimmed and organellar-filtered sequence data (Table S1).
Genome sequencing, assembly, and annotation Sequencing data for the ME034V genome assembly consisted of 10 Gb of ONT long reads (699,624 reads with an N50 of 41 kb) and 45 Gb of 150 bp paired-end Illumina short reads. These data amount to 23-26x ONT long read coverage and 104-115x coverage with Illumina short reads assuming the maximum and minimum k-mer estimated genome size (Table S1). We then established a multistage de novo genome assembly workflow (Figure 1). The initial assembly was performed using minimap2 and miniasm with parameter settings optimized for long, noisy ONT reads (Li 2016). In order to find the best initial assembly for polishing and scaffolding, a range of miniasm parameter combinations were executed, and each resulting assembly was evaluated for total contig count and length (assembly v0.1; Figure 1). We error corrected this initial assembly via three rounds of polishing with ONT reads (assembly v0.2; Figure 1) followed by two rounds of polishing with Illumina reads (assembly v0.3; Figure 1). The resulting assembly v0.3 consisted of 48 contigs spanning 397.7 Mb, with an N50 of 19.5 Mb. Contigs were scaffolded into pseudochromosomes using the A10 (v2) reference genome to yield assembly ME034V v0.4 (Figure 1). Of the 48 total contigs, 44 correspond to A10 nuclear genome sequence (Table 1), with the remaining four contigs consisting of chloroplast genome sequence (File S1). Using the chloroplast genomes of Sorghum bicolor and Zea mays as references, we determined that each chloroplast-derived contig contained a full-length chloroplast genome consisting of both short and large single copy loci and ribosomal DNA inverted repeats ( Figure S2). The four chloroplast contigs are .99.9% similar and largely syntenic, with the exception of the short single copy locus in utg000045l that is inverted with respect to the other three ( Figure S2). Multiple chloroplast sequences is suggestive of heteroplasmy, which has been documented in Z. mays and other plant chloroplasts (Oldenburg and Bendich 2004;Bendich 2007). Due to ambiguity as to the true chloroplast genome sequence, we excluded the four chloroplast-derived contigs from the final assembly (ME034V v1.0) as well as all downstream analyses. Through a combination of RepeatModeler and RepeatMasker annotations, 46.02% of assembly bases were flagged as repetitive elements (Table 1; Table S4), an estimate that matches the overall S. italica repeat content (Zhang et al. 2012). As a proportion of bases masked, the most abundant classified group of mobile elements belong to the long terminal repeat (LTR) retrotransposons, constituting over one quarter of the genome (27.5%). Of these, 64,897 gypsy-like and 20,632 copia-like elements were predicted (Table S4). The ratio of approximately 3:1 gypsy-like to copia-like elements was also observed in the S. italica genome ( (Table S4). To facilitate comparisons, we also processed other grass genomes through our annotation pipeline. The repeat content of sorghum and maize was 62.7% and 80.8%, respectively (Table S5). These results are consistent with the pattern that millets have less repetitive sequence than the nuclear genomes of other grasses (Haberer et al. 2005;McCormick et al. 2018).
Protein-coding genes were identified through a combination of ab initio, homology-based, and transcriptome-based prediction methods. A total of 37,908 gene models encoding 49,829 transcripts were predicted, with an average of 1.31 transcripts per gene ( Table 2). The average protein-coding gene was 2,436 bp long and contained 4.06 exons. These numbers are comparable to the primary gene count of A10 (N = 38,334) and S. italica (N = 34,584) (Zhang et al. 2012;Mamidi et al. 2020). We observed that gene density was highest near the ends of chromosomes and generally replete in repeat-dense regions (Figure 2a). A notable exception was the gene sparse Chr08, which had the largest number of gaps (N = 12), likely due to its high repeat content (56.6%; Table 1).
TEs can have profound effects on gene family evolution by altering protein-coding regions as well as gene transcriptional levels and regulation (Feschotte and Pritham 2007;Feschotte 2008 In ME034V, long interspersed nuclear elements (LINEs) make up only 1.4% of the total nuclear genome assembly; nevertheless, LINEs showed an insertion bias for genic regions, representing 5.1% of all repeats that intersect genes compared to only 2.4% of non-genic insertions (Figure 2b). In contrast, LTR elements such as copia and gypsy were nearly 3.6-fold more common in non-genic than genic space (40.7% vs. 11.3%, respectively) ( Figure 2b). While LINEs, LTRs, and most other complex repeat classes did not exhibit preferred insertional strand bias, rolling-circle (RC) elements (e.g., helitrons) were nearly twice as likely to be inserted into the same strand as a gene (Figure 2b). Functional annotations were assigned to the majority of predicted proteins (File S2). In total, 60.41% of the final 37,908 genes were assigned at least one Pfam (El-Gebali et al. 2019) domain, of which the majority of these domains were protein kinases PF00069 (12.06% of genes), WD40 repeats PF00400 (7.84%), and pentatricopeptide repeats PF01535 (19.34%) and PF13041 (14.89%). Gene Ontology (GO) (Gene Ontology Consortium 2004) associations were also common, with 47.75% of genes assigned to at least one GO category. Additional functional annotations were assigned to 76.75%, 67.61%, 19.19%, 5.04%, 3.92% of genes using the PANTHER (Mi et al. 2013), InterPro (Jones et al. 2014), Trans Membrane (TMHMM) (Krogh et al. 2001), KEGG (Kanehisa et al. 2008), and plantiSMASH (Kautsar et al. 2017) databases, respectively.

Quality assessment
To evaluate the completeness and coverage of the assembly, we aligned the Illumina gDNA and RNA reads to the ME034V genome assembly. The alignment rate of the ME034V gDNA reads was 99.14%, 98.43%, and 98.39% against the ME034V, A10, and S. italica genomes, respectively. We extended this assessment to samples from additional S. viridis and S. italica accessions obtained from the SRA. Of the 235 different accessions, 143 (60.9%) aligned best to the ME034V assembly, 36.6% aligned best to the S. italica assembly, and 2.56% aligned best to the A10 assembly (Table S3). Although long-read genome assemblies can exhibit collapse of repetitive or highly similar sequences (Vollger et al. 2019), the high alignment rate suggests minimal missing sequence and that the amount of collapsed regions in our ME034V genome assembly is minimal.
Many of the de novo predicted genes in ME034V showed evidence of in silico expression. Over 62% of the final gene models had expression support (.1 TPM) across the merged RNA-Seq sample set from ME034V leaf, root, and sheath samples ( Table 2, Table S2). The average read alignment rate across all RNA-Seq libraries was 44.4% (Table S2). Lastly, we used BUSCO (Waterhouse et al. 2018) to assess the completeness of the ME034V predicted proteome. Within the ME034V protein-coding gene set, 277 of 303 conserved eukaryotic genes (91.4%) were identified as complete, of those 70.6% were present in single-copy and 20.8% were duplicated.

Comparison of assemblies
Cumulative lengths of the nuclear chromosomes among the three Setaria genomes are highly comparable. S. italica has the longest assembly at 401.3 Mb (excluding unplaced contigs; Ensembl release 45), A10 has the smallest at 395.1 Mb (Mamidi et al. 2020), and ME034V is in between at 397.0 Mb. The ME034V assembly is largely syntenic with both A10 and S. italica genome assemblies, as revealed by whole genome alignments (Figure 3; Figure S3). Overall, our ME034V genome had fewer genome-specific variants when compared to A10 than to S. italica. However, a few large-scale variations in chromosomal structure are shared between A10 and S. italica that differentiate these assemblies from ME034V. These include a 500 kb n■  gap on Chr01 ( Figure S3a), a 2.5 Mb inversion on Chr02 ( Figure S3b), complex rearrangements on Chr03 ( Figure S3c), an inverted rearrangement on Chr05 ( Figure S3e) and two inversions on Chr09 ( Figure S3i). As a result of improved sequencing and assembling technologies since the completion of the S. italica assembly (Bennetzen et al. 2012;Zhang et al. 2012), large assembly gaps (greater than 10 bp) are far less prevalent in the ME034V (N = 35) and the A10 (N = 61) genomes than the S. italica genome (N = 6,158). We performed a series of permutation tests to evaluate patterns of GC bias and repeat abundance in regions near gaps in our ME034V assembly. Sequences directly adjacent (50 bp flanking) to gaps in the ME034V assembly had significantly lower average GC content (p-value = 0.035) than randomized shuffling of sequences of similar lengths ( Figure S4, Table S6). At 1,500 bp from the gap edge, the flanking sequence was significantly more likely to contain repetitive elements than expected by chance (p-value = 0.025). The reason significant repeat abundance was reached 1.5 kb away, rather than closer to the gap edge, is likely an artifact of annotation; if a repeat element were to extend into a gap, the truncation of the sequence could have caused repeat masking software to not call the element. Together, these data illustrate that despite the increased read-length of ONT sequences, limitations in contiguous assembly remain at genome positions of reduced nucleotide complexity and repetitive elements.

Structural variation
Short-read alignments from over 200 Setaria accessions (Table S3) against the ME034V assembly were utilized to increase resolution for identifying structural variants (SVs). This sample set is of sufficient size and genetic diversity (Mamidi et al. 2020) that even rare structural polymorphisms could be identified. Bioinformatic SV predictions are made through detection of anomalous paired read alignments. Insert sizes that are too large or too small are called as deletions or insertions, respectively, whereas read pairs with inverted orientations (e.g., +/+ or 2/2) indicate inversions. From this analysis, we compiled a list of curated SVs less than 5 Mb in length, which contained 150 deletions, 186 duplications, and 85 inversions with read support (.5 paired-reads) at predicted variant breakpoints (Table S7). Of these, 91 deletions, 107 duplications, and 50 inversions were present in the sample set with minor allele frequencies of at least 0.01 (Table S7). The median variant lengths for deletions was 6,630.5 bp and 2,120.0 bp for tandem duplications. The median variant length for inversions were significantly larger at 148,918 bp.
Our SV calling pipeline identified several indels containing TEs that are polymorphic within the sample population, which is suggestive of relatively recent TE activity. The most abundant Setaria TE class, the LTR retrotransposons copia and gypsy, were among these polymorphic variants. The size distribution of small deletions (,10 kb) showed several peaks that co-occurred with    Chr03:33,177,245,787 in ME034V that is missing in the homologous locus in A10,window Chr03:34,798,868,544 (see also Figure S5; Figure S6) b) Copia insertion unique to A10 (window Chr_03:28,599,645,328), and absent in ME034V (window Chr03:29,091,132,959) (see also Figure S7; Figure S8). c) Presence of a gypsy element shared between ME034V (window Chr02:32,419,465,022) and A10 (window Chr_02:31,649,696,029) is indicated by near-perfect alignment in the synteny plots (see also Figure S9; Figure S10). For all synteny plots, blue-gray bars connect the two genomes when DNA sequence with .70% identity is observed (red line indicates threshold). Blue and cyan bars above the top track indicate sequence identity along the chromosomal segment from 0-100%, with the color indicating wither single copy (blue) or double copy (cyan) matches. Purple rectangles indicate genes and green rectangles indicate LTRs (alternating hues aid in distinguishing between unique elements). The strandedness of the genes and LTRs is indicated by placing elements encoded on the forward strand higher relative to elements encoded on the reverse strand. Putative target site duplications (TSD) are indicated in collinear regions in (a) and (b). Orange rectangles in part c indicate the 1:1 homologous region absent in accessions Estep_ME018 and Feldman_ MF156; support for this deletion is visualized by split reads (cyan) at the left (d) and right (e) breakpoint of the read alignment. Read pairs are connected with gray lines, and reads on the forward and reverse strand are colored pink and purple, respectively.
ME034V copia (peaks around 3 and 13 kb) and gypsy (peaks around 3 and 8 kb) lengths (Figure 4). We then used synteny analyses coupled with visualization of read alignments at predicted SV breakpoints to confirm the presence of select SVs. A deletion of ME034V sequence around Chr03:33.2 Mb (DEL00053315) was predicted in five Setaria accessions (Estep_ME062, Huang_TX01, Huang_MO13, Feldman MF131, Estep_ME050V; Table S7) and confirmed through breakpoint assessments ( Figure S5). Synteny analyses of this region in ME034V and its homologous locus in S. italica (chr3:35.5 Mb) revealed that the adjacent regions are not identical in sequence but are both LTR-rich with 10 kb unique sequence in ME034V ( Figure S6). Pairwise alignments of A10 to both ME034V and S. italica illustrate that the A10 assembly (Chr_ 03:34.8 Mb) is fully missing the locus (Figure 5a; Figure S6). Furthermore, co-linear overlaps at the apparent A10 indel is suggestive of a target site duplication (TSD) (Figure 5a), which is a signal of a retrotransposon insertion through target-primed reverse transcription (Ewing 2015). A second example includes an insertion of a copia element in A10 (DEL00051066) that is absent from both ME034V and S. italica (Figure 5b; Figure S7), which also has synteny at the putative indel breakpoint, resembling a possible TSD (Figure 5b). Read alignment patterns at the putative breakpoint indicate a homozygous insertion for four (Estep_ME005, Estep_ ME009, Estep_ME061V, Estep_ME059V) and heterozygous insertion for two (Feldman_MF136, Estep_ME010) Setaria accessions ( Figures S8; Table S7). Lastly, we identified an apparent deletion (DEL00033261) containing a gypsy element that is present in all three reference genomes (ME034V, A10 and S. italica). All three Setaria assemblies are syntenic at this locus (Figure 5c; Figure S9), yet three accessions (Feldman_MF137, Feldman_MF156, and Estep_ME018) have apparent homozygous deletions based on breakpoints in their read alignments (Figure 5d and e; Figure S10). Figure 6 Analysis of gene families in Setaria. a) Comparison of orthogroups in ME034V, A10, and S. italica. Conserved orthogroups (green) were present one or more times in all 27 genomes in the analysis. Monocot-specific orthogroups (blue) were present in two or more monocot genomes and absent from all others. b) The species phylogeny was taken from the PLAZA 4.0 monocots online database. Branch thicknesses and colors are scaled based on the number of predicted duplicated events to have occurred at the descendant node; thinner, blue branches indicate fewer duplications; thicker, red branches indicate more (see Table S10). The 742 duplications predicted at the Setaria ancestral node are indicated with the gray arrow. Tests for enrichment of functional categories was performed on this gene set: c) top ten most significantly enriched GO categories (see Table S11); d) all significantly enriched plantiSMASH specialized metabolism enzyme classes (see Table S12).

Gene family analysis
To investigate the evolution of different gene families, including those that may be unique or expanded in Setaria species, we performed an OrthoFinder (Emms and Kelly 2015) analysis using the proteincoding genes of ME034V and 26 other eudicot genomes, including A10 and S. italica (Table S8). The OrthoFinder analysis identified 28,055 unique orthogroups (predicted gene families) consisting of two or more species in the analysis (Table S9). Of the 18,973 orthogroups containing one or more ME034V sequences, 4,135 orthogroups (21.79%) were present in all species in the analysis, and 7,390 (38.95%) were found only in monocots (Figure 6a). The total number of ME034V orthogroups was comparable to the other sequenced genomes of Setaria, which ranged from 20,620 in A10 to 17,253 in S. italica. The proportion of genome-specific orthogroups was more similar between S. italica (6.30%) and ME034V (7.77%) than A10 (12.75%). In total, 36,521 of 37,908 ME034V proteins (96.34%) were assigned to an orthogroup containing sequence from one or more additional Poaceae (File S3).
To identify orthogroups that may have expanded in the ancestor of the three Setaria genomes, we parsed the number of OrthoFinder predicted gene duplications at each node of the inferred species tree (Figure 6b; Table S10). The average number of orthogroups that duplicated one or more times at a given node was 1541.53 and ranged from only 2 duplications at internode 16 (the common ancestor of Phyllostachys and the Pooideae) to 12,238 duplications in the hexaploid Triticum aestivum (Figure 6b; Table S10). A total of 742 orthogroups duplicated in the last common ancestor of the three Setaria (Figure 6b; Table S10), and the ME034V genes that duplicated at this internode were enriched in 153 Gene Ontology (GO) categories (Benjamini-Hochberg adjusted p-value , 0.05; Table S11). The most significantly enriched GO categories included those for ADP and metal binding (GO:0043531; GO:0030145; GO:0005507), nutrient reservoir activity (GO:0045735), defense response (GO:0006952), extracellular region (GO:0005576), and cysteine-type peptidase activity (GO:0008234) (Figure 6c; Table  S11). In addition, we checked for enrichment of enzyme families typically associated with specialized metabolic (SM) processes (Kautsar et al. 2017) and found that 11 SM enzyme families were enriched in the set of genes that duplicated in the ancestor of Setaria (Benjamini-Hochberg adjusted p-value , 0.05; Table S12) including those for the production of terpenes, strictosidines, chalcones, and stilbenes ( Figure 6c).

DISCUSSION
Many publications reference a genome size estimate of 490 Mb for S. italica and S. viridis, based on C-values derived from flow cytometry data (Le Thierry D'Ennequin et al. 1998;Bennett et al. 2000) and later at 485 Mb based on k-mer analysis of Illumina sequencing data (Zhang et al. 2012). Despite this, both S. italica and A10 reference assemblies are significantly smaller at 405.7 Mb and 395.7 Mb, respectively (Bennetzen et al. 2012;Mamidi et al. 2020). Similarly, our ME034V genome assembly totals 397 Mb. The k-mer based genome size estimate for our assembly suggests the true S. viridis genome size is closer to the assembled genome sizes of 400 Mb. However, it is likely that some repetitive regions of the ME034V genome have been collapsed, as is seen in other complex genomes (Vollger et al. 2019). This could explain some but not all of the disagreement in genome size. Disconnect between flow cytometry and k-mer based genome size estimates has been documented by others (Pflug et al. 2020), and should be investigated in more detail in future analyses.
Although the ME034V assembly is largely syntenic with the two other sequenced Setaria genomes from S. italica and A10, several SVs were identified in ME034V. Validation of many SVs by read mapping against the Setaria diversity panel indicates that the SVs are unlikely to be the result of misassembly and instead represent true genome variation in the species. Identification of this genome variation, thanks in large part to the high contiguity of the ME034V assembly, illustrates the utility of ultra-long DNA sequencing data to improve genetic resources for emerging model systems. Preliminary surveys of the ME034V assembly has revealed a repeat-rich landscape, with some transposable element classes displaying compelling patterns of recent mobility. Structural variant predictions in large sample sets can facilitate the identification of rare deletions or insertions. Identified from the sample set of hundreds of Setaria accessions, we have presented evidence of LTR retrotransposons whose insertions are either genome-specific or completely absent in a subset of samples. Further analyses are required to validate these bioinformatic predictions in the population, assess the completeness and age of these putative TE insertions (Bennetzen et al. 2017), as well as evaluate their abundance in a phylogenetic context.
Lastly, our analysis of gene family evolution in Setaria identified hundreds (n = 742) of orthogroups that likely duplicated in a common ancestor of the three genomes analyzed (ME034V, A10 and S. italica). These duplicated gene families appear to be enriched in processes related to specialized metabolism, nutrient acquisition, and defense response, which is consistent with previous observations that these gene families are some of the most likely to undergo frequent duplication in plants (Pichersky and Lewinsohn 2011;Chae et al. 2014).
Altogether, our assembly of the Setaria viridis ME034V genome constitutes an essential resource for monocot research and further establishes Setaria as an ideal model plant system. Combined with the high A. tumefaciens transformation rate of ME034V, the assembly and annotation described here will further aid in genetic manipulations, securing ME034V as the preferred S. viridis reference accession.