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. 2025 Dec 15;117(3):600–610. doi: 10.1093/jhered/esaf105

“A genome assembly of the California flannelbush, Fremontodendron californicum”

William T McMahan 1, Merly Escalona 2, Reed Kenny 3, Mohan P A Marimuthu 4, Oanh Nguyen 5, Colin W Fairbairn 6, William Seligmann 7, Courtney Miller 8, Howard Bradley Shaffer 9, Shannon Still 10, Daniel Potter 11,✉
PMCID: PMC13147169  PMID: 41395837

Abstract

Fremontodendron (Malvaceae) is a genus of shrubs native to the California Floristic Province (CFP) with dense, stellate trichomes on their leaves (hence the common name “flannelbush”). The current treatment of the genus includes the widespread and morphologically variable species Fremontodendron californicum, along with the rare F. decumbens and F. mexicanum. While F. californicum is spread across several ecoregions of the CFP, F. decumbens and F. mexicanum are highly restricted. Here, we introduce the first genome-scale resource with which to study this important genus, a de novo, scaffold scale assembly of an individual of F. californicum. Following the overall strategy of the California Conservation Genomics Project (CCGP), we used Pacific Biosciences HiFi long reads and Omni-C chromatin mapping to produce an assembly of the nuclear and plastid (chloroplast) genomes. The nuclear assembly consists of two, phased haplotypes, haplotypes one and two, with similar sizes of around 1.2 Gb, similar scaffold N50s around 26 Mb, and each with a Benchmarking Universal Single-Copy Ortholog (BUSCO) completeness score of 99.4%. This assembly will be a valuable resource for understanding the distribution, genetic variation, and species delimitation of this California genus of conservation value, as well as a tool to further investigate the complex evolutionary history of Malvaceae s.l.

Keywords: California Conservation Genomics Project, CCGPCalifornia floristic province, conservation, Malvaceae

Introduction

We present the nuclear and chloroplast genome assemblies of Fremontodendron californicum (Torr.) Coville (Malvaceae), “California flannelbush.” These assemblies were produced as part of the California Conservation Genomics Project (CCGP), an initiative to document the landscape genomics of the animals and plants which span the range of ecological and phylogenetic diversity of the state of California (Shaffer et al. 2022; Toffelmier et al. 2022). In addition to compiling a genome assembly for each taxon, the project will resequence up to 150 different individuals of each taxon across their geographic range to generate a profile of their genetic diversity which can be used to inform conservation and management decisions. The taxa, some of which are key species and some of which are endangered or threatened, are found in marine, freshwater, and terrestrial habitats. Compiling their genomic information will reveal trends not only at the intraspecific level but also at the community and ecological level, allowing insights into biodiversity hotspots, potential blockages to gene flow, and challenges regarding their preservation and adaptation to climate change.

In general, species of Fremontodendron are shrubs which bear lobed leaves with tawny stellate pubescence on the abaxial surfaces (see Table 1). Nine taxa have been recognized, at the specific or infraspecific level, within Fremontodendron (Table 1). In the current treatments for the Jepson e-flora (Preston et al. 2012) and the Flora of North America (Hanes 2015), three species are recognized, and no formal taxonomic recognition is given to morphological variants within the most widespread species, Fremontodendon californicum (Torr.) Coville. The other two species both have highly restricted distributions and are listed at the state (rare) and federal (endangered) levels (CNDDB 2020), with California Rare Plant Ranks of 1B (“rare, threatened, or endangered in California and elsewhere”; CNPS 2020). F. mexicanum Davidson, “Mexican flannelbush” (rank 1B.1), is found only in southern California and Baja California, Mexico, with only one confirmed natural occurrence in each area, the former in San Diego County (Kelman 1991). F. decumbens R.M. Lloyd, “Pine Hill flannelbush” (rank 1B.2), is known only from a few populations in the Pine Hill area (El Dorado County) of the Sierra Nevada foothills (Kelman et al. 2006). In contrast, F. californicum is found throughout the California floristic province, occurring naturally in 12 of the 19 United States Department of Agriculture (USDA) ecoregions within California and extending into Oregon, Arizona, and northern Baja California, Mexico. It grows in a range of habitats including coastal scrub, oak/pine woodlands, chaparral, and arid high desert. It occurs at elevations ranging from 180 to 2320 m, spanning the Coast Ranges to the Western Sierra Nevada foothills (Preston et al. 2012). It is valuable to animals and insects as a source of both nectar and seed and is considered to be a fire recruiter and fire-dependent given its ability to survive and regenerate as well as sprout from seed in post-fire environments. Its drought resistance and showy yellow calyces have made it a favorite of California native plant horticulture (Abrahamson 2021). Individuals of F. californicum are generally erect shrubs, 2 to 5 m in height, and are highly morphologically variable, both between and within populations (Preston et al. 2012).

Table 1.

Taxa which have been recognized within the genus Fremontodendron. Those marked with * were originally described under the generic name Fremontia, but this name is considered illegitimate due to having been previously applied (Torrey 1843) to a species later determined to be a synonym of Batis vermiculates Hook. (Chenopodiaceae; Torrey 1854), now treated as Sacrobatus vermiculatus (Hook.) Torr. (Sarcobataceae).

Taxon Also treated as Distinguishing features Distribution Currently treated as
F. californicum (Torr.) Coville, 1893* F. californicum var. californicum, F. californicum subsp. californicum Sepals yellow; sepal pits with hairs; seeds hairy, with appendage California, Oregon, Arizona, Baja California F. californicum
F. californicum subsp. crassifolium (Eastw.) J.H. Thomas, 1955 Fremontia crassifolia Eastw., 1934* Thicker leaves and larger flowers than typical F. californicum Santa Cruz Mountains, California F. californicum
F. californicum subsp. napense (Eastw.) Munz, 1964 Fremontia napensis Eastw., 1934* Smaller, thinner leaves and smaller flowers than typical F. californicum Napa and Counties, California F. californicum
F. californicum subsp. obispoense (Eastw.) Munz, 1964 Fremontia obispoensis Eastw., 1934*, F. californicum var. obispoense (Eastw.) Hoover, 1970 Small, generally unlobed leaves San Luis Obispo County, California F. californicum
Fremontia californica var. diegensis M. Harv., 1943* Leaves unlobed, petioles > blade length San Diego County, California. F. californicum
Fremontia californica var. integra M. Harv., 1943* Leaves unlobed, petioles < or = ½ blade length Tulare and Kern Counties, California. F. californicum
Fremontia californica var. viridis M. Harv., 1943* Leaf hairs whitish, not tawny. Tehama County, California F. californicum
F. decumbens R.M. Lloyd, 1964 F. californicum subsp. decumbens (R.M. Lloyd) A.E. Murray, 1982 Decumbent habit; sepals orange; sepal pits with hairs; seeds hairy, with appendage Northern Sierra Nevada foothills F. decumbens
F. mexicanum Davidson, 1917 F. californicum subsp. mexicanum (Davidson) Munz, 1968 Erect habit; sepals orange; sepal pits lacking hairs; seed glabrous, lacking appendage Southern California, Baja California F. mexicanum
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Despite the well-studied variability in F. californicum and the conservation status of F. mexicanum and F. decumbens, there have been few studies of their genetic diversity, and few genomic resources are available to help evaluate the status of ambiguous individuals or to guide the management of populations of concern. The chromosome number for F. californicum is reported at n = 20 (Lenz 1950), and the only study of genetic diversity in the genus relied on amplified fragment length polymorphisms (Kelman et al. 2006). Here, we present both the first scaffold-scale nuclear genome assembly and a chloroplast genome assembly for F. californicum. In collaboration with and under the guidance of the CCGP, these assemblies are based on Pacific Biosciences (PacBio) HiFi long read sequencing, and the nuclear assembly was scaffolded de novo using Omni-C data. These genomic resources for F. californicum will form the basis of studies to determine species evolution, delimitation, and diversity within this important California native plant.

Methods

Biological materials

We selected a naturally occurring individual of F. californicum as the basis for the genome assemblies. The individual is located in Napa County, California, along the Knoxville-Berryessa Road, approximately 0.5 miles south of the border with Lake County (38.85135°N, 122.39593°W, datum WGS84). Leaves for extraction and enough plant material suitable for a herbarium voucher were collected on 31 October 2020. The corresponding herbarium voucher is deposited at the UC Davis Center for Plant Diversity (Acc. # DAV 238751, Fig. 1A).

Fig. 1.

Fig. 1

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Images of the California flannelbush (Fremontodendron californicum). A: Image: https://cch2.org/imglib/cch2/DAV/DAV238/DAV238751_lg.jpg. (A) The herbarium specimen of the individual used to make the assemblies presented here, collected in Napa County, CA. B: Image: SMS_90568_2021-04-19.jpeg. (B) An adult individual of F. californicum, exhibiting the characteristic upright, shrubby growth habit. C: Image: IMG_6965.jpeg. (C) Leaves of F. californicum with stellate pubescence visible. Photo credit for (B, C): Shannon Still.

Nucleic acid library preparation and sequencing

Pacific Biosciences HiFi Library

We extracted high-molecular-weight (HMW) genomic DNA from 500 mg of young leaves (Sample ID: 1011) using the cetyltrimethylammonium bromide (CTAB) method as described in Inglis et al. (2018), with the following modifications: (i) we used sodium metabisulfite (1% w/v) instead of 2-mercaptoethanol (1% v/v) in the sorbitol wash buffer and CTAB solutions; (ii) we repeated the tissue homogenate wash steps until the supernatant turned clear; (iii) we performed the CTAB lysis step at 45 °C and (iv) the chloroform extraction step twice using ice-cold chloroform. The DNA purity was estimated by absorbance ratios (260/280 = 1.80 and 260/230 = 2.16) measured using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, MA). The DNA yield (13.2 μg) was quantified using a Quantus Fluorometer (QuantiFluor ONE dsDNA Dye assay; Promega, WI), and the size distribution of the DNA was estimated using the Femto Pulse system (Genomic DNA 165 kb kit, Agilent, CA), where 71% of the DNA fragments were found to be 30 kb or longer.

The HiFi SMRTbell library was constructed using the SMRTbell Express Template Prep Kit v2.0 (PacBio Cat. #100-938-900) according to the manufacturer’s instructions. HMW genomic DNA was sheared to a target DNA size distribution between 15 and 18 kb using Diagenode’s Megaruptor 3 system (Diagenode, Belgium; cat. B06010003). The sheared gDNA was concentrated using 0.45× of AMPure PB beads (PacBio CA; Cat. #100-265-900) for the removal of single-strand overhangs at 37 °C for 15 min, followed by further enzymatic steps of DNA damage repair at 37 °C for 30 min, end repair and A-tailing at 20 °C for 10 min and 65 °C for 30 min, and ligation of overhang adapters v3 at 20 °C for 60 min. The SMRTbell library was purified and concentrated with 1× AMPure PB beads for nuclease treatment at 37 °C for 30 min and size selection using the PippinHT system (Sage Science, MA; Cat #HPE7510) to collect fragments greater than 7 to 9 kb. The 15 to 20 kb average HiFi SMRTbell library was sequenced at UC Davis DNA Technologies Core (Davis, CA) using one 8 M SMRT cell, Sequel II sequencing chemistry 2.0, and 30-h movies each on a PacBio Sequel II sequencer.

Omni-C library

The Omni-C library was prepared using the Dovetail Omni-C Kit (Dovetail Genomics, CA) according to the manufacturer’s protocol with slight modifications. First, specimen tissue (leaves, ID: 1101C) was thoroughly ground with a mortar and pestle while cooled with liquid nitrogen. Nuclear isolation was then performed using published methods (Workman et al. 2018). Subsequently, chromatin was fixed in place in the nucleus and digested under various conditions of DNase I until a suitable fragment length distribution of DNA molecules was obtained. Chromatin ends were repaired and ligated to a biotinylated bridge adapter, followed by proximity ligation of adapter containing ends. After proximity ligation, crosslinks were reversed and the DNA was purified from proteins. Purified DNA was treated to remove biotin which was not internal to ligated fragments. A Next Generation Sequencing (NGS) library was generated using an NEB Ultra II DNA Library Prep kit (New England Biolabs, MA) with an Illumina-compatible y-adaptor. Biotin-containing fragments were then captured using streptavidin beads. The postcapture product was split into two replicates prior to Polymerase Chain Reaction (PCR) enrichment to preserve library complexity with each replicate receiving unique dual indices. The library was sequenced at Vincent J. Coates Genomics Sequencing Lab (Berkeley, CA) on an Illumina NovaSeq platform (Illumina, CA) to generate approximately 100 million 2 × 150 bp read pairs per gigabase of genome size.

Nuclear genome assembly

We assembled the genome of the F. californicum individual following the CCGP assembly pipeline Version 5.0, as outlined in Table 2, listing the tools and nondefault parameters used in the assembly process. We removed the remnant adapter sequences from the PacBio HiFi dataset using HiFiAdapterFilt (Sim et al. 2022) and generated an initial diploid phased assembly using HiFiasm (Cheng et al. 2021) in HiC mode with the filtered PacBio HiFi reads and the Omni-C short-reads, a process which generates two assemblies, one per haplotype. We then aligned the Omni-C data to both assemblies following the Arima Genomics Mapping Pipeline (https://github.com/ArimaGenomics/mapping_pipeline) and then scaffolded both assemblies with SALSA (Ghurye et al. 2017; Ghurye et al. 2019).

Table 2.

Assembly pipeline and software used.

Assembly step Software and any nondefault options Version Reference
Initial assembly
Filtering PacBio HiFi adapters HiFiAdapterFilt Commit 64d1c7b Sim et al. 2022
K-mer counting Meryl (k = 21) 1 https://github.com/marbl/meryl
Estimation of genome size and heterozygosity GenomeScope 1 Vurture et al. 2017
De novo assembly (contiging) HiFiasm (Hi-C Mode, −primary, output hic.hap1.p_ctg, hic.hap2.p_ctg) 0.16.1-r375 Cheng et al. 2021
Scaffolding
Omni-C data alignment Arima Genomics Mapping Pipeline Commit 2e74ea4 https://github.com/ArimaGenomics/mapping_pipeline
Arima Genomics Mapping Pipeline (AGMP) BWA-MEM 0.7.17-r1188 Li 2013
samtools 1.11 Danecek et al. 2021
filter_five_end.pl (AGMP) Commit 2e74ea4 https://github.com/ArimaGenomics/mapping_pipeline
two_read_bam_combiner.pl (AGMP) Commit 2e74ea4 https://github.com/ArimaGenomics/mapping_pipeline
picard 2.27.5 https://broadinstitute.github.io/picard/
Omni-C Scaffolding SALSA (-DNASE, -i 20, -p yes) 2 Ghurye et al. 2017, Ghurye et al. 2019
Omni-C Contact map generation
Short-read alignment BWA-MEM (-5SP) 0.7.17-r1188 Li 2013
SAM/BAM processing samtools 1.11 Danecek et al. 2021
SAM/BAM filtering pairtools 0.3.0 Open2C et al. 2024
Pairs indexing pairix 0.3.7 Lee et al. 2022
Matrix generation cooler 0.8.10 Abdennur and Mirny 2020
Matrix balancing hicExplorer (hicCorrectmatrix correct --filterThreshold -2 4) 3.6 Ramírez et al. 2018
Contact map visualization HiGlass 2.1.11 Kerpedjiev et al. 2018
PretextMap 0.1.4 https://github.com/wtsi-hpag/PretextView
PretextView 0.1.5 https://github.com/wtsi-hpag/PretextMap
PretextSnapshot 0.0.3 https://github.com/wtsi-hpag/PretextSnapshot
Manual curation tools Rapid curation pipeline (Wellcome Trust Sanger Institute, Genome Reference Informatics Team) Commit 7acf220c https://gitlab.com/wtsi-grit/rapid-curation
Genome quality assessment
Basic assembly metrics QUAST (−-est-ref-size) 5.0.2 Gurevich et al. 2013
Assembly completeness BUSCO (−m geno, −l embryophyta) 5.0.0 Manni et al. 2021
Merqury 2020 January 29 Rhie et al. 2020
Repeat content analysis RepeatModeler 2.0.5 Smit and Hubley 2015
RepeatMasker 4.1.5 Smit et al. 2015
Contamination screening
Local alignment tool BLAST+ (-db nt, -outfmt ‘6 qseqid staxids bitscore std’, -max_target_seqs 1, -max_hsps 1, -evalue 1e-25) 2.15 Camacho et al. 2009
General contamination screening BlobToolKit (HiFi coverage, BUSCO = embryophyta, NCBI Taxa ID = 93 767) 2.3.3 Challis et al. 2020
Chloroplast genome assembly
de novo genome assembler Oatk (−c 50) 1.0 Zhou et al. 2024
Graph visualizer Bandage 2022.8-dev Wick et al. 2015
Annotation GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) 2021 Tillich et al. 2017
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The assemblies for both haplotypes were manually curated by iteratively generating and analyzing their corresponding Omni-C contact maps. Briefly, to generate the contact maps we aligned the Omni-C data with BWA-MEM (Li 2013), identified ligation junctions, and generated Omni-C pairs (Lee et al. 2022) using pairtools (Open2C et al. 2023). Then, we generated multi-resolution Omni-C matrices with cooler (Abdennur and Mirny 2020) and balanced them with hicExplorer (Ramírez et al. 2018). We used HiGlass (Kerpedjiev et al. 2018) and the PretextSuite (https://github.com/wtsi-hpag/PretextView;  https://github.com/wtsi-hpag/PretextMap;  https://github.com/wtsi-hpag/PretextSnapshot) to visualize the contact maps. We identified misassemblies and misjoins in these contact maps and modified the assemblies using the Rapid Curation pipeline from the Wellcome Trust Sanger Institute, Genome Reference Informatics Team (https://gitlab.com/wtsi-grit/rapid-curation). Some of the remaining gaps (joins generated during scaffolding and/or curation) were closed using the PacBio HiFi reads and YAGCloser (https://github.com/merlyescalona/yagcloser). We checked for contamination using the BlobToolKit Framework (Challis et al. 2020).

Genome quality assessment

We generated k-mer counts from the PacBio HiFi reads using meryl (https://github.com/marbl/meryl). The k-mer counts were then used in GenomeScope (Vurture et al. 2017) to estimate genome features including genome size, heterozygosity, and repeat content. To obtain general contiguity metrics, we ran QUAST (Gurevich et al. 2013). To evaluate genome quality and functional completeness, we used BUSCO (Manni et al. 2021) with the Embryophyta ortholog database (Embryophyta_odb10) which contains 1614 genes. Assessment of base level accuracy (QV) and k-mer completeness was performed using the previously generated meryl database and merqury (Rhie et al. 2020). We further estimated genome assembly accuracy via BUSCO gene set frameshift analysis using the pipeline described in Korlach et al. (2017). Measurements of the size of the phased blocks are based on the size of the contigs generated by HiFiasm in HiC mode. We follow the quality metric nomenclature established by Rhie et al. (2021), with the genome quality code x.y.P.Q.C, where, x = log10[contig NG50]; y = log10[scaffold NG50]; P = log10 [phased block NG50]; Q = Phred base accuracy QV (quality value); C = % genome represented by the first “n” scaffolds, following a karyotype of 2n = 92 for this species, estimated as a mode from ancestral species number of chromosomes (Genome on a Tree—GoaT; tax_name(F. californicum); Challis et al. 2023). Quality metrics for the notation were calculated using the haplotype one assembly. We further assessed the size and classification of repeat elements within the nuclear assembly by using RepeatModeler and RepeatMasker (Smit and Hubley 2015; Smit et al. 2015).

Chloroplast genome assembly

We used the Oatk pipeline (https://github.com/c-zhou/oatk;  Zhou et al. 2024) to generate a chloroplast assembly from the PacBio HiFi reads, with coverage set to 50. We visualized the Oatk output in Bandage (Wick et al. 2015) to verify the completeness of the assembly. Bandage generated an expected chloroplast graph pattern with one small single copy, one large single copy, and two inverted repeat regions. We annotated the chloroplast assembly using GeSeq (Tillich et al. 2017).

Results

Sequencing data

The Omni-C library generated 146.99 million read pairs, and the PacBio HiFi library generated 2.45 million reads. The PacBio HiFi sequences yielded ~57× genome coverage and had an N50 read length of 13,916 bp; a minimum read length of 65 bp; a mean read length of 13,063 bp; and a maximum read length of 54,523 bp. Based on the PacBio HiFi data, Genomescope estimated a genome size of 883.75 Mb, a 0.132% sequencing error rate, and 1.01% heterozygosity. The k-mer spectrum shows a bimodal distribution with a major peak at ~25-fold coverage and a minor peak ~13-fold coverage (Fig. 2A).

Fig. 2.

Fig. 2

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A: Image: GenomeScope Profile. (A) K-mer spectrum output of the PacBio HiFi data, trimmed of adapters, using GenomeScope2.0. The bimodal distribution indicates a diploid genome. K-mers with lower coverage and low frequency represent differences between haplotypes, whereas higher coverage and higher frequency k-mers represent similarities between the two haplotypes. B: Image: Haplotype 1 Blob ToolKit Snail plot. (B) A graphical representation of the quality metrics of haplotype 1 (rendered numerically in Table 3) represented as a BlobToolKit Snail plot. The plot circle represents the full size of the assembly. From the inside out, the central plot covers length-related metrics. The longest line represents the size of the longest scaffold; all other scaffolds are arranged in size order, moving clockwise around the plot, and start from the outside of the central plot. The arcs show the scaffold N50 and scaffold N90 values. The central light-gray spiral shows the cumulative scaffold count with a white line at each order of magnitude. White regions in this area reflect the proportion of Ns in the assembly. The dark vs. light-blue area around it shows mean, maximum, and minimum GC vs. AT content at 0.1% intervals (Challis et al. 2020). C: Image: Haplotype 1 contact map. D: Image: Haplotype 2 contact map. E: Image: Haplotype 1 contact map of the 20 largest scaffolds. F: Image: Haplotype 2 contact map of the 20 largest scaffolds. Caption: (C–F) Omni-C contact maps for the haplotype 1 (C) and haplotype 2 (D) nuclear assembly generated with PretextSnapshot. The same contact maps but for the 20 largest scaffolds are presented in (E) and (F), respectively. Contact maps translate proximity of genomic regions in 3D space to contiguous linear organization. Each cell in the contact map corresponds to sequencing data supporting the linkage (or join) between two regions of the genome assembly. Scaffolds are separated by black lines and higher density corresponds to higher levels of fragmentation.

Nuclear genome assembly

The final genome assembly (ddFreCali1) consists of two phased haplotypes. Both assemblies are similar in size, and they are also similar in size but not equal to the estimated genome size from GenomeScope, as has been observed in other taxa (see Pflug et al. 2020, for example).

The haplotype one assembly (ddFreCali1.0.hap1) consists of 389 scaffolds spanning 1.23 Gb with a contig N50 of 11.83 Mb, a scaffold N50 of 23.3 Mb, the largest contig size of 42.29 Mb, and the largest scaffold size of 44.02 Mb. The haplotype two assembly (ddFreCali1.0.hap2) consists of 202 scaffolds spanning 1.22 Gb with a contig N50 of 13.98 Mb, a scaffold N50 of 26.7 Mb, the largest contig size of 28.83 Mb, and the largest scaffold size of 44.01 Mb.

The haplotype one assembly has a BUSCO completeness score for the Embryophyta gene set of 99.4%, a base pair quality value (QV) of 66.07, a kmer completeness of 83.96%, and a frameshift indel QV of 45.37. The haplotype two assembly has a BUSCO completeness score for the Embryophyta gene set of 99.4%, a base pair QV of 66.17, a kmer completeness of 84.06%, and a frameshift indel QV of 46.31.

During manual curation, we made a total of 154 joins (77 per haplotype) and 45 breaks (24 on haplotype one and 21 on haplotype two) based on the Omni-C contact map signal. We closed a total of 40 gaps (22 on haplotype one and 18 on haplotype two). No other contigs were modified or removed. The Omni-C contact maps show highly contiguous assemblies, with chromosome-length scaffolds (Fig. 2B). Assembly statistics are reported in Table 3 and represented graphically in Fig. 2C. We have deposited the genome assembly on NCBI GenBank (see Table 3 and Data Availability for details).

Table 3.

Assembly statistics.

Bio Projects
& vouchers		 CCGP NCBI BioProject PRJNA720569
Genera NCBI BioProject PRJNA765826
Species NCBI BioProject PRJNA765826
NCBI BioSample SAMN35735723
Specimen identification D. Potter 1011
NCBI Genome accessions Haplotype 1 Haplotype 2
Assembly accession JAULJK000000000 JAULJL000000000
Genome sequences GCA_030761205.1 GCA_030761185.1
Genome Sequence PacBio HiFi reads Run 1 PACBIO_SMRT (Sequel IIe) run: 2.5 M spots, 32G bases, 18.4 Gbytes
Accession SRX25150516
Omni-C Illumina reads Run 2 ILLUMINA (Illumina NovaSeq 6000) runs: 147 M spots, 44.4G bases, 15.9 Gbytes
Accession SRX25150517-8
Genome Assembly Quality Metrics Assembly identifier (quality codea) ddFreCali1(7.7.P7.Q66.C95)
HiFi read coverageb 57.39X
Haplotype 1 Haplotype 2
Number of contigs 505 320
Contig N50 (bp) 11 831 472 13 982 549
Contig NG50b 22 154 149 20 612 997
Longest contigs 42 296 852 28 838 739
Number of scaffolds 389 202
Scaffold N50 26 303 202 26 782 959
Scaffold NG50b 37 899 299 36 980 028
Largest scaffold 44 025 563 44 017 524
Size of final assembly 1 233 188 417 1 222 422 046
Phased block NG50b 21 039 688 20 612 997
Gaps per Gbp (# Gaps) 94(116) 97(118)
Indel QV (Frame shift) 45.37399284 46.31611688
Base pair QV 66.1746 66.1746
Full assembly = 66.1283
k-mer completeness 83.966 84.069
Full assembly = 98.8237
BUSCO completeness
(embryophyta) n = 1614		 C c S c D c F c M c
H1d 99.40% 61.90% 37.50% 0.20% 0.40%
H2d 99.40% 62.50% 36.90% 0.20% 0.40%
Organelles Complete chloroplast sequence PV236216
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a

Assembly quality code x.y.P.Q.C derived notation, from (Rhie et al. 2021). x = log10[contig NG50]; y = log10[scaffold NG50]; P = log10 [phased block NG50]; Q = Phred base accuracy QV (quality value); C = % genome represented by the first “n” scaffolds, following a karyotype of 2n = 92 for this species, estimated as a mode from ancestral species number of chromosomes (Genome on a Tree—GoaT; tax_name(Fremontodendron californicum); Challis et al. 2023). Quality code for all the assembly denoted by haplotype one assembly (ddFreCali1.0.hap1).

b

Read coverage and NGx statistics have been calculated based on the estimated genome size of 883.75 Mb.

c

BUSCO Scores. Complete BUSCOs (C). Complete and single-copy BUSCOs (S). Complete and duplicated BUSCOs (D). Fragmented BUSCOs (F). Missing BUSCOs (M).

d

(H1) Haplotype 1 and (H2) Haplotype 2 assembly values.

De novo repeat libraries proposed by RepeatModeler were used to mask repeat elements in each assembly with RepeatMasker. Haplotype 1 and haplotype 2 are similar in terms of repeat element content with 71.87% and 72.06% masked, respectively (Table 4). Retroelements comprise most of the previously described repeat element content (about 32% for each haplotype) followed by DNA transposons (2% for each haplotype). Long terminal repeats and especially the Gypsy/DIRS-1 group of LTRs make up most of the retroelements (roughly 26% per haplotype), while the MULE-MuDR family makes up most of the DNA transposons (at 1.5%). Almost half of the masked repeat elements are designated as unclassified by RepeatMasker at around 33% per haplotype.

Table 4.

Results of RepeatMasker for both the haplotype 1 and haplotype 2 assemblies. Custom repeat libraries for RepeatMasker for each assembly were built de novo using RepeatModeler.

Haplotype 1 (ddFreCali1.0.hap1) Haplotype 2 (ddFreCali1.0.hap2)
Number of elements Length occupied (bp) % of sequence Number of elements Length occupied (bp) % of sequence
Retroelements 237,048 403,922,529 32.75 217,571 391,630,342 32.03
SINEs: 91 21 994 0.00 676 107,715 0.01
Penelope: 340 313,297 0.03 0 0 0.00
LINEs: 15,953 14,778,940 1.20 14,006 14,599,861 1.19
CRE/SLACS 0 0 0.00 0 0 0.00
L2/CR1/Rex 0 0 0.00 0 0 0.00
R1/LOA/Jockey 420 57,348 0.00 0 0 0.00
R2/R4/NeSL 0 0 0.00 0 0 0.00
RTE/Bov-B 433 238,308 0.02 954 348,927 0.03
L1/CIN4 15,100 14,483,284 1.17 13,052 14,250,934 1.17
LTR elements: 221,004 389,121,595 31.55 202,889 376,922,766 30.83
BEL/Pao 13,129 14,428,258 1.17 4468 3,200,323 0.26
Ty1/Copia 39,916 50,787,200 4.12 42,885 50,177,934 4.10
Gypsy/DIRS1 153,121 314,257,337 25.48 151,285 321,818,303 26.32
Retroviral 12,613 8,390,902 0.68 239 191,896 0.02
DNA transposons 48,246 30,040,759 2.44 39,739 24,818,635 2.03
hobo-Activator 8385 2,254,647 0.18 3587 1,271,713 0.10
Tc1-IS630-Pogo 548 192,830 0.02 0 0 0.00
En-Spm 0 0 0.00 0 0 0.00
MULE-MuDR 16,873 18,016,929 1.46 21,361 19,085,571 1.56
PiggyBac 0 0 0.00 0 0 0.00
Tourist/Harbinger 705 349,504 0.03 647 389,942 0.03
Other (Mirage, P-element, Transib) 0 0 0.00 0 0 0.00
Rolling-circles 6004 2,601,743 0.21 9851 3,898,411 0.32
Unclassified: 1,056,958 417,975,707 33.89 1,093,401 436,438,733 35.70
Total interspersed repeats: 852,252,292 69.11 852,887,710 69.76
Small RNA: 23 915 13,830,187 1.12 1584 8,880,462 0.73
Satellites: 322 156,719 0.01 0 0 0.00
Simple repeats: 300,908 14,429,618 1.17 287,131 12,493,773 1.02
Low complexity: 56,915 3,016,923 0.24 54,726 2,797,318 0.23
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Chloroplast genome assembly

The chloroplast assembly size is 160,827 bp, with 36.75% GC content. The large single copy (LSC) is 89,841 bp long, the small single copy (SSC) is 20,317 bp long, and the inverted repeat (IR) is 25,333 bp long. This plastome contains a total of 290 genes including rRNAs and tRNAs (not counting the duplication of the IR).

Discussion

The nuclear genome assembly of F. californicum is the first of its kind for this genus and will be an invaluable resource to evaluate the intraspecific and interspecific diversity of this important constituent of the California flora. It will also expand our genomic knowledge of the family Malvaceae, which contains several taxa of tremendous economic importance and has also been the subject of much evolutionary research. The precise phylogenetic position of Fremontodendron within the family has not been established with certainty, but the most recent evidence indicates it is a basal member of the Malvatheca, a clade which encompasses the subfamilies Bombacoideae and Malvoideae (Nyffeler et al. 2005). Thus, this genome assembly should be of considerable interest beyond its significance for conservation efforts within California.

The number of chromosomes as indicated by the Omni-C contact map (n = 46, Fig. 2) does not match the karyotype as reported from a camera lucida study where n = 20 (Lenz 1950), which was based on an individual of F. californicum from Tehama County. It’s unlikely that the individual of F. californicum used in our study is an auto or allopolyploid as the GenomeScope Profile’s bimodal distribution indicates a diploid genome (Fig. 2). The taxonomic treatment of F. decumbens estimated the chromosome number of that species to be n = 49 (Lloyd 1965), which more closely aligns with our finding. It’s possible that the camera lucida study vastly underestimated the number of chromosomes, given the nature of that technique; it’s also possible that there is variation in chromosome number within F. californicum, as it’s currently described, which warrants further investigation.

Other published assemblies for genera in Malvaceae (s.l.) include Gossypium, Hibiscus, and Abelmoschus in subfamily Malovideae; Bombax and Adansonia in Bombacoideae; Durio in Helicteroideae; Firmiana in Sterculioideae; Theobroma in Byttnerioideae; and Microcos and Corchorus in Grewioideae (following the circumscriptions as proposed by Cvetković et al. 2021). The Fremontodendron assembly has a similar chromosome number but about two-thirds the size of another basal member of the Malvatheca, Ochroma pyramidale, where n = 42 with a genome size of 1884 Mb (Sahu et al. 2023). The Fremontodendron assembly, while not at the chromosome scale of the previously discussed assemblies, demonstrates a comparable scaffold N50, suggesting similar contiguity with other published genomes in Malvaceae such as Theobroma cacao = 36.4 Mb and Hibiscus sabdariffa = 26.25 Mb (Kim et al. 2024).

The total percentage of repeat elements is higher than similar analyses of T. cacao genomes (52.3% to 61.5%), with the same study suggesting that domesticated genotypes have lower repeat content than wild counterparts (Nousias et al. 2024). F. californicum’s repeat content is similar to that of a recent assembly of H. sabdariffa (74.48% masked), a domesticated tetraploid species (Kim et al. 2024). These results indicate that F. californicum’s genome has undergone a complex evolutionary history, maintaining a large amount of repetitive elements, especially a relatively high percentage of as-of-yet unclassified elements (33.89% and 35.7% in haplotype 1 and haplotype 2, respectively). Whole genome multiplications (WGMs) have been documented throughout Malvaceae s.l. with a notable allopolyploid multiplication occurring in the common ancestor of the Malvatheca (Conover et al. 2019). This multiplication may be best described as a decaploidization of the Malvaceae ancestral karyotype (n = 11) resulting from an allotetraploid hybridizing with an allohexaploid, which was also the common ancestor of the Helicteroideae. In addition to WGMs, chromosomal structural changes, including reciprocal translocations and chromosomal fusions, have played a considerable role in shaping the genomic architecture of the Malvatheca (Sun et al. 2024). These findings may help to explain why F. californicum’s diploid genome has a relatively high chromosome count (n = 46), a large volume of repetitive elements, and observed duplications among the BUSCO genes (Table 3). This assembly will be a tool to help further clarify Malvaceae s.l.’s complex evolutionary history.

The genome assembly of F. californicum will also become the basis for future studies to understand the genomic and morphological variation within Fremontodendorn, which will have consequences for the taxonomic identity and status as rare of different populations throughout California. Establishing the taxonomic identities of morphologically ambiguous populations will clarify the precise distribution of the widespread and morphologically variable F. californicum and its rare and endangered sister taxa, F. decumbens and F. mexicanum. This, in turn, will have important management and conservation implications. This genomic resource furthers the goals of the CCGP to generate genomic data across multiple ecoregions, and between widespread and restricted/rare taxa, within California. This genome assembly, in combination with subsequent resequencing data funded through the CCGP, will provide a rich source of information which can guide current and future conservation strategies in the state.

Acknowledgments

PacBio Sequel II/IIe library prep and sequencing were carried out at the DNA Technologies and Expression Analysis Core at the UC Davis Genome Center, supported by NIH Shared Instrumentation Grant 1S10OD010786-01. Deep sequencing of Omni-C libraries used the Novaseq S4 sequencing platforms at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumentation Grant. We thank the staff at the UC Davis DNA Technologies and Expression Analysis Core and the UC Santa Cruz Paleogenomics Laboratory for their diligence and dedication to generating high-quality sequence data. Support from the UC Davis Agricultural Experiment Station (project number CA-D-PLS-6273-H) is gratefully acknowledged.

Contributor Information

William T McMahan, Department of Plant Sciences, University of California, Davis, CA, United States.

Merly Escalona, Department of Biomolecular Engineering, University of California, Santa Cruz, CA, United States.

Reed Kenny, Department of Plant Sciences, University of California, Davis, CA, United States.

Mohan P A Marimuthu, DNA Technologies and Expression Analysis Core Laboratory, Genome Center, University of California, Davis, CA, United States.

Oanh Nguyen, DNA Technologies and Expression Analysis Core Laboratory, Genome Center, University of California, Davis, CA, United States.

Colin W Fairbairn, Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, CA, United States.

William Seligmann, Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, CA, United States.

Courtney Miller, Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA, United States.

Howard Bradley Shaffer, Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA, United States.

Shannon Still, Department of Plant Sciences, University of California, Davis, CA, United States.

Daniel Potter, Department of Plant Sciences, University of California, Davis, CA, United States.

Author contributions

William T. McMahan (Formal analysis, Project administration, Writing—original draft, Writing—review & editing), Merly Escalona (Data curation, Formal analysis, Methodology, Project administration, Software, Visualization, Writing—original draft, Writing—review & editing), Reed Kenny (Data curation, Formal analysis, Methodology), Mohan P.A. Marimuthu (Data curation, Methodology, Writing—original draft), Oanh Nguyen (Data curation, Methodology, Writing—original draft), Colin W. Fairbairn (Data curation, Methodology), William Seligmann (Data curation, Methodology, Writing—original draft), Courtney Miller (Project administration, Resources), H. Bradley Shaffer (Funding acquisition, Project administration, Resources, Supervision), Shannon Still (Conceptualization, Data curation, Funding acquisition, Writing—original draft), and Daniel Potter (Conceptualization, Data curation, Funding acquisition, Project administration, Supervision, Writing—original draft)

Funding

This work was supported by the California Conservation Genomics Project, with funding provided to the University of California by the State of California, State Budget Act of 2019 [UC Award ID RSI-19-690224].

Data availability

Data generated for this study are available under NCBI BioProject PRJNA1126700. Raw sequencing data for sample Potter1011 (NCBI BioSample SAMN35735723) are deposited in the NCBI Short Read Archive (SRA) under SRR29646341 for PacBio HiFi sequencing data, and SRR29646339, SRR29646340 for the Omni-C Illumina sequencing data. GenBank accessions for both primary and alternate assemblies are GCA_030761205.1 and GCA_030761185.1; and for genome sequences JAULJK000000000 and JAULJL000000000. Assembly scripts and other data for the analyses presented can be found at the following GitHub repository: www.github.com/ccgproject/ccgp_assembly

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data generated for this study are available under NCBI BioProject PRJNA1126700. Raw sequencing data for sample Potter1011 (NCBI BioSample SAMN35735723) are deposited in the NCBI Short Read Archive (SRA) under SRR29646341 for PacBio HiFi sequencing data, and SRR29646339, SRR29646340 for the Omni-C Illumina sequencing data. GenBank accessions for both primary and alternate assemblies are GCA_030761205.1 and GCA_030761185.1; and for genome sequences JAULJK000000000 and JAULJL000000000. Assembly scripts and other data for the analyses presented can be found at the following GitHub repository: www.github.com/ccgproject/ccgp_assembly

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