Chromosome-level genome assembly of the threatened resource plant Cinnamomum chago.

Cinnamomum chago is a tree species endemic to Yunnan province, China, with potential economic value, phylogenetic importance, and conservation priority. We assembled the genome of C. chago using multiple sequencing technologies, resulting in a high-quality, chromosomal-level genome with annotation information. The assembled genome size is approximately 1.06 Gb, with a contig N50 length of 92.10 Mb. About 99.92% of the assembled sequences could be anchored to 12 pseudo-chromosomes, with only one gap, and 63.73% of the assembled genome consists of repeat sequences. In total, 30,497 genes were recognized according to annotation, including 28,681 protein-coding genes. This high-quality chromosome-level assembly and annotation of C. chago will assist us in the conservation and utilization of this valuable resource, while also providing crucial data for studying the evolutionary relationships within the Cinnamomum genus, offering opportunities for further research and exploration of its diverse applications.

Gap-free X and Y chromosome assemblies of Salix arbutifolia reveal an evolutionary change from male to female heterogamety in willows, without a change in the position of the sex-determining locus.

In the Vetrix clade of Salix, a genus of woody flowering plants, sex determination involves chromosome 15, but an XY system has changed to a ZW system. We studied the detailed genetic changes involved. We used genome sequencing, with chromosome conformation capture (Hi-C) and PacBio HiFi reads to assemble chromosome level gap-free X and Y of Salix arbutifolia, and distinguished the haplotypes in the 15X- and 15Y-linked regions, to study the evolutionary history of the sex-linked regions (SLRs). Our sequencing revealed heteromorphism of the X and Y haplotypes of the SLR, with the X-linked region being considerably larger than the corresponding Y region, mainly due to accumulated repetitive sequences and gene duplications. The phylogenies of single-copy orthogroups within the SLRs indicate that S. arbutifolia and Salix purpurea share an ancestral SLR within a repeat-rich region near the chromosome 15 centromere. During the change in heterogamety, the X-linked region changed to a W-linked one, while the Z was derived from the Y.

High-quality genome assembly enables prediction of allele-specific gene expression in hybrid poplar.

Poplar (Populus) is a well-established model system for tree genomics and molecular breeding, and hybrid poplar is widely used in forest plantations. However, distinguishing its diploid homologous chromosomes is difficult, complicating advanced functional studies on specific alleles. In this study, we applied a trio-binning design and PacBio high-fidelity long-read sequencing to obtain haplotype-phased telomere-to-telomere genome assemblies for the 2 parents of the well-studied F1 hybrid "84K" (Populus alba × Populus tremula var. glandulosa). Almost all chromosomes, including the telomeres and centromeres, were completely assembled for each haplotype subgenome apart from 2 small gaps on one chromosome. By incorporating information from these haplotype assemblies and extensive RNA-seq data, we analyzed gene expression patterns between the 2 subgenomes and alleles. Transcription bias at the subgenome level was not uncovered, but extensive-expression differences were detected between alleles. We developed machine-learning (ML) models to predict allele-specific expression (ASE) with high accuracy and identified underlying genome features most highly influencing ASE. One of our models with 15 predictor variables achieved 77% accuracy on the training set and 74% accuracy on the testing set. ML models identified gene body CHG methylation, sequence divergence, and transposon occupancy both upstream and downstream of alleles as important factors for ASE. Our haplotype-phased genome assemblies and ML strategy highlight an avenue for functional studies in Populus and provide additional tools for studying ASE and heterosis in hybrids.

The chromosome-scale genome of Magnolia sinica (Magnoliaceae) provides insights into the conservation of plant species with extremely small populations (PSESP).

Magnolia sinica (Magnoliaceae) is a highly threatened tree endemic to southeast Yunnan, China. In this study, we generated for the first time a high-quality chromosome-scale genome sequence from M. sinica, by combining Illumina and ONT data with Hi-C scaffolding methods. The final assembled genome size of M. sinica was 1.84 Gb, with a contig N50 of ca. 45 Mb and scaffold N50 of 92 Mb. Identified repeats constituted approximately 57% of the genome, and 43,473 protein-coding genes were predicted. Phylogenetic analysis shows that the magnolias form a sister clade with the eudicots and the order Ceratophyllales, while the monocots are sister to the other core angiosperms. In our study, a total of 21 individuals from the 5 remnant populations of M. sinica, as well as 22 specimens belonging to 8 related Magnoliaceae species, were resequenced. The results showed that M. sinica had higher genetic diversity (θw = 0.01126 and θπ = 0.01158) than other related species in the Magnoliaceae. However, population structure analysis suggested that the genetic differentiation among the 5 M. sinica populations was very low. Analyses of the demographic history of the species using different models consistently revealed that 2 bottleneck events occurred. The contemporary effective population size of M. sinica was estimated to be 10.9. The different patterns of genetic loads (inbreeding and numbers of deleterious mutations) suggested constructive strategies for the conservation of these 5 different populations of M. sinica. Overall, this high-quality genome will be a valuable genomic resource for conservation of M. sinica.

Genomic analyses provide insights into sex differentiation of tetraploid strawberry (Fragaria moupinensis).

The strawberry genus, Fragaria, exhibits a wide range of sexual systems and natural ploidy variation. Nearly, all polyploid strawberry species exhibit separate sexes (dioecy). Research has identified the sex-determining sequences as roughly conserved but with repeatedly changed genomic locations across octoploid strawberries. However, it remains unclear whether tetraploid wild strawberries evolved dioecy independently or shared a common origin with octoploid strawberries. In this study, we investigated the sex determinants of F. moupinensis, a dioecious plant with heterogametic females (ZW). Utilizing a combination of haplotype-resolved genome sequencing of the female F. moupinensis, k-mer-based and coverage-based genome-wide association studies (GWAS), and transcriptomic analysis, we discovered a non-recombining, approximately 33.6 kb W-specific region on chromosome 2a. Within this region, only one candidate sex-determining gene (FmoAFT) was identified. Furthermore, an extensive resequencing of the entire Fragaria genus indicated that the W-specific region displays conservative female specificity across all tetraploid species. This observation suggests that dioecy evolved independently in tetraploid and octoploid strawberries. Moreover, employing virus-induced gene silencing (VIGS), we knocked down the expression of the FmoAFT homologue transcript in cultivated strawberries, revealing its potential role in promoting female functions during early carpel development. We also applied DNA affinity purification sequencing (DAP-seq) and yeast one-hybrid assays to identify potential direct targets of FmoAFT. These insights shed new light on the genetic basis and evolutionary history of sex determination in strawberries, thereby facilitating the formulation of strategies to manipulate sex determination in breeding programs.

A chromosome-level genome assembly for Onobrychis viciifolia reveals gene copy number gain underlying enhanced proanthocyanidin biosynthesis.

Sainfoin (Onobrychis viciifolia), which belongs to subfamily Papilionoideae of Leguminosae, is a vital perennial forage known as "holy hay" due to its high contents of crude proteins and proanthocyanidins (PAs, also called condensed tannins) that have various pharmacological properties in animal feed, such as alleviating rumen tympanic disease in ruminants. In this study, we select an autotetraploid common sainfoin (2n = 4x = 28) and report its high-quality chromosome-level genome assembly with 28 pseudochromosomes and four haplotypes (~1950.14 Mb, contig N50 = 10.91 Mb). The copy numbers of genes involved in PA biosynthesis in sainfoin are significantly greater than those in four selected Fabales species, namely, autotetraploid Medicago sativa and three other diploid species, Lotus japonicus, Medicago truncatula, and Glycine max. Furthermore, gene expansion is confirmed to be the key contributor to the increased expression of these genes and subsequent PA enhancement in sainfoin. Transcriptomic analyses reveal that the expression of genes involved in the PA biosynthesis pathway is significantly increased in the lines with high PA content compared to the lines with medium and low PA content. The sainfoin genome assembly will improve our understanding of leguminous genome evolution and biosynthesis of secondary metabolites in sainfoin.

Chromosome-scale genome assembly of sweet tea (Lithocarpus polystachyus Rehder).

Lithocarpus, with >320 species, is the second largest genus of Fagaceae. However, the lack of a reference genome limits the molecular biology and functional study of Lithocarpus species. Here, we report the chromosome-scale genome assembly of sweet tea (Lithocarpus polystachyus Rehder), the first Lithocarpus species to be sequenced to date. Sweet tea has a 952-Mb genome, with a 21.4-Mb contig N50 value and 98.6% complete BUSCO score. In addition, the per-base consensus accuracy and completeness of the genome were estimated at 60.6 and 81.4, respectively. Genome annotation predicted 37,396 protein-coding genes, with repetitive sequences accounting for 64.2% of the genome. The genome did not undergo whole-genome duplication after the gamma (γ) hexaploidy event. Phylogenetic analysis showed that sweet tea diverged from the genus Quercus approximately at 59 million years ago. The high-quality genome assembly and gene annotation resources enrich the genomics of sweet tea, and will facilitate functional genomic studies in sweet tea and other Fagaceae species.

Haplotype-resolved chromosomal-level genome assembly of Buzhaye (Microcos paniculata).

Microcos paniculata is a shrub used traditionally as folk medicine and to make herbal teas. Previous research into this species has mainly focused on its chemical composition and medicinal value. However, the lack of a reference genome limits the study of the molecular mechanisms of active compounds in this species. Here, we assembled a haplotype-resolved chromosome-level genome of M. paniculata based on PacBio HiFi and Hi-C data. The assembly contains two haploid genomes with sizes 399.43 Mb and 393.10 Mb, with contig N50 lengths of 43.44 Mb and 30.17 Mb, respectively. About 99.93% of the assembled sequences could be anchored to 18 pseudo-chromosomes. Additionally, a total of 482 Mb repeat sequences were identified, accounting for 60.76% of the genome. A total of 49,439 protein-coding genes were identified, of which 48,979 (99%) were functionally annotated. This haplotype-resolved chromosome-level assembly and annotation of M. paniculata will serve as a valuable resource for investigating the biosynthesis and genetic basis of active compounds in this species, as well as advancing evolutionary phylogenomic studies in Malvales.

Telomere-to-telomere and haplotype-resolved genome assembly of the Chinese cork oak (Quercus variabilis).

The Quercus variabilis, a deciduous broadleaved tree species, holds significant ecological and economical value. While a chromosome-level genome for this species has been made available, it remains riddled with unanchored sequences and gaps. In this study, we present a nearly complete comprehensive telomere-to-telomere (T2T) and haplotype-resolved reference genome for Q. variabilis. This was achieved through the integration of ONT ultra-long reads, PacBio HiFi long reads, and Hi-C data. The resultant two haplotype genomes measure 789 Mb and 768 Mb in length, with a contig N50 of 65 Mb and 56 Mb, and were anchored to 12 allelic chromosomes. Within this T2T haplotype-resolved assembly, we predicted 36,830 and 36,370 protein-coding genes, with 95.9% and 96.0% functional annotation for each haplotype genome. The availability of the T2T and haplotype-resolved reference genome lays a solid foundation, not only for illustrating genome structure and functional genomics studies but also to inform and facilitate genetic breeding and improvement of cultivated Quercus species.

A haplotype-resolved genome assembly of Rhododendron vialii based on PacBio HiFi reads and Hi-C data.

Rhododendron vialii (subgen. Azaleastrum) is an evergreen shrub with high ornamental value. This species has been listed as a plant species with extremely small populations (PSESP) for urgent protection by China's Yunnan provincial government in 2021, due to anthropogenic habitat fragmentation. However, limited genomic resources hinder scientifically understanding of genetic threats that the species is currently facing. In this study, we assembled a high-quality haplotype-resolved genome of R. vialii based on PacBio HiFi long reads and Hi-C reads. The assembly contains two haploid genomes with sizes 532.73 Mb and 521.98 Mb, with contig N50 length of 35.67 Mb and 34.70 Mb, respectively. About 99.92% of the assembled sequences could be anchored to 26 pseudochromosomes, and 14 gapless assembled chromosomes were included in this assembly. Additionally, 60,926 protein-coding genes were identified, of which 93.82% were functionally annotated. This is the first reported genome of R. vialii, and hopefully it will lay the foundations for further research into the conservation genomics and horticultural domestication of this ornamentally important species.

Haplotype-resolved genome assembly of Coriaria nepalensis a non-legume nitrogen-fixing shrub.

Coriaria nepalensis Wall. (Coriariaceae) is a nitrogen-fixing shrub which forms root nodules with the actinomycete Frankia. Oils and extracts of C. nepalensis have been reported to be bacteriostatic and insecticidal, and C. nepalensis bark provides a valuable tannin resource. Here, by combining PacBio HiFi sequencing and Hi-C scaffolding techniques, we generated a haplotype-resolved chromosome-scale genome assembly for C. nepalensis. This genome assembly is approximately 620 Mb in size with a contig N50 of 11 Mb, with 99.9% of the total assembled sequences anchored to 40 pseudochromosomes. We predicted 60,862 protein-coding genes of which 99.5% were annotated from databases. We further identified 939 tRNAs, 7,297 rRNAs, and 982 ncRNAs. The chromosome-scale genome of C. nepalensis is expected to be a significant resource for understanding the genetic basis of root nodulation with Frankia, toxicity, and tannin biosynthesis.

The chromosome-level genome assembly and genes involved in biosynthesis of nervonic acid of Malania oleifera.

Nervonic acid (C24:1 Δ15, NA) is a very long-chain monounsaturated fatty acid, a clinically indispensable resource in maintaining the brain and nerve cells development and regeneration. Till now, NA has been found in 38 plant species, among which the garlic-fruit tree (Malania oleifera) has been evaluated to be the best candidate for NA production. Here, we generated a high-quality chromosome-scale assembly of M. oleifera employing PacBio long-read, short-read Illumina as well as Hi-C sequencing data. The genome assembly consisted of 1.5 Gb with a contig N50 of ~4.9 Mb and a scaffold N50 of ~112.6 Mb. ~98.2% of the assembly was anchored into 13 pseudo-chromosomes. It contains ~1123 Mb repeat sequences, and 27,638 protein-coding genes, 568 tRNAs, 230 rRNAs and 352 other non-coding RNAs. Additionally, we documented candidate genes involved in NA biosynthesis including 20 KCSs, 4 KCRs, 1 HCD and 1 ECR, and profiled the expression patterns of these genes in developing seeds. The high-quality assembly of the genome provides insights into the genome evolution of the M. oleifera genome and candidate genes involved in NA biosynthesis in the seeds of this important woody tree.

Evolutionary history of two evergreen Rhododendron species as revealed by chromosome-level genome assembly.

Background: The genus Rhododendron (Ericaceae), a species-rich and widely distributed genus of woody plants, is distinguished for the beautiful and diverse flowers. Rhododendron delavayi Franch. and Rhododendron irroratum Franch., are highly attractive species widely distributed in south-west China and abundant new varieties have been selected from their genetic resources. Methods: We constructed chromosome-scale genome assemblies for Rhododendron delavayi and Rhododendron irroratum. Phylogenetic and whole-genome duplication analyses were performed to elucidate the evolutionary history of Rhododendron. Further, different types of gene duplications were identified and their contributions to gene family expansion were investigated. Finally, comprehensive characterization and evolutionary analysis of R2R3-MYB and NBS-encoding genes were conducted to explore their evolutionary patterns. Results: The phylogenetic analysis classified Rhododendron species into two sister clades, 'rhododendrons' and 'azaleas'. Whole-genome duplication (WGD) analysis unveiled only one WGD event that occurred in Rhododendron after the ancestral γ triplication. Gene duplication and gene family expansion analyses suggested that the younger tandem and proximal duplications contributed greatly to the expansion of gene families involved in secondary metabolite biosynthesis and stress response. The candidate R2R3-MYB genes likely regulating anthocyanin biosynthesis and stress tolerance in Rhododendron will facilitate the breeding for ornamental use. NBS-encoding genes had undergone significant expansion and experienced species-specific gain and loss events in Rhododendron plants. Conclusions: The reference genomes presented here will provide important genetic resources for molecular breeding and genetic improvement of plants in this economically important Rhododendron genus.

Gapless genome assembly of azalea and multi-omics investigation into divergence between two species with distinct flower color.

The genus Rhododendron (Ericaceae), with more than 1000 species highly diverse in flower color, is providing distinct ornamental values and a model system for flower color studies. Here, we investigated the divergence between two parental species with different flower color widely used for azalea breeding. Gapless genome assembly was generated for the yellow-flowered azalea, Rhododendron molle. Comparative genomics found recent proliferation of long terminal repeat retrotransposons (LTR-RTs), especially Gypsy, has resulted in a 125 Mb (19%) genome size increase in species-specific regions, and a significant amount of dispersed gene duplicates (13 402) and pseudogenes (17 437). Metabolomic assessment revealed that yellow flower coloration is attributed to the dynamic changes of carotenoids/flavonols biosynthesis and chlorophyll degradation. Time-ordered gene co-expression networks (TO-GCNs) and the comparison confirmed the metabolome and uncovered the specific gene regulatory changes underpinning the distinct flower pigmentation. B3 and ERF TFs were found dominating the gene regulation of carotenoids/flavonols characterized pigmentation in R. molle, while WRKY, ERF, WD40, C2H2, and NAC TFs collectively regulated the anthocyanins characterized pigmentation in the red-flowered R simsii. This study employed a multi-omics strategy in disentangling the complex divergence between two important azaleas and provided references for further functional genetics and molecular breeding.

Subgenome phasing for complex allopolyploidy: case-based benchmarking and recommendations.

Accurate subgenome phasing is crucial for understanding the origin, evolution and adaptive potential of polyploid genomes. SubPhaser and WGDI software are two common methodologies for subgenome phasing in allopolyploids, particularly in scenarios lacking known diploid progenitors. Triggered by a recent debate over the subgenomic origins of the cultivated octoploid strawberry, we examined four well-documented complex allopolyploidy cases as benchmarks, to evaluate and compare the accuracy of the two software. Our analysis demonstrates that the subgenomic structure phased by both software is in line with prior research, effectively tracing complex allopolyploid evolutionary trajectories despite the limitations of each software. Furthermore, using these validated methodologies, we revisited the controversial issue regarding the progenitors of the octoploid strawberry. The results of both methodologies reaffirm Fragaria vesca and Fragaria iinumae as progenitors of the octoploid strawberry. Finally, we propose recommendations for enhancing the accuracy of subgenome phasing in future studies, recognizing the potential of integrated tools for advanced complex allopolyploidy research and offering a new roadmap for robust subgenome-based phylogenetic analysis.

A chromosome-level genome assembly of the Chinese cork oak (Quercus variabilis).

Quercus variabilis (Fagaceae) is an ecologically and economically important deciduous broadleaved tree species native to and widespread in East Asia. It is a valuable woody species and an indicator of local forest health, and occupies a dominant position in forest ecosystems in East Asia. However, genomic resources from Q. variabilis are still lacking. Here, we present a high-quality Q. variabilis genome generated by PacBio HiFi and Hi-C sequencing. The assembled genome size is 787 Mb, with a contig N50 of 26.04 Mb and scaffold N50 of 64.86 Mb, comprising 12 pseudo-chromosomes. The repetitive sequences constitute 67.6% of the genome, of which the majority are long terminal repeats, accounting for 46.62% of the genome. We used ab initio, RNA sequence-based and homology-based predictions to identify protein-coding genes. A total of 32,466 protein-coding genes were identified, of which 95.11% could be functionally annotated. Evolutionary analysis showed that Q. variabilis was more closely related to Q. suber than to Q. lobata or Q. robur. We found no evidence for species-specific whole genome duplications in Quercus after the species had diverged. This study provides the first genome assembly and the first gene annotation data for Q. variabilis. These resources will inform the design of further breeding strategies, and will be valuable in the study of genome editing and comparative genomics in oak species.

Deferoxamine/magnesium modified ß-tricalcium phosphate promotes the bone regeneration in osteoporotic rats.

Recently, Deferoxamine (DFO) and magnesium (Mg) have been identified as critical factors for angiogenesis and bone formation. However, in current research studies, there is a lack of focus on whether DFO plus Mg can affect the regeneration of ß-tricalcium phosphate (ß-TCP) in osteoporosis and through what biological mechanisms. Therefore, the present work was aimed to preparation and evaluate the effect of Deferoxamine/magnesium modified ß-tricalcium phosphate promotes (DFO/Mg-TCP) in ovariectomized rats model and preliminary exploration of possible mechanisms. The MC3T3-E1 cells were co-cultured with the exudate of DFO/Mg-TCP and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Counting Kit-8(CCK-8), Alkaline Phosphatase (ALP) staining, Alizarin Red Staining (RES) and Western Blot. In vitro experiments, CCK-8, ALP and ARS staining results show that the mineralization and osteogenic activity of MC3T3-E1increased significantly after intervention by DFO/Mg-TCP, as well as a higher levels of protein expressions including VEGF, OC, Runx-2 and HIF-1α. In vivo experiment, Micro-CT and Histological analysis evaluation show that DFO/Mg-TCP treatment presented the stronger effect on bone regeneration, bone mineralization and biomaterial degradation, when compared with OVX+Mg-TCP group and OVX+TCP group, as well as a higher VEGF, OC, Runx-2 and HIF-1α gene expression. The present study indicates that treatment with DFO/Mg-TCP was associated with increased regeneration by enhancing the function of osteoblasts in an OVX rat.

Repetitive Elements, Sequence Turnover and Cyto-Nuclear Gene Transfer in Gymnosperm Mitogenomes.

Among the three genomes in plant cells, the mitochondrial genome (mitogenome) is the least studied due to complex recombination and intergenomic transfer. In gymnosperms only ∼20 mitogenomes have been released thus far, which hinders a systematic investigation into the tempo and mode of mitochondrial DNA evolution in seed plants. Here, we report the complete mitogenome sequence of Platycladus orientalis (Cupressaceae). This mitogenome is assembled as two circular-mapping chromosomes with a size of ∼2.6 Mb and which contains 32 protein-coding genes, three rRNA and seven tRNA genes, and 1,068 RNA editing sites. Repetitive sequences, including dispersed repeats, transposable elements (TEs), and tandem repeats, made up 23% of the genome. Comparative analyses with 17 other mitogenomes representing the five gymnosperm lineages revealed a 30-fold difference in genome size, 80-fold in repetitive content, and 230-fold in substitution rate. We found dispersed repeats are highly associated with mitogenome expansion (r = 0.99), and most of them were accumulated during recent duplication events. Syntenic blocks and shared sequences between mitogenomes decay rapidly with divergence time (r = 0.53), with the exceptions of Ginkgo and Cycads which retained conserved genome structure over long evolutionary time. Our phylogenetic analysis supports a sister group relationship of Cupressophytes and Gnetophytes; both groups are unique in that they lost 8-12 protein-coding genes, of which 4-7 intact genes are likely transferred to nucleus. These two clades also show accelerated and highly variable substitution rates relative to other gymnosperms. Our study highlights the dynamic and enigmatic evolution of gymnosperm mitogenomes.

Cultivated hawthorn (Crataegus pinnatifida var. major) genome sheds light on the evolution of Maleae (apple tribe).

Cultivated hawthorn (Crataegus pinnatifida var. major) is an important medicinal and edible plant with a long history of use for health protection in China. Herein, we provide a de novo chromosome-level genome sequence of the hawthorn cultivar "Qiu Jinxing." We assembled an 823.41 Mb genome encoding 40 571 genes and further anchored the 779.24 Mb sequence into 17 pseudo-chromosomes, which account for 94.64% of the assembled genome. Phylogenomic analyses revealed that cultivated hawthorn diverged from other species within the Maleae (apple tribe) at approximately 35.4 Mya. Notably, genes involved in the flavonoid and triterpenoid biosynthetic pathways have been significantly amplified in the hawthorn genome. In addition, our results indicated that the Maleae share a unique ancient tetraploidization event; however, no recent independent whole-genome duplication event was specifically detected in hawthorn. The amplification of non-specific long terminal repeat retrotransposons contributed the most to the expansion of the hawthorn genome. Furthermore, we identified two paleo-sub-genomes in extant species of Maleae and found that these two sub-genomes showed different rearrangement mechanisms. We also reconstructed the ancestral chromosomes of Rosaceae and discussed two possible paleo-polyploid origin patterns (autopolyploidization or allopolyploidization) of Maleae. Overall, our study provides an improved context for understanding the evolution of Maleae species, and this new high-quality reference genome provides a useful resource for the horticultural improvement of hawthorn.