Haplotype-based pangenomes reveal genetic variations and climate adaptations in moso bamboo populations.

Moso bamboo (Phyllostachys edulis), an ecologically and economically important forest species in East Asia, plays vital roles in carbon sequestration and climate change mitigation. However, intensifying climate change threatens moso bamboo survival. Here we generate high-quality haplotype-based pangenome assemblies for 16 representative moso bamboo accessions and integrated these assemblies with 427 previously resequenced accessions. Characterization of the haplotype-based pangenome reveals extensive genetic variation, predominantly between haplotypes rather than within accessions. Many genes with allele-specific expression patterns are implicated in climate responses. Integrating spatiotemporal climate data reveals more than 1050 variations associated with pivotal climate factors, including temperature and precipitation. Climate-associated variations enable the prediction of increased genetic risk across the northern and western regions of China under future emissions scenarios, underscoring the threats posed by rising temperatures. Our integrated haplotype-based pangenome elucidates moso bamboo's local climate adaptation mechanisms and provides critical genomic resources for addressing intensifying climate pressures on this essential bamboo. More broadly, this study demonstrates the power of long-read sequencing in dissecting adaptive traits in climate-sensitive species, advancing evolutionary knowledge to support conservation.

Dynamics of rhizosphere microbial structure and function associated with the biennial bearing of moso bamboo.

Moso bamboo (Phyllostachys edulis) is a valuable nontimber forestry product with a biennial cycle, producing abundant bamboo shoots within one year (on-year) and few shoots within the following year (off-year). Moso bamboo plants undergo clonal reproduction, resulting in similar genetic backgrounds. However, the number of moso bamboo shoots produced each year varies. Despite this variation, the impact of soil nutrients and the root microbiome on the biennial bearing of moso bamboo is poorly understood. We collected 139 soil samples and determined 14 major physicochemical properties of the rhizosphere, rhizoplane, and bulk soil in different seasons (i.e., the growing and deciduous seasons) and different years (i.e., on- and off-years). Based on 16S rRNA and metagenomic sequencing, major variations were found in the rhizospheric microbial composition during different seasons and years in the moso bamboo forest. Environmental driver analysis revealed that essential nutrients (i.e., SOC, TOC, TN, P, and NH4+) were the main drivers of the soil microbial community composition and were correlated with the on- and off-year cycles. Moreover, 19 MAGs were identified as important biomarkers that could distinguish on- and off-years. We found that both season and year influenced both the microbial community structure and functional pathways through the biosynthesis of nutrients that potentially interact with the moso bamboo growth rhythm, especially the on-year root-associated microbiome, which had a greater abundance of specific nutrients such as gibberellins and vitamin B6. This work provides a dynamic perspective of the differential responses of various on- and off-year microbial communities and enhances our understanding of bamboo soil microbiome biodiversity and stability.

The Global Atlas of Bamboo and Rattan (GABR) Phase II: new resources for sustainable development.

Bamboo, the fast-growing grass plant, and rattan, the spiky climbing palm, are both essential forest resources that have been closely linked with human lives, livelihoods and material culture since ancient times. To promote genetic and genomic research in bamboo and rattan, a comprehensive and coordinated international project, the Genome Atlas of Bamboo and Rattan (GABR), was launched in 2017. GABR achieved great success during Phase I (2017-2022). We will focus on investigating and protecting bamboo and rattan germplasm resources in Phase II ( 2022-2027). Here, we briefly review the achievements of Phase I and introduce the goals of Phase II.

Analyzing lignin biosynthesis pathways in rattan using improved co-expression networks of NACs and MYBs.

BACKGROUND: The rattan is a valuable plant resource with multiple applications in tropical forests. Calamus simplicifolius and Daemonorops jenkinsiana are the two most representative rattan species, supplying over 95% of the raw materials for the rattan industry. Hence, the wood properties of both rattans have always attracted researchers' attention. RESULTS: We re-annotated the genomes, obtained 81 RNA-Seq datasets, and developed an improved pipeline to increase the reliability of co-expression networks of both rattans. Based on the data and pipeline, co-expression relationships were detected in 11 NACs, 49 MYBs, and 86 lignin biosynthesis genes in C. simplicifolius and four NACs, 59 MYBs, and 76 lignin biosynthesis genes in D. jenkinsiana, respectively. Among these co-expression pairs, several genes had a close relationship to the development of wood properties. Additionally, we detected the enzyme gene on the lignin biosynthesis pathway was regulated by either NAC or MYB, while LACCASES was regulated by both NAC and MYB. For D. jenkinsiana, the lignin biosynthesis regulatory network was characterized by positive regulation, and MYB possible negatively regulate non-expressed lignin biosynthesis genes in stem tissues. For C. simplicifolius, NAC may positively regulate highly expressed genes and negatively regulate non-expressed lignin biosynthesis genes in stem tissues. Furthermore, we established core regulatory networks of NAC and MYB for both rattans. CONCLUSIONS: This work improved the accuracy of rattan gene annotation by integrating an efficient co-expression network analysis pipeline, enhancing gene coverage and accuracy of the constructed network, and facilitating an understanding of co-expression relationships among NAC, MYB, and lignin biosynthesis genes in rattan and other plants.

The flying spider-monkey tree fern genome provides insights into fern evolution and arborescence.

To date, little is known about the evolution of fern genomes, with only two small genomes published from the heterosporous Salviniales. Here we assembled the genome of Alsophila spinulosa, known as the flying spider-monkey tree fern, onto 69 pseudochromosomes. The remarkable preservation of synteny, despite resulting from an ancient whole-genome duplication over 100 million years ago, is unprecedented in plants and probably speaks to the uniqueness of tree ferns. Our detailed investigations into stem anatomy and lignin biosynthesis shed new light on the evolution of stem formation in tree ferns. We identified a phenolic compound, alsophilin, that is abundant in xylem, and we provided the molecular basis for its biosynthesis. Finally, analysis of demographic history revealed two genetic bottlenecks, resulting in rapid demographic declines of A. spinulosa. The A. spinulosa genome fills a crucial gap in the plant genomic landscape and helps elucidate many unique aspects of tree fern biology.

Analysis of 427 genomes reveals moso bamboo population structure and genetic basis of property traits.

Moso bamboo (Phyllostachys edulis) is an economically and ecologically important nontimber forestry species. Further development of this species as a sustainable bamboo resource has been hindered by a lack of population genome information. Here, we report a moso bamboo genomic variation atlas of 5.45 million single-nucleotide polymorphisms (SNPs) from whole-genome resequencing of 427 individuals covering 15 representative geographic areas. We uncover low genetic diversity, high genotype heterozygosity, and genes under balancing selection underlying moso bamboo population adaptation. We infer its demographic history with one bottleneck and its recently small population without a rebound. We define five phylogenetic groups and infer that one group probably originated by a single-origin event from East China. Finally, we conduct genome-wide association analysis of nine important property-related traits to identify candidate genes, many of which are involved in cell wall, carbohydrate metabolism, and environmental adaptation. These results provide a foundation and resources for understanding moso bamboo evolution and the genetic mechanisms of agriculturally important traits.

Integrative lncRNA landscape reveals lncRNA-coding gene networks in the secondary cell wall biosynthesis pathway of moso bamboo (Phyllostachys edulis).

BACKGROUND: LncRNAs are extensively involved in plant biological processes. However, the lack of a comprehensive lncRNA landscape in moso bamboo has hindered the molecular study of lncRNAs. Moreover, the role of lncRNAs in secondary cell wall (SCW) biosynthesis of moso bamboo is elusive. RESULTS: For comprehensively identifying lncRNA throughout moso bamboo genome, we collected 231 RNA-Seq datasets, 1 Iso-Seq dataset, and 1 full-length cDNA dataset. We used a machine learning approach to improve the pipeline of lncRNA identification and functional annotation based on previous studies and identified 37,009 lncRNAs in moso bamboo. Then, we established a network of potential lncRNA-coding gene for SCW biosynthesis and identified SCW-related lncRNAs. We also proposed that a mechanism exists in bamboo to direct phenylpropanoid intermediates to lignin or flavonoids biosynthesis through the PAL/4CL/C4H genes. In addition, we identified 4 flavonoids and 1 lignin-preferred genes in the PAL/4CL/C4H gene families, which gained implications in molecular breeding. CONCLUSIONS: We provided a comprehensive landscape of lncRNAs in moso bamboo. Through analyses, we identified SCW-related lncRNAs and improved our understanding of lignin and flavonoids biosynthesis.

Refinement of bamboo genome annotations through integrative analyses of transcriptomic and epigenomic data.

Bamboo, one of the most crucial nontimber forest resources worldwide, has the capacity for rapid growth. In recent years, the genome of moso bamboo (Phyllostachys edulis) has been decoded, and a large amount of transcriptome data has been published. In this study, we generated the genome-wide profiles of the histone modification H3K4me3 in leaf, stem, and root tissues of bamboo. The trends in the distribution patterns were similar to those in rice. We developed a processing pipeline for predicting novel transcripts to refine the structural annotation of the genome using H3K4me3 ChIP-seq data and 29 RNA-seq datasets. As a result, 12,460 novel transcripts were predicted in the bamboo genome. Compared with the transcripts in the newly released version 2.0 of the bamboo genome, these novel transcripts are tissue-specific and shorter, and most have a single exon. Some representative novel transcripts were validated by semiquantitative RT-PCR and qRT-PCR analyses. Furthermore, we put these novel transcripts back into the ChIP-seq analysis pipeline and discovered that the percentages of H3K4me3 in genic elements were increased. Overall, this work integrated transcriptomic data and epigenomic data to refine the annotation of the genome in order to discover more functional genes and study bamboo growth and development, and the application of this predicted pipeline may help refine the structural annotation of the genome in other species.

A bamboo leaf-specific aquaporin gene PePIP2;7 is involved in abiotic stress response.

KEY MESSAGE: PePIP2;7, a leaf-specific aquaporin gene in bamboo, is upregulated under abiotic stresses. Overexpressing PePIP2;7 confers abiotic stresses tolerance in transgenic Arabidopsis plant and yeast. Aquaporins (AQPs) participate in the regulation of water balance in plants. However, the function of AQPs in bamboo remains unclear. Here, PePIP2;7 was identified as a leaf-specific aquaporin gene in moso bamboo based on the expression analysis of transcriptome data and PCR. In situ hybridization further indicated that PePIP2;7 was mainly expressed in mesophyll cells of mature leaves, while in immature leaves it was dominant in blade edge cells followed by mesophyll cells. Interestingly, PePIP2;7 was strongly expressed in the mesophyll cells near bulliform cells of immature leaves, suggesting that PePIP2;7 might function in water transport and contribute to leaf unfolding. The transient expression assay showed that PePIP2;7 was a plasma membrane intrinsic protein. Furthermore, PePIP2;7 was upregulated under abiotic stresses such as high light, drought, and NaCl. Compared with Col-0, transgenic Arabidopsis plants overexpressing PePIP2;7 had better seed germination rate, longer taproot length, higher SOD activity, and lower MDA content under abiotic stresses. Besides, yeasts expressing PePIP2;7 also had higher tolerance to stress compared to the control. Taken together, our results show that PePIP2;7 is leaf-specific and involved in stress response, which provides new insights into aquaporin function in bamboo.

Targeted enrichment of novel chloroplast-based probes reveals a large-scale phylogeny of 412 bamboos.

BACKGROUND: The subfamily Bambusoideae belongs to the grass family Poaceae and has significant roles in culture, economy, and ecology. However, the phylogenetic relationships based on large-scale chloroplast genomes (CpGenomes) were elusive. Moreover, most of the chloroplast DNA sequencing methods cannot meet the requirements of large-scale CpGenome sequencing, which greatly limits and impedes the in-depth research of plant genetics and evolution. RESULTS: To develop a set of bamboo probes, we used 99 high-quality CpGenomes with 6 bamboo CpGenomes as representative species for the probe design, and assembled 15 M unique sequences as the final pan-chloroplast genome. A total of 180,519 probes for chloroplast DNA fragments were designed and synthesized by a novel hybridization-based targeted enrichment approach. Another 468 CpGenomes were selected as test data to verify the quality of the newly synthesized probes and the efficiency of the probes for chloroplast capture. We then successfully applied the probes to synthesize, enrich, and assemble 358 non-redundant CpGenomes of woody bamboo in China. Evaluation analysis showed the probes may be applicable to chloroplasts in Magnoliales, Pinales, Poales et al. Moreover, we reconstructed a phylogenetic tree of 412 bamboos (358 in-house and 54 published), supporting a non-monophyletic lineage of the genus Phyllostachys. Additionally, we shared our data by uploading a dataset of bamboo CpGenome into CNGB ( https://db.cngb.org/search/project/CNP0000502/ ) to enrich resources and promote the development of bamboo phylogenetics. CONCLUSIONS: The development of the CpGenome enrichment pipeline and its performance on bamboos recommended an inexpensive, high-throughput, time-saving and efficient CpGenome sequencing strategy, which can be applied to facilitate the phylogenetics analysis of most green plants.

Coexpression Analysis Reveals Dynamic Modules Regulating the Growth and Development of Cirri in the Rattans (Calamus simplicifolius and Daemonorops jenkinsiana).

Rattan is regarded as one of the major non-timber forest products, second only to wood and bamboo, worldwide. Although the published genomes of Calamus simplicifolius and Daemonorops jenkinsiana have facilitated genome-wide gene functional analyses, coexpression networks (CENs) provide more comprehensive and complete annotations of gene function at the transcriptome level. Thus, we analyzed the CENs of the two rattans, C. simplicifolius and D. jenkinsiana, by integrating the genome sequences and analyzing in-house transcriptome data from different development stages of their cirri using a well-developed strategy. A total of 3,504 and 3,027 functional modules were identified in C. simplicifolius and D. jenkinsiana, respectively, based on a combination of CENs, gene family classification, and function enrichment tools. These modules covered the major developmental processes, including photosynthesis, lignin biosynthesis, flavonoid biosynthesis, and phenylpropanoid biosynthesis. Reference annotations were refined using CENs and functional modules. Moreover, we obtained novel insights into the regulation of cirrus growth and development in rattans. Furthermore, Rattan-NET (http://rattan.bamboogdb.org/), an online database with analysis tools for gene set enrichment analysis, module enrichment, network comparison analysis, and cis-element analysis, was constructed for the easy analysis of gene function and regulation modules involved in the growth and development of cirri in rattans.

Genome-wide identification and expression analysis of brassinosteroid action-related genes during the shoot growth of moso bamboo.

Brassinosteroids (BRs) are a group of plant steroid hormones that play crucial roles in a range of plant growth and development processes. BR action includes active BR formation by a complex biosynthesis process and driving BR biological function through signal transduction. Although the characterization of several BR action-related genes has been conducted in a few model plants, systematic information about these genes in bamboo is still lacking. We identified 64 genes related to BR action from the genome of moso bamboo (Phyllostachys edulis), including twenty that participated in BR biosynthesis and forty-four involved in BR signal transduction. The characteristics of all these candidate genes were identified by bioinformatics methods, including the gene structures, basic physical and chemical properties of proteins, conserved domains and evolutionary relationships. Based on the transcriptome data, the candidate genes demonstrated different expression patterns, which were further validated by qRT-PCR using templates from bamboo shoots with different heights. Thirty-four positive and three negative co-expression modules were identified by 44 candidate genes in the newly emerging bamboo shoot. The gene expression patterns and co-expression modules of BR action-related genes in bamboo shoots indicated that they might function to promote bamboo growth through BR biosynthesis and signal transduction processes. This study provides the first step towards the cloning and functional dissection of the role of BR action-related genes in moso bamboo, which also presents an excellent opportunity for genetic engineering using the candidate genes to improve bamboo quantity and quality.

Genome-wide identification and expression analysis of the MYB transcription factor in moso bamboo (Phyllostachys edulis).

The MYB family, one of the largest transcription factor (TF) families in the plant kingdom, plays vital roles in cell formation, morphogenesis and signal transduction, as well as responses to biotic and abiotic stresses. However, the underlying function of bamboo MYB TFs remains unclear. To gain insight into the status of these proteins, a total of 85 PeMYBs, which were further divided into 11 subgroups, were identified in moso bamboo (Phyllostachys edulis) by using a genome-wide search strategy. Gene structure analysis showed that PeMYBs were significantly different, with exon numbers varying from 4 to 13. Phylogenetic analysis indicated that PeMYBs clustered into 27 clades, of which the function of 18 clades has been predicted. In addition, almost all of the PeMYBs were differently expressed in leaves, panicles, rhizomes and shoots based on RNA-seq data. Furthermore, qRT-PCR analysis showed that 12 PeMYBs related to the biosynthesis and deposition of the secondary cell wall (SCW) were constitutively expressed, and their transcript abundance levels have changed significantly with increasing height of the bamboo shoots, for which the degree of lignification continuously increased. This result indicated that these PeMYBs might play fundamental roles in SCW thickening and bamboo shoot lignification. The present comprehensive and systematic study on the members of the MYB family provided a reference and solid foundation for further functional analysis of MYB TFs in moso bamboo.

Co-expression Gene Network Analysis and Functional Module Identification in Bamboo Growth and Development.

Bamboo is one of the fastest-growing non-timber forest plants. Moso bamboo (Phyllostachys edulis) is the most economically valuable bamboo in Asia, especially in China. With the release of the whole-genome sequence of moso bamboo, there are increasing demands for refined annotation of bamboo genes. Recently, large amounts of bamboo transcriptome data have become available, including data on the multiple growth stages of tissues. It is now feasible for us to construct co-expression networks to improve bamboo gene annotation and reveal the relationships between gene expression and growth traits. We integrated the genome sequence of moso bamboo and 78 transcriptome data sets to build genome-wide global and conditional co-expression networks. We overlaid the gene expression results onto the network with multiple dimensions (different development stages). Through combining the co-expression network, module classification and function enrichment tools, we identified 1,896 functional modules related to bamboo development, which covered functions such as photosynthesis, hormone biosynthesis, signal transduction, and secondary cell wall biosynthesis. Furthermore, an online database (http://bioinformatics.cau.edu.cn/bamboo) was built for searching the moso bamboo co-expression network and module enrichment analysis. Our database also includes cis-element analysis, gene set enrichment analysis, and other tools. In summary, we integrated public and in-house bamboo transcriptome data sets and carried out co-expression network analysis and functional module identification. Through data mining, we have yielded some novel insights into the regulation of growth and development. Our established online database might be convenient for the bamboo research community to identify functional genes or modules with important traits.

Whole-Genome and Expression Analyses of Bamboo Aquaporin Genes Reveal Their Functions Involved in Maintaining Diurnal Water Balance in Bamboo Shoots.

Water supply is essential for maintaining normal physiological function during the rapid growth of bamboo. Aquaporins (AQPs) play crucial roles in water transport for plant growth and development. Although 26 PeAQPs in bamboo have been reported, the aquaporin-led mechanism of maintaining diurnal water balance in bamboo shoots remains unclear. In this study, a total of 63 PeAQPs were identified, based on the updated genome of moso bamboo (Phyllostachys edulis), including 22 PePIPs, 20 PeTIPs, 17 PeNIPs, and 4 PeSIPs. All of the PeAQPs were differently expressed in 26 different tissues of moso bamboo, based on RNA sequencing (RNA-seq) data. The root pressure in shoots showed circadian rhythm changes, with positive values at night and negative values in the daytime. The quantitative real-time PCR (qRT-PCR) result showed that 25 PeAQPs were detected in the base part of the shoots, and most of them demonstrated diurnal rhythm changes. The expression levels of some PeAQPs were significantly correlated with the root pressure. Of the 86 sugar transport genes, 33 had positive co-expression relationships with 27 PeAQPs. Two root pressure-correlated PeAQPs, PeTIP4;1 and PeTIP4;2, were confirmed to be highly expressed in the parenchyma and epidermal cells of bamboo culm, and in the epidermis, pith, and primary xylem of bamboo roots by in situ hybridization. The authors' findings provide new insights and a possible aquaporin-led mechanism for bamboo fast growth.

Chromosome-level reference genome and alternative splicing atlas of moso bamboo (Phyllostachys edulis).

Background: Bamboo is one of the most important nontimber forestry products worldwide. However, a chromosome-level reference genome is lacking, and an evolutionary view of alternative splicing (AS) in bamboo remains unclear despite emerging omics data and improved technologies. Results: Here, we provide a chromosome-level de novo genome assembly of moso bamboo (Phyllostachys edulis) using additional abundance sequencing data and a Hi-C scaffolding strategy. The significantly improved genome is a scaffold N50 of 79.90 Mb, approximately 243 times longer than the previous version. A total of 51,074 high-quality protein-coding loci with intact structures were identified using single-molecule real-time sequencing and manual verification. Moreover, we provide a comprehensive AS profile based on the identification of 266,711 unique AS events in 25,225 AS genes by large-scale transcriptomic sequencing of 26 representative bamboo tissues using both the Illumina and Pacific Biosciences sequencing platforms. Through comparisons with orthologous genes in related plant species, we observed that the AS genes are concentrated among more conserved genes that tend to accumulate higher transcript levels and share less tissue specificity. Furthermore, gene family expansion, abundant AS, and positive selection were identified in crucial genes involved in the lignin biosynthetic pathway of moso bamboo. Conclusions: These fundamental studies provide useful information for future in-depth analyses of comparative genome and AS features. Additionally, our results highlight a global perspective of AS during evolution and diversification in bamboo.

The chromosome-level genome assemblies of two rattans (Calamus simplicifolius and Daemonorops jenkinsiana).

Background: Calamus simplicifolius and Daemonorops jenkinsiana are two representative rattans, the most significant material sources for the rattan industry. However, the lack of reference genome sequences is a major obstacle for basic and applied biology on rattan. Findings: We produced two chromosome-level genome assemblies of C. simplicifolius and D. jenkinsiana using Illumina, Pacific Biosciences, and Hi-C sequencing data. A total of ∼730 Gb and ∼682 Gb of raw data covered the predicted genome lengths (∼1.98 Gb of C. simplicifolius and ∼1.61 Gb of D. jenkinsiana) to ∼372 × and ∼426 × read depths, respectively. The two de novo genome assemblies, ∼1.94 Gb and ∼1.58 Gb, were generated with scaffold N50s of ∼160 Mb and ∼119 Mb in C. simplicifolius and D. jenkinsiana, respectively. The C. simplicifolius and D. jenkinsiana genomes were predicted to harbor 51,235 and 53,342 intact protein-coding gene models, respectively. Benchmarking Universal Single-Copy Orthologs evaluation demonstrated that genome completeness reached 96.4% and 91.3% in the C. simplicifolius and D. jenkinsiana genomes, respectively. Genome evolution showed that four Arecaceae plants clustered together, and the divergence time between the two rattans was ∼19.3 million years ago. Additionally, we identified 193 and 172 genes involved in the lignin biosynthesis pathway in the C. simplicifolius and D. jenkinsiana genomes, respectively. Conclusions: We present the first de novo assemblies of two rattan genomes (C. simplicifolius and D. jenkinsiana). These data will not only provide a fundamental resource for functional genomics, particularly in promoting germplasm utilization for breeding, but also serve as reference genomes for comparative studies between and among different species.

Expression and functional analysis of two PsbS genes in bamboo (Phyllostachys edulis).

Higher plants have an array of photoprotection mechanisms alleviating the harmful effects of light. Non-photochemical quenching (NPQ) is one of the photoprotective mechanisms, which dissipates the excess of light energy absorbed in the light-harvesting complexes (LHCs) into thermal energy. The photosystem II subunit S (PsbS), a member of the LHC family thought to be present exclusively in higher plants, is supposed to activate NPQ through interactions with antenna proteins. However, the roles of PsbS in bamboo remain unclear. Here, two genes of bamboo (Phyllostachys edulis), PePsbS1 and PePsbS2, are investigated and functionally analyzed. PePsbS1 and PePsbS2 have a similar gene structure with three introns separated by two exons, which encode 269 and 268 amino acid residues, respectively. Tissue-specific analysis showed that PePsbS1 and PePsbS2 are highly expressed in leaf blade. Besides, they are both upregulated in the leaf blade when plantlets are submitted to an increased and prolonged light intensity, suggesting that they are light-induced. Western blot analysis indicated that the accumulation level of total PePsbSs is consistent with what obtained by quantitative real-time polymerase chain reaction for PePsbS1 and PePsbS2. Transgenic Arabidopsis plants overexpressing PePsbS1 and PePsbS2 both displayed an enhanced photoprotection. Moreover, the expression of PePsbS1 and PePsbS2 could both rescue the NPQ of Arabidopsis npq4 mutant, indicating that the PsbSs are functionally conserved between monocots and dicots. These results indicated that both PePsbS1 and PePsbS2 could circumvent photoinhibition and enhance photoprotection, which are key factors for bamboo's adaptation to different light environment.