Targeted Enrichment of Novel Chloroplast-Based Probes Reveals a Large-Scale Phylogeny of 412 Bamboos

Targeted Enrichment of Novel Chloroplast-Based Probes Reveals a Large-Scale Phylogeny of 412 Bamboos | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Targeted Enrichment of Novel Chloroplast-Based Probes Reveals a Large-Scale Phylogeny of 412 Bamboos Jiongliang Wang, Weixue Mu, Ting Yang, Yue Song, YinGuang Hou, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-58636/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Feb, 2021 Read the published version in BMC Plant Biology → Version 1 posted 9 You are reading this latest preprint version Abstract 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 15M 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. Plant Physiology and Morphology Plant Molecular Biology and Genetics Bambusoideae Chloroplast Probe Targeted enrichment Bamboo phylogeny. Figures Figure 1 Figure 2 Figure 3 Background The subfamily Bambusoideae belongs to the grass family Poaceae and exhibits substantial phenotypic diversity, with 1,642 species in 125 genera, three tribes, and 15 subtribes, which have been classified into ~ 75 clades 1 . The Bambuseae consists of tropical woody bamboos (Bambuseae), temperate woody bamboos (Arundinarieae) and herbaceous bamboo tribe (Olyreae). Bambusoideae predominantly distributed in the Old World, such as China, Japan, Thailand, Indonesia, and the countries of Southeast Asian. As one of the most ecologically and industrially valuable tribes of Bambusoideae, woody bamboos were used for furniture, paper, fiber textiles, and fuel 2 . In total, about 500 bamboos are distributed in Asia, spanning a wide geographic and temperature range. However, infrequent, incongruent, and unpredictable flowering events as well as unstable vegetative characteristics, severely restricted the identification and classification of woody bamboos. The phylogenetic relationships based on more massive amounts of woody bamboos remain elusive due to the lack of extensive and high-quality genomic resources. The chloroplast genome (CpGenome) is an essential resource for the study of plant evolution 3 . This high-copy organelle is one of the most technically accessible regions of the genome. The chloroplast genomic DNA of green plants commonly exhibits a conserved genome structure that contains two copies of inverted repeat (IR) separating the small single-copy region (SSC) and the large single-copy region (LSC) 2 , 4 , 5 . The CpGenome has been a popular source of reconstructing the phylogeny of green plants, and many chloroplast DNA loci are contributing to the development of plant taxonomy. To obtain chloroplast DNA suitable for whole chloroplast genome sequencing, it can be traditionally enriched by using the sucrose gradient centrifugation method 6 , the high salt method 7 , long PCR technology by using primers 8 . The characters of the strategies above are the use of physical methods to extract chloroplast DNA or the need for high quality, sufficiently extracted cellar DNA and the appropriate primers. With the development of sequencing technology, next-generation sequencing (NGS) has the advantageous characteristics of high-throughput and efficient, resulting in a rapid increase in the amount of sequencing data. Chloroplast DNA generally accounts for only about 0.5–13% of the whole genome 9 . But, the chloroplast DNA sequencing data from the whole genome sequencing (WGS) data produced a lot of “useless” data except for “useful” ones, consuming much of the sequencing capacity and reducing the efficiency of parallelly chloroplast sequencing. The above methods for obtaining chloroplast DNA sequencing data cannot meet the needs of large-scale CpGenome sequencing, which significantly restricts and hinders the in-depth research of plant genetics and evolution. In this study, the main goals were: (1) To develop and evaluate a pipeline to target-enrich and assembly the chloroplast data of bamboos. (2) To obtain high-quality and high coverage of bamboo CpGenomes by the pipeline, to reconstruct a phylogenetic tree, and to promote phylogenetic knowledge of bamboo. (3) To share the new sequenced bamboo CpGenomes, allowing researchers to quickly compare suspect chloroplast data and explore the bamboo CpGenomes. Methods Species selection for probe design and evaluation To improve the variability and versatility of the probes, we selected 567 representative species from the 3,654 published CpGenomes species (collected from NCBI, Released Dec 2018) to design and evaluate probes for a targeted enrichment strategy of CpGenomes (Supplementary Table S1 and S2). Among the 567 species, 22 are bamboo species. For data preprocessing, we elucidated our approach in a flow chart (Supplementary Figure S1). A phylogenetic tree (Supplementary Figure S2) was constructed based on the 567 complete CpGenomes, which spanned the phylogenetic diversity of 7 major clades, including 40 orders and 57 families. The model species in each clade were selected as core candidates. Thus, a total of 99 CpGenomes, including 6 bamboo CpGenomes, were chosen as the representative species for the probe design (Table 1 ), and the remaining (468 CpGenomes) were chosen as test data further to assess the efficiency of the probes for chloroplast capture. The species for probe design and the species for probe evaluation were different genera but belong to the same family (e.g., Danthonia and Chionochloa , both are Poaceae). Table 1 The taxonomic composition of the chloroplast genome sequences which used for design probes NCBI species ID classfy order family genus Species NC_010093.1 Monocots Acorales Acoraceae Acorus Acorus americanus NC_022133.1 Monocots Poales Poaceae Aegilops Aegilops tauschii NC_023934.1 Monocots Poales Poaceae Arundinaria Arundinaria appalachiana NC_012927.1 Monocots Poales Poaceae Bambusa Bambusa oldhamii NC_011032.1 Monocots Poales Poaceae Brachypodium Brachypodium distachyon NC_025663.1 Monocots Asparagales Orchidaceae Corallorhiza Corallorhiza wisteriana NC_021432.1 Monocots Asparagales Orchidaceae Cymbidium Cymbidium tracyanum NC_025232.1 Monocots Poales Poaceae Danthonia Danthonia californica NC_009601.1 Monocots Dioscoreales Dioscoreaceae Dioscorea Dioscorea elephantipes NC_024715.1 Monocots Poales Poaceae Fargesia Fargesia nitida NC_019648.1 Monocots Poales Poaceae Festuca Festuca altissima NC_024728.1 Monocots Liliales Liliaceae Fritillaria Fritillaria cirrhosa NC_024720.1 Monocots Poales Poaceae Indocalamus Indocalamus wilsonii NC_022926.1 Monocots Zingiberales Musaceae Musa Musa textilis NC_001320.1 Monocots Poales Poaceae Oryza Oryza sativa Japonica NC_017609.1 Monocots Asparagales Orchidaceae Phalaenopsis Phalaenopsis equestris NC_023245.1 Monocots Poales Poaceae Pharus Pharus lappulaceus NC_013991.2 Monocots Arecales Arecaceae Phoenix Phoenix dactylifera NC_015817.1 Monocots Poales Poaceae Phyllostachys Phyllostachys edulis NC_022850.1 Monocots Poales Poaceae Setaria Setaria italica NC_008602.1 Monocots Poales Poaceae Sorghum Sorghum bicolor NC_002762.1 Monocots Poales Poaceae Triticum Triticum aestivum NC_015894.1 Monocots Alismatales Araceae Wolffiella Wolffiella lingulata NC_024725.1 Monocots Poales Poaceae Yushania Yushania levigata NC_001666.2 Monocots Poales Poaceae Zea Zea mays NC_005086.1 Basal angiosperms Amborellales Amborellaceae Amborella Amborella trichopoda NC_006050.1 Basal angiosperms Nymphaeales Nymphaeaceae Nymphaea Nymphaea alba NC_023242.1 Magnoliidae Magnoliales Magnoliaceae Magnolia Magnolia sprengeri NC_008457.1 Magnoliidae Piperales Piperaceae Piper Piper cenocladum NC_026690.1 Eudicots Ericales Actinidiaceae Actinidia Actinidia chinensis NC_009265.1 Eudicots Brassicales Brassicaceae Aethionema Aethionema cordifolium NC_015621.1 Eudicots Asterales Asteraceae Ageratina Ageratina adenophora NC_022412.1 Eudicots Myrtales Myrtaceae Angophora Angophora costata NC_000932.1 Eudicots Brassicales Brassicaceae Arabidopsis Arabidopsis thaliana NC_009268.1 Eudicots Brassicales Brassicaceae Arabis Arabis hirsuta NC_022810.1 Eudicots Apiales Araliaceae Aralia Aralia undulata NC_021121.1 Eudicots Ericales Primulaceae Ardisia Ardisia polysticta NC_025910.1 Eudicots Asterales Asteraceae Artemisia Artemisia montana NC_022432.1 Eudicots Gentianales Asclepiadaceae Asclepias Asclepias syriaca NC_016734.1 Eudicots Brassicales Brassicaceae Brassica Brassica napus NC_024541.1 Eudicots Ericales Theaceae Camellia Camellia crapnelliana NC_010323.1 Eudicots Brassicales Caricaceae Carica Carica papaya NC_014674.1 Eudicots Fagales Fagaceae Castanea Castanea mollissima NC_011163.1 Eudicots Fagales Fagaceae Cicer Cicer arietinum NC_025642.1 Eudicots Lamiales Orobanchaceae Cistanche Cistanche phelypaea NC_008334.1 Eudicots Sapindales Rutaceae Citrus Citrus sinensis NC_008535.1 Eudicots Gentianales Rubiaceae Coffea Coffea arabica NC_022409.1 Eudicots Myrtales Myrtaceae Corymbia Corymbia eximia NC_007144.1 Eudicots Cucurbitales Cucurbitaceae Cucumis Cucumis sativus NC_009963.1 Eudicots Solanales Convolvulaceae Cuscuta Cuscuta exaltata NC_014569.1 Eudicots Geraniales Geraniaceae Erodium Erodium texanum NC_022396.1 Eudicots Myrtales Myrtaceae Eucalyptus Eucalyptus aromaphloia NC_015206.1 Eudicots Rosales Rosaceae Fragaria Fragaria vesca NC_007942.1 Eudicots Fabales Fabaceae Glycine Glycine max NC_016668.1 Eudicots Malvales Malvaceae Gossypium Gossypium raimondii NC_024732.1 Eudicots Asterales Campanulaceae Hanabusaya Hanabusaya asiatica NC_023110.1 Eudicots Asterales Asteraceae Helianthus Helianthus decapetalus NC_026726.1 Eudicots Solanales Solanaceae Iochroma Iochroma loxense NC_009808.1 Eudicots Solanales Convolvulaceae Ipomoea Ipomoea purpurea NC_026677.1 Eudicots Fabales Fabaceae Libidibia Libidibia coriaria NC_024064.1 Eudicots Malpighiales Chrysobalanaceae Licania Licania alba NC_002694.1 Eudicots Fabales Fabaceae Lotus Lotus japonicus NC_023090.1 Eudicots Fabales Fabaceae Lupinus Lupinus luteus NC_010433.1 Eudicots Malpighiales Euphorbiaceae Manihot Manihot esculenta NC_003119.6 Eudicots Fabales Fabaceae Medicago Medicago truncatula NC_012615.1 Eudicots Ranunculales Ranunculaceae Megaleranthis Megaleranthis saniculifolia NC_008359.1 Eudicots Rosales Moraceae Morus Morus indica NC_025339.1 Eudicots Proteales Nelumbonaceae Nelumbo Nelumbo nucifera NC_010358.1 Eudicots Myrtales Onagraceae Oenothera Oenothera argillicola NC_013707.2 Eudicots Lamiales Oleaceae Olea Olea europaea NC_006290.1 Eudicots Apiales Araliaceae Panax Panax ginseng NC_009259.1 Eudicots Fabales Fabaceae Phaseolus Phaseolus vulgaris NC_009143.1 Eudicots Malpighiales Salicaceae Populus Populus trichocarpa NC_014697.1 Eudicots Rosales Rosaceae Prunus Prunus persica NC_015996.1 Eudicots Rosales Rosaceae Pyrus Pyrus pyrifolia NC_016736.1 Eudicots Malpighiales Euphorbiaceae Ricinus Ricinus communis NC_026722.1 Eudicots Malpighiales Salicaceae Salix Salix purpurea NC_026202.1 Eudicots Lamiales Scrophulariaceae Scrophularia Scrophularia takesimensis NC_023085.1 Eudicots Saxifragales Crassulaceae Sedum Sedum sarmentosum NC_016730.1 Eudicots Caryophyllales Caryophyllaceae Silene Silene latifolia NC_008096.2 Eudicots Solanales Solanaceae Solanum Solanum tuberosum NC_014676.2 Eudicots Malvales Malvaceae Theobroma Theobroma cacao NC_024034.1 Eudicots Fabales Fabaceae Trifolium Trifolium grandiflorum NC_021449.1 Eudicots Lamiales Lentibulariaceae Utricularia Utricularia gibba NC_021091.1 Eudicots Fabales Fabaceae Vigna Vigna angularis NC_007957.1 Eudicots Vitales Vitaceae Vitis Vitis vinifera NC_023259.1 Eudicots Geraniales Vivianiaceae Viviania Viviania marifolia NC_013086.1 Lycopodiophyta Selaginellales Selaginellaceae Selaginella Selaginella moellendorffii NC_008829.1 Moiliformopses Marattiales Marattiaceae Angiopteris Angiopteris evecta NC_014699.1 Moiliformopses Equisetales Equisetaceae Equisetum Equisetum arvense NC_014348.1 Moiliformopses Dennstaedtiales Dennstaedtiaceae Pteridium Pteridium aquilinum NC_016063.1 Gymnosperms Pinales Cephalotaxaceae Cephalotaxus Cephalotaxus wilsoniana NC_009618.1 Gymnosperms Cycadales Cycadaceae Cycas Cycas taitungensis NC_026301.1 Gymnosperms Gnetales Gnetaceae Gnetum Gnetum gnemon NC_024022.1 Gymnosperms Pinales Cupressaceae Juniperus Juniperus monosperma NC_021456.1 Gymnosperms Pinales Pinaceae Picea Picea abies NC_011153.4 Gymnosperms Pinales Pinaceae Pinus Pinus contorta NC_023805.1 Gymnosperms Pinales Podocarpaceae Podocarpus Podocarpus lambertii NC_016065.1 Gymnosperms Pinales Cupressaceae Taiwania Taiwania cryptomerioides Construction of non-redundant chloroplast reference Using the CpGenome of Arabidopsis thaliana as the initial reference sequence (as a database sequence), other selected CpGenomes (as query sequences) were aligned to the database sequence by BLAST + v2.2.25 software with default parameters. The sequences with more than 90% identity were masked from the query sequences. Then, the resulting sequences were subjected to a secondary round masking of redundant sequences, which were identified by an all-against-all BLAST+. Finally, a non-redundant chloroplast reference, as a pan-chloroplast genome (pan-CpGenome), was obtained by iterative analysis. Sequences with high similarity ( > = 90%) were masked with “Ns”, and others were highly divergent sequences in the pan-CpGenome (Supplementary File F1). The visualization of the alignment of 98 CpGenomes to Arabidopsis thaliana CpGenome was conducted by BLAST Ring Image Generator (BRIG V0.9) 10 with default parameters. Universal probes designed for bamboo CpGenomes The regions of the pan-CpGenome sequences which have not been masked to “Ns” were extended by 40 bp on both sides for the design of the probes. Each region was divided into K -mers of 90 bp in length and the melting temperatures of the K -mers were calculated 11 . A comprehensive score of uniqueness, frequency, melting temperature, and GC content was calculated for each probe by Primer3 v2.4.0 12 . The probes with the highest comprehensiveness scores were selected in 20 bp window and slid along the target region at the fixed interval. For ensuring high coverages of the probe sequences in the target region, the target region was covered at least 2 times by these selected probes. Finally, a total of 180,519 DNA oligonucleotides were synthesized by a CustomArray B3 Synthesizer (CustomArray, Washington, DC, USA) according to the manufacturer’s instructions and dissolved in 10 × TE buffer (pH = 8.0). Taxa sampling All sampled species covering more than 30 genera (Supplementary Table S4) were collected in spring 2015 and 2016 under the permission of four main bamboo gardens in China:(1) Taiping base of ICBR: N:30°20'57.03", E:118°01'30.21", 150 M, (2) WangJianglou Park, Chengdu: N:30°37'54.85", E:104°05'23.84", 150 M, (3) Yunnan Pu'er Asia Bamboo and Rattan Exposition Garden: N:22°41'24.67", E:100°56'26.51", 1000 M, and (4) BaiMa base of Nanjing Forestry University: N:31°36'35.62", E:119°10'34.29", 50 M. During the sampling process, identification services of bamboo samples were provided by related taxonomists at each bamboo garden. Totally, 358 bamboo samples, mainly from young leaves, were collected. All samples were frozen in liquid nitrogen immediately and were preserved in ultra-low temperature refrigerator at -80 °C, followed by DNA extraction. DNA extraction and target enrichment sequencing for bamboos A total of 358 woody bamboo samples were sampled and sequenced in this study (Supplementary Table S4), as a practical application of target enrichment sequencing and an evaluation of the capture efficiency. Genomic DNA from each sample was extracted using the CTAB method 13 and fragmented to a peak size of 200 bp using a Covaris E220 sonicator (Covaris, Woburn, Massachusetts, USA), followed by the end-repair, addition of base “A”, and adapter ligation. DNA fragments of the desired size (200 bp) were selected on an agarose gel and hybridized to the probes for 72 h. The probes captured DNA fragments were recycled by magnetic beads coated with streptavidin, which interacted with the biotin on the probes to wash away the uncaptured DNA fragments. The captured DNA fragments were sequenced on the BGISEQ-500 platform at Beijing Genomics Institute, Shenzhen, China. High-quality reads ranging from 1 Gb to 9 Gb with 100 bp paired-end were acquired for each sample. For data preprocessing, we illuminated our method in a flow chart (Supplementary Figure S1). SOAPfilter (v2.2) 14 was applied to remove low-quality reads and adaptors in the following criteria (1) reads with > 10% base of N; (2) reads with > 40% of low-quality reads (value < = 10); (3) reads contaminated with adaptors and produced by PCR duplication. A CpGenome of Phyllostachys edulis (downloaded from NCBI, accession number: HQ337796.1) was used as a reference for assembly using MITObim (V1.8) 15 . In this way, we finally recovered the complete CpGenomes of all 358 samples. Additionally, the plastid genomes were annotated in the current standard web-based program DOGMA 16 ( http://dogma.ccbb.utexas.edu/ ,). Phylogenetic analysis of woody bamboos We downloaded previously published CpGenomes of 71 bamboo species from NCBI (released May 2020) to amplify the sampling of the species tree (Supplementary Table S5). Redundancy sequences were removed, resulting in 412 non-redundant bamboo CpGenomes (Supplementary Table S6). The CDS sequences of each gene family were aligned using MAFFT (V7.017) 17 with default parameters based on the corresponding protein sequences, and then sequences were concatenated to produce 54,078 nucleotide positions. A maximum likelihood (ML) species tree was constructed with IQ-TREE (V1.6.12) 18 with parameters: -m MFP, -B 1000, -bnni, -alrt 1000. Sharing the bamboo CpGenome dataset All 358 woody bamboo CpGenomes provided in Supplementary Table S4 were deposited in China National GeneBank (CNGB) ( https://db.cngb.org/blast/blast/blastn/ ), with the database named “Chinese Bamboo Database”. The CNGB developed BLAST+ (version 2.6.0) service to allow public searches against the bamboo CpGenomes. Results Development of universal chloroplast probes for bamboos From the 3,654 CpGenomes collected from NCBI, 567 high-quality CpGenomes were selected for probe development and divided into two datasets, with 99 CpGenomes for probe design and 468 CpGenomes for probe evaluation. Considering the applicability and robustness of the probes designing for bamboos, and the diversity of CpGenomes, the 99 CpGenomes were selected from different families. Details of the related methods were provided in Supplementary Figure S1. A 15 Mb pan-CpGenome was assembled based on the alignment to Arabidopsis thaliana (Supplementary File F1). The comparison analysis showed the CpGenomes had great variations across species (Fig. 1 ). Lycophytes CpGenome showed the greatest gaps in the alignment, followed by Ferns, Horsetails, and Gymnosperm. Eudicots and some of Monocots had the highest integrity of CpGenomes. Compared to Eudicots, some of Monocots, Gymnosperm, Ferns, Horsetails, and Lycophytes had large gaps at 146–150 kb, 124–129 kb, and 88–92 kb. According to the mapping depth, the depth of probe coverage at 100–110 kb, 35–42 kb, and 130–140 kb were rather lower than at other sites. For evaluating the quality of the pan-CpGenome, we calculated the coverage of the probes designed for the 99 complete CpGenomes. Alignment with the 99 reference CpGenomes showed an average coverage of 88.2% and an average base depth of 9.04×. In bamboos, the corresponding average coverage and average base depth were 99.6% and 8.43×, respectively (Fig. 2 A). A total of 180,519 (21,842,799 bp) probes, covering 92.04% of target regions, were designed and showed high consistency in their theoretical melting temperatures and GC contents (Supplementary Table S3). The probes sequences were available in Supplementary File F2. All the designed probes had excellent uniqueness, with an average 1 time while being aligned with the pan-genome. The probes were mostly distributed in the range of 70–80% melting temperatures and 30–40% GC content (Supplementary Figure S3). To assess the broad spectrum of the probes, the BLAST + program was employed to align the probes to the 468 complete CpGenomes for evaluating the probes. The average coverage ratio in the 468 complete CpGenomes was 90.54% (Supplementary Table S8). In bamboos, the coverage ratio was all over 93.00%, with an average coverage of 94.78% (Fig. 2 B and Supplementary Table S8). Moreover, some orders such as Magnoliales, Pinales, Poales also had high coverage. Probe-based targeted enrichment and assembly of bamboo CpGenomes A total of 358 fresh woody bamboo samples collected from China were included (Supplementary Table S4) and used to evaluate capture efficiency. A total of 1G-9G raw reads were obtained, and low-quality reads and adaptors were filtered in data preprocessing (Fig. 2 C and Supplementary Table S9). Clean and high-quality reads were used for reference-guided assemblies by MITObim and recovered nearly complete CpGenomes for the 358 bamboo species. The assembled CpGenomes ranged from 139,664 to 140,064 base pairs (bp), and the LSC regions varied from 83,496 bp to 83,845 bp in length (Supplementary Table S9). The CpGenomes were annotated with approximately 121 genes, including around 113 unique genes encoding 80 proteins, 4 ribosomal RNAs, and 29 transfer RNAs, exhibiting a higher degree of conservation. We detected 15 overlapped bamboo CpGenomes that were present in both the in-house and published data (Fig. 2 D). To assess the target enrichment, we mapped the raw reads to the corresponding CpGenome released previously and compared assembled bamboo CpGenome to corresponding released ones. The results showed more than 45.77% in average of the raw reads from in-house bamboo CpGenomes can be mapped to the corresponding published CpGenomes, and the mapping depth was higher than 1200×. Alignment with the published CpGenomes, the coverage of assembled CpGenomes was greater than 98.59% (Fig. 2 D and Supplementary Table S10). A phylogenomic relationship based on 412 bamboo CpGenomes For comprehensively collecting bamboo CpGenomes, 71 bamboo CpGenomes from NCBI were acquired, resulting in a total of 412 non-redundant bamboo CpGenomes after removing redundancy (Supplementary Table S6). We reconstructed a phylogenetic tree of bamboos based on the concatenated sequences of 75 protein-coding genes in the 412 bamboo CpGenomes. Phylogenetic analyses supported the relationship of (Arthrostylidiinae (Bambusinae, Olyreae)). We classified different clades in the phylogenetic tree based on previous studies 19 , 20 . The pattern of (XI((VIII, IV)VI)((IX, III)(VII, V))) was provided in Arthrostylidiinae (Supplementary Figure S4). Most of the newly sequenced species distributed in Clade V, Clade VI, and Clade Paleotropical. Clade XI ( Ampelocalamus calcareus ) was the earliest diverging Arthrostylidiinae species. The Phyllostachys was a representative genus in bamboo, with the clade embed into Clade V, which was the sister clade of Bashania fargesii . There are some non- Phyllostachys species were found in Phyllostachys genus clade. The Phyllostachys genus clade was divided into two groups based on the phylogenetic tree. Phyllostachys edulis , the most planted bamboo in China, distributed in Phy-II (Fig. 3 ). The sequences from NCBI clustered with corresponding in-house sequences. For example, Phyllostachys edulis sequence from NCBI clustered with in-house sequences of Phyllostachys edulis f epruinosa, Phyllostachys edulis f exaurita, Phyllostachys edulis f flexuosa, et al . China Bamboo Database in CNGB We uploaded the bamboo CpGenomes sequenced in this study to CNGB to facilitate the accumulation of knowledge on bamboo phylogeny. Researchers can download the raw data and assembled CpGenome sequences from CNGB through Project ID: CNP0000502 ( https://db.cngb.org/search/project/CNP0000502/ ). Moreover, researchers can search for all assembled bamboo plastid genomes in this study through web-based BLAST + service ( https://db.cngb.org/blast/ ). The available plastid genome sequences of bamboos and the corresponding BLAST + server can promote researchers to explore the complex and elusive history of bamboo evolution. Discussion CpGenome provides an essential resource for plant evolution As an essential component of plant organelles and photosynthesis organs, chloroplasts have a simple structure, the small genome size (~ 110–165 kb) containing ~ 90–110 protein-coding genes 21 and highly conserved gene region across species, due to their non-recombinant, haploid and uniparentally 22 . The genomic characterization of various aspects of chloroplasts has led to an important role in the research of plant origin, evolution and phylogenetic analysis relationship between different plant species 23 , 24 . Many studies had been reported using chloroplast genes to construct phylogenetic trees of plants. For example, Jansen et al 25 used 81 chloroplast genes to estimate relationships among the major angiosperm clades; Saarela et al 26 found weak support for Amborella as the basal-most angiosperm lineage using 17 plastid genes and the nuclear gene phytochrome C ( PHYC ). With the deepening of chloroplast research, more and more researchers are focusing on the complete chloroplast sequence 27 – 29 . Kane et al 30 suggested that the whole CpGenome could serve as an ultra-barcode for identifying plant varieties. Hybridization-based probes for target enrichment in large-scale CpGenome sequencing Chloroplast DNA can be traditionally acquired by the sucrose gradient centrifugation method 6 or the high salt method 7 . Another method was to amplify the entire chloroplast DNA from the whole cellular DNA base on a long PCR technology by primers, which were designed on conserved sequences 8 . These methods were not suitable for large-scale samples due to the large amount of labor and material resources required to obtain chloroplast DNA, and the labor-intensive method used to prepare chloroplast DNA. Chloroplast reads also can be identified from WGS reads by aligning the WGS data with the reference CpGenome. It is a demanding bioinformatics technique and requires a closely related reference CpGenome. The method was not suitable for the species that are not closely related or have poor quality reference genome sequences. Moreover, to assemble only CpGenome based on this method, a great deal of useless sequencing data was thus generated, consuming much of the sequencing capacity and reducing the efficiency of parallelly chloroplast sequencing, since the chloroplast DNA sequencing data represents only a small fraction of WGS. Therefore, most existing methods for obtaining DNA and sequencing data suitable for whole CpGenomes cannot meet the needs of large-scale CpGenome sequencing, greatly limiting and hindering the in-depth research of plant genetics and evolution. Target enrichment before sequencing is a useful method that allow for in-depth analysis of specific portions of the genome. Moreover, a group of universal probes covering whole CpGenome in a tribe species can make target enrichment strategy exert it’s advantages. Large scale CpGenomes target enrichment by universal probes can provide cost-effective, high density, and high coverage. Efficiency target enrichment and comparative analysis of CpGenomes for different clades More than 3,000 chloroplast genomes have been released recently 31 , since the first reported sequencing of the complete CpGenome of Nicotiana tabacum 32 . We chose the 99 representative CpGenomes, including 6 bamboo CpGenomes from 3,654 CpGenomes published to design probes. These vascular plants included 7 clades (Lycopodiophyta, Moiliformopses, Gymnosperms, Basal angiosperms, Monocots, Eudicot, and Magnoliidae), belonging to 57 families and 40 orders. The alignment of the CpGenomes of 7 clades to Arabidopsis thaliana CpGenome may show the CpGenome structure variation during evolution and indicating differences among different clades (Fig. 1 ). Structure variation indicated the pan-CpGenome derived from CpGenomes of distinct clades was essential for constructing greater applicability of pan-CpGenome with more divergent sequences. In 146–150 kb, 124–129 kb, and 88–92 kb, Poaceae had alignment gaps compared to the rest of Monocots, ANA grade, Magnoliids, and Eudicots. Moreover, Ferns, Horsetails, Gymnosperm, and Lycophytes indicated fragment sequences at the corresponding positions. It may suggest the corresponding CpGenome regions completed in angiosperm during evolution and uniquely lost in Poaceae after Angiosperm. However, the phenomenon should be further tested on the basis of broad-spectrum reference and amplification samplings. In pan-CpGenome construction, unique sequences were selected, and the final pan-CpGenome size was ~ 15 Mb. A total of 180,519 probes were designed and synthesized using a new hybridization-based approach to enrich chloroplast DNA fragments. Evaluation of the quality of the probes and pan-CpGenome showed a high mapping ratio, which was stable and efficient in bamboo CpGenomes. Besides bamboos, the amplified plant CpGenomes expanded variational sequences and universality of the probes in the pan-genome construction step. Thus, the probes also had high mapping rates in some orders, such as Malvales, Rosales, Pinales and Poales, et al , and indicated the applicability of the probes in these clades. Conversely, lower mapping rates were found in Nymphaeales, Solanales, Schizaeales, Lamiales, et al , which may due to inadequate and poor corresponding CpGenomes materials in pan-Genome constructing. It can be solved by amplifying corresponding CpGenomes to expand divergent sequences in pan-CpGenome or decreasing parameter restriction. Comparing of the assembled CpGenome with its published counterparts demonstrated a mapping coverage of over 98%, further confirming the efficiency of the probes in enriching chloroplast DNA fragments. In general, this pipeline of pan-CpGenome construction, pan-CpGenome-based probes design, and CpGenome enrichment showed its performance in bamboo CpGenomes and recommended a strategy of large-scale CpGenomes acquiring to green plants. Bamboo CpGenomes could provide additional information on large-scale phylogenetic relationships There are more than 500 bamboo species in China, which play significant roles in economy, ecology, culture, aesthetics, and technology 33 , 34 . Bambusoideae is one of three subfamilies in Poaceae known as the BEP clade 35 . Bamboo remains one of the most challenging groups for plant taxonomists and field botanists 36 , due to infrequent, incongruent, unpredictable flowering events, and diversity vegetative characters, which may result from frequent hybridization occurred in bamboos 36 , 37 . As a useful strategy in phylogenetics and classification of species, phylogenetic analysis based on sequences has been performed in bamboos over the past decades. Extensive sampling and sequencing of the plastid genome has been a remarkable effort in genetic, phylogenetic, and classification analysis of bamboo. We have constructed a phylogenetic tree of 412 samples, covering more than 300 species, 40 genera, which is the largest sampling project of bamboo in China and provides a large-scale phylogenetic tree of bamboos. According to the phylogenetic tree, XI ( Ampelocalamus calcareus ) is the earliest diverging Arthrostylidiinae species, consistent with previous studies 19 , 20 , 38 . The phylogenetic tree supports (Arundinarieae (Bambuseae, Olyreae)) pattern ,and the pattern is consistent with previous studies based on smaller-scale plastid sequences, suggesting a non-monophyletic lineage of woody bamboos 35,39−41 . The results also showed the stability of the pattern, which may no change under amplified sampling. Differently, phylogenetic trees using nuclear sequences suggested the basal position of Olyreae in Bambusoideae and showed a monophyletic origin of the woody characteristic of bamboo 36 , 42 . For clarifying the confliction, the analysis should focus on changes in gene duplications and genome structure caused mainly by multiple hybridizations in bamboo, by performing largely amplified sampling and genome-wide sequences. Additionally, there is a fundamental demand for bamboo life trees, especially in China, which has the world’s largest areas of bamboo plantation 43 . The Phyllostachys genus, with 59 species, is the most economically important among bamboos 44 – 46 . Phyllostachys edulis is the most significant Phyllostachys species, accounting for ∼73.8% bamboo-growing regions in China (4.43 million ha), and is the most abundant non-wood resource 34 . This study included 102 Phyllostachys CpGenome sequences, covering more than 90% Phyllostachys species, and provides an unprecedented opportunity to expand taxonomic knowledge of Phyllostachys genus. Traditionally, Phyllostachys genus can be divided into two groups, P. sect. Phyllostachys and P. sect. Heteroclada , based on morphological features such as inflorescences and rhizomes et al 47 , 48 . But there is a controversy in this classification due to some in-between morphological features of two groups 44 , 47 . Compared to the traditional taxonomy, the species tree we constructed exhibited different phylogenetic relationships in P. sect. Phyllostachys and P. sect. Heteroclada , specifically the two groups of species intermixed in the species tree. Incongruence between morphological taxonomy and the phylogenetic tree may be due to complex evolutionary processes or taxonomic treatments. Totally, 13 non- Phyllostachys species, such as Indocalamus pedalis , Oligostachyum oedogonatum , Pleioblastus solidus, et al were found in Phyllostachys genus Clade. They are all scattered in Phy-II. The existence of numerous non- Phyllostachys species may indicate non-monophyly of the Phyllostachys genus. It is supporting the non-monophyly thesis of Phyllostachys genus based on previous studies of plastid sequences 37 , 49 , 50 and conflicting with previous results based on non-genome wide nuclear sequences or morphological features 44 , 47 , 48 . The classification should be treated carefully because of the evolutionary complexity of bamboos. Moreover, The incongruence between plastid and nuclear gene phylogenies in Arundinarieae was found in the previous study 19 . Though the species tree we constructed supports more than 90% species coverage of Phyllostachys , the taxonomy of Phyllostachys clade should be further tested within the phylogenies based on genome-wide nuclear genes. Conclusions A practical and large-scale approach to CpGenome acquisition will promote plant genetics and phylogenetics. We recommend a universal probe-based CpGenome enrichment pipeline, which successfully applied to bamboo CpGenomes, and 358 woody bamboo CpGenomes were acquired. Moreover, the universal probes we designed for bamboo exhibited a broad spectrum, which may also be applicable in Magnoliales, Pinales, Poales et al . We also reconstructed a phylogenetic tree of bamboos in China based on CpGenomes which supported the non-monophyly of the genus Phyllostachys . For promoting evolution, phylogenetic and population studies, we uploaded the sequences to CNGB to provide a BLAST + server. For further research, we will explore many divergent hotspot regions associated with repeat sequences of LSC regions, such as tRNA clusters, which can be used as genetic markers for phylogenetic studies. Abbreviations CNGB, China National GeneBank CpGenome, chloroplast genome IR, inverted repeat IRA, Inverted Repeat A IRB, Inverted Repeat B LSC, large single-copy region NGS, next-generation sequencing pan-CpGenome, pan-chloroplast genome PHYC, phytochrome C SSC, single-copy region WGS, whole genome sequencing Declarations Ethics approval and consent to participate Not applicable. Consent for publication All authors consent to publish. Availability of data and materials The datasets supporting the conclusions of this article are available in the CNGB repository, https://db.cngb.org/search/project/CNP0000502/ . Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Sub-Project of the National Science and Technology Support Plan of the Twelfth Five-Year Plan in China (grant numbers 2015BAD04B03 and 2015BAD04B01). The funding numbers provided the financial support to the research programs, but didn’t involve in work design, data collection, analysis and preparation of the manuscript. Authors’ Contributions Conceptualization, HZ, TY, WM, HL; Data curation, HZ, ZG, TY, YS, HL; Formal analysis, JW, TY, WM, YS, YH, YW; Funding acquisition, HZ; Investigation, HZ, JW, TY, WM, XL, HL; Project administration, HZ, TY, HL; Resources, HZ, ZG, TY, HL; Supervision, HZ, HL; Visualization, JW, WM; Writing - original draft, HZ, JW, TY; Writing - review & editing, HZ, JW; All authors have read and approved the manuscript. Acknowledgements We wish to acknowledge the GABR Consortium members, partners, advisors, and supporters who have helped the GABR project run smoothly. Authors' Information Not applicable. References Soreng RJ, et al. A worldwide phylogenetic classification of the Poaceae (Gramineae) II: An update and a comparison of two 2015 classifications. Journal of Systematics Evolution. 2017;55:259–90. Horn T, Häser A. Bamboo tea: reduction of taxonomic complexity and application of DNA diagnostics based on rbcL and matK sequence data. PeerJ. 2016;4:e2781. Twyford AD, Ness RW. Strategies for complete plastid genome sequencing. Mol Ecol Resour. 2017;17:858–68. doi: 10.1111/1755-0998.12626 . Sungkaew S, Stapleton CM, Salamin N, Hodkinson TR. Non-monophyly of the woody bamboos (Bambuseae; Poaceae): a multi-gene region phylogenetic analysis of Bambusoideae ss. Journal of plant research. 2009;122:95. Stapleton C, Chonghaile GN, Hodkinson TR. 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Cite Share Download PDF Status: Published Journal Publication published 05 Feb, 2021 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Major revision 29 Oct, 2020 Review # 2 received at journal 28 Oct, 2020 Review # 1 received at journal 26 Oct, 2020 Reviewer # 2 agreed at journal 09 Oct, 2020 Reviewer # 1 agreed at journal 08 Oct, 2020 Reviewers invited by journal 17 Sep, 2020 Editor assigned by journal 03 Sep, 2020 Submission checks completed at journal 02 Sep, 2020 Editor invited by journal 02 Sep, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-58636","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":2379720,"identity":"dae8d21d-37c0-424a-9c34-eee12951e0ed","order_by":0,"name":"Jiongliang Wang","email":"","orcid":"","institution":"International Center for Bamboo and Rattan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiongliang","middleName":"","lastName":"Wang","suffix":""},{"id":2379721,"identity":"999c7856-0e0f-482b-a911-76d5bff9c0ea","order_by":1,"name":"Weixue Mu","email":"","orcid":"","institution":"Bhabha Group of Institutions","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weixue","middleName":"","lastName":"Mu","suffix":""},{"id":2379722,"identity":"46ebccee-1122-41d9-8f05-d46e576289ba","order_by":2,"name":"Ting Yang","email":"","orcid":"","institution":"Bhabha Group of Institutions","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Yang","suffix":""},{"id":2379723,"identity":"82604437-038f-47bf-b4f6-b44d75fc8c05","order_by":3,"name":"Yue Song","email":"","orcid":"","institution":"Bhabha Group of Institutions","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Song","suffix":""},{"id":2379724,"identity":"ad42d981-ae34-49ec-8fef-e51a807be6e1","order_by":4,"name":"YinGuang Hou","email":"","orcid":"","institution":"International Center for Bamboo and Rattan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"YinGuang","middleName":"","lastName":"Hou","suffix":""},{"id":2379725,"identity":"f63e7c87-848b-4f31-aa7d-af295723effd","order_by":5,"name":"Yu Wang","email":"","orcid":"","institution":"International Center for Bamboo and Rattan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Wang","suffix":""},{"id":2379726,"identity":"b5895100-692a-4eab-b45c-8f2dfbea0248","order_by":6,"name":"Zhimin Gao","email":"","orcid":"","institution":"International Center for Bamboo and Rattan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhimin","middleName":"","lastName":"Gao","suffix":""},{"id":2379727,"identity":"4a157b4b-8890-4fd4-9c07-478f833cda3b","order_by":7,"name":"Xin Liu","email":"","orcid":"","institution":"International Center for Bamboo and Rattan","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Liu","suffix":""},{"id":2379728,"identity":"0e023e20-2be4-4be5-890c-e654c9faa23b","order_by":8,"name":"Huan Liu","email":"","orcid":"","institution":"Bhabha Group of Institutions","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Liu","suffix":""},{"id":2379729,"identity":"9012a5f6-756c-4c80-87e6-92489c5edb63","order_by":9,"name":"Hansheng Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACCQYGZhBtACI+GDAkkKaFcQbJWph5GIjQIj+7+eHjgpo79ubsvYdf2xTY5Zk3MD/8wFBzB6cWxjnHjI1nHHuWuLPnXJp1jkFyscwBNmMJhmPPcGphlkgwk+ZhO5xgcCPHzDjH4EDiDAYGMwbGhsM4tbBJpH+T5vl32N7g/hszYwuwFvZveLXwSOSYSfO2HWbccIPH+DEDWAsPflskJHKKjXn7DiduOJNjxtgD9IsEM0+xRMIx3FrkZ6RvfMzzDeiw42eMP/z4Y5cnwd6+8cOHGtxaUP0FpkDRlECUBqDaD0QqHAWjYBSMghEGANZGT/zFabBkAAAAAElFTkSuQmCC","orcid":"","institution":"International Center for Bamboo and Rattan","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hansheng","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2020-08-13 10:14:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-58636/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-58636/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-020-02779-5","type":"published","date":"2021-02-05T15:01:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":2453943,"identity":"872055ad-a4a8-4b78-b4e5-b6a43994e436","added_by":"auto","created_at":"2020-09-17 13:47:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":212933,"visible":true,"origin":"","legend":"The circle of alignment and depth sketch of a core CpGenome by BRIG. The CpGenome of Arabidopsis thaliana with a length of 154,478 bp was used as the core sequence of pan-genome. Please see the details for Methods. The inner circles show the alignment of 7 clade CpGenomes to A. thaliana using BLAST+. The black circle indicates gene positions, and adjacent colorful circles manifest the genome structure of A. thaliana. Based on DOGMA, the CpGenome was divided into four sections: Inverted Repeat A (IRA), Small Single Copy (SSC), Inverted Repeat B (IRB), and Large Single Copy (LSC). The outer circle shows the depth of the probes mapping to A. thaliana.","description":"","filename":"Onlinefloatimage1.Png","url":"https://assets-eu.researchsquare.com/files/rs-58636/v1/Onlinefloatimage1.Png"},{"id":2453944,"identity":"d740f32c-d2e0-4ce1-9140-cbc54474e468","added_by":"auto","created_at":"2020-09-17 13:47:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49038,"visible":true,"origin":"","legend":"Evaluation of the pipeline performance in woody bamboos. (A) A dot plot provides the average depth (×) and coverage ratio of the 99 plant CpGenomes used to design the probes. The red and blue dots represent bamboos and other plant species, respectively. The black lines represent the average depth (×) and a coverage ratio of the bamboo species. (B) A dot plot provides log10(cover length) and the coverage ratio of the 468 plant CpGenomes used to evaluate the probes. The red and blue dots represent bamboos and other plant species, respectively. The black lines represent log10(cover length) and the coverage ratio of the bamboo species, respectively. (C) A box plot of gene number, genome size, and raw bases (bp) of the sequenced bamboos CpGenomes in this study. (D) Evaluation of mapping and coverage of the probes compared to the in-house and released bamboo CpGenomes. The mapping ratio represents the proportion of reads obtained by the probes aligned with the released bamboo CpGenomes. Mapping coverage represents the proportion of the assembled CpGenomes based on the probes aligned with the released bamboo CpGenomes.","description":"","filename":"Onlinefloatimage2.Png","url":"https://assets-eu.researchsquare.com/files/rs-58636/v1/Onlinefloatimage2.Png"},{"id":2453945,"identity":"8fd04d77-9883-4261-9f97-60c727ef3239","added_by":"auto","created_at":"2020-09-17 13:47:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":140942,"visible":true,"origin":"","legend":"A species tree of Phyllostachys clade based on 75 chloroplast genes. The species tree divided into 2 parts, which labeled with different background colors. Numbers at the node indicated the bootstrap values and bootstrap values lower than 80 were concealed. The red, purple, grey, and blue blocks in the tree represented P. sect. Heteroclada species, P. sect Phyllostachys species, unlabeled and non-Phyllostachys species, respectively. Name with ‘LOC’ stands represented newly sequenced sequences in this study.","description":"","filename":"Onlinefloatimage3.Png","url":"https://assets-eu.researchsquare.com/files/rs-58636/v1/Onlinefloatimage3.Png"},{"id":13594010,"identity":"4c4c858c-f58f-4391-8a8b-a5a7666a36a8","added_by":"auto","created_at":"2021-09-17 05:18:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2526002,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-58636/v1/f67f06c9-1114-4ed1-aa94-27590fc98154.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTargeted Enrichment of Novel Chloroplast-Based Probes Reveals a Large-Scale Phylogeny of 412 Bamboos\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eThe subfamily Bambusoideae belongs to the grass family Poaceae and exhibits substantial phenotypic diversity, with 1,642 species in 125 genera, three tribes, and 15 subtribes, which have been classified into ~\u0026thinsp;75 clades\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The Bambuseae consists of tropical woody bamboos (Bambuseae), temperate woody bamboos (Arundinarieae) and herbaceous bamboo tribe (Olyreae). Bambusoideae predominantly distributed in the Old World, such as China, Japan, Thailand, Indonesia, and the countries of Southeast Asian. As one of the most ecologically and industrially valuable tribes of Bambusoideae, woody bamboos were used for furniture, paper, fiber textiles, and fuel\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In total, about 500 bamboos are distributed in Asia, spanning a wide geographic and temperature range. However, infrequent, incongruent, and unpredictable flowering events as well as unstable vegetative characteristics, severely restricted the identification and classification of woody bamboos. The phylogenetic relationships based on more massive amounts of woody bamboos remain elusive due to the lack of extensive and high-quality genomic resources.\u003c/p\u003e \u003cp\u003eThe chloroplast genome (CpGenome) is an essential resource for the study of plant evolution\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This high-copy organelle is one of the most technically accessible regions of the genome. The chloroplast genomic DNA of green plants commonly exhibits a conserved genome structure that contains two copies of inverted repeat (IR) separating the small single-copy region (SSC) and the large single-copy region (LSC)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The CpGenome has been a popular source of reconstructing the phylogeny of green plants, and many chloroplast DNA loci are contributing to the development of plant taxonomy. To obtain chloroplast DNA suitable for whole chloroplast genome sequencing, it can be traditionally enriched by using the sucrose gradient centrifugation method\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, the high salt method\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, long PCR technology by using primers\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The characters of the strategies above are the use of physical methods to extract chloroplast DNA or the need for high quality, sufficiently extracted cellar DNA and the appropriate primers. With the development of sequencing technology, next-generation sequencing (NGS) has the advantageous characteristics of high-throughput and efficient, resulting in a rapid increase in the amount of sequencing data. Chloroplast DNA generally accounts for only about 0.5\u0026ndash;13% of the whole genome\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. But, the chloroplast DNA sequencing data from the whole genome sequencing (WGS) data produced a lot of \u0026ldquo;useless\u0026rdquo; data except for \u0026ldquo;useful\u0026rdquo; ones, consuming much of the sequencing capacity and reducing the efficiency of parallelly chloroplast sequencing. The above methods for obtaining chloroplast DNA sequencing data cannot meet the needs of large-scale CpGenome sequencing, which significantly restricts and hinders the in-depth research of plant genetics and evolution.\u003c/p\u003e \u003cp\u003eIn this study, the main goals were: (1) To develop and evaluate a pipeline to target-enrich and assembly the chloroplast data of bamboos. (2) To obtain high-quality and high coverage of bamboo CpGenomes by the pipeline, to reconstruct a phylogenetic tree, and to promote phylogenetic knowledge of bamboo. (3) To share the new sequenced bamboo CpGenomes, allowing researchers to quickly compare suspect chloroplast data and explore the bamboo CpGenomes.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSpecies selection for probe design and evaluation\u003c/h2\u003e \u003cp\u003eTo improve the variability and versatility of the probes, we selected 567 representative species from the 3,654 published CpGenomes species (collected from NCBI, Released Dec 2018) to design and evaluate probes for a targeted enrichment strategy of CpGenomes (Supplementary Table S1 and S2). Among the 567 species, 22 are bamboo species. For data preprocessing, we elucidated our approach in a flow chart (Supplementary Figure S1). A phylogenetic tree (Supplementary Figure S2) was constructed based on the 567 complete CpGenomes, which spanned the phylogenetic diversity of 7 major clades, including 40 orders and 57 families. The model species in each clade were selected as core candidates. Thus, a total of 99 CpGenomes, including 6 bamboo CpGenomes, were chosen as the representative species for the probe design (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and the remaining (468 CpGenomes) were chosen as test data further to assess the efficiency of the probes for chloroplast capture. The species for probe design and the species for probe evaluation were different genera but belong to the same family (e.g., \u003cem\u003eDanthonia\u003c/em\u003e and \u003cem\u003eChionochloa\u003c/em\u003e, both are Poaceae).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe taxonomic composition of the chloroplast genome sequences which used for design probes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCBI species ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eclassfy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eorder\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003efamily\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003egenus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_010093.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAcorales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAcoraceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAcorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e 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colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eArundinaria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eArundinaria appalachiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_012927.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e 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colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eLiliaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eFritillaria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eFritillaria cirrhosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_024720.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIndocalamus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eIndocalamus wilsonii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_022926.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eZingiberales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMusaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eMusa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eMusa textilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_001320.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eOryza\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eOryza sativa Japonica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_017609.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAsparagales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eOrchidaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePhalaenopsis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePhalaenopsis equestris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_023245.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePharus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePharus lappulaceus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_013991.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eArecales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eArecaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePhoenix\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePhoenix dactylifera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_015817.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePhyllostachys\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePhyllostachys edulis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_022850.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSetaria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSetaria italica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_008602.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSorghum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSorghum bicolor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_002762.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eTriticum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eTriticum aestivum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_015894.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAlismatales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAraceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eWolffiella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eWolffiella lingulata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_024725.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eYushania\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eYushania levigata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_001666.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonocots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePoales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePoaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eZea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eZea mays\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_005086.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBasal angiosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAmborellales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAmborellaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAmborella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAmborella trichopoda\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_006050.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBasal angiosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eNymphaeales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eNymphaeaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eNymphaea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eNymphaea alba\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_023242.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMagnoliidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMagnoliales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMagnoliaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eMagnolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eMagnolia sprengeri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_008457.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMagnoliidae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePiperales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePiperaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePiper\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePiper cenocladum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_026690.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEricales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eActinidiaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eActinidia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eActinidia chinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_009265.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBrassicales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eBrassicaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAethionema\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAethionema cordifolium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_015621.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAsterales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAsteraceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAgeratina\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAgeratina adenophora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_022412.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMyrtales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMyrtaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAngophora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAngophora costata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_000932.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBrassicales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eBrassicaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eArabidopsis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_009268.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBrassicales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eBrassicaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eArabis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eArabis hirsuta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_022810.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eApiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAraliaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAralia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAralia undulata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_021121.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEricales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePrimulaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eArdisia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eArdisia polysticta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_025910.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAsterales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAsteraceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eArtemisia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eArtemisia montana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_022432.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGentianales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAsclepiadaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAsclepias\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAsclepias syriaca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_016734.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBrassicales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eBrassicaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eBrassica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eBrassica napus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_024541.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEricales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTheaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCamellia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCamellia crapnelliana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_010323.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBrassicales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaricaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCarica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCarica papaya\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_014674.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFagales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFagaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCastanea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCastanea mollissima\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_011163.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFagales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFagaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCicer\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCicer arietinum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_025642.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLamiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eOrobanchaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCistanche\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCistanche phelypaea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_008334.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSapindales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRutaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCitrus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_008535.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGentianales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRubiaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCoffea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCoffea arabica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_022409.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMyrtales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMyrtaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCorymbia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCorymbia eximia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_007144.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCucurbitales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCucurbitaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCucumis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCucumis sativus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_009963.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSolanales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eConvolvulaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCuscuta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCuscuta exaltata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_014569.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGeraniales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eGeraniaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eErodium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eErodium texanum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_022396.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMyrtales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMyrtaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEucalyptus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eEucalyptus aromaphloia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_015206.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRosales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRosaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eFragaria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eFragaria vesca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_007942.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFabales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFabaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eGlycine\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eGlycine max\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_016668.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMalvales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMalvaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eGossypium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eGossypium raimondii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_024732.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAsterales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCampanulaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eHanabusaya\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eHanabusaya asiatica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_023110.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAsterales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAsteraceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eHelianthus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eHelianthus decapetalus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_026726.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSolanales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSolanaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIochroma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eIochroma loxense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_009808.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSolanales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eConvolvulaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIpomoea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eIpomoea purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_026677.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFabales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFabaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eLibidibia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLibidibia coriaria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_024064.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMalpighiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eChrysobalanaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eLicania\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLicania alba\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_002694.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFabales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFabaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eLotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLotus japonicus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_023090.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFabales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFabaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eLupinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLupinus luteus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_010433.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMalpighiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eEuphorbiaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eManihot\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eManihot esculenta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_003119.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFabales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFabaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eMedicago\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eMedicago truncatula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_012615.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRanunculales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRanunculaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eMegaleranthis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eMegaleranthis saniculifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_008359.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRosales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMoraceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eMorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eMorus indica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_025339.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eProteales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eNelumbonaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eNelumbo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eNelumbo nucifera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_010358.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMyrtales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eOnagraceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eOenothera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eOenothera argillicola\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_013707.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLamiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eOleaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eOlea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eOlea europaea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_006290.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eApiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAraliaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePanax\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePanax ginseng\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_009259.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFabales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFabaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePhaseolus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_009143.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMalpighiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSalicaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePopulus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePopulus trichocarpa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_014697.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRosales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRosaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePrunus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePrunus persica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_015996.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRosales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRosaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePyrus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePyrus pyrifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_016736.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMalpighiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eEuphorbiaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRicinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eRicinus communis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_026722.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMalpighiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSalicaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSalix\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSalix purpurea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_026202.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLamiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eScrophulariaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eScrophularia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eScrophularia takesimensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_023085.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSaxifragales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCrassulaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSedum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSedum sarmentosum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_016730.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCaryophyllales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaryophyllaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSilene\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSilene latifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_008096.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSolanales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSolanaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSolanum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSolanum tuberosum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_014676.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMalvales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMalvaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eTheobroma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eTheobroma cacao\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_024034.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFabales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFabaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eTrifolium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eTrifolium grandiflorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_021449.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLamiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eLentibulariaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eUtricularia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eUtricularia gibba\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_021091.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFabales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFabaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eVigna\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eVigna angularis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_007957.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eVitales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eVitaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eVitis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eVitis vinifera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_023259.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEudicots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGeraniales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eVivianiaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eViviania\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eViviania marifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_013086.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLycopodiophyta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSelaginellales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSelaginellaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eSelaginella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSelaginella moellendorffii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_008829.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMoiliformopses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMarattiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMarattiaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eAngiopteris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAngiopteris evecta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_014699.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMoiliformopses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEquisetales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eEquisetaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eEquisetum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eEquisetum arvense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_014348.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMoiliformopses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDennstaedtiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDennstaedtiaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePteridium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePteridium aquilinum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_016063.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGymnosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePinales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCephalotaxaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCephalotaxus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCephalotaxus wilsoniana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_009618.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGymnosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCycadales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCycadaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCycas\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCycas taitungensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_026301.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGymnosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGnetales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eGnetaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eGnetum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eGnetum gnemon\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_024022.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGymnosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePinales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCupressaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eJuniperus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eJuniperus monosperma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_021456.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGymnosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePinales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePinaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePicea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePicea abies\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_011153.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGymnosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePinales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePinaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePinus contorta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_023805.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGymnosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePinales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePodocarpaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePodocarpus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePodocarpus lambertii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNC_016065.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGymnosperms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePinales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCupressaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eTaiwania\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eTaiwania cryptomerioides\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of non-redundant chloroplast reference\u003c/h2\u003e \u003cp\u003eUsing the CpGenome of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e as the initial reference sequence (as a database sequence), other selected CpGenomes (as query sequences) were aligned to the database sequence by BLAST\u0026thinsp;+\u0026thinsp;v2.2.25 software with default parameters. The sequences with more than 90% identity were masked from the query sequences. Then, the resulting sequences were subjected to a secondary round masking of redundant sequences, which were identified by an all-against-all BLAST+. Finally, a non-redundant chloroplast reference, as a pan-chloroplast genome (pan-CpGenome), was obtained by iterative analysis. Sequences with high similarity (\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;90%) were masked with \u0026ldquo;Ns\u0026rdquo;, and others were highly divergent sequences in the pan-CpGenome (Supplementary File F1). The visualization of the alignment of 98 CpGenomes to \u003cem\u003eArabidopsis thaliana\u003c/em\u003e CpGenome was conducted by BLAST Ring Image Generator (BRIG V0.9)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e with default parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eUniversal probes designed for bamboo CpGenomes\u003c/h2\u003e \u003cp\u003eThe regions of the pan-CpGenome sequences which have not been masked to \u0026ldquo;Ns\u0026rdquo; were extended by 40\u0026nbsp;bp on both sides for the design of the probes. Each region was divided into \u003cem\u003eK\u003c/em\u003e-mers of 90\u0026nbsp;bp in length and the melting temperatures of the \u003cem\u003eK\u003c/em\u003e-mers were calculated\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. A comprehensive score of uniqueness, frequency, melting temperature, and GC content was calculated for each probe by Primer3 v2.4.0\u003csup\u003e12\u003c/sup\u003e. The probes with the highest comprehensiveness scores were selected in 20\u0026nbsp;bp window and slid along the target region at the fixed interval. For ensuring high coverages of the probe sequences in the target region, the target region was covered at least 2 times by these selected probes. Finally, a total of 180,519 DNA oligonucleotides were synthesized by a CustomArray B3 Synthesizer (CustomArray, Washington, DC, USA) according to the manufacturer\u0026rsquo;s instructions and dissolved in 10\u0026thinsp;\u0026times;\u0026thinsp;TE buffer (pH\u0026thinsp;=\u0026thinsp;8.0).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eTaxa sampling\u003c/h2\u003e \u003cp\u003eAll sampled species covering more than 30 genera (Supplementary Table S4) were collected in spring 2015 and 2016 under the permission of four main bamboo gardens in China:(1) Taiping base of ICBR: N:30\u0026deg;20'57.03\", E:118\u0026deg;01'30.21\", 150\u0026nbsp;M, (2) WangJianglou Park, Chengdu: N:30\u0026deg;37'54.85\", E:104\u0026deg;05'23.84\", 150\u0026nbsp;M, (3) Yunnan Pu'er Asia Bamboo and Rattan Exposition Garden: N:22\u0026deg;41'24.67\", E:100\u0026deg;56'26.51\", 1000\u0026nbsp;M, and (4) BaiMa base of Nanjing Forestry University: N:31\u0026deg;36'35.62\", E:119\u0026deg;10'34.29\", 50\u0026nbsp;M. During the sampling process, identification services of bamboo samples were provided by related taxonomists at each bamboo garden. Totally, 358 bamboo samples, mainly from young leaves, were collected. All samples were frozen in liquid nitrogen immediately and were preserved in ultra-low temperature refrigerator at -80\u0026nbsp;\u0026deg;C, followed by DNA extraction.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and target enrichment sequencing for bamboos\u003c/h2\u003e \u003cp\u003eA total of 358 woody bamboo samples were sampled and sequenced in this study (Supplementary Table S4), as a practical application of target enrichment sequencing and an evaluation of the capture efficiency. Genomic DNA from each sample was extracted using the CTAB method\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and fragmented to a peak size of 200\u0026nbsp;bp using a Covaris E220 sonicator (Covaris, Woburn, Massachusetts, USA), followed by the end-repair, addition of base \u0026ldquo;A\u0026rdquo;, and adapter ligation. DNA fragments of the desired size (200\u0026nbsp;bp) were selected on an agarose gel and hybridized to the probes for 72\u0026nbsp;h. The probes captured DNA fragments were recycled by magnetic beads coated with streptavidin, which interacted with the biotin on the probes to wash away the uncaptured DNA fragments.\u003c/p\u003e \u003cp\u003eThe captured DNA fragments were sequenced on the BGISEQ-500 platform at Beijing Genomics Institute, Shenzhen, China. High-quality reads ranging from 1\u0026nbsp;Gb to 9\u0026nbsp;Gb with 100\u0026nbsp;bp paired-end were acquired for each sample. For data preprocessing, we illuminated our method in a flow chart (Supplementary Figure S1). SOAPfilter (v2.2)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e was applied to remove low-quality reads and adaptors in the following criteria (1) reads with \u0026gt;\u0026thinsp;10% base of N; (2) reads with \u0026gt;\u0026thinsp;40% of low-quality reads (value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;10); (3) reads contaminated with adaptors and produced by PCR duplication. A CpGenome of \u003cem\u003ePhyllostachys edulis\u003c/em\u003e (downloaded from NCBI, accession number: HQ337796.1) was used as a reference for assembly using MITObim (V1.8)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In this way, we finally recovered the complete CpGenomes of all 358 samples. Additionally, the plastid genomes were annotated in the current standard web-based program DOGMA\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dogma.ccbb.utexas.edu/\u003c/span\u003e\u003c/span\u003e,).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis of woody bamboos\u003c/h2\u003e \u003cp\u003eWe downloaded previously published CpGenomes of 71 bamboo species from NCBI (released May 2020) to amplify the sampling of the species tree (Supplementary Table S5). Redundancy sequences were removed, resulting in 412 non-redundant bamboo CpGenomes (Supplementary Table S6). The CDS sequences of each gene family were aligned using MAFFT (V7.017)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e with default parameters based on the corresponding protein sequences, and then sequences were concatenated to produce 54,078 nucleotide positions. A maximum likelihood (ML) species tree was constructed with IQ-TREE (V1.6.12)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e with parameters: -m MFP, -B 1000, -bnni, -alrt 1000.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSharing the bamboo CpGenome dataset\u003c/h2\u003e \u003cp\u003eAll 358 woody bamboo CpGenomes provided in Supplementary Table S4 were deposited in China National GeneBank (CNGB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.cngb.org/blast/blast/blastn/\u003c/span\u003e\u003c/span\u003e), with the database named \u0026ldquo;Chinese Bamboo Database\u0026rdquo;. The CNGB developed BLAST+ (version 2.6.0) service to allow public searches against the bamboo CpGenomes.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of universal chloroplast probes for bamboos\u003c/h2\u003e \u003cp\u003eFrom the 3,654 CpGenomes collected from NCBI, 567 high-quality CpGenomes were selected for probe development and divided into two datasets, with 99 CpGenomes for probe design and 468 CpGenomes for probe evaluation. Considering the applicability and robustness of the probes designing for bamboos, and the diversity of CpGenomes, the 99 CpGenomes were selected from different families. Details of the related methods were provided in Supplementary Figure S1. A 15\u0026nbsp;Mb pan-CpGenome was assembled based on the alignment to \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Supplementary File F1). The comparison analysis showed the CpGenomes had great variations across species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Lycophytes CpGenome showed the greatest gaps in the alignment, followed by Ferns, Horsetails, and Gymnosperm. Eudicots and some of Monocots had the highest integrity of CpGenomes. Compared to Eudicots, some of Monocots, Gymnosperm, Ferns, Horsetails, and Lycophytes had large gaps at 146\u0026ndash;150\u0026nbsp;kb, 124\u0026ndash;129\u0026nbsp;kb, and 88\u0026ndash;92\u0026nbsp;kb. According to the mapping depth, the depth of probe coverage at 100\u0026ndash;110\u0026nbsp;kb, 35\u0026ndash;42\u0026nbsp;kb, and 130\u0026ndash;140\u0026nbsp;kb were rather lower than at other sites. For evaluating the quality of the pan-CpGenome, we calculated the coverage of the probes designed for the 99 complete CpGenomes. Alignment with the 99 reference CpGenomes showed an average coverage of 88.2% and an average base depth of 9.04\u0026times;. In bamboos, the corresponding average coverage and average base depth were 99.6% and 8.43\u0026times;, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA total of 180,519 (21,842,799\u0026nbsp;bp) probes, covering 92.04% of target regions, were designed and showed high consistency in their theoretical melting temperatures and GC contents (Supplementary Table S3). The probes sequences were available in Supplementary File F2. All the designed probes had excellent uniqueness, with an average 1 time while being aligned with the pan-genome. The probes were mostly distributed in the range of 70\u0026ndash;80% melting temperatures and 30\u0026ndash;40% GC content (Supplementary Figure S3). To assess the broad spectrum of the probes, the BLAST\u0026thinsp;+\u0026thinsp;program was employed to align the probes to the 468 complete CpGenomes for evaluating the probes. The average coverage ratio in the 468 complete CpGenomes was 90.54% (Supplementary Table S8). In bamboos, the coverage ratio was all over 93.00%, with an average coverage of 94.78% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Supplementary Table S8). Moreover, some orders such as Magnoliales, Pinales, Poales also had high coverage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProbe-based targeted enrichment and assembly of bamboo CpGenomes\u003c/h2\u003e \u003cp\u003eA total of 358 fresh woody bamboo samples collected from China were included (Supplementary Table S4) and used to evaluate capture efficiency. A total of 1G-9G raw reads were obtained, and low-quality reads and adaptors were filtered in data preprocessing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Supplementary Table S9). Clean and high-quality reads were used for reference-guided assemblies by MITObim and recovered nearly complete CpGenomes for the 358 bamboo species. The assembled CpGenomes ranged from 139,664 to 140,064 base pairs (bp), and the LSC regions varied from 83,496\u0026nbsp;bp to 83,845\u0026nbsp;bp in length (Supplementary Table S9). The CpGenomes were annotated with approximately 121 genes, including around 113 unique genes encoding 80 proteins, 4 ribosomal RNAs, and 29 transfer RNAs, exhibiting a higher degree of conservation.\u003c/p\u003e \u003cp\u003eWe detected 15 overlapped bamboo CpGenomes that were present in both the in-house and published data (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). To assess the target enrichment, we mapped the raw reads to the corresponding CpGenome released previously and compared assembled bamboo CpGenome to corresponding released ones. The results showed more than 45.77% in average of the raw reads from in-house bamboo CpGenomes can be mapped to the corresponding published CpGenomes, and the mapping depth was higher than 1200\u0026times;. Alignment with the published CpGenomes, the coverage of assembled CpGenomes was greater than 98.59% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and Supplementary Table S10).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eA phylogenomic relationship based on 412 bamboo CpGenomes\u003c/h2\u003e \u003cp\u003eFor comprehensively collecting bamboo CpGenomes, 71 bamboo CpGenomes from NCBI were acquired, resulting in a total of 412 non-redundant bamboo CpGenomes after removing redundancy (Supplementary Table S6). We reconstructed a phylogenetic tree of bamboos based on the concatenated sequences of 75 protein-coding genes in the 412 bamboo CpGenomes. Phylogenetic analyses supported the relationship of (Arthrostylidiinae (Bambusinae, Olyreae)). We classified different clades in the phylogenetic tree based on previous studies\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The pattern of (XI((VIII, IV)VI)((IX, III)(VII, V))) was provided in Arthrostylidiinae (Supplementary Figure S4). Most of the newly sequenced species distributed in Clade V, Clade VI, and Clade Paleotropical. Clade XI (\u003cem\u003eAmpelocalamus calcareus\u003c/em\u003e) was the earliest diverging Arthrostylidiinae species. The \u003cem\u003ePhyllostachys\u003c/em\u003e was a representative genus in bamboo, with the clade embed into Clade V, which was the sister clade of \u003cem\u003eBashania fargesii\u003c/em\u003e. There are some non-\u003cem\u003ePhyllostachys\u003c/em\u003e species were found in \u003cem\u003ePhyllostachys\u003c/em\u003e genus clade. The \u003cem\u003ePhyllostachys\u003c/em\u003e genus clade was divided into two groups based on the phylogenetic tree. \u003cem\u003ePhyllostachys edulis\u003c/em\u003e, the most planted bamboo in China, distributed in Phy-II (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The sequences from NCBI clustered with corresponding in-house sequences. For example, \u003cem\u003ePhyllostachys edulis\u003c/em\u003e sequence from NCBI clustered with in-house sequences of \u003cem\u003ePhyllostachys edulis\u003c/em\u003e f epruinosa, \u003cem\u003ePhyllostachys edulis\u003c/em\u003e f exaurita, \u003cem\u003ePhyllostachys edulis\u003c/em\u003e f flexuosa, \u003cem\u003eet al\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChina Bamboo Database in CNGB\u003c/h2\u003e \u003cp\u003eWe uploaded the bamboo CpGenomes sequenced in this study to CNGB to facilitate the accumulation of knowledge on bamboo phylogeny. Researchers can download the raw data and assembled CpGenome sequences from CNGB through Project ID: CNP0000502 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.cngb.org/search/project/CNP0000502/\u003c/span\u003e\u003c/span\u003e). Moreover, researchers can search for all assembled bamboo plastid genomes in this study through web-based BLAST\u0026thinsp;+\u0026thinsp;service (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.cngb.org/blast/\u003c/span\u003e\u003c/span\u003e). The available plastid genome sequences of bamboos and the corresponding BLAST\u0026thinsp;+\u0026thinsp;server can promote researchers to explore the complex and elusive history of bamboo evolution.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003e \u003cb\u003eCpGenome provides an essential resource for plant evolution\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs an essential component of plant organelles and photosynthesis organs, chloroplasts have a simple structure, the small genome size (~\u0026thinsp;110\u0026ndash;165\u0026nbsp;kb) containing\u0026thinsp;~\u0026thinsp;90\u0026ndash;110 protein-coding genes\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and highly conserved gene region across species, due to their non-recombinant, haploid and uniparentally\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The genomic characterization of various aspects of chloroplasts has led to an important role in the research of plant origin, evolution and phylogenetic analysis relationship between different plant species\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Many studies had been reported using chloroplast genes to construct phylogenetic trees of plants. For example, Jansen \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e used 81 chloroplast genes to estimate relationships among the major angiosperm clades; Saarela \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e found weak support for \u003cem\u003eAmborella\u003c/em\u003e as the basal-most angiosperm lineage using 17 plastid genes and the nuclear gene phytochrome C (\u003cem\u003ePHYC\u003c/em\u003e). With the deepening of chloroplast research, more and more researchers are focusing on the complete chloroplast sequence\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Kane \u003cem\u003eet al\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e suggested that the whole CpGenome could serve as an ultra-barcode for identifying plant varieties.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHybridization-based probes for target enrichment in large-scale CpGenome sequencing\u003c/b\u003e \u003c/p\u003e \u003cp\u003eChloroplast DNA can be traditionally acquired by the sucrose gradient centrifugation method\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e or the high salt method\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Another method was to amplify the entire chloroplast DNA from the whole cellular DNA base on a long PCR technology by primers, which were designed on conserved sequences\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These methods were not suitable for large-scale samples due to the large amount of labor and material resources required to obtain chloroplast DNA, and the labor-intensive method used to prepare chloroplast DNA. Chloroplast reads also can be identified from WGS reads by aligning the WGS data with the reference CpGenome. It is a demanding bioinformatics technique and requires a closely related reference CpGenome. The method was not suitable for the species that are not closely related or have poor quality reference genome sequences. Moreover, to assemble only CpGenome based on this method, a great deal of useless sequencing data was thus generated, consuming much of the sequencing capacity and reducing the efficiency of parallelly chloroplast sequencing, since the chloroplast DNA sequencing data represents only a small fraction of WGS. Therefore, most existing methods for obtaining DNA and sequencing data suitable for whole CpGenomes cannot meet the needs of large-scale CpGenome sequencing, greatly limiting and hindering the in-depth research of plant genetics and evolution.\u003c/p\u003e \u003cp\u003eTarget enrichment before sequencing is a useful method that allow for in-depth analysis of specific portions of the genome. Moreover, a group of universal probes covering whole CpGenome in a tribe species can make target enrichment strategy exert it\u0026rsquo;s advantages. Large scale CpGenomes target enrichment by universal probes can provide cost-effective, high density, and high coverage.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEfficiency target enrichment and comparative analysis of CpGenomes for different clades\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMore than 3,000 chloroplast genomes have been released recently\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, since the first reported sequencing of the complete CpGenome of \u003cem\u003eNicotiana tabacum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We chose the 99 representative CpGenomes, including 6 bamboo CpGenomes from 3,654 CpGenomes published to design probes. These vascular plants included 7 clades (Lycopodiophyta, Moiliformopses, Gymnosperms, Basal angiosperms, Monocots, Eudicot, and Magnoliidae), belonging to 57 families and 40 orders. The alignment of the CpGenomes of 7 clades to \u003cem\u003eArabidopsis thaliana\u003c/em\u003e CpGenome may show the CpGenome structure variation during evolution and indicating differences among different clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Structure variation indicated the pan-CpGenome derived from CpGenomes of distinct clades was essential for constructing greater applicability of pan-CpGenome with more divergent sequences. In 146\u0026ndash;150\u0026nbsp;kb, 124\u0026ndash;129\u0026nbsp;kb, and 88\u0026ndash;92\u0026nbsp;kb, Poaceae had alignment gaps compared to the rest of Monocots, ANA grade, Magnoliids, and Eudicots. Moreover, Ferns, Horsetails, Gymnosperm, and Lycophytes indicated fragment sequences at the corresponding positions. It may suggest the corresponding CpGenome regions completed in angiosperm during evolution and uniquely lost in Poaceae after Angiosperm. However, the phenomenon should be further tested on the basis of broad-spectrum reference and amplification samplings.\u003c/p\u003e \u003cp\u003eIn pan-CpGenome construction, unique sequences were selected, and the final pan-CpGenome size was ~\u0026thinsp;15\u0026nbsp;Mb. A total of 180,519 probes were designed and synthesized using a new hybridization-based approach to enrich chloroplast DNA fragments. Evaluation of the quality of the probes and pan-CpGenome showed a high mapping ratio, which was stable and efficient in bamboo CpGenomes. Besides bamboos, the amplified plant CpGenomes expanded variational sequences and universality of the probes in the pan-genome construction step. Thus, the probes also had high mapping rates in some orders, such as Malvales, Rosales, Pinales and Poales, \u003cem\u003eet al\u003c/em\u003e, and indicated the applicability of the probes in these clades. Conversely, lower mapping rates were found in Nymphaeales, Solanales, Schizaeales, Lamiales, \u003cem\u003eet al\u003c/em\u003e, which may due to inadequate and poor corresponding CpGenomes materials in pan-Genome constructing. It can be solved by amplifying corresponding CpGenomes to expand divergent sequences in pan-CpGenome or decreasing parameter restriction. Comparing of the assembled CpGenome with its published counterparts demonstrated a mapping coverage of over 98%, further confirming the efficiency of the probes in enriching chloroplast DNA fragments. In general, this pipeline of pan-CpGenome construction, pan-CpGenome-based probes design, and CpGenome enrichment showed its performance in bamboo CpGenomes and recommended a strategy of large-scale CpGenomes acquiring to green plants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBamboo CpGenomes could provide additional information on large-scale phylogenetic relationships\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThere are more than 500 bamboo species in China, which play significant roles in economy, ecology, culture, aesthetics, and technology\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Bambusoideae is one of three subfamilies in Poaceae known as the BEP clade\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Bamboo remains one of the most challenging groups for plant taxonomists and field botanists\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, due to infrequent, incongruent, unpredictable flowering events, and diversity vegetative characters, which may result from frequent hybridization occurred in bamboos\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. As a useful strategy in phylogenetics and classification of species, phylogenetic analysis based on sequences has been performed in bamboos over the past decades. Extensive sampling and sequencing of the plastid genome has been a remarkable effort in genetic, phylogenetic, and classification analysis of bamboo. We have constructed a phylogenetic tree of 412 samples, covering more than 300 species, 40 genera, which is the largest sampling project of bamboo in China and provides a large-scale phylogenetic tree of bamboos. According to the phylogenetic tree, XI (\u003cem\u003eAmpelocalamus calcareus\u003c/em\u003e) is the earliest diverging Arthrostylidiinae species, consistent with previous studies\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The phylogenetic tree supports (Arundinarieae (Bambuseae, Olyreae)) pattern ,and the pattern is consistent with previous studies based on smaller-scale plastid sequences, suggesting a non-monophyletic lineage of woody bamboos\u003csup\u003e35,39\u0026minus;41\u003c/sup\u003e. The results also showed the stability of the pattern, which may no change under amplified sampling. Differently, phylogenetic trees using nuclear sequences suggested the basal position of Olyreae in Bambusoideae and showed a monophyletic origin of the woody characteristic of bamboo\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. For clarifying the confliction, the analysis should focus on changes in gene duplications and genome structure caused mainly by multiple hybridizations in bamboo, by performing largely amplified sampling and genome-wide sequences. Additionally, there is a fundamental demand for bamboo life trees, especially in China, which has the world\u0026rsquo;s largest areas of bamboo plantation\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePhyllostachys\u003c/em\u003e genus, with 59 species, is the most economically important among bamboos\u003csup\u003e\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePhyllostachys edulis\u003c/em\u003e is the most significant \u003cem\u003ePhyllostachys\u003c/em\u003e species, accounting for \u0026sim;73.8% bamboo-growing regions in China (4.43\u0026nbsp;million ha), and is the most abundant non-wood resource\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This study included 102 \u003cem\u003ePhyllostachys\u003c/em\u003e CpGenome sequences, covering more than 90% \u003cem\u003ePhyllostachys\u003c/em\u003e species, and provides an unprecedented opportunity to expand taxonomic knowledge of \u003cem\u003ePhyllostachys\u003c/em\u003e genus. Traditionally, \u003cem\u003ePhyllostachys\u003c/em\u003e genus can be divided into two groups, P. sect. \u003cem\u003ePhyllostachys\u003c/em\u003e and P. sect. \u003cem\u003eHeteroclada\u003c/em\u003e, based on morphological features such as inflorescences and rhizomes \u003cem\u003eet al\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. But there is a controversy in this classification due to some in-between morphological features of two groups\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Compared to the traditional taxonomy, the species tree we constructed exhibited different phylogenetic relationships in P. sect. \u003cem\u003ePhyllostachys\u003c/em\u003e and P. sect. \u003cem\u003eHeteroclada\u003c/em\u003e, specifically the two groups of species intermixed in the species tree. Incongruence between morphological taxonomy and the phylogenetic tree may be due to complex evolutionary processes or taxonomic treatments. Totally, 13 non-\u003cem\u003ePhyllostachys\u003c/em\u003e species, such as \u003cem\u003eIndocalamus pedalis\u003c/em\u003e, \u003cem\u003eOligostachyum oedogonatum\u003c/em\u003e, \u003cem\u003ePleioblastus solidus, et al\u003c/em\u003e were found in \u003cem\u003ePhyllostachys\u003c/em\u003e genus Clade. They are all scattered in Phy-II. The existence of numerous non-\u003cem\u003ePhyllostachys\u003c/em\u003e species may indicate non-monophyly of the \u003cem\u003ePhyllostachys\u003c/em\u003e genus. It is supporting the non-monophyly thesis of \u003cem\u003ePhyllostachys\u003c/em\u003e genus based on previous studies of plastid sequences\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and conflicting with previous results based on non-genome wide nuclear sequences or morphological features\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The classification should be treated carefully because of the evolutionary complexity of bamboos. Moreover, The incongruence between plastid and nuclear gene phylogenies in Arundinarieae was found in the previous study\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Though the species tree we constructed supports more than 90% species coverage of \u003cem\u003ePhyllostachys\u003c/em\u003e, the taxonomy of \u003cem\u003ePhyllostachys\u003c/em\u003e clade should be further tested within the phylogenies based on genome-wide nuclear genes.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eA practical and large-scale approach to CpGenome acquisition will promote plant genetics and phylogenetics. We recommend a universal probe-based CpGenome enrichment pipeline, which successfully applied to bamboo CpGenomes, and 358 woody bamboo CpGenomes were acquired. Moreover, the universal probes we designed for bamboo exhibited a broad spectrum, which may also be applicable in Magnoliales, Pinales, Poales \u003cem\u003eet al\u003c/em\u003e. We also reconstructed a phylogenetic tree of bamboos in China based on CpGenomes which supported the non-monophyly of the genus \u003cem\u003ePhyllostachys\u003c/em\u003e. For promoting evolution, phylogenetic and population studies, we uploaded the sequences to CNGB to provide a BLAST\u0026thinsp;+\u0026thinsp;server. For further research, we will explore many divergent hotspot regions associated with repeat sequences of LSC regions, such as tRNA clusters, which can be used as genetic markers for phylogenetic studies.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eCNGB, China National GeneBank\u003c/p\u003e \u003cp\u003eCpGenome, chloroplast genome\u003c/p\u003e \u003cp\u003eIR, inverted repeat\u003c/p\u003e \u003cp\u003eIRA, Inverted Repeat A\u003c/p\u003e \u003cp\u003eIRB, Inverted Repeat B\u003c/p\u003e \u003cp\u003eLSC, large single-copy region\u003c/p\u003e \u003cp\u003eNGS, next-generation sequencing\u003c/p\u003e \u003cp\u003epan-CpGenome, pan-chloroplast genome\u003c/p\u003e \u003cp\u003ePHYC, phytochrome C\u003c/p\u003e \u003cp\u003eSSC, single-copy region\u003c/p\u003e \u003cp\u003eWGS, whole genome sequencing\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eAll authors consent to publish.\u003c/p\u003e\n\u003ch3\u003eAvailability of data and materials\u003c/h3\u003e\n\u003ch3\u003eThe datasets supporting the conclusions of this article are available in the CNGB repository, \u003ca href=\"https://db.cngb.org/search/project/CNP0000502/\"\u003ehttps://db.cngb.org/search/project/CNP0000502/\u003c/a\u003e.\u003c/h3\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis work was supported by the Sub-Project of the National Science and Technology Support Plan of the Twelfth Five-Year Plan in China (grant numbers 2015BAD04B03 and 2015BAD04B01). The funding numbers provided the financial support to the research programs, but didn\u0026rsquo;t involve in work design, data collection, analysis and preparation of the manuscript.\u003c/p\u003e\n\u003ch3\u003eAuthors\u0026rsquo; Contributions\u003c/h3\u003e\n\u003cp\u003eConceptualization, HZ, TY, WM, HL; Data curation, HZ, ZG, TY, YS, HL; Formal analysis, JW, TY, WM, YS, YH, YW; Funding acquisition, HZ; Investigation, HZ, JW, TY, WM, XL, HL; Project administration, HZ, TY, HL; Resources, HZ, ZG, TY, HL; Supervision, HZ, HL; Visualization, JW, WM; Writing - original draft, HZ, JW, TY; Writing - review \u0026amp; editing, HZ, JW;\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the manuscript.\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eWe wish to acknowledge the GABR Consortium members, partners, advisors, and supporters who have helped the GABR project run smoothly.\u003c/p\u003e\n\u003ch3\u003eAuthors' Information\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eSoreng RJ, et al. 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The floral transcriptomes of four bamboo species (Bambusoideae; Poaceae): support for common ancestry among woody bamboos. BMC Genom. 2016;17:384. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12864-016-2707-1\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eJiang Z. Bamboo and rattan in the world. (2007).\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eZhang LN, et al. Using nuclear loci and allelic variation to disentangle the phylogeny of Phyllostachys (Poaceae, Bambusoideae). Mol Phylogenet Evol. 2019;137:222\u0026ndash;35. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ympev.2019.05.011\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eZhao H, et al. Developing genome-wide microsatellite markers of bamboo and their applications on molecular marker assisted taxonomy for accessions in the genus Phyllostachys. Sci Rep. 2015;5:8018. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/srep08018\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eCanavan S, et al. The global distribution of bamboos: assessing correlates of introduction and invasion. AoB Plants. 2016. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/aobpla/plw078\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eWang CP, et al. A taxonomical study of Phyllostachys, China. \u003cem\u003eActa Phytotaxonomica Sinica\u003c/em\u003e (1980).\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eHong DY. Flora reipublicae Popularis Sinicae. \u003cem\u003eScience Press\u003c/em\u003e 73 (1983).\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003ePeng S, Yang H-Q, Li D-Z. Highly heterogeneous generic delimitation within the temperate bamboo clade (Poaceae: Bambusoideae): evidence from GBSSI and ITS sequences. Taxon. 2008;57:799\u0026ndash;810.\u003c/span\u003e \u003c/li\u003e \u003cli\u003e \u003cspan\u003eZeng CX, Zhang YX, Triplett JK, Yang JB, Li DZ. Large multi-locus plastid phylogeny of the tribe Arundinarieae (Poaceae: Bambusoideae) reveals ten major lineages and low rate of molecular divergence. Molecular Phylogenetics Evolution. 2010;56:821\u0026ndash;39.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bambusoideae, Chloroplast, Probe, Targeted enrichment, Bamboo phylogeny.","lastPublishedDoi":"10.21203/rs.3.rs-58636/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-58636/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e 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.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e 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 15M 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 \u003cem\u003eet al\u003c/em\u003e. Moreover, we reconstructed a phylogenetic tree of 412 bamboos (358 in-house and 54 published), supporting a non-monophyletic lineage of the genus\u003cem\u003e Phyllostachys\u003c/em\u003e. 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.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe 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.\u003c/p\u003e","manuscriptTitle":"Targeted Enrichment of Novel Chloroplast-Based Probes Reveals a Large-Scale Phylogeny of 412 Bamboos","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-09-17 13:47:07","doi":"10.21203/rs.3.rs-58636/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-10-30T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-10-28T12:00:00+00:00","index":2,"fulltext":"Recommendation: Accept after discretionary revisions\nForm responses:\n---\n\nComments to Author:\n---\nIn this report, the authors used 99 high-quality CpGenomes assembled\n15M pan-chloroplast genome, and to develop a set of bamboo hybridization-based targeted enrichment probe for chloroplast capture, and then this method successfully used in 358 woody bamboo CpGenomes sequencing. After reviewing this paper, the experiments were generally well designed and performed, I think the scientific value of this work is very high, this is an high-throughput and efficient approach for most green plant large-scale CpGenome sequencing and recommend for publication.\nMinor questions:\n1. In figure S1, 358 plus 71, and minus 15 overlapped samples is 414? Where is the another two CpGenome?\n2. In line 59, high‐ copy organelle should be corrected.\n3. In line 223-225, when construct the NJ-tree, why not choose the all 81 protein-coding genes?\n4. In figure S4, in order to better understanding the phylogenetic tree for readers, the author should use an arrow to point out the group of different color of word.\n5. After reading the manuscript. I have an problem, can this method be applied in mitochondrial genome? If yes, maybe the conflict of non-monophyletic and monophyletic lineage of the genus Phyllostachys can be verified by probe of mitochondrial genome.\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **No**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **No**\n"},{"type":"editorInvitedReview","content":"","date":"2020-10-26T12:00:00+00:00","index":1,"fulltext":"Recommendation: Accept after discretionary revisions\nForm responses:\n---\n\nComments to Author:\n---\nThe author developed a set of bamboo probes by using 99 high-quality CpGenomes with 6 bamboo CpGenomes as representative species and 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.This study constructed a 354 phylogenetic tree of 412 samples, covering more than 300 species, 40 genera, which 355 is the largest sampling project of bamboo in China and provides a large-scale 356 phylogenetic tree of bamboos. The dataset is large and the result promoted evolution, phylogenetic and population studies. a little revision need to be checked:\nTable 1: in taxonmy, the \"order\"-ales and \"family\"-aceae usually are not italic.\nFigure 1: the font size is too small to read in the picture, please change it.\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **No**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"reviewerAgreed","content":"","date":"2020-10-09T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-10-08T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-09-17T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-09-03T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-09-02T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-09-02T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"812effb3-9726-4a81-9aa7-f37118ee1a41","owner":[],"postedDate":"September 17th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":536533,"name":"Plant Physiology and Morphology"},{"id":536534,"name":"Plant Molecular Biology and Genetics"}],"tags":[],"updatedAt":"2021-02-07T15:03:26+00:00","versionOfRecord":{"articleIdentity":"rs-58636","link":"https://doi.org/10.1186/s12870-020-02779-5","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2021-02-05 15:01:44","publishedOnDateReadable":"February 5th, 2021"},"versionCreatedAt":"2020-09-17 13:47:07","video":"","vorDoi":"10.1186/s12870-020-02779-5","vorDoiUrl":"https://doi.org/10.1186/s12870-020-02779-5","workflowStages":[]},"version":"v1","identity":"rs-58636","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-58636","identity":"rs-58636","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}