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Communication

De Novo Assembly of an Allotetraploid Artemisia argyi Genome

by
Qiming Mei
1,2,3,
Hanxiang Li
1,2,
Yanbin Liu
4,
Feng Wu
4,
Chuang Liu
4,
Keya Wang
1,2,
Hongjun Liu
5,
Cheng Peng
1,2,6,
Zhengfeng Wang
1,2,
Long Wang
1,2,
Zhanfeng Liu
1,2,
Junhua Yan
1,2 and
Wei Zhang
1,2,*
1
Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
2
South China National Botanical Garden, Guangzhou 510650, China
3
Guangzhou Linfang Ecological Technology Co., Ltd., Guangzhou 510520, China
4
Tea Bureau of Tongbai County, Nanyang 474750, China
5
People’s Government of Tongbai County, Nanyang 474750, China
6
College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(2), 436; https://doi.org/10.3390/agronomy13020436
Submission received: 5 December 2022 / Revised: 18 January 2023 / Accepted: 30 January 2023 / Published: 1 February 2023
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
Review Reports Versions Notes

Abstract

:
The Chinese mugwort (Artemisia argyi Lév. et Vaniot) is an important traditional Chinese medicine plant that is ubiquitously disturbed in Asia. However, the molecular mechanisms that reflect the natural evolution of Artemisia argyi remain unclear. In this study, a high-quality draft assembly of the allotetraploid A. argyi (ArteW1-Tongbai) was conducted utilizing PacBio long-read sequencing and Hi-C technologies. The assembly is about 7.20 Gb with a contig N50 length of 0.87 Mb. The allotetraploid genome of ArteW1-Tongbai is highly heterozygous and rich in repeat sequences (the heterozygous ratio is 1.36%, and the repeat rate is 86.26%). A total of 139,245 protein-coding genes were identified. The KEGG enrichment analysis revealed that 846 species-specific genes were related to the biosynthesis of secondary metabolites. The plants with allopolyploid genomes can potentially exhibit a better adaptive capacity to environmental stresses and accumulation of secondary metabolites. Therefore, the genome assembly serves as a valuable reference for Artemisia, the genus characterized by species richness and diverse specialized metabolites.

1. Introduction

The Artemisia argyi H. Lév. et Vaniot (Asteraceae), also known as Chinese mugwort or Aicao, is ubiquitously distributed in Asia, and is an important traditional Chinese medicine (TCM) herb. Tongbai County, Henan Province of Central China, is one of the main places for producing A. argyi. The cultivated and wild A. argyi growing in Tongbai are named “Tongbai Ai”. Dried A. argyi leaves are the original material for moxibustion. Previous phytochemical studies reported that the main components present in A. argyi leaves, such as flavonoids, polysaccharides, terpenoid, polyketides, and phenolic acids, exhibit antioxidant, anti-cancer, antimicrobial, and neuroprotective activities [1]. Recently, the terpenoid biosynthesis pathway was analyzed by transcriptome profiling [2]. However, the molecular mechanisms underlying the biological synthesis of medicinal components of A. argyi are still largely unknown.
Polyploidy changes the quantitative and qualitative patterns of secondary metabolite production in plants [3]. Polyploidy, or whole-genome duplication (WGD), is one of the main evolutionary forces enhancing the adaptive potential of organisms [4]. There are two types of polyploidies in plants: allopolyploidy and autopolyploid. The formation of allopolyploidy involving interspecific hybridization and genome duplication is more prevalent than autopolyploidization in nature [5]. Allopolyploidation may be the most common mechanism of sympatric speciation and promotes adaptation in plants [6]. Many Asteraceae species are allopolyploid because of interspecific hybridization [7]. The Artemisia is a large genus of the Asteraceae family that consists of about 380 species [8]. The chromosome number of Artemisia species was identified mainly to be x = 9, which is prevalent; or x = 8, which is less frequent [9,10].
In the last decade, a dozen Artemisia chloroplast genomes were completely characterized, which provide a valuable resource for phylogenetic analysis [11,12]. Recently, the genome assembly of A. annua set an excellent model system for studying the artemisinin synthesis and evolution of Artemisia [13]. In this study, the high-quality assembly and annotation of an allopolyploid genome of A. argyi were conducted utilizing PacBio long-read sequencing and Hi-C technologies. The results provide fundamental information for illustrating the structure and organization of the highly heterozygous genome. In addition, the genome assembly of A. argyi is a useful tool for dissecting phytochemical component synthesis pathways and drug discovery.

2. Materials and Methods

2.1. Plant Sample, Sequencing, and Genome Survey

In September 2019, a wild individual of A. argyi (ArteW1-Tongbai) was collected from the “Protected Area of Tongbai Ai” located at Huaiyuan Town, Tongbai County, Henan Province, China (32°29′201″ N, 113°15′12″E) at altitudes of 287.67 m (Figure 1a,b). The samples were sent to the commercial genome research organization Frasergen (Wuhan, China) for DNA extraction and sequencing. The total genomic DNA was extracted from fresh leaves utilizing a modified CTAB method, in which LiCl and polyvinylpyrrolidone (PVP) were added to the extraction buffer to remove the high level of polysaccharides, polyphenolics, and secondary metabolites [14]. RNA contaminant was removed by RNaseA. Then, the quality of the DNA was checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) as well as agarose gel electrophoresis. Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the DNA.
Four libraries with insert sizes larger than 15 kb were constructed according to the SMRTbell Express Template Prep kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). Then, single-molecule DNA sequencing was conducted on the PacBio Sequel II platform (Pacific Biosciences, Menlo Park, CA, USA). A total of 570.70 Gb (80× of the estimated genome size) bases were generated. The raw reads were filtered by HTQC v.1.92.310 to remove adapters and low-quality sequences (read pairs with an average quality lower than 20 and with any end shorter than 75 bp) [15]. In addition, two libraries with an insertion length of 150 bp were generated and sequenced on the HiSeq X Ten platform (Illumina, San Diego, CA, USA). A total of 162.63 Gb of short-read sequences were generated for the genome survey. Then, the genome size, the level of heterozygosity, and repeat content of the genome were estimated utilizing the k-mer method. The 17-mer survey was conducted by GCE software [16].

2.2. Genome Assembly and Quality Evaluation

Clean data generated by the PacBio Sequel II platform were processed to genome assembly after filtering by fastp v.0.12.6 [17]. The draft genome was assembled using mecat2 with default parameters [18]. Then, the genome was polished by using the arrow pipeline from the SMRT link 4 toolkit to correct errors in the initial genome assembly. Finally, the short reads derived from the Illumina sequencer were utilized to correct the remaining errors by pilon v.1.22 [19].
In this study, the Hi-C technique was employed to construct chromosome-level assemblies. Fresh leaves of ArteW1-Tongbai were fixed in formaldehyde to create DNA–protein bonds. Then, the restriction enzyme MboI was utilized to digest the chromatin. After re-ligation, the DNA was sheared into 300–500 bp fragments by sonication. Then, the Hi-C library was prepared following a standard procedure and sequenced on the IlluminaHiSeq X Ten platform under the PE150 model. A total of 381 Gb data were generated from the Hi-C library (50× of the estimated genome size). Next, the raw data were filtered to remove self-ligation, non-ligation, and other invalid reads by fastp v.0.12.6 [17]. The clean reads were mapped to the polished genome using BWAv.0.7.16a (r1181) with default parameters [20].
The 3d-DNA pipeline was used to cluster, order, and orient the Hi-C contigs [21]. The 3d-DNA pipeline anchored and oriented 7.20 Gb (98.98%) contigs into 32 superscaffolds (named aar1 to aar32) according to the syntenic relationship (Supplementary Table S1). Then, a contact map was plotted using Juicer [22], and visualized and corrected by Juicebox [23]. Finally, the quality of pseudo-chromosome assembly was assessed by BUSCO v.3.0.2 with the embryophyta_odb10 dataset [24].

2.3. De Novo Genome Annotation

The gene structural annotation of A. argyi was conducted following three strategies: (1) de novo prediction performed by AUGUSTUS v.3.3.1 [25] and GENSCAN [26]; (2) homology-based annotation using Exonerate v.2.2.0 with the default parameters [27]; and (3) finding coding regions in RNA-Seq transcripts by Cufflinks v.2.2.1 [28]. Finally, the results of the three methods were combined into gene models by MAKER [29].
The predicted genes were further functionally annotated according to the best match of the alignments with Lactuca saligna, Mikania micrantha, Cynara cardunculus var. scolymus, Artemisia annua, and Helianthus annuus. The sequences were queried against databases including the National Center for Biotechnology Information (NCBI) Non-Redundant (NR) database [30], TrEMBL [31], Swiss-Prot [32], and the Kyoto Encyclopedia of Genes and Genomes (KEGG) [33] database by blasstp (e-value = 1 × 10−5) [34]. In addition, InterProScan v.5.35–74.0 [35] and PfamScan [36] were used to annotate the protein domains based on the InterPro [37] and Pfam [38] databases. Gene Ontology (GO) [39] IDs for predicted genes were obtained from Blast2GO [40]. Finally, Benchmarking Universal Single-Copy Orthologs (BUSCO) was used to validate the gene annotations with the embryophyta_odb10 and default parameters [24].

2.4. Evolutionary Analysis

The genomes of six Asteraceae species including L. sativa, M. micrantha, C. cardunculus var. scolymus, A. annua, and H. annuus, as well as A. argyi were included in the phylogenetic analysis. The Solanum tuberosum was used as the outgroup. OrthoFinder v.2.3.1 was used for the identification of orthologous groups of the A. argyi genome with an e-value of 10−5 [41]. Gene families unique for each species were extracted from the clustering result. Expansion and contraction analysis of gene families was conducted using CAFE v.2.2 [42]. The enrichment of KEGG and Gene Ontology terms for the expansion, contraction, and lineage-specific gene families was performed using goseq [43].
Orthologous groups (OGs) with single-copy genes were used in the phylogenetic analysis. Protein sequences from each OG were aligned using MUSCLE v.3.8.31 [44]. Ambiguous sites were removed manually by trimAI [45]. The final dataset was generated by concatenating alignments. The phylogenetic tree was constructed using IQ-TREE software with maximum likelihood (ML) algorithm. The divergence time of species using MCMCTree was implemented in PAML v.4.9 package [46]. The tree was calibrated by Asteraceae crown age (95–106 million years ago, Mya) and split time between C. cardunculus var. scolymus and L. sativa (27–40 Mya). The time-calibrating points were obtained from the timetree website accessed on 14 July 2020 (timetree.org).
We utilized the wgd software to calculate Ks distribution (ranging from 0.05 to 3) among paralogs from A. argyi [47]. The paralogs were pruned on the basis of co-linearity analysis using i-ADHoRe [48]. Then, we fitted the Ks distribution of the paralogs from each hypothesized WGD peak according to the fitted mixture model (BGMM in ‘wgd’).

3. Results

3.1. Genomic Characteristics of Wild A. argyi

The A. argyi (ArteW1-Tongbai) used in this study is native to the semi-arid hilly region of Huaiyuan Town, Tongbai County, Henan Province, China (32°29′201″ N, 113°15′12″ E) at altitudes of 287.67 m (Figure 1a,b). The karyotype analysis revealed that the nuclear genome of ArteW1-Tongbai comprises 34 chromosomes (2n = 34).
The genome size of ArteW1-Tongbai was estimated through k-mer analysis using Illumina short reads at a k-mer size of 17. As shown in Figure 1c, the frequency distribution of k-mer manifested two clear peaks at depths of 17 and 34, corresponding to the heterozygous and homozygous reads, respectively. Then, the haploid genome size of ArteW1-Tongbai was estimated to be about 3.81 Gb based on the homozygous peak depth, and the diploid genome size was predicted to be about 7.62 Gb heterozygous based on the heterozygous peak depth.
The k-mer results indicated that the heterozygous ratio of ArteW1-Tongbai is 1.36%, and the repeat rate is 86.26% (Figure 1c). Both the chromosome number and genome size were different from the diploid A. argyi (2n = 2x = 18, genome size = 4.3 Gb) [10], suggesting that ArteW1-Tongbai possesses an allotetraploid genome. The genome size of ArteW1-Tongbai is approximately four-fold higher than the closely-related species A. annua (1.74 Gb) [13].

3.2. De Novo Assembly and Annotation of the A. argyi Genome

In this study, an assembly of an allopolyploid A. argyi genome was conducted utilizing PacBio long-read sequencing and Hi-C technologies. The 7.20 Gb genome is assembled into eight main clusters of superscaffolds and further divided into 32 pseudochromosomes (numbered aar1–32) with contig N50 of 0.87 Mb and scaffold N50 of 215.81 Mb. The size of superscaffolds ranged from 175.86 Mb to 372.72 Mb. In addition, the 32 pseudochromosomes formed eight main clusters, and each cluster contained two “haplotype-fused” homologous chromosomes (Supplementary Figure S1 and Supplementary Table S1). The number of pseudochromosomes (32) did not match the karyotype result (34), because four pairs of them are fused and very difficult to isolate. The Hi-C assembly contained 98.98% of the assembled sequences. The completeness of the genome assembly was qualified by BUSCO [24]. In total, 1572 out of 1614 BUSCOs were identified as complete (97.40%).
Genes were predicted in the allotetraploid A. argyi genome by a pipeline integrating de novo, homology-based, and RNA-Seq methods. A total of 139,245 protein-coding gene models were identified. Next, the gene models were evaluated using the same methods as gene prediction. A total of 133,932 (96.18%) predicted genes were supported by two or three methods. In addition, BUSCO was utilized to validate the annotation results: 1607 of 1614 (99.6%) BUSCOs were complete. Then, the gene models were further functionally annotated by blasting against databases. A total of 134,956 genes were successfully annotated, corresponding to 96.92% of the predicted genes.

3.3. Comparative Genome Analysis of Asteraceae Species

In this study, the genome of A. argyi was compared to the other five Asteraceae species with genomic assemblies (A. annua, H. annuus, L. sativa, C. var. scolymus, and M.micrantha), and the asteroid S. tuberosum was used as the outgroup of the phylogenetic analysis. A total of 139,245 genes belonging to 27,279 families in the A. argyi genome were identified (Table 1).
The phylogeny of A. argyi and other Asteraceae species was reconstructed using a concatenated sequence alignment of 24 single-copy genes shared by A. argyi and another six plant species (Figure 2a). The A. argyi was clustered with the closely related A. annua, as expected. The divergent time of A. argyi and A. annua was estimated to be around 18.28 Mya. When compared to A. annua, a total of 9119 gene families were expanded in A. argyi, whereas 401 were contracted (Figure 2a). moreover, 4697 gene families were shared by A. argyi and A. annua, whereas 4858 gene families were specific to A. argyi (Figure 2b). The KEGG enrichment analysis indicated that the number of genes involved in secondary metabolites biosynthesis (846) was notably higher than the species-specific genes of other pathways, for example, the biosynthesis of amino acids (256) (Figure 2c). Most expanded gene families were associated with secondary metabolites, such as isoquinoline alkaloid biosynthesis (ko00950), tropane, piperidine, and pyridline alkaloid biosynthesis (ko00960), monobactam biosynthesis (ko00261), betalain biosynthesis (ko00965), and glucosinolate biosynthesis (ko00966) (Figure 2c). Additionally, some key enzymes of secondary metabolites biosynthesis pathways were expanded in A. argyi. For example, the polyphenol oxidase (K00422) primary-amine oxidase (K00276) catalyzed the synthesis of main metabolic intermediates of various isoquinoline alkaloids. Isoquinoline alkaloids were enriched in herbal plants that have been used for their anti-inflammatory, antimicrobial, and analgesic effects [49].
Moreover, the distribution of synonymous substitutions per synonymous site (Ks) revealed the whole-genome duplication (WGD) events that occurred in the A. argyi genome. The peak observed from the lg(Ks) = −3, demonstrating a most recent genome duplication, and peak at lg(Ks) = 0 represented an older WGD that occurred in the evolution of Artemisia (Figure 3).

4. Discussion

A. argyi is an important traditional Chinese medicine plant. In this study, we conducted high-quality assembly and annotation on an allopolyploid genome of wild A. argyi. The results showed that the allotetraploid genome of ArteW1-Tongbai is highly heterozygous and rich in repeat sequences. The A. argyi genome contains a larger number of gene families compared to other Asteraceae species (Table 1) [13,50,51,52,53]. The Ks results indicated that a duplication event occurred recently and led to the synthesis of the allotetraploid A. argyi genome (Figure S1). The plants with allopolyploid genomes tend to exhibit a better adaptive capacity to environmental stresses and the accumulation of secondary metabolites [6]. A total of 846 species-specific genes were related to the biosynthesis of secondary metabolites. The results of the genome characteristics are consistent with the results reported recently (high heterozygosity and high repetitive sequences), but vary in terms of gene numbers (62,844–279,294) and genome size (7.20 Gb in this study, and 7.44–7.87 Gb in previous reports) [54,55]. In addition, all three assemblies demonstrate chromosome fusion events by synteny analysis (Figure S1a).
A high-quality reference genome assembly serves as a fundamental resource for research on polyploid plant genomes. On the other hand, the high ratio of repeat sequences and heterozygosity are the main obstacles to the assembly of polyploid plant genomes [56]. In this case, the A. argyi genome is highly heterozygous (heterozygosity = 1.36%) and contains a high ratio of repeat sequences (86.26%) (Figure 1c). Therefore, we could not derive a haplotype-resolved assembly from the allopolyploid genome despite utilizing long-read sequencing and Hi-C assembly technologies. For further study, we plan to conduct the genome sequencing of diploid A. argyi and provide an updated assembly of the allopolyploid genome.
Polyploidization is common in plants. The increase in gene numbers facilitates the production of secondary metabolites [3]. For example, the tetraploid Echinacea purpurea has a higher abundance of cichoric acid than diploid [57]. Moreover, hybridization enhances the variation of secondary metabolites and herbivore resistance [58]. The allopolyploidy contains two sets of genes involved in secondary metabolite biosynthesis inherited from parental species. Recent research reported that the hybrid cultivar of oolong tea (Camellia sinensis) exhibited structural variations and the expansion of terpene synthase gene families, which contributed to the high aroma and stress tolerance [59]. As a result, the allotetraploid A. argyi showed great potential for metabolic engineering.
The A. argyi genome contains a larger number of gene families compared to other Asteraceae species (Table 1). The Artemisia species synthesized diverse secondary metabolites [60]. In this study, the functional annotation and KEGG enrichment analysis revealed that many secondary metabolites synthesis gene families are unique to A. argyi. Our study provides fundamental resources for analyzing diverse specialized metabolites in Artemisia.

5. Conclusions

In summary, we assembled and annotated a high-quality genome of the genus Artemisia. The genome data can be used in studying gene functions, the genome evolution of Asteraceae, and Artemisia taxonomy. The results showed that A. argyi diverged from other Artemisia species very recently (at ~8.12 Mya) and experienced two rounds of WGD events. We also found that the genes of secondary metabolites biosynthesis expanded. These results can provide insight into thedrug discovery of Artemisia plants. The findings suggest that the allotetraploid A. argyi can be used as a useful tool for dissecting phytochemical component synthesis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13020436/s1, Figure S1: De novo assembly of the A. argyi genome; Table S1: Estimation of the genome size of ArteW1-Tongbai based on k-mer analysis.

Author Contributions

Conceptualization, Q.M. and W.Z.; methodology, H.L. (Hanxiang Li); software, Z.W.; validation, L.W. and Z.L.; formal analysis, Q.M.; investigation, Y.L. and C.L.; resources, F.W. and K.W.; writing—original draft preparation, Q.M.; writing—review and editing, Q.M. and W.Z.; visualization, C.P.; supervision, J.Y.; project administration, W.Z.; funding acquisition, F.W. and H.L. (Hongjun Liu). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a grant from Tongbai County Government, Henan Province of China (Y841191001), and the National Natural Science Foundation of China (3160020257).

Data Availability Statement

The A. argyi genome assembly was submitted to the National Genomics Data Centerof China (accession number CRX552966). The raw sequencing reads and Hi-C data were deposited in the NCBI Sequence Read Archive (SRA) under the BioSample SAMN20447770. The annotation file is available at https://figshare.com/articles/dataset/Gene_annotation_of_Artemisia_argyi/16621711 (uploaded on 15 September 2021).

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 13 00436 g001 550
Figure 1. Location of the study site and genomic characteristic of the wild A. argyi. (a) Sampling site of ArteW1-Tongbai (Google Earth, earth.google.com/web/, accessed on 30 April 2020). (b) Picture of the ArteW1-Tongbai growing in “Protected Area of wild A. argyi”. (c) Estimation of the genome size of ArteW1-Tongbai based on k-mer analysis.
Figure 1. Location of the study site and genomic characteristic of the wild A. argyi. (a) Sampling site of ArteW1-Tongbai (Google Earth, earth.google.com/web/, accessed on 30 April 2020). (b) Picture of the ArteW1-Tongbai growing in “Protected Area of wild A. argyi”. (c) Estimation of the genome size of ArteW1-Tongbai based on k-mer analysis.
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Figure 2. Evolutionary analysis of A. argyi genome. (a) Phylogenetic tree of Asteraceae species. Branch and taxa labels are numbers of gene families manifesting expansion (green) and contraction (red). Node labels are divergence times estimated by maximum likelihood (ML) algorithm. (b) Overlap of gene families of four Asteraceae species and Solanum. (c) KEGG enrichment analysis of expansion gene families in the A. argyi genome.
Figure 2. Evolutionary analysis of A. argyi genome. (a) Phylogenetic tree of Asteraceae species. Branch and taxa labels are numbers of gene families manifesting expansion (green) and contraction (red). Node labels are divergence times estimated by maximum likelihood (ML) algorithm. (b) Overlap of gene families of four Asteraceae species and Solanum. (c) KEGG enrichment analysis of expansion gene families in the A. argyi genome.
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Figure 3. Distribution of synonymous substitutions per site (Ks) (a) and lg(Ks) (b) inferring WGD events.
Figure 3. Distribution of synonymous substitutions per site (Ks) (a) and lg(Ks) (b) inferring WGD events.
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Table
Table 1. Comparison of gene number of A. argyi and other species.
Table 1. Comparison of gene number of A. argyi and other species.
SpeciesGenesFamiliesClustered GenesUnclustered GenesSpecific GenesSpecific Families
A. argyi139,24527,279123,51215,73318,5764858
A. annua66,91823,93561,118580075472030
C. cardunculus38,40616,74937,5678391695333
H. annuus44,14416,81840,078406659091656
L. sativa45,24317,97243,605163856121000
M. micrantha46,35117,55443,088326380591522
S. tuberosum37,96715,60235,723224475631541
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Mei, Q.; Li, H.; Liu, Y.; Wu, F.; Liu, C.; Wang, K.; Liu, H.; Peng, C.; Wang, Z.; Wang, L.; et al. De Novo Assembly of an Allotetraploid Artemisia argyi Genome. Agronomy 2023, 13, 436. https://doi.org/10.3390/agronomy13020436

AMA Style

Mei Q, Li H, Liu Y, Wu F, Liu C, Wang K, Liu H, Peng C, Wang Z, Wang L, et al. De Novo Assembly of an Allotetraploid Artemisia argyi Genome. Agronomy. 2023; 13(2):436. https://doi.org/10.3390/agronomy13020436

Chicago/Turabian Style

Mei, Qiming, Hanxiang Li, Yanbin Liu, Feng Wu, Chuang Liu, Keya Wang, Hongjun Liu, Cheng Peng, Zhengfeng Wang, Long Wang, and et al. 2023. "De Novo Assembly of an Allotetraploid Artemisia argyi Genome" Agronomy 13, no. 2: 436. https://doi.org/10.3390/agronomy13020436

APA Style

Mei, Q., Li, H., Liu, Y., Wu, F., Liu, C., Wang, K., Liu, H., Peng, C., Wang, Z., Wang, L., Liu, Z., Yan, J., & Zhang, W. (2023). De Novo Assembly of an Allotetraploid Artemisia argyi Genome. Agronomy, 13(2), 436. https://doi.org/10.3390/agronomy13020436

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