Transcriptome analysis of Sacha Inchi (Plukenetia volubilis L.) seeds at two developmental stages
© Wang et al.; licensee BioMed Central Ltd. 2012
Received: 7 September 2012
Accepted: 16 December 2012
Published: 20 December 2012
Sacha Inchi (Plukenetia volubilis L., Euphorbiaceae) is a potential oilseed crop because the seeds of this plant are rich in unsaturated fatty acids (FAs). In particular, the fatty acid composition of its seed oil differs markedly in containing large quantities of α-linolenic acid (18C:3, a kind of ω-3 FAs). However, little is known about the molecular mechanisms responsible for biosynthesis of unsaturated fatty acids in the developing seeds of this species. Transcriptome data are needed to better understand these mechanisms.
In this study, de novo transcriptome assembly and gene expression analysis were performed using Illumina sequencing technology. A total of 52.6 million 90-bp paired-end reads were generated from two libraries constructed at the initial stage and fast oil accumulation stage of seed development. These reads were assembled into 70,392 unigenes; 22,179 unigenes showed a 2-fold or greater expression difference between the two libraries. Using this data we identified unigenes that may be involved in de novo FA and triacylglycerol biosynthesis. In particular, a number of unigenes encoding desaturase for formation of unsaturated fatty acids with high expression levels in the fast oil accumulation stage compared with the initial stage of seed development were identified.
This study provides the first comprehensive dataset characterizing Sacha Inchi gene expression at the transcriptional level. These data provide the foundation for further studies on molecular mechanisms underlying oil accumulation and PUFA biosynthesis in Sacha Inchi seeds. Our analyses facilitate understanding of the molecular mechanisms responsible for the high unsaturated fatty acids (especially α-linolenic acid) accumulation in Sacha Inchi seeds.
KeywordsTranscriptome Unsaturated fatty acids Omega-3 fatty acids Triacylglycerols Gene expression
Sacha Inchi (Plukenetia volubilis L.), also known as Inca Inchi or mountain peanut, belongs to the family Euphorbiaceae. It is a perennial oleaginous woody vine indigenous to tropical Peruvian jungles that lie at altitudes between 200 and 1500 m. This legume has a star-shaped fruit, which contains dark oval seeds characterized by high levels of protein (ca. 30%) and oil (ca. 50%) [1, 2]. Its flour and oil are consumed by Peruvian Indians, who have used them to prepare foods and beverages for hundreds of years . The fatty acid (FA) composition of Sacha Inchi seed oil is distinctive because its seeds contain a large amount of polyunsaturated fatty acids (PUFAs) comprising about 93% of total FAs. Specifically, α-linolenic acid (18C:3 cis Δ9, 12, 15, a kind of ω-3 FAs) comprises ca. 50% of the FAs content, and linoleic acid (18C:2 cis Δ9, 12, a kind of ω-6 FAs) comprises ca. 35%. Other FAs such as palmitic acid (16C:0), stearic acid (18C:0), and oleic acid (18C:1cisΔ9) are present in small proportions [1, 2].
Because PUFAs such as α-linolenic and linoleic acids cannot be synthesized in mammals, they are essential FAs (i.e., required in the diet) [4, 5]. These PUFAs not only exert a hypocholesterolemic effect against coronary heart disease and hypertension when used as food supplements, but also are critical for the development of infants during pregnancy and breastfeeding periods [6–8]. The recommended ω-6: ω-3 FA ratio in the human diet is approximately 2:1 to 6:1 [9, 10]; however, traditional oil seed crops (e.g., soybean, peanut, maize, sunflower, and rape) have relatively low α-linolenic content (below 10%). The high ω-6: ω-3 FA ratio in our typical diet (approximately 15:1) is thought to be a major contributor to cardiovascular disease . In contrast, Sacha Inchi seed oil has an approximately 7:10 ratio of ω-6: ω-3 FAs. The physiological mechanisms responsible for ω-3 FAs biosynthesis in the seeds of Sacha Inchi are unknown. Understanding the molecular mechanisms underlying seed development and lipid biosynthesis in Sacha Inchi may provide new insights for isolating novel genes and serve the use of genetic engineering to increase the α-linolenic content of traditional vegetable oils.
Generally, two main pathways are involved in storage lipid accumulation: FA biosynthesis and triacylglycerol (TAG) assembly. In all plants studied, acyl carrier protein (ACP)-dependent de novo FA synthesis occurs almost exclusively in plastids [11, 12]. FAs are synthesized from acetyl-CoA in a three-step process: (a) irreversible carboxylation of acetyl-CoA by acetyl-CoA carboxylase to form malonyl-CoA; (b) repeated condensation of malonyl-CoA with a growing ACP-bound acyl chain by FA synthase complex, consecutively adding two carbon units to form 16:0-ACP; and (c) elongation and desaturation of 16:0-ACP to form 18:0-ACP and 18:1-ACP, respectively . Desaturation of 18:0-ACP to 18:1-ACP catalyzed by a stromal stearoyl-ACP desaturase is one of the key steps regulating unsaturated FA levels in the cell. In oilseeds, more than 95% of newly synthesized FAs [18C:1 > > 16C:0 > 18C:0] are exported from plastids as CoA thioesters to enter the eukaryotic glycerolipid synthesis pathway [14, 15]. In the endoplasmic reticulum, acyl-CoAs can be used for the sequential sn-1 and sn-2 acylation of glycerol-3-phosphate to produce phosphatidic acid, and are then converted to 1, 2-sn-diacylglycerol (DAG) by phosphatidate phosphatase. The third acyl-CoA-dependent acylation catalyzed by DAG acyltransferase leads to the production of TAG (i.e., FA incorporation onto the glycerol backbone known as the Kennedy pathway) . TAG can also be formed by acyl-CoA-independent reactions using DAG and phosphatidylcholine (PC) as acyl donors [17, 18]. Phospholipid:DGAT (diacylglycerol acyltransferase) catalyzes the acyl transfer from PC to DAG to form lysophosphatidylcholine and TAG, whereas diacylglycerol transacylase catalyzes the transfer of an acyl moiety between two DAG molecules to form TAG and monoacylglycerol . In the mature oilseed, TAG is stored in densely packed oil bodies surrounded by oleosin protein [19, 20]. Usually, oil accumulation in developing seeds is associated with an increase in the number of oil bodies.
In oil-accumulating cells of plant seeds, 18C:1 is desaturated to 18C:2 and 18C:3 by two microsomal desaturase enzymes, FAD2 and FAD3 [21, 22]. Extraplastidic desaturation of 18C:1 to 18C:2 and 18C:3 arises while the FAs are bound to PC , the major site of FA desaturation in eukaryotes . In current models of seed lipid biosynthesis, 18C:1 is incorporated into PC by one of two routes. Direct incorporation from 18C:1-CoA exported by the plastids likely occurs through the action of acyl-CoA: lysophosphatidylcholine acyltransferase [25, 26]. Alternatively, 18C:1 may be incorporated into DAG by the Kennedy pathway, after which 18C:1-DAG is converted to PC by cytidine 5'-diphosphocholine: diacylglycerol cholinephosphotransferase [27–29]. Oil-accumulating tissues use different strategies to enrich PUFAs in TAG. For example, converting PC to DAG (for TAG synthesis) may be accomplished by the reverse action of cholinephosphotransferase or by phosphatidylcholine: diacylglycerol cholinephosphotransferase [29–31]. Alternatively, PUFAs may be enriched in TAG by PC acyl editing that incorporates nascent 18C:1 into PC and releases PUFAs, generating an acyl-CoA pool of newly synthesized FAs and further-modified FAs for glycerolipid synthesis in the endoplasmic reticulum . Mechanisms for PUFA biosynthesis and accumulation are not known for most oilseeds, nor are the enzymes/genes that differ between the pathways . Because Sacha Inchi seed oil contains uncommonly high levels of ω-3 FAs, it is a good model to dissect metabolic pathways involved in oil and PUFA biosynthesis.
The development of high-throughput sequencing technologies has enabled the efficient and economical resequencing of entire genomes or sampling of entire transcriptomes. For example, Illumina sequencing technology produces millions of short cDNA reads from a single instrument run, which consists of the transcription level for each gene. It determines absolute rather than relative gene expression, thus providing greater insight and accuracy than microarray analysis [34–36]. De novo transcriptome sequencing and characterization based on Illumina second-generation sequencing technology has enabled the rapid identification and profiling of differentially expressed genes in the sweet potato , eucalyptus tree , chickpea , orchid , taxus , sesame , and peanut . However, transcriptomic information is lacking for Sacha Inchi. We were interested in identifying genes involved in PUFA biosynthesis (especially α-linolenic acid) in Sacha Inchi seeds during the period of fast oil accumulation; therefore, transcriptome analysis was carried out during two stages of seed development by using an Illumina paired-end sequencing strategy. In this study, we generated more than four billion bases of high-quality DNA sequence and obtained 70,392 unigenes from the Sacha Inchi seed transcriptome. Among them, 22,179 unigenes showed a 2-fold or greater expression difference between the two seed development stages. The assembled, annotated transcriptome sequences and gene expression profiles provide useful information for the identification of genes involved in unsaturated FAs and TAG biosynthesis.
Results and Discussion
Illumina paired-end sequencing and de novo assembly
Summary statistics of clean reads in two cDNA libraries from Sacha Inchi seeds
S1 Initial stage (5–10 DAP) library
S2 Fast stage (50–65 DAP) library
Total Nucleotides (nt)
High-quality reads (%)
Low quality reads
Low quality reads (%)
GC percentage of high-quality reads (%)
Characterization of the nonredundant unigenes of Sacha Inchi
Of the 70,392 nonredundant unigenes, sequence directions of 44,762 unigenes were determined by performing BLASTX searches against NCBI nonredundant, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and Clusters of Orthologous Groups protein databases with an E-value cut-off of 10-5 and by using ESTScan software . In addition, the protein coding regions of 43,608 unigenes were predicted.
Functional classification of Sacha Inchi unigenes by Gene Ontology, Clusters of Orthologous Groups, and KEGG
Pathway-based analysis can further our understanding of the biological functions and interactions of genes. A total of 20,142 unigenes were assigned to 119 pathways in the KEGG database. The most represented pathways included “metabolic pathways” (4,360 unigenes), “biosynthesis of secondary metabolites pathways” (2,348 unigenes) and “plant-pathogen interaction pathways” (1,711 unigenes) (see Additional file 1). Notably, some pathways are closely linked to changes in oil content and composition that take place during Sacha Inchi seed ripening, such as “fatty acid biosynthesis pathway” (89 unigenes), “glycerolipid metabolism pathway” (146 unigenes), “linoleic acid metabolism pathway” (114 unigenes), and “α-linolenic acid metabolism pathway” (180 unigenes) (see Additional file 1). These lipid unigenes identified provided critical clues to clone and identify key functional genes involved in unsaturated FA and TAG biosynthesis in Sacha Inchi seeds.
Analysis of differentially expressed genes at the two developmental stages
Most highly expressed transcripts in the initial stage (S1) of Sacha Inchi seed development
S1 Initial stage
S2 Fast stage
UV excision repair protein
EXORDIUM LIKE 2
Low temperature inducible
Splicing factor 1
Conserved hypothetical protein
Iron transport protein 2
EXORDIUM LIKE 2
Omega-3 fatty acid desaturase, chloroplast precursor
Heat shock cognate 70 kDa protein
Proline rich protein
Major latex-like protein
Gibberellin regulated protein
Most highly expressed transcripts in the fast oil accumulation stage (S2) of Sacha Inchi seed development
S1 Initial stage (RPKM)
S2 Fast stage (RPKM)
Ricin-agglutinin family protein
Microsomal omega-3 fatty acid desaturase
BURP domain-containing protein
Lipid transfer protein (LTP) family protein
Ricin-agglutinin family protein
Lipid transfer protein (LTP) family protein
Glutamyl-tRNA(Gln) amidotransferase subunit A
Seed maturation protein PM35
60S ribosomal protein
Thiazole biosynthetic enzyme
Conserved hypothetical protein
Major latex allergen Hev b
Endoplasmic reticulum 18:2 desaturase
Amidase family protein
Aspartyl-tRNA(Asn)/glutamyl-tRNA (Gln) amidotransferase subunit A
Lipid transfer protein (LTP) family protein
Extensin like protein
To further understand the biological functions of differentially expressed genes, we mapped these genes to the KEGG database. In the KEGG Orthology enrichment, the most represented pathways included metabolic pathways (1,999 enzymes), biosynthesis of secondary metabolites (1,184), and plant-pathogen interactions (1,028) (see Additional file 3). Of these, pathways closely related to seed oil biosynthesis, including α-linolenic acid metabolism, linoleic acid metabolism, biosynthesis of unsaturated FAs, FA biosynthesis, and FA metabolism (see Additional file 3) may provide valuable information for the identification of novel genes involved in α-linolenic acid synthesis.
Identification and characterization of lipid genes involved in fatty acids and triacylglycerols biosynthesis in Sacha Inchi seeds
Sacha Inchi seeds have potential applications in food and pharmaceutical industries through its ability to biosynthesize and accumulate considerable amounts of linolenic acid. The main objective of this research is to understand the mechanisms underlying lipid biosynthesis in Sacha Inchi seeds and to identify key genes involved in the biosynthesis of unsaturated FAs serving genetic engineering practice for improving seed oil quantity and quality in traditional oil crops. Expression level analysis of lipid genes between the initial and fast oil accumulation stages of Sacha inchi seed could reveal the potential critical genes underlying unsaturated FAs biosynthesis in Sacha Inchi seeds. Based on the annotation results, we summarized and compared expression levels of unigenes involved in FAs and TAG biosynthesis pathways in our two transcriptome libraries.
In the pathway of TAG assembly (Kennedy pathway), 77 unigenes were identified, including 14 unigenes encoding glycerol-3-phosphate acyltransferase (GPAT, which catalyzes the initial step of the Kennedy pathway), 14 unigenes encoding lysophosphatidic acid acyltransferase (LPAAT, which catalyzes the acylation of sn-1-acyl lysophosphatidic acid to form phosphatidic acid), 25 unigenes encoding phosphatidic acid phosphatase (PAP, which converts phosphatidic acid to sn-1, 2-diacylglycerol), 13 unigenes encoding acyl-CoA: diacylglycerol acyltransferase (DGAT, which transfer an acyl group from acyl-CoA to the sn-3 position of sn-1, 2-diacylglycerol to form TAG) (see Figure 8B). Besides, 11 unigenes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPDH, which catalyzes sn-glycerol 3-phosphate, an initial substrate for Kennedy pathway) were identified (see Figure 8B). In the fast oil accumulation stage, expression of unigene21846 and unigene33663 encoding GPDH were upregulated more than 8-fold; expression of unigene280 encoding PAP was upregulated 7-fold; expression of unigene30542 encoding DGAT1 was upregulated 7-fold; expression of all unigenes encoding DGAT2 were substantially upregulated (see Additional file 4, Kennedy pathway). These results strongly implied that these genes upregulated in the fast oil accumulation stage might play a major role in TAG assembly. After biosynthesis, pools of TAG usually are stored in the form of oil bodies surrounded by oleosin or steroleosin proteins in seeds. Generally, the expression of oleosin genes is tightly associated with oil accumulation in developing seeds . Seven unigenes encoding oil-body oleosins and seven unigenes encoding steroleosins (see Figure 8B) were identified with high expression in the fast oil accumulation stage (see Additional file 4, Oil accumulation), suggesting their involvement in oil accumulation in Sacha Inchi seeds.
For the formation of unsaturated FAs, 21 unigenes encoding fatty acid desaturase were identified, including three unigenes encoding stearoyl-ACP desaturase [SAD, which removes two hydrogen atoms from stearic acid (18C:0) to form oleic acid (18C:1)] (Figure 8A), eight unigenes encoding oleate desaturase [two for FAD6 and six for FAD2, both of which remove two hydrogen atoms from oleic acid to form linoleic acid (18C:2)] (Figure 8A,B), and ten unigenes encoding linoleate desaturase [(eight for FAD7 and two for FAD3, both of which remove two hydrogen atoms from linoleic acid to form linolenic acid (18C:3)] (Figure 8A,B). Because the oleic acid, linoleic acid and linolenic acid are the major constituents of PUFAs in Sacha Inchi seeds, the function of 21 unigenes identified may underlie the molecular basis of PUFAs formation in Sacha Inchi seeds. Expression analysis of the 21 unigenes between the initial and the fast oil accumulation stages showed that all unigenes encoding FAD6 and FAD7 genes (both of which were located and expressed in plastids in plant cells) were weakly expressed in the fast oil accumulation stage, while most of unigenes encoding SAD, FAD2 and FAD3 genes were highly expressed in the fast oil accumulation stage (see Additional file 4, Desaturation). In particular, the expression of unigene 15582 encoding FAD2 was upregulated 29-fold, and expression of unigene 17787 and unigene 54197 encoding FAD3 were upregulated 265-fold and 287-fold respectively (see Additional file 4, Desaturation). Genes FAD2 and FAD3 were both located and expressed in ER in cells in association with storage lipid accumulation in cells . These results indicated that genes FAD2 and FAD3 might play a major role in the formation of PUFAs in Sacha Inchi seeds. Further cloning and functional analyses for unigenes 15582, 17787 and 54197 would probably reveal the molecular mechanisms underlying PUFAs biosynthesis in Sacha Inchi seeds. In addition, Acyl-CoA independent TAG assembly pathway including acyl editing and PC-DAG interconversion was believed to facilitate the incorporation of polyunsaturated FAs into TAG in some plant species . 97 unigenes encoding phospholipases and 25 unigenes related to the Acyl-CoA independent TAG biosynthesis pathway were identified (see Figure 8B and Additional file 4). All of the twenty-five unigenes, however, have low transcript levels in the fast oil accumulation stage (see Additional file 4, Acyl editing/alternative TAG synthesis), and transcripts coding for phosphatidylcholine: diacylglycerol cholinephosphotransferase (PDCT), a key enzyme in PUFA synthesis in the Acyl-CoA independent TAG-synthetic pathway , was not detected in our libraries. These results implied that the Acyl-CoA independent TAG biosynthesis pathway might not be an active pathway in TAG biosynthesis in Sacha Inchi seeds. For the 97 unigenes encoding phospholipases, most of the unigenes have relatively higher transcript levels in the initial stage of seed development compared to the fast oil accumulation stage (see Additional file 4, Phospholipases). These unigenes may be related to the formation of membrane lipids and cell division. Lipases that arose in developing seeds of castor bean may be involved in re-modelling of TAGs after synthesis [54, 55]. Seventeen unigenes encoding lipases (nine for monoglyceride lipase and eight for triacylglycerol lipase) were identified from our libraries. The function of these lipase genes in Sacha Inchi developing seeds is unclear. The unigenes 11440 and 1193 encoding TAG lipases were upregulated 13-fold and 7-fold in the fast oil accumulation stage, respectively (see Additional file 4, TAG lipases). It appears reasonable to assume these lipase unigenes were involved in re-modelling of TAGs after synthesis in Sacha Inchi seeds.
We report here the first comprehensive dataset derived by using high-throughput sequencing technology (Illumina) for Sacha inchi. Transcriptome analyses from two developmental stages (the initial stage of seed development and the fast oil accumulation stage) of Sacha Inchi seeds revealed 70,392 unigenes. Expression of 22,179 unigenes differed at least 2-fold between the two libraries. Transcriptome sequences were annotated with Gene Ontology and KEGG Orthology identifiers. Functional categorization of differentially expressed genes reflected a number of important pathways, including the de novo FA biosynthesis, TAG assembly and the formation of PUFAs. Focusing on lipid genes, we identified 397 unigenes associated with the de novo FA biosynthesis, TAG assembly and the formation of PUFAs from Sacha Inchi seeds. These unigenes provide the foundation for further studies on molecular mechanisms underlying oil accumulation and PUFA biosynthesis in Sacha Inchi seeds. In particular, those candidate genes (such as SAD, FAD2, FAD3) associated with PUFA biosynthesis would provide critical clues to reveal the molecular mechanisms underlying the high levels of α-linolenic acid biosynthesis in Sacha Inchi seeds.
Biological material and RNA preparation
The two-year-old Sacha Inchi trees introduced from Peru by seeds were grown at Xishuangbanna Tropical Botanical Garden (21°56′ N, 101°15′ E, 600 m asl) at the Chinese Academy of Sciences, Yunnan, China under natural climate conditions. We observed the development process of Sacha Inchi fruits and seeds from female flowers pollinated to mature seeds in March-October, 2011. Mature female flowers were tagged and hand-pollinated by the time when the stigma was fully expanded, and the tagging dates were recorded as 0 day after pollination (DAP). Capsules at different development stages were harvested and dissected, and the oil content of developing seeds were measured as described previously by Xu et al. . Based on the changes of oil accumulation in developing seeds (data not shown), we determined two stages for transcriptome sampling, i.e. the initial stage of seed development (S1, 5–10 days DAP) and in the fast oil accumulation stage (S2, 50–65 DAP). The developing seeds did not start to accumulate TAG in S1 stage and fast accumulated TAG in S2 stage (Figure 1). For the transcriptome sampling, the seeds at two developing stages were collected from the same individual at the same time (at ca 10:00 a.m.) of a day (on September 12, 2011). The collected samples were flash frozen in liquid nitrogen and stored at −80°C until further use. Total RNA from the two seed groups was prepared using Trizol reagent (Invitrogen, USA) and purified by using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. RNA integrity was evaluated with a 1.0% agarose gel stained with Goldview. Then RNA samples were quantified and examined for protein contamination (A260/A280) and reagent contamination (A260/A230) by using a NanoDrop ND-1000 spectrophotometer. The RNA integrity number determined by the Agilent 2100 Bioanalyzer was greater than 9.0 for both samples.
cDNA library preparation and sequencing
Beads with oligo (dT) were used to isolate poly (A) mRNA from total RNA, according to the Illumina manufacturer’s instructions. Fragmentation buffer was added to disrupt the mRNA into short fragments. Using these short fragments as templates, random hexamers were used as primers to synthesize first-strand cDNA. Second-strand cDNA was synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. Short fragments were purified with QIAquick PCR extraction kit and resolved with EB buffer for end repair and poly (A) addition. Then, the short fragments were connected with sequencing adapters. After agarose gel electrophoresis, suitable fragments were selected as templates for PCR amplification. The cDNA library was sequenced at The Beijing Genome Institute (Shenzhen, China) using an Illumina HiSeq™ 2000 sequencing system.
Analysis of Illumina sequencing results
Sequencing-received raw image data were transformed by base calling into sequence data. Raw reads with only 3' adaptor fragments were removed before data analysis. Clean reads were assembled using the short reads assembly program SOAPdenovo  and clustered using TGI Clustering tools . Unigenes were aligned by BLASTX (e-value < 0.00001) to the NCBI nonredundant, SWISS-PROT, and KEGG protein databases. Unigenes that could not be aligned with any database were scanned by ESTScan . We used the Blast2GO program  to determine Gene Ontology annotation of unigenes. After annotating every unigene, we used WEGO software  to determine the Gene Ontology-based functional classification for all unigenes. KEGG Orthology annotations of the unigenes were determined using InterProScan software.
Differential expression of unigenes
Set RPKM (A) as the expression of unigene A, C as the number of reads uniquely aligned to unigene A, N as the total number of reads uniquely aligned to all unigenes, and L as the number of bases in unigene A. The RPKM method eliminates the influence of gene length and sequencing level on the calculation of gene expression. The calculated gene expression can thus be directly used to compare gene expression between samples.
Gene Ontology and KEGG Orthology enrichment analyses for differentially expressed unigenes
In this equation, N represents the number of genes with Gene Ontology/KEGG Orthology annotation, n represents the number of differentially expressed genes in N, M represents the number of genes in each Gene Ontology/KEGG Orthology term, and m represents the number of differentially expressed gene in each term. For Gene Ontology enrichment analysis, all P-values were adjusted with the Bonferroni correction. We selected the corrected P-value 0.05 as the threshold to determine significant enrichment of the gene sets. For KEGG Orthology enrichment analysis, we used the false discovery rate 0.05 as the threshold to determine significant enrichment of the gene sets.
Acyl carrier protein
Polyunsaturated fatty acid
Days after pollination
National Center for Biotechnology Information
Kyoto Encyclopedia of Genes and Genomes
Reads per kb per million reads
3-ketoacyl-acyl carrier protein synthase
Lysophosphatidic acid acyltransferase
This work was jointly supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KSCX2-EW-Z-15) and the “Hundreds of Talents” program of the Chinese Academy of Sciences (AL).
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