Transcriptomic and functional analyses unveil the role of long non-coding RNAs in anthocyanin biosynthesis during sea buckthorn fruit ripening

Transcriptomic and functional analyses unveil the role of long non-coding RNAs in anthocyanin... Fruit ripening is a developmental process regulated by a complex network of endogenous and exogenous cues. Sea buckthorn is an excellent material for fruit ripening studies due to its dra- matic ripening process and high contents of nutritional and anti-oxidant compounds in berries. Here, the whole transcriptome of sea buckthorn fruit at three development stages were ana- lysed using multiple high-throughput sequencings. We assembled and annotated 9,008 long non-coding RNAs (lncRNAs) in sea buckthorn fruits, and identified 118 differentially expressed lncRNAs (DE-lncRNAs) and 32 differentially expressed microRNAs in fruit developmental process. In addition, we predicted 1,061 cis-regulated and 782 trans-regulated targets of DE-lncRNAs, and these DE-lncRNAs are specifically enriched in the biosynthesis of ascorbic acid, carotenoids and flavonoids. Moreover, the silencing of two lncRNAs (LNC1 and LNC2) in vivo and expression analysis revealed that LNC1 and LNC2 can act as endogenous target mimics of miR156a and miR828a to reduce SPL9 and induce MYB114 expression, respectively, which lead to increased and decreased anthocyanin content as revealed by high-performance liquid chromatography analysis. Our results present the first global functional analysis of lncRNA in sea buckthorn and provide two essential regulators of anthocyanin biosynthesis, which provides new insights into the regulation of fruit quality. Key words: anthocyanin biosynthesis, endogenous target mimics, fruit ripening, long non-coding RNAs, sea buckthorn 1. Introduction of anti-oxidant compounds, such as carotenoids, anthocyanins and Fruits are unique plant developmental systems representing an im- flavonoids, and can therefore be used as dietary nutraceutics for hu- 2,3 portant constituent of human and animal diets due to their high min- man health. The textures, flavours and nutritional qualities of 1 4 eral, vitamin and sugar contents. In addition, fruits are rich sources fruits are determined at the ripening stage. In fruit trees and crops, V C The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 465 Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 466 lncRNAs in sea buckthorn fruit various genes and regulators have been identified as encoding or reg- expression of SPL9 and MYB114, respectively, to regulate anthocya- ulating enzymes in the biosynthetic pathways of fruit components nin biosynthesis and accumulation in sea buckthorn fruit. during ripening. For example, in tomato, MADS-box genes, ripen- ing inhibitor (RIN) and agamous-like 1 (TAGL1) dramatically affect 6,7 2. Materials and methods fruit ripening. However, fruit ripening is a developmental process characterized by a series of transitions that are coordinated by a net- 2.1. Plant materials work of interacting genes and signalling pathways, and thus, the ge- The sea buckthorn plant ‘hongguo’ (H. rhamnoides L. subsp. netic and molecular factors that regulate ripening must be Mongolica Rousi  chinensis Rousi) was used in this study. All sea deciphered to improve fruit quality at the genome-wide level. buckthorn plants were planted in the desert forest experimental cen- With the rapid development of transcriptome sequencing, a large tre in Inner Mongolia, China. Healthy fresh sea buckthorn fruits proportion of non-coding RNAs (ncRNAs) have been found in the were harvested at 46, 63 and 76 days post-anthesis, which respec- genome. Regulatory ncRNAs, including small ncRNAs and long tively considered as MG, BR and RR stages. The whole fruit was ncRNAs (lncRNAs), play vital roles in plant growth and develop- quick-frozen in liquid nitrogen and stored at -80 C. The sea buck- ment. In the past decade, the functions of small ncRNAs including thorn ‘hongguo’ was also planted for virus-induced gene silencing microRNAs and small interfering RNAs in plant development have (VIGS) in fruits. been intensively studied. For example, in transcript regulation, sly- miR1534 plays a major role in the synthesis of plant hormones in 2.2. RNA extraction, library construction tomato. Although many studies on fruit ripening have been per- and sequencing 13–15 formed, only a few studies have reported on the role of Total RNA was isolated from the MG, BR and RR fruits of ‘hon- lncRNAs in fruit qualities and fruit pigmentation. Generally, gguo’ (three biological replicates per stages from two fruits each) lncRNAs are >200 nucleotides in length and mainly transcribed by using TRIzol reagent (Invitrogen, USA) according to the manufac- RNA polymerase II, and they are involved in the regulation of vari- turer’s protocol. Agarose gel electrophoresis was used for checking ous biological processes, including plant growth and development the RNA integrity. Then, total RNA was treated to remove rRNA us- 16–18 and the response to stress. According to their positions with re- ing a kit. An Agilent 2100 Bioanalyzer was used to measure the spect to protein-coding genes, lncRNAs can be classified into inter- quantity and quality of retaining RNA without rRNA. Then, nine genic lncRNAs (lincRNAs), anti-sense lncRNAs (lncNATs) and 19 transcriptome libraries were constructed using the Ribo-Zero Kit intronic lncRNAs. Currently, lncRNAs are regarded as star RNAs (Illumina, USA) for mRNA and lncRNAs sequencing. In addition, to- in many respects, including the regulation of gene expression, protein tal RNA was also isolated from the MG, BR and RR fruits of ‘hon- binding and the maintenance of chromosome stability. LncRNAs gguo’ using TRIzol reagent (Invitrogen, USA). And, small RNA also function as precursors and endogenous target mimics (eTMs) library construction of each stage and Illumina high-throughput for certain miRNAs, providing a new mechanism for the regulation 21 sequencing were performed according to past research. These librar- of miRNA activity. With the development of next-generation se- ies were run on an Illumina HiSeq 2500 sequencer (Illumina, USA). quencing, thousands of lncRNAs have been identified in a small 17 22 23 number of plants, such as Arabidopsis, tomato, Populus, 24 25 2.3. Transcriptome assembly and lncRNA identification wheat and maize. Recently, the regulatory mechanism of vernali- zation in Arabidopsis by COOLAIR (cool-assisted intronic ncRNA) The raw reads were trimmed and quality-filtered using FastQC tools (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Each and COLDAIR (cold-assisted intronic ncRNA), two lncRNAs tran- scribed from Flowering Locus C, has been illustrated. However, RNA-seq sequence dataset was aligned to the whole sea buckthorn the functions of the majority of lncRNAs have not been fully studied genome (unpublished) by using TopHat2. The transcripts from to date. each dataset were assembled using the Cufflinks 2.0 program. Sea buckthorn (Hippophae rhamnoides L.) is a pioneer plant for Cuffmerge was used to pool and merge the final transcripts. Then, land reclamation, and its fruits have been used for nutritional pur- the abundance of all transcripts from the BAM output files, esti- poses for many centuries in Russia, Europe and Asia due to their mated using fragments per kilobase of transcript per million mapped high contents of fat-soluble vitamins (A, K and E), fatty acids, amino reads (FPKM) values, were calculated using Cuffdiff. acids, vitamins C, B1 and B2, folic acid and phenols. The berries of On the basis of the merged results, the remaining 654, 750 tran- sea buckthorn contain high amounts of natural anti-oxidants includ- scripts were used to identify the intergenic lncRNAs (lincRNAs), ing ascorbic acid (ASA), tocopherols, carotenoids and flavonoids, anti-sense lncRNAs (lncNATs) and intronic ncRNAs. The process was as follows: (i) Transcripts with an FPKM score <2 in a single which are known to have beneficial effects on human health. Because of its various beneficial contents and significant ecological exon or 0.5 in multiple exons in at least one sample were discarded; value, sea buckthorn has become an ideal material for both basic re- (ii) Transcripts with a length longer than 200 bp and an open reading search and application. However, the number, expression pattern, frame (ORF) length shorter than 100 aa were retained; (iii) The CPC and characteristics of lncRNAs in sea buckthorn remain largely un- program was used to calculate the coding potential of the retained transcripts, and the transcripts with CPC scores >0 were dis- known. Therefore, it is necessary and urgent to identify novel lncRNAs as well as their functions in sea buckthorn fruit carded; (iv) HMMER was employed to scan the protein coding po- development. tential of each transcript against the Pfam protein family database Here, the whole transcriptome of three development stages fruit, with the transcript length >200 nucleotides, and no ORF encoding including mature green (MG), breaker (BR) and red-ripe (RR), were >100 amino acids; (v) The transcripts were compared against the analysed using multiple high-throughput sequencings and diverse NCBI non-redundant (NR) protein database, the Kyoto bioinformatic platforms. Function and expression analysis revealed Encyclopedia of Genes and Genomes (KEGG) database and the that the lncRNAs LNC1 and LNC2 can reduce and induce the Swiss-Protein database (Swiss-Prot) by BLASTX (E-value ¼ 1e-10, Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 G. Zhang et al. 467 coverage> 85%, and identity> 95%) to retain transcripts without lncRNA–miRNA–mRNA network and visualized using Cytoscape significant homology to known proteins. The raw reads generated software. have been deposited in the NCBI SRA database under accession numbers SRP124171 for the miRNA-Seq reads and the ssRNA-Seq 2.8. qRT-PCR analysis reads. The total RNA from sea buckthorn fruit (MG, BR, RR) was used for quantitative PCR analysis. The RNA was then reverse transcribed to 2.4. Differential expression of lncRNAs and mRNAs cDNA using ReverTra Ace reverse transcriptase (Toyobo). qRT-PCR of the randomly selected DE-lncRNAs was performed using a Bio- Differentially expressed lncRNAs (DE-lncRNAs) between the sam- TM Rad CFX96 Touch RealTime PCR Detection System (Bio-Rad) ples at different developmental stages were identified using Cuffdiff. with SYBR Green RealTime PCR Master Mix (ABI) according to the Any lncRNA exhibiting a log -transformed jfold changej 1 and ad- standard protocol. Specific primers for DE-lncRNAs and DE- justed P-value< 0.05 was selected as a DE-lncRNA. On the basis of miRNAs are listed in Supplementary Table S1. The 18S rRNA and the features of the lncRNAs, their localization and abundance were U6 were used as the internal control genes for DE-lncRNAs and DE- shown using Circos. miRNAs in these experiments. All reactions were conducted in tripli- –DDCT cate for both technical and biological repetitions. The 2 method 2.5. Prediction and functional analysis of DE-lncRNA was used to calculate the relative gene expression levels. target To explore the functions of lncRNAs, we first predicted their cis and 2.9. Subcellular localization of LNC1/2 trans targets. In the cis role, lncRNAs act on neighbouring target The subcellular localization of two sea buckthorn DE-lncRNAs was genes. In this study, we searched for coding genes 100 kb upstream examined using fluorescence in situ hybridization (FISH). Sea buck- and downstream of a lncRNA. The trans role refers to the influence thorn (‘hongguo’) fruits at RR stage were collected for FISH. We se- of lncRNAs on other genes at the expression level. Here, we calcu- lected three healthy fresh fruits for subcellular localization of LNC1/ lated Pearson’s correlation coefficients between the expression levels 2. The pipeline of subcellular localization was followed as previously of lncRNAs and mRNAs with custom scripts (jrj> 0.95). Then, we described, including probe synthesis, sample fixation and probe hy- performed functional enrichment analysis of the target genes of bridization. Firstly, the whole fruit are fixed with fixative: n-Heptan 34 35 lncRNAs by using goseq and KOBAS. (1:1) solution. Then, to preserve tissue morphology, samples were rinsed in 75% (v/v) ethanol, 50% (v/v) ethanol/PBS and 25% (v/v) 2.6. Bioinformatics analysis of small RNAs ethanol/PBS each for 10 min. Tissues are incubated in Hybridization After small RNA sequencing, low-quality reads, adapter- Mix at 52 C. Afterwards, samples were rinsed in Washing Solution contaminating tags and reads with lengths smaller than 18 nt were to remove non-specific and/or repetitive RNA hybridization, and discarded. All unique clean reads were considered for ncRNA analysed by microscopy. Two probe sequences are listed in (rRNA, scRNA, snoRNA, snRNA and tRNA) identification in a Supplementary Table S2. BLAST all search against the Rfam (version 10.1) database. Next, the remaining reads were compared with known miRNAs of plants 2.10. VIGS of sea buckthorn fruits deposited in miRbase. Then, the secondary structures of these According to previous study, VIGS of sea buckthorn fruit was per- miRNAs were predicted by RNAfold. The frequency of the miRNAs formed using tobacco rattle virus (TRV). To avoid off-target silenc- was normalized to transcripts per million (TPM) in each sample. 16 ing, we analysed LNC1 and LNC2 using the VIGS tool. A pTRV2- Differentially expressed miRNAs were identified by log -transformed LNC1/2 construct was generated by inserting the EcoRI-digested jfold changej> 1 and P-value< 0.05. To explore whether mRNAs PCR fragment of lncRNA into the pTRV2 vector. Agrobacterium functioned as targets of miRNAs, the transcripts and identified strain GV3101 constructs containing pTRV1, pTRV2 and pTRV2- miRNAs were submitted to psRobot with an expectation  2. LNC1/LNC2 vectors were grown at 28 C in Luria Broth medium Transcripts containing a total of no more than four mismatches and containing 10 mM MES, 20 lM acetosyringone, 50 lgml kanamy- G/U pairs within the complementary regions were considered as cin and 50 lgml rifampicin. After shaking for 12 h, agrobacterium miRNA targets. The target mimics were predicted using cells were harvested through centrifugation, resuspended in infiltra- psRNATarget combined with local scripts and the rules established tion buffer (10 mM MgCl , 200 lM acetosyringone and 5% sucrose) by Wu et al. to a final OD600 of 1.0. Resuspensions of pTRV1 and pTRV2 or pTRV2-lncRNA were mixed at a ratio of 1:1. After 3 h of incuba- tion, the agrobacterium was infiltrated into the fruits with a micro 2.7. Construction of DE-lncRNA-miRNA-mRNA network syringe. Sea buckthorn fruits infiltrated with pTRV1 and pTRV2 To infer the functions of lncRNAs, networks were constructed based were used as controls (CK). Three different plants from each sample on the complementary pairs of miRNAs-lncRNAs and miRNAs- were used for infiltration. When the VIGS phenotype was visible, the mRNAs. The DE-lncRNA–miRNA–mRNA network was recon- sea buckthorn fruits were collected and stored at –80 C. structed based on ceRNA theory. First, expression correlation between lncRNAs and mRNAs was evaluated using the Pearson cor- 2.11. qRT-PCR of lncRNAs, miRNAs and genes relation coefficient (PCC). The lncRNA-mRNA pairs were consid- ered as co-expression pairs with threshold PCC> 0.9 and P< 0.05. involved in anthocyanin biosynthesis Then, for a lncRNA-mRNA pair, both mRNA and lncRNA, which Total RNA was extracted from the CK, TRV-LNC1 and TRV- as common miRNA targets and co-expressed negatively with this LNC2 fruits using the Total RNA Kit (Aidlab, Beijing, China). TM miRNA, were selected as co-expression competing triplet. Finally, all Briefly, the first cDNA strand was obtained using the Prime Script these co-expression competing triplets were used for constructing RT Master Mix (Takara, Dalian, China). All primers used in this Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 468 lncRNAs in sea buckthorn fruit study are listed in Supplementary Table S1. qRT-PCR was per- indicated that lncRNAs might have a different expression pattern TM formed on the Bio-Rad CFX96 Touch RealTime PCR Detection from protein-coding genes. Only two and 36 DE-lncRNAs were System using a standard SYBR Green PCR Kit (Bio-Rad). The condi- expressed specifically in the MG and RR stage, respectively tions for quantitative PCR and the primers for the lncRNAs and (Fig. 1D). In contrast to MG, 38 and 72 lncRNAs were upregulated genes are listed in Supplementary Table S1. Sample cycle threshold in BR or RR, and the remaining 44 and 46 lncRNAs were (Ct) values were determined and standardized relative to the endoge- downregulated. –DDCT nous control gene 18S rRNA, and the 2 method was used to calculate the relative changes in gene expression based on the qRT- PCR data. All reactions were carried out in three biological repeti- 3.2. Functional analysis of DE-lncRNAs tions. qRT-PCR was used to validate the sequencing results of two To investigate the functions of DE-lncRNAs, we predicted the poten- miRNAs and the genes involved in anthocyanin biosynthesis. The tial cis and trans targets of the lncRNAs. For the cis action of quantitative PCR conditions and the primers for the miRNAs and lncRNAs, computational prediction identified 1,061 potential target genes are listed in Supplementary Table S1. genes for 84 DE-lncRNAs (Supplementary Table S6). Gene Ontology (GO) analysis revealed that cis lncRNA targets significantly repre- 2.12. Determination of anthocyanins by high- sented in the developmental process (Supplementary Fig. S2). performance liquid chromatography analysis Moreover, pathway analysis showed that these cis target genes of Sea buckthorn fruit from CK, TRV-LNC1 and TRV-LNC2 plants lncRNAs were enriched in plant hormone signal transduction, fatty was collected to quantify the content of anthocyanins. Each group acid and unsaturated fatty acid metabolism, carotenoid biosynthesis included three biological repetitions. The method of anthocyanin de- and flavonoid biosynthesis (Supplementary Table S7). On the other termination was described in previous research. hand, the trans functions of 67 DE-lncRNAs were examined based on their expression correlation coefficients (jPearson correlationj 0.95) with protein-coding genes. A total of 21,772 interactions were 3. Results detected in trans between 67 DE-lncRNAs and 782 protein-coding 3.1. Identification and characterization of sea buckthorn genes in the sea buckthorn genome (Supplementary Table S8). lncRNAs Functional analysis showed that the trans target genes were also By integrating lncRNA computational identification methods, we de- enriched in a variety of biological processes. Importantly, we observed veloped a de novo lncRNA prediction pipeline using the resulting a few plant growth terms, including the regulation of growth, cell wall strand-specific RNA-seq (ssRNA-seq) data sets from three develop- organization or biogenesis, and regulation of cellular component mental stages of sea buckthorn (Supplementary Table S3; organization. Of the identified 110 KEGG pathways, five were associ- Supplementary Fig. S1). After mapping the RNA-seq data to the ated with fruit development, namely, ascorbate and aldarate whole genome of sea buckthorn, we identified 9,008 lncRNAs in the metabolism, flavonoid biosynthesis and brassinosteroid biosynthesis fruits. The obtained lncRNAs were further classified into three types: (Supplementary Table S9). lincRNAs, lncNATs and intronic lncRNAs, based on their locations to the protein coding genes. Whereas the majority of all lncRNAs (6,750) was located in intergenic regions, only 13.4% and 11.7% of 3.3. DE-lncRNAs participate in miRNA-lncRNA-mRNA all lncRNAs were intronic lncRNAs and lncNATs of protein-coding genes, respectively. A circos plot clearly showed that sea buckthorn networks lncRNAs were not evenly distributed across chromosomes (Fig. 1E), Recent evidence suggested the roles of miRNAs and lncRNAs in reg- 43–45 similar to the distribution of lncRNAs observed in other spe- ulating the expression of mRNAs in many species, but the com- 18,23,42 cies. In addition, the expression level of lncRNAs varied be- prehensive patterns of miRNA and lncRNAs in sea buckthorn tween chromosomes. For instance, the average expression levels of remain unknown. We predicted 68 conserved miRNAs and 79 pre- lncRNAs generated from chromosomes 4 and 7 were 67.69 and miRNAs, which were identified as belonging to 31 known miRNA 2.1415, respectively. families deposited in miRBase. A total of 32 DE-miRNAs were pre- We compared the lengths of transcript and ORF and exon counts dicted to target 67 genes in this study (Supplementary Table S10). of the 9,008 putative lncRNAs with identified mRNAs. The length The target prediction analysis revealed that 22 lncRNAs may act as distribution of the identified lncRNAs (median 271 bp; average eTMs of 25 DE-miRNAs (Supplementary Table S11). Recently, 463.28 bp) was shorter than that of the mRNAs (median 714 bp; av- some researches have shown that lncRNAs can affect the regulation erage 976.08 bp), while the length distribution showed no significant 21,46 of miRNAs as eTMs in plants. To investigate the function of differences among intergenic lncRNAs, intronic lncRNAs and lncRNAs as miRNA targets, a comprehensive genome-wide network lncNATs (Fig. 1A). The exon counts of the putative lncRNAs (aver- mediated by miRNAs was constructed. The network consisted of age 1.56 exons per transcript) were also lower than those of the 109 nodes and 201 edges, including 13 DE-miRNAs, 10 DE- mRNAs (average 4.19 exons per transcript), while the exon counts lncRNAs and 87 DE-mRNAs (Fig. 2A). The majority of the nodes in of the three kinds of lncRNA showed no differences (Fig. 1B). We the network belonged to the miR396 family, which regulates growth found the ORF lengths of the majority of lncRNAs were shorter than by targeting growth regulation factors. Then, the function of each mRNAs (Fig. 1C). lncRNA was inferred based on the functions of the connected A total of 118 known lncRNAs and 108 novel transcripts were mRNAs. We found that lncRNAs mainly participate in cellular, met- differentially expressed in the three samples (Supplementary abolic and some biological processes, especially highly enriched in Table S4). We also found fewer DE-lncRNAs (2.46%) than cellular component, binding including ‘ion binding’, ‘nucleotide DE-mRNAs (5.64%, total mRNAs 45,546) in fruits at different de- velopment stages (Supplementary Table S5). This distinction binding’ and ‘small molecule binding’ (Supplementary Table S12). Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 G. Zhang et al. 469 Figure 1. Characteristics of sea buckthorn lncRNAs. (A) Length distributions of lncRNAs and transcripts. (B) Number of exons per lncRNA and transcript. (C) Distribution of the number of open reading frame (ORFs) in lncRNAs and transcripts. (D) A Venn diagram showing DE-lncRNAs that are commonly expressed in the mature green (MG), breaker (BR) and red-ripe (RR) stages as well as those specifically expressed in one but not another. (E) The expression levels of lncRNAs (log FPKM) along the twelve sea buckthorn chromosomes (generated using Circos). Each of the three concentric rings corresponds to different sam- ples. In the second, third and fourth track (outer to inner), each vertical line reports the expression of lncRNAs in sea buckthorn fruit in the MG, BR andRR stages, respectively. 3.4. Analysis of lncRNAs related to the anthocyanin To validate the putative relationships between the miRNAs and biosynthesis of fruits lncRNAs, their expression levels were examined by qRT-PCR According to previous studies, the module miR156-SPL affects an- (Fig. 3). The qRT-PCR results were consistent with our RNA-seq thocyanin biosynthesis in plants. Additionally, R2R3-MYBs data regarding lncRNA and miRNA expression, suggesting that (MYB114) are known to combine with bHLHs and WDR to form a these two lncRNAs identified by next-generation sequencing might ternary complex that activates late biosynthesis genes and transfers have negative regulation relation with miR156a and miR828a. protein genes. Here, based on the miRNA-lncRNA-mRNA net- Distribution analysis is an important step towards functional re- work, we identified two anthocyanin biosynthesis-related lncRNAs search on lncRNAs as eTMs. To demonstrate the subcellular locali- (TCONS_00694050 and TCONS_00438839, termed LNC1 and zation of LNC1 and LNC2, FISH was performed in fruits using a LNC2) for further analysis (Fig. 2). On the basis of the network, we fluorescence probe specific to LNC1 and LNC2. In combination with found that LNC1 and LNC2 may regulate the expression of SPL9 the DAPI signal, the probe showed that these two lncRNAs were and MYB114 by acting as eTMs of miR156a and miR828a. mainly enriched in cytoplasm (Fig. 4A). Genome chromosomal Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 470 lncRNAs in sea buckthorn fruit Figure 2. Analysis of lncRNA-miRNA-mRNA networks. (A) The networks of lncRNA-miRNA-mRNA. The triangle represents lncRNAs, the circle with label repre- sents miRNAs, and the circle without label represents mRNAs. (B) LNC1/2 as endogenous target mimics (eTMs) of miRNAs. Secondary structure of miRNAs pre- cursors and the basepairing relationship between lncRNA and miRNAs. The predicted secondary structure was generated using RNAfold. (C) Sub interaction networks of LNC1/2 from lncRNA-miRNA-mRNA networks. The ellipse represents lncRNAs, the triangle represents miRNAs, and the rectangle represents mRNAs. location analyses revealed that both LNC1 and LNC2 reside in chro- in sea buckthorn fruits. Compared with that in TRV control fruits, mosome 1 and lack homologs compared with other plants (Fig. 4B). the transcript level of LNC1 in TRV-LNC1 fruits was dramatically decreased by 96% (Fig. 5C). Similarly, the level of LNC2 in TRV- LNC2 fruits decreased by 63.3% compared with that in TRV con- 3.5. LNC1 and LNC2 regulate anthocyanin biosynthesis trol fruits (Fig. 5C). Consistently, the anthocyanin content of TRV- in fruits LNC1 fruit increased by 25.23% with respect to TRV control To test the regulation role of LNC1 and LNC2 in anthocyanin bio- fruits, and the anthocyanin content of TRV-LNC2 fruit decreased synthesis, VIGS was performed to silence LNC1 and LNC2 in sea by 39.12% with respect to TRV control fruits (Fig. 5C). Previous buckthorn fruits (Fig. 5A). Intriguingly, two or three weeks after in- studies have reported that SPL and MYB transcription factors can filtration, anthocyanin contents of sea buckthorn fruits injected affect anthocyanin content by regulating the expression of genes in- with TRV-LNC1 or TRV-LNC2 showed significant difference com- volved into anthocyanin biosynthesis pathway, such as ANS, DFR, pared with that of control fruits injected with TRV (Fig. 5B). Semi- F3 H, LDOX, UGT78D2 and UGT75C1. Similarly, three genes quantitative PCR analysis suggested that a recombinant virus could involved in anthocyanin biosynthesis were increased or decreased spread from carpogonia to fruits, resulting in the VIGS of lncRNAs in TRV-LNC1 compared with their levels in TRV control sea Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 G. Zhang et al. 471 Figure 3. qRT-PCR validation of putative lncRNAs and miRNAs in sea buckthorn. The expression levels of lncRNAs (A) and miRNAs (B) in three developmental stages: mature green (MG), breaker (BR) and red-ripe (RR). 4. Discussion buckthorn fruits (Fig. 5C). In contrast, DFR, F3 H and LDOX were decreased in TRV-LNC2 compared with their levels in TRV control 4.1. A reliable list of lncRNAs from sea buckthorn fruits sea buckthorn fruits (Fig. 5C). These results strongly suggested the Recently, plenty of studies have revealed the crucial roles of 17,45,49 essential role of the two novel lncRNAs in the regulation of the an- lncRNAs in various biological processes in plants. Although thocyanin accumulation of sea buckthorn fruits. many lncRNAs have been identified in model plants, such as Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 472 lncRNAs in sea buckthorn fruit Figure 4. Subcellular localization and chromosome location of LNC1 and LNC2 in sea buckthorn fruit. (A) Visualization of LNC1 and LCN2 in sea buckthorn fruit by RNA fluorescence in situ hybridization using an antisense probe. Scale bar, 20 lm. (B) Location regions of LNC1 and LNC2 on chromosome. 50 51 25 Arabidopsis, rice, and maize, little research has been performed the most important reason for the pigmentation of red fruit in sea on sea buckthorn. In this study, a total of 9008 lncRNA loci were buckthorn. Therefore, these results revealed that lncRNAs may play identified in the fruit of sea buckthorn, an ideal material for the roles in the development of sea buckthorn fruit pigmentation. study of fruit development. Furthermore, structural analysis Sea buckthorn fruit contains abundant ASA and is known as the showed that the mean length of lncRNAs was 1.1-fold shorter than king of vitamin C, and ASA is a prominent product of the ascorbate that of coding transcripts, and the lncRNA expression level was and aldarate metabolism pathway. In this pathway, we found seven lower than that of coding transcripts, in agreement with previous lncRNAs targeting GDP-mannose-epimerase (GME) and SKS12, 16,42,52 studies. LncRNAs also have specific expression patterns in which encodes L-ascorbate oxidase. Several studies have revealed tissue types and subcellular compartments. In our study, among the that GME and SKS12 are associated with the accumulation of ASA significant DE-lncRNAs, we found five expression patterns in MG, in the fruits of many plants, including blueberry, Arabidopsis, to- 58–61 BR and RR. mato and peach. For instance, the importance of GME in the major ascorbate biosynthesis pathway in tomato was confirmed us- ing gene-silencing technology. Thus, our results provide a resource 4.2. DE-lncRNAs contribute to fruit development of sea for further analysis of the regulation of ASA biosynthesis in sea buck- buckthorn fruit thorn fruit. Transcriptomic sequencing of different varieties of sea buckthorn 28,53 Flavonoids are a large group of natural products that are widely revealed a lot of ncRNAs in many biological processes. In this present in the leaves, flowers, fruits and seeds of various plants. study, we predicted potential cis and trans target genes based on According to their basic chemical structures, flavonoids can be fur- physical location and expression relationships with mRNA, respec- ther divided into different subgroups, including flavonols, anthocya- tively. Although the functions of the majority of lncRNAs are un- nins and proanthocyanidins. Our results consistent with those of known, previous studies have suggested that lncRNAs may play previous studies, reveal that sea buckthorn fruit contains various fla- different roles in a variety of biological processes. According to pre- vonoids. In the flavonoid biosynthesis pathway, chalcone synthase vious study, we analysed the functions of DE-lncRNAs based on (CHS) is the first committed enzyme that facilitates the stepwise syn- their target mRNAs, which involved in many processes, including ca- thesis of chalcone, and it is also pivotal for the biosynthesis of flavo- rotenoid, ASA, flavonoid, sugar and hormone pathways. noid and anthocyanin pigments in plants. TCONS_00085219 was We identified 10 DE-lncRNAs involved in carotenoid biosynthesis predicted to target CHS, indicating a possible regulatory relationship pathways, including b-carotenoid and lycopene biosynthesis. Among between lncRNA and CHS (Fig. 6). Another important enzyme in- these DE-lncRNAs, one lncRNA (TCONS_00082246) targeted the volved in this pathway is flavanone 3-hydroxylase (F3H), which was phytoene synthase (PSY) gene, which is involved in the first commit- reported in rice. TCONS_01039552 was predicted to target F3H, ted step in carotenoid biosynthesis (Supplementary Table S12). This implying a potential role in flavonol and anthocyanin biosynthesis reaction has been reported to be the rate-limiting step controlling the through the regulation of F3H. In addition to CHS and F3H, other metabolic flux to carotenoid biosynthesis in many plants. For ex- important genes, such as the gene encoding flavonol synthase 1, were ample, the PSY gene plays an important role in carotenoid biosynthe- sis in tomato. Therefore, this study opened the door to predicted as target genes of two lncRNAs (TCONS_00061167 and TCONS_00061354) identified in our study. All these results implied understanding the role of lncRNAs in the regulation of genes in- volved in carotenoid biosynthesis in sea buckthorn fruit. Recently, the widespread involvement of lncRNAs in the regulation of many studies have shown that carotenoids are considered as the flavonoid biosynthesis and fruit pigment in the development of sea main fruit pigments in tomato, bilberry, citrus, papaya, watermelon buckthorn fruit, which will provide novel insight into the regulation 55–57 and sea buckthorn. In addition, lycopene accumulation is of flavonoid biosynthesis in other plants. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 G. Zhang et al. 473 Figure 5. Silencing of LNC1/2 in sea buckthorn fruit. (A) Sea buckthorn fruits (3 weeks after infiltration). CK: sea buckthorn fruits infiltrated with pTRV1 and pTRV2; TRV-LNC1: sea buckthorn infiltrated with pTRV1 and pTRV2-LNC1; TRV-LNC2: sea buckthorn infiltrated with pTRV1 and pTRV2-LNC2. (B) High-perfor- mance liquid chromatography analysis of total anthocyanin content in sea buckthorn fruits. (C) qRT -PCR analysis of the expression of related genes in sea buckthorn fruits. The expression of 18S rRNA was used as an internal reference. The relative level was normalized to that in TRV control plants. Error bars indi- cate SD of three biological replicates, each sample measured in three replicates. Asterisks indicate a significant difference as determined by Student’s t-test (*P< 0.01). Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 474 lncRNAs in sea buckthorn fruit Figure 6. Model of lncRNA regulation in sea buckthorn fruit. This pathway begins with the general phenylpropanoid metabolism, and subsequent steps are cat- alyzed by a series of structural enzymes, leading to the biosynthesis of flavonols and anthocyanins. The expression of the late biosynthetic genes (LBGs) requires the transcriptional activation activity of the R2R3-MYB/bHLH/WDR (MBW) complex. LncRNAs target FLS and CHS: functions in trans regulation. 0 0 0 LncRNAs target F3H and F3 H: functions in cis regulation. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3 H, flavanone 3 - hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase. regulated through the LNC1-induced downregulation of SPL9, 4.3. LNC1 and LNC2 regulate anthocyanin biosynthesis which affects the stability of the MYB, bHLH and WDR (MBW) in the fruit development of sea buckthorn by eTMs complex, thus promoting anthocyanin biosynthesis in sea buckhorn Previous studies suggest that lncRNAs function via interacting with fruit. In contrast, the LNC2-induced downregulation of MYB114 re- miRNAs. The reverse miRNA-lncRNA regulation, first discovered duced anthocyanin biosynthesis in sea buckhorn fruit. Thus, this in Arabidopsis, was termed target mimicry. Previous research sys- study illuminated the complex regulation of fruit anthocyanin bio- tematically identified 36 eTMs for 11 Arabidopsis miRNAs and 189 21,51 synthesis, which might instigate more comprehensive studies on sea target mimics for 19 rice miRNAs. In Populus, seven miRNAs target TCONS_00013609, including pto-miR6462a/b/c. buckthorn lncRNAs. Furthermore, the functional motifs and target Therefore, the prediction and analysis of miRNAs that interact with genes of lncRNAs in trees need to be investigated further to fully elu- lncRNAs provide a useful way to explore the functions of the corre- cidate the regulatory mechanisms of lncRNAs in trees. sponding lncRNAs. In this study, we identified 19 DE-lncRNA eTMs for 23 DE-miRNAs (seven families). These results first showed that miRNAs target lncRNAs in sea buckthorn. Furthermore, our investi- Conflict of interest gation constructed a comprehensive RNA-mediated network, includ- None declared. ing DE-miRNA–DE-lncRNA and DE-miRNA–DE-mRNA interactions. However, a mechanistic understanding of the roles of lncRNAs in Funding plants, especially trees, has remained extremely lacking. This study was supported by grants from Special Fund for Forest Scientific lncRNA1459 and lncRNA1840 were found to regulate the ripening Research in the Public Welfare (201504103) and the National Natural Science of tomato. For mammal, lnc-mg was identified to promote myo- Foundation of China (31470616). genesis by functioning as a ceRNA/eTM for microRNA-125b to con- trol the protein abundance of insulin-like growth factor 2. In Arabidopsis, a representation of the classical TM model, miR399: Supplementary data IPS1, was discovered by Franco-Zorrilla. Few studies have reported on the mechanism of lncRNA involvement in anthocyanin Supplementary data are available at DNARES online. biosynthesis during fruit development. In this study, we identified two lncRNAs (LNC1 and LNC2) involved in the anthocyanin bio- References synthesis pathway (Fig. 6). Additionally, we first used FISH and VIGS for function analysis of lncRNAs in sea buckthorn. The VIGS 1. Giovannoni, J.J. 2003, Genetic regulation of fruit development and ripen- results provide strong evidence that these two lncRNAs function in ing, Plant Cell, 16(Suppl.), S170. fruit anthocyanin biosynthesis. When LNC1 and LNC2 expression is 2. Winkel-Shirley, B. 2001, It takes a garden. How work on diverse plant reduced, anthocyanin accumulation is increased or decreased, respec- species has contributed to an understanding of flavonoid metabolism, tively. This mechanism shows that anthocyanin accumulation is Plant Physiol., 127, 1399–404. 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Transcriptomic and functional analyses unveil the role of long non-coding RNAs in anthocyanin biosynthesis during sea buckthorn fruit ripening

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Abstract

Fruit ripening is a developmental process regulated by a complex network of endogenous and exogenous cues. Sea buckthorn is an excellent material for fruit ripening studies due to its dra- matic ripening process and high contents of nutritional and anti-oxidant compounds in berries. Here, the whole transcriptome of sea buckthorn fruit at three development stages were ana- lysed using multiple high-throughput sequencings. We assembled and annotated 9,008 long non-coding RNAs (lncRNAs) in sea buckthorn fruits, and identified 118 differentially expressed lncRNAs (DE-lncRNAs) and 32 differentially expressed microRNAs in fruit developmental process. In addition, we predicted 1,061 cis-regulated and 782 trans-regulated targets of DE-lncRNAs, and these DE-lncRNAs are specifically enriched in the biosynthesis of ascorbic acid, carotenoids and flavonoids. Moreover, the silencing of two lncRNAs (LNC1 and LNC2) in vivo and expression analysis revealed that LNC1 and LNC2 can act as endogenous target mimics of miR156a and miR828a to reduce SPL9 and induce MYB114 expression, respectively, which lead to increased and decreased anthocyanin content as revealed by high-performance liquid chromatography analysis. Our results present the first global functional analysis of lncRNA in sea buckthorn and provide two essential regulators of anthocyanin biosynthesis, which provides new insights into the regulation of fruit quality. Key words: anthocyanin biosynthesis, endogenous target mimics, fruit ripening, long non-coding RNAs, sea buckthorn 1. Introduction of anti-oxidant compounds, such as carotenoids, anthocyanins and Fruits are unique plant developmental systems representing an im- flavonoids, and can therefore be used as dietary nutraceutics for hu- 2,3 portant constituent of human and animal diets due to their high min- man health. The textures, flavours and nutritional qualities of 1 4 eral, vitamin and sugar contents. In addition, fruits are rich sources fruits are determined at the ripening stage. In fruit trees and crops, V C The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 465 Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 466 lncRNAs in sea buckthorn fruit various genes and regulators have been identified as encoding or reg- expression of SPL9 and MYB114, respectively, to regulate anthocya- ulating enzymes in the biosynthetic pathways of fruit components nin biosynthesis and accumulation in sea buckthorn fruit. during ripening. For example, in tomato, MADS-box genes, ripen- ing inhibitor (RIN) and agamous-like 1 (TAGL1) dramatically affect 6,7 2. Materials and methods fruit ripening. However, fruit ripening is a developmental process characterized by a series of transitions that are coordinated by a net- 2.1. Plant materials work of interacting genes and signalling pathways, and thus, the ge- The sea buckthorn plant ‘hongguo’ (H. rhamnoides L. subsp. netic and molecular factors that regulate ripening must be Mongolica Rousi  chinensis Rousi) was used in this study. All sea deciphered to improve fruit quality at the genome-wide level. buckthorn plants were planted in the desert forest experimental cen- With the rapid development of transcriptome sequencing, a large tre in Inner Mongolia, China. Healthy fresh sea buckthorn fruits proportion of non-coding RNAs (ncRNAs) have been found in the were harvested at 46, 63 and 76 days post-anthesis, which respec- genome. Regulatory ncRNAs, including small ncRNAs and long tively considered as MG, BR and RR stages. The whole fruit was ncRNAs (lncRNAs), play vital roles in plant growth and develop- quick-frozen in liquid nitrogen and stored at -80 C. The sea buck- ment. In the past decade, the functions of small ncRNAs including thorn ‘hongguo’ was also planted for virus-induced gene silencing microRNAs and small interfering RNAs in plant development have (VIGS) in fruits. been intensively studied. For example, in transcript regulation, sly- miR1534 plays a major role in the synthesis of plant hormones in 2.2. RNA extraction, library construction tomato. Although many studies on fruit ripening have been per- and sequencing 13–15 formed, only a few studies have reported on the role of Total RNA was isolated from the MG, BR and RR fruits of ‘hon- lncRNAs in fruit qualities and fruit pigmentation. Generally, gguo’ (three biological replicates per stages from two fruits each) lncRNAs are >200 nucleotides in length and mainly transcribed by using TRIzol reagent (Invitrogen, USA) according to the manufac- RNA polymerase II, and they are involved in the regulation of vari- turer’s protocol. Agarose gel electrophoresis was used for checking ous biological processes, including plant growth and development the RNA integrity. Then, total RNA was treated to remove rRNA us- 16–18 and the response to stress. According to their positions with re- ing a kit. An Agilent 2100 Bioanalyzer was used to measure the spect to protein-coding genes, lncRNAs can be classified into inter- quantity and quality of retaining RNA without rRNA. Then, nine genic lncRNAs (lincRNAs), anti-sense lncRNAs (lncNATs) and 19 transcriptome libraries were constructed using the Ribo-Zero Kit intronic lncRNAs. Currently, lncRNAs are regarded as star RNAs (Illumina, USA) for mRNA and lncRNAs sequencing. In addition, to- in many respects, including the regulation of gene expression, protein tal RNA was also isolated from the MG, BR and RR fruits of ‘hon- binding and the maintenance of chromosome stability. LncRNAs gguo’ using TRIzol reagent (Invitrogen, USA). And, small RNA also function as precursors and endogenous target mimics (eTMs) library construction of each stage and Illumina high-throughput for certain miRNAs, providing a new mechanism for the regulation 21 sequencing were performed according to past research. These librar- of miRNA activity. With the development of next-generation se- ies were run on an Illumina HiSeq 2500 sequencer (Illumina, USA). quencing, thousands of lncRNAs have been identified in a small 17 22 23 number of plants, such as Arabidopsis, tomato, Populus, 24 25 2.3. Transcriptome assembly and lncRNA identification wheat and maize. Recently, the regulatory mechanism of vernali- zation in Arabidopsis by COOLAIR (cool-assisted intronic ncRNA) The raw reads were trimmed and quality-filtered using FastQC tools (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Each and COLDAIR (cold-assisted intronic ncRNA), two lncRNAs tran- scribed from Flowering Locus C, has been illustrated. However, RNA-seq sequence dataset was aligned to the whole sea buckthorn the functions of the majority of lncRNAs have not been fully studied genome (unpublished) by using TopHat2. The transcripts from to date. each dataset were assembled using the Cufflinks 2.0 program. Sea buckthorn (Hippophae rhamnoides L.) is a pioneer plant for Cuffmerge was used to pool and merge the final transcripts. Then, land reclamation, and its fruits have been used for nutritional pur- the abundance of all transcripts from the BAM output files, esti- poses for many centuries in Russia, Europe and Asia due to their mated using fragments per kilobase of transcript per million mapped high contents of fat-soluble vitamins (A, K and E), fatty acids, amino reads (FPKM) values, were calculated using Cuffdiff. acids, vitamins C, B1 and B2, folic acid and phenols. The berries of On the basis of the merged results, the remaining 654, 750 tran- sea buckthorn contain high amounts of natural anti-oxidants includ- scripts were used to identify the intergenic lncRNAs (lincRNAs), ing ascorbic acid (ASA), tocopherols, carotenoids and flavonoids, anti-sense lncRNAs (lncNATs) and intronic ncRNAs. The process was as follows: (i) Transcripts with an FPKM score <2 in a single which are known to have beneficial effects on human health. Because of its various beneficial contents and significant ecological exon or 0.5 in multiple exons in at least one sample were discarded; value, sea buckthorn has become an ideal material for both basic re- (ii) Transcripts with a length longer than 200 bp and an open reading search and application. However, the number, expression pattern, frame (ORF) length shorter than 100 aa were retained; (iii) The CPC and characteristics of lncRNAs in sea buckthorn remain largely un- program was used to calculate the coding potential of the retained transcripts, and the transcripts with CPC scores >0 were dis- known. Therefore, it is necessary and urgent to identify novel lncRNAs as well as their functions in sea buckthorn fruit carded; (iv) HMMER was employed to scan the protein coding po- development. tential of each transcript against the Pfam protein family database Here, the whole transcriptome of three development stages fruit, with the transcript length >200 nucleotides, and no ORF encoding including mature green (MG), breaker (BR) and red-ripe (RR), were >100 amino acids; (v) The transcripts were compared against the analysed using multiple high-throughput sequencings and diverse NCBI non-redundant (NR) protein database, the Kyoto bioinformatic platforms. Function and expression analysis revealed Encyclopedia of Genes and Genomes (KEGG) database and the that the lncRNAs LNC1 and LNC2 can reduce and induce the Swiss-Protein database (Swiss-Prot) by BLASTX (E-value ¼ 1e-10, Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 G. Zhang et al. 467 coverage> 85%, and identity> 95%) to retain transcripts without lncRNA–miRNA–mRNA network and visualized using Cytoscape significant homology to known proteins. The raw reads generated software. have been deposited in the NCBI SRA database under accession numbers SRP124171 for the miRNA-Seq reads and the ssRNA-Seq 2.8. qRT-PCR analysis reads. The total RNA from sea buckthorn fruit (MG, BR, RR) was used for quantitative PCR analysis. The RNA was then reverse transcribed to 2.4. Differential expression of lncRNAs and mRNAs cDNA using ReverTra Ace reverse transcriptase (Toyobo). qRT-PCR of the randomly selected DE-lncRNAs was performed using a Bio- Differentially expressed lncRNAs (DE-lncRNAs) between the sam- TM Rad CFX96 Touch RealTime PCR Detection System (Bio-Rad) ples at different developmental stages were identified using Cuffdiff. with SYBR Green RealTime PCR Master Mix (ABI) according to the Any lncRNA exhibiting a log -transformed jfold changej 1 and ad- standard protocol. Specific primers for DE-lncRNAs and DE- justed P-value< 0.05 was selected as a DE-lncRNA. On the basis of miRNAs are listed in Supplementary Table S1. The 18S rRNA and the features of the lncRNAs, their localization and abundance were U6 were used as the internal control genes for DE-lncRNAs and DE- shown using Circos. miRNAs in these experiments. All reactions were conducted in tripli- –DDCT cate for both technical and biological repetitions. The 2 method 2.5. Prediction and functional analysis of DE-lncRNA was used to calculate the relative gene expression levels. target To explore the functions of lncRNAs, we first predicted their cis and 2.9. Subcellular localization of LNC1/2 trans targets. In the cis role, lncRNAs act on neighbouring target The subcellular localization of two sea buckthorn DE-lncRNAs was genes. In this study, we searched for coding genes 100 kb upstream examined using fluorescence in situ hybridization (FISH). Sea buck- and downstream of a lncRNA. The trans role refers to the influence thorn (‘hongguo’) fruits at RR stage were collected for FISH. We se- of lncRNAs on other genes at the expression level. Here, we calcu- lected three healthy fresh fruits for subcellular localization of LNC1/ lated Pearson’s correlation coefficients between the expression levels 2. The pipeline of subcellular localization was followed as previously of lncRNAs and mRNAs with custom scripts (jrj> 0.95). Then, we described, including probe synthesis, sample fixation and probe hy- performed functional enrichment analysis of the target genes of bridization. Firstly, the whole fruit are fixed with fixative: n-Heptan 34 35 lncRNAs by using goseq and KOBAS. (1:1) solution. Then, to preserve tissue morphology, samples were rinsed in 75% (v/v) ethanol, 50% (v/v) ethanol/PBS and 25% (v/v) 2.6. Bioinformatics analysis of small RNAs ethanol/PBS each for 10 min. Tissues are incubated in Hybridization After small RNA sequencing, low-quality reads, adapter- Mix at 52 C. Afterwards, samples were rinsed in Washing Solution contaminating tags and reads with lengths smaller than 18 nt were to remove non-specific and/or repetitive RNA hybridization, and discarded. All unique clean reads were considered for ncRNA analysed by microscopy. Two probe sequences are listed in (rRNA, scRNA, snoRNA, snRNA and tRNA) identification in a Supplementary Table S2. BLAST all search against the Rfam (version 10.1) database. Next, the remaining reads were compared with known miRNAs of plants 2.10. VIGS of sea buckthorn fruits deposited in miRbase. Then, the secondary structures of these According to previous study, VIGS of sea buckthorn fruit was per- miRNAs were predicted by RNAfold. The frequency of the miRNAs formed using tobacco rattle virus (TRV). To avoid off-target silenc- was normalized to transcripts per million (TPM) in each sample. 16 ing, we analysed LNC1 and LNC2 using the VIGS tool. A pTRV2- Differentially expressed miRNAs were identified by log -transformed LNC1/2 construct was generated by inserting the EcoRI-digested jfold changej> 1 and P-value< 0.05. To explore whether mRNAs PCR fragment of lncRNA into the pTRV2 vector. Agrobacterium functioned as targets of miRNAs, the transcripts and identified strain GV3101 constructs containing pTRV1, pTRV2 and pTRV2- miRNAs were submitted to psRobot with an expectation  2. LNC1/LNC2 vectors were grown at 28 C in Luria Broth medium Transcripts containing a total of no more than four mismatches and containing 10 mM MES, 20 lM acetosyringone, 50 lgml kanamy- G/U pairs within the complementary regions were considered as cin and 50 lgml rifampicin. After shaking for 12 h, agrobacterium miRNA targets. The target mimics were predicted using cells were harvested through centrifugation, resuspended in infiltra- psRNATarget combined with local scripts and the rules established tion buffer (10 mM MgCl , 200 lM acetosyringone and 5% sucrose) by Wu et al. to a final OD600 of 1.0. Resuspensions of pTRV1 and pTRV2 or pTRV2-lncRNA were mixed at a ratio of 1:1. After 3 h of incuba- tion, the agrobacterium was infiltrated into the fruits with a micro 2.7. Construction of DE-lncRNA-miRNA-mRNA network syringe. Sea buckthorn fruits infiltrated with pTRV1 and pTRV2 To infer the functions of lncRNAs, networks were constructed based were used as controls (CK). Three different plants from each sample on the complementary pairs of miRNAs-lncRNAs and miRNAs- were used for infiltration. When the VIGS phenotype was visible, the mRNAs. The DE-lncRNA–miRNA–mRNA network was recon- sea buckthorn fruits were collected and stored at –80 C. structed based on ceRNA theory. First, expression correlation between lncRNAs and mRNAs was evaluated using the Pearson cor- 2.11. qRT-PCR of lncRNAs, miRNAs and genes relation coefficient (PCC). The lncRNA-mRNA pairs were consid- ered as co-expression pairs with threshold PCC> 0.9 and P< 0.05. involved in anthocyanin biosynthesis Then, for a lncRNA-mRNA pair, both mRNA and lncRNA, which Total RNA was extracted from the CK, TRV-LNC1 and TRV- as common miRNA targets and co-expressed negatively with this LNC2 fruits using the Total RNA Kit (Aidlab, Beijing, China). TM miRNA, were selected as co-expression competing triplet. Finally, all Briefly, the first cDNA strand was obtained using the Prime Script these co-expression competing triplets were used for constructing RT Master Mix (Takara, Dalian, China). All primers used in this Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 468 lncRNAs in sea buckthorn fruit study are listed in Supplementary Table S1. qRT-PCR was per- indicated that lncRNAs might have a different expression pattern TM formed on the Bio-Rad CFX96 Touch RealTime PCR Detection from protein-coding genes. Only two and 36 DE-lncRNAs were System using a standard SYBR Green PCR Kit (Bio-Rad). The condi- expressed specifically in the MG and RR stage, respectively tions for quantitative PCR and the primers for the lncRNAs and (Fig. 1D). In contrast to MG, 38 and 72 lncRNAs were upregulated genes are listed in Supplementary Table S1. Sample cycle threshold in BR or RR, and the remaining 44 and 46 lncRNAs were (Ct) values were determined and standardized relative to the endoge- downregulated. –DDCT nous control gene 18S rRNA, and the 2 method was used to calculate the relative changes in gene expression based on the qRT- PCR data. All reactions were carried out in three biological repeti- 3.2. Functional analysis of DE-lncRNAs tions. qRT-PCR was used to validate the sequencing results of two To investigate the functions of DE-lncRNAs, we predicted the poten- miRNAs and the genes involved in anthocyanin biosynthesis. The tial cis and trans targets of the lncRNAs. For the cis action of quantitative PCR conditions and the primers for the miRNAs and lncRNAs, computational prediction identified 1,061 potential target genes are listed in Supplementary Table S1. genes for 84 DE-lncRNAs (Supplementary Table S6). Gene Ontology (GO) analysis revealed that cis lncRNA targets significantly repre- 2.12. Determination of anthocyanins by high- sented in the developmental process (Supplementary Fig. S2). performance liquid chromatography analysis Moreover, pathway analysis showed that these cis target genes of Sea buckthorn fruit from CK, TRV-LNC1 and TRV-LNC2 plants lncRNAs were enriched in plant hormone signal transduction, fatty was collected to quantify the content of anthocyanins. Each group acid and unsaturated fatty acid metabolism, carotenoid biosynthesis included three biological repetitions. The method of anthocyanin de- and flavonoid biosynthesis (Supplementary Table S7). On the other termination was described in previous research. hand, the trans functions of 67 DE-lncRNAs were examined based on their expression correlation coefficients (jPearson correlationj 0.95) with protein-coding genes. A total of 21,772 interactions were 3. Results detected in trans between 67 DE-lncRNAs and 782 protein-coding 3.1. Identification and characterization of sea buckthorn genes in the sea buckthorn genome (Supplementary Table S8). lncRNAs Functional analysis showed that the trans target genes were also By integrating lncRNA computational identification methods, we de- enriched in a variety of biological processes. Importantly, we observed veloped a de novo lncRNA prediction pipeline using the resulting a few plant growth terms, including the regulation of growth, cell wall strand-specific RNA-seq (ssRNA-seq) data sets from three develop- organization or biogenesis, and regulation of cellular component mental stages of sea buckthorn (Supplementary Table S3; organization. Of the identified 110 KEGG pathways, five were associ- Supplementary Fig. S1). After mapping the RNA-seq data to the ated with fruit development, namely, ascorbate and aldarate whole genome of sea buckthorn, we identified 9,008 lncRNAs in the metabolism, flavonoid biosynthesis and brassinosteroid biosynthesis fruits. The obtained lncRNAs were further classified into three types: (Supplementary Table S9). lincRNAs, lncNATs and intronic lncRNAs, based on their locations to the protein coding genes. Whereas the majority of all lncRNAs (6,750) was located in intergenic regions, only 13.4% and 11.7% of 3.3. DE-lncRNAs participate in miRNA-lncRNA-mRNA all lncRNAs were intronic lncRNAs and lncNATs of protein-coding genes, respectively. A circos plot clearly showed that sea buckthorn networks lncRNAs were not evenly distributed across chromosomes (Fig. 1E), Recent evidence suggested the roles of miRNAs and lncRNAs in reg- 43–45 similar to the distribution of lncRNAs observed in other spe- ulating the expression of mRNAs in many species, but the com- 18,23,42 cies. In addition, the expression level of lncRNAs varied be- prehensive patterns of miRNA and lncRNAs in sea buckthorn tween chromosomes. For instance, the average expression levels of remain unknown. We predicted 68 conserved miRNAs and 79 pre- lncRNAs generated from chromosomes 4 and 7 were 67.69 and miRNAs, which were identified as belonging to 31 known miRNA 2.1415, respectively. families deposited in miRBase. A total of 32 DE-miRNAs were pre- We compared the lengths of transcript and ORF and exon counts dicted to target 67 genes in this study (Supplementary Table S10). of the 9,008 putative lncRNAs with identified mRNAs. The length The target prediction analysis revealed that 22 lncRNAs may act as distribution of the identified lncRNAs (median 271 bp; average eTMs of 25 DE-miRNAs (Supplementary Table S11). Recently, 463.28 bp) was shorter than that of the mRNAs (median 714 bp; av- some researches have shown that lncRNAs can affect the regulation erage 976.08 bp), while the length distribution showed no significant 21,46 of miRNAs as eTMs in plants. To investigate the function of differences among intergenic lncRNAs, intronic lncRNAs and lncRNAs as miRNA targets, a comprehensive genome-wide network lncNATs (Fig. 1A). The exon counts of the putative lncRNAs (aver- mediated by miRNAs was constructed. The network consisted of age 1.56 exons per transcript) were also lower than those of the 109 nodes and 201 edges, including 13 DE-miRNAs, 10 DE- mRNAs (average 4.19 exons per transcript), while the exon counts lncRNAs and 87 DE-mRNAs (Fig. 2A). The majority of the nodes in of the three kinds of lncRNA showed no differences (Fig. 1B). We the network belonged to the miR396 family, which regulates growth found the ORF lengths of the majority of lncRNAs were shorter than by targeting growth regulation factors. Then, the function of each mRNAs (Fig. 1C). lncRNA was inferred based on the functions of the connected A total of 118 known lncRNAs and 108 novel transcripts were mRNAs. We found that lncRNAs mainly participate in cellular, met- differentially expressed in the three samples (Supplementary abolic and some biological processes, especially highly enriched in Table S4). We also found fewer DE-lncRNAs (2.46%) than cellular component, binding including ‘ion binding’, ‘nucleotide DE-mRNAs (5.64%, total mRNAs 45,546) in fruits at different de- velopment stages (Supplementary Table S5). This distinction binding’ and ‘small molecule binding’ (Supplementary Table S12). Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 G. Zhang et al. 469 Figure 1. Characteristics of sea buckthorn lncRNAs. (A) Length distributions of lncRNAs and transcripts. (B) Number of exons per lncRNA and transcript. (C) Distribution of the number of open reading frame (ORFs) in lncRNAs and transcripts. (D) A Venn diagram showing DE-lncRNAs that are commonly expressed in the mature green (MG), breaker (BR) and red-ripe (RR) stages as well as those specifically expressed in one but not another. (E) The expression levels of lncRNAs (log FPKM) along the twelve sea buckthorn chromosomes (generated using Circos). Each of the three concentric rings corresponds to different sam- ples. In the second, third and fourth track (outer to inner), each vertical line reports the expression of lncRNAs in sea buckthorn fruit in the MG, BR andRR stages, respectively. 3.4. Analysis of lncRNAs related to the anthocyanin To validate the putative relationships between the miRNAs and biosynthesis of fruits lncRNAs, their expression levels were examined by qRT-PCR According to previous studies, the module miR156-SPL affects an- (Fig. 3). The qRT-PCR results were consistent with our RNA-seq thocyanin biosynthesis in plants. Additionally, R2R3-MYBs data regarding lncRNA and miRNA expression, suggesting that (MYB114) are known to combine with bHLHs and WDR to form a these two lncRNAs identified by next-generation sequencing might ternary complex that activates late biosynthesis genes and transfers have negative regulation relation with miR156a and miR828a. protein genes. Here, based on the miRNA-lncRNA-mRNA net- Distribution analysis is an important step towards functional re- work, we identified two anthocyanin biosynthesis-related lncRNAs search on lncRNAs as eTMs. To demonstrate the subcellular locali- (TCONS_00694050 and TCONS_00438839, termed LNC1 and zation of LNC1 and LNC2, FISH was performed in fruits using a LNC2) for further analysis (Fig. 2). On the basis of the network, we fluorescence probe specific to LNC1 and LNC2. In combination with found that LNC1 and LNC2 may regulate the expression of SPL9 the DAPI signal, the probe showed that these two lncRNAs were and MYB114 by acting as eTMs of miR156a and miR828a. mainly enriched in cytoplasm (Fig. 4A). Genome chromosomal Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 470 lncRNAs in sea buckthorn fruit Figure 2. Analysis of lncRNA-miRNA-mRNA networks. (A) The networks of lncRNA-miRNA-mRNA. The triangle represents lncRNAs, the circle with label repre- sents miRNAs, and the circle without label represents mRNAs. (B) LNC1/2 as endogenous target mimics (eTMs) of miRNAs. Secondary structure of miRNAs pre- cursors and the basepairing relationship between lncRNA and miRNAs. The predicted secondary structure was generated using RNAfold. (C) Sub interaction networks of LNC1/2 from lncRNA-miRNA-mRNA networks. The ellipse represents lncRNAs, the triangle represents miRNAs, and the rectangle represents mRNAs. location analyses revealed that both LNC1 and LNC2 reside in chro- in sea buckthorn fruits. Compared with that in TRV control fruits, mosome 1 and lack homologs compared with other plants (Fig. 4B). the transcript level of LNC1 in TRV-LNC1 fruits was dramatically decreased by 96% (Fig. 5C). Similarly, the level of LNC2 in TRV- LNC2 fruits decreased by 63.3% compared with that in TRV con- 3.5. LNC1 and LNC2 regulate anthocyanin biosynthesis trol fruits (Fig. 5C). Consistently, the anthocyanin content of TRV- in fruits LNC1 fruit increased by 25.23% with respect to TRV control To test the regulation role of LNC1 and LNC2 in anthocyanin bio- fruits, and the anthocyanin content of TRV-LNC2 fruit decreased synthesis, VIGS was performed to silence LNC1 and LNC2 in sea by 39.12% with respect to TRV control fruits (Fig. 5C). Previous buckthorn fruits (Fig. 5A). Intriguingly, two or three weeks after in- studies have reported that SPL and MYB transcription factors can filtration, anthocyanin contents of sea buckthorn fruits injected affect anthocyanin content by regulating the expression of genes in- with TRV-LNC1 or TRV-LNC2 showed significant difference com- volved into anthocyanin biosynthesis pathway, such as ANS, DFR, pared with that of control fruits injected with TRV (Fig. 5B). Semi- F3 H, LDOX, UGT78D2 and UGT75C1. Similarly, three genes quantitative PCR analysis suggested that a recombinant virus could involved in anthocyanin biosynthesis were increased or decreased spread from carpogonia to fruits, resulting in the VIGS of lncRNAs in TRV-LNC1 compared with their levels in TRV control sea Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 G. Zhang et al. 471 Figure 3. qRT-PCR validation of putative lncRNAs and miRNAs in sea buckthorn. The expression levels of lncRNAs (A) and miRNAs (B) in three developmental stages: mature green (MG), breaker (BR) and red-ripe (RR). 4. Discussion buckthorn fruits (Fig. 5C). In contrast, DFR, F3 H and LDOX were decreased in TRV-LNC2 compared with their levels in TRV control 4.1. A reliable list of lncRNAs from sea buckthorn fruits sea buckthorn fruits (Fig. 5C). These results strongly suggested the Recently, plenty of studies have revealed the crucial roles of 17,45,49 essential role of the two novel lncRNAs in the regulation of the an- lncRNAs in various biological processes in plants. Although thocyanin accumulation of sea buckthorn fruits. many lncRNAs have been identified in model plants, such as Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 472 lncRNAs in sea buckthorn fruit Figure 4. Subcellular localization and chromosome location of LNC1 and LNC2 in sea buckthorn fruit. (A) Visualization of LNC1 and LCN2 in sea buckthorn fruit by RNA fluorescence in situ hybridization using an antisense probe. Scale bar, 20 lm. (B) Location regions of LNC1 and LNC2 on chromosome. 50 51 25 Arabidopsis, rice, and maize, little research has been performed the most important reason for the pigmentation of red fruit in sea on sea buckthorn. In this study, a total of 9008 lncRNA loci were buckthorn. Therefore, these results revealed that lncRNAs may play identified in the fruit of sea buckthorn, an ideal material for the roles in the development of sea buckthorn fruit pigmentation. study of fruit development. Furthermore, structural analysis Sea buckthorn fruit contains abundant ASA and is known as the showed that the mean length of lncRNAs was 1.1-fold shorter than king of vitamin C, and ASA is a prominent product of the ascorbate that of coding transcripts, and the lncRNA expression level was and aldarate metabolism pathway. In this pathway, we found seven lower than that of coding transcripts, in agreement with previous lncRNAs targeting GDP-mannose-epimerase (GME) and SKS12, 16,42,52 studies. LncRNAs also have specific expression patterns in which encodes L-ascorbate oxidase. Several studies have revealed tissue types and subcellular compartments. In our study, among the that GME and SKS12 are associated with the accumulation of ASA significant DE-lncRNAs, we found five expression patterns in MG, in the fruits of many plants, including blueberry, Arabidopsis, to- 58–61 BR and RR. mato and peach. For instance, the importance of GME in the major ascorbate biosynthesis pathway in tomato was confirmed us- ing gene-silencing technology. Thus, our results provide a resource 4.2. DE-lncRNAs contribute to fruit development of sea for further analysis of the regulation of ASA biosynthesis in sea buck- buckthorn fruit thorn fruit. Transcriptomic sequencing of different varieties of sea buckthorn 28,53 Flavonoids are a large group of natural products that are widely revealed a lot of ncRNAs in many biological processes. In this present in the leaves, flowers, fruits and seeds of various plants. study, we predicted potential cis and trans target genes based on According to their basic chemical structures, flavonoids can be fur- physical location and expression relationships with mRNA, respec- ther divided into different subgroups, including flavonols, anthocya- tively. Although the functions of the majority of lncRNAs are un- nins and proanthocyanidins. Our results consistent with those of known, previous studies have suggested that lncRNAs may play previous studies, reveal that sea buckthorn fruit contains various fla- different roles in a variety of biological processes. According to pre- vonoids. In the flavonoid biosynthesis pathway, chalcone synthase vious study, we analysed the functions of DE-lncRNAs based on (CHS) is the first committed enzyme that facilitates the stepwise syn- their target mRNAs, which involved in many processes, including ca- thesis of chalcone, and it is also pivotal for the biosynthesis of flavo- rotenoid, ASA, flavonoid, sugar and hormone pathways. noid and anthocyanin pigments in plants. TCONS_00085219 was We identified 10 DE-lncRNAs involved in carotenoid biosynthesis predicted to target CHS, indicating a possible regulatory relationship pathways, including b-carotenoid and lycopene biosynthesis. Among between lncRNA and CHS (Fig. 6). Another important enzyme in- these DE-lncRNAs, one lncRNA (TCONS_00082246) targeted the volved in this pathway is flavanone 3-hydroxylase (F3H), which was phytoene synthase (PSY) gene, which is involved in the first commit- reported in rice. TCONS_01039552 was predicted to target F3H, ted step in carotenoid biosynthesis (Supplementary Table S12). This implying a potential role in flavonol and anthocyanin biosynthesis reaction has been reported to be the rate-limiting step controlling the through the regulation of F3H. In addition to CHS and F3H, other metabolic flux to carotenoid biosynthesis in many plants. For ex- important genes, such as the gene encoding flavonol synthase 1, were ample, the PSY gene plays an important role in carotenoid biosynthe- sis in tomato. Therefore, this study opened the door to predicted as target genes of two lncRNAs (TCONS_00061167 and TCONS_00061354) identified in our study. All these results implied understanding the role of lncRNAs in the regulation of genes in- volved in carotenoid biosynthesis in sea buckthorn fruit. Recently, the widespread involvement of lncRNAs in the regulation of many studies have shown that carotenoids are considered as the flavonoid biosynthesis and fruit pigment in the development of sea main fruit pigments in tomato, bilberry, citrus, papaya, watermelon buckthorn fruit, which will provide novel insight into the regulation 55–57 and sea buckthorn. In addition, lycopene accumulation is of flavonoid biosynthesis in other plants. Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 G. Zhang et al. 473 Figure 5. Silencing of LNC1/2 in sea buckthorn fruit. (A) Sea buckthorn fruits (3 weeks after infiltration). CK: sea buckthorn fruits infiltrated with pTRV1 and pTRV2; TRV-LNC1: sea buckthorn infiltrated with pTRV1 and pTRV2-LNC1; TRV-LNC2: sea buckthorn infiltrated with pTRV1 and pTRV2-LNC2. (B) High-perfor- mance liquid chromatography analysis of total anthocyanin content in sea buckthorn fruits. (C) qRT -PCR analysis of the expression of related genes in sea buckthorn fruits. The expression of 18S rRNA was used as an internal reference. The relative level was normalized to that in TRV control plants. Error bars indi- cate SD of three biological replicates, each sample measured in three replicates. Asterisks indicate a significant difference as determined by Student’s t-test (*P< 0.01). Downloaded from https://academic.oup.com/dnaresearch/article-abstract/25/5/465/5033008 by guest on 17 October 2018 474 lncRNAs in sea buckthorn fruit Figure 6. Model of lncRNA regulation in sea buckthorn fruit. This pathway begins with the general phenylpropanoid metabolism, and subsequent steps are cat- alyzed by a series of structural enzymes, leading to the biosynthesis of flavonols and anthocyanins. The expression of the late biosynthetic genes (LBGs) requires the transcriptional activation activity of the R2R3-MYB/bHLH/WDR (MBW) complex. LncRNAs target FLS and CHS: functions in trans regulation. 0 0 0 LncRNAs target F3H and F3 H: functions in cis regulation. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3 H, flavanone 3 - hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase. regulated through the LNC1-induced downregulation of SPL9, 4.3. LNC1 and LNC2 regulate anthocyanin biosynthesis which affects the stability of the MYB, bHLH and WDR (MBW) in the fruit development of sea buckthorn by eTMs complex, thus promoting anthocyanin biosynthesis in sea buckhorn Previous studies suggest that lncRNAs function via interacting with fruit. In contrast, the LNC2-induced downregulation of MYB114 re- miRNAs. The reverse miRNA-lncRNA regulation, first discovered duced anthocyanin biosynthesis in sea buckhorn fruit. Thus, this in Arabidopsis, was termed target mimicry. Previous research sys- study illuminated the complex regulation of fruit anthocyanin bio- tematically identified 36 eTMs for 11 Arabidopsis miRNAs and 189 21,51 synthesis, which might instigate more comprehensive studies on sea target mimics for 19 rice miRNAs. In Populus, seven miRNAs target TCONS_00013609, including pto-miR6462a/b/c. buckthorn lncRNAs. Furthermore, the functional motifs and target Therefore, the prediction and analysis of miRNAs that interact with genes of lncRNAs in trees need to be investigated further to fully elu- lncRNAs provide a useful way to explore the functions of the corre- cidate the regulatory mechanisms of lncRNAs in trees. sponding lncRNAs. In this study, we identified 19 DE-lncRNA eTMs for 23 DE-miRNAs (seven families). These results first showed that miRNAs target lncRNAs in sea buckthorn. Furthermore, our investi- Conflict of interest gation constructed a comprehensive RNA-mediated network, includ- None declared. ing DE-miRNA–DE-lncRNA and DE-miRNA–DE-mRNA interactions. However, a mechanistic understanding of the roles of lncRNAs in Funding plants, especially trees, has remained extremely lacking. This study was supported by grants from Special Fund for Forest Scientific lncRNA1459 and lncRNA1840 were found to regulate the ripening Research in the Public Welfare (201504103) and the National Natural Science of tomato. For mammal, lnc-mg was identified to promote myo- Foundation of China (31470616). genesis by functioning as a ceRNA/eTM for microRNA-125b to con- trol the protein abundance of insulin-like growth factor 2. In Arabidopsis, a representation of the classical TM model, miR399: Supplementary data IPS1, was discovered by Franco-Zorrilla. 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Journal

DNA ResearchOxford University Press

Published: Oct 1, 2018

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