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High throughput sequencing of small RNAs reveals dynamic microRNAs expression of lipid metabolism during… Feng, Jin-Ling; Yang, Zhi-Jian; Chen, Shi-Pin; El-Kassaby, Yousry A; Chen, Hui Jul 20, 2017

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RESEARCH ARTICLEHigh throughput sequencACl-KBackgroundC. oleifera and C. meiocarpa belong to the camellia family,Camellia oil aids in cholesterol reduction, resistance tostress, oxidation reduction, reduced inflammation, andfundamentaltity [6–8].ry moleculeswide range ofon of specificaries demon-lism in plantsn [12]; wheatFeng et al. BMC Genomics  (2017) 18:546 DOI 10.1186/s12864-017-3923-zexpressed with different seed oil content and composition.1College of Forestry, Fujian Agriculture and Forestry University, Fuzhou350002, China[13]; Jatropha [14]; Arabidopsis [15]), animals [16, 17],and insects [18]). Different oil crops were differentially2Department of Forest and Conservation Sciences, Faculty of Forestry,University of British Columbia, Forest Sciences Centre, 2424 Main Mall,Vancouver, BC V6T 1Z4, Canadathat is dubbed as the Eastern olive oil [1, 2]. Some of thecamellia species are ancient oilseed plants with history ofcultivation for over two thousand years [3]. Camellia oil isknown for its edible and medicinal uses with an oleic acidcontent reaching above 80%, a high content of monoun-saturated lipid, and the lowest level of saturated fats [4].of camellia seed prior to oil extraction is afactor affecting its fatty acid quality and quanMicroRNAs (miRNAs) are small regulatothat have been shown to be involved in abiological pathways by modulating expressimRNAs [9]. Sequencing small RNA librstrated the role of miRNAs in lipid metabo(safflower [10]; Camelina sativa [11]; soybea* Correspondence: y.el-kassaby@ubc.ca; zjchchenh@163.comTheaceae, and are known for their high quality oilseed improved human immunity [5]. The drying managementAbstractBackground: Camellia species are ancient oilseed plants with a history of cultivation over two thousand years.Prior to oil extraction, natural seed drying is often practiced, a process affecting fatty acid quality and quantity.MicroRNAs (miRNA) of lipid metabolism associated with camellia seed natural drying are unexplored. To obtaininsight into the function of miRNAs in lipid metabolism during natural drying, Illumina sequencing of C. oleiferaand C. meiocarpa small-RNA was conducted.Results: A total of 274 candidate miRNAs were identified and 3733 target unigenes were annotated by performing aBLASTX. Through integrated GO and KEGG function annotation, 23 miRNA regulating 131 target genes were identifiedas lipid metabolism, regulating fatty acid biosynthesis, accumulation and catabolism. We observed one, two, and fourmiRNAs of lipid metabolism which were specially expressed in C. Meiocarpa, C. oleifera, and the two speciescollectively, respectively. At 30% moisture contents, C. meiocarpa and C. oleifer produced nine and eight significantdifferentially expressed miRNAs, respectively, with high fatty acid synthesis and accumulation activities. Across the twospecies, 12 significant differentially expressed miRNAs were identified at the 50% moisture content.Conclusions: Sequencing of small-RNA revealed the presence of 23 miRNAs regulating lipid metabolism in camelliaseed during natural drying and permitted comparative miRNA profiles between C. Meiocarpa and C. oleifera. Furthermore,this study successfully identified the best drying environment at which the quantity and quality of lipid in camellia seedare at its maximum.Keywords: miRNAs, Lipid metabolism, Natural drying, Camellia meiocarpa, Camellia oleiferareveals dynamic microRNlipid metabolism duringand C. meiocarpa seed naJin-Ling Feng1, Zhi-Jian Yang1, Shi-Pin Chen1, Yousry A. E© The Author(s). 2017 Open Access This articInternational License (http://creativecommonsreproduction in any medium, provided you gthe Creative Commons license, and indicate if(http://creativecommons.org/publicdomain/zeOpen Accessing of small RNAss expression ofamellia oleiferatural dryingassaby2* and Hui Chen1*le is distributed under the terms of the Creative Commons Attribution 4.0.org/licenses/by/4.0/), which permits unrestricted use, distribution, andive appropriate credit to the original author(s) and the source, provide a link tochanges were made. The Creative Commons Public Domain Dedication waiverro/1.0/) applies to the data made available in this article, unless otherwise stated.and miR166a, miR2910, miR824, miR414, and miR5206Feng et al. BMC Genomics  (2017) 18:546 Page 2 of 14were identified to encode fatty acid oxidase [11, 13, 14, 21],which regulate fatty acid catabolism. As above, the samefatty acid protein can be regulated by different miRNA, andthe same miRNA can encode different fatty acid protein. InC. Meiocarpa and C. oleifera, the presence of differentmiRNAs expression pattern at different environmentsand, in particular, in dry seed suggest that miRNAs mayplay critical roles in lipid metabolism during naturalseed drying [22–27].In order to explore the potential role of miRNAs inlipid metabolism during C. Meiocarpa and C. oleiferanatural drying, miRNA expression profiles of seed sam-ples at different moisture content (10, 20, 30, 40, and50%) were investigated using high throughput nextgeneration small RNA sequencing technology, so thedifferentially expressed miRNAs are unraveled to helpunderstand their involvement in lipid metabolism.MethodsPlant materialIn 2012, mature fruits of C. meiocarpa and C. oleiferawere collected from the four cardinal directions of 3superior trees’ crowns per species. The trees are growingat the Minhou Tongkou State Forest Farm (26°05′ N,119°17′ E), Fujian Province, China. The collected fruitswere placed in a ventilated room until they naturallycracked and seeds were extracted by manual shell cut-ting. The seed moisture content at the time of extractionwas closed to 50%. Seeds were naturally dried and theirmoisture content was determined daily. Over time, seedsamples were collected at moisture content of 50, 40, 30,20, and 10% and were sequentially identified as S01 toS05 and S06 and S10 for C. meiocarpa and C. oleifera,respectively, and sampled three times, then were flashThere were 28 miRNAs regulated lipid metabolism fromsoybean [12], 30 miRNAs from Camelina sativa [11], and13 miRNAs differentially expressed from two safflowergenotypes that have difference to regulate oleic acid accu-mulation [10]. So there may be different miRNA expres-sion between C. Meiocarpa and C. oleifera.Recent research uncovered miRNAs regulation of lipidmetabolism, which related to diverse pathways, such asfatty acid synthesis, accumulation, and catabolism. Forexample, miR159b, miR5026, and miR2911 were identi-fied to encode Δ12-desaturase (FAD2) [10, 15, 19, 20],miR408a and miR159b for Fatty acid elongase (FAE1)[11, 15, 44], which related to fatty acid synthesis. Add-itionally, miR319a, miR001 and miR007 were identifiedto encode 1-acylglycerol-3-phosphocholine acyltransferase(LPCAT) [11, 14], which involved in fatty acid accumulationfrozen in liquid nitrogen and stored at −80 °C until RNAextraction.Oil content analysisTo obtain the camellia seed oil content, the Soxhletmethod was used by a fatty acid instrument (SZF-06A,Shanghai Hongji Instrument Equipment company) [28].Camellia seeds at different moisture content were cutinto thin slices, dried by silica gel and liquid nitrogen,milled into a powder by a pulverizer (FW100, TianjinTaisite Instrument company). About 2 g of powder wereweighed out (M1), packed in a folded filter paper bagand bound with a skim cotton thread, placed into theSoxhlet extraction thimble with 15 mL of petroleumether for 1 h. Soxhlet extraction bottle were weight (M2)into which extraction thimble was placed, extracted for5 h using 70 ml petroleum ether (75 °C). After extrac-tion, the solvent was evaporated in Soxhlet extractionbottle and dried at 75 °C drying oven. Then Soxhletextraction bottle was weighed again (M3).Seed oil content ¼ M3−M2ð Þ=M1 100%All experiments were carried out at least in triplicateand data were analyzed using the SPSS statistics 17.0software.RNA isolationTotal RNA was extracted from the seed samples of thetwo camellia species using RNA kit (RNA simply totalRNA kit, Tiangen, Beijing, China) according to the man-ufacturer’s instructions. The purity and quality of theRNA were determined by assessing the absorbance ratioOD260/280 using NanoDrop ND1000 Spectrophotometer(NanoDrop, Wilmington, DE). The RNA was quantifiedwith a Qubit 2.0 Fluoremeter (Invitrogen Corporation,Carlsbad, CA, USA) in accordance with the manufac-turer’s instructions. The integrity of the RNA samples wasverified using an Agilent 2100 Bioanalyzer (Agilent, SantaClara, CA, USA).Small RNA library construction and sequencingEqual amount of total RNA from the three samples at eachmoisture content of the two camellia species, was mixed toconstruct a transcriptome library using an Illumina TruSeqRNA Sample PrepKit following the manufacturer’s instruc-tions. Small RNAs of 18–30 nt in length were separatedand purified by denatureing polyacrylamide gel electro-phoresis. After dephosphorylation and ligation of a pair ofSolexa adaptors to their 5′ and 3′ ends, the products werereverse-transcribed and amplified by RT-PCR and gel puri-fication. After the library was constructed, the Qubit 2.0Fluorometer (Invitrogen Corporation, Carlsbad, CA, USA)were used to calculate the molar concentration and confirmthe insert size. The cDNA libraries were sequenced usingthe Illumina HiSeq2500 Genome analyzer (Illumina Inc.,San Diego, CA, USA).NAs and used for further analysis to identify known anddatabases based on an Evalue of less than 10−5 [45]. GeneFeng et al. BMC Genomics  (2017) 18:546 Page 3 of 14novel miRNAs [33, 34]. Known miRNAs were annotatedby identifying their homologous imRNAs in miRBase data-base (http://www.mirbase.org/) using the following criteria:1) seed region, nucleotides 2–7 must be identical; and 2)the remainder of the sequence alignment must contain nomore than two mismatches [35, 36]. The putative miRNAsproduced by miRDeep2 analysis were regarded as conserva-tive miRNAs when it met the above criteria. Those miR-NAs produced by miRDeep2 analysis that did not meet theabove criteria were considered as novel miRNAs. In orderto predict novel miRNAs with high confidence, only thosewith a miRDeep-P score higher than ≥0 were retained astrue novel miRNAs [34, 37].Screening of differentially expressed miRNAsDifferentially expressed miRNAs were identified usingthe TPM [38] and IDEG6 software [39]. TPM (TagsPer Million reads) is a standardized method for calcu-lating miRNA expression levels. TPM values were cal-culated using the following equation: TPM = numberof mapped miRNA reads/number of clean samplereads × 106. In order to calculate the levels of differen-tial expressed miRNAs, normally the value was set as0.01 by default when the sequencing read is 0 (noreads) [40]. We calibrated miRNA expression levelsusing multiple hypothesis tests with a false discoveryrate (FDR) ≤0.01, performed generalized chi-squaretests for differential miRNA expression using theIDEG6 software (http://telethon.bio.unipd.it/bioinfo/IDEG6/), and screened the miRNAs for those with P-values less than 0.01 or TPM ratios between samplesthat were greater than 1 (fold change ≥1) or FDR ≤0.01.miRNA-Seq data analysisAfter sequencing, the raw reads (FASTQ files) were proc-essed into clean reads, then the adaptor sequences, poy(A)tails and the inserted tag were removed, followed by filter-ing the low-quantity reads (ambiguous bases ‘N’ ≥ 10%and more than 20% with Quality Score < 30 bases), theclean 18–30 nt sRNAs were mapped to GenBank (http://www.ncbi.nlm.nih.gov/), Rfam (version 10.1) database(http://rfam.sanger.ac.uk), tRNAdb [29], SILVA rRNA [30]and Repbase (http://www.girinst.org/), and rRNA, tRNA,snRNA, and snoRNA were discarded from the sRNAreads using bowtie2 software with perfect matches (0 mis-matches) used for further analysis [31].The unannotated sequences were then analyzed bymiRDeep-P software package to predict miRNAs, whichwas developed by modifying miRDeep2 [32]. All maturesequences, star sequences and precursor sequences coredby miRDeep2 were retained and regarded as putative miR-The miRNAs that met these criteria were identified asbeing differentially expressed [41].ontology (GO) and Kyoto encyclopedia of genes andgenomes (KEGG) enrichment analysis were performedwith package GO stats (http://www.geneontology.org/)of P value <0.05 was set as the cut-off to select out sig-nificantly enriched terms [46].Quantitative real-time PCR analysis of miRNAsqRT-PCR was used to validate the miRNAs identifiedusing deep sequencing and to analyze their expressionpatterns. Total RNA of C. meiocarpa and C. oleifera seedssamples, was extracted using TRUzol Universal Reagentaccording to the manufacturer’s protocol. They were thenreverse-transcribed into cDNA using the microRNAcDNA First Strand cDNA Synthesis Kit (CWBIO, Beijing,China) according to the manufacturer’s instructions. ThecDNA was quantified by microRNA Real-Time PCRAssay Kit (CWBIO, Beijing, China) using a 20 μL reactionmixture, which consisted of 1 μL of diluted cDNA,0.25 μM forward and reverse primer, and 10 μL of2 × SYBR Green PCR Master Mix (CWBIO, Beijing,China). All reactions were performed under the followingconditions: 95 °C for 5 min, 40 cycles of 95 °C for 15 S,62 °C for 45 S. Melting curve analysis was performed toverify specific amplification (from 72 to cycles at 60 °C for15 S). Each sample was processed in triplicate, and 5.8SrRNA was used as an internal control [47, 48]. The equa-tion ratio 2 -ΔΔCT was applied to calculate the relativeexpression level of miRNAs. The qRT-PCR primers aremiRNA target predictionThe putative target sites of miRNAs were identified byaligning mature miRNA sequences with a draft genomesequence using TargetFinder, (http://carringtonlab.org/re-sources/targetfinder). miRNA targets were computationallypredicted essentially as described [38, 42, 43]. Briefly,potential targets from FASTA searches were scored usinga position-dependent, mispair penalty system. Penaltieswere assessed for mismatches, bulges, and gaps (+1 perposition) and G:U pairs (+0.5 per position). Penaltieswere doubled if the mismatch, bulge, gap, or G:U pairoccurred at positions 2 to 13 relative to the 59 end ofthe miRNAs. Only one single-nucleotide bulge or single-nucleotide gap was allowed, and targets with penalty scoresof four or less were considered to be putative miRNA tar-gets [42, 44].Functional annotation of predicted target genes wasassigned using Nr (non-redundant protein database, NCBI),Nt (non-redundant nucleotide database, NCBI), Swiss-Prot,GO (gene ontology, http://www.geneontology.org/) andCOG (clusters of orthologous groups) databases. BLASTXwas employed to identify related sequences in the proteinlisted in file (Additional file 1: Table S1) and Ct value of5.8S (Additional file 1: Table S2).ResultsOil content of two camellia species during natural seeddryingCamellia oil content were determined by Soxhlet extrac-tion method during natural seed drying (Table 1). Thetwo species showed increased in oil content at 50 to 30%moisture contents followed by a slow decline at 30 to10% moisture content, with 30% moisture content pro-Feng et al. BMC Genomics  (2017) 18:546 Page 4 of 14ducing the highest oil content. Oil content accumulationwas largest when the seed moisture content droppedfrom 50 to 40% and 40 to 30% for C. meiocarpa and C.oleifera, respectively. At 30% moisture content, C. meio-carpa and C. oleifera showed increase in their seed oilcontent of 8.80 and 10.23%, respectively, indicating thatthe effect of appropriate seed natural drying on oil accumu-lation can be great. While the percentages of oil content in-crease seem different between the two species, the relativeincrease amounted to 4.40%, indicating that natural dryingcan promote oil accumulation in both camellia specie.Sequence analysis of small RNAsTo obtain a comprehensive profile of the sRNAs involvedin natural drying, camellia seed from both species weresubjected to Solexa high-throughput sequencing, with 5libraries for each species corresponding to the sampledmoisture contents. Average total of 30,641,435 and31,012,304 reads were generated from C. meiocarpaand C. oleifera, respectively (Table 2). After filtering the lowquality reads, such as 3′ insert null, poly(A), length < 18 ntor length > 30 nt, and other artifacts, the majority ofthe small RNAs were 21 to 24 nt in length. A total of24,070,601 and 21,653,584 clean reads of 18–30 nt wereobtained for C. meiocarpa and C. oleifera, respectively(Table 2). GC content of clean reads were more than47.75 and 48.99 and the Q30 (meaning 1 error in 1000reads) of clean reads were more than 85.29 and 85.25%for C. meiocarpa and C. oleifera, respectively (Table 2).Through quality control, each sample of clean data weregreater than 19.40 M, indicating the high sRNA quality(Tables 2, 3 and 4).After searching against GenBank, Rfam, tRNAdb, Silva,and Repbase database for small RNA sequences by bowtie2software, rRNA (3,224,963), snRNA (4741), snoRNA (151),Table 1 Seed oil content during camellia seed natural dryingMoisture content(%)Seed oil content (%)C. meiocarpa C. oleifera50 33.41 ± 0.001d 37.66 ± 0.117e40 39.91 ± 0.002b 38.72 ± 0.116d30 42.21 ± 0.001a 47.89 ± 0.001a20 40.35 ± 0.002b 44.25 ± 0.001b10 37.83 ± 0.001c 42.09 ± 0.001cdifferent letters indicate significant at P < 0.05tRNA (364,615), Repbase-associated sRNAs (12,366) wereannotated and removed, and other unannotated RNAs(20,463,766) were obtained for C .meiocarpa on average(Table 3). The same process was conducted for C. oleiferaand rRNA (4,402,729), snRNA (5316), snoRNA (572), tRNA(557,421), Repbase-associated sRNAs (13,857) were an-notated and removed, and other unannotated RNAs(16,673,689) were obtained for C. oleifera on average(Table 4). The unannotated RNAs were subjected tofurther analyses for miRNA identification.The majority of small RNAs were 21 to 24 nt inlength, producing similar length distributions in bothspecies (Fig. 1). The 24 nt small RNAs were the mostabundant representing 35.07 and 27.85% of smallRNAs in C. meiocarpa and C. oleifera, respectively, thesecond was 21 nt representing 17.72 and 18.85%, thirdwas 22 nt representing 16.35 and 17.97% (Additionalfile 1: Tables S3 and S4). The 40% moisture level (sampleS02) for C. meiocarpa, produced the highest 24-nt RNApeak while 10% moisture level (sample S05) produced thehighest 21-nt and 22-nt RNA peaks among the studiedmoisture content levels (Additional file 1: Table S3),conversely, 50% moisture (sample S06) produced thehighest 24-nt, 21-nt, and 22-nt RNA peaks in C. oleifera(Additional file 1: Table S4).Identification of miRNAs during natural dryingWe used the software miRDeep2 to map the retainedsequence reads to identify candidate miRNAs. Acrossthe five moisture content levels, C. meiocarpa producedsuccessful 2,355,539 reads (11.51%) that were mapped tothe plant genomes, of which the 10 and 50% moisture con-tent levels (S05 and S01) produced the most (2,724,598)and least (1,902,986) mapped reads, respectively. Similarly,on average, C. oleifera produced 2,396,805 (14.37%) suc-cessful mapped reads, of which 50 and 20% moisture con-tent levels (S06 and S09) produced most (2,803,519) andleast (2,121,984) mapped reads. In total, 2,288,508 (12.80%)mapped reads were successfully annotated (Table 5).A total of 274 candidate miRNAs, 248 and 246 uniquesequences were assigned to C. meiocarpa and C. oleifera,respectively (Additional file 1: Table S5). Out of theidentified candidate miRNA sequences, 110 were identi-fied to 64 families, 57 families belonging to each of C.meiocarpa (99 out of 248 candidate miRNA) and C.oleifera (98 out of 246 candidate miRNA) (Additionalfile 1: Tables S5, S6 and S7). The diversity of miRNAfamilies could be determined from their members’ number.As shown, MIR482 families were the largest with 10 mem-bers, followed by MIR159 and MIR535 with 5 members,and MIR160, MIR169_2 and MIR5272 with 4 members(Additional file 1: Table S6). Most of the conserved miRNAfamilies had one member (65.63%) (Additional file 1: TableS6). The miRNAs sequences ranged in length from 18 toTable 2 Statistics relating to C. meiocarpa and C. oleifera sRNA sequences produced by Solexa sequencingSpecies Samples Raw reads <18 nt reads >30 nt reads Clean reads GC(%) Q20(%) CycleQ20(%) Q30(%)C. meiocarpa S01 29,906,668 1,658,148 7,025,688 20,417,236 49.32 93.1 100 86.98S02 32,844,767 1,179,743 3,844,931 27,795,553 47.75 91.9 100 86.17S03 28,888,019 1,676,660 3,899,605 23,290,661 48.54 91.42 100 85.47S04 28,599,822 1,902,807 3,697,488 2,2978,269 48.32 91.22 100 85.29S05 32,967,901 2,333,282 4,738,442 25,871,287 48.67 91.24 100 85.36Average 30,641,435 1,750,128 4,641,231 24,070,601 48.52 91.78 100 85.86467071Feng et al. BMC Genomics  (2017) 18:546 Page 5 of 1425 nt, with a peak of 24 nt (Additional file 1: Figure S1).The miRNA first nucleotide preference distributions areshow in Fig. 2a. miRNAs of 24 nt tended to start with 5′-A while the 21 nt tended with 5′-U (Fig. 2a). Tall miRNAstended to start with 5′-U and not 5′-G (Fig. 2b). Duringthe seed natural drying process, the number of miRNAsacross moisture content levels showed a decreasing trendwhich was followed by increase with a peak at 40% mois-ture content (S02) for C. meiocarpa (Table 6). C. oleifera,on the other hand, showed a trend of reduction, followedby increase, followed by reduction in the number of miR-NAs across the studied moisture content levels with apronounced peak at 50% moisture content (S06) (Table 6).Next we conducted sequence homology search of thesecandidate miRNAs against known mature miRNAs in miR-Base. Any miRNA that met the sequence homology criteriaof Yang et al. [35] and Jain et al. [36] was considered a con-served miRNA gene. Through this analysis, we identified aC. oleifera S06 34,169,238 4,386,045 4,606,66S07 31,865,014 1,751,974 9,877,43S08 30,501,297 2,833,009 4,644,10S09 31,087,840 3,519,595 5,720,36S10 27,438,132 3,369,136 3,659,23Average 31,012,304 3,171,952 5,701,56total of 151 conserved miRNAs which belonging to 47miRNA families in both camellia species (Additional file 1:Table S6). miRDeep2 score above 1.0 resulted in 98 pre-miRNAs (64.90%) of which 35 with a read count rangingbetween 10 and 100 (23.19%), 41 with read count above100 (27.15%), and 75 with read count below 10 (49.67%)(Additional file 1: Table S6).Table 3 Distribution of small RNAs among different categories durinCategory S01 S02 S03rRNA 2,538,153 2,904,882 3,126snRNA 3532 6061 3311snoRNA 161 194 118tRNA 337,182 247,988 383,8Repbase 9927 9090 15,48Unannotated 17,528,281 24,627,338 19,76Total 20,417,236 27,795,553 23,29Those miRNA sequences, which met the threshold ofmiRDeep2 analysis but did not have any known homolo-gous miRNA gene families in miRBase, were further ana-lyzed to predict novel miRNAs in the two camellia species.The remaining miRNAs, which met the total score of >0,were considered to be true novel miRNAs. A total 123novel mature miRNAs sequences were discovered andbelonged to 36 miRNA families in both camellia species(Additional file 1: Table S7). miRDeep2 score above 1.0 had87 pre-miRNAs (70.73%). The majority of pre-miRNA (69)read count ranged from 10 to 100 (56.10%), followed by 35precursors in above 100 (28.45%) and 19 precursors below10 (15.45%) (Additional file 1: Table S7).Prediction and annotation of miRNAs target genesTo better understand the functions of the identifiedmiRNAs, putative target genes were predicted usingTargetFinder software [42]. A total of 6250 target genes25,151,946 49.13 91.23 100 85.2519,405,898 49.28 93.21 100 87.0223,001,131 48.99 91.35 100 85.4321,016,629 49.7 93.05 100 86.7419,692,318 49.55 93.09 100 86.8621,653,584 49.33 92.39 100 86.26were identified (Table 6). Only 3733 target unigenes(59.73%) were annotated by performing a BLASTX searchagainst diverse protein databases, revealing that 1368(21.89%), 2190 (35.04%), 901 (14.42%), 2160 (34.56%),2730 (43.68%), 2735 (43.76%), and 3718 (59.49%) unigeneshave significant matches with sequences in COG, GO,KEGG, KOG, Pfam, Swissprot, and nr protein databasesg natural drying in C. meiocarpaS04 S05 Average,948 3,295,085 4,259,745 3,224,9635799 5000 4741141 140 15189 368,857 485,157 364,6155 12,733 14,597 12,3660,910 19,295,654 21,106,648 20,463,7660,661 22,978,269 25,871,287 24,070,601respectively (Table 7). A total of 2743 (73.48%) annotatedtarget genes had the length of ≥1000 (Table 7).To evaluate the potential functions of these miRNAtarget genes, we next applied gene ontology (GO) andregulating 131 target genes that were annotated as lipidmetabolism (Additional file 1: Table S10). These miRNAsregulated the changes of seed oil content at differentmoisture content levels. There were high correlationsTable 4 Distribution of small RNAs among different categories during natural drying in C. oleiferaCategory S06 S07 S08 S09 S10 AveragerRNA 4,061,785 3,880,373 4,384,408 5,417,932 4,269,148 4,402,729snRNA 5381 4753 6280 6407 3761 5316snoRNA 376 255 652 351 1224 572tRNA 368,965 437,180 641,000 662,085 677,875 557,421Repbase 18,113 11,547 11,476 14,753 13,396 13,857Unannotated 20,697,326 15,071,790 17,957,315 14,915,101 14,726,914 16,673,689Total 25,151,946 19,405,898 23,001,131 21,016,629 19,692,318 21,653,584Feng et al. BMC Genomics  (2017) 18:546 Page 6 of 14KEGG pathway analyses to categorize the miRNA targets.The miRNA target genes were categorized according tobiological process, cellular component and molecularfunction by Go analysis (Fig. 3). A total of 1860 targetgenes were categorized into 19 biological process. Basedon molecular function, 1722 target genes were classifiedto 14 categories. A total of 1212 target genes were catego-rized as cellular components. Target genes related to lipidmetabolism were found among 51 GO terms, in which 18GO terms are related to biological process and 33 relatedto molecular function. There were 31 miRNAs involved inlipid metabolism and targeted 148 unigenes (Additionalfile 1: Table S8). The KEGG enrichment analysis revealed12 pathways related to lipid metabolism, involved in 15miRNA targeted 93 unigenes. There were 19 target genesin Glycolysis/Gluconeogenesis pathway, 12 in Fatty acidbiosynthesis pathway, and 7 in biosynthesis of unsaturatedfatty acids pathway (Additional file 1: Table S9). IntegratedGO and KEGG function annotation, identified 23 miRNAFig. 1 Length distributions of small RNAs in two camellia species. Data of Ccontent (50, 40, 30, 20 and 10%) for each species separatelybetween transcript abundance with added value of oilcontent, for example MIR482 (Additional file 1: TableS11). Finally, miRNA of lipid metabolism only expressedwere identified in C. Meiocarpa (Group1_Unigene_BMK.30485_635795) and C. oleifera (Group1_Unigene_BMK.37364_696840 and Group1_Unigene_BMK.38037_703962)(Table 8).Differentially expressed miRNAs of lipid metabolismduring natural dryingThe differences between the miRNA profiles of the twocamellia species are possibly related to differences intheir responses to natural drying and were investigatedusing the IDEG6 software. For C. Meiocarpa, miRNAabundance pairwise analysis between the different mois-ture content libraries, indicated that 40, 46, 63, and 38 sig-nificantly differentially expressed miRNAs were identifiedbetween 40 and 50% (S01 vs. S02), 30–40% (S02 vs. S03),20–30% (S03 vs. S04), and 10–20% (S04 vs. S05) moisture. meiocarpa and C. oleifera were averaged across five level moisturecontents, respectively. The highest number of significantlydifferentially expressed miRNAs was observed between 20The highest number of significantly differentially expressedmiRNAs was observed between 40 and 50% (S06 vs. S07)moisture contents, with the highest up- and lowest down-regulated numbers of 61 and 10, respectively (Table 9), with8 significantly different miRNAs involved in lipid metabol-ism (Tables 11 and S13).Comparing across the two species, the average numberof miRNAs in C. oleifera seeds was higher than that ofC. Meiocarpa (Table 9). Pairwise analysis of miRNA abun-dance between the two species for the same moisture levellibraries indicated that there were 78, 51, 44, 58, and 61 sig-nificant differentially expressed miRNAs for the 50, 40, 30,20, and 10% moisture contents, respectively. There werethree differentially expressed miRNAs of lipid metabolismduring the seed natural drying process of the studied twocamellia species (Group1_Unigene_BMK.37987_703484,Group2_Unigene_BMK.25259_1025465, and Group2_Unigene_BMK.25259_1025498) (Tables 12 and S14). The high-est up- (69) and lowest down-regulated number (9) of sig-nificant differentially expressed miRNAs were detected for50% moisture contents (S06 vs. S01) (Table 9). This indi-Table 5 Alignment information statistics with the plant genomesSpecies Sample Unannotated Mapped reads PercentC. meiocarpa S01 17,528,281 1,902,986 10.86S02 24,627,338 2,509,074 10.19S03 19,760,910 2,288,508 11.58S04 19,295,654 2,352,529 12.19S05 21,106,648 2,724,598 12.91Average 20,463,766 2,355,539 11.51C. oleifera S06 20,697,326 2,803,519 13.55S07 15,071,790 2,295,301 15.23S08 17,957,315 2,527,567 14.08S09 14,915,101 2,121,984 14.23S10 14,726,914 2,235,656 15.18Average 16,673,689 2,396,805 14.37Two species 18,568,728 2,376,172 12.80Feng et al. BMC Genomics  (2017) 18:546 Page 7 of 14and 30% (S03 vs. S04) moisture contents, with the 1owestup- and highest down-regulated numbers of 10 and 53,respectively (Table 9), with 9 significantly differentmiRNAs involved in lipid metabolism (Table 10 andAdditional file 1: Table S12). C. oleifera produced simi-lar results with 71, 54, 36, and 56 significantly differen-tially expressed miRNAs observed between 40 and 50%(S06 vs. S07), 30–40% (S07 vs. S08), 20–30% (S08 vs. S09),and 10–20% (S09 vs. S10) moisture contents, respectively.Fig. 2 First nucleotide (a) and position nucleotide (b) biases of miRNA in tcated that the greatest difference between the two specieswas observed at the 50% moisture content, with 12 signifi-cant differentially expressed miRNAs regulating lipid me-tabolism during seed natural drying (Tables 12 and S14).Validation of the expression patterns of differentiallyexpressed miRNAs related lipid metabolism by RT-qPCRTo validate the data obtained from the high-throughput se-quencing, four significantly differentially expressed miRNAswo camellia speciesof the two camellia species were regulated by miRNA dur-ing the seed natural drying process.DiscussionmiRNAs related to the lipid metabolism of camellia speciesRecently, sequencing of oil crops produced a large amountof miRNA associated with lipid metabolism-related genes(e.g., 97, 30, 10, and 4 miRNAs targeting 89, 133, 21, and4 lipid biosynthesis genes were reported for soybean [12],Camelina sativa [11], castor bean [49], and peanut [19],Table 6 Sum of annotated miRNA during camellia seed naturaldryingSpecies Sample All miRNA miRNA with target Target geneC. meiocarpa S01 169 169 3541S02 196 196 5936S03 190 190 3768S04 183 183 3311S05 173 173 3523Total 248 248 6057Feng et al. BMC Genomics  (2017) 18:546 Page 8 of 14(Group1_Unigene_BMK.45675_802511, Group2_Unigene_BMK.63506_1315063, Group1_Unigene_BMK.37987_703484, and Group2_Unigene_BMK.38504_1137258) werepredicted to target genes involved in lipid metabolism andtheir expression levels were quantified using stem-loopqRT-PCR (Fig. 4). The results were consistent with deepsequencing data (Table 8) and the majority of analyzedmiRNAs showed moisture content- and species-specificexpression. For C. meiocarpa, Group1_Unigene_BMK.45675_802511 and Group2_Unigene_BMK.63506_1315063peaked at 10 and 30% moisture content while the othertwo miRNAs (Group1_Unigene_BMK.37987_703484, andC. oleifera S06 191 191 3738S07 173 173 3397S08 177 177 3616S09 178 178 3451S10 169 169 3545Total 246 246 4532Two species 274 274 6250Group2_Unigene_BMK.38504_1137258) peaked at 20%moisture content. Additionally, all four miRNAs had differ-ent lowest point (Fig. 4). C. oleifera showed four miRNAspeaked at 50% moisture content but had different lowestpoint at 10 (Group1_Unigene_BMK.45675_802511 andGroup2_Unigene_BMK.63506_1315063), 20 (Group2_Unigene_BMK.38504_1137258), and 40% (Group1_Unigene_BMK.37987_703484) moisture content (Fig. 4).These expression patterns indicate that lipid metabolismTable 7 Functional annotation of the two camellia speciesDatabase Annotated Number 300 ≤ lengCOG_Annotation 1368 174 (12.72%GO_Annotation 2190 469 (21.42%KEGG_Annotation 901 198 (21.98%KOG_Annotation 2160 430 (19.91%Pfam_Annotation 2730 424 (15.53%Swissprot_Annotation 2735 584 (21.35%nr_Annotation 3718 977 (26.28%All_Annotated 3733 990 (26.52%respectively). In the present study, we detected 23 pre-miRNAs targeting 131 candidate genes regulating lipidmetabolism functions during camellia seed natural drying(Additional file 1: Table S10). Of these pre-miRNAs, 5,5, and 11 regulated fatty acid biosynthesis, accumula-tion, and catabolism, respectively, and additional 2(Group1_Unigene_BMK.30485_635795 and Group1_Unigene_BMK.45675_802511) regulated not only fattyacid biosynthesis and accumulation, but also fatty acidcatabolism (Additional file 1: Table S10). Group1_Unigene_BMK.30485_635795 and Group1_Unigene_BMK.45675_802511 encode 56 and 41 target genes involved in fattyacid biosynthesis, accumulation, and catabolism, respect-ively (Additional file 1: Table S10).Concurring with the above, the most abundant miRNAs(osa-miR2118e: Group1_Unigene_BMK.37987_703484,Group2_Unigene_BMK.25259_1025465, and Group2_-Unigene_BMK.25259_1025498 (Additional file 1: Table S10and Table 8)) that target genes encoding a long-chain-alcohol oxidase involving fatty acid-oxidation [50]. Thesecond abundant miRNAs (Group2_Unigene_BMK.63506_1315063) targeting diacylglycerol kinase, a lipidkinase converting diacylglycerol to phosphatidic acid,regulating balance of diacylglycerol and phosphatidicacid [51], and potentially involved in regulating lipiddeposition [52]. The third, Group2_Unigene_BMK.38504_1137258 (Additional file 1: Table S10 and Table 8),targeting lipases/acylhydrolase, fatty acid hydroxylase,and carboxylesterase, which are hydrolytic enzymes in-volved in degradation of fatty acid [53–55], collectivelysuggesting that these miRNAs may play important rolesth < 1000 length ≥ 1000 %Annotated target gene) 1194 (87.28%) 21.89) 1721 (78.58%) 35.04) 703 (78.02%) 14.42) 1730 (80.09%) 34.56) 2306 (84.47%) 43.68) 2151 (78.65%) 43.76) 2741 (73.72%) 59.49) 2743 (73.48%) 59.73Table 8 miRNA of lipid metabolism transcript abundance in the two camellia species during seed natural dryingPre-miRNA miRNA C. meiocarpa C. oleiferaS01 S02 S03 S04 S05 Average S06 S07 S08 S09 S10 AverageCL22479Contig1_234494 Unkown 0 0 0 1 0 0.2 1 0 1 0 1 0.6CL22738Contig1_61999 Unkown 0 0 1 0 0 0.2 0 0 0 0 1 0.2Group1_Unigene_BMK.23434_588836 MIR5067 20 21 33 25 24 24.6 19 20 15 9 10 14.6Group1_Unigene_BMK.30485_635795 Unkown 0 1 0 0 0 0.2 0 0 0 0 0 0.0Group1_Unigene_BMK.37364_696840 Unkown 0 0 0 0 0 0.0 1 2 0 0 0 0.6Group1_Unigene_BMK.37987_703484 MIR2118 302 563 747 807 602 604.2 650 280 434 325 359 409.6Group1_Unigene_BMK.38037_703962 Unkown 0 0 0 0 0 0.0 0 0 0 0 1 0.2Group2_Unigene_BMK.25259_1025465 MIR482 302 563 747 807 602 604.2 650 280 434 325 359 409.6Group2_Unigene_BMK.25259_1025498 MIR482 302 563 747 807 602 604.2 650 280 434 325 359 409.6Group2_Unigene_BMK.34335_1,093,229 MIR5067 20 21 33 25 24 24.6 19 20 15 9 10 14.6Group2_Unigene_BMK.50808_1268477 Unkown 0 0 0 1 0 0.2 1 0 0 1 0 0.4Group2_Unigene_BMK.9543_1378570 Unkown 15 22 11 26 21 19.0 17 10 15 11 7 12.0CL19455Contig1_54014 Unkown 26 26 23 22 27 24.8 40 18 28 25 14 25.0CL19777Contig1_314088 Unkown 23 34 30 29 26 28.4 35 18 31 26 20 26.0CL2440Contig1_359627 MIR1861 2 6 13 5 4 6.0 5 1 5 3 4 3.6CL4378Contig1_191450 Unkown 11 8 10 14 15 11.6 35 13 22 17 12 19.8CL9644Contig1_380257 Unkown 26 24 21 21 18 22.0 20 12 18 10 24 16.8Group1_Unigene_BMK.45675_802511 Unkown 4 4 8 1 10 5.4 9 4 4 5 3 5.0Group2_Unigene_BMK.24252_1018543 Unkown 1 1 1 0 0 0.6 1 2 2 1 0 1.2Group2_Unigene_BMK.38504_1137258 Unkown 35 31 29 40 33 33.6 45 25 24 13 25 26.4Group2_Unigene_BMK.38504_1137263 Unkown 6 3 4 3 4 4.0 2 0 2 2 1 1.4Group2_Unigene_BMK.63506_1315063 Unkown 58 47 89 49 53 59.2 96 49 34 50 20 49.8Group2_Unigene_BMK.39605_1150110 Unkown 0 1 1 0 0 0.4 2 1 0 1 0 0.8Fig. 3 GO categories and distribution of miRNA targets in two camellia speciesFeng et al. BMC Genomics  (2017) 18:546 Page 9 of 14Table 9 Comparison of the number of differentially expressedmiRNAs between samples with different moisture content withinand across the two camellia species during seed natural dryingType Compared type Number Up-regulatedDown-regulatedC. meiocarpa S01 vs. S02 40 21 19S02 vs. S03 46 20 26S03 vs. S04 63 10 53S04 vs. S05 38 29 9Average 47 20 27C. oleifera S06 vs. S07 71 61 10S07 vs. S08 54 9 45S08 vs. S09 36 19 17S09 vs. S10 56 16 40Feng et al. BMC Genomics  (2017) 18:546 Page 10 of 14in decreasing the rate of lipid breakdown during camel-lia seed natural drying. This can be confirmed by theAverage 54 26 28Between species S06. vs. S01 78 69 9S07 vs. S02 51 22 29S08 vs. S03 44 27 17S09 vs. S04 58 19 39S10 vs. S05 61 44 17Average 58 36 224% increase in oil content in two camellia seed throughseed nature drying (Table 1).miRNAs regulate lipid metabolism during camellia seednatural dryingSeveral studies have shown that miRNAs are differentiallyregulated in response to stress [56] and that this differen-tial regulation varied among different plant species [57].For example, distinct responses to drought stresses wereTable 10 Significant differentially expressed miRNAs of lipidmetabolism during C. meiocarpa seed natural drying acrossdifferent moisture content levels (%)1Pre-miRNA 50 vs.40%40 vs.30%30 vs.20%20 vs.10%Group2_Unigene_BMK.25259_1025498 1 1 1 0Group1_Unigene_BMK.37987_703484 1 1 1 0Group2_Unigene_BMK.34335_1,093,229 0 0 1 0CL2440Contig1_359627 0 0 1 0Group1_Unigene_BMK.23434_588836 0 0 1 0CL19777Contig1_314088 0 0 1 0Group2_Unigene_BMK.25259_1025465 1 1 1 0Group1_Unigene_BMK.45675_802511 0 0 1 1Group2_Unigene_BMK.63506_1315063 0 1 1 01 Represents significant DEG and 0 represents non-significant DEG1significance at P < 0.05reported for miRNAs in Arabidopsis [58], switchgrass[59], Populus [60] and Caragana intermedia [61]. Espe-cially, drought stress in switchgrass [59] and Populus [60]were regulated by miRNAs related to lipid metabolism;however, the linkage between drought responses and lipidmetabolism miRNAs changes is not established.In the present study, C. Meiocarpa produced 9 signifi-cant differentially expressed miRNAs regulating lipidmetabolism with only 4 at 20–30% moisture contents.These pre-miRNAs belonged to Group2_Unigene_BMK.34335_1,093,229, Group1_Unigene_BMK.23434_588836,CL19777_Contig1_314088, and CL2440_Contig1_359627,with the former three showing more than 100 TPM(Table 10 and Additional file 1: Table S12) and target-ing 3-ketoacyl-CoA synthase III, which catalyze the ini-tial elongation step of fatty acid biosynthetic process[62] and glycerol-3-phosphate transporter, a precursorprotein for phospholipid biosynthesis [63]. It is interestingTable 11 Significant differentially expressed miRNAs of lipidmetabolism during C. oleifera seed natural drying acrossdifferent moisture content levels (%)1Pre-miRNA 50 vs.40%40 vs.30%30 vs.20%20 vs.10%Group2_Unigene_BMK.63506_1315063 1 1 0 1CL19455Contig1_54014 0 0 0 1Group1_Unigene_BMK.37987_703484 1 1 0 1Group1_Unigene_BMK.23434_588836 1 1 0 0Group2_Unigene_BMK.25259_1025498 1 1 0 1Group2_Unigene_BMK.25259_1025465 1 1 0 1Group2_Unigene_BMK.38504_1137258 1 1 0 0Group2_Unigene_BMK.34335_1,093,229 1 1 0 01 Represents significant DEG and 0 represents non-significant DEG1significance at P < 0.05to note that these three pre-miRNAs were down-regulatedresulting in a reduction in the fatty acid biosynthesis, soseeds with 30% moisture content have high fatty acid syn-thesis and accumulation activities (Table 1).Similarly, C. oleifera produced 8 significant differentiallyexpressed miRNAs during seed natural drying, with up-and down-regulated for the 40–50 and 30–40% moisturecontents, respectively (Tables 11 and S13). The largest foldchanges were observed for Group1_Unigene_BMK.23434_588836 and Group2_Unigene_BMK.34335_1,093,229,which target 3-ketoacyl-CoA synthase III (Additional file 1:Table S10 and Table S13). These pre-miRNAs regulatefatty acid biosynthesis with seeds at 40–50% moisture con-tent showing low fatty acid biosynthesis activities whilethose at 30–40% moisture content exhibiting high activities(Table S13). This can be confirmed by the observed 1.06and 9.17% increase in oil content at 40–50 and 30–40%moisture contents, and reaching the highest point at 30%moisture content (Table 1). So seeds with 30% moisturecontent have also high fatty acid synthesis and accumula-tion activities.Comparing the significant differentially expressed miR-NAs of C. meiocarpa (9) with those of C. oleifera (8),indicated that the two species share 6 miRNAs involvedin lipid metabolism, with unique miRNAs belonging toeach species (CL19455Contig1_54014 and Group2_Unigene_BMK.38504_1137258 in C. oleifera, and CL2440Contig1_359627, CL19777Contig1_314088 and Group1_Unigene_BMK.45675_802511 in C. meiocarpa) (Tables 10Table 12 Significant differentially expressed miRNAs of lipid metabolism during the two camellia species seed natural drying acrossthe same moisture content level (C. oleifera vs. C. meiocarpa MC%)1Pre-miRNA 50 vs. 50% 40 vs. 40% 30 vs. 30% 20 vs. 20% 10 vs. 10%CL19777Contig1_314088 1 0 0 0 0CL19455Contig1_54014 1 0 0 0 0Group2_Unigene_BMK.25259_1025465 1 1 1 1 1Group2_Unigene_BMK.9543_1378570 1 0 0 0 0CL9644Contig1_380257 1 0 0 0 0Group2_Unigene_BMK.25259_1025498 1 1 1 1 1Group2_Unigene_BMK.38504_1137258 1 0 0 0 0Group2_Unigene_BMK.34335_1,093,229 1 0 1 0 0Group2_Unigene_BMK.63506_1315063 1 0 1 1 1Group2_Unigene_BMK.38504_1137263 1 0 0 0 0Group1_Unigene_BMK.23434_588836 1 0 1 0 0Group1_Unigene_BMK.37987_703484 1 1 1 1 11Represents significant DEG and 0represents non-significant DEG1significance at P < 0.05Feng et al. BMC Genomics  (2017) 18:546 Page 11 of 14Fig. 4 qRT-PCR validation of miRNA in C. meiocarpa and C. oleifera. Relative expgene was 5.8 rRNA. Normalized miRNA in 50% moisture content of C. oleifera wression of miRNA in 10, 20, 30, 40, and 50% moisture contents. Referenceere arbitrarily set to 1. Error bars were calculated based on three replicatesFeng et al. BMC Genomics  (2017) 18:546 Page 12 of 14and 11). Group2_Unigene_BMK.38504_1137258 andCL2440Contig1_359627 regulated fatty acid catabolism,CL19455Contig1_54014 and CL19777Contig1_314088regulated fatty acid accumulation, and Group1_Unigene_BMK.45675_802511 regulated fatty acid synthe-sis, accumulation, and catabolism (Additional file 1:Table S10). These indicate that oil content differencesbetween the two camellia species are mainly due totheir differential abilities miRNAs of fatty acid accu-mulation and catabolism.Collectively, the studied two camellia species produced12 significant differentially expressed miRNAs to regulatelipid metabolism during seed natural drying (Tables 12and S14). These pre-miRNAs indicated that C. meiocarpahas higher activities to regulate the lipid metabolism andthis can be confirmed by its lower oil content as comparedto C. oleifera (Table 1). It should be stated that all these12 differentially expressed miRNAs were significantlyexpressed in the 50% moisture content stage (Tables 12and S14). Seeds with 50% moisture content had only signifi-cant differentially expressed pre-miRNAs (CL19777Contig1_314088, CL19455Contig1_54014, Group2_Unigene_BMK.9543_1378570, CL9644Contig1_380257, Group2_Unigene_BMK.38504_1137258 and Group2_Unigene_BMK.38504_1137263) (Tables 8 and S14). CL19777Contig1_314088 and CL19455Contig1_54014 targetglycerol-3-phosphate transporter (Additional file 1: TableS10). Group2_Unigene_BMK.9543_1378570 target acetyl-CoA-carboxylase (Additional file 1: Table S10), whichcatalyze the ATP-dependent carboxylation of acetyl-CoAto form malonyl-CoA, the rate limiting and first commit-ted reaction in fatty acid synthesis [64]. So these threepre-miRNA control different fatty acid synthesis and accu-mulation in the two camellia species. For all significantdifferentially expressed miRNAs, the largest fold changewas observed for Group2_Unigene_BMK.38504_1137263(Fatty acid hydroxylase), followed by CL9644Contig1_380257 (Carboxylesterase), and Group1_Unigene_BMK.23434_588836 (3-ketoacyl-CoA synthase III) and Group2_Unigene_BMK.34335_1,093,229 (3-ketoacyl-CoA synthaseIII) (Additional file 1: Table S10 and Table S14). So the oilcontent of C. oleifera is higher than C. meiocarpa and thisis attributable to four pre-miRNAs, of which Group2_Unigene_BMK.38504_1137263 and CL9644Contig1_380257regulating fatty acid catabolism and Group1_Unigene_BMK.23434_588836 and Group2_Unigene_BMK.34335_1,093,229 regulating fatty acid synthesis.ConclusionThe present study identified 274 candidate miRNAs, with248 and 246 unique sequences to C. meiocarpa and C.oleifera, respectively. Integrated GO and KEGG functionannotation, produced 23 miRNAs regulating 131 targetgenes, all were annotated as lipid metabolism, regulatingfatty acid biosynthesis, accumulation and catabolism dur-ing seed natural drying. Lipid metabolism primarilyfocused on fatty acid catabolism, with five miRNAs(Group1_Unigene_BMK.37987_703484, Group2_nigene_BMK.25259_1025465 Group2_Unigene_BMK.25259_1025498, Group2_Unigene_BMK.63506_1315063, andGroup2_Unigene_BMK.38504_1137258) playing import-ant roles in decreasing the rate of lipid breakdown andadditional two miRNAs (Group2_Unigene_BMK.34335_1,093,229 and Group1_Unigene_BMK.23434_588836)regulating fatty acid synthesis. Across the two species, fourpre-miRNAs were identified to regulate lipid metabolism,with Group2_Unigene_BMK.38504_1137263 and CL9644Contig1_380257, and Group1_Unigene_BMK.23434_588836 and Group2_Unigene_BMK.34335_1,093,229regulating fatty acid catabolism and synthesis, respectively.To our knowledge, this work provides the first smallRNA expression analysis of lipid metabolism in camelliaseed during natural drying as well as the first comparativemiRNA profiling analysis between C. Meiocarpa andC. oleifera that exhibit significantly different fatty acidprofiles.Additional fileAdditional file 1: Figure S1. Length distributions of miRNAs in twocamellia species. Table S1. qRT-PCR primer sequences. Table S2. Lengthdistributions of small RNAs in C. meiocarpa during seed natural drying.Table S3. Length distributions of small RNAs in C. oleifera during seednatural drying. Table S4. miRNAs transcript abundance in the two camelliaspecies during seed natural drying. Table S5. All conservative miRNAdiscovered in the two camellia species during seed natural drying.Table S6. All novel miRNA discovered in the two camellia speciesduring seed natural drying. Table S7. GO terms related with the lipidmetabolism in the two camellia species during seed natural drying.Table S8. KEGG enrichment related with the lipid metabolism in thetwo camellia species during seed natural drying. Table S9. miRNA of lipidmetabolism targets and their putative functions. Table S10. Differentiallyexpressed miRNAs of lipid metabolism during C. meiocarpa seed naturaldrying. Table S11. Differentially expressed miRNAs of lipid metabolismduring C. oleifera seed natural drying. Table S12. Differentially expressedmiRNAs of lipid metabolism between two camellia species during seednatural drying. (DOCX 168 kb)AcknowledgmentsWe thank Minhou Tongkou State Forest Farm of Fujian Province forproviding access to research material. We also thank Professor Bruce CLarson, University of British Columbia, for helpful suggestions. This work wassupported by grants received under the Doctoral Fund of the Ministry ofEducation of China (K4112020A) and Fujian Province Major Science andTechnology Project (2013NZ0207).Availability of data and materialsThe data supporting this study are available in the Dryad Digital Repository,doi: 10.5061/dryad.1mj01Authors’ contributionsJ-LF, HC, YAK conceived and designed the experiment, J-LF, Z-JY performedthe experiment, J-LF, Z-JY, S-P C, collected the data collection and preparedthe figures, J-LF performed the data analysis, and J-LF, YAK wrote themanuscript. All authors read and approved the final manuscript.Feng et al. BMC Genomics  (2017) 18:546 Page 13 of 14Ethics approval and consent to participateNot required.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Received: 7 December 2016 Accepted: 4 July 2017References1. Lee CP, Yen GC. Antioxidant activity and bioactive compounds of tea seed(C.oleifera Abel.) oil. J Agric Food Chem. 2006;54(3):779–84.2. Ma J, Ye H, Rui Y, Chen G, Zhang N. Fatty acid composition of C.oleifera oil.J Verbr Lebensm. 2011;6(1):9–12.3. Yang C, Liu X, Chen Z, Lin Y, Wang S. Comparison of oil content andfatty acid profile of ten new C. oleifera cultivars. Journal of lipids. 2016.doi:10.1155/2016/3982486.4. Haiyan Z, Bedgood DR, Bishop AG, Prenzler PD, Robards K. Endogenousbiophenol, fatty acid and volatile profiles of selected oils. Food Chem.2007;100(4):1544–51.5. 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