Integrated mRNA and miRNA transcriptome analysis on the regulational of mechanism tuber expansion in Yam

doi:10.21203/rs.2.9777/v1

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Integrated mRNA and miRNA transcriptome analysis on the regulational of mechanism tuber expansion in Yam

https://doi.org/10.21203/rs.2.9777/v1

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published 03 Feb, 2020

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Background

Tuber is the storage organ of yam derived from modified stems. The development of tube expansion is a complex process and depends on the expression of genes that are involved by environmental and endogenous factors. However, little is known about the mechanism that regulates tuber expansion. In this study, in order to identify genes and miRNAs involvd in tuber enlargement, we examined the transcriptomes and small RNA in tuber initiation and expansion stages.

Results

A total of 14238 transcripts expressed differentially in the expansion stage were firstly identified using transcriptome technology. Of these, 5723 and 8515 genes were up and down regulated, respectively. The functional analysis revealed the coordination of tuber plant involved in processes of cell events, metabolism, biosynthesis and signalling transduction pathways at the transcriptional level, suggesting these DEGs are somehow involved in the responses of tuber expansion. In additional, 536 transcription factor genes were found differently expression in expansion stage at the transcript level. REVEILLE 6-like is identified to be up regulated in circadian rhythm pathway. REVEILLE 6-like LHY protein, zinc finger CCCH domain-containing protein 14, and DELLA genes were involved up regulated in expansion stage. Moreover, these genes were respectively involved in circadian rhythm pathway, starch and sucrose metabolism pathway, and GA pathway by KEGG analysis. Noteworthy, the analysis of the data showed that there were 23 known tuber miRNAs belonged to 11 miRNA families, and 50 novel miRNAs, miRNA160 miRNA396, and miR535 may be involved in complexity network to regulate cell division and differentiation in the expansion stage in yam.

The integrated analysis of miRNA-mRNA were identified to be preferentially expressed in hormone signalling in expansion stage, like the ARFs by miRNA160 highlighting the involvement of miRNA-mRNA in the regulation hormone of tuber expansion in yam.

Conclusion

The transcriptome and miRNA datasets presented here identified a subset of candidate genes and miRNAs putatively associated with tuber expansion in yam, thus a hypothetical model of genetic regulatory network associated with tuber expansion in yam was put forward, which may provide a foundation for molecular regulatory mechanism researching on tuber expansion in Dioscorea species.

Epigenetics & Genomics

Yam

Expansion

Transcriptome

Small RNA

Yams are monocotyledonous plant belonging to the family of Dioscoreaceae, and the tuber is its harvested organ. Tuber originates from the expansion of underground stem, which is suitable for storage of nutrients, with lots of large parenchyma cells. Tuber morphogenesis, which involves three universal process: induction, initiation and formation; starch and stored protein of accumulation are two main processes in tuber growth[ 1]. The tuber morphogenesis of yam could be divided into three periods: initiation stage, expansion stage, and maturation stage, and the expansion stage could be divided into three periods: early expansion stage, middle expansion stage, late expansion stage[ 2, 3].Tuber morphogenesis is a complex physiological process regulated by hereditary, environment, hormones,etc[ 4]. Great efforts have been made to the explore the physiological factors affecting tuber morphogenesis of yam, especially the initiation stage developing into expansion stage, short days promoting tuber expansion at the initiation tuber stage of water yam (D. alata), whereas long days inhibiting tuber expansion[ 5, 6]. Endogenous hormones played an important role in tuber morphogenesis, for example gibberellins(GA), acetic acid(IAA) and abscisic acid(ABA) playing a key role at the beginning of tuber expansion stage, and trans-zeatin(tZ), jasmonic acid(JA) were also involved in tuber expansion[ 2, 7, 8].To date, exogenous hormones have been used to study tuber expansion, GAs could promote tuber expansion and yield by treatment in vitro and in vivo[ 9, 10].Exogenous GA combined with ABA promoted microtuber growth and expansion[ 11].Exogenous JA was found to be essential for yam tuberization and induced an increase of tuber number in vitro and in vivo[ 12, 13]. However, fundamental knowledge about endogenous metabolic networks is utterly lacking in tuber expansion.

The induction and growth of microtubers in vitro in yam have been found to be under the control of nutrients, sucrose concentration was the most important factor affecting tuberization and frequency of proliferation[ 7, 14]. Yam tuber morphology were significantly correlated with nutrient accumulation and enzymatic activity, sucrose, soluble sugars,proteins are significantly increased in tuber expansion stage, subsequently decreased in maturity stage, starch content was increased in the whole tuber morphogenesis, and sucrose synthase, sucrose phosphate synthase, and AGPase were significantly correlated with these nutrient accumulation[ 15]. Although many DNA molecular markers were used to uncover the genetic diversity and relationship among yam germplasms [ 16-18], it is little known about specific genes involving tuber morphogenesis. The sucrose synthase 4 and sucrose-phosphate synthase 1 were associated with the earliest stages of starch biosynthesis and storage, a SCARECROW-LIKE gene was involved in formation of adventitious roots[ 19]. PE2.1 and PE53 are the members of pectinesterase(PE) superfamily, which are likely to be associated with the regulation of starch and sucrose metabolism and signalling pathways. Therefore, they may play important roles in microtuber formation[ 20]. The tuber morphogenesis is a complex biological process involving many specific genes and proteins, especially yam tuber expansion. Transcriptome techniques can efficiently find and detect these genes and proteins. Potato is a tuber crop, many transcriptome analysis revealed that there were many genes were regulated in early stages of stolon to tuber transitions or tuberization by nutrients, photoperiodic conditions, exogenous hormones and stress[

                 21-24
              </a>]. Former transcriptome study revealed that some putative gens were involved in dioscin
           biosynthesis[<a href="#_ENREF_25">
              
                 25
              </a>], and chalcone isomerase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3'-monooxygenase(F3'H),
           dihydroflavonol 4-reductase(DFR), leucoanthocyanidin dioxygenase(LDOX), and flavonol
           3-O-glucosyltransferase(UF3GT) were significantly expressed in flavonoid biosynthesis[<a href="#_ENREF_26">
              
                 26
              </a>]. However, there are not the reports on the transcriptome study on tuber expansion.
           Thus, to investigate the key genes involved in the metabolism of tuber expansion in
           yam will provide a clear network to elucidate the molecular mechanism of tuber growth
           by transcriptome.</p>

microRNAs (miRNAs) are endogenous small non-coding RNA with important functions in many biological processes, such as the regulations of growth and development, stress response, and metabolism. Many studies have indicated that miRNAs play important roles in root and tuber formation or development[ 27-29].MiR165/166 regulated root growth through determining the fate of root cells combined with phytohormone crosstalk in Arabidopsis，by negative regulating its target genes auxin response factor ARF10, ARF16 and ARF17[ 30]. miRNA 172 and miR156 were involved in tuberization process, acting either as a component or a regulator of long distance gibberellin signaling pathways[ 31, 32]. Potato specific miRNA193, miRNA152, and the conserved miR172-1, miRNA172-5 showed significant expression during developmental stages of tuberization[

                 28
              </a>]. Although a number of studies have been identified that miRNAs are involved in tuber
           and root development, the miRNA-mediated regulatory network during tuber expansion
           is still unclear.</p>

Although the whole genome sequencing of the heterozygous diploid Guinea yam(D. rotundata) had been done for sex determination at the seedling stage[

                 33
              </a>], a more detailed comparative transcriptome and miRNA analysis in the expansion stage
           should be detected. In this study, both transcriptome and small RNA sequencing were
           carried out to thoroughly investigate the crucial genes and miRNA in tuber expansion stages in yam.</p>

Overview of transcriptome dynamics and small RNA sequencing

In order to identify transcriptome and miRNAs co-regulated regulation in tuber expansion, we examined the transcriptomes and small RNA in tuber initiation and expansion stages(Fig.1). Meanwhile, the transcriptome library was constructed from mixed RNA pools consisting of initiation and expansion stages in order to construct small RNA and transcriptomes. Approximately 6.67Gb bases in total on BGISEQ-500 sequencing platform were gained by transcriptome de novo analysis(Additional file 1:Table S1). After filtering low-quality reads and adaptor sequences, 7.7% of the total unigenes longer than 2000bp in the length of distribution of all the assembled yams transcripts were gained, which indicated that it is difficult to calculate the number of genes in yams due to the deficiency information of its genome dates(Fig.2). A total of 32207 genes was performed functional annotation with 7 functional database (NR, NT, GO, KOG, KEGG, SwisProtand, InterPpro), 25694(79.78%), 16891(52.45%), 17603(54.66%), 19472(60.46%), 20191(62.69)%, 22159(68.80%), 8270(25.68%) reads were annotated function, respectively(Fig.3). 13566 genes were commonly annotated in NR, KOG, KEGG, SwissProt and InterPpro databases. Based on the NR database function annotation results(Fig.4), the species distributions of the best matches for each sequence matched the genes of Elaeis guineensis (33.21%), followed by Phoenix dactylifera (23.35%) , Ananas comosus (7.47%), Musa acuminate subsp.malaccensis (7.31%).

A total of 32026 genes were detected, accounting for approximately 6.6 G of clean reads of each mRNA library by RNA-seq analysis(Additional file 1:Table S1). The average mapping rate of transcriptome library was 82.57%(Additional file 1:Table S1). Both the tuber initiation and expansion stages samples showed good correlations in each other based on the Pheatmap cluster analysis(Fig.5).

Furthermore, the corresponding small RNA libraries at the three time points were also constructed for deep sequencing (Additional file 1:Table S1). Subsequently, 4593044(17.48%), 5032588(18.58%), 4642869(17.58%) tags in tuber initiation and 6388211 (25.11%), 5872589(22.36%), 6086348(22.98%) in tuber expansion were mapped to sRNA database, repectively. These tags were clustered into RNA classes(including miRNA, sncRNA), genome and unannotated group.

Differentially expressed genes annotation by GO term and KEGG pathway

To identify the genes regulated differentially in tuber expansion stage, DEseq software was used to compare the gene expression between initiation and expansion stages. Of these, 5765 and 8515 genes were up and down regulated, respectively(Additional file 1:Table S2).

To better understand the function of the DEGs, 44 significant GO categories were identified. For cellular component, 15 GO categories including cell, membrane and membrane part, organelle, and organelle part were enriched among DEGs(Fig.6). For biological processes, DEGs related to cellular process, metabolic process and biological regulation were enriched in expansion stage(Fig.6). The molecular functions of the DEGs were mainly associated with catalytic activity, binding, transporter activity, structural molecular activity. Among the significant GO term anaylsis, 15 genes were enriched in cell wall polysaccharide metabolic process, 15 genes were involved in hemicellulose metabolic process, and 13 genes were related to xyloglucan metabolic process related to cell wall formation in expansion stage(Table 1). Moreover, the results also showed a number of significantly expression genes involved in tissue development, root morphogenesis, root system development, root development(Table 2). KEGG pathways analysis revealed that plant hormone signal transduction,biosynthesis of amino acids were enriched with DEGs in expansion stage(Fig .7,Fig.8). The other pathways like MAPK signalling pathway, starch and sucrose metabolism and carbon metabolism were also expressed in KEGG pathway. These metabolic pathways may be closely related to the development of tuber expansion and bioactive compound synthesis.

Comprehensive analysis of differentially expressed genes in tuber initiation and expansion stages

Compared with expansion stage, there were large number of DETs in tuber initiation stages using NR, GO and KEGG annotation. Signal transduction, cell wall, cell division, starch and sucrose metabolism were selected for profiling during the expansion of yam tuber(Additional file 1: Table S3). 242 DETs were identified to show high similarity with many plant hormone signals, inincluding 131 down-regulated and 111 up-regulated DETs. Interestingly, the majority of plant hormone related genes in GA, BRs, IAA, ABA signal pathways were found in expansion stage(Fig.9). 14 mitogen activated protein kinases(MAPK) genes showed up-regulated in expansion stage, while some others including MAPKKK17_18, MPK6 and MPK8 were down-regulated. In all, 48 DETs were homologous with calcium signal-related genes, including 27 calcium dependent protein kinases(CDPKs), 16 calcium binding proteins(CBPs), and 5 Calreticulins(CBL).

A total of 98 transcripts homologous to the genes associated with cell wall and cell cycle were observed as differentially regulated during expansion stage , including Xyloglucan endotransglucosylase/hydrolase(XTH), expansin, extension, cyclin-dependent kinases(CKS), cell division protease (ftsHs), cell division cycle 5-like protein (CDC5), cell division control protein (CDC), cyclin-dependent kinases (CDKs), cyclin-dependent kinase inhibitor (CDKIs). All of the expansin, extension, cell wall synthesis and CKS genes were down-regulated in expansion stage. Meanwhile, the majority of cell cytoskeleton and XTH were down-regulated in expansion stage in yam.

The major constituents of starch and sucrose metabolism in expansion are sucrose synthase genes (SuSy), sucrose phosphate synthase genes(SPS), starch synthase(SS) and andinvertase genes(INV)( Additional file 1: Table S3). Among them, the majority of SPS were up-regulated, and SuSy were down-regulated in expansion stage. Interestingly, dioscorins, the major storage proteins in yam tubers, were significantly up-regulated in expansion stage(Additional file 1: Table S3). These results indicated that many functional genes were involved in expansion stage in yam.

Transcription factor analysis

Transcription factors are widely involved in various biological processes. 1254 TF-encoding genes were found and classified into 55 different families in yam tuber, of which MYB, MYB-related and AP2-EREP were enriched in tuber(Fig.10). Further analysis revealed that 541TF-encoding genes belong to 48 TF families were differentially expressed between initiation and expansion stage. There were 286 and 255 TF-encoding genes were up and down regulated, respectively(Additional file 1: Table S4). The TF gene families with the highest number were express by heat map in expansion stage (Fig.11). Interestingly, REVEILLE 6-like LHY protein (Unigene12250_Total_1), zinc finger CCCH domain-containing protein 14 (CL256.Contig1_Total_1),and DELLA genes(Unigene5584_Total_1, Unigene22110_Total_1, CL1653.Contig2_Total_1, CL1340.Contig2_Total_1)were involved up regulated in expansion stage(Additional file 1: Table S2).Moreover, these genes were respectively involved in circadian rhythm pathway, starch and sucrose metabolism pathway and GA pathway by KEGG analysis(Additional file 1: Table S2).

Detection of known and novel miRNAs expressed in tuber initiation and expansion stages

The investigation of both known miRNA and novel putative miRNAs was performed by miRDeep2 program. This program combined the position and frequency of small RNAs with the secondary structure of miRNA precursors to provide novel miRNA which may be specifically in tuber. Data analysis showed that there were 23 known miRNAs(21 and 20 in tuber initiation and expansion stage, respectively ) and 50 novel miRNAs in yam tuber (Additional file 1: Table S5).

Further analysis revealed that 23 known miRNAs belonged to 11 miRNA families, miRNA168, miRNA159 and novel miRNA16 were the most extensively represented families. All miRNAs were analyzed to detect differential miRNA expressions (DEMs). The results showed that miRNAs expression was dynamically regulated in expansion stages. A total of 44 differentially expressed miRNAs were identified including 11 known and 33 novel miRNAs, It showed that, 40 were down regulated, and 4 were up regulated in the expansion stage(Table 3). It is interesting that miR160 family(miRNA160,miRNA160a-5p, miRNA160b-1, miRNA160h-1) and miR535a_1 were down-regulated in expansion stage, miRNA396b and miRNA168 were up-regulated compared to their miRNA families(Table 3).

Identification of target genes of differentially expressed miRNA in tuber expansion compared with initiation stage

The miRNA regulates target mRNA through translational repression or mRNA degradation. To identify the correlation between the expression of DEMs and DEGs, a total of 11 DEMs were putatively targeted 34 DEGs(Additional file 1: Table S6). Furthermore, based on GO and KEGG analysis of the targets, it revealed that several key genes involved in expansion stage were completely regulated by miRNAs. GO analysis revealed differentially expressed miRNAs regulated in expansion stage by binding, cell, membrane, organelle, membrane part(Fig.12). KEGG pathways analysis were involved in transport and catabolism, signal transduction, biosynthesis of other secondary metabolites, energy metabolism, carbohydrate metabolism, lipid metabolism. It is notable that 3 genes which were targets of 2 differentailly expressed miRNAs(miR535a_1, miR160) were identified in the pathway of plant hormone signal transduction (Fig.13). miRNA160 was down regulated in expansion stage, ARF18(CL2135.Contig1_Total_1), ARF17(Unigene5660_Total_1) were up regulated in auxin signal transduction and Log2(GH16-E/GH16-I) of three miRNAs and their target genes showed not only all of them were differentially expressed significantly but also these miRNAs regulated the expression of their targets in expansion stage. miR535a_1 was involved in brassinosteroid signal transduction pathway by targeting SBP. Moreover, miR396b and miR396a-5b were involved in other metabolism processes and functional proteins by targeting genes, including IST1-like protein, growth-regulating factor 4, photosystem II oxygen-evolving enhancer protein 2, acetyl-CoA carboxylase biotin carboxyl carrier protein(Fig.13, Additional file 1: Table S6).

Some target genes and corresponding miRNA were involved in cell event, regulation of plant hormone levels, carbohydrate metabolism, and other metabolism, implying that they may play crucial roles during the tuber expansion stage(Fig .14).

Validation of the DEGs and DEMs data using RT-qPCR

To identify the accuracy and reliability of transcriptome and miRNA data, the RT-qPCR was used to measure the expressions of some DEGs and DEMs, including 17 mRNAs, and 11 miRNAs, and specific primers were designed (Additional file 1: Tables S6 and S7). All of mRNA and miRNA expressions were confirmed to the accuracy of the RNA-seq data. Overall, these results indicated that 16 out of 17 mRNAs showed similar expression patterns compared to DEGs analysis(Fig.15), and 9 out of 11 miRNAs also showed very similar patterns compared to DEMs analysis(Fig.16).

In stem tuber plant, the formation of tuber is based on successive gene expression during development, and the expression of genes depends on specific tissues or developmental stage by transcriptome analyze, such as Solanum tuberosum, Miscanthus lutarioriparius, Raphanus sativus, Nelumbo nucifera and Aconitum heterophyllum[ 28, 34-37]. Yam is one of the most marketable stem tuber crops. Tuber expansion stage in yam is attractive theoretical model to study the development of underground organs because it is the main storage organ for reservoirs of nutrients. Thus, the understanding of candidate genes involved in the expansion stage is of predominant importance. In this study, 6.6G clean reads were generated and 14238transcripts were identified as significantly differentially expressed in the expansion stage by transcriptome.

Multiple signalling pathways regulation

KEGG enrichment analysis revealed that a large number of genes were involved in the pathways related to MAPK signalling pathway, plant hormone signal transduction, starch and sucrose metabolism and carbon metabolism(Fig.8), which are involved in cell wall biosynthesis, cell proliferation and expansion, nutrient accumulation, primary metabolism and hormone signal transduction[ 34, 38, 39]. Cellular processes are triggered by specific stimuli and hormones involving a series of signal pathways. Calcium signal is a major essential nutrient required for normal growth and development of plants. As a second messenger, it plays a role in a number of fundamental cellular processes like cytoplasmic streaming, thigmotropism, gravitropism, cell division, cell differentiation, photomorphogenesis, plant defense and various stress responses [ 40-42]. A potato Ca²⁺-dependent protein kinase, StCDPK1, which is reported to be expressed in tuberization, was possible involved in potato tuberization by transcriptional activation of some of the genes[ 40, 43]. In addition, the StCDPK1 was impacted by the treatments with JA, ABA, GA[ 43-45],suggested the CDPK is a possible mediator of the response to tuberization stimuli by hormone. Moreover, majority of studies have demonstrated that mRNA abundance of CBL, CBP, GaM were impacted in cell division in the swelling of potato tubers[ 40, 43, 46]. In this study, there was an increased in expansion stage in the expressions of calcium signal related genes, including 27 CDPKs and16 CBPs. Interesting, CBLs were down-regulated(Additional file 1: Table S3). The role of CBL in the root development displayed a strong gene expression in the cell proliferation in root, its expression in root was regulated by auxin and cytokinin[ 47, 48]. These results demonstrated that calcium signal is regulated in expansion stage. In addition, MAPK signal pathways are known to play a central role in cell proliferation, differentiation and hormone, but it is not known to be involved in tuber or root formation[ 49]. In this study, MAPK signal related genes were up-regulated in expansion stage(Additional file 1: Table S3). Cell number and cell size are two key determinants of plant and organ size, while involved in cell division, cell expansion and cell cycles[ 50, 51]. In this study, XTH, expansin, extension, CKS, ftsHs, CDC5, CDC, CDKs, CDKIs involved in cell division, cell expansion and cell cycles were differentially regulated in expansion stage(Additional file 1: Table S3). These differentially regulated genes involved in cell wall and cell cycle metabolism were identified in Arabidopsis, Rehmannia glutinosa, radish[

                 35
              </a>, <a href="#_ENREF_36">
              
                 36
              </a>, <a href="#_ENREF_52">
              
                 52
              </a>].</p>

Transcription factors regulation

The transcription factors have been identified to play an important role in regulation of plant growth, development and secondary metabolism. Majority of transcription factors have been identified to play critical roles in the formation of organ, including MADS, bHLH, GRAS. In sweetpotato, MADS were preferentially expressed in root at the initiation of tuberization, and in the vascular cambium region where the active cell proliferation regulated by jasmonic acid, cytokinins and stress response[ 53, 54]. In Arabidopsis, bHLH family, like PIF3, PIF4, PIF5, key regulators for cell elongation during seedling development, is antagonistically regulated by light and gibberellins [ 55]. GRAS transcription factor family specifically regulated the initiation of axillary meristem in Arabidopsis, like SCARECROW (SCR) was expressed in cortex/endodermal initial cells in root system and played key role in regulating the radial organization of the root[ 56]. DELLA, such as RGA and GAI controlled cell expansion and cell division in hypocotyl, shoot and root, and floral induction[ 57]. In this study, a lot of transcription factor genes were found differently expression in expansion stage at the transcript level, including 66MYB, 64AP2-EREBP, 52bHLH, 37WRKY(Fig.10), and REVEILLE 6-like were identified to up regulated in circadian rhythm pathway. Zinc finger CCCH domain-containing protein 14 is involved up regulation in starch and sucrose metabolism pathway(Fig.10) ,which play a variety of important roles in growth and development, hormone response, and response to biotic and biotic stresses[

                 58
              </a>]. 3 DELLA genes were up regulated  in GA pathway(Fig.10), which may play key regulatory
           functions in expansion stage, and are involved in cell division and expansion[<a href="#_ENREF_57">
              57</a>]. All the data suggest that these transcription factors may be potentially involved
           in the expansion stage.</p>

Hormonal signaling regulation

A set of transcriptome evidences indicated that hormone signal pathway related-genes were enriched in regulation of the onset and formation storage organ in sweetpotato, potato, ridish, lotus and carrot, while may be potentially involved in cell division, differentiation and expansion[ 1, 35, 37, 59, 60]. In KEGG pathway annotation, the hormone signal pathway was the most enrich one, which was involved in 8 signal pathways (Fig.8). In radish, most Aux/IAA ,ARFs and SAUR genes were abundant expressed in the root cortex splitting and expanding stage, implying that these transcripts may be involved in cell expansion in the cambium[ 35].Among auxin signal related genes, Aux/IAA, ARFs were highly expressed in expansion stage in this study(Additional file 1: Table S2), and auxin has been identified to regulate cell division and expansion by changing genes expression[ 1, 61], implying that Aux/IAA, ARFs may be provided excellent candidates in expansion stage.

Interestingly, in this study, DELLA genes were up regulated, while GIDI, like GID1 and GID2 were down regulated in expansion stage(Additional file 1: Table S3). DELLA proteins are nuclear transcriptional regulators, which repress GA signal and restrict plant growth presumably by causing transcriptional reprogramming. Binding of GA to GID1 enhances the interaction between GID1 and DELLA, resulting in rapid degradation of DELLAs via the ubiquitin proteasome pathway. The GA-GID1-DELLA signal controleds hypocotyl land root elongation by reducing GA levels, DELLA interacted directly with PIF4, PIF3 and SCL to mediate crosstalk between GA and light signal[ 55, 62]. The DELLA were significantly expressed in rhizome development processed by GA mediated, implying these DELLA could regulated rhizome expansion [

                 37
              </a>].Moreover, GA, IAA and ethylene affected cell growth in the root by DELLA proteins[<a href="#_ENREF_57">
              
                 57
              </a>].Overall, these results imply that hormone signal related genes have complexity of
           regulatory networks involved in the expansion stage of tuber.</p>

Starch and sucrose metabolism regulation

Starch and sucrose is considered as one of the major carbohydrates, which the expansion tuber is high coordination with starch and sucrose metabolism genes in potato,radish and lotus by transcriptome analyze [

              22
           </a>, <a href="#_ENREF_35">
           
              35
           </a>, <a href="#_ENREF_37">
           
              37
           </a>], In this study, alpha-amylase, beta-amylase and isoamylase were up-regulated(Additional
           file 1: Table S3) ,this results are similar to that beta-amylase activity was increased in the swelling taproot in the radish[<a href="#_ENREF_35">
              
                 35
              </a>],where starch content decreased in tuber expansion stage[<a href="#_ENREF_15">
              
                 15
              </a>], and SS was down-regulated in the expansion stage(Additional file 1: Table S3).
           Some sucrose metabolism genes were detected in expansion stage, including SuSy, SPS,
           INV and invertase inbibitor(Additional file 1: Table S3). Evidence shows that sucrose
           could invert into starch in storge root by SuSy and AGPase, implying that they play
           a key role in the early expansion stage in radish[<a href="#_ENREF_35">
              
                 35
              </a>]. The SuSy and SPS genes were down and up regulated in the expansion stage, respectively.
           The results were similar  to taproot expansion stage in radish [<a href="#_ENREF_35">
              
                 35
              </a>],implying they may play a major in the tuber expansion stage in yam. Yam tuber morphology
           were significantly correlated with sucrose and starch in tuber expansion stage[<a href="#_ENREF_15">
              15</a>]. Therefore, these starch and sucrose metabolism genes were required for the tuber
           expansion development.</p>

The regulation miRNAs by targeting potential genes

miRNA mediated gene regulation has been extensively studied in root and tuber development by transcriptional and posttranscriptional levels, which is better understanding of the molecular regulatory networks in tuber expansion stage. In this study, majority of miRNA were down regulated in expansion stage(Table 3). In maize leaf, miR160 were significantly up regulated in the meristem relative to the elongation and mature zones[

                 63
              </a>], however, miR160 was down regulated in yam tuber expansion stage(Table 3). In general,
           miR160 expression forms are differently in different plant. In this study, some miRNA-mRNA pairs were observed in expansion stage(Fig.13), miR160 was involved in root cell
           division and differentiation by regulating auxin response factors(ARFs), and influenced
           root development in Arabidopsis thaliana[<a href="#_ENREF_30">
              
                 30
              </a>]. miRNA160  was down regulated in expansion stage, auxin response factor ARF18 andARF17
           were up regulated in auxin signal transduction(Fig.13), while over expressed miR160
           in transgenic rice not only were down regulated the expressions of ARF10,ARF16, and
           ARF17,but also inhibited root cap cell differentiation, lost control of cell division
           and led to ectopic expansion of apical stem cell populations[<a href="#_ENREF_64">
              
                 64
              </a>]. Former study revealed that the ARFs targeted by miRNA160, were identified to be
           involved in cell expansion and cell differentiation in radish root and potato tuber[<a href="#_ENREF_28">
              28</a>]. In addition, miRNA396 played important roles in root growth and inhibited leaf
           cell division by UV-B radiation[<a href="#_ENREF_65">
              65</a>, <a href="#_ENREF_66">
              66</a>]. miR535 were expressed  in fruit development[<a href="#_ENREF_67">
              67</a>]. miRNA160, miRNA396 and miR535 may be involved in complexity network to regulate
           cell division and differentiation in expansion stage. </p>

The regulatory networks associated with tuber expansion

Tuber development and response to environment are involved in gene regulatory networks[

                 21-24
              </a>, <a href="#_ENREF_34">
              
                 34-37
              </a>]. miRNA and target genes has been extensively studies in tuber development[<a href="#_ENREF_21">
              
                 21
              </a>, <a href="#_ENREF_28">
              
                 28
              </a>], which greatly advanced our understanding of molecular regulatory networks underlying
           the tuber expansion development in yam. In  this study, a putative model of regulatory
           network associated with yam tuber expansion was proposed according to our transcriptome
           and small RNA analysis, previous other tuber crop transcriptomics analysis and research
           results(Fig 13). In detail, the CBL, CBP, GaM were found to be impacted in cell division
           and  proliferation in the expansion of potato tubers[<a href="#_ENREF_40">
              40</a>, <a href="#_ENREF_43">
              43</a>, <a href="#_ENREF_46">
              46</a>], The role of CBL and <em>StCDPK1</em> expressions were regulated by hormone in taproot development [<a href="#_ENREF_43">
              43-45</a>, <a href="#_ENREF_47">
              47</a>, <a href="#_ENREF_48">
              48</a>]. In this study, CDPKs, CBPs and CBLs were extensively expressed in the expansion
           stage. MAPK signal related genes were up-regulated in the expansion stage. MAPK signal
           pathway increased PI3K pathway, and then led to the increase of GA levels, generating
           calcium signal[<a href="#_ENREF_68">
              68-70</a>]. Moreover, MAPK signal pathways were known to play a central role in cell proliferation,
           differentiation and hormone [<a href="#_ENREF_50">
              50</a>, <a href="#_ENREF_51">
              51</a>]. XTH, expansin, extension, CKS, ftsHs, CDC5, CDC, CDKs, CDKIs involved in cell division,
           cell expansion and cell cycles were differentially regulated in expansion stage, the
           finding is similar that these differentially regulated genes involved in cell wall
           and cell cycle metabolism were identified in Arabidopsis, <em>Rehmannia glutinosa</em>, radish[<a href="#_ENREF_35">
              
                 35
              </a>, <a href="#_ENREF_36">
              
                 36
              </a>, <a href="#_ENREF_52">
              
                 52
              </a>]. Evidence shows that DELLA interacted directly with bHIH and SCL to mediate cell
           division in hypocotyl, shoot and root, and floral crosstalk between hormone and signal[<a href="#_ENREF_55">
              
                 55
              </a>, <a href="#_ENREF_62">
              
                 62
              </a>].The DELLA were significantly expressed in rhizome development processed by GA mediated,
           implying these DELLA could regulated rhizome expansion[<a href="#_ENREF_37">
              
                 37
              </a>]. DELLA can interact with PIF3, PIF4 to impacted the expansion genes expression in
           hypocotyl elongation [<a href="#_ENREF_71">
              
                 71
              </a>]. In sweetpotato, MADS were preferentially expressed in root at the initiation of
           tuberization, and in the vascular cambium region where the active cell proliferation
           regulated by jasmonic acid, cytokinins and stress response[<a href="#_ENREF_53">
              53</a>, <a href="#_ENREF_54">
              54</a>]. Among auxin signal related genes, Aux/IAA, ARFs were enriched express in the enlargement
           stage in this study. most Aux/IAA, ARFs and  SAUR genes were enrich expressed in the
           root cortex splitting and expanding stage in radish, implying that these transcripts
           may be involved in cell expansion in the cambium[<a href="#_ENREF_35">
              35</a>]. SuSy, SPS, INV, SS and beta-amylase, were high correlated with in the expansion
           tuber, like potato, radish and lotus[<a href="#_ENREF_22">
              
                 22
              </a>, <a href="#_ENREF_35">
              
                 35
              </a>, <a href="#_ENREF_37">
              
                 37
              </a>]. dioscorinns, the major storage proteins in yam tubers, were significantly up-regulated
           in expansion stage. In this study, some miRNA and target genes were observed in tuber
           expansion stage in yam, the ARF(target by miR396), the bHLH(target by miR5021), the
           SBP(target by miR535a), and the ARFs targeted by miRNA160, were identified to be involved
           in cell expansion and cell differentiation in radish root and potato tuber[<a href="#_ENREF_28">
              28</a>, <a href="#_ENREF_35">
              35</a>].To sum up, environmental factors is the first signal to stimulate the  tuber growth,
           the signal transcduction pathways(hormone, calcium and MAPK signalling ) and metabolism
           possesses(cell wall, starch and sucrose metabolism) are attributed to cell  differentiation
           division, expansion by certain specific genes. Taken together, the result implied
           that these DEGs could play key roles in the regulatory network of tuber expansion.
           </p>

A total of 14238 transcripts expressed differentially in expnasion stage of yam were firstly identified using transcriptome technology. These results reveal the coordination of the tuber involved in the processes of cell events, metabolism, biosynthesis and signal transduction pathways at the transcriptional level. Noteworthy, the integrated analysis of miRNA-mRNA were identified to be preferentially expressed in auxin signal in expansion stage, highlighting the involvement of miRNA-mRNA in the hormone regulation of tuber expansion development. Taken together, the transcriptome and miRNA datasets presented here identified that a subset of candidate genes and miRNA putatively were assocaited with tuber enlargement in yam, thus a hypothetical model of genetic regulatory network associated with tuber expansion in yam was put forward, which may provide a foundation for molecular regulatory mechanism researching on enlargement development in Dioscorea species.

Plant Material

D. opposite cultivar Guihuai 16 was planted at the farm of Guangxi University. Tuber samples were collected when tuber initiation after field planting approximately 40 days(initiation stage), additional samples were collected at 60days(expansion stage ). Two types of tuber developmental stages including initiation and expansion stage were used for the construction of various libraries, the three types of libraries were constructed, including one transcriptome denovo analysis libraries for mixed sample of initiation and expansion stage(named Total_1), six RNA-seq analysis libraries(initiation stage named GH16_I, expansion stage named GH16_E), and small RNA analysis(initiation stage named C, and expansion stage named T in original data, in order to be consistent with the transcriptome data analyse, we were rename GH16_I, GH16_E for initiation and expansion stage,respectively). For three biological replicates of each of initiation and expansion stage were studied, respectively.

Construction and sequencing of transcriptome de novo, RNA-seq and small RNA libraries

For information about sample pooling, It can found the subsections of plant materials above. Total RNA were isolated from tuber samples (initiation and expansion stage) using MiniBEST reagent(TaKaRa), and RNA integrity was assessed by an Agilent 2100 BioAnalyzer. Construction of the transcriptome de novo and RNA-seq libraries were followed the manufacturer’s protocols. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, fragmented, and reverse transcribed into cDNA, and cDNA were enriched by cycles of linear PCR to obtain final cDNA libraries. Finally, sequencing by synthesis was performed using a BGISEQ-500 sequencing platform. For the small RNA libraries, RNA bands of around 18-30nt in length were isolated. Libraries were prepared according to the small sample preparation protocol, and were sequenced with BGISEQ-500 sequencing platform. The raw reads from mRNA and small RNA were subjected to quality control(QC) to harvest high quality of sequencing data. Raw reads were further filtered by a few of steps to obtain clean reads.

Processing and mapping of mRNA-Seq and small RNA libraries sequencing data

After filtering, the clean reads were performed de novo assembly to obtain unigenes, and functional databases are NT, NR, GO, KOG, KEGG, SwissProt and InterPro used to annotate unigene function, Blastn, Blastx, Diamond ,Blast2GO and InterProScan5 were aligned unigenes to these functional data. RNA-seq reads were mapped to the transcriptome de novo analysis data using bowtie2 results with default parameters after preprocessing mRNA-seq data [ 72]. Gene expression levels were presented as FPKM values [ 73], genes with expression levels >1 FPKM were retained for further analysis. Small RNA reads also were screened from raw sequencing reads by removing adaptors, poly A sequences, and low quality bases, sequences shorter than 18nt or longer than 32nt, after trimming, were removed. The high quality clean reads were mapped to the reference genome and to other sRNA databases by Bowtie, Cmsearch and Rfam [ 74].

Identification of known and novel miRNAs, and prediction of the targets of miRNAs

To identify the isoforms of known miRNA, miRProf tool was run to with clean sRNA reads, and the conserved miRNAs were identified with the known plant miRNAs registered in miRBase[ 75]. To identify novel miRNAs and their precursors, the unique reads were submitted to the RIPmiR[ 76]. The potential miRNA targets were predicted using the psRoot and Target Finder[ 77].

Identification and function annotation of differentially expressed genes and miRNAs

The expression levels of mRNA transcripts were measured as fragments per kilobase of exon per million fragments mapped(FPKM), whereas miRNA read counts were normalized to tags per million(TRM). The differentially expressed genes(DEGs) and miRNAs(DEMs) were identified by DEGseq[ 78]. Therefore, DEGs and DEMs were identified by stringent threshold(|log2 (ratio) | >1). All DEGs were subjected to gene ontology and KEGG pathway analysis. Each DEG in tuber was predicted by aligning the sequences of genes against the Plant Transcriptional Factor Database. The DEGs were classified according to their TF families.

Validation of the DEGs and DEMs data using RT-qPCR

To identify the accuracy and reliability of transcriptome and miRNA data, RT-qPCR was used to measure the expressions of DEGs and DEMs. Total RNA used for RNA-seq and small RNA analysis previously was reversely transcribed into cDNA with PrimeScript™ RT reagent kit and Mir-X™ miRNA first strand synthesis kit (Takara, Dalian, China), separately, according to the manufacture methods. RT-qPCR were performed using primers (Table S6, S7) on real-time PCR detection system (BIO-RAD). For DEGs quantification expression detected by the TB Green^™ Premix Ex Taq^™ II (Tli RNaseH Plus) (Takara, Dalian, China), 10 μl reaction solution containing 1x Green TB Green TB Premix Ex Taq II, 10μM primer, one-third dilution of the cDNA sample, and then the reaction conditions were: 30s at 95°C followed by 40 cycles of 30 s at 95°C, and 30 s at 60°C. For DEMs quantification expression were detected by the Mir-X miRNA qRT-PCR SYBR kit, 25μl reaction solution containing 2X SYBR advantage premix, 50XROX dye, 10μM miRNA-specific Primer, 2μl cDNA sample, and then the reaction conditions were: 10s at 95°C followed by 40 cycles of 5s at 95°C, and 20s at 60°C. All mRNAs and miRNA expressions had three biological replicates with three technical replicates for each of biological replicate, ACTIN and U6 genes were used as reference genes, then relative expression levels were collected by the comparative Ct protocol.

DEG: Differentially expressed gene; DEM: Differentially expressed microRNA; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; miRNA: MicroRNA; qRT-PCR: Real-time quantitative RT-PCR; RNA-Seq: RNA sequencing; TF: Transcription factor

Acknowledgements

Not applicable

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81860670), the Guangxi Innovation Team Project of Tubers of Modern Agricultural Industrial Technology System of China(nycytxgxcxtd-11-01), and the Guangxi Natural Science Foundation of China (Grant No. 2018GXNSFDA281029).

Availability of data and materials

The materials of this study were provided by College of Agriculture at Guangxi University. Correspondence and requests for materials should be addressed to Long-Fei He ([email protected]). The raw sequencing data have been submitted to the NCBI SRA database (PRJNA533985).

Authors'contributions.

DX and LFH designed experiments. YYZ and SZL analyzed the sequencing data for transcriptome assembly. JZ and AQW developed the qPCR experiments. YYZ and LFH wrote the manuscript

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

We declare that the research was conducted in the absence of any commercial or financial relationships that could be a potential conflict of interest.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author details

ACollege of Agriculture, Guangxi University, Nanning 530004, PR China. BGuangxi Key Laboratory for Agro-Environment and Agro-Product Safety, Nanning 530004, PR China. CGuangxi Colleges and Universities Key Laboratory of Crop Cultivation and Tillage, Nanning 530004, PR China

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Table 1 GO enricher analysis of DEGs

Type	ID	Term	Gene Number	Rich Ratio	P value
Molecular function	GO:0005198	Structural molecule activity	183	0.62	1.83867E-07
	GO:0003735	Structural constituent of ribosome	143	0.61	1.71036E-05
	GO:0003700	DNA binding transcription factor activity	116	0.59	0.001099451
Cellular component	GO:0005576	Extracellular region	91	0.70	2.22612E-07
	GO:0043228	Non-membrane-bounded organelle	285	0.58	1.68443E-06
	GO:0043232	Intracellular non-membrane-bounded organelle	285	0.58	1.68443E-06
	GO:0005840	Ribosome	157	0.61	1.90586E-05
	GO:0005618	Cell wall	49	0.72	4.36888E-05
	GO:0030312	External encapsulating structure	49	0.72	4.36888E-05
	GO:0048046	Apoplast	32	0.78	0.000075892
	GO:0022625	Cytosolic large ribosomal subunit	22	0.81	0.000355453
Biological process	GO:0044262	Cellular carbohydrate metabolic process	77	0.69	1.68372E-05
	GO:0005975	Carbohydrate metabolic process	171	0.60	0.00025216
	GO:0044264	Cellular polysaccharide metabolic process	53	0.70	0.000285538
	GO:0045786	Negative regulation of cell cycle	11	1.00	0.000452873
	GO:0006073	Cellular glucan metabolic process	50	0.69	0.000495823
	GO:0010383	Cell wall polysaccharide metabolic process	15	0.88	0.001069447
	GO:0010410	Hemicellulose metabolic process	15	0.88	0.001069447
	GO:0010411	Xyloglucan metabolic process	13	0.93	0.000841262

Table 2 Functional classification and pathway assignment of differentially expressed DEG by GO in expansion stage

Tissue development [GO:0009888]	Gene ID	log2(GH16_E/GH16_I)
Actin-related protein 3	CL1179.Contig2_Total_1	-2.00
Anaphase-promoting complex subunit 10	CL1997.Contig4_Total_1	1.82
Alpha-tubulin	CL28.Contig3_Total_1	-5.08
Tubulin alpha chain	CL3054.Contig1_Total_1	-1.81
Glutamine synthetase nodule isozyme	Unigene128_Total_1	-4.98
Phosphoenolpyruvate carboxylase 2	Unigene13453_Total_1	-1.23
Phosphoenolpyruvate carboxylase 2	Unigene13455_Total_1	1.32
Anaphase-promoting complex subunit 10	Unigene18039_Total_1	-1.15
Anaphase-promoting complex subunit 6	Unigene2600_Total_1	-1.06
Actin-related protein 2	Unigene4771_Total_1	-1.10
ATPase ASNA1 homolog	Unigene5972_Total_1	-1.32
Homeobox protein knotted-1-like 3	Unigene6778_Total_1	3.48
Root morphogenesi,root system development,root development [GO:0009888,GO:0022622,GO:0048364]
Cytoplasmic tRNA 2-thiolation protein 2	CL1237.Contig1_Total_1	-1.02
Cytoplasmic tRNA 2-thiolation protein 2	CL1237.Contig2_Total_1	-2.28
Mediator of RNA polymerase II transcription subunit 32	CL2787.Contig2_Total_1	2.02
Succinate dehydrogenase assembly factor 2	CL3034.Contig1_Total_1	-1.04
Guanine nucleotide-binding protein	Unigene18752_Total_1	-3.18
Enhanced ethylene response protein 5	Unigene2193_Total_1	-1.75
ATPase ASNA1	Unigene5972_Total_1	-1.32

Table 3 The differentially expressed miRNAs involved in expansion stage

miRNA id	Read count(GH16_I)	Read count(GH16_E)	Expression(GH16_I)	Expression(GH16_E)	log2Ratio(GH16_E/GH16_I)	P value
novel_mir43	54974	33041	3682.196667	1731.716667	-1.97	0
novel_mir35	52437	32873	3515.556667	1728.51	-1.91	0
miR160a-5p	18032	121	1171.813333	6.423333333	-8.45	0
miR168a-5p	55653	59949	3708.9	3153.816667	-1.13	0
miR168	20208	106873	1334.103333	5618.38	1.17	0
novel_mir1	16275	7760	1085.586667	407.58	-2.30	0
miR396b	1097	21915	72.84333333	1176.08	3.09	0
miR535a_1	24978	25221	1654.31	1315.066667	-1.22	0
miR396a-5p	12295	7317	816.34	374.44	-1.98	0
novel_mir33	6408	4269	425.4933333	224.5533333	-1.82	0
novel_mir15	9666	10433	642.81	549.1166667	-1.12	0
novel_mir11	4852	3769	323.0733333	199.1266667	-1.60	0
novel_mir25	5833	6318	386.43	333.3366667	-1.12	0
novel_mir32	3196	2381	212.7166667	125.7166667	-1.66	0
novel_mir5	2039	1094	135.3133333	58.02666667	-2.13	0
novel_mir50	2777	2281	183.85	120.27	-1.52	0
novel_mir10	3197	3009	213.0166667	158.4866667	-1.32	0
novel_mir44	3381	3299	224.6533333	173.7233333	-1.27	0
novel_mir36	1859	1246	124.13	65.29333333	-1.81	#######
novel_mir31	1632	1049	108.56	55.05666667	-1.87	#######
novel_mir18	1701	1305	114.0466667	68.55	-1.62	#######
novel_mir19	1491	1071	99.14666667	56.34666667	-1.71	#######
novel_mir12	1666	1308	111.28	69.05	-1.58	#######
novel_mir45	1478	1093	99.12	57.63	-1.67	#######
miR160	926	451	62.61	24.59	-2.27	#######
novel_mir48	1107	690	74.52	36.20666667	-1.92	#######
novel_mir26	1488	1201	99.78666667	63.01	-1.54	#######
miR160b_1	463	30	30.26666667	1.616666667	-5.18	#######
novel_mir46	1268	926	83.82	48.75333333	-1.69	#######
novel_mir42	694	275	46.22333333	14.49666667	-2.57	#######
novel_mir49	676	265	45.07333333	13.82333333	-2.59	#######
novel_mir24	1045	779	69.79	40.94	-1.66	#######
novel_mir23	377	109	25.15666667	5.676666667	-3.03	#######
miR168a-3p	1330	1489	87.83	78.66333333	-1.07	#######
novel_mir20	374	2206	24.76333333	116.7666667	1.33	#######
novel_mir30	964	999	63.39	52.50333333	-1.18	#######
novel_mir37	173	45	11.51666667	2.37	-3.18	#######
novel_mir47	90	10	6.033333333	0.504	-4.40	#######
novel_mir4	394	447	25.80333333	23.69666667	-1.05	#######
novel_mir34	10	200	0.677333333	10.7	3.09	#######
novel_mir8	197	207	13.12	10.72366667	-1.16	#######
miR168_1	30	5	1.976666667	0.260333333	-3.82	#######
novel_mir9	123	142	8.243333333	7.476666667	-1.03	#######
miR160h_1	7	0	0.463333333	0.001	-5.04	0.00041

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Integrated mRNA and miRNA transcriptome analysis on the regulational of mechanism tuber expansion in Yam

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