Genome-wide analysis of small RNAs reveals eight fiber elongation-related and 257 novel microRNAs in elongating cotton fiber cells
© Xue et al.; licensee BioMed Central Ltd. 2013
Received: 5 February 2013
Accepted: 6 July 2013
Published: 17 September 2013
MicroRNAs (miRNAs) and other types of small regulatory RNAs play critical roles in the regulation of gene expression at the post-transcriptional level in plants. Cotton is one of the most economically important crops, but little is known about the roles of miRNAs during cotton fiber elongation.
Here, we combined high-throughput sequencing with computational analysis to identify small RNAs (sRNAs) related to cotton fiber elongation in Gossypium hirsutum L. (G. hirsutum). The sequence analysis confirmed the expression of 79 known miRNA families in elongating fiber cells and identified 257 novel miRNAs, primarily derived from corresponding specific loci in the Gossypium raimondii Ulbr. (G. raimondii) genome. Furthermore, a comparison of the miRNAomes revealed that 46 miRNA families were differentially expressed throughout the elongation period. Importantly, the predicted and experimentally validated targets of eight miRNAs were associated with fiber elongation, with obvious functional relationships with calcium and auxin signal transduction, fatty acid metabolism, anthocyanin synthesis and the xylem tissue differentiation. Moreover, one tasiRNA was also identified, and its target, ARF4, was experimentally validated in vivo.
This study not only facilitated the discovery of 257 novel low-abundance miRNAs in elongating cotton fiber cells but also revealed a potential regulatory network of nine sRNAs important for fiber elongation. The identification and characterization of miRNAs in elongating cotton fiber cells might promote the further study of fiber miRNA regulation mechanisms and provide insight into the importance of miRNAs in cotton.
KeywordsCotton Comparative miRNAome analysis Fiber cell elongation High-throughput sequencing miRNAs tasiRNA
Cotton (Gossypium hirsutum L.) is one of the most economically important crops and provides the largest renewable source of material for the textile industry. Cotton fiber is a single-cell trichome derived from the epidermal cells of the ovule, and its morphogenesis is comprised of four overlapping developmental stages: initiation, elongation, secondary wall thickening, and maturation . After initiation on the day of anthesis, single-celled fibers undergo rapid elongation (approximately 15 days) and secondary wall deposition (approximately 20 days), followed by maturation into spinnable fibers . One of the most significant features in fiber development is that the rate of fiber elongation and the length of the mature fiber are much greater than those commonly observed for plant cells; the cotton fiber is likely the fastest growing and the longest single cell in higher plants [3, 4]. Thus, the cotton fiber is an excellent model in which to study the molecular mechanisms of plant cell elongation without the interference of cell division and multicellular development. Over the past decade, a number of studies have reported that several important proteins, such as sucrose/K+ transporters , sucrose synthase , vacuolar invertase , kinesin-like calmodulin-binding protein  and calcium dependent protein kinase , play critical roles in the process of rapid fiber cell elongation. In addition to calcium signal transduction, the important second messenger molecule H2O2 might function as a termination signal in cotton fiber cell elongation . Furthermore, recent pioneering experiments revealed that plant hormones play important roles in fiber elongation. Genes involved in auxin and brassinosteroid (BR) signaling are expressed at higher rates in G. barbadense than in G. hirsutum, and these expression patterns have been associated with the development of longer fibers , while the overexpression of the gibberellin 20-oxidase gene (GhGA20ox1) might promote the initiation and elongation of cotton fibers through the regulation of gibberellin (GA) synthesis . Interestingly, many genes encoding these proteins and phytohormone-responsive factors might be potential targets for new types of small molecule miRNAs.
Small RNAs (sRNAs), which range in size from 18 to 24 nt, can regulate gene expression at the transcriptional and/or post-transcriptional level . In plants, there are two classes of endogenous sRNAs: small interfering RNAs (siRNAs) and miRNAs. Endogenous siRNAs consist of repeat-associated siRNAs (rasiRNAs), trans-acting siRNAs (tasiRNAs), and natural antisense siRNAs (nat-siRNAs). Of these, tasiRNAs are in-phase-generated sRNAs that depend on the miRNA-mediated cleavage of transcripts of tasiRNA genes for their production. Both miRNAs and tasiRNAs can trigger the cleavage of mRNAs containing complementary sequences in plants. As a result, miRNAs play fundamental regulatory roles in various aspects of plant development and adaptive responses to stresses, including plant organ development , auxin signaling , growth phase changes , and disease resistance . Similarly, miRNAs have also been implicated in the development of cotton fibers .
A total of 31 miRNA families, including 27 conserved and four novel miRNA families, have been identified in developing cotton ovules using a high-throughput sequencing approach, which showed that the general repression of miRNAs in fibers is associated with the upregulation of a dozen validated miRNA targets that are highly expressed in cotton fibers . Another investigation into cotton fiber sRNAs has also identified more than 100 conserved miRNAs, representing 33 families . In our previous studies, we characterized the dynamic changes of cotton ovule miRNAomes during initiation, ranging from −3 to +3 days pre-/post-anthesis (dpa), and identified seven fiber initiation-related miRNAs . These studies explored the miRNAs that are differentially expressed in the cotton ovules of wild type upland cotton and a fiberless mutant, and the results suggested potential regulatory functions for these miRNAs in cotton fiber initiation. However, there have been no systematic studies of miRNAs expressed during fiber rapid elongation, and the role of miRNAs during cotton fiber elongation remains elusive. A total of 675 plant miRNAs have been identified in the model species Oryza sativa (462) and Arabidopsis thaliana (213) ; however, only 43 miRNAs from Gossypium spp. have been registered in miRBase (version 19.0). It is obvious that there are many miRNAs to be identified and much to learn about the specific roles of miRNAs in cotton fiber cells. Furthermore, the recently published genome sequence of the diploid cotton G. raimondii has facilitated the identification of sRNAs, particularly miRNAs in this genus [22, 23].
In the present study, we performed high-throughput sequencing of cotton fiber sRNAs to identify novel and potential fiber elongation-related miRNAs. Four small RNA libraries were constructed from cotton fibers sampled at four different time points from 5 to 20 dpa, and more than 18 million short RNA sequence reads were generated for each library. The comparative miRNAomic analysis revealed 257 novel miRNAs and nine fiber elongation-associated sRNAs, including eight miRNAs and one tasiRNA, in allotetraploid cotton fiber cells. The predicted targets of the nine sRNAs were experimentally validated and implicated in different cellular responses and metabolic processes, demonstrating the regulatory importance of miRNAs and tasiRNAs in cotton fiber elongation. Thus, this study advances our understanding of the important regulation of miRNAs and tasiRNAs in allotetraploid cotton through the identification of new miRNAs and a putative miRNA-mediated regulatory network supporting cotton fiber cell elongation.
Results and discussion
High-throughput sequencing data overview
Distribution of sequence reads in elongating cotton fiber small RNA libraries
The clean reads were mapped to the diploid cotton G. raimondii genome, generating 4983316, 7113695, 7107883 and 8871116 genome-matched reads from the 5, 10, 15 and 20 dpa fiber libraries, respectively (Table 1). These small RNA reads were grouped into several RNA classes, such as known miRNAs, rRNA, tRNA, snRNA, snoRNA, protein-coding genes and repeats. The remaining unclassified small RNA reads had the highest fraction of unique and total clean reads and likely included new types of regulatory small RNAs and novel miRNAs. For example, in the 10 dpa library, the unclassified small RNA reads accounted for 95.8% and 81.7% of the unique and total clean reads, respectively (Table 1).
Known miRNAs in elongating cotton fiber cells
Currently, miRBase (version 19.0, released on August 1, 2012) lists just 43 mature miRNA genes belonging to 21 miRNA gene families as cloned or predicted to be expressed in cotton. Based on our previous work, 36 new miRNAs from cotton ovules were officially named by miRBase and included in the subsequent release . To identify the known miRNAs, the clean reads were subjected to a BLASTn search for known miRNA sequences deposited in miRBase (version 19.0) and the newly identified cotton miRNAs . Nearly 6000 unique sequences of known miRNAs belonging to 79 families were obtained from these four sRNA libraries (Additional file 1: Table S1). In addition to the 21 known cotton miRNAs families in miRBase (version 19.0) and 23 miRNAs families described in the previous study , 35 known miRNA families were also identified based on their conservation in other plant species. In total, the 79 miRNA families included not only nearly all the known miRNA families previously identified in cotton [18, 20] but also all 24 miRNA families that are conserved between eudicotyledons and monocotyledons .
To accurately measure miRNA read abundances, the abundances of known miRNAs were normalized to reads per ten million (RPTM). The four miRNA families with the greatest abundance were 167 (164706 RPTM), 156/157 (86430 RPTM), 165/166 (71375 RPTM), and 894 (39985 RPTM), accounting for 41.3%, 21.7%, 17.9% and 10.0% of all the known miRNA reads, respectively. The miRNA families 164, 168, 3476, 7508, 172, 390, 535, 159/319, 2911, 2950 and 396-3p also exhibited average abundances of more than 1000 RPTM. In contrast, certain miRNA families were expressed at low abundance (Additional file 1: Table S1). The varied abundances of the miRNAs in different families suggested that these miRNA genes are differentially transcribed during fiber elongation.
A total of 213 potential miRNA gene loci, belonging to 48 miRNA families, were identified based on the EST sequences (CGI11), genome survey sequences (GSS) and G. raimondii genome sequences using mireap_0.2 (Additional file 2: Table S2), comprising 37 of the 43 cotton miRNA genes in miRBase (version 19.0). Of the 213 potential loci, 174 originated from the G. raimondii genome sequence, including 32 of the 43 Gossypium precursors deposited in miRBase (19 version); 34 loci originated from CGI11; and six loci originated from GSS, suggesting that many miRNA genes with low expression were absent from the ESTs database (CGI11) and that the G. raimondii genome sequences could be used for the analysis of miRNA precursors identified in G. hirsutum high-throughput small RNA sequencing (Additional file 2: Table S2). Furthermore, as shown in Additional file 3: Figure S1, 139 novel precursors of known miRNAs (i.e., precursors not present in miRBase version 19) were identified.
Of the 48 known families with precursors, 26 had miRNA* sequences, providing evidence for a DCL1-processed stem-loop (Additional file 1: Table S1 and Additional file 2: Table S2). As expected, the majority of miRNAs showed dominant abundance of one arm of the stem loop, suggesting that the miRNA* on the complementary arm was degraded quickly after biogenesis . However, for miR162, miR169, miR2111 and miR393, similar numbers of 5p and 3p reads were observed. Furthermore, miR160-3p and miR396-3p were even more abundant than miR160-5p and miR396-5p (Additional file 1: Table S1); similar phenomena were also reported in a study of the small RNAs in Brassica napus.
The sequence of the most abundant miRNA in each miRNA family (i.e. the representative miRNA sequence) is shown in the “Sequence” column of Additional file 1: Table S1. However, a subset of the representative sequences diverged among the different time-course small RNA libraries (superscript d to j in Additional file 1: Table S1). In fact, many known miRNA families not only had multiple loci but also certain members that varied by few nucleotides (“location” and “miRNA sequences” columns in Additional file 2: Table S2). The miR156 (six members), miR159/319 (four members), miR165/166 (five members), miR167 (three members), miR169_1 (six members), miR169_2 (five members), miR172 (five members), miR396 (three members), miR399 (six members), miR477 (six members), miR482 (seven members) and miR7504b (three members) families all contained more than two distinct members (“member” column in Additional file 2: Table S2). The representative sequence of miR167 from 5–15 dpa was “UGAAGCUGCCAGCAUGAUCUCA” and contained an adenylation modification at the 3’ end of the mature miRNA compared to the representative sequence of miR167 shown in Additional file 1: Table S1. This adenylation might protect the miRNA from degradation . Hence, the presence of this highly abundant and stable form of miR167 might indicate a crucial role for miR167 in cotton fiber elongation. Similarly, the representative sequences of miR172 and 397 differed between 5–15 dpa and 20 dpa. The representative sequences of miR172 and miR397 at 5–15 dpa were “AGAAUCCUGAUGAUGCUGCAG” (83 in “index” column in Additional file 2: Table S2) and “UCAUUGAGUGCAGCGUUGAUG” (118 in “index” column in Additional file 2: Table S2), whereas the representative sequences at 20 dpa were “AGAAUCUUGAUGAUGCUGCAU” (79 and 81 in “index” column in Additional file 2: Table S2) and “UUGAGUGCAGCGUUGAUGAGC” (45 in “index” column in Additional file 2: Table S2), respectively. This observation suggested that the members of a known miRNA family are expressed differentially during different stages of cotton fiber elongation.
Newly identified miRNAs in elongating cotton fibers
The 5’ terminal nucleotides and lengths of the 257 novel cotton miRNAs
miRNA length (nt)
Cotton fiber elongation-related miRNAs
A newly identified tasiRNA in cotton
Similar to the gene structure in Arabidopsis, the tasiRNA-generating locus in G. hirsutum is flanked by two miR390 binding sites (Figure 3A), and most of the tasiRNAs were 21 nt long (Additional file 9: Figure S6). The most abundant sense strand-produced sRNAs were produced at the 5’D7 (+) position (Figure 3A), similar to the pattern observed in Arabidopsis, suggesting that the tasiRNA biogenesis pathway is highly conserved between plant species.
We analyzed the expression patterns of DW503626 5’D7 (+), considered a homolog of functional tasiRNAs in Arabidopsis, during fiber development based on the sequence number. The accumulation of tasiRNA increased from 5 to 10 dpa and decreased from 10 to 20 dpa (Figure 3B). Target prediction revealed that genes of the Auxin Response Factor (ARF) family, such as ARF2 (TC239405), ARF3 (TC232322), and ARF4 (TC251617), were putative targets of tasiRNAs. The in vivo cleavage of ARF4 (TC251617) transcripts was detected using RNA ligase-mediated (RLM) 5’ RACE (Rapid Amplification of cDNA Ends) (5’-RLM-RACE) (Figure 3C). Furthermore, the expression of ARF4, as determined using quantitative RT-PCR (qRT-PCR), was negatively correlated with tasiRNA accumulation (Figure 3B), suggesting that its expression was strongly regulated by tasiRNA during fiber development.
tasiR-ARF, a conserved tasiRNA that targets ARFs in plant species, was required for proper leaf polarity development [31, 32] and the suppression of the juvenile-to-adult phase transition in Arabidopsis . In developing cotton fibers, tasiRNA repressed ARF4 during the rapid elongation phase (Figure 3B). It remains unclear whether this regulatory pathway contributes to fiber elongation, although ARF genes are crucial components of the auxin-signaling pathway and have been suggested to play an important role in fiber development . Nevertheless, this represents the first identification of a tasiRNA in G. hirsutum, which expands the plant tasiRNA family and demonstrates its conservation among different species.
Target prediction, validation, and expression correlated with miRNAs
To characterize the functions of miRNAs with differential RPTM in cotton fiber elongation, we first predicted and experimentally validated their targets. Using the psRNATarget Web server (http://plantgrn.noble.org/psRNATarget/), the targets of the miRNAs shown in Figure 2A were determined (Additional file 10: Table S4 and Additional file 11: Table S5). Subsequently, 5’-RLM-RACE was performed to obtain the target cleavage transcripts. To maximize the detection efficiency of miRNA-triggered target cleavage, RNAs extracted from 5, 10, 15 and 20 dpa fibers were separately ligated to a 5’ RACE adaptor and used to construct RACE libraries. The RACE library with the greatest abundance of a particular miRNA (according to sequencing reads) was used to validate the corresponding target.
As shown in Figure 4A, the cleavage products of TC241907 (ARF8) and TC236756 (MYB) were precisely terminated at the tenth position from the 5’ end of the complementary region of each associated miRNA. However, the TC246633 (LRR-RLK) sequences were cleaved three nucleotides downstream of the predicted site (Figure 4A), which could reflect further nuclease-mediated degradation of the targets after miRNA-mediated cleavage . Two cleavage sites were found for TC229767 (ACCD), TC259543 (CaMBP), and TC251689 (CIBP). One cleavage site was located downstream of the predicted site. As described for the cleavage product of LRR-RLK, these cleavage products may be further degraded after silencing by the corresponding miRNAs . The other cleavage site was located upstream of the predicted site. This type of cleavage was also found in many other 5’-RLM-RACE experiments [35, 36], indicating that the potential function of the cleavage by these miRNAs warrants further investigation.
The expression of validated miRNA target genes was examined using qRT–PCR to determine whether there was a negative correlation between the accumulation of miRNAs and their targets at various periods during development. As shown in Figure 4B, for the target of Type A GhmiRNAs, TC272934 (GhSPL9) transcript levels were negatively correlated with the accumulation of GhmiR156/157. In contrast to GhmiR156/157 expression, which increased from 5 to 20 dpa, TC272934 (GhSPL9) expression was reduced from 5 to 20 dpa. For the targets of Type B GhmiRNAs, which increased from 5 dpa and reached peak levels at 10 or 15 dpa, the expression level was reduced from 5 dpa to 10 or 15 dpa and subsequently upregulated at 20 dpa. These data suggested that the accumulation of these GhmiRNAs was negatively correlated with the expression of their targets in elongating fibers and showed that the target genes were strictly controlled through these GhmiRNAs. Additionally, MYB, which is a target of miR399, was expressed at low levels and did not meet MIQE guidelines ; thus, no qRT-PCR analysis was performed for this gene.
A potential functional network of fiber elongation-related tasiRNA and miRNAs
For a cotton fiber cell, the precise control of the elongation period is critical. It is not known how the fiber cell knows when to start or end the rapid elongation stage. However, an H2O2 burst at 20 dpa may serve as a termination signal for rapid fiber elongation . Anthocyanins are widely identified in plant species to provide flowers and fruits with color  and protect the plants from damage by active oxygen species . Previous studies reported that the SPL9, targeted by miR156, negatively regulates anthocyanin accumulation by directly preventing the expression of the anthocyanin biosynthetic genes DFR, F3H, and ANS. In this study, the reads of GhmiR156/157 increased gradually from 5 dpa to 20 dpa and were negatively correlated with the level of GhSPL9 expression (Figure 4B). However, the expression levels of the anthocyanin biosynthetic homologous genes in cotton, GhDFR, GhANS, GhF3H, and the levels of anthocyanin in developing fibers were reduced from 5 to 20 dpa, similar to the expression pattern of GhSPL9, suggesting that GhSPL9 might play a positive role in regulating the expression of these genes through an unknown mechanism that might be distinct from that in Arabidopsis (Additional file 12: Figure S7A and B). Hence, GhmiR156/157, via its mediation of the cleavage of GhSPL9 mRNA, might negatively regulate the expression of the anthocyanin biosynthetic genes GhDFR, GhF3H, and GhANS. This in turn may lead to a strong decline in anthocyanin synthesis at 20 dpa, which may be crucial for the H2O2 signaling in fiber elongation termination.
Calcium-mediated signal transduction plays crucial roles in a wide array of growth and developmental processes. In plants, changes in cellular Ca2+ concentration are transmitted through several calcium sensors or calmodulin (CaM) . The obvious inhibition of fiber growth was observed when ovules were cultured in the presence of the CaM antagonist TFP . Here, we identified the calcium sensor CIBP as a target of GhmiR396-5p. Therefore, we speculated that GhmiR396-5p, via its mediation of the cleavage of CIBP mRNA, might promote fiber rapid elongation from 5 dpa. Furthermore, we also identified the calmodulin (CaM)-binding protein CaMBP as a target of GhmiRnC. CaM isoforms, which might bind CaMBP, show high affinity to the kinesin-like CaM-binding motor protein (KCBP), which regulates cotton fiber elongation [8, 42]. Therefore, we speculated that the regulation of CaMBP through miRNAs could play important roles in cotton fiber elongation through Ca2+ signal transduction.
Various studies on cotton fiber cell development have identified plant hormones as critical regulators of fiber development. Auxin promotes the fiber cell development of in vitro cultured ovules, and a positive correlation between final fiber length and in vivo-quantified IAA levels has also been observed . ARF8, a target of GhmiR167, was involved in IAA signal transduction . Previous evidence showed that an exogenous auxin signal was transduced sequentially through miR167 and ARF8 . The reduced expression of GhARF8, which is regulated by GhmiR167 during rapid fiber elongation (Figure 4B), was consistent with the negative roles of AtARF8 in cell expansion during hypocotyl and lateral root elongation [46, 47]. Hence, GhmiR167, via its mediation of the cleavage of ARF8 mRNA, might be critical for rapid fiber elongation. The accumulation of tasiR-ARF exhibited dynamics similar to those of GhmiR167, and its target, ARF4, also showed an expression pattern similar to that of ARF8, which is targeted by GhmiR167 (Figure 3B, 4B). This observation suggests that tasiR-ARF might be involved in fiber elongation, similar to GhmiR167. In addition to auxin, BR has been suggested to promote cotton fiber elongation. Cotton fiber responses to BR are mediated through a plasma membrane-bound leucine-rich (LRR) receptor-like protein kinase known as BRI, which has an expression pattern and domain structure similar to that of LRR-RLK, which is targeted by GhmiR7505 (Figure 4B) . Moreover, we detected significantly higher LRR-RLK expression in G. hirsutum compared with G. arboreum, which has shorter fibers than does G. hirsutum, throughout the elongation phase (Additional file 13: Figure S8). The expression of LRR-RLK was high in 5 to 10 dpa and sharply declined at 15 dpa (Figure 4B), suggesting that GhmiR7505-regulated LRR-RLK might play a positive role in initiating rapid fiber elongation.
The rapid elongation of cotton fiber cells requires substantial lipid synthesis to support the developing organelles and membranes. ACCD, the target of GhmiRnE, encodes the β-carboxyl transferase subunit of acetyl-CoA carboxylase (ACCase, EC 18.104.22.168), which is a regulatory enzyme in fatty acid synthesis . However, fatty acid synthesis should be regulated in the rapid elongation stage of the cotton fiber. A recent report showed that the down-regulation of WRI1, a positive regulator of lipid biosynthesis, reduced carbon flow into fatty acid biosynthesis, including the expression of ACCase; meanwhile, the fixed carbon was channeled into fiber growth, resulting in longer fibers . Hence, the GhmiRnD promoted cotton fiber elongation by mediating the decrease in ACCD expression and diverting more carbon into fiber growth. Unlike many other plant cells, cotton fibers contain little to no lignin in their secondary cell walls [4, 51]. Lignin is primarily deposited into the cell walls of xylem vessel elements, and irregular xylem4 (irx4) mutant plants have 50% less lignin than wild-type plants . The ectopic expression of ATHB-8 in Arabidopsis could promote vascular cell differentiation, specifically the differentiation of xylem tissue [53, 54]. miR166 exhibited strong downregulation in high-lignin tissues (wood) in Acacia mangium, and the expression of the miR166 target HD-ZIP 3 is much higher in high-lignin tissue (compression wood) . The number of reads of GhmiR165/166 in rapid elongation fibers (10 and 15 dpa) was much higher than that in 15 dpa seeds (Additional file 14: Table S6), and the expression of GHHB8 at this stage was clearly repressed (Figure 4B). We speculated that the lower GHHB8 expression mediated by GhmiR165/166 might contribute to fiber elongation by inhibiting the differentiation of high-lignin tissues, such as xylem, because a number of lignification mutants revealed the link bewteen cell expansion and xylem tissue differentiation [56, 57].
In this study, 46 GhmiRNAs were differentially expressed during the cotton fiber elongation progress, 40 (Type of A and B GhmiRNAs) of which were dramatically upregulated during rapid fiber cell elongation (10 and 15 dpa). In Arabidopsis, miRNAs are loaded into AGO1, which acts an RNA slicer and is the target of miR168 [58, 59]. Although no AGO1 (CO084924) cleavage site was detected in the complementary region of GhmiR168 (data not shown), we speculated that GhmiR168 might cleave AGO1 mRNA. According to the tendency of GhmiR168 to decrease from 5 to 20 dpa, the expression of AGO1 mRNA should be upregulated. The increased expression of AGO1, mediated through GhmiR168, might facilitate the gene silencing of the type A and B GhmiRNA targets discussed above.
Furthermore, the abundance of GhmiRNAs in this network accounted for approximately 80% of the total miRNA reads. As shown in Additional file 14: Table S6, the read numbers of Type A and B miRNAs were increased at 10 and 15 dpa (rapid elongation) compared with 5 dpa, and the reads of Type A and B (except for Ghmi167) were higher in 15 dpa fibers than in 15 dpa seeds. Similarly, the Type C read numbers were decreased at 10 and 15 dpa compared with 5 dpa, and the Type C reads numbers were lower in 15 dpa fibers than in 15 dpa seeds. These trends indicated that the miRNAs in this regulatory network played special roles in cotton fiber cell elongation to some extent. Taken together, the data provided in this study showed that miRNAs are precisely regulated during fiber elongation and play important roles in various biochemical processes that promote fiber elongation. Hence, we propose the miRNA-mediated response plays an important role in fiber elongation, opening new avenues for the functional study of miRNAs in fibers and even plant cell elongation.
A total of 79 known miRNA families and 257 novel miRNAs from cotton fibers were identified through the high-throughput sequencing of sRNAs isolated from 5, 10, 15 and 20 dpa cotton fibers. This expands the population of known G. hirsutum miRNAs and confirmed the existence of ta-siRNAs in G. hirsutum. Comparisons of the miRNAomes of each stage revealed that 46 miRNAs were differentially expressed during the cotton fiber elongation; further, the target genes of eight miRNAs and one ta-siRNA were validated through 5’-RLM-RACE. The detailed analysis of the expression of their targets at different developmental time points hinted at their varied roles in the regulation of fiber elongation in G. hirsutum.
Upland cotton (Gossypium hirsutum cv. CRI35) was field grown under normal agronomic conditions. The Cotton Research Institute, Chinese Academy of Agricultural Sciences, kindly provided these seeds. The flowers were bagged one day prior to anthesis to prevent hybridization with other varieties and tagged on the day of anthesis after the removal of the bag. Normal bolls were harvested at 5, 10, 15 and 20 dpa and temporarily stored on ice. The young seeds with fibers were stripped of hulls, frozen in liquid nitrogen, and stored at −80°C until further use.
Small RNA library construction and Solexa sequencing
Total RNA was extracted from the frozen 5, 10, 15 and 20 dpa fibers and the 15 dpa young seeds (as a reference) using the PureLink™ Plant RNA Reagent kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA quality was examined on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) (Additional file 15: Figure S9). The sequencing libraries were constructed as previously described . Briefly, sRNAs of 15–30 nt in length were isolated using 15% PAGE (7 M urea). After ligation to both 5’ and 3’ RNA adaptors, the purified RNAs were reverse transcribed using primers with partial complementarity to the adaptors. The DNA pool amplified from the first-strand cDNA was subsequently sequenced using Hiseq 2000 (Illumina, San Diego, CA) at the Beijing Genomics Institute (BGI), Shenzhen.
Analysis of sequencing data
Sequences were trimmed to eliminate low-quality reads and other contaminants from the adaptor tags. Repeats and protein coding sequences derived small RNAs were identified using a BLAST search against the annotated G. raimondii genome (http://cgp.genomics.org.cn/page/species/index.jsp). The rRNA, tRNA, snRNA, and snoRNA sequences were analyzed with a BLAST search against Sanger Rfam data (http://ftp.sanger.ac.uk/pub/databases/Rfam/). Known miRNA family sequences were analyzed through BLAST analysis against miRBase (version 19, http://microrna.sanger.ac.uk). Sequences that were identical or related to known miRNA sequences (two or fewer nucleotide substitutions) were considered as known miRNAs (Additional file 1: Table S1). Then, the G. raimondii (http://cgp.genomics.org.cn/page/species/index.jsp) sequences matching known miRNAs, rRNA, tRNA, snRNA, and snoRNA (Table 1) were obtained using the BLAST method. To identify novel miRNAs, the remaining sequences were mapped to the cotton nucleotide database, including the cotton genome survey sequences (GSS) from NCBI, ESTs from the cotton gene index (CGI, http://occams.dfci.harvard.edu/pub/bio/tgi/data/Gossypium/CGI.release_11.zip), and the G. raimondii genome (http://cgp.genomics.org.cn/page/species/index.jsp). Potential pre-miRNAs and secondary structures from the corresponding ESTs and genomic sequences were examined using mireap_0.2 (http://sourceforge.net/projects/mireap). After this analysis, candidate miRNAs that satisfied the following rules were accepted as novel miRNAs: (1) The set of reads from the candidate miRNA locus should account for more than 95% of all the precursor-mapped small RNA reads, and reliable candidate miRNA reads (five reads at least) should account for more than 75% of the corresponding set of reads; (2) the miRNA* reads should have two-nucleotide 3’ overhangs; or (3) the base-paring mismatch between the miRNA and the other arm of the hairpin, or between the miRNA* sequence and the other arm, should be less than five nt in length, meanwhile, no asymmetric bulges larger than two nucleotides and no more than two asymmetric bulges should be present within the miRNA/miRNA* duplex . A previously reported strategy for TAS3 homolog identification was used to identify the cotton TAS3 locus .
Identification of elongation-related miRNAs and target prediction
The abundances of known and novel miRNAs in each library were reported as normalized reads (reads per ten million, RPTM). The miRNA families with reads greater than 100 RPTM in at least one library were subjected to Student’s t-test to assess the statistical significance of the differences between the highest and lowest abundance levels of each miRNA, as previously described . miRNAs with statistically significant differences in expression were considered cotton fiber elongation-related miRNAs. A complete linkage hierarchical cluster analysis of fiber elongation-related miRNAs in cotton fibers (5–20 dpa) was performed through a comparison of the RPTM of each miRNA to the average value in the four libraries , and the fiber elongation-related miRNA families were further subjected to target prediction.
The psRNATarget web server (http://plantgrn.noble.org/psRNATarget/) was used for target prediction. The most abundant sequence from each miRNA family served as the query, and CGI Release 11 served as the database. To annotate the potential target ESTs, a BLASTx search was performed against the NCBI non-redundant protein database of flowering plants (tax id: 3398). The top three hits (P < 0.001) were chosen to annotate the potential function of each predicted target EST (Additional file 10: Table S4 and Additional file 11: Table S5).
5’-RACE and quantitative RT-PCR
To validate the predicted cotton miRNA target genes in vivo, RNA Ligase-Mediated 5’-RACE (RLM-RACE) was performed using the First Choice RLM-RACE Kit (Ambion, Austin, TX), as previously described . Quantitative RT-PCR of targets was also performed as previously described . Briefly, cDNA was synthesized from 2 μg of total RNA using the TaKaRa RNA PCR Kit (TaKaRa), and the mRNA for UBQ10 was amplified in parallel reactions to normalize the cDNA quantity added to each reaction . Quantitative RT-PCR of three mature miRNAs was assayed by stem–loop reverse transcription-PCR . DNA-free RNA (2 μg) was reverse-transcribed using miRNA-specific stem-loop primers with the TaKaRa RNA PCR Kit (TaKaRa), and U6 was used as an internal control miRNA. Quantitative RT–PCR was performed on an iCycler iQ5 Multicolor real-time PCR detection system (Bio-Rad) using the Power SYBR Green PCR MasterMix (Applied Biosystems). The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed to ensure the reliability of the obtained results . All primers are listed in Additional file 16: Table S7.
A previously described method  was used to measure the anthocyanin levels. The (A535-A650) per gram of cotton fiber was used to determine the amount of anthocyanin in the cotton fibers,. All experiments were repeated at least three times.
Auxin response factor
Small interfering RNA
- tasiRNAs or TAS:
natural antisense siRNAs
small nuclear ribonucleic acid
small nucleolar RNA
Reads per ten million
Cotton Gene Index
Genome survey sequences
- G. raimondii:
Gossypium raimondii Ulbr.
- G. hirsutum:
Gossypium hirsutum L.
- G. arboretum:
Gossypium arboretum L.
- G. herbaceum:
Gossypium herbaceum L.
- RACE Rapid:
Amplification of cDNA Ends
Squamosa promoter-binding protein-like 9
class III HD-Zip protein 8
Acetyl-CoA carboxylase beta subunit
Calmodulin binding protein
Leucine-rich repeat receptor-like protein kinase
Calcium ion binding protein
Growth regulating factor
Indole acetic acid.
The authors would like to thank Yu Zhang for the bioinformatics analysis. This work was supported by grants from the State Key Basic Research and Development Plan (2010CB126003) and the National Transgenic Animals and Plants Research Project (2011ZX08005-003 and 2011ZX08009-003).
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