Degradome Libraries Research Articles

Comprehensive analysis of hairpin small RNAs involved in resistance and pathogenesis during wheat-Puccinia triticina interactions.

Wheat leaf rust, caused by the fungus Puccinia triticina (Pt), severely affects the grain quality and quantity of bread wheat (Triticum aestivum L.). Hairpin small(s)RNAs, like micro(mi)RNAs and their variants [including isomiRNAs (isomiRs) and microRNA-like RNAs (milRNAs)], along with their corresponding target genes, bestow leaf rust disease resistance, development and progression from both interacting species. However, the regulatory networks remain inadequately understood. Thirteen differentially expressed novel miRNAs, including two isomiRs and three milRNAs were discerned from induced reads of wheat sRNA libraries, and a further 5,393 and 1,275 candidate target genes were predicted in wheat and Pt, respectively. Functional annotation divulged that wheat-originated miRNAs/isomiRs were involved in resistance, while Pt-derived milRNAs imparted pathogenesis. The identified milRNAs- Tae-Pt-milR5, Tae-Pt-milR12, and Tae-Pt-milR14b and their cleavage sites on Pt target gene MEP5 were confirmed through degradome library screening, suggesting cross-kingdom translocation of Pt virulent genes in wheat host. Co-expression analysis of miRNAs/isomiRs-target genes provided insights into combating leaf rust disease, while co-expression analysis of milRNAs-target gene pairs reflected the extent of pathogenicity exerted by Pt with varied expression levels at the analyzed time points. The analysis pinpointed leaf rust-responsive candidate hairpin sRNAs- Tae-miR8, Tae-Pt-miR12, Tae-Pt-miR14a, and Tae-Pt-miR14b in wheat and Tae-Pt-milR12 in Pt. This study provides new insights into the hairpin sRNAs involved in the resistance and pathogenesis of wheat and Pt, respectively. Furthermore, crucial hairpin sRNAs and their promising targets for future biotechnological interventions to augment stress resilience have been identified.

Identification of the Coexisting Virus-Derived siRNA in Maize and Rice Infected by Rice Black-Streaked Dwarf Virus.

Rice black-streaked dwarf virus is transmitted by small brown planthoppers, which causes maize rough dwarf disease and rice black-streaked dwarf disease. This virus leads to slow growth or death of the host plants. During the coevolutionary arms race between viruses and plants, virus-derived small interfering RNAs (vsiRNAs) challenge the plant's defense response and inhibit host immunity through the RNA silencing system. However, it is currently unknown if rice black-streaked dwarf virus can produce the same siRNAs to mediate the RNA silencing in different infected species. In this study, four small RNA libraries and four degradome libraries were constructed by extracting total RNAs from the leaves of the maize (Zea mays) inbred line B73 and japonica rice (Oryza sativa) variety Nipponbare exposed to feeding by viruliferous and nonviruliferous small brown planthoppers. We analyzed the characteristics of small RNAs and explored virus-derived siRNAs in small RNA libraries through high-throughput sequencing. On analyzing the characteristics of small RNA, we noted that the size distributions of small RNAs were mainly 24 nt (19.74 to 62.00%), whereas those of vsiRNAs were mostly 21 nt (41.06 to 41.87%) and 22 nt (39.72 to 42.26%). The 5'-terminal nucleotides of vsiRNAs tended to be adenine or uracil. Exploring the distribution of vsiRNA hot spots on the viral genome segments revealed that the frequency of hotspots in B73 was higher than those in Nipponbare. Meanwhile, hotspots in the S9 and S10 virus genome segments were distributed similarly in both hosts. In addition, the target genes of small RNA were explored by degradome sequencing. Analyses of the regulatory pathway of these target genes unveiled that viral infection affected the ribosome-related target genes in maize and the target genes in the metabolism and biosynthesis pathways in rice. Here, 562 and 703 vsiRNAs were separately obtained in maize and rice and 73 vsiRNAs named as coexisting vsiRNAs (co-vsiRNAs) were detected in both hosts. Stem-loop PCR and real-time quantitative PCR confirmed that co-vsiRNA 3.1 and co-vsiRNA 3.5, derived from genome segment S3, simultaneously play a role in maize and rice and inhibited host gene expression. The study revealed that rice black-streaked dwarf virus can produce the same siRNAs in different species and provides a new direction for developing new antiviral strategies.

Genome-Wide Identification and Functional Analysis of Salvia miltiorrhiza MicroRNAs Reveal the Negative Regulatory Role of Smi-miR159a in Phenolic Acid Biosynthesis.

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs in plants. They play critical functions in various biological processes during plant growth and development. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant with significant medicinal, economic, and academic values. In order to elucidate the role of miRNAs in S. miltiorrhiza, six small RNA libraries from mature roots, young roots, stems, mature leaves, young leaves and flowers of S. miltiorrhiza and one degradome library from mixed tissues were constructed. A total of 184 miRNA precursors, generating 137 known and 49 novel miRNAs, were genome-widely identified. The identified miRNAs were predicted to play diversified regulatory roles in plants through regulating 891 genes. qRT-PCR and 5' RLM-RACE assays validated the negative regulatory role of smi-miR159a in SmMYB62, SmMYB78, and SmMYB80. To elucidate the function of smi-miR159a in bioactive compound biosynthesis, smi-miR159a transgenic hairy roots were generated and analyzed. The results showed that overexpression of smi-miR159a caused a significant decrease in rosmarinic acid and salvianolic acid B contents. qRT-PCR analysis showed that the targets of smi-miR159a, including SmMYB62, SmMYB78, and SmMYB80, were significantly down-regulated, accompanied by the down-regulation of SmPAL1, SmC4H1, Sm4CL1, SmTAT1, SmTAT3, SmHPPR1, SmRAS, and SmCYP98A14 genes involved in phenolic acid biosynthesis. It suggests that smi-miR159a is a significant negative regulator of phenolic acid biosynthesis in S. miltiorrhiza.

Genome-wide expression analysis of novel heat-responsive microRNAs and their targets in contrasting wheat genotypes at reproductive stage under terminal heat stress.

Heat stress at terminal stage of wheat is critical and leads to huge yield losses worldwide. microRNAs (miRNAs) play significant regulatory roles in gene expression associated with abiotic and biotic stress at the post-transcriptional level. In the present study, we carried out a comparative analysis of miRNAs and their targets in flag leaves as well as developing seeds of heat tolerant (RAJ3765) and heat susceptible (HUW510) wheat genotypes under heat stress and normal conditions using small RNA and degradome sequencing. A total of 84 conserved miRNAs belonging to 35 miRNA families and 93 novel miRNAs were identified in the 8 libraries. Tae-miR9672a-3p, tae-miR9774, tae-miR9669-5p, and tae-miR5048-5p showed the highest expression under heat stress. Tae-miR9775, tae-miR9662b-3p, tae-miR1120a, tae-miR5084, tae-miR1122a, tae-miR5085, tae-miR1118, tae-miR1130a, tae-miR9678-3p, tae-miR7757-5p, tae-miR9668-5p, tae-miR5050, tae-miR9652-5p, and tae-miR9679-5p were expressed only in the tolerant genotype, indicating their role in heat tolerance. Comparison between heat-treated and control groups revealed that 146 known and 57 novel miRNAs were differentially expressed in the various tissues. Eight degradome libraries sequence identified 457 targets of the differentially expressed miRNAs. Functional analysis of the targets indicated their involvement in photosynthesis, spliceosome, biosynthesis of nucleotide sugars and protein processing in the endoplasmic reticulum, arginine and proline metabolism and endocytosis. This study increases the number of identified and novel miRNAs along with their roles involved in heat stress response in contrasting genotypes at two developing stages of wheat.

Unraveling the role of microRNA-like RNAs in enhancing Puccinia triticina pathogenicity in wheat by high-throughput sequencing data

Wheat (Triticum aestivum L.), a staple crop cultivated across an estimated 215 million hectares worldwide, encounters significant yield reductions due to the leaf rust disease, instigated by the fungus Puccinia triticina (Pt). Control strategies for this pathogen are limited. Research on wheat-leaf rust interaction in the past few years has mainly focused on characterizing and modulating the performance of resistance (R) and other defense genes of the host plant, but rarely targeted to decipher the network of molecular strategies employed by the pathogen except secretome analysis. In wheat plants, pathogen may induce a greater diversity of small RNAs (sRNAs) to manipulate the host's gene expression and suppress defense responses. In the present study, Pt microRNA-like-RNAs (milRNAs) were screened and identified from sRNA libraries prepared from leaf rust pathogen-infected leaves of wheat near isogenic lines (NILs). Pathogenesis engendering Pt target genes were validated through degradome libraries. Our study indicates that milRNAs enter the host system to suppress its immunity and promote symbiosis-like association to maintain a longer virulence span. Through RT‒qPCR expression analysis, it was suggested that milRNAs create an environment to enhance symbiosis via cross-kingdom silencing of host disease resistance gene encoding proteins. We found that seven out of eight milRNAs effectively regulate pathogenesis-related genes and repressed the disease-resistance genes in host wheat. Functional annotation of target genes of Pt reveals regulation of vesicle synthesis, transport, and fusion, which represents cross-kingdom vesicle-mediated export of milRNAs. Our results also manifest an integrative view of how virulence-associated milRNAs endeavor to stamp out PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity) to increase susceptibility of the host. This study epitomizes one of the tactics developed by Pt employing milRNAs in regulating virulence during leaf rust infection and will usher to design strategies to manage pathogenesis to protect wheat immunity against leaf rust disease.

Comparative Study of Small-RNA and Degradome Sequencing Reveals Role of Novel stu-miR8006 in Regulating Root Development in Solanum tuberosum L.

MicroRNAs are a class of endogenous, non-coding, small-RNA molecules with important functions in plant development and stress response processes. Root systems are important because they allow plants to absorb nutrients and water from the soil and are fundamental for anchoring the plant and responding to environmental signals. However, the roles of miRNAs underlying root development remain poorly investigated in potato. In this research, small-RNA sequencing was performed to thoroughly detect underlying miRNAs and their roles in regulating root development between the early root (ER) stage and the mature root (MR) stage of potato roots. A total of 203 known and 137 novel miRNAs were obtained, and 64 differentially expressed miRNAs (DEMs) were identified between the ER and MR stages. The expression patterns of 12 DEMs were also determined via qRT-PCR. In addition, a mixed degradome library was constructed from the ER and the MR stages to identify the targets of the identified miRNAs, and 2400 target genes were verified to be the targets of 131 miRNAs. Based on target annotation, we identified that nine target genes of six DEMs were probably involved in potato root development, and eight targets of six DEMs were validated via 5’-RLM-RACE assays. These targets may participate in root development by regulating cell proliferation, root cultures (PGSC0003DMT400013837), root meristem growth (PGSC0003DMT400079970), root morphogenesis (PGSC0003DMT400040282), post-embryonic root development (PGSC0003DMT400021612), root hair elongation (PGSC0003DMT400034518), cell wall repair (PGSC0003DMT400074930), and auxin polar transport (PGSC0003DMT400079970), and by negatively regulating cell proliferation (PGSC0003DMT400009997) and cell growth (PGSC0003DMT400003464). The qRT-PCR analysis indicated that most miRNAs have opposing expression patterns to their targets. It is widely accepted that potato root development is regulated by miRNAs, among which stu-miR8006-p5-1ss9AT is substantially down-regulated during root development. We show here that the suppression of stu-miR8006-p5-1ss9AT led to an alteration in the potato root architecture and that it targeted auxin induction in the root culture protein 12-encoding gene that is potentially involved in the regulation of root development. In addition, the suppression of stu-miR8006-p5-1ss9AT led to a significant alteration in the potato root architecture. Altogether, our results might provide some useful insights into stu-miR8006-p5-1ss9AT and the crucial role that it plays in potato root development; they could also facilitate the molecular genetic breeding of potato.

Poplar coma morphogenesis and miRNA regulatory networks by combining ovary tissue sectioning and deep sequencing

Poplar coma, commonly referred to as "seed hairs", is a tuft of trichomes attached to the seed coat that helps seed dispersal. However, they can also trigger health impacts for humans, including sneezing, shortness of breath, and skin irritation. Despite efforts to study the regulatory mechanism of herbaceous trichome formation, poplar coma remains poorly understood. In this study, we showed that the epidermal cells of the funiculus and placenta are the origin of poplar coma based on observations of paraffin sections. Small RNA (sRNA) and degradome libraries were also constructed at three stages of poplar coma development, including initiation and elongation stages. Based on 7,904 miRNA-target pairs identified by small RNA and degradome sequencing, we constructed a miRNA-transcript factor and a stage-specific miRNA regulatory network. By combining paraffin section observation and deep sequencing, our research will provide greater insight into the molecular mechanisms of poplar coma development.

Genome-Wide Analysis of microRNA Expression Profile in Roots and Leaves of Three Wheat Cultivars under Water and Drought Conditions

Wheat is one of the most important food sources on Earth. MicroRNAs (miRNAs) play important roles in wheat productivity. To identify wheat miRNAs as well as their expression profiles under drought condition, we constructed and sequenced small RNA (sRNA) libraries from the leaves and roots of three wheat cultivars (Kukri, RAC875 and Excalibur) under water and drought conditions. A total of 636 known miRNAs and 294 novel miRNAs were identified, of which 34 miRNAs were tissue- or cultivar-specific. Among these, 314 were significantly regulated under drought conditions. miRNAs that were drought-regulated in all cultivars displayed notably higher expression than those that responded in a cultivar-specific manner. Cultivar-specific drought response miRNAs were mainly detected in roots and showed significantly different drought regulations between cultivars. By using wheat degradome library, 6619 target genes were identified. Many target genes were strongly enriched for protein domains, such as MEKHLA, that play roles in drought response. Targeting analysis showed that drought-downregulated miRNAs targeted more genes than drought-upregulated miRNAs. Furthermore, such genes had more important functions. Additionally, the genes targeted by drought-downregulated miRNAs had multiple interactions with each other, while the genes targeted by drought-upregulated miRNAs had no interactions. Our data provide valuable information on wheat miRNA expression profiles and potential functions in different tissues, cultivars and drought conditions.

Comprehensive, integrative genomic analysis of microRNA expression profiles in different tissues of two wheat cultivars with different traits.

Wheat is one of the most important food sources on Earth. MicroRNAs (miRNA) play important roles in wheat productivity. To identify wheat miRNAs, we constructed and sequenced sRNA libraries from leaves and roots of two wheat cultivars (RAC875 and Kukri) with many different traits. Given that available miRNA wheat complement in the plant-specific database PmiREN ( https://pmiren.com ) does not include root tissues and root-associated miRNAs might thus be missing, we performed first the prediction of novel miRNAs using the sRNAbench tool. We found a total of 150 putatively novel miRNA genes with expression of both arms from 289 unique mature sequences and nearly 30% of all miRNA reads in roots corresponded to novel miRNAs. In contrast, this figure in leaves dropped to under 3%, confirming the undersampling of roots in the complement of known miRNAs. By using 120 publicly available wheat datasets, 598 Zea mays small RNA libraries, 64 plant species genomes, wheat degradome library, and functional enrichment analysis, a subset of novel miRNAs were confirmed as bona-fide miRNAs. Of the total 605 miRNAs identified in this study inclusive of 316 known miRNAs, 528 miRNAs were shared by both cultivars, 429 miRNAs were shared by both root tissues and 329 miRNAs were shared by both leaf tissues. In addition, 32 miRNAs were specific to Kukri while 45 miRNAs were specific to RAC875. These miRNAs had diverse functions, such as regulation of gene transcription, protein translation, energy metabolism, and cell cycle progression. Our data provide a genome-wide miRNA expression profile in these two wheat cultivars and help functional studies of wheat genomics.

Species-specific regulatory pathways of small RNAs play sophisticated roles in flower development in Dimocarpus longan Lour.

Flower development plays vital role in horticultural plants. Post-transcriptional regulation via small RNAs is important for plant flower development. To uncover post-transcriptional regulatory networks during the flower development in Dimocarpus longan Lour. ‘Shixia’, an economically important fruit crop in subtropical regions, we collected and analyzed sRNA deep-sequencing datasets and degradome libraries Apart from identifying miRNAs and phased siRNA generating loci (PHAS loci), 120 hairpin loci, producing abundant sRNAs, were identified by in-house protocols. Our results suggested that 56 miRNA-target pairs, 22 21-nt-PHAS loci, and 111 hairpin loci are involved in post-transcriptional gene silencing during longan reproductive development. Lineage-specific or species-specific post-transcriptional regulatory modules have been unveiled, including miR482-PHAS and miRN15. miR482-PHAS might be involved in longan flower development beyond their conserved roles in plant defense, and miRN15 is a novel miRNA likely associated with a hairpin locus (HPL-056) to regulate strigolactone receptor gene DWARF14 (D14) and the biogenesis of phasiRNAs from D14. These small RNAs are enriched in flower buds, suggesting they are likely involved in post-transcriptional regulatory networks essential for longan flower development via the strigolactone signaling pathway.

MiR319 and its target TCP4 involved in plant architecture regulation in Brassica napus

Plant architecture is a collection of genetically controlled crop productivity and adaptation. MicroRNAs (miRNAs) have been proved to function in various biological processes, but little is known about how miRNA regulates plant architecture in rapeseed (Brassica napus L.). In this study, four small RNA libraries and two degradome libraries from shoot apex of normal and rod-like plants were sequenced. A total of 639 miRNA precursors and 16 differentially expressed miRNAs were identified in this study. In addition, 322 targets were identified through degradome sequencing. Among them, 14 targets were further validated via RNA ligase-mediated 5′ rapid amplification of cDNA ends. Transgenic approach showed that increased TCP4 activity in Arabidopsis resulted in premature onset of maturation and reduced plant size along with early flowering and shortened flowering time. miR319-OE lines in Brassica napus exhibited serrated leaves and abnormal development of shoot apical meristem (SAM), which led to the deformed growth of stem and reduced plant height. In conclusion, our study lays the foundation for elucidating miRNA regulate plant architecture and provides new insight into the miR319/TCP4 module regulates plant architecture in rapeseed.

Small RNA and degradome analyses reveal regulatory roles of miRNAs in vascular cambium development in Cryptomeria fortunei

The vascular cambium activity of trees directly affects the yield and quality of forest products . Therefore, studying the laws governing vascular cambium activity in Cryptomeria fortunei , a species mainly used for wood production, is important. miRNAs regulate target gene expression and play an important role in plant growth and development, but the role of miRNAs in C. fortunei vascular cambium growth remains poorly understood. Here, small RNA (sRNA) and degradome libraries were constructed for C. fortunei vascular cambium at five different growth stages. A total of 1064 miRNAs (859 known and 205 novel miRNAs), including 266 differentially expressed miRNAs (DEMs), were identified by sRNA sequencing , and 4215 targets of 843 miRNAs were identified by degradome analysis. These targets were enriched in metabolism (e.g., phenylpropanoid biosynthesis), genetic information processing and signal transduction (e.g., plant hormone signal transduction) pathways during vascular cambium development. We obtained 365 differentially expressed target pairs and constructed a regulatory network of 111 annotated negatively regulated target pairs. The blue module was identified as a key module through weighted DEM coexpression network analysis, and seven negatively regulated target pairs (pab-miR159g/aly-miR397a-5p/pab-miR397a/novel23_mature- MYB , novel12_star- UGT72B1 and smo-miR1083/pab-miR1083- GARP-ARR-B ) were considered as key regulators of C. fortunei vascular cambium growth. This study provides important miRNA and target data related to C. fortunei vascular cambium growth. These results increase the understanding of the posttranscriptional regulatory mechanisms of vascular cambium development in conifers and provide important references for the genetic improvement of wood production in plants. • In Chinese cedar vascular cambium, 859 known and 205 novel miRNAs were identified. • A degradome analysis identified 4215 targets of 843 miRNAs. • Differentially expressed miRNAs and target pairs were identified. • A negatively regulated target pair network was constructed. • A key blue module was obtained through WGCNA, and key target pairs were identified.

Identification of microRNAs and their target genes related to the accumulation of anthocyanin in purple potato tubers (Solanum tuberosum).

MicroRNAs (miRNAs) are types of endogenous non‐coding small RNAs found in eukaryotes that are 18–25 nucleotides long. miRNAs are considered to be key regulatory factors of the expression of target mRNA. The roles of miRNAs involved in the regulation of anthocyanin accumulation in pigmented potatoes have not been systematically reported. In this study, the differentially expressed miRNAs and their target genes involved in the accumulation of anthocyanin during different developmental stages in purple potato ( Solanum tuberosum L.) were identified using small RNA (sRNA) and degradome sequencing. A total of 275 differentially expressed miRNAs were identified in the sRNA libraries. A total of 69,387,200 raw reads were obtained from three degradome libraries. The anthocyanin responsive miRNA–mRNA modules were analyzed, and 37 miRNAs and 23 target genes were obtained. Different miRNAs regulate the key enzymes of anthocyanin synthesis in purple potato. The structural genes included phenylalanine ammonia lyase, chalcone isomerase, flavanone 3‐hydroxylase, and anthocyanidin 3‐O‐glucosyltransferase. The regulatory genes included WD40, MYB, and SPL9. stu‐miR172e‐5p_L‐1R‐1, stu‐miR828a, stu‐miR29b‐4‐5p, stu‐miR8019‐5p_L‐4R‐3, stu‐miR396b‐5p, stu‐miR5303f_L‐7R + 2, stu‐miR7997a_L‐3, stu‐miR7997b_L‐3, stu‐miR7997c_L + 3R‐5_2ss2TA3AG, stu‐miR156f‐5p_L + 1, stu‐miR156a, stu‐miR156a_R‐1, stu‐miR156e, stu‐miR858, stu‐miR5021, stu‐miR828 and their target genes were validated by qRT‐PCR. They play important roles in the coloration and accumulation of purple potatoes. These results provide new insights into the biosynthesis of anthocyanins in pigmented potatoes.

Integrated small RNA profiling and degradome analysis of Anthurium andraeanum cultivars with different-colored spathes

MicroRNAs (miRNAs) are known to play vital roles in coloration of leaves, flowers, and fruits in plants. However, their functions in spathe coloration are poorly known. Anthurium andraeanum is a popular ornamental plant with various spathe colors. In this study, small RNA and degradome libraries from three A. andraeanum cultivars with different-colored spathes were constructed and sequenced. Illumina sequencing resulted in 94 conserved miRNAs, and 34 novel miRNAs in total were then identified based on precursor sequences and hairpin structures. Differential expression analysis showed that 52, 51, and 49 miRNAs were differentially expressed in comparisons of orange- versus white-colored spathe, purple- versus white-colored spathe, and purple- versus orange-colored spathe, respectively. The expression patterns of miRNAs and their corresponding targets involved in spathe coloration were further analyzed, and displayed that miR156b and miR529 were highly abundant in the spathes with higher anthocyanin content. These two miRNAs co-targeted a gene encoding SPL17, which may function as a negative regulator in anthocyanin accumulation. In addition, miR408 was also abundantly expressed in purple- and orange-colored spathes, and its typical targets were also identified. This comprehensive integrated analysis provides insight into the miRNA-mediated genetic regulation in spathe coloration of A. andraeanum.

Identification of anthocyanin-related microRNAs in ornamental kale (Brassica oleracea L. var. acephala) by high throughput sequencing

Anthocyanins have been found to be responsible for the pigmentation of red, purple and blue colors in plants. MicroRNA (miRNA) plays an important role in the regulation of anthocyanin biosynthesis in plants. However, few miRNAs and their corresponding functions related to anthocyanin biosynthesis in ornamental kale (Brassica oleracea var. acephala) have been reported. In this study, small RNA (sRNA) and degradome libraries from inner and outer leaves (IL and OL) of ornamental kale line “Pigeon Red” were constructed, respectively. Totally, 139 known and 14 novel miRNAs were identified by sRNA sequencing, and 304 target genes cleaved by 92 bo-miRNAs were identified by degradome sequencing. Of these, 5 miRNAs and their corresponding targets were potentially involved in the anthocyanin biosynthesis. And 35 miRNAs were differently expressed between IL and OL. Integrated analysis of sRNA-Seq, degradome sequencing and RNA-Seq revealed that bo-miR828 and its targets potentially play a key role in the regulation of anthocyanin biosynthesis in ornamental kale leaves. Finally, an inverse correlation between the expression of bo-miR828 and its targets was revealed by both qRT-PCR and high-throughput sequencing. These results represented a comprehensive expression profiling of miRNAs related to anthocyanin accumulation in B.oleracea var. acephala and provided important clues for understanding the regulatory network of anthocyanin biosynthesis mediated by miRNA in Brassica crops.

Comparative Transcriptome Analysis Uncovers the Regulatory Roles of MicroRNAs Involved in Petal Color Change of Pink-Flowered Strawberry.

The pink-flowered strawberry is popular in China due to its high ornamental value. In the present study, sRNAome, transcriptome, and degradome sequencing were performed to understand the functions of microRNAs (miRNAs) and their target genes during flower development in pink-flowered strawberry. Nine small RNA libraries and a mixed degradome library from flower petals at different developmental stages were constructed and sequenced. A total of 739 known miRNAs and 964 novel miRNAs were identified via small RNA sequencing, and 639 miRNAs were identified to cleave 2,816 target genes based on the degradome data. Additionally, 317 differentially expressed miRNAs among the various stages of flower development were identified, which regulated 2,134 differentially expressed target genes. These target genes were significantly enriched in the transcriptional regulation, phenylpropanoid biosynthesis, and plant hormone signal transduction pathways. Furthermore, integrated microRNAomic and transcriptomic analyses suggested that 98 miRNAs targeted several transcription factors, including MYBs (26), bHLHs (12), NACs (14), and SPLs (19), related to anthocyanin accumulation. In addition, 27 differentially expressed miRNAs might affect anthocyanin biosynthesis by regulating 23 targets involved in the hormone signal transduction pathway. The quantitative real-time PCR (qRT-PCR) analysis confirmed the expression changes of 21 miRNA-target pairs. Furthermore, the transient expression of candidate miRNAs was performed in the pink-flowered strawberry cultivar “Fenyun” at the bud stage. Introduction of FamiR156a, FamiR396e, and FamiR858_R-2 in the “Fenyun” increased flower color intensity, while transient expression of FamiR828a decreased flower color intensity. Overall, the present study uncovers the regulatory functions of microRNAs, including anthocyanin biosynthesis, hormone signaling, and regulation factors during flower development and coloration in pink-flowered strawberry. This work expands the knowledge of miRNAs affecting coloration in strawberry and provides rich resources for future functional studies.

Genome-wide identification of MITE-derived microRNAs and their targets in bread wheat

BackgroundPlant miRNAs are a class of small non-coding RNAs that can repress gene expression at the post-transcriptional level by targeting RNA degradation or promoting translational repression. There is increasing evidence that some miRNAs can derive from a group of non-autonomous class II transposable elements called Miniature Inverted-repeat Transposable Elements (MITEs).ResultsWe used public small RNA and degradome libraries from Triticum aestivum to screen for microRNAs production and predict their cleavage target sites. In parallel, we also created a comprehensive wheat MITE database by identifying novel elements and compiling known ones. When comparing both data sets, we found high homology between MITEs and 14% of all the miRNAs production sites detected. Furthermore, we show that MITE-derived miRNAs have preference for targeting degradation sites with MITE insertions in the 3’ UTR regions of the transcripts.ConclusionsOur results revealed that MITE-derived miRNAs can underlay the origin of some miRNAs and potentially shape a regulatory gene network. Since MITEs are found in millions of insertions in the wheat genome and are closely linked to genic regions, this kind of regulatory network could have a significant impact on the post-transcriptional control of gene expression.

Identification of microRNAs Responding to Aluminium, Cadmium and Salt Stresses in Barley Roots.

Plants are frequently exposed to various abiotic stresses, including aluminum, cadmium and salinity stress. Barley (Hordeum vulgare) displays wide genetic diversity in its tolerance to various abiotic stresses. In this study, small RNA and degradome libraries from the roots of a barley cultivar, Golden Promise, treated with aluminum, cadmium and salt or controls were constructed to understand the molecular mechanisms of microRNAs in regulating tolerance to these stresses. A total of 525 microRNAs including 198 known and 327 novel members were identified through high-throughput sequencing. Among these, 31 microRNAs in 17 families were responsive to these stresses, and Gene Ontology (GO) analysis revealed that their targeting genes were mostly highlighted as transcription factors. Furthermore, five (miR166a, miR166a-3p, miR167b-5p, miR172b-3p and miR390), four (MIR159a, miR160a, miR172b-5p and miR393) and three (miR156a, miR156d and miR171a-3p) microRNAs were specifically responsive to aluminum, cadmium and salt stress, respectively. Six miRNAs, i.e., miR156b, miR166a-5p, miR169a, miR171a-5p, miR394 and miR396e, were involved in the responses to the three stresses, with different expression patterns. A model of microRNAs responding to aluminum, cadmium and salt stresses was proposed, which may be helpful in comprehensively understanding the mechanisms of microRNAs in regulating stress tolerance in barley.

Integration of mRNA and miRNA Analysis Reveals the Molecular Mechanism of Cotton Response to Salt Stress.

To identify the regulatory network of known and novel microRNAs (miRNAs) and their targets responding to salt stress, a combined analysis of mRNA libraries, small RNA libraries, and degradome libraries were performed. In this study, we used unique molecular identifiers (UMIs), which are more sensitive, accurate, and reproducible than traditional methods of sequencing, to quantify the number of molecules and correct for amplification bias. We identified a total of 312 cotton miRNAs using seedlings at 0, 1, 3, and 6 h after NaCl treatment, including 80 known ghr-miRNAs and 232 novel miRNAs and found 155 miRNAs that displayed significant differential expression under salt stress. Among them, fifty-nine differentially expressed miRNAs were simultaneously induced in two or three tissues, while 66, 11, and 19 were specifically expressed in the roots, leaves, and stems, respectively. It is indicated there were different populations of miRNAs against salt stress in roots, leaves and stems. 399 candidate targets of salt-induced miRNAs showed significant differential expression before and after salt treatment, and 72 targets of 25 miRNAs were verified by degradome sequencing data. Furthermore, the regulatory relationship of miRNA-target gene was validated experimentally via 5′RLM-RACE, proving our data reliability. Gene ontology and KEGG pathway analysis found that salt-responsive miRNA targets among the differentially expressed genes were significantly enriched, and mainly involved in response to the stimulus process and the plant hormone signal transduction pathway. Furthermore, the expression levels of newly identified miRNA mir1 and known miRNAs miR390 and miR393 gradually decreased when subjected to continuous salt stress, while overexpression of these miRNAs both increased sensitivity to salt stress. Those newly identified miRNAs and mRNA pairs were conducive to genetic engineering and better understanding the mechanisms responding to salt stress in cotton.

Genome-Wide Analysis of Alternative Splicing and Non-Coding RNAs Reveal Complicated Transcriptional Regulation in Cannabis sativa L.

It is of significance to mine the structural genes related to the biosynthetic pathway of fatty acid (FA) and cellulose as well as explore the regulatory mechanism of alternative splicing (AS), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the biosynthesis of cannabinoids, FA and cellulose, which would enhance the knowledge of gene expression and regulation at post-transcriptional level in Cannabis sativa L. In this study, transcriptome, small RNA and degradome libraries of hemp ‘Yunma No.1’ were established, and comprehensive analysis was performed. As a result, a total of 154, 32 and 331 transcripts encoding key enzymes involved in the biosynthesis of cannabinoids, FA and cellulose were predicted, respectively, among which AS occurred in 368 transcripts. Moreover, 183 conserved miRNAs, 380 C. sativa-specific miRNAs and 7783 lncRNAs were predicted. Among them, 70 miRNAs and 17 lncRNAs potentially targeted 13 and 17 transcripts, respectively, encoding key enzymes or transporters involved in the biosynthesis of cannabinoids, cellulose or FA. Finally, the crosstalk between AS and miRNAs or lncRNAs involved in cannabinoids and cellulose was also predicted. In summary, all these results provided insights into the complicated network of gene expression and regulation in C. sativa.