Small RNA Libraries Research Articles

First report of Capsicum chlorosis virus naturally infecting Ageratum conyzoides in China.

Capsicum chlorosis virus (CaCV; family Tospoviridae, genus Orthotospovirus) was first reported to infect capsicum (Capsicum annuum) and tomato (Solanum lycopersicum) in Australia in 2002 (McMichael et al., 2002). Subsequently, its infection was detected in different plants including waxflower (Hoya calycina Schlecter) in the United States (Melzer et al. 2014), peanut (Arachis hypogaea) in India (Vijayalakshmi et al. 2016), and spider lily (Hymenocallis americana) (Huang et al. 2017), Chilli pepper (Capsicum annuum) (Zheng et al. 2020), and Feiji cao (Chromolaena odorata) (Chen et al. 2022) in China. Ageratum conyzoides L. (commonly known as goat weed, family Asteraceae) is a natural weed in crop fields distributed in subtropical and tropical areas and a reservoir host of numerous plant pathogens (She et al. 2013). In April 2022, we observed that 90% of plants of A. conyzoides in maize fields in Sanya, Hainan province, China, exhibited typical virus-like symptoms of vein yellowing, leaf chlorosis, and distortion (Fig. S1 A-C). Total RNA was extracted from one symptomatic leaf of A. conyzoides. Small RNA libraries were constructed using the small RNA Sample Pre Kit (Illumina, San Diego, USA) for sequencing with an Illumina Novaseq 6000 platform (Biomarker Technologies Corporation, Beijing, China). A total 15,848,189 clean reads were obtained after removing low-quality reads. Quality-controlled qualified reads were assembled into contigs using Velvet 1.0.5 software with a k-mer value of 17. One hundred contigs shared nucleotide identity ranging from 85.7% to 100% with the CaCV using BLASTn searches online (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). Numerous contigs (45, 34, and 21) obtained in this study were mapped to the L, M, and S RNA segments of the CaCV-Hainan isolate (GenBank accession no. KX078565- KX078567) from spider lily (Hymenocallis americana) in Hainan province, China, respectively. The full-length of L, M, and S RNA segments of CaCV-AC were determined to be 8,913, 4,841, and 3,629 bp, respectively (GenBank accession no. OQ597167- OQ597169). Furthermore, five symptomatic leaf samples were tested to be positive for CaCV using a CaCV enzyme-linked immunosorbent assay (ELISA) kit (MEIMIAN, Jiangsu, China) (Fig. S1-D). Total RNA from these leaves was amplified by RT-PCR with two sets of primer pairs. Primers CaCV-F (5'-ACTTTCCATCAACCTCTGT-3') and CaCV-R (5'-GTTATGGCCATATTTCCCT-3') were used for the amplification of 828 bp fragment from nucleocapsid protein (NP) on CaCV S RNA. While another, primers gL3637 (5'-CCTTTAACAGTDGAAACAT-3') and gL4435c (5'-CATDGCRCAAGARTGRTARACAGA-3') were used for the amplification of 816 bp fragment from RNA-dependent RNA polymerase (RdRP) on CaCV L RNA (Fig. S1-E and -F) (Basavaraj et al. 2020). These amplicons were cloned into the pCE2 TA/Blunt-Zero vector (Vazyme, Nanjing, China) and three independent positive colonies of Escherichia coli DH5α carrying each viral amplicon were sequenced. These sequences were deposited in the GenBank database under accession nos. OP616700-OP616709. Pairwise sequence comparison revealed that nucleotide sequences of NP and RdRP genes of the five CaCV isolates shared 99.5% (812 bp out of 828 bp) and 99.4% (799 bp out of 816 bp) nucleotide identities, respectively. They showed 86.2-99.2% and 86.5-99.1% nucleotide identities with corresponding nucleotide sequences of other CaCV isolates derived from GenBank database, respectively. The highest nucleotide sequence identity (99%) of the CaCV isolates obtained in the study was observed with the CaCV-Hainan isolate. Phylogenetic analysis based on NP amino acid demonstrated that six CaCV isolates (this study = 5 and NCBI database = 1) clustered into one distinct clade (Fig. S2). Our data confirmed for the first time the presence of CaCV naturally infecting A. conyzoides plant in China, which enriches information on the host range and will be helpful for disease management.

First report of cucumber mosaic virus infecting Azorina vidalii in Azores Islands.

Virus diseases that occur in crops pose a major threat not only to global food security but also to wild plant communities growing in natural ecosystems (Jones, 2020, and references therein). In Azores (Portugal) little is known about viruses present on native flora and therefore, they have not been taken into consideration in conservation programs. Considering this, we selected Azorina vidalii (Campanulaceae), an endangered (IUCN) plant, endemic to Azores (Bilz, 2011), to survey for plant viruses. A. vidalii, the sole species of its genus, is often found in crevices with no soil accumulation on coastal cliffs, exposed to storms and sea spray, and is used as an ornamental. Leaves from 53 plants of A. vidalii from three populations from Terceira Island and three populations from Flores Island were randomly collected without obvious symptoms of virus infection, between the summer of 2021 and fall of 2022. RNA extraction was performed using the Plant/Fungi Total RNA Purification Kit (Norgen Biotek, Canada). RNA extracts from each population were pooled into six distinct composite samples (AvT1, AvT2, AvT3, AvF1, AvF4 and AvF5) and sent to Lexogen (Austria) for small RNA library preparation and High-Throughput Sequencing. Single-end RNA sequencing using Illumina NextSeq2000 system yielded between 10.1 M and 33.8 M raw reads. Adaptors and low-quality reads were removed with Trim Galore! and PRINSEQ. Trimmed reads were mapped to the genome phylogenetically nearest to A. vidalii available at the NCBI database (Adenophora triphylla). The resulting 2.5 M - 13.5 M unmapped reads were analysed with VirusDetect online version (database v248) (Zheng et al., 2017) for virus detection and identification. Sequences of cucumber mosaic virus (CMV) (contigs of up to 3045 nt for RNA1, 2917 nt for RNA2 and 2086 nt for RNA3) were identified in five (AvT1, AvT2, AvT3, AvF1 and AvF5) of the six composite samples and CMV satellite sequences (two contigs with 145 and 197 nt) were identified in only one (AvT1) of the composite samples. To confirm the presence of CMV, all samples were tested by two-step RT-PCR using primers targeting CMV-specific RdRp gene (513 bp) (Grieco et al., 2000), which retrieved 18 positive samples (34%). Nine samples were selected for Sanger sequencing (six out of 13 from Terceira and three out of five from Flores) based on digestion profile obtained with AluI and MboI. The resulting sequences (OQ176229-OQ176233, OQ732757-OQ732760) share an identity of 97.2-100% and BLASTn showed them to have 98.3-99.6% identity to CMV strain TN (AB176848). A Neighbour-Joining tree (Supplementary material) inferred in MEGA11 (Tamura et al., 2021) with 237 additional CMV-RdRp sequences, showed that A. vidalii CMV-derived isolates clustered together with reference strains of subgroup II, as those used by Roossinck (2002) for phylogenetic analysis of the 2a ORF. Besides CMV, tomato spotted wilt virus and polerovirus-associated RNAs sequences were found in one of the A. vidalii populations, but with lower coverage, and need to be further investigated. To the best of our knowledge, this is the first report of CMV infecting A. vidalli. CMV, genus Cucumovirus, is an agriculturally important virus and one of the most successful viruses known, infecting over 1,200 species of plants (Palukaitis & García-Arenal, 2003). In addition to A. vidalii being a CMV reservoir, which may have implications on adjacent crop fields, further research is needed to investigate the impact of CMV on A. vidalli fitness.

Small RNA-Seq to Unveil the miRNA Expression Patterns and Identify the Target Genes in Panax ginseng.

Panax ginseng, renowned for its medicinal properties, relies on adventitious roots and hairy roots as crucial sources for the production of ginsenosides. Despite the widespread utilization of ginseng, investigations into its miRNAs have remained scarce. To address this gap, two samples of ginseng adventitious roots and ginseng hairy roots were collected, and subsequent construction and sequencing of small RNA libraries of ginseng adventitious roots and hairy roots were performed using the Illumina HiSeq X Ten platform. The analysis of the sequencing data unveiled total miRNAs 2432. The miR166 and miR396 were the most highly expressed miRNA families in ginseng. The miRNA expression analysis results were used to validate the qRT-PCR. Target genes of miRNA were predicted and GO function annotation and KEGG pathway analysis were performed on target genes. It was found that miRNAs are mainly involved in synthetic pathways and biological processes in plants, which include metabolic and bioregulatory processes. The plant miRNAs enriched KEGG pathways are associated with some metabolism, especially amino acid metabolism and carbohydrate metabolism. These results provide valuable insights miRNAs and their roles in metabolic processes in ginseng.

First Report of Cotton Leafroll Dwarf Virus infection of Malvaviscus arboreus in China.

Cotton leafroll dwarf virus (CLRDV, genus Polerovirus, family Solemoviridae) has been reported to infect cotton in Brazil, Argentina, India, Thailand and Timor-Leste (Agrofoglio YC et al. 2017; Corrêa RL et al. 2005; Mukherjee et al. 2012; Ray et al. 2016; Sharman et al. 2015), and in the United States (Ali and Mokhtari et al. 2020; Avelar et al. 2019). It has also been recently reported to infect Cicer arietinum (chickpea) in Uzbekistan and Hibiscus syriacus in Korea (Igori et al. 2022; Kumari et al. 2020). In China, the natural infection of plants by CLRDV has not been reported previously. In August 2017, leaf samples were collected from a wild plant of Malvaviscus arboreus (Malvaceae) exhibiting symptoms including leaf yellowing and distorting in Tengchong County of Yunnan Province. Leaves were used for total RNA extraction using TRIzol Reagent (Invitrogen, USA). Small RNA library construction and deep sequencing was performed on illumina HiSeqTM 2000 platform by Novogene Bioinformatic Technology Co., Ltd (Beijing, China). A total of 11, 525, 708 raw reads were obtained and further computationally analyzed by Perl scripts. The adaptors were removed and the obtained 7, 520, 902 clean reads with size of 18- to 26-nucleotide (nt) were aligned with the GenBank virus RefSeq database using Bowtie software. These reads were mainly mapped to the genomes of hibiscus bacilliform virus (genusBadnavirus,familyCaulimoviridae), hibiscus chlorotic ringspot virus (genus Betacarmovirus, family Procedovirinae), hibiscus latent Singapore virus (genus Tobamovirus, family Virgaviridae) and CLRDV isolate ARG (accession no. GU167940). The average coverage depth of clean reads mapped to CLRDV genome was 97.76%. Contigs greater than 50 nt were used to search for similar sequences by BLASTx, and 107 contigs were annotated as homologous to CLRDV isolates. To confirm CLRDV infection, reverse transcription polymerase chain reaction (RT-PCR) was performed using the specific primer pair CLRDV-F (5'-TCCACAGGAAGTATCACGTTCG-3') and CLRDV-R (5'-CCTTGTGTGGTTTGATTCGTGA-3') designed based on two of these contigs well-aligned to the genome of CLRDV isolate ARG. An amplicon of 1095-bp size was amplified, and was sequenced by Sanger sequencing (TsingKe Biological Technology, Chengdu, China), and BLASTn search results showed a maximum nucleotide identity of 95.45% with CLRDV isolate CN-S5, an isolate obtained from soybean aphid host in China (accession no. KX588248). To obtain more information on this CLRDV isolate, four primer pairs were designed and used for RT-PCR amplification (TableS1). The amplicons with size of about 860-, 1400-, 3200- and 1100-bp, were obtained separately and assembled into a complete genome sequence up to 5, 865-nt in length (isolate YN, deposited under GenBank accession no. MN057665). BLASTn showed the highest nucleotide similarity of 94.61% with CLRDV isolate CN-S5. From 2018 to 2022, additional M. arboreus samples with leaf yellowing or curling symptoms (9 from Shapingba District in Chongqing City, 5 from Nanchong City in Sichuan Province, 9 from Kunming City and 12 from Tengchong County in Yunnan Province) were collected and tested for CLRDV by RT-PCR using primer pairs CLRDV-F/CLRDV-R. The nucleotide sequences of the CLRDV P0 gene in two samples from Tengchong County were obtained by Sanger sequencing and deposited under GenBank (CLRDV isolate TCSL1 P0 gene, accession no. OQ749809; CLRDV isolate TCSW2 P0 gene, accession no. OQ749809). To our knowledge, this is the first report of CLRDV naturally infecting Malvaviscus arboreus in China, thus extending the information on its geographical distribution and host range. Malvaviscus arboreus is a widely cultivated ornamental plant in Yunnan Province, China. The natural occurrence of CLRDV not only affects the ornamental value of Malvaviscus arboreus, but also poses a potential threat to cotton production in China. This study will assist further surveillance of CLRDV infection and future development of effective protection strategies against CLRDV in China.

Time-resolved small RNA transcriptomics of the ichthyosporean Sphaeroforma arctica.

Ichthyosporea, a clade of holozoans, represent a clade closely related to animals, and thus hold a key phylogenetic position for understanding the origin of animals. We have previously discovered that an ichthyosporean, Sphaeroforma arctica, contains microRNAs (miRNAs) as well as the miRNA processing machinery. This was the first discovery of miRNAs among the closest single-celled relatives of animals and raised intriguing questions about the roles of regulatory small RNAs in cell development and differentiation in unicellular eukaryotes. Like many ichthyosporeans, S. arctica also undergoes a transient multicellular developmental life cycle. As miRNAs are, among other roles, key regulators of gene expression during development in animals, we wanted to investigate the dynamics of miRNAs during the developmental cycle in S. arctica. Here we have therefore collected a comprehensive time-resolved small RNA transcriptome linked to specific life stages with a substantially higher sequencing depth than before, which can enable further discovery of functionally relevant small RNAs. The data consists of Illumina-sequenced small RNA libraries from two independent biological replicates of the entire life cycle of S. arctica with high temporal resolution. The dataset is directly linked and comes from the same samples as a previously published mRNA-seq dataset, thus enabling direct cross-functional analyses.

Differentially Expressed Conserved Plant Vegetative Phase-Change-Related microRNAs in Mature and Rejuvenated Silver Birch In Vitro Propagated Tissues.

In plants, phase change from the juvenile stage to maturity involves physiological and anatomical changes, which are initiated and controlled by evolutionary highly conserved microRNAs. This process is of particular significance for the in vitro propagation of woody plant species, as individuals or tissues that have undergone the transition to vegetative maturity are recalcitrant to propagation. Conserved miRNAs differentially expressed between juvenile (including rejuvenated) and mature silver birch tissues were identified using high-throughput sequencing of small RNA libraries. Expression of some miR156 isoforms was high in juvenile tissues and has been previously reported to regulate phase transitions in a range of species. Additional miRNAs, such as miR394 and miR396, that were previously reported to be highly expressed in juvenile woody plant tissues were also differentially expressed in this study. However, expression of miR172, previously reported to be highly expressed in mature tissues, was low in all sample types in this study. The obtained results will provide insight for further investigation of the molecular mechanisms regulating vegetative phase change in silver birch and other perennial woody plant species, by analysing a wider range of genotypes, tissue types and maturation stages. This knowledge can potentially assist in identification of rejuvenated material at an earlier stage than currently possible, increasing the efficiency of silver birch in vitro propagation.

Identification of miRNAs Involved in Male Fertility and Pollen Development in Brassica oleracea var. capitata L. by High-Throughput Sequencing

MicroRNAs (miRNAs) are endogenously expressed, non-coding RNAs that play essential roles in numerous developmental and physiological processes in plants. However, there is a lack of data available on regulating fertility in cabbage (Brassica oleracea var. capitata L.). This study examined the expression of miRNAs during cabbage pollen development using high-throughput sequencing. Two small RNA libraries were constructed from flower buds from the cytoplasmic male-sterile (CMS) line and its maintainer fertile (MF) linee of cabbage, resulting in over 10 million sequence reads. A total of 211 new miRNAs and 7 conserved miRNAs were discovered. Eight randomly chosen miRNAs were found to have differential expression between CMS and MF, as determined by stem-loop quantitative real-time PCR (qRT-PCR). The expression patterns were similar to those detected with Illumina sequencing. Moreover, the possible targets of these putative miRNAs were inferred using a BlastX search and Gene Ontology (GO) analysis. The majority of targets have been indicated to be essential for the development of pollen or other plant parts. The discovery and characterization of miRNAs involved in pollen development in CMS and MF cabbage lines might drive research on the regulatory mechanisms of male sterility, leading to a better understanding of the crucial roles performed by miRNAs in this process.

Characterization of microRNAs and Target Genes in Musa acuminata subsp. burmannicoides, var. Calcutta 4 during Interaction with Pseudocercospora musae

Endogenous microRNAs (miRNAs) are small non-coding RNAs that perform post-transcriptional regulatory roles across diverse cellular processes, including defence responses to biotic stresses. Pseudocercospora musae, the causal agent of Sigatoka leaf spot disease in banana (Musa spp.), is an important fungal pathogen of the plant. Illumina HiSeq 2500 sequencing of small RNA libraries derived from leaf material in Musa acuminata subsp. burmannicoides, var. Calcutta 4 (resistant) after inoculation with fungal conidiospores and equivalent non-inoculated controls revealed 202 conserved miRNAs from 30 miR-families together with 24 predicted novel miRNAs. Conserved members included those from families miRNA156, miRNA166, miRNA171, miRNA396, miRNA167, miRNA172, miRNA160, miRNA164, miRNA168, miRNA159, miRNA169, miRNA393, miRNA535, miRNA482, miRNA2118, and miRNA397, all known to be involved in plant immune responses. Gene ontology (GO) analysis of gene targets indicated molecular activity terms related to defence responses that included nucleotide binding, oxidoreductase activity, and protein kinase activity. Biological process terms associated with defence included response to hormone and response to oxidative stress. DNA binding and transcription factor activity also indicated the involvement of miRNA target genes in the regulation of gene expression during defence responses. sRNA-seq expression data for miRNAs and RNAseq data for target genes were validated using stem-loop quantitative real-time PCR (qRT-PCR). For the 11 conserved miRNAs selected based on family abundance and known involvement in plant defence responses, the data revealed a frequent negative correlation of expression between miRNAs and target host genes. This examination provides novel information on miRNA-mediated host defence responses, applicable in genetic engineering for the control of Sigatoka leaf spot disease.

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.

Identification of regulatory pathways of miRna and phasiRna related to flax lignan synthesis

ABSTRACT Lignan is an important functional health care substance. At present, research on the molecular mechanism of flax lignan biosynthesis is primarily focused at the transcriptional level, with no research on the regulatory mechanism of small RNAs, particularly phased small interfering RNAs (phasiRNAs) related to lignan content. In this study, we used flax seeds at different developmental stages as materials to construct small RNA libraries and degradation libraries. Eighteen known and 4 new miRNA families containing 40 and 4 miRNA members, respectively, were identified. A total of 104 target genes were identified. Using small RNA data for a genome-wide search, a total of twenty-two 21-nt PHAS loci and twenty-eight 24-nt PHAS loci were obtained. Five miRNAs capable of triggering target genes to produce phasiRNAs were identified. These 5 miRNAs triggered their target genes to produce 39 phasiRNAs. Three complete miRNA-PHAS loci-phasiRNA-target gene functional regulatory pathways were identified. Furthermore, qRT‒PCR confirmed an obvious regulatory relationship among the regulatory pathways. In this study, the molecular regulatory network among flax miRNAs, phasiRNAs and target genes was described from the perspective of lignan synthesis, providing an important scientific basis for the study of flax molecular breeding for high lignan content.

Identification and characterization of immune-related microRNAs in hybrid snakehead(Channa maculata♀ × Channa argus♂)after treated by Echinacea purpurea (Linn.) Moench

Echinacea purpurea (Linn.) Moench (EP) is a globally popular herbal medicine, which showed effects on growth promotion, antioxidant and immunomodulatory activities in fish culture world widely. However, there are few studies about the effects on miRNAs by EP in fish. The hybrid snakehead fish (Channa maculate♀ × Channa argus ♂) was new important economic specie of freshwater aquaculture in China with high market value and demand while there were only a few reports about its miRNAs. To overview immune-related miRNAs of the hybrid snakehead fish and to further understand the immune regulating mechanism of EP, we herein constructed and analyzed three small RNA libraries of immune tissues including liver, spleen and head kidney of the fish with or without EP treatment via Illumina high-throughput sequencing technology. Results showed that EP can affect the immune activities of fish by the miRNA-regulated ways. Totally, 67 (47 up and 20 down) miRNAs in liver, 138 (55 up and 83 down) miRNAs in spleen, and 251 (15 up and 236 down) miRNAs in spleen were detected, as well as 30, 60, 139 kinds of immune-related miRNAs belonging to 22, 35 and 66 families of the three tissues respectively. The expressions of 8 immune-related miRNA family members were found in all the three tissues, including miR-10, miR-133, miR-22 and etc. Some miRNAs have been identified involved in the innate and adaptive immune responses, such as the miR-125, miR-138, and miR-181 family. Ten miRNA families with antioxidant target genes were also discovered, including miR-125, miR-1306, and miR-138, etc. Results from Gene Ontology (GO) and KEGG pathway analysis further confirmed there are a majority immune response targets of the miRNAs involved in the EP treatment process. Our study deepened understanding roles of miRNAs in fish immune system and provides new ideas for the study of immune mechanism of EP.

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.

Co-expression analysis of long non-coding RNAs and mRNAs involved in intramuscular fat deposition in Muchuan black-bone chicken

ABSTRACT 1. The intramuscular fat (IMF) content in meat products is positively correlated with meat quality, making it an important consumer trait. Long non-coding RNAs (lncRNAs) play central roles in regulating various biological processes, but little is currently known about the mechanisms by which they regulate IMF deposition in chickens. 2. This study sampled the breast muscles of chickens with high (H) and low (L) IMF content and constructed six small RNA libraries. High-throughput sequencing technology was used to profile the breast muscle transcriptome (lncRNA and mRNA) and to identify the differentially expressed lncRNAs (DELs) and mRNAs (DEGs) between the H and L groups. In total, 263 DELs (118 up-regulated and 145 down-regulated lncRNAs) and 443 DEGs (203 up-regulated and 240 down-regulated genes) were identified between the two groups. 3. To analyse the DELs-DEGs interaction network, co-expression analysis was conducted to identify lncRNA-mRNA pairs. In total, 19270 lncRNA/mRNA pairs were identified, including 16 398 significant correlation pairs that presented as positive and 2872 pairs that presented as negative. The lncRNA – mRNA network comprised 263 lncRNA nodes and 440 mRNA nodes. 4. Pathway analysis, using the Kyoto Encyclopedia of Genes and Genomes, indicated pathways associated with fat deposition and lipid metabolism such as the MAPK, PPAR, GnRH, ErbB and calcium signalling pathways, fatty acid elongation and fatty acid metabolism. Overall, the study identified potential candidate lncRNAs, genes and regulatory networks associated with chicken IMF deposition. These findings provide new insights to help clarify the regulatory mechanisms of IMF deposition in chickens which can be used to improve the IMF content in poultry.

PeGRF6-PeGIF1 complex regulates cell proliferation in the leaf of Phalaenopsis equestris

Phalaenopsis equestris is an ornamental plant with very large leaves. In this study, we identified genes related to the regulation of leaf development in Phalaenopsis and explored their mechanism of action. Sequence alignment and phylogenetic analyses revealed that PeGRF6 in the PeGRF family of P. equestris has similarities with the Arabidopsis genes AtGRF1 and AtGRF2, which are known to be involved in the regulation of leaf development. Among the PeGRFs, PeGRF6 was continuously and stably expressed at various stages of leaf development. The functions of PeGRF6 and of its complex formed with PeGIF1 in leaf development were verified by virus-induced gene silencing (VIGS) technology. The results show that the PeGRF6-PeGIF1 complex forms in the nucleus and positively regulates leaf cell proliferation via influencing cell size. Interestingly, VIGS suppression of PeGRF6 resulted in anthocyanin accumulation in Phalaenopsis leaves. Analyses of the regulatory mechanism of the miR396-PeGRF6 model based on the P. equestris small RNA library constructed here suggested that PeGRF6 transcripts are cleaved by Peq-miR396. These results show that, compared with PeGRF6 or PeGIF1 alone, the PeGRF6-PeGIF1 complex plays a more important role in the leaf development of Phalaenopsis, possibly by regulating the expression of cell cycle-related genes.

Malus asiatica as a natural host of apple scar skin viroid in China.

Malus asiatica (Rosaceae, Malus) is a small deciduous tree, which has been cultivated in China more than 450 years (Jin, 2019). M. asiatica is deeply favored by consumers because of its sweet taste and high nutritional attributes, rich in vitamins, minerals and dietary fiber (Xue et al, 2013). Although the M. asiatica annual output is nearly 30 000 kg, it still cannot meet the market demand in China (Jin, 2019). In August 2021, the virus-like symptom such as colored spots on fruit epidermis of M. asiatica were observed in an orchard of Langfang (38°42'16.88″N, 116°39'15.23″E) of Hebei province, China. To investigate whether this symptom is related to virus infection, the symptomatic sample was subjected to small RNA sequencing. Total RNA was extracted from branch bark of a symptomatic tree using an RNAprep Pure Plant Kit (TianGen, China), The extracted RNA was used to construct a small RNA library using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1), (NEB, USA), then the resulting library was sequenced using Illumina novoseq 6000 (Illumina, USA) at Tianjin Novogene company (China). A total of 14,685,616 sequence reads were obtained. After filtering the low-quality reads, polyA, adaptor contaminants, fragments < 18 nt and > 26 nt, and reads matching apple genome, the number of reads reduced to 392,883. Finally, assembly of these clean reads generated 225 non-redundant contigs with Velvet software and 55 assembled contigs were aligned to Refseq viral database of NCBI by Bowtie software. One viral contig with length of 329 nt showed 98.48% significant similarity to genome sequences of Hohhot isolate of ASSVd (ASSVd-Hohhot) (GenBank Accession No. MZ476527.1) (Yuan et al, 2022). We then used a specific primer pair (ASSVd-F: 5'-G G T A A A C A C C G T G C G G T T C C-3'; ASSVd-R: 5'-G G G A A A C A C C A A T T G T G T T T T A-3') for reverse transcription (RT)-PCR to amplify the genome sequence of ASSVd. A 330 bp amplified product was cloned into the pGEM-T easy vector (Promega, USA), then sequenced by Sanger sequencing using T7 primer by Sangon Biotech (Shanghai) Co., Ltd. in China. The sequence of ASSVd has been deposited in the GenBank datebase (GenBank Accession No. ON093255). Blast analysis showed that the sequence had highest identity (326/330, 98.79%) with ASSVd-Hohhot (GenBank Accession No. MZ476527.1) (Yuan et al, 2022). To confirm the pathogenicity of ASSVd, fifteen healthy cucumber seedlings were inoculated mechanically with the extracts of ASSVd-infected branch bark of M. asiatica. There were no obvious symptoms were observed at 14 days post inoculation (dpi), however, the result of RT-PCR and Sanger sequencing showed four cucumber samples were positive for ASSVd. In addition, another 19 randomly collected M. asiatica samples with or without clear symptoms from Langfang were detected by RT-PCR, and ten (52.6%) of them were confirmed the presence of ASSVd. And all ten positive samples were symptomatic, while nine nonsymptomatic M. asiatica samples tested negative. The positive amplicons were cloned into the pGEM-T easy vector and sequenced using T7 primer by Sanger sequencing. All of the sequences were essentially identical to one another (GenBank Accession No. ON093255), which indicates that the positive samples are indeed ASSVd infected. To the best of our knowledge, this is the first report of ASSVd infection in M. asiatica, which expands our understanding of the host range of ASSVd.

Exploring conserved and novel MicroRNA-like small RNAs from stress tolerant Trichoderma fusants and parental strains during interaction with fungal phytopathogen Sclerotium rolfsii Sacc.

The study investigated potential microRNA-like small RNAs (milRNAs) from multi-stress-tolerant Tricho-fusants and parental strains (P1- Trichoderma virens NBAIITvs12 and P2- Trichoderma koningii MTCC796) for antagonistic activity during interaction with phytopathogen Sclerotium rolfsii. The Trichoderma was cultured in-vitro, with and without antagonism, against the pathogen and total RNA was extracted followed by small RNA library construction and sequencing. The milRNAs were identified by mapping high-quality unique reads against a reference genome. The milRNAs were recognized higher in antagonist Trichoderma during interaction with test pathogen compared to normal growth. The novel milRNAs candidates were found to vary during interaction with the pathogen and normal growth. The gene ontology and functional analysis illustrated that a total of 5828 potential targeted genes were recognized for 93 milRNAs of potent Fu21_IB and 3053 genes for 62 milRNAs of least fusant Fu28_IL. Functional annotation of milRNA-predicted genes integrating KEGG pathways indicates new insights into regulatory mechanisms, by interfering with milRNAs, associated with signal transduction, amino sugar metabolism, benzoate degradation, amino acid metabolism, and steroid and alkaloid metabolism for potential biocontrol of stress-tolerant Tricho-fusant FU21 during interaction with S. rolfsii. The present investigation is the first report of conserved and novel milRNAs from Tricho-fusants and parental strains interacting with S. rolfsii.

Characterization and identification of grapevine heat stress-responsive microRNAs revealed the positive regulated function of vvi-miR167 in thermostability

High temperature stress is one of the primary abiotic stresses that restrict fruit tree production. Grapevine (Vitis vinifera) with high economic value throughout the world is a cultivated fruit crop, and its growth and development is often influenced by high temperature stress. Studying the heat stress-response mechanism of grapevine has great significance for understanding the acclimation to heat stress. In this study, we identified a series of heat stress responsive miRNAs and analyzed their function during the heat tolerance response. CK (control group, 25 °C) and heat treatment stress (TS, 45 °C) small RNA (sRNA) libraries were constructed and sequenced by high-throughput sequencing in ‘Thompson seedless’ grapevine. 873 known-miRNAs and 86 novel-miRNAs were identified, of which 88 known and three novel miRNAs were expressed differentially under heat stress. 322 genes were predicted to be targeted by the miRNAs. Eight selected miRNAs and its targets were confirmed by real time quantitative PCR (RT - qPCR), indicating that these “miRNA - target” were responsive to heat stress. In addition, most of the predicted target genes were negatively regulated by corresponding miRNAs. Gene function and pathway analyses indicated that these genes probably play crucial roles in heat stress tolerance. Vvi-miR167b transiently overexpression in grapevine leaves decreased target gene vvARF6, vvARF6-like and vvARF8 expression. The function of vvi-miR167 was verified by ectopic transformation in Arabidopsis thaliana, and the heat tolerance in transgenic lines was enhanced significantly, suggesting that the vvi-miR167 plays a positive regulatory role in grape thermostability. Comparison of miRNA expression patterns between heat treatment stress and CK can help elucidate the heat stress response and resistance mechanisms in grapes. In conclusion, these results gave us useful information to better understand the heat stress-response during domestication as well as for breeding new cultivars with heat stress resistance in fruit trees.

Multiomics analysis revealed miRNAs as potential regulators of the immune response in Carassius auratus gills to Aeromonas hydrophila infection.

The gill of fish is an important immune organ for pathogen defense, but its microRNA (miRNA) expression and regulatory mechanism remain unclear. In this study, we report on the histopathological and immunohistochemical features of the gills of the crucian carp Carassius auratus challenged with Aeromonas hydrophila. Small RNA libraries of the gills were constructed and sequenced on the Illumina HiSeq 2000 platform. A total of 1,165 differentially expressed miRNAs (DEMs) were identified in gills, of which 539 known and 7 unknown DEMs were significantly screened (p<0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes/proteins were primarily involved in 33 immune-related pathways, in which the inflammatory responses were focused on the Toll-like receptor (TLR), mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB) signaling pathways. Moreover, the expression levels of 14 key miRNAs (e.g., miR-10, miR-17, miR-26a, miR-144, miR-145, and miR-146a) and their target genes (e.g., TNFα, TLR4, NF-κB, TAB1, PI3K, and IRAK1) were verified. In addition, the protein levels based on isobaric tags for relative and absolute quantification (iTRAQ) were significantly associated with the results of the quantitative real-time PCR (qRT-PCR) analysis (p<0.01). miR-17/pre-miR-17 were identified in the regulation expression of the NF-κB target gene, and the phylogenetic tree analysis showed that the pre-miR-17 of C. auratus with the closest similarity to the zebrafish Danio rerio is highly conserved in teleosts. This is the first report of the multi-omics analysis of the miRNAs and proteins in the gills of C. auratus infected with A. hydrophila, thus enriching knowledge on the regulation mechanism of the local immune response in Cyprinidae fish.

Abstract 103: Isomir Diversity Is Increased In Peripheral Blood Following Ischemic Stroke And May Represent Shifts In Regulation Of Neuroinflammatory Response

MicroRNA (miRNA) expression is altered following ischemic stroke. Advances in RNA sequencing and bioinformatic tools allow study of isomiRs, isoforms of miRNA, which can have altered regulatory actions from canonical parent miRNA due to base modifications within the molecule or on the flanking regions. Here, we characterize isomiRs in the blood of ischemic stroke patients compared to vascular risk factor controls (VRFCs) and elucidate diversity of the post-stroke miRNA environment. Total RNA was isolated from peripheral blood of 47 ischemic stroke patients and 31 VRFCs using PAXgene protocol. Small RNA libraries were prepared using QIAseq miRNA Library Kit and sequenced to 14±2 M 1x75 bp reads. Raw sequence reads were trimmed, de-duplicated, and profiled using isomiRmap , which separates isomiRs from canonical miRNA sequences based on 3’ and 5’ miRNA modifications. Analysis of differentially expressed isomiRs was performed with DESeq2. A total of 74,562 unique isomiRs were identified, the majority expressed at very low level. Filtering for low abundance left 2,133 isomiRs (from 335 canonical miRNA) expressed in all subjects. Of these, 505 isomiRs (from 129 canonical miRNA) were differentially expressed (LFC &gt; |0.5|, p adj &lt; 0.05) between stroke and VRFCs. These isomiRs represent significant elevations in 3’ modifications (68.3% of DE isomiRs), 5’ modifications (20.2%) and non-template additions (34.3%). miRNA previously implicated in stroke had diverse isomiR profiles including cases where all isomiRs within a miRNA group were differentially expressed (e.g. let-7i) and cases where multiple isomiRs were present but not differentially expressed (e.g. miR-363). Four of 28 differentially expressed let-7i isomiRs have changes to the seed region that interacts with mRNA. Thus, miRNA-mRNA interactions distinct from the canonical let-7i seed may exist and play an altered role in stroke pathology and neuroinflammation. Analysis of small noncoding RNA sensitive to base-specific variations detects many differentially expressed isomiRs and highlights the complexity of the post-stroke miRNA environment. Further study of these isomiRs within mRNA regulatory networks will deepen our understanding of stroke related neuroinflammation and neural repair.

Identification and prediction of functions for drought-responsive microRNAs in sugar beet (Beta vulgaris)

Context Drought is a common abiotic stress affecting crop yield and quality worldwide. Sugar beet (Beta vulgaris L.) is a temperate crop that contributes greatly to world sucrose production and is affected by frequent drought. MicroRNAs (miRNAs) have been demonstrated to play an important role in plant abiotic stress responses. Protein-coding genes associated with drought resistance have been identified in sugar beet; however, studies on miRNAs involved in drought stress response are lacking. Aims The present study focused on analysing miRNAs in sugar beet and their roles in drought stress response. Method Small-RNA libraries were constructed from leaves of plants subjected to drought stress and well-watered conditions. High-throughput sequencing and bioinformatics analysis were used to investigate the genome-wide quantity of miRNAs and identify possible drought response regulatory effects. Key results Deep sequencing identified 49 known miRNAs and 59 new miRNAs. According to the Kyoto Encyclopaedia of Genes and Genomes (KEGG), the sulfur relay system was significantly enriched under drought stress. Co-regulated pairs between miR156a-5p, novel_18 and novel_41, and their target genes BVRB_6g136190, BVRB_009610 and BVRB_6g136680 were observed, suggesting a negative feedback modulation involved in the miRNA pathways. Conclusions Our results indicate that certain metabolic pathways such as the sulfur relay system are activated under drought conditions. Implications The results aid understanding of the mechanisms of drought response at the molecular level and may enable tools to be devised that enhance drought resistance in sugar beet.