Comparative expression profiling of miRNA during anther development in genetic male sterile and wild type cotton [small RNA]
ABSTRACT: Genetic male sterility (GMS) in cotton (Gossypium hirsutum) plays an important role in the utilization of hybrid vigor. However, the molecular mechanism of the GMS is still unclear. While numerous studies have demonstrated that microRNAs (miRNA) regulate flower and anther development, whether different small RNA regulations exist in GMS and its wild type is unclear. To investigate the global expression and complexity of small RNAs during cotton anther development, three small RNA libraries were constructed from the anthers of three development stages each from fertile wild type (WT) and its GMS mutant cotton. Examination of different miRNA profiles in 2 lines.
Project description:In this study, in order to identify miRNA targets, a degradome library derived from anthers of the WT and GMS (Genetic Male Sterility) mutant representing three stages of development was constructed and sequenced, resulting in the generation of 24.6 million raw reads. After removal of low quality sequences and adapter sequences, 24.4 million clean reads were obtained and 98% were 20 or 21 nt in length as expected in that normally length distribution peak of degradome fragment is between 20 and 21 nt [Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ: Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol 2008, 18:758-762]. Identification of miRNA targets in the WT and GMS muant anthers. Anthers of the WT and GMS mutant representing three stages of development [the meiosis stage (WT: Mar-F-1; mutant: Mar-S-1) and tetrad stage (WT: Mar-F-2; mutant: Mar-S-2), together with the uninucleate microspore stage (WT: Mar-F-3; mutant: Mar-S-3) from the GMS ‘Dong A’ mutant and its fertile wild type] were collected during early mornings.
Project description:MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are short (19–25 nucleotides) non-coding RNA molecules that have large-scale regulatory effects on development and on stress responses in plants.The objective of this study is to investigate the transcriptional profile of miRNAs and other small non-coding RNAs in Verticillium–inoculated cotton roots. Four small RNA libraries were constructed from mocked and infected roots of two cotton cultured species which are with different Verticillium tolerance (‘Hai-7124’, Gossypium barbadense L., a Verticillium-tolerant cultivar, and ‘Yi-11’, Gossypium hirsutum L. a Verticillium-sensitive cultivar). The length distribution of obtained small RNA pools was significantly different among libraries. A total of 215 conserved miRNA families were identified in the two cotton species, of them 14 are novel. There were >65 families with different expression between two libraries. We also identified two ta-siRNAs and thousands of endogenous siRNA candidates, and hundred of them exhibited altered expression after inoculation of Verticillium. The profiling of these miRNAs and other small non-coding RNAs lay the foundation for further understanding of small RNAs function in the regulation of Verticillium defence responses in cotton roots. Examination of 2 different traetments in 2 cotton types.
Project description:To identify potential miRNAs involved in fiber development and elucidate their expression differences between G. barbadense and G. hirsutum, we constructed two small RNA libraries, Gb10 and Gh10, prepared from fibers of 3-79 (G. barbadense) and TM-1 (G. hirsutum) collected at 10 days post-anthesis (DPA). We identified 28 conserved miRNA families, including 24 that exactly match known plant miRNA families in miRBase. With MIREAP and newly developed software miRsearcher, 7 candidate-novel miRNAs were found. 5 candidate-novel miRNAs were expressed in both species, 2 candidate-novel miRNAs were expressed only in one species. Moreover, 4 miRNA families showed significant expression differences between sea-island cotton and upland cotton in 10 DPA fibers. two examples including 3-79 and TM-1 10 DPA fibers
Project description:Background: Dwarf cottons are more resistant to damage from wind and rain and associated with stable, increased yields, and also desirable source for breeding the machine harvest varieties. In an effort to uncover the transcripts and miRNA networks involved in plant height, the transcriptome and small RNA sequencing were performed based on dwarf mutant Ari1327 (A1), tall-culm mutant Ari3697 (A3) and wild type Ari971 (A9) in Gossypium hirsutum. Results: The transcriptome sequencing analysis showed that the enriched pathways of top 3 differentially expressed genes (DEGs) were categorized as carotenoid biosynthesis, plant-pathogen interaction and plant hormone signal transduction in both A1-A9 and A3-A9. The ABA and IAA related factors were differentially expressed in the mutants. Importantly, we found the lower expressed SAUR and elevated expressed GH3, and ABA related genes such as NCED and PP2C maybe relate to reduced growth of the plant height in Ari1327 which is consistent with the higher auxin and ABA content in this mutant. Furthermore, miRNA160 targeted to the auxin response factor (ARF) and miRNA166 (gma-miR166u and gma-miR166h-3p) targeted to ABA responsive element binding factor were related to the mutation in cotton. We have noticed that the cell growth related factors (smg7 targeted by gra-miR482 and 6 novel miRNAs and Pectatelyases targeted by osa-miR159f), the redox reactions related factors (Cytochrome P450 targeted by miR172) and MYB genes targeted by miR828, miR858 and miR159 were also involved in plant height of the cotton mutants. A total of 226 conserved miRNAs representing 32 known miRNA families were obtained, and 38 novel miRNAs corresponding to 23 unique RNA sequences were identified. Total 531 targets for 211 conserved miRNAs were obtained. Using PAREsnip, 27 and 29 miRNA/target conserved interactions were validated in A1-A9 and A3-A9, respectively. Furthermore, miRNA160, miRNA858 and miRNA172 were validated to be up-regulated in A1-A9 but down-regulated in A3-A9, whereas miRNA159 showed the opposite regulation. Conclusions: This comprehensive interaction of the transcriptome and miRNA at tall-culmand dwarf mutant led to the discovery of regulatory mechanisms in plant height. It also provides the basis for in depth analyses of dwarf mutant genes for further breeding of dwarf cotton. Total RNA was purified from stem apexes of three samples at the fifth true leaf stages and sequenced deeply using Illumina HiSeq 2000 system.
Project description:MicroRNAs play critical roles in various biological and metabolic processes. The function of miRNAs has been widely studied in model plants such as Arabidopsis and rice. However, the number of identified miRNAs and related miRNA targets in peach (Prunus persica) is limited. To understand further the relationship between miRNAs and their target genes during tissue development in peach, a small RNA library and three degradome libraries were constructed from three tissues for deep sequencing. We identified 117 conserved miRNAs and 186 novel miRNA candidates in peach by deep sequencing and 19 conserved miRNAs and 13 novel miRNAs were further evaluated for their expression by RT-qPCR. The number of gene targets that were identified for 26 conserved miRNA families and 38 novel miRNA candidates, were 172 and 87, respectively. Some of the identified miRNA targets were abundantly represented as conserved miRNA targets in plant. However, some of them were first identified and showed important roles in peach development. Our study provides information concerning the regulatory network of miRNAs in peach and advances our understanding of miRNA functions during tissue development. To identify more conserved and peach-speciﬁc miRNAs and their target genes and to understand further the mechanism of miRNA-regulated target genes during tissue development in peach, a small RNA library and three degradome libraries were constructed from three different tissues for deep sequencing.
Project description:Cotton is the most important economic crop that provides natural fibre and by-products such as oil and protein. The global gene expression could provide insight into the biological processes underlying growth and development, which involving suites of genes expressed with temporal and spatial controls by regulatory networks. Improvement of cotton fiber in yield and quality is the main goal for molecular breeding, but many previous research have been largely focused on identifying genes only in fibres, so that we ignore seed which may play an important role in the development of fibers. In this study, we constructed and systematically analyzed twenty-one strand-specific RNA-Seq libraries on Gossypium hirsutum L. covering different tissues, organs and development stages, of which approximately 970 million reads were generated. In total, 5,6754 transcripts derived from 2,9541 unigenes were obtained to provide a global view of gene expression for cotton development. Hierarchical clustering of transcriptional profiles suggests that transcriptomes among tissues or organs corresponded well to their developmental relatedness. The organ (tissue)-specific gene expressions were investigated efficiently and provided further insight into the dynamic programming of the transcriptome, in particularly for coordinating development between fiber cell and seed (ovule). We identified series of transcription factors and seed-specific genes, which as the candidate genes should help elucidate key mechanisms and regulatory networks that underlie fiber and seed development. This report identified comprehensive transcriptome changes in different stage of cotton development and will serves as a valuable genome-wide transcriptome resource for cotton breeding. Examination of transcriptome of cotton
Project description:MicroRNAs (miRNAs) are endogenous non-coding ~21 nucleotide (nt) RNAs that regulate gene expression at transcriptional and post-transcriptional levels in plants and animals. They play an important role in development, abiotic stress responses or pathogen responses. miRNAs with their related target genes have been widely studied in model plants，and increasing studies have been performed on some crops，however, the number of identified miRNAs in cotton was limited, and global identification of related targets through degradome sequencing has not been developed previously. In this study, we globally identified small RNAs and their related target genes during cotton somatic embryogenesis by the high throughput small RNA and degradome sequencing technology using fresh hypocotyls and EC (embryogenic calli) of Gossypium hirsutum YZ1. A total of 36 differentially expressed conserved miRNA families of which 19 miRNA families represented by 29 precursors and 25 novel miRNAs were identified, with star sequences of 20 known miRNAs and 2 novel miRNAs discovered. 234 genes in EC and 322 genes in CK were identified as targets of 23 and 30 known miRNA families, and 16 genes were found as targets of 8 novel miRNAs. The expression profiles of several miRNAs and their targets were verified by qRT-PCR and 5’RACE were further used to validate the sliced sites of the targets. Interestingly, four TAS3 D6 and D7 were also found in both degradome libaries which can perfectly match their precursors. The profiling of the miRNAs and their target genes provides more information about the regulatory network of miRNAs during somatic embryogenesis in cotton. small RNA and degradome sequencing of CK(fresh hypocotyls) and EC (embryogenic calli) of Gossypium hirsutum YZ1
Project description:The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNA) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 81 unique miRNA sequences from 30 families, and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing datasets and TaqMan qPCR results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, expressions of cold-stress responsive smRNAs integrated in the auxin-signaling pathway and their target genes were largely anticorrelated. We investigated tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway, and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress. Examination of 7 small RNA libraries in spike tissues during cold and control condition
Project description:The small RNA transcriptomes of bread wheat (Triticum aestivum L.) and its emerging model (Brachypodium distachyon (L.) Beauv) were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNA) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of ESTs, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5' RACE experiments demonstrated their capability to cleave predicted target genes including three disease resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in Brachypodium distachyon. Keywords: Small RNA One wheat small RNA library (Tae) and two Brachypodium small RNA libraries (BdR and BdV) were sequenced.
Project description:We profiled and quantitated miRNAs in two skin tumors (Basal cell carcinoma and Merkel cell carcinoma) and identified tumor-specific miRNAs. We used these tumor-specific miRNAs to guide development of miRNA fluorescence in situ hybridization. 2 barcoded sequencing runs, including 40 unique samples (36 used in manuscript). The details of each sample can be found in Supplementary Tables S1 and S2.