Conceived and designed the experiments: YJ XP. Performed the experiments: Analyzed the data: QY FW. Wrote the paper: Revised the text: Six-hundred sixty-one rice miRNAs are known that are important in plant development. It was approved by supervision department of Guangdong wild rice protection. We analyzed flowering-related miRNAs in O. Three O. Differential expression analysis of flowering-related miRNAs showed that: Real-time PCR revealed the same expression patterns as deep sequencing.
This is the first investigation of flowering miRNAs in wild rice. The result indicates that variation in miRNAs occurred during rice domestication and lays a foundation for further study of phase change and mir159 abandoned in O. Complicated regulatory networks mediated by multiple miRNAs regulate the expression of flowering genes that control the induction of flowering.
MicroRNAs miRNAspresent widely in eukaryotes, are a class of endogenous, non-coding small RNAs 20—24 nt that regulate gene expression by targeted protein-coding gene mRNA sequence-specific cleavage, translation repression or DNA methylation at the post-transcriptional level, often resulting in gene silencing  — .
Studies in Arabidopsis rice Ipomoea nil and the early-flowering mir159 abandoned of trifoliate orange  have shown that miRNAs, such as miR and miR, regulate the expression of developmental factors involved in flowering.
MiR, one of the most highly conserved plant microRNAs, is part of an intrinsic pathway for controlling the transition from vegetative growth to flowering in plants. The regulation of this pathway is based on changes in the mir159 abandoned content, miR declines from the vegetative stage to the reproductive stage, but the levels of its targets, SPL Squamosa Promoter Binding Protein Like transcriptional factors, increase during the same period.
However, SPLs control the transition between the juvenile and flowering stages by regulating the expression of a class of MADS box genes, which induced flowering  — . Like AP2 mutants, over-expressing of miR plants flower earlier and produce abnormal floral organs. The roles of miR and AP2 -like genes are important in controlling flowering time in plants miR is relatively well conserved in different plant species such Arabidopsismaize and rice.
A recent study has shown that miR acts downstream of miR, and its expression is regulated by miR MiR and miR have inverse patterns of expression, miR declines while miR increases during the plant life cycle, and the miR target SPL9 promotes transcription of miRb .
MiR degrades its target MYB33 in the process of gibberellin-induced flowering in Arabidopsisresulting in non-expression of LFY, delayed flowering in short days and male sterility  — .
However, over-expression of miR, a sequence highly similar to miR, decreases the levels of TCP transcription factors, then delays flowering in long days, stamen morphogenesis, and causes abortive stamens . MiR represses C gene expression in sepals and mir159 abandoned that target the nuclear transcription factor NF-YA, may promote drought stress and induce Arabidopsis early flowering in long days .
Silencing these genes can lead to miRNA loss, which further emphasizes their important functions during growth and development in rice. Some of these mir159 abandoned have been function analyzed in tissues such as inflorescence, stalks and mir159 abandoned seeds and spikelets .
Rice plants over-expressing osa-miRc or osa-miR show decreased salt and alkali stress tolerance . Over-expression of miR in rice can lead to spikelet loss, abnormal floral organs, and reduced fertility . The perennial O.
Very few investigations into genes involved in flowering have been conducted in common wild rice. Hagiwara et al. Furthermore, polymorphism in RFT1 at all sites mir159 abandoned higher than Hd3a. Our previous analysis of genetic diversity in the photoperiod genes Heading date 1 Hd1Earlyheading date1 Ehd1 and OsGI in common wild rice populations from different regions of China showed variation in OsGI introns between O.
Flowering is one of the most important agronomical traits. In a survey of O. Most common wild rice plants flower once a year, mir159 abandoned some populations flower twice a year. Within individual populations of common wild rice in Guangxi and Guangdong, there are both single-flowering plants and double-flowering plants. Transplanting these plants to the experimental base in Lingshui, Hainan Province mir159 abandoned a growth chamber in the laboratory indicated that the flowering mir159 abandoned was genetically stable .
In this study, common wild rice plants with different flowering times were collected from a single population growing in Gaozhou, Guangdong Province. There are not field trials in this study, we only survey flowering time of wild rice in the original habitat. There were two groups of plants that differed in their flowering times in the same population, the single-flowering group flowered from Mir159 abandoned to December, and the double-flowering group flowered initially in April to Mir159 abandoned, and again from October to December.
In late April, two libraries, CWR-V1 and CWR-V2, were constructed from leaves sampled in the vegetative stage of the single-flowering group and the double-flowering group, respectively. Library CWR-F2 was constructed from leaves in the flowering stage of the double-flowering group.
Genomic sequences nt upstream and nt downstream approximately bp surrounding each unannotated sRNA were extracted for secondary structures analysis by MIREAP https: The essential criteria were used for identifying new miRNA candidates in common wild rice .
Furthermore, KEGG pathway was used to further confirm the metabolic and signal transduction pathways . The mir159 abandoned expression of flowering-related miRNAs between pairs of libraries was demonstrated mir159 abandoned plotting the relative fold-change against the identified miRNAs as follows:.
Fold-change formula: Approximately 2. Quantitative real-time PCR assays were performed to investigate the expression of new miRNAs in roots, stems, leaves and young mir159 abandoned at the flowering stage. All reactions were performed using one biological sample with three technical replicates.
Sequencing of the CWR-V1, CWR-V2, and CWR-F2 libraries generated 23,, 21, and 24, raw reads, respectively, of which 20,, 21, and 20, corresponding genome-matched clean reads from the same three libraries, respectively Table mir159 abandoned. Similar to previous reports in rice the numbers of known rice miRNA reads were 3,, 4,, and 2,, respectively, accounting for samsung c3303 usb driver We hypothesize that highly expressed miRNAs benefit normal development in O.
Furthermore, many sRNAs were unannotated because of spatio-temporal-specific expression, genome differences between wild rice and cultivated rice, sequencing errors, etc. A sequence similarity search in miRBase The remaining known mir159 abandoned miRNAs were undiscovered in miRBase, and possible reasons for this are as follows: However, for flowering mir159 abandoned, 22 miRNAs were expressed only in the single-flowering group, 70 miRNAs were expressed only in the double-flowering group, and miRNAs were expressed in both groups.
Analyse-it adobe to previous deep sequencing reports in rice the expression of conserved miRNAs was considerably higher than that of non-conserved miRNAs. The most highly expressed miRNA, the miR family, mir159 abandoned for In addition, the expression of diverse members of the same family was very different. For example, the expression of miRa-miRj was in the millions of reads, while there were only 15 and reads specific for miRk and miRl, respectively.
The number of miRa and miRd-specific reads were andbut the maximum expression of miRb was 74 reads, and there were only three miRc-specific reads.
The expression of the other non-conserved miRNA families is irregular, with the expression of highly expressed miRNAs, such as miR, miR, in the thousands of reads. Conserved mir159 abandoned generally had different levels of expression in the two species, the most highly expressed member of a given family might play mir159 abandoned leading functional role in regulating one or more genes or gene families. The high sensitivity of the deep sequencing technology makes it feasible to investigate the distributions and numbers of the unique sequences along the miRNA precursors.
The same phenomena was also observed for miRd, miR, miR, and others. Perhaps this simply indicates that annotated miRNAs sequences are not necessarily real rice miRNAs, and that there is a complex mechanism of miRNAs biogenesis that includes multiple enzymes. Secondary analysis showed that the new miRNA precursors formed specific stem-loop structures Figure 2and the lengths varied mir159 abandoned 68 to bp, with an average of bp.
The new miRNA precursors were located in vastly different positions, and the number mir159 abandoned precursors on the sense strand of the genome wasmir159 abandoned located on the antisense strand. Also, 46 new miRNAs have been located in the introns of protein-coding genes, these intronic miRNAs could be mirtrons that do not mir159 abandoned on DCL cleavage, but rather are spliced and debranched by lariat debranching enzyme Ldbrafter which they fold into pre-miRNA hairpins and are processed to form mature miRNAs, a known example in rice is miR Most new miRNAs originate from single genetic loci, with only a single precursors present in the genome.
However, 20 new miRNAs were found to be originated from more than one genetic locus, 16 of these new miRNAs were mir159 abandoned expressed in our libraries. Of these, oru-miRNAs were expressed in a games for qmobile noir a10 library, 19 oru-miRNAs were expressed in all three libraries, and the remaining 38 were expressed in two of the libraries.
With respect to developmental stages, oru-miRNAs were expressed during the vegetative stage, 85 oru-miRNAs were expressed during the flowering stage, and the other 57 oru-miRNAs were expressed in both stages. For flowering times, 68 mir159 abandoned were expressed in the single-flowering group, oru-miRNAs were expressed in the double-flowering group, and 34 oru-miRNAs were expressed in both groups.
The miRNA sequences were consistent with the deep sequencing results. Expression analysis of new miRNAs in roots, stems, leaves, and booting panicles in the flowering stage indicated that most oru-miRNAs are mainly expressed in leaves Figure 4. Oru-miR was mainly expressed in roots, expressed at lower levels in leaves, and barely expressed in stems and booting panicles.
These results indicated that new oru-miRNAs had tissue-specific patterns of expression in O. OSActin was used as a housekeeping gene control. Four-hundred fifty-four of known rice miRNAs and of new oru-miRNAs had a total of 8, potential targets, with an average of Most miRNA families have multiple target sites, indicating mir159 abandoned these miRNAs play more than one role in plant development. These results suggest that miRNAs are involved in a wide range of physiological functions, and some miRNA targets associated with flowering.
Cellular Component categories indicated that the miRNA targets were related to seven cellular parts Figure 5Band mir159 abandoned three most frequent terms were cellcell part and organelle The three most highly represented GO terms out of six categories in Molecular Function were bindingcatalytic activity and transcription regulator activity Figure 5C.
In addition, these targets were further annotated using KEGG pathway analysis. A total of pathways were found, some of which mir159 abandoned consistent with biological processes revealed by GO. The five most frequent pathways were metabolic pathways, biosynthesis of secondary metabolites, plant-pathogen interaction, RNA degradation, and ribosome biogenesis in eukaryotes. Importantly, we found that 32 miRNA targets were involved in the rice circadian rhythm in our KEGG pathway analysis, the pathways that contained rhythmic regulated genes included UV-B protection, cell elongation, photoperiodic flowering, and antenna proteins.
Only seven out of 32 targets regulated the process of UV-B protection, the other 25 targets acted upstream of photoperiodic flowering Figure S3. Functional annotation and sequence homology analysis indicated that 54 miRNAs mir159 abandoned were directly associated with flowering in rice. For example, Os07gthe target of both miR and miR, was predicted to encode a putative RFT1 protein, which was reported to be a rice florigen that promoted flowering in rice . The target of miR The target of miR, Os02g