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Identification and expression analysis of microRNAs at the grain filling stage in rice(Oryza sativa L.)via deep sequencing.

Yi R, Zhu Z, Hu J, Qian Q, Dai J, Ding Y - PLoS ONE (2013)

Bottom Line: A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain.In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally.Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Hybrid Rice, Department of Genetics, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACT
MicroRNAs (miRNAs) have been shown to play crucial roles in the regulation of plant development. In this study, high-throughput RNA-sequencing technology was used to identify novel miRNAs, and to reveal miRNAs expression patterns at different developmental stages during rice (Oryza sativa L.) grain filling. A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that 161 known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from 5 to 7 days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling.

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miRNA editing analysis.(A) Summary of the nucleotide substitution types observed in each library. (B) Summary of nucleotide substitution positions among miRNAs. (C) Validation of the editing sites inferred from deep sequencing via Sanger sequencing. Sequencing chromatogram traces from four miRNA sequences are shown. The edited positions are highlighted with yellow shading. The top trace is genomic DNA (gDNA), and the bottom trace is cDNA.
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pone-0057863-g005: miRNA editing analysis.(A) Summary of the nucleotide substitution types observed in each library. (B) Summary of nucleotide substitution positions among miRNAs. (C) Validation of the editing sites inferred from deep sequencing via Sanger sequencing. Sequencing chromatogram traces from four miRNA sequences are shown. The edited positions are highlighted with yellow shading. The top trace is genomic DNA (gDNA), and the bottom trace is cDNA.

Mentions: Through analyzing the sequencing data from the four small RNA libraries produced in this study, a large number of miRNA editing events were found (Table S10). The sequencing results revealed that miRNAs and their variants exhibit many types of site-specific nucleotide substitutions and the average rate of each type of nucleotide substitutions found in each library was similar (Fig. 5A). In contrast to the four most common nucleotide substitutions observed in miRNAs from Arabidopsis, which are C to U (9.7%), A to G (16.4%), G to A (24.2%) and U to C (12.6%) [35], in the present study, the most dominant nucleotide substitution type was A to U (over 60% in each library) because the mutation rate of A to U at position 14 of the miR156k variant was much higher compared to other forms of nucleotide substitutions (Table S10 and Fig. 5A). To understand whether the detected miRNA editing occurs in vivo, or was caused by DNA SNPs and mismatches that took place during data analysis and other technical artifacts, pre-miRNAs sequence cloned from rice genomic DNA were compared to mature miRNAs obtained from rice cDNA. Two types of nucleotide substitutions, high-level editing (90%–100%) and median-level editing (60%–70%), were chosen to be tested in the 5 DAF sample. pre-miRNAs and mature miRNAs for osa-miR164c, osa-miR166j, osa-miR166m and a 3′-end deletion variant of osa-miR1861a (the most dominant small RNA forms in our study) were selected to confirm the miRNA editing events observed in this study. The miRNA RT-PCR sequencing results showed that all four of these edited miRNAs or variants exhibit the same nucleotide substitutions at the same sites as in the deep sequencing data. The DNA sequencing results showed there were no SNPs detected between the reference and miRNA genes, except for the osa-miR1861a variant (Fig. 5C). These results indicate that the miRNA editing results obtained from Solexa sequencing are reliable, and the detected miRNA editing is genuine and occurs during the grain filling process.


Identification and expression analysis of microRNAs at the grain filling stage in rice(Oryza sativa L.)via deep sequencing.

Yi R, Zhu Z, Hu J, Qian Q, Dai J, Ding Y - PLoS ONE (2013)

miRNA editing analysis.(A) Summary of the nucleotide substitution types observed in each library. (B) Summary of nucleotide substitution positions among miRNAs. (C) Validation of the editing sites inferred from deep sequencing via Sanger sequencing. Sequencing chromatogram traces from four miRNA sequences are shown. The edited positions are highlighted with yellow shading. The top trace is genomic DNA (gDNA), and the bottom trace is cDNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585941&req=5

pone-0057863-g005: miRNA editing analysis.(A) Summary of the nucleotide substitution types observed in each library. (B) Summary of nucleotide substitution positions among miRNAs. (C) Validation of the editing sites inferred from deep sequencing via Sanger sequencing. Sequencing chromatogram traces from four miRNA sequences are shown. The edited positions are highlighted with yellow shading. The top trace is genomic DNA (gDNA), and the bottom trace is cDNA.
Mentions: Through analyzing the sequencing data from the four small RNA libraries produced in this study, a large number of miRNA editing events were found (Table S10). The sequencing results revealed that miRNAs and their variants exhibit many types of site-specific nucleotide substitutions and the average rate of each type of nucleotide substitutions found in each library was similar (Fig. 5A). In contrast to the four most common nucleotide substitutions observed in miRNAs from Arabidopsis, which are C to U (9.7%), A to G (16.4%), G to A (24.2%) and U to C (12.6%) [35], in the present study, the most dominant nucleotide substitution type was A to U (over 60% in each library) because the mutation rate of A to U at position 14 of the miR156k variant was much higher compared to other forms of nucleotide substitutions (Table S10 and Fig. 5A). To understand whether the detected miRNA editing occurs in vivo, or was caused by DNA SNPs and mismatches that took place during data analysis and other technical artifacts, pre-miRNAs sequence cloned from rice genomic DNA were compared to mature miRNAs obtained from rice cDNA. Two types of nucleotide substitutions, high-level editing (90%–100%) and median-level editing (60%–70%), were chosen to be tested in the 5 DAF sample. pre-miRNAs and mature miRNAs for osa-miR164c, osa-miR166j, osa-miR166m and a 3′-end deletion variant of osa-miR1861a (the most dominant small RNA forms in our study) were selected to confirm the miRNA editing events observed in this study. The miRNA RT-PCR sequencing results showed that all four of these edited miRNAs or variants exhibit the same nucleotide substitutions at the same sites as in the deep sequencing data. The DNA sequencing results showed there were no SNPs detected between the reference and miRNA genes, except for the osa-miR1861a variant (Fig. 5C). These results indicate that the miRNA editing results obtained from Solexa sequencing are reliable, and the detected miRNA editing is genuine and occurs during the grain filling process.

Bottom Line: A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain.In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally.Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Hybrid Rice, Department of Genetics, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACT
MicroRNAs (miRNAs) have been shown to play crucial roles in the regulation of plant development. In this study, high-throughput RNA-sequencing technology was used to identify novel miRNAs, and to reveal miRNAs expression patterns at different developmental stages during rice (Oryza sativa L.) grain filling. A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that 161 known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from 5 to 7 days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling.

Show MeSH