IC4R003-miRNA-2011-21791435

Project Title

Differential expression of the microRNAs in superior and inferior spikelets in rice (Oryza sativa)

The Background of This Project

Figure 1. Figure. 1. Superior and inferior spikelets in rice.

• Rice (Oryza sativa) is an important model species of monocotyledon plants with the smallest genome among the major cereals. As one of the world’s most important staple foods, rice is essential to food security, especially in populous countries of Asia and Africa. In the field of crop science, the product of yield sink capacity and filling efficiency is equal to grain yield in rice. To increase yield, a series of potential high yield rice varieties, such as New Plant Type rice (from the International Rice Research Institute, IRRI) and ‘super’ rice (being developed in China), have been selected and cultivated successively by traditional breeding technology. Although these cultivars generally have a higher yield potential from their larger panicles and numerous spikelets per panicle compared with conventional varieties, often this high yield potential is not realized completely because of poor grain-filling of inferior spikelets. In general, the later-flowering inferior spikelets of rice are located on secondary rachis branches of the lower primary rachis, while earlier-flowering superior spikelets are located on upper primary rachis branches. Inferior spikelets usually exhibit a slower rate of increase in dry weight during grain development and a lower grain weight than superior spikelets. Currently, improvement of the grain-filling rate and grain weight in inferior spikelets is one of the major tasks to enhance rice yield.
• Endogenous small RNAs (sRNAs) such as microRNAs(miRNAs) and short-interfering RNAs (siRNAs) play critical roles in endonucleolytic cleavage and translational inhibition at the post-transcriptional level and have been widely recognized as essential and effective regulators in diverse aspects in many eukaryotic organisms. Since the first miRNA was discovered in rice, many new miRNAs in rice have been identified using high-throughput sequencing technology. Some miRNAs are preferentially expressed at different stages of rice seed development, indicating the regulation by miRNAs during rice seed development. A rice miRNA, miR167, targeted ARF6 and ARF8, which might be involved in rice seed development through regulation of auxin signalling. miR156 has been predicted and confirmed to regulate OsSPL14, which contributes to panicle branching and higher grain productivity in rice. Overexpression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice

Plant Culture & Treatment

• Oryza sativa spp. japonica cv. Xinfeng 2 was planted from the same seed lot and grown in the field under non-stressed conditions. Superior and inferior spikelets (n¼35), sampled at 5 d intervals from fertilization to 45 DAF, were used to measure dry grain chimeric oligonucleotide adaptors, reverse transcribed, and amplified with 15 cycles of PCR to produce sequencing libraries. Finally, high-throughput sequencing was employed to sequence the sRNAs from the two rice grain samples at the Beijing Genomics Institute according to the manufacturer’s protocols. Image analysis, sequencing quality evaluation, and data production summarization were subsequently conducted with Solexa (Illumina, San Diego, CA, USA). All sRNA sequences were submitted to the Gene Expression Omnibus of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE28028.

Research Findings

• The grain weight of caryopses located on the upper primary rachis branches (superior spikelets) was significantly greater than that of those located on the secondary rachis branches of the lower primary rachis (inferior spikelets; Figure. 1). The grain weight of superior spikelets increased rapidly at 5–20 DAF, while that of inferior spikelets increased at 15–30 DAF (Figure. 1C, D). The size and appearance of brown rice from superior spikelets were better than those from inferior spikelets (Figure. 1B, C).
• Gene ontology (GO), the de facto standard for gene functionality descriptions, is widely used in functional annotation and enrichment analysis. Here, potential miRNA targets were subjected to functional enrichment analysis against a GO biological process terms database using AgriGO with default parameters. TIGR locus version 091211 was then selected as the suggested background for selecting references. To understand the function of these targets fully, a flash bar chart of overrepresented GO terms in the biological process category was generated by singular enrichment analysis.

Figure 2.

• Six known miRNAs (miR156a-j, miR164e, miR167d,f-h,j, miR397a,b, miR1861b,f,i,l, and miR1861e,g,k,m) and two newly identified miRNAs (miR5337 and miR5338) were validated using QRT-PCR (Figure. 2). In the reverse transcription reaction, mature miRNA was reverse transcribed into cDNAs using an miRNA-specific stem loop reverse transcription primer and a reverse transcriptase enzyme (Promega, Madison, WI, USA). In the QRT-PCR, the expression levels of the previously known and newly identified miRNAs were analysed using miRNA-specific primers (forward and reverse primers). Real-time quantitative PCR was conducted using an ABI PRISM ? 7500 Sequence Detection System, and all the primers used are listed in Supplementary Table S1 at JXB online. For each reaction, 5 ll of 1:20 diluted template cDNA was mixed with 10 ll of 2 3 SYBR green reaction mix (SYBR ? Green QRT-PCR Master Mix; Toyobo, Osaka, Japan), and 0.5 ll each of the forward and reverse primers and 4 ll of diethylpyrocarbonate (DEPC)-treated dH 2 O were added to make a final volume of 20 ll. The amplification program was as follows: 95 ?C for 5 min, followed by 15 s at 95 ?C, 15 s at 65 ?C, and 32 s at 72 ?C for 40 cycles.

Labs working on this Project

• Research Center for Rice Engineering and Key Laboratory of Physiology, Ecology and Genetic Improvement of Food Crops in Henan Province, Henan Agricultural University, Zhengzhou, 450002, China
• Henan University of Science and Technology, Luoyang, 471003, China

Corresponding Author

• Quanzhi Zhao(qzzhaoh@126.com)