IC4R006-lncRNA-2015-26387578

Project Title

• Analysis of non-coding transcriptome in rice and maize uncovers roles of conserved lncRNAs associated with agriculture traits

The Background of This Project

• Long non-coding RNAs (lncRNAs) have recently been found to widely exist in eukaryotes and play important roles in key biological processes. To extend the knowledge of lncRNAs in crop plants, the researchers performed both non-directional and strand-specific RNA-sequencing experiments to profile non-coding transcriptomes of various rice and maize organs at different developmental stages.

Plant Culture & Treatment

• Seeds from the cultivated rice subspecies Oryza sativa L. ssp. Japonica cultivar Nipponbare were grown in a greenhouse in Singapore under natural light conditions. Flower buds were collected before flowering and flowers were collected at the flowering day. Flag leaves and roots were collected at both the before- and afterflowering stage. The before-flowering sample was defined as a mixture of different stages in a period from panicle initiation to 1 day before flowering. The after-flowering sample was defined as a mixture of different stages after the flowering day. Milk grains and mature seeds were also collected. Maize (Zea mays L. ssp. mays) B73 seeds were germinated on wetted paper towel in plates for 2 days and then transferred to soil and grown for 2 weeks under 26°C and 16 h light and 8 h dark condition in a growth chamber at The Rockefeller University. Shoot and root tissues were separately collected. All samples were frozen in liquid nitrogen.

Research Findings

• Analysis of more than 3 billion reads identified 22 334 long intergenic non-coding RNAs (lincRNAs) and 6673 pairs of sense and natural antisense transcript (NAT).

• To see whether lincRNAs idenrified there are usually expressed at specific developmental stages or tissues with an organ preferenceit, the researchers compared expression levels of each transcript among all sequenced samples and found lincRNAs displayed more expression variation than mRNA in rice (P < 2.29x 10-16, Kolmogorov– Smirnov test, Figure 1a). Similar results were seen in maize. They found that the largest number of rice lincRNAs showed expression peaks in mature seeds followed by flower buds (Figure 1b). Several hundred organ-specific rice lincRNAs were identified in flowers, leaves or roots. We also found some developmental stage-specific lincRNAs (Figure 1c).

• Many lincRNA genes were associated with epigenetic marks. Expression of rice lincRNA genes was significantly correlated with that of nearby protein-coding genes(Figure 1e). A set of NAT genes also showed expression correlation with their sense genes(Figure 1f).

Figure 1 Temporal-spatial expression of lincRNAs and NATs in rice and maize. (a) Heat maps of rice lincRNA and mRNA expression levels (log2 of FPKM value). (b) Organ distribution of expression peaks of rice mRNAs and lincRNAs. (c) Three groups of organ- and/or developmental stage-specific rice lincRNAs. Y-axis gives the log2 value of FPKMs for lincRNAs. The total number of lincRNAs in each group is also given. (d) Validation of differentially expressed lincRNAs by qRT-PCR. Y-axis gives the relative expression levels. Error bars represent standard errors (n = 3). (e) Expression correlation of rice lincRNAs and flanking protein-coding genes. Y-axis gives Pearson product-moment correlation coefficient. Error bars represent standard deviations (n = 3). (f) Number of rice positively correlated and negatively correlated NAT pairs identified from comparison between any two samples. S1, milk grains. S2, mature seeds. R1, roots sampled before flowering. L1, leaves sampled before flowering. F1, flower buds. R2, roots sampled after flowering. L2, leaves sampled after flowering. F2, flowers.

• The researchers found 264 rice lincRNAs displaying sequence conservation with maize lincRNAs and more than half of rice lincRNAs (1177 out of 2281 lincRNAs, 51.6%) showing sequence conservation to maize mRNAs (Figure 2a). To further uncover lincRNAs located in conserved genomic regions but with limited sequence conservation, the researchers performed synteny search and found many more lincRNAs with positional conservation. Around 26.4% of rice (2965 out of 11 229) and 23.3% of maize lincRNAs (2589 out of 11 105) were embedded in synteny blocks (Figures 2b). Results of a chi-squared test showed that the number of lincRNA genes with positional conservation was significantly greater than that of lincRNA genes containing homologous sequences (P-value <0.001)(Figure 2c). They found the synteny blocks carrying lincRNA homologs were usually enriched for genes encoding transcription factors; these genes are involved in several important biological processes, such as responses to stress and developmental processes (Figure 2d).

Figure 2 Sequence and positional conservation of rice lncRNAs. (a) Sequence conservation of rice lincRNAs based on results of whole-genome alignment. (b) Positional conservation of rice lincRNA genes based on analysis of synteny blocks. (c) An example of a positionally conserved rice lincRNA. Gene structures are shown in solid color. lincRNA gene and high scoring pair (HSP) of synteny block are marked by diagonals and vertical lines. (d) Functional enrichment of flanking genes of positionally conserved rice lincRNAs.

• Integrating previous genome-wide association studies (GWAS), the researchers found that hundreds of lincRNAs contain trait-associated SNPs (single nucleotide polymorphisms [SNPs])(Figure 3) suggesting their putative contributions to developmental and agriculture traits.

Figure 3 LincRNA genes containing agriculture trait-associated SNPs. (a) A 20-kb flanking region of a rice lincRNA gene carrying a casual locus related to panicle number per plant. (b) Validation of a rice lincRNA gene containing a trait-associated SNP by qRT-PCR. Error bars give standard errors, n = 3. (c, d) Conserved lincRNAs associated with agronomic traits. (e) Organ-specific expression of conserved lincRNAs with potential functions. Y-axis gives FPKM values of lincRNAs. Agronomic trait-associated SNPs are indicated by triangles. Gene structures are shown in solid color. lincRNA locus and HSP of synteny block are marked by diagonals and vertical lines.

Labs working on this Project

• Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA, and
• Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore City 117604, Singapore

Corresponding Author

• chua@mail.rockefeller.edu