Functional & Integrative Genomics

, Volume 15, Issue 3, pp 323–348 | Cite as

Novel and conserved heat-responsive microRNAs in wheat (Triticum aestivum L.)

  • Ranjeet Ranjan KumarEmail author
  • Himanshu Pathak
  • Sushil Kumar Sharma
  • Yugal Kishore Kala
  • Mahesh Kumar Nirjal
  • Gyanendra Pratap Singh
  • Suneha Goswami
  • Raj Deo Rai
Original Paper


MicroRNAs (miRNAs) are small endogenous RNAs of ~22 nucleotides that have been shown to play regulatory role by negatively affecting the expression of genes at the post-transcriptional level. Information of miRNAs on some important crops like soybean, Arabidopsis, and rice, etc. are available, but no study on heat-responsive novel miRNAs has yet been reported in wheat (Triticum aestivum L.). In the present investigation, a popular wheat cultivar HD2985 was used in small RNA library construction and Illumina HiSeq 2000 was used to perform high-throughput sequencing of the library after cluster generation; 110,896,604 and 87,743,861 reads were generated in the control (22 °C) and heat-treated (42 °C for 2 h) samples, respectively. Forty-four precursor and mature miRNAs were found in T. aestivum from miRBase v 19. The frequencies of the miRNA families varied from 2 (tae-miR1117) to 60,672 (tae-miR159b). We identify 1052 and 902 mature miRNA sequences in HD2985 control and HS-treated samples by mapping on reference draft genome of T. aestivum. Maximum identified miRNAs were located on IWGSC_CSS_3B_scaff (chromosome 3B). We could identify 53 and 46 mature miRNA in the control and HS samples and more than 516 target genes by mapping on the reference genome of Oryza sativa, Zea mays, and Sorghum bicolor. Using different pipelines and plant-specific criteria, 37 novel miRNAs were identified in the control and treated samples. Six novel miRNA were validated using qRT-PCR to be heat-responsive. A negative correlation was, however, observed between the expression of novel miRNAs and their targets. Target prediction and pathway analysis revealed their involvement in the heat stress tolerance. These novel miRNAs are new additions to miRNA database of wheat, and the regulatory network will be made use of in deciphering the mechanism of thermotolerance in wheat.


Heat stress Heat-responsive Illumina HiSeq miRNA Next-generation sequencing qRT-PCR Stress-associated proteins Transcriptome Target genes Triticum aestivum Survey sequence Genome 

List of abbreviations


Next-generation sequencing


RNA-induced silencing complex




Heat stress


Heat shock factor


Heat shock protein


Calcium-dependent protein kinase


Quantitative real-time PCR


Stress-associated genes


Stress-associated proteins



Financial assistance provided by the Indian Council of Agriculture Research (ICAR) under the National Initiative for Climate Resilient Agriculture (NICRA) project (Project 12/115 TG3079) is highly acknowledged. We also thank Director, Indian Agricultural Research Institute (IARI) for providing all the logistic support required for executing the research work.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

Experiments comply with the current laws of the country in which they were performed.

Availability of NGS raw data

The data sets supporting the results of this article are available in the National Center for Biotechnology Information (NCBI) repository [BioProject Database: PRJNA172054;].

Supplementary material

10142_2014_421_MOESM1_ESM.docx (158 kb)
ESM_1.tif Flow chart of the work plan undertaken for the identification of conserved and novel heat-responsive miRNA in control and heat stress treated samples of HD2985 (thermotolerant) cultivar of wheat (Triticum aestivum L.). (DOCX 158 kb)
10142_2014_421_MOESM2_ESM.docx (45 kb)
ESM_2.tif Work-flow for novel miRNA discovery using the raw reads from the sequenced libraries of control and heat stress treated samples of HD2985 cultivar of wheat (Triticum aestivum L.); different pipeline analyzers were used for the identification of novel heat-responsive miRNAs from the potential candidates. (DOCX 45 kb)
10142_2014_421_MOESM3_ESM.xls (39 kb)
ESM_3.xls List of primers used for the comparative expression analysis of mature miRNAs and their target genes in control and heat stress treated samples of HD2985 (thermotolerant) cultivar of wheat (Triticum aestivum). (XLS 39 kb)
10142_2014_421_MOESM4_ESM.xls (44 kb)
ESM_4.xls List of miRNAs identified in control and heat stress treated samples of HD2985 (thermotolerant) cultivar using the reference draft genome of wheat (Triticum aestivum). (XLS 44 kb)
10142_2014_421_MOESM5_ESM.xls (50 kb)
ESM_5.xls A list of candidate miRNAs, identified targets and their functional annotation based on the reference draft genome of Triticum aestivum (retrieved from (XLS 50 kb)
10142_2014_421_MOESM6_ESM.xls (48 kb)
ESM_6.xls List of miRNAs identified in control and heat stress treated samples of HD2985 (thermotolerant) cultivar of wheat based on homology search using the reference genome of rice (Oryza sativa). (XLS 48 kb)
10142_2014_421_MOESM7_ESM.xls (42 kb)
ESM_7.xls List of miRNAs identified in control and heat stress treated samples of HD2985 (thermotolerant) cultivar of wheat based on homology search using the reference genome of maize (Zea mays). (XLS 42 kb)
10142_2014_421_MOESM8_ESM.xls (42 kb)
ESM_8.xls List of miRNAs identified in control and heat stress treated samples of HD2985 (thermotolerant) cultivar of wheat based on homology search using the reference genome of sorghum (Sorghum bicolor). (XLS 41 kb)
10142_2014_421_MOESM9_ESM.xls (230 kb)
ESM_9.xls List of target genes of mature miRNAs identified by homology based search using the reference genome of T. aestivum, O. sativa, Z. mays and S. bicolor. (XLS 229 kb)
10142_2014_421_MOESM10_ESM.docx (96 kb)
ESM_10.tif Secondary structure analysis of validated heat-responsive miRNAs from wheat (Triticum aestivum); Flicker v3.0 was used for the prediction of secondary structure and structure analysis was performed using Zuker’s algorithm. (DOCX 95 kb)
10142_2014_421_MOESM11_ESM.xls (122 kb)
ESM_11.xls Novel miRNAs (identified based on homology search using the reference genome of O. sativa, Z. mays and S. bicolor) and their respective target genes identified in control sample of HD2985 cultivar of wheat. (XLS 122 kb)
10142_2014_421_MOESM12_ESM.xls (60 kb)
ESM_12.xls Novel miRNAs (identified based on homology search using the reference genome of O. sativa, Z. mays and S. bicolor) and their target genes identified in heat shock treated samples of HD2985 cultivar of wheat. (XLS 60 kb)
10142_2014_421_MOESM13_ESM.xls (335 kb)
ESM_13.xls List of mature miRNAs (identified in present investigation) involved in thermotolerance pathways along with their target genes. (XLS 335 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Ranjeet Ranjan Kumar
    • 1
    Email author
  • Himanshu Pathak
    • 2
  • Sushil Kumar Sharma
    • 1
  • Yugal Kishore Kala
    • 3
  • Mahesh Kumar Nirjal
    • 1
  • Gyanendra Pratap Singh
    • 3
  • Suneha Goswami
    • 1
  • Raj Deo Rai
    • 1
  1. 1.Division of BiochemistryIndian Agricultural Research InstituteNew DelhiIndia
  2. 2.Division of Environmental ScienceIndian Agricultural Research InstituteNew DelhiIndia
  3. 3.Division of GeneticsIndian Agricultural Research InstituteNew DelhiIndia

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