Protocol of miRNA-based marker assay.
The high conservation of miRNA sequences provided an opportunity to develop an effective type of markers that is useful not only for genetic diversity study but also as potential biomarkers in plant stress responses. The fundamental potential of miRNA-based markers relies on the primer design based on the sequences of mature miRNAs, which are part of the step-loop structures. The advantages of this marker system include high polymorphism, reproducibility, and transferability across species. The abundance of mature miRNAs, which is linked to the expression of MIRNA genes, varies greatly among miRNAs, tissue types, or developmental stages, indicating the spatially and temporally regulated expression patterns of plant miRNAs. The results confirm the significance, reliability, and the position of miRNA-based markers as stress-sensitive biomarkers, indicating their potential in a wide range of applications of agricultural research.
- molecular markers
Systematic documentation and evaluation of plant genome based on molecular markers that capture variability at the level of DNA or its protein products is essential for plant genetic resources management. These markers complement the morphological, agronomic, and other characteristics necessary to classify and identify plant genotypes within the species. Their importance and benefits are significant both for research and breeding, and for practice [1, 2]. The study and comparison of molecular information of individual organisms involves the search for DNA polymorphisms . Polymorphisms are differences in DNA sequence caused by mutations. Molecular techniques in which polymorphisms can be visualized without sequencing are called molecular marker techniques . A molecular marker is a specific fragment of DNA that can be identified within the whole genome and that is transmitted to the next generation following the standard rules of inheritance (Mendel’s Laws). Marker may be located in or close to a gene or in noncoding regions.
The presence of polymorphisms between individuals will lead to a different pattern of markers after electrophoresis. These patterns are comparable with a “fingerprint”; therefore, these techniques are referred to as fingerprinting techniques. These patterns reveal the DNA polymorphisms between the studied individuals. The more the individuals are related, the more their fingerprint will match. The level of polymorphisms in a group of individuals reveals the genetic diversity within this group .
There are different types of molecular markers. Molecular markers can be divided into two groups (a) biochemical markers, which detect variation at the gene product level such as changes in proteins and amino acids and (b) molecular markers, which detect variation at the DNA level such as nucleotide changes: deletion, duplication, inversion, and/or insertion. DNA markers are based on hybridization or PCR (amplification of DNA) . The multiplex ratio is defined as the amount of markers generated with one single reaction (e.g., one PCR, one hybridization reaction). The obtained pattern can be simple (one or a few bands, low multiplex ratio) or complex (a high number of bands, high multiplex ratio).
Molecular marker methods are either dominant or co-dominant. Using co-dominant marker techniques, the different genotype combinations can be distinguished from each other at the study locus (or multiple loci). This means that homozygous (two identical alleles at a certain DNA locus) and heterozygous (two different alleles at a certain DNA locus) individuals will be identifiable. In the case of a dominant technique, it is not possible to detect the alleles that are present at a certain locus/loci, so homozygotes are not distinguishable from heterozygotes [6, 7].
The high conservation of microRNA sequences provided an opportunity to develop miRNA-based marker system referred to as stable, polymorphic, functional, and transferable genotyping technique.
2. The role of miRNAs in plant genome response to abiotic stress
The plant organism has to cope with the environmental stress in natural and agricultural conditions. The genetic background of the plant organism allows it to adapt and defend itself through different mechanisms at a molecular level.
RNA interference represents the plant immune and defense system. It is a conserved mechanism induced by double-stranded RNA (dsRNA) or hairpin-structured RNA (hpRNA). One of the modules of RNA interference is provided by the microRNA (miRNA) molecules [8, 9, 10], which are capable to form double-stranded hairpin-like structures referred to as pre-miRNA. These small molecules have significant regulatory potential in the genetic and epigenetic control of gene expression. They are one of the key players in plant genome response to abiotic and biotic stress factor(s). Especially, deeply conserved miRNA families are integral components of many regulatory networks in plant organism [11, 12].
In general, the function of miRNAs molecules in plant organisms is defined as regulatory in the following processes:
plant growth and development
leaf morphology and plant polarity
processes of transition from embryogenic to vegetative phase
flowering time, formation of flower organs, and reproduction
defense mechanisms through transferring of signaling molecules.
Plant adaptation mechanism requires complex modifications of gene expression machinery at the transcriptional and posttranscriptional level. Detailed studies of posttranscriptional gene regulation allow identifying stress-responsive miRNAs, which are differentially regulated under various stress factors. In plants, various abiotic stress-regulated miRNAs have been identified and characterized [13, 14, 15, 16, 17].
Certain families of miRNAs are either under or over-expressed, or new types of miRNAs can be synthesized under stress [13, 18]. Regulation of target genes expression by miRNA molecules is mediated by hybridization between miRNA sequences and their nascent reverse complementary sequences of mRNAs, which leads to their degradation or translational repression [19, 20, 21]. Because of their mode of action, they are generally referred to as negative regulators of gene expression.
Plant adaptation mechanism to environmental conditions includes minimization of their growth rates and reorganizing their resources. The primary focus of the adjustment is cell cycle, cell division, and cell wall constitution ; it means developmental processes regulated by conserved families of microRNAs.
3. MicroRNA-based markers
Genomic conservation of miRNA sequences and especially the stem-loop region of precursor molecules of miRNA (pre-miRNA) provided an opportunity to develop a novel type of molecular markers.
MicroRNA-based genotyping technique as a novel type of marker system was published in 2013 by authors Fu et al. . Since then, this system has been applied to genotyping applications of Setaria italica and in related grass species . The structure analysis of miRNA genes revealed that repetitive sequences are part of them, which led to development of miRNA-based microsatellite markers in Oryza sativa [25, 26] and Medicago truncatula and related legume species . Given the origin of sequences of this type of markers, they can be considered as functional markers at the DNA levels [23, 24, 28].
good stability due to a direct PCR-based marker system
improved reproducibility and sequence specificity due to high annealing temperature (more than 60°C) and the use of “touchdown” PCR approach
relatively high polymorphism because of possible random combinations of primers
putative functionality due to their polymorphism nature and the ability to predict phenotypes controlled by miRNAs
cross-genera transferability potential because of the conservation level of miRNAs between species and the way of deriving markers from the consensus sequences of miRNAs.
3.1 MicroRNA-based marker assay
Following subsection provides the approach of microRNA-based marker assay applied in our laboratory. As referred in Table 1, the following procedure has been applied in several plant species for different research purposes.
|PCR component||Concentration||Final concentration|
|PCR buffer KCl, (NH4)2SO4, 20 mmol × dm−3 MgCl2||10×||1×|
|dNTP mix||2.5 mmol × dm−3||0.8 mmol × dm−3|
|Primer forward||100 μmol × dm−3||10 pmol × dm−3|
|Primer reverse||100 μmol × dm−3||10 pmol × dm−3|
|Taq polymerase||5 U||2 U|
The genomic DNA isolation protocol is based on the type of plant biological material (in terms of secondary metabolite content or other aspects). The primers for the miRNA-based markers are designed according to the mature or precursors sequences (pre-miRNAs) available on the miRBase database (
The amplification protocol has originated from methodologies [23, 24] and was modified  (Tables 1 and 2). The total volume of PCR was 20 μl and the DNA concentration was 70 ng μl−1. Amplification products are separated on 15% TBE-urea polyacrylamide (PAGE) gels, running in 1× TBE running buffer at constant power 180 V, 30 mA for 90 min. The gels are stained with PAGE GelRed™ Nucleic Acid Gel stain and are visualized on G-Box Syngene electrophoresis documentation system. For the recording of loci number and their position, as well as the identification of unique fragments, the gels are analyzed by GeneTools software (Syngene) (Figure 1).
|Amplification process||Temperature||Time period||Number of cycles|
|Initial denaturation||94°C||5 min||1 cycle|
|Denaturation||94°C||30 s||5 cycles|
|Annealing||64°C (with temperature reduction of 1°C per cycle)||45 s|
|Denaturation||94°C||30 s||30 cycles|
|Final polymerization||72°C||10 min|
4. Contribution of miRNA-based markers on plant genome response to abiotic stress and for genotyping applications
Genomic polymorphism of plants is the basis of their survival and ability to different climatic conditions. The cognition and mapping of plant genome variability using molecular markers is a prerequisite for extending the genetic base of crops to reduce their susceptibility to adverse environmental conditions . An ideal molecular marker should be polymorphic, stable, reproducible, providing sufficient resolution, fast, and with fairly low cost . The miRNA-based marker system is characterized by relatively high polymorphism, reproducibility, transferability across species, and ease of use with putative functionality . The high level of transferability demonstrates the usability of miRNA-based markers for comparative genome mapping and phylogenetic studies .
5. MiRNA-based markers in genotyping applications
Recognizing relationships between species or within species help to focus more closely on a wide range of human interests, from basic description and disaggregation, through efficient resource genetic management to the production of quality and safe food, whether plant or animal origin .
Within our research, we focused on the use of miRNA-based markers to highlight their broad spectrum of regulatory impact activities in different plant species of nutritional and pharmaceutical uses (Table 3): flax (Linum usitatissimum L.), medlar (Messpilus germanica L.), milk thistle (Silybum marianum (L.) Gaertn.), ginkgo (Ginkgo biloba L.), common ivy (Hedera helix L.), avocado (Persea americana Mill), and ribwort plantain (Plantago lanceolata L.). A total of 13 miRNA-based markers were applied of which 28 primer combinations were made.
|Research purpose||Plant species||Reference|
|Genome profiling with regard to genotype origin||Linum usitatissimum|||
|Spatial and temporal abundance of individual miRNA markers||Linum usitatissimum||[33, 34]|
|Functional markers of commercial type of the crop||Linum usitatissimum||[35, 36]|
|Genomic authentication of varieties||Silybum marianum|||
|Genotyping||Ginkgo biloba||[38, 39]|
Useful molecular markers produce fragments between 150 and 500 bp in length, as this size of fragments can easily be distinguished using agarose or PAGE gels . In our experiments, this size of fragments varied predominantly from 40 to 300 bp and could be clearly identified on agarose gels (Figure 2).
This range is due to variability of stem-loop structure of which the mature miRNA sequences are part of and the length of stem-loop structure ranges from less than 100 to over 900 nt. The primers’ design based on miRNAs sequences can be linked to different places of the same stem-loop structure. Another possibility is that primers amplify regions between neighboring miRNAs .
The level of polymorphism varied from 70 to 90%. Mature miRNAs are expressed as small 21–24 nt endogenous molecules. As a result of different transcriptional activity among MIRNA genes, the miRNAs abundance in the cell varies greatly in dependence of miRNA family . It should be noted that the level of polymorphism depended on the effectiveness of primer combination as well as the level of marker transferability.
The results of the studies have repeatedly confirmed the following:
MicroRNA-based markers show the cross-genera transferability potential.
MicroRNA-based markers display sufficient level of polymorphism in analyzed genotypes and are suitable to differentiate within genotypes of one specimen.
MicroRNA-based markers provide genotype-specific profile of miRNA loci.
The abundance of selective miRNA-based markers is tissue specific and developmental specific.
5.1 MiRNA-based plant genome response to abiotic stress conditions
Different mechanism of stress response contributes to stress tolerance or resistance at different morphological, biochemical, and molecular level . Many stress-regulated genes are found to be regulated by miRNAs.
In the flax study, we applied nutritional stress factor under in vitro conditions . The genome response of the flax genotype CDC Bethune was analyzed under five variants (including control variant) of Murashige-Skoog  medium by two miRNA-based markers, lus-miR395 and lus-miR399 [16, 17]. The results show that flax genome responds to the nutritional stress stimulus. Our results have supported the capability of miRNA-based molecules as potential biomarkers of abiotic stress factors.
Another study was conducted in order to test the ultrasound-induced oxidative stress in lettuce (Lactuca sativa L.) tissues by miRNA-based markers . We have confirmed that reactive oxygen species (ROS), caused by sonication treatment, induced the polymorphism at the molecular level detected by miRNA-based stress markers. We have observed the statistically significant differences (p ≤ 0.01) in miRNA markers ability to detect this polymorphism. The response of miR168 marker was statistically more sensitive in comparison with miR156 marker as a result of their specific regulatory nature.
The aim of research into the impact of soil compaction was to identify the barley (Hordeum vulgare L.) genome response by stress-responsive miRNA-based markers. A prerequisite for the research was that the plants are exposed to a lack of soil moisture and nutrients due to soil compaction. The effect of soil compaction was analyzed by four different miRNA-based markers (hvu-miR156, hvu-miR399, hvu-miR408, and hvu-miR827), within the leaf, stem, and root tissues of barley plants. We can state that due to soil compaction, the barley plants were exposed to the lack of moisture which subsequently affected the intake and utilization of nutrients from the soil and showed lower plant growth parameters and reduced the yields. Moreover, this genome response was tissue specific. The roots were most affected by dehydration, and the nutrient deficiency was the most pronounced on leaves. The number of amplified miRNA loci was statistically significantly dependent on the stress-sensitive marker applied.
We have conducted experiments in connection with research on the drought resistance of wheat (Triticum aestivum L.). Genomes of susceptible and drought-resistant genotypes were screened by stress-sensitive miRNA-based markers (hvu-miR408 and hvu-miR827). Genotypes were tested under in vitro conditions on Murashige-Skoog culture medium with different concentrations (0, 5, 10, 15, and 20%) of polyethylene glycol (PEG 6000) to induce dehydration stress. Drought-resistant wheat genotypes responded to dehydration stress, by significantly higher activity of hvu-miR408 biomarker in comparison with susceptible genotypes. This response points to a better genome adaptation ability of the resistant genotypes to abiotic stress. By using the conserved type marker hvu-miR156, which is involved in the regulation of plant growth and development processes, a reduced activity of this type of marker was observed, both in susceptible and resistant genotypes, indicating that the adaptation mechanism of plants to cope with stress conditions is implemented at the expense of growth processes.
It can be summarized that the marker system based on microRNA molecules represents (a) flexible marker system based on sequences of regulatory molecules, (b) species-transfer system due to the conserved nature of mature sequences of miRNAs, (c) functionally potential markers, where observed polymorphism points to changes in miRNA loci sequences evoking changes in the target gene regulatory model, (d) tissue-specific and development-specific characters of markers, and (e) screening tool of genome adaptation changes to induced abiotic stress referred to as stress-sensitive biomarkers. It should be noted that in selecting suitable type(s) of miRNA markers for a particular type of study, it is necessary to know the regulatory background, regulation mechanism, and target sequences of particular type of miRNA molecules.
This work was supported by the project of Slovak Scientific Agency of Ministry of Education of the Slovak Republic, VEGA, No. 1/0246/18 and Slovak Research and Development Agency APVV-15-0156.
Conflict of interest
The authors declare no conflict of interest.