miRNA-based simple sequence repeats markers for DNA fingerprinting of plants
MicroRNAs (miRNAs) are new generation genomic resources. They are small, endogenously expressed, non-coding RNA molecules that are present in the genomes of many organisms. Initially a large transcript is encoded by the enzyme RNA Pol II from the genomic region (DNA) which is known as primary transcript. This primary transcript undergoes processing and produced shorter (around 200 nt) single stranded transcript, known as precursor transcript (pre-mRNA) by a RNA Pol III/DCL1 protein. This pre-mRNA further processed to produce mature miRNA sequences of 20-24 nt length (Mondal et al., 2015). These mature miRNAs then either cleave to some protein coding gene or do methylation to the cytosine bases present in the promoter sequence of protein-coding genes. Both the ways, miRNAs ultimately silence the gene expression. This broadly denote as epigenetic gene-regulation. Since 2002, when the first plant miRNA was reported, numerous new miRNAs have been discovered via high-throughput sequencing and computational approaches. Today it has proven well that miRNAs play important roles for gene regulation in every domain of life such as abiotic stress, biotic stress, quality trait and growth as well as development. However, there is paucity in understanding the genetic variation of miRNA genes owing to the lack of miRNA-specific DNA markers. miRNA marker system is a novel functional marker system which is predominantly explored in the animal systems, where these genetic elements have shown a promise as potential biomarkers for the classification and prognostication of a variety of diseases including cancer. However, in plants this marker system is very poorly understood and the markers based on miRNAs are reported in very few plant species. In plants. the DNA markers are particularly useful for the breeding purposes to improve many traits. Development of miRNA-based molecular markers linked to any trait will form a new type of marker repository for breeders and researchers from where they can decide onthe particular candidate marker(s) for their downstream research. Though profoundly exploited in the animal systems, the miRNA based markers are currently absorbing the attentions of the plant researchers across the world to developing such a marker system in plants too. The miRNA based marker system are non-coding in nature and has advantages over the other marker systems due to their high polymorphism, reproducibility and cross species transferability (Mondal and Ganie 2014). Molecular markers, especially simple sequence repeat (SSR) markers, play an important role in marker-assisted breeding of plant species including the model monocot staple crop rice. Although a large number of SSRs have been discovered in the rice, the most of them are either from protein-coding regions or untranslated regions of the rice genome. SSRs from the non-coding miRNA genes of rice genome are almost untapped. The discovery of new SSR markers has always been a challenge to the molecular breeders. The high conservation of miRNA gene sequences provides an opportunity to develop a novel molecular marker type. The development of novel markers from the conserved regions of different genomes will thus be useful for studying the genetic diversity of closely related species or self-pollinated species.
Thus, a genome-wide mining of SSR markers from the miRNA genes of rice by us (Ganie and Mondal, 2015) has made a good source of markers available to plant breeder community. The rice researchers can utilize these markers for the different purposes of crop improvement programmes. From this reservoir of miRNA based markers, we selected the markers from the salt responsive miRNAs and validated them among the contrasting panels of salt tolerant as well as susceptible rice genotypes (Mondal and Ganie, 2014). Twelve such miRNA-SSR markers were found which could efficiently differentiate the salt tolerant and susceptible genotypes. It was found that miRNA genes were more diverse in susceptible genotypes than the tolerant ones. The more repeat variation of the salt responsive miRNA genes among the susceptible rice genotypes was supposed to interfere with the formation the proper stem-loop structure of premature miRNA and the subsequent synthesis of corresponding mature miRNAs in susceptible genotypes. Later, this study was carried out by analysing the variations in the microsatellite repeats proximal and distal to the precursor sequences (Ganie et al., 2015). It was found that microsatellite repeats that are close/proximal to precursor-miRNA sequence are conserved than the distal repeats among the two contrasting panels of rice differing in salinity response. This conservation is logical from the view point of stability of stem loop formation and subsequent the formation of mature miRNA sequence. Thus, miRNA-based marker system provides a novel and economical genotyping technique with high efficiency, reproducibility, stability and good transferability.
1. Mondal TK, Ganei SA, Debnath A B (2015) Identification of novel and conserved microRNAs related to salinity stress of halophyte, Oryza coarctata, a wild relative of rice PLOS one. 10(10): e0140675. doi:10.1371/journal.pone.0140675
2. Ganie SA and Mondal TK (2015) Genome-wide development of novel miRNA-based microsatellite markers of rice (Oryza sativa) for genotyping applications. Molecular Breeding. 35: 51-60
3. Mondal TK and Ganie SA. (2014) Identification and characterization of salt responsive miRNA-SSR markers in rice (Oryza sativa). Gene. 535:204-209.
4. Ganie A, Molla SA, Mondal TK (2015) Microsatallite repeats proximal to pre-miRNA are more conserved than the distal repeats among the salinity responsive miRNA genes of rice (Oryza sativa). Inter J Trop Agri. 33:1405-1409
About Author / Additional Info:
I am working as Senior Scientist (Biotechnology) in the Division of Genomic Resources, National Bureau of Plant Genetic Research, New Delhi, India