Extrapolative microRNA precursor based SSR mining from tea EST database in respect to agronomic traits

Tea (Camellia sinensis) is most popularly consumed beverage across the world and being a cash-crop, it receives much attention to the scientific community. The vibrant research interest basically stands for its massive demand to health conscious people for its antioxidant and a broad spectrum therapeutic potentiality [1, 2]. China is the largest tea producer as well as exporter preceding by India and Sri Lanka [3]. The fermented tea or black tea is the most common among all different types of tea consumed, although antioxidant and other health benefit properties lies maximum on non-fermented green tea [4]. As a result, demand for quality tea has increased much to the end users and tea planters. Challenges for exploring superior cultivars with better agronomic traits are still point of interest to the researchers.

Molecular marker assisted technique in breeding programme for the selection or development of cultivars with desired trait from a large population is well established [5]. Among different markers used in crop improvement and molecular breeding technique, microsatellite markers are profoundly used for its reliability and time saving method. Moreover, due to being co-dominant, abundant, hyper-variable and co-operative to high-throughput analysis, microsatellite markers are considered as ideal for plant genetic linkage mapping, physical mapping, population studies, genotype identification and crop improvement [6]. Predominant of such markers like SSR, ISSR, EST-SSR have been effectively utilized in several crop improvement program [7–11]. MicroRNA precursor based SSR markers are very recently incorporated in this chapter and mostly utilized in marker trait association analysis in several species [12–15]. These micro RNAs are short, non-coding RNA molecules, involved in post transcriptional mode of gene regulation and [16] thus effects on related phenotype [17, 18]. There are a large number of molecular markers available for tea so far [19], however a very few are reported to be linked with some specific trait [20]. Therefore exploration and characterization of novel and already available markers are of prime point of interest. Considering the above, present study aims to identify the microsatellite motifs within the reported and predicted miRNA precursors that are effectively followed by designing of primers for PCR validation.

In the present study, previously reported miRNA sequences were utilized to find their homolog ones from tea as it is already known that plant mature miRNAs are highly conserved within the plant kingdom, and miRNA genes in one species may exist as orthologs or homologs in other species [30, 37]. With the help of this hypothesis, known miRNAs were utilized to discover novel potential miRNAs in tea. All 8442 miRNAs reported from viridiplantae so far were utilized and after elimination of 3676 exact duplicates using bioinformatics tool, a non-redundant collection was taken for further analysis. Similarly, tea EST collection of 49,670 sequences were made into a non-redundant 40,686 numbers. The BLAST search could fish out 52 number of ESTs with required level of homology i.e. minimum 18nt length similarity with not more than 3 mismatches. A total 15 of them already reported earlier as pre-miRNA [21–23]. Leftover candidates were analyzed through Mfold program as the miRNA precursors should be able to form stem-loop hairpin in their secondary structure for processing by Dicer enzyme [38] and subsequently possible false miRNA precursors were manually removed. In present study two more potential pre-miRNA (Fig. 1) were identified with accession JK478587.1 and FS955851.1 for having miR1533 and miR8002-3p respectively.

Plant microRNAs do regulate the transcripts expression for growth, development and stress responses by altering leaf morphology and polarity, organ development, lateral root formation, hormone signalling, cell death, signal transduction, cell differentiation and proliferation, transition from juvenile to adult vegetative phase, vegetative to flowering phase, flowering time, floral organ identity and reproduction [41, 43–46]. Meanwhile, miRNAs are involved in the regulation of gene expression through mRNA cleavage or translational inhibition [47] has been reported, therefore obtaining information about target genes of potential microRNAs was an essential part of the study. Since the full genomic information is lacking in case of tea, DFCI Gene Index of Arabidopsis thaliana, Glycine max, and Zea mays, Solanum tuberosum etc. were used as target database. Among the presently reported two microRNAs, miR1533 were not found to have any target similarity with significant threshold values of input parameter. The other one, miR8002-3p were predicted to have cleavage activity on General transcription factor 2-related zinc finger protein (Target Accession: AT1G42710.1), phosphoglycerate mutase (Target Accession: TC194811). In addition the target genes of other predicted miRNAs of tea were explored from the literature to elucidate their role in pre-miRNA SSR based polymorphism assessment. Zhu and Luo [23] reported that their predicted miRNA target genes encoded transcriptional factors, involved in stress response, transmembrane transport, and signal transduction and transcription regulation. Target genes encoding transcription factors and cell integrity maintenance machinery during stress response was also mentioned by Prabu and Mandal [21]. Multiple target of a single miRNA is the system biology network, when they control expression of different transcription factors which in turn regulates specific genes for different metabolism [48].

Microsatellite markers are widely used tool for estimating the genetic variation and especially used for construction linkage map, understanding of marker trait association, identification of disease resistant loci [14, 49]. The distribution of simple sequence repeats or SSRs in the genome is inherently unstable and therefore highly polymorphic [50]. People assumed that increment of the repeat unit and repeat tracts gives rise the chances of the mutation rate [51]. Some reports have already established the fact that SSR expansions or contractions within genome sequences can affect functions of these sequences and even lead to phenotypic changes [17]. Evidences have shown the effect of SSR unit variation within protein coding regions. However, the consequences of the same in non-coding transcripts are less studied. A very few reports demonstrated the significance of analysis of SSRs in non-coding miRNA [12–15, 49]. In current study, 13 of 33 total predicted pre-miRNAs had one or more SSR motifs (Table 1). A total of 9 sequences had SSR motif in a single region, whereas 3 sequences contained SSR motifs in two locations and 1 sequence was with 3 different SSR motifs. Trinucleotide motifs were most abundant among all followed by dinucleotides. There was one each of penta and hexa-nucleotide motifs. Forward and reverse primers from the each microsatellite motifs flanking region could be generated in all members excluding only one where SSR motif present toward the terminal end. The microsatellite motifs may be conserved among group or become signature [13] which might be used in studies of genetic fingerprinting work of tea. Some traits rely on specific repeats of microsatellites and their numbers. Such advantage has been efficiently employed by rice researchers when miRNA-SSR markers were employed to differentiate the salt tolerant and susceptible genotypes [15]. They found more repeat variation of the salt responsive miRNA genes among the susceptible rice genotypes than tolerant one. Such extensive work can be applied in tea to distinguish the cultivars with varying agronomic traits on the basis of miRNA-SSR polymorphism.

Finally, newly predicted microRNAs in tea would enrich the assemblage in absence of whole genomic information of tea and make easier subsequent studies for experimental validation. Some of them might be related to certain metabolic functions thereby phenotypes as well. Excavating of microsatellite motifs from predicted microRNA precursors and designing primers from SSR flanking regions would pave the way in developing novel markers for tagging tea specific agronomic traits as well as accelerating non-conventional breeding program. This can freely be followed by genetic diversity assessment of tea cultivars with varying characters.