Application of the RAPD and miRNA markers in the genotyping of Silybum marianum (L.) Gaertn.

Abstract

Two types of molecular markers based on randomly amplified DNA by RAPD-based assay and amplified microRNA by miRNA-based assay were applied for the genotyping of five accessions of Silybum marianum (L.) Gaertn. It was also verified the effectiveness of the isolation of genomic DNA by three commercially available isolation kits and two similar complex methods and their applicability for this medicinal herb. None of the commercial isolation kits provided the genomic DNA of sufficient quality and quantity. Applied originally designed CTAB method and modified CTAB method for purpose of DNA isolation from medicinal plants allowed to isolate DNA of required quality and of sufficient yield. RAPD-based DNA fingerprints, allowed to distinguish the individual genotypes of Silybum marianum. MicroRNA-based markers showed the cross-genera transferability potential and displayed sufficient level of polymorphism. Both types of molecular markers could be used as suitable tool for genotyping of milk thistle. However, because of the size of miRNA amplicons is the efficiency of miRNA-based markers PCR amplifications preferred and consequently less DNA quality-dependent which may be advantageous in view of the content of undesirable secondary metabolites in medicinal plants.

Genotyping of Flax Genetic Resources by Mirna-Based Molecular Markers and Morphology

“…For the assay, we selected two types of miRNA-based primers: gm-miR156b and lus-miR408a. The effectiveness and transferability of the gm-miR156b primer have been confirmed in previous studies (Hlavačková et al 2015;Ražná et al 2015).…”

MicroRNA-Based Markers in Plant Genome Response to Abiotic Stress and Their Application in Plant Genotyping

“…Genome profiling with regard to genotype origin Linum usitatissimum [30] Spatial and temporal abundance of individual miRNA markers Linum usitatissimum [33,34] Functional markers of commercial type of the crop Linum usitatissimum [35,36] Genotyping Mespilus germanica [36] Genomic authentication of varieties Silybum marianum [37] Genotyping Ginkgo biloba [38,39] Genotyping Hedera helix [40] Genotyping Persea americana -Genotyping Plantago lanceolata - Table 3.…”

The Applicability of Genetic Markers Based on Molecules Micro RNA in Agricultural Research

“…Due to the species transferability of miRNA-based primers was possible to confirmed single forward primers with each other. The effectiveness and species transferability of used primers was in previous studies [9,10,[14][15][16].…”

“…The miRNA-based molecular markers have been reported in very few plants; Brassica species [4], Setaria italica L. [7] and Oryza sativa L. [8,6]. The advantages of microRNA-based marker system including high polymorphism and reproducibility, transferability across species and putative functionality [4,9,10].…”

“…Therefore, the specificity of these markers relies on the capability of miRNA-based primers to amplify different parts of specific stem-loop structure as well as variable length polymorphism of regions between neighboring miRNAs molecules. In addition, miRNA-based primers can be combined randomly (it means combinations of various forward primers or forward and reverse primers) which it is not possible in the case of SSR primers [4,9]. However, miRNA-based microsatellite markers have been developed from salt responsive miRNA genes for genetic diversity analysis of salt responsive miRNAs among the salt tolerant and sensitive rice genotypes of rice [6,8].…”