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Stevia rebaudiana germplasm characterization using microsatellite markers and steviol glycosides quantification by HPLC.

ABSTRACT: Stevia rebaudiana Bertoni, Asteraceae, is an herbaceous perennial plant native to Paraguay. This species is considered since ancient times a medicinal plant with important bioactive compounds and pharmacologic and food properties, namely diterpenes glycosides. The high natural sweetener potential stevioside and rebaudioside A produced by S. rebaudiana plants are suitable sucrose substitutes, and their obtention is influenced by environmental, phytosociological, and genetic factors. The plants' genetic profile and sweet potential depiction are needed for suitable plant selection for improvement and deployment. Thirty-one S. rebaudiana accessions grown in the same plot where leaves samples were collected in early 2019, were genotyped using six microsatellite markers, including two steviol glycosides biosynthesis functionally involved markers. Additionally, an aqueous extract of each sample was obtained in a water bath and purified by SPE for stevioside and rebaudioside A quantification by normal phase HPLC. Stevioside and rebaudioside A contents varied between 0.53-7.36% (w w^-1) and 0.37-3.60% (w w^-1), respectively. Two genotypes displayed interesting ratios of rebaudioside A/stevioside (number 3 and 33). The level of genetic similarity between genotypes was tested through a pairwise similarity coefficient, and two groups of individuals had the same fingerprinting. Strong relatedness was found within genotypes, possibly due to cloning, thus, influx of new germplasm ought to be made to prevent mating between relatives, and for further selection and genetic improvement.

SUBMITTER: Ribeiro MM

PROVIDER: S-EPMC8060219 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundmiRNAs are major regulators of gene expression and have proven their role in understanding the genetic regulation of biosynthetic pathways. Stevioside and rebaudioside-A, the two most abundant and sweetest compounds found in leaf extract of Stevia rebaudiana, have been used for many years in treatment of diabetes. It has been found that the crude extract is more potent than the purified extract. Stevioside, being accumulated in higher concentration, imparts licorice like aftertaste. Thus, in order to make the sweetener more potent and palatable, there is a need to increase the intrinsic concentration of steviol glycosides and to alter the ratio of rebaudioside-A to stevioside. Doing so would significantly increase the quality of the sweeteners, and the potential to be used on a wider scale. To do so, in previous report, miRNAs associated with genes of steviol glycosides biosynthetic pathway were identified in S. rebaudiana. In continuation to that in this study, the two miRNAs (miR319g and miRStv_11) targeting key genes of steviol glycosides biosynthetic pathway were modulated and their impact was evaluated on steviol glycosides contents.ResultsThe over-expression results showed that miRStv_11 induced, while miR319g had repressive action on its target genes. The knock-down constructs for miR319g and miRStv_11 were then prepared and it was demonstrated that the expression of anti-miR319g produced inhibitory effect on its target miRNA, resulting in enhanced expression of its target genes. On the other hand, anti-miRStv_11 resulted in down-regulation of miRStv_11 and its target gene. Further miRStv_11 and anti-miR319gwere co-expressed which resulted in significant increase in stevioside (24.5%) and rebaudioside-A (51%) contents.ConclusionIn conclusion, the role of miR319g and miRStv_11 was successfully validated in steviol gycosides biosynthetic pathway gene regulation and their effect on steviol gycosides contents. In this study, we found the positively correlated miRNA-mRNA interaction network in plants, where miRStv_11 enhanced the expression of KAH gene. miRNAs knock-down was also successfully achieved using antisense precursors. Overall, this study thus reveals more complex nature and fundamental importance of miRNAs in biosynthetic pathway related gene networks and hence, these miRNAs can be successfully employed to enhance the ratio of rebaudioside-A to stevioside, thus enhancing the sweetening indices of this plant and making it more palatable.

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Stevia rebaudiana germplasm characterization using microsatellite markers and steviol glycosides quantification by HPLC.

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