Genetic diversity in Sickleweed (Falcaria vulgaris) and using stepwise regression to identify marker associated with traits.

One of the well-known medicinal plants in the Falcaria genus is Sickleweed. Falcaria species exhibit a high degree of genetic variability, posing challenges in the examination of genetic diversity due to the significant potential for hybridization and introgression among them. Utilizing morphological traits and molecular markers may prove to be a valuable approach in evaluating and harnessing germplasm, considering the current obstacles faced in breeding this medicinal herb. In 2021, fifteen Sickleweed populations were cultivated in pots under field conditions, employing a randomized complete block design with three replications. This aimed to assess genetic diversity and conduct marker-trait association analyses utilizing morpho-physiological characteristics and SSR markers. The Sickleweed populations displayed considerable genetic diversity across all traits. Through cluster analysis of traits and the utilization of the UPGMA method based on the Gower distance matrix, the population was classified into three distinct clusters. Upon examining all genotypes, 52 polymorphic bands were detected, with an average of 8.68 bands per primer. The average expected heterozygosity across all loci was 0.864, while the average PIC was 0.855. Molecular data analysis employing the Jaccard similarity index and UPGMA method revealed the division of Sickleweed populations into two major groups. Furthermore, the results of molecular variance analysis indicated that variation within the population exceeded that between populations. Thirty-two SSR fragments were found to be significantly associated with genomic regions controlling the studied traits, determined through the application of stepwise regression. Selection based on molecular markers offers a rapid method for breeding programs, with the genetic information obtained from these markers playing a crucial role. Therefore, alongside traits, selecting superior genotypes and populations of high value in breeding programs becomes feasible. The findings highlight that certain markers are linked to multiple traits, emphasizing the critical importance of this characteristic in plant breeding for the simultaneous improvement of numerous traits. The study's insights regarding markers hold potential for application in Sickleweed breeding programs.

Antioxidant gene expression analysis and evaluation of total phenol content and oxygen-scavenging system in tea accessions under normal and drought stress conditions.

BACKGROUND: Abiotic and biotic stresses induce oxidative processes in plant cells that this process starts with the production of ROSs which cause damage to the proteins. Therefore, plants have increased their antioxidant activity to defend against this oxidative stress to be able to handle stress better. In this research, 14 different tea accessions in a randomized complete block design with two replications were evaluated in two normal and drought stress conditions, and their antioxidant activity was measured by DPPH-free radicals' assay and gene expression analysis. RESULTS: The results of gene expression analysis showed that the 100 and 399 accessions and Bazri cultivar had high values for most of the antioxidant enzymes, ascorbate peroxidase, superoxide dismutase, catalase, and peroxidase under drought stress conditions while the 278 and 276 accessions had the lowest amount of antioxidant enzymes in the same situation. Results showed that the IC50 of the BHT combination was 90.12 µg/ ml. Also, The IC50 of accessions ranged from 218 to 261 µg/ml and 201-264 µg/ml at normal and drought stress conditions, respectively. The 100 and 399 accessions showed the lowest IC50 under normal and drought stress conditions, while 278 and 276 accessions had the highest value for IC50. The antioxidant activity of tea accession extracts under normal conditions was ranged from 25 to 69% for accessions 278 and 100, respectively. While, the antioxidant activities of extracts under drought stress condition was 12 to 83% for accessions 276 and 100, respectively. So, according to the results, 100 and 399 accessions exhibited the least IC50 and more antioxidant activity under drought stress conditions and were identified as stress-tolerant accessions. However, 278 and 276 accessions did not show much antioxidant activity and were recognized as sensitive accessions under drought stress conditions. CONCLUSIONS: These results demonstrate that total phenol content, antioxidant activity, and the oxygen-scavenging system can be used as a descriptor for identifying drought-tolerant accessions.

The expression of monoterpene synthase genes and their respective end products are affected by gibberellic acid in Thymus vulgaris.

Thymus vulgaris L. (Lamiaceae), a well-known aromatic medicinal herb, has many important essential constituents in its oil, including γ-terpinene, carvacrol, thymol, and p-cymene. Gibberellins comprise hundreds of components, which regulate several various growths and underlying developmental processes, such as cell division and elongation, shoot elongation, seed germination, and gene expression. In this study, we investigated the influence of sprayed gibberellic acid (GA3) treatments on the internode length, leaf morphology, length of new shoot, expression of monoterpene synthase genes and monoterpenes content during two plant growth stages. Our results showed that increasing of internode length was a clear effect of GA3 that was varied with internode position. The results also showed that all internodes displayed a dramatic increase in the highest concentration of GA3. Also, the foliar application of GA3 resulted in not only an increased expression level of monoterpene synthase genes, but also the improved production of a monoterpene, especially in the moderate concentration of GA3 that they were up-regulated. In the lowest GA3 concentrations, relative expression levels were similar or lower than the control plants and a notable downregulation in those genes was observed in the application of the highest concentration of GA3 rather than the moderate concentrations. Overall, the expression of two out of five monoterpene synthase genes, TPS and CYP71D181, showed a correlation with the level of γ-terpinene and carvacrol, respectively, indicating that they are regulated at the transcriptional levels.

An efficient protocol for isolation of inhibitor-free nucleic acids even from recalcitrant plants.

For fast and easy isolation of inhibitor-free genomic DNA even from the toughest plant leaf samples, including those high in polyphenols and polysaccharides, a protocol has been developed. To prevent the solubility of polysaccharides in the DNA extract, high salt concentration (1.4 M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) was used for the removal of polyphenols as polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by proteinase K and removed by centrifugation from plant extracts during the isolation process resulting in pure DNA and RNA ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA and RNA isolated from leaves and roots of recalcitrant plants which was free from contamination and color. The average yields of total RNA from roots and shoot of Betula and Grape ranged from 285 to 364 ng/µl with A260/A280 between 1.9 and 2.08. The RNA isolated with this protocol was verified to be suitable for PCR, quantitative real-time PCR, semi-quantitative reverse transcription polymerase chain reaction, cDNA synthesis and expression analysis. This protocol shown here is reproducible and can be used for a broad spectrum of plant species which have polyphenols and polysaccharide compounds.