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Article - Biological and Applied Sciences • Braz. arch. biol. technol. 67 • 2024 • https://doi.org/10.1590/1678-4324-2024231177 linkcopy

Open-access Determination of Genetic Diversity of Salvia viridis L. var. horminum with TU-DAMD Markers

AuthorshipSCIMAGO INSTITUTIONS RANKINGS

Abstract

Salvia is the largest and taxonomically complex genus of Lamiaceae family of which Salvia viridis L. var. horminum species (annual clary, orval) is an annual ornamental and medicinal plant. Little attention has been paid to describe its genetic diversity worldwide. Touch-Up Directed Amplification of Minisatellite DNA (TU-DAMD) marker has been employed to assess DNA polymorphism of wild S. viridis L. var. horminum species grown in coastal regions of Syria. TU-DAMD marker highlighted a total of 217 DNA fragments of which 198 (91.2%) were polymorphic with a mean polymorphic information content (PIC) and marker index (MI) average of 0.265 and 3.527, respectively. Unweighted Pair Group Mean Arithmetic average (UPGMA) cluster analysis revealed that S. viridis (SV) samples were divided in two main clusters, the first cluster involved SV1, SV2 and SV3 genotypes; whereas, the second one involved the remaining other studied S. viridis (SV4-SV19) samples. Overall, the current study suggests that the SV1, SV2 and SV3 genotypes clustered in one cluster are genetically distinct from the remaining other studied S. viridis (SV) genotypes based on the estimated percent disagreement value (PDV) average of 0.238. The current study highlighted high genetic diversity (P% of 91.2%) among the studied S. viridis samples. Expanding the employment of the TU-DAMD for genetic diversity of other plants species in order to discover its effectives in molecular studies is requested.

Keywords:
Cluster analysis; Genetic diversity; Molecular marker; Salvia viridis L; Touch-up directed amplification of minisatellite DNA (TU-DAMD)

HIGHLIGHTS

Genetic diversity of 19 natural Salvia viridis L. var. horminum samples (SV1-SV19), has been assessed with the help of TU-DAMD markers.

.SV1, SV2 and SV3 S. viridis L. var. horminum genotypes clustered in one cluster are genetically distinct from the remaining other studied S. viridis (SV) genotypes based on the estimated percent disagreement value (PDV) average of 0.238.

The current study highlighted high genetic diversity (P% of 91.2%) among the studied S. viridis genotypes.

INTRODUCTION

Salvia is the largest and taxonomically complex genus of Lamiaceae family, includes approximately 1000 species [1]. Mouterde [2] reported that this genus represented in 28 species in Syria, grown along the Syrian coastal regions at different sea altitudes up to 900 m.

Salvia viridis L. (annual clary, orval) is an annual plant native to an area extending from the Mediterranean to the Crimea and into Iran. It distributed in Spain, Syria, France, Italy, Germany, Central of Europe, Russia, Turkey and Iran [2].

Salvia viridis L. var. horminum (Red topped sage) exhibited pharmacological properties including antioxidant, anti-inflammatory, antiallergic, antibacterial, antiviral and antiseptic activities [3, 4].

Many molecular PCR-based marker types were successfully employed for genetic studies of plant species. In Salvia genus, these researches in majority focused on genetic diversity at Salvia species level (among species); e.g. recently, touch-down directed amplification of minisatellite DNA (TD-DAMD) marker for genetic diversity of 10 Salvia species [5], large single copy (LSC) and small single copy regions (SSC) analyses for plastid genome structure and phylogeny of 19 Salvia species [6] and chloroplast genomes among Salvia species [7].

As for Salvia subspecies level (within same species), many studies have been published; e.g. random amplified polymorphic DNA (RAPD) for Salvia miltiorrhiza and its related species molecular characterization [8]; sequenced multiple DNA regions (ITS, trnL-trnF, and psbA-trnH) for Salvia divinorum phylogenetic study [9] and ribulose bisphosphate carboxylase large subunit (rbcL) gene for the same species identification [10]; inter simple sequence repeat (ISSR) for Salvia lachnostachys [11], Salvia hispanica [12] and Salvia nemorosa genetic diversity ([13]; trnL-trnF region marker for Neotropical sages [(Salvia subg. Calosphace)] [14] and plastome and nrDNA data analysis of the same Salvia subg. Calosphace phylogenomic study [15]; TD-DAMD marker for Salvia tomentosa species genetic diversity [16]; amplified fragment length polymorphism (AFLP) marker among Salvia spinosa and Salvia syriaca species [17] and for Salvia officinalis [18] genetic diversity; internal transcribed spacer (ITS) and the plastid regions trnL-trnF and trnH-psbA markers for molecular characterization of a new Salvia gavilanensis sp. nov species [19]. Recently, Shakoor and coauthors [20] reported Salvia viridis polymorphism using sequence-related amplified polymorphism (SRAP) marker.

Moreover, Saleh [21] recently applied TU-DAMD for Salvia judaica and Salvia palaestina species molecular characterization. Recently, Saleh [22] applied similar technique for Origanum syriacum L. (Lamiaceae) species DNA polymorphism assessment and more recently in S. officinalis L. (Lamiaceae) and its related species genetic diversity [23].

Prior to our knowledge, little attention has been given to describe S. viridis L. var. horminum genetic diversity worldwide and particularly in Syria. Thereby, the present study focused on its genetic diversity investigation using TU-DAMD marker.

MATERIAL AND METHODS

Nineteen Salvia viridisi L. var. horminum genotypes leaf samples of natural Salvia viridis population were collected from their natural habitats from Lattakia city - Syria and its rural regions (Table 1, Figure 1). Salvia tomentosa (Lamiaceae) species from Lattakia was included as out-group reference. Sampling has been carried out during blooming stage. Samples were kept at -80 °C in liquid nitrogen for DNA extraction.

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Table 1
Collection sites of the studied natural S. viridis population.

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Figure 1
Distribution map of the studied natural S. viridis population.

DNA Isolation

Total genomic DNA of S. viridis var. horminum along with S. tomentosa species was performed according to CTAB (cetyltrimethylammonium bromide) protocol as previously described by Doyle and Doyle [24]. DNA quantification was performed by DNA fluorimeter (GeneQuant-Amersham, Biosciences - England, Ser 802111-98/88411) and kept at -80°C until use.

TU-DAMD test and data analysis

Fifteen DAMD primers (Table 2) were tested to assess genetic diversity in S. viridis var. horminum through Touch-Up Directed Amplification of Minisatellite DNA (TU-DAMD) test.

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Table 2
DAMD primers employed in the present study.

PCR amplification reactions were performed in 25 μL total volume in a T-gradient thermal cycler (Bio-Rad, Hercules, USA) according to Saleh [21].

Final PCR products were separation on a 2 % ethidium bromide-stained agarose (Bio-Rad) in 0.5× Tris-borate-EDTA (TBE) buffer. Electrophoresis was performed at 85 V for 2.5 h and visualized with a UV transilluminator. A VC 100bp Plus DNA Ladder (Vivantis) ladder standard was used to determine molecular weight of TU-DAMD products size.

TU-DAMD data were transformed into binary matrix; where, the presence or absence of each band size was manually scored as 1 or 0, respectively. The Unweighted Pair Group Mean Arithmetic average (UPGMA) using Statistica program [25], was constructed based on percent disagreement values (PDV). Whereas, polymorphic information content (PIC) was calculated according to the formula:

P I C = 1 − Σ ( P i j ) 2

Where Pij is the frequency of the ith pattern revealed by the jth primer summed across all patterns revealed by the primers [26]. Moreover, marker index (MI) was calculated as described by Powell and coauthors [27] according to the formula:

M I = P I C × η β

Where PIC is the mean PIC value, η the number of bands, and β is the proportion of polymorphic.

Genetic similarity (GS) among the studied S. viridis samples was determined according to Jaccard's similarity matrix [28].

RESULTS

TU-DAMD makers showed that PCR products size ranged between 100-1500 bp. TU-DAMD polymorphism profile among the studied S. viridis samples produced by URP1F, URP2R and OGRB01 DAMD primers was illustrated in Figure 2.

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Figure 2
TU-DAMD polymorphism profile among the studied S. viridis genotypes produced by URP1F (a), URP2R (b) and OGRB01 (c) DAMD primers. 1-19 lanes: S. viridis genotypes and 20 lane: S. tomentosa as an outside reference. M: A VC100bp Plus DNA Ladder (Vivantis) ladder standard.

In the current study, all the 15 tested DAMD primers were found to be informative. Employed TU-DAMD marker gave total bands (TB) number ranged between 9 (URP30F) and 24 (URP1F) with a mean average of 14.5 bands/primer (Table 3). Whereas, the polymorphic bands (PB) number ranged between 8 (URP13R) and 22 (OGRB01) with a mean average of 13.2 polymorphic bands/primer (Table 3). Three DAMD [URP2R, HVR(-) and URP30F] primers among the 15 DAMD tested primers, successfully produced polymorphism level of 100%. Whereas, for the remaining primers, this value ranged between 80.0% (URP13R) and 95.7% (OGRB01) with a mean average of 90.8% (Table 3). Moreover, PIC value ranged between 0.159 (URP38F) and 0.365 (URP2R) with a mean average of 0.265 (Table 3). As for MI, it ranged between 2.008 (URP13R) and 6.930 (OGRB01) with a mean average of 3.527 (Table 3).

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Table 3
Banding pattern of TU-DAMD amplified fragments scored.

To construct the cluster degree among the studied S. viridis samples, Unweighted Pair Group Mean Arithmetic average (UPGMA) cluster analysis using Statistica program and percent disagreement values (PDV) were employed. UPGMA data showed that S. tomentosa species was genetically so far from the studied S. viridis samples (Figure 3).

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Figure 3
UPGMA cluster analysis revealing genetic diversity of natural S. viridis population through TU -DAMD marker. SV1: S. viridis genotype1 - SV19: S. viridis genotype19 and ST20: S. tomentosa20.

Whereas, the studied S. viridis samples are divided into two main groups (Figure 3); the first group involved SV1, SV2 & SV3 genotypes of which SV1 & SV2 genotypes were closed with PDV of 0.194 (GS of 0.622) (Tables 4 - 5). Whereas, the second one involved the remaining S. viridis genotypes of which SV14 & SV15 were the closest with PDV of 0.115 (GS of 0.737) (Tables 4 - 5). Overall, the closest samples were SV14 & SV15 exhibiting the lowest PDV value of 0.115 and the highest similarity value of 0.737 (Tables 4 - 5). Whereas, the most distant samples were SV1 & SV7 (PDV of 0.355 and GS of 0.384) and SV3 & SV17 (PDV of 0.355 and GS of 0.394) (Tables 4 - 5).

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Table 4
Percent disagreement values (PDV) in natural S. viridis population yielded by TU-DAMD data based on UPGMA routine in statistical program.
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Table 5
Jaccard's similarity matrix (GS) in natural S. viridis population yielded by TU-DAMD data.

DISCUSSION

Genetic diversity of natural S. viridis population includes 19 samples, has been assessed using TU-DAMD markers. Recently, Shakoor and coauthors [20] reported DNA polymorphism among 30 Salvia species grown in Iran; they reported P% of 66.50% in S. viridis and that P% ranged between 35.68% (S. urmiensis) - 69.50% (S. limbata), using SRAP marker.

In the current study, S. viridis population revealed high P% of 91.2% using TU-DAMD marker. Similar findings have been reported in other Lamiaceae species; e.g. P% was recorded to be 90.7% in Iranian Nepeta kotschyi Boiss using RAPD marker [29], 91.638% in Origanum vulgare using selectively amplified microsatellite polymorphic loci (SAMPL) marker [30], 92.9% in Iranian Teucrium (Teucrium polium L.) using ISSR marker [31], 94.4% in Korean Mint Agastache rugosa (Fisch & Meyer) Kuntze (Lamiaceae) using ISSR marker [32], 95.6% in Brazilian S. lachnostachys using ISSR marker [11], 98.3% in Origanum acutidens using inter-primer binding sites (iPBS) marker [33] and 99.3% in Turkish Teucrium leucophyllum Montbret & Aucher ex Bentham. using ISSR marker [34].

Whereas, moderate P% has been reported in other Lamiaceae species; e.g. P% was recorded to be 80.851% in O. syriacum L. species using TU-DAMD marker [22], 82.911% in S. tomentosa Miller using TD-DAMD marker [16] and 83.47% in S. miltiorrhiza using RAPD marker [8].

While, low P% genetic diversity level has been recorded in other Salvia species, e.g. P% was recorded to be 66.50% in S. viridis and that P% ranged between 35.68% (S. urmiensis) - 69.50% (S. limbata), using SRAP marker [20]; 63.54% in S. officinalis using AFLP marker [18]; 60.18% and 45.89% in S. spinosa and S. syriaca, respectively using AFLP marker [17]; 48.681% in S. nemorosa using ISSR marker [13]; 40.45 and 42.31% in S. judaica and S. palaestina species, respectively using TU-DAMD marker [21] and 16.656% in S. hispanica using ISSR marker [12].

More recently, Feng and coauthors [35] reported rich genetic diversity of the cultivated S. miltiorrhiza populations in China using four intergenic spacer sequences, ETS (psbA-trnH, trnL-trnF, and ycf1-rps15).

Overall, the current study suggests that the SV1, SV2 and SV3 genotypes clustered in one cluster are genetically distinct from the remaining other studied S. viridis (SV) genotypes based on the estimated PDV average of 0.238.

The high genetic diversity observed in S. viridis population could be attributed to one or more following factors: Outcrossing process as similarly reported for S. tomentosa Miller [16], or due to spontaneous hybrids occurred either in the wild or cultivated Salvia species [36] or to reproductive biology, gene flow, seed dispersal and nature selection in S. lachnostachys [11]; or to an interspecifc hybrid as similarly reported in S. divinorum [37]. Furthermore, the presence of several main roads crossing the collection sites could be act as corridors for insects, the main pollinator of Salvia species [11]. Thereby, these events encouraged efficient gene flow, leading finally to heterozygosity and thereby, genetic diversity expansion.

In the current study, TU-DAMD markers highlighted high genetic diversity (P% of 91.2%) among the studied S. viridis genotypes. This variation could be exploited in looking for determinant factors responsible for improvement of essential oils content in the studied genotypes. The elites genotypes could be later selected to introduce them in the future improvement breeding programs of this species.

CONCLUSION

Employed TU-DAMD marker in the current study showed high P% of 91.2% in the natural S. viridis population. Due to the recent TU-DAMD successful as a new marker for assessment of genetic diversity of S. judaica, S. palaestina, O. syriacum L. and S. officinalis L. (Lamiaceae) and its related species; it is worth noting to expanding its employment in molecular studies in order to discovery its effectiveness in genetic diversity assessment of other plants species. Furthermore, the high genetic diversity observed in the natural S. viridis population could serve in its future breeding programs.

Acknowledgments

I thank Dr. I. Othman (Director General of AECS) and Dr. N. Mirali (Head of Molecular Biology and Biotechnology Department in AECS) for their support, and the Plant Biotechnology group for technical assistance.

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  • Funding:
    This study was completely supported by the Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria, P.O. Box 6091, Damascus, Syria.

Edited by

  • Editor-in-Chief:
    Paulo Vitor Farago
  • Associate Editor:
    Jane Manfron Budel

Publication Dates

  • Publication in this collection
    15 Nov 2024
  • Date of issue
    2024

History

  • Received
    28 Feb 2024
  • Accepted
    11 Aug 2024
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