Morphometric analysis and sequence related amplified polymorphism determine genetic diversity in Salvia species

Salvia species is a member of the Lamiaceae family, and it also possesses medicinal and horticulture values. The genetic diversity was assessed through sequence-related amplified polymorphism. To uncover genetic diversity and species characteristics in Salvia species were studied through a combination of morphological and molecular data. One hundred forty-five individuals related to 30 Salvia were collected in 18 provinces. A total of 157 (Number of total loci) (NTL) DNA bands were produced through polymerase chain reaction (PCR) from 30 Salvia species. These bands were produced with the combinations of 10 selective primers. The total number of amplified fragments ranged from 10 to 20. The predicted unbiased heterozygosity (H) varied between 0.11 (Salvia urmiensis) and 0.31 (Salvia limbata). High Shannon’s information index was detected in Salvia limbata. The genetic similarities between 30 species are estimated from 0.46 to 0.91. Clustering results showed two major clusters. According to the SRAP (Sequence-related amplified polymorphism) markers analysis, Salvia hydrangea and Salvia sharifii had the lowest similarity. Salvia bracteata and Salvia suffruticosa were genetically dissimilar to each other. This study also detected a significant signature of isolation by distance. Present results showed that sequence-related amplified polymorphism has the potential to decipher genetic affinity between Salvia species. Current results have implications in biodiversity and conservation programs. Besides this, present results could pave the way for selecting suitable ecotypes for forage and pasture purposes in Iran.


Introduction
Salvia species is a member of the Lamiaceae family, and the high diversity of Salvia L. has been reported in Western Asia, Eastern Asia, and South America (Walker et al., 2004). Iran is considered one of Salvia's 2 important regions, and 19 species are endemic to Iran (Jamzad, 2013). The majority of species are aromatic and possess medicinal and horticultural values (Safaei et al., 2016). Some of the Salvia species are also pharmacologically important and possess anti-inflammatory (Hosseinzadeh et al., 2003) and gastro-protective properties (Mayer et al., 2009). Scientific data shows that member of Salvia species, such as Salvia miltiorrhiza, is widely used in folk and traditional medicine and used in cardiovascular treatment (Wang et al., 2009). Several systematics and genetic diversity studies were carried out via molecular markers to address population structure, genetic diversity, and phylogenetic relations between the species (Robarts and Wolfe, 2014). Some experiments have been performed to determine genetic diversity within the Salvia genus (Song et al., 2010;Erbano et al., 2015). In the past, molecular methods such as Inter Simple Sequence Repeats (ISSR) and Random Amplification Polymorphic DNA (RAPD) have been used to study genetic diversity in Salvia (Song et al., 2010;Javan et al., 2012;Peng et al., 2014;Erbano et al., 2015). Sequence-related amplified polymorphism (SRAP) is PCR -based marker system. It is an efficient and simple marker system to study gene mapping and gene tagging in plant species (Li and Quiros, 2001). SRAP are potential markers to assess plant systematics and genetic diversity studies (Robarts and Wolfe, 2014). Previously, Wu et al. (2010) assessed genetic diversity and population structure in Pogostemon cablin with SRAP markers' aid. SRAP markers were successfully implemented in Lamiaceae family to study natural populations and variations within the family (Saebnazar and Rahmani, 2013;Talebi et al., 2015). These past studies showed that molecular markers, including SRAP markers, are efficient in investigating genetic diversity analyses and the phylogenetic relationship among Salvia species in the Lamiaceae family. Indeed, molecular markers are efficient methods to study genetic diversity . Genetic diversity helps plant species to survive and adapt against constantly changing environmental conditions (Pauls et al., 2013).
In order to develop conservation strategies and proper utilization of plant genetic resources, it is crucial to characterize plant species based on genetic studies (Kharazian et al., 2015), particularly this approach may assist in understanding genotypes of the geographically differentiated genus, such as Salvia (Song et al., 2010;Erbano et al., 2015). In Iran, most of the studies have been conducted to assess the medicinal potential of the Salvia species. The majority of the studies have addressed phytochemical and antimicrobial aspects. Therefore, we implemented the morphometric analysis and Sequence related amplified polymorphism to study genetic diversity. According to current knowledge, this is the first study that successfully documented genetic diversity based on novel sequence-related amplified polymorphism markers.
The present study investigated the molecular variation of 30 Salvia species in Iran. The study's objectives were: estimate genetic diversity; evaluate population relationships using WARD approaches. Current results have implications in breeding and conservation programs.

Plants collection
One hundred forty-five (145) individuals of Salvia were sampled. These individuals were recorded based on eco-geographical different features. Thirty Salvia species in East Azerbaijan, Lorestan, Kermanshah, Guilan, Mazandaran, Golestan, Yazd, Esfahan, Tehran, Arak, Hamadan, Kurdistan, Ilam, Bandar Abbas, Ghazvin, Khorasan, and Ardabil Provinces of Iran were selected and sampled during July-August 2017-2019 (Table 1). Morphometric and SRAP analyses on 145 plant accessions were carried out. One to twelve samples from each population belonging to 30 different species were selected based on other eco-geographic characteristics. Samples were stored at -20 °C till further use. Detailed information about samples locations and geographical distribution of species are mentioned (Table 1 and Figure 1).

Morphological studies
Each species was subjected to morphometric analysis, and twelve samples per species were processed. Qualitative (9) and quantitative (13) morphological characters were studied. Data were transformed before calculation. Different morphological characters of flowers, leaves, and seeds were studied. Ordination analyses were conducted while using Euclidean distance (Podani, 2000).

Sequence-related amplified polymorphism method
Fresh leaves from one to twelve plants, were randomly used. These were dried with silica gel powder.
Genomic DNA was extracted while following the previous protocol (Esfandani-Bozchaloyi et al., 2019). SRAP assay was performed as described previously (Li and Quiros, 2001). Ten SRAP in different primer combinations (PCs) were used. A 25μl volume containing ten mM of Tris-HCl buffer at pH 8; 50 mM of KCl; 1.5 mM of MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of single primer; 20 ng of genomic DNA and 3 U of Taq DNA polymerase (Bioron, Germany) were subjected to PCR reactions. The overall reaction volume consisted of 25 μl. For the PCR reaction, the Techne thermocycler (Germany) was used. In the initial denaturation step, the temperature was kept at 94 °C for 5 minutes. It was followed by 40 cycles for 1 minute at 94 °C, 1 minute at 52-57 °C, and 2 minutes at 72 °C. In the final extension step, the cycle duration was kept 7-10 min at 72 °C. Staining was performed with the aid of ethidium bromide. DNA bands/fragments were compared against a 100 bp molecular size ladder (Fermentas, Germany).

Data analyses
In the current study, the UPGMA (Unweighted paired group using average) ordination method was implemented to assess morphological characters. ANOVA (Analysis of variance) was conducted to assess morphological differences among species. Principle coordinates analysis (PCoA) was implemented to identify variable morphological characters in Salvia species. Multivariate statistical analyses, i.e., PCoA analysis, were performed in PAST software version 2.17 (Hammer et al., 2001).

Molecular analyses
Sequence-related amplified polymorphism (SRAP) bands were recorded. These brands' presence and absence were scored based on the present (1) and absent (0). Total loci (NTL) and the number of polymorphism loci (NPL) for each primer were calculated. Furthermore, the polymorphic ratio was assessed based on NPL/NTL values. Polymorphism information content was calculated as previously suggested by Roldan-Ruiz et al. (2000). Resolving power for individual marker system was calculated as Rp = ΣIb. Ib (band informativeness) was estimated while following equation: proposed as: Ib= 1 -[2 x (0.5-p)]. In the equation, p indicates the presence of bands (Prevost and Wilkinson, 1999). The pairwise genetic similarity of the marker system was evaluated (Jaccard, 1908). Unbiased expected heterozygosity and Shannon information index were calculated in GenAlEx 6.4 software (Peakall and Smouse, 2006). Gene flow was conducted in POPGENE software, version 1.32 (Yeh et al., 1999). Analysis of molecular variance test was conducted in GenAlEx (Peakall and Smouse, 2006). Mantel test was performed with 5000 permutations in the PAST, version 2.17 (Hammer et al., 2001).

Morphometry
The ANOVA findings showed substantial differences (p<0.01) between the species in terms of quantitative morphological characteristics. Principle coordinates analysis results explained 67% cumulative variation. The first PCoA axis explained 53% of the total variation. The correlation (> 0.7) was shown by morphological characters such as seed form, calyx shape, calyx length, bract length, and basal leaf shape. The morphological characters of Salvia species are shown in the PCoA plot ( Figure 2). Each species formed separate groups based on morphological characters. The morphometric analysis showed a clear difference among Salvia species and separated each group.  Species identification and genetic diversity Ten (10) suitable primer combinations (PCs) were screened out of 25 PCs in this research. Figure 3 illustrates the banding pattern of Em4-Me1 primer by the SRAP marker profile. One hundred and forty-four (144) amplified polymorphic bands (number of polymorphic loci) were produced. These bands (fragments) had different ranges i.e., 100bp to 3000 bp. Maximum and minimum numbers of polymorphic bands were 20 and 10 for Em2-Me5 and 10 Em1-Me1, respectively. Each primer produced 14.4 polymorphic bands on average. The PIC ranged from 0.35 (Em2-Me5) to 0.51 (Em1-Me1) for the 10 SRAP primers, with an average of 0.45 per primer. The resolving power (Rp) of the primers ranged from 22.87 (Em2-Me2) to 44.23 (Em1-Me4), with an average of 35.98 per primer (Figure 3, Table 2).  Analysis of molecular variance (AMOVA) results showed significant genetic difference (p = 0.01) among Salvia species. AMOVA findings revealed that 73% of the total variation was between species, and comparatively less genetic variation was recorded at the species level (Table 4). Genetic difference between   In the present study, a strong correlation between geographical and genetic distances (r = 0.19, p=0.0002) and a gene flow (Nm) score of 0.287 was reported among species. Detailed information about genetic distances and genetic identity (Nei's) are described (Table S1, Supplementary Table). The findings suggested the significantly highest degree of genetic similarity (0.91) between S. suffruticosa and S. bracteata. On the other hand, S. hydrangea and S. sharifii (0.46) had the lowest genetic resemblance.

Discussion
We used morphological and molecular (SRAP) data to evaluate Salvia species relationships in the present study. Morphological analyses of Salvia species showed that quantitative indicators (ANOVA test results) and qualitative characteristics are well differentiated. PCoA analysis suggests that morphological characters such as bract length, stipule length, bract shape, calyx shape, petal shape, length have the potentials to identify and delimitate Salvia species. Multivariate statistical analysis results suggest the utilization of morphological characters to identify and delimitate Salvia Species. Morphological characters, comprising stipule length and petal shapes, play an important role in plant systematics and taxonomy. Our work also highlighted the significance of morphological characters and molecular data to identify and study species genetic diversity in Salvia species. Past research conducted on Salvia species has successfully linked morphological characters with molecular data to assess genetic diversity and species delimitation (Safaei et al., 2016). Current morphometric and molecular methods have been implemented to study genetic diversity in Salvia species. For instance, morphometric analysis, coupled with molecular markers, revealed genetic diversity in Salvia species (Radosavljevic et al., 2019). Genetic diversity is usually an important feature that helps plant species adapt to harsh environments (Tomasello et al., 2015). Current Salvia species showed high genetic diversity; therefore, it could be argued that Salvia species can withstand the changing environment. In general, genetic relationships obtained from SRAP data coincides with morphometric results. This finding is in accordance with the parameters of AMOVA and genetic diversity results. SRAP molecular markers detected apparent genetic differences among species. These results indicate that SRAP has the potentials to study plant systematics and taxonomy in Salvia members. Given the negative impact of biodiversity threats and overexploitation of Salvia plant species in Iran, it is necessary to conduct genetic diversity studies on Salvia species. Genetic diversity-based studies pave our understanding to develop conservation strategies . Genetic diversity studies are conducted through an appropriate selection of primers and indexes, including Polymorphic information content (PIC) and marker index (MI). These are important indexes and primers to fathom genetic variation in species (Sivaprakash et al., 2004). Common logic suggests that different makers have different abilities to assess genetic diversity, and usually, genetic diversity is linked with polymorphism (Sivaprakash et al., 2004).
In this research, we reported PIC values of SRAP primers from 0.35 to 0.51, with a mean value of 0.45. PIC values indeed show low and high genetic diversity among genotypes. Values are ranging from zero to 0.25 show low genetic diversity; in contrast to this, 0.25 to 0.50 highlight mid-level of genetic diversity. In addition to this, values higher than 0.5 are associated with high genetic diversity (Tams et al., 2005). Present results highlighted the efficiency of SRAP markers to estimate genetic diversity in Salvia species. In our study, SRAP markers detected an average percentage of polymorphism (93%). Current research results also described average PIC values of SRAP makers (0.45) and average RP (resolving power) values i.e. 35.98 of SRAP markers.
These current reported values are higher than other reported markers on Salvia species (Wang et al., 2009;Song et al., 2010;Yousefiazar et al., 2016;Etminan et al., 2018). In the recent study, low gene flow (Nm) was detected among Salvia species. Despite the presence of limited gene flow in Salvia species, two distinct ecotypes were reported previously. These ecotypes were formed due to reproductive isolation caused by altitude gradients and different niches (Moein et al., 2019). The present study also depicted a significant correlation between genetic and geographical distances. Our findings revealed that isolation by distance (IBD) existed between Salvia species (Mantel test results). Several mechanisms, such as isolation, local adaptation, and genetic drift, shape the species or population differentiation (Frichot et al., 2013;De Kort et al., 2014). The magnitude of variability among Na, Ne, H, and I indices demonstrated a high level of genetic diversity among Salvia species. Dendrogram and principal coordinates analysis results showed a clear difference among Salvia species. This result indicates the high utilization of the SRAP technique to identify Salvia species. Our results have implications for conservation and breeding programs. Furthermore, it may identify suitable ecotypes for forage and pasture.

Conclusions
The present study investigated the molecular variation of 30 species. The molecular and morphometric analysis confirmed the morphological and genetic differences among Salvia species. This study was the first attempt to assess genetic diversity through Sequence-related amplified polymorphism and morphometrics analysis in Iran. The current study reported two major clusters, and these two major groups were different from each other due to differences in morphological and genetic characters. The genetic similarities between 30 species were estimated from 0.46 to 0.91. SRAP (Sequence-related amplified polymorphism) markers analysis showed that Salvia hydrangea and Salvia sharifii had the lowest similarity. The current study also reported the correlation between genetic and geographical distances, which indicated the isolation mechanism involved in the Salvia species ecology. Present results indicated the potential of sequence-related amplified polymorphism to assess genetic diversity and genetic affinity among Salvia species. Current results have implications in biodiversity and conservation programs. Besides this, present results could pave the way for selecting suitable ecotypes for forage and pasture purposes in Iran. Future studies might include other environmental variables, including soil chemistry and geology data, to disentangle the diversity and ecology of Salvia species. However, this study focused on genetic diversity while incorporating morphometric analysis and a novel SRAP marker system.

Authors' Contributions
A.S -Conceptualization, analyzed the data, mathematical processing, designed the study, prepared the initial draft; F.Z -Contributed to the conceptualization of ideas, the methodology, and the manuscripts' review, supervision, project administration, and funding acquisition. G.Z. -Genetic analysis, writing; curation, visualization, review, and editing; W.L -Literature review and contributed in mathematical processing, design; X.L -Contributed in the collection of data and analysis; S.E-B -Contributed in study design and reviewed the initial draft. All authors have read and agreed to the published version of the manuscript. All authors read and approved the final manuscript.