Leaf Protein Patterns: a Windows for Assessment of Genetic Diversity of Melissa officinalis Accessions

It has been suggested that the lemon balm (Melissa officinalis L.) is one of the most important medicinal herbs in the family Lamiaceae. Numerous studies have reported this species includes three subspecies: M. officinalis ssp. altissima, M. officinalis ssp. inodora and M. officinalis ssp. officinalis, however, only ssp. officinalis has commercial and medicinal value [1]. Reviewed from previous literatures the plant mainly grown in the Mediterranean regions such as Turkey, Southern Europe and Northern Africa, and Northern Iran [2,3]. In Iran, the plant has a broad distribution in Northern, Northeast (Golestan forest, Mazandaran, Haraz Valley), and West region of Kermanshah and Rijab, Tehran (Tochal altitudes) and between Qazvin and Karaj [4].

gene effects Nakamura [11], while molecular markers such as protein markers reflect the genotype more directly, independent of environmental influences [12,13]. Protein markers are useful tools to identify cultivar, registration of new varieties and classification of crop species to study genetic diversity, thereby improving the efficiency of plant breeding programs [14][15][16]. Among biochemical techniques, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) due to its simplicity, minimum cost in time and labor, and effectiveness is commonly used in the studies of plants genetic diversity [17][18][19][20].
With respect to genetic diversity, several polymorphic proteins have been reported in the genus Mentha Hassan et al. [21], Ocimum species Mustafa et al. [22], legume Boulter et al. [23], wheat El-Bakatoushi [24] and Brassicaceae [25]. An enormous lack of information related to the genetic diversity of M. officinalis is tangible. Therefore, the objective of present study was investigation on genetic diversity of M. officinalis accessions from different countries using protein analysis, which might increase efficiency of conservation of germplasm in order to utilize in breeding programs.

Plant materials
A total of 25 accessions of M. officinalis were collected from different countries (England, Germany, Iran, Italy, Japan and Majarestan) ( Table 1). The seeds were germinated into the growth chamber at 28 °C. After 48h, the germinated seeds were transferred into the pots with mixture of top soil and sand (2:1 v/v) in the greenhouse conditions with a temperature of 25 °C and irrigation every 48h at Medicinal Plants Research Center, Shahed University, Tehran. The leaves were harvested from the 45 days seedling to extract the total protein.

Leaf protein extraction
One gram of fresh leaf tissue from three samples of each accession was grounded in liquid nitrogen using pre-chilled mortar and pestle to obtain a fine powder. One g of the powder was homogenized and mixed with 2.5mL of the HEPES/KOH buffers according to the method of Talei et al. [26].

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Each sample was vortexed for 5min and then centrifuged at 20,000rpm for 20min at 4 °C using centrifuge (Model Sigma 3-30K) for 2-3 times. Then ¼ volume supernatants, chloroform was added to each sample and was then mixed and kept at room temperature for 5min. Finally, the samples were centrifuged for 15min at 16,000rpm at 4 °C. The supernatants were collected and stored at -20 °C. The total protein concentration was determined by the Bradford method (1976) at 595nm, using a spectrophotometer (Lambda 25, UV/VIS) [26].

Statistical analysis
The SPSS software version 22 was used for variance analyses and the NTSYS-pc version 2.1 software was used to calculate the Jaccard coefficient and cophenetic coefficient [28]. The cluster analysis was performed based on protein bands using weighted pair group method arithmetic averages (WPGMA) of DARwin 5 software and PCoA [29]. In practice, the seed protein analysis of the 25 accessions of Melissa officinalis unveiled the presence of 16 different types of proteins ranged from 10 to 100kDa, yet, only six of them with low molecular weight were found polymorphic ( Figure 1). The results indicated that most of the bands were similar in all accessions. Potentially, these protein bands can also serve as useful markers to hybridization and breeding programs in future studies. The analysis of variance indicated that there were significant differences among the accessions in terms of protein content (P≤0.01). The highest protein content (0.588mg/g) and the lowest protein content (0.240mg/g) were observed in Karaj -Dehkadeh and England accessions, respectively (Table 2).

Results
Jaccard's similarity coefficients were computed for all possible pairs of electropherograms among the 25 accessions (Table 3). The highest genetic similarity was observed between Germany and Malard accessions with a value of 1.0 and the lowest one's was obtained between Italy and N. Khorasan accessions with a value of 0.5.

Discussion
In this study we investigated the pattern and variability of M. officinalis is an important medicinal plant and characterization of genetic diversity in plants is susceptible to ontogeny and environmental condition. We hypothesized protein markers were used to distinguish genetic diversity among 25 accessions of M. officinalis. The gel analysis resulted in the detection of 18 protein bands with 59.09 percentage polymorphism. The results indicated that the main bands (common proteins) were same in all the accessions and the fundamental differences were for minor bands.
The results provided some interesting findings regarding to Jaccard's similarity coefficient results, the highest genetic distance was between Italy and N. Khorasan accessions. The results matched up well with the findings of Radwan et al. [19] that show crosses between accessions with high polymorphism could create more genetic diversity. Genetic diversity among the accessions was independent in terms of the collecting region. Some accessions with near geographically relationships were really unlike in their leaf protein bands, while some distant geographically accessions were too similar in their leaf proteins.
Dendrograms based on the Jaccard index and the WPGMA method are used to evaluate the degree of plants genetic diversity [30]. In the current study, the WPGMA cluster analysis of 25 accessions of M. officinalis based on the protein profiles generated three clusters. In summary, this study indicated that the ability to select leaf protein pattern for study of variability of officinalis accessions and showed that some Iranian accessions belonged to the first cluster were geographically close intervals, while Germany and Japan accessions with high geographical distance were geographically more distant from each other. The sources of the Pakan Bazr Company may be being collected at the close or the same original population of accessions segregation in the first group. The accessions of the second groups were collected from different region far apart from each other, which may be diverging from the same original population. These accessions could be mixed from different genotypes, or random mating populations with the same alleles but differing allele frequencies and probability due to open pollination of M. officinalis. Our results matched up well with the findings of Aharizad et al. [31], who reported that the morphological and essential oil content data of different M. officinalis accessions with high geographical distances were placed in the same group. In addition, Ghaffariyan et al. [32] in a study on genetic diversity of the 12 ecotypes of lemon balm using IRAP markers, based on long terminal repeats (LTRs) of barley retrotransposons showed that the average PIC and the average marker index between ecotypes were 0.27 and 14.36, respectively. Molecular analysis of variance indicated that the variance within population is greater than the variance between the populations, and finally, the populations were grouped into three clusters. Mustafa et al. [22] reported that the cluster analysis based on morphological trait, isozyme and seed protein data demonstrated genetic diversity among and within population of two species of Ocimum that may be due to environmental modification and natural hybridization. Sharma & Krishna [16] in a study on 52 accessions of cowpea (Vigna unguiculata) reported that SDS -PAGE approach is a powerful tool to identify diverse genotypes of cowpea. SDS-PAGE analysis of eleven samples of leaf protein in Ocimum showed that the method was adequate to determine genetic diversity Bompalli & Nallabilli [33].
Protein diversity is not random, since they associated with the genome expression and supply additional opportunities for polymorphism that noticed the presence of important genes for breeding purposes. Improvement in plant breeding program through selection is possible, especially if we extend the genetic basis from various habitats to comprise most of the genetic determinants of a desirable trait, such as; yield, a biotic stress tolerance, and quality Ghafoor & Arshad [34]. This result is reasonable given that reasonable genetic variation in the leaf protein pattern in 25 accessions of M. officinalis. This variation might depend on environmental conditions such as geographic regions, season of culturing, altitude, annual rainfall, temperature, land fertility and genotype variation [35].

Conclusion
This present study demonstrated the protein variation among the M. officinalis populations can be seriously taken into account in molecular studies. Furthermore, the electrophoresis of leaf proteins can be utilized as an effective strategy in the programs involved with M. officinalis conservation [15]. The observed differences among the accessions would be of immediate importance for development of the M. officinalis gene bank and may be used in hybridization and breeding programs to identify diverse parental combinations and creating segregating progeny with high genetic diversity [36]. Such a strategy in its turn could potentially lead to improving the phytochemical content of the plant [37]. However, future investigations into the limitations research and some other research questions to improve our knowledge are necessary.