Genetic structure, molecular and phytochemical analysis in Iranian populations of Ruscus hyrcanus (Asparagaceae)

https://doi.org/10.1016/j.indcrop.2020.112716Get rights and content

Highlights

  • Ruscus hyrcanus is a rich source of medicinally important steroidal saponins.

  • Genetic structure of Ruscus hyrcanys populations was studied for the first time.

  • ISSR markers showed a significant difference among the twelve studied populations.

  • The most genetic diversity was intra-populational (Hs = 0.17).

  • The populations of RH6 and RH9 showed high pharmaceutical potential for further exploitation.

Abstract

Ruscus hyrcanus Woronow a commercially important ornamental and medicinal plant belonging to the Asparagaceae family, has various applications in Iranian traditional medicine due to its diuretic, appetizing, cathartic, vasoconstrictor, anti-hemorrhage, ant-nephritis, anti-infection, aperient and anti-varicose properties. In the present study, morphological, phytochemical and molecular markers were investigated in twelve R. hyrcanus (RH) populations collected from the Hyrcanian ecoregion in the north of Iran. Phytochemical characteristics were remarkably variable among the studied populations, but little morphological variation was observed. The highest contents of ruscogenin and neoruscogenin in the underground parts of the plant were recorded in populations RH9 (Abbasabad 3.41 mg/g DW) and RH6 (Darab Kola 1.44 mg/g DW), respectively. A total of 66 bands were obtained from 11 inter-simple sequence repeat (ISSR) markers, 62 (93.94 %) of which were polymorphic. Although the ISSR evaluation indicated an inter-population genetic differentiation (GST = 0.27), most of the genetic diversity was intra-populational (PPB = 32.26–75.81 %, He = 0.11−0.24, I = 0.17−0.36). A UPGMA cluster analysis of 120 samples of R. hyrcanus was carried out to categorize the studied populations, based on a molecular data matrix calculated with Jaccard’s similarity coefficient. A multiple regression analysis of the relationships between the ISSR bands and morphological and phytochemical traits demonstrated that eight ISSR fragments were related to ruscogenin in the plant materials. Several ISSR markers showed association with some morphological traits. This study provided the comprehensive preliminary data on morphological, phytochemical, and genetic characteristics of R. hyrcanus populations in order to exploit genetic reserves conservation programs.

Introduction

The genus Ruscus L., recently classified in the Asparagus family, comprises around 7–10 species. Distributed from Europe to Iran, these species are evergreen perennial shrubs with thick crawling rhizomes and stems up to 1 m high. Ruscus plants are a rich source of steroid saponins (SSs) in glycoside form, whose aglycone derivatives have a triterpenoid or steroidal structure (Masullo et al., 2016). Although the SSs have the same biosynthetic origin as triterpenoids through the mevalonic acid (MVA) pathway (Fig. 1) (Dewick, 2002), they are mainly classified based on the number of attached sugar moieties and their conjugation point to aglycone (Pengelly, 2004). Ruscogenin (RG) (diol aglycones (25R)-spirost-5-en-1β,3β-diol) and neoruscogenin (NRG) (spirost-5,25(27)-dien-1β,3β-diol) are well-known SSs, which were first isolated from the underground parts of R. aculeatus L. in 1957 (Sannie and Lapin, 1957). These aglycones, including spirostanols and furostanols, have also been isolated from the other Ruscus species. Therapeutic properties of RG and NRG have been reported, including for venous insufficiency (Vanscheidt et al., 2002), varicocele and hemorrhoids (Masullo et al., 2016), interdict cerebral ischemic injury (Cao et al., 2016) and liver injury (Wu et al., 2001), inhibition of acute lung injury (Sun et al., 2012), and anti-inflammatory (Huang et al., 2008), and antithrombotic activities (Kou et al., 2006). Several SSs with spirostanol derivatives have been isolated so far from the aerial and underground parts of R. hypoglossum L., R. colchicus Yeo., R. poticus Woronow, and R. hypophyllum L. (De Combarieu et al., 2002; Perrone et al., 2009; Pkheidze et al., 1971). RuscoVen, Arkocaps Rusco, Fragon, Butcher's-Broom, and Rosakalm are the most important formulated medicinal products from plant materials of Ruscus species. The demand for rhizomes of these plants has recently increased and the main suppliers are France and Turkey.

Ruscus hyrcanus Woronow, known as “Kooleh Khas” in Persian, is distributed across the Hyrcanian ecoregion, including the forests and wet valleys of the marginal Caspian Sea (Rechinger et al., 1990). Various applications of the plant, such as diuretic, appetizing, cathartic, vasoconstrictor, anti-hemorrhage, anti-nephritis, anti-infection, aperient and anti-varicose, are mentioned in the traditional medicine of Iran (Dehghan et al., 2016). According to local and unofficial reports, a massive volume of the plant rhizomes is collected annually from the forests of northern Iran and exported to other countries, especially Turkey. Therefore, before further germplasm depletion, a study of the plant population diversity is needed for conservation, domestication, breeding and commercial exploitation programs.

The evaluation of genetic diversity in wild accessions is the first important step in identifying, preserving and maintaining genetic reserves (Rao and Hodgkin, 2002). Inter-simple sequence repeat (ISSR) markers are extensively used for genetic diversity and population structure studies, because of their high polymorphism, reproducibility, and comparatively low cost. Moreover, sequence data are not needed to synthesize the primers, and microsatellite marker specificity is retained (Uysal et al., 2010). In fact, this technique is also suitable for investigating the genetic diversity of plant species without available sequencing information (Godwin et al., 1997). Inter-simple sequence repeat markers are a useful tool for estimating genetic variation within populations (Wolfe and Liston, 1998) and have been widely used in genetic studies of medicinal plants such as Satureja rechingeri Jamzad (Hadian et al., 2014), Artemisia dracunculus L. (Karimi et al., 2015), Thymus daenensis Celak. (Heydari et al., 2019), Salvia miltiorrhiza Bunge (Song et al., 2010), and Verbascum songaricum Schrenk (Selseleh et al., 2019).

The possible factors influencing phenotypic and phytochemical variation in different genotypes are genetics and the environment. Varieties with a genetic origin are more important for plant breeding, and the breeder utilizes the gene pool or available germplasm according to the specific breeding purpose (Heywood, 2000). The study of different natural populations of medicinal plants and the selection of individuals with superior growth characteristics and phytochemical traits is an important phase for their protection, domestication and breeding (Hadian et al., 2017). Although most of the medicinal plant varieties cultivated today originate from simple selection methods among wild populations or indigenous accessions, selection efficiency depends on the precise recognition of variation (De Vicente et al., 2004). Recently, SS variability in the different R. hyrcanus plant organs authenticated by molecular techniques has been reported by our group (Ghorbani et al., 2020). A literature survey revealed that phenotypic, genetic and phytochemical diversity of R. hyrcanus (RH) populations has not been previously reported. The present study aimed to characterize the genetic structure and variation of RH populations growing in Iran based on morphological, phytochemical, and molecular characteristics for the first time. These data can be useful for further in situ and ex situ domestication, breeding and sustainable exploitation programs of SSs-rich R. hyrcanus genotypes to meet the growing market and pharmaceutical industry demand for these plants.

Section snippets

Plant material

In total, 120 individual plants from the twelve RH populations were collected on 1–20 November, 2018, from their natural habitats in the northern forests of Iran at Golestan, Mazandran, and Guilan Provinces. The geographical characteristics of each collection site were recorded by a digital global positioning system (GPS) (Table 1, Fig. 2). Individuals were selected from plants of the same age. The distance between the sampled individuals in each collection site and between each population was

Habitat characterization

As can be seen in Table 1 and Fig. 2, the studied R. hyrcanus populations (RH1-RH12) are geographically distributed between the latitude of 36˚ 10′ 50.4ʺ N and 37˚ 47′ 13.6ʺ N and longitude of 48˚ 52′ 55.9ʺ E and 54˚ 89′ 00.3ʺ E, at altitudes ranging from 154 m (RH8, Sonbolrood) to 857 m (RH2, Shast Kalateh P32) (Table 1). All the plant populations are scattered across the Hyrcanian ecoregion, which has wet and Mediterranean climates with a mean rainfall of 498.8–1388.3 mm/year. Accompanying

Conclusion

Indigenous germplasm is considered to be the national treasure of each country. To protect the genetic resources of any species and landraces requires awareness of genetic variation and the relationship between species traits and genotypes. Therefore, a general understanding of inter and intra-population genetic diversity is essential to safeguard biodiversity. The highest contents of the medically important RG and NRG were found in the underground parts of plants collected from Abbasabad (RH9)

CRediT authorship contribution statement

Salimeh Ghorbani: Conceptualization, Investigation. Hassan Esmaeili: Methodology. Samad Nejad Ebrahimi: Writing - review & editing. Javier Palazón: Writing-review & editing. Mohammad Hossein Mirjalili: Supervision, Methodology, Validation, Formal analysis, Writing-review & editing.

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgements

The authors thank the Research Council of Shahid Beheshti University, Tehran, Iran for their financial support. We also wish to thank Mr. Hamid Ahadi for his kind help in HPLC analysis. This work is part of Salimeh Ghorbani MSc’s thesis.

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