INTRODUCTION

Cassia angustifolia Vahl. which belongs to the family Fabaceae is a small, annual, or perennial, branched under-shrub grown for its medicinal value of leaves and pods which contain sennosides, rhein, aloe-amine, kaempferein, and iso-rhein in free and glycosides forms [1]. Cassia angustifolia is found growing in the wild in north African countries, including Ethiopia and Sudan [2]. It is native to Yemen, Somalia, and Saudi Arabia, and is now grown globally. It is used in recent and other folk medicinal systems. Its therapeutic properties help relieve constipation [3].

Medicinal plants have a long history in the Middle East and are important elements in prophetic medicine. C. angustifolia is found in Saudi Arabia's mountains [4]. Mistletoe is a medicinal plant with great therapeutic potential [5]. The use of polyphenol-rich plants to prevent thrombus formation is of interest and it has anti-inflammatory effect [6, 7]. Lately, a new combination therapy used metals and plants as nano-antibiotics [8]. Cassia angustifolia Vahl, Senna, or Sonamukhi., it is also called Tinevelly Senna. It was brought to Tirunelvelly district in Tamil Nadu from Europe in the mid-18^th century, hence the name Tinnevelly Senna. The laxative properties come from sennosides A, B, C, and D found in the leaves and pods [1, 9]. Senna is a multi-habitat plant that has successfully colonized a wide range of habitats in various climates and latitudes. Eighty percent of the 350 species currently assigned to the genus are found in North America, while the rest are found in tropical Africa, Madagascar. Using DNA profiling techniques to identify genotypes has several advantages over using morphological data. DNA sequences are independent of environmental conditions and can identify plants at any stage of growth [10-12]. PCR has enabled the development of simple and rapid DNA profiling methods [13]. Molecular markers are the best tools for genetic analysis. Most RFLP (restriction fragment length polymorphism) issues were resolved with the advent of PCR-based RAPD and SSR (microsatellites). RAPD markers allow rapid screening of the genome for genetic polymorphisms. RAPD gained popularity due to its simplicity, efficiency, ease of use, and lack of sequence information. RAPD has been used to identify and analyze genetic variation in Cassia species [14]. RAPD markers are a useful tool for detecting polymorphism and identifying chromosome-specific DNA fragments. RAPD reveals one allele per locus, corresponding to the amplification product. RAPD should not detect heterozygous loci. RAPD has limitations here. When a single primer generates at least one complementary, it can identify heterozygotes. This marker identifies polymorphic amplification products from each parent, species, and population loci. RAPD is a useful tool for predicting areas of maximum diversity and estimating genetic variability in natural populations. The accessions were genetically clustered into seven clusters. Identical accessions were found to be the best for hybridization and producing productive recombinants for Senna genetic improvement [15]. The current study aimed to compare Cassia angustifolia planted in two different areas in morphology and molecular level using the RAPD technique.

MATERIALS AND METHODS

Samples collection

 The seeds of Cassia angustifolia were collected from Jadah western (A) and Riyadh (B) Saudi Arabia and sowed in the trial field of the Botanical Garden of Biology Department, College of Science, Imam Mohammad Ibn Saud Islamic University, and the plants were morphologically analyzed, young leaves were kept it in a clean container under -20 until used for DNA extraction.

Morphology character measurements

 In this study digital Vernia Guage tool was used to measure the length and width of stems, leaves, and seeds.

DNA extraction

Samples preparation

Half of a young dry leaf was taken and cut it into small pieces then grinded using a porcelain mortar and pestle.

Extraction method

The Invitrogen™ PureLink™ DNA Kit (Thermo Fisher Scientific, USA)  was used for high-quality plant DNA from a wide variety of sample types. The kit uses proven PureLink™ spin-column technology for robust yields of DNA that is ready for downstream PCR, sequencing, and other applications. Typical DNA recovery is 5–20 µg from 1 mL of the plant.

RAPD analysis and primer selection

PCR amplification of genomic DNA using random primers was performed (RAPD). 25ng of genomic DNA was amplified using four primers: OPA03, AGTCAGCCAC, OPA09, GGGTAACGCC, UBC-101, GCGGCTGGAG, UBC-104, GGGCAATGAT. The standard William et al., [14]. The RAPD-PCR method was used by Hamza et al.; Kumar [2, 16].

  A 25ng template DNA (1μl), 0.5U Taq polymerase (0.5μl), 10 picomoles random primers (2μl), 100mm each dNTP(2,5μl), 50mm MgCl₂(1.5 μl/ml) and 1X PCR reaction buffer (2,5μl) were used to make 20 μl PCR reaction mixtures each. Double distilled water was used to make up to 20μl. Thermocycler optimized PCR mixtures. PCR had three steps: 5min initial denaturation at 94°C. Step 2: Run 40 cycles of denaturation at 94°C, annealing at 36°C, and extension at 72°C. Step 3: 7-minute final extension cycle at 72°C. To keep the product at 40°C, the PCR thermocycler was tuned. Electrophoresis was done using ethidium bromide-stained and 2% agrose gel.

 Statistical analysis

 The data was statistically analyzed by using excel 2010 for descriptive data as graphs. DNA analysis using ethidium bromide-stained from PCR products by gel electrophoresis.

RESULTS AND DISCUSSION

Morphological characterization

The study showed that when comparing Cassia angustifolia collected from the two areas, the length of plants was 78.60±0.65 cm (65- 78 cm) in seeds from the western area while in seeds from Riyadh was 55.36±0.57 cm (44- 55cm) (Figure 1). The length of the leaf was 5.54±0.24 cm (4-6.9 cm) and 3.80±0.32 cm (2- 6.6cm) in the western area and Riyadh, respectively (Figure 2). The leaf width was 1.60±0.07 cm (0.9-1.9 cm) and 0.84±0.08 cm (0.5- 1.6cm) in western area and Riyadh, respectively (Figure 3). The length of the fruit long was 6.56±0.29 cm (5.6-7.5cm) in the western area and 4.00±0.25 cm (2.5- 5.2cm) in Riyadh (Figure 4). The width of fruit was 2.33±0.11 cm (1.8-3cm) in the western area while in Riyadh was 1.30±0.05 cm (1- 1.5cm) (Figure 5). All morphological parameters were statistically significant (P ≤ 0.05) when comparing Cassia angustifolia plants from the two areas. The variation in morphology was attributed to the different environments in which samples were cultured.

Molecular results (genetics)

Four random APOM and UBC primers (APOM3, APOM9, UBC-101, UBC-104) were used for RAPD analysis of the two Cassia angustifolia populations to detect polymorphism. The primers screened, (APOM3, APOM9, UBC-101, UBC-104) showed amplification in all subjected samples. The sizes of the ladder DNA were 1 kb. The 1, 2, 3, and Four primers yielded ten RAPD bands. The observed bands ranged in size from 100 to 600 Pb, with one to three bands per primer. Ten DNA fragments were polymorphic bands between both the two plants were confirmed in phonomertic characters observed in this study (Figure 6 and Table 1).

Table 1. Primers showing polymorphism in two Saudi Arabian Cassia angustifolia populations from Jadh and Riyadh.