Detection of Genetic Polymorphisms using Random Amplified Polymorphic DNA (RAPD)-PCR in Fenugreek (Trigonella foenum-graecum) Plants after Seed Treatment with Biotic and Abiotic Agents

Trigonella foenum-graecum L. is a widely used herb in traditional medicine. The aim of this study was to evaluate the genetic polymorphisms in fenugreek plants following the treatment of fenugreek seeds with different combinations of biotic and abiotic agents using the random amplified polymorphic DNA (RAPD)-PCR technique. We assessed the effects of two strains of the fungus Trichoderma harzianum (Th-1 and Th-2), methyl jasmonate (MeJA), and Aloe vera gel (AVG) on growth parameters of fenugreek plants. Combinations of Th-1, MeJA, AVG significantly increased fenugreek root length, shoot length, shoot fresh weight, number of true leaves, and chlorophyll content. The Th-2 isolate, on the other hand, markedly slowed plant development (except for root length which was not affected significantly). In contrast, the combination with MeJA had no considerable effect on all growth measures, whereas the combination with VAG resulted in a substantial drop in shoot height and chlorophyll content when compared to other growth parameters that were unaffected. The present study has shown that the PCR amplification of DNA, using five primers for RAPD analysis, produced 62 DNA fragments that could be scored in all genotypes. The total number of polymorphic bands was 26, and the average percentage of polymorphism was 54.21%. The RAPD-PCR results showed that the treatment of fenugreek seeds with Th-1 alone or in combination with MeJA and AVG induced polymorphisms in fenugreek leaves.

MeJA may be considered a promising elicitor for diosgenin production in fenugreek plants 15 . Simultaneously, Jasmonic Acid (JA) has synergistic and antagonistic effects with abscisic acid (ABA), ethylene (ET), salicylic acid (SA), and other plant hormones in the process of resisting environmental stress 16 . Aloe leaf extract (ALE) has been used to improve the vegetative growth of Abelmoschuses culentus, Oenothera biennis and Majorana hortensis 17 . El Sherif 18 suggested that ALE is an efficient alternative source to improve the growth of Populus trees grown under in vitro conditions. Many biologically active chemicals can be found in aloe vera, more scientific research into this medicinal plant would be beneficial, as would promoting its widespread use 19 . Mirihagalla and Fernando 20 reported that aloe vera could be used in developing natural root inducing substance and resolving and reducing the risk of chemical toxicity in plants due to plant growth regulators, since, aloe vera leaves produce more rooting hormones after separated from the plant.
Fenugreek seed quantity and quality can be improved via cultivation, irrigation, and harvest management practices 14 . However, the potential for genetic advancement and successful conservation and management of germplasm resources in Trigonella sp. is limited due to a lack of knowledge on genetic variation and intra-specific relatedness 21 . Random amplified polymorphic DNA (RAPD) is a PCR-based DNA amplification technique that is widely used in the field of applied plant breeding. RAPD is a simple and efficient tool that does not require full sequence information 21 . Karp et al 22 , and can be used for genetic diversity analyses 23 . Furthermore, RAPD techniques are advantageous because they require only a small quantity of DNA and can uncover a large number of polymorphisms 24 . The relative genetic distances across fenugreek types did not always correspond to the geographic distance of their growth. And the genotypes that fall into distinct groupings show that there is genetic variation among the members of different clusters. Because the members of distinct clusters are genetically diverse, they can be crossed for future crop improvement 25 . Other studies, reported that the SRAP technique is effective in detecting genetic diversity and population structure of Iranian fenugreek varieties 26 .
The aim of the present study was to conduct random amplified polymorphic DNA (RAPD)-PCR to investigate the extent of genetic polymorphisms and to correlate genetic relatedness in fenugreek plants following seed treatments with various combinations of T. harzianum, MeJA, and AVG.

MATeRIAL AND MeThoDS
Fenugreek seed preparation T. foenum-graecum L. seeds were purchased from the local market in Al-Hilla City, Babylon Province, Iraq. We selected homogeneous one-year-old fenugreek seeds with no cracks or other visible deformations. Seeds were suspended in distilled water to assess viability; seeds that settled to the bottom were selected and surface sterilized using 2% NaOCl for 5 min. Seeds were rinsed several times with sterile distilled water and then dried on sterile filter paper (filter papers were sterilized by enveloping them in foil and placed in an autoclave basket at 121°C for 15 minutes) under a laminar flow hood for 30 min (Allegrucci et al) 27 .

Trichoderma harzianum source and culture
Two T. harzianum isolates, Th-1 and Th-2 (distinguished by phenotypic characteristics), were obtained from Dr. Jawad K. Abood Aljanabi, Proffesor of Advanced Mycology, College of Science, the University of Babylon in September 2020.
To prepare T. harzianum cultures, autoclaved Potato Dextrose Agar medium (PDA)manufacture by Titan Biotech Limited/ India, was poured into sterilized test tubes and allowed to solidify at an angle. Loopful inoculums of T. harzianum isolates were transferred to PDA slants and incubated at 25°C for 7 d. Pure cultures were maintained at 4°C until use 28 .
To prepare spore suspensions, T. harzianum isolates were cultured on Petri plates containing sterilized PDA, incubated in the dark at 26°C for 7 days, and then placed under continuous light to promote sporulation 29 . Fungal spores were obtained by adding 10 ml of sterilized distilled water containing 0.1% Tween 80 (v/v) to each Petri dish to decrease the surface tension and promote the release of fungal spores (conidia). The colony surfaces were scraped gently with a sterile lancet 30 to prepare the suspensions, and the mixture was filtered through a three-layer muslin cloth. The filtrate was collected, and the spore suspension concentration was adjusted to 1.0 × 10 8 conidia per milliliter using a hemocytometer under a light microscope 31,32 .

Soil preparation and planting
Peat moss and sand were mixed 1:1, wet with distilled water, and packed in appropriate plastic bags for autoclaving. Approximately 50 g of soil was transferred into each plastic container (3.5 cm diameter at the bottom and 5.5 cm diameter at the top; 5.5 cm depth).

Seed treatment
A preliminary experiment was conducted with and without 2% Arabic Gum to ensure the gum did not affect seed viability using three replicates. No differences in percentage germination of fenugreek seeds were recorded. Ten treatments were prepared using two isolates of T. harzianum (Th-1 and Th-2) at concentration shown in 2.2, while 100 µL/L (0.01%) was used from methyl jasmonate (95% MeJA; Sigma-Aldrich and 100% from fresh-natural Aloe vera gel (AVG) was prepared. Fenugreek seeds were dipped separately (25 seeds each) for two hours in each of the follows treatments: Control, distilled water; Th-1; Th-2; 20 µM MeJA; Th-1 + 10 µM MeJA; Th-2 + 10 µM MeJA; AVG (100% concentration); AVG (50%) + 10 µM MeJA; Th-1 + AVG; Th-2 + AVG. In all experimental formulations, 2% Arabic Gum was used to ensure the adhesion of T. harzianum conidia to fenugreek seeds. All seeds were drained and dried in a laminar airflow hood for two hours 33,34 .
Five fenugreek seeds were sown per mini pots in a growth chamber at 25±1°C temperature and at a relative humidity of 70%. After two weeks, germination took place within three days of sowing, and the standard germination rate was 98%.

experimental treatment and design
Germinated seeds were transferred after two weeks to plastic pots (12 cm diameter at the top and 7 cm diameter at the bottom; 12 cm depth) filled with 200 g of autoclaved soil, as five seedlings per pot, in a greenhouse with 17hour light periods and a light intensity of 200 mol quanta m-2 s-1, and day/night temperatures of 25°C/18°C. In four replicates, the pot experiment was set up in a completely randomized designs following 35,36 with modification. A total of 40 pots, each containing four plants, were used in the experiment. Seedlings were watered with an equal amount of tap water when required

Growth parameters
Fenugreek plants were harvested soon after flowering at 45 days old. Plants were removed from the pots, and the shoots and roots were separated and washed with distilled water three times, dried, and weighed. The number of leaves, chlorophyll content (using a self-calibrating chlorophyll meter-model SPAD 502), shoot length, and fresh weight of the shoots were all measured. The length of the roots and shoots was measured from the soil line to the top of the shoot 37 and then weighed using a sensitive balance.

Genotypic identification Sampling and DNA extraction
Two plants were used for DNA extraction to ascertain the changes in genetic diversity in fenugreek using RAPD markers. DNA from fenugreek plants was obtained and purified using an extraction and purification kit (Wizbio, South Korea).

Primers
Five arbitrary or "random" primers were obtained from Bioneer (Integrated DNA Technologies, USA). Fenugreek plant DNA was tested for single primers for RAPD-PCR amplification (Table 1).

RAPD-PCR amplification
Reaction mixtures with a final volume of 30 μl were prepared with 5 μl of a single primer, 12.5 µl of Green Master Mix, 3 μl of genomic DNA, and also the reaction volume was rounded to 30 µl with the addition of 8.5 µl of nucleasefree water. The reaction mixture was dispensed in a thermo-cycler (Eppendorf) programmed for 3 min at 94°C, followed by 42 cycles of 1 min at 94°C, 1 min at 42°C, and 3 min at 72°C; then a final extension of 7 min at 72°C. Amplification products were electrophoresed in 2% agarose gels and visualized via staining with ethidium bromide. Standard molecular markers were conjointly enclosed in every electrophoresis run. Images of the ultraviolet trans-illuminated gels were captured (Gel documentation system, UV source\ Cleaver Scientific -UK)

Phylogenetic analysis
Locations of scorable RAPD bands were remodeled to a matrix of binary characters ("1" for the existence of a band at a certain location; "0" for the absence of a band at the location). Cluster analysis was conducted based on Jaccard's similarity coefficient matrices, calculated from RAPD data to create a dendrogram of fenugreek genotypes. A phylogenetic tree was generated from the similarity matrices using the un-weighted pair-group method arithmetic (UPGMA) average cluster analysis (Program \ past3).

Statistical analysis
Data were subjected to mono-factorial or bi-factorial analysis of variance (ANOVA) depending upon the experimental design. Treatment means were compared using a least significant difference range test at P < 0.05 with standard error.

Fenugreek growth
Fenugreek plant growth varied among treatments ( Table 2). The results are expressed as the mean of four replicates revealed that treatment with Th-1, MeJA, AVG, and their combinations enhanced the growth capacity of fenugreek plants. According to our findings, the Th-1 and Th-2 isolates reacted differently to the growth of the fenugreek plant.

Root length
Root length was significantly (P < 0.

Shoot height
The shoot height was significantly (P < 0.001) taller when seeds were treated with Th-1 alone or in combination with MeJA and AVG. The tallest plant height was recorded in Th-1 + AVG (33.75), followed by AVG + MeJA (32.32 cm), Th-1 (32.00 cm), AVG (31.92 cm), Th-1 + MeJA (30.12 cm), and MeJA (29.77 cm), which were all significantly taller than the control. The minimum height was observed in Th-2 (19.57cm) and in Th-2 + AVG (20.25 cm), which both produced shorter plants than the control (23.30 cm).

RAPD analysis of fenugreek
In the RAPD analysis, five RAPD primers were screened, and amplified bands were selected for further study. The size of the PCR products ranged from 100 bp to 1500 bp; the average number of polymorphic bands for each primer was 5.1 (Table 3). The total number of polymorphic bands was 26, and the average percentage of polymorphism was 54.21%. Cluster analyses were conducted using RAPD data and the similarity matrices were used to generate a dendrogram using the UPGMA method (Table 2). RAPD profiles detect alterations in genomic DNA with the use of arbitrarily primed PCR reactions, with the potential to detect treatment-induced DNA effects. However, RAPD is a qualitative method through which the nature and amount of DNA can only be speculated. The results revealed substantial effect of Th-1 of MeJA and AVG treatments and their combination on RAPD profiling in fenugreek ( Fig. 1-6). UPGMA analysis for the dendrogram made based on the RAPD data generated by primers (OPX-8, OPW-2, OPA-18, OPA-5, OPF-2) were performed and shown in Fig. (7). Analysis   PCR amplification of DNA using the five primers for RAPD analysis produced 62 DNA fragments that could be scored in all genotypes.

disCussiON
Trichoderma is one of the most economically important microorganisms in the agro-ecosystem industry, influencing soil health and crop productivity. The anti-pathogenic, plantgrowth promoting, and bioremediating effects of Trichoderma increase crop yield in terms of both biological and economic output 38 .
The present experiment demonstrated the substantial effects of T. harzianum on the growth of fenugreek plants. However, the effects of T. harzianum were dependent on the isolate applied, since differential reaction patterns were observed between the two T. harzianum isolates toward the growth parameters of the studied plant. The Th-1 strain, alone and in combination with MeJA and AVG, produced an increase in fenugreek root length, shoot height, shoot fresh weight, number of true leaves, and chlorophyll content, reflecting growth improvement consistent with previous reports 39, 40 . Several mechanisms for plant growth stimulation via Trichoderma have been described. When root endophytic fungi in the genus Trichoderma colonize the roots of crop plants, upregulation of genes and pigments that enhance the photosynthesis is observed 41 . It was reported 42 that the release of soluble compounds by Trichoderma may exert beneficial effects on microbial groups cohabiting the rhizosphere, compounding the positive impact on plants. Moreover, Trichoderma produces auxins that can stimulate plant growth and root development 43 . Trichoderma can also improve plant uptake of copper, phosphorus, iron, cobalt, arsenic, cadmium, zinc, boron, aluminum, manganese, and sodium 44 by increasing the bioavailability of these nutrients 45 . It is most likely that Trichoderma stimulates growth by influencing the balance of hormones such as IAA, gibberellic acid and ethylene 46 .
Conversely, the Th-2 strain negatively impacted fenugreek growth, the Authors have no explanation for this phenomenon which never previously demonstrated. Our findings demonstrate that combination treatments with Th-1 and MeJA or AVG produced significantly higher fenugreek growth than Th-1 alone. Li et al 47 , reported increases in plant height and biomass as a result of MeJa application in Helianthus annuus (sunflower), Solanum lycopersicum (tomato), and Glycine max (soybean). Another study demonstrated that MeJA has an impact on gene expression and stomatal opening 48 . The present study revealed a substantial effect of AVG on the growth of fenugreek whether used alone or in combination with MeJA or Th-1 which possibly due to its content of β-polysaccharides, amino acids, vitamins, glycoproteins, soluble sugars, anthraquinones, and enzymes 49,50 . El Sherif 16 concluded that the leaf extract of Aloe Vera is an efficient alternative source to improve the growth of Populus clones. T. harzianum plays an important role in inducing the expression of defense genes in infected plants 51 . They used biotic and abiotic agents to suppress Fusarium oxysporum, which infects tomato plants.
The observed DNA profile modifications, such as changes in band intensity and loss of bands, may be due to the changes in oligonucleotide priming sites due to genomic rearrangements, DNA damage in the primer binding sites, or the presence of DNA photoproducts, which can block or reduce the polymerization of DNA in the PCR reaction 52 .
RAPD markers have been used to determine genetic variation and relationships among many plants 53 . RAPD molecular markers are useful tools for assessing genetic variability because they are abundant, very reproducible, highly polymorphic, highly informative, and simple 54,55 . In this study, the genetic diversity among some Iraqi populations of fenugreek was investigated using RAPD markers.
The five selected primers are sensitive enough to evaluate the diversity and intergenic or intragenic phylogenetic relationship among fenugreek plants. These markers can help in understanding the population structure, variation in genetic diversity within the species related to the geographic range, mode of reproduction, mating system, seed dispersal, and fecundity 56 . The genetic variability that detected in the present studies may be due to these prevalent background factors as the genotype of fenugreek. Dangi et al 57 , reported 70-72% of polymorphisms using RAPD and ISSR markers to analyze fenugreek collected from different countries. Other researchers 58 , observed an average of 76.78% polymorphism using RAPD in the 30 accessions collected from a western part of India.
Finally, the investigation has demonstrated that cluster analysis could be profitably used in unraveling the genetic variation within the accessions and the two molecular markers, Journal of Pure and Applied Microbiology RAPD could be used as an effective tool to evaluate genetic diversity and assess the genetic relationship in fenugreek.

CONClusiONs
The positive impact of combinations of T. harzianum, VAG, and MeJA on fenugreek plant growth was not previously reported, according to the researcher's knowledge. Treatments with strain Th-1 produced higher plant shoot and root growth and increased biomass and chlorophyll production. However, Th-2 negatively impacted plant growth. Some combinations show potential as fertilizer alternatives to stimulate fenugreek growth and production; however, our results demonstrate the importance of T. harzianum strain selection. Moreover, our findings show that RAPD can detect treatment-induced differences in fenugreek.
The current study also revealed that some primers failed to give different bands to the treatments under study, which could be due to a lack of suitable conditions for the operation of the polymerase chain reaction, such as temperature or nucleic acid quantity, GC ratio to primers, or the number of cycles used.