Introduction

Olive trees are recognized as subtropical trees with long lifespans of a few decades. The evergreen olive (Olea europaea L.) tree has been domesticated for over 7000 years. These trees are one of the most valued agricultural resources for good health1. Olives are widely cultivated for their significant nutritional, antioxidant, and secondary metabolite content. Many olive varieties are grown all over the world. Still, around 98% of the world’s olive trees, which are grown on more than eight million acres, are restricted to the Mediterranean region, which has the most significant number of olive-growing regions2,3. They are widely cultivated in Spain, Greece, Türkiye, Syria, Jordan, Tunisia, Morocco, Algeria, Libya, Egypt, the United States, Australia, Argentina, and Chile4. The vast gene pool of the wild olive and other olive subspecies represents plantations in traditional and emerging olive-growing nations. Cross-breeding between olive cultivars and wild olive with certain traits may be a successful plant breeding strategy and for biodiversity conservation5.Different markers are used to analyze the genetic pool of olive-cultivated varieties. Molecular markers offer trustworthy measurements that go beyond diversity6,7. Genetic diversity in the olive collection has been identified using different molecular markers such as Random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), single-nucleotide polymorphism (SNP), restriction fragment length polymorphism (RFLP), single-nucleotide polymorphism8, simple sequence repeats5 and chloroplast DNA (Table S1).

RAPD markers are frequently utilized as a promising marker system9,10. RAPDs have been used to establish genetic links among cultivars, differentiate among olive cultivars, examine inter- or intra-cultivar gene variation, and study genetic variations in the olive tree11. Genetic diversity using these markers for germplasm characterization in olive has been reported in olive cultivars growing in Israel12, Tunisia10, Spain13, Portugal14, Türkiye15, Greece16, and Italy17. The olive trees are not indigenous to India. It has been acclimatized and now cultivated. The cultivars grown in India were primarily imported from Israel and were planted in different climatic zones in Rajasthan, India, in 2006, in collaboration with Israeli partners. Arbequina, Barnea, Cortina, Frantoio, Koroneiki, Picholine, and Picual are the cultivars currently flowering in different climatic zones of Rajasthan. They were selected for their robust morphology, resistance to diseases, and resistance to the damaging effects of high and low temperatures and high fruit production. Detailed research on the olive plant grown in Rajasthan can help understand the genetic diversity of the cultivars. Considering the above research9,10,11,12,13,14,15,16,17, the current study analyses olive cultivars’ genetic diversity and phylogenetic relationships using the RAPD markers. Olive diversity is usually characterized by assessments of morphological trait analysis18,19,20, biochemical markers21,22, proximate composition23,24. Some studies have also considered the antioxidant activity21,25, and the antimicrobial activity25,26 to assess the genetic diversity among the cultivars of olive. Phenolic compounds and flavonoids are well-known for their antioxidant activity. These compounds also differ based on the solvents in which they are extracted. The amount of phenolics, flavonoids, and antioxidants are potential markers for distinguishing different cultivars of olive that differ among themselves and act as biochemical markers. The diversity among the cultivars based on the antioxidant activity can be related to their geographic origin. As all the cultivars grown in Barore farm had diverse geographic origins, it was interesting to understand their diversity and interrelationship among themselves. Thus, to understand this genetic diversity and phylogenetic relationship of all the olive cultivars growing in Barore, Rajasthan, India, a cumulative approach was studied using RAPD markers, considering the morphological, biochemical, proximate analysis, and their antioxidant and antimicrobial properties of the cultivars (Fig. 1). This is the first time that the genetic behavior of Israeli origin cultivars under Indian climatic conditions has been analyzed comprehensively. These cultivars are now climate resilient species that are growing luxuriantly under the concept of sustainable agriculture to provide food and oil for all.

Fig. 1
figure 1

Olives cultivars from Isreal were established in India and an effort to understand their molecular diversity considering a combinatorial approach was undertaken.

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Materials and methods

Plant material

The olive plant (Olea europaea L.) herbarium was identified and authenticated by the Department of Botany, University of Rajasthan27, and assigned the voucher accession number RUBL211669, dated March 6, 2018.

Morphological traits analysis of different cultivars of olives

Seven morphologically diverse olive cultivars from Israel were introduced to Indian soil in 2007 and planted in different locations in Rajasthan. Barore farms have shown luxuriant growth in olive trees, flowering, fruiting, and oil yield since 2012. This study was focused on this farm situated in Ganganagar (29.903839′′N latitude, 73.877190′′E longitude), Rajasthan, in northwest India. The cultivars were healthy, in complete flowering stage, and between 10 and 12 years old.

A survey on these olive cultivars was conducted with special emphasis on the leaves and fruits to get a general impression of the variability among the olive cultivars grown on the Barore farm. All the olive cultivars were tested for six morphological traits vis-à-vis. habit, height of plant (PH), number of primary branches per plant, leaf [shape of leaf (LS), length of leaf (LL), width of leaf (LW), area of leaf (LA)], flowering, fruit [shape of fruit (FS), length of fruit (FL), and width of fruit (FW), area if fruit (FA); and fruit production per plant (FP)]. A visual assessment of the data was followed by random sampling to measure the plants and their corresponding plant parts. Sorting was done among the samples collected, and only intact plant parts were taken. Data considered was in triplicate for all the morphological characteristics under investigation.

Assessment of bioactive compounds encompassing antioxidant and antimicrobial properties of the Olive leaf extract from different cultivars

The biochemical parameters and morphological parameters were also considered to understand the diversity among the different cultivars grown in Barore, Rajasthan. The shade-dried leaves from olive cultivars were ground to a fine powder (diameter < 250 μm) and stored at 37 °C. To make the olive leaf extract, 50 g of crushed olive leaves were combined with 1000 mL Milli-Q water, heated for 6 h at 60 ℃, and filtered to get the olive leaf extract28. Care was taken so that there was no intermixing of the leaves.

Phenolics content

The total amount of phenolic compounds of Olive leaf extract of seven different cultivars was quantified in both polar (ethanol, methanol, water) and non-polar (hexane, and chloroform) solvents using the Folin-Ciocalteu assay29 method. The results were expressed in milligrams of GAE per gram of dry matter. The comparison of the cultivars was also performed for the phenolic compounds using the Fourier transform infra-red (FTIR) spectroscopy in an FTIR analyser (Bruker Alpha, Germany), ranging from 4000 cm− 1 to 400 cm− 130.

Flavonoids content

Methanolic extracts were filtered through a 0.45 μm sterile syringe filter, and with the filtrate, HPTLC analysis was performed using quercetin (0.1 mg/mL) as a standard. The spots were applied using a CAMAG Linomat-5 TLC Sampler 4 (ATS 4) with nitrogen flow and a 150 nl/s delivery speed from the application syringe31. The flavonoid concentration in the methanol extract of Olive was measured using a slightly modified version of the aluminium trichloride (AlCl3) procedure32. A comparison of the cultivars was also performed with the flavonoid compounds using FTIR spectroscopy30.

Antioxidant analysis

The total antioxidant capacity of the different cultivars of olive leaf extract was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity method33. During the current research, the antioxidant activity of Olive leaf extracts of seven different cultivars in five different solvents, viz. ethanolic, methanolic, aqueous, hexane, and chloroform, was performed as per our earlier report34. The DPPH radical scavenging activity was calculated as percentage inhibition by the formula given below:

$$\:Inhibition\:\left(\%\right)=\frac{Acontrol-Asample}{Acontrol}*100$$

The IC50 values (mg/mL) were calculated using the concentration-response curves35.

Antimicrobial assay

The nonpathogenic bacterial isolates, Staphylococcus aureus (MTCC No. 9542), Escherichia coli (ATCC 25923), Bacillus subtilis (MZ683213.1), and Pseudomonas aeruginosa (MZ348930) were administered to the antimicrobial assay. Antimicrobial assay of crude plant extract fractions was performed using nutrient agar media through the agar-well diffusion method34 using olive leaf extract of Arbequina, Barnea, Coratina, Frantoio, Koroneiki, Picual, Picholine cultivars, and Amikacin as positive control. They were allowed to diffuse for one hour at room temperature and later left undisturbed in an incubator at room temperature for the whole night36. The zone of inhibition (mm) was measured after every 24 h for 3 days.

Proximate analysis in the Olive leaf extract of different cultivars

Olive leaf extract was analyzed using the Standard Procedure (AOAC, 1995) to evaluate its moisture, crude fiber, protein, ash, and fat levels37,38. The comparison of the cultivars was also performed for the primary metabolites related to proximate analysis using the FTIR spectroscopy30.

Genomic DNA extraction and PCR amplification

This study thoroughly rinsed olive leaves with running water and gently washed them with sterile distilled water. The genomic DNA from the olive leaves of the different cultivars was extracted using the modified CTAB process39. This genomic DNA was screened using 8 decamer primers (Table 1). The RAPD primer sequences used in the present study were obtained from The Operon Technologies, USA (publicly available), and the primers were synthesized by M/s Bangalore Genei Pvt. Ltd., India.

Table 1 RAPD primers used to check polymorphism among 7 Olive cultivars.
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The PCR amplification was performed using double-distilled water, 0.5 mM dNTP mix, 2.5 mM MgCl2, 1 U of Taq polymerase, 8 times reaction buffer, 1.0 µM primer, and 25 ng of genomic DNA. Using a BioRad C1000 Touch thermal cycler (Bio-Rad Laboratories), the amplification was carried out under reaction conditions consisting of a 3-minute pre-denaturation step at 94 °C for 3 min, 45 cycles of denaturation at 94 °C for 30 s, 1 min of annealing at 45 °C, and 1.5 min of extension at 72 °C. The final extension occurred at 72 °C for 7 min, with a hot temperature of 4 °C. Electrophoresis was performed on 0.8% agarose gels stained with ethidium bromide (1 µg/ml). The voltage of the gel was maintained at 90 V/cm until the loading dye/bromophenol blue reached the other end of the gel.The transparent gel was photographed using a gel documentation system.

Data analysis

Amplified bands generated from RAPD amplification were scored based on the presence (1) or absence (0) of bands in primer and were used to calculate a SM similarity matrix among seven cultivars, triple readings for each of the variables using NTSYS-pc version 2.140. Cluster analysis was based on morphological, biochemical, antimicrobial, and molecular data. Similarity matrices were created for all pairings of accessions using SM similarity coefficients. Jaccard’s similarity coefficient was used to calculate the similarity coefficient between seven olive cultivars. Cluster analysis was performed using the Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) analysis, and Dendrograms were also created. Principal component analysis (PCA) was also used with the NTSYS-pc software to discover any genetic relationship between the genotypes. The matrix measuring similarity and variation was made using the simple matching (SM) similarity coefficient, which was calculated using NTSYS-pc software (Numerical Taxonomy & Multivariate Analysis System; Version 2.1, Exeter Software, Applied Biostatistics Inc., New York), and the dendrogram was created using this matrix40.

Statistical analysis

The morphological and biochemical data were subjected to mean values and relative standard deviations. One-way analysis of variance (ANOVA) was carried out for the statistical evaluation of the results using Microsoft Excel’s Data Analysis tool, with a significance level (p ≤ 0.05). The F-test was used to validate the quantitative attributes displaying substantial variation among the populations. All morphological and biochemical data were analyzed to determine their mean values and relative standard deviations. The analytical results were used for statistical comparisons, and all assays were performed in triplicate.

Results and discussion

Morphological markers of the different Olive cultivars

The different cultivars showed variations in habit (canopy) of the olive tree and trunk pattern, as shown in Fig. 2. It is believed that evolution gave rise to the tree’s mechanically efficient shape. Arbequina (origin Spain) is one of the most productive olive cultivars, and it is cultivated in Spain and Argentina. The Arbequina tree showed maximum plant height, while the Coratina and Picholine had the lowest height (Table 2). The evergreen tree of Arbequina (origin Spain) showed an upright spreading habit of growth with medium-to-dense branching structure (Fig. 2A-B), and less refined foliage. The Barnea (origin Israel) cultivar is a vigorously growing tree cultivated in Israel. It showed an erect habit with less branching and produced thin, narrow gray-green leaves (Fig. 2C-D). Coratina (origin Italy) tree is a quick-growing Italian olive cultivar with a generally dense and erect growth habit. As with most trees, regular pruning promotes the formation of an even thicker canopy (Fig. 2E-F). The fruit has dark green foliage and small, white blossoms with a vast root system. The cultivar Frantoio (origin Italy) tree grows slowly, with medium vigour and less branching (Fig. 2G-H). The Koroneiki, Greece’s most common oil cultivar, showcases moderate growth and an open, expansive habit, a bushy tree with a delicate canopy (Fig. 2I-J). Picholine (origin France) cultivar has a medium vigour, spreading semi-dense canopy, and an open habit (Fig. 2K-L). The tree of Picual (origin Spain) cultivar of olive is recognized for its upright growth pattern, healthy foliage with long, thin, and dark green leaves (Fig. 2M-N).

Table 2 Olive leaf and fruit characteristics.
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Fig. 2
figure 2

Tree and Trunk pattern of olive cultivars growing in Rajasthan (A, B) Arbequina; (C, D) Barnea, (E, F) Coratina, (G, H) Frantoio, (I, J) Koroneiki, (K, L) and Picholine, (M, N) Picual.

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The number of primary branches per plant was lowest in Barnea and Coratina (˂ 7 no) and the greatest in Picholine and Picual (˂ 9 no), respectively (Fig. 2I-J). Olive fruits and leaves despite the challenging environmental circumstances in which they develop showed diversity considering the various morphological factors, height of the plant, leaf shape, length, width, shape of the fruit, length, width, number of primary branches per plant, and fruit yield per plant (Table 2; Figure S1). The leaves of Coratina and Barnea fruits were the largest among all cultivars studied (Figure S1). Arbequina and Barnea types had elliptical leaves; Picual had long leaves; and Coratina, Frantoio, Koroneiki, and Picholine had elliptical-lanceolate leaves. The length of the leaves shows significant differences. They ranged from 4.6 to 10.1 cm, while the width of the leaves ranged from 0.6 to 1.6 cm. Coratina (16.1 cm) had the highest average leaf size, while Koroneiki (2.7 cm) had the lowest (Table 2).

The reproductive structures of olive trees were the axillary buds found on the leaves of the previous year’s shoots. In olive cultivars grown in Rajasthan, India, inflorescence development (Fig. 3A-U) was observed from mid-February, when the trees are subjected to sufficiently cold temperatures for a prolonged period. Their floral buds finally emerge from hibernation, and after bud break occurs. The Arbequina olive tree produces small, white flowers with four petals, measuring 1–2 cm in length (Fig. 3A-C). These delicate, feathery blooms appear in late spring or early summer, growing in clusters and emitting a fragrant scent. Arbequina is partially self-fertile, capable of producing fruit without cross-pollination41. However, having a pollinator variety nearby, such as ‘Koroneiki’ or ‘Frantoio’, can enhance fruit set and yield by 10–20%42. Despite this, Arbequina remains a productive variety even without cross-pollination. The fruit of the Arbequina olive is distinctive, with a spherical shape that sets it apart from other cultivars. Fruit sizes range from 1.4 to 2.5 cm in length and 0.6 to 1.0 cm in width. The area of fruit sizes ranged from 0.9 to 2.25 cm (Table 2). Within cultivars, olive fruit production per plant also varies. The annual production of Arbequina fruit was 20–25 kg, whereas the plant’s annual production of Barnea fruit was nearly 55 kg/ plant per year. Similarly, the yearly fruit yield per plant was Coratina 30 kg, Frantoio 5 kg, Koroneiki 10–12 kg, Picholine 5 kg, and Picual, respectively. The morphological characteristics, specifically the fruits and leaves of four olive cultivars grown in Greece43 and 28 different olive cultivars from Turkey44, were investigated and found to vary in shape and size. In another study from Iran45, fruit was also a good marker for distinguishing 243 different cultivars of olive. In addition to leaf and fruit, the bark of 25 Italian olive cultivars was also reported46,47 as a morphological marker.

Fig. 3
figure 3

Budding, flowering, and fruiting stages of different cultivars of olive [A, C] Arbequina, [D, F] Barnea, [G, I] Coratina, [J, L] Frantoio, [M, O] Koroneiki, [P, R] Picholine, [S, U] Picual.

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Molecular analysis of polymorphism among different cultivars of Olive growing in Barore forms in Rajasthan

Establishing a fingerprint for each species is necessary to identify polymorphic DNA using RAPD markers. The presence or absence of bands resulting from the multiplication of specific plant genome segments was used to analyze the genetic relationship in the study’s results. The molecular weights of those bands depended on the quantity and locations of the prefix sequences on the DNA template, and very light bands were ignored. Divergence based on molecular marker (RAPD) profiling was performed. Based on the eight primers selected for RAPD analysis, based on their reproducibility and banding patterns, a total of 51 bands were generated from 8 RAPD primers. Forty-five bands were polymorphic (56.25%), averaging 6.37 polymorphic bands per primer. The OPA-2 and OPB-2 primers amplified the highest number of amplicons (7) with 100% polymorphism, whereas the OPB-1 primer generated the lowest number of amplicons (2). A dendrogram has been generated using NTSYS-pc version 2.148, considering morphological (leaf and fruit) parameters using UPGMA cluster analysis based on Jaccard’s similarity coefficient value, which revealed that the average similarity percentage was 62% (Fig. 4A-H).

Fig. 4
figure 4

RAPD Fingerprinting of Olive Cultivars. Agarose gel electrophoresis of RAPD fragments amplified using different primers: [A–D]: OPA primers (OPA_1 to OPA_4; [E–H]: OPB primers (OPB_1 to OPB_4); Lane Details: Lane 1: Molecular Marker (10 kb ladder with fragments ranging from 1000 to 50 kb); Lanes 1–7: Olive cultivar samples.

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Diversity analysis of Olive using RAPD markers and clustering (Fig. 5) revealed two significant clusters with 59% similarities. The genetic similarity values ranged from 0.52 to 0.73 based on morphological and RAPD markers. Based on these data, this similarity coefficient amongst the seven olive cultivars showed an average of 0.62. All seven cultivars were differentiated and clustered using a dendrogram based on RAPD data into two major clusters and two subclusters Fig. 5A-B. First cluster A was the smallest and comprised only two cultivars consist, Picholine and Frantoio, while second cluster B was the biggest and comprised five olive cultivars, Arbequina, Barnea, Coratina, Picual, and Koroneiki. Subclusters B-I and B-II were created from Cluster B. Subcluster B-I comprised Koroneiki, Picual, and Coratina cultivars. In contrast, subcluster B-II was made up of Arbequina and Barnea. Picual and Coratina’s cultivars had the highest similarity coefficient (0.73%), and Koroneiki had a 0.65% similarity coefficient in subcluster B1. Cultivar Picholine and Frantoio had cluster A’s lowest similarity coefficient (0.57%). The second subcluster B2 included two distinct cultivars, Arbequina and Barnea, with a similarity coefficient of 0.68%, and Koroneiki with a similarity coefficient of 0.65. This study demonstrated the ability of RAPD indicators to differentiate between seven olive cultivars on the Barore farm in Rajasthan olive cultivars under investigation, India, and identify the genetic diversity and degree of proximity between them. The correlation coefficient (r) was calculated using Mantel Z-statistics49 and estimated at 0.73. This number was regarded as a good fit for the data by the UPGMA cluster pattern. The observed morphological variations among the cultivars could be attributed to genetic differences, environmental influences, or a combination. For instance, plant height and leaf shape differences might be related to genetic variations in growth habit and leaf morphology. Similarly, the fruit size and shape variations could be influenced by environmental factors such as climate, soil, and water availability.

Fig. 5
figure 5

RAPD analysis based on morphometric data of 7 olive cultivars [A] 3-D Principal component analysis plot [B] 2-D dendrogram.

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Using RAPD markers13 used the primers, OPK16, OPA19, OPF06, OPX09, and OPZ11, to discriminated 103 olive Spanish cultivars. Later, they found that a combination of the primers, OPK-16, OPA-19, and OPX-09, was most favourable and consistent in distinguishing most cultivars. Inter simple sequence repeats (ISSRs) and start codon targeted (SCoT) markers have recently been used to evaluate the genetic polymorphism among the olive cultivars Arbequina, Rosana, Aski, Coratina, Frantoio, Koroneiki, Manzanillo, and Picual from Saudi Arabia50. Earlier investigations51 from Portugal compared 17 ISSR and 20 RAPD markers to identify genetic variation and distances across wild olives and reported 73 to 100% polymorphism. RAPD, ISSR, and SSR molecular markers depicted a 95% polymorphism among the 101 olive Greek cultivars16. RAPD and ISSR marker was used to evaluate the relationship between polymorphic markers and olive oil properties, of nineteen Tunisian olive cultivars and noted 80% polymorphic bands using RAPD markers52. According to the study made by Abood and his colleagues53, 95.9% polmorphism was reported among the olive cultivars, where a cluster study based on RAPD revealed that Arbequina, Picual, and Arbosana were aggregating and forming one cluster based on their geographical origin. Still, the wild accession and Koroneiki cultivar failed to create a cluster and were individually separated. Spanish olive cultivars from the Extremadura region were documented and identified using three RAPD primers (OPF-6, OPA-8, and OPK-16) and morphological traits54. The findings showed a relationship between cultivars in terms of geographical origin. The results of our RAPD analysis motivated us to analyze the diversity considering a combination of markers related to its quantitative assessment of different primary and secondary metabolites and their related antioxidant and antimicrobial characteristics.

Biochemical markers as a toolkit for deciphering the genetic diversity among the different cultivars of Olive

Qualitative assessment of the primary and secondary metabolites in different cultivars of Olive

The qualitative analysis of the phenolic components present in the olive leaves from different cultivars was distinguished using FTIR spectroscopy (Fig. 6A-G). The present study noted the presence of a broad absorption band aligning with the functional group, primary amine (N-H), at 3310 cm− 1 in all seven olive cultivars. O-H stretching bands at 2929 cm− 1 and 2936 cm− 1, indicating the occurrence of the aromatic ring and alkyl group. In Picholine and Coratina cultivars, the 2929 and 2936 cm− 1 bands were absent. Peaks at 681, 519, 525, 539, and 573 cm− 1 may indicate C-I or C-Br stretching, suggesting the presence of halogenated compounds. Our FTIR results align with previous studies, associated with phenolic compounds in various reports for different olive cultivars55,56,57,58,59,60, specifically for the cultivars , Arbequina, Arbosana (Chile), Kalamata, Koroneiki, Picual (Spain), Koroneiki (Greece), Picholine (Morocco), at 3310 cm− 1, 1612.02 cm− 1, 1386.23 cm− 1 and 1230 cm− 1, respectively.

Fig. 6
figure 6

Qualitative and quantitative analysis of secondary metabolites in leaf extract of different Olive cultivars [A–G], FTIR analysis of [A] Arbequina [B] Barnea [C] Coratina, [D] Frantoio [E] Koroneiki [F] Picholine [G] Picual; [H] Total phenolics content present in the leaves of different olive cultivars extracted using different solvents.

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The qualitative analysis of the flavonoid component present in the olive leaves from different cultivars was distinguished using FTIR spectroscopy (Figs. 6A-G). In the current research, the absorption peak for the flavonoid (C = C) functional group at 1636 cm− 1 was present in all the cultivars except in Coratina. C = C stretching, bands at 1636 cm− 1, indicates the presence of amide and alkene group occurrence. According to a report from Chile, the spectrum areas between 1600 –1500 cm− 1 indicate the detection of flavonoid compounds in Arbequina and Arbosana Olive cultivars58. Previous research61 reported that the peak between 1400 and 1650 cm− 1 is attributed to the stretching vibration of C = C groups from aromatic compounds in olive Picual and Koroneiki olive leaves harvested from Spain and Greece61 and in leaves of the Neb jmel olive from Tunisia62.

Using HPTLC, the derivatized samples showed the presence of the flavonoids as light greenish-blue luminous zones under UV 366 nm 30. To find the spectral peak, samples of olive leaf extract from all seven kinds were scanned at 254 and 366 nm wavelengths. The spectral peak indicating the presence of flavonoids in olive methanolic extracts was detected at 254 nm (Fig. 7A) and 366 nm. The Rf value of quercetin was determined at a value of 0.30. Coratina, Frantoio, Picholine, and Koroneiki show a Quercetin peak at 0.03 Rf value, and Arbequina, Barnea, and Picual show a quercetin peak at 0.02 Rf at 254 nm. While Coratina shows Quercetin peak at 0.02 and 0.03 Rf, Frantoio at 0.18 Rf, Picholine 0.02 and 0.18 Rf, Picual 0.18 Rf, Arbequina and Koroneiki 0.03 Rf, and Barnea shows zero peak at 366 nm. The present investigation exhibits more peaks and area in Picholine and Coratina cultivars detected at 366 nm as compared to 254 nm (Fig. 7B-D). Alvarez and Gutierrez reported58 that the phenolic substances in olives are linked to high antioxidant and anti-inflammatory capabilities. The presence of the phenolic compound and flavonoid can be directly correlated with the antioxidant property of the different cultivars of the olives63,64,65,66,67.

Fig. 7
figure 7

Flavonoid analysis of leaf extract of Olive cultivars using qualitative and quantitative analysis [A] HPTLC fingerprinting at [a-b] 254nm, [c and d], 366 nm. [B] HPTLC densitogram of [a, aʹ] Quercetin, [b, bʹ] (Coratina), [c, cʹ] Frantoio, [d, dʹ] Barnea, [e, eʹ] Picholine, [f, fʹ] Picual, [g, g’] Arbequina, [h, hʹ] Koroneiki, at 254 nm at 366 nm; [C, D] Densitometry analysis compared to quercetin at [C] 254 nm and [D] 366 nm [E]; Quantitative analysis using AlCl₃ method.

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The qualitative analysis of the primary metabolites related to the nutritional content in the olive leaves from different cultivars was distinguished using FTIR spectroscopy (Fig. 6A-G). The prominent peak between 1030 and 1070 cm− 1 was attributed to polysaccharides, aromatic ethers, and carbohydrates68,69. In a current report, the broad absorption band for the protein with the functional group (C = C) at 1636 cm− 1 was present in all the cultivars except Coratina. C-O stretching, bands at 1055 and 1061 cm− 1  were denoted for the presence ofcarbohydrate. Leaves have a small amount of fats, so fatty acid peaks are absent in all seven cultivars of olive.

Quantitative assessment of the primary and secondary metabolites in different cultivars of Olive

The significant components of food, such as moisture, ash, crude fiber, total carbohydrate, protein, fat, and calories, are known as proximate analysis, as shown in Figure S2. In the present study, a proximate analysis of the leaves of olive cultivars showed ash content varying between 0.87 and 10.93%, and moisture content between 0.86 and 6.99%, respectively. Coratina cultivars have a higher moisture and ash content. Koroneiki is 6.99% and 15.49%, respectively, and Koroneiki cultivars have the least moisture and ash content, 0.86%, respectively. Among the primary metabolites, carbohydrate content in all cultivars varied between 76.19% and 97.85%, and total energy ranged from 300 to 400 kcal. Therefore, it can be said that the concentration of different nutritional parameters related to proximate analysis of the olive leaves differs among cultivars. Eventually, in 100 g of olive leaf extract, 0.86 g to 6.99 g of moisture, 0.15 g to 0.84 g of fat, and 0.17 g to 2.35 g of protein was noted. On analysis of one-way ANOVA, there was a significant difference (p = 0.0002) among the cultivars considering these characteristics. Carbohydrate content was found to range from 88 to 91%, although crude fiber was absent. According to a study from Portugal, the nutritional assessment of the leaves of olive cultivars, Madural, Verdeal, and Cobrançosa, was noted in the following trend: total carbohydrate > moisture > total protein > ash > total fat70. The nutritional content of Koroneiki olive leaves from Brazil showed protein ranging from 10.5 to 13.1 g/100 whereas the total carbohydrate ranged from 8.74 to 32.63%71. The nutritional composition of Egyptian olive leaves also reported that the protein ranged from 10.6 to 10.7 g/100 and total carbohydrate content was 74.5 to 74.7%, respectively72. This type of result was also observed in a study from Portugal37,73 where the protein was 8.02 g/100 dry leaves, and carbohydrate content was 37.65 g/100 dry leaves.

In the present study , the total amount of total phenolic content in olive leaves [extracted using different solvents, aqueous, ethanolic, methanolic, hexane, and chloroform] ranged from 31.6 to 93.23 GAE mg/g, respectively (Fig. 6H). The total phenolic content in different cultivars of olive leaves extracted using methanol was highest and ranged from 44.27 GAE mg/g to 90.05 GAE mg/g, respectively. The results were found to be significant (p-value 0.0036) according to one-way ANOVA analysis. The significant difference in total phenolic content among the cultivars suggests that some olive leaf cultivars may have higher antioxidant potential than others. The chloroform leaf extract of the Picual cultivar had the least total phenolic content in comparison to other cultivars. The methanolic extract of the leaves of all the cultivars showed high phenolic content (49 to 95 mg GAE/g DE) as evaluated using leaf extracts from different cultivars. In a previous study74, the high phenolic content of the methanolic olive leaf extract (131.7 ± 9.4 mg GAE/g dw) was noted from olive leaves growing in Brazil. A recent study75 reported that the ethanolic leaf extract of Tunisian, Sigoise, and Rougette olive cultivars had the highest phenolic concentration of 161.54 ± 0.99 and 160.53 ± 1.17 mg GAE/g DE, respectively. In contrast, the hexanolic leaf extract from olive cultivars75, Octoubri, Verdal, and Sofiana, showed low phenolic values (17.12 ± 0.77 mg GAE/g DE, 18.48 ± 0.50 mg GAE/g DE, and 18.53 ± 1.41 mg GAE/g DE. The effect of using different polar and non-polar solvents was also reported65,76 on Kalamon, Koroneiki and Megaritiki olive cultivars from Greece in different solvents.

Earlier study77 found that the leaves of 17 cultivars of olive viz. Manzanilla, Conservolea, Arbequina, Mishen, Coratina, Roghani, Kalamon, Amphissis, Yellow, Amigdalifolia, Mary, Leccino, Shenge, Gordal, Sevillenca, Fishomi, and Beleidi, growing in the farms of Iran showed that the methanolic extract of cultivars, Kalamon, Gordal, and Coratina had the highest concentration of total phenolic compounds ranging from 155.91 ± 0.06 mg to 190.65 ± 0.03 GAE/g extract, respectively. It was found that the methanolic extract of the Koroneiki cultivar of olive showed the highest total phenolic content, and the Kalamon cultivar showed the least phenolic content. On examination of the total phenolics in the methanolic extract of the Moroccan Meknès region78, cultivars viz. Arbequina, Hojiblanca, Frantoio, Koroneiki, Lechín, Luque, Manzanilla, Picholine de Languedoc, Picholine Marocaine, Picual, and Verdal, it was found that the olive cultivar Picholine showed the highest total phenolic content (44 g/kg dry weight). Picual showed the lowest amount at 11 g/kg dry weight. In the present study, the methanolic extract showing high total phenolic content can be considered a potential biochemical marker for the analysis of the genetic diversity.

According to the findings, the flavonoid content in different cultivars of olive leaves extracted using methanol ranged from 10.58 to 20.16 mg QE/g, respectively, as shown in Fig. 7E. The least flavonoid content was 10.58 mg QE/g in the Frantoio cultivar, and the highest flavonoid content was observed at 20.16 mg QE/g in the Picholine cultivar. The highest total flavonoid concentration of the olive cultivar growing in Spain37 was found to be 20.45 mg QE/g dried extract. In a recent report from Egypt33, Picual showed the highest total flavonoid content of 59.00 ± 1.00 µg CE/g, and Shamlali had the lowest 42.00 ± 0.60 µg CE/g of total flavonoids, respectively. According to research done on five olive leaf cultivars (Zarrazi, Limouni, Chemlali Tataouine, Dhokkar, and Toffehi) from Tunisia, the Tofffehi methanolic extracts had the highest flavonoid content of 42.754 ± 0.674 mg RE/g of dry extract79. The flavonoid concentration of Brazilian olive leaves was 19.4 ± 1.3 mg QE/g DW75. The flavonoid concentration of a methanolic extract of the Chemlali variety from Tunisia70, at two distinct harvesting stages, October and January, was between 98.4 and 377.06 mg CE/100 g DE.

Antioxidant analysis among different cultivated cultivars of Olive growing in Barore forms in Rajasthan

Their phenolic component content is the primary factor for olive leaves’ antioxidant potential. To find the activity of scavenging free radicals or other reactive species is measured by its antioxidant activity. Presuming that the antioxidant potential also differs among the different cultivars as phenolic content, this potential of the Olive was also considered a biochemical marker. Using varying ascorbic acid concentrations as standard as discussed in the earlier study34, the values of DPPH were estimated. The result displayed the ability of ethanol, methanol, aqueous, hexane, and chloroform leaf extracts of olive to scavenge this radical. In the current study, there was a high inhibition activity found in the ethanolic Arbequina leaf extracts at 94%. The lowest inhibition activity was represented by chloroform Picholine leaf extract and hexanoic Koroneiki at 89% in Fig. 8A. In all types of solvent extracts, the activity was not variable for all the cultivars, ranging from 89%to 94%. IC50 values of olive leaf extract were determined to be 0.38 to 0.41 mg/ml at different concentrations (0.2 to 0.6 mg/ml) respectively in Fig. 8B. The observed significant differences in the IC50 values (p = 0.46) among all the cultivars of olive in our present study can be related to the biochemical profiles and structural composition of the extracts. For instance, Barnea chloroform, and Frantoio ethanolic extracts exhibited the lowest IC50 values (IC50 = 0.38 mg mL− 1), can be related to less amount of the phenolic compounds.

Fig. 8
figure 8

Antioxidant activity in the leaf extracts of Olive cultivars in different solvents [A] DPPH radical scavenging activity [B] DPPH IC50.

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The DPPH scavenging among all these cultivars were found to be significantly different (p = 0.015) on ANOVA analysis. Figure 8B depicts a graphical representation of the DPPH% inhibition in several solvents (aqueous, ethanol, methanol, chloroform, and hexane) ranging from 89 to 94%. The ethanolic Frantoio extract exhibited the least activity (IC50 = 0.38 mg mL− 1), whereas the aqueous Arbequina extract had the highest IC50 value 0.41 mg mL− 1. According to earlier studies35, the DPPH IC50 activity differed among the cultivars Picual, Tofahi, and Shemlali cultivars from Alexandria, respectively, whereas Picual shows the highest antioxidant activity. Previous report78 showed that the DPPH radical scavenging activity of olive leaf extract for different solvents ranged from 22.72 to 69.81%. In another similar study79, DPPH radical scavenging activity of olive leaf in different cultivars were Arbequina 43.7, Arbosana 36.9, Changlot Real 46.8, Frantoio 41.2, Koroneiki 36.7, Picual 33.03, and Sikitita 45.83 mg of TE g− 1 dry weight respectively.

DPPH IC50 activity of aqueous extract of Picual leaves was reported as 48.14 ± 0.15 µg/mL63. Among the different solvents (ethanol, methanol, acetone, and water) in which olive leaf extract was extracted, the antioxidant activity of methanolic extracts (80%) of the cultivar Kalamata showed the highest antioxidant activity (28.09 µg/mL)64. While the antioxidant activity value reported in the Kalamata cultivar is notable, it is essential to recognize that activity can vary significantly depending on factors like extraction methods, solvent composition, and plant cultivar80. Further studies are needed to fully understand the range of antioxidant activity in olive leaf extracts. The methanolic extract of the following cultivars, Arbequina, Hojiblanca, Frantoio, Koroneiki, Lechín, Luque, Manzanilla, Picholine de Languedoc, Picholine Marocaine, Picual, and Verdal, reported that the antioxidant activity (DPPH IC50) ranged from 33 ± 04 TE g− 1 d.w. to 46.8 ± 0.2 mg of TE g − 1 d.w81.

Antimicrobial activity of the different cultivars of Olive leaf extracts

The antimicrobial activity of olive leaf extract against specific nonpathogenic bacteria (B. subtilis, P. aeruginosa, S. aureus, and E. coli) was obtained by the well diffusion method illustrated in Fig. 9A-D. According to the present study, it was found that the olive leaf extracts from different cultivars effectively inhibited the different test organisms. Results indicated that B. subtilis was the microorganism that was most responsive to Olive leaf extract, whereas E. coli, P. aeruginosa, and S. aureus were the least susceptible. It was also statistically verified that all the cultivars showcased significant differences (p = 0.002) of inhibition of microbial growth. Similar results were also observed by previous researchers,82,83,84,85,86. The macerated extracts of olive leaves showed potent antibacterial action against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Listeria monocytogenes. In an interesting study82, the antimicrobial activities of olive leaf aqueous extracts against S. aureus and foodborne pathogens were also evident. According to another recent study83, the olive leaf ethanolic leaf extract demonstrated strong antibacterial activity against five pathogenic bacteria (Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Listeria monocytogenes). It was reported36 that olive leaf aqueous extract can inhibit B. subtilis, P. aeruginosa, S. aureus, and E. coli.

Fig. 9
figure 9

Antimicrobial activity of Olive leaf extract against [A] P.aeruginosa, [B] B. subtilis, [C] S.aureus, [D] E.coli.

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According to our results there was an impact on the metabolic content based on the solvent used, thereby this had an profound role in contribution of the functional role in imparting their antioxidant and antimicrobial activity. The observed significant differences in the IC50 values (p = 0.46) among all the cultivars of olive in our present study can be related to the biochemical profiles and structural composition of the extracts. For instance, chloroform extract of Barnea, and ethanolic extract of Frantoio exhibited low IC50 value of 0.38 mg mL− 1. It can be related to less amount of the phenolic compounds. The methanolic extract of Frantoio showed 20.16 QE/g flavonoid content and 44.27 GAE.mg/g phenolic content, while the same methanolic extract of anther olive cultivar, Barnea, showed 20.09 mg QE/g. Flavonoid content and 76.01 GAE.mg/g phenolic content. The same methanolic extract of Frantoio and Barnea showcased an IC50 activity of 0.4 mg mL− 1 and hence conclude to have high antioxidant activity. The methanolic extract of Barnea and Frantoio exhibited good antimicrobial activity in comparison to olive leaf methanolic extracts of other cultivars, Picholine, Picual and Koroneiki. Hence, both cultivars exhibited profound biological functions (antioxidant and antimicrobial activity) due to the presence of the hydrophilic compounds like flavonoids and phenols extracted in polar solvents. When the extract was prepared in hexanolic solvent (non-polar), the antioxidant and antimicrobial activity was minimal, as both phenols and flavonoids were absent in these non-polar extracts.

Cumulative data analysis of morphological, biochemical, nutritional, antimicrobial, and molecular data using the UPGMA method

Using all the above data from the morphological, biochemical, nutritional, and antimicrobial studies, the current study predicted genetic variability among olive cultivars (genotypes) growing in the Barore farm in Rajasthan, India. The earlier morphological characterization of polymorphisms was the foundation for adaptability to various climatic situations. Morphological and biochemical assessment using several features indicated high variation in leaf, fruit, and stem properties, indicating that olive cultivars have the potential to be used in plant breeding and improvement. All these cultivars contain a reasonably high quantity of glucose, potassium, magnesium, and calcium, and hence might be used as a nutritional source. Based on morphological, biochemical, nutritional, and antimicrobial variables, the seven genotypes showed pairwise similarity ranging from 0.03 to 0.12 with an average of 0.9 (Fig. 9). The seven olive genotypes show a comparatively low degree of genetic similarity, with an average of 0.9 and pairwise similarity indices ranging from 0.03 to 0.12. This implies that the genotypes are genetically varied, which aligns with our conclusions drawn from the antimicrobial, nutritional, biochemical, and morphological traits.

All seven cultivars were differentiated and clustered using a dendrogram based on the cumulative (morphological, biochemical, nutritional, and antimicrobial) data into two major clusters, A and B, and the A cluster was divided into three subclusters. In contrast, the B cluster had only 1 subcluster. The first sub cluster included two genotypes: Arbequina, and Picholine (0.8%) similarity index. In the present study, Arbequina showed high phenolic content, and Picholine showed high flavonoid content. This can be related to their high antioxidant activity and ability to inhibit microorganisms. Moreover, as per the previous reports, Arbequina and Picholine are self-incompatible (cannot pollinate themselves) and also cross-compatible (can pollinate each other, and they were grouped in the G1 group based on single-nucleotide polymorphisms85). A genetic system probably prevents self-fertilization. This confirms the genetic relationship between Arbequina and Picholine, as evidenced by the present study.

The second sub-cluster included two germplasms, Barnea and Picual, showing a 0.8% similarity index. The flavonoid content in both cultivars was almost the same, thus confirming a similar high DPPH activity. Using RAPD and ISSR markers85. It was reported that Barnea and Picual are closely related based on the morphological traits of the fruits. The final third sub-cluster has two cultivars, Frantoio and Koroneiki. The highest similarity (0.12%) was seen in two genotypes of this cluster, Frantoio and Koroneiki. In the present study, the antioxidant activity of both cultivars was similar due to their high flavonoid and phenolic content. Similar evidence also found similarity among the olive cultivars Frantoio and Koroneiki in the context of their high phenolic content in the olive oil86,87.

Arbequina, Picholine, and Barnea, Picual had the similarity index of 8%. In comparison, Frontoio and Koroneiki had the highest (0.12%) and B cluster has one subcluster with one genotype Coratina, has the lowest similarity (0.04%). In Fig. 10, the 3-D PCA plot and the 2-D dendrogram based on molecular similarity between various genotypes showed that Coratina is the most diverse species since it has not formed any group with other genotypes. The genotypes are exotic arrivals from Israel that might have experienced genetic drift, mutation, or selection during their adaption to the Indian environment, may be the cause of the low similarity indices. The prospective use of these similarity indicators in olive breeding programs gives them biological significance88. Additionally, the low similarity indices imply that the genotypes might have unique adaptive strategies, which could help us comprehend how they react to various environmental circumstances. The variability of some olive cultivars using microsatellite markers and morphological trait was studied and found that Coratina and Frantoio were both monoclonal and similar 88,89. But in the present context the fruit morphology differed, and all the biochemical parameters were also different. Hence on RAPD analysis, this cultivar (genotype) was found to be the most diverse among the others. The average similarity across all germplasm was 0.07, showing a low level of genetic similarity between genotypes.

Fig. 10
figure 10

RAPD analysis based on the combined morphometric and biochemical data of 7 olive cultivars [A] Principal component analysis plot [B] Dendrogram.

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According to a recent study, several marker attributes, including polymorphism percentage, polymorphism information content, effective multiple ratio, marker index, and resolving power for RAPD markers, were utilized to evaluate the informativeness of the primer sets. The mean polymorphic information content (PIC) of 0.20 was noted10. In the present study on the olive cultivars growing on Barore farm, the RAPD study showed that primer OPB-2 had the highest PIC value (0.40), whereas primer OPB-01 had the lowest (0.24) value. EMR readings varied from 0.48 to 2.87, with a mean of 2.0. The average PIC value for RAPD, a measure of allelic diversity, was 0.34. The resolving power (Rp) of the evaluated RAPD markers ranged from 1.42 to 3.42, with an average value of 2.73 (Table 3). The highest resolving power (3.42) was found in RAPD for OPA-1 and OPA-3. Marker Index varied from 0.11 to 1.16, with an average of 0.72, and the correlation coefficient (r) was estimated as 0.67. The r value of 0.67 was considered a good fit of the UPGMA cluster pattern to the data.

Table 3 Marker’s efficiency parameter.
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In the present study, a comprehensive analysis of the genetic diversity and the relationship of RAPD molecular markers considering the morphological, biochemical, antioxidant, and antimicrobial properties in seven Indian acclimatized olive cultivars showed distinct differences between them due to diverse chemical characteristics, in addition to geographical provenance. Since RAPD is associated with a few limitations, like dominant nature, less reproducibility, etc., for further breeding programs, the SSR and SNP markers based on the advancement of genomic facilities need to be developed and validated for olive cultivars growing in India.

Conclusion

Finally, the findings show that olive cultivars show genetic diversity among seven olive cultivars from Barore farm. This study demonstrated the effectiveness of the RAPD technique in differentiating between the analyzed olive types as well as identifying the genetic distance and degree of closeness between them. Using RAPD primers, OPA1, OPA2, OPA3, OPA4, OPB1, OPB2, OPB3, and OPB4 produced distinct, reproducible bands that could be evaluated. The OPB1 gene produced the 7 amplicons and the OPA1 gene produced the fewest 2 amplicons. Olive cultivars (leaves) showed variations among nutritive value and were rich in phenolics and flavonoids showcasing high antioxidant, and antimicrobial activity. Arbequina leaf extract showed the greatest phenolic content whereas the highest flavonoid content was observed in the Picholine cultivar. Arbequina showed the highest radical scavenging activity and effectively inhibited microorganisms. A high degree of genetic variation was observed, and four distinct groups were noted. Both Picholine and Arbequina cultivar can be used for both table olives and olive oil production. They showed certain similarities that helped to group both of them in one cluster, yet they showed differences among themselves. On the basis of the morphology, Arbequina and Barnea were placed in the same cluster. Arbequina is often not a compatible pollinator for Barnea, meaning they do not effectively cross-pollinate each other; in some cases, Barnea is even considered partially self-incompatible when pollinated with Arbequina pollen. Barnea and Picual also showed similarity in secondary metabolite content, yet it is noted that Picual is primarily used for olive oil production due to its high oil content, whereas Barnea can also be used for table olives. Frantoio is one of the hardy cultivars and can resist very low temperatures. Although both Frantoio and Koroneiki show similarities in metabolite content and antioxidant activities, and they were clustered as one group. Yet in context to morphological characteristics, the fruits of Frantoio are bigger than those of Koroneiki. It was evident that the claim for phylogenetic relationship was pronounced by studying the morphological, biochemical, and antimicrobial properties, correlating them to molecular differences among the olive cultivars. This study used marker systems to better understand the performance of the olive cultivars, despite their geographically disparate origins. This information can be helpful for olive breeding programs and cultivation in India.