1. INTRODUCTION

Mango (Mangifera indica L.) belonging to the family Anacardiaceae, with the botanical classification Mangifera genus, producing a drupe-type fruit with a panicle inflorescence is one of the most important crops grown in tropical regions [1]. According to the Ministry of Agriculture and Rural Development of Vietnam, ranks 13th globally in mango production. Mekong Delta is the largest mango cultivation area, accounting for 48% of the 87,000 hectares nationwide with many cultivars including Hoa Loc, Cat Chu, Chau Nghe, Thai, and Taiwan [2]. Additionally, mangoes are packed with essential nutritional elements like vitamin C, vitamin A, potassium, and dietary fiber, along with powerful phytochemicals such as mangiferin, beta-carotene, polyphenols, and flavonoids, all contributing to their health-promoting benefits. The appropriateness of climate and alluvial soil make a contribution to the abundance of different cultivars belonging to M. indica L. in the Mekong Delta. However, reports that shed light on the authentication of mango cultivars and a clear trademark system for them have been limited. Therefore, it is an urgent demand in the assessment of cultivar identity, genetically diverse level, and parental selection for the breeding program of M. indica L. in Vietnam [3].

Molecular assessment is superior to morphological and chemical characterization as DNA-based markers are stable and detectable in all tissues, regardless of any developmental stage of the cell, from growth to differentiation or state of defense. Another advantage of molecular markers is that they are not affected by environmental, pleiotropic, or epistatic factors [4]. Inter simple sequence repeats (ISSRs) are based on regions between adjacent, oppositely oriented microsatellites. They are non-specific markers so no requirement for sequence information for primer construction is needed [5]. For genetic identification, ISSRs were informative in categorizing Thai mango accessions when over 80% of the bands were polymorphic among the 78 bands generated [6]. ISSR markers revealed high polymorphism among mango cultivars from ten provinces in Vietnam [7].

DNA barcoding is a leading-edge molecular system that utilizes different short, standardized DNA fragments for species authentication. Thus, DNA barcoding has been proposed as a prospective candidate for the evaluation of species identity, the evolution of ecological populations, protective status, and biodiversity [8]. In terrestrial plants, the plastid genome contains two core barcodes for DNA barcoding: rbcL and matK, while intergenic regions within the chloroplast (trnH-psbA) and nuclear genome internal transcribed spacer (ITS) would serve as the accompanying sources for the construction of DNA barcoding database [9]. rbcLmainly encodes for the key photosynthesis enzyme ribulose bisphosphate carboxylase (RuBisCo), while matK codes for a mature enzyme that is responsible for type II intron splicing throughout RNA transcripts [10]. trnH-psbA is considered an efficient tool in species discrimination owing to its genetic variability. However, it is uncomplicated to design trnH-psbA as a universal primer due to the 75-bp conserved regions at two ends of this intergenic region [11]. The internal transcribed spacer (ITS) regions are non-coding, highly variable sequences surrounding the 5.8S ribosomal RNA gene. In the plastid genome, ycf1b is one of two regions belonging to gene ycf1 that are necessary for plant viability [12]. In addition, two non-coding plastid regions (atpF–atpH and psbK–psbI) are commonly combined with two core barcodes in species identification and phylogenetic construction. Multi-locus DNA barcode was applied to identify different taxa of a desert plant called Rhazya stricta, in which the plastid spacers psbK-psbI and atpF-atpH revealed highly diverse among tested taxa compared to that of coding regions matK, rbcL [13]. Regions of the plastid genome (rbcL, matK, and trnL-F) and nuclear genome (ITS) were assigned as DNA barcodes to authenticate 14 grass accessions [14].

Both DNA-based markers and DNA barcodes are virtually exempt from the stage of development, physiological state, and environmental factors, thereby being used as quick, reliable techniques in species identification, phylogenetic construction, and genetic relationships. Therefore, this study utilized ten ISSR markers to evaluate the genetic relatedness of three mango cultivars (Chau Nghe, Hoa Loc, and Cat Chu). Additionally, we created a barcode database using regions from the nuclear genome (ITS) and plastid genome (ycf1b, trnH-psbA, and atpF-atpH) and assessed the genetic relationship among different accessions of observed mango cultivars.

2. MATERIALS AND METHODS

2.2. Methods

2.2.1. DNA extraction

The extraction of DNA from dried mango leaves was performed according to the cetyltrimethylammonium bromide (CTAB) method described by Rogers and Bendich [15]. The DNA samples were stored at −20°C until use. DNA quality was evaluated using electrophoresis on 1% agarose gel in TBE 1X buffer, stained with SafeView. The result was observed under ultraviolet light by Bio-rad UV2000. DNA concentrations were determined by Nanodrop One (America).

2.2.2. ISSR amplification and analysis

The nucleotide sequences of ten ISSR primers used [16-17] are described in Table 1. The components of PCR consisted of 10 μl 2X Master Mix, 50 ng DNA, 20 pmol/μl primer, and ultrapure water for a final volume of 25 μl. The thermal cycling conditions of ISSR procedure were conducted as follows: 4 minutes for initial denaturation at 94°C; then 35 cycles of 1 minute at 94°C, 45 seconds at 50°C, and 2 minutes at 72°C and 7 minutes for final extension at 72°C. The PCR procedure was performed with MultiGene™ OptiMax Thermal Cyclers (USA).

Table 1. List of ISSR markers. [Click here to view]

Next, the quality of PCR products was evaluated by electrophoresis on 2% agarose gel in TBE 1X buffer and stained with SafeView. The result was detected under ultraviolet light by Bio-rad UV2000. The presence or absence of bands was recorded in binary code as 1 or 0, respectively. Matrices of similarity and pairwaise distance were analyzed, thereby creating a dendrogram according to cluster analysis by using NTSYSpc 2.1 program based on character differences. The software tool iMEC was used to determine the Polymorphic Information Content (PIC) [18].

2.2.3. DNA barcode amplification and analysis

Sequences of the primers for mango barcoding evaluation were sourced from boldsystems.org, as indicated in Table 2. The PCR composition for DNA barcoding amplification included 20 μl of Master Mix, 50 ng of DNA, 1 μl of forward primer, and 1 μl of reverse primer. Ultrapure water was added to achieve a final volume of 50 μl. The thermal cycling conditions for the PCR procedure are shown in Table 2.

Table 2. List of DNA barcoding markers and PCR cycling for DNA barcoding sequences. [Click here to view]

PCR products that exhibited clear bands on a 2% agarose gel were sent to a DNA sequencing company. Tax code: 1801742446. Address: U34C Six street, Hung Phu residential area, Hung Thanh ward, Cai Rang district, Can Tho city, Vietnam.

The sequences and compositions of DNA fragments for each mango genotype were analyzed using BioEdit version 7.0.5.3 software. The selection of consensus sequences (common ones) was based on the alignment of DNA segments within the same species. Afterward, the variable sites were determined by nucleotide comparison of these consensus sequences with one another on the MEGA-11 program.

4. RESULTS

4.1. ISSR Amplification and Sequencing Analysis

The data obtained (Table 3) indicated that a total of 76 bands were generated, of which 47 (61.84%) showed polymorphism.

Table 3. Description for amplified result of ISSR marker. [Click here to view]

The size of amplified bands was between 200 and 1500 base pairs (bps). The polymorphic percentage ranged from 17% (marker UBC809) to 100% (marker ISSR827) with an average of 61.9%. The PIC value ranged from 0.32 (ISSR826 and ISSR827) to 0.37 (UBC840, UBC855, and UBC888). The resolving power (RP) value varied from 4.3 (marker UBC809) to 16.3 (marker ISSR827). These data illustrated that ISSR markers used are suitable for amplifying regions with highly genetic variation between mango cultivars and accessions.

Markers ISSR811, ISSR818, and ISSR818 generated unique bands for discrimination between different accessions of the same cultivars as well as between different cultivars In contrast, remaining primer pairs used just could be used for cultivar characterization especially between Chau Nghe mango and two other mango cultivars when each marker gave at least one fragment that only appears in Chau Nghe accessions.

Among all the mango samples observed, two accessions, namely L1 and L3, did not amplify with all ISSR markers used.

Based on the Unweighted Pair Group Method with Arithmetic Mean cluster analysis (Fig. 1), 28 mango cultivars and accessions were separated into two major clusters composed of four groups with a similarity coefficient of 0.44. First, 9 out of 10 Chau Nghe mango accessions belonged to the same group. Among these, accessions N5 and N6 exhibited the highest similarity coefficient of 100%. At a cut-off value of 0.89, accession N2 was separated from the remaining Chau Nghe samples. It means that N2 is genetically most distantly related to other Chau Nghe accessions observed. Group 3 consisted of five accessions from Loc mango (L2, L5, L7, L8, and L9) and four accessions from Cat Chu mango (C1, C6, C7, and C9) with a similarity coefficient of 0.64. Regarding group 4, C5 had the highest coefficient with C8 (0.99), while C3 illustrated the farthest genetic distance compared with other samples of the same group with a similarity coefficient of 0.8.

Figure 1. Dendrogram of the ISSR markers for 28 mango genotypes. [Click here to view]

The result of gel electrophoresis showed that ITS, ycf1b, trnH-psbA, and atpF-atpH have fragment lengths of 700 bp, 800 bp, and 500 bp for the two latter, respectively, which was equal to the expected band sizes. Figure 2 shows that most band patterns of mango genotypes with trnH-psbA were clearly visible on a 2% agarose gel. There was a missing band at N2 genotype and smeared DNA bands at N5 and N7 samples. The missing band of mango samples could be due to the variation of mango genotypes, thereby unsuccessfully amplifying these genotypes with barcoding markers. Among 30 mango samples used, trnH-psbA and atpF-atpH (50%) had the highest success rate of PCR amplification, while the success rate of PCR amplification for ITS was the lowest (43.33%). For DNA sequencing, atpF-atpH demonstrated the highest success rate at 50%, followed by ITS at 26.67%, and trnH-psbA at 23.33%.

Figure 2. PCR products with trnH-psbA primer on agarose gel. [Click here to view]

The alignment results indicated that all observed mango genotypes displayed a high level of genetic conservation (Table 4). As a result, M. indica L. cultivars indicated their close genetic relatedness. However, the genetetic diversity of the studied mango fruit cultivars and accessions was evidenced by the presence of SNPs and indel mutations. The nuclear ITS marker exhibited numerically superior variability compared to other spacers used with 78 variable sites. However, trnH-psbA and atpF-atpH markers occupied the largest parsimony informative sites among the variable sites detected. Regarding indel mutations, trnH-psbA revealed their diversity among mango samples used, followed by ITS and ycf1b, with six and three mutations detected, respectively.

Table 4. Characteristics of aligned sequences of four DNA barcode candidates. [Click here to view]

Regarding the ITS spacer, the difference between N3, N7, and other samples was observed due to an indel mutation of the C nucleotide and a SNP (A/G), respectively (Fig. 3). Moreover, N1 was distinguished from other mango samples by an indel mutation of G nucleotide and a SNP as C/G. The discrimination between Chau Nghe accessions and a Loc mango accession (L7) was revealed by 76 SNPs and an indel mutation of C nucleotide at locus 646. Regarding ycf1b, accession N9 differed from other mango samples by a SNP (T/G), at loci 195 and 199. The difference of L5 was successfully identified by an indel mutation of T nucleotide and five substitution mutations, including A/G, T/G, G/A, and T/A (Fig. 4). The divergence of L10 from other mango samples was detected through 23 SNPs and two indel mutations of A nucleotide at loci 328 and 365. From the alignment result, it could be concluded that ycf1b is an effective locus for discrimination between Chau Nghe mango and some accessions of Hoa Loc mango. In contrast, the divergence between Chau Nghe and Cat Chu mango could not be distinguished using this barcoding gene.

Figure 3. Variable sites and indel mutations of nuclear fragment ITS. [Click here to view]

Figure 4. Variable sites and indel mutations of ycf1b gene. [Click here to view]

In terms of trnH-psbA, serial positions from 8 to 14 and 16–21 were the most informative variable sites for discrimination between mango cultivars (Chau Nghe and Cat Chu mango) and Chau Nghe accessions (Fig. 5). Six singleton sites (14, 21, 42, 47, 51, and 52) and an insertion of nucleotides at loci 27 and 31 were detected, contributing to the identification of accession N1 from other mango samples. The difference between accession C1 and other mango samples was identified through sequences of SNPs, namely TCTTAA and ATTACAAA. In addition, a serial deletion of five nucleotides AAAAA or AAATA was found on a Cat accession (C6), thereby distinguishing it from other mango samples observed.

Figure 5. Variable sites and indel mutations of trnH-psbA spacer. [Click here to view]

From the alignment results, atpF-atpH was considered the most informative and effective marker for discriminating between mango cultivars and accessions belonging to Mangifera indica L. in Vietnam (Fig. 6). Accession N10 was distinguished from other mango samples by an indel mutation of G nucleotide and SNPs substitution mutations, including A/G, C/A, A/T, and G/T. Five Chau Nghe samples (N5, N6, N8, N9, and N10) exhibited a close relationship with a Loc accession (L2) based on G and A nucleotides at four loci (259, 261, 266, and 268). N1 was clearly different from other mango samples through substitution mutations, including A/T, T/C, G/T, G/A, and G/C.

Figure 6. Variable sites and indel mutations of atpF- atpH spacer. [Click here to view]

Using standard nucleotide BLAST from NCBI, it was founded that four mango genotypes from India, M. indica (MF444902.1, OL960664.1, and AB598049.1), Mangifera odorata (MF444901.1) showed the identity percentage from 98.68% to 99.55% compared with Mangifera indica L. tested with nuclear gene fragment ITS in our study (Table 5). Similarly, Mangifera indica L. samples tested with other DNA barcoding genes revealed the identity percentage from 96.25% to 100% compared with the following accessions including M. indica (MN711724.1, KX871231.1, NC_035239.1) and M. sylvatica (MN786795.1) from three different countries. The high identity percentage demonstrated genetic relatedness between three cultivars of M. indica L., with those accessions available on NCBI.

Table 5. Nucleotide polymorphism between M. indica L. and other Mangifera genotypes on NCBI database. [Click here to view]

5. DISCUSSION

Overall, the percentage of polymorphic bands reported here was higher than those described in previous studies on mango fruit by Kheshin et al. [19], Hidayat et al. [20], and Ghounim et al. [21], but lower than the figures reported by Ganogpichayagrai et al. [6] and Ho and Tu [7].

All of the markers used in this study showed a moderate PIC value, meaning that ISSR markers are appropriate for assessing genetic relatedness in mango fruit, according to Botstein et al. [22]. In addition, the RP index reflects the discriminatory proficiency of a primer to differentiate a genotype or individual [23].

The result of ISSR analysis is consistent with the findings reported by Ho and Tu [7], in which Hoa Loc mango and Cat Chu mango belonged to two different groups of the same cluster. The phylogenetic result indicated that the studied mango accessions from Hoa Loc and Cat Chu cultivars were not classified as geographical distributions where samples were accumulated. This may be attributed to the exchange of accessions among local mango cultivation areas. In addition, hybridization between closely related genotypes among Mangifera indica accessions in Brazil was demonstrated by the high level of endogamy with an index value of 0.6 [24].

6. CONCLUSION

The biological significance of ISSR markers for studying the genetic diversity and genetic relationship among three M. indica L. cultivars (Chau Nghe, Hoa Loc, and Cat Chu) was determined by the obtained data. A phylogenetic tree constructed by ISSR markers indicated that Chau Nghe mango has distant genetic relatedness with other cultivars of Mangifera indica L. In addition, markers ITS and ycf1b successfully discriminated between different cultivars of mango fruit, while other plastid DNA barcode regions (trnH-psbA and atpF-atpH) clearly and effectively identified mango accessions. This study demonstrated that molecular markers and DNA barcode sequences are efficient tools for detection and discrimination at the species level of mango fruit. These findings are an indispensable breakthrough for the assessment of genetic characteristics and sub-species classification in the conservation and breeding of Vietnam’s mango germplasm.

7. ACKNOWLEDGMENTS

This research was fully funded by Tra Vinh University under grant contract number 74/2022/HD.HDKH&DT-DHTV. Other support was provided by the Tra Vinh Science and Technology Department, which facilitated the collection of plant material for this study.

8. AUTHOR CONTRIBUTIONS

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines

9. CONFLICTS OF INTEREST

The authors report no financial or any other conflicts of interest in this work.

10. ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.

11. DATA AVAILABILITY

All the data is available with the authors and shall be provided upon request.

12. PUBLISHER’S NOTE

All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

13. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY

The authors declares that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.

REFERENCES

1. Derese S, Guantai EM, Souaibou Y, Kuete V. Mangifera indica L. (Anacardiaceae). In: Kuete V (ed.). Medicinal spices and vegetables from Africa, Academic Press, Cameroon, pp 451 -83, 2017; CrossRef

2. Lam DH, Truong KH, Vo SM, Doan NH, Ly KN, Tran MT, et al. Allocative efficiency of Chu-Mango farms in Dong Thap, Vietnam. Kurd Stud 2023;11(2):3315–25; CrossRef

3. Vu HT, Le TN, Nguyen N, Tran BH, Tran HD. Evaluation of genetic variations of Mangifera sp. in southern Vietnam. Asian J Plant Sci 2022;21(2):169–82; CrossRef

4. Singh U, Deb R, Alyethodi RR, Alex R, Kumar S, Chakraborty S, et al. Molecular markers and their applications in cattle genetic research: a review. Biomark Genom Med 2014;6(2):49–58; CrossRef

5. Gemmill CEC, Grierson ERP. Inter-simple sequence repeats (ISSR), microsatellite-primed genomic profiling using universal primers. Methods Mol Biol 2021;2222:249–62; CrossRef

6. Ganogpichayagrai A, Rungsihirunrat K, Palanuvej C, Ruangrungsi N. Characterization of Mangifera indica cultivars in Thailand based on macroscopic, microscopic, and genetic characters. J Adv Pharm Technol Res 2016;7(4):127–33; CrossRef

7. Ho TV, Tu TN. Genetic characterization of mango accessions through RAPD and ISSR markers in Vietnam. SABRAO J Breed Genet 2019;51(3):252–65.

8. Jin L, Shi HY, Li T, Zhao N, Xu Y, Xiao TW, et al. A DNA barcode library for woody plants in tropical and subtropical China. Sci Data 2023;10(1):819; CrossRef

9. Wang J, Yan Z, Zhong P, Shen Z, Yang G, Ma L. Screening of universal DNA barcodes for identifying grass species of Gramineae. Front Plant Sci 2022;13:998863; CrossRef

10. Kang Y, Deng Z, Zang R, Long W. DNA barcoding analysis and phylogenetic relationships of tree species in tropical cloud forests. Sci Rep 2017;7:12564; CrossRef

11. Mir RA, Bhat KA, Rashid G, Ebinezer LB, Masi A, Rakwal R, et al. DNA barcoding: a way forward to obtain deep insights about the realistic diversity of living organisms. Nucleus 2021;64:157–65; CrossRef

12. Dong W, Xu C, Li C, Sun J, Zuo Y, Shi S, et al. ycf1, the most promising plastid DNA barcode of land plants. Sci Rep 2015;5:8348; CrossRef

13. Khan SA, Baeshen MN, Ramadan HA, Baeshen NA. Emergence of plastidial intergenic spacers as suitable DNA barcodes for arid medicinal plant Rhazya stricta. Am J Plant Sci 2017;8:1774–89; CrossRef

14. Rogers SO, Bendich AJ. Extraction of DNA from plant tissues. In: plant molecular biology manual. Springer, Dordrecht, The Netherlands, pp 73–83, 1988; CrossRef

15. He XH, Li YR, Guo YZ, Tang ZP. Genetic analysis of 23 mango cultivar collection in Guangxi province revealed by ISSR. Mol Plant Breed 2005;3:829–34; CrossRef

16. He XH, Guo YZ, Li YR, Ou SJ. Assessment of the genetic relationship and diversity of mango and its relatives by cpISSR marker. Agr Sci China 2007;6(2):137–42; CrossRef

17. Huang GD, Luo C, He XH, Ou SJ, Zhou JA. Analysis on the genetic relationship of Gui Re Mango (Mangifera indica L.) cultivars or lines. J Trop Subtropical Bot 2008;16:129–34; CrossRef

18. Amiryousefi A, Hyvönen J, Poczai P. iMEC: online marker efficiency calculator. Appl Plant Sci 2018;6(6):e01159; CrossRef

19. Kheshin MAE, Sayed HA, Allati AMA. Morphological and molecular analysis of genetic diversity among some ‘Sukkary’ mango (Mangifera indica L.) genotypes. J Hortic Sci Ornam Plants 2016;8(1):01–10; CrossRef

20. Hidayat A, Rahayu ES, Abdullah M, Retnoningsih A. Microsatellites to reveal genetic diversity and to distinguish four mangoes of Tegal District, Central Java, Indonesia. Biodiversitas 2021;22:3467–347; CrossRef

21. Ghounim IES, Mustafa MR, Hmmamn I. Molecular evaluation of selected mango genotypes grown in Egypt using SCoT and ISSR markers. Hortic Sci (Prague) 2022;49(4):213–24; CrossRef

22. Botstein D, White R.L, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 1980;32(3):314–31.

23. Sabreena, Nazirb M, Mahajana R, Hashimd MJ, Iqbale J, Alyemenif MN, et al. Deciphering allelic variability and population structure in buckwheat: an analogy between the ef?ciency of ISSR and SSR markers. Saudi J Biol Sci 2021;28(11):6050–6; CrossRef

24. Souza IGB, Valente SES, Britto FB, de Souza VAB, Lima PSC. RAPD analysis of the genetic diversity of mango (Mangifera indica) germplasm in Brazil. GMR 2011;10(4):3080–9; CrossRef

25. Ashour BM, Rabiei M, Shiran B. Intraspecific identification of some pomegranate (Punica granatum L.) genotypes based on DNA barcoding and morpho-biochemical characteristics. Trees 2023;37:1435–42; CrossRef

26. Do TK, Tran GH, Nguyen HT, Phan TTL, Sau TAT, Nguyen TC, et al. DNA barcode: a potential tool for identifying ‘Hoa Loc’ mango cultivar in Vietnam. Asian J Agric Biol 2021;2021(2):202005272; CrossRef

27. Ho TV, Tu TN, Nguyen AT. DNA fingerprinting and molecular characterization of mango (Mangifera spp.) cultivars in Vietnam using ITS DNA barcode. Bulg J Agric Sci 2021;27(1):128–34.

28. Fazeli-Nasab B, Sayyed RZ, Farsi M, Ansari S, El-Enshasy HA. Genetic assessment of the internal transcribed spacer region (ITS1.2) in Mangifera indica L. landraces. Physiol Mol Biol Plants 2021;26(1):107–17; CrossRef

29. Saha K, Dholakia BB, Sinha RK, Sinha S. DNA barcoding of selected Zingiberaceae species from North-East India. J Plant Biochem Biotechnol 2020;29:2–3; CrossRef

30. Kim M, Kim TJ. Genetic species identification using ycf1b, rbcL, and trnH-psbA in the genus Pinus as a complementary method for anatomical wood species identification. Forests 2023;14:1095; CrossRef

31. Balkanska R, Stefanova K, Stoikova-Grigorova R, Ignatova M. A preliminary assessment of trnH-psbA as DNA barcode for botanical identification of polyfloral honey samples and comparison with rbcL marker. Bulg J Agric Sci 2020;26(1):238–42.

32. Feng S, Jiao K, Zhu Y, Wang H, Jiang M, Wang H. Molecular identification of species of Physalis (Solanaceae) using a candidate DNA barcode: the chloroplast psbA–trnH intergenic region. Genome 2017;61(1):15–20; CrossRef

33. Kumar S, Kaushik RA, Jain D, Saini VP, Babu SR, Choudhary R, et al. Genetic diversity among local mango (Mangifera indica L.) germplasm using morphological, biochemical and chloroplast DNA barcodes analyses. Mol Biol Rep 2022;49:3491–501; CrossRef

34. Hassan AHM. DNA barcode trnH-psbA is a promising candidate gene for efficient identification of bitter and sweet almond and related species. Egyptian J Desert Res 2023;73(1):265–81.

35. Thakur VV, Tiwari S, Tripathi N, Tiwari G. Molecular identification of medicinal plants with amplicon length polymorphism using universal DNA barcodes of the atpF–atpH, trnL and trnH–psbA regions. Biotech 2019;9(5):188; CrossRef

36. Matra DD, Fathoni MA, Majiidu M, Wicaksono H, Sriyono A, Gunawan G, et al. The genetic variation and relationship among the natural hybrids of Mangifera casturi Kosterm. Sci Rep 2021;11:19766; CrossRef

37. Muthukumar M, Bajpai A, Rajan S. Chloroplast genes reveal hybridity in mango (Mangifera indica L.). J Appl Hortic 2018;20(1):55–9; CrossRef