INTRODUCTION

Mango (Mangifera indica L.), belonging to the Anacardiaceae family, is one of the most important fruit crops in the world (Azim et al. 2014, Jena & Chand 2021). According to FAO (2022), mango continued to be the most significantly traded commodity, in terms of exported quantities, in 2021, with an increase of approximately 3 %, or 75,000 tons from the previous year. Currently, mango is commercially grown in over 100 tropical and subtropical countries, e.g., India, Indonesia, China, Pakistan, Mexico, Brazil and Nigeria (Jena & Chand 2021, WPR 2023).

Indonesia is the second-leading mango producer in the world today, with a production of about 3.6 million tons (WPR 2023). This success is inseparable from the ideal climate and ample farmland to cultivate and harvest the crops (WPR 2023), including the variety of cultivars and the presence of wild relatives of mangoes with their unique characteristics (Anggraheni & Mulyaningsih 2021). Examples include M. caesia (‘Binjai’), M. foetida (‘Hambawang’), M. odorata (‘Kueni’) and M. casturi (‘Kasturi’) (Ariffin et al. 2015). Some species have been cultivated by local farmers, although not intensively, whereas others are wild in the forest (Ariffin et al. 2015).

The Mangifera genus has approximately 69 species globally, with 30 endemic to Indonesia (Hidayat et al. 2011, Anggraheni & Mulyaningsih 2021). According to Fitmawati et al. (2017), the high diversity of Mangifera is essential for future mango breeding programs. This is because wild mango relatives provide many beneficial genes (agronomic traits) for breeding, such as having good resistance to biotic and abiotic stressors (Fitmawati et al. 2017), as reported by Ledesma et al. (2017). However, their economic importance, genetic diversity and phylogenetic relationships are poorly understood (Salma et al. 2010) due to the complexity of vegetative and reproductive organs (Hidayat et al. 2011, Ariffin et al. 2015).

The key to success in mango breeding is determining the genetic diversity within and among species (Anggraheni & Mulyaningsih 2021). For years, morphological traits have been applied in many phylogenetic studies, including mango (Majumder et al. 2013, Mohamed et al. 2015, Toili et al. 2016, Fitmawati et al. 2020, Zhang et al. 2020). However, these traits have certain limitations due to being time consuming and strongly influenced by environmental conditions. Presently, molecular marker utilization is more comprehensive to support and strengthen the morphological data or facilitate the phylogenetic resolution of the germplasm (Fitmawati et al. 2017).

In general, molecular marker applications are fast, neutral and not influenced by environmental factors, unlike morphological traits (Ariffin et al. 2015). During the last few years, several molecular markers have been applied to address the genetic diversity and relationship between mango and wild relatives, e.g., random amplified polymorphic DNA or RAPD (Fitmawati et al. 2010, Anggraheni & Mulyaningsih 2021), microsatellite or inter simple sequence repeat (ISSR) (Ravishankar et al. 2011, Shamili et al. 2012, Nazish et al. 2017) and chloroplast DNA (cpDNA) markers (Fitmawati & Hartana 2010, Azim et al. 2014, Fitmawati et al. 2017, Rafidah et al. 2019).

This study aimed to determine the genetic diversity and relationship of endemic mango and its wild relatives (Mangifera spp.) from Borneo Island, Indonesia, using leaf morphology and internal transcribed spacer (ITS) region. According to Senavirathna et al. (2020), the ITS is the nuclear molecular marker that is useful in determining the genetic diversity and phylogeny of germplasm. This is due to the high mutation rate in this region (Lee et al. 2017). In addition, ITS provides universality and simplicity in its application and has been successfully applied in some plants, e.g., Acanthopanacis (Zhao et al. 2015), Anoectochilus (Chen & Shiau 2015, Thinh et al. 2020), Dioscorea (Purnomo et al. 2017), Uncaria (Zhu et al. 2018) and Zanthoxylum (Zhao et al. 2018, Suriani et al. 2021).

MATERIAL AND METHODS

The study was conducted at the University of Lambung Mangkurat, Indonesia, from November 2021 to May 2022. Covering 12 Mangifera species, 15 plant samples were used (Table 1). The samples were collected from eight locations, being six in South Borneo and two in Central Borneo, Indonesia (Figure 1). All leaf samples were taken to the laboratory to be morphologically and molecularly prepared. Morphological characterization was performed using leaf characteristics only (IPGRI 2006, Hasim et al. 2016).

Molecular characterization begins with DNA extraction using leaf samples and the Geneaid commercial kit (GP100, UK). In this stage, 50 g of leaf samples were crushed and prepared according to the manufacturer’s instructions until pure DNA was obtained. Before amplification, DNA (genome) samples were quantified using spectrophotometric methods. DNA amplification was performed using a PCR machine (MultiGene Optimax, Labnet International Inc., Nort Carolina, USA) with a total volume of 25 µL and the following reaction conditions: initial denaturation (94 ºC for 5 min); denaturation (94 ºC for 30 s); annealing (48 ºC for 30 s); extension (72 ºC for 45 s); and final extension (72 ºC for 7 min) (Mursyidin et al. 2021). The PCR reaction consisted of 22.0 μL PCR mix [containing DNA polymerase, dNTP and MgCl₂ (Bioline, USA), DNA template (2 μL) and primary DNA (1 μL; 10 μM)]. The primers used in the study were for ITS: forward (5’-TCGTAACAAGGTTTCCGTAGGTG-3) and reverse (5’-TCCTCCGCTTATTGATATGC-3)’. The amplification results were visualized using agarose gel electrophoresis (2 %) and a UV transilluminator, and then documented with a digital camera. Sequencing was carried out at 1st Base Ltd. (Malaysia), using the Sanger method, bi-directionally, with an ABI PRISM 377 DNA sequencer (Applied Biosystems, Massachusetts, USA).

The leaf morphological data were analyzed using a multivariate approach in MVSP version 3.1 (Kovach 2007). The Shannon diversity index (H’) was applied to determine the genetic diversity of this germplasm, with high (H′ > 0.60), moderate (0.40 ≤ H′ ≤ 0.60) and low (H′ < 0.40) criteria (Mursyidin & Khairullah 2020). For the molecular analysis, the ITS regions were aligned and analyzed using the MEGA 11 software (Tamura et al. 2021), to determine the genetic diversity, GC content, variable sites (including informative parsimony and singleton sites) and phylogenetic relationships. In this case, genetic diversity was carried out using the nucleotide diversity index (π) method (Nei & Li 1979). Meanwhile, the genetic relationship was reconstructed using the unweighted pair group method with arithmetic average (UPGMA) and maximum likelihood (ML) methods (Lemey et al. 2009). The dendrogram and phylogram were then evaluated with bootstrap statistics (1,000 replicates) and confirmed using principal component analysis or PCA (Mursyidin et al. 2022). Tree diagrams were evaluated visually (Baum 2008).

RESULTS AND DISCUSSION

Morphologically, endemic Mangifera has a unique leaf shape. In general, this germplasm showed six different leaf shapes: elliptical, lanceolate, linear, oblong-lanceolate, oblong and ovate (Figure 2). In this case, unique leaves were shown by ‘Tambusui’ (M. macrocarpa) with the linear form and ‘Binjai’ (M. caesia) with the oblong-lanceolate form (see samples M2 and M10, respectively, in Figure 2). Based on leaf texture, most Mangifera leaves were coriaceous, except for membranous for ‘Tambusui’ (M. macrocarpa). In addition, five Mangifera samples, including ‘Tambusui’ (M. macrocarpa), ‘Kasturi’ and ‘Kasturi Mawar’ (M. casturi), ‘Samputar’ (M. torquenada) and ‘Pauh’ (M. quadrifida), had leaf pubescence or a glabrous character (Table 2). Other leaf characteristics of this germplasm are provided in Table 2.

According to Kole (2021), most Mangifera leaves are oblong, as in M. indica, M. laurina and M. foetida. According to Zumajo-Cardona et al. (2019), the leaf morphology may correspond to evolution through independent processes, such as branching, planation, webbing and overtopping. Based on these traits, this germplasm has low diversity (Table 3).

Based on the ITS sequence, Mangifera endemic to Borneo had a genetic diversity of 0.069 (Table 4). According to Jagadeesh et al. (2018), the value of such diversity is relatively high. The genetic diversity of Mangifera was also higher, if compared to other studies with similar markers, such as Soumnya & Nair (2017), who showed a genetic diversity of 0.035 in Averrhoa, and Haque et al. (2009), who found a genetic diversity of 0.039 in Commiphora wightii. A similar value of 0.068 was reported by Jagadeesh et al. (2018) for Magnaporthe oryzae. According to Mursyidin et al. (2021), a high genetic diversity in germplasm is closely related to mutations that occur in the sequences studied.

In this case, the ITS sequence of Mangifera had a total variable number of 215 bp, of which 110 bp were singleton sites, 89 were informative parsimony and 41 were indels (Table 4). Figure 3 shows more clearly the mutations that occurred. Based on this figure, substitutions were higher than indels. Furthermore, this sequence had a GC content of 58.64 %, with bias values and ratios of transition/transversion of 1.70 and 1.84, respectively.

According to Lee et al. (2017), the ITS region shows a high mutation rate. Compared to other studies, this number was higher than the results of Soumnya & Nair (2017) in the ITS Averrhoa region, with as many as 54 variable characteristics and 33 parsimony informative sites. Even in Trichogramma, the ITS region has only 14 variable sites (Viana et al. 2021). According to Drábková et al. (2009), the high mutation event on ITS is linked to hybridization. In this context, differences in ITS sequences may be met after hybridization and become homogenized after a time, but the latter may not be consistent among descendant lineages (Soltis & Soltis 2009).

Related to indels, this gene mutation is essential, since it determines which part of the protein is affected, and not all amino acids are necessary for a proper protein function (Rodriguez-Murillo & Salem 2013). According to Ludwig (2016), several mechanisms generate indels, e.g., complete and partial chromosomal duplication, proliferation of transposable elements, replication errors and unequal crossover. These diverse mutational mechanisms of indel production contribute to single locus and total DNA size variation in the genome. In this case, however, deletions were more frequent than insertions. Furthermore, the genomic prevalence of indels declines with length, and this decline is faster for insertions than deletions (Ludwig 2016).

Apart from mutation, genetic diversity is necessary for breeding and conservation programs. For plant breeding, genetic diversity is essential to promote the adaptability of populations to environmental changes and to preserve large gene pools for future genetic breeding (Govindaraj et al. 2015). In other words, knowledge on the genetic and population diversity of germplasm collections serves as a solid foundation for crop breeding (Dwivedi et al. 2017). In this case, defining the population diversity within the germplasm is beneficial to avoid false associations while performing association mapping studies (Jena & Chand 2021). Furthermore, knowledge on the genetic background of parents is a necessary start to developing new varieties endowed with high-yielding features of fruit and more adapted to constantly changing climatic conditions (Govindaraj et al. 2015).

For conservation, genetic diversity is critical to long-term survival, sustainable productivity and genetic enhancement of commercially profitable genotypes (Rachmat et al. 2016). In this context, the assessment of the level of genetic diversity of a species or population helps to ascertain its current status and threat (Graudal et al. 2014). Thus, the information can provide a basis for adopting appropriate scientific management policies and devising effective conservation strategies (Jena & Chand 2021). In other words, information on this parameter is essential for the effective and efficient management of conservation and the prospective utilization of biodiversity in any crop species (Gavin et al. 2018). In particular, genetic diversity is an essential precursor in studying a species (Wu et al. 2020). This is because the range and magnitude of heterogeneity in the species or population greatly influence its evolutionary potential (Jena & Chand 2021).

In addition to genetic diversity, determining the phylogenetic relationship among cultivated varieties, including their wild relatives, is also necessary (Skuza et al. 2019). In this study, Mangifera was grouped into three clusters for morphological traits (Figure 4) and four clades for ITS (Figure 5). Different groupings were shown by the PCA analysis, where six groups were for morphological (Figure 6A) and seven for molecular (Figure 6B) traits. Following the similarity coefficient (Figure 7A), the furthest relationship is shown by ‘Hampalam’ (M. laurina) and ‘Tambusui’ (M. macrocarpa) at 0.095. The nearest is by popular mango (M. indica) and ‘Asam Buluh’ (M. laurina).

Based on its coefficient of divergence (Figure 7B), the furthest genetic relationship was shown by ‘Rawa-Rawa’ (M. similis) with ‘Samputar’ (M. torquenda), while the closest relationship was shown by ‘Samputar’ (M. torquenda) with ‘Pauh’ (M. quadrifida). In particular, the popular mango (M. indica) was closely related to M. laurina, i.e., ‘Hampalam’ and ‘Asam Buluh’, at divergence coefficients of 0.010 and 0.022, respectively. However, this mango was distantly related to M. similis (‘Rawa-Rawa’), with a divergence coefficient of 0.084 (Figure 7B).

Following the literature, M. similis has elliptical fruits about 75 mm long and 65 mm wide, sometimes even up to 100 mm in diameter (Atmoko et al. 2016). When ripe, the taste of this fruit tends to be sweet, with a slight sensation of refreshing sourness (Lim 2012a). Meanwhile, M. laurina has a drupe-like small mango, obliquely oblong, and a small fruit (Lim 2012b). While the fruit is less delicious because it is very sour, this species has a potential as a genetic resource, because it is resistant to the anthracnose disease caused by Colletotrichum gloeosporioides (Verheij & Coronel 1991). In this case, interspecific hybridization between a popular mango (M. indica) and its wild relatives, e.g., M. rubrapetala (‘Raba’), M. casturi (‘Kasturi’) and M. lalijiwa (honey mango), has been reported by Ledesma et al. (2017).

In short, wild relatives provide many essential genes that are beneficial in breeding programs, such as resistance to pests and diseases (Migicovsky & Myles 2017). Thus, well-to-type multiplication for the preservation of endemic mango populations is essential to maintain the diversity present in local landraces, to prevent the extinction of elite genotypes available in these areas, and to reduce the risk of loss of desired characteristics (such as fruit quality) due to uncontrolled depression of natural inbreeding (Jena & Chand 2021).

CONCLUSIONS

Based on leaf morphology, endemic mango (Mangifera spp.) from Borneo, Indonesia, had a low diversity. In contrast, following the internal transcribed spacer (ITS) region, this germplasm had a high level of genetic diversity. The phylogenetic analysis showed that Mangifera was grouped into three clusters for morphological traits and four clades for molecular traits (ITS). In this case, the furthest relationships were between ‘Hampalam’ (M. laurina) and ‘Tambusui’ (M. macrocarpa) and between ‘Rawa-Rawa’ (M. similis) and ‘Samputar’ (M. torquenda). In contrast, the closest relationships were shown for ‘Hambawang Damar’ and ‘Hambawang Puntara’ (M. foetida), and for ‘Samputar’ (M. torquenda) and ‘Pauh’ (M. quadrifida).

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