Genetic diversity and relationship of mango and its wild relatives (Mangifera spp.) based on morphological and molecular markers

ABSTRACT Mango and its wild relatives (Mangifera spp.) are essential for future mango breeding, including preservation programs, because they provide many beneficial genes (agronomic traits), particularly those related to resistance to biotic and abiotic stressors. However, there is a limited understanding of the genetic diversity and relationships of this germplasm. This study aimed to determine the diversity and relationship between endemic mango and its wild relatives (Mangifera spp.) from Borneo Island, Indonesia, using leaf morphology and the internal transcribed spacer (ITS) region. Fifteen samples of Mangifera, covering 12 species, were used. Morphologically, the endemic Mangifera had a low diversity of only 0.22. Based on the ITS sequence, Mangifera endemic to Borneo had a high level of genetic diversity (0.069). In addition, this sequence had a total variable number of 215 bp, of which 110 bp were singleton sites, 89 informative parsimony and 41 indels. Phylogenetic analysis showed that Mangifera was grouped into three clusters for leaf morphological traits and four clades for the ITS region. In this case, the furthest relationship was pointed out by ‘Hampalam’ (M. laurina) and ‘Tambusui’ (M. macrocarpa), as well as by ‘Rawa-Rawa’ (M. similis) and ‘Samputar’ (M. torquenda). In contrast, the closest relationship was shown by ‘Hambawang Damar’ (M. foetida) and ‘Hambawang Puntara’ (M. foetida), including ‘Samputar’ (M. torquenda) and ‘Pauh’ (M. quadrifida). In particular, the common mango (M. indica) was closely related to ‘Asam Buluh’ and ‘Hampalam’ (M. laurina) and distantly related to ‘Pauh’ (M. quadrifida) and ‘Rawa-Rawa’ (M. similis).

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).
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.

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).
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).
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  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, Genetic diversity and relationship of mango and its wild relatives (Mangifera spp.) based on... 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).
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).

Figure 1 .
Figure 1.Map of sampling location, where 15 samples of Mangifera were collected, including South and Central Kalimantan (Borneo), Indonesia.See Table1for detailed information on sample identity.

Figure 3 .
Figure 3. Multiple sequence alignment showing some mutational events on the internal transcribed spacer (ITS) region in Mangifera endemic to Borneo, Indonesia.

Figure 4 .
Figure 4. Dendrogram showing the grouping of Mangifera endemic to Borneo, Indonesia, into three clusters.

Figure 5 .
Figure 5. Phylogram showing the grouping of Mangifera endemic to Borneo, Indonesia, into four clades.The number on the node is a bootstrap value of 1,000 times (above 50).

Figure 6 .
Figure 6.Principal component analysis (PCA) showing the grouping of Mangifera endemic to Borneo, Indonesia, into six groupsfor morphological traits (A) and seven for molecular traits (B).In this case, the total variability was 35.024 % for PC1 and 64.066 % for PC2.

Figure 7 .
Figure 7. Heatmap showing the similarity coefficient (A) and genetic divergence (B), representing the genetic relationship for Mangifera endemic to Borneo, Indonesia.

Table 1 .
Samples of the Mangifera used in this study, including local names and origins.

Table 3 .
Shannon diversity index of Mangifera leaf characteristics.