Assessing diversity and phylogeny of Indonesian breadfruit (Artocarpus spp.) using internal transcribed spacer (ITS) region and leaf morphology

Abstract

Background

Breadfruit (Artocarpus spp.) is the main genus of Moraceae with multipurpose benefits, both ecologically and economically important, e.g., food ingredients, building materials, traditional medicine, and natural insecticides. However, most endemic Artocarpus have been threatened due to natural disasters and habitat degradation. The objective of our study was to determine the genetic diversity and relationships of endemic Artocarpus from South Borneo, Indonesia, using an internal transcribed spacer (ITS) region and leaf morphology.

Results

Morphologically, endemic Artocarpus endemic to South Borneo, Indonesia, has a different leaf shape, i.e., narrow-obovate to broad-elliptic, from simple to deeply dissected. Following the ITS region, this germplasm has a moderate level of nucleotide diversity (0.069). The phylogenetic analysis revealed Artocarpus into four (4) main clades, where the nearest is shown by the ‘Puyian’ (Artocarpus rigidus) and ‘Binturung’ (Artocarpus odoratissimus) at a coefficient divergence of 0.027, whereas the furthest by ‘Kulur’ (A. camansi) and ‘Tiwadak’ (A. integer) at a coefficient of 0.132.

Conclusion

In brief, although an endemic Artocarpus of South Borneo, Indonesia, has a moderate level of nucleotide diversity, this germplasm also shows a unique phylogenetic relationship. Thus, this information is urgent in supporting the future Artocarpus breeding and preservation programs, mainly to save this germplasm from being threatened.

Background

Breadfruit (Artocarpus spp.) is the main genus of Moraceae with multipurpose benefits, both ecologically and economically important, e.g., food ingredients, building materials, traditional medicine, and natural insecticides [1,2,3]. For example, Artocarpus altilis (‘sukun’— Java), Artocarpus heterophyllus (‘nangka’ — Java), Artocarpus integer (‘cempedak’ — Java), Artocarpus lakoocha (‘Mahat’ — Thai), Artocarpus lanceifolius (‘keledang’ — Banjar), and Artocarpus kemando (‘cempedak air’ — Malay) are six species of Artocarpus whose fruits are edible or can made for foodstuffs [4]. The bark of A. lanceifolius, A. integer, and A. heterophyllus can be used as building materials because they are permanent and durable [2]. The leaves of A. altilis are known to be efficacious in traditional medicine, especially for treating cirrhosis of the liver, hypertension, and diabetes [2].

Globally, Artocarpus has high genetic diversity, consisting of 70 species spread throughout the world, mainly in southern and southeastern Asia, including India, Sri Lanka, Pakistan, China, Malaysia, and Indonesia, as well as the Solomon Islands [1, 5]. For Indonesia, Artocarpus has recorded as many as 30 species and is present on five large islands, including Java, Sumatra, Borneo (Kalimantan), Sulawesi, and Maluku [6]. However, Borneo is the largest, with 23 species, and is estimated to be the center of the world’s Artocarpus diversity [1]. However, due to the deforestation or over-conversion of land, especially for agricultural, plantation, and settlement purposes, part of Artocarpus began to be scarce and difficult to find in the wild [7].

IUCN [8] reported seven Artocarpus with vulnerable status, i.e., Artocarpus rubrovenus, Artocarpus anisophyllus, Artocarpus tamaran, Artocarpus treculianus, Artocarpus hypargyreus, Artocarpus nobilis, and Artocarpus blancoi. Meanwhile, A annulatus and A. nanchuanensis are critically endangered [8]. Consequently, conservation or preservation, including their breeding and cultivation efforts to save various species of Artocarpus, is urgent. Conceptually, conservation activities aim to ensure the continuity of species, habitats, and biological communities, as well as interactions between species, including between species and their ecosystems [7]. Meanwhile, breeding and cultivation efforts aim to preserve essential genes with superior properties for future use [9].

On the other hand, the genetic characterization of germplasm using molecular markers is currently becoming alternative and complementary to the morphological characterization commonly used, as reported in Artocarpus by Jones et al. [10], Estalansa et al. [11], Karunarathne et al. [12], and Daley et al. [13]. In general, although quite expensive, molecular markers provide high speed and accuracy in the genetic characterization of germplasm [14]. In contrast, morphological ones tend to be time-consuming and are strongly influenced by environmental factors. Internal transcribed spacer (ITS) is the nuclear molecular marker that is useful in determining genetic diversity and the relationship of Artocarpus, including its taxonomy [5, 15].

Unlike chloroplast DNA with inherited maternally, the ITS is inherited biparentally [16]. Besides, due to the universality and simplicity of amplifying and showing a high mutation rate, the ITS provides better resolution in the estimation of the genetic diversity of most plants [16, 17]. Some plants that successfully verified using this marker are as follows: Acanthopanacis [18], Anoectochilus [17], Dioscorea [19], Litsea [20], Uncaria [21], and Zanthoxylum [22].

This study aimed to determine the genetic diversity and relationships of endemic Artocarpus to South Borneo, Indonesia, using ITS marker and leaf morphology. Our results provide essential information for future Artocarpus preservation and breeding tasks, especially in Indonesia.

Materials and methods

Plant materials

In this study, we used 14 (leaf) samples of breadfruit (Artocarpus spp.) collected from a part of Borneo Island, especially three regencies in South Borneo, Indonesia (Fig. 1). All were characterized morphologically and molecularly. Table 1 provides complete information about the Artocarpus samples used in the study.

Fig. 1

The map of South Borneo, Indonesia, shows the sampling location and the number of samples collected each. For more information about those samples, see Table 1

Table 1 Breadfruit species (Artocarpus spp.) used in the study, including their origin and genetic status

Morphological analysis

Morphological analysis was conducted by leaf characteristics only. It was performed descriptively using the guidance of Ragone and Wiseman [23].

Molecular analysis

All leaf samples of Artocarpus were extracted by a commercial DNA extraction kit (GP100) from Geneaid Biotech Ltd., Taiwan. The concentration and purity of DNAs were determined by UV-VIS spectrophotometry. The DNAs were then amplified by ITS primers [24] (forward: 5′-TCGTAACAAGGTTTCCGTAGGTG-3′; reverse: 5′-TCCTCCGCTTATTGATATGC-3′), using these reactions: initial denaturation (94 °C for 5 m); denaturation (94 °C for 30 s); annealing (48 °C for 30 s); extension (72 °C for 45 s); and final extension (72 °C for 7 m) [25]. The PCR was employed using 25 μl of a total volume, comprising of 20 ng DNA template (2 μl), 0.2 μmol forward and reverse primers (0.5 μl each), and 22 μl of MyTaq HS Red PCR Mix (Bioline, UK). The PCR product (DNA target) was visualized by electrophoresis of agarose gel (2%) and a UV transilluminator and then sequenced bidirectionally using the Sanger method (1st Base Ltd., Malaysia).

Data analysis

The data (ITS sequences of Artocarpus) were reconstructed and analyzed manually by the MEGA 11 [26]. All were then aligned using MultAlin [27] to find out some mutational events, both substitution (transition-transversion) and indels (insertion-deletion). The genetic diversity (π) was determined using the Nei and Li [28] criteria, i.e., low (≤ 0.04), moderate (0.05–0.07), and height (≥ 0.08), whereas haplotype (h) and haplotype diversity (Hd) by DnaSP ver. 6.0 [29]. The phylogenetic tree was reconstructed by the maximum likelihood (ML) with a Kimura-2 parameter and a nearest-neighbor-interchange (NNI) model [26, 30]. The phylogenetic tree was evaluated by bootstrap statistics (1000 replicates) and confirmed using principal component analysis or PCA [31]. The secondary structure ITS was also analyzed and reconstructed using the online software of ITS2 Workbench (http://its2.bioapps.biozentrum.uni-wuerzburg.de/) [32].

Results

Morphologically, endemic Artocarpus has a different leaf shape, namely narrow-obovate to broad-elliptic, from simple to deeply dissected (Fig. 2). The simple narrow-obovate leaf was shown by ‘Tiwadak’ or A. integer (A2), whereas the broad-elliptic by ‘Puyian’ or A. rigidus (A13). Besides, the narrow deeply dissected leaf was shown by ‘Mantiwadaka’ or A. kemando (A7), whereas the broad one was by ‘Kulur’ or Artocarpus camansi (A3) (see Fig. 2).

Fig. 2

Leaf morphology of endemic breadfruit (Artocarpus spp.) from South Borneo, Indonesia, shows two different forms, i.e., narrow-obovate to broad-elliptic, from simple to shallowly dissected. The name of each sample is provided in Table 1

The sequencing results showed that endemic Artocarpus had different partial ITS sequence lengths, between 640 and 727 bp (Table 2). In this case, the shortest ITS sequence is shown by ‘Binturung-2’ (A. odoratissimus), whereas the longest is by ‘Karusung’ (Artocarpus anysophyllus). After being aligned, several mutation events are seen in the ITS sequence, especially substitutions and indels or insertions-deletions (Fig. 3). Table 3 presents various genetic characteristics of ITS Artocarpus sequences, from mutation events and GC content to nucleotide or molecular diversity. Meanwhile, Fig. 4 shows the substitution pattern of the ITS Artocarpus sequence in more detail. When reconstructed, these sequences have unique secondary structures (Fig. 5). However, only 11 of the 14 samples had predictable ones. Meanwhile, ‘Anjani’ (Artocarpus hirsutus) and ‘Tarap’ (Artocarpus sericicarpus) have a similar ITS secondary structure (see Fig. 5).

Table 2 Endemic breadfruit species (Artocarpus spp.) used in the study, including the length of ITS sequences

Fig. 3

Multiple sequence alignments, showing two mutational events, i.e., substitutions and indels, from ITS sequence of endemic breadfruit (Artocarpus spp.) from South Borneo, Indonesia

Table 3 The genetic information of ITS sequence of endemic breadfruit (Artocarpus spp.) from South Borneo, Indonesia^a

Fig. 4

Nucleotide substitution patterns of endemic breadfruit (Artocarpus spp.) from South Borneo, Indonesia

Fig. 5

The secondary structures of the breadfruit (Artocarpus spp.) ITS sequence endemic to South Borneo, Indonesia, shows different forms. In this case, ‘Anjani’ (A. hirsutus) and ‘Tarap’ (A. sericicarpus) have similar

Following Fig. 3 and Table 3, the total mutation or polymorphic (segregating) site of Artocarpus ITS is 215 bp, of which substitution (174 bp) is more than indels (53 bp). In addition, singleton mutations (110 bp) are also higher than parsimony informative sites (89 bp) (see Table 3). Referring to the similar table, Artocarpus has a nucleotide diversity of 0.069, a haplotype diversity of 1.000, with a GC content of 58.64%. Furthermore, based on Fig. 4, the ITS Artocarpus sequence has the highest substitution pattern for the transition of 23.8 for the change of T to C, while the transversion is 5.3 for G to C and C to A, respectively.

Analysis of genetic relationship used the maximum likelihood method, separating endemic Artocarpus into four (4) main clades, of which three clades (I, II, and IV) consist of 4 samples or members while the remaining (clade III) are only two (Fig. 6). However, the principal component (PCA) analysis confirmed the grouping of Artocarpus into five groups, where the first was the largest with nine samples (Fig. 7).

Fig. 6

The phylogenetic tree shows the grouping of endemic breadfruit (Artocarpus spp.) from South Borneo, Indonesia, into four main clades. Values and letters (nucleotides) on nodes indicate the bootstrap analysis 1000 times and possible ancestors, respectively

Fig. 7

Grouping of endemic breadfruit (Artocarpus spp.) from South Borneo, Indonesia, into five main groups based on principal component analysis (PCA)

Based on Fig. 6, Artocarpus was split into two large groups polyphyletically, of which clades 1 and 2 became the first group with G ancestor, while clades 3 and 4 were the second groups (GA ancestor). In other words, the first group, consisting of clades 1 and 2, is monophyletic, as are clades 3 and 4, because they have the same ancestry, respectively. In this case, ‘Tiwadak’ or breadfruit (A. integer) has the closest relationship to ‘Nangka’ or jackfruit (A. heterophyllus), similarly ‘Tampang Susu’ (A. limpato) with ‘Tampang’ (A. primackii) (see Fig. 6).

Further analysis (Fig. 8) shows that the relationship of the nearest Artocarpus belongs to the ‘Puyian’ (A. rigidus) with the ‘Binturung_2’ (A. odoratissimus) at a coefficient divergence of 0.027. In contrast, the furthest relationship is between ‘Kulur’ (A. camansi) and ‘Tiwadak’ (A. integer) at a coefficient of 0.132 (Fig. 8). In addition, the relationship of ‘Tiwadak’ (A. integer) with ‘Jackfruit’ (A. heterophyllus) is recorded at coefficient 0.044, as for ‘Tampang Susu’ (A. limpato) and ‘Tampang’ (A. primackii) at 0.111 (see Fig. 8).

Fig. 8

The heatmap shows the relationship among endemic breadfruit (Artocarpus spp.) from South Borneo, Indonesia, based on the coefficient divergence value. In this case, the lower the coefficient value, the closer the genetic relationship would be

Discussion

Based on morphological observations, endemic breadfruit (Artocarpus spp.) from South Borneo, Indonesia, shows a different leaf shape, i.e., simple narrow-obovate to broad-elliptic deeply dissected (Fig. 2). According to Doyle [33], simple leaves exhibit primitive evolution rather than more complex dissected. It corresponds to the ‘telome’ theory, which leaves are proposed to have evolved through independent processes of branching, planation, webbing, and overtopping [34]. Referring to Harrison and Morris [35], initial constraints to leaf evolution probably involved several factors, e.g., high atmospheric global temperatures, low stomatal densities, and low water uptake capacities before root evolution.

Following the Nei and Li [28] criteria, this germplasm has a moderate level of genetic diversity (see Table 3). It is quite an encouraging thing. In plant breeding activities, information about genetic diversity is indispensable. According to Witherup et al. [36], maintaining crop diversity is urgent in retaining their capability for future adaptation. In other words, insufficient genetic diversity may cause crop failures. Also, maintaining genetic diversity is essential to support the current and upcoming crop breeding initiatives.

In concept, mutations are the main factor giving rise to genetic diversity. In other words, mutations are genetic changes that occur spontaneously in an allele or chromosome in which genetic diversity appears [37]. In this study, the ITS sequence of Artocarpus showed several mutations, especially substitutions and indels (see Fig. 3). According to Chen and Shiau [38], this region has a higher nucleotide substitution rate, making it suitable for genetic diversity and the related study between germplasm [19]. Besides, the gene occasionally exhibits entire codon insertions or deletions, showing a conservative pattern of nucleotide replacement [39]. However, although this mutation has significant benefits remains the question in some plant families of whether a gene can maintain the stable structure and function of the protein produced [40].

Apart from the presence of mutations in the ITS sequence of Artocarpus, using wild or underutilized populations (minor crops) with certain important traits may broaden the genetic diversity level and improve agricultural quality for both pests and diseases resistance; drought, salinity, and other abiotic stresses tolerance; and gain higher quality and yield ability. Witherup et al. [36] also state that most underutilized or minor crops provide a unique opportunity to quantify genetic diversity baselines before bottleneck selection is present and to implement practice learning from other crops that have previously suffered losses. Although most studies focus on the diversity loss of primary crops, many minor crops are now systematic improvement targets for the development of uniform lines and industrialization [9].

In particular, the development of Artocarpus is currently directed toward the dwarf cultivars assembling. For example, breadfruit (A. altilis), a traditional staple crop for starchy in the tropics, is modified to be dwarf due to vulnerability to wind damage [40]. In this case, dwarfing rootstocks of breadfruit may come from wild relatives, for example, A. anisophyllus, A. hirsutus, A. camansi, A. nitidus, Artocarpus mariannensis, A. integer, A. heterophyllus, A. lakoocha, Artocarpus petelotii, and Artocarpus xanthocarpus. The last two species may be good candidates for dwarfism because they have a height of about 10 and 8 m, respectively [41].

Thus, information about genetic relationships is also essential to consider. In this study, the nearest Artocarpus is shown by the ‘Puyian’ (A. rigidus) and ‘Binturung’ (A. odoratissimus) at a coefficient divergence of 0.027, whereas the furthest by ‘Kulur’ (A. camansi) and ‘Tiwadak’ (A. integer) at a coefficient of 0.132 (Fig. 8). According to Mursyidin and Makruf [7], Artocarpus exhibits a complex phylogenetic relationship. For example, using nucleus genes, Zerega and Gardner [3] reported the close relationship between Artocarpus nitidus, A. heterophyllus, and A. camansi. Following microsatellite markers, Zerega et al. [36] revealed the closeness of A. camansi to A. mariannensis. Meanwhile, based on the complete chloroplast gene, Li and Song [42] reported the genetic proximity between A. heterophyllus and Artocarpus nanchuanensis.

According to Acquaah [43], when two individuals with far relationships cross, the genetic diversity of the offspring may be extant. Conversely, if the two closely related crosses, it will produce offspring with narrow genetic diversity. However, for the latter case, crosses between such elders tend to be avoided as they can result in inbreeding descendants. Referring to de Los Reyes [44], inbreeding will increase susceptibility to disease and stress, including decreasing crop yields.

Conclusion

The results showed that breadfruit (Artocarpus spp.) from South Borneo, Indonesia, has two types of leaf morphology with moderate genetic diversity at the nucleotide level, amounting to 0.069. Meanwhile, the genetic relationship analysis revealed that the nearest Artocarpus is shown by the ‘Puyian’ (A. rigidus) and ‘Binturung’ (A. odoratissimus) at a coefficient divergence of 0.027, whereas the furthest by ‘Kulur’ (A. camansi) and ‘Tiwadak’ (A. integer) at a coefficient of 0.132. This information is urgent in supporting the future Artocarpus breeding and preservation programs.