The rbcL region of the durian
The ribulose-1, 5-bisphosphate carboxylase/oxygenase, or rbcL, is a functional gene in the chloroplast genome engaged primarily in plant photosynthesis . This gene is found in the chloroplast genome’s large single-copy (LSC) region and exhibits high similarity across plant germplasm . According to Singh and Banerjee , this gene has a 600–800 nucleotide intergenic spacer. The rbcL gene contains around 1400 nucleotides that code for the large subunit protein, and the length varies significantly among flowering plants or Angiosperm .
In this study, the rbcL region of durians has different lengths, ranging from 527 to 578 bp (Table 2). These differences, both partial and complete, have been reported by several researchers. For example, Kumekawa et al.  have reported that durian (D. zibethinus) has a partial rbcL of 250 bp, and Amandita et al.  about 500 bp. In complete, this germplasm has the rbcL sequence of 1428 bp .
Further, a new DNA barcoding motif was discovered in the multiple sequence alignment of the rbcL of durians, in which a conserved region is introduced by polymorphism or other mutational events (Fig. 3). Based on Table 2, the rbcL durians of Kalimantan showed 23 variable sites or mutational events, and all are substitutions (transition-transversion), and no indels are present. According to Clegg , complete codon insertions/deletions are occasionally found in the gene, demonstrating a conservative pattern of nucleotide replacement. In general, grasses and other plant species such as Orchidales, Liliales, Bromeliales, and Arecales have a >5-fold differential in rbcL substitution rate .
According to Dong et al. , this gene represents distinctions in molecular evolution mode and tempo in angiosperms, monocotyledons, Gramineae, and Elymus. In another study, the inter/intrageneric levels of rbcL were highly efficient in Cornaceae, Cupressaceae, Ericaceae, and Graniaceae . The rbcL gene evolved more quickly in annual plants, particularly in the Asteridae and Poaceae families, and was dubbed “most morphologically advanced forms” in these families .
Genetic diversity and its benefits
In this study, exotic durian (Durio spp.) germplasm originally from Kalimantan, Indonesia, has a low genetic diversity, shown by nucleotide diversity (π%) of 0.24 (Table 2). The low level of genetic diversity may be attributed to a combination of founder effects and subsequent bottlenecks encountered in its short domesticated history . While the founder effect is a ubiquitous domestication bottleneck, millennia of cultivation and dissemination into new habitats have provided a considerable opportunity in selecting novel diversity in most crops .
Referred to Teixeira and Huber , low genetic diversity is often interpreted as an indicator of inbreeding depression and increased genetic drift. In other words, inbreeding, genetic drift, restricted gene flow, and small population size contribute to a genetic diversity reduction. Accordingly, populations lacking genetic diversity often exhibit an increased extinction rate . Ujvari et al.  also reported that a decline in genetic diversity is linked to an increased risk of inbreeding depression, resulting in decreased growth rate, fertility, fecundity, and offspring viability, as well as in increased vulnerability to pathogens. Furthermore, a loss of genetic diversity would harm individual fitness with increased susceptibility to disease and parasites  and limits a population’s ability to respond to threats in reduced long- and short-term survival of endangered species .
Compared to other studies with similar markers used, durian (Durio spp.) germplasm from this region has a high diversity. For example, tidal swamp rice (Oryza sativa) shows a genetic diversity of 0.086. According to Teixeira and Huber , high levels of genetic diversity are beneficial to promoting population survival and guaranteeing the adaptive potential of natural populations in the face of rapidly changing environmental pressures. These principles are reflected in strategies such as genetic rescue, where the genetic diversity of a threatened or endangered population is increased by facilitating gene flow from a population with high levels of diversity .
However, emerging genetic diversity strongly correlated with the polymorphic or mutation found in a target region. According to Frankham et al. , genetic diversity and mutational events are two things that are related. In this study, the rbcL region of the durian germplasm has generated 23 variable sites with a transition/transversion (Ti/Tv) bias value of 1.00 (Table 2). Multiple alignments revealed that transversion is more than transition (Fig. 3 and Table 3). Guo et al.  have reported that the first mutation is a more frequent encounter in this sequence and has higher regulatory effects than transitions. However, a pattern of the last mutation is favored several times over transversions is commonly occur in molecular evolution [49, 50].
Regardless of the presence of mutations in the rbcL sequence of durians, genetic diversity is essential for plant genetic resources conservation, breeding practices, and preventing genetic basis erosion of breeding populations . For these purposes, examining genetic diversity is essential in managing threatened species or taxa . According to Teixeira and Huber , conservation genetic practice rests on the assumption that measured levels of diversity provide a direct indicator of the degree to which genetic factors contribute to the risk of extinction. For crop improvement, genetic diversity is beneficial for parental selection  or selecting parents with genetically divergent . In this context, determining populations with a high level of genetic diversity will become a valuable resource for broadening the genetic base or gene pool of germplasm, as this enables the identification of superior alleles for several traits .
Following the AMOVA (Table 4), the durian germplasm has a higher variation (94.38%) at the intra-species level than the inter-species one (5.62%). It means that the future durian breeding program can be oriented to outcrossing, as was done by Hariyati et al.  and Prihatini et al. . According to Uji , several wild durian species, except D. zibethinus, have potential genes that can be incorporated into this program, such as being resistant to diseases and having a high tolerance for environmental challenges.
Genetic relationship and divergence
The phylogenetic study or genetic relationships is also beneficial for plant genetic conservation and breeding practices . For the first program, this study can be applied in inferring species and their evolutionary history, including species delimitation, genetic differentiation, and gene flow . In other words, this information is given the objective metrics for conservation purposes in the past evolution history, genetic status of species in the present time, and management program for future ones . For the second or last purposes, information of this relationship is usable in predicting the genetic diversity of the offspring when individuals or populations cross .
In this study, the durian (Durio spp.) germplasm from Kalimantan, Indonesia, shows unique relationships, mainly based on the number and composition of durian members in each clade or group formed. In general, following the maximum likelihood (ML) and neighbor-joining (NJ) methods, this germplasm is grouped into four main clades (Figs. 4 and 5, respectively). According to the PCA, this germplasm was separated into six groups (Fig. 6). Interestingly, both for ML and NJ, most of the durian samples were consistent in the same clade, except for Durian Si Japang (D. zibethinus), which belongs to Clade II in ML and Clade I in NJ (Table 5). Briefly, these phylogenetic trees (Figs. 4 and 5) and grouping illustrated the closeness and distant relationship between the samples.
The divergence analysis (Table 6) showed that by species group, D. zibethinus was very closely related to D. exleyanus. Meanwhile, the farthest shows by D. lowianus and D. excelsus. By ITS and ndhF markers, Nyffeler and Baum [53, 54] reported a close relationship between D. zibethinus and D. oxleyanus. Such relationships were also stated by Santoso et al.  using RFLP, Santoso et al.  by microsatellite, and Santoso et al.  with ITS.
However, within individuals (Table 7), 29 durian pairs are very closely related, and the farthest shown by Durian Burung (D. acutifolius) and Kalih Haliyang (D. kutejensis), and Pampaken Burung Kecil (D. kutejensis) with Durian Burung (D. acutifolius) as well, at a divergence coefficient of 0.011. Following the Pearson correlation analysis, only 20 pairs of individual durians have a strong relation, for example, Maharawin Hamak and Durian Burung as well as Mantuala Batu Hayam and Durian Burung Besar (Fig. 7). According to Acquaah , crossing individuals with distant relationships may generate high genetic diversity in the offspring. Conversely, crossing individuals with very close related may result in offspring with a low or narrow genetic diversity. In general, crossing individuals with a very close relationship is tends to avoid, as inbreeding occurs in the offspring . Thus, our results are essential in supporting the future durian genetic conservation and breeding practices.