Molecular Diversity of The Tidal Swamp Rice ( Oryza sativa L . ) Germplasm of The South Kalimantan , Indonesia

Tidal swamp rice has long been cultivated by the local people of the South Kalimantan, Indonesia. This germplasm possess some important traits for adapted to a wide range of abiotic and biotic stresses. In this study, a total of sixteen cultivars of this germplasm, consisting of fifteen of the South Kalimantan Province and one of Sumatera Island, Indonesia (an outgroup) were analyzed, phylogenetically based on the chloroplast trnL-F and nuclear intergenic spacer region (IGS). The results showed that this germplasm has a relatively more extraordinary genetic diversity than other local rice germplasm. In a nucleotide level, this germplasm showed a genetic diversity of 0.61 for nuclear IGS and 0.58 for trnL-F. The phylogenetic reconstruction also exhibited that this germplasm has the unique illustration of phylogenetic trees, particularly for the combined sequence datasets. Thus, the results of our study would provide useful information for further understanding of evolutionary relationships of this germplasm and facilitate the efficient utilization of valuable genes for genetic improvement, particularly in the tidal swamp areas.


Introduction
The tidal swamps are a potential area to be developed into future agricultural farming in many Asian countries because still widely available with a total of 20.1 million hectares [1].While the region considers the limitations as fragile or marginal areas, primarily due to natural problems, it reserves an excessive amount of indigenous rice germplasm [1].Hundreds of the tidal swamp rice landraces have been found in the region, and show some beneficial agronomic traits.By the result of domestication process and through the natural selection for a long-time, this germplasm shows a better adaptation to the local conditions and cultures [2].This germplasm also exhibited both a tremendous genetic diversity and the substantial genes in promoting the development of new rice cultivars in the future [3][4][5].
In South Kalimantan for example, one of the biggest rice producers in Indonesia, there is about 70 tidal swamp rice cultivars which still found and preserved by the local farmers [6].Nevertheless, most of this germplasm has not adequately understood genetically and not incorporated optimally into the rice breeding programs [7,8].In general, characterization of rice germplasm has been undertaken based on a morphological marker [9,10].However, this marker has some disadvantages, such as time-consuming, unreliable in discriminating species with closely related taxa [10], and strongly influenced by the environmental factors [11].
The molecular marker application may contribute in-depth information on the potential and genetic diversity of the rice germplasm [12].This marker has more effective, efficient and accurate, and stable to environmental changes than morphological marker [11].DNA sequencing is a part of molecular applications that constitute a substantial contribution to the study of phylogenetic, whether in plants with distant or close relationships [13,14].In this case, the chloroplast trnL-F genes and nuclear intergenic spacer (IGS) region sequences could be used for that purpose [15,16].It is because both have an elevated mutation rate, as well as the genetic diversity [17][18][19][20].Some of the plant germplasm have revealed by these markers, like Brassica [21], Oryza [14,22], tomato [23], and Annonaceae [24].
This study directed to investigate and determine the genetic diversity and relationship of the tidal swamp rice germplasm of the South Kalimantan Province, Indonesia using the chloroplast trnL-F and nuclear IGS sequences.In a local scale, such studies could reveal the complex interaction between germplasm and farmers cultivation practices in good view [25].Hence, the results of this study expected to be used as a reference in the rice genetic improvement and conservation program in the future.

Plant Materials
A total of sixteen cultivars of the tidal swamp rice germplasm, consisting of fifteen of the South Kalimantan Province and one of Sumatera Island, Indonesia (an outgroup) sampled for this study (Table 1, Figure 1).An outgroup cultivar obtained from The Indonesian Agriculture of The Tidal Swamp Research Institute, South Kalimantan, Indonesia.The seeds germinated after being soaked in a fungicide solution and rinsed thoroughly in warm water (30-35•C).Three-week-old seedlings transplanted into pots with soil collected from a paddy field.The pots were 30 cm in diameter and 20 cm in height and placed in a greenhouse.For further analyses, one seedling from each cultivar used.

DNA isolation, amplification, and sequencing
Isolation of genomic DNA using three-weeks-old of each rice leaf sample, following the Mini Kit of DNA Extraction protocol (Geneid, UK).The genomic DNA isolated then measured using UV-VIS spectrophotometer (NanoVue, GE Healthcare, UK), at wavelengths of 260 nm and 280 nm [26].The isolated DNA samples were then amplified using the chloroplast trnL-F and nuclear IGS primers (Table 2)

Data Analysis
The DNA sequences from the two datasets were separately aligned using the Clustal X version 2.0 [27] and refined manually.Indels (insertion or deletion) introduced into the alignment coded in the following ways.Shared indels treated as single characters.Indels of uniform length coded as absence (1) or presence (0) characters independent of the indel length.The gapped regions in the alignment excluded from subsequent analysis unless some position included nucleotide diversity.Gaps treated as missing entries [28].Maximum Likelihood (ML) analysis performed by heuristic search with Nearest-Neighbor-Interchange (NNI) options, strong branch swap filter, and initial tree automatically (Maximum Parsimony).The sequence data were also analyzed with a neighbor-joining method using the Kimura 2-parameter distance estimates [29].Topological robustness assessed by bootstrap analysis with 1000 replicates using simple taxon addition [30].The characters weighted equally in all phylogenetic analyses.The phylogenetic analyses conducted by a software of MEGA7 [31].

Sequence characteristics and the genetic diversity
Approximately 0.89 kilobase pairs (kb) of two sets of DNA fragments from the chloroplast trnL-F and nuclear IGS genomes sequenced separately from sixteen of the tidal swamp rice germplasm.The phylogenetic information generated by both sequences given in Table 3.In general, the aligned sequences of nuclear IGS indicate a more extraordinary value of the number of segregating sites, Bayesian Information Criterion (BIC), and Akaike Information Criterion (AICc) than chloroplast trnL-F.Similarly, GC content and nucleotide diversity were higher in IGS sequences than the chloroplast trnL-F.However, both sequences have a similar value of Ti/Tv ratio (1.13) (see Table 3).Moreover, the Tajima's test of neutrality shows that these sequences have a progressive value of D greater than zero (D>0).

Phylogenetic relationship based on the chloroplast trnL-F spacer
The phylogenetic tree was obtained for the chloroplast trnL-F sequences from the tidal swamp rice germplasm (Figure 2).In general, four separate groups with relatively high values of bootstrap support (bs) formed on this tree.Based on this tree, the Lakatan Pacar was closely related to Sardani, an outgroup, with a bootstrap value of 100%.Similarly, the Pandak Laut was closely related to Ciherang, an improved cultivar, at bs=68%.However, the Sardani has far related to Pandak Putih (bs=99%).

Phylogenetic relationship based on the nuclear IGS region
The phylogenetic relationship obtained for the IGS sequences from the tidal swamp rice germplasm.The IGS tree shown in Figure 3.In general, two independent groups of the tidal swamp rice germplasm resolved by the IGS sequences.Differ from the trnL-F, the IGS was able to combine the Sardani with three tidal swamp rice germplasms of the South Kalimantan, such as Lakatan Pacar, Banih Putih, and Ganal Perak, at a bootstrap value of 96%.Whereas, the Ciherang clustered with Pandak Putih, not with Pandak Laut, in a similar group (bs=100%) as well.In this tree, the far related showed by Lakatan Pacar and Sardani.

Phylogenetic relationship of combined chloroplast and nuclear datasets
One most phylogenetic tree reconstructed based on the combined of two individual sequencing datasets (Figure 4).This combined dataset produced many changes in a phylogenetic tree of the tidal swamp rice germplasm, including their bootstrap values.In general, two independent groups formed on this tree, and relatively similar to the topology of the IGS phylogenetic tree.In this case, Lakatan Pacar has closely related to Sardani at 100% bootstrap support, whereas Ciherang to Lakatan Wangi and Pandak Laut (bs=100%).Based on this tree, Sardani and Ganal Perak were two cultivars with considering the farthest relationship.

Discussion
Genetic diversity manifested by differences in many characters, such as morphology, anatomy, and in the proteins, enzymes and DNA sequences of almost all organisms [32].It required for populations to adapt the environmental changes [32].In our results, the aligned sequences of nuclear IGS show a relatively higher genetic diversity than chloroplast trnL-F, indicated both by nucleotide diversity and the number of segregating sites (Table 1).Moreover, this region strongly influenced by some mutations, like deletion and substitution, as reported by Cordesse et al. [33].In the Oryza genus, the IGS shows a high genetic diversity as well [33].According to Chung et al. [34], nuclear IGS have been changing quickly, both in the number of copies and its chromosome segments.Furthermore, Chung et al. [34] reported that this region on some chromosomes of 10 genera of Oryza has an excessive number of repeated elements, located at the end of the short arm of chromosome 9 [34].
Based on the result, while the chloroplast trnL-F has relatively lower genetic diversity than IGS, it could be useful for phylogenetic studies [20].Drábková et al. [35] reported that trnL-F has a higher substitution rate than an insertion-deletion (indel), making it a valuable tool for studying short-term evolutionary processes.Furthermore, Bayer and Starr [36] confirmed that this region is a primary tool for phylogenetic studies because of mutation rates of insertion-deletion.Thus, both sequences (trnL-F and IGS) have a good chance to apply in the future rice breeding program or developing new rice cultivars.
Overall, the tidal swamp rice of the South Kalimantan, Indonesia demonstrated a relatively more extraordinary genetic diversity than other local rice germplasm.For instance, in this study, our rice germplasm showed a genetic diversity by 0.61 for nuclear IGS and 0.58 for trnL-F, whereas the Bangladesh local rice, using SSR markers, only had 0.46-0.53,as reported by Wang et al. [37].Similarly, using SSR markers, the South Korean local rice showed a lower genetic diversity (0.035) than our studied rice [22].Thus, this indicates that the tidal swamp rice germplasm experienced a long-term period of domestication process [38].In fact, this process may generate the changes of an individual genome [39].Furthermore, the Tajima test of neutrality indicated that selection balance (there are no rare alleles) has occurred in this rice population [40].Thus, the more accurate application of other molecular markers, such as single nucleotide polymorphism (SNP), to study the local rice population is profoundly important to do.
Further, studies on phylogenetic relationships are indispensable to support the rice breeding programs in the future [41], particularly in choosing the parents for developing new rice cultivars.These studies are extensively conducted by many researchers, such as Kim et al. [22], Choudhury et al. [42], Liu et al. [11], Zhang et al. [43], etc., using different molecular markers, both in a local and a global scale.Nevertheless, such studies are still limited to the tidal swamp rice germplasm.
Our results revealed the phylogenetic relationships of the tidal swamp rice germplasm of the South Kalimantan, Indonesia based on the chloroplast trnL-F and nuclear IGS sequences.Both reflected the differences in clustering when analyzed separately, four groups for trnL-F (Figure 2) and two for IGS (Figure 3).While these trees have generated by two aligned sequences, both have a low resolution, indicated by the average of bootstrap values for each tree.Such condition can be caused by the weakness of sequence variation used [14].For this reason, Anderberg et al. [44] suggested the combined sequence data use in increasing the phylogenetic tree resolution.
In this study, the combined sequences of both regions provided a better resolution of a phylogenetic tree than sequence data used separately (Figure 4) but generated a similar pattern of a tree by IGS.This tree, however, could not show the germplasm groupings based on the geographical origin as well.Using the combined sequences data of chloroplast trnL-F, nuclear ITS, and mitochondrial nad1 of the Oryza genus, Duan et al. [14] reported a different result of this study, where the phylogenetic tree has a high-resolution with a geographical origin associated.
Regardless of there is no association of geographical origin, the phylogenetic trees provided a unique illustration, where rice germplasm has a monophyletic divergence (derived from a common ancestor).Using the chloroplast trnL-F sequences, Hall et al. [45] succeeded to show and reconstruct the genetic relationships of Capparaceae and Brassicaceae and concluded that their genetic divergences are monophyletic.Such cases are also reported on Diseae (Orchidoideae: Orchidaceae) [46].However, in Annonaceae, Pirie et al. [24] revealed a different phylogenetic signal to this marker.According to Pirie et al. [24] this probably due to the genes paralogy, caused by duplicating events, in the past, where the second copy of trnL-F diverged from the first trnL-F directly from Annonaceae ancestor.
Using the chloroplasts and nuclear genome on clustering, Kim et al. [22] succeeded to classify and reconstruct the phylogenetic relationships of nine species of Oryza into three different groups.The same grouping is also shown by Duan et al. [14] when analyzing the Oryza genus using a combined sequence data of chloroplast trnL-F and nuclear ITS, as well as mitochondrial nad1.Thus, the markers can be used in other phylogenetic studies [47].Whereas, this result may provide a particular opportunity to explore phylogenetic relationships of the tidal swamp rice germplasm from different angles in the future.

Conclussion
The tidal swamp rice of the South Kalimantan, Indonesia demonstrated a relatively more extraordinary genetic diversity than other local rice germplasm.In this study, this germplasm showed a genetic diversity of 0.61 for nuclear IGS and 0.58 for trnL-F.The phylogenetic reconstruction also exhibited that this germplasm has the unique illustration of phylogenetic trees, particularly for the combined sequences datasets.Thus, the results of our study would provide useful information for further understanding of evolutionary relationships of this germplasm and facilitate the efficient utilization of valuable genes for genetic improvement, particularly in the tidal swamp areas.

Figure 1 .
Figure 1.A map of Indonesia showing three collecting sites of the tidal swamp rice germplasm in the South Kalimantan Province, Indonesia: Tanah Laut (A), Banjar (B), and Barito Kuala (C).
. The reaction was carried out at a total volume of 25 μL, consisted of 1.25 μl of 10 ng DNA template, 4.75 μl of the PCR grade water, 1.25 μl each primer (0.5 μM), and 16.50 μl master mix PCR (Kapabiosystem).The master mix PCR containing 50 mmol KCl, 10 mmol Tris-HCl pH 8.8, 0.1% Triton-X, 1.5 mmol MgCl2, 200 μmol dNTP, and 0, 5 U Taq DNA polymerase.All amplification performed in a Thermal Cycler PCR (Biorad T100, USA) by 35 cycles, and programmed with the following conditions [14]: (1) An initial denaturation for 3 min at 95 o C; (2) Denaturation for 30 sec at 95 o C, (3) Annealing for 30 sec at 55 o C for trnL-F and 53 o C for IGS; (4) Extension for 1 min at 72 o C; and (5) The final extension for 5 min at 72 o C. PCR products separated on 1.5% agarose gels.The amplified DNA fragments which corresponding to the expected size then collected from the gels, purified and sequenced by Integrated DNA Technologies, Singapore.

Figure 2 .
Figure 2. Phylogenetic tree of the tidal swamp rice germplasm based on the chloroplast trnL-F sequences.The numbers above branches indicate bootstrap values inferred from 1000 replicates ( a an outgroup).

Figure 3 .
Figure 3. Phylogenetic tree of the tidal swamp rice germplasm based on the nuclear IGS sequences.The numbers above branches indicate bootstrap values inferred from 1000 replicates ( a an outgroup).

Figure 4 .
Figure 4. Phylogenetic tree of the tidal swamp rice germplasm inferred from the combined of chloroplast trnL-F and nuclear IGS sequences.The numbers above branches indicate bootstrap values inferred from 1000 replicates ( a an outgroup).

Table 1 .
List of the tidal swamp rice germplasm employed in this study.

Name of cultivars Genetic status Grain type Sub-Species Origin
1An outgroup.

Table 2 .
Primers used for PCR amplification and for cycle sequencing.

Table 3 .
Comparison of phylogenetic information for the tidal swamp rice genome from two datasets 1 .