Phylogenetic analysis of 23 accessions of Indonesian banana cultivars based on Internal Transcribed Spacer 2 (ITS2) region

Pisang Kepok (Musa spp. [ABB ’Saba’ subgroup]) has several unique characteristics, such as tolerance to drought and Fusarium Foc (TR4) disease. Currently, the genetic diversity of Pisang Kepok in Indonesia is not well identified, although it is widely cultivated. Information on genetic diversity is essential for developing breeding strategies to achieve efficient cultivar improvement in the future. Aims of this research were to analyze the genetic variation of Pisang Kepok from some islands in Indonesia and to determine the genetic relationship between Pisang Kepok and other accessions banana cultivars based on ITS2 region, as a basis for future research in improving banana quality through molecular breeding. We have conducted the multiple sequence alignment and built the phylogenetic tree analysis using the Bayesian Inference Phylogeny method of one million generations (ngen = 1,000,000). The ITS2 region showed two clade ingroups: first clade consists of banana with B genome (balbisiana), while the second clade consists of banana with only A genome (acuminata). In general, all accessions of Pisang Kepok cultivars were clustered in the B genome of bananas cultivars. In addition, the ITS2 sequences and secondary structures among Pisang Kepok from various regions are identical, suggesting that there was no genetic variation in the ITS2 region of Pisang Kepok from multiple areas in Indonesia.


Introduction
The increasing global population demands a dependable source of food that is highly nutritious, cheap, and easy to cultivate in most developing countries. Banana is consid ered as a promising crop due to its adaptability in a wide range of climate, which currently is the staple for hun dreds of millions in the tropics and subtropics. The high est banana consumption per capita is recorded in Uganda, Rwanda, and Cameroon, exceeding 200 kg of bananas and plantains. In the rural areas of these countries, ba nana supplies up to 25% of the daily calorie intake. In the Philippines and Brazil, the population consumes around 60 kg/year (FAO 2019) either from local production or im ported.
Humans have been cultivating bananas since the emer gence of agriculture (Denham et al. 2003), and in 2017, global production reached 144 million tons . The domestication of the Musa genus originated in South east Asia, as inferred by the chloroplast and nuclear genes Li et al. (2013), resulting in a large variety of plantains and bananas. Indonesia is considered as the center of origin of a diverse banana species (Simmonds 1962), highly rich in genetic resources of both wild and cultivated bananas Daniells et al. (2001). There are at least 325 recorded Musa cultivars in Indonesia (Valmayor et al. 2000), those are widespread in Sumatra, Bali, Nusa Tenggara, Java, Kalimantan, Sulawesi, Maluku, and Papua.
Bananas play multiple roles in the lives of Indonesian, supplying nutrition when consumed as fresh or cooked, and also serving as a key element in their culture, such as for religious ceremonies in Bali (Hidayat et al. 2018). Un fortunately, banana production in Indonesia is sometimes troublesome that decreases both the quality and quantity of harvest due to biotic and abiotic stresses. For many years, the banana plantation in Indonesia has been struck by Tropical Race 4 (TR4) of the Fusarium wilt fungus (Molina et al. 2009; Jumjunidang and Soemargono 2012; Maryani et al. 2019. A soil pathogen that attacks vascu lar tissue and inhibits transport of water and sugars . The increasing heat and diminishing rainfall in tropics and subtropics also threaten the production of this Musa spp. ( (Baral 2019). Thus, the selec tion of banana cultivar with a high tolerance to Fusarium and drought is required. One of the most cultivated bananas in the tropics is Pisang Kepok or Musa spp. (ABB) subgroup 'Saba'. In Indonesia, the cultivation is mostly done in traditional farms that are widely dispersed in their archipelago. Saba banana has been long serving as an important source of nu trition for the locals. In addition to that, from the molecu lar breeding standpoint, Saba banana is a valuable genetic resource for generating cultivars tolerant to drought (Ravi et al. 2013) and/or Fusarium wilt disease (Foc TR 1) culti vated in the greenhouse (Rebouças et al. 2018). Pisang Ke pok or Saba banana is a triploid of the hybridized result of Musa balbisiana (BB genome) and Musa acuminata (AA genome) (Ploetz et al. 2007).
Currently, the genetic diversity of Pisang Kepok in Indonesia is not well identified. Genetic diversity in formation is required in developing breeding strategies to achieve efficient cultivar improvement in the future (Opara et al. 2010). Poerba and Ahmad (2010) identified the genetic variability of cooking bananas and plantains by RAPD and ISSR markers from 36 accessions, with 12 of them were described as local Pisang Kepok. Ahmad et al. (2014) also studied the genetic diversity of M. balbisiana Colla in Indonesia based on the AFLP marker. The cul tivar is wellknown for its resistance to pest, disease, and drought (Morpurgo et al. 1994; Boonruangrod et al. 2008). However, the fruit contains big and plenty of seeds that make it hardly edible, locally it is also known as Pisang Batu or "stone fruit banana".
The DNA marker of Internal Transcribed Spacer 2 (ITS2) is a potential universal marker for plants and an imals. It has sufficient variability to identify a broader range of plant taxa and even to distinguish closely related species . The success rate of identification, based on ITS2 region, at the species level of Musa is up to 82.5% . This DNA marker also resolves the minimal genetic variation in the East African highland bananas (Němečková et al. 2018) and contributes to revealing the disjunct genome group of Musa ornata in Mexico and Asia (BurgosHernández et al. 2017). Further, the identification of ITS secondary struc ture improves discrimination interspecies (Hřibová et al. 2011; Zhang et al. 2015, enabling us to select superior cul tivars based on their ITS2 region. In this presented study, we analyzed the sequence of ITS2 region and secondary structure of Pisang Kepok or Saba banana from West Java, Bali, Flores, and Papua to determine the genetic variability inferred from the dispersal and years of cultivation in the subjected areas. Further, the expected variation might also indicate phenotypic modification that could be beneficial, such as stress tolerance and disease resistance.

Plant Materials and DNA Extraction
Seven cultivars of banana were collected from six lo cations dispersed in four islands of Indonesia (Table 1). Cigar leaves were sampled and placed in a 50 mL conical tube containing a sachet of 2 g of silica gel. Prior to this, the cutter blade was wiped with antiseptic tissue contain ing 70% alcohol. Within three to four days, after reaching our laboratory at School of Life Sciences and Technology ITB, the sachet of silica gel was removed, and the leaf sam ples in the conical tube were immediately dipped in liquid nitrogen and stored at 80 ºC until subsequent process.
The DNA was extracted by using CTAB method (Doyle 1990) with a modification to scale down the vol ume. Also, precipitation step with cold isopropanol (35%) was made in a shorter incubation time within one or two hours. White DNA strands were apparent in most of the extractions from Saba banana, allowing spooling with a glass hook prior to the final protein precipitation step by using ammonium acetate 5 M (wash buffer). If this was not the case, then the supernatant was poured gently into a fresh 1.5 mL microtube, and wash buffer was added. Quality of the extracted DNAs was anal ysed by gel electrophoresis, while quantification by spec trophotometer analysis (Eppendorf Biospectrometer Ki netic, Germany). Extracted DNAs with a thick and intact band, and with a ratio of A 260/280nm 1.8 from each sampled cultivar were selected for the next steps.

Amplification of ITS2 Region and Sequence Analysis
Amplification of ITS2 region was done by polymerase chain reaction (PCR) using the universal primer pair as described in Chen et al. (2010). The reaction was set for denaturation at 94 ºC for 30 s, specific primer an nealing at 55 ºC for 30 s, and elongation at 72 ºC for 30 s for a total of 40 cycles. A predenaturation reac tion was made at 94 ºC for 1 min at the beginning and postelongation at 72 ºC for 1 min prior to concluding the PCR. The 50 μL of PCR reaction comprised 5 μL of DNA samples and mixed with 25 μL of GoTaq Green Master Mix (Promega Co., USA); 15 μL of NucleaseFree Water; and 2.5 μL of each of the primer set ITSS2F and ITS S3R. The primer sequences used in the reaction were ITS S2F: 5'ATGCGATACTTGGTGTGAAT3' and ITSS3R: 5'GACGCTTCTCCAGACTACAAT3' (Gu2). The PCR products were then purified by using Wiz ard SV® Gel and PCR Cleanup System (Promega, USA) and sequenced at Macrogen (South Korea) using pairend Sanger sequencing platform. The contigs of sequencing results were constructed using CodonCode Aligner soft ware (CodonCode Corporation, Dedham, Massachusetts), and ITS2 region was annotated based on ITS2 database (Merget et al. 2012). Following this, annotated sequences were analyzed for identity against GenBank data by using BLASTn (Altschul et al. 1990). The sequencing results are presented in Suppl. Table 1.

Phylogenetic Tree Construction and Secondary Structure Analysis
Multiple sequence alignment was created by using ClustalX (Thompson et al. 1997) and submitting se quences of ITS2 from the seven cultivars of banana. Then, the phylogenetic tree was built on MrBayes version 3.2 (Ronquist and Huelsenbeck 2003) by using the model of general timereversible (rates= gamma, nst=mixed). MarkovChain MonteCarlo (MCMC) reconstruction method was set to perform repetitions as many as 1 million generations (ngen=1,000,000). Prediction of the secondary structure of the analyzed banana ITS2 was made in ITS2 ribosomal DNA database,  (Koetschan et al. 2010). Evalue less than 0.01 and Viridiplantae model was se lected for ITS2 bio apps motif prediction, while the sec ondary structure was also predicted in the same program with evalue cutoff < 1e16 and model possessing the high est transfer helices was depicted (Koetschan et al. 2010). ITS2 sequences were aligned with the ClustalO algorithm (Sievers and Higgins 2014) and integrated within SeaView 4 viewer (Gouy et al. 2010). Consensus representation of multiple sequence alignment was transformed using We bLogo 3 (http://weblogo.threeplusone.com/) (Crooks et al. 2004) and then was manually juxtaposed according to its motif prediction.

Results and Discussion
The 23 accessions in this study were the representation of AA, AAA, AB, BB, and ABB genome of bananas. Ba nanas and plantains are known to have originated in Papua New Guinea and East of Indonesia, which then further generated a diverse species and cultivars of Musa as dis persing to South East Asia and China (Simmonds 1962; De Langhe 2009). Currently, these regions have the great est banana diversity in the world. Cavendish banana is the most popular globally, with most are imported from the tropics and subtropics (FAO 2020). However, this cultivar has a very narrow genetic pool as it has been propagated through a vegetative manner in agricultural practices. The cultivar is susceptible to a wide range of pathogens and to breed in a generative manner is problematic (Daniells et al. 2001(Daniells et al. , 2013. Pisang Kepok (ABB genome) exhibits superior traits compared to Cavendish (AAA genome), which are toler ant to drought and Fusarium wilt disease (Foc TR1). Tra ditionally, Pisang Kepok or Saba banana has also been pro viding nutrition to the rural populations in Indonesia and The Philippines. Thus, improving the quality and quantity of Pisang Kepok production could be a promising alterna tive to diminish the dominance of Cavendish banana in the global market, which is also expected to be more sustain able although this would need more effort to also educate the consumers since Pisang Kepok requires a cooking pro cess to be more tasteful. However, almost all bananas in the local market came from backyard farming with poor management. The production scale of these smallholding farming is usually quite large, collectively, but often do not meet the standards for export. Resolving the genetic vari ance in local banana cultivars would assist the selection of superior cultivars inbreeding, that further will help the local farmers by providing seedlings with better qualities.

Morphological characteristics of the collected Pisang Kepok
The most striking morphological character of M. acumi nata and M. balbisiana is the color of bract that touches the younger layer or facing downward. Cultivars with B genome tend to display red color from the base to the tip, while those with A genome exhibits a yellowish or cream color at the base. Further, the petiole leaves canal of ba nanas containing A genome, are usually widely open but closed or overlap in bananas containing B genome. The general morphologies of the collected samples are compli ant with the description of Pisang Kepok or Saba banana. They possess a pseudostem with a height of 3 m or higher, waxy and greenyellowish in color, and the leaves are in termediate upright. The petiole margins are enclosed, not winged, and clasping the pseudostem, and the base of the second leaf is rounded on the sides. The rachis of Pisang Kepok is perpendicularly hanging with an ovoid inflores cence. The fruits are usually 2125 cm in lengths, with a rounded tip and a persistent style of the stamen. In this study, four accessions of Pisang Kepok were collected from Bandung (West Java), Denpasar (Bali), Flores (East Nusa Tenggara), and Sorong (Papua). The detachment style of bractea between Pisang Kepok from West Java and Bandung was quite distinctive. In Flores, the bractea rolled up prior to detaching, while in Bandung, they opened upward then detached. This may not be a re sult of genetic variance but simply a response to tempera ture in their respective habitat. The higher temperature in  Flores could prompt more rapid transpiration compared to the colder temperature in Bandung. Bractea tip is prone to turgor loss, hence curling up, as it is the farthest from watersupplying xylem in the pseudostem. Thus, we did not find any significant differences in the morphology of Pisang Kepok from West Java, Bali, East Nusa Tenggara, and Papua.

DNA extraction, ITS2 amplification, and sequence analysis
The concentration of acquired DNA was approximately 300 1000 ng/µL. Extracted DNA from all accessions was visualized in electrophoresis gel and resulted in a single band at length longer than 14 kb ( Figure 1A, 1B). How ever, some shorter smeared bands were also detected that might be contamination of RNA. This was not a problem because the products of misannealed primer would be vis ible in an electrophoresis gel. The fragment of ITS2 was detected at a length of approximately 500 bp ( Figure 1C, 1D), and no shorter band was visible, indicating that am plification was made based on the DNA template. The re sulted fragments were longer than banana ITS2 analyzed by Hřibová et al. (2011), which were in a range of 205 -250 bp. This was due to the amplified region of ITS2 in this study also included parts of the 5.8S and 28S regions of the ribosomal RNA genes. Following this, the ampli fied fragments from each accession were sequenced and analyzed using BLAST (Altschul et al. 1990). The results showed that all submitted sequences have 99100% query cover and identity to ITS2 region of Musa cultivars with AA, AB, B, AAA, and ABB genome ( Table 2). The length of amplified ITS2 fragments was ranging from 218 to 222 after removing the 5.8S and 28S regions from the sequence (Table 3). These results were compara ble to Hřibová et al. (2011), documenting that Musa ITS2 regions were 205250 bp long. The GC content of Musa ITS2 fragments in this study was in the range of 69.27% 71.56%, which was slightly higher than the results of Hři bová et al. (2011) of 56.11% 70.97%. We also found that Ambon Hijau, Ambon Lumut, and Cavendish possess diverse length and GC content of ITS2, although these three cultivars are classified as M. acuminata (AAA) sub group Cavendish. However, this was not documented in Musa spp. (ABB) subgroup Saba, with identical length and GC content from all accessions. In general, the ABB and BB bananas in this study have ITS2 region compris ing of 220 nucleotides and 70.36% GC content. Based on the multiple sequence alignment, Pisang Kepok from West Java, Bali, Flores, and Papua also exhibited an identical se quence of ITS2 regions (Figure 2). De Langhe (2009) sug gests that the variance of length and GC content within a Musa species could be a consequence of mutation. Several types of mutation that possibly caused this variance are the insertion or deletion of a single nucleotide, nucleotide sub stitution, transition mutation of selfreplacement to purine or pyrimidine, or transverse mutation of crossreplacement to purine or pyrimidine (Campbell and Reece 2000).

Phylogenetic analysis
The phylogenetic tree was constructed by using the Bayesian method, involving the search of the most repre sentative tree by generating up to 88% 90% of highly probable trees (Xiong 2006). Statistically, Bayesian is highly correlated to maximum likelihood, focusing on the prior probability and likelihood (Holder and Lewis 2003).  (2002) suggested that morphologically Pisang Ke pok is tightly related to Klutuk Wulung and cultivars with ABB genome. Pisang Kepok and other ABB cultivars are grouped with Klutuk Wulung (BB genome) due to their dominant characters such as the ovoid shape of the in florescence, ovulum are grouped in four per locus, and the detachment of bractea in an opening pattern (Siddiqah 2002). Clade I display polytomy as indicated by the insuf ficient data to display an additional branch. This suggested that there was no genetic variance in bananas with AB and BB genome, which was also confirmed the identical length and GC content of their respective ITS2 sequence. All accessions of Pisang Kepok are grouped together with bananas of genome AB, and ABB generated a polytomy (Dover 1982), which was a consequence of the dominance of one particular type of rDNA sequence in the hybrid of M. acuminata x balbisiana (Kovarik et al. 2004). Fur ther, this might have led to the complete homogenization of rDNA locus from M. balbisiana (B genome). This also could be a consequence of chromosome replacement be tween two genomes (A and B genome) (Wang et al. 2019). Bile (AB genome) generated a sister clad with Bali (ABB genome) (PP = 0.69) in clade I. The reconstruction of phy logenetic tree by using Bayesian analysis showed that M. balbisiana is separated from the other taxa and created a polytomy.

The separation of ingroup and outgroup in Bayesian anal
Accessions

Predicted secondary structure of ITS2
Although the comparison of ITS2 sequences has been ac knowledged as a powerful tool in discriminating taxa, it still needs improvement to identify a broad range of species. One of the strategic means is by analyzing the sec ondary structure of ITS2, as this does not increase the num ber of sequenced area and may also infer function (Zhang et al. 2015). Generally, RNAs activities in cells are deter mined by their secondary structures, although the defini tive role of ITS2 RNA in molecular regulation is still yet to reveal. ITS2 secondary structure is highly conserved in plant DNA (Jobes and Thien 1997). It consists of four he lices structured spacers that are unique and useful to im prove discrimination intraspecies. Helix I and IV have higher diversity and only helix II and III that could be iden tified in general to all eukaryotes (Coleman 2007). He lix II could be determined by the content of pyrimidine pyrimidine nodules, as for Helix III, are characterized by its content, which is the most conserved primer on 5' with UGGU sequence (Schultz et al. 2005).
The predicted secondary structures of ITS2 from all accessions in this study are presented in Table 3. Pisang Kepok from West Java, Bali, East Nusa Tenggara, and Papua exhibit the identical secondary structure of ITS2 as characterized by three bubbles in helix I, a hooklike structure between helix I and II, four bubbles in helix III, and three bubbles in helix IV (Figure 4). The identical structure suggests that these accessions are the same culti var with no genetic variation. It was interesting that the identical secondary structure of ITS2 was also detected interspecies in M. ornata and M. velutina, although that was not the case with their sequence length and identity (Suppl. Figure 2). In this case, the RNA of ITS2 in M. ornata and M. velutina might be playing a consistent role. The hooklike structure between helix I and II was almost constantly found in accessions comprising B genome, but none in cultivars with only A genome. Ambon Hijau and FIGURE 4 Prediction of secondary structure of ITS2 RNA from Pisang Kepok, and also is representing genome AB. The model is identical for the four accessions from West Java, Bali, East Nusa Tenggara, and Papua. It contains four conserved motifs of UGGU, U-U mismatch in between helix I and II, and U-U mismatch in between helix II and III.
Ambon Kuning also possess the identical secondary struc ture of ITS2, consisting of four bubbles in helix I, one in helix II, three in helix III, and also three bubbles in helix IV (Figure 4). However, this was slightly different from the secondary structure of ITS2 in Ambon Lumut that pos sesses only two bubbles in helix I (Figure 3) The formation of bubbles in the secondary structure is a consequence of mismatch pairings of the nucleotide bases on the ITS2 RNA, subsequent to the generation of helices. These mismatch pairings also present in the four specific and conserved motifs of ITS2 RNA, which were also found in the secondary structure of ITS2 from Pisang Kepok ( Figure 4) and all the other bananas analyzed in this study (Suppl. Figure 13). The motifs are the UGGU (III, 5' side), UU mismatch (II, left), and UU mismatch (II, right) with AAA (btw. II, III). These motifs are pre dicted by 14 unique bases from the sequences of ITS2 RNA. Generally, the UGGU motif precedes the apex of the helix III or a pyrimidinepyrimidine mismatch in he lix II (Merget et al. 2012). Our data showed that UGGU motif was found in helix III in all secondary structures of ITS2 from the 23 accessions, starting from base 118, 119, or 120. We also found that each UGGU motif contains four UU mismatches, one preceding, and another one fol lowing. The UU mismatch (II, left) was consistently pre dicted between helix I and II from base 53, 54, or 55, with the two UU mismatch positions were separated by two nu cleotides on each ITS2 RNA sequence. Lastly, UU mis match (II, right) with AAA (btw. II, III) in accessions was predicted to be positioned between helix II and III with insertion of G in the AAA.
ITS sequences in plants are associated with environ mental adaptation such as shade, salinity, and drought tol erance, although much remains unknown (Whittle 2006). The predicted secondary structures of ITS2 in banana cul tivars implies variation in the sequences of ITS2 is not always supported by their secondary structure. Hence, analyzing nucleotide sequence and secondary structure is recommended to reveal the phylogenetic relationship be tween banana cultivars. Our data suggested that the four accessions of Pisang Kepok are not genetically distinct based on their ITS2 RNA sequence and secondary struc ture. Hence, they could be categorized as the same culti var.
As the national effort to support the improvement of Indonesia bananas production, the characterization of superior cultivars is a prerequisite. This study showed that Pisang Kepok from Bandung (West Java), Denpasar (Bali), Flores (East Nusa Tenggara), and Sorong (Papua) possess identical ITS2 sequence, which indicates that they may also exhibit tolerance to Fusarium (Foc TR1) and drought. Hence, this could be one of the foundations fur ther to analyze the genetic basis of these advantageous traits. This needs more research to inquire about the no tion given that the analysis was performed based on ITS2 in which does not correlate to disease resistance pheno type and adaptation to a hostile environment. Traditional or molecular breeding of Fusarium and/or drought suscep tible cultivars with Pisang Kepok could be performed im mediately. However, an extended time frame is necessary if we are to transfer the responsible genes through genetic engineering methods.
An alternative meaning to improve export and increase the price of our local bananas in the international market is to investigate cultivars that are tolerant to postharvest diseases. Currently, the local production of bananas in In donesia is considered one of the highest in Asia, following The Philippines and India. However, Indonesian bananas are mostly unable to penetrate the global market due to the poor quality of the harvest. The seeking of these desirable traits, with the fruits that are able to endure long posthar vest processing and distribution, could also be performed by the analysis of ITS2 RNA. In this case, we would need to determine the cultivar(s) that exhibit those traits, then compare their ITS2 RNA sequence and secondary struc tures with other candidates. If the ITS2 RNA of the can didates is highly identical, then they could be further ana lyzed, as mentioned earlier.

Conclusions
We conclude that Pisang Kepok from Bandung (West Java), Denpasar (Bali), Flores (East Nusa Tenggara), and Sorong (Papua) are the same cultivar as indicated by the identical ITS2 length, structure, and secondary structure. This also suggests that the distribution of Pisang Kepok to these four islands in Indonesia took place a short time ago and possibly was introduced by humans for agricul tural purposes. Furthermore, it indicates that they might have the identical superior traits of Fusarium and drought tolerance, which are useful for the improvement of other banana cultivars. ITS2 has been illustrated in many studies as a robust molecular marker to some degree, particularly in this research, in clustering the bananas based on their genome group. Nevertheless, the use of ITS2 in the minor taxonomical groups such as cultivar could be meticulous and needs extra details. Provided that cultivar 'Kepok' might possess desirable traits such as tolerance to drought and disease such as in Foc TR1, with more research, it assumed that these superior traits might provide the im provement of other banana cultivars.