Comparative Chloroplast Genomics and Maternal Lineage Diversity in Oil Palm (Elaeis guineensis)

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Ardie, Willy B. Suwarno, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8662850/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 16 You are reading this latest preprint version Abstract Background Oil palm ( Elaeis guineensis ) is the world’s most important source of vegetable oil, yet comparative studies of its chloroplast genomes remain limited, particularly for African accessions. Understanding plastome diversity and maternal lineage variation is critical for tracing domestication history and developing molecular markers for breeding and conservation. Results We report the first complete chloroplast genomes from Tanzanian Dura and Pisifera, Cameroonian Dura, and the ancestral Deli Dura from Indonesia, analyzed alongside 44 additional plastomes. Comparative analyses revealed highly conserved genome organization and gene content, with subtle differences in IR–SC boundaries and intergenic spacers. Simple sequence repeat (SSR) profiling confirmed the dominance of A/T mononucleotide repeats but highlighted lineage-specific variation, including a markedly reduced genic SSR density in Pisifera-Tz, while total SSR counts remained stable across accessions (≈ 859–860; Supplementary Table S3 ). Phylogenetic reconstruction clustered Pisifera-Tz with Dura-Cmr, while Dura-Tz and Dura-BBG formed distinct clades, reflecting multiple maternal lineages. Haplotype network analysis identified 14 distinct haplotypes, revealing moderate but structured plastome diversity. Benchmarking of sequencing platforms demonstrated complementary strengths: MGI provided deep coverage, while PacBio improved assembly contiguity and resolution of repetitive regions. Conclusion This study expands plastome resources for E. guineensis , generating the first Tanzanian and Cameroonian chloroplast genomes and uncovering lineage-specific SSR variation. Integration of comparative genomics, phylogenetics, and haplotype analysis advances understanding of oil palm domestication and supports the development of plastome-based molecular markers for breeding, conservation, and genetic resource management. Oil palm chloroplast genome plastome diversity SSR variation phylogenetics maternal lineage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Oil palm ( Elaeis guineensis Jacq.) is the most productive source of vegetable oil worldwide, contributing more than one-third of the global supply (1). Its high oil yield per unit area has made it central to food security, bioenergy, and industrial applications (2,3). However, the rapid expansion of oil palm cultivation has raised concerns about deforestation, biodiversity loss, and sustainability (4,5). In addition, the limited genetic base of current breeding populations constrains progress in developing resilience traits, yield stability, and climate adaptation (6,7). The narrow genetic base of Southeast Asian oil palm populations largely stems from a small number of African introductions during the colonial era (7). This founder effect has restricted genetic variation in commercially important lines, particularly the Deli Dura population in Indonesia (8). In contrast, African wild populations harbor significant allelic richness and represent untapped reservoirs of genetic diversity (8,9). Expanding cytoplasmic and nuclear variation is therefore critical for sustainable cultivation and long-term breeding success (10). Chloroplast genomes (plastomes) are powerful tools for studying plant diversity, evolution, and adaptation (11,12). Plastomes play essential roles in photosynthesis, energy metabolism, and biosynthesis of amino acids, fatty acids, and pigments. Their conserved quadripartite structure, uniparental inheritance, and relatively low mutation rate make them valuable for phylogenetics, species differentiation, and lineage tracing (13). In flowering plants, plastomes typically range from 120 to 160 kb and encode 110 to 130 genes involved in transcription, translation, and photosynthesis (14,15). Despite their conservation, single-nucleotide polymorphisms, indels, and structural variations in non-coding regions provide informative markers for evolutionary research, germplasm authentication, and lineage tracing (16,17). Previous studies of oil palm plastomes revealed highly conserved gene content and organization, consistent with other members of the palm family (Arecaceae) (18). The first complete E. guineensis plastome established a baseline structure of ~ 156,973 bp (18), and subsequent work confirmed its stability across the species (19,20). Nevertheless, comparative analyses identified nucleotide variations concentrated in single-copy regions and intergenic spacers, offering opportunities to differentiate closely related lineages (21). Commercial planting materials are derived from hybridization between Dura (thick-shelled) and Pisifera (shell-less) types to produce the high-yielding Tenera hybrid (22). Tracing maternal lineages is therefore essential for germplasm authentication and understanding cytoplasmic genetic effects (23,24) Given the narrow maternal base of commercially dominant Deli Dura lines (16), resolving plastome diversity across wild and cultivated accessions is critical for breeding programs (25,26). While nuclear genomic studies of E. guineensis and E. oleifera are extensive (27–30), detailed comparative analyses of intraspecific plastome diversity remain limited. Even small numbers of variable sites within plastomes can be powerful for differentiating lineages and mapping cytoplasmic diversity (31). Characterizing plastome variation across diverse African accessions is thus essential for conservation, breeding authentication, and marker development (32). Tanzania and Cameroon represent centers of origin and diversity for oil palm, harboring ancient lineages and wild populations that could significantly enrich global breeding programs (9,32). In contrast, Indonesian Dura populations descend from a narrow genetic base introduced from Africa in the early 20th century (16). Comparative plastome analyses across African regions, therefore, offer unique insights into domestication history, adaptation mechanisms, and maternal lineage divergence (27) Recent advances in sequencing technologies have transformed plastome research (33). Short-read platforms such as Illumina and MGI DNBSEQ provide cost-effective accuracy for sequencing and variant detection but struggle with repetitive regions and structural variations (34). Long-read platforms such as PacBio HiFi and Oxford Nanopore generate reads spanning thousands of bases, enabling resolution of repetitive elements and structural variants (35). Long reads improve assembly contiguity, facilitate haplotype phasing, and enable precise detection of complex rearrangements (35). Comparing short- and long-read assemblies provides critical validation for plastome reconstruction, particularly in complex plant lineages such as oil palm (34). However, systematic comparisons of sequencing platform performance in oil palm plastomes remain scarce. This study addresses these gaps by sequencing and analyzing chloroplast genomes of Tanzanian Dura (Dura-Tz) and Pisifera (Pisifera-Tz) accessions. We integrate these with previously sequenced Cameroonian Dura (Dura-Cmr), the ancestral Deli Dura from Indonesia (Dura-BBG), 29 Indonesian breeding lines, and 15 plastome accessions from public databases. By benchmarking MGI and PacBio sequencing platforms, we highlight their complementary strengths for plastome assembly and variant detection. Collectively, this work provides new insights into plastome diversity in E. guineensis, advances understanding of domestication history, and supports the development of plastome-based molecular markers for breeding, conservation, and genetic resource management. Methods Oil palm chloroplast genome samples Two chloroplast genomes were sequenced in this study, including one Tanzanian Dura type (Dura-Tz) and one Tanzanian Pisifera type (Pisifera-Tz). The selected plant materials was identified by Mr. Basil E. Kavishe, Research Assistant at Tanzania Agricultural Research Institute (TARI). No voucher specimen was deposited in publicly accessible herbarium. However, the plant materials were photographed during sample collection, and they are now monitored by TARI for reference and future studies. Young leaves were collected from the Kwitanga plantation, Kigoma, Tanzania. Samples were cleaned, cut into ~ 20 cm pieces, sterilized with 70% ethanol, air-dried, sealed in airtight plastic bags, and transported in a cold box to PT Genetika Science, Indonesia, for sequencing. Two representative wild oil palm samples, one Cameroonian Dura-type (Dura-Cmr) and one Deli Dura progenitor from Bogor Botanical Garden, Indonesia (Dura-BBG), were sequenced in a previous project at PMB Lab, IPB University (NCBI accession numbers are on Progress). Moreover, as many as twenty-nine (29) samples were also from previously sequenced chloroplast genomes and obtained from Dr. Azis Natawijaya (PT Bumitama Gunajaya Agro Company). Inquiries for these 29 sequences should be directly forwarded to Dr. Azis Natawijaya. Other cpDNAs were obtained from NCBI GenBank DNA Database ( https://ncbi.nlm.nih.gov/ ). All chloroplast genome sequences generated in this study, including their associated genome annotations, are publicly available in the NCBI database under BioProject accession number PRJNA1416287 . Chloroplast genome sequencing and assembly Short-read sequencing of Dura-Tz and Pisifera-Tz total genomes was performed using the MGI DNBSEQ-G400 platform (34) ( https://mgi-tech.eu/sequencing-products/dnbseq-g400 ) (PE150) with MGIEasy FS DNA Library Prep Set. Long-read sequencing of Dura-Tz total genomes was conducted using the PacBio Vega platform with HiFi Plex Prep Kit 96 (PacBio, 103-122-800) (36) ( https://www.pacb.com/wp-content/uploads/Insert-HiFi-plex-prep-kit-96 ). Raw reads of Dura-Tz and Pisifera-Tz were processed with Fastp v0.20.1 (37) to trim low-quality sequences and adaptor contamination. De novo assembly was performed using NOVOPlasty v4.3.5 (38), and the assemblies were polished using EnsemblPlants56 ( https://plants.ensembl.org ). Sequencing depth and coverage of the Dura-Tz and Pisifera-Tz chloroplast genomes were evaluated by mapping raw reads back to the assembled plastomes. Depth profiles were generated across the entire genome length (0–156 kb) for both Dura-Tz and Pisifera-Tz accessions. Coverage plots were inspected to confirm uniformity across large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions. Chloroplast genome annotation Assembled genomes in FASTA format were annotated using GeSeq within CHLOROBOX (39) and the Dual Organellar Genome Annotator (DOGMA) with default parameters—annotation employed 95% sequence similarity thresholds for protein-coding genes, rRNAs, and tRNAs. BLASTX and BLASTN were used to identify coding genes and rRNAs, while tRNAscan-SE v2.0.7 was used for tRNA annotation (40). Intron boundaries, start, and stop codons were manually curated by comparison with a combination of many related species of Arecaceae plastomes. Genome maps were visualized using OGDRAW (41). Comparative analysis of Plastome structure and Synteny Using assembled chloroplast genomes, the base composition and frequencies of the LSC, SSC, and IR regions were determined in Geneious Prime v2024.0.7 (42). Two multiple progressive sequence alignments were conducted; the first one included the two new plastomes plus Dura-BBG and Dura-Cmr. Multiple sequence alignments of the plastomes were generated using MAFFT v7.490 (43). IR boundary variation was visualized using IRscope (44). The second analysis was carried out using the two new plastomes plus Dura-BBG and Dura-Cmr and six Indonesian breeding lines (Dura-Id_574, Dura-Id_101128179, Pisifera-Id_221, Pisifera-Id_1169, Tenera-Id_1093, Tenera-Id_15730) and six plastomes that were available on NCBI (ON248756, NC_017602, OR125034, OR125040, OR125048, OR125054) in Mauve v2.4.0 (45). Simple sequence repeats (SSRs) Pairwise multiple alignments of cp genomes were performed using MAFFT v7.490 (43) in Geneious Prime. Simple sequence repeats (SSRs) containing 1–6 nucleotides were identified using the Phobos v3.3.12 (46). The following configuration was adopted to search for SSR motifs: SSR of one to six nucleotides long, with a minimum repeat number of 10, 5, and 4 units for mono-, di-, and trinucleotide SSRs, respectively, and three units for tetra-, penta-, and hexanucleotide SSRs. Phylogenetic and haplotype analysis Phylogenetic analysis included 48 E. guineensis cp genomes. Multiple sequence alignments were generated using MAFFT v7.490 (43). Phylogenetic trees were constructed using the Maximum Likelihood (ML) method with the Tamura-Nei (TN93/TNG93 + G + I) genetic distance model, and 1000 bootstrap replications (47,48). Haplotype networks were constructed from 48 E. guineensis cp genomes, including 19 PT Bumitama Gunajaya Agro accessions, 25 NCBI accessions, and 4 genomes sequenced in the PMB lab at IPB University. Alignments were performed in Geneious Prime, and haplotype networks were generated using NETWORK software (49). Results Chloroplast Genome Sequencing Sequencing output is summarized in Table 1 . For the Dura‑Tz and Pisifera‑Tz accessions sequenced with MGI, approximately 242 million reads were generated per sample, corresponding to ~ 36 Gb of total bases. In contrast, PacBio sequencing of Dura‑Tz produced only 5.95 million reads but yielded a substantially higher total base output of 63.33 Gb (Table 1 ). Table 1 Sequencing data summary for Tanzanian Dura (Dura-Tz) and Pisifera (Pisifera-Tz) Sample name Sequencing Platform Total reads (M.) Total bases (Gb) Dura-Tz MGI 241.78 36.27 PacBio 5.95 63.33 Pisifera-Tz MGI 242.00 36.30 These results highlight the complementary strengths of the two platforms. MGI provides deep coverage through massive read counts, ensuring reliable variant detection and uniform base representation. PacBio, despite generating fewer reads, delivers long sequences that enhance assembly contiguity and resolve repetitive regions more effectively. Together, the datasets demonstrate that combining short‑read and long‑read technologies offers both depth and structural accuracy for plastome reconstruction. Sequencing Depth and Coverage Robust sequencing coverage was achieved across the chloroplast genomes of both Dura-Tz and Pisifera-Tz accessions. The Dura-Tz assembly reached depths of up to ~ 3,500 reads (Fig. 1 A), while Pisifera-Tz attained nearly 7,000 reads (Fig. 1 B), reflecting its substantially higher sequencing throughput. Coverage profiles were generally uniform across the plastome, with expected peaks in the inverted repeat (IR) regions due to duplication and troughs in AT-rich or non-coding regions that are inherently more challenging to sequence. The overall consistency of coverage confirms the completeness and reliability of both assemblies. Significantly, the greater depth observed in Pisifera-Tz enhances confidence in variant detection and structural resolution, thereby providing a stronger foundation for downstream comparative and phylogenetic analyses. Chloroplast Genome Assembly and Structure The complete chloroplast genomes of Dura‑Tz (PX957236) and Pisifera‑Tz (PX957237) were assembled into circular maps of 156,883 bp, each displaying the typical quadripartite organization comprising large single‑copy (LSC), small single‑copy (SSC), and inverted repeat (IR) regions (Fig. 2 A, 2 B). Comparative analysis with previously sequenced accessions (Dura‑Cmr and Dura‑BBG) revealed highly conserved structural and compositional features (Table 2 ). These findings are consistent with earlier oil palm plastome characterizations (23,50) and broader plastid genome studies in seed plants (11,13,14, 23). Table 2 Summary of general features of 4 accessions of Elaeis guineensis chloroplast-genomes Attribute Dura-Tz Pisifera-Tz Dura-Cmr Dura-BBG Genome Size (bp) 156,983 156,983 156,982 156,990 GC content (%) 37.4 37.4 37.4 37.4 LSC size (bp) 85,194 85,194 85,193 85,198 SSC size (bp) 17,643 17,643 17,643 17,644 IRs size (bp) 27,073 27,073 27,073 27,074 LSC GC content (%) 35.5 35.5 35.5 35.5 SSC GC content (%) 31.0 30.9 31.0 31.0 IRs GC content (%) 42.5 42.5 42.5 42.5 Number of genes 115 115 115 115 Number of protein-coding genes 79 80 80 80 Number of tRNA 32 31 31 31 Number of rRNA 4 4 4 4 Chloroplast Genome Annotation and Gene Content Annotation identified a consistent set of 115 genes across all accessions, comprising 79–80 protein‑coding genes, 31–32 tRNAs, and 4 rRNAs. Dura-Tz contained 79 protein-coding genes and 32 tRNAs, compared with 80/31 in others (Table 2 ). This subtle difference may reflect lineage-specific variation in gene annotation or minor events of tRNA duplication or loss, possibly involving ycf1 or related loci. Overall, gene composition remains highly stable, underscoring the structural conservation of E. guineensis plastomes. Such uniformity highlights the evolutionary stability of the chloroplast genome, while slight differences in gene counts may serve as useful markers for lineage differentiation and comparative genomics. Functional categorization revealed a conserved organization across all four accessions, including genes encoding photosystem components, ribosomal proteins, ATP synthase, NADH dehydrogenase, Rubisco, RNA polymerase, and proteins involved in DNA replication and repair. Transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were distributed throughout the plastome, ensuring its translational capacity. Hypothetical chloroplast reading frames (ycf) were also identified, consistent with their conserved presence in angiosperm plastomes. Gene transcription orientation was bidirectional, with genes on the outer ring transcribed clockwise and those on the inner ring transcribed counterclockwise. Core functional categories—photosynthesis, transcription, translation, and genome maintenance—were consistently represented, in line with previous annotations of oil palm plastomes (10,17,19). Together, these findings confirm the high degree of conservation in gene content and organization among oil palm plastomes. The stability observed between Dura‑Tz and Pisifera‑Tz, along with other accessions, provides a reliable foundation for comparative and evolutionary analyses. At the same time, minor lineage‑specific variations can serve as potential markers of genetic differentiation. SSR Distribution and Motif Composition Analysis of simple sequence repeats (SSRs) across the four accessions—Dura‑BBG, Dura‑CMR, Pisifera‑Tz, and Dura‑Tz—revealed a highly conserved repeat landscape (Fig. 3 , Supplementary Table 1). Total SSR counts ranged narrowly from 859 to 860 per plastome, indicating strong structural stability across lineages. Mononucleotide repeats dominated the SSR profile, averaging 338.5 per accession, with a pronounced bias toward A/T motifs. Each accession contained approximately 317 A/T mononucleotide repeats, accounting for over 93% of the total, while G/C repeats were rare (22 per plastome). This A/T enrichment reflects the low GC content and mutational bias typical of plastid genomes (10,17) Dinucleotide repeats were equally conserved, with 58 per accession. AT and TA motifs were the most abundant, comprising 144 of the 232 total dinucleotide repeats (62%). Other motifs such as AG, GA, TC, and CT were evenly distributed across accessions, with no lineage-specific enrichment. Trinucleotide and tetranucleotide motifs also showed uniform distribution, with 71 and 95 repeats per accession, respectively. Trinucleotide motifs were dominated by A/T-rich sequences such as TAT, AAG, and AAC, while tetranucleotide repeats included AAAT, AATA, ATTA, and TTTA. These patterns reinforce the A/T bias observed in shorter motifs and suggest that even longer SSRs retain compositional consistency across accessions. Pentanucleotide repeats were similarly conserved, with 134 motifs per accession. Dominant sequences included AAAAT, AATTT, TTTTA, and TTAAA, all of which are rich in A/T bases. The recurrence of these motifs across all accessions further supports the plastome’s mutational bias and structural stability. No accession-specific expansion or contraction of pentanucleotide motifs was observed. Hexanucleotide repeats showed slightly more diversity, with counts ranging from 157 to 158 per accession. Despite the broader sequence range, dominant motifs remained A/T-rich, including TTCTTT, TTTTAT, and TTTTTT. The consistent counts and motif composition across accessions suggest that even the longest SSRs are evolutionarily stable and not subject to lineage-specific expansion. Hexanucleotide repeats showed minor variation (157–158), likely reflecting annotation differences or sequencing noise rather than true biological divergence. Although Pisifera-Tz exhibited a marginally higher total SSR count (860 vs. 859 in other accessions), its genic SSR density was substantially reduced (277 vs. ~681–682). This indicates that while overall SSR abundance remains stable, Pisifera-Tz plastomes have undergone lineage-specific contraction in genic regions (see Supplementary Table S3 ).” Overall, the uniformity of SSR types and motif composition across all accessions underscores the evolutionary stability of the oil palm plastome. While SSR variability is limited, conserved tetranucleotide, pentanucleotide, and hexanucleotide motifs—particularly those rich in A/T bases—may still serve as neutral molecular markers for population genetics, germplasm authentication, and comparative genomics. This conserved SSR profile highlights plastome stability and mirrors SSR distributions reported in other palm species (8,51,52). SSR marker development in oil palm has similarly emphasized A/T-rich motifs (53,54). Genic SSRs Across Accessions Comparative analysis of genic SSRs revealed both broad conservation and striking lineage‑specific differences among the four E. guineensis chloroplast genomes (Table 3 ). Dura‑Cmr and Dura‑BBG each contained 682 genic SSRs, while Dura‑Tz harbored 681, reflecting near‑identical SSR abundance and distribution across genes associated with photosynthesis, replication, and genome maintenance. This consistency underscores the evolutionary stability of genic SSRs in most oil palm accessions, with plastome function preserved through balanced representation across essential pathways. Table 3 Summary of genic SSRs in four Elaeis guineensis chloroplast genomes Genome Total genic SSRs Gene categories with SSRs Dura-Cmr/Dura-BBG/Dura-Tz 681–682 Photosynthesis : atp (B/E/F/I), ccsA , cemA , ndH (A/B/D/E/H/I/K), paf (I/II), pet (A/B/D), psa (A/B/J), psb (A/B/C/F/I/K/L/M/T/Z), rbcL. Maintenance : accD , clpP1 , matK , rpl (2/16/22/32/33), rps (3/4/7/11/12/14/15/16/18/19), ycf (1/2) Replication : rpo A/B/C1/C2 Pisifera-Tz 277 Photosynthesis : atp (B/E/F/I), cemA , ndhK , paf I/II, pet A/B/D, psa A/B/I/J, psb (A/B/C/F/I/K/L/M/T/Z), rbcL . Maintenance : accD , clpP1 , matK , rpl (16/22/33), rps (3/4/11/12/14/16/18) Replication : rpo A/B/C1/C2 In contrast, Pisifera‑Tz exhibited a markedly reduced genic SSR count (277), representing a substantial contraction relative to the other accessions. Despite this reduction, SSRs in Pisifera‑Tz were still distributed across key functional genes, including rbcL, matK, psa, psb, and rpo, ensuring that core photosynthetic and transcriptional processes remained represented. However, the absence of SSRs in several maintenance‑related loci—including ycf1, ycf2, and members of the ndh family—suggests lineage‑specific loss or reduced SSR density in regions typically associated with genome stability and stress response. This pattern highlights two complementary insights: first, the conserved SSR profiles in Dura accessions reflect plastome resilience and functional redundancy across diverse lineages; second, the reduced SSR density in Pisifera‑Tz may indicate either structural streamlining or selective pressures that minimized repetitive elements in its plastome. From a practical perspective, these differences provide opportunities for marker development: conserved genic SSRs in Dura accessions can serve as stable lineage markers. At the same time, the unique contraction in Pisifera‑Tz offers diagnostic motifs for distinguishing maternal lineages in breeding programs. Despite this contraction, SSRs remained present in key functional genes, including rbcL , matK , psa , psb , and rpo . The absence of SSRs in ycf1 , ycf2 , and ndh family genes suggests lineage-specific streamlining, consistent with plastome variation observed in Pisifera lineages (55,56) Plastome Structure and Synteny Multiple genome alignment of 16 Elaeis guineensis chloroplast genomes—representing Dura, Tenera, and Pisifera—revealed a high degree of structural conservation across accessions (Fig. 4 ). Using the Mauve algorithm, plastome sizes ranged narrowly from 153,000 to 158,000 bp, with most accessions exhibiting uniform syntenic block arrangements and stable genome architecture. This consistency reflects strong evolutionary constraints on plastome organization, likely driven by the essential roles of chloroplast genes in photosynthesis and cellular metabolism. Notably, two Indonesian Pisifera accessions (Pisifera‑Id_221 and Pisifera‑Id_101128179) displayed structural rearrangements in the 140–145 kb region, characterized by translocations and/or inversions (highlighted in red blocks). These localized disruptions suggest lineage‑specific plastome remodeling, potentially arising from recombination events or selective pressures unique to the Pisifera background. While the overall plastome structure remains conserved, such rearrangements may influence gene regulation or plastome stability, warranting further investigation into their functional consequences. Complementary mapping of the 16 plastomes confirmed the conservation of junction architecture between the large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions (Fig. 5 ). All accessions shared nearly identical genome sizes (~ 156.98 kb) and consistent placement of key genes at junctions, including rps19, rpl2, psbA, ndhF, ycf1, and trnN. Minor differences in junction lengths (25–65 bp) were observed but did not affect gene orientation or overall structural layout. These findings reinforce the evolutionary stability of plastome boundaries in E. guineensis and validate their utility for comparative and phylogenetic analyses. The conserved junctions and syntenic blocks across most accessions provide a robust framework for plastome‑based lineage tracing. In contrast, the structural variation in select Pisifera accessions offers a unique opportunity to explore plastome dynamics and their potential role in fruit form differentiation or adaptation. Such rearrangements have been linked to recombination events in plastomes of palms and other monocots (20,24,45,57) Comparative mapping of LSC, SSC, and IR junctions confirmed consistent placement of key genes ( rps19 , rpl2 , psbA , ndhF , ycf1 , trnN ) and minor junction length variation (25–65 bp), reinforcing plastome boundary stability [54](44). Phylogenetic Relationships Phylogenetic analysis based on complete chloroplast genome sequences revealed distinct clustering patterns among the four E. guineensis accessions (Fig. 6 ). Pisifera-Tz and Dura-Cmr formed a closely related clade, suggesting high genetic similarity and a likely shared maternal lineage. In contrast, Dura-Tz and Dura-BBG grouped separately, indicating moderate divergence that may reflect differences in evolutionary origin, geographic adaptation, or breeding history. Despite these variations, all accessions clustered within a unified species group, confirming their overall genomic relatedness and supporting their classification within E. guineensis . This pattern underscores the utility of chloroplast genomes for resolving maternal lineages while reinforcing the evolutionary cohesion of the species. These results align with previous phylogenetic studies that resolved maternal lineages in oil palm using plastome data (12,17,18). Haplotype Diversity Median-joining haplotype network analysis identified 14 distinct plastome haplotypes (HAP1–HAP14) among 48 accessions (Supplementary table 2 ). The network displayed short mutational distances and interconnected haplotypes, reflecting low divergence and shared evolutionary origin. Median vectors (mv1–mv5) represented inferred ancestral or intermediate haplotypes, providing insight into mutational pathways and lineage continuity. This moderate but structured plastome diversity supports the evolutionary stability of the plastome and offers practical utility for germplasm management and conservation of cytoplasmic diversity (Fig. 7 ). The presence of median vectors (mv1–mv5) suggests inferred ancestral haplotypes, consistent with haplotype diversity patterns reported in oil palm germplasm collections (9,28,49,51,52) Discussion The assemblies confirm the evolutionary stability of oil palm plastomes, with conserved genome sizes and GC content across accessions. This mirrors findings from broader palm plastome studies (20,24,58) and emphasizes strong selective constraints on chloroplast architecture (10,11,59). Annotation results reinforce this resilience, with highly conserved gene repertoires across accessions, consistent with previous genome sequencing efforts (10,17,60) SSR analysis revealed conserved motif landscapes across accessions, dominated by A/T mononucleotide repeats. This pattern reflects mutational bias typical of plastid genomes (14,15) and parallels SSR distributions reported in oil palm (53,54). Genic SSR analysis highlighted Pisifera-Tz as an outlier, with reduced SSR density and absence in ycf and ndh loci, paralleling reports of cytoplasmic variation in oil palm (61,62). Comparative synteny analysis confirmed conserved plastome architecture across most accessions, with structural rearrangements observed only in Indonesian Pisifera genomes. These rearrangements are consistent with recombination-driven plastome variation in palms (20,45,57). Phylogenetic analysis resolved maternal lineages, clustering Pisifera-Tz with Dura-Cmr, while haplotype network analysis (Fig. 7 ) revealed moderate diversity but strong connectivity, consistent with maternal inheritance and evolutionary stability (12,17,49). Together, these findings highlight a dual narrative: deep conservation of plastome structure and gene content across E. guineensis, coupled with subtle lineage-specific variation in SSR density, Synteny, and haplotype distribution. This balance of stability and divergence provides a robust framework for phylogenetic inference, germplasm authentication, and breeding strategies aimed at conserving cytoplasmic diversity in oil palm (8,28,52,63). Conclusions Comparative analysis of four Elaeis guineensis chloroplast genomes revealed a highly conserved plastome structure, with only minor variation in junction lengths and genome sizes. Despite this overall stability, Pisifera-Tz exhibited a markedly reduced genic SSR abundance, highlighting its distinct genomic profile. Phylogenetic and haplotype analyses further demonstrated close evolutionary relationships among accessions, while uncovering moderate chloroplast valuable diversity for lineage tracing, population genetics, and applied breeding. Together, these findings provide valuable genomic resources that strengthen evolutionary understanding and support future conservation and crop improvement strategies in oil palm. Declarations Authors' contributions : AJM and SS: Research conceptualization, implementation, data analysis, original manuscript preparation, and reviewing. RA: Data analysis and manuscript data preparation SWA and WBS: Supervising and reviewing the manuscript. AN and GIL: Providing the chloroplast genome of 19 oil palm germplasm accessions from Indonesia and reviewing the manuscript. Data availability All data supporting the findings of this study are available within the paper and its Supplementary Information. All chloroplast genome sequences generated in this study, including their associated genome annotations, are publicly available in the NCBI database under BioProject accession number PRJNA1416287 . Ethics, Consent to Participate, and Consent to Publish declarations Not applicable. Acknowledgments We appreciate the management of Bogor Botanical Gardens and the National Research and Innovation Agency (BRIN) for giving access to the Dura-BBG sample. This manuscript is part of the AJM's master's (MSc) thesis. We acknowledge the support of Revocatus Petro Mushumbusi, PhD, the Director of Tanzania Forestry Research Institute (TAFORI), and his support for AJM's graduate program at IPB University, Bogor, Indonesia. AI was used to polish the English language quality in the manuscript preparation. Funding: Ally Juma Mkude, Rare Forest Profession, Tanzania Forest Fund Redi Aditama, The Oil Palm Research Grant, the Indonesian Palm Oil Fund Management Agency (BPDPKS), GRS_20220227110350 Sudarsono Sudarsono, The Oil Palm Research Grant, the Indonesian Palm Oil Fund Management Agency (BPDPKS), GRS_20220227110350 References Qaim M, Sibhatu KT, Siregar H, Grass I. Environmental, Economic, and Social Consequences of the Oil Palm Boom. Annu Rev Resour Econ [Internet]. 2020 Oct 6;12(1):321–44. Available from: https://www.annualreviews.org/doi/10.1146/annurev-resource-110119-024922 Meijaard E, Brooks TM, Carlson KM, Slade EM, Garcia-Ulloa J, Gaveau DLA, et al. The environmental impacts of palm oil in context. Nat Plants [Internet]. 2020 Dec 7;6(12):1418–26. Available from: https://www.nature.com/articles/s41477-020-00813-w Murphy DJ, Goggin K, Paterson RRM. Oil palm in the 2020s and beyond: challenges and solutions. CABI Agric Biosci [Internet]. 2021 Oct 11;2(1):39. 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Supplementary Files AllyMkudeSupplementaryTableS1.docx AllyMkudeSupplementaryTableS2.docx AllyMkudeSupplementaryTableS3.docx AllyMkudeSupplementaryTableS4.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 19 Mar, 2026 Reviews received at journal 05 Mar, 2026 Reviews received at journal 01 Mar, 2026 Reviews received at journal 27 Feb, 2026 Reviews received at journal 24 Feb, 2026 Reviews received at journal 24 Feb, 2026 Reviewers agreed at journal 23 Feb, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers invited by journal 18 Feb, 2026 Editor assigned by journal 18 Feb, 2026 Editor invited by journal 06 Feb, 2026 Submission checks completed at journal 06 Feb, 2026 First submitted to journal 06 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8662850","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594827976,"identity":"0b1babfa-d639-4993-97db-f3143c9c214c","order_by":0,"name":"Ally Juma Mkude","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDACHgYGCQYeGzkwiwQtMmnGpGqxOZTYQLQW3Z4zhjc+5BxI33D87MEHHxjs5HQbCGgxO9tjbDnjzJ3cDWfykg1nMCQbmx0gpOU8j5k0b8+z3A0HcsykeRgOJG4jSsvff4fTDc6/IVbL2R4zaQaewwkGN4i25cyxYssenjTDmTfeGBvOMCDGL2eSN974wWMjz3c+x/DBhwo7OYJa4EABrNKAWOUgIN9AiupRMApGwSgYUQAAWKtEh1Lzu0AAAAAASUVORK5CYII=","orcid":"","institution":"Tanzanian Forestry Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Ally","middleName":"Juma","lastName":"Mkude","suffix":""},{"id":594827977,"identity":"2480a8e5-9eab-4ba6-90b7-8cbed16fa269","order_by":1,"name":"Redi Aditama","email":"","orcid":"","institution":"IPB University","correspondingAuthor":false,"prefix":"","firstName":"Redi","middleName":"","lastName":"Aditama","suffix":""},{"id":594827978,"identity":"76b5086d-83da-4fd6-99f5-13ef8357847d","order_by":2,"name":"Sintho W. 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(B) Pisifera‑Tz assembly reaching ~7,000 reads, reflecting higher sequencing throughput. Coverage is generally uniform, with peaks in inverted repeat (IR) regions and troughs in AT‑rich or non‑coding regions. Greater depth in Pisifera‑Tz enhances confidence in variant detection and structural resolution.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/9f703fda248ace34ef2c3e4c.png"},{"id":103262447,"identity":"3055a613-e016-4329-bc58-c623adaacf2b","added_by":"auto","created_at":"2026-02-23 18:32:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":235241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCircular maps of Dura‑Tz (PX957236) and Pisifera‑Tz (PX957237) chloroplast genomes.\u003c/strong\u003e Genome organization showing large single‑copy (LSC), small single‑copy (SSC), and inverted repeat (IR) regions. Gene content and order are highly conserved, with minor differences in intergenic spacers and IR–SC boundaries.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/5806845c5047248cc025d433.png"},{"id":103505263,"identity":"140a1fee-bd71-480a-aea7-b9f66397618c","added_by":"auto","created_at":"2026-02-26 13:29:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of simple sequence repeats (SSRs) across chloroplast genomes.\u003c/strong\u003e Comparison of SSR types and densities among Dura‑Tz, Pisifera‑Tz, Dura‑Cmr, and Dura‑BBG. Mononucleotide A/T repeats dominate across all plastomes, with Pisifera‑Tz showing markedly reduced genic SSR density.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/00e1774dff3203a48110b404.png"},{"id":103262449,"identity":"6d66d086-6987-4a75-97b5-2680fe158f9f","added_by":"auto","created_at":"2026-02-23 18:32:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":278350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultiple genome alignment of 16 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eElaeis guineensis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plastomes\u003c/strong\u003e. Showing structural conservation and localize rearrangements. The alignment was performed using the Mauve algorithm to identify Local Collinear Blocks (LCBs). Each horizontal track represents an individual accession, including Dura, Pisifera, and Tenera fruit forms, with the reference genome NC-017602 included for comparison. The green LCB (left) corresponds to the Large Single Copy (LSC) region, while the teal LCB (right) represents the Small Single Copy (SSC) and Inverted Repeat regions. The vertical height of the colored profiles indicates the degree of sequence conservation. Colored lined connecting the tracks denote synteny regions; diagonal red lines and displaced red blocks in accessions Pisifera-Id_221 and pisifera-Id_1109 highlight structural variations, including translocations and inversions relative to the reference. Scale bar at the top indicates genomic position in base pairs.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/6f27cba25ce50feb9848c349.png"},{"id":103506104,"identity":"a1b8f0cd-e9c1-4ec8-a05a-56dad502082c","added_by":"auto","created_at":"2026-02-26 13:34:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":512500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of inverted repeat (IR) boundary regions among16 oil palm (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eElaeis guineensis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) plastomes using IRscope\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/ac580e44d2290c3d0dfd2d96.png"},{"id":103262456,"identity":"01028557-7291-4b80-a0ff-bd2a105c8e59","added_by":"auto","created_at":"2026-02-23 18:32:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":219068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic relationships among oil palm accessions inferred using the Maximum Likelihood method and Tamura -Nei (TN93+G+I) model based on\u003c/strong\u003e \u003cstrong\u003elikelihood tree based on complete plastome sequences\u003c/strong\u003e. Pisifera‑Tz clusters with Dura‑Cmr, while Dura‑Tz and Dura‑BBG form distinct clades, reflecting multiple maternal lineages.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/d6f180b81534dc097e3c9fd1.png"},{"id":103505636,"identity":"6c4fda53-d3a2-44ed-9a30-aa758f93a145","added_by":"auto","created_at":"2026-02-26 13:32:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":133246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe fourteen haplotypes from 48 Elaeis guineensis\u003c/em\u003eplastome genomes, with circle sizes proportional to haplotype frequency and red squares (mv1–mv5) denoting inferred median vectors.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/3e8d4e2c7703609c1cc3b24f.png"},{"id":103510940,"identity":"92de3f7d-3745-48b6-b78d-3c6b8b65c38d","added_by":"auto","created_at":"2026-02-26 14:07:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2907215,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/311e9432-4143-437b-9e8e-bc024af0b895.pdf"},{"id":103506217,"identity":"27148d5c-2393-43a9-99a6-3d78de91426f","added_by":"auto","created_at":"2026-02-26 13:34:39","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15431,"visible":true,"origin":"","legend":"","description":"","filename":"AllyMkudeSupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/de6b8a431048ae846f2e655e.docx"},{"id":103506203,"identity":"4389fa8b-e747-4d3a-84f3-a5eaff234457","added_by":"auto","created_at":"2026-02-26 13:34:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":61590,"visible":true,"origin":"","legend":"","description":"","filename":"AllyMkudeSupplementaryTableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/1f2a0cef9e29a283b3c7f00d.docx"},{"id":103505122,"identity":"ddacbd8d-e046-465b-bb12-68e1de36603f","added_by":"auto","created_at":"2026-02-26 13:24:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18657,"visible":true,"origin":"","legend":"","description":"","filename":"AllyMkudeSupplementaryTableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/5b65ddc370e5bcf00fdbf2db.docx"},{"id":103506543,"identity":"366c5e9a-0fa0-442e-8401-bb6a8c20726e","added_by":"auto","created_at":"2026-02-26 13:37:31","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14544,"visible":true,"origin":"","legend":"","description":"","filename":"AllyMkudeSupplementaryTableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-8662850/v1/e9fc45e252438e05004b3a31.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Chloroplast Genomics and Maternal Lineage Diversity in Oil Palm (Elaeis guineensis)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOil palm (\u003cem\u003eElaeis guineensis\u003c/em\u003e Jacq.) is the most productive source of vegetable oil worldwide, contributing more than one-third of the global supply (1). Its high oil yield per unit area has made it central to food security, bioenergy, and industrial applications (2,3). However, the rapid expansion of oil palm cultivation has raised concerns about deforestation, biodiversity loss, and sustainability (4,5). In addition, the limited genetic base of current breeding populations constrains progress in developing resilience traits, yield stability, and climate adaptation (6,7).\u003c/p\u003e \u003cp\u003eThe narrow genetic base of Southeast Asian oil palm populations largely stems from a small number of African introductions during the colonial era (7). This founder effect has restricted genetic variation in commercially important lines, particularly the Deli Dura population in Indonesia (8). In contrast, African wild populations harbor significant allelic richness and represent untapped reservoirs of genetic diversity (8,9). Expanding cytoplasmic and nuclear variation is therefore critical for sustainable cultivation and long-term breeding success (10).\u003c/p\u003e \u003cp\u003eChloroplast genomes (plastomes) are powerful tools for studying plant diversity, evolution, and adaptation (11,12). Plastomes play essential roles in photosynthesis, energy metabolism, and biosynthesis of amino acids, fatty acids, and pigments. Their conserved quadripartite structure, uniparental inheritance, and relatively low mutation rate make them valuable for phylogenetics, species differentiation, and lineage tracing (13). In flowering plants, plastomes typically range from 120 to 160 kb and encode 110 to 130 genes involved in transcription, translation, and photosynthesis (14,15). Despite their conservation, single-nucleotide polymorphisms, indels, and structural variations in non-coding regions provide informative markers for evolutionary research, germplasm authentication, and lineage tracing (16,17).\u003c/p\u003e \u003cp\u003ePrevious studies of oil palm plastomes revealed highly conserved gene content and organization, consistent with other members of the palm family (Arecaceae) (18). The first complete \u003cem\u003eE. guineensis\u003c/em\u003e plastome established a baseline structure of ~\u0026thinsp;156,973 bp (18), and subsequent work confirmed its stability across the species (19,20). Nevertheless, comparative analyses identified nucleotide variations concentrated in single-copy regions and intergenic spacers, offering opportunities to differentiate closely related lineages (21). Commercial planting materials are derived from hybridization between Dura (thick-shelled) and Pisifera (shell-less) types to produce the high-yielding Tenera hybrid (22). Tracing maternal lineages is therefore essential for germplasm authentication and understanding cytoplasmic genetic effects (23,24)\u003c/p\u003e \u003cp\u003eGiven the narrow maternal base of commercially dominant Deli Dura lines (16), resolving plastome diversity across wild and cultivated accessions is critical for breeding programs (25,26). While nuclear genomic studies of E. guineensis and E. oleifera are extensive (27\u0026ndash;30), detailed comparative analyses of intraspecific plastome diversity remain limited. Even small numbers of variable sites within plastomes can be powerful for differentiating lineages and mapping cytoplasmic diversity (31). Characterizing plastome variation across diverse African accessions is thus essential for conservation, breeding authentication, and marker development (32).\u003c/p\u003e \u003cp\u003eTanzania and Cameroon represent centers of origin and diversity for oil palm, harboring ancient lineages and wild populations that could significantly enrich global breeding programs (9,32). In contrast, Indonesian Dura populations descend from a narrow genetic base introduced from Africa in the early 20th century (16). Comparative plastome analyses across African regions, therefore, offer unique insights into domestication history, adaptation mechanisms, and maternal lineage divergence (27)\u003c/p\u003e \u003cp\u003eRecent advances in sequencing technologies have transformed plastome research (33). Short-read platforms such as Illumina and MGI DNBSEQ provide cost-effective accuracy for sequencing and variant detection but struggle with repetitive regions and structural variations (34). Long-read platforms such as PacBio HiFi and Oxford Nanopore generate reads spanning thousands of bases, enabling resolution of repetitive elements and structural variants (35). Long reads improve assembly contiguity, facilitate haplotype phasing, and enable precise detection of complex rearrangements (35). Comparing short- and long-read assemblies provides critical validation for plastome reconstruction, particularly in complex plant lineages such as oil palm (34). However, systematic comparisons of sequencing platform performance in oil palm plastomes remain scarce.\u003c/p\u003e \u003cp\u003eThis study addresses these gaps by sequencing and analyzing chloroplast genomes of Tanzanian Dura (Dura-Tz) and Pisifera (Pisifera-Tz) accessions. We integrate these with previously sequenced Cameroonian Dura (Dura-Cmr), the ancestral Deli Dura from Indonesia (Dura-BBG), 29 Indonesian breeding lines, and 15 plastome accessions from public databases. By benchmarking MGI and PacBio sequencing platforms, we highlight their complementary strengths for plastome assembly and variant detection. Collectively, this work provides new insights into plastome diversity in E. guineensis, advances understanding of domestication history, and supports the development of plastome-based molecular markers for breeding, conservation, and genetic resource management.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOil palm chloroplast genome samples\u003c/h2\u003e \u003cp\u003eTwo chloroplast genomes were sequenced in this study, including one Tanzanian Dura type (Dura-Tz) and one Tanzanian Pisifera type (Pisifera-Tz). The selected plant materials was identified by Mr. Basil E. Kavishe, Research Assistant at Tanzania Agricultural Research Institute (TARI). No voucher specimen was deposited in publicly accessible herbarium. However, the plant materials were photographed during sample collection, and they are now monitored by TARI for reference and future studies. Young leaves were collected from the Kwitanga plantation, Kigoma, Tanzania. Samples were cleaned, cut into ~\u0026thinsp;20 cm pieces, sterilized with 70% ethanol, air-dried, sealed in airtight plastic bags, and transported in a cold box to PT Genetika Science, Indonesia, for sequencing. Two representative wild oil palm samples, one Cameroonian Dura-type (Dura-Cmr) and one Deli Dura progenitor from Bogor Botanical Garden, Indonesia (Dura-BBG), were sequenced in a previous project at PMB Lab, IPB University (NCBI accession numbers are on Progress). Moreover, as many as twenty-nine (29) samples were also from previously sequenced chloroplast genomes and obtained from Dr. Azis Natawijaya (PT Bumitama Gunajaya Agro Company). Inquiries for these 29 sequences should be directly forwarded to Dr. Azis Natawijaya. Other cpDNAs were obtained from NCBI GenBank DNA Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All chloroplast genome sequences generated in this study, including their associated genome annotations, are publicly available in the NCBI database under BioProject accession number \u003cb\u003ePRJNA1416287\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChloroplast genome sequencing and assembly\u003c/h3\u003e\n\u003cp\u003eShort-read sequencing of Dura-Tz and Pisifera-Tz total genomes was performed using the MGI DNBSEQ-G400 platform (34) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mgi-tech.eu/sequencing-products/dnbseq-g400\u003c/span\u003e\u003cspan address=\"https://mgi-tech.eu/sequencing-products/dnbseq-g400\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (PE150) with MGIEasy FS DNA Library Prep Set. Long-read sequencing of Dura-Tz total genomes was conducted using the PacBio Vega platform with HiFi Plex Prep Kit 96 (PacBio, 103-122-800) (36) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pacb.com/wp-content/uploads/Insert-HiFi-plex-prep-kit-96\u003c/span\u003e\u003cspan address=\"https://www.pacb.com/wp-content/uploads/Insert-HiFi-plex-prep-kit-96\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRaw reads of Dura-Tz and Pisifera-Tz were processed with \u003cb\u003eFastp v0.20.1\u003c/b\u003e (37) to trim low-quality sequences and adaptor contamination. De novo assembly was performed using \u003cb\u003eNOVOPlasty v4.3.5\u003c/b\u003e (38), and the assemblies were polished using \u003cb\u003eEnsemblPlants56\u003c/b\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org\u003c/span\u003e\u003cspan address=\"https://plants.ensembl.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSequencing depth and coverage of the Dura-Tz and Pisifera-Tz chloroplast genomes were evaluated by mapping raw reads back to the assembled plastomes. Depth profiles were generated across the entire genome length (0\u0026ndash;156 kb) for both Dura-Tz and Pisifera-Tz accessions. Coverage plots were inspected to confirm uniformity across large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions.\u003c/p\u003e\n\u003ch3\u003eChloroplast genome annotation\u003c/h3\u003e\n\u003cp\u003eAssembled genomes in FASTA format were annotated using \u003cb\u003eGeSeq\u003c/b\u003e within CHLOROBOX (39) and the \u003cb\u003eDual Organellar Genome Annotator (DOGMA)\u003c/b\u003e with default parameters\u0026mdash;annotation employed 95% sequence similarity thresholds for protein-coding genes, rRNAs, and tRNAs. \u003cb\u003eBLASTX\u003c/b\u003e and \u003cb\u003eBLASTN\u003c/b\u003e were used to identify coding genes and rRNAs, while \u003cb\u003etRNAscan-SE v2.0.7\u003c/b\u003e was used for tRNA annotation (40). Intron boundaries, start, and stop codons were manually curated by comparison with a combination of many related species of \u003cem\u003eArecaceae\u003c/em\u003e plastomes. Genome maps were visualized using \u003cb\u003eOGDRAW\u003c/b\u003e (41).\u003c/p\u003e\n\u003ch3\u003eComparative analysis of Plastome structure and Synteny\u003c/h3\u003e\n\u003cp\u003eUsing assembled chloroplast genomes, the base composition and frequencies of the LSC, SSC, and IR regions were determined in \u003cb\u003eGeneious Prime v2024.0.7\u003c/b\u003e (42). Two multiple progressive sequence alignments were conducted; the first one included the two new plastomes plus Dura-BBG and Dura-Cmr. Multiple sequence alignments of the plastomes were generated using \u003cb\u003eMAFFT v7.490\u003c/b\u003e (43). IR boundary variation was visualized using \u003cb\u003eIRscope\u003c/b\u003e (44). The second analysis was carried out using the two new plastomes plus Dura-BBG and Dura-Cmr and six Indonesian breeding lines (Dura-Id_574, Dura-Id_101128179, Pisifera-Id_221, Pisifera-Id_1169, Tenera-Id_1093, Tenera-Id_15730) and six plastomes that were available on NCBI (ON248756, NC_017602, OR125034, OR125040, OR125048, OR125054) in Mauve v2.4.0 (45).\u003c/p\u003e\n\u003ch3\u003eSimple sequence repeats (SSRs)\u003c/h3\u003e\n\u003cp\u003ePairwise multiple alignments of cp genomes were performed using \u003cb\u003eMAFFT v7.490\u003c/b\u003e (43) in Geneious Prime. Simple sequence repeats (SSRs) containing 1\u0026ndash;6 nucleotides were identified using the \u003cb\u003ePhobos v3.3.12\u003c/b\u003e (46). The following configuration was adopted to search for SSR motifs: SSR of one to six nucleotides long, with a minimum repeat number of 10, 5, and 4 units for mono-, di-, and trinucleotide SSRs, respectively, and three units for tetra-, penta-, and hexanucleotide SSRs.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic and haplotype analysis\u003c/h2\u003e \u003cp\u003ePhylogenetic analysis included 48 E. guineensis cp genomes. Multiple sequence alignments were generated using \u003cb\u003eMAFFT v7.490\u003c/b\u003e (43). Phylogenetic trees were constructed using the \u003cb\u003eMaximum Likelihood\u003c/b\u003e (ML) method with the \u003cb\u003eTamura-Nei (TN93/TNG93\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I)\u003c/b\u003e genetic distance model, and \u003cb\u003e1000 bootstrap replications\u003c/b\u003e (47,48). Haplotype networks were constructed from 48 E. guineensis cp genomes, including 19 PT Bumitama Gunajaya Agro accessions, 25 NCBI accessions, and 4 genomes sequenced in the PMB lab at IPB University. Alignments were performed in Geneious Prime, and haplotype networks were generated using \u003cb\u003eNETWORK software\u003c/b\u003e (49).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eChloroplast Genome Sequencing\u003c/h2\u003e\n \u003cp\u003eSequencing output is summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. For the Dura‑Tz and Pisifera‑Tz accessions sequenced with MGI, approximately 242 million reads were generated per sample, corresponding to ~\u0026thinsp;36 Gb of total bases. In contrast, PacBio sequencing of Dura‑Tz produced only 5.95 million reads but yielded a substantially higher total base output of 63.33 Gb (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSequencing data summary for Tanzanian Dura (Dura-Tz) and Pisifera (Pisifera-Tz)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSequencing Platform\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal reads (M.)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal bases (Gb)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDura-Tz\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMGI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e241.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePacBio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePisifera-Tz\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMGI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e242.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThese results highlight the complementary strengths of the two platforms. MGI provides deep coverage through massive read counts, ensuring reliable variant detection and uniform base representation. PacBio, despite generating fewer reads, delivers long sequences that enhance assembly contiguity and resolve repetitive regions more effectively. Together, the datasets demonstrate that combining short‑read and long‑read technologies offers both depth and structural accuracy for plastome reconstruction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eSequencing Depth and Coverage\u003c/h2\u003e\n \u003cp\u003eRobust sequencing coverage was achieved across the chloroplast genomes of both Dura-Tz and Pisifera-Tz accessions. The Dura-Tz assembly reached depths of up to ~\u0026thinsp;3,500 reads (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), while Pisifera-Tz attained nearly 7,000 reads (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), reflecting its substantially higher sequencing throughput. Coverage profiles were generally uniform across the plastome, with expected peaks in the inverted repeat (IR) regions due to duplication and troughs in AT-rich or non-coding regions that are inherently more challenging to sequence.\u003c/p\u003e\n \u003cp\u003eThe overall consistency of coverage confirms the completeness and reliability of both assemblies. Significantly, the greater depth observed in Pisifera-Tz enhances confidence in variant detection and structural resolution, thereby providing a stronger foundation for downstream comparative and phylogenetic analyses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eChloroplast Genome Assembly and Structure\u003c/h2\u003e\n \u003cp\u003eThe complete chloroplast genomes of Dura‑Tz (PX957236) and Pisifera‑Tz (PX957237) were assembled into circular maps of 156,883 bp, each displaying the typical quadripartite organization comprising large single‑copy (LSC), small single‑copy (SSC), and inverted repeat (IR) regions (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Comparative analysis with previously sequenced accessions (Dura‑Cmr and Dura‑BBG) revealed highly conserved structural and compositional features (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings are consistent with earlier oil palm plastome characterizations (23,50) and broader plastid genome studies in seed plants (11,13,14, 23).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary of general features of 4 accessions of \u003cem\u003eElaeis guineensis\u003c/em\u003e chloroplast-genomes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAttribute\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDura-Tz\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePisifera-Tz\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDura-Cmr\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDura-BBG\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGenome Size (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156,983\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156,983\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156,982\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e156,990\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGC content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLSC size (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85,194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85,194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85,193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85,198\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSSC size (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17,643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17,643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17,643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17,644\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIRs size (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27,073\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27,073\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27,073\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27,074\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLSC GC content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSSC GC content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIRs GC content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e115\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of protein-coding genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of tRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of rRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eChloroplast Genome Annotation and Gene Content\u003c/h2\u003e\n \u003cp\u003eAnnotation identified a consistent set of 115 genes across all accessions, comprising 79\u0026ndash;80 protein‑coding genes, 31\u0026ndash;32 tRNAs, and 4 rRNAs. Dura-Tz contained 79 protein-coding genes and 32 tRNAs, compared with 80/31 in others (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). This subtle difference may reflect lineage-specific variation in gene annotation or minor events of tRNA duplication or loss, possibly involving \u003cem\u003eycf1\u003c/em\u003e or related loci. Overall, gene composition remains highly stable, underscoring the structural conservation of E. guineensis plastomes. Such uniformity highlights the evolutionary stability of the chloroplast genome, while slight differences in gene counts may serve as useful markers for lineage differentiation and comparative genomics.\u003c/p\u003e\n \u003cp\u003eFunctional categorization revealed a conserved organization across all four accessions, including genes encoding photosystem components, ribosomal proteins, ATP synthase, NADH dehydrogenase, Rubisco, RNA polymerase, and proteins involved in DNA replication and repair. Transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were distributed throughout the plastome, ensuring its translational capacity. Hypothetical chloroplast reading frames (ycf) were also identified, consistent with their conserved presence in angiosperm plastomes. Gene transcription orientation was bidirectional, with genes on the outer ring transcribed clockwise and those on the inner ring transcribed counterclockwise. Core functional categories\u0026mdash;photosynthesis, transcription, translation, and genome maintenance\u0026mdash;were consistently represented, in line with previous annotations of oil palm plastomes (10,17,19).\u003c/p\u003e\n \u003cp\u003eTogether, these findings confirm the high degree of conservation in gene content and organization among oil palm plastomes. The stability observed between Dura‑Tz and Pisifera‑Tz, along with other accessions, provides a reliable foundation for comparative and evolutionary analyses. At the same time, minor lineage‑specific variations can serve as potential markers of genetic differentiation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eSSR Distribution and Motif Composition\u003c/h2\u003e\n \u003cp\u003eAnalysis of simple sequence repeats (SSRs) across the four accessions\u0026mdash;Dura‑BBG, Dura‑CMR, Pisifera‑Tz, and Dura‑Tz\u0026mdash;revealed a highly conserved repeat landscape (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Table 1). Total SSR counts ranged narrowly from 859 to 860 per plastome, indicating strong structural stability across lineages.\u003c/p\u003e\n \u003cp\u003eMononucleotide repeats dominated the SSR profile, averaging 338.5 per accession, with a pronounced bias toward A/T motifs. Each accession contained approximately 317 A/T mononucleotide repeats, accounting for over 93% of the total, while G/C repeats were rare (22 per plastome). This A/T enrichment reflects the low GC content and mutational bias typical of plastid genomes (10,17)\u003c/p\u003e\n \u003cp\u003eDinucleotide repeats were equally conserved, with 58 per accession. AT and TA motifs were the most abundant, comprising 144 of the 232 total dinucleotide repeats (62%). Other motifs such as AG, GA, TC, and CT were evenly distributed across accessions, with no lineage-specific enrichment. Trinucleotide and tetranucleotide motifs also showed uniform distribution, with 71 and 95 repeats per accession, respectively. Trinucleotide motifs were dominated by A/T-rich sequences such as TAT, AAG, and AAC, while tetranucleotide repeats included AAAT, AATA, ATTA, and TTTA. These patterns reinforce the A/T bias observed in shorter motifs and suggest that even longer SSRs retain compositional consistency across accessions.\u003c/p\u003e\n \u003cp\u003ePentanucleotide repeats were similarly conserved, with 134 motifs per accession. Dominant sequences included AAAAT, AATTT, TTTTA, and TTAAA, all of which are rich in A/T bases. The recurrence of these motifs across all accessions further supports the plastome\u0026rsquo;s mutational bias and structural stability. No accession-specific expansion or contraction of pentanucleotide motifs was observed.\u003c/p\u003e\n \u003cp\u003eHexanucleotide repeats showed slightly more diversity, with counts ranging from 157 to 158 per accession. Despite the broader sequence range, dominant motifs remained A/T-rich, including TTCTTT, TTTTAT, and TTTTTT. The consistent counts and motif composition across accessions suggest that even the longest SSRs are evolutionarily stable and not subject to lineage-specific expansion.\u003c/p\u003e\n \u003cp\u003eHexanucleotide repeats showed minor variation (157\u0026ndash;158), likely reflecting annotation differences or sequencing noise rather than true biological divergence. Although Pisifera-Tz exhibited a marginally higher total SSR count (860 vs. 859 in other accessions), its genic SSR density was substantially reduced (277 vs. ~681\u0026ndash;682). This indicates that while overall SSR abundance remains stable, Pisifera-Tz plastomes have undergone lineage-specific contraction in genic regions (see Supplementary Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u0026rdquo;\u003c/p\u003e\n \u003cp\u003eOverall, the uniformity of SSR types and motif composition across all accessions underscores the evolutionary stability of the oil palm plastome. While SSR variability is limited, conserved tetranucleotide, pentanucleotide, and hexanucleotide motifs\u0026mdash;particularly those rich in A/T bases\u0026mdash;may still serve as neutral molecular markers for population genetics, germplasm authentication, and comparative genomics. This conserved SSR profile highlights plastome stability and mirrors SSR distributions reported in other palm species (8,51,52). SSR marker development in oil palm has similarly emphasized A/T-rich motifs (53,54).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eGenic SSRs Across Accessions\u003c/h2\u003e\n \u003cp\u003eComparative analysis of genic SSRs revealed both broad conservation and striking lineage‑specific differences among the four E. guineensis chloroplast genomes (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Dura‑Cmr and Dura‑BBG each contained 682 genic SSRs, while Dura‑Tz harbored 681, reflecting near‑identical SSR abundance and distribution across genes associated with photosynthesis, replication, and genome maintenance. This consistency underscores the evolutionary stability of genic SSRs in most oil palm accessions, with plastome function preserved through balanced representation across essential pathways.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary of genic SSRs in four \u003cem\u003eElaeis guineensis\u003c/em\u003e chloroplast genomes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenome\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal genic SSRs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene categories with SSRs\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDura-Cmr/Dura-BBG/Dura-Tz\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e681\u0026ndash;682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhotosynthesis\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eatp\u003c/em\u003e (B/E/F/I), \u003cem\u003eccsA\u003c/em\u003e, \u003cem\u003ecemA\u003c/em\u003e, \u003cem\u003endH\u003c/em\u003e (A/B/D/E/H/I/K), \u003cem\u003epaf\u003c/em\u003e (I/II), \u003cem\u003epet\u003c/em\u003e (A/B/D), \u003cem\u003epsa\u003c/em\u003e (A/B/J), psb (A/B/C/F/I/K/L/M/T/Z), \u003cem\u003erbcL.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMaintenance\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003eclpP1\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003erpl\u003c/em\u003e (2/16/22/32/33), \u003cem\u003erps\u003c/em\u003e (3/4/7/11/12/14/15/16/18/19), \u003cem\u003eycf\u003c/em\u003e (1/2)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eReplication\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003e\u003cem\u003erpo\u003c/em\u003e A/B/C1/C2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePisifera-Tz\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e277\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhotosynthesis\u003c/strong\u003e: \u003cem\u003eatp\u003c/em\u003e (B/E/F/I), \u003cem\u003ecemA\u003c/em\u003e, \u003cem\u003endhK\u003c/em\u003e, \u003cem\u003epaf\u003c/em\u003e I/II, \u003cem\u003epet\u003c/em\u003e A/B/D, \u003cem\u003epsa\u003c/em\u003e A/B/I/J, \u003cem\u003epsb\u003c/em\u003e (A/B/C/F/I/K/L/M/T/Z), \u003cem\u003erbcL\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMaintenance\u003c/strong\u003e: \u003cem\u003eaccD\u003c/em\u003e, \u003cem\u003eclpP1\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003erpl\u003c/em\u003e (16/22/33), \u003cem\u003erps\u003c/em\u003e (3/4/11/12/14/16/18)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eReplication\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003e\u003cem\u003erpo\u003c/em\u003e A/B/C1/C2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eIn contrast, Pisifera‑Tz exhibited a markedly reduced genic SSR count (277), representing a substantial contraction relative to the other accessions. Despite this reduction, SSRs in Pisifera‑Tz were still distributed across key functional genes, including rbcL, matK, psa, psb, and rpo, ensuring that core photosynthetic and transcriptional processes remained represented. However, the absence of SSRs in several maintenance‑related loci\u0026mdash;including ycf1, ycf2, and members of the ndh family\u0026mdash;suggests lineage‑specific loss or reduced SSR density in regions typically associated with genome stability and stress response.\u003c/p\u003e\n \u003cp\u003eThis pattern highlights two complementary insights: first, the conserved SSR profiles in Dura accessions reflect plastome resilience and functional redundancy across diverse lineages; second, the reduced SSR density in Pisifera‑Tz may indicate either structural streamlining or selective pressures that minimized repetitive elements in its plastome. From a practical perspective, these differences provide opportunities for marker development: conserved genic SSRs in Dura accessions can serve as stable lineage markers. At the same time, the unique contraction in Pisifera‑Tz offers diagnostic motifs for distinguishing maternal lineages in breeding programs. Despite this contraction, SSRs remained present in key functional genes, including \u003cem\u003erbcL\u003c/em\u003e, \u003cem\u003ematK\u003c/em\u003e, \u003cem\u003epsa\u003c/em\u003e, \u003cem\u003epsb\u003c/em\u003e, and \u003cem\u003erpo\u003c/em\u003e. The absence of SSRs in \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003eycf2\u003c/em\u003e, and \u003cem\u003endh\u003c/em\u003e family genes suggests lineage-specific streamlining, consistent with plastome variation observed in Pisifera lineages (55,56)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003ePlastome Structure and Synteny\u003c/h2\u003e\n \u003cp\u003eMultiple genome alignment of 16 Elaeis guineensis chloroplast genomes\u0026mdash;representing Dura, Tenera, and Pisifera\u0026mdash;revealed a high degree of structural conservation across accessions (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Using the Mauve algorithm, plastome sizes ranged narrowly from 153,000 to 158,000 bp, with most accessions exhibiting uniform syntenic block arrangements and stable genome architecture. This consistency reflects strong evolutionary constraints on plastome organization, likely driven by the essential roles of chloroplast genes in photosynthesis and cellular metabolism.\u003c/p\u003e\n \u003cp\u003eNotably, two Indonesian Pisifera accessions (Pisifera‑Id_221 and Pisifera‑Id_101128179) displayed structural rearrangements in the 140\u0026ndash;145 kb region, characterized by translocations and/or inversions (highlighted in red blocks). These localized disruptions suggest lineage‑specific plastome remodeling, potentially arising from recombination events or selective pressures unique to the Pisifera background. While the overall plastome structure remains conserved, such rearrangements may influence gene regulation or plastome stability, warranting further investigation into their functional consequences.\u003c/p\u003e\n \u003cp\u003eComplementary mapping of the 16 plastomes confirmed the conservation of junction architecture between the large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). All accessions shared nearly identical genome sizes (~\u0026thinsp;156.98 kb) and consistent placement of key genes at junctions, including rps19, rpl2, psbA, ndhF, ycf1, and trnN. Minor differences in junction lengths (25\u0026ndash;65 bp) were observed but did not affect gene orientation or overall structural layout.\u003c/p\u003e\n \u003cp\u003eThese findings reinforce the evolutionary stability of plastome boundaries in E. guineensis and validate their utility for comparative and phylogenetic analyses. The conserved junctions and syntenic blocks across most accessions provide a robust framework for plastome‑based lineage tracing. In contrast, the structural variation in select Pisifera accessions offers a unique opportunity to explore plastome dynamics and their potential role in fruit form differentiation or adaptation. Such rearrangements have been linked to recombination events in plastomes of palms and other monocots (20,24,45,57) Comparative mapping of LSC, SSC, and IR junctions confirmed consistent placement of key genes (\u003cem\u003erps19\u003c/em\u003e, \u003cem\u003erpl2\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e, \u003cem\u003endhF\u003c/em\u003e, \u003cem\u003eycf1\u003c/em\u003e, \u003cem\u003etrnN\u003c/em\u003e) and minor junction length variation (25\u0026ndash;65 bp), reinforcing plastome boundary stability [54](44).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003ePhylogenetic Relationships\u003c/h2\u003e\n \u003cp\u003ePhylogenetic analysis based on complete chloroplast genome sequences revealed distinct clustering patterns among the four E. guineensis accessions (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Pisifera-Tz and Dura-Cmr formed a closely related clade, suggesting high genetic similarity and a likely shared maternal lineage. In contrast, Dura-Tz and Dura-BBG grouped separately, indicating moderate divergence that may reflect differences in evolutionary origin, geographic adaptation, or breeding history. Despite these variations, all accessions clustered within a unified species group, confirming their overall genomic relatedness and supporting their classification within \u003cem\u003eE. guineensis\u003c/em\u003e. This pattern underscores the utility of chloroplast genomes for resolving maternal lineages while reinforcing the evolutionary cohesion of the species. These results align with previous phylogenetic studies that resolved maternal lineages in oil palm using plastome data (12,17,18).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eHaplotype Diversity\u003c/h2\u003e\n \u003cp\u003eMedian-joining haplotype network analysis identified 14 distinct plastome haplotypes (HAP1\u0026ndash;HAP14) among 48 accessions (Supplementary table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The network displayed short mutational distances and interconnected haplotypes, reflecting low divergence and shared evolutionary origin. Median vectors (mv1\u0026ndash;mv5) represented inferred ancestral or intermediate haplotypes, providing insight into mutational pathways and lineage continuity. This moderate but structured plastome diversity supports the evolutionary stability of the plastome and offers practical utility for germplasm management and conservation of cytoplasmic diversity (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). The presence of median vectors (mv1\u0026ndash;mv5) suggests inferred ancestral haplotypes, consistent with haplotype diversity patterns reported in oil palm germplasm collections (9,28,49,51,52)\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe assemblies confirm the evolutionary stability of oil palm plastomes, with conserved genome sizes and GC content across accessions. This mirrors findings from broader palm plastome studies (20,24,58) and emphasizes strong selective constraints on chloroplast architecture (10,11,59). Annotation results reinforce this resilience, with highly conserved gene repertoires across accessions, consistent with previous genome sequencing efforts (10,17,60)\u003c/p\u003e \u003cp\u003eSSR analysis revealed conserved motif landscapes across accessions, dominated by A/T mononucleotide repeats. This pattern reflects mutational bias typical of plastid genomes (14,15) and parallels SSR distributions reported in oil palm (53,54). Genic SSR analysis highlighted Pisifera-Tz as an outlier, with reduced SSR density and absence in \u003cem\u003eycf\u003c/em\u003e and \u003cem\u003endh\u003c/em\u003e loci, paralleling reports of cytoplasmic variation in oil palm (61,62).\u003c/p\u003e \u003cp\u003eComparative synteny analysis confirmed conserved plastome architecture across most accessions, with structural rearrangements observed only in Indonesian Pisifera genomes. These rearrangements are consistent with recombination-driven plastome variation in palms (20,45,57). Phylogenetic analysis resolved maternal lineages, clustering Pisifera-Tz with Dura-Cmr, while haplotype network analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) revealed moderate diversity but strong connectivity, consistent with maternal inheritance and evolutionary stability (12,17,49).\u003c/p\u003e \u003cp\u003eTogether, these findings highlight a dual narrative: deep conservation of plastome structure and gene content across E. guineensis, coupled with subtle lineage-specific variation in SSR density, Synteny, and haplotype distribution. This balance of stability and divergence provides a robust framework for phylogenetic inference, germplasm authentication, and breeding strategies aimed at conserving cytoplasmic diversity in oil palm (8,28,52,63).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eComparative analysis of four \u003cem\u003eElaeis guineensis\u003c/em\u003e chloroplast genomes revealed a highly conserved plastome structure, with only minor variation in junction lengths and genome sizes. Despite this overall stability, Pisifera-Tz exhibited a markedly reduced genic SSR abundance, highlighting its distinct genomic profile. Phylogenetic and haplotype analyses further demonstrated close evolutionary relationships among accessions, while uncovering moderate chloroplast valuable diversity for lineage tracing, population genetics, and applied breeding. Together, these findings provide valuable genomic resources that strengthen evolutionary understanding and support future conservation and crop improvement strategies in oil palm.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAJM and SS: Research conceptualization, implementation, data analysis, original manuscript preparation, and reviewing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRA: Data analysis and manuscript data preparation\u003c/p\u003e\n\u003cp\u003eSWA and WBS: Supervising and reviewing the manuscript.\u003c/p\u003e\n\u003cp\u003eAN and GIL: Providing the chloroplast genome of 19 oil palm germplasm accessions from Indonesia and reviewing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information. All chloroplast genome sequences generated in this study, including their associated genome annotations, are publicly available in the NCBI database under BioProject accession number \u003cstrong\u003ePRJNA1416287\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate, and Consent to Publish declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the management of Bogor Botanical Gardens and the National Research and Innovation Agency (BRIN) for giving access to the Dura-BBG sample. This manuscript is part of the AJM's master's (MSc) thesis. We acknowledge the support of Revocatus Petro Mushumbusi, PhD, the Director of Tanzania Forestry Research Institute (TAFORI), and his support for AJM's graduate program at IPB University, Bogor, Indonesia. AI was used to polish the English language quality in the manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eAlly Juma Mkude, Rare Forest Profession, Tanzania Forest Fund\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eRedi Aditama, The Oil Palm Research Grant, the Indonesian Palm Oil Fund Management Agency (BPDPKS), GRS_20220227110350\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSudarsono Sudarsono, The Oil Palm Research Grant, the Indonesian Palm Oil Fund Management Agency (BPDPKS), GRS_20220227110350\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQaim M, Sibhatu KT, Siregar H, Grass I. Environmental, Economic, and Social Consequences of the Oil Palm Boom. Annu Rev Resour Econ [Internet]. 2020 Oct 6;12(1):321\u0026ndash;44. 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Available from: https://jms.mabjournal.com/index.php/mab/article/view/2975\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Oil palm, chloroplast genome, plastome diversity, SSR variation, phylogenetics, maternal lineage","lastPublishedDoi":"10.21203/rs.3.rs-8662850/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8662850/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOil palm (\u003cem\u003eElaeis guineensis\u003c/em\u003e) is the world\u0026rsquo;s most important source of vegetable oil, yet comparative studies of its chloroplast genomes remain limited, particularly for African accessions. Understanding plastome diversity and maternal lineage variation is critical for tracing domestication history and developing molecular markers for breeding and conservation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe report the first complete chloroplast genomes from Tanzanian Dura and Pisifera, Cameroonian Dura, and the ancestral Deli Dura from Indonesia, analyzed alongside 44 additional plastomes. Comparative analyses revealed highly conserved genome organization and gene content, with subtle differences in IR\u0026ndash;SC boundaries and intergenic spacers. Simple sequence repeat (SSR) profiling confirmed the dominance of A/T mononucleotide repeats but highlighted lineage-specific variation, including a markedly reduced genic SSR density in Pisifera-Tz, while total SSR counts remained stable across accessions (\u0026asymp;\u0026thinsp;859\u0026ndash;860; Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Phylogenetic reconstruction clustered Pisifera-Tz with Dura-Cmr, while Dura-Tz and Dura-BBG formed distinct clades, reflecting multiple maternal lineages. Haplotype network analysis identified 14 distinct haplotypes, revealing moderate but structured plastome diversity. Benchmarking of sequencing platforms demonstrated complementary strengths: MGI provided deep coverage, while PacBio improved assembly contiguity and resolution of repetitive regions.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study expands plastome resources for \u003cem\u003eE. guineensis\u003c/em\u003e, generating the first Tanzanian and Cameroonian chloroplast genomes and uncovering lineage-specific SSR variation. Integration of comparative genomics, phylogenetics, and haplotype analysis advances understanding of oil palm domestication and supports the development of plastome-based molecular markers for breeding, conservation, and genetic resource management.\u003c/p\u003e","manuscriptTitle":"Comparative Chloroplast Genomics and Maternal Lineage Diversity in Oil Palm (Elaeis guineensis)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 18:32:08","doi":"10.21203/rs.3.rs-8662850/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-19T20:09:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-05T10:29:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-01T21:29:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-27T14:15:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T03:22:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T03:13:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117843563695271243294070940411679095060","date":"2026-02-24T03:27:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41294436877869304934275901962533582021","date":"2026-02-19T13:07:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280809109341340313500433158698014268886","date":"2026-02-19T12:16:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109284986128218017380306639830053353562","date":"2026-02-19T08:32:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331250881095119248581629524168550007697","date":"2026-02-19T03:35:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-19T03:23:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-19T03:21:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-06T19:55:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T17:10:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2026-02-06T16:58:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e4f42baa-3ac8-4b54-abc1-7910954d4fb8","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-19T20:23:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 18:32:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8662850","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8662850","identity":"rs-8662850","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}