GWAS determined genetic loci associated with callus induction in oil palm tissue culture

E�cient callus induction is vital for successful oil palm tissue culture, yet identifying genomic loci and markers for early detection of genotypes with high potential of callus induction remains unclear. In this study, immature male in�orescences from 198 oil palm accessions (dura, tenera and pisifera) were used as explants for tissue culture. Callus induction rates were collected at one, two-and three-months after inoculation (C1, C2 and C3) as phenotypes. Resequencing generated 11,475,258 high quality single nucleotide polymorphisms (SNPs) as genotypes. GWAS was then performed and correlation analysis revealed a positive association of C1 with both C2 (R=0.81) and C3 (R=0.50), indicating that C1 could be used as the major phenotype for callus induction rate. Therefore, only signi�cant SNPs in C1 (including samples with and without callus) were identi�ed to develop markers for screeningindividuals with high potential of callus induction. Among 21 signi�cant SNPs (P ≤ 0.05) in C1, LD block analysis revealed 6 SNPs on chromosome 12 (Chr12) potentially linked to callus formation. Subsequently, 13 SNP markers were identi�ed from these loci and electrophoresis results showed that marker C-12 at locus Chr12_12704856 can be used effectively to distinguish the GG allele, which showed the highest probability (69%) of callus induction. Furthermore, a rapid SNP variant detection method without electrophoresis was established via qPCR-based melting curve analysis. Our �ndings facilitated marker-assisted selection for speci�c palms with high potential of callus induction using immature male in�orescence as explant, aiding ortet palm selection in oil palm tissue culture.


Introduction
Oil palm (Elaeis guineensis Jacq.) is one of the most important tropical oil crops in the world.With the growing global population, ensuring the sustainable development of the palm oil industry is crucial to meet the increasing demand for palm oil consumption (Wang et al. 2019).However, the sustainability and productivity of oil palm plantations, crucial for the global vegetable oil industry, face signi cant challenges.The growth in yield is hampered by shrinking planted areas and reduced arable land.Several factors, such as crop loss to pests and diseases, impact productivity.Unpredictable weather patterns due to climate change affect fruiting cycles and overall productivity.Maintaining soil fertility and moisture content, especially in peatland areas, remains a persistent challenge, resulting in substantial yield losses (Parveez et al. 2023).The use of tissue culture to produce oil palm clones enables rapid multiplication of genetically superior palms while maintaining their desirable traits.Tissue culture accelerates propagation, preserves genetic purity, and ensures uniform and predictable plantations.Furthermore, it integrates genetic engineering and molecular breeding, enhancing the overall genetic quality of oil palms.The challenges faced by oil palm plantations demand a comprehensive approach that combines traditional breeding with modern biotechnological advancements, essential for developing varieties with desired traits and ensuring the industry's long-term sustainability (Low et al. 2008;Sahara et al. 2023).Somatic embryogenesis (SE) is an effective technique for the mass plant production, particularly in perennial crops like oil palm (Zhang et al. 2021).Despite its importance, a notable obstacle in oil palm tissue culture lies in the restricted rate of somatic embryogenesis (Ooi et al. 2021;Sahara et al. 2023).
The successful development of somatic embryos relies on e cient embryogenic callus induction, making it a critical step for subsequent somatic embryo development.In the aspect of improving phenotypic traits through genetic engineering, it is important for the callus derived from recalcitrant plant species to be generable to intact plant (Tuskan et al. 2018).
Recent research conducted by (Ong-Abdullah and Siew Eng 2007) has identi ed key genes, such as EgPER1, EgHOX1, and EgPK1, that distinguish between embryogenic and non-embryogenic calli in oil palm tissue culture.In a study by (Low et al. 2008), candidate genes were pinpointed as being differentially expressed during callogenesis and embryogenesis in oil palm.These genes include lipid transfer proteins, metallothionein-like proteins, glutathione S-transferase, and dehydrin-like protein.Ooi et al. (2021) proposed in their article that genes linked to owering time, light response, photosynthesis, and stress response may be correlated with somatic embryogenesis potential in oil palm.Sahara et al. (2023) reported the transcriptome pro ling of high-and low-embryogenic ortets of oil palm at the callus and somatic embryoid stages using RNA-seq.This article contributes potential differentially expressed genes (DEGs) for high-embryogenic ortets, thereby enhancing the e ciency of oil palm micropropagation.
Recent research on maize has highlighted the genotype-dependent nature of embryogenic callus induction (Liang et al. 2023).Several genes responsible for callus induction have been identi ed in numerous plant species.For instance, the WAK gene family in Chinese cabbage has shown potential involvement in callus cell growth and reproduction (Zhang et al. 2020a).In Arabidopsis, LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes play a key role in callus induction (Fan et al. 2012a).The AtbZIP59-LBD complex is crucial in regulating auxin-induced callus formation (Xu et al. 2018), while CYCLIN D3 (CYCD3) is involved in wounding-induced callus formation (Ikeuchi et al. 2017).Moreover, NAC DOMAIN CONTAINING PROTEIN71 (ANAC071) and AP2/ERF transcription factor RAP2.6L have been identi ed as essential regulators during wound-induced callus formation process in Arabidopsis (Ikeuchi et al. 2013).Studies in cotton have indicated that the WOX genes play a pivotal role in callus induction (Muhammad Tajo et al. 2022).Overexpression of ZmBBM2 promotes callus formation (Du et al. 2019), and ZmMYB138 transcription factor being another promoter of callus formation via GA signal transduction in maize (Ge et al. 2016).Additionally, ZmARF23 mediates callus induction by binding to the ZmSAUR15 promoter and enhancing its transcriptional expression (Liang et al. 2023).In Panax ginseng, silencing the PgWRKY6 gene reduced the rate of embryogenic callus induction (Yang et al. 2020).As genetic factors play a crucial role in callus induction and proliferation, identifying genes and regulatory elements responsible for controlling callus formation can provide valuable insights into developing in vitro systems for recalcitrant plant species especially in oil palm (Tuskan et al. 2018;Weckx et al. 2019).
Genome-wide association study (GWAS) has emerged as an effective tool to investigate the associations between genotypes and phenotypes, facilitating the identi cation of single nucleotide polymorphisms (SNPs) linked to phenotypic variation within a population (Alqudah et al. 2020).GWAS have been conducted in oil palm to identify markers and candidate genes associated with various traits.These studies have used genotyping-by-sequencing (GBS) and SNP analysis to identify SNPs that are signi cantly associated with traits such as height (Babu et al. 2019b;Somyong et al. 2022), morphological and yield-related traits (Osorio-Guarín et al. 2019), fatty acid composition (Xia et al. 2019), and leaf area, rachis length and total dry weight (Babu et al. 2019a).These ndings have the potential to improve the e ciency and effectiveness of oil palm breeding programs through marker-assisted selection (MAS) and targeted functional analyses.The method's adaptability across different plant species, including oil palm (Babu et  In this study, callus induction rates at 1-, 2-, and 3-months after inoculation (C1, C2 and C3) were observed in immature male in orescences of 198 oil palm accessions for phenotyping, along with genotyping by resequencing.GWAS was then conducted accordingly to identify SNP loci linked to callus induction rates.Subsequently, a specialized SNP marker, designed to distinguish alleles with a high potential for callus induction, was developed to facilitate the process of ortet palm selection at early stages.This strategic marker not only expedites the identi cation of promising ortets but also addresses challenges related to the e cient and accurate selection of ortet palm.The issues encountered by the selection of ortet palms, such as variability in callus induction potential and genotypic complexities, are mitigated by the precision and discriminatory power of the developed SNP marker.The utilization of this SNP marker in the early stages allows for rapid and targeted identi cation of ortets with high potential of callus induction, signi cantly enhancing the e ciency of oil palm breeding programs.

Plant materials
Oil palm trees were planted in the National Germplasm Nursery for Tropical Palms in Wenchang, Hainan, China.A total of 198 oil palm trees (including dura, tenera and pisifera) around 12 years old were selected in the present study, and immature male in orescence located at 12th -16th fronds from each accession was collected for callus induction and resequencing.Sampling period was in the local drought season from March to September of the same year.
Subculturing was conducted for treatments C2 and C3, with intervals set at one month after C1 (resulting in C2) and another month after C2 (resulting in C3).Callus induction rates of 198 accessions were calculated simultaneously during each subculturing event.The callus induction rate of each accession was recorded using the formula: The phenotyping of callus morphology was conducted through visual scoring, involving a qualitative assessment of key morphological features, such as color, texture, and structure, as illustrated in Fig. 1a.
The patterns depicted in Fig. 1a was de ned as callus and used for calculating the callus induction rate.Subsequently, samples with and without callus at C1, C2 and C3 stages of all 198 accessions were collected, immediately frozen in liquid nitrogen and stored at -80˚C for subsequent analyses.

Genotyping
The DNA extraction and puri cation was performed using the CTAB method according to the BGI (Beijing Genomics Institute) manufacturer's protocol.The DNA quantity and quality were measured with a DS-11 Spectrophotometer (DeNovix, USA) and agarose gel electrophoresis.Whole genome resequencing for association mapping was performed using whole genome sequencing library preparation method (DNBSEQ, BGI, China).Resequencing data was ltered using SOAPnuke software (v1.5.6) (Chen et al.

Population genetic analysis
The genetic assignment based on the SNP pro les were performed using ADMIXTURE (v1.3) (Alexander et al. 2009).This unlinked SNP set was selected from ltered SNPs by removing SNPs with linkage disequilibrium (LD, r 2 ) above 0.2 using plink (v1.9) (Chang et al. 2015).The ADMIXTURE was run with the cross validation (CV) ag specifying from K = 1 to K = 9 clusters, the lowest cross-validation error was chosen as the best K.The R package pophelper (v2.

GWAS for callus induction
The GWAS for callus induction trait was performed using Gemma software (v0.98.1) (Zhou and Stephens 2012) to implement the calculation of four models: General Linear Model (GLM), GLM with population structure (GLM-Q), Mixed linear model (MLM) with kinship matrix (MLM-K), and MLM with both population structure and kinship matrix (MLM-QK).The population structure matrix corresponding to the optimal K value of Admixture is used as the Q matrix of the corresponding model, and the inter-sample a nity matrix calculated by the GCTA software is used as the K matrix of the corresponding model.The R package ggplot2 (Wickham 2016) was used to visualize the Manhattan and quantile quantile (QQ) plots.
Bonferroni correction threshold (p-value = 0.01/marker number or 0.05/marker number) was used to identify signi cant associations.Subsequently, to gain biological insights into the potential functional relevance of the identi ed associations, candidate genes in proximity to the signi cant markers were identi ed.To achieve this, a genomic window extending 50kb both upstream and downstream of each signi cant associated marker was focused.Within these anking regions, we systematically identi ed candidate genes based on their proximity to the signi cant markers.The rationale behind this approach is to consider genes that may be under the in uence of regulatory elements or genetic variants in close genomic proximity.This strategy aims to prioritize genes that could be functionally linked to the observed associations and provides a biologically relevant context for the identi ed genetic markers.All signi cant SNPs (P ≤ 0.5) obtained from MLM(Q + K) model GWAS were used to identify potential candidate genes associated with callus induction trait.The functional annotation information of candidate genes was obtained from National Center for Biotechnology Information Database (NCBI, https://www.ncbi.nlm.nih.gov/).The LD was visualized and haplotype blocks were constructed using the LDBlockShow software with the following parameter: -SeleVar 2 (Dong et al. 2021), and the correlation coe cient (R 2 ) was calculated to determine pairwise LD decay.Peak SNPs in LD regions were used to predict candidate genes.Subsequently, we performed comparative analyses of callus induction rate in accessions that possessed signi cant SNPs, which we will designate as peak SNPs.The callus induction rate is a measure of the percentage of accessions within each SNP group that successfully formed callus.Then, the probability of callus induction (PCI) for each haplotype was observed using the formula: The PCI for each haplotype was calculated by dividing the total number of accessions forming callus in each haplotype by the total number of accessions in that haplotype, and the result was multiplied by 100 to express it as a percentage.Comparisons were then conducted among the PCI values of different haplotypes, and a haplotype was considered to have a high potential of callus induction, if its PCI was greater than that of the other haplotypes.

Development of SNP primers
Isolated DNA samples were further diluted to a working concentration of 100ng/µl with Tris-EDTA (TE) buffer and used for SNP marker analysis.Allele speci c primers were designed using WASP: a Webbased Allele-Speci c PCR assay designing tool (https://bioinfo.biotec.or.th/WASP) (Wangkumhang et al. 2007) with melting temperatures 55-65°C (with a maximum difference of 3°C in the Tm's of the two primers), primer length 20-27bp, and GC content 50-85%.To enhance reaction speci city, a mismatched arti cial base pair consisting of C/T and G/A was added at the penultimate position from the 3´end (SNP site) (Table 1).This particular mismatch has a strong destabilizing effect, which helps to improve the e ciency of the ampli cation reaction (Wangkumhang et al. 2007).Ampli cation of SNP primers was performed on Biometra Thermal Cycler (Analytik Jena, Germany).The PCR reaction contained 1µL DNA, 10µL of 2X M5 HiPer plus Taq HiFi PCR mix (with blue dye) (Mei5 Biotechnology, Co., Ltd, Beijing, China) and 0.5µL each of 10µM forward and reverse primer in a nal volume of 20µL.PCR parameters were as follows: initial denaturation of 95°C for 3 min, 32 cycles of ampli cation (94°C for 25s, 53°C for 25s and 72°C for 5s) and a nal extension at 72°C for 5 min.Agarose gel electrophoresis was employed to detect the polymorphism assay of SNP primers.Quantitative PCR (qPCR)-based melting curve analysis In our study, we conducted qPCR to assess the differentiation e ciency of the SNP marker.The objective of employing qPCR with the designated primers was to analyze the melting peaks associated with different alleles.The qPCR analysis was performed on qTOWER3 G (Analytik Jena AG, Germany) under the following conditions: initial denaturation at 95˚C for 5min, followed by 40 cycles of denaturation process at 95˚C for 30s, annealing at 53˚C for 30s, and extension at 72 ˚C for 30s.Subsequently, the melting curve analysis was performed immediately at melting rate value of 5˚C/s, from 60-95 ˚C.Each reaction mixture contained 1µL of 100ng/µL DNA, 5µL MonAmp™ ChemoHS qPCR Mix, 0.2µL of each 10µM forward and reverse primer in a nal volume of 10µL.Actin, known for its stable and constitutive expression across diverse cell types, ensures the accuracy and reliability of the melting curve analysis results.By providing a consistent baseline, Actin facilitates the precise interpretation of melting curves.Therefore, to enhance the robustness of our ndings and enable a more accurate interpretation of the melting curve analysis, we utilized the housekeeping gene Actin as an endogenous control.The qPCRsoft 4.1 software developed by Analytik Jena AG was used to analyze the melting curve.

Genetic variation
Totally 11,624,016 SNPs were generated by resequencing.After ltering low quality SNPs (minor allele frequency < 5%, missing data > 20%, and heterozygosity > 80%), a total of 11,475,258 high quality SNPs from both samples with callus and those without callus were conserved for subsequent analysis.Additionally, the distribution of high-quality SNPs was investigated, and the results showed that the highquality SNPs were evenly distributed in 16 chromosomes of oil palm (Fig. 2).This indicates that the SNPs used in the subsequent analysis represent a comprehensive coverage of the oil palm genome.

Population structure and LD decay analysis
A total of 11,475,258 high quality SNPs obtained after screening were utilized for population structure, PCA, and phylogenetic analysis.Dynamic changes in population structure were explored using different K values (K = 2 to 9) (Fig. S1a), revealing the smallest cross-validation error (CV error) at K = 3, indicating the presence of three subpopulations among the 198 oil palm accessions (Fig. S1b).Neighbour-joining, kinship and PCA analyses further demonstrated distant relationships among the majority of accessions (Fig. S1c, d and e), indicating the collected accessions' diversity.These ndings collectively supported the suitability of the selected population for GWAS analysis.Furthermore, LD decay analysis revealed that r 2 decreased to half of its maximum value at approximately 50kb (Fig. S1f), suggesting that genes located within the 50kb region around SNPs could be potential candidate genes associated with callus induction.

GWAS analysis
To explore the genetic factors associated with callus induction, GWAS analysis was performed using four linear regression models: GLM, GLM-Q, MLM-K, and MLM-QK (Fig. S2).After generating QQ plots for each of the four GWAS models, the model that exhibited the best t between the expected and observed values was selected as the optimal model.This model was then used in subsequent analyses.The Q + K mixed linear model (MLM) is widely recognized as the most popular method for GWAS (Wang and Xu 2019).
Our ndings con rm the suitability of the MLM-QK model for GWAS analysis (Fig. S2), consequently leading us to employ this model for further analyses.

Analysis of SNP loci linked to callus induction
In our study, the positive associations between callus induction rates at different stages, such as the strong correlation between C1 and C2 (R = 0.81) and C1 and C3 (R = 0.50) (Fig. 1c), suggest that callus induction at the early stage (C1) serves as a reliable predictor for subsequent stages (C2 and C3).This high correlation indicates that the callus formation observed at C1 is signi cantly linked to the later stages, implying that the genetic factors in uencing callus induction are established early on.As a result, only SNPs in C1, encompassing both samples with callus and those without callus, were identi ed to develop markers for screening accessions capable of callus induction at early stage.Results showed that a total of 21 high quality SNPs were signi cantly associated with C1.These SNPs were distributed across Chr2, Chr5, Chr6, Chr7, Chr8, Chr9, Chr12, Chr14 and Chr16 (Table S1).Furthermore, 35 promising candidate genes associated with C1 were identi ed (Table S2).The analysis showed that these SNPs contributed to a phenotypic variation (R 2 ) ranging from 11-22% (Table S1), with Chr12 contained largest number of high-quality SNPs (7 SNPs), including Chr12_12696848, Chr12_12704827, Chr12_12704830, Chr12_12704835, Chr12_12704836, Chr12_12704839, and Chr12_12704856 (Fig. 3a, Table S1).The LD block analysis showed a high level of linkage relationship among six of these SNP loci, except for Chr12_12696848 (Fig. 3b).The allelic variants of those six SNPs are "T/C", "A/G", "T/C", "C/A", "C/T" and "A/G" (Table S1), and there are three haplotypes (Hap1, Hap2 and Hap3) in each SNP locus.Genotype analyses revealed that accessions carrying Hap1 were associated with low callus induction potential when explants were cultured on Y3 medium, whereas the accessions of Hap3 demonstrated a higher potential for callus induction.Callus induction rates of the accessions with Hap2 was between those of Hap1 and Hap3 (Fig. 3c).Further analysis revealed that all six SNP loci were closely located at approximately 25kb downstream region of LOC105054851, which was annotated as wall-associated receptor kinase 2-like (WAK2) (Fig. 3d).

Analysis of SNP primers for allele discrimination
The allele discrimination e ciency of 13 SNP primers (Table 1) was assessed using 2% agarose gel.Among the developed primers, the C-12 derived from SNP Chr12_12704856 effectively differentiated the GG from both AG and AA alleles.In accessions carrying either AG or AA alleles, a visible band of 212bp was ampli ed, whereas accessions possessing the GG allele did not exhibit any observable band (Fig. 4a).Further analysis revealed variations in the probability of callus induction (PCI) across the different genotypes at this locus.The GG genotypes exhibited a markedly higher PCI, with a rate of 69%, compared to AG and AA genotypes (Fig. 4b).Notably, accessions S30 and S46, which consistently exhibited high callus induction rates (> 50%) from C1 to C3 (Fig. 1b), possessed the GG allele.
In this study, qPCR analysis was performed to detect allelic variants of SNP locus Chr12_12704856.The results demonstrated successful ampli cation of the target samples using the C-12 marker (Fig. 5a).
Thereafter, the melting peaks corresponding to different alleles were determined.During the melting curve analysis in qPCR, the temperature is gradually increased, causing double-stranded DNA to dissociate into single strands.This dissociation of melt curves is monitored in real-time by measuring the uorescence of the DNA-binding dye.Each genotype exhibits a unique melting pro le due to variations in the DNA sequence, leading to distinct melting temperatures (Tm) for AG, AA and GG.For AG and AA, the Tm values were determined to be 78.1˚C and 78˚C, respectively, while GG exhibited melt peaks at temperatures of 80.7˚C and 86.6˚C (Fig. 5b).As the temperature increases during the melting curve analysis, the DNA strands separate, and the uorescence decreases.The point at which each DNA sequence dissociates is re ected in the melting curve as a speci c peak or transition.Analyzing these melt curves allows us to identify the distinct peaks or patterns corresponding to speci c genotypes present in the sample.Therefore, the dissociation of melt curves is instrumental in discriminating between AG, AA and GG alleles, providing a reliable and effective means of genotyping based on the unique characteristics of their melt curves.

Discussion
Callus induction is a critical process in plant tissue culture, where cells undergo dedifferentiation to form a mass of undifferentiated cells known as callus.This process is essential for plant regeneration, as it allows for the formation of new plantlets from these callus tissues (Fan et  ) identi ed tissue-speci c WAK genes that potentially contribute to callus cell growth and reproduction.This observation suggests that certain WAK genes are expressed in a tissuespeci c manner, a phenomenon that could be crucial for the initiation and maintenance of callus formation.This intricate network of molecular events underscores the complexity and importance of callus induction in plant tissue culture.Effective callus induction emerges as a critical factor for plant regeneration, especially for species like oil palm, where tissue culture recalcitrance presents a major bottleneck (Weckx et al. 2019;Zhang et al. 2020b).Identifying molecular markers associated with callus induction could facilitate early determination of oil palm accessions suitable for callus induction.
Utilizing these markers for the identi cation of ortet palms amenable to inoculation and tissue culture has the potential to signi cantly enhance the e ciency of callus induction and mass propagation in oil palm.Despite the importance, relevant studies in this area remain sparse.
Genome-wide association study (GWAS) has been widely used to identify genetic variants linked to agronomic traits across various plant species (Yano et  analyses suggested the diversity within the sampled population.These ndings support the suitability of the selected population for conducting GWAS analysis.Subsequently, GWAS was conducted to explore the loci associated with callus induction in oil palm.The correlation coe cient analysis revealed a positive correlation of C1 with both C2 (R = 0.81) and C3 (R = 0.5), implying that the initiation of callus at the early stage (C1) effectively predicts subsequent stages (C2 and C3).This pronounced correlation suggests that the callus formation observed during C1 is markedly associated with the later phases, which implies that the genetic elements affecting callus induction are determined in the early stages.
Hence, we focused on identifying signi cant SNPs (P ≤ 0.5) only in C1 to develop markers for screening accessions capable of callus induction at early stage.By conducting GWAS, a total of 21 signi cant SNPs associated with C1 were identi ed.Based on LD decay distance, genes located within the 50kb region around SNPs associated with callus induction were identi ed as potential candidate genes.Consequently, 35 promising candidate genes were identi ed in C1.Notably, six SNPs on chromosome 12 formed a high-linkage LD block, near the gene encoding wall-associated receptor kinase 2-like (WAK2).
The wall-associated kinase (WAK) gene, a subfamily of the receptor-like kinase (RLK) gene family, is known to be associated with the plant cell wall and plays a crucial role in cell expansion (Zhang et al. 2020a).Previous study conducted on Nicotiana benthamiana have identi ed various cis-acting elements within the promoter regions of wall-associated kinases (WAKs) and WAK-like kinases (WAKLs).These cisacting elements were found to be associated with phytohormone and/or stress responses (Zhong et al. 2023).This suggests that these genes play a role in coordinating plant responses to phytohormones and stress factors.Another study conducted on Chinese cabbage has suggested the potential importance of the WAK-like genes in callus formation (Zhang et al. 2020a).Our nding supports the hypothesis that WAK2 likely play an important role in callus induction.Further research may provide insights into the speci c mechanisms by which WAK2 regulate cell proliferation and differentiation during callus induction.
Genotype analysis in this study revealed that the callus induction potential of Hap3 was higher than Hap1 and Hap2, suggesting that genotype plays a critical role in determining callus induction e ciency.Further analysis of allele discrimination using SNP primers showed that the marker (C-12) effectively distinguished GG allele.Melting curve analysis by qPCR is a powerful and reliable technique for variant scanning and genotyping (Wittwer 2009; Hung et al. 2011).Our previous study on date palm(Wang et a l.2020) utilized qPCR melting curve analysis to determine sex alleles (X and Y).This technique doesn't need the electrophoresis steps and reduce the time.The entire process, including ampli cation and melting curve analysis, can be completed in a single run within a short period of time.Thus, qPCR analysis was performed in the current study to analyze the melting curve of different alleles.Notably, the GG allele, associated with the highest probability (69%) of callus induction, exhibited a distinctive melting pro le characterized by the presence of two melting peaks.It's possible that the GG allele has a unique sequence context that results in a different melting pro le compared to other alleles.This could be due to variations in the anking regions of the SNP, which can affect the overall stability of the DNA double helix.The e ciency of the qPCR assay was evident because it allows rapid and precise genotyping without the need for traditional gel electrophoresis, streamlining the work ow and reducing analysis time signi cantly.Among the tested oil palm accessions, S30 and S46 consistently showed high callus induction rates (> 50%) from C1 to C3.Further analysis revealed that these accessions possessed the GG allele.Thus, the early detection of the GG allele is crucial as it serves as a predictive indicator for callus induction e ciency in oil palm tissue culture.This early identi cation facilitates the selection of plant materials with a high potential of callus induction, which could bene t oil palm tissue culture by enhancing callus induction rate.

Conclusions
GWAS was applied to investigate the associations between SNPs and callus induction rates of immature male in orescences harvested from 198 oil palm accessions.A total of 21 signi cant SNPs were identi ed in C1, in which 6 SNPs potentially linked to callus induction were further revealed by LD block analysis.A total of 13 primers targeting on the selected 6 SNP alleles were then assessed accordingly.
The primer C-12 from the locus Chr12_12704856 able to distinguish palms with GG allele, which showed the highest probability (69%) of callus induction.The marker-assisted identi cation of palms with high potential of callus induction could greatly enhance the selection e ciency for donor plants in oil palm tissue culture.Our work not only contributes to the production of clone seedling for elite oil palm varieties, but also provides research outlines for other traits, such as long stalk and high oleic acid content.
contributed to material preparation.DZ and YW revised the manuscript.All authors read and approved the nal manuscript.The GG genotypes i.e., S30, S46, S2, S229, S48, were associated with the highest PCI at 69%, followed by AG at 25% and AA at 20%.

Funding
The research was supported by the National Natural Science Foundation of China (32071740), the postdoc project of Hainan Yazhou Bay Seed Laboratory (B21Y10301/B22C10303), the National Key Research and Development Program of China (2023YFD2200703) and the earmarked fund for China Agriculture Research System (CARS-14).

Figures
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Figure 1 Evaluation
Figure 1

Figure 2 Distribution
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Table 1
Primer sequences of targeted SNP region used in this study al. 2016; Qu et al. 2017; Zhao et al. 2021; Kim et al. 2022; Li et al. 2022; Ahn et al. 2023).Similar marker association with tissue culture related traits have been explored in rose (Nguyen et al. 2020), Populus (Tuskan et al. 2018; Zhang et al. 2020b), soybean (Yang et al. 2011a), rice(Zhang et al. 2019b) and maize(Ma et al. 2018) for callus induction, yet oil palm research is limited in this aspect.In our current study, callus induction rates at 1-, 2-, and 3-months after inoculation (C1, C2 and C3) in 198 oil palm accessions were investigated as phenotypes.A total of 11,475,258 high quality single nucleotide polymorphisms (SNPs) obtained from all samples with and without callus at C1, C2 and C3 through resequencing were employed as genotypes.The noticeable variations among the 198 oil palm accessions observed through phylogenetic and population structure