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High-throughput and high-density (HD) genetic marker genotyping systems are critical to optimize the efficiency of oil palm breeding and improvement programmes. This study reports the development of the 78 K Infinium® HD customized SNP array, which was used to genotype a thousand palms of a commercial Deli dura x AVROS pisifera family. A total 64,108 (∼82.0%) polymorphic SNP markers were identified of which, 57, 465 (89.6%) were mapped onto the genetic map that has the largest number of markers published so far in oil palm and holds 14,781 SNPs on 2,363 orphan scaffolds (whose chromosomal locations were unknown), which will improve the existing oil palm reference genome (EG5.1). The SNPs were highly informative based on the parent-to-progeny allelic inheritance analysis. The data demonstrated that 4.3% of the progeny resulted from unintentional self-fertilization of the dura female parent. These unintended ‘selfs’ are highly inbred, which will affect their yield. This study also for the first time, describes the homozygosity in the Deli dura and AVROS pisifera, two important parental lines widely used in commercial seed production. As expected, both the parental palms were highly homozygous, having 138 Mb homozygous regions in common, with 70.3% identical alleles. Such a detailed genetic analysis of the individual palm has been made possible with this customized HD SNP array, which will be a valuable tool for routine application in oil palm improvement programmes. The strategy used to design and apply the array will also be of interest for wider scientific research.

Sustainable production of palm oil can be boosted by the use of appropriate technological tools to improve productivity within existing acreage. The discovery of the SHELL gene and subsequent development of a diagnostic assay to differentiate oil palm’s three fruit forms set the stage for the application of such technological tools by commercial estates prior to planting. The use of DNA-testing can ensure that new planting and re-planting programmes exploit the hybrid vigour expected from selected maternal (dura) and paternal (pisifera) lines. An expanded nationwide survey of more than a million seeds and seedlings not only confirmed the robustness and scalability of SHELL DNA-testing, but more importantly revealed a 12.8 per cent level of undesired planting material across the Malaysian supply chain, which far exceeds that allowed by the national standard. If SHELL testing had been deployed in the past, the value of crude palm oil (CPO) and crude palm kernel oil (CPKO) production in recent years would have increased by $1.63 billion USD in 2021 (or $276/ha/yr), resulting in up to $2.17 billion per year in increased economic activity (or $367/ha/yr), and up to $0.52 billion in additional tax collection by the Malaysian central government (or $88/ha/yr). Compared to the cost of testing of $4 per hectare per year, if testing had been done in the past, each dollar spent on testing would have returned $69.0 in additional production and $91.8 in economic gains. This indicates that the adoption of DNA-testing to manage the oil palm seed and seedling supply chain is necessary if the industry hopes to increase sustainability, productivity, and profitability in the long-term. Keywords: Sustainable palm oil, SHELL DNA testing, non-tenera contamination, seed testing, supply chain quality.

Transcript profiling during the early induction phase of oil palm tissue culture and RNAi studies in a model somatic embryogenesis system showed that EgENOD93 expression is essential for somatic embryogenesis. Micropropagation of oil palm through tissue culture is vital for the generation of superior and uniform elite planting materials. Studies were carried out to identify genes to distinguish between leaf explants with the potential to develop into embryogenic or non-embryogenic callus. Oil palm cDNA microarrays were co-hybridized with cDNA probes of reference tissue, separately with embryo forming (media T527) and non-embryo (media T694) forming leaf explants sampled at Day 7, Day 14 and Day 21. Analysis of the normalized datasets has identified 77, 115 and 127 significantly differentially expressed genes at Day 7, Day 14, and Day 21, respectively. An early nodulin 93 protein gene (ENOD93), was highly expressed at Day 7, Day 14, and Day 21 and in callus (media T527), as assessed by RT-qPCR. Validation of EgENOD93 across tissue culture lines of different genetic background and media composition showed the potential of this gene as an embryogenic marker. In situ RNA hybridization and functional characterization in Medicago truncatula provided additional evidence that ENOD93 is essential for somatic embryogenesis. This study supports the suitability of EgENOD93 as a marker to predict the potential of leaf explants to produce embryogenic callus. Crosstalk among stresses, auxin, and Nod-factor like signalling molecules likely induces the expression of EgENOD93 for embryogenic callus formation.

Several hypomethylated sites within the Karma region of EgDEF1 and hotspot regions in chromosomes 1, 2, 3, and 5 may be associated with mantling. One of the main challenges faced by the oil palm industry is fruit abnormalities, such as the “mantled” phenotype that can lead to reduced yields. This clonal abnormality is an epigenetic phenomenon and has been linked to the hypomethylation of a transposable element within the EgDEF1 gene. To understand the epigenome changes in clones, methylomes of clonal oil palms were compared to methylomes of seedling-derived oil palms. Whole-genome bisulfite sequencing data from seedlings, normal, and mantled clones were analyzed to determine and compare the context-specific DNA methylomes. In seedlings, coding and regulatory regions are generally hypomethylated while introns and repeats are extensively methylated. Genes with a low number of guanines and cytosines in the third position of codons (GC3-poor genes) were increasingly methylated towards their 3′ region, while GC3-rich genes remain demethylated, similar to patterns in other eukaryotic species. Predicted promoter regions were generally hypomethylated in seedlings. In clones, CG, CHG, and CHH methylation levels generally decreased in functionally important regions, such as promoters, 5′ UTRs, and coding regions. Although random regions were found to be hypomethylated in clonal genomes, hypomethylation of certain hotspot regions may be associated with the clonal mantling phenotype. Our findings, therefore, suggest other hypomethylated CHG sites within the Karma of EgDEF1 and hypomethylated hotspot regions in chromosomes 1, 2, 3 and 5, are associated with mantling.

A high-quality reference genome is an important resource that can help decipher the genetic basis of traits in combination with linkage or association analyses. The publicly available oil palm draft genome sequence of AVROS pisifera (EG5) accounts for 1.535 Gb of the 1.8 Gb oil palm genome. However, the assemblies are fragmented, and the earlier assembly only had 43% of the sequences placed on pseudo-chromosomes. By integrating a number of SNP and SSR-based genetic maps, a consensus map (AM_EG5.1), comprising of 828.243 Mb genomic scaffolds anchored to 16 pseudo-chromosomes, was generated. This accounted for 54% of the genome assembly, which is a significant improvement to the original assembly. The total length of N50 scaffolds anchored to the pseudo-chromosomes increased by ∼18% compared to the previous assembly. A total of 139 quantitative trait loci for agronomically important quantitative traits, sourced from literature, were successfully mapped on the new pseudo-chromosomes. The improved assembly could also be used as a reference to identify potential errors in placement of specific markers in the linkage groups of the genetic maps used to assemble the consensus map. The 3422 unique markers from five genetic maps, anchored to the pseudo-chromosomes of AM_EG5.1, are an important resource that can be used preferentially to either construct new maps or fill gaps in existing genetic maps. Synteny analysis further revealed that the AM_EG5.1 had high collinearity with the date palm genome cultivar 'Barhee BC4' and shared most of its segmental duplications. This improved chromosomal-level genome is a valuable resource for genetic research in oil palm.

Optimal oil palm (Elaeis guineensis) crop yields rely on the purity of the tenera fruit form. The high yielding hybrid tenera fruit form is the consequence of heterozygosity for one of nine genetic variants within the SHELL gene. High-throughput genotyping allows cost-efficient screening prior to planting to decrease unintentional non-tenera palm cultivation. We present a paradigm for dramatically reducing non-tenera cultivation by SHELL genotyping a ~ 10% sampling of seeds per seed production fruit bunch. Identification and seed supply chain removal of bunches above a predetermined non-tenera threshold represent a new paradigm for applying SHELL genetic testing in the industry. In a demonstration involving 121,896 embryos from 1304 independent dura x pisifera controlled crosses from two independent seed production units, we found that 38.4% of bunches achieved a 100% pure tenera prediction rate. The remaining bunches (61.6%) had predicted non-tenera contamination ranging from 1.0 to 89.6%, with an overall average of 3.32% seeds per bunch. SHELL genotyping of expected tenera embryos identified rare aneuploid embryos, confirmed by whole genome sequencing-based heterozygosity and copy number analyses.

Main conclusion: Karyotyping using high-density genome-wide SNP markers identified various chromosomal aberrations in oil palm (Elaeis guineensis Jacq.) with supporting evidence from the 2C DNA content measurements (determined using FCM) and chromosome counts. Abstract: Oil palm produces a quarter of the world’s total vegetable oil. In line with its global importance, an initiative to sequence the oil palm genome was carried out successfully, producing huge amounts of sequence information, allowing SNP discovery. High-capacity SNP genotyping platforms have been widely used for marker–trait association studies in oil palm. Besides genotyping, a SNP array is also an attractive tool for understanding aberrations in chromosome inheritance. Exploiting this, the present study utilized chromosome-wide SNP allelic distributions to determine the ploidy composition of over 1,000 oil palms from a commercial F1 family, including 197 derived from twin-embryo seeds. Our method consisted of an inspection of the allelic intensity ratio using SNP markers. For palms with a shifted or abnormal distribution ratio, the SNP allelic frequencies were plotted along the pseudo-chromosomes. This method proved to be efficient in identifying whole genome duplication (triploids) and aneuploidy. We also detected several loss of heterozygosity regions which may indicate small chromosomal deletions and/or inheritance of identical by descent regions from both parents. The SNP analysis was validated by flow cytometry and chromosome counts. The triploids were all derived from twin-embryo seeds. This is the first report on the efficiency and reliability of SNP array data for karyotyping oil palm chromosomes, as an alternative to the conventional cytogenetic technique. Information on the ploidy composition and chromosomal structural variation can help to better understand the genetic makeup of samples and lead to a more robust interpretation of the genomic data in marker–trait association analyses.

Acyl-CoA-binding proteins (ACBPs) are involved in binding and trafficking acyl-CoA esters in eukaryotic cells. ACBPs contain a well-conserved acyl-CoA-binding domain. Their various functions have been characterized in the model plant Arabidopsis and, to a lesser extent, in rice. In this study, genome-wide detection and expression analysis of ACBPs were performed on Elaeis guineensis (oil palm), the most important oil crop in the world. Seven E. guineensis ACBPs were identified and classified into four groups according to their deduced amino acid domain organization. Phylogenetic analysis showed conservation of this family with other higher plants. All seven EgACBPs were expressed in most tissues while their differential expression suggests various functions in specific tissues. For example, EgACBP3 had high expression in inflorescences and stalks while EgACBP1 showed strong expression in leaves. Because of the importance of E. guineensis as an oil crop, expression of EgACBPs was specifically examined during fruit development. EgACBP3 showed high expression throughout mesocarp development, while EgACBP1 had enhanced expression during rapid oil synthesis. In endosperm, both EgACBP1 and EgACBP3 exhibited increased expression during seed development. These results provide important information for further investigations on the biological functions of EgACBPs in various tissues and, in particular, their roles in oil synthesis.

Oil palm produces two types of edible oil, namely palm oil and palm kernel oil that are important sources of food and raw materials for industries, such as cosmetics, biofuels, pharmaceutical and oleochemicals. It is harvested all year round and has the highest oil yield per hectare compared to other edible oil crops. Nevertheless, its yields have stagnated for the past three decades and continuous improvements are required to keep up with future demands of the growing world population. The need to improve productivity motivated the research community and the oil palm industry to jointly embrace the latest biotechniques to study and understand the crop. This partnership paved the way for the development of more comprehensive research programmes that resulted in the release of the assembled oil palm genome sequence, identification of genes/markers associated with traits of interest, and development of new tools. The most important successes to date are the identification of two genes linked to important agronomic traits, i.e. shell phenotype and fruit colour, and a gene that causes the abnormal mantled somaclonal variant. These discoveries have since been translated into diagnostic assays that can be used to screen and select planting materials as early as at the seed or nursery stage, allowing planters to only plant palms with the desired traits. This chapter explores the developments leading to the sequencing of the oil palm genome and its subsequent application in the development of new knowledge and tools that will help shape the next generation of planting materials.

Oil palm breeding involves crossing dura and pisifera palms to produce tenera progeny with greatly improved oil yield. Oil yield is controlled by variant alleles of a type II MADS-box gene, SHELL, that impact the presence and thickness of the endocarp, or shell, surrounding the fruit kernel. We identified six novel SHELL alleles in noncommercial African germplasm populations from the Malaysian Palm Oil Board. These populations provide extensive diversity to harness genetic, mechanistic and phenotypic variation associated with oil yield in a globally critical crop. We investigated phenotypes in heteroallelic combinations, as well as SHELL heterodimerization and subcellular localization by yeast two-hybrid, bimolecular fluorescence complementation and gene expression analyses. Four novel SHELL alleles were associated with fruit form phenotype. Candidate heterodimerization partners were identified, and interactions with EgSEP3 and subcellular localization were SHELL allele-specific. Our findings reveal allele-specific mechanisms by which variant SHELL alleles impact yield, as well as speculative insights into the potential role of SHELL in single-gene oil yield heterosis. Future field trials for combinability and introgression may further optimize yield and improve sustainability.

A set of Elaeis guineensis genes had been generated by combining two gene prediction pipelines: Fgenesh++ developed by Softberry and Seqping by the Malaysian Palm Oil Board. PalmXplore was developed to provide a scalable data repository and a user-friendly search engine system to efficiently store, manage and retrieve the oil palm gene sequences and annotations. Information deposited in PalmXplore includes predicted genes, their genomic coordinates, as well as the annotations derived from external databases, such as Pfam, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Information about genes related to important traits, such as those involved in fatty acid biosynthesis (FAB) and disease resistance, is also provided. The system offers Basic Local Alignment Search Tool homology search, where the results can be downloaded or visualized in the oil palm genome browser (MYPalmViewer). PalmXplore is regularly updated offering new features, improvements to genome annotation and new genomic sequences. The system is freely accessible at http://palmxplore.mpob.gov.my.

The first and most crucial step of all molecular techniques is to isolate high quality and intact nucleic acids. However, DNA and RNA isolation from fungal samples are usually difficult due to the cell walls that are relatively unsusceptible to lysis and often resistant to traditional extraction procedures. Although there are many extraction protocols for Ganoderma species, different extraction protocols have been applied to different species to obtain high yields of good quality nucleic acids, especially for genome and transcriptome sequencing. Ganoderma species, mainly G. boninense causes the basal stem rot disease, a devastating disease that plagues the oil palm industry. Here, we describe modified DNA extraction protocols for G. boninense, G. miniatocinctum and G. tornatum, and an RNA extraction protocol for G. boninense. The modified salting out DNA extraction protocol is suitable for G. boninense and G. miniatocinctum while the modified high salt and low pH protocol is suitable for G. tornatum. The modified DNA and RNA extraction protocols were able to produce high quality genomic DNA and total RNA of ~ 140 to 160 µg/g and ~ 80 µg/g of mycelia respectively, for Single Molecule Real Time (PacBio Sequel® System) and Illumina sequencing. These protocols will benefit those studying the oil palm pathogens at nucleotide level.

The diacylglycerol acyltransferases (DGAT) (diacylglycerol:acyl-CoA acyltransferase, EC 2.3.1.20) are a key group of enzymes that catalyse the final and usually the most important rate-limiting step of triacylglycerol biosynthesis in plants and other organisms. Genes encoding four distinct functional families of DGAT enzymes have been characterised in the genome of the African oil palm, Elaeis guineensis. The contrasting features of the various isoforms within the four families of DGAT genes, namely DGAT1, DGAT2, DGAT3 and WS/DGAT are presented both in the oil palm itself and, for comparative purposes, in 12 other oil crop or model/related plants, namely Arabidopsis thaliana, Brachypodium distachyon, Brassica napus, Elaeis oleifera, Glycine max, Gossypium hirsutum, Helianthus annuus, Musa acuminata, Oryza sativa, Phoenix dactylifera, Sorghum bicolor, and Zea mays. The oil palm genome contains respectively three, two, two and two distinctly expressed functional copies of the DGAT1, DGAT2, DGAT3 and WS/DGAT genes. Phylogenetic analyses of the four DGAT families showed that the E. guineensis genes tend to cluster with sequences from P. dactylifera and M. acuminata rather than with other members of the Commelinid monocots group, such as the Poales which include the major cereal crops such as rice and maize. Comparison of the predicted DGAT protein sequences with other animal and plant DGATs was consistent with the E. guineensis DGAT1 being ER located with its active site facing the lumen while DGAT2, although also ER located, had a predicted cytosol-facing active site. In contrast, DGAT3 and some (but not all) WS/DGAT in E. guineensis are predicted to be soluble, cytosolic enzymes. Evaluation of E. guineensis DGAT gene expression in different tissues and developmental stages suggests that the four DGAT groups have distinctive physiological roles and are particularly prominent in developmental processes relating to reproduction, such as flowering, and in fruit/seed formation especially in the mesocarp and endosperm tissues.

Palm oil has a balanced fatty acid composition and has no trans fat. As a result, its use in food has increased as food-labeling laws have changed to specify trans fat content. Increasing oil production is the main goal in oil palm breeding. Genetic mapping and genomic studies in palm trees are necessary to understand the genetic architecture of economic traits of importance for palm oil production. To help achieve this, we sampled 422 oil palms from MPOB (Malaysian Palm Oil Board)­Angola germplasm collection and measured 13 economic traits from these palms. Multi-locus genome-wide association studies (GWAS) were conducted using least absolute shrinkage and selection operator (LASSO) and genome-wide efficient mixed model analysis. We identified 19 quantitative trait loci (QTLs) for 8 traits. Of these, four Angola-specific QTLs associated with bunch components were detected on chromosomes 4, 8, and 11. These QTLs are potentially useful for introgression of desirable genes from the Angola palms to advanced breeding populations for improvement of bunch and oil yield traits. The majority of the QTLs were detected by LASSO-A, in which the p values of individual markers were calculated based on bootstrapped standard errors. Many of the detected QTLs are nearby known QTLs detected from linkage studies reported by other research groups. We also conducted genomic selection (GS) for the 13 traits and concluded that GS can be an effective tool for oil palm breeding. This is the first GWAS and GS study conducted on oil palm germplasm from Angola, and the results can be very useful in oil palm genetic studies and breeding.

Oil palm (Elaeis guineensis Jacq.) is an outbreeding perennial tree crop with long breeding cycles, typically 12 years. Molecular marker technologies can greatly improve the breeding efficiency of oil palm. This study reports the first use of the DArTseq platform to genotype two closely related self-pollinated oil palm populations, namely AA0768 and AA0769 with 48 and 58 progeny respectively. Genetic maps were constructed using the DArT and SNP markers generated in combination with anchor SSR markers. Both maps consisted of 16 major independent linkage groups (2n = 2× = 32) with 1399 and 1466 mapped markers for the AA0768 and AA0769 populations, respectively, including the morphological trait “shell-thickness” (Sh). The map lengths were 1873.7 and 1720.6 cM with an average marker density of 1.34 and 1.17 cM, respectively. The integrated map was 1803.1 cM long with 2066 mapped markers and average marker density of 0.87 cM. A total of 82% of the DArTseq marker sequence tags identified a single site in the published genome sequence, suggesting preferential targeting of gene-rich regions by DArTseq markers. Map integration of higher density focused around the Sh region identified closely linked markers to the Sh, with D.15322 marker 0.24 cM away from the morphological trait and 5071 bp from the transcriptional start of the published SHELL gene. Identification of the Sh marker demonstrates the robustness of using the DArTseq platform to generate high density genetic maps of oil palm with good genome coverage. Both genetic maps and integrated maps will be useful for quantitative trait loci analysis of important yield traits as well as potentially assisting the anchoring of genetic maps to genomic sequences.

Gene prediction is one of the most important steps in the genome annotation process. A large number of software tools and pipelines developed by various computing techniques are available for gene prediction. However, these systems have yet to accurately predict all or even most of the protein-coding regions. Furthermore, none of the currently available gene-finders has a universal Hidden Markov Model (HMM) that can perform gene prediction for all organisms equally well in an automatic fashion. We present an automated gene prediction pipeline, Seqping that uses self-training HMM models and transcriptomic data. The pipeline processes the genome and transcriptome sequences of the target species using GlimmerHMM, SNAP, and AUGUSTUS pipelines, followed by MAKER2 program to combine predictions from the three tools in association with the transcriptomic evidence. Seqping generates species-specific HMMs that are able to offer unbiased gene predictions. The pipeline was evaluated using the Oryza sativa and Arabidopsis thaliana genomes. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis showed that the pipeline was able to identify at least 95% of BUSCO’s plantae dataset. Our evaluation shows that Seqping was able to generate better gene predictions compared to three HMM-based programs (MAKER2, GlimmerHMM and AUGUSTUS) using their respective available HMMs. Seqping had the highest accuracy in rice (0.5648 for CDS, 0.4468 for exon, and 0.6695 nucleotide structure) and A. thaliana (0.5808 for CDS, 0.5955 for exon, and 0.8839 nucleotide structure). Seqping provides researchers a seamless pipeline to train species-specific HMMs and predict genes in newly sequenced or less-studied genomes. We conclude that the Seqping pipeline predictions are more accurate than gene predictions using the other three approaches with the default or available HMMs.

The genome sequence of the African oil palm, the main source of oil production, is used to predict at least 34,802 genes, including oil biosynthesis genes; comparison with the draft sequence of the South American oil palm reveals that the two species may have diverged in the New World and that segmental duplications of chromosome arms define the palaeotetraploid origin of palm trees. Two papers published in this issue of Nature deal with the genetics of two variants of one of the most important crops in use today — the African oil palm Elaeis guineensis and its South American cousin Elaeis oleifera. Palm oil accounts for almost half the edible oil consumed worldwide and is also a biofuel, although not without controversy, as in many areas palm oil monoculture has replaced valuable natural forest. Analyses of the 1.8-gigabase genome sequence of E. guineensis and draft sequence of E. oleifera provide insights into oil biosynthesis genes and their regulators, and a record of genome evolution. A key event in the domestication and breeding of the oil palm was loss of the thick, coconut-like shell. The second of the two papers identifies mutations the SHELL gene that specify the different fruit forms found in the oil palm and shows that SHELL gene mutations that originated in pre-colonial Africa are responsible for the single gene hybrid vigour and high yields attained by the oil palm. Oil palm is the most productive oil-bearing crop. Although it is planted on only 5% of the total world vegetable oil acreage, palm oil accounts for 33% of vegetable oil and 45% of edible oil worldwide, but increased cultivation competes with dwindling rainforest reserves. We report the 1.8-gigabase (Gb) genome sequence of the African oil palm Elaeis guineensis, the predominant source of worldwide oil production. A total of 1.535 Gb of assembled sequence and transcriptome data from 30 tissue types were used to predict at least 34,802 genes, including oil biosynthesis genes and homologues of WRINKLED1 (WRI1), and other transcriptional regulators1, which are highly expressed in the kernel. We also report the draft sequence of the South American oil palm Elaeis oleifera, which has the same number of chromosomes (2n = 32) and produces fertile interspecific hybrids with E. guineensis2 but seems to have diverged in the New World. Segmental duplications of chromosome arms define the palaeotetraploid origin of palm trees. The oil palm sequence enables the discovery of genes for important traits as well as somaclonal epigenetic alterations that restrict the use of clones in commercial plantings3, and should therefore help to achieve sustainability for biofuels and edible oils, reducing the rainforest footprint of this tropical plantation crop.

Genetic mapping and whole-genome sequencing studies identify the SHELL gene (a homologue of Arabidopsis SEEDSTICK) as responsible for the three different fruit forms in oil palm (Elaeis guineesis); this has important economic implications for modulating SHELL activity to breed desired fruit forms and enhance oil yields. Two papers published in this issue of Nature deal with the genetics of two variants of one of the most important crops in use today — the African oil palm Elaeis guineensis and its South American cousin Elaeis oleifera. Palm oil accounts for almost half the edible oil consumed worldwide and is also a biofuel, although not without controversy, as in many areas palm oil monoculture has replaced valuable natural forest. Analyses of the 1.8-gigabase genome sequence of E. guineensis and draft sequence of E. oleifera provide insights into oil biosynthesis genes and their regulators, and a record of genome evolution. A key event in the domestication and breeding of the oil palm was loss of the thick, coconut-like shell. The second of the two papers identifies mutations the SHELL gene that specify the different fruit forms found in the oil palm and shows that SHELL gene mutations that originated in pre-colonial Africa are responsible for the single gene hybrid vigour and high yields attained by the oil palm. A key event in the domestication and breeding of the oil palm Elaeis guineensis was loss of the thick coconut-like shell surrounding the kernel. Modern E. guineensis has three fruit forms, dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), a hybrid between dura and pisifera1,2,3,4. The pisifera palm is usually female-sterile. The tenera palm yields far more oil than dura, and is the basis for commercial palm oil production in all of southeast Asia5. Here we describe the mapping and identification of the SHELL gene responsible for the different fruit forms. Using homozygosity mapping by sequencing, we found two independent mutations in the DNA-binding domain of a homologue of the MADS-box gene SEEDSTICK (STK, also known as AGAMOUS-LIKE 11), which controls ovule identity and seed development in Arabidopsis. The SHELL gene is responsible for the tenera phenotype in both cultivated and wild palms from sub-Saharan Africa, and our findings provide a genetic explanation for the single gene hybrid vigour (or heterosis) attributed to SHELL, via heterodimerization. This gene mutation explains the single most important economic trait in oil palm, and has implications for the competing interests of global edible oil production, biofuels and rainforest conservation6.

Background Oil palm is an important source of edible oil. The importance of the crop, as well as its long breeding cycle (10-12 years) has led to the sequencing of its genome in 2013 to pave the way for genomics-guided breeding. Nevertheless, the first set of gene predictions, although useful, had many fragmented genes. Classification and characterization of genes associated with traits of interest, such as those for fatty acid biosynthesis and disease resistance, were also limited. Lipid-, especially fatty acid (FA)-related genes are of particular interest for the oil palm as they specify oil yields and quality. This paper presents the characterization of the oil palm genome using different gene prediction methods and comparative genomics analysis, identification of FA biosynthesis and disease resistance genes, and the development of an annotation database and bioinformatics tools. Results Using two independent gene-prediction pipelines, Fgenesh++ and Seqping, 26,059 oil palm genes with transcriptome and RefSeq support were identified from the oil palm genome. These coding regions of the genome have a characteristic broad distribution of GC3 (fraction of cytosine and guanine in the third position of a codon) with over half the GC3-rich genes (GC3 ≥ 0.75286) being intronless. In comparison, only one-seventh of the oil palm genes identified are intronless. Using comparative genomics analysis, characterization of conserved domains and active sites, and expression analysis, 42 key genes involved in FA biosynthesis in oil palm were identified. For three of them, namely EgFABF, EgFABH and EgFAD3, segmental duplication events were detected. Our analysis also identified 210 candidate resistance genes in six classes, grouped by their protein domain structures. Conclusions We present an accurate and comprehensive annotation of the oil palm genome, focusing on analysis of important categories of genes (GC3-rich and intronless), as well as those associated with important functions, such as FA biosynthesis and disease resistance. The study demonstrated the advantages of having an integrated approach to gene prediction and developed a computational framework for combining multiple genome annotations. These results, available in the oil palm annotation database (http://palmxplore.mpob.gov.my), will provide important resources for studies on the genomes of oil palm and related crops. Reviewers This article was reviewed by Alexander Kel, Igor Rogozin, and Vladimir A. Kuznetsov. Electronic supplementary material The online version of this article (doi:10.1186/s13062-017-0191-4) contains supplementary material, which is available to authorized users.

Oil palm (Elaeis guineensis) is the most productive oil bearing crop worldwide. It has three fruit forms, namely dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), which are controlled by the SHELL gene. The fruit forms exhibit monogenic co-dominant inheritance, where tenera is a hybrid obtained by crossing maternal dura and paternal pisifera palms. Commercial palm oil production is based on planting thin-shelled tenera palms, which typically yield 30% more oil than dura palms, while pisifera palms are female-sterile and have little to no palm oil yield. It is clear that tenera hybrids produce more oil than either parent due to single gene heterosis. The unintentional planting of dura or pisifera palms reduces overall yield and impacts land utilization that would otherwise be devoted to more productive tenera palms. Here, we identify three additional novel mutant alleles of the SHELL gene, which encode a type II MADS-box transcription factor, and determine oil yield via control of shell fruit form phenotype in a manner similar to two previously identified mutant SHELL alleles. Assays encompassing all five mutations account for all dura and pisifera palms analyzed. By assaying for these variants in 10,224 mature palms or seedlings, we report the first large scale accurate genotype-based determination of the fruit forms in independent oil palm planting sites and in the nurseries that supply them throughout Malaysia. The measured non-tenera contamination rate (10.9% overall on a weighted average basis) underscores the importance of SHELL genetic testing of seedlings prior to planting in production fields. By eliminating non-tenera contamination, comprehensive SHELL genetic testing can improve sustainability by increasing yield on existing planted lands. In addition, economic modeling demonstrates that SHELL gene testing will confer substantial annual economic gains to the oil palm industry, to Malaysian gross national income and to Malaysian government tax receipts.

Comparative genomics and transcriptomic analyses were performed on two agronomically important groups of genes from oil palm versus other major crop species and the model organism, Arabidopsis thaliana. The first analysis was of two gene families with key roles in regulation of oil quality and in particular the accumulation of oleic acid, namely stearoyl ACP desaturases (SAD) and acyl-acyl carrier protein (ACP) thioesterases (FAT). In both cases, these were found to be large gene families with complex expression profiles across a wide range of tissue types and developmental stages. The detailed classification of the oil palm SAD and FAT genes has enabled the updating of the latest version of the oil palm gene model. The second analysis focused on disease resistance (R) genes in order to elucidate possible candidates for breeding of pathogen tolerance/resistance. Ortholog analysis showed that 141 out of the 210 putative oil palm R genes had homologs in banana and rice. These genes formed 37 clusters with 634 orthologous genes. Classification of the 141 oil palm R genes showed that the genes belong to the Kinase (7), CNL (95), MLO-like (8), RLK (3) and Others (28) categories. The CNL R genes formed eight clusters. Expression data for selected R genes also identified potential candidates for breeding of disease resistance traits. Furthermore, these findings can provide information about the species evolution as well as the identification of agronomically important genes in oil palm and other major crops.

A total of 5,521 expressed sequence tags (ESTs) from oil palm were used to search for type and frequency of simple sequence repeat (SSR) markers. Dimeric repeat motifs appeared to be the most abundant, followed by tri-nucleotide repeats. Redundancy was eliminated in the original EST set, resulting in 145 SSRs in 136 unique ESTs (114 singletons and 22 clusters). Primers were designed for 94 (69.1%) of the unique ESTs (consisting of 14 consensus and 80 singletons). Primers for 10 EST-SSRs were developed and used to evaluate the genetic diversity of 76 accessions of oil palm originating from seven countries in Africa, and the standard Deli dura population. The average number of observed and effective alleles was 2.56 and 1.84, respectively. The EST-SSR markers were found to be polymorphic with a mean polymorphic information content value of 0.53. Genetic differentiation (FST) among the populations studied was 0.2492 indicating high level of genetic divergence. Moreover, the UPGMA (unweighted pair-group method with arithmetic mean) analysis revealed a strong association between genetic distance and geographic location of the populations studied. The germplasm materials exhibited higher diversity than Deli dura, indicating their potential usefulness in oil palm improvement programmes. The study also revealed that the populations from Nigeria, Congo and Cameroon showed the highest diversity among the germplasm evaluated in this study. The EST-SSRs further demonstrated their worth as a new source of polymorphic markers for phylogenetic analysis, since a high percentage of the markers showed transferability across species and palm taxa.

Somaclonal variation arises in plants and animals when differentiated somatic cells are induced into a pluripotent state, but the resulting clones differ from each other and from their parents. In agriculture, somaclonal variation has hindered the micropropagation of elite hybrids and genetically modified crops, but the mechanism responsible remains unknown. The oil palm fruit 'mantled' abnormality is a somaclonal variant arising from tissue culture that drastically reduces yield, and has largely halted efforts to clone elite hybrids for oil production. Widely regarded as an epigenetic phenomenon, 'mantling' has defied explanation, but here we identify the MANTLED locus using epigenome-wide association studies of the African oil palm Elaeis guineensis. DNA hypomethylation of a LINE retrotransposon related to rice Karma, in the intron of the homeotic gene DEFICIENS, is common to all mantled clones and is associated with alternative splicing and premature termination. Dense methylation near the Karma splice site (termed the Good Karma epiallele) predicts normal fruit set, whereas hypomethylation (the Bad Karma epiallele) predicts homeotic transformation, parthenocarpy and marked loss of yield. Loss of Karma methylation and of small RNA in tissue culture contributes to the origin of mantled, while restoration in spontaneous revertants accounts for non-Mendelian inheritance. The ability to predict and cull mantling at the plantlet stage will facilitate the introduction of higher performing clones and optimize environmentally sensitive land resources.

Oil palm, a plantation crop of major economic importance in Southeast Asia, is the predominant source of edible oil worldwide. We report the identification of the VIRESCENS (VIR) gene, which controls fruit exocarp colour and is an indicator of ripeness. VIR is a R2R3-MYB transcription factor with homology to Lilium LhMYB12 and similarity to Arabidopsis PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1). We identify five independent mutant alleles of VIR in over 400 accessions from sub-Saharan Africa that account for the dominant-negative virescens phenotype. Each mutation results in premature termination of the carboxy-terminal domain of VIR, resembling McClintock’s C1-I allele in maize. The abundance of alleles likely reflects cultural practices, by which fruits were venerated for magical and medicinal properties. The identification of VIR will allow selection of the trait at the seed or early-nursery stage, 3-6 years before fruits are produced, greatly advancing introgression into elite breeding material., Fruit colour is a trait that affects the harvesting, and therefore oil yield, of the economically important oil palm. Here, the authors identify a gene that may control fruit colour in the oil palm and suggest that selection for this gene during early development could advance the breeding potential of this important crop.

Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

The palm family, consisting of over 2,500 species arrayed among ca. 200 genera, is the third most economically important family of plants after the grasses and legumes. Three palm species account for the large majority of the family’s economic importance: coconut (Cocos nucifera), African oil palm (Elaeis guineensis), and date palm (Phoenix dactylifera). Of the three, genomics has been least developed in the coconut, where molecular tools have largely been used to characterize germplasm, and, to a lesser extent, develop quantitative trait loci (QTL). Both date palm and oil palm have recently had their genomes sequenced. The application of genomic tools to these palm species will result in enormous advances in the genetic improvement of all three crops.

Publisher Summary Plant genomics is reversing the previous paradigm of identifying genes behind biological functions and instead focuses on finding biological functions behind genes. The oil palm belongs to the genus Elaeis, which contains two species, E. Guineensis and E. oleifera. It is regarded as the most efficient oil-bearing crop in the world compared to other crops of the same genre in terms of land use. However, the oil yield of oil palm has reached stagnation of late and there is an urgent need for improvement. Hence, this chapter discusses the current trend of leveraging genomics in the production of new and improved planting materials. The genomic platforms form the resource toward achieving the target of producing superior oil palms. As with other plant genomes, the oil palm has now been sequenced and with the plummeting price of sequencing as time goes by, more genomes will follow suit. Malaysian Palm Oil Board (MPOB) is known to have the largest oil palm germplasm collection in the world. That is an ideal source for identifying new alleles essential for breeding.

This study reports on the detection of additional expressed sequence tags (EST) derived simple sequence repeat (SSR) markers for the oil palm. A large collection of 19243 Elaeis guineensis ESTs were assembled to give 10258 unique sequences, of which 629 ESTs were found to contain 722 SSRs with a variety of motifs. Dinucleotide repeats formed the largest group (45.6%) consisting of 66.9% AG/CT, 21.9% AT/AT, 10.9% AC/GT and 0.3% CG/CG motifs. This was followed by trinucleotide repeats, which is the second most abundant repeat types (34.5%) consisting of AAG/CTT (23.3%), AGG/CCT (13.7%), CCG/CGG (11.2%), AAT/ATT (10.8%), AGC/GCT (10.0%), ACT/AGT (8.8%), ACG/CGT (7.6%), ACC/GGT (7.2%), AAC/GTT (3.6%) and AGT/ACT (3.6%) motifs. Primer pairs were designed for 405 unique EST-SSRs and 15 of these were used to genotype 105 E. guineensis and 30 E. oleifera accessions. Fourteen SSRs were polymorphic in at least one germplasm revealing a total of 101 alleles. The high percentage (78.0%) of alleles found to be specific for either E. guineensis or E. oleifera has increased the power for discriminating the two species. The estimates of genetic differentiation detected by EST-SSRs were compared to those reported previously. The transferability across palm taxa to two Cocos nucifera and six exotic palms is also presented. The polymerase chain reaction (PCR) products of three primer-pairs detected in E. guineensis, E. oleifera, C. nucifera and Jessinia bataua were cloned and sequenced. Sequence alignments showed mutations within the SSR site and the flanking regions. Phenetic analysis based on the sequence data revealed that C. nucifera is closer to oil palm compared to J. bataua; consistent with the taxanomic classification.

Background Marker Assisted Selection (MAS) is well suited to a perennial crop like oil palm, in which the economic products are not produced until several years after planting. The use of DNA markers for selection in such crops can greatly reduce the number of breeding cycles needed. With the use of DNA markers, informed decisions can be made at the nursery stage, regarding which individuals should be retained as breeding stock, which are satisfactory for agricultural production, and which should be culled. The trait associated with oil quality, measured in terms of its fatty acid composition, is an important agronomic trait that can eventually be tracked using molecular markers. This will speed up the production of new and improved oil palm planting materials. Results A map was constructed using AFLP, RFLP and SSR markers for an interspecific cross involving a Colombian Elaeis oleifera (UP1026) and a Nigerian E. guinneensis (T128). A framework map was generated for the male parent, T128, using Joinmap ver. 4.0. In the paternal (E. guineensis) map, 252 markers (199 AFLP, 38 RFLP and 15 SSR) could be ordered in 21 linkage groups (1815 cM). Interval mapping and multiple-QTL model (MQM) mapping (also known as composite interval mapping, CIM) were used to detect quantitative trait loci (QTLs) controlling oil quality (measured in terms of iodine value and fatty acid composition). At a 5% genome-wide significance threshold level, QTLs associated with iodine value (IV), myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2) content were detected. One genomic region on Group 1 appears to be influencing IV, C14:0, C16:0, C18:0 and C18:1 content. Significant QTL for C14:0, C16:1, C18:0 and C18:1 content was detected around the same locus on Group 15, thus revealing another major locus influencing fatty acid composition in oil palm. Additional QTL for C18:0 was detected on Group 3. A minor QTL for C18:2 was detected on Group 2. Conclusion This study describes the first successful detection of QTLs for fatty acid composition in oil palm. These QTLs constitute useful tools for application in breeding programmes.

Demand for palm oil has been increasing by an average of ∼8% the past decade and currently accounts for about 59% of the world's vegetable oil market. This drives the need to increase palm oil production. Nevertheless, due to the increasing need for sustainable production, it is imperative to increase productivity rather than the area cultivated. Studies on the oil palm genome are essential to help identify genes or markers that are associated with important processes or traits, such as flowering, yield and disease resistance. To achieve this, 294,115 and 150,744 sequences from the hypomethylated or gene-rich regions of Elaeis guineensis and E. oleifera genome were sequenced and assembled into contigs. An additional 16,427 shot-gun sequences and 176 bacterial artificial chromosomes (BAC) were also generated to check the quality of libraries constructed. Comparison of these sequences revealed that although the methylation-filtered libraries were sequenced at low coverage, they still tagged at least 66% of the RefSeq supported genes in the BAC and had a filtration power of at least 2.0. A total 33,752 microsatellites and 40,820 high-quality single nucleotide polymorphism (SNP) markers were identified. These represent the most comprehensive collection of microsatellites and SNPs to date and would be an important resource for genetic mapping and association studies. The gene models predicted from the assembled contigs were mined for genes of interest, and 242, 65 and 14 oil palm transcription factors, resistance genes and miRNAs were identified respectively. Examples of the transcriptional factors tagged include those associated with floral development and tissue culture, such as homeodomain proteins, MADS, Squamosa and Apetala2. The E. guineensis and E. oleifera hypomethylated sequences provide an important resource to understand the molecular mechanisms associated with important agronomic traits in oil palm.

Background Oil palm (Elaeis guineensis Jacq.) is one of the most important oil bearing crops in the world. However, genetic improvement of oil palm through conventional breeding is extremely slow and costly, as the breeding cycle can take up to 10 years. This has brought about interest in vegetative propagation of oil palm. Since the introduction of oil palm tissue culture in the 1970s, clonal propagation has proven to be useful, not only in producing uniform planting materials, but also in the development of the genetic engineering programme. Despite considerable progress in improving the tissue culture techniques, the callusing and embryogenesis rates from proliferating callus cultures remain very low. Thus, understanding the gene diversity and expression profiles in oil palm tissue culture is critical in increasing the efficiency of these processes. Results A total of 12 standard cDNA libraries, representing three main developmental stages in oil palm tissue culture, were generated in this study. Random sequencing of clones from these cDNA libraries generated 17,599 expressed sequence tags (ESTs). The ESTs were analysed, annotated and assembled to generate 9,584 putative unigenes distributed in 3,268 consensi and 6,316 singletons. These unigenes were assigned putative functions based on similarity and gene ontology annotations. Cluster analysis, which surveyed the relatedness of each library based on the abundance of ESTs in each consensus, revealed that lipid transfer proteins were highly expressed in embryogenic tissues. A glutathione S-transferase was found to be highly expressed in non-embryogenic callus. Further analysis of the unigenes identified 648 non-redundant simple sequence repeats and 211 putative full-length open reading frames. Conclusion This study has provided an overview of genes expressed during oil palm tissue culture. Candidate genes with expression that are modulated during tissue culture were identified. However, in order to confirm whether these genes are suitable as early markers for embryogenesis, the genes need to be tested on earlier stages of tissue culture and a wider range of genotypes. This collection of ESTs is an important resource for genetic and genome analyses of the oil palm, particularly during tissue culture development.