Leslie Cheng-Li Ooi

Oil palm genome sequence reveals divergence of interfertile species in Old and New worlds

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 regulators, 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. guineensis 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 plantings, and should therefore help to achieve sustainability for biofuels and edible oils, reducing the rainforest footprint of this tropical plantation crop.

The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK

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 pisifera. 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 Asia. 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 conservation.

Oil palm (Elaeis guineensis Jacq.) tissue culture ESTs: identifying genes associated with callogenesis and embryogenesis.

BackgroundOil 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.ResultsA 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.ConclusionThis 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.

The oil palm VIRESCENS gene controls fruit colour and encodes a R2R3-MYB

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.

Normalized embryoid cDNA library of oil palm (Elaeis guineensis)

A normalized embryoid cDNA library (EON) was constructed based on reassociation kinetics reaction. Results from dot blot hybridization and sequencing of EON cDNA clones clearly indicated that the normalization process reduced the frequency of high abundance transcripts and increased the frequency of low abundance gene transcripts. A total of 553 non-redundant expressed sequence tags (ESTs) were identified, 325 of these were not observed in the standard oil palm cDNA libraries sequenced previously. A total of 10 EON cDNA clones were chosen for expression profiling across samples from different stages of the tissue culture process. Two of the genes exhibited promising expression patterns for predicting the embryogenic potential in callus. Some of these genes were also differentially expressed in the various tissues of oil palm. This study showed that normalization of the existing embryoid library improved the chances of identifying transcripts not captured in the standard libraries, some of which could be associated with embryogenesis. This collection of ESTs is particularly well suited for use as candidate genes for development of an oil palm DNA chip, which can be used to obtain a more comprehensive view of the molecular mechanism associated with oil palm tissue culture.

Non-tenera Contamination and the Economic Impact of SHELL Genetic Testing in the Malaysian Independent Oil Palm Industry

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.

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

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.

Coconut, Date, and Oil Palm Genomics

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.