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.

Analyses of hypomethylated oil palm gene space.

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.

Normalized embryoid cDNA library of oil palm (Elaeis guineensis) / Biblioteca ADNc normalizada de embrioides en aceite de palma (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.

Mapping quantitative trait loci (QTLs) for fatty acid composition in an interspecific cross of oil palm.

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.

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

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.