High-resolution genetic linkage map and height-related QTLs in an oil palm (Elaeis guineensis) family planted across multiple sites.

A refined SNP array containing 92,459 probes was developed and applied for chromosome scanning, construction of a high-density genetic linkage map and QTL analysis in a selfed Nigerian oil palm family (T128). Genotyping of the T128 mapping family generated 76,447 good quality SNPs for detailed scanning of aberration and homozygosity in the individual pseudo-chromosomes. Of them, 25,364 polymorphic SNPs were used for linkage analysis resulting in an 84.4% mapping rate. A total of 21,413 SNPs were mapped into 16 linkage groups (LGs), covering a total map length of 1364.5 cM. This genetic map is 16X denser than the previous version used to establish pseudo-chromosomes of the oil palm reference genome published in 2013. The QTLs associated with height, height increment and rachis length were identified in LGs TT05, 06, 08, 15 and 16. The present QTLs as well as those published previously were tagged to the reference genome to determine their chromosomal locations. Almost all the QTLs identified in this study were either close to or co-located with those reported in other populations. Determining the QTL position on chromosomes was also helpful in mining for the underlying candidate genes. In total, 55 putative genes and transcription factors involved in the biosynthesis, conjugation and signalling of the major phytohormones, especially for gibberellins and cell wall morphogenesis were found to be present in the identified genomic QTL regions, and their potential roles in plant dwarfism are discussed. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01360-2.

Loss of Karma transposon methylation underlies the mantled somaclonal variant of oil palm.

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.

Variation for heterodimerization and nuclear localization among known and novel oil palm SHELL alleles.

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.

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 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.

Chromosome-scale Elaeis guineensis and E. oleifera assemblies: Comparative genomics of oil palm and other Arecaceae.

Elaeis guineensis and E. oleifera are the two species of oil palm. E. guineensis is the most widely cultivated commercial species, and introgression of desirable traits from E. oleifera is ongoing. We report an improved E. guineensis genome assembly with substantially increased continuity and completeness, as well as the first chromosome-scale E. oleifera genome assembly. Each assembly was obtained by integration of long-read sequencing, proximity ligation sequencing, optical mapping and genetic mapping. High interspecific genome conservation is observed between the two species. The study provides the most extensive gene annotation to date, including 46,697 E. guineensis and 38,658 E. oleifera gene predictions. Analyses of repetitive element families further resolve the DNA repeat architecture of both genomes. Comparative genomic analyses identified experimentally validated small structural variants between the oil palm species and resolved the mechanism of chromosomal fusions responsible for the evolutionary descending dysploidy from 18 to 16 chromosomes.

Cysteine 64 of Ref-1 is not essential for redox regulation of AP-1 DNA binding.

Ref-1 participates in DNA repair as well as in redox regulation of transcription factor function. The redox function of Ref-1 involves reduction of oxidized cysteine residues within the DNA binding domains of several transcription factors, including Fos and Jun. Reduction of these residues is required for DNA binding, providing a redox-dependent mechanism for regulation of target gene expression. Previous in vitro studies implicated cysteine 65 of human Ref-1 (cysteine 64 of mouse Ref-1) as the redox catalytic site. We analyzed the in vivo role of cysteine 64 in redox regulation of AP-1 activity by introducing a cysteine-to-alanine point mutation into the endogenous mouse Ref-1 gene (ref-1(C64A)). Unlike Ref-1 null mice, which die very early in embryonic development, homozygous ref-1(C64A) mice are viable, they survive to normal life expectancy, and they display no overt abnormal phenotype. Although Ref-1 provides the major AP-1-reducing activity in murine cells, ref-1(C64A) cells retain normal levels of endogenous AP-1 DNA binding activity in vivo as well as normal Fos- and Jun-reducing activity in vitro. These results demonstrate that Ref-1 cysteine 64/65 is not required for redox regulation of AP-1 DNA binding in vivo, and they challenge previous hypotheses regarding the mechanism by which Ref-1 regulates the redox-dependent activity of specific transcription factors.

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.

Transcription repression in oncogenic transformation: common targets of epigenetic repression in cells transformed by Fos, Ras or Dnmt1.

Fos and Ras function in both dependent and independent signal transduction pathways, and sustained activity of either oncogene is sufficient to induce cell transformation and tumorigenesis. Increased DNA (cytosine-5) methyltransferse (Dnmt1) activity is involved in the mechanism of transformation by both oncogenes, suggesting that inappropriate epigenetic transcription regulation may be a common route of oncogenesis, and that cell transformation may model aspects of the epigenetic deregulation that often occurs in tumors. Here, we have taken a microarray-based gene expression approach to identify differentially expressed genes in cells transformed by c-fos, v-fos, ras or Dnmt1. The cohort of genes differentially expressed in all four transformation systems includes an over-representation of repressed genes, many of which have been functionally implicated in the suppression of transformation or tumorigenesis. Furthermore, we identified four potential tumor suppressor genes subject to epigenetic transcriptional repression in transformed cells. The results emphasize the role of transcription repression in oncogenesis, and they provide insights into the potential common epigenetic mechanisms impacting cell transformation.

A transcriptome map of cellular transformation by the fos oncogene.

BACKGROUND: The c-fos gene was originally identified as the cellular homolog of the oncogene v-fos carried by the Finkel-Biskis-Jenkins and Finkel-Biskis-Reilly murine osteogenic sarcoma retroviruses. Sustained expression of fos is sufficient to induce cellular transformation in vitro and tumorigenesis in vivo. Fos functions as a component of the AP-1 transcription factor complex to regulate gene transcription and several differentially expressed genes have been identified in cells transformed by fos. We have extended these studies by constructing a cellular system for conditional transformation by v-fos. Using Affymetrix-based DNA microarray technology, we analyzed transcriptional changes over the course of transformation and reversion in an inducible v-fos system. RESULTS: Microarray analyses of temporal gene expression during the process of v-fos mediated cellular transformation and morphological reversion revealed a remarkably dynamic transcriptome. Of the more than 8000 genes analyzed in this study, 3766 genes were categorized into 18 gene-expression patterns by using self-organizing map analysis. By combining the analysis of gene expression profiles in stably transformed cells with the analysis of sequential expression patterns during conditional transformation, we identified a relatively small cohort of genes implicated in v-fos mediated cellular transformation. CONCLUSION: This approach defines a general conditional cell transformation system that can be used to study the endogenous transcription regulatory mechanisms involved in transformation and tumorigenesis. In addition, this study is the first reported analysis of dynamic changes in gene expression throughout experimentally controlled morphological transformation mediated by v-fos.

Hprt(CAG)146 mice: age of onset of behavioral abnormalities, time course of neuronal intranuclear inclusion accumulation, neurotransmitter marker alterations, mitochondrial function markers, and susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

We reported previously a model of polyglutamine repeat disorders with insertion of 146 CAG repeats into the murine hypoxanthine phosphoribosyl transferase locus (Hprt(CAG)146; Ordway et al. [1997] Cell 91:753-763), which does not normally contain polyglutamine repeats. These mice develop an adult-onset neurologic phenotype of incoordination, involuntary limb clasping, seizures, and premature death. Histologic analysis demonstrates widespread ubiquinated neuronal intranuclear inclusions (NIIs). We now report characterization of the age of onset of behavioral abnormalities, correlated with the time course of occurrence of NIIs in several brain regions, and the occurrence of NIIs in non-neuronal tissues. Onset of behavioral abnormalities occurred at approximately 22 weeks of age. There was variable time course of expression of NIIs in several brain regions. Assessment of several non-neuronal tissues revealed nuclear inclusions in hepatocytes and choroid plexus epithelium. Gamma-aminobutyric acid (GABA)/benzodiazepine receptors, dopamine D1-like and D2-like receptors, and type 2 vesicular monoamine transporter (VMAT2) binding sites were assayed before and after the onset of behavioral abnormalities. GABA/benzodiazepine receptors were unchanged either before or after the onset of behavioral abnormalities in any region analyzed, whereas striatal D1-like and D2-like receptors were diminished after but not before the onset of symptoms. Dorsal striatal VMAT2 binding sites were decreased before the onset of behavioral changes. Mitochondrial electron transport chain components were assayed with histochemical methods before and after the onset of behavioral changes. There was no change in behaviorally presymptomatic or symptomatic animals. Hprt(CAG)146 mice did not exhibit increased susceptibility to the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Hprt(CAG)146 mice are a useful model for studying polyglutamine repeat disorders.

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.

DNA methylation in personalized medicine.

The importance of epigenetics in normal development and tissue-specific gene expression, as well as in diseases such as cancer, is well established. DNA methylation is a primary epigenetic modification that is directly linked to the genome itself. Here, we review evidence supporting the promise of DNA methylation-based biomarkers in personalized medicine, discuss standard and emerging technologies for profiling DNA methylation on a genome-wide scale, and forecast how these approaches will be used in parallel to better understand the epigenetics of health and disease and apply that knowledge to advance the field of personalized medicine.

Comprehensive high-throughput arrays for relative methylation (CHARM).

DNA methylation (DNAm) is a term used to describe the heritable covalent addition of a methyl group to cytosines at CpG dinucleotides in mammals. While methods for examining DNAm status at specific loci have existed for several years, recent technological advances have begun to enable the examination of DNAm across the genome. In this unit, we describe comprehensive high-throughput arrays for relative methylation (CHARM), a highly sensitive and specific approach to measure DNA methylation across the genome. This method makes no assumptions about where functionally important DNAm occurs, i.e., CpG island or promoter regions, and includes lower-CpG-density regions of the genome. In addition, it uses a novel genome-weighted smoothing algorithm to correct for CpG density and fragment biases present in methyl-enrichment or methyl-depletion DNA-fractionation methods. It can be applied to studying epigenomic changes in DNAm for normal and diseased samples.

Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA.

Cytosine-methylation changes are stable and thought to be among the earliest events in tumorigenesis. Theoretically, DNA carrying tumor-specifying methylation patterns escape the tumors and may be found circulating in the sera from cancer patients, thus providing the basis for development of noninvasive clinical tests for early cancer detection. Indeed, using methylation-specific PCR-based techniques, several groups reported the detection of tumor-associated methylated DNA in the sera from cancer patients with varying clinical success. However, by design, such analytical approaches allow assessment of the presence of molecules with only one methylation pattern, leaving the bigger picture unexplored. The limited knowledge about circulating DNA methylation patterns hinders the efficient development of clinical methylation tests and testing platforms. Here, we report the results of a comprehensive methylation pattern analysis from breast cancer clinical tissues and sera obtained using massively parallel bisulphite pyrosequencing. The four loci studied were recently discovered by our group, and demonstrated to be powerful epigenetic biomarkers of breast cancer. The detailed analysis of more than 700,000 DNA fragments derived from more than 50 individuals (cancer and cancer-free) revealed an unappreciated complexity of genomic cytosine-methylation patterns in both tissue derived and circulating DNAs. Both tumor and cancer-free tissues (as well as sera) contained molecules with nearly every conceivable cytosine-methylation pattern at each locus. Tumor samples displayed more variation in methylation level than normal samples. Importantly, by establishing the methylation landscape within circulating DNA, this study has better defined the development challenges facing DNA methylation-based cancer-detection tests.

Identification of novel high-frequency DNA methylation changes in breast cancer.

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.