A practical genome-enabled legitimacy assay for oil palm breeding and seed production.

BACKGROUND: Legitimacy in breeding and commercial crop production depends on optimised protocols to ensure purity of crosses and correct field planting of material. In oil palm, the presence of three fruit forms permits these assumptions to be tested, although only after field planting. The presence of incorrect fruit forms in a cross is a clear sign of illegitimacy. Given that tenera forms produce 30% more oil for the same weight of fruit as dura, the presence of low levels of dura contamination can have major effect during the economic lifespan of an oil palm, which is around 25 years. We evaluated two methods for legitimacy test 1) The use of SHELL markers to the gene that determines the shell-thickness trait 2) The use of SNP markers, to determine the legitimacy of the cross. RESULTS: Our results indicate that the SHELL markers can theoretically reduce the major losses due to dura contamination of tenera planting material. However, these markers cannot distinguish illegitimate tenera, which reduces the value of having bred elite tenera for commercial planting and in the breeding programme, where fruit form is of limited utility, and incorrect identity could lead to significant problems. We propose an optimised approach using SNPs for routine quality control. CONCLUSIONS: Both dura and tenera contamination can be identified and removed at or before the nursery stage. An optimised legitimacy assay using SNP markers coupled with a suitable sampling scheme is now ready to be deployed as a standard control for seed production and breeding in oil palm. The same approach will also be an effective solution for other perennial crops, such as coconut and date palm.

Evaluation of methods and marker Systems in Genomic Selection of oil palm (Elaeis guineensis Jacq.).

BACKGROUND: Genomic selection (GS) uses genome-wide markers as an attempt to accelerate genetic gain in breeding programs of both animals and plants. This approach is particularly useful for perennial crops such as oil palm, which have long breeding cycles, and for which the optimal method for GS is still under debate. In this study, we evaluated the effect of different marker systems and modeling methods for implementing GS in an introgressed dura family derived from a Deli dura x Nigerian dura (Deli x Nigerian) with 112 individuals. This family is an important breeding source for developing new mother palms for superior oil yield and bunch characters. The traits of interest selected for this study were fruit-to-bunch (F/B), shell-to-fruit (S/F), kernel-to-fruit (K/F), mesocarp-to-fruit (M/F), oil per palm (O/P) and oil-to-dry mesocarp (O/DM). The marker systems evaluated were simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). RR-BLUP, Bayesian A, B, Cπ, LASSO, Ridge Regression and two machine learning methods (SVM and Random Forest) were used to evaluate GS accuracy of the traits. RESULTS: The kinship coefficient between individuals in this family ranged from 0.35 to 0.62. S/F and O/DM had the highest genomic heritability, whereas F/B and O/P had the lowest. The accuracies using 135 SSRs were low, with accuracies of the traits around 0.20. The average accuracy of machine learning methods was 0.24, as compared to 0.20 achieved by other methods. The trait with the highest mean accuracy was F/B (0.28), while the lowest were both M/F and O/P (0.18). By using whole genomic SNPs, the accuracies for all traits, especially for O/DM (0.43), S/F (0.39) and M/F (0.30) were improved. The average accuracy of machine learning methods was 0.32, compared to 0.31 achieved by other methods. CONCLUSION: Due to high genomic resolution, the use of whole-genome SNPs improved the efficiency of GS dramatically for oil palm and is recommended for dura breeding programs. Machine learning slightly outperformed other methods, but required parameters optimization for GS implementation.

Development of Nuclear DNA Markers for Applications in Genetic Diversity Study of Oil Palm-Pollinating Weevil Populations.

The oil palm-pollinating weevil (Elaeidobius kamerunicus Faust) was introduced from Cameroon, West Africa, to Malaysia in 1981, and subsequently, to other oil palm-growing countries as well. This study aims to develop a set of robust E. kamerunicus-specific nuclear DNA markers to directly assess the genetic diversity of the weevil populations. A total of 19,148 SNP and 223,200 SSR were discovered from 48 weevils representing three origins (Peninsular Malaysia, Sabah, and Riau) using RAD tag sequencing. Subsequent filtering steps further reduced these to 1000 SNP and 120 SSR. The selected 220 SNP exhibited a polymorphism information content (PIC) of 0.2387 (±0.1280), and 8 SSR had the PIC of 0.5084 (±0.1928). These markers were found to show sufficient polymorphism, making it possible to assign 180 weevils into three major clusters from Ghana, Cameroon, and Southeast Asia (mainly in Malaysia and Indonesia). These DNA markers successfully confirmed the Cameroon origin of the Southeast Asian cluster. However, the presence of null alleles in the SSR markers, due to limited flexibility of the probe design on the short RAD tags, led to an underestimation of heterozygosity within the populations. Hence, the developed SNP markers turned out to be more efficient than the SSR markers in the genetic diversity assessment of the E. kamerunicus populations. The genetic information provides useful insight into developing guidelines for the genetic monitoring and conservation planning of E. kamerunicus.

A combination of evolutionary trace method, molecular surface accessibility and hydrophobicity analysis to design a high hydrophobicity laccase.

Laccases are industrially attractive enzymes and their applications have expanded to the field of bioremediation. The challenge of today's biotechnology in enzymatic studies is to design enzymes that not only have a higher activity but are also more stable and could fit well with the condition requirements. Laccases are known to oxidize non-natural substrates like polycyclic aromatic hydrocarbons (PAHs). We suppose by increasing the hydrophobicity of laccase, it would increase the chance of the enzyme to meet the hydrophobic substrates in a contamination site, therefore increasing the bioremediation efficacy of PAHs from environment. In this attempt, the applications of evolutionary trace (ET), molecular surface accessibility and hydrophobicity analysis on laccase sequences and laccase's crystal structure (1KYA) are described for optimal design of an enzyme with higher hydrophobicity. Our analysis revealed that Q23A, Q45I, N141A, Q237V, N262L, N301V, N331A, Q360L and Q482A could be promising exchanges to be tested in mutagenesis experiments.

Major QTLs for Trunk Height and Correlated Agronomic Traits Provide Insights into Multiple Trait Integration in Oil Palm Breeding.

Superior oil yield is always the top priority of the oil palm industry. Short trunk height (THT) and compactness traits have become increasingly important to improve harvesting efficiency since the industry started to suffer yield losses due to labor shortages. Breeding populations with low THT and short frond length (FL) are actually available, such as Dumpy AVROS pisifera (DAV) and Gunung Melayu dura (GM). However, multiple trait stacking still remains a challenge for oil palm breeding, which usually requires 12-20 years to complete a breeding cycle. In this study, yield and height increment in the GM × GM (GM-3341) and the GM × DAV (GM-DAV-3461) crossing programs were evaluated and palms with good yield and smaller height increment were identified. In the GM-3341 family, non-linear THT growth between THT_2008 (seven years old) and THT_2014 (13 years old) was revealed by a moderate correlation, suggesting that inter-palm competition becomes increasingly important. In total, 19 quantitative trait loci (QTLs) for THT_2008 (8), oil per palm (O/P) (7) and FL (4) were localized on the GM-3341 linkage map, with an average mapping interval of 2.01 cM. Three major QTLs for THT_2008, O/P and FL are co-located on chromosome 11 and reflect the correlation of THT_2008 with O/P and FL. Multiple trait selection for high O/P and low THT (based on the cumulative effects of positive alleles per trait) identified one palm from 100 palms, but with a large starting population of 1000-1500 seedling per cross, this low frequency could be easily compensated for during breeding selection.

An Improved Oil Palm Genome Assembly as a Valuable Resource for Crop Improvement and Comparative Genomics in the Arecoideae Subfamily.

Oil palm (Elaeis guineensis Jacq.) is the most traded crop among the economically important palm species. Here, we report an extended version genome of E. guineensis that is 1.2 Gb in length, an improvement of the physical genome coverage to 79% from the previous 43%. The improvement was made by assigning an additional 1968 originally unplaced scaffolds that were available publicly into the physical genome. By integrating three ultra-dense linkage maps and using them to place genomic scaffolds, the 16 pseudomolecules were extended. As we show, the improved genome has enhanced the mapping resolution for genome-wide association studies (GWAS) and permitted further identification of candidate genes/protein-coding regions (CDSs) and any non-coding RNA that may be associated with them for further studies. We then employed the new physical map in a comparative genomics study against two other agriculturally and economically important palm species-date palm (Phoenix dactylifera L.) and coconut palm (Cocos nucifera L.)-confirming the high level of conserved synteny among these palm species. We also used the improved oil palm genome assembly version as a palm genome reference to extend the date palm physical map. The improved genome of oil palm will enable molecular breeding approaches to expedite crop improvement, especially in the largest subfamily of Arecoideae, which consists of 107 species belonging to Arecaceae.

Linkage-based genome assembly improvement of oil palm (Elaeis guineensis).

Meiotic crossovers in outbred species, such as oil palm (Elaeis guineensis Jacq., 2n = 32) contribute to allelic re-assortment in the genome. Such genetic variation is usually exploited in breeding to combine positive alleles for trait superiority. A good quality reference genome is essential for identifying the genetic factors underlying traits of interest through linkage or association studies. At the moment, an AVROS pisifera genome is publicly available for oil palm. Distribution and frequency of crossovers throughout chromosomes in different origins of oil palm are still unclear. Hence, an ultrahigh-density genomic linkage map of a commercial Deli dura x AVROS pisifera family was constructed using the OP200K SNP array, to evaluate the genetic alignment with the genome assembly. A total of 27,890 linked SNP markers generated a total map length of 1,151.7 cM and an average mapping interval of 0.04 cM. Nineteen linkage groups represented 16 pseudo-chromosomes of oil palm, with 61.7% of the mapped SNPs present in the published genome. Meanwhile, the physical map was also successfully extended from 658 Mb to 969 Mb by assigning unplaced scaffolds to the pseudo-chromosomes. A genic linkage map with major representation of sugar and lipid biosynthesis pathways was subsequently built for future studies on oil related quantitative trait loci (QTL). This study improves the current physical genome of the commercial oil palm, and provides important insights into its recombination landscape, eventually unlocking the full potential genome sequence-enabled biology for oil palm.

Key glycolytic branch influences mesocarp oil content in oil palm.

The fructose-1,6-bisphosphate aldolase catalyzed glycolysis branch that forms dihydroxyacetone phosphate and glyceraldehyde-3-phosphate was identified as a key driver of increased oil synthesis in oil palm and was validated in Saccharomyces cerevisiae. Reduction in triose phosphate isomerase (TPI) activity in a yeast knockdown mutant resulted in 19% increase in lipid content, while yeast strains overexpressing oil palm fructose-1,6-bisphosphate aldolase (EgFBA) and glycerol-3-phosphate dehydrogenase (EgG3PDH) showed increased lipid content by 16% and 21%, respectively. Genetic association analysis on oil palm SNPs of EgTPI SD_SNP_000035801 and EgGAPDH SD_SNP_000041011 showed that palms harboring homozygous GG in EgTPI and heterozygous AG in EgGAPDH exhibited higher mesocarp oil content based on dry weight. In addition, AG genotype of the SNP of EgG3PDH SD_SNP_000008411 was associated with higher mean mesocarp oil content, whereas GG genotype of the EgFBA SNP SD_SNP_000007765 was favourable. Additive effects were observed with a combination of favourable alleles in TPI and FBA in Nigerian x AVROS population (family F7) with highest allele frequency GG.GG being associated with a mean increase of 3.77% (p value = 2.3E-16) oil content over the Family 1. An analogous effect was observed in yeast, where overexpressed EgFBA in TPI - resulted in a 30% oil increment. These results provide insights into flux balances in glycolysis leading to higher yield in mesocarp oil-producing fruit.

Characterizing haploinsufficiency of SHELL gene to improve fruit form prediction in introgressive hybrids of oil palm.

The fundamental trait in selective breeding of oil palm (Eleais guineensis Jacq.) is the shell thickness surrounding the kernel. The monogenic shell thickness is inversely correlated to mesocarp thickness, where the crude palm oil accumulates. Commercial thin-shelled tenera derived from thick-shelled dura × shell-less pisifera generally contain 30% higher oil per bunch. Two mutations, sh MPOB (M1) and sh AVROS (M2) in the SHELL gene - a type II MADS-box transcription factor mainly present in AVROS and Nigerian origins, were reported to be responsible for different fruit forms. In this study, we have tested 1,339 samples maintained in Sime Darby Plantation using both mutations. Five genotype-phenotype discrepancies and eight controls were then re-tested with all five reported mutations (sh AVROS , sh MPOB , sh MPOB2 , sh MPOB3 and sh MPOB4 ) within the same gene. The integration of genotypic data, pedigree records and shell formation model further explained the haploinsufficiency effect on the SHELL gene with different number of functional copies. Some rare mutations were also identified, suggesting a need to further confirm the existence of cis-compound mutations in the gene. With this, the prediction accuracy of fruit forms can be further improved, especially in introgressive hybrids of oil palm. Understanding causative variant segregation is extremely important, even for monogenic traits such as shell thickness in oil palm.

Genomic Selection in Commercial Perennial Crops: Applicability and Improvement in Oil Palm (Elaeis guineensis Jacq.).

Genomic selection (GS) uses genome-wide markers to select individuals with the desired overall combination of breeding traits. A total of 1,218 individuals from a commercial population of Ulu Remis x AVROS (UR x AVROS) were genotyped using the OP200K array. The traits of interest included: shell-to-fruit ratio (S/F, %), mesocarp-to-fruit ratio (M/F, %), kernel-to-fruit ratio (K/F, %), fruit per bunch (F/B, %), oil per bunch (O/B, %) and oil per palm (O/P, kg/palm/year). Genomic heritabilities of these traits were estimated to be in the range of 0.40 to 0.80. GS methods assessed were RR-BLUP, Bayes A (BA), Cπ (BC), Lasso (BL) and Ridge Regression (BRR). All methods resulted in almost equal prediction accuracy. The accuracy achieved ranged from 0.40 to 0.70, correlating with the heritability of traits. By selecting the most important markers, RR-BLUP B has the potential to outperform other methods. The marker density for certain traits can be further reduced based on the linkage disequilibrium (LD). Together with in silico breeding, GS is now being used in oil palm breeding programs to hasten parental palm selection.

Genome-wide association study identifies three key loci for high mesocarp oil content in perennial crop oil palm.

GWAS in out-crossing perennial crops is typically limited by insufficient marker density to account for population diversity and effects of population structure resulting in high false positive rates. The perennial crop oil palm is the most productive oil crop. We performed GWAS for oil-to-dry-mesocarp content (O/DM) on 2,045 genotyped tenera palms using 200K SNPs that were selected based on the short-range linkage disequilibrium distance, which is inherent with long breeding cycles and heterogeneous breeding populations. Eighty loci were significantly associated with O/DM (p ≤ 10(-4)) and three key signals were found. We then evaluated the progeny of a Deli x AVROS breeding trial and a 4% higher O/DM was observed amongst those having the beneficial genotypes at two of the three key loci (p < 0.05). We have initiated MAS and large-scale planting of elite dura and pisifera parents to generate the new commercial tenera palms with higher O/DM potential.

Development and Validation of a High-Density SNP Genotyping Array for African Oil Palm.

High-density single nucleotide polymorphism (SNP) genotyping arrays are powerful tools that can measure the level of genetic polymorphism within a population. To develop a whole-genome SNP array for oil palms, SNP discovery was performed using deep resequencing of eight libraries derived from 132 Elaeis guineensis and Elaeis oleifera palms belonging to 59 origins, resulting in the discovery of >3 million putative SNPs. After SNP filtering, the Illumina OP200K custom array was built with 170 860 successful probes. Phenetic clustering analysis revealed that the array could distinguish between palms of different origins in a way consistent with pedigree records. Genome-wide linkage disequilibrium declined more slowly for the commercial populations (ranging from 120 kb at r(2) = 0.43 to 146 kb at r(2) = 0.50) when compared with the semi-wild populations (19.5 kb at r(2) = 0.22). Genetic fixation mapping comparing the semi-wild and commercial population identified 321 selective sweeps. A genome-wide association study (GWAS) detected a significant peak on chromosome 2 associated with the polygenic component of the shell thickness trait (based on the trait shell-to-fruit; S/F %) in tenera palms. Testing of a genomic selection model on the same trait resulted in good prediction accuracy (r = 0.65) with 42% of the S/F % variation explained. The first high-density SNP genotyping array for oil palm has been developed and shown to be robust for use in genetic studies and with potential for developing early trait prediction to shorten the oil palm breeding cycle.

Evolutionary trace analysis at the ligand binding site of laccase.

Laccase belongs to the family of blue multi-copper oxidases and are capable of oxidizing a wide range of aromatic compounds. Laccases have industrial applications in paper pulping or bleaching and hydrocarbon bioremediation as a biocatalyst. We describe the design of a laccase with broader substrate spectrum in bioremediation. The application of evolutionary trace (ET) analysis of laccase at the ligand binding site for optimal design of the enzyme is described. In this attempt, class specific sites from ET analysis were mapped onto known crystal structure of laccase. The analysis revealed 162PHE as a critical residue in structure function relationship studies.