Construction of high density genetic linkage maps and fine mapping of quantitative trait loci associated with yield components and fatty acid composition in oil palm

The African oil palm (Elaeis guineensis Jacq.) is a highly valued oil producing perennial crop with a productive lifespan of about 25 years. Although benefiting from a long productivity period, oil palm genetic improvement is a slow and tedious process which requires between 10 to 12 years to complete one selection cycle. As such, marker-assisted selection (MAS) is an invaluable tool for yield improvement in oil palm. In particular, MAS could be used to facilitate efforts to increase the unsaturated fatty acid content of palm oil by introgression of desired alleles from the American species, E. oleifera which produces highly unsaturated oil. In order to help achieve this, high density genetic linkage maps were constructed for a commercial Deli dura (maternal) x Yangambi pisifera (paternal) breeding population (P2) and a Colombian E. oleifera (maternal) x Nigerian E. guineensis (paternal) interspecific hybrid population (OxG). The P2 and OxG mapping populations were screened with approximately 700 oil palm SSR primer-pairs and genotyped with a 4.5K customized oil palm SNP array to identify informative markers. The P2 and OxG populations consisted of 87 and 108 palms, respectively. Genetic linkage maps were first constructed for the individual parental palms of the mapping populations and followed by integration of the two parental maps. A P2 integrated map with 1,331 markers spanning 1,867 cM over the 16 linkage groups was constructed, representing the 16 chromosome pairs in oil palm. This study for the first time reports the genetic map for the Colombian E. oleifera, although the map only comprises of ten linkage groups with 65 markers spanning 471 cM. The Colombian E. oleifera map was also successfully integrated with the Nigerian E. guineensis parental map, resulting in a partial integrated map for OxG. The genetic maps and the available phenotypic data were successfully used to identify 22 quantitative trait loci (QTLs) associated with various yield components (YC) in the P2 population and 12 QTLs for fatty acid composition (FAC) in the OxG population. The QTLs identified for FAC include the total unsaturation levels via iodine value (IV), myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) acids. On the P2 integrated map, QTLs for YC were associated with mean bunch number (MBN), mean fruit weight (MFW), oil/bunch ratio (OTB), oil/wet mesocarp ratio (OTWP), oil/dry mesocarp ratio (OTDP), mean mesocarp weight (MPW), wet mesocarp weight (WPWT), kernel yield (KY), mean kernel weight (MKW), mean shell weight (MSW), shell to fruit (STF), total oil (TOT), oil yield (OY) and dry mesocarp weight (DPWT). The closely linked markers demonstrated significant allelic effects associated with the YC and FAC phenotypes analysed. The genetic effects (estimated by G Model) for the identified QTLs can be ranked from high for C16:0, C18:1 and IV (ranging from 2.16 – 2.46) to medium for OTWP, DPWT, MPW, WPWT, OTB, MFW, OTDP, MBN and STF (0.50 – 2.14) and low (< 0.5) for KY, MKW, MSW, C18:2, C18:0, C16:1 and C14:0. The three major QTLs for C16:0, C18:1 and IV were also successfully cross-mapped on two interspecific BC2 populations, which adds confidence on the association of the markers with the traits concerned and reflecting their potential utility in a MAS programme. This study also identified candidate regulatory genes and transcription factors (TFs) within the QTL confidence intervals by aligning to the oil palm (EG5) genome build. A set of five candidate genes (HIBCH, PATE/FATB, BASS2, LACS4 and DGAT1) and a TF (WRI1) were identified within the QTL confidence interval associated with genetic effects for C16:0, C18:1, C14:0, C18:0 and IV in LGOT1, which was supported by the significant differential expression patterns observed for the candidate genes and TF in RNA sequencing (RNA-seq) and real-time PCR (qRT-PCR) experiments. The RNA-seq and qRT-PCR experiments included palms from OxG and two independent interspecific backcross populations. Clear differential expression patterns were observed for some of the genes and their putative isoforms, which requires further validation in future studies. The high-density SNP and SSR-based genetic maps developed in this study have greatly improved marker density and genome coverage in comparison with the first reference map based on AFLP, RFLP and SSR markers. The improved maps with reduced gap between markers were aligned to the EG5 genome build, which proved useful for mining of candidate genes associated with the QTLs from the targeted regions. The closely linked markers and candidate genes associated with FAC and YC provide a good starting point for other genetic improvement studies in oil palm including whole genome association mapping studies. The candidate gene approach as used in the present study is useful for identifying the potential causal genes linked to QTLs. Finally, the markers closely linked to specific traits, specially IV, C16:0 and C18:1, MFW, MPW and OTDP have great potential and should be prioritized for further validation in the effort towards their adoption for MAS to introduce greater unsaturation and improve oil yield in commercial oil palm

Statistical mapping of quantitative trait loci controlling the time to first callusing in oil palm (elaeis guineensis Jacq.) tissue culture

An additional 400 genetic markers (126 RFLPs, 274 AFLPs) were successfully mapped on the earlier developed linkage maps using 87 F1 progenies derived from Deli dura X Yangambi pisifera cross. This resulted in a denser map with coverage length of 1, 714cM and 1,225cM for pisifera and dura, respectively. Further exploration to searchfor quantitative trait loci (QTL) associated with time to first callusing (TFC) was carried out by Kosambi Interval Mapping using the computer program MapQTL Version 4. O. The tissue culture trait data showed a continuous distribution. In this paper; three likelihood QTLs were detected in pisifera and two QTLs in dura at 99% and 95% significant thresholds. These QTL locations can be designated as statistically significant for contributing to the variation of TFG. Therefore, the information points to a genomic loci affecting tissue culturability in oil palm

Candidate genes linked to QTL regions associated with fatty acid composition in oil palm

© 2020, Institute of Molecular Biology, Slovak Academy of Sciences. The present study searched for candidate genes in five linkage groups (LGs) - T2, T3, OT4, OT6 and T9 hosting the QTLs associated with iodine value (IV) and fatty acid composition (FAC) in an oil palm interspecific hybrid population. Each of the five LGs was successfully anchored to its corresponding chromosomal segment where, a wider repertoire of candidate genes was identified. This study further revealed a total of 19 candidate genes and four transcription factors involved in biosynthesis of fatty acids, lipids (including triacylglycerol) and acetyl-CoA, glycosylation and degradation of fatty acids. Their possible involvement in regulating the levels of saturation are discussed. In addition, 22 candidate genes located outside the QTL intervals were also identified across the interspecific hybrid genome. A total of 92 SSR markers were developed to tag the presence of these candidate genes and 50 were successfully mapped onto their respective positions on the genome. The data obtained here complements the previous studies, and collectively, these QTL-linked candidate gene markers could help breeders in more precisely selecting palms with the desired FAC

SSR mining in oil palm EST database: application in oil palm germplasm diversity studies.

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

Genetic Linkage Map of a High Yielding FELDA Deli×Yangambi Oil Palm Cross

Enroute to mapping QTLs for yield components in oil palm, we constructed the linkage map of a FELDA high yielding oil palm (Elaeis guineensis), hybrid cross. The parents of the mapping population are a Deli dura and a pisifera of Yangambi origin. The cross out-yielded the average by 8–21% in four trials all of which yielded comparably to the best current commercial planting materials. The higher yield derived from a higher fruit oil content. SSR markers in the public domain - from CIRAD and MPOB, as well as some developed in FELDA - were used for the mapping, augmented by locally-designed AFLP markers. The female parent linkage map comprised 317 marker loci and the male parent map 331 loci, both in 16 linkage groups each. The number of markers per group ranged from 8–47 in the former and 12–40 in the latter. The integrated map was 2,247.5 cM long and included 479 markers and 168 anchor points. The number of markers per linkage group was 15–57, the average being 29, and the average map density 4.7 cM. The linkage groups ranged in length from 77.5 cM to 223.7 cM, with an average of 137 cM. The map is currently being validated against a closely related population and also being expanded to include yield related QTLs

Expression of fatty acid and triacylglycerol synthesis genes in interspecific hybrids of oil palm

© 2020, The Author(s). Evaluation of transcriptome data in combination with QTL information has been applied in many crops to study the expression of genes responsible for specific phenotypes. In oil palm, the mesocarp oil extracted from E. oleifera × E. guineensis interspecific hybrids is known to have lower palmitic acid (C16:0) content compared to pure African palms. The present study demonstrates the effectiveness of transcriptome data in revealing the expression profiles of genes in the fatty acid (FA) and triacylglycerol (TAG) biosynthesis processes in interspecific hybrids. The transcriptome assembly yielded 43,920 putative genes of which a large proportion were homologous to known genes in the public databases. Most of the genes encoding key enzymes involved in the FA and TAG synthesis pathways were identified. Of these, 27, including two candidate genes located within the QTL associated with C16:0 content, showed differential expression between developmental stages, populations and/or palms with contrasting C16:0 content. Further evaluation using quantitative real-time PCR revealed that differentially expressed patterns are generally consistent with those observed in the transcriptome data. Our results also suggest that different isoforms are likely to be responsible for some of the variation observed in FA composition of interspecific hybrids

Comparison of quantitative trait loci (QTLs) associated with yield components in two commercial Dura × Pisifera breeding crosses

The high yielding tenera is the commercial oil palm planting material of choice in Southeast Asia. Notwithstanding this, there is continuous effort to further improve the yield and one way to do this is by addressing the yield components (YCs ). Using 4,451 SNP and over 600 SSR markers , this study revealed quantitative trait loci (QTL) associated with YCs in two breeding populations, a Deli dura x Yangambi pisifera (P2) and a Deli dura x AVROS pisifera (KULIM DxP). Thirteen and 29 QTLs were identified in P2 and KULIM DxP, respectively . They were compared to other YC-linked QTLs reported previously for different genetic backgrounds by mapping the QTL-linked markers to the oil palm genome . The comparison revealedfour common chromosomes containing QTLs influencing various YCs . The results reveal the possible presence of closely linked loci or pleiotropic genes influencing YCs in oil palm. Exploiting the genome data has also facilitated the discovery of candidate genes within or near the QTL regions including those related to glycosylation, fatty acid and oil biosynthesis, and development of flower, seed and fruit

Identification of Quantitative Trait Loci for Tissue Culture Amenability Traits in a Dura X Pisifera F1 Oil Palm Population

Tissue culture is employed for the large scale production of elite planting materials in the oil palm plantation industry of Malaysia. The advantage of this technique is the homogeneous quality that can be produced across the clones. However, the production cost is high compared to conventional seedlings due to some unpredictable constraints. One of the major problems that has been reported is the average low rate of regeneration among the clones. Inconsistencies have been reported in that both callogenesis and embryogenesis varied among the lines. Attempts were made to generate restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP). These DNA markers were used for constructing genetic linkage maps for two commercial palms namely, Ulu Remis Deli dura (ENL48) and Yangambi pisifera (ML161). Both maps were used as frameworks to detect quantitative trait loci (QTL) associated with the traits of tissue culture amenability. The generation of DNA markers involved the genotyping of 87 F1 palms using 70 cDNA probes and 16 EcoRI/MseI and 8 PstI/MseI primer-pairs. This produced a total of 36 RFLPs and 43 AFLPs for ENL48 and 66 RFLPs and 58 AFLPs for ML161. The data were integrated into the existing database for the mapping analysis by using JoinMap®3.0 at LOD 4.0 and recombination frequency θ <0.400. The ENL48 linkage map was constructed by using 87 RFLPs and 123 AFLPs. These markers were grouped into 25 linkage groups covering a total genetic distance of 1,136.3cM. A denser map of ML161 was obtained with 147 RFLPs and 225 AFLPs which resulted in 16 linkage groups with a total map length of 1,755.7cM. The QTL for tissue culture amenability were determined using interval mapping and multiple quantitative mapping (MQM) with the help of the MapQTL® 4.0 software. The LOD thresholds were estimated using the permutation test. The QTLs for the callusing rate (CR) were detected at linkage groups D9 and P7 on the ENL48 and the ML161 maps, respectively. For embryogenesis rate (Er_ex), QTL was only detected on the ML161 genetic map at linkage group P13. All the detected QTLs were confirmed in a second experiment where the tissue culture data was collected using different media compositions. These improved confidence in the QTLs detected for tissue culture amenability

Construction of high density genetic linkage maps and fine mapping of quantitative trait loci associated with yield components and fatty acid composition in oil palm

The African oil palm (Elaeis guineensis Jacq.) is a highly valued oil producing perennial crop with a productive lifespan of about 25 years. Although benefiting from a long productivity period, oil palm genetic improvement is a slow and tedious process which requires between 10 to 12 years to complete one selection cycle. As such, marker-assisted selection (MAS) is an invaluable tool for yield improvement in oil palm. In particular, MAS could be used to facilitate efforts to increase the unsaturated fatty acid content of palm oil by introgression of desired alleles from the American species, E. oleifera which produces highly unsaturated oil. In order to help achieve this, high density genetic linkage maps were constructed for a commercial Deli dura (maternal) x Yangambi pisifera (paternal) breeding population (P2) and a Colombian E. oleifera (maternal) x Nigerian E. guineensis (paternal) interspecific hybrid population (OxG). The P2 and OxG mapping populations were screened with approximately 700 oil palm SSR primer-pairs and genotyped with a 4.5K customized oil palm SNP array to identify informative markers. The P2 and OxG populations consisted of 87 and 108 palms, respectively. Genetic linkage maps were first constructed for the individual parental palms of the mapping populations and followed by integration of the two parental maps. A P2 integrated map with 1,331 markers spanning 1,867 cM over the 16 linkage groups was constructed, representing the 16 chromosome pairs in oil palm. This study for the first time reports the genetic map for the Colombian E. oleifera, although the map only comprises of ten linkage groups with 65 markers spanning 471 cM. The Colombian E. oleifera map was also successfully integrated with the Nigerian E. guineensis parental map, resulting in a partial integrated map for OxG. The genetic maps and the available phenotypic data were successfully used to identify 22 quantitative trait loci (QTLs) associated with various yield components (YC) in the P2 population and 12 QTLs for fatty acid composition (FAC) in the OxG population. The QTLs identified for FAC include the total unsaturation levels via iodine value (IV), myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) acids. On the P2 integrated map, QTLs for YC were associated with mean bunch number (MBN), mean fruit weight (MFW), oil/bunch ratio (OTB), oil/wet mesocarp ratio (OTWP), oil/dry mesocarp ratio (OTDP), mean mesocarp weight (MPW), wet mesocarp weight (WPWT), kernel yield (KY), mean kernel weight (MKW), mean shell weight (MSW), shell to fruit (STF), total oil (TOT), oil yield (OY) and dry mesocarp weight (DPWT). The closely linked markers demonstrated significant allelic effects associated with the YC and FAC phenotypes analysed. The genetic effects (estimated by G Model) for the identified QTLs can be ranked from high for C16:0, C18:1 and IV (ranging from 2.16 – 2.46) to medium for OTWP, DPWT, MPW, WPWT, OTB, MFW, OTDP, MBN and STF (0.50 – 2.14) and low (< 0.5) for KY, MKW, MSW, C18:2, C18:0, C16:1 and C14:0. The three major QTLs for C16:0, C18:1 and IV were also successfully cross-mapped on two interspecific BC2 populations, which adds confidence on the association of the markers with the traits concerned and reflecting their potential utility in a MAS programme. This study also identified candidate regulatory genes and transcription factors (TFs) within the QTL confidence intervals by aligning to the oil palm (EG5) genome build. A set of five candidate genes (HIBCH, PATE/FATB, BASS2, LACS4 and DGAT1) and a TF (WRI1) were identified within the QTL confidence interval associated with genetic effects for C16:0, C18:1, C14:0, C18:0 and IV in LGOT1, which was supported by the significant differential expression patterns observed for the candidate genes and TF in RNA sequencing (RNA-seq) and real-time PCR (qRT-PCR) experiments. The RNA-seq and qRT-PCR experiments included palms from OxG and two independent interspecific backcross populations. Clear differential expression patterns were observed for some of the genes and their putative isoforms, which requires further validation in future studies. The high-density SNP and SSR-based genetic maps developed in this study have greatly improved marker density and genome coverage in comparison with the first reference map based on AFLP, RFLP and SSR markers. The improved maps with reduced gap between markers were aligned to the EG5 genome build, which proved useful for mining of candidate genes associated with the QTLs from the targeted regions. The closely linked markers and candidate genes associated with FAC and YC provide a good starting point for other genetic improvement studies in oil palm including whole genome association mapping studies. The candidate gene approach as used in the present study is useful for identifying the potential causal genes linked to QTLs. Finally, the markers closely linked to specific traits, specially IV, C16:0 and C18:1, MFW, MPW and OTDP have great potential and should be prioritized for further validation in the effort towards their adoption for MAS to introduce greater unsaturation and improve oil yield in commercial oil palm

Putative regulatory candidate genes for QTL linked to fruit traits in oil palm (Elaeis guineensis Jacq.)

Palm oil is among the most important vegetable oils, contributing to a quarter of the world’s oils and fats market. The oil palm (Elaeis guineensis Jacq.) fruitlets, which are the source of palm oil, vary from 8eight to 20 g in weight. Palm oil content in the fruitlets is approximately 45 – 50 % by weight and an increase in the percentage of mesocarp-to-fruit is likely to have a positive effect on oil yield. In this study, we report a quantitative trait loci (QTL) associated with two yield related components, namely fruit and mesocarp content in a commercial breeding population (Deli dura x Yangambi pisifera). The QTL confidence interval of about 12 cM (~6.7 Mbp) was fine-mapped with 31 markers (17 SNPs and 14 SSRs) consisting of 20 nuclear markers derived from the maternal parent, six paternal and five co-segregating markers. Interestingly, inheritance of the paternal alleles leads to a larger difference in both fruit and mesocarp weight, when comparing genotypes in the progeny palms. Candidate genes and transcription factors were mined from the QTL region by positioning markers on the oil palm EG5 genome build. Putative genes and transcription factors involved in various biological processes including flower organ development, flowering, photosynthesis, microtubule formation, nitrogen and lipid metabolism were identified within this QTL interval on pseudo-chromosome 3. This genome-based approach allowed us to identify a number of potential candidate gene markers associated with oil palm fruit and mesocarp weight which can be further evaluated for potential use in marker-assisted breeding