Developing functional markers for vitamin E biosynthesis in oil palm.

Vitamin E is essential for human health and plays positive roles in anti-oxidation. Previously, we detected large variation in vitamin E content among 161 oil palm accessions. In this study, twenty oil palm accessions with distinct variation in vitamin E contents (171.30 to 1 258.50 ppm) were selected for genetic variation analysis and developing functional markers associated with vitamin E contents. Thirty-seven homologous genes in oil palm belonging to vitamin E biosynthesis pathway were identified via BLASTP analysis, the lengths of which ranged from 426 to 25 717 bp (average 7 089 bp). Multiplex PCR sequencing for the 37 genes found 1 703 SNPs and 85 indels among the 20 oil palm accessions, with 226 SNPs locating in the coding regions. Clustering analysis for these polymorphic loci showed that the 20 oil palm accessions could be divided into five groups. Among these groups, group I included eight oil palm accessions whose vitamin E content (mean value: 893.50 ppm) was far higher than other groups (mean value 256.29 to 532.94 ppm). Correlation analysis between the markers and vitamin E traits showed that 134 SNP and 7 indel markers were significantly (p < 0.05) related with total vitamin E content. Among these functional markers, the indel EgTMT-1-24 was highly correlated with variation in vitamin E content, especially tocotrienol content. Our study identified a number of candidate function associated markers and provided clues for further research into molecular breeding for high vitamin E content oil palm.

High-quality reference genome sequences of two coconut cultivars provide insights into evolution of monocot chromosomes and differentiation of fiber content and plant height.

BACKGROUND: Coconut is an important tropical oil and fruit crop whose evolutionary position renders it a fantastic species for the investigation of the evolution of monocot chromosomes and the subsequent differentiation of ancient plants. RESULTS: Here, we report the assembly and annotation of reference-grade genomes of Cn. tall and Cn. dwarf, whose genome sizes are 2.40 Gb and 2.39 Gb, respectively. The comparative analysis reveals that the two coconut subspecies diverge about 2-8 Mya while the conserved Arecaceae-specific whole-genome duplication (ω WGD) occurs approximately 47-53 Mya. It additionally allows us to reconstruct the ancestral karyotypes of the ten ancient monocot chromosomes and the evolutionary trajectories of the 16 modern coconut chromosomes. Fiber synthesis genes in Cn. tall, related to lignin and cellulose synthesis, are found at a higher copy number and expression level than dwarf coconuts. Integrated multi-omics analysis reveals that the difference in coconut plant height is the result of altered gibberellin metabolism, with both the GA20ox copy number and a single-nucleotide change in the promoter together leading to the difference in plant height between Cn. tall and Cn. dwarf. CONCLUSION: We provide high-quality coconut genomes and reveal the genetic basis of trait differences between two coconuts through multi-omics analysis. We also reveal that the selection of plant height has been targeted for the same gene for millions of years, not only in natural selection of ancient plant as illustrated in coconut, but also for artificial selection in cultivated crops such as rice and maize.

Coconut genome assembly enables evolutionary analysis of palms and highlights signaling pathways involved in salt tolerance.

Coconut (Cocos nucifera) is the emblematic palm of tropical coastal areas all around the globe. It provides vital resources to millions of farmers. In an effort to better understand its evolutionary history and to develop genomic tools for its improvement, a sequence draft was recently released. Here, we present a dense linkage map (8402 SNPs) aiming to assemble the large genome of coconut (2.42 Gbp, 2n = 32) into 16 pseudomolecules. As a result, 47% of the sequences (representing 77% of the genes) were assigned to 16 linkage groups and ordered. We observed segregation distortion in chromosome Cn15, which is a signature of strong selection among pollen grains, favouring the maternal allele. Comparing our results with the genome of the oil palm Elaeis guineensis allowed us to identify major events in the evolutionary history of palms. We find that coconut underwent a massive transposable element invasion in the last million years, which could be related to the fluctuations of sea level during the glaciations at Pleistocene that would have triggered a population bottleneck. Finally, to better understand the facultative halophyte trait of coconut, we conducted an RNA-seq experiment on leaves to identify key players of signaling pathways involved in salt stress response. Altogether, our findings represent a valuable resource for the coconut breeding community.

Genome-wide discovery and characterization of long noncoding RNAs in African oil palm (Elaeis guineensis Jacq.).

Long noncoding RNAs (lncRNAs) are an important class of genes and play important roles in a range of biological processes. However, few reports have described the identification of lncRNAs in oil palm. In this study, we applied strand specific RNA-seq with rRNA removal to identify 1,363 lncRNAs from the equally mixed tissues of oil palm spear leaf and six different developmental stages of mesocarp (8-24 weeks). Based on strand specific RNA-seq data and 18 released oil palm transcriptomes, we systematically characterized the expression patterns of lncRNA loci and their target genes. A total of 875 uniq target genes for natural antisense lncRNAs (NAT-lncRNA, 712), long intergenic noncoding RNAs (lincRNAs, 92), intronic-lncRNAs (33), and sense-lncRNAs (52) were predicted. A majority of lncRNA loci (77.8%-89.6%) had low expression in 18 transcriptomes, while only 89 lncRNA loci had medium to high expression in at least one transcriptome. Coexpression analysis between lncRNAs and their target genes indicated that 6% of lncRNAs had expression patterns positively correlated with those of target genes. Based on single nucleotide polymorphism (SNP) markers derived from our previous research, 6,882 SNPs were detected for lncRNAs and 28 SNPs belonging to 21 lncRNAs were associated with the variation of fatty acid contents. Moreover, seven lncRNAs showed expression patterns positively correlated expression pattern with those of genes in de novo fatty acid synthesis pathways. Our study identified a collection of lncRNAs for oil palm and provided clues for further research into lncRNAs that may regulate mesocarp development and lipid metabolism.

Alternative splicing of flowering time gene FT is associated with halving of time to flowering in coconut.

Coconut palm has two distinct types-"tall" and "dwarf"-which differ morphologically. Tall coconut varieties need 8-10 years to start flowering, while dwarf coconut varieties only require 3-5 years. We compared seedling and reproductive stage transcriptomes for both coconut types to determine potential molecular mechanisms underlying control of flowering time in coconut. Several key genes in the photoperiod pathway were differentially expressed between seedling and reproductive leaf samples in both tall and dwarf coconut. These genes included suppressor of overexpression of constans (SOC1), flowering locus T (FT), and Apetala 1 (AP1). Alternative splicing analysis of genes in the photoperiod pathway further revealed that the FT gene produces different transcripts in tall compared to dwarf coconut. The shorter alternative splice variant of FT [which included a 6 bp deletion, alternative 3' splicing sites (A3SS)] was found to be exclusively present in dwarf coconut varieties but absent in most tall coconut varieties. Our results provide a valuable information resource as well as suggesting a probable mechanism for differentiation of flowering time onset in coconut, providing a target for future breeding work in accelerating time to flowering in this crop species.

iTRAQ-based comparative proteomic analysis of two coconut varieties reveals aromatic coconut cold-sensitive in response to low temperature.

Coconut (Cocos nucifera L.) is an important economic fruit and oil crop largely cultivated in humid and sub-humid tropical coastal zones worldwide. To date proteomic profile analysis of coconut under cold stress yet not been conducted. In order to understand the cold stress tolerance in coconut, the iTRAQ approach was employed to dissect proteomic response of two coconut varieties Hainan Tall, BenDi (BD) and Aromatic coconut, XiangShui (XS) under cold stress. Under cold treatment at (8 °C) for 2 days, 193 up and 134 down-regulated in BD (Cn-DB-0_VS_Cn-DB-2) and 140 up and 155 down-regulated DEPs in XS (Cn-XS-0_VS_Cn-XS-2) were identified. The 5 days post cold treatment also identified increased abundance of up-regulated proteins in BD compared to XS. The 5 days post treatment (dpt) depicted 172-up/127-down and 108-up/134-down accumulated proteins for BD (Cn-DB-0_VS_Cn-DB-5) and XS (Cn-XS-0_VS_Cn-XS-5) respectively. A total of 22, 12 and 14 DEP categories were enriched in biological process, cellular component and molecular function respectively in Gene Ontology (GO) analysis of two coconut varieties. Metabolic and biosynthesis of secondary metabolites pathways were highly enriched in KEGG pathway analysis of DEPs between two varieties. Twenty-two different functional classes revealed differentially expressed proteins in two varieties. Among those, four major categories involved in metabolism, stress response, photosynthesis and respiration related DEPs increased abundance in two varieties. However, general function perdition only (GFPO) and stress-responsive proteins were greatly up-regulated in BD than XS. Increased abundance of stress response related proteins up-regulation under cold stress suggested that BD is cold-tolerant variety. Collectively, iTRAQ-based coconut leaf proteomic analysis showed that XS (aromatic) coconut variety is cold-sensitive compared to BD (Hainan Tall) variety. This study provided a basis for further functional analyses to understand the molecular mechanisms of tropical crops adapting to cold stress. SIGNIFICANCE: Leaf proteomic approach determines the role of differentially expressed proteins (DEPs) under cold stress in crops. However, cold stress could damage the coconut fruit lead to decrease in crop yield during winter in China. Here, we report the first ever iTRAQ-based proteomic analysis of two coconut varieties in response to cold stress. The study identified the proteins involved in biosynthesis of secondary metabolites, photosynthesis, respiration, biotic and abiotic stresses under cold stress in two coconut varieties. Moreover, the increased abundance of stress-responsive and general function proteins in BD under cold stress suggested that Hainan Tall is cold-tolerant compared to aromatic coconut variety. Inhibition abundance of photosynthesis related proteins may reduce photodamage owing to the over energized state of thylakoid membrane lead to ROS generation during oxidative stress. This could be the reason for adaption of BD to low temperature stress. Nonetheless, further research may insight the mechanism involved in cold tolerance/sensitive in coconut in response to low temperature.

Identifying Vitamin E Biosynthesis Genes in Elaeis guineensis by Genome-Wide Association Study.

Elaeis guineensis is a tropical oil crop and has the highest oil yield per unit area. Palm oil has high palmitic acid content and is also rich in vitamins, including vitamin E. We conducted genome-wide association studies in a diversity panel of 161 E. guineensis accessions to identify single-nucleotide polymorphisms (SNPs) linked with vitamin E and validated candidate genes in these marker-associated intervals. Based on the SNPs reported in our previous research, 47 SNP markers were detected to be significantly associated with the variation of tocopherol and tocotrienol content at a cutoff P value of 6.3 × 10-7. A total of 656 candidate genes in the flanking regions of the 47 SNPs were identified, followed by pathway enrichment analysis. Of these candidate genes, EgHGGT (homogentisate geranylgeranyl transferase) involved in the biosynthesis of tocotrienols had a higher expression level in the mesocarp compared to other tissues. Expression of the EgHGGT gene was positively correlated with the variation in α-tocotrienol content. Induced overexpression of the gene in Arabidopsis caused a significant increase in vitamin E content and production of α-tocotrienols compared to wild Arabidopsis.

Correlation analysis of cold-related gene expression with physiological and biochemical indicators under cold stress in oil palm.

Oil palm (Elaeis guineensis Jacq.) is a representative tropical oil crop that is sensitive to low temperature. Oil palm can experience cold damage when exposed to low temperatures for a long period. During these unfavorable conditions, a series of gene induction/repression and physico-chemical changes occur in oil palm. To better understand the link between these events, we investigated the expression levels of various genes (including COR410, COR413, CBF1, CBF2, CBF3, ICE1-1, ICE1-2, ICE1-4, SIZ1-1, SIZ1-2, ZAT10, ZAT12) and the accumulation of osmolytes (proline, malondialdehyde and sucrose). Likewise, the activity of superoxide dismutase (SOD) in oil palm under cold stress (4°C, 8°C and 12°C) was examined. The results showed a clear link among the expression of CBFs (especially CBF1 and CBF3) and the all genes examined under cold stress (12°C). The expression of CBF1 and CBF2 also exhibited a positive link with the accumulation of sucrose and proline under cold stress in oil palm. At 4°C, the proline content exhibited a very significant correlation with electrolyte leakage in oil palm. The results of this study provide necessary information regarding the mechanism of the response and adaption of oil palm to cold stress. Additionally, they offer clues for the selection or development of cold-tolerant cultivars from the available germplasms of oil palm.

Genetic control of fatty acid composition in coconut (Cocos nucifera), African oil palm (Elaeis guineensis), and date palm (Phoenix dactylifera).

MAIN CONCLUSION: Predominant gene isoforms and expression bias in lipid metabolism pathways are highly conserved between oil-producing Arecaceae crop species coconut and oil palm, but diverge in non-oil-producing species date palm. Coconut (Cocos nucifera), African oil palm (Elaeis guineensis) and date palm (Phoenix dactylifera) are three major crop species in the Arecaceae family for which genome sequences have recently become available. Coconut and African oil palm both store oil in their endosperms, while date palm fruits contain very little oil. We analyzed fatty acid composition in three coconut tissues (leaf, endosperm and embryo) and in two African oil palm tissues (leaf and mesocarp), and identified 806, 840 and 848 lipid-related genes in 22 lipid metabolism pathways from the coconut, African oil palm and date palm genomes, respectively. The majority of lipid-related genes were highly homologous and retained in homologous segments between the three species. Genes involved in the conversion of pyruvate to fatty acid had a five-to-sixfold higher expression in the coconut endosperm and oil palm mesocarp than in the leaf or embryo tissues based on Fragments Per Kilobase of transcript per Million mapped reads values. A close evolutionary relationship between predominant gene isoforms and high conservation of gene expression bias in the lipid and carbohydrate gene metabolism pathways was observed for the two oil-producing species coconut and oil palm, differing from that of date palm, a non-oil-producing species. Our results elucidate the similarities and differences in lipid metabolism between the three major Arecaceae crop species, providing important information for physiology studies as well as breeding for fatty acid composition and oil content in these crops.

The genome draft of coconut (Cocos nucifera).

Coconut palm (Cocos nucifera,2n = 32), a member of genus Cocos and family Arecaceae (Palmaceae), is an important tropical fruit and oil crop. Currently, coconut palm is cultivated in 93 countries, including Central and South America, East and West Africa, Southeast Asia and the Pacific Islands, with a total growth area of more than 12 million hectares [1]. Coconut palm is generally classified into 2 main categories: "Tall" (flowering 8-10 years after planting) and "Dwarf" (flowering 4-6 years after planting), based on morphological characteristics and breeding habits. This Palmae species has a long growth period before reproductive years, which hinders conventional breeding progress. In spite of initial successes, improvements made by conventional breeding have been very slow. In the present study, we obtained de novo sequences of the Cocos nucifera genome: a major genomic resource that could be used to facilitate molecular breeding in Cocos nucifera and accelerate the breeding process in this important crop. A total of 419.67 gigabases (Gb) of raw reads were generated by the Illumina HiSeq 2000 platform using a series of paired-end and mate-pair libraries, covering the predicted Cocos nucifera genome length (2.42 Gb, variety "Hainan Tall") to an estimated ×173.32 read depth. A total scaffold length of 2.20 Gb was generated (N50 = 418 Kb), representing 90.91% of the genome. The coconut genome was predicted to harbor 28 039 protein-coding genes, which is less than in Phoenix dactylifera (PDK30: 28 889), Phoenix dactylifera (DPV01: 41 660), and Elaeis guineensis (EG5: 34 802). BUSCO evaluation demonstrated that the obtained scaffold sequences covered 90.8% of the coconut genome and that the genome annotation was 74.1% complete. Genome annotation results revealed that 72.75% of the coconut genome consisted of transposable elements, of which long-terminal repeat retrotransposons elements (LTRs) accounted for the largest proportion (92.23%). Comparative analysis of the antiporter gene family and ion channel gene families between C. nucifera and Arabidopsis thaliana indicated that significant gene expansion may have occurred in the coconut involving Na+/H+ antiporter, carnitine/acylcarnitine translocase, potassium-dependent sodium-calcium exchanger, and potassium channel genes. Despite its agronomic importance, C. nucifera is still under-studied. In this report, we present a draft genome of C. nucifera and provide genomic information that will facilitate future functional genomics and molecular-assisted breeding in this crop species.

Genome-Wide Identification and Transferability of Microsatellite Markers between Palmae Species.

The Palmae family contains 202 genera and approximately 2800 species. Except for Elaeis guineensis and Phoenix dactylifera, almost no genetic and genomic information is available for Palmae species. Therefore, this is an obstacle to the conservation and genetic assessment of Palmae species, especially those that are currently endangered. The study was performed to develop a large number of microsatellite markers which can be used for genetic analysis in different Palmae species. Based on the assembled genome of E. guineensis and P. dactylifera, a total of 814 383 and 371 629 microsatellites were identified. Among these microsatellites identified in E. guineensis, 734 509 primer pairs could be designed from the flanking sequences of these microsatellites. The majority (618 762) of these designed primer pairs had in silico products in the genome of E. guineensis. These 618 762 primer pairs were subsequently used to in silico amplify the genome of P. dactylifera. A total of 7 265 conserved microsatellites were identified between E. guineensis and P. dactylifera. One hundred and thirty-five primer pairs flanking the conserved SSRs were stochastically selected and validated to have high cross-genera transferability, varying from 16.7 to 93.3% with an average of 73.7%. These genome-wide conserved microsatellite markers will provide a useful tool for genetic assessment and conservation of different Palmae species in the future.

RNA-Seq analysis of Cocos nucifera: transcriptome sequencing and de novo assembly for subsequent functional genomics approaches.

BACKGROUND: Cocos nucifera (coconut), a member of the Arecaceae family, is an economically important woody palm grown in tropical regions. Despite its agronomic importance, previous germplasm assessment studies have relied solely on morphological and agronomical traits. Molecular biology techniques have been scarcely used in assessment of genetic resources and for improvement of important agronomic and quality traits in Cocos nucifera, mostly due to the absence of available sequence information. METHODOLOGY/PRINCIPAL FINDINGS: To provide basic information for molecular breeding and further molecular biological analysis in Cocos nucifera, we applied RNA-seq technology and de novo assembly to gain a global overview of the Cocos nucifera transcriptome from mixed tissue samples. Using Illumina sequencing, we obtained 54.9 million short reads and conducted de novo assembly to obtain 57,304 unigenes with an average length of 752 base pairs. Sequence comparison between assembled unigenes and released cDNA sequences of Cocos nucifera and Elaeis guineensis indicated that the assembled sequences were of high quality. Approximately 99.9% of unigenes were novel compared to the released coconut EST sequences. Using BLASTX, 68.2% of unigenes were successfully annotated based on the Genbank non-redundant (Nr) protein database. The annotated unigenes were then further classified using the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. CONCLUSIONS/SIGNIFICANCE: Our study provides a large quantity of novel genetic information for Cocos nucifera. This information will act as a valuable resource for further molecular genetic studies and breeding in coconut, as well as for isolation and characterization of functional genes involved in different biochemical pathways in this important tropical crop species.

[Generation of selectable marker-free and vector backbone sequence-free Xa21 transgenic rice].

The dominant gene Xa21 with broad-spectrum and high resistance to Xanthomonas oryzae pv. oryzae (Xoo) was transferred into C418, an important restorer line of japonica hybrid rice in China using double right-border (DRB) T-DNA binary vector through Agrobacterium-mediated transformation. 17 transgenic lines were Xa21-positive with high resistance to the race P6 of Xoo through PCR analysis and resistance identification, among the total 27 independent primary transformants (T0) obtained. The subsequent analysis of the T1 progenies of these 17 T0 lines through PCR-assisted selection and resistance investigation showed that four Xa21 transgenic T0 lines could produce selectable marker-free (SMF) progenies. The frequency of primary transformants producing SMF progenies was 15%. In addition, PCR analysis also revealed these SMF progenies did not contain vector backbone sequence, and they were named as SMF and vector backbone sequence-free (SMF-VBSF) Xa21 transgenic plants. The further molecular and phenotypic analysis of the T2 and T3 progenies testified the homozygous SMF-VBSF Xa21 transgenic plants were obtained with high resistance to Xoo.