Whole-genome sequencing of Ganoderma boninense, the causal agent of basal stem rot disease in oil palm, via combined short- and long-read sequencing.

The hemibiotrophic Basidiomycete pathogen Ganoderma boninense (Gb) is the dominant causal agent of oil palm basal stem rot disease. Here, we report a complete chromosomal genome map of Gb using a combination of short-read Illumina and long-read Pacific Biosciences (PacBio) sequencing platforms combined with chromatin conformation capture data from the Chicago and Hi-C platforms. The genome was 55.87 Mb in length and assembled to a high contiguity (N50: 304.34 kb) of 12 chromosomes built from 112 scaffolds, with a total of only 4.34 Mb (~ 7.77%) remaining unplaced. The final assemblies were evaluated for completeness of the genome by using Benchmarking Universal Single Copy Orthologs (BUSCO) v4.1.4, and based on 4464 total BUSCO polyporales group searches, the assemblies yielded 4264 (95.52%) of the conserved orthologs as complete and only a few fragmented BUSCO of 42 (0.94%) as well as a missing BUSCO of 158 (3.53%). Genome annotation predicted a total of 21,074 coding genes, with a GC content ratio of 59.2%. The genome features were analyzed with different databases, which revealed 2471 Gene Ontology/GO (11.72%), 5418 KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthologous/KO (25.71%), 13,913 Cluster of Orthologous Groups of proteins/COG (66.02%), 60 ABC transporter (0.28%), 1049 Carbohydrate-Active Enzymes/CAZy (4.98%), 4005 pathogen-host interactions/PHI (19%), and 515 fungal transcription factor/FTFD (2.44%) genes. The results obtained in this study provide deep insight for further studies in the future.

Identification of oil palm cis-regulatory elements based on DNA free energy and single nucleotide polymorphism density.

Transcription control through cis-regulatory elements (CREs) is one of important regulators of gene expression. This study aimed to identify the location of CREs in oil palm (Elaeis guineensis Jacq.) using the combination of DNA free energy and single nucleotide polymorphism (SNP) density approaches. Promoter region sequences were extracted oil palm genome spanning from 1500 nucleotides (nt) upstream to 1000 nt downstream of every annotated transcription start sites (TSS). Free energy profiles of each promoter region were calculated using PromPredict software. Raw reads from the deep sequencing of 59 oil palm origins were used to calculate SNP density of each promoter region. The result showed that the average free energy (AFE) on the upstream region of TSS is about 1.5 kcal/mol higher compared to the downstream region. Using DNA free energy method, 16,281 regions of CREs were predicted. Most of predicted CREs was located between 1 and 500 nt upstream of TSS. Anti-correlation pattern between free energy and SNP density was observed on the predicted regions of CREs. This anti-correlated pattern was also observed on an experimentally determined promoter of the oil palm metallothionein gene, EgMSP1. Considering the increasing use of promoter information on plant biotechnology, an easy and accurate promoter prediction using the combination of free energy and SNP density method could be recommended.

Identification of microRNAs involved in the Phosphate starvation response in Oil Palm (Elaeis guineensis Jacq.).

BACKGROUND: Plant microRNA, often known as miRNA, is a novel form of gene expression regulator that is known to play a significant role in phosphate starvation. The identification of microRNAs involved in the response to phosphate starvation in oil palms is beneficial for breeding programs. METHOD: The main nursery stage seedlings of two oil palm progenies were treated with three different fertiliser namely: complete fertiliser with urea, P2O5, K2O, and MgO based on the standard procedure as a control (C); fertiliser with urea, K2O, MgO without P2O5 (P0); and no fertiliser (F0) for 24 weeks. A total of six oil palm roots were subjected to RNA isolation, followed by miRNA sequencing using the Illumina HiSeq 4000 platform, and all reads were computationally analysed. RESULTS: In total, 119 potential miRNAs related to 5,891 genes were identified. The P-specific miRNAs were assumed based on the miRNAs that identified without P fertilizer treatment, resulted of twenty miRNA sequences in the treatment comparison of (C vs P0) vs (C vs F0). Those 20 miRNA sequences were grouped into 9 families, namely EgmiR319; EgmiR399; EgmiR396; EgmiR172; EgmiR156; EgmiR157; miR5648; miR5645; and EgmiRNA_unidentified. Two miRNAs were selected for RT-qPCR validation, namely EgMir399 and EgMir172. Their expression pattern was similar with the RNA sequencing results and shown opposite expression pattern with their target genes, UBC E2 24 and APETALA2, respectively. CONCLUSIONS: The nine micro RNA families was identified in oil palm root tissue at phosphate starvation.

Transcriptome profiling of high and low somatic embryogenesis rate of oil palm (Elaeis guineensis Jacq. var. Tenera).

Oil palm micropropagation through tissue culture is a technique to provide elite oil palms to meet the desired traits. This technique is commonly carried out through somatic embryogenesis. However, the oil palm's somatic embryogenesis rate is quite low. Several approaches have been made to overcome this problem, including transcriptome profiling through RNA-seq to identify key genes involved in oil palm somatic embryogenesis. RNA sequencing was applied in high- and low-embryogenic ortets of Tenera varieties based on the somatic embryoid rate at the callus, globular, scutellar, and coleoptilar embryoid stages. Cellular analysis of embryoid inductions and proliferations showed that high-embryogenic ortets resulted in higher embryoid proliferation and germinations than low-embryogenic ortets. Transcriptome profiling showed that there are a total of 1,911 differentially expressed genes (DEGs) between high- and low-embryogenic ortets. ABA signaling-related genes such as LEA, DDX28, and vicilin-like protein are upregulated in high-embryogenic ortets. Furthermore, DEGs associated with other hormone signaling, such as HD-ZIP associated with brassinosteroids and NPF associated with auxin, are upregulated in high-embryogenic ortets. This result suggests a physiological difference between high- and low-embryogenic ortets that is connected to their capacity for somatic embryogenesis. These DEGs will be used as potential biomarkers for high-embryogenic ortets and will be validated in further studies.

Whole-Genome Sequence of Oryctes rhinoceros Nudivirus from Riau Province, Indonesia.

A double-stranded DNA virus, Oryctes rhinoceros nudivirus (OrNV), was detected in the total DNA of diseased larvae of O. rhinoceros in Riau Province, Indonesia. The complete genome sequence was 124,926 bp long and encodes 123 open reading frames (ORFs). This strain belongs to the family Nudiviridae and was designated LiboV.

CROP: a CRISPR/Cas9 guide selection program based on mapping guide variants.

The off-target effect, in which DNA cleavage was conducted outside the targeted region, is a major problem which limits the applications of CRISPR/Cas9 genome editing system. CRISPR Off-target Predictor (CROP) is standalone program developed to address this problem by predicting off-target propensity of guide RNAs and thereby allowing the user to select the optimum guides. The approach used by CROP involves generating substitution, deletion and insertion combinations which are then mapped into the reference genome. Based on these mapped variants, scoring and alignment are conducted and then reported as a table comprising the off-target propensity of all guide RNAs from a given gene sequence. The Python script for this program is freely available from: https://github.com/vaprilyanto/crop .

Complete Mitochondrial Genome Sequence of the Phytopathogenic Basidiomycete Ganoderma boninense Strain G3.

Here, we report the complete sequence of the mitochondrial (mt) genome of Ganoderma boninense that assembled as a single circular double-stranded DNA (dsDNA) of 86,549 bp with a G+C content of 26.81%. Genome annotation identified genes that encode 15 conserved proteins, 27 tRNAs, small and large rRNA subunits, 4 hypothetical proteins, and 5 homing endonucleases.

Draft Genome Sequence of the Phytopathogenic Fungus Ganoderma boninense, the Causal Agent of Basal Stem Rot Disease on Oil Palm.

Ganoderma boninense is the dominant fungal pathogen of basal stem rot (BSR) disease on Elaeis guineensis We sequenced the nuclear genome of mycelia using both Illumina and Pacific Biosciences platforms for assembly of scaffolds. The draft genome comprised 79.24 Mb, 495 scaffolds, and 26,226 predicted coding sequences.