DOI: 10.5061/dryad.t4b8gtj42
title: Genome assembly of Olea europaea subsp. cuspidate

description: Background: The Olive complex, comprised of six subspecies, are very
 valuable plants for global trade, human health, and food safety. However,
 only one subspecies (Olea europaea subsp. europaea,
 OE) and its wild form (Olea europaea subsp. europaea var.
 sylvestris, OS) have genomic references, hindering our
 understanding on the evolution of this species. Results: By utilizing a
 hybrid approach to incorporate Illumina, Nanopore, and Hi-C
 technology, we obtained by far the best reference genome
 assembly among wild olive subspecies for African olive, Olea
 europaea subsp cuspidate (OC) with contig and scaffold
 N50 values 3.83 Mb and 38.04 Mb, respectively. The assessment of
 protein-coding gene completeness revealed the high integrity of OC, which
 is at the similar level as OE assembly reported previously and much higher
 than that of OS. The divergence time between OC and the last common
 ancestor of OE and OS was estimated to be 4.21 Mya (95% CI: 1.43 - 7.31
 Mya). The pathways of positively selected genes of OC are related to
 metabolism of cofactors and vitamins, indicating the potential medical and
 economic values of OC for further utilizing and research. The gene
 origination analyses revealed a substantial outburst (19.5%) of gene
 transposition events in the common ancestor of olive subspecies,
 suggesting the importance of olive speciation in shaping the new gene
 evolution of OC subspecies. Conclusions: In this study, we constructed the
 de novo assembly and protein-coding gene pool for Olea europaea
 subsp cuspidate (OC), which may facilitate the medical
 and breeding utilizations of this widely distributed olive close relative.
description: The sequencing for DNA and RNA molecules was based on an
 individual of OC sampled from Kunming Arboretum (N 25°9′13″, E 102°45′9″),
 Yunnan Academy of Forestry and Grassland, Yunnan province of China.
 Yong-Kang Sima identified it. Voucher specimen (Wu20056) was deposited in
 the Herbarium of Yunnan Academy of Forestry and Grassland. The standard
 preparing procedures before sequencing, including DNA and RNA extraction,
 Hi-C library construction, etc., were based on the requirements of
 specific sequencers. Totally, five tissues including leaves, roots, twigs,
 bark, and fruits, were used for RNAseq sequencing in Illumina platform.
 For DNAseq, ~50x genome short reads (300 bp PE) and ~70x Nanopore
 long-reads were obtained from DNBSEQ-T7 and PromethION platform,
 respectively. The raw reads were filtered using the fastp preprocessor. To
 achieve chromosome-level assembly, we further generated ~130Gb data of the
 paired-end Hi-C reads (150bp) from DNBSEQ-T7 platform (MGI). We conducted
 the karyotyping of OC to determine the number of chromosomes using
 cultivated root, which has active meristems of mitosis suitable for
 detecting clear chromosomes. Cells were treated with Nitrous Oxide to
 obtained sufficient cells at mitosis metaphase for staining with DAPI and
 telomere repetitive sequences (TTTAGGG)6.
 The basecalling output from PromethION platform was treated using
 Guppy. Only the reads with mean quality scores &gt;7 were retained and
 further corrected using the NextDenovo software with parameters
 "reads_cutoff:2k, seed_cutoff:18k"
 (https://github.com/Nextomics/NextDenovo). The assembling processes
 include the correction module using NextCorrect and the assemble module
 using NextGraph with default parameters. Subsequently, the Nextpolish
 software was used to polish genome with short-reads four times and
 long-reads three times (sgs_options = -max_depth 100). The paired-end Hi-C
 reads were filtered by fastp to remove adapter and low-quality reads
 (Phred Score &gt; 15, and 5 &gt; number of Ns in the reads). The
 clean reads and draft genome were analyzed using LACHESIS with parameters
 "CLUSTER MIN RE SITES = 100；CLUSTER MAX LINK DENSITY=2.5；CLUSTER
 NONINFORMATIVE RATIO = 1.4". The RepeatMasker was
 used for repeats annotation following the manual recommended parameters.
 To aid gene annotation, totally ~75 Gb RNA-sequencing (RNA-Seq) clean
 pair-ended reads from five tissues, including leaves, roots, twigs, bark,
 and fruits, were generated using Illumina HiSeq platform. All libraries
 were de novo assembled separately and subsequently merged using TransABySS
 manual pipeline. The protein-coding and non-coding gene structural
 annotation was conducted using MAKER pipeline by incorporating
 transcriptome mapping,<em> de novo </em>gene predictions, and
 homology predictions.