The genome sequence of the European crab apple, Malus sylvestris (L.) Mill., 1768

We present a genome assembly from an individual Malus sylvestris (the European or 'wild' crab apple; Streptophyta; Magnoliopsida; Rosales; Rosaceae). The genome sequence is 642 megabases in span. Most of the assembly (99.98%) is scaffolded into 17 chromosomal pseudomolecules. The mitochondrial and chloroplast genomes were also assembled, with respective lengths of 396.9 kilobases and 160.0 kilobases.


Background
Malus sylvestris (European wild or crab apple) belongs to the Rose family (Rosaceae) and is a small tree native across Europe. It reaches its north-western distribution limit in Scotland and Norway and is probably the UK's least understood and most under-appreciated tree, as even foresters sometimes do not seem to be aware of its native status. There is evidence of its longstanding use, as wild apples have been found at Neolithic and Bronze Age archaeological sites across Europe (Cornille et al. 2014). Additionally, without the European wild apple, the eating apples so many people love would not be quite the same. It is one of the main contributors to the domesticated apple, M. domestica (Cornille et al., 2014;Cornille et al., 2019). The widely planted cultivated varieties of M. domestica continue to hybridise with the wild apple trees, causing concern that the genetic integrity of M. sylvestris might be eroded in the long run (Cornille et al., 2013;Feurtey et al., 2017;Ruhsam et al., 2019;Schnitzler et al., 2014).
In a recent study, nearly 30% of the wild apple trees genotyped turned out to be of hybrid origin (Ruhsam et al., 2019). As morphological identification of hybrid trees is difficult, access to the full genome of both M. sylvestris and M. domestica will facilitate the development of genome-wide species-specific markers, enabling the reliable assessment of levels of introgression in the European wild apple.
The genome of the European crab apple, M. sylvestris, was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

Genome sequence report
The genome was sequenced from a single M. sylvestris (hermaphroditic) (Figure 1), collected from Glen Falloch, Scotland, UK. A total of 25-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 87-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 64 missing/misjoins and removed 10 haplotypic duplications, reducing the assembly size by 0.17% and the scaffold number by 36.0%.
The final assembly has a total length of 641 Mb in 32 sequence scaffolds with a scaffold N50 of 36.9 Mb ( Table 1). Most of the assembled sequence (99.98%) was assigned to 17 chromosomallevel scaffolds numbered by synteny based on Malus domestica (apple) GCA_004115385.1 (Figure 2- Figure 5; Table 2).

Sample acquisition and nucleic acid extraction
Leaf samples from a single M. sylvestris specimen (drMalSylv7; genome assembly, Hi-C) were collected from Glen Falloch, Scotland, UK (latitude 56.343794, longitude -4.7078054) by Markus Ruhsam (Royal Botanic Garden Edinburgh), who also identified the specimen. The leaf samples were picked by hand, cut and put into FluidX tubes, then snap-frozen in liquid nitrogen.
The drMalSylv7 leaf sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Leaf tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. High molecular weight (HMW) DNA was extracted using the Qiagen Plant MagAttract HMW DNA extraction kit. Low molecular weight DNA was removed from a 200 ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing. HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30. Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics Chromium read cloud sequencing libraries were constructed according to the manufacturers' instructions. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (10X) instruments. Hi-C data were generated in the Tree of Life laboratory from remaining leaf tissue of drMalSylv7 using the Arima v2 kit and sequenced on a NovaSeq 6000 instrument. Standard read sequencing   libraries were generated using an Illumina NovaSeq 6000 (10X) instrument, as per the manufacturer's instructions.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with freebayes (Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using SALSA2 (Ghurye et al., 2019. The assembly was checked for contamination as described previously (Howe et al., 2021). Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022). The mitochondrial and plastid genomes were assembled using MBG (Rautiainen & Marschall, 2021) from PacBio HiFi reads mapping to related genomes. A representative circular sequence was selected for each from the graph based on read coverage. The genome was analysed and BUSCO scores were generated within the BlobToolKit environment (Challis et al., 2020). Table 3  The genome sequence is released openly for reuse. The M. sylvestris genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. The genome will be annotated and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.
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