DOI: 10.5061/dryad.5hqbzkh9f
title: Assembly and annotation of eleven Salix (shrub willow) genomes

description: The shrub willows (Salix section Vetrix) are an emerging bioenergy crop in
 North America and Eurasia. However, genomics resources in this section are
 still quite limited, with only a few reference genomes available, despite
 many species in use in breeding programs. Here we present de novo
 assemblies and annotations of eleven shrub willow genomes from six
 species. Copy number variation of candidate sex determination genes within
 each genome was characterized and revealed remarkable differences in
 putative master regulator gene duplication and deletion. We also analyzed
 copy number and expression of candidate genes involved in floral secondary
 metabolism and identified substantial variation across genotypes, which
 can be used for parental selection in breeding programs. Lastly, we report
 on a genotype that produces only female descendants and identified gene
 presence/absence variation in the mitochondrial genome that may be
 responsible for this unusual inheritance.
description: <em>DNA Sequencing</em> Fresh
 young leaf tissue for all 11 <em>Salix</em> genotypes was
 collected and ground in liquid nitrogen. DNA extraction was performed
 using a modified CTAB based protocol. For long read sequencing, 1 μg of
 DNA was used as input to Oxford Nanopore’s genomic DNA by ligation
 sequencing kit (SQK-LSK109) and the subsequent library was sequenced on a
 R.9.4.1 flow cell. Short read sequencing of the same samples was performed
 on the Illumina HiSeq X Ten platform. <em>RNA
 Sequencing</em> RNA was extracted from eight
 tissues (root, xylem, internode, node, young leaf, mature leaf, petiole,
 and young stem) for all 11 genotypes, as well as fasciated shoot tissue
 from 04-BN-051. Strand-specific RNA-Seq libraries were prepared by BGI and
 sequenced on the DNB-Seq platform, which generated paired-end 150 bp
 reads. The same RNA preps from mature leaves and roots were also sequenced
 on the Oxford Nanopore MinION platform, with the exception of ‘Jorr’,
 which failed quality control. The SQK-PCB109 PCR-based cDNA library kit
 was used to generate sequencing libraries for leaf and root tissue for all
 11 genotypes and were sequenced on R.9.4.1 flow cells.
 <em>Hi-C library preparation</em>
 Hi-C libraries were prepared with the Phase Genomics Proximo
 Plant Hi-C kit. Hi-C libraries were sequenced on the Illumina NovaSeq 6000
 instrument which generated paired-end 150 bp reads. The sequencing data of
 each Hi-C library underwent quality control with the phase genomics
 hic_qc.py script (https://github.com/phasegenomics/hic_qc, accessed Nov. 15, 2021) to ensure a sufficient number of informative Hi-C reads were present in each library. <em>Genome Assembly</em> Assembly was performed with Oxford Nanopore reads using Flye 2.8.3. Illumina short reads were mapped to the assembled contigs with BWA-MEM. Pilon and a custom python script were used to generate the corrected draft assembly with the Illumina data. Assembled contigs were scaffolded using Hi-C reads with Falcon and Juicer Hi-C to generate phased genome assemblies. A BUSCO search of the Eudicot core genes was performed against each assembly to assess the quality and completeness of each genome. One assembly, 04-FF-016, produced two chimeric contigs, HiC_scaffold_5 and HiC_scaffold_6, each spanning the entire length of several chromosomes. BLASTN analysis was used to determine alignment to specific chromosomes and each chimeric contig was manually cut at the approximate site where mapping behavior became abnormal. Resulting scaffolds were appended with a letter (e.g. a, b, c, etc.) to denote their origin from the original chimeric scaffold. Annotation Genome annotation was performed with the LoReAn v2.5 pipeline, which utilized both Oxford Nanopore and Illumina RNA-Seq, along with protein models from the JGI <em>Populus</em> <em>trichocarpa</em> v4.1, <em>Populus deltoides</em> v2.1, and <em>Populus</em> <em>nigra</em> x <em>maximowiczii</em> v1.1 reference genome annotations obtained from Phytozome (https://phytozome-next.jgi.doe.gov accessed March 21, 2022), followed by Augustus ab initio gene prediction. BLASTN analysis was performed for each annotated transcript for every genome against the <em>S</em>. <em>purpurea</em> v5.1 annotation on Phytozome (https://phytozome-next.jgi.doe.gov accessed July 6, 2022) to identify homologous gene models. Functional prediction of mRNAs in each annotation was performed using interproscan 5.52-86.0. The estimated number of missing genes from each annotation was determined by performing a BLAST analysis of all <em>S.</em> <em>purpurea</em> v5.1 CDS sequences against all annotated genes for each genome and identifying those <em>S</em>. <em>purpurea</em> v5.1 genes without a match in each genome. <em>Data Availability</em> All raw sequencing data have been deposited at the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra). Raw Illumina and nanopore DNA sequencing data can be accessed with the BioProject ID PRJNA827350. The raw Illumina RNA-Seq data can be accessed with the BioProject ID PRJNA827350. Nanopore RNA-Seq data can be accessed with the BioProject ID PRJNA888070. 
description: Data include fasta files of genome assemblies, gff3 annotation
 files, and text files with genome annotation info. All files can be opened
 in any text editor in principle, but fasta files are large may be too
 large to open on smaller memory machines or may take considerable time to
 load and should be viewed in a command line. Assemblies and annotations
 can be loaded into IGV to view the assembly and annotation in browser
 form. 
description: # Assembly and Annotation of Eleven Salix (shrub willow) genomes
 Full assemblies and annotations of eleven shrub willow (Salix) genomes.
 Assemblies were performed using long read Oxford nanopore and short read
 illumina data and scaffolded with HiC. Annotation was performed using
 LoReAn software and illumina short read and Oxford Nanopore long read
 RNA-Seq from eight tissues from each genotype. Raw sequencing reads are
 available on NCBI SRA. ## Description of the Data and file structure The
 prefixes for each of the eleven genomes are as follows: 94006, a Salix
 purpurea female 94001, a Salix purpurea male P63, a Salix suchowensis male
 P294, a Salix suchowensis female P295, a Salix suchowensis female P336, a
 Salix integra female SH3, a Salix koriyanagi female 04-FF-016, a Salix
 koriyanagi male 07-MBG-5027, a Salix viminalis female Jorr, a Salix
 viminalis male 04-BN-051, a Salix udensis male Each genome contains five
 files, as described below: *_hap.FINAL.fasta ; This is the assembled
 genome in fasta format *.gff3 ; This is the annotation file describing the
 location and relationship of each feature *_mRNA_info.txt ; This file
 contains additional information about each transcript. Column one is the
 mRNA as described in the gff file, Column two is the PANTHER ID for the
 transcript, column three is the PANTHER ID description, column four is the
 closest BLAST hit in the JGI v5.1 S. purpurea genome, and column five is
 the description of the closest BLAST hit. *_transcripts.fa ; This file is
 the nucleotide fasta sequence of each transcript, as described in the gff
 file *_protein.fa ; This file is the protein fasta sequence of each
 peptide, as described in the gff file. All files can be viewed on any
 standard text editor (e.g. Atom, notepad, textedit, sublime, etc.) IGV can
 be used the view the genome and annotation in browser form with the
 assembly and gff3 files. ## Sharing/access Information Raw DNA and RNA
 sequence data used to generate these assemblies and annotations are
 available on the NCBI sequence read archive. **April Version
 changes**: "Salix_genomes_contigs2chrs.csv"was added, which
 describes which chromosome number each scaffold maps to (based on the
 standard Populus and Salix chromosome numbering system).