DOI: 10.5061/dryad.c2fqz61cr
title: Genomic characterization of a wild-like tomato accession from Arizona

description: Tomato domestication history has been revealed to be a highly complex
 story. A major contributor to this complexity is an evolutionary
 intermediate group (Solanum lycopersicum var. cerasiforme (Alef.) Voss;
 SLC) between the cultivated tomato (Solanum lycopersicum var. lycopersicum
 L.; SLL) and its wild relative (Solanum pimpinellifolium L.; SP). SLC
 includes accessions with a broad spectrum of genomic and phenotypic
 characteristics. Some of the SLC accessions were previously hypothesized
 to be spreading northward from South America into Mesoamerica and that
 migration probably entailed reversal to wild-like phenotypes such as
 smaller fruits. Prior to this study, the northernmost confirmed extension
 of the SLC was limited to northern Mexico. In this study, we employed
 genomic methods to investigate the origin of a wild-like tomato found in a
 garden in Scottsdale Arizona, USA. The so-called “Arizona tomato” featured
 a vigorous growth habit and carried small fruits weighing 2-3 grams. Our
 phylogenomic analyses revealed the identity of the Arizona tomato as a
 member of the Mexican SLC population (SLC MEX). To our knowledge, this is
 the first report of an SLC accession, confirmed using genomics, growing
 spontaneously in Arizona. This finding could have implications for
 conservation biology as well as agriculture.
description: <strong>Plant collection and voucher
 specimens</strong>: The "Arizona tomato" was discovered
 growing naturally in a suburban yard in Scottsdale, Arizona, USA in
 January 2021 by April Kuipers. All plant materials used in this study were
 obtained with permission from Ms. Kuipers, who found the plant on her
 personal property. She collected seeds from the fruits and sent them to
 the University of Massachusetts Amherst, where plants were grown in a
 greenhouse kept at 22 ℃ (with a few degrees of day/night fluctuations)
 under ambient daylight conditions from February - August 2021. Seeds were
 soaked in 2% sodium hypochlorite bleach solution, rinsed with distilled
 water, then placed ~ 0.5 cm deep in ProMix BX potting medium, resulting in
 100% germination rate (nine out of nine seeds). Two plants were grown to
 maturity, and two specimens from each plant were preserved on June 30,
 2021, and deposited in the University of Massachusetts Herbarium (MASS) as
 vouchers.  <strong>Genome
 sequencing</strong>: Genomic DNA was extracted from a plant grown at
 Cornell University. Approximately 4 grams of leaf samples from the
 youngest leaves were used.  Leaf samples were frozen and ground in liquid
 nitrogen. The DNA purification was conducted using NucleoBond® HMW DNA kit
 (from TaKaRa) and the purification steps were followed exactly as
 described in the protocol accompanying the kit. The total DNA yield from
 the leaf samples was 500 ng per microliter, of which 600 ng was used as
 input for DNA sequencing library preparation. The sequencing library
 preparation was conducted using New England Biolab FS DNA Library Prep Kit
 (E7805, E6177). A paired-end library was made with fragment sizes of
 100-300 bp. The resulting library was visualized on a 0.4% agarose gel to
 confirm the expected spectrum of fragment sizes. The library was sequenced
 with Illumina NextSeq technology using the paired-end mode (2 × 150 bp) at
 the Genomics Facility of the Cornell Institute of
 Biotechnology.  <strong>Sequence read alignment and variant
 calling</strong>: The reads obtained through genome sequencing were
 filtered by removing low-quality reads and the universal Illumina adaptors
 using cutadapt (v. 2.1; Martin 2011) with the default options. The
 remaining reads were checked for quality with FastQC (v. 0.11.8;
 https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and aligned to
 the most recent version of the cultivated tomato reference genome (SL4.0)
 using <em>mem</em> from bwa (v. 0.7.17; Li 2013). Similarly,
 genome alignments of SP, SLC, and SLL accessions created in previous
 studies (Razifard et al. 2020 and Pereira et al. 2021) were also included
 to create a variants dataset. A combined dataset of 21,358,376 variants
 including SNPs and indels (insertions and deletions &lt; 10 bp) was
 made using <em>mpileup</em> from bcftools (v. 1.14; Danecek et
 al. 2021) software package. From this dataset, rare alleles (minimum
 allele frequency &lt; 0.02) were removed using vcftools (v. 0.1.17;
 Danecek et al. 2011).  <strong>Phylogenetic
 reconstruction</strong>: A SNP phylogeny was built using only
 four-fold degenerate SNPs ("4D SNPs”), i.e. SNPs at the third codon
 positions in which changes to all four base-pairs do not affect the
 translated amino acid from those codons. Such codon positions are
 considered to be under less selective pressure, thus more suitable for
 studying phylogenetic relationships. The input dataset for the
 phylogenetic analyses was made as follows: A custom script (available from
 https://github.com/hrazif) was developed in R (v. 4.0.5; https://www.r-project.org/) to extract 4D SNPs from the variants dataset described above. From the resulting 4D SNPs dataset, we kept only those with no missing data and minimum allele frequency &gt; 0.012 (to keep 4D SNPs with alternate alleles in at least two homozygous accessions, four heterozygous accessions, or some other genotype combination). These steps resulted in 41,787 4D SNPs. For drawing a phylogenetic tree using the 4D SNPs, we used the coalescent method of SVDQuartets (Chifman and Kubatko, 2014) included in PAUP (v. 4a168; Swofford DL. 2003). The “exhaustive” search option, i.e. including all possible quartets, was chosen for the SVDQuartets analysis.  As an alternative method, we also generated a phylogeny based on a genome-wide dissimilarity matrix calculated from 13,956,415 variants (all variants except those with minimum allele frequency &lt; 0.012 and missing data &gt; 10%) in all accessions using SNPRelate (v1.10.2; Zheng et.al.,2012) in R. We then used the resulting distance matrix to create the phylogeny, based on the default “complete linkage” method.