DOI: 10.5061/dryad.6wwpzgmwg
title: Onion (Allium cepa) pseudoreference genome

description: Onion (Allium cepa) is not highly tractable for development of molecular
 markers due to its large (16 gigbases per 1C) nuclear genome. 
 Single nucleotide polymorphisms (SNPs) are useful for genetic
 characterization and marker-aided selection of onion because of
 codominance and common occurrence in elite germplasm.  We
 completed genotyping by sequencing (GBS) to identify SNPs in onion using
 46 F2 plants, parents of the F2 plants (Ailsa Craig 43 and Brigham Yellow
 Globe 15-23), two doubled haploid (DH) lines (DH2107 and DH2110), and
 plants from 94 accessions in the USDA National Plant Germplasm System
 (NPGS).  SNPs were called using the TASSEL 3.0 Universal Network
 Enabled Analysis (UNEAK) bioinformatics pipeline.  Sequences from
 the F2 and DH plants were used to construct a pseudo-reference genome
 against which genotypes from all accessions were scored.  Quality
 filters were used to identify a set of 284 high quality SNPs which were
 placed onto an existing genetic map for the F2 family. 
 Accessions showed a moderate level of diversity (mean He = 0.341) and
 evidence of inbreeding (mean F = 0.592).  GBS is promising for
 SNP discovery in onion, although lack of a reference genome required
 extensive custom scripts for bioinformatics analyses to identify high
 quality markers.
description: 46 F2 plants and parents of the onion (<em>Allium
 cepa</em>) mapping population Brigham Yellow Globe 15-23 x Ailsa
 Craig 43 were genotyped, as well as two doubled haploid (DH) onion lines
 DH2107 and DH2110 which were used as completely homozygous controls.
 Genotyping by sequencing (GBS) was performed using an Illumina HiSeq 2000
 on two to four replicates of every DNA sample.  GBS libraries were
 prepared at Cornell University’s Genomic Diversity Facility using the
 restriction enzyme EcoT22I and assayed in 96-plex format using standard
 protocols.   SNP calling on the 46 F2 plants, two parents, and the two DH
 lines was performed using TASSEL 3.0 Universal Network Enabled Analysis
 (UNEAK) bioinformatics pipeline, which does not require a reference
 genome.  Over 70,000 raw SNPs were scored in these samples.  Quality
 filters were then applied to SNPs as follows:  not heterozygous in either
 DH line, minor allele frequency greater than or equal to 30%, minimum
 genotypic read depth of seven, maximum missing data of 10%, and conforming
 to the expected 1:2:1 segregation ratio (goodness-of-fit &gt; 0.01)
 within the F2 family.  For the resulting 752 SNPs, MSTMap software tool
 was used to construct a genetic linkage map using a grouping LOD criteria
 of <i>p</i> &lt; 1 x 10<sup>-7</sup>.  This
 gave 701 SNPs in 15 linkage groups (LG) with ≥ 15 markers each (the
 remaining 51 markers were not placed on a linkage group).  The number of
 SNPs per LG ranged from 15 – 90, and the estimated size of LGs ranged from
 52 to 327 cM. Because UNEAK treats redundant, reverse complement tags from
 opposite strands as separate markers, 171 redundant tag pairs were
 eliminated from this linkage map.  A pseudo-reference genome was
 constructed consisting of one tag from each of the 530 non-redundant,
 mapped tag pairs concatenated together into a single pseudo-molecule. To
 prevent spurious alignment across two distinct pseudo-reference tags, each
 tag in the pseudo-reference was separated by a span of at least 32 A
 nucleotides. The purpose of the pseudo-reference was to allow discovery of
 additional SNPs within each tag pair locus in 94 diverse onion accessions
 that were not segregating in the mapping population, thereby reducing the
 ascertainment bias that would result from using only SNPs discovered in
 only one F2 family in a population survey.