DOI: 10.5061/dryad.hhmgqnkgg
title: Variant discovery in full-sibling families of Pinus taeda L

description: Fusiform rust disease, caused by the endemic fungus Cronartium quercuum f.
 sp. fusiforme, is the most damaging disease affecting economically
 important pine species in the southeast United States. In this report, we
 detail the genomic localization and sequence-level discovery of candidate
 race-nonspecific broad-spectrum fusiform rust resistance genes in Pinus
 taeda L. Two full-sib families, each with ~1000 progeny, were challenged
 with a complex inoculum consisting of over 150 pathogen isolates.
 High-density linkage mapping revealed three QTL distributed on two linkage
 groups. The two QTL on linkage group 2 were additive with respect to their
 effects on the probability of disease outcome. All three QTL were
 validated using a population of 2057 cloned pine genotypes in a
 six-year-old multi-environmental field trial. As a complement to the QTL
 mapping approach, bulked  segregant RNAseq analysis revealed a
 small number of candidate nucleotide binding leucine rich repeat genes
 harboring SNP significantly associated with disease resistance. The
 results of this study demonstrate that single qualitative resistance genes
 can confer effective resistance against genetically diverse mixtures of an
 endemic pathogen.
description: A total of 15 samples had adequate quantity and quality of RNA
 for library preparation. Each sample was sequenced on two lanes of an S2
 flow cell of the NovaSeq6000 Illumina sequencer, resulting in
 7.6x10<sup>9</sup> 50bp paired-end reads. For each sample,
 reads originating from lanes 1 and 2 were combined into a single
 <i>fastq</i> file for each mate, and aligned to the PacBio
 reference transcriptome using <b>bwa mem</b> with the default
 options (Heng Li, 2013). Around 70% of the sequences from each sample had
 both mate pairs properly mapped to the transcriptome, with an average
 quality score of 35.8, an average insert size of ~275bp, and an average
 depth of 163x. Following alignment, variants were
 called using <b>Freebayes </b>version 0.9.6 (Garrison
 & Marth, 2012). Each <i>bam</i> file from a single
 family was combined in a variant discovery run. Since each sample
 represented a bulk of 100 (for the random) or 50 (for the disease status)
 individuals, the population model was specified as ‘pooled’ using the ‘-J’
 qualifier. Complex alleles of up to 25bp were allowed using the
 ‘—max-complex-gap’ qualifier.   Biallelic SNP with a minimum of 10
 observations of the alternate allele were considered for downstream
 analysis.
description: Sample ID's appearing in the .vcf files are described
 below.<br> <br> <strong>Family
 E4</strong><br> S15 random bulk collected prior to inoculation
 (100 full-sib)<br> S11 non-diseased bulk collected 7 months
 post-inoculation (50 full-sib)<br> S19 non-diseased bulk collected
 10 months post-inoculation (100 full-sib)<br> S2 non-diseased bulk
 collected 7 months post-inoculation (50 full-sib)<br> S18 diseased
 bulk collected 10 months post-inoculation (100 full-sib)<br> S1
 diseased bulk collected 7 months post-inoculation (50 full-sib)<br>
 S5 diseased bulk collected 7 months post-inoculation (50
 full-sib)<br> <br> <strong>Family
 E9</strong><br> S16 random bulk collected prior to inoculation
 (100 full-sib)<br> S12  non-diseased bulk collected 7 months
 post-inoculation (50 full-sib)<br> S17 non-diseased bulk collected
 10 months post-inoculation (100 full-sib)<br> S3 non-diseased bulk
 collected 7 months post-inoculation (50 full-sib)<br> S7
 non-diseased bulk collected 7 months post-inoculation (50
 full-sib)<br> S20 diseased bulk collected 10 months post-inoculation
 (100 full-sib)<br> S4 diseased bulk collected 7 months
 post-inoculation (50 full-sib)<br> S8 diseased bulk collected 7
 months post-inoculation (50 full-sib)