DOI: 10.5061/dryad.j3tx95xc0
title: Malinae481 exonic probe set

description: The subtribe Malinae (Rosaceae) comprises close to 1,000 species
 and up to 30 genera. It is defined, among other characters, by a
 derived base chromosome number of x = 17. A parsimonious pattern of
 chromosome breakage and fusion explains the derivation of the x = 17
 karyotype from a polyploidisation event of two x = 9 genomes. High
 collinearity between the genetic and physical maps of Pyrus and Malus as
 well as their identical karyotypes suggest that the
 reorganization of the Malinae genome occurred before the
 divergence of the two genera, and presumably before speciation of the
 whole subtribe. The preservation of extensive duplicated chromosomal
 segments in the genome of members of the Malinae, dating back to the
 genome-wide duplication, necessitates an assessment of the homology of
 markers used in phylogenetic studies. First, we identified
 single-copy orthologs and relatively recently diverged
 paralogs in the genome of Malus domestica. Average
 sequence divergence among orthologs was 3.65% in a genome-wide comparison
 of Malus and Pyrus, which is about one third of
 the average sequence divergence of 9.36% between Maleae and Amygdaleae
 orthologs and around half of the divergence based on fourfold degenerate
 site transversion of 8% between paralogs from the most recent WGD within
 each of these genomes. Based on this information, we developed a strategy
 to identify in the Malus genome (1) single-copy loci
 (presumably located within non-duplicated regions, or hidden paralogs due
 to differential gene loss) and (2) loci only duplicated once (one paralog
 only present in the genome; avoidance of multi-gene families). We further
 constrained our locus selection by imposing the minimum divergence between
 them to 6% as minimum sequence divergence between orthologs and paralogs.
 Specifically, we blasted 28,695 mRNAs (queries thereafter) in the range of
 800–3,000 bp from the Malus domestica ‘Golden
 Delicious’ (GDDH13) genome v.1.1, downloaded from the Genome
 Database for Rosaceae, against the Malus
 domestica GDDH13 genome v.1.1 (subject thereafter) using the
 nucleotide BLAST search. Default settings were used except for the
 e-value, which was lowered to 0.00001. In an initial filtering step, we
 only kept the hits exceeding 70 bp and 10% of query length with ≥80%
 sequence similarity between query and subject. We then assigned the hits
 (usually corresponding to exons) to loci based on the criterion that the
 length of introns separating the hits does not exceed 10,000 bp. Queries
 that showed hits with &gt;6 loci were not taken into account. In a
 second, refined filtering step we only kept loci that fulfilled the
 following criteria: length cover and sequence similarity of the sum of all
 hits for a particular locus ≥90%, length of single hits ≥100 bp (in
 accordance with the bait length of 100 bp), intron length ≤1,200 bp,
 number of loci per query ≤2, sequence divergence among loci ≥6%. We then
 blasted the 1,280 Malus queries that fulfilled our
 selection criteria against the Pyrus communis Bartlett
 DH genome v.2.0 and applied the same selection criteria. 616 of these
 queries fulfilled them. Subject sequences from
 the Malus (799) and Pyrus (764)
 genomes, matching chosen mRNAs and representing full loci, were then
 extracted and exon-intron boundaries inferred based on the
 alignments together with the blasted queries. After filtering for
 identical numbers of loci in both genomes, sequence divergence between
 exons of single-copy loci
 in Malus and Pyrus ≤15% and exon
 length ≥80 bp, we ended up with 713 loci (481 loci if pairs of paralogous
 loci are treated as single loci), which corresponded to the 546
 mRNAs. Extracted sequences were collapsed at ≥95% similarity and
 used for bait design. The final exonic probe set covers 2,008,479 bp in
 total. This way, we expect to get baits with good correspondence
 to the Malinae genomes, i.e. both orthologs and paralogs are
 targeted.