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Scheunert, Agnes [1], Lautenschlager, Ulrich [1], Oberprieler, Christoph [1].

In-silico identification of low-copy nuclear loci for your plant group of interest, based on published target capture probe sets and genomes.

Sequencing collections of nuclear loci using NGS methods has become an important tool for molecular systematics of plants. Target enrichment can be achieved by in-solution hybridization using pre-designed probes, which cover a set of conserved loci. However, there are situations when use of expensive commercial probe kits might be uneconomical, or when analyzing a small number of loci is more appropriate, e.g., when allele phasing is intended to reconstruct histories of hybridization / polyploidization. Here, we describe a novel method which uses a commercial probe set for in-silico mining of suitable low-copy loci within the genus Leucanthemum Mill. (Anthemideae, Compositae). The pipeline, mainly consisting of several shell scripts, was constructed for use with the "CompCOS loci" probe set of 1,061 conserved orthologous loci designed for the Compositae (Mandel et al., 2014), but can be adapted to any exonic target capture kit as long as the genetic sequences used for bait generation are available (e.g., ESTs). While the approach is easy to use, it allows the identification of promising single-/low-copy loci at essentially no cost. It consists of four main steps:
1) BLAST search of the genetic sequences the probe kit was designed from, against an annotated genome related to the plant group of interest
2) Extraction of a list of introns from the annotation of the reference genome
3) Filtering of sequences with two or more BLAST hits, according to a set of pre-defined criteria (, excluding loci with hits on different chromosomes or strands, and those with several hits on the same chromosome (all suggesting possible duplication of the underlying gene)
4) Alignment of remaining BLAST hits (representing exons of the loci of interest) to the reference genome (, while only considering loci comprising at least one intron and highlighting the position of the latter in the reference genome sequence; output as a folder containing one FASTA file per input sequence
As alignments comprise exonic probe fragments beside reference intronic regions, they can easily be examined for loci with the desired intron properties, marker length etc., and primer design is straightforward. In Leucanthemum, the pipeline resulted in the identification of 40 loci, of which 20 were chosen for primer design. Twelve of these loci were successfully amplified and sequenced, with variability ranging from approx. 0.3 to 3.94%. Even better results might be achieved, depending on the degree of relatedness of the reference genome and the bait kit used.

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1 - University of Regensburg, Institute of Plant Sciences, Biology and Pre-Clinical Medicine, Universitätsstraße 31, Regensburg, D-93053, Germany

target capture
in-silico loci selection
low-copy nuclear loci
CompCOS loci
shell script

Presentation Type: Poster
Session: P3, Phylogenomics Posters
Location: Virtual/Virtual
Date: Wednesday, July 21st, 2021
Time: 5:00 PM(EDT)
Number: P3PL017
Abstract ID:578
Candidate for Awards:None

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