Abstract Detail

Population Genetics/Genomics

Barrett, Craig [1], Sinn, Brandon [2], Simon, Sandra [1], Difazio, Stephen [1], Santee , Mathilda Viola [1], Fama, Nicole [1].

Flexible, economical methods for genome-scale variant discovery using ISSR sequencing.

The capability to generate densely sampled single nucleotide polymorphism (SNP) data is essential in diverse subdisciplines of plant biology, including crop breeding, pathology, forensics, forestry, ecology, evolution, and conservation. However, access to the expensive equipment and bioinformatics infrastructure required for genome-scale sequencing is still a limiting factor in the developing world and for institutions with limited resources. Here we present ‘ISSRseq,’ a PCR-based method for reduced representation of genomic variation using inter-simple sequence repeats (ISSR). Briefly, ISSR regions are amplified with single primers, pooled, and to construct Illumina libraries using a low-cost, efficient commercial kit, and sequenced on the Illumina MiSeq platform. A similar, recently published method (MIG-seq), uses a highly multiplex, two-step, tailed amplicon approach to ISSR sequencing. Several potential issues exist with MIG-seq: 1) lack of repeatability; 2) high up-front costs associated with synthesizing long, single-barcoded primers; 3) potential multimerization of low complexity primers and tails, leading to unpredictable annealing; and 4) reduced power to detect paralogous loci and adaptive variants due to short sequences from fragment-end sequencing. We provide an approach that is highly repeatable, flexible in terms of the number of single primers used (based on the needs of a particular study), low-cost (a few cents per PCR reaction with single primers and < $10 USD per library prep), and robust to the potential biases of MIGseq. We further present a flexible UNIX bioinformatic pipeline that assembles, conservatively filters, calls variants with GATK, formats data outputs, and conducts population analyses using R. We tested ISSRseq in a mycoheterotrophic, IUCN Red-Listed, genetically depauperate Corallorhiza bentleyi. Based on 20 individual ISSR primers, we generated ~2.3 Mb of post-filtered sequence, with 46,853 SNPs in 6,734 loci among 37 individuals on a single MiSeq run. 128 loci were significant outliers displaying evidence of local adaptation based on analysis in PCAdapt. Discriminant Analysis of PCA and coalescent analysis with SVDquartets yielded two major clusters of ancestry from six of the ~10 known localities for this species, largely corresponding to those localities with cleistogamous vs. chasmogamous forms. The level of variation detected exceeds published levels from MIG-seq by orders of magnitude and is comparable to those typical of various RAD-seq approaches, at a low cost per sample. ISSRseq represents a straightforward approach to SNP genotyping in non-model organisms that we predict will be useful for K-12, undergraduate, and graduate research, and more broadly by those lacking access to expensive resources.

1 - West Virginia University, Biology, 53 Campus Drive, Morgantown, WV, 26506, United States
2 - Otterbein University, Biology and Earth Science, 1 South Grove Street, Westerville, OH, 43081, USA

Keywords:
population genomics
Conservation genomics
reduced representation
single nucleotide polymorphism
Corallorhiza bentleyi
non-model
bioinformatics pipeline.

Presentation Type: Oral Paper
Session: POPGEN2, Population Genetics/Genomics II
Location: Virtual/Virtual
Date: Thursday, July 30th, 2020
Time: 3:45 PM
Number: POPGEN2004
Abstract ID:452
Candidate for Awards:None