Genetic transformation is a powerful means for the improvement of crop plants, but requires labor‐ and resource‐intensive methods. An efficient method for identifying single‐copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (
The capability to generate densely sampled single nucleotide polymorphism (SNP) data is essential in diverse subdisciplines of biology, including crop breeding, pathology, forensics, forestry, ecology, evolution and conservation. However, the wet‐laboratory expertise and bioinformatics training required to conduct genome‐scale variant discovery remain limiting factors for investigators with limited resources. Here we present ISSRseq, a PCR‐based method for reduced representation of genomic variation using simple sequence repeats as priming sites to sequence inter simple sequence repeat (ISSR) regions. Briefly, ISSR regions are amplified with single primers, pooled, used to construct sequencing libraries with a commercially available kit, and sequenced on the Illumina platform. We also present a flexible bioinformatic pipeline that assembles ISSR loci, calls and hard filters variants, outputs data matrices in common formats, and conducts population analyses using R. Using three angiosperm species as case studies, we demonstrate that ISSRseq is highly repeatable, necessitates only simple wet‐laboratory skills and commonplace instrumentation, is flexible in terms of the number of single primers used, and can generate genomic‐scale variant discovery on par with existing RRS methods which require more complex wet‐laboratory procedures. ISSRseq represents a straightforward approach to SNP genotyping in any organism, and we predict that this method will be particularly useful for those studying population genomics and phylogeography of non‐model organisms. Furthermore, the ease of ISSRseq relative to other RRS methods should prove useful to those lacking advanced expertise in wet‐laboratory methods or bioinformatics.
- NSF-PAR ID:
- 10446826
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Methods in Ecology and Evolution
- Volume:
- 13
- Issue:
- 3
- ISSN:
- 2041-210X
- Page Range / eLocation ID:
- p. 668-681
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Basic Protocol 1 : A fast genomic DNA preparation method from genome edited plantsBasic Protocol 2 : Barcoding the amplicons of edited regions from each individual by two rounds of PCRBasic Protocol 3 : Bioinformatics analysis
