ISSRseq: an extensible method for reduced representation sequencing

Sinn, Brandon T.; Simon, Sandra; Santee, Mathilda V.; DiFazio, Stephen P.; Fama, Nicole M.; Barrett, Craig F.

doi:10.1101/2020.12.21.423774

Cited by 2 publications

(1 citation statement)

References 46 publications

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“…Raw sequencing reads for all samples are available via NCBI BioProject PRJNA771539. Data matrices, scripts used and their usage instructions in wiki format, as well as the wet-laboratory protocol, are available via GitHub (www.github.com/btsin n/ISSRseq) or Sinn et al (2021).…”

Section: Stimulatementioning

confidence: 99%

ISSRseq: An extensible method for reduced representation sequencing

et al. 2021

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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.

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Section: Stimulatementioning

confidence: 99%

ISSRseq: An extensible method for reduced representation sequencing

et al. 2021

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Integrating Genetics, Morphology, and Fungal Host Specificity in Conservation Studies of a Vulnerable, Selfing, Mycoheterotrophic Orchid (Corallorhiza bentleyi Freudenst.)

2021

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Mycoheterotrophic plants derive most or all carbon and nutrients from fungal partners and represent poorly understood components of forest biodiversity. Many are rare or endangered yet can be ecological indicators of forest ecosystem function due to their often highly specific fungal host requirements. One such species is the IUCN red-listed ('vulnerable'), fully mycoheterotrophic orchid, Corallorhiza bentleyi. This recently described species is among the rarest plants in Appalachia, known from five counties in Virginia and West Virginia, USA. The species has a restricted range, small population size, and is self-pollinating. Here we take an integrative approach to conservation genetic assessment in C. bentleyi using floral morphometrics, simple-sequence repeats, and fungal host DNA to characterize variation within and among sampling localities. Morphology reveals some differentiation among individuals from six sampling localities. Surprisingly, most genetic variation is found within localities, contra to the expectation for a selfing species. Fungal host DNA reveals extreme specificity upon a few genotypes of a single ectomycorrhizal host species, Tomentella fuscocinerea, across all localities.We discuss the conservation implications of morphological, genetic, and symbiotic diversity in this vulnerable species, and recommend additional assessment of conservation status based on: an obligate reproductive mode of selfing, preventing benefits of outcrossing among genetically non-identical individuals; extreme host specificity, severely restricting niche space; and highly fragmented habitat under threat from anthropogenic disturbance. This study underscores the importance of integrative conservation assessment, analyzing multiple data sources, and reveals patterns not readily apparent from census-based assessments alone.

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ISSRseq: an extensible method for reduced representation sequencing

Cited by 2 publications

References 46 publications

ISSRseq: An extensible method for reduced representation sequencing

ISSRseq: An extensible method for reduced representation sequencing

Integrating Genetics, Morphology, and Fungal Host Specificity in Conservation Studies of a Vulnerable, Selfing, Mycoheterotrophic Orchid (Corallorhiza bentleyi Freudenst.)

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