title: Development of Transcriptomic Markers for Population Analysis Using Restriction Site Associated RNA Sequencing (RARseq).
abstract: We describe restriction site associated RNA sequencing (RARseq), an RNAseq-based genotype by sequencing (GBS) method. It includes the construction of RNAseq libraries from double stranded cDNA digested with selected restriction enzymes. To test this, we constructed six single- and six-dual-digested RARseq libraries from six F2 pitcher plant individuals and sequenced them on a half of a Miseq run. On average, the de novo approach of population genome analysis detected 544 and 570 RNA SNPs, whereas the reference transcriptome-based approach revealed an average of 1907 and 1876 RNA SNPs per individual, from single- and dual-digested RARseq data, respectively. The average numbers of RNA SNPs and alleles per loci are 1.89 and 2.17, respectively. Our results suggest that the RARseq protocol allows good depth of coverage per loci for detecting RNA SNPs and polymorphic loci for population genomics and mapping analyses. In non-model systems where complete genomes sequences are not always available, RARseq data can be analyzed in reference to the transcriptome. In addition to enriching for functional markers, this method may prove particularly useful in organisms where the genomes are not favorable for DNA GBS.
authors.affiliation: Department of Plant Biology, University of Georgia, Athens, GA, 30604, United States of America.
authors.forename: Magdy S
authors.initials: MS
authors.lastname: Alabady
authors.affiliation: Department of Plant Biology, University of Georgia, Athens, GA, 30604, United States of America.
authors.forename: Willie L
authors.initials: WL
authors.lastname: Rogers
authors.affiliation: Department of Plant Biology, University of Georgia, Athens, GA, 30604, United States of America.
authors.forename: Russell L
authors.initials: RL
authors.lastname: Malmberg
mesh_terms: D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D015723:Gene Library; D005819:Genetic Markers; D014644:Genetic Variation; D060005:Genotyping Techniques; D006239:Haplotypes / Q000235:genetics; D006824:Hybridization, Genetic; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D015183:Restriction Mapping; D052583:Sarraceniaceae / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome
chemical_list: D018076:DNA, Complementary; D018744:DNA, Plant; D005819:Genetic Markers; D012333:RNA, Messenger; D018749:RNA, Plant