Not surprisingly, although all worked independently, many of the same regions were explored ( Appendix 2) and, in some cases, identical or nearly identical primers were designed. There are now at least 150 primer pairs available to amplify almost every intergenic, intron, and exon region of the chloroplast genome, including portions of the inverted repeats, thanks to the efforts of Shaw et al. They examined sequence diversity for 107 single-copy noncoding regions, providing the most comprehensive analysis to date. ( 2014) go one step further, comparing complete chloroplast genome sequences for 25 (primarily congeneric) sister species pairs. ( 2007) took an even more applied approach when they examined sequences for three different taxon pairs ( Atropa/ Nicotiana, Lotus/Medicago, and Saccharum/Oryza), specifically searching for faster-evolving regions. ( 2001) and I designed primers, but Shaw et al. These near-universal primers were designed based on the complete chloroplast genome sequences of 16 flowering plant species (see Appendix 1). Around the same time, I developed nondegenerate primers for 36 noncoding regions in the large and small single-copy regions of the chloroplast genome (published here). They were the first to take advantage of the new genomic data boom, providing a set of 20 universal chloroplast primers designed around the complete chloroplast data from seven flowering plant species. ( 2001) were visionary when they moved beyond analyzing regions commonly being used to design primers for lesser-known and potentially faster-evolving regions of the chloroplast genome. In the mid-2000s, a small number of complete chloroplast genome sequences were available for land plants and some of those were not annotated (e.g., Medicago truncatula Gaertn. The Tortoise and the Hare II paper was revolutionary in assessing sequence variability for all regions studied across a broad diversity of flowering plants, and providing a ranking of that variability. Our laboratory was also compiling such information, as were many others. The work presented here was inspired by “The Tortoise and the Hare II” ( Shaw et al., 2005), which was the first study to pull together information on a large number of regions commonly in use (at that time) for plant phylogenetics. Hundreds of primer sequences have been published, many designed for specific taxonomic groups. The second critical question to “which markers” is “which primers?”. These studies are exceedingly valuable, demonstrating there is no one-size-fits-all answer to the question “which markers?”. Information for model organisms abounds (e.g., grasses Saski et al., 2007 Bortiri et al., 2008 Leseberg and Duvall, 2009), and a few studies have specifically screened the same set of markers across a diversity of plant groups, ranking the utility of these markers either explicitly or implicitly ( Shaw et al., 2005, 2007, 2014). Identifying the most appropriate markers to employ has been challenging. Whole genome sequencing is more available and less expensive than ever before, yet most scientists continue to rely on targeted, comparative sequencing for phylogenetics and phylogeography.
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