Genetic variation was surveyed at nine microsatellite loci and the mitochondrial control region (868 bp) to test for the presence of genetic stock structure in young-of-the-year Atlantic bluefin tuna (Thunnus thynnus thynnus) from the Mediterranean Sea. Bluefin tuna were sampled over a period of 5 years from the Balearic and Tyrrhenian seas in the western basin of the Mediterranean Sea, and from the southern Ionian Sea in the eastern basin of the Mediterranean Sea. Analyses of multilocus microsatellite genotypes and mitochondrial control region sequences revealed no significant heterogeneity among collections taken from the same location in different years; however, significant spatial genetic heterogeneity was observed across all samples for both microsatellite markers and mitochondrial control region sequences (FST=0.0023, P=0.038 and PhiST=0.0233, P=0.000, respectively). Significant genetic differentiation between the Tyrrhenian and Ionian collections was found for both microsatellite and mitochondrial markers (FST=0.0087, P=0.015 and PhiST=0.0367, P=0.030, respectively). These results suggest the possibility of a genetically discrete population in the eastern basin of the Mediterranean Sea.

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

Keywords: 3RAD, dual digest RADseq, ddRAD, DNA libraries, multiplexing, NGS. Bldg., University of Georgia, Athens, GA 30602, USA; 3 2 E-mail: njbayona@uga.edu . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/205799 doi: bioRxiv preprint first posted online Oct. 19, 2017; 2 Running Title: 3RAD -triple-enzyme RADseq digestion of genomic DNA to sample consistent portions of the genome from individuals being 4 0 studied (e.g., RADseq, GBS). However, researchers often find the existing methods expensive 4 1 to initiate and/or difficult to implement consistently. Here, we present a low-cost and highly integrating adapter designs that can be used interchangeably; 6) incorporating variable-length 4 9 internal tags within the adapters to increase the scope of sample tagging and facilitate pooling 5 0 while also increasing sequence diversity; 7) maintaining compatibility with universal dual- discover thousands of variable loci in each species.

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