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Using antisera to analyze the expression of specific gene products is a common procedure. However, in multigene families, such as the beta-keratins of the avian integument where strong homology exists among the scale (ScbetaK), claw (ClbetaK), feather (FbetaK), and feather-like (FlbetaK) subfamilies, determining the cellular and tissue expression patterns of the subfamilies is difficult because polyclonal antisera produced from any one protein recognize all family members. Traditionally, researchers produced and screened multiple monoclonal antisera produced from the proteins of interest until an antiserum with sufficient specificity could be obtained. Unfortunately, this approach requires a lot of effort, and once obtained, such antisera may have limited applications. Here, we present procedures by which comparative DNA sequences of members from the beta-keratin multigene family were translated and aligned to identify amino acid domains that were conserved within the FbetaK subfamily, but which were divergent from the other subfamilies. A synthetic 23-mer peptide with the conserved amino acid sequence was generated and used to produce a polyclonal antiserum that recognizes only the FbetaK subfamily of proteins. Western blot analysis and confocal microscopy with this antiserum are now providing valuable new insights concerning the developmental and evolutionary relationships between the scale, claw, and feather proteins found in birds. This represents a powerful new approach combining techniques from molecular evolution and developmental biology to study the expression and evolution of specific members of multigene families.

A series of techniques are presented to construct genomic DNA libraries highly enriched for microsatellite DNA loci. The individual techniques used here derive from several published protocols but have been optimized and tested in our research laboratories as well as in classroom settings at the University of South Carolina and University of Georgia, with students achieving nearly 100% success. Reducing the number of manipulations involved has been a key to success, decreasing both the failure rate and the time necessary to isolate loci of interest. In our lab during the past 3 years alone, these protocols have been successfully used to isolate microsatellite DNA loci from at least 55 species representing three kingdoms. These protocols have made it possible to reduce the time to identify candidate loci for primer development from most eukaryotic species to as little as 1 week.

To provide context for the diversification of archosaurs-the group that includes crocodilians, dinosaurs, and birds-we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs.

To better determine the history of modern birds, we performed a genome-scale phylogenetic analysis of 48 species representing all orders of Neoaves using phylogenomic methods created to handle genome-scale data. We recovered a highly resolved tree that confirms previously controversial sister or close relationships. We identified the first divergence in Neoaves, two groups we named Passerea and Columbea, representing independent lineages of diverse and convergently evolved land and water bird species. Among Passerea, we infer the common ancestor of core landbirds to have been an apex predator and confirm independent gains of vocal learning. Among Columbea, we identify pigeons and flamingoes as belonging to sister clades. Even with whole genomes, some of the earliest branches in Neoaves proved challenging to resolve, which was best explained by massive protein-coding sequence convergence and high levels of incomplete lineage sorting that occurred during a rapid radiation after the Cretaceous-Paleogene mass extinction event about 66 million years ago.

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. [ABSTRACT FROM AUTHOR]

Ligating adapters with unique synthetic oligonucleotide sequences (sequence tags) onto individual DNA samples before massively parallel sequencing is a popular and efficient way to obtain sequence data from many individual samples. Tag sequences should be numerous and sufficiently different to ensure sequencing, replication, and oligonucleotide synthesis errors do not cause tags to be unrecoverable or confused. However, many design approaches only protect against substitution errors during sequencing and extant tag sets contain too few tag sequences. We developed an open-source software package to validate sequence tags for conformance to two distance metrics and design sequence tags robust to indel and substitution errors. We use this software package to evaluate several commercial and non-commercial sequence tag sets, design several large sets (maxcount = 7,198) of edit metric sequence tags having different lengths and degrees of error correction, and integrate a subset of these edit metric tags to polymerase chain reaction (PCR) primers and sequencing adapters. We validate a subset of these edit metric tagged PCR primers and sequencing adapters by sequencing on several platforms and subsequent comparison to commercially available alternatives. We find that several commonly used sets of sequence tags or design methodologies used to produce sequence tags do not meet the minimum expectations of their underlying distance metric, and we find that PCR primers and sequencing adapters incorporating edit metric sequence tags designed by our software package perform as well as their commercial counterparts. We suggest that researchers evaluate sequence tags prior to use or evaluate tags that they have been using. The sequence tag sets we design improve on extant sets because they are large, valid across the set, and robust to the suite of substitution, insertion, and deletion errors affecting massively parallel sequencing workflows on all currently used platforms. [ABSTRACT FROM AUTHOR]

The diversity of available 2nd and 3rd generation DNA sequencing platforms is increasing rapidly. Costs for these systems range from <$100 000 to more than $1 000 000, with instrument run times ranging from minutes to weeks. Extensive trade-offs exist among these platforms. I summarize the major characteristics of each commercially available platform to enable direct comparisons. In terms of cost per megabase (Mb) of sequence, the Illumina and SOLiD platforms are clearly superior (≤$0.10/Mb vs. >$10/Mb for 454 and some Ion Torrent chips). In terms of cost per nonmultiplexed sample and instrument run time, the Pacific Biosciences and Ion Torrent platforms excel, with the 454 GS Junior and Illumina MiSeq also notable in this regard. All platforms allow multiplexing of samples, but details of library preparation, experimental design and data analysis can constrain the options. The wide range of characteristics among available platforms provides opportunities both to conduct groundbreaking studies and to waste money on scales that were previously infeasible. Thus, careful thought about the desired characteristics of these systems is warranted before purchasing or using any of them. Updated information from this guide will be maintained at: and . [ABSTRACT FROM AUTHOR]

Microsatellite DNA loci have emerged as the dominant genetic tool for addressing questions associated with genetic diversity in many wildlife species, including crocodilians. Despite their usefulness, their isolation and development can be costly, as well as labour intensive, limiting their wider use in many crocodilian species. In this study, we investigate the cross-species amplification success of 82 existing microsatellites previously isolated for the saltwater crocodile (Crocodylus porosus) in 18 non-target crocodilian species; Alligator sinensis, Caiman crocodylus, Caiman latirostris, Caiman yacare, Melanosuchus niger, Paleosuchus palpebrosus, Crocodylus acutus, Mecistops cataphractus, Crocodylus intermedius, Crocodylus johnstoni, Crocodylus mindorensis, Crocodylus moreletii, Crocodylus niloticus, Crocodylus novaeguineae, Crocodylus palustis, Crocodylus rhombifer, Crocodylus siamensis, and Osteolaemus tetraspis. Our results show a high level of microsatellites crossamplification making available polymorphic markers for a range of crocodilian species previously lacking informative genetic markers. [ABSTRACT FROM AUTHOR]

Genomic elucidation and mapping of novel organisms requires the generation of large genetic resources. In this study, 253 novel and polymorphic microsatellite loci were isolated and characterized for the saltwater crocodile (Crocodylus porosus) by constructing libraries enriched for microsatellite DNA. All markers were evaluated on animals obtained from Darwin Crocodile Farm in the Northern Territory, Australia, and are intended for future use in the construction of a genetic-linkage map for the saltwater crocodile. The 253 loci yielded an average of 4.12 alleles per locus, and those selected for mapping had an average polymorphic information content (PIC) of 0.425. [ABSTRACT FROM AUTHOR]

We describe polymerase chain reaction primers and amplification conditions for 13 microsatellite DNA loci isolated from two bisexual species of whiptail lizards Aspidoscelis costata huico and Aspidoscelis inornata. Primers were tested on either 16 or 48 individuals of A. c. huico and/or 26 individuals of A. inornata. Ten of the 13 primers were also tested against a panel of 31 additional whiptail taxa. We detected three to nine alleles per locus in A. c. huico and four to 19 alleles per locus in A. inornata, with observed heterozygosity ranging from 0.60 to 0.87 and from 0.15 to 1.00, respectively. These primers will be an important resource for surveys of genetic variation in these lizards. [ABSTRACT FROM AUTHOR]

We isolated and characterized 10 polymorphic microsatellite loci in Drosophila hydei. The number of alleles per locus ranged from 3 to 8 ( N = 23 individuals). Polymorphic information content ranged from 0.316 to 0.750 and observed heterozygosity from 0.261 to 0.913. These markers will be valuable in studies of sexual selection and parental investment in D. hydei. [ABSTRACT FROM AUTHOR]

Eight polymorphic microsatellite DNA loci were isolated from the flightless cormorant ( Phalacrocorax harrisi) for future population genetic studies. Genetic variability was assessed using at least 38 individuals from two populations. Allele numbers ranged from three to nine per locus. Mean observed heterozygosity varied from 0.27 to 0.78. No locus deviated from Hardy–Weinberg (HW) or linkage equilibria in either population. The high levels of detected polymorphism indicate the utility of these markers for population genetic studies of this Galápagos species. [ABSTRACT FROM AUTHOR]

We have developed eight high-quality microsatellite DNA loci for the saltmarsh sharp-tailed sparrow and one additional locus with evidence of null alleles. In a sample of 250–350 individuals, the average number of alleles per locus was 14.7 and average observed heterozygosity was 0.80. These loci were tested in three additional species of emberizid sparrows, indicating that more than half of the loci could be useful in other sparrows. [ABSTRACT FROM AUTHOR]

Twelve polymorphic microsatellite loci were isolated from polyploid alpine wood-sorrel, Oxalis alpina (Oxalidaceae), and optimized for future studies of its breeding system. The loci were screened for variability among 72 individuals from Pinos Altos, New Mexico. The primers amplified loci with allele number ranging from two to 17 per locus and with estimates of Nei's genetic diversity varying from 0.10 to 0.99. These primers provide an opportunity to use polymorphic DNA markers to study the causes of breeding system variability in this species. [ABSTRACT FROM AUTHOR]

Fifteen polymorphic microsatellite DNA loci were isolated from barn swallow ( Hirundo rustica) and optimized for future studies of radiation-induced mutations in populations from Ukraine. The loci were screened for variability among 25 individuals from two populations. The primers amplified loci with relatively high numbers of alleles ranging from five to 32 alleles per locus and polymorphic information content from 0.481 to 0.951. Observed heterozygosity varied from 0.458 to 0.960. None of the loci showed deviations from Hardy–Weinberg equilibrium in either population. [ABSTRACT FROM AUTHOR]

Twelve polymorphic microsatellite DNA loci were isolated from saltgrass ( Distichlis spicata) and optimized for future studies of its breeding system. The loci were screened for variability among 24 individuals from two populations. The primers amplified loci with numbers of alleles ranging from four to 14 per locus and polymorphic information content from 0.481 to 0.951. Observed heterozygosity varied from 0.227 to 0.958. [ABSTRACT FROM AUTHOR]

Phylogenomics offers the potential to fully resolve the Tree of Life, but increasing genomic coverage also reveals conflicting evolutionary histories among genes, demanding new analytical strategies for elucidating a single history of life. Here, we outline a phylogenomic approach using a novel class of phylogenetic markers derived from ultraconserved elements and flanking DNA. Using species-tree analysis that accounts for discord among hundreds of independent loci, we show that this class of marker is useful for recovering deep-level phylogeny in placental mammals. In broad outline, our phylogeny agrees with recent phylogenomic studies of mammals, including several formerly controversial relationships. Our results also inform two outstanding questions in placental mammal phylogeny involving rapid speciation, where species-tree methods are particularly needed. Contrary to most phylogenomic studies, our study supports a first-diverging placental mammal lineage that includes elephants and tenrecs (Afrotheria). The level of conflict among gene histories is consistent with this basal divergence occurring in or near a phylogenetic "anomaly zone" where a failure to account for coalescent stochasticity will mislead phylogenetic inference. Addressing a long-standing phylogenetic mystery, we find some support from a high genomic coverage data set for a traditional placement of bats (Chiroptera) sister to a clade containing Perissodactyla, Cetartiodactyla, and Carnivora, and not nested within the latter clade, as has been suggested recently, although other results were conflicting. One of the most remarkable findings of our study is that ultraconserved elements and their flanking DNA are a rich source of phylogenetic information with strong potential for application across Amniotes. [ABSTRACT FROM AUTHOR]