SNP discovery and characterisation in White Rhino (Ceratotherium simum) with application to parentage assignment

Abstract The white rhino is one of the great success stories of modern wildlife conservation, growing from as few as 50-100 animals in the 1880s, to approximately 20,000 white rhinoceros remaining today. However, illegal trade in conservational rhinoceros horns is adding constant pressure on remaining populations. Captive management of ex situ populations of endangered species using molecular methods can contribute to improving the management of the species. Here we compare for the first time the utility of 33 Single Nucleotide Polymorphisms (SNPs) and nine microsatellites (MS) in isolation and in combination for assigning parentage in captive White Rhinoceros. We found that a combined dataset of SNPs and microsatellites was most informative with the highest confidence level. This study thus provided us with a useful set of SNP and MS markers for parentage and relatedness testing. Further assessment of the utility of these markers over multiple (> three) generations and the incorporation of a larger variety of relationships among individuals (e.g. half-siblings or cousins) is strongly suggested

Methylation-based markers for the estimation of age in African Cheetah, Acinonyx jubatus

Age is a key demographic in conservation biology where individual age classes show diffuse differences in terms of important population dynamics metrics such as morbidity and mortality. Furthermore, several traits including reproductive potential show clear senescence with aging. Thus, the ability to estimate the ages for the individuals of a population as part of age class assignment is critical in understanding both the current population structure as well as in modelling and predicting the future survival of species. This study explored the utility of age-related changes in methylation for six candidate genes, EDARADD, ELOVL2, FHL2, GRIA2, ITGA2B, and PENK, to create an age estimation model in captive cheetah. Gene orthologues between humans and cheetah were retrieved containing a hundred CpG’s. Target regions were assayed for differential methylation and fragmentation patterns in fifty samples using mass array technology for a total of seventy-seven CpG clusters. Correlation analyses between CpG methylation and chronological age identified six CpG’s with an age relationship, of which four were hypomethylated and two were hypermethylated. Regression models, fitted for different combinations of CpG’s, indicated that age models using four and six CpG’s were most accurate, with the six CpG model having superior correlation and predictive power (R2 = 0.70, Mean Absolute Error = 25 months). This model was more accurate than previous attempts using methylation sensitive Polymerase Chain Reaction and performed similarly to models created using a candidate gene approach in several other mammal species, making methylation a promising tool of age estimation in cheetah

Heritage Matters- January 2007

INSIDE THIS ISSUE Conferences upcoming, p. 10 Grant applications, p. 11 Internships, p. 3 National Register listings, p. 5 Tribal Preservation Officer list, p. 9 CONTENTS NPS ACTIVITIES, pages 2-4 Inupiaq Heritage in Northwestern Alaska, p. 2 Cultural Resources Diversity Internship Program, p. 3 Historic Trees, the National Park Service, and History, p. 3 STATE INITIATIVES, pages 5-8 National Register Nominations, p. 5 • Ridge Trail Historic District, Walsh and Pembina Counties, North Dakota • Emery Hall, Wilberforce University, Green County, Ohio • Ponca Agency, Boyd County, Nebraska • Ohio Theatre, Toledo, Ohio • Hydaburg Totem Park, Hydaburg, Alaska • Sunrise Mine Historic District, eastern Wyoming • Pilsen Historic District, Chicago, Illinois TRIBAL INITIATIVES, pages 8-9 NAGPRA Develops New Outreach Initiatives, p. 8 Current THPO Listings, p. 9 CONFERENCES, ANNOUNCEMENTS, AND EVENTS, pages 10-11 Conferences, p. 10 Vernacular Architectural Forum Annual Meeting in Savannah, p. 10 Organization of American Historians Annual Meeting in Minneapolis, p. 10 US/ICOMOS International Symposium on Heritage Tourism in San Francisco, p. 10 Arkansas Historical Association Conference in Little Rock, p. 10 American Association of Museums Annual Meeting in Chicago, p. 11 National Trust for Historic Preservation National Preservation Conference in St. Paul, p. 11 Announcements, p. 11 Preserve America Grant Applications, p. 11 Save America’s Treasures Grant Applications, p. 11 Events, p. 11 Preservation-related Commemorations, p. 1

Heritage Matters- January 2007

INSIDE THIS ISSUE Conferences upcoming, p. 10 Grant applications, p. 11 Internships, p. 3 National Register listings, p. 5 Tribal Preservation Officer list, p. 9 CONTENTS NPS ACTIVITIES, pages 2-4 Inupiaq Heritage in Northwestern Alaska, p. 2 Cultural Resources Diversity Internship Program, p. 3 Historic Trees, the National Park Service, and History, p. 3 STATE INITIATIVES, pages 5-8 National Register Nominations, p. 5 • Ridge Trail Historic District, Walsh and Pembina Counties, North Dakota • Emery Hall, Wilberforce University, Green County, Ohio • Ponca Agency, Boyd County, Nebraska • Ohio Theatre, Toledo, Ohio • Hydaburg Totem Park, Hydaburg, Alaska • Sunrise Mine Historic District, eastern Wyoming • Pilsen Historic District, Chicago, Illinois TRIBAL INITIATIVES, pages 8-9 NAGPRA Develops New Outreach Initiatives, p. 8 Current THPO Listings, p. 9 CONFERENCES, ANNOUNCEMENTS, AND EVENTS, pages 10-11 Conferences, p. 10 Vernacular Architectural Forum Annual Meeting in Savannah, p. 10 Organization of American Historians Annual Meeting in Minneapolis, p. 10 US/ICOMOS International Symposium on Heritage Tourism in San Francisco, p. 10 Arkansas Historical Association Conference in Little Rock, p. 10 American Association of Museums Annual Meeting in Chicago, p. 11 National Trust for Historic Preservation National Preservation Conference in St. Paul, p. 11 Announcements, p. 11 Preserve America Grant Applications, p. 11 Save America’s Treasures Grant Applications, p. 11 Events, p. 11 Preservation-related Commemorations, p. 1

ABI Sanger Sequencing of Avian Clock genes to elucidate markers for Migration Phenology

This protocol follows up on "PCR Amplification of Clock genes with EmeraldAmp® GT PCR Master Mix in Avian species" and is intended to provide the next steps used in the sanger sequencing of the produced amplicons. This protocol uses ABI BigDye reagents (but suitable alternatives exist). The same primers used to produce the PCR products, of the respective clock genes, are used individually in a forward and reverse sequencing reaction. Cycling conditions mimic those used for PCR. Sequencing reactions are purified and subsequently resolved on an ABI Genetic Analyzer. The sequence read data was used in a BLAST search and confirmed to be the genes and regions of interest for all tested species

Heritage Matters- July 2005

INSIDE THIS ISSUE Conferences upcoming, p. 17 National Historic Landmark designations, p. 3 National Register nominations, p. 8 Publication of note, p. 18 CONTENTS NPS ACTIVITIES, pages 1–5 Cultures of a Country, p. 1 National Historic Landmarks designated, p. 3 Preserve and Play Conference, p. 4 PARTNERS’ ACTIVITIES, pages 6–7 Seminar, Public Meaning of Archeological Heritage, p. 6 AAM Annual Meeting 2005 report, p. 7 STATE ACTIVITIES, pages 8-15 National Register Nominations, p. 8 • Pascua Cultural Center, Tucson, Arizona • Town Doctor’s House and Site, Southold, New York • Louis J. Bailey Branch Library—Gary International Institute, Gary, Indiana • Our Savior’s Scandinavian Lutheran Church, Kenaston, North Dakota • B’nai Jacob Synagogue, Ottumna, Iowa • Whitepath and Fly Smith Gravesite, Hopkinsville, Kentucky • Winnemucca Hotel, Humboldt County, Nevada • Government Boarding School at Lac de Flambeau, Wisconsin • Ivey Delph Apartments, New York City • Edificio Patio Español, San Juan, Puerto Rico POINT OF VIEW, page 16 The Original Mason-Dixon Line, p. 16 CONFERENCES AND NOTICES, pages 17–19 Conferences, p. 17 • Multicultural Days: An International Perspective Conference, St. Catherines, Ontario, Canada • 5th International Conference on Diversity in Organizations, Communities and Nations, Bejing, China • 2005 Pecos Conference, White Rock, New Mexico • Graduate Association for African-American History at the University of Memphis, Memphis, Tennessee • SACRPH Biennial Conference, Coral Gables, Florida • American Studies Association Annual Conference, Washington, DC • Call for Papers: Mid-Atlantic Popular/American Culture Association Annual Conference, New Brunswick, New Jersey • Call for Papers: Association for Museum History Annual Conference, Washington, DC Notices, p. 18 Asian Reflections on the American Landscape Available Assistance Needed for Untold Stories and Unsung Heroes in the Making of the National Park

PCR Amplification of Clock and Adcyap1 genes with EmeraldAmp® GT PCR Master Mix in Avian species for polymorphism elucidation

This PCR protocol is used to amplify Clock and Adcyap1 gene regions in avian species which have previously shown polymorphisms, such as poly-Q runs, that correlated to migration phenology. It was tested and optimized in Woodlands kingfisher (Halcyon senegalensis) and Diederik cuckoos (Chrysococcyx caprius). The primers were designed based on those previously used by Johnson et al. (2007) and Steinmeyer et al. (2009) by comparing the relevant gene sequences for chickens (Galus galus) with several other available avian species to select primers that would account for the most common variations in primer regions, enabling more universal amplification. Individual clock gene sequences were retrieved from Genbank and aligned in BioEdit 7.2. Primers were then selected based on the annotated regions. The assay was designed using 25 μL (half) reactions of EmeraldAmp® GT PCR Master Mix, which is premixed with loading buffer for easy gel loading following PCR and does not require a long initial denaturation step (thereby shortening the run time). Gel electrophoresis was able to confirm successful amplification of a product ±280 bp long in both species. The same primers were subsequently used for sanger sequencing. A BLAST search of the resulting sequences confirmed the identity of the amplified regions

Designing an EpiTYPER bisulfite sequencing assay for age estimation in Acinonyx jubatus based on human orthologues

Age is key factor in animal ecology as it can be used to assign animals to important age classes, ranging from immature young to reproductive adults and eventual old age and fragility. Different groups contribute to different aspects that need to be considered when modeling current and future population dynamics as part of continued conservation efforts. Due to the need of an accurate molecular method for assigning age, several studies have explore various aspects of epigenetic clocks. Epigenetics is a collective term for mechanisms that modify DNA and DNA packaging, independent of genetic sequence. One widely studied epigenetic feature is DNA methylation; a process that adds a methyl group to the 5’ cytosine of Cytosine-Guanine pairs (CpG’s). Studies have revealed that within genes, nearly a third of all CpG sites are influenced by age. Given its consistency, the epigenetic clock is a promising avenue of chronological age prediction which has been illustrated in many human studies. This protocol illustrates how CpG's with known age-correlations from human studies can be used to (1) identify orthologous regions in other species and (2) design primers to assay differential methylation using EpiTYPER mass array technology.<br/