Who’s Your Daddy?Who’s Your Daddy?

Determination of Maternity and Determination of Maternity and Paternity in a family of box turtles Paternity in a family of box turtles

((Terrapene carolinaTerrapene carolina))Rebecca StewartRebecca Stewart

Capstone 490 Capstone 490

OverviewOverview

• IntroductionIntroduction– GoalsGoals– BackgroundBackground– ProceduresProcedures– ResultsResults

IntroductionIntroduction

• Dr. McShaffrey has Dr. McShaffrey has been keeping box been keeping box turtles for some turtles for some timetime

• In 1999 some of In 1999 some of the turtles he the turtles he keeps had their keeps had their first successful first successful breeding eventbreeding event

IntroductionIntroduction

• Of the two clutches Of the two clutches laid by either of two laid by either of two different females different females (Terry & Carol) one (Terry & Carol) one hatchling survived.hatchling survived.

• In 2003, another In 2003, another clutch was laid, clutch was laid, containing 5 eggs, containing 5 eggs, all of which all of which survived.survived.

Photos copyright Dave Photos copyright Dave McShaffreyMcShaffrey

IntroductionIntroduction

• Both the 1999 hatchling (Dylan), and the 2003 Both the 1999 hatchling (Dylan), and the 2003 hatchlings (Riker, Geordi, Data, Worf, and Picard) hatchlings (Riker, Geordi, Data, Worf, and Picard) have the same suspected father (Linus)have the same suspected father (Linus)

• However, due to sperm storage, the father could However, due to sperm storage, the father could be any male the females had contact with up to 4 be any male the females had contact with up to 4 years prior to laying the eggs (Ewing 1943, 113)years prior to laying the eggs (Ewing 1943, 113)

Photos Photos copyright copyright Dave Dave McShaffreMcShaffreyy

IntroductionIntroduction

• Whether the mother Whether the mother of Dylan or the 2003 of Dylan or the 2003 hatchlings is Terry hatchlings is Terry or Carol is unknownor Carol is unknown

• So, Dr. McShaffrey So, Dr. McShaffrey found a student found a student (me) to do a (me) to do a capstone on capstone on determining the determining the heritage of the box heritage of the box turtlesturtles

Copyright Dave Copyright Dave McShaffreyMcShaffrey

IntroductionIntroduction

• More information about these turtles and More information about these turtles and Dr. McShaffrey’s box turtle observation Dr. McShaffrey’s box turtle observation project can be found at: project can be found at:

http://www.marietta.edu/~mcshaffd/boxt/http://www.marietta.edu/~mcshaffd/boxt/boxt.htmlboxt.html

OverviewOverview

– IntroductionIntroduction

• GoalsGoals– BackgroundBackground– ProceduresProcedures– ResultsResults

GoalsGoals

• Ultimately to create the beginning of Ultimately to create the beginning of a pedigree for these turtlesa pedigree for these turtles

• To do that…To do that…– Must first establish a set of successful Must first establish a set of successful

procedures and find primers that are procedures and find primers that are specific enough for paternity specific enough for paternity comparisonscomparisons

OverviewOverview

– IntroductionIntroduction– GoalsGoals

• BackgroundBackground– ProceduresProcedures– ResultsResults

BackgroundBackground

• The literature does not show that The literature does not show that anyone has ever done paternity anyone has ever done paternity testing with box turtles beforetesting with box turtles before

• However, paternity testing was However, paternity testing was performed on painted turtles in a performed on painted turtles in a study done by Pearse et al. (2001)study done by Pearse et al. (2001)

• They used PCR followed by gel They used PCR followed by gel electrophoresis to determine electrophoresis to determine paternitypaternity

And now for a crash And now for a crash course in gel course in gel

electrophoresiselectrophoresis

OverviewOverview

– IntroductionIntroduction– GoalsGoals– BackgroundBackground

• ProceduresProcedures– ResultsResults

ProceduresProcedures

• Very basically…Very basically…– The samples were blinded to me by Dr. The samples were blinded to me by Dr.

McShaffreyMcShaffrey– DNA was extracted using procedures DNA was extracted using procedures

modified from Cline et al. (2003, 3)modified from Cline et al. (2003, 3)– DNA was amplified using a PCR DNA was amplified using a PCR

thermocycler and protocols taken from thermocycler and protocols taken from Pearse et al. (2001, 379) and Kuo et al. Pearse et al. (2001, 379) and Kuo et al. (2004, 427) and primers taken from (2004, 427) and primers taken from Pearse et al.’s study and Rothuizen et al. Pearse et al.’s study and Rothuizen et al. (1994, 15)(1994, 15)

ProceduresProcedures

• More basics…More basics…– DNA was then electrophoresed using DNA was then electrophoresed using

agarose gels and later polyacrylamide agarose gels and later polyacrylamide gels with silver staining for better claritygels with silver staining for better clarity

– Lastly, the resulting bands were Lastly, the resulting bands were compared in order to try to determine compared in order to try to determine maternity and paternitymaternity and paternity

ProceduresProcedures

• Began with the painted turtle primers Began with the painted turtle primers (Pearce et al.)and PCR (Pearce et al.)and PCR (Polymerase Chain (Polymerase Chain Reaction)Reaction) protocols . protocols .

• Did this instead of beginning with Did this instead of beginning with RAPDRAPD(Random Amplification of Polymorphic DNA)(Random Amplification of Polymorphic DNA)– Lower chance of bacterial contamination Lower chance of bacterial contamination

with specific primerswith specific primers– Thought that the primers might cross Thought that the primers might cross

species if in the same genusspecies if in the same genus

ProceduresProcedures

• Primers worked when tested on painted Primers worked when tested on painted turtle DNA (control), but not on box turtlesturtle DNA (control), but not on box turtles

• Tried changing annealing temperaturesTried changing annealing temperatures– 5252oo C original temp C original temp– Tried 48Tried 48oo, 50, 50oo, and 54, and 54oo

• Banding at 48Banding at 48oo

ProceduresProcedures

• Why not continue Why not continue with the painted with the painted turtle primers?turtle primers?– The resulting bands The resulting bands

were not polymorphicwere not polymorphic– Using such a low Using such a low

annealing annealing temperature for those temperature for those primers results in primers results in non-specific bindingnon-specific binding• Probably unreliableProbably unreliable

ProceduresProcedures

• Why RAPD primers?Why RAPD primers?– They are much shorter than specific They are much shorter than specific

primers (13 base pairs opposed to 23 primers (13 base pairs opposed to 23 bp)bp)

– Will easily cross speciesWill easily cross species– Much greater chance of successful Much greater chance of successful

amplification ~1in 67 million instead of amplification ~1in 67 million instead of ~1 in 70 trillion~1 in 70 trillion

ProceduresProcedures

• Switched to 6 RAPD primers taken Switched to 6 RAPD primers taken from a study performed on purebred from a study performed on purebred dogs (Rothuizen)dogs (Rothuizen)

• Used PCR protocols later found in Used PCR protocols later found in ornate box turtle study (Kuo), but ornate box turtle study (Kuo), but adapted for RAPD primersadapted for RAPD primers– Specific primers available (King et al. Specific primers available (King et al.

2004, 721-2) but success still less likely 2004, 721-2) but success still less likely than with RAPD primersthan with RAPD primers

ProceduresProcedures

• Had to use toenail samples instead of Had to use toenail samples instead of oral swabs to reduce chances of oral swabs to reduce chances of bacterial contaminationbacterial contamination

• Each of the primers had a different ideal Each of the primers had a different ideal annealing temperature as calculated by annealing temperature as calculated by NetPrimer (www.premierbiosoft.com)NetPrimer (www.premierbiosoft.com)

• After testing, all of the primers seemed After testing, all of the primers seemed to perform at an annealing temperature to perform at an annealing temperature of 30of 30oo C, with the exception of #6, which C, with the exception of #6, which was then excluded.was then excluded.

ProceduresProcedures

• Also switched to Also switched to a silver stained a silver stained polyacrylamide polyacrylamide gelgel– Better clarity Better clarity

and separation and separation of bandsof bands

– Added control Added control group to check group to check for for contaminationcontamination

OverviewOverview

– IntroductionIntroduction– GoalsGoals– BackgroundBackground– ProceduresProcedures

• ResultsResults

ResultsResults

• During the process of purifying my During the process of purifying my DNA samples after extraction, I DNA samples after extraction, I managed to mix two of the samples managed to mix two of the samples togethertogether

• So I lost one of the maternal So I lost one of the maternal samples, making results of band samples, making results of band comparison less certaincomparison less certain

ResultsResults

• Fortunately, I got bands on the gelFortunately, I got bands on the gel

• Of the bands that were comparable, Of the bands that were comparable, one was maternal and two were one was maternal and two were offspringoffspring

Special Thanks:Special Thanks:• Dr. McShaffrey for providing me with Dr. McShaffrey for providing me with

an interesting capstone and for all of an interesting capstone and for all of his help sampling.his help sampling.

• Dr. Spilatro for making sure I cleaned Dr. Spilatro for making sure I cleaned up after myselfup after myself

• Mrs. Jarrel for ordering phenol at the Mrs. Jarrel for ordering phenol at the last minutelast minute

• And especially to my advisor Dr. And especially to my advisor Dr. Brown for his immense help and Brown for his immense help and patience in making sure it didn’t patience in making sure it didn’t make too many dumb mistakes.make too many dumb mistakes.

Literature CitedLiterature CitedAnonymous. (Anonymous. (Premier Biosoft InternationalPremier Biosoft International). “Netprimer launch.” ). “Netprimer launch.”

(http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html) (http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html) no date given.no date given.

Cline RE; Laurent NM; Foran DR. 2003. The fingernails of Mary Sullivan: Cline RE; Laurent NM; Foran DR. 2003. The fingernails of Mary Sullivan: developing reliable methods for selectively Isolating Endogenous and developing reliable methods for selectively Isolating Endogenous and Exogenous DNA from Evidence. Journal of Forensic Science 48(2): 1-6.Exogenous DNA from Evidence. Journal of Forensic Science 48(2): 1-6.

Ewing HE. 1943. Continued fertility in female box turtles following mating. Ewing HE. 1943. Continued fertility in female box turtles following mating. Copeia 1943(2): 112-14.Copeia 1943(2): 112-14.

King TL; Julian SE. 2004. Conservation of microsatellite DNA flanking sequence King TL; Julian SE. 2004. Conservation of microsatellite DNA flanking sequence across 13 Emydid genera assayed with novel bog turtle (across 13 Emydid genera assayed with novel bog turtle (Glyptemys Glyptemys muhlenbergiimuhlenbergii) loci. Conservation Genetics 5: 719-725.) loci. Conservation Genetics 5: 719-725.

Kuo CH; Janzen FJ. 2004. Genetic effects of a persistent bottleneck on a Kuo CH; Janzen FJ. 2004. Genetic effects of a persistent bottleneck on a natural population of ornate box turtles (natural population of ornate box turtles (Terrapene ornataTerrapene ornata). Conservation ). Conservation Genetics 5: 425-437.Genetics 5: 425-437.

McShaffrey, Dave. (McShaffrey, Dave. (Marietta CollegeMarietta College). “Box turtle observation project.” ). “Box turtle observation project.” (http://www.marietta.edu/~mcshaffd/boxt/boxt.html). March 2004(http://www.marietta.edu/~mcshaffd/boxt/boxt.html). March 2004

Pearse DE; Janzen FJ; Avise JC. 2001. Genetic markers substantiate long-term Pearse DE; Janzen FJ; Avise JC. 2001. Genetic markers substantiate long-term storage and utilization of sperm by female painted turtles. Heredity 86: storage and utilization of sperm by female painted turtles. Heredity 86: 378-384.378-384.

Rothuizen J; Wolferen MV. 1994. Randomly amplified DNA polymorphisms in Rothuizen J; Wolferen MV. 1994. Randomly amplified DNA polymorphisms in dogs are reproducible and display Mendelian transmission. Animal dogs are reproducible and display Mendelian transmission. Animal Genetics. 25: 13-18.Genetics. 25: 13-18.

All photographs are copyright Rebecca Stewart unless otherwise indicated.All photographs are copyright Rebecca Stewart unless otherwise indicated.

Questions?Questions?