GENETIC DIVERSITY OF WHITE TIGERS AND GENETIC FACTORS ... ?· GENETIC DIVERSITY OF WHITE TIGERS AND…

  • Published on
    02-Jul-2018

  • View
    212

  • Download
    0

Embed Size (px)

Transcript

  • GENETIC DIVERSITY OF WHITE TIGERS AND GENETIC

    FACTORS RELATED TO COAT COLOR

    Approved by: Research Advisor: Dr. Jan Janecka

    Major: Biomedical Sciences Wildlife and Fisheries Sciences

    May 2013

    Submitted to Honors and Undergraduate Research Texas A&M University

    in partial fulfillment of the requirements for the designation as

    UNDERGRADUATE RESEARCH SCHOLAR

    An Undergraduate Research Scholars Thesis

    by

    SARA ELIZABETH CARNEY

  • 1

    TABLE OF CONTENTS

    Page

    TABLE OF CONTENTS .....................................................................................................1

    ABSTRACT .........................................................................................................................2

    ACKNOWLEDGEMENTS .................................................................................................4

    CHAPTER

    I INTRODUCTION .......................................................................................5

    Candidate genes for the white coat phenotype ................................8 Use of microsatellites to determine genetic diversity ......................9 II METHODS ................................................................................................11

    Samples ..........................................................................................11 PCR methodology ..........................................................................11 Microsatellite analysis ...................................................................12 MC1R analysis ...............................................................................13

    III RESULTS ..................................................................................................14

    IV DISCUSSION ............................................................................................19

    V CONCLUSION ..........................................................................................22

    REFERENCES ..................................................................................................................24

  • 2

    ABSTRACT

    Genetic Diversity of White Tigers and Genetic Factors Related to Coat Color. (May 2013)

    Sara Elizabeth Carney Department of Veterinary Medicine and Biomedical Sciences

    Texas A&M University

    Research Advisor: Dr. Jan Janecka Department of Veterinary Medicine and Biomedical Sciences

    White tigers are greatly cherished by the public, making them valuable to zoos and breeders.

    Unfortunately, a number of health issues have occasionally surfaced within some of the white

    tiger population such as neurological and facial defects. There is interest amongst private tiger

    breeders to determine if these maladies are associated with the coat color or breeding practices,

    and to find ways to prevent these health issues. The genes involved in producing the white

    phenotype and the disease phenotype are currently unknown. Furthermore, the relationship

    between the genes associated with coat color and levels of inbreeding also remain unknown.

    Microsatellites are a tool frequently used within by geneticists and ecologists alike. These

    segments of highly repeatable DNA mutate frequently and are variable in length. Thus

    microsatellites can be used to determine heterozygosity within a population by detecting the

    alleles present at the loci of interest. The amount of heterozygosity within a population can be

    indicative of the amount of inbreeding present and overall levels of genetic diversity. A panel of

    twelve microsatellites was used to analyze heterozygosity, thus inferring the levels of genetic

  • 3

    diversity present. Among the tigers sampled, estimated heterozygosity was determined to be

    0.761 in white tigers and 0.772 in orange tigers.

    The genes Melanocortin-1-Receptor (MC1R) and Agouti Signaling Protein (ASIP) have been

    found to affect coat color phenotypes in other species similar to that of the white tiger, making

    them ideal candidates for this project. These genes work antagonistically to each other in

    production of melanin. MC1R is responsible for the production of -melanocyte stimulating

    hormone (-MSH) while ASIP silences this activity. Thus, a loss-of-function associated with

    MC1R or a gain-of-function associated with ASIP could lead to reduced pigment production.

    This study continues the initial investigation by focusing on sequencing MC1R. Differences in

    the nucleotides and amino acids of the sequences were compared though alignment in

    Sequencher. At this time a causal mutation has not been found within exons 1 and 2 of ASIP or

    MC1R.

  • 4

    Acknowledgements

    First, I would like to thank everyone in the Texas A&M Molecular Cytogenetics and Genomics

    Lab for providing a helpful and friendly environment for me to begin exploring the scientific

    process. I would also like to thank Dr. Jan Janecka, Dr. Bhanu Chowdhary and Dr. Samantha

    Steelman for allowing me the opportunity to embark on such an exciting project and for

    providing ample guidance and encouragement. I am also grateful to my friend Emilee Larkin,

    who sparked my interest in the subject and was always available to answer questions and provide

    support. I would like to thank my friends and family for their enthusiasm and support.

    I would also like to express my gratitude to those who provided samples for this project

    including the San Francisco Zoo, In-sync Exotics (Vicky Keahey), Big Cat C.A.R.E, (Heidi

    Riggs Berry), Sierra Endangered Cat Haven (Dale Anderson), Big Cat Rescue, Tiger Creek, the

    Exotic Feline Rescue Center, Ferdinand and Antonin Fercos, T.I.G.E.R.S. (Doc Antle) and

    REXANO (Zuzana Kukol). Your generosity has not only helped us begin to understand the

    genetics of white tigers, but it has also provided me with the opportunity explore the world of

    research and for that, I thank you.

  • 5

    CHAPTER I

    INTRODUCTION

    To many the white tiger, Panthera tigris, has been a source of awe, combining the power and

    grace exhibited by the standard orange tiger with the rare beauty from its unusual coat color.

    Though many find the white tiger to be inspiring, this is not a universally held opinion. Critics

    contend that the white tiger is a detriment to tiger conservation, claiming that the tigers must be

    inbred in order for the white coat to be present. Furthermore, they attribute the ailments faced by

    some white tigers (eg. crossed-eyes and cleft palates) (Roychoudhury and Sankhala 1978) to the

    white coat trait, believing it to be inseparable from inbreeding.

    In light of this controversy, it is important to determine the white tigers role in conservation of

    the species. Though some do not place priority on the preservation of the white tiger, it is evident

    that the species as a whole is facing the threat of extinction. Three of the original eight tiger

    subspecies, Bali (Panthera tigris balica), Caspian, (Panthera tigris virgata), and Javan

    (Panthera tigris sondaica), have recently become extinct (Luo et al. 2004). The tiger population

    has faced recent rapid decline. Within the last 100 years the wild tigers habitat has been reduced

    to only 7% of the land in once roamed (Dinerstein et al. 2007). Poaching as well as habitat loss

    and fragmentation poses the greatest threat to the wild tiger population. Deforestation has

    significantly impacted the wildlife present in these areas particularly the tiger and its prey

    (Kinnaird et al. 2003). The tiger faces additional risks associated with its dwindling population,

    primarily decreased genetic diversity. Frequently, populations facing significant decline may

    resort to inbreeding, potentially leading to inbreeding depression (Hedrick and Kalinowski

    2000). Consequently, deleterious homozygotic traits that were once masked in a healthy

  • 6

    population of heterozygotes may become rampant in a genetically isolated population. Thus, this

    genetically compromised population becomes increasingly vulnerable to disease (Lynch 1977).

    While the wild tiger population faces steady decline, the captive population has successfully

    propagated. Tigers have relatively few complications associated with reproduction, which often

    plagues captive breeding programs. Additionally, captive-bred populations are protected from

    many of the threats that face their wild counterparts, such as habitat degradation, disease and

    poaching. Though the captive tiger has escaped many of these issues, loss of genetic diversity is

    still a present concern within segments of the population (Lacy 1987). The white tiger is

    particularly vulnerable to increased homozygosity due to selection for this phenotype. In many

    ways the captive environment has allowed rare coat color polymorphisms such as that of the

    white tiger to persist.

    Though there are early reports of white tigers in India, the first lineage of captive white tigers

    originated in what was known at the time as Rewa, (which is now Madhya Pradesh), from a

    single male known as Mohan who was captured in 1951 (Thorton et al. 1966). The first breeding

    of Mohan to Belgum, a wild orange female, was unsuccessful in producing a white offspring.

    Mohan was subsequently bred to his daughter, Radha, produced from the previous cross. This

    resulted in four litters, all producing white offspring (Thorton et al. 1966). It can be inferred that

    Rewa, an F1, was heterozygous for the white coat allele. Thus the Rewa-Mohan cross gave

    offspring of the union a 50% chance of being homozygous for and therefore expressing the white

    coat allele. The white coat polymorphism is an autosomal characteristic inherited in a

    Mendelian-recessive fashion (Thorton et al. 1966). Although inbreeding was prevalent in early

  • 7

    breeding of white tigers, it is not essential to produce a white tiger. Because the trait follows a

    Mendelian inheritance pattern, the coat can be propagated given that both parents are carriers of

    the allele.

    Although the white coat polymorphism can be obtained without inbreeding, it can be challenging

    to manage inbreeding levels while also selecting for the white phenotype. Because of this

    breeders often resort to inbreeding to ensure that the trait is maintained. Mismanaged breeding

    practices have reportedly led to an increase in health problems in some white tigers, such as

    strabismus, facial deformities and neurological defects (Roychoudhury and Sankhala 1978).

    However, it remains unclear to what extent these abnormalities are due to inbreeding. Some have

    suggested that some of these health concerns may be linked to the white phenotype itself. For

    example, strabismus, which is caused by retinal nerve fibers connecting at the opposite side of

    the brain rather that the same side, is found in carnivores that are homozygous for an allele

    within the albino series such as Siamese cats (Gulliery and Kaas 1973). Examination of a white

    tigers lateral geniculate nucleus of the brain, (a region involved in processing visual information

    gathered by the retina), revealed a defect of the A1 layer similar to, though less severe than that

    of the Siamese (Gulliery and Kaas 1973). Therefore, determination of the degree of involvement

    of the white phenotype versus inbreeding is essential in order to develop a scientifically based

    breeding strategy for white tigers.

    Though pigmentation and neurological development may seem unrelated, they are both derived

    from the neural crest during the embryonic development of vertebrates (Rawles 1947).

    Melanocyte precursors develop from the neural crest and spread to the hair and skin and

  • 8

    synthesize melanin (Rawles 1947). There are 2 forms of melanin: pheomelanin which produces

    red or yellow pigment and eumelanin responsible for producing black or brown pigment

    (Pawelek et al. 1982). These 2 types of melanin are structurally distinct; melanocytes producing

    eumelanin tend to be more rounded than those producing eumelanin (Pawelek et al. 1982). White

    tigers lack function in melanocytes producing pheomelanin, causing them to lack pigment where

    other tigers would be orange. They carry pigment in their stripes which are gray or chocolate and

    their eyes are blue. Therefore, white tigers are not albinos, though the coat of the white tiger is

    due to an autosomal recessive mutation of the chinchilla allele, cch, and that locus is near the

    albino locus (Robinson 1968).

    Candidate genes for the white coat phenotype

    Melanocortin-1-receptor (MC1R) and Agouti Signaling Protein (ASIP)

    MC1R is responsible for regulating the hormone -melanocyte stimulating hormone, (-MSH),

    which is involved in pigment production (Barsh 1996). MC1R has known effects on pigments in

    many animals. In jaguars, Panthera onca, and jaguarundis, Puma yagouaroundi, a dominant

    mutation of MC1R is responsible for melanism, the overproduction of eumelanin (Eizrik et al.

    2003). However, melanism due to MC1R in domestic cats, Felis catus, follows a recessive

    inheritance pattern (Eizrik et al. 2003). By contrast, repression of MC1R can lead to lack of

    pigment, as in the case of the Kermode bear, Ursus americanus kermodei, which is a white color

    morph of the black bear (Ritland et al. 2001). The lack of eumelanin is caused by a recessive

    mutation at codon 298, replacing tyrosine with cytosine (Ritland et al. 2001). As with the case of

    the white tiger, the Kermode bear is not an albino.

  • 9

    ASIP was also selected as a candidate gene due to antagonistic relationship to MC1R, silencing

    the effects of -MSH (Rieder, et al. 2001). Like MC1R, mutations in ASIP also affect some felid

    species. Leopards, Panthera pardus, and Asian golden cats, Pardofelis temminckii, appear

    melanistic due to single nucleotide polymorphism, (SNP), mutations in ASIP that cause ASIP to

    loose function (Schneider et al. 2012). In this case, a loss-of-function has led to melanistic

    individual. In contrast, a gain of function in ASIP could lead to an individual with reduced

    pigment production. The ASIP gene contains three coding exons, the first two of which were

    sequenced by former undergraduate research scholar, Emilee Larkin in an earlier phase of this

    project (Larkin 2012).

    Use of microsatellites to determine genetic diversity

    Short tandem repeats of DNA known as microsatellites have shown to be invaluable in the

    assessment of genetic diversity within a population or species. The high mutation rate of

    microsatellites makes them ideal for individual identification and tracing evolution within a

    population. Microsatellites are present within the non-coding regions of DNA and the mutations

    that affect them influence the length of the microsatellite (Ellegren 2004). Because many

    microsatellites are found within the non-coding region of DNA, they do not experience the same

    evolutionary pressures found in genes within the coding segments of DNA (Ellegren 2004).

    Additionally, the microsatellites are positioned next to a highly conserved region, known as the

    flanking r...

Recommended

View more >