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Novel Polymorphisms of ZRSR2 and GPM6B Gene Homologs and Their Use in Sex Identification of Bovine and Porcine Species
Evan K. Peterson Utah State University
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This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. NOVEL POLYMORPHISMS OF ZRSR2 AND GPM6B GENE HOMOLOGS AND
THEIR USE IN SEX IDENTIFICATION OF BOVINE AND PORCINE SPECIES
by
Evan K. Peterson
A thesis submitted in partial fulfillment of the requirements for the degree
of
MASTER OF SCIENCE
in
Animal, Dairy, and Veterinary Science (Animal Molecular Genetics)
Approved:
______Lee F. Rickords, Ph.D. Aaron Thomas, Ph.D. Major Professor Committee Member
______S. Clay Isom, Ph.D. Janis L. Boettinger, Ph.D. Committee Member Acting Vice Provost of Graduate Studies
UTAH STATE UNIVERSITY Logan, Utah
2020 ii
Copyright © Evan K. Peterson
All Rights Reserved
iii
ABSTRACT
Novel Polymorphisms of ZRSR2 and GPM6B Gene Homologs and their use in
Sex Identification of Bovine and Porcine Species
by
Evan K. Peterson, Master of Science
Utah State University, 2020
Major Professor: Dr. Lee F. Rickords Department: Animal, Dairy, and Veterinary Science
Bovine and porcine DNA-based sex identification is of importance to livestock producers. Currently, the most efficient sex identification methods use PCR and qPCR to determine genotypic sex based on the presence or absence of Y-chromosome specific genes in a DNA sample. Zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 (ZRSR2) in Bos taurus (European domestic cattle) and glycoprotein M6B
(GPM6B) in Sus scrofa (domestic pigs) are X-chromosome specific genes with Y- chromosome specific homologs. These Y-chromosome homologs contain deletion regions that were targeted in order to create amplicons of differing length for use in sex identification.
The Bos taurus ZRSR2 gene and its Y-specific homolog (ZRSR2Y) were aligned and two adjacent deletions on ZRSR2Y (totaling 33 bp) were identified. PCR primers were designed to flank these deletions and create amplicons 149 bp and 116 bp long from iv
the X- and Y-chromosomes respectively. These primers were then tested in PCR and
qPCR reactions with DNA from Bos taurus, Bos indicus (African domestic cattle), Bison
bison (bison), Capra hircus (domestic goat), Ovis aries (domestic sheep), and Bos grunniens (domestic yak). Results from both PCR and qPCR indicate that the primer regions exist in all six bovine species tested as do Y-specific deletion regions of an equivalent length. Additionally, melting curve analysis during qPCR indicates that the two fragments have unique melting temperatures which can be used for rapid sex identification of a DNA sample.
Sus scrofa GPM6B and its Y-specific pseudogene were aligned and several adjacent deletions were identified (totaling 43 bp). PCR primers were designed to flank the deletion regions and create X- and Y-specific amplicons 244 bp and 201 bp long respectively. Testing confirmed the presence of the Y-specific deletion regions as well as the presence of a 16 bp intron deletion variant (rs1111696537) in a subset of the population tested. The ability to identify sex using this primer pair in porcine samples was also confirmed.
(103 Pages) v
PUBLIC ABSTRACT
Novel Polymorphisms of ZRSR2 and GPM6B Gene Homologs and their use in
Sex Identification of Bovine and Porcine Species
Evan K. Peterson
Accurate and cost-effective PCR based sex identification is important in animal production because it gives producers the ability to determine the sex of embryos prior to transfer, saving time and money. The most efficient PCR sex identification assays work by using a single primer pair to amplify a specific target region located on the Y- chromosome and a second, separate target region on the X-chromosome.
This thesis reports the design of two novel assays. The first assay was designed to target the Zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 (ZRSR2) gene found on the X-chromosome and its Y-chromosome homolog, ZRSR2Y, in
European domestic cattle (Bos taurus). It creates X- and Y-specific amplicons of 149 bp and 116 bp long respectively and is effective for sex identification of Bos taurus, African domestic cattle (Bos indicus), bison (Bison bison), domestic goat (Capra hircus), domestic sheep (Ovis aries), and domestic yak (Bos grunniens).
The second assay was designed to target the glycoprotein M6B (GPM6B) gene found on the X-chromosome in domestic pigs (Sus scrofa) and a homologous pseudogene on the Y-chromosome. It creates X- and Y-specific amplicons of 244 bp and 201 bp long, respectively. The presence of a 16 bp intron deletion variant (rs1111696537) on the X- specific amplicon in a subset of the population was confirmed. Despite the variant, the primer pair is effective for sex identification of domestic pigs. vi
ACKNOWLEDGMENTS
The results presented in this thesis are a result of a collaborative effort. This research was made possible through a grant from the Utah Agricultural Research Station
(project #1330) and the kind efforts of many people. Those I mention here are not the only ones who helped me, but they are the primary ones. Each is responsible, in their own way, for this thesis and all have my deepest and most profound gratitude.
First and foremost, I’d like to thank Dr. Lee Rickords. I met him outside the university as an undergraduate student, when I was unable to find a biology lab that would even let me wash their glassware. He offered me a spot in his lab or his help getting into or any other lab I was interested in. That chance meeting and conversation has changed my life! In the seven years since then, Dr. Rickords has been my P.I., instructor, mentor, major professor, editor, advocate, sounding board, greatest frustration, best chance at self-improvement, and my friend. He has always given me the freedom to choose my own path and has also stepped in (more than once) to stop me from wandering too far afield. He encouraged me to pursue graduate school and has pushed me to keep working when I didn’t think I could finish.
My committee members Drs. Clay Isom and Aaron Thomas, also deserve a special thanks. They have provided crucial guidance at critical moments in the processes of research and data analysis. Additionally, both have spent hours proofreading and helping me make edits during my writing process.
My undying gratitude goes out to Miriam Laker. She started this project and laid
all the foundations for this work. She has been my second brain during all of this and my vii
first contact for every project related question. When I have felt totally inadequate, she
has always reminded me that: this is lab work and that means that no matter how careful
and skilled you are, sometimes things are just not going to work the way they should (or
the way they have the last 40 times).
My family deserves more praise than will fit in these pages. Everything good at
the core of who I am I owe to my parents. Together they have been my best and greatest instructors. My siblings and their spouses and children have supported me physically, spiritually, mentally, and emotionally. Whether I needed someone to tell me I could do
hard things, someone to smile and nod while I babbled about science (allowing me to
remind myself that I love it), someone to distract me from worries and remind me that
there is more to life than grad school, or even just a healthy meal and a free place to do laundry; they have always been there right when I needed them most!
I’d like to thank Marcy Labrum, I owe many of my good lab related habits to her teaching, and her patience. I sincerely appreciate Dr. Abby Benninghoff for teaching me much of what I know about professional presenting. I’m grateful for Sherri Cox, Laurel
Hancey, Charles Delaney, Karma Wood, Cara Allen, and the many other members of the
ADVS staff past and present, I don’t know what most of them did/do, but I know my life would have been a lot harder without them. I’d like to thank my fellow graduate students, those who are finished gave me hope that it was possible and those who are not done yet reminded me that we are all in this together!
Finally, a very special thanks to my beautiful new bride. Since we began dating, she has believed in me far more than I believed in myself and has seen in me potential I viii didn’t think possible. She inspires me and makes me want to be better every day. She wasn’t with me at the beginning, but I wouldn’t have made it to the end without her!
Evan K. Peterson ix
CONTENTS
Page
ABSTRACT ...... iii
PUBLIC ABSTRACT ...... v
ACKNOWLEDGMENTS ...... viii
LIST OF TABLES ...... xiii
LIST OF FIGURES ...... xiv
LIST OF ABBREVIATIONS AND DEFINITIONS ...... xv
CHAPTER
1. LITERATURE REVIEW ...... 1
Introduction ...... 1
Sex Determination ...... 1
Mammalian Sex Identification ...... 7
Current PCR/qPCR Gene Targets ...... 26
Conclusion ...... 32
2. SEX IDENTIFICATION OF BOVID SPECIES USING ZRSR2 & ZRSR2Y
GENE HOMOLOGS ...... 34
Abstract ...... 34
Introduction ...... 35
Materials and Methods ...... 37
Results ...... 41
Discussion ...... 50 x
3. SEX IDENTIFICATION OF PIGS USING GPM6B & ITS Y-CHROMOSOME
HOMOLOGS ...... 52
Abstract ...... 52
Introduction ...... 53
Materials and Methods ...... 54
Results ...... 57
Discussion ...... 58
REFERENCES ...... 61 xi
LIST OF TABLES
Table Page
1 Bovid Tissues Used ...... 38
2 ZRSR2/ZRSR2Y Targeting Primer Pair Results Summary ...... 44
3 Summary of X-Amplicon Sizes Observed ...... 57 xii
LIST OF FIGURES
Figure Page
1 Portion of ZRSR2-ZRSR2Y alignment in Bos taurus ...... 39
2 NCBI BLAST alignment for the amplified region of ZRSR2 ...... 42
3 NCBI BLAST alignment for the amplified region of ZRSR2Y ...... 43
4 Gels showing representative PCR results for bovid species tested...... 45
5 Graph of melting curve of excised X- and Y-specific fragments ...... 46
6 Graphs of melting curves for a subset of Bos taurus samples ...... 47
7 Graphs of melting curves for a subset of Bos indicus samples ...... 48
8 Graphs of melting curves of Bos taurus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens samples ...... 49
9 Portion of Sus scrofa GPM6B and its Y-chromosome pseudogene aligned ...... 55
10 A representative gel showing the amplicons produced by the primer pair targeting GPM6B and its Y-specific homologue ...... 58
11 Sequencing data from long and short X-amplicon generated from GPM6B primer pair compared to the Sus scrofa reference sequence (AC_000187.1 bases 11387210 to 11387329) and to the rs1111696537 deletion ...... 59
xiii
LIST OF ABBREVIATIONS AND DEFINITIONS
AMELX: Amelogenin gene. X-linked gene important in enamel production in mammals.
AMELY: Amelogenin-like gene. Y-linked gene that plays a minor role in enamel
production in mammals.
Amplicon: A piece of DNA produced by PCR or qPCR replication.
APRT: Adenine phosphoribosyl transferase. Enzyme produced by the autosomal gene
APRT.
ASW: Avian sex-specific W-linked. W-linked gene in avian species that is, in part,
responsible for inducing female development of embryos.
Bison bison: American bison, bison, or American buffalo.
BLAST: Basic local alignment search tool. A sequence alignment algorithm and program
for rapid comparison of a query sequence against a sequence database or library.
Bos grunniens: Domestic yak.
Bos indicus: African domestic cattle.
Bos taurus: European domestic cattle.
Capra hircus: Domestic goat.
CDC: Centers for Disease Control and Prevention.
CT value: Cycle threshold. The qPCR cycle at which fluorescence rises above the
threshold differentiating it from the background signal.
Cytogenetic sex identification: Genotypic sex identification method in which a karyotype
is created to determine if the individual has two X-chromosomes (female) or an
X- and a Y-chromosome (male). xiv
DDX3X: DEAD-box helicase 3 X-linked. X-linked member of the DEAD-box gene
family.
DDX3Y: DEAD-box helicase 3 Y-linked. Y-linked member of the DEAD-box gene
family.
Direct sex identification: A category of sex identification methods that rely on direct
examination of some aspect of an individual in order to identify its sex.
DMRT1: Doublesex and mab-3 related transcription factor 1. Z-linked gene involved in
the male sex determination in chickens.
Sex identification by DNA probe of Y-specific targets: Genotypic sex identification
method in which the binding of fluorescent Y-specific probes indicates the
presence of a Y-chromosome.
Eutherian: Mammal of the major group Eutheria, which contains only placental
mammals.
FET1: Female Expressed Transcript 1. W-linked gene in avian species that is, in part,
responsible for inducing female development of embryos.
G6PD: Glucose 6-phosphate dehydrogenase. X-linked enzyme produced by the gene
G6PD.
Gender: The subjective perception of an individual’s own sex and sexual orientation.
Genital ridge: The portion of a developing embryo that is the precursor to the gonads
(also known as the gonadal ridge).
Genotypic sex: The sex of an individual based on the sex chromosomes they possess (XX
is female, XY is male, for example). xv
GPM6B: Glycoprotein M6B. An X-linked gene that codes for a membrane glycoprotein
of the proteolipid protein family.
Haplodiploidy: A mechanism of sex determination based on whether the individual is
haploid or diploid, with males being haploid and females being diploid (also
known as arrhenotoky).
HMG Box: High Mobility Group Box. A protein domain with DNA binding activity.
HPRT: Hypoxanthine phosphoribosyl transferase. X-linked enzyme produced by the gene
HPRT.
H-Y antigen detection sex identification: Genotypic sex identification method in which
binding of H-Y antibody indicates the presence of a Y-chromosome in the cell.
H-Y antigen: A male specific antigen.
IETS: The International Embryo Transfer Society
Indirect sex identification: A category of sex identification methods in which a secondary
indicator (often the binding of a fluorescently labeled antibody or probe) is used
to indicate the sex of an individual.
Intersex: A general term used to describe an individual whose genotypic sex and
phenotypic sex do not fully match (hermaphrodite, true hermaphrodite,
pseudohermaphrodite, male pseudohermaphrodite, sex regressed, congenital
eunuch, etc. are other terms for intersex individuals)
IVF: In vitro fertilization.
IVP: In vitro-produced. Refers to embryos created through in vitro fertilization. xvi
MCA: Melting curve analysis. A qPCR analysis method that involves the slow raising of
the reaction temperature while fluorescence is being measured. Used to determine
the melting temperature and generate melting curves.
Oligo: Oligonucleotide. A very short strand of DNA.
Osteological sex identification: Phenotypic sex identification method that compares the
measurements of bones that are highly variable in size between sexes against a
population average in order to determine the sex of a given individual in that
population.
Ovis aries: Domestic sheep.
PAR: Pseudoautosomal region. Homologous regions on the X- and Y-chromosomes
capable of crossing over between the sex chromosomes. They also allow for the
sex chromosomes to properly segregate during Meiosis in males.
PCR: Polymerase chain reaction.
PCR fragment: See amplicon.
PCR primer: Oligonucleotide that serves (as part of a pair) to determine which section(s)
of the template DNA are replicated during PCR.
PCR/qPCR sex identification: Genotypic sex identification which uses PCR to create an
amplicon from a portion of the Y-chromosome (and often a different size
amplicon from the X-chromosome). Presence of the Y-specific amplicon indicates
the presence of a Y-chromosome (i.e. a genotypic male). Absence of the Y-
amplicon indicates a lack of Y-chromosome (i.e. genotypic female). xvii
Phenotypic sex: The sex of an individual based on the internal and external genitalia they
possess, as well as expression of secondary sex characteristics.
PAGE: Polyacrylamide gel electrophoresis. Type of electrophoresis that uses a
polyacrylamide gel, generally offers a higher resolution than agarose gels.
Primary sex characteristics: The internal and external genitalia of an individual.
qPCR: Quantitative polymerase chain reaction (also known as real-time polymerase chain
reaction). PCR with the addition of any of several types of fluorescent chemistries
and measurements of fluorescence during the reaction, providing real-time and
potentially quantitative data.
RFLP: Restriction fragment length polymorphism. Method of determining the presence
of polymorphisms based on the length of fragments produced by a restriction
enzyme digestion. rs1111696537: A 16 base pair long intron deletion variant on the X-chromosome in Sus
scrofa.
SE47/SE48: A primer pair developed for PCR sex identification in cattle.
Secondary Sex Characteristics: Characteristics of sex that appear in individuals as the
near sexual maturity.
Sex Determination: The process by which sex is selected and develops in individuals of a
given species.
Sex Identification: Any method by which humans ascertain the sex of an individual.
SF1: Steroidogenic factor 1. An orphan nuclear receptor that acts in conjunction with SRY
to upregulate SOX9 expression in the process of sex determination. xviii
Sox9: SRY-related HMG box 9. A gene activated and upregulated by SRY gene activity.
Important in maintaining the process of male sex development in sex
determination.
SRY: Sex-determining region Y. Y-linked gene responsible for initiating the cascade that
results in male sex development during embryonic development of therian
mammals.
Sus scrofa: Domestic pigs.
TDF: Testes determining factor. Prior to the discovery of the SRY gene. This was the
name given to the theoretical gene suspected to be responsible for sex
determination in therian mammals.
Therian: Mammal of the major group Theria. Includes all marsupials and placental
mammals.
Ultrasound sex identification: Phenotypic sex identification method that uses ultrasound
technology to view the genital structures of a developing fetus in utero.
Visual sex identification: Phenotypic sex identification method in which sex of an
individual is ascertained by visual examination of primary sex characteristics.
X-linked enzymatic sex identification: Genotypic sex identification method in which the
relative activity of an X-linked enzyme prior to X-inactivation is used to
determine if an embryo’s cells contain one or two X-chromosomes.
ZFX: Zinc finger protein X-linked. Gene on the X-chromosome that codes for a DNA
binding protein. A target for some PCR sex identification assays. xix
ZFY: Zinc finger protein Y-linked. Gene on the Y-chromosome that appears to play a role
in normal male sex development and spermatogenesis. A target for some PCR sex
identification assays.
ZRSR2: Zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2. An X-
linked gene that encodes for U2 small nuclear ribonucleoprotein auxiliary 35 kDa
subunit-related protein 2. A target of the primer pair described in chapter two for
the purpose of sex identification in bovid species.
ZRSR2Y: Zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 Y-
linked. A Y-linked, bovid specific, gene whose functions are not clearly
understood. A target of the primer pair described in chapter two for the purpose of
sex identification in Bovid species. CHAPTER 1:
LITERATURE REVIEW
Introduction
Sex is an important defining characteristic of sexually reproducing animals. This review of literature will discuss the most common of the various methods by which sex is determined. As the focus of the research is on eutherian species (placental mammals), special attention will be given to the way in which sex is determined in them (X/Y sex determination). This will include a history of the discovery of the so-called testes determining factor (TDF), sex-determining region Y (SRY), on the Y-chromosome and the way in which this gene induces male embryonic development. Discussion will also include the reasons why sex identification of eutherians is important and the various direct and indirect methods by which sex identification has and is accomplished. PCR and qPCR methods will be discussed in detail as they are the methods used in the research presented in the following chapters. The review will conclude with a discussion of the details of several of the specific genes that are currently targets for PCR/qPCR sex identification.
Sex Determination
Sex and Sex Determination
Within the animal kingdom, the vast majority of species undergo sexual reproduction during which haploid cells of individuals of different sexes merge to create a new diploid organism1. The sex of an individual is generally determined during 2
embryonic development, though the exact mechanism by which sex is determined varies
significantly. Methods of sex determination include chromosomal sex determination,
temperature-dependent sex determination2, location or contact-dependent sex determination3, and sequential hermaphroditism4. The most studied of these methods is
chromosomal sex determination. Within chromosomal sex determination, different
species accomplish sex determination through many mechanisms including
haplodiploidy, ZW sex chromosomes, XX/XO determination, and XY sex chromosomes
(including both X/A ratio, Y chromosome determination methods).
Haplodiploidy (also known as arrhenotoky) is the mechanism of sex
determination in all insects of the orders Hymenopterida and Thysanoptera.
Haplodiploidy determines sex based on whether the individual is haploid or diploid, with
males being born from unfertilized (haploid) eggs, and females being born from fertilized
(diploid) eggs5,6. As a result, males cannot have direct male progenitors or descendants.
Additionally, female offspring with the same male and female parents will have a
relatedness coefficient of 0.75 with one another and 0.50 with their mother (on average
they share 75% and 50% of their genomes respectively), in contrast with the standard
0.50 between all siblings and between parents and offspring as seen in species that
reproduce using other means. This altered ratio of relatedness is used to explain the
eusocial behavior of species that exhibit haplodiploidy in sex determination, as the
offspring will have more of their own genes expressed if they help their parent produce
more female offspring than if they were to produce their own offspring, in an
evolutionary strategy known as kin selection7. 3
ZW sex determination is a category of methods by which sex is determined in
birds, as well as some fish, insects, and reptiles. In this system, there are a pair of dissimilar sex chromosomes called the Z and W chromosomes. The Z chromosome is larger than the W chromosome and contains more genes allowing for a visible difference between the sex chromosomes. There is a large amount of variability in the mechanisms by which sex is determined in this system, so much so that the only truly defining characteristic of the system is that heterogametic (or monogametic in some species as described below) individuals (ZW or Z0 respectively) are female, while homogametic individuals (ZZ) are male8,9.
In some species, the presence of the W-chromosome specific genes are needed for
the embryo to develop into a female. For example, in birds the genes FET1 and ASW
(both found on the W-chromosome) are responsible for directing development toward the
female sex10–12. In contrast, in chickens, the double dosage of a Z-chromosome gene
(DMRT1) as a result of being homogametic also induces development of ZZ embryos into males13. Within invertebrates, some species have an altered mechanism in which
they do not directly rely on the W chromosome for determination, as is the case for some
members of the order Lepidoptera that have an altered form of ZW determination in
which ZO and ZZW also generate females9. The inconsistency in method is likely due to
the ZW system evolving more than once, as has been shown with the divergent origins of reptile and bird Z and W chromosomes14.
The last major chromosomal sex determination mechanism is the XX/XY system.
The XY system is similar to the ZW system in the amount of variability and the types of 4
variability in the mechanisms. They are also similar in one sex usually being heterochromatic and the other being homochromatic. Indeed, early research reports used
the terms interchangeably,9 and there has been some debate as to whether the two should
be given separate designations at all15–17. The main difference is that in the XX/XY
system, the male is heterochromatic (XY) and the female is homochromatic (XX). As
with ZW, there are several specific ways in which sex is determined. For example,
Drosophila melanogaster sex is dependent on the ratio of X to autosomal chromosomes
(X:A ratio), such that 1:1 X:A ratio individuals develop female and 1:2 X:A ratio
individuals develop male18,19. Within therians, nearly all species determine sex based on
the presence of a Y-chromosome and the SRY gene on the Y-chromosome in particular,
as will be described below. For simplicity, hereafter all further references to mammals
should be taken to be in reference specifically to therian mammals unless specifically
described otherwise.
Discovery of Mammalian Sex Determination
In most mammals, generation of biological sex is caused by the XY sex-
determination system. In this system, females are most often homogametic, having two
X-chromosomes, while males are heterogametic, having one X- and one Y-chromosome.
All initial research of mammalian sex determining mechanisms was primarily
accomplished studying humans and mice as described here. For the sake of simplicity,
human protein and gene naming conventions will be used hereafter regardless of species.
When the X- and Y-chromosomes were first discovered in humans in 192320 it
was believed that they influenced sex determination via the same mechanism as 5
Drosophila melanogaster, namely the X:autosome ratio. This was believed until 1959
when aneupliod individuals in the form of both XXY individuals having testes 21 and XO
individuals with ovaries22 were discovered. These discoveries meant that human sex was
determined by some element within the Y-chromosome and not in the same manner as
Drosophila melanogaster, as the X:autosome ratio would have created phenotypes opposite of what was observed. In 1947, castration of fetal rabbits prior to sexual differentiation was found to cause both XX and XY rabbits to develop female ducts and external genitalia, leading to the realization that 1) the default state of development without gonads is female and 2) development of either testes or ovaries determines sex23.
The three decades following the discovery of the Y-chromosome’s role in determining testes development were spent trying to identify which gene on the Y- chromosome initiated the change in gonadal fate. Studies to find this testes determining factor gene (TDF) were carried out largely by examining the X- and Y-chromosomes of individuals expressing aberrant sex genotype by phenotype interactions (i.e. intersex individuals, XX males, XY females, etc.). After numerous false starts and misidentifications of TDF (including H-Y24, ZFY125, AND ZFY225), the location of the
gene was found to lie within a 60 kb region of the Y-chromosome. This was determined
through analysis of one XX intersex and three XX male humans that carried only a 60 kb
section of the Y-chromosome in common26. Shortly thereafter, another laboratory
identified a gene in this area which they termed SRY (sex-determining region Y)27. The
role of the SRY gene in sex determination of mammals was then confirmed by further
studies of the SRY gene in both humans28,29, and in mice25,30,31. 6
How SRY Induces Sexual Differentiation
The SRY protein is a transcription factor that contains a conserved domain similar to DNA-binding domains known as high mobility group (HMG) boxes27. Many related
transcription factors have been discovered and are termed the SRY-related HMG box
protein (SOX) family32,33. This family of proteins operates by binding the minor groove of DNA at the consensus sequence (A/T)ACAA(T/A)34 and bending the target DNA
sharply at a 60-85° angle35,36. Outside of the HMG domain, no other highly conserved
functional regions have been identified in the SRY protein.
In mice, the SRY gene drives sexual differentiation through expression in early
gonad development. It follows a wave-like pattern beginning in the center of the genital
ridges, in pre-Sertoli cells, and rapidly extending out to the poles37. Expression is rapidly
extinguished following a similar pattern, reducing in expression in the center first, then
moving toward the poles. This occurs with the extreme posterior pole persisting in SRY
expression somewhat longer than the anterior pole37. This same pattern is seen in the
expression of the SRY protein in the genital ridges as well, though delayed by 6-8
hours38. The timing of SRY protein expression is critical for proper sexual
development39–41. Expression of SRY need only be brief (~2 days in mice37,38), though prolonged expression is seen in some mammals including humans42,43.
Across animal species, the SOX9 gene is a major target of SRY proteins, with its expression initiation pattern in the genital ridges closely following the wave pattern of
SRY described in the previous paragraph38,40,44–46. However, unlike SRY, SOX9 expression is maintained once initiated44, and there are indications that this continued expression is 7
needed to prevent Sertoli cells from reverting to their nondifferentiated state, which
would lead to sex regression47. SRY, in conjunction with an orphan nuclear receptor
called steroidogenic factor 1 (SF1), upregulates SOX9 gene expression in gonadal tissue48. SOX9 operates with SF1 to create a feedback loop increasing and maintaining its
own expression level independent of SRY48. Beyond maintaining its own expression
level, SOX9 appears to target several other genes in a cascade effect that results in the development of Sertoli cells from pre-Sertoli cells, as well as development of testes, and
ultimately the male sex (for a more complete review on what is known about this complex cascade, see references 47,49–53). While the entirety of the mechanism of mammalian sex determination is not known, and there may be additional direct ways in which SRY influences sex, its role as a switch to begin the cascade of male sex determination via SOX9 activation is clear.
Mammalian Sex Identification
Purpose
Sex identification is important for many applications in humans and other mammals. Major uses include in vitro fertilization (IVF)/embryo transfer, prenatal sex
identification, population ecological studies, lab sample controls, archeology, and various
forensic uses.
The CDC reports that 197,706 assisted reproductive technology procedures (99% of which are in vitro fertilization) were performed in 2016 with the intent of transferring at least one embryo54. Of those procedures, 154,439 (78.1%) led to the actual transfer of 8
an embryo and 65,964 (42.7%) resulted in a live birth54. Hamilton and McManus report
that the average cost to the patient is $10,000-15,000 per attempted IVF treatment55.
Given the low success rates and high costs, sex identification of embryos prior to transfer is a potentially large boon in cases where a child of a particular sex is desired.
The International Embryo Transfer Society (IETS) Data Retrieval Committee reported that, worldwide, during 2018, among cattle, horses, sheep, and goats, a total of
434,137 in vivo-derived (IVD) embryos were transferred (386,133, 22,495, 17,510, and
7,999 respectively) and 744,504 in vitro-produced (IVP) embryos were transferred
(742,908, 1,080, 358, and 158 respectively)56. Given the high cost of these procedures
(highly variable, but generally ranging from $1,000-5,000 per animal born), paying to transplant embryos of an undesired sex is prohibitive, and thus the low cost of embryonic sex identification prior to transfer is preferable.
Sex identification in prenatal fetuses is also beneficial in both humans and other mammals (particularly livestock). In humans this is primarily used to sate the curiosity of the parents57, however, it can also be used to reduce invasive prenatal testing for X-linked
genetic disorders58–60. The impact in animals is more significant economically, as it can
be used by producers to make informed decisions earlier than if sex could not be
determined until parturition. Examples of decisions include potential presale of animals,
sale of pregnant animals dependent on the sex of the fetus, determining if contracts are
completed or if more embryos should be produced, and selective termination of
pregnancies61,62.
In population ecology, sex identification is useful in monitoring populations, 9
estimating sex ratios, determining range and distribution, and understanding the roles of
each sex within an organism’s social structure63–65. Of particular use are sex identification
methods that can be used non-invasively so as to not disrupt the normal interactions of
the population.
Chromosomal based sex identification methods can be used as a primary control
against mislabeled or potentially contaminated mammalian samples. A simple way of
detecting if a biological sample has been contaminated, mislabeled or damaged severely
is to use one of the several chromosomal based sex identification methods that exist66,67.
While normal results do not confirm the identity or purity of a sample, aberrant sex
results are a strong indication that the sample is contaminated or mislabeled.
Archaeology has similar needs to ecology with regard to sex identification. The
main difference is that while ecologists use special methods for sex identification to
avoid disturbing the natural habits of the population, archaeologists need special methods
because they are working with a skeletal record68,69.
Finally, sex identification is used in forensic science in several ways. Perhaps the
most obvious of these is determining the sex of a potential suspect in a criminal
investigation based on the analysis of tissue or DNA70. In situations where hunting one
sex of animal (usually the male) is legal, but it is illegal to take the other, poachers may
strip the meat from the carcass leaving behind all easily identifiable markers of sex71. In
such situations, other sex identification methods may be used to identify game animals
taken illegally71. A similar method is used in countries, such as India, where slaughter of livestock may be illegal depending on the sex or where the price of meat is dependent on 10
the sex of the animal it came from72,73.
Methods of Mammalian Sex Identification
A multitude of different methods of sex identification exist for mammals. These methods can be divided into two broad categories, namely: direct and indirect identification methods. Direct methods are those that look directly at some aspect of the animal to determine sex, and include methods such as visual detection of physical sex
characteristics, osteological sex identification, ultrasound viewing of fetal genital structures, and karyotyping. Indirect methods rely on a secondary indicator to determine sex. These methods include H-Y antigen detection, X-linked enzymatic identification,
DNA probes of Y-specific targets, and PCR/qPCR sex identification.
It is important to recognize that there are three distinct ways sex can be perceived: genotypic sex, phenotypic sex, and gender74. Genotypic sex refers to the sex
chromosomes possessed by an individual (XX is female, XY is male for example).
Phenotypic sex refers to the internal and external genitalia of an individual, as well as
expression of secondary sex characteristics. Gender is the subjective perception of an
individual’s own sex and sexual orientation. As gender is only applicable in humans, and
is far beyond the scope of this work, no more will be said on the subject of gender.
Genotypic and phenotypic sex are generally the same, however, in a small portion of individuals (which varies between species) genotypic and phenotypic sex do not match. In such cases the individual is considered to be intersex (other terms include hermaphrodite, true hermaphrodite, pseudohermaphrodite, male pseudohermaphrodite, sex regressed, congenital eunuch, etc.). Intersex individuals are generally rare; however, 11
they are worth mentioning here as all sex identification methods currently identify either
genotypic or phenotypic sex, but not both. The description of each method, given below
will also note whether it identifies genotypic or phenotypic sex.
Direct Methods
Visual detection of sex characteristics
Visual detection of physical sex characteristics is the easiest, simplest, and
cheapest method of sex identification and is the most common method of identifying phenotypic sex, in many situations. This involves identifying the presence or absence of primary and secondary sex characteristics in the individual. Secondary sex characteristics
appear as the animal reaches sexual maturity and are highly variable from species to
species, though they often include subtle differences in size, bone structure, and
musculature. Primary sex characteristics are generally more consistent between species
and the external genitalia can be used for visual identification in many instances. For
males, the presence of a penis structure is the primary external indicator of sex.
Additionally, in many creatures, testicles outside of the abdominal cavity also indicate the male sex. In females, presence of the vaginal opening is generally the external indicator of phenotypic sex.
For many applications, this is not a viable method of sex identification as it requires the individual to be present and to have genitalia developed enough to be seen.
Meaning, it cannot be used for forensic purposes or for sex identification of embryos or of fetuses in utero and it tends to be an invasive process for ecological purposes. Further, 12
special training or species familiarity may be required in species that are particularly
difficult, or do not follow normal patterns. One such species is the spotted hyena, in which females are generally larger and more muscular than males (opposite from the pattern most commonly observed in mammals), and they have a pseudo-penis75 which
can be confused with the male penis by those unfamiliar with the species.
Osteological
Osteological sex identification uses skeletal measurements of animal remains in
order to identify the phenotypic sex of the individual. As with visual inspection of the animal, osteological sex identification is easier after the individual has reached maturity and secondary sex characteristics are present. This is because as mentioned above,
skeletal changes occur as animals reach sexual maturity. Even before the animal reaches
maturity however, it may be possible to determine sex, though accuracy is generally
much lower.
These changes are often different between species requiring a specific knowledge
of each skeletal sexual dimorphisms in any given species before sex can be determined.
In humans for example, females have smaller and more delicate (lighter) bones and these
differences between the sexes are most easily detected in the nuchal crest, mastoid
process, supraorbital margin, supraorbital ridge/glabella, and mental eminence68. In
contrast, the breadth measurement of metacarpal bones is used to determine sex in
cattle76.
While potentially reliable, interpopulational variance requires means to be
determined for each population before sex identification can be performed with any real 13 degree of accuracy. Additionally, because the osteological features of each sex vary within a given population and the variances overlap, the accuracy in the portion of the population that contains this overlap is decreased68. The “nontrivial error rate” of osteological sex identification has led researchers to recommend use of DNA based sex identification techniques whenever possible68 as those methods have a much higher accuracy. The high error rate and the need for the presence of intact bone limits the usefulness of the method to archaeological applications where DNA sex identification is not possible.
Ultrasound
Ultrasound (sonography or ultrasonography) sex identification uses the emission and detection of high frequency (1-18 MHz) sound waves to determine the phenotypic sex of an animal in utero. The sound waves are produced by a piezoelectric or capacitive transducer, both of which convert electrical energy into high frequency sound emissions.
The emitted sound is partially reflected back to a sensor on the probe with different tissues and structures reflecting and absorbing the sound in different ways. The sensor works in the reverse of the transducer, converting sound into electrical current. The signals coming in from the transducer are then analyzed based on how long it took the echo to return and how strong it was. The scanner uses this information to determine which pixels of the image to light up and to what intensity and this information is used to display the image.
The first published data on the use of ultrasound for fetal imaging was in 195877 and the first published data on fetal sex identification using ultrasound was in 197757. 14
Methods of ultrasound for fetal sex identification vary by animal, for example, in humans
the ultrasound is performed through the abdominal wall, while in cattle transrectal
ultrasound is used. This imaging method uses the same basic premise as visual
inspection, in that both involve observing the genital structures of the subject. The major
difference is that the error rate is higher in ultrasound as it can be difficult to get a good
image of the genitals, though training and experience significantly improve accuracy.
This method of sex identification has the advantage of being rapid and relatively
inexpensive; however, it is of course only useful for in utero sex identification.
Additionally, successful identification is only likely after the point in the pregnancy when
primary sex characteristics have developed.
Cytogenetic
Cytogenetic sex identification is a method for determining the genotypic sex of an
individual. This method works in the same way as generating a karyotype namely,
inducing cell replication, arresting cell replication in metaphase, hypotonic shock,
fixation, staining, and microscopic visualization, primarily looking for the presence of the
Y-chromosome. This methodology has been used for sexing of embryos by taking a
biopsy of the embryos prior to transfer78–81 and by collecting and culturing amniotic fluid
cells82. It can also be used on somatic cells83 so long as they are living and capable of
replication.
The requirement, for cells to be living and replicating, limits the uses of this methodology such that it is not used except for sex identification of embryos or of fetuses in utero and in such cases it is often not the most efficient method. When sexing embryos 15
in this way, it can be difficult to get sufficient numbers of cells in metaphase and of a
high enough quality in a reasonable amount of time. The best way to work around the
lack of cells is to take a larger sample, but this can cause different issues. For example, by
waiting until bovine embryos reach day 14 or day 15, a larger sample may be taken78
without, in theory, compromising the ability of the embryo to develop normally84.
Unfortunately, this late in development, the embryo has hatched and if frozen and thawed
for later implantation the success rate of transfer drops significantly, meaning that the
embryo may need to be transferred before the sexing is actually completed. In contrast,
day-6 or day-7 bovine embryos can still be frozen and thawed but have only a limited
number of cells that can be biopsied without damaging the embryo’s developmental
potential79–81. The only advantage of this method currently is that it does not require
information regarding the genomic sequence of the species, as methods such as DNA probes and PCR methods do.
Indirect Methods
Antigen detection
Detection of antigens such as the Histocompatibility-Y (H-Y) antigen have been
used to identify the genotypic sex of embryos prior to transfer by using the presence of
Y-specific proteins to identify Y-chromosome containing samples. With H-Y antigen this is done by either arresting the development of male embryos through culturing in the presence of H-Y antiserum and complement which causes H-Y positive cells to lyse85 or
by indirect immunofluorescence assays using polyclonal or monoclonal antibodies86,87. 16
By culturing cells with the antiserum and complement, cells containing the
antigen will experience antibody binding followed by the complement cascade, which
will ultimately lead to cell death in any cells containing the Y-specific antigen. In
contrast, the immunofluorescence assay uses binding of a fluorescently tagged secondary
antibody to bind to the primary antibody. Thus when viewed under a fluorescent
microscope, cells expressing the antigen will fluoresce making them potentially easy to
identify. Unfortunately, H-Y antigen detection is not highly accurate, which is thought to be a result of the highly subjective nature of the immunofluorescence assays86. This
subjectivity seems to be due to altered fluorescence depending on the quality of the
embryo and to the inability to detect the antigen at different stages in embryonic
development86.
X-linked enzyme methods
In mammals, females have a higher copy number of all genes on the X-
chromosome because they carry two copies of the chromosome while males only carry
one. This difference in copy number requires that females inactivate one X-chromosome as a form of dosage compensation in order to allow for normal development. While the exact timing of X-inactivation varies between species, there is a short period in the early development of all species where both X-chromosomes are active. X-linked enzymatic sex identification takes advantage of this brief period of doubled X-linked gene products in females to differentiate between the genotypic sexes of embryos by measuring the relative activity of an X-linked enzyme.
Two different X-linked enzymes have been used for this, glucose 6-phosphate 17
dehydrogenase (G6PD)88 and hypoxanthine phosphoribosyl transferase (HPRT)89. In T.J.
Williams’s 1986 report, the sex of 64% of mouse embryos successfully carried to term
was determined accurately88. This was done using a vital colorimetric assay. Briefly,
morula to blastocyst stage embryos were incubated in a medium containing glucose 6-
phosphate, NaDP, and brilliant cresyl blue stain. Embryos were then graded semi-
quantitatively based on the amount of staining. Results showed a bimodal distribution of
staining and embryos with a high staining score were assumed female and low staining
were assumed male, with the result that 72% of the predicted females were sexed
correctly and 57% of the predicted males were sexed correctly, for an overall accuracy of
64%88.
A study conducted by M. Monk and A. H. Handyside used the expression of
HPRT in single mouse blastocysts separated from zona-free eight-cell embryos to determine sex. In addition to the X-linked enzyme, HPRT, they also examined the activity of the autosomal-linked enzyme adenine phosphoribosyl transferase (APRT) for use as a control of base cellular activity. They found a bimodal distribution of
HPRT:APRT ratios 89, similar to the findings of Williams88, and determined that those
with a low ratio were male while those with a high ratio were female. Subsequent
examination of the fetuses indicated 91% (11/12) were accurately sexed as female, and
100% (3/3) were accurately sexed as male89.
As stated above, this method is limited to early embryos as X-inactivation corrects for the double dosage. Significant testing would be required to adapt the method for each new species, because X-inactivation does not occur at a consistent 18
developmental stage across species. An increased mortality rate in extreme stained
embryos88 indicates that there is still some toxicity to the process, but in some species separation of single blastocysts is not as feasible as it is in mice. Taken together, this is a suboptimal form of embryonic sex identification.
DNA probe
DNA probe methods for genotypic sex identification use Southern blotting or dot
blotting of DNA or in situ hybridization of cells with a probe consisting of a relatively
short DNA sequence specific to a conserved region of the Y-chromosome labeled with
either a radioactive or fluorescent tag90–93. These methods detect the presence of the Y-
chromosome in a DNA sample or cell of any kind making this one of the most broadly
useful and accurate (95-99%)92,93 methods of sex identification.
Use of DNA probes for genotypic sex identification came from the use of probes
in attempts to identify TDF in humans94. In searching for TDF, Y-specific sequences
were isolated, those sequences were then replicated, tagged, and used to test individuals
whose genotypic sex did not match their phenotypic sex. This was done to determine
which parts of the Y-chromosome were present or absent in XX males and XY females
respectively95–97.
In the early days of DNA probe sex identification, this method (especially
creating new probes) was a difficult, time and labor intensive process (see references 93,94
for more information). Modern sequence databases and the ability to perform BLAST
searches and order premade labeled probes has significantly reduced the difficulty of
making DNA probes for most species. Despite these advances, use of DNA probes is still 19
a time and labor-intensive method for sex identification, especially compared to PCR and
qPCR methods.
PCR and qPCR
Overview of PCR and qPCR
Polymerase chain reaction (PCR) and quantitative PCR (qPCR) methods of sex
identification both use PCR amplification to identify genotypic sex by either determining
whether a Y-chromosome or two X-chromosomes are present in a DNA sample. Since
PCR and qPCR can be used in a multitude of ways for sex identification and are of
primary importance to the research presented in this thesis; a more detailed explanation of the methods, chemistries, and applications of these techniques will be discussed. An
overview of the methods will be discussed prior to their use in sex determination
procedures.
PCR and qPCR differ mainly in the methods of detection and data analysis. Both
PCR and qPCR operate on the principle of massively replicating a DNA sequence (or
sequences) from the sample DNA template. A pair of short oligonucleotides, called
primers, determines the replicated sequence, called an amplicon. Amplicons created
using PCR are frequently detected and analyzed using gel electrophoresis and a DNA
binding stain. This separates amplicons by size allowing users to determine the
approximate size of the amplicon and determine if amplicons of different sizes are
present.
In qPCR more analysis options are available in addition to gel electrophoresis, 20
including real-time data analysis as the reaction is occurring (qPCR is also known as real-
time PCR). The collection of real-time data is dependent on the use of fluorophores in the reaction that only fluoresce under certain conditions. Throughout the reaction (usually at the end of each cycle), the fluorescence of the fluorophore(s) is measured and recorded.
There are two main classes into which the fluorescent chemistries can be divided, specific and non-specific. Specific chemistries use oligonucleotides with a fluorophore and a quencher bound in proximity to each other (called probes). Under the excitatory wavelength of light, the fluorophore’s emission is absorbed, or quenched, by the adjacent quencher molecule. During the reaction, the probe will bind to a specific sequence in the amplicon and will be digested by the 5’ to 3’ exonuclease of the polymerase allowing the fluorophore and quencher to separate, thus as more copies of the amplicon are made fewer fluorophores are quenched, and greater fluorescent emission is detected.
In contrast are non-specific chemistries. In these methods, the fluorophore only fluoresces when bound to double stranded DNA (dsDNA). As more amplicons are generated, there is more dsDNA and thus more fluorescence with each cycle of the reaction.
Both methods of the chemistry provide fluorescence data, which can be used to quantify the absolute or relative number of copies of the amplified region in the original sample. Absolute quantification is done by comparing the fluorescence of the sample of unknown concentration to a calibration curve, created using DNA standards generated by the same primers, in order to calculate the exact number of sequence copies initially. For this calculation to be accurate, the amplification efficiency (also known as primer or 21
qPCR efficiency) of the sample and the standards needs to be the same.
Amplification efficiency is calculated using serial dilutions of the sample as
follows. qPCR is done on the serial dilutions and the cycle numbers at which the
fluorescence rises above the detection threshold (the Ct values) are graphed against the
log of the sample dilution. The slope of that line is then used in the following equation to
calculate the percent efficiency of the amplification.
An efficiency of 100% means that for every round of replication the number of
copies of PCR product doubles. This is not often the case, but an efficiency of 90-110%
is generally considered adequate for replication, but for accurate quantification data the
efficiencies of the samples and standards must be similar (for a review of the error
introduced to quantitative results by differing efficiencies see reference 98).
Relative quantification is measured in a different way (specifically using delta-
delta-Ct method) and while efficiency is important here as well, small differences in
efficiency between target and reference efficiencies do not introduce as much error as in
absolute quantification98. Quantitative analysis is used for gene expression analysis as a way of determining copy number of cDNA derived from reverse transcription. This can also be used to determine degree of infection in virally infected tissue.
In addition to the quantitative capabilities of qPCR, it can also be used in a
deterministic fashion. For example, using primers that only generate an amplicon when a
certain mutation is present, or when DNA from a particular bacterial or viral strain is
found in the template DNA, the generation of any amplicon (indicated by an increase in
fluorescence) is sufficient to determine its presence. 22
A final method of analysis (and of particular importance in chapter two of this thesis) is melting curve analysis (also called fusion or melting temperature analysis) which allows for the differentiation of amplicons based on their melting temperature.
Melting curve analysis requires a special measurement step after the final extension of the reaction. Traditionally, after the final extension the reaction is heated and cooled to the denaturation and annealing temperatures respectively, as though beginning another cycle of replication. However, once the DNA is annealed, the reaction is slowly heated back up to the melting temperature as frequent fluorescence measurements are taken at regular intervals. This is usually done using an intercalating dye, which is only able to fluoresce when bound to dsDNA. As the DNA melts, separating into single strand DNA
(ssDNA), the amount of fluorescence will decrease. As the melting temperature of a particular amplicon is reached, the rapid conversion of dsDNA to ssDNA will create a correlated rapid decrease in the amount of fluorescence detected. The change in fluorescence is then graphed against the temperature and is known as the melting curve.
A number of factors including GC content, fragment length, and sequence structure influences DNA melting temperatures, thus the presence of multiple peaks in the melting curve analysis indicates the presence of multiple unique amplicons.
Use in sex identification
Sex identification using PCR and qPCR technology can be accomplished using a variety of subtle modifications. Here, detection of the Y-chromosome without a positive control, multiplex and inherent positive controls, differences in techniques depending on the detection mechanism in qPCR, and a qPCR method sex identification based on the 23
relative number of X-chromosomes will be briefly discussed.
Non-quantitative PCR sex identification is accomplished by detecting the
presence of a Y-chromosome, through amplification of a specific portion of the Y-
chromosome, then determining if the amplification occurred and if the Y-chromosome
specific amplicon is present. The major variation in these methods is 1) the chromosomal
region amplified, 2) the absence or presence (and form) of a positive reaction control, and
3) the method by which the amplicon is determined to be present and specific to the Y-
chromosome.
A large variety of Y-chromosomal regions have been targeted for sex
determination and an exhaustive list would be impractical to give, however, these targets
can generally be placed into one of two groups 1) microsatellites/highly repetitive
regions99,100 and 2) gene specific targets. Microsatellites and highly repetitive regions were mainly used in the early years of PCR sex identification, but has fallen out of common use. Gene specific targets are still routinely used.
If the primer only amplifies a target on the Y-chromosome (ex. those targeting the
SRY gene), there can be an increased risk of a false negative (incorrectly determining that a male is a female) because there will be no difference between a failed reaction and an
X-chromosome only containing female sample101–103. Multiplexing (usually duplexing)
with other primer pairs that target the X-chromosome or an autosome can mitigate this
risk somewhat. As a multiplexed reaction where the non-Y-specific amplicon is present, but the Y-specific amplicon is not, indicates that the reaction conditions do allow PCR to proceed104–107. Having a multiplexed reaction can complicate the assay development 24
process however, as it requires consideration of multiple additional factors such as
matching primer annealing temperatures and checking for primer dimer formation among
twice as many primers. In addition to these complications, it does not act as a control for
the Y-specific primers, thus if the primers fail either because they were not added in
proper concentrations or they have been compromised, this will not be identifiable. A
more efficient control can be achieved by selecting a primer that creates two amplicons of
differing sizes, one from the Y-chromosome and one from either the X-chromosome or
an autosomal chromosome108–110,63,64. This eliminates the complications of multiplexing
multiple primer pairs, and if there is a problem with either of the primers it will be detected through this inherent control.
Amplicons are generally visualized by gel electrophoresis and DNA staining with ethidium bromide or other less toxic stains such as Sybr Safe™ (Invitrogen, Life
Technologies, Carlsbad, CA). While visualization is consistent across methodologies, the way in which the amplicon is determined is variable. By amplifying only a Y- chromosomal region, presence of the Y-chromosome can be determined simply by visualizing an amplicon of the expected length111,112. As mentioned above, the use of a
single Y-chromosome specific primer pair has the problem of females and failed reactions being indistinguishable. A different method of identifying the Y-chromosome,
that was briefly used, is through Restriction Fragment Length Polymorphism (RFLP).
RFLP methods create Y-specific and non-Y-specific amplicons during PCR which are then digested using restriction enzymes. Due to differences in the amplicon sequences the
restriction enzyme digestion creates fragments of a length unique to the origin of the 25
amplicon allowing for easy identification of the presence of the Y-chromosome based on
the length of the fragments99,113,114. Relative to methods that create amplicons of differing
length during PCR, RFLP methods add extra work with no benefit. The final and most
important method for identifying Y-specific amplicons, relative to this body of work, is
through generation of a Y-specific and a non-Y-specific amplicon through the PCR that
differ in length64,104–108,110. This method includes the reaction control in each sample and
requires no added steps between PCR and visualization in order to allow for
differentiation between the amplicons.
Due to the genetic variance between species, there is a high probability that
different primer sets will need to be designed for species that are not closely related,
increasing the labor of establishing a working test in a new species.
qPCR can be used to identify genotypic sex by either determining the presence of a Y-chromosome or determining if more than one X-chromosome is present115. Methods that identify sex by determining if a Y-chromosome is present may use the same primers that are used in PCR. An intercalating dye may also be used, in conjunction with melting curve analysis, to determine if the Y-chromosome is present so long as the amplicons differ enough in melting temperature to be distinguished from one another116. Use of a single primer pair that creates amplicons of differing sizes (as described in the PCR
section above) is accomplished in the same ways as multiplexing, however, if the two
amplicons are highly homologous (which is likely), special care must be taken in probe
selection to ensure that probes bind only to their intended amplicon.
qPCR methods that determine if there is more than one X-chromosome rely on 26
the assumption that equivalent quantities of genomic DNA will have near equivalent
chromosomal copy numbers. With this assumption then, a relative copy number of X-
chromosomes can be determined based on CT values using an intercalating dye. This
technique uses the 2 –ΔΔCT method to determine relative copy number of an X-linked amplicon and by extension the genotypic sex of the individual115. This technique will
inaccurately predict phenotypic sex in XO (Turner syndrome) and XXY (Klinefelter
syndrome) individuals which occurs in 1 in 1893117 to 5000118 and 1 in 500118 to 650119
humans respectively. Thus, on average such a test in humans would be expected to not be
an accurate representation of phenotypic sex 0.17%-0.25% of the time. In contrast, PCR
and qPCR techniques that identify Y-chromosome will only have the genotype and
phenotype misalign in cases where an XY individual is phenotypically female or a XX
individual is phenotypically male. This only occurs in about 1 in 80000120 and 1 in
20000121 humans respectively, making PCR and qPCR sex identification based on the
presence of a Y-chromosome far more likely to accurately predict phenotypic sex.
Current PCR/qPCR Gene Targets
Common Origin of X & Y
As most PCR and qPCR sex identification of mammals is done by targeting genes
on the Y-chromosome and a homologous region on the X-chromosome, a brief overview of the evolutionary history of mammalian sex chromosomes is appropriate.
In the 1960s it was proposed that the vertebrate X- and Y-chromosomes were originally a pair of homologous autosomal chromosomes that evolved over time122. This 27
evolution was such that the proto-Y-chromosome developed the genes needed to be the
chromosome of the heterogametic sex (males) and progressively deteriorated until it
could not exchange most of its genetic material with its original homolog, this while the
proto-X-chromosome remained largely the same with the exception of developing
methods of dosage compensation122. The exact way and order in which this occurred is
still not fully known and many different hypotheses have been put forward to explain it
(for a more information on the proposed evolution of sex chromosomes see references
122–128).
Despite this, it is generally agreed that the X- and Y-chromosomes were
originally homologous chromosomes that lost the ability to recombine after the proto-Y-
chromosome gained a sex determining role. Interestingly, the Y-chromosome has become subject to erosion such that it is almost entirely devoid of genes, relative to the X- chromosome; yet it contains an abundance of repetitive DNA sequences124,127,128. The inability of most of the Y-chromosome to recombine is believed to be a major cause for its erosion and loss of function. In contrast, the X-chromosome retains much of its original structure due to its ability to recombine with other X-chromosomes in females.
The erosion of the Y-chromosome in therians throughout the ongoing processes of evolution and speciation has led to a number of species specific differences in the Y- chromosome. These differences range from point mutations between some species to entirely different genes being present. The presence, absence, and variations of genes on the Y-chromosome between genes can possibly give indications of the historical stability of particular regions as well as the evolutionary history of certain species. This can be 28
seen in the examples of gene targets described below. While, the same gene is targeted in
many species, the primer pairs are often not targeting the region of the gene. This is because while different species may have the same X-homologs on the Y-chromosome, the Y-chromosome has eroded differently during the evolution of each species. This lends additional importance to the cross-species comparison of the X- and Y- chromosomal regions detailed here, as the variations between species may help answer questions regarding the past and future evolution of the Y-chromosome.
The Y-chromosome in most mammals has developed such that, outside of a pseudoautosomal region (PAR) located on the distal end of the short arm of the Y- chromosome, which is homologous between X- and Y-chromosomes (allowing for X-Y pairing and gene crossover in meiosis), there is very little homology between the X- and
Y-chromosomes. The few regions of homology between the sex chromosomes that persist outside of the PAR are very unlikely to cross over which is of great importance in
PCR/qPCR methods of sex identification designed to identify the presence of the Y- chromosome and include an inherent reaction control.
The genes described in the remainder of this section, have been, or are currently, used as targets for PCR or qPCR sex identification in various species. With the exception of the SRY gene, all of the genes have a homolog on the X-chromosome. Assays targeting those genes with an X-chromosome homolog have been designed to take advantage of this potential for an inherent positive reaction control as described earlier in the chapter.
Lacking an X-chromosome homolog, the SRY gene must use duplex reactions to provide a positive reaction control. 29
SRY
As described above, the SRY gene is found exclusively on the Y-chromosome in
mammals and is responsible for initiating the change from female (the default sex) to
male in early embryonic development. Its presence always indicates a male regardless of
whether the individual has a Y-chromosome or a translocation event involving the SRY
gene has occurred, making it a reasonable target gene for sex identification.
ZFX/ZFY
In humans, the zinc finger protein X-linked (ZFX) gene codes for a member of the
krueppel C2H2-type zinc-finger protein family. ZFX is a DNA binding protein with a
transcriptional activation domain, a nuclear localization domain, and a DNA binding
domain present in the full-length protein. In mice ZFX is a required transcriptional
regulator needed for self-renewal of embryonic and adult hematopoietic stem cells,
though it is not involved in their growth or differentiation129. Multiple isoforms of ZFX
have been identified and are produced by alternatively spliced transcript variants, the full- length nature of all of these variant isoforms has not yet been determined130. Also, ZFX
escapes X inactivation131.
ZFY may act as a transcription factor in mammals and was once a preeminent
candidate of being the testis-determining factor and was in fact named TDF for a short
time (the term TDF is no longer in common use and is now used in connection with the
SRY gene if at all). In mice, there is evidence that correct ZFY function is required for
proper spermatogenesis, as improper expression of either of the two gene splice variants leads to apoptosis at the mid-pachytene checkpoint132. In humans ZFY is mainly produced 30
in the testis and prostate, though it is also found in several other tissues in the urinary and
digestive systems.
ZFX and ZFY are targets for sex identification in a number of species including humans, cattle, sheep, goats, red deer, and elk107,113.
Amelogenin/Amelogenin-like
The amelogenin gene (AMELX, formerly AMG or AMGX) is an X-linked gene
with a Y-linked paralogue termed amelogenin-like (AMELY, formerly AMGL or AMGY).
Both genes produce functional isoforms of the amelogenin protein, though expression of
AMELY is only ~10% of AMELX expression in humans133. Both AMELX and AMELY
are key extracellular proteins in the process of amelogenesis, through which enamel is
produced133. Amelogenin, operates in tandem with ameloblastins, enamelins, and tuftelins in the process of mineralization in tooth development, with amelogenin making up 50-90% of the proteins present during the stages of enamel development134.
Amelogenin self assembles in developing teeth into nanosphere structures which create
the structure around which early enamel crystallites form135,136.
The chromosomal locations of both AMELX and AMELY were first identified in
humans and mice and were found to be on the short arm of the X-chromosome and near the centromere of the Y-chromosome respectively137. Soon after discovery of their
locations, both AMELX and AMELY were first sequenced in humans from genomic
DNA138. Prior to this, the only sequences of amelogenin came from cloned murine and
bovine cDNA respectively139,140 and the chromosomal origins were not specifically
known. The sequencing of AMELX and AMELY by Nakahori et al identified a significant 31
deletion region on AMELY relative to AMELX, which was able to be used as a target for
sex identification in humans141. The common presence of AMELX and AMELY in mammals and the frequency in which significant deletion regions are seen in AMELY has made it a common target for sex identification in a variety of different species including: humans109,142, nonhuman primates63,143,144, various ungulates (including cattle, sheep,
goats, bison, yaks, pigs, deer, moose, and caribou)102,103,108,110,145–148, cetaceans146,
felids149, and bears65,150.
DEAD-Box Protein (DDX3X/DDX3Y) Gene
DEAD-Box helicase 3 X-linked (DDX3X also called DBX; DDX3; HLP2;
DDX14; CAP-Rf; MRX102) is a member of the DEAD-Box protein family. The DEAD-
Box family of proteins is the largest member of helicase superfamily 2 (SF-2)151,152 and
have nine conserved sequence motifs153,154. Motif II (or Walker B motif) of DEAD-Box
proteins has the amino acid sequence D-E-A-D and is the source of the protein family’s name153. DEAD-Box proteins are RNA helicases with ATPase activity and are involved
in transcription155, pre-mRNA splicing156, ribosome biogenesis157, nuclear export158, translation initiation159,160, RNA degradation161, and organelle gene expression162. For a
more detailed review of the DEAD-Box family of proteins and their functions, see
references 151,163–166.
DDX3X was first identified as a homologue of DDX3Y (initially termed DBY)
that escapes X-inactivation167. As with other DEAD-Box proteins, DDX3X is an RNA
helicase. It has been shown to have DNA helicase activity, a high level of RNA- independent ATPase activity, and ATPase activity appears to be stimulated by both DNA 32
and RNA which is unusual among DEAD-Box proteins168,169. It is expressed ubiquitously
in all tissues170. Haploinsufficiency is linked to intellectual disabilities171. It is directly
targeted by many viruses and has been suggested as the target of a broad spectrum of
antiviral drugs172,173.
DDX3Y is the Y-specific homolog of DDX3X. It was discovered in a systematic
search of the human Y-chromosome and was useful in the discovery of DDX3X167.
Transcription of DDX3Y is broad, but not ubiquitous and is generally lower in most
tissues than DDX3X, with the highest expression of DDX3Y transcription in bone marrow
and testes170. Despite the widespread transcription of the gene, DDX3Y protein is only
produced in testicular tissue174. Unsurprisingly, mutations in DDX3Y are associated with
male infertility ranging from reduced production of germ cells to Sertoli-cell-only (SCO) syndrome175.
DDX3X and DDX3Y are targets for sex identification in a number of species
including American bison, European cattle, sheep, pigs, Japanese weasels, Siberian
weasels, and Japanese martens72,176,177.
Conclusion
Sex determination involves complex mechanisms and is highly variable between
species. In therian species sex determination is controlled by the SRY gene located on the
Y-chromosome, which acts as a switch when present, pushing embryonic development
into the path of male phenotypic development.
Sex identification of humans and animals has a number of important applications 33
including embryo transfer, ecology population studies, lab sample controls, archeology,
and use in criminal justice. Over time, various methods of sex identification have been
developed in order to meet these complex needs. These include direct methods of sex
identification such as visual inspection, ultrasound, and cytogenetic identification of sex
chromosomes by karyotyping, as well as indirect methods such as Y-specific antigen
detection, enzyme based X-chromosome copy number detection, Y-specific DNA probes,
and PCR/qPCR. For determining genotypic sex, PCR and qPCR are the fastest, most
accurate, and most effective methods.
PCR and qPCR sex identification is done in a wide variety of species using primer
pairs that exploit the common ancestry of the X- and Y-chromosomes. This is most often
done by targeting Y-chromosomal regions that contain deletions relative to a largely
homologous X-chromosomal region. Much is still unknown about the evolution of the
sex chromosomes, and the information produced by this method of sex identification can
increase the knowledge of the process by creating direct comparisons of homologous
regions of the X- and Y-chromosomes within a species as well as between species.
The remainder of this thesis reports the development of two new pairs of
PCR/qPCR primers. The first primer pair described creates amplicons from ZRSR2 and
ZRSR2Y which are X- and Y-specific respectively. The second creates amplicons from
the X-specific GPM6B and its Y-specific pseudogene. In both cases, the Y-specific
amplicon contains deletions relative to its X-specific counterpart making it possible to distinguish the amplicons via gel electrophoresis and/or melting curve analysis. Chapter 2 focuses on the ZRSR2 targeting primer pair and demonstrates its efficacy in sex 34
identification of Bos taurus, Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens. Chapter 3 deals with the GPM6B targeting primer pair and shows its efficacy in sex identification in pigs. 35
CHAPTER 2:
SEX IDENTIFICATION OF BOVID SPECIES USING ZRSR2 & ZRSR2Y GENE
HOMOLOGS
Abstract
A single PCR primer pair designed to produce an X-chromosome specific
amplicon and a separate Y-chromosome specific amplicon has a distinct advantage in sex
identification assays. In this case, the single primer pair has a built-in control for the PCR
reaction and eliminates duplex PCR reaction inefficiencies. Primer pairs of this kind
generally target gene regions on the X-chromosome that have homologs on the Y-
chromosome containing deletions or insertions. One example is the amelogenin (AMELX)
gene. Primers spanning deletions in the amelogenin-like gene (AMELY) found on the Y-
chromosome have commonly been used for sex identification108,141,143.
The ZRSR2 and ZRSR2Y genes are shown here to be alternative sex identification primer targets in Bos taurus (European cattle), Bos indicus (African cattle), Bison bison
(American bison), Capra hircus (domestic goat) Ovis aries (domestic sheep), and Bos grunniens (domestic yak). Comparison of the Bos taurus ZRSR2 and ZRSR2Y gene sequences yielded multiple deletion regions located within the ZRSR2Y sequence.
Primers were then designed to flank two of these deletions. DNA samples from the aforementioned species were tested with these primers and proved to be highly accurate in sex identification of all samples tested. Female samples produced a single X-
chromosome specific ZRSR2 amplicon, 149 bp in length, whereas male samples produced 36
two amplicons, one X-chromosome specific ZRSR2 amplicon (149 bp) and one Y-
chromosome specific ZRSR2Y specific amplicon, 116 bp in length. This assay provides
an efficient alternative assay for sex identification in domestic cattle species.
Introduction
Sex identification of DNA samples is an important component of preimplantation
embryo diagnosis, animal identification, and forensic biology. PCR methods are often
used in determining sex ratios in a wildlife population when the only samples available
are fecal, hair or tissue. When sex identification of subjects is an important research
parameter, costs may be reduced through testing and excluding samples of the undesired
sex. Another benefit of sex identification via PCR is the ability to examine population
structure as well as dynamics related to sex in a noninvasive manner.
Amplification of the Y-chromosome specific SRY gene and any one of a number
of X-chromosome specific or autosomal genes in a multiplex reaction is a common
practice in determining the presence of a Y-chromosome in a DNA sample. This method,
however, leaves the potential for false negatives in the event of failure of the primers
targeting SRY. For this reason, the development of assays that target Y-chromosome specific genes with X-chromosome analogs are preferable because careful design allows a single primer pair to create X- and Y-chromosome specific amplicons. Such a design
creates a built in positive reaction control in each reaction. By designing the primer pair to span a deletion unique to one of the genes, two amplicons of unique size can be produced which can be separated via gel electrophoresis. Alternately, real time PCR 37
using either melting curve analysis or X- and Y-chromosome specific probes can provide
equivalent information. The amelogenin gene and its Y-specific analog have frequently
been targeted for this method of single primer pair sex identification in a variety of
different species.
Here a novel alternative primer pair useful in sex determination of several
members of the family Bovidae is presented. The primer pair targets the X-chromosome
specific ZRSR2 gene and a deletion-containing portion of its Y-chromosome specific homolog ZRSR2Y. In humans, the ZRSR2 gene encodes for U2 small nuclear ribonucleoprotein auxiliary 35 kDa subunit-related protein 2. This protein is a critical component in the structure of the spliceosome and plays an important role in eukaryotic precursor messenger RNA splicing and mRNA assembly. It is involved in the recognition of the 3’ splice site and assists in positioning the nucleotide favorably for splicing178.
ZRSR2Y appears to be bovid specific179 and its function has not yet been determined.
Deletions have been found in Bos taurus ZRSR2Y relative to ZRSR2. The primers
described here were designed to produce an X-chromosome specific amplicon from the
ZRSR2 gene, while also spanning two deletions (6 bp and 27 bp) found in ZRSR2Y. PCR
amplification using this primer pair results in a single X-chromosome specific amplicon
of 149 bp in both male and female samples and an additional Y-chromosome specific
amplicon of 116 bp in males. When the amplicons are separated using gel
electrophoresis, female samples reveal a single distinguished band, while male samples
reveal two distinct bands.
A melting curve analysis (MCA) protocol is also described here, which can be 38
used in lieu of gel electrophoresis for the purpose determining the presence or absence of
the aforementioned amplicons based on the presence or absence of melting curve peaks
unique to the X- and Y-specific amplicons. Initial testing indicates that these primers can
be used for Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens sex
identification in addition to Bos taurus.
Materials and Methods
Sample Collection and DNA Extraction
A variety of tissues including blood, ear notch, hair, and muscle were collected
from Bos taurus, Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens as indicated in Table 1. Several DNA extraction kits were used including Agencourt
DNAdvance Genomic DNA Isolation kit (Beckman Coulter Life Sciences, Indianapolis,
IN), PureLink™ Genomic DNA kit (ThermoFisher Scientific, USA), Dneasy Blood and
Tissue kit (Qiagen, USA), Gentra Puregene Tissue kit (Qiagen, USA), Promega Genomic
DNA kit (Promega, USA), and Viogene Blood & Tissue kit (Viogene, Taipei, Taiwan).
In addition to the kits used, modified Laird’s Buffer DNA isolation protocols180 were also
used. Some samples were received as already purified DNA and no information was
available on the tissue or extraction method. The method of DNA extraction had no effect on results. Extracted DNA was stored at -80° Celsius (or C).
Primer Design
To find appropriate primers, the Bos taurus ZRSR2 (XM_003584349.4) and
ZRSR2Y (GQ426330.1) sequences were aligned using Ensemble’s EMBOSS Needle 39
Nucleotide Alignment software. An area with adjacent 6 and 27 base pair deletions in the
Y-chromosome was identified and primers were designed to flank the two deletions (Fig.
1). The forward and reverse primer sequences are 5’-AGTTCAGTCTCTGCTCCC-3’ and 5’-GGGGAAGGCACAGTTC-3’ respectively. The positions of the 5’ base of the forward and reverse primers on the X-chromosome are 134994806 and 134994954 respectively (AC_000187.1). The primers produce amplicons 149 bp long from the X- chromosome and 116 bp long from the Y-chromosome in Bos taurus. A NCBI Primer-
BLAST search was run with the designed primers, which confirmed a minimal likelihood of off target amplification from the Bos taurus genome.
Table 1 | Bovid Tissues Used Species Tissue Number of samples Bos taurus Blood 10 Bos taurus Ear Notch 59 Bos taurus Hair 24 Bos taurus Muscle 50 Bos indicus Muscle 5 Bos indicus Dry Blood 11 Bos indicus Hair 10 Bison bison Unknown 5 Capra hircus Blood 2 Capra hircus Unknown 2 Ovis aries Unknown 22 Bos grunniens Hair 6 Counts of bovid samples obtained from different species and tissue types.
Cross-Species Analysis of Amplified Regions
The X- and Y-specific sequences predicted to be amplified in Bos taurus were
used as query sequences and compared to the reference genomes of Bos indicus
(taxid:9915), Bison bison (taxid:9901), Capra hircus (taxid:9925), Ovis aries 40
(taxid:9940), and Bos grunniens (taxid:30521) to determine the level of conservation of the amplicon in these bovid species. The query sequences were compared to the NCBI
RefSeq Reference and Representative genomes nucleotide database for each species using the NCBI BLAST tool with the megablast program181 to optimize the search for highly similar sequences.
Fig. 1 | Portion of ZRSR2/ZRSR2Y alignment in Bos taurus. Forward and reverse primer locations are indicated by orange and blue bold text respectively. Red dashes indicate deletions on ZRSR2Y.
PCR Amplification
Testing for the presence of the deletion regions in ZRSR2Y and the efficacy of the primers for use in sex identification was performed in Bos taurus, Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens via PCR and gel electrophoresis. PCR amplification was carried out in 15 µl reactions with 0.5 µM of each primer, 20 ng/reaction of template DNA, 1x GoTaq G2 Green Master Mix (Promega, USA, cat.
M7122), and nuclease free water. Amplification was done on Bio-Rad iCycler (Bio-Rad,
Hercules, CA). Thermocycler parameters were as follows: initial denaturation at 95°C for
2 minutes followed by 30 cycles of amplification, each containing denaturation at 95°C 41
for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 15 seconds.
After the final cycle, a final extension was performed at 72°C for 5 minutes.
Visualization of PCR Product
Each amplified product was subjected to electrophoresis, with Forever Ladder 100 bp molecular weight standard (Seegene, Seoul, South Korea). Electrophoresis was performed on a 6% non-denaturing polyacrylamide gel (73 x 82 x 0.75 mm) using Mini-
PROTEAN® 3 Cell apparatus (Bio-Rad, Hercules, CA) at room temperature for 45-60
minutes at 100 volts using 1x TBE running buffer. The gels were then stained in either a
0.5 μg/ml ethidium bromide solution or a 1X SYBR Safe (Invitrogen, Life Technologies,
Carlsbad, CA) solution for 30 minutes prior to visualization under UV light.
Retesting Using SE47/SE48 Primers
All Bos taurus samples whose amplification did not correlate with the sex on
record (i.e. samples labeled male showing female amplification pattern, and vice versa)
were retested using the ZRSR2/ZRSR2Y targeting primer pair to determine if reaction
contamination was at fault. These samples were also tested using the primers SE47 and
SE48108, which generates X- and Y-specific amplicons from Bos taurus AMELX and
AMELY respectively. This was done to confirm the presence or absence of the Y- chromosome in the original sample and determine if the sample was mislabeled or the sex incorrectly identified at the time of collection.
Bos taurus X and Y Controls for qPCR
X- and Y-chromosome associated amplicons were obtained from a male Bos 42 taurus sample, for use as positive controls in qPCR and MCA. This was done by excising the associated bands from a PAGE gel after electrophoresis and visualization as detailed above. The excised bands were placed in different 0.5 ml tubes each containing 100 μl of
1x TE buffer and incubated at 4°C for 48 hours to allow the amplicons to diffuse into the buffer. After the incubation, the supernatant was moved to new 0.5 ml tubes and stored at
-80°C. The controls were tested using the melting curve analysis (MCA) as described below.
qPCR & Melting Curve Analysis
qPCR amplification was carried out in 10 μl reactions with 0.5 µM of each primer and 10 ng/reaction of template DNA, 1x iTaq Universal SYBR Green Supermix (Bio-
Rad, Hercules, CA) or 1x Perfecta SYBR Green SuperMix (Quanta Biosciences, Inc.,
Beverly, MA), and nuclease free water. qPCR amplification and MCA were performed using an Eppendorf mastercycler ep realplex4 gradient S thermocycler and Realplex 2.2 software (Eppendorf, Hamburg, Germany) was used for analysis. Thermocycler parameters were as follows: initial denaturation at 95°C for 2 minutes followed by 35 cycles of amplification, each containing denaturation at 95°C for 30 seconds, annealing at
59°C (as was determined to be the optimal annealing temperature from the qPCR optimization) for 30 seconds, and extension at 72°C for 15 seconds. After the final cycle a final extension was performed at 72°C for 5 minutes. The MCA consisted of an initial denaturation at 95°C for 15 seconds, a melting curve start phase at 59°C for 15 seconds, followed by temperature increase up to 95°C over the course of 20 minutes with continual fluorescence measurement. 43
Results
Alignment and BLAST Results
The results of the Primer-BLAST search indicated that there would be amplification from Bos taurus with low risk of off target amplification. Figs. 2 and 3
show alignments from the BLAST searches performed in Bos indicus, Bison bison,
Capra hircus, Ovis aries, and Bos grunniens using the predicted Bos taurus X- and Y-
specific amplicon sequences as the query sequence respectively. The X- and Y-specific sequences produced no alignment with Bos grunniens and the Y-specific sequences also produced no alignment from Capra hircus, or Ovis aries. As the PCR results will show, the lack of produced alignment was not a result of the sequence being absent in Capra hircus, Ovis aries, and Bos grunniens.
The Bison bison alignments from both the X- and the Y-specific sequence queries come from unplaced genomic scaffolds. The Capra hircus X-specific query alignment is from an unlocalized X-chromosomal genomic scaffold. All other alignments are localized within the chromosome with all alignments from the X-specific query coming from X- chromosomes and the Bos indicus alignment with the Y-specific query coming from the
Y-chromosome.
PCR Results
All samples tested using PCR and primer pair designed to target ZRSR2 and
ZRSR2Y matched either the recorded sex or the sex as determined by testing with
SE47/SE48 primers in the process of retesting. A summary of result details is found in 44
Table 2 for Bos taurus, Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens. Of the 143 Bos taurus samples tested, 24 had an incorrectly recorded sex
(based on sex identification performed using SE47/SE48 primers). ZRSR2/ZRSR2Y sex identification agreed with all SE47/SE48 test results in all cases.
Query 1 AGTTCAGTCT CTGCTCCCTG ACCTCCTCCC GCCATGGCTC 40 Bos i. 79805303 AGTTCAGTCT CTGCTCCCTG ACCTCCTCCC GCCATGGCNC 79805342 Bison b. 1897 AGTTCAGTCT CTGCTCCCTG ACCTCCTCCC GCCATGGCTC 1936 Capra h. 54474614 AGTTCAGTCT CTGCTCCCTG ACCTCCTCCC GCCTCGGCTC 54474653 Ovis a. 14686911 AGTTCAGTCT CTGCTCCCTG ACCTCCTCCC GCCACGGCTC 14686872
Query 41 CTGGACCTGG AGGAGCTCTG GCTGCGGCTC CTGCGGCTGT 80 Bos i. 79805343 CTGGACCTGG AGGAGCTCTG GCTGCGGCTC CTGCGGCTGT 79805382 Bison b. 1937 CTGGACCTGG AGGAGCTCTG GCTGCGGCTC CTGCGGCTGT 1976 Capra h. 54474654 CTGGACCTGG AGGAGCTCTG GCTGCGGCTC CTGCGGCTAT 54474693 Ovis a. 14686871 CTGGACCTGG AGGAGCTCTG GCTGCGGCTC CTGCGGCTAT 14686832
Query 80 GGCTCCGGCT GCCCCGACCA CGGATGCGGT CCCTTTTTCT 120 Bos i. 79805383 GGCTCCGGCT GCCCCGACCA CGGATGCGGT CCCTTTTTCT 79805422 Bison b. 1977 GGCTCCGGCT GCCCCGACCA CGGATGCGGT CCCTTTTTCT 2016 Capra h. 54474694 GGCTCCGGCT GCCCCGTCCG CGGCTGCGGT CCCTTTTTCT 54474733 Ovis a. 14686831 GGCTCCGGCT GCCCCGTCCG CGGCTGCGGT CCCTTTTTCT 14686792
Query 121 TCCTCTGCTT CGGGAACTGT GCCTTCCCC 149 Bos i. 79805423 TCCTCTGCTT CGGGAACTGT GCCTTCCCC 79805451 Bison b. 2017 TCCTCTGCTT CGGGAACTGT GCCTTCCCC 2045 Capra h. 54474734 TCCTCTGCTT CGGGAACTGT GCCTTCCCC 54474762 Ovis a. 14686791 TCCTCTGCTT CGGGAACTGT GCCTTCCCC 14686763
Fig. 2 | NCBI BLAST alignment for the amplified region of ZRSR2. The predicted X- specific Bos taurus amplicon sequence was used as the query sequence. Query was compared to Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens RefSeq Reference and Representative genomes. Highlighted bases are those which differ from the query sequence. No Bos grunniens alignment was returned from the search.
The 24 samples with an incorrectly recorded sex were obtained as part of a group of 41 samples from a slaughterhouse. Sex identification was performed based on visual identification of secondary sex characteristics of the hanging carcass at the time of collection. These 41 samples were the only samples in which sex identification was not performed via visual inspection of primary sex characteristics of a living animal. Fig. 4 45 shows representative images of the gels produced from ZRSR2/Y amplification of Bos taurus, Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens samples.
Query 1 AGTTCAGTCT CTGCTCCCCA ACCTCCTCCC ACCACGGCTC 40 Bos i. 644465 AGTTCAGTCT CTGCTCCCCA ACCTCCTCCC ACCACGGCTC 644504 Bison b. 13809 AGTTCAGTCT CTGCTCCCCA ACCTCCTCCC ACCACGGCTC 13770
Query 41 ATGGAGGAGC TCTGGCTGCT GTTCCTGCTG CTGTGGTCCC 80 Bos i. 644505 ATGGAGGAGC TCTGGCTGYT GTTCCTGCTG CTGTGGTCCC 644544 Bison b. 13769 ATGGAGGAGC TCTGGCTGCT GTTCCTGCTG CTGTGGTCCC 13730
Query 81 TTTTTCTTCC GCTGCTTTGA GAACTGTGCC TTCCCC 116 Bos i. 644545 TTTTTCTTCC GCTGCTTTGA GAACTGTGCC TTCCCC 644580 Bison b. 13729 TTTTTCTTCC GCTGCTTTGA GAACTGTGCC TTCCCC 13694
Fig. 3 | NCBI BLAST alignment for the amplified region of ZRSR2Y. The predicted Y-specific Bos taurus amplicon sequence was used as the query sequence. Query was compared to Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens RefSeq Reference and Representative genomes. The highlighted base is the only one which differed from the query sequence. No Capra hircus, Ovis aries, or Bos grunniens alignments were returned from the search.
Melting Curve Analysis Results
MCA of the excised X- and Y-specific fragments using the ZRSR2/ZRSR2Y targeting primers revealed that each fragment had a single melting curve peak and that the peaks were unique from one another (Fig. 5). Analysis of whole genome Bos taurus and Bos indicus samples produced both X- and Y-specific peaks from male samples and only X-specific peaks from female samples, as expected (Figs. 6 and 7). MCA of Bison bison, Capra hircus, Ovis aries, and Bos grunniens samples gave results similar to those of the Bos taurus and Bos indicus with males producing X- and Y-specific peaks and females producing only an X-specific peak (Fig. 8).
46
Table 2 | ZRSR2/ZRSR2Y Targeting Primer Pair Results Summary Bos Bos Bison Capra Ovis Bos Total taurus indicus bison hircus aries grunniens Number of Samples 143 26 5 4 22 6 206 Number of Males 91 16 3 2 11 4 127 (Recorded) Number of Females 52 10 2 2 11 2 79 (Recorded) Number of Males 103 ------103 (Corrected)* Number of Females 40 ------40 (Corrected)* Results Match 119 26 5 4 22 6 182 Recorded Sex Results Match 24 0 0 0 0 0 24
PCR SE47/SE48 Sex Accuracy of ZRSR2/Y 100 100 100 100 100 100 100 Primers (%)** Results Match 119 26 5 4 22 6 182
Record** Results Match 24 0 0 0 0 0 24
qPCR SE47/SE48 Sex** Accuracy of ZRSR2/Y 100 100 100 100 100 100 100 Primers (%)** *Corrected numbers of males and females was determined based on results of sex identification done using SE47/SE48 primers in samples where ZRSR2/Y primers produced results contrary to those expected based on the recorded sex. **Results and percent accuracy represent data after retesting protocol was completed. Data for each species tested including total number of samples, recorded number of male and female samples, corrected sex information of samples (if applicable), and accuracy information for both PCR and qPCR testing.
47
a b
c d
e f
Fig. 4 | Gels showing representative PCR results for bovid species tested. The size marker in all gels is Forever Ladder molecular weight standard (100-500 bp in 100 bp increments) (Seegene, Seoul, South Korea). NTC is the negative control in each gel. (a) Bos taurus male (lanes 2-5) and female (lanes 6-9). (b-f) Gels of Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens samples, respectively. The right-most two occupied lanes of each gel (b-f) contains Bos taurus male and female PCR products (from left to right respectively) as a reference for the amplicon size in each other species. 48
Fig. 5 | Graph of melting curve of excised X- and Y-specific fragments. The Y- specific fragment peaks at 84.1°C the X-specific fragment peaks at 88.5°C. 49
a
b
Fig. 6 | Graphs of melting curves for a subset of Bos taurus samples. (a) Female Bos taurus samples which, are distinguished by a single melting curve peak. (b) Male Bos taurus samples which, are distinguished by the presence of two melting curve peaks.
50
a
b
Fig. 7 | Graphs of melting curves for a subset of Bos indicus samples. (a) Female Bos indicus samples, which are distinguished by a single melting curve peak. (b) Male Bos indicus samples, which are distinguished by the presence of two melting curve peaks.
51
a
b
Fig. 8 | Graphs of melting curves of Bos taurus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens samples. (a) A single female of each species. (b) A single male of each species. All species present a nearly identical melting curve to Bos taurus.
52
Discussion
The data from the BLAST searches did not indicate that amplification from the
Bos grunniens X-chromosome, or from the Capra hircus, Ovis aries, or Bos grunniens Y- chromosomes should be expected. However, the PCR and qPCR tests created X- and Y- specific amplicons in all species. Indicating that the reference genomes for Capra hircus,
Ovis aries, and Bos grunniens are incomplete in the regions where ZRSR2Y, and ZRSR2
in the case of Bos grunniens, are located. Given the difficulty of sequencing sex
chromosomes (particularly the Y-chromosome) it is unsurprising that these regions were missing from the reference genome databases. Additionally, it appears, based on the cross-species alignment data and the PCR and qPCR results that the Bison bison alignments matching the X- and Y-specific Bos taurus query sequences belong to the bison X- and Y-chromosomes respectively. Further, the BLAST results combined with the PCR and qPCR results indicates that these regions are conserved on both the X- and
Y-chromosomes between these species and may be largely conserved throughout their respective sub families (Bovidae and Caprinae) if not the whole family Bovidae.
The 100% sex identification accuracy in all Bos taurus, Bos indicus, Bison bison,
Capra hircus, Ovis aries, and Bos grunniens DNA samples tested to date, using either
PCR or qPRC, indicates that the ZRSR2/ZRSR2Y targeting primer pair developed is a viable for use in sex identification in these species. The ZRSR2 and ZRSR2Y genes appear
to be fairly stable with a low likelihood of mutation, as indicated by the conservation of
these genes between species. The apparent stability of these gene regions is a further
indicator that the designed primer pair has a low likelihood of failure due to mutations 53
within a species. Together, this strongly suggests that the primer pair will be useful for
rapid and cost effective sex identification of DNA samples.
In conclusion, the deletion regions identified in the Bos taurus ZRSR2Y gene are
present in Bos taurus, Bos indicus, Bison bison, Capra hircus, Ovis aries, and Bos grunniens. These deletions can be readily identified compared to the homologous region of ZRSR2 by the use of PCR and gel electrophoresis analysis or qPCR and melting curve analysis using the primer pair developed. In addition to confirming the presence of the deletions, a sex identification assay using these same primers that is rapid and highly effective in a variety of bovid species has been developed.
54
CHAPTER 3:
SEX IDENTIFICATION OF PIGS USING GPM6B & ITS Y-CHROMOSOME
HOMOLOG
Abstract
As described previously (see Chapters 1 and 2), PCR and qPCR sex identification
assays that use a single primer pair to generate X- and Y-chromosome specific amplicons
have a distinct advantage over duplexed methods. The presence of a built-in (or inherent) reaction control without the reaction inefficiencies of duplex PCR is the main advantage afforded. Such advantageous primer pairs generally target gene regions on the X-
chromosome with homologs on the Y-chromosome that contain deletions or insertions.
One example is the amelogenin (AMELX) gene, which is targeted for PCR and qPCR sex
identification in various species108,141,143.
The GPM6B gene located on the X-chromosome and its Y-chromosome
pseudogene are shown here to be viable gene targets PCR sex identification in Sus scrofa.
Comparison of the homologous sequences in the Sus scrofa genome showed multiple
deletion regions located within the Y-chromosome pseudogene sequence. A single primer pair flanking several of these deletions was designed, resulting in a Y-chromosome specific amplicon 43 bp shorter than the X-chromosome specific GPM6B amplicon produced in the same reaction. Utilizing this primer pair, correct identification of sex was obtained in 100% of samples tested. In addition to the ability to determine the sex of individuals, a deletion variant in the GPM6B gene present in a portion of the population 55
was also confirmed. Female samples produced a single X-chromosome specific GPM6B amplicon, either 244 bp or 228 bp in length, whereas male samples produced two amplicons, one X-chromosome specific GPM6B amplicon (244 or 228 bp) and one Y- chromosome pseudogene specific amplicon, 201 bp in length.
Introduction
As demonstrated in chapters 1 and 2, sex identification of DNA samples is important in a variety of applications. ZRSR2 and ZRSR2Y were previously investigated as potential targets for a PCR sex identification assay in bovid species, resulting in the development of a novel primer pair useful in this method of sex identification (Chapter
2). Testing of this ZRSR2/ZRSR2Y targeting primer pair in Sus scrofa (domestic pigs) produced no amplification. Subsequent analysis of the genome revealed mismatches between the Bos taurus and Sus scrofa genomes in the primer regions (unpublished data).
A search of literature identified GPM6B as a porcine gene on the X-chromosome with a highly homologous region (a pseudogene) on the Y-chromosome182, making it a
probable target for designing primers capable of being used for sex identification in Sus
scrofa. In humans, the GPM6B gene encodes for a membrane glycoprotein of the
proteolipid protein family183. It is expressed throughout most regions of the brain, and is
believed to function in membrane trafficking and cell-to-cell communication. It is also
involved in the regulation of osteoblast function and bone formation184.
Alignment of the homologs allowed for the identification of deletions on the Y-
specific homolog relative to GPM6B. From this, a single primer pair, was designed to 56
produce an X-chromosome specific amplicon from the GPM6B gene, and an amplicon
spanning several deletions (totaling 43 bp) from the Y-chromosome homolog in Sus
scrofa. PCR using the designed primer pair resulted in an X-chromosome specific
amplicons 244 and/or 228 bp long in female samples and a Y-chromosome specific amplicon 201 bp long (in addition to one of the X-specific amplicons) in males.
The possibility of different sized X-specific amplicons is due to a 16 bp intron deletion variant (rs1111696537) in the amplified region, which is found in a portion of the population. When the amplicons are separated using gel electrophoresis, female samples reveal either a single band or two bands very close together (difficult to distinguish without a lengthened running time on a high-resolution gel), while male samples reveal two distinct bands with greater separation than seen between the possible
X-amplicons.
Materials and Methods
Sample Collection and DNA Extraction
A variety of tissues including blood, hair, muscle, and tail were collected from 59 pigs. Several DNA extraction kits were used including PureLink™ Genomic DNA kit
(ThermoFisher Scientific, USA), Dneasy Blood and Tissue kit (Qiagen, USA), Gentra
Puregene Tissue kit (Qiagen, USA). In addition, modified Laird’s Buffer DNA isolation180 and phenol/chloroform extraction protocols were also used. The method of
DNA extraction had no effect on results. Extracted DNA was stored at -80° C.
57
Primer Design
To find appropriate primers, the Sus scrofa GMP6B gene (NC_010461.4 base
pairs 11383241 to 11388803) and its Y-specific pseudogene (NC_010462.3 base pairs
10459005 to 10464501) were aligned using NCBI’s BLAST tool. From the alignment, a
section containing multiple deletions on the pseudogene was selected (Fig. 9) and primers
were created to flank the region containing the deletions. The forward and reverse primer
sequences are 5’- GGAAAAGCTGGCAAGG -3’ and 5’- CGTTGCACATCTGTGTG -
3’ respectively. The positions of the 5’ base of the forward and reverse primers on the X-
chromosome are 11387210 and 11387453 respectively (AC_000187.1). The primers
were predicted to produce amplicons 244 bp long from the X-chromosome and 201 bp
long from the Y-chromosome in Sus scrofa.
Fig. 9 | Portion of Sus scrofa GPM6B and its Y-chromosome pseudogene aligned. Forward and reverse primer locations are indicated by orange and blue bold text respectively. Red dashes indicate deletions on the pseudogene.
58
PCR Amplification
Testing to identify the presence of the deletion regions in the Y-specific
pseudogene and the efficacy of the primers for use in sex identification was performed in
Sus scrofa via PCR amplification. PCR amplification was carried out in 15 µl reactions with 0.5 µM of each primer, 20 ng/reaction of template DNA, 1x GoTaq G2 Green
Master Mix (Promega, USA, cat. M7122), and nuclease free water. Amplification was done on Bio-Rad iCycler (Bio-Rad, Hercules, CA). Thermocycler parameters were as follows: initial denaturation at 95°C for 2 minutes followed by 30 cycles of amplification, each containing denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 15 seconds. After the final cycle, a final extension was performed at 72°C for 5 minutes.
Visualization of PCR Product
Each PCR product was subjected to electrophoresis, with Forever Ladder 100 bp molecular weight standard (Seegene, Seoul, South Korea). Electrophoresis was done on a
6% non-denaturing polyacrylamide gel (73 x 82 x 0.75 mm) using Mini-PROTEAN® 3
Cell apparatus (Bio-Rad, Hercules, CA) at room temperature for 45-60 minutes at 100 volts using 1x TBE running buffer. The gels were then stained in either a 0.5 μg/ml ethidium bromide solution or a 1X SYBR Safe (Invitrogen, Life Technologies, Carlsbad,
CA) solution for 30 minutes prior to visualization under UV light.
X-Amplicon Sequencing Analysis
Sequencing analysis of long and short X-amplicons identified during the PCR 59
analysis was done by the Utah State University Center for Integrated Biosystems core genomics lab. PCR product from a female homozygous for the long X-amplicon and a
female homozygous for the short X-amplicon were sequenced on an ABI PRISM™ 3730
DNA Analyzer using the GPM6B targeting reverse primer as sequencing primer (the
reverse primer was chosen as the expected deletion was near the forward primer). The
resulting sequencing data from both the long and short amplicons was then aligned
against the reference genome and the suspected deletion sequence (rs1111696537) using
BioEdit185.
Results
The PCR analysis of porcine samples using the GPM6B targeting primer pair
described above allowed for accurate sex identification of all 59 samples tested (100%
accuracy). A subset of eight individuals expressed at least one shortened X-specific
fragment. Of these animals, two females were heterozygous for the mutation creating the
long and short X-amplicon, making it possible to estimate that the short fragment was 15
to 20 bases shorter than the longer fragment. All 51 other samples displayed only the
longer amplicon. With the exception of the eight samples just mentioned, all female
samples tested showed a single amplicon. All male samples displayed two amplicons
with the Y-amplicon being approximately 201 bp long and the X-amplicon being either
approximately 244 bp or the shortened X-amplicon. Fig. 10 shows a representative gel
image of the PCR results, including the long and short X-specific amplicons. Table 3
gives a summary of the sex and X-amplicon sizes produced. 60
Table 3 | Summary of X-Amplicon Sizes Observed Sex | Amplicon Size Number of Samples Male | Normal X-amplicon 27 Male | Short X-amplicon 3 Female | Both Normal X-amplicon 24 Female | Both Short X-amplicon 3 Female | Mixed X-amplicons 2 Counts of Sus scrofa samples based on sex and the size(s) of amplicons created using the primer pair designed to target GPM6B and its Y-chromosme homolog.
Fig. 10 | A representative gel showing the amplicons produced by the primer pair targeting GPM6B and its Y-specific homologue. The size marker is Forever Ladder molecular weight standard (Seegene, Seoul, South Korea). ♂ Sus scrofa 1 and 2 are from males with normal (longer) X-amplicons. ♂ Sus scrofa 3 and 4 are examples of males with a short X-amplicon. ♀ Sus scrofa 1 and 4 are female samples in which fragments from both X-chromosomes produce normal (long) amplicons. ♀ Sus scrofa 2 is an example of a female with a short X-amplicon. ♀ Sus scrofa 3 is a female with a normal (long) and a short X-fragment. NTC is the no template (negative) control.
Examination of variants within the amplified region indicated that a 16 bp intron variant, rs1111696537, was likely to be the reason for the shortened X-amplicons. This was confirmed by the alignment of the long and short X-amplicons, the relevant portion 61
of which is shown in Fig. 11. The alignment clearly shows a deletion in the same region
as the deletion variant rs1111696537.
X-RefSeq 1 GGAAAAGCTG GCAAGGACTC GGTGCTGAGC ACAGGCAATT 40 Long Fragment 1 ...... 40 Short Fragment 1 ...... ----- 35 rs1111696537 1 CAATT 5
X-RefSeq 41 TCCCCCTCCC TCAATTTCCC CCTCCCTCTA CCCATCAGAT 80 Long Fragment 41 ...... 80 Short Fragment 36 ------...... 64 rs1111696537 6 TCCCCCTCCC T 16
X-RefSeq 81 GTCTGGAAAC CACAAGCCAC ACCTGCCCAA GTTACACATA 120 Long Fragment 81 ...... 120 Short Fragment 65 ...... 104
Fig. 11 | Sequencing data from long and short X-amplicon generated from GPM6B primer pair compared to the Sus scrofa reference sequence (AC_000187.1 bases 11387210 to 11387329) and to the rs1111696537 deletion. Dots represent bases conserved with reference sequence. Orange text represents the forward primer. Red dashes indicate a deletion relative to the reference sequence.
Discussion
Despite the differences in X-amplicon sizes, sex was easily able to be determined based on electrophoresis data. Use of male and female control samples allowed for easy differentiation between male samples and female samples with mixed X-amplicon sizes.
This allowed for 100 % accuracy (59/59 samples) in identifying sex in porcine samples using the primer pair designed to target GPM6B and its Y-chromosome homolog. It is not currently known how broadly useful the designed primer pair is, as it has not been tested extensively in species outside of Sus scrofa.
Further investigation of the amplified region revealed that the shortened X- amplicons were likely the result of the 16 bp intron deletion variant, rs1111696537, 62 resulting in a 228bp X-chromosome fragment. This was confirmed by comparing sequencing data from the long and short X-specific amplicons. Interestingly, the 16 bp region missing in the shorter fragment appears twice in the longer fragment indicating that the variant came about through a duplication event not a deletion.
In summary, a primer pair has been created for use in Sus scrofa, which targets
GPM6B and a section its Y-chromosome homolog that contains deletion regions amounting to a 43 bp or 27 bp difference in length. The section of GPM6B amplified contains a 16 bp deletion variant (rs1111696537) in a portion of the species. This deletion variant is not large enough to complicate PCR sex determination so long as male and female positive control samples are also run on the gel. Altogether, this indicates that the
GPM6B targeting primer pair designed is viable in PCR based sex identification of Sus scrofa.
63
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