Paternal transmission of mitochondrial DNA in ...

Paternal transmission of mitochondrial DNA in interspecific crosses of Drosophila simulans

Mohammad Michael Nafisinia

A thesis submitted in fulfilment of the requirement for Master of Philosophy (MPhil)

Supervisor: Professor Bill Ballard Co-supervisor: Associate Professor Andrew Brown

School of Biotechnology and Biomolecular Sciences The University of New South Wales, Sydney, Australia

2011

Abstract Arguably, the three central pillars mitochondrial DNA (mtDNA) evolution that make it a powerful tool for evolutionary studies are maternal inheritance, lack of recombination and high copy number. While it is likely that these three pillars cooccur in most higher eukaryotes it is important to consider mechanisms causing these mainstays to fail. One such mechanism causing failure of strict maternal inheritance is paternal leakage. The occurrence of paternal leakage followed by its transmission to offspring may bias the interpretation of mtDNA as a molecular marker by introducing additional haplotypes into the mtDNA pool of a single population and create individuals with more than one type of mtDNA (heteroplasmy). The presence of paternal mtDNA can potentially affect a variety of disciplines including evolutionary genetics, molecular ecology, biogeography, mitochondrial medicine, and forensic science. Here, I examined the frequency of paternal mtDNA transmission in intraspecific crosses of Drosophila simulans harbouring distinct mtDNA haplotypes. First I optimized two primer sets. In initial optimization studies I could detect as little as 0.1% paternal mtDNA in an individual. Second I assayed a total of 33 individuals from each of the 62 intraspecific crosses. Two crosses and six individuals present strong evidence for mtDNA paternal leakage indicating the paternal leakage was in the order of 0.3%. The main limitations of this study were the detection levels of the specific primers and the need to complete the reciprocal cross to corroborate the results presented. This experiment clearly showed the notable contribution of paternal mtDNA leakage to the inheritance of mitochondria. Further study regarding estimation of sperm/oocyte content and the mechanisms leading to elimination of paternal mtDNA such as ubiquitination of sperm mitochondria in different species may lead to better understanding of this mechanism.

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Acknowledgements First and foremost, my sincere gratitude goes to my supervisor, Professor Bill Ballard in helping me shape my thoughts and ideas to come up with this thesis. His wise and expert guidance, timeless effort, vast academic knowledge, and consistent patience throughout my entire study are invaluable. My special words of thanks and respect go to my co−supervisor, Associate Professor Andrew Brown. I’m also grateful to Dr Jonici Wolff. His sparkling ideas, valuable suggestions and technical support have made this project feasible. I would also like to thank my committee members in the University of New South Wales Associate Professor Bill Sherwin and Associate Professor Alan Wilton, who graciously agreed to serve on my committee.

My deepest thanks go to Dr. Martin Horan, Kylie Cairns, Carolina Correa, and Pann Pann Chung for reading my thesis. I also thank my special study friend Wen Chyuan Aw and the people in Wilton labs.

Last but not least, I would like to thank my beautiful lady Maryam and my children Setare and Dana for inspiring and supporting me. Thank you so much!!!

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Table of Contents Abstract …………………………………………………………………………..i Acknowledgements ……………………………………………………………...ii Table of Contents ……………………………………………………………….iii List of Figures ……………………………………………………………………v List of Tables ……………………………………………………………………vii List of Abbreviations …………………………………………………………..viii

Chapter 1: Introduction …………………………………………………………1

Chapter 2: Materials and methods …………………………………………….11 2.2.1 Fly Lines and crosses …..…………………………………..........12 2.2.2 DNA extraction .............................................................................13 2.2.3 Allele Specific Polymerase Chain Reaction (AS-PCR) ………....14 2.2.4 Determining frequency of paternal leakage………………………….18 2.2.5 Confirmation of the suggested cases of mtDNA paternal leakage...19 2.2.6 Calculating the frequency of paternal leakage……………………….20 2.2.7 Testing for NUMTs ………………………………………………20

Chapter 3: Results ……………………………………………………………...21 3.3.1 Allele Specific Polymerase Chain Reaction (AS-PCR)……………...22 3.3.2 Determining frequency of paternal leakage……………………………27 3.3.3 Confirmation of mtDNA paternal leakage………………………………30

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3.3.4 Calculation the maximum frequency of the paternal leakage……....30

Chapter 4:

Discussion …………………………………..…………………….. 33

References ……………………………………………………………….....…….. 42 Appendix 1 ………………………………………………………………..……….. 50 Appendix 2 ……………………………………………………………………..….61 Appendix 3 ………………………………………………………………………… 64 Appendix 4 ………………………………………………………………………… 71 Appendix 5 ………………………………………………………………………… 77

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List of Figures

Figure 2-1

Predicted primer locations for two AS-PCR assays ……………….16 distinguishing flies harbouring distinct mtDNA haplotgroups of siII (2-KY0418) and siIII (3-KY0410)

Figure 3-1

PCR dilution test with different ratios of experimentally ………….23 mixed 3-KY0410: 2-KY0418 amplified with Dean primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe

Figure 3-2

Complementary PCR dilution test with different ratios …………....24 of experimentally mixed 3-KY0410: 2-KY0418 amplified with Dean primer set and visualized after electrophoresis using a1.5% agarose gel stained with Sybrsafe

Figure 3-3

PCR dilution test with different ratios of experimentally ………….25 mixed 3-KY0410 : 2-KY0418 amplified with 1432+/231 &1793- primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe

Figure 3-4

Complementary PCR dilution test with different volume ………….26 ratios of 1/1000, 1/2000, 1/4000, 1/8000, and 1/10000, of experimentally mixed 3-KY0410: 2-KY0418 with 1432+ /2314- &1793- primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe

Figure 3-5

Evidence for paternal leakage using Dean primer set

Figure 3-6

Evidence for paternal leakage using primer set 1432+ …………....29

……………28

/2314- &1793-

Figure 3-7 Chromatograms belonging to the cases 4:1 and 4:4 at nucleotide ……32 position 5269 show one replacement case (4:1) and one heteroplasmic case (4:4) in comparison to their parents. Figure A3.1 Results from the Dean primer (after optimization of dNTPs) ……66 on 50/50 mixture 3-KY0410 and 2-KY0418 mtDNA template. Figure A3.2 Optimization of the 1432+/2314- & 1793- primer set using MgCl2...68 gradient (1.5 mM (A), 2.0 mM (B) and 2.5 mM (C)) in temperature gradient 55°C -70°C (55, 57.4, 59.2, 61.4, 63.9, 66.1, 68.9 and 70 °C ) on 3-KY0410 mtDNA template. v

Figure A3.3 Optimization of the 1432+/2314- & 1793- primer set using ……….69 MgCl2 gradient (1.5 mM (A), 2.0 mM (B) and 2.5 mM (C)) in temperature gradient 55°C -70°C (55, 57.4, 59.2, 61.4, 63.9, 66.1, 68.9 and 70 °C ) on 2-KY0418 mtDNA template. Figure A3.4 Optimization of the 1432+/2314- & 1793- primer set using primer….70 2314- gradient (3-(A), 5- (B), and 10 (C) pmol) in temperature gradient 55°C -70°C (55, 57.4, 59.2, 61.4, 63.9, 66.1, 68.9 and 70 °C ) on 2-KY0418 mtDNA template. Figure A4-1

PCR dilution test with different ratios of experimentally …………73 mixed 2-KY0418: 3-KY0410 amplified with Dean primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe

Figure A4-2 Complementary PCR dilution test with different ratios of …………74 experimentally mixed 2-KY0418: 3-KY0410 amplified with Dean primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe Figure A3-3

PCR dilution test with different ratios of experimentally ………….75 mixed 2-KY0418: 3-KY0410 (maternal/paternal) amplified with 1432+/2314-&1793- primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe

Figure A4-4

Complementary PCR dilution test with different volume …………76 ratios of 1/1000, 1/2000, 1/4000, 1/8000, and 1/10000, of experimentally mixed 2-KY0418: 3-KY0410 with 1432+/2314- &1793- prime set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe

Figure A5-2. Chromatogram comparison between a replacement case and a…….…80 heteroplasmic case belong to 4:4 against their parents in nucleotide position 1712.

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List of Tables

Table 2.1

Primer sequences that were used for differentiation of flies…………16 harbouring distinct mtDNA haplogroups of siII (2-KY0418) and siIII (3-KY0410).

Table 3.1

Intraspecific variation in two positive cases of mtDNA paternal………31 leakage in comparison with their parents and the reference sequences.

Table 3-2

Chromatogram comparison between replacement mtDNA cases……...32 and heteroplasmy mtDNA case belong to 4:4 against their parents in nucleotide position 5267

Table A5.1 Intra-specific variation in six positive cases of mtDNA paternal……...79 leakage in comparison with their parents and reference sequences.

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List of Abbreviations bp

base pair

C

Celsius

cm

centimeter (10-2)

d

day

DNA

deoxyribonucleic acid

g

gram

h

hour

L

liter

μg

microgram (10-6)

μL

microliter (10-6)

MgCl2

magnesium chloride

M

molar (mole per liter)

mg

milligram (10-3)

mL

milliliter (10-3)

mM

milimolar (10-3)

mm

millimeter(10-6)

min

minute

mtDNA

mitochondrial deoxyribonucleic acid

ng

nanogram (10-9)

nmol

nanomole (10-9)

pmol

picomole (10-12)

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1 Introduction

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The focus of this thesis is to study one of the central pillars of mitochondrial DNA (mtDNA) evolution. Arguably, mtDNA has three evolutionary pillars that make it a powerful tool for studies of evolutionary genetics and phylogeography (McBride et al. 2006, Bromilow and Sperling 2011). These central pillars include maternal inheritance, lack of recombination and high copy number in cells (Ingman et al. 2000, Lee et al. 2006, Gissi et al. 2008). While it is likely that these pillars of mitochondrial evolution co-occur in the majority of organisms it is important to consider when and where they fail to comply with the generally accepted dogma. The aim of this thesis is to test the assumption of maternal inheritance and study the rates of paternal leakage in a common fly.

The assumption of maternal inheritance influences a variety of parameters inferred from mtDNA including mutation rates and divergences times (Posada et al. 2002, Rokas et al. 2003, Gantenbein et al. 2005, Lanier and Olson 2009, Brandley et al. 2011). The occurrence of paternal leakage also creates individuals with more than one type of mtDNA (heteroplasmy) (Rokas et al. 2003) and potentially enables recombination between distinct mtDNA haplotypes (Eyre-Walker 2000, Rokas et al. 2003). Accordingly, the co-occurrence of paternal leakage with mtDNA recombination may bias the interpretation of data gathered from mtDNA unless its influences is fully understood (Saville et al. 1996, Tsaousis et al. 2005, Wolff et al. 2008).

Uniparental transmission is the commonly accepted mechanism of mtDNA inheritance in eukaryotes and a majority of studies have failed to detect paternal mtDNA (Dawid and Blackler 1972, Hutchison et al. 1974, Hayashi et al. 1978, Kroon

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et al. 1978, Avise et al. 1979, Francisco et al. 1979, Giles et al. 1980, Reilly and Thomas 1980). In the case of uniparental inheritance mtDNA is inherited maternally and transmitted homoplasmically (St John and Schatten 2004). A consequence of this uniparental maternal transmission is that mtDNA genes are selected to increase female fitness whereas nuclear genes are selected to increase male and female fitness equally (Hoekstra 2000, Ballard and James 2004, Fujita et al. 2007, Frank and Crespi 2011).

Failures to detect paternal mtDNA cannot completely exclude the potential presence of paternal leakage due to the low sensitivity of techniques used and the small sample sizes tested (Kondo et al. 1992, Kaneda et al. 1995). Biparental transmission occurs when mtDNA is transmitted from both parents to offspring (Schwartz and Vissing 2002). Paternal mtDNA has been detected in the offspring of at least 15 species with the rate of biparental mtDNA inheritance varying widely. The most extreme case of bi-parental transmission of mtDNA occurs within species of the families Mytilidae (sea mussles) and Unionidae (fresh water mussles). Within these species mtDNA is transmitted from father to sons and from mother to both sons and daughters (Dalziel and Stewart 2002, Kenchington et al. 2009, Kyriakou et al. 2010). This system of biparental mtDNA transmission is termed doubly uniparental inheritance (DUI) (Sano et al. 2010, Ghiselli et al. 2011). Other species where paternal mtDNA has been detected in offspring include mammals (Gyllensten et al. 1991, Magoulas and Zouros 1993, Shitara et al. 1998, Kajander et al. 2001, Schwartz and Vissing 2002, Piganeau et al. 2004, Zhao et al. 2004, Zsurka et al. 2005), fish (Magoulas and Zouros 1993, Hoarau et al. 2002, Ciborowski et al. 2007, Guo et al. 2007), birds (Kvist et al. 2003), reptiles (Ujvari et al. 2007), arthropods (Kondo et al. 1990, Meusel and Moritz 1993,

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Gantenbein et al. 2005, Arunkumar et al. 2006, Sherengul et al. 2006, Fontaine et al. 2007), flatworms (Lunt and Hyman 1997, Armstrong et al. 2007) and molluscs (Zouros and al. 1992, Ladoukakis and Zouros 2001, Theologidis et al. 2007). Apart from mussels (Dalziel and Stewart 2002, Kenchington et al. 2009, Kyriakou et al. 2010) and a case in an exercise intolerant human (Schwartz and Vissing 2003), the majority of studies have employed backcrossing to estimate the frequency of paternal leakage (Gyllensten et al. 1985, Kondo et al. 1990, Shitara et al. 1998, Sherengul et al. 2006). This strategy enables the accumulation of paternal mtDNA such that it is more detectable (Kondo et al. 1992, Kaneda et al. 1995). However, it pools two important and distinct evolutionary parameters: the rate of paternal leakage and the rate of loss of paternally acquired mtDNA. In this study, I quantify paternal mtDNA in the multiple F1 adults.

Additional studies have detected paternal leakage but the frequency was not robustly estimated due to the small sample size tested (Kondo et al. 1990, Gyllensten et al. 1991, Magoulas and Zouros 1993, Meusel and Moritz 1993, Shitara et al. 1998, Hoarau et al. 2002, Schwartz and Vissing 2002, Kvist et al. 2003, Zhao et al. 2004, Sherengul et al. 2006, Ciborowski et al. 2007, Fontaine et al. 2007, Guo et al. 2007, Theologidis et al. 2007). Samples of at least 300 progeny are required to estimate its frequency to 1% accuracy (Milligan 1992). In combination, these data strongly suggest that the frequency of paternal leakage is greater than currently estimated. In this theses tested a total of 33 individuals from each of the 62 intraspecific crosses for paternal mtDNA.

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Paternal leakage of mtDNA The major theoretical hypothesis that has been proposed to explain why mtDNA is most frequently inherited maternally focuses on the idea of selfish mtDNA – that is, uniparental mitochondrial inheritance is a system evolved by the nucleus to ensure mitochondrial quality (Birky 2001, Goddard et al. 2006, Agnati et al. 2009). The theoretical difficulty with this hypothesis is that the population must be polymorphic for mitochondria varying in degrees of selfishness in order to select for nuclear modifiers of mitochondrial inheritance, and in simple models the spread of selfish mitochondria does not easily evolve to a stable polymorphism – either they are lost or go to fixation (Burt and Trivers 2006).

The machineries that prevent paternal mtDNA transmission in animals are not well understood but four mechanisms are proposed. The first mechanism hypothesized to prevent paternal mtDNA transmission is the ubiquitination of paternal sperm mitochondria upon fertilization (Cummins 2000, Sutovsky et al. 2000, Sutovsky 2003, Thompson et al. 2003, Hayashida et al. 2005, Ciborowski et al. 2007). Ubiquitination is a universal marker of proteolysis and protein recycling in the cytoplasm of eukaryote cells from plants to mammals where the protein molecule of ubiquitin binds to the target and labels it for degradation (Fried and Smith 1989, Hershko and Ciechanover 1998). Three enzymes are involved in activation of ubiquitin molecules; ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and the ubiquitin protein ligase (E3) (Myung et al. 2001). In mammals, it is hypothesized that the recognition of labelled paternal mtDNA breaks down in hybrid zones and in cases where the paternal and maternal mtDNA haplotypes diverge by more than about 2% (Sutovsky et al. 2000, Sherengul et al. 2006). The role of

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ubiquitination in non-mammalian species has yet to be confirmed by independent investigation.

The second hypothesis proposes that high sperm motility causes oxidative damage to mtDNA damage resulting in the accumulation harmful mutations by the time that sperm reach the egg (Rand 1994, Kumar et al. 2009, Venkatesh et al. 2009). Conversely, maternal mtDNA is less likely to be damaged and more likely selected (Bromham et al. 2003). This hypothesis intrinsically assumes that a mechanism for detecting and excluding damaged mtDNA exists in the zygote.

A third mechanism proposed to limit paternal leakage of mtDNA is the presence of genetic bottleneck that may eliminate paternal mtDNA as minor allele contributions (Cummins 2000, Shoubridge and Wai 2007, Wai et al. 2008, White et al. 2008). Despite a high mutation rate and the high amount of mtDNA in majority of cells, which leads to accumulation of mutations (and therefore creation of heteroplasmy) over time, heteroplasmy is lower than expected in most species. Hauswirth and Laipis (1982) confirm this inconsistency by showing a rapid shift in mtDNA variants between heteroplasmic Holstein cows indicating replacement of single haplotypes within a few generations in some individuals. This classic study led to the proposal that mtDNA is subject to a genetic bottleneck during early embryogenesis, oogenesis and/or postnatal oocyte maturation (Hauswirth and Laipis 1982, Jansen and de Boer 1998, Cao et al. 2009). The bottleneck is also thought to act as a mechanism against the accumulation of deleterious mutations.

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A fourth mechanism proposed for the maternal inheritance of mtDNA is simply the dilution of paternal mtDNA in the shaft of the sperm by maternal mtDNA in the zygote (Shitara et al. 1998, Cummins 2000, Schwartz and Vissing 2003, White et al. 2008, Wolff and Gemmell 2008, Moriyama and Kawano 2010). To my knowledge, estimates for the dilution effect are limited to studies in human, mouse and teleost but is likely to be taxon specific relating to such factors as gametal investment, gametal shape and reproductive pattern such as internal and external fertilization (Hecht et al. 1984, Shitara et al. 2000, Steuerwald et al. 2000, Reynier et al. 2001, Barritt et al. 2002, May-Panloup et al. 2003, Chan et al. 2005, May-Panloup et al. 2005, Santos et al. 2006, Amaral et al. 2007, Wolff and Gemmell 2008). The paternal to maternal mtDNA ratio is between 1:5.68x105 and 1: 2.84x104 in human zygotes, 1:1.6x104 to 1:2.1x103 in mouse zygotes and 1:7.35x108 ± 4.67x108 in salmon zygotes (Wolff and Gemmell 2008).

If sperm mitochondria successfully enter the oocyte without any degradation or elimination paternal leakage of mtDNA may occur, however, its frequency may change during development (Meusel and Moritz 1993, Kaneda et al. 1995, Fontaine et al. 2007). For example, Kaneda et al. (1995) showed that paternally derived mtDNA can only be detected in early pronucleus stage in intra-specific hybrids of Mus musculus while it was detectable in 54- 72% of various developmental stages to neonates in inter-specific hybrids between Mus musculus and Mus spretus. Meusel and Moritz (1993) performed a paternal leakage experiments during developmental stages of Apis mellifera carnica × Apis mellifera capensis using autoradiographs. They detected up to 27% paternal mtDNA in 12 hours fertilized eggs but it slowly decreased until hatching of the larvae to 2.4 ±1.6% (Meusel and Moritz 1993).

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Conversely, Fontaine et al. (2007) failed to detect any paternal mtDNA in 1 day old eggs but paternal leakage was successfully detected from other developmental stages.

In the following section I review the choice of the model organism selected for my study. Drosophila simulans was selected because heteroplamic flies have been collected in nature (Satta et al. 1988, Matsuura et al. 1991, Dean et al. 2003) and three previous studies have previously documented the presence of paternal leakage (Kondo et al. 1990; Kondo et al. 1992, Sherengul et al. 2006). In this thesis I improved upon previous studies by testing for paternal leakage in the F1 offspring of specific crosses using two Allele Specific polymerase chain reaction (AS-PCR) sets and then corroborate the results by DNA sequencing. Further I assay a total of 2,046 flies so that the frequency of paternal leakage can be accurately quantified.

Drosophila simulans as the experimental model D. simulans is a cosmopolitan species with three distinct mitochondrial haplogroups, siI, -II, and –III, that differ by 3% (de Stordeur et al. 1989, Ballard and James 2004). The siI mitotype is an island endemic while the siII type has a cosmopolitan distribution. Flies with siIII mtDNA occur in east Africa and Reunion Island where they are sympatric with flies harbouring siII mtDNA.

Heteroplasmy has been documented in field-collected flies. In these flies the frequency of heteroplasmy is a function of the rate of paternal leakage and the maternal transmission of heteroplasmy. The initial studies suggested that 6-12% of individuals collected from east Africa and Reunion were heteroplasmic and harboured siII and siIII mtDNA (Satta et al. 1988, Matsuura et al. 1991). Employing AS-PCR

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this estimate was raised slightly to 15% (Dean et al. 2003). However, to my knowladge, no studies have quantified whether the levels of heteroplasmy changes over time or space.

Three studies have quantified paternal mtDNA leakage in Drosophila (Kondo et al. 1990, Kondo et al. 1992, Sherengul et al. 2006). Kondo et al. (1990) performed both intra- and inter-specific paternal leakage studies. In the inter-specific study they crossed 331 D.simulans × D. mauritiana lines reciprocally for ten generations. They observed paternal leakage in four lines and reported 0.1% of inter-specific paternal leakage (Kondo et al. 1990). Among 100 intra-specific crosses between D. simulans flies harbouring siIII and those with siII mtDNA no paternally derived mtDNA was detected using southern blotting and hybridization with a reported detection limit of 0.03% (Kondo et al. 1990). Kondo et al. (1992) used PCR to increase the sensitivity of their assays and reported a detection limit of this their technique to be 0.017%. Sherengul et al. (2006), employed specific primers with sensitivity in the range of 10-3 to 10-6 to quantify paternal leakage in two intra-specific and three three inter-specific crosses. More than 100 offspring were tested and paternal leakage rate was detected in approximately 20 - 60 % of the backcrosses (Sherengul et al. 2006). A limitation of these studies is that they employed backcrossing to quantify paternal leakage so that it is difficult to accurately determine the rate leakage in each generation and to distinguish that from the maternal transmission rate of heteroplasmy.

MtDNA types in D. simulans have the rank order of fitness siII > siIII> siI. de Stordeur et al. (1997) conducted a series of studies where mtDNA from one mitochondrial type were microinjected into a zygote that naturally harboured a second

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mtDNA type (de Stordeur 1997). In this study, he observed that microinjected siII mtDNA typically outcompeted siIII mtDNA and always outcompeted siI mtDNA. Further

microinjected

siIII

always

outcompeted

siI

mtDNA.

Reciprocal

microinjections confirmed that these data were robust. Ballard and James (2004) corroborated this result and found this same rank order of fitness in perturbation cage experiments. It is clear, however, that siII mtDNA does not out complete siIII mtDNA in all cases (and/ or quickly) as siII / siIII heteroplasmy has been observed to be maintained in two isofemale lines for six years (Satta et al. 1988).

In this thesis females collected from Kenya harbouring the siII mtDNA type were crossed with males harbouring siIII mtDNA. In total, paternal leakage was assayed in a total of 2046 flies using AS-PCR. The presence of paternal mtDNA was detected in 6 individuals from two crosses and the result confirmed by direct sequencing.

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2 Material and Methods

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2.1 Fly Lines and crosses The D. simulans flies used in this study were collected as single females in Nairobi (Kenya) during November 2004 (Dean and Ballard 2005). These flies were 2-KY0418 and 3-KY0410. The 2 or 3 before the hyphen denotes the mtDNA haplogroup (siII or siIII respectively). The 04 refers to the year of collection and the last two numbers indicates the specific line numbers (Melvin et al. 2008). These flies were tested for Wolbachia infection and were not naturally infected.

Flies were maintained in the laboratory at 23◦C, 60% relative humidity, and with a 12 hour light/dark (LD) cycle (Katewa and Ballard 2007). To confirm the identity and purity of experimental flies in each vial, four individual flies from each vial (total of twenty flies from five vials belong to five lines) were subjected to DNA extraction, PCR amplification and DNA sequencing. These sequences were compared to the D. simulans mtDNA reference sequences (Appendix 1) to explicitly distinguish fly lines using single nucleotide polymorphisms (SNPs).

In this study, females of 2-KY0418 were mated with males of 3-KY0410. The reciprocal cross will need to be completed prior to publishing these results. Experimental 2-KY0418 flies were established by placing approximately 100 flies in egg collection bottles (Appendix 2A). Eggs were allowed to hatch and virgin flies were collected within 3 hours after eclosion. Females were stored in vials containing fly food for a week. If larvae were observed in a specific vial all females in that vial were discarded as at least one had mated (Ballard et al. 1996, Bubliy et al. 2001). After 1 week, each virgin female was transferred to a new vial containing a single 3KY0410 male and fresh medium.

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In total, 200 pairs of flies were placed in individual vials (one male and one female in each vial). Each pair was allowed five days to mate. After mating and egg deposition by females, each pair was placed into a 1.5 ml eppendorf tube and stored at -20 ⁰C.

2.2 DNA extraction DNA was extracted in 96 well-plate format (James and Ballard 2003). For each plate, 33 flies belonging to each of 2 crosses were included. The DNA extraction protocol was adapted from the Genera Puregene® Cell Kit (Qiagen Sciences, MD, USA). Briefly, extractions were homogenised and then incubated for 1.5 h with 0.2 µL of 20 mg/mL Proteinase K at 55 ⁰C (Thermo Fisher Scientific Inc, Espoo, Finland) to achieve a complete digest. The samples were cooled to room temperature and 8 µL

protein precipitation solution (Qiagen Sciences, MD, USA) was added to each well. The plate was incubated on ice for 5 min. Extractions were then spun at 4000 rcf for 15 min. The supernatant was immediately pipetted out, without disturbing the pellet, and added to 20 µL of 100% isopropanol in a new 96-well PCR plate. Extractions were sealed, mixed and spun at 4000 rcf for 15 min. Supernatants were discarded and the pellet was washed by adding 20 µL of 70% ethanol. The plate was then spun at 4000 rcf for 15 min. The ethanol was discarded and the plate was allowed to air-dry for 10-15 min. To protect DNA from degradation, the DNA pellet was dissolved in 40 µL 1X TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA) and stored at -20 ⁰C (see

Appendix 2B). The purity and quantity of the extracted DNA was quantified on a Nanodrop® spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, USA).

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2.3 Allele Specific Polymerase Chain Reaction (AS-PCR) The first step for detecting paternal mtDNA leakage was to develop a highly specific and sensitive AS-PCR assay. In this study, I include two sets of primers. This protocol serves three purposes. First, it can be used to independently corroborate the results for one primer set. Second, independent primer sets may have different efficiencies and the detection of paternal mtDNA by one set and not another has the potential to give insight into the ratio of maternal compared with paternal mtDNA. Third, different results between primer sets have the potential to be a first indicator of recombination between different mtDNA types.

The first set of three primers was taken from the study of Dean et al. (2008) (Table 2.1, Fig. 2.1). These primers will henceforth be referred to as the Dean primers. This set of primers has specific forward primers (4726+ & 5183+) and one common reverse primer (5983-). The primer names refer to the 3' end of the primer on the mtDNA genome. In this case the PCR amplicons differ by 457 bp with the siIII specific amplicon being the longer of the two.

I designed a new primer set using the Primer 3 software (Rozen & Skaletsky 1999) (Table 2.1, Fig. 2.1). This primer set has a single common forward primer (1432+) and haplotype specific revers primers (1793- & 2314-). These primers will be referred to as the 1432+/2314- & 1793- primer set. The PCR amplicons in this second set differ by 521 bp. In this case the siII specific band is the larger of the two.

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Table 2.1 Primer sequences that were used for differentiation of flies harbouring distinct mtDNA haplogroups of siII (2-KY0418) and siIII (3-KY0410). Primers

Direction

Sequences (5'-3')

Assay 1: Dean primers 4726+

Foward (3-KY0410, siIII)

TATTTGCTGTATTAAGAACTTTAT

5183+

Foward (2-KY0418, siII)

TTCAGGAGTTACTGTAACC

5983-

Reverse primer

TATTCCTTGATTTCATTCATG

Assay 2: 1432+/2314- & 1793- primer set 1432+

Forward primer

GAATTAGGACATCCTGGAGCAT

2314-

Reverse (2-KY0418, siII)

ATTGAGGAAATTCCCGCTAA

1793-

Reverse (3-KY0410, siIII)

GAGTATCAACGTCTATTCCAACTGTG

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Fig. 2.1 Predicted primer locations for the two allele specific-PCR assays distinguishing flies harbouring distinct mtDNA haplotgroups of siII (2-KY0418) and siIII (3-KY0410).

Product sizes siII

siIII

800 bp

1257 bp

Dean primer set

4726+ siIII 47+

5183+ siII

5983-

1432+/2314- & 1793- primer set

882 bp

1432+

1793siIII

2314siII

16

361 bp

The most straightforward ways of optimizing an AS-PCR with a given set of primers is to change the reaction component concentrations, annealing temperature or time. In this study, the Dean primer set was tested on both 2-KY0418 and 3-KY0410 templates using AS-PCR. Once optimized (see Appendix 3), PCR reactions were carried out in 25 μl reactions containing 30 ng DNA, 4.25 pmol of the primer 4726+, 4 pmol of 5983-, 2.25 pmol of 5183+, 3 mM MgCl2 (New England BioLabs Inc., MA, USA), 1x Crimson polymerase buffer (New England BioLabs Inc., MA, USA), 0.2 mM dNTPs (Bioline, London, UK), and 0.5 U Taq DNA polymerase (New England BioLabs Inc., MA, USA). PCRs were run on a Bio-Rad® Dyad Peltier Thermal Cycler ( Bio-Rad® Laboratories, Hercules, USA) with initial temperature of 95 °C for 2 min followed by 95 °C for 15 sec, 54 °C for 10 sec and 68 °C for 60 sec for 35 cycles. Negative and positive controls were included in all reactions. The negative control contained no added DNA. The positive controls consisted of one well with siII mtDNA only and a second well containing only siIII mtDNA. These positive controls ensured that the AS-PCR reactions were capable of detecting each mtDNA haplotype in each reaction. An additional positive control may have been to use mtDNA mixture that contained half of each haplotype.

The 1432+/2314- & 1793- primer set (Table 2.1) was also optimised for the both 2KY0418 and 3-KY0410 (see Appendix 3). PCR reactions were carried out in 25 μl reactions containing 30 ng DNA, 10 pmol of the primer 1432+, 10 pmol of the primer 1793-, and 3 pmol of the primer 2314-, 2 mM MgCl2 (New England BioLabs Inc., MA, USA), 1x Crimson polymerase buffer (New England BioLabs Inc., MA, USA), 0.2 mM dNTPs (Bioline, London, UK), and 0.5 U Taq DNA polymerase (New England BioLabs Inc., MA, USA). PCRs were subjected to initial temperature of 95

17

°C

for 2 min followed by 95 °C for 20 sec, 57 °C for 20 sec and 68 °C for 60 sec for 35

cycles. Negative and positive controls were included in each set of reactions.

Next, I evaluated the detection limit (sensitivity) of both primer sets using a nine step dilution series. Specifically, extracted DNA from 2-KY0418: 3-KY0410 was diluted in dilution ratios: 1:0 (control for 2-KY0418) , 1:1, 1:5, 1:10, 1:20, 1:100, 1:1000, 1: 10000, 0:1 (control for 3-KY0410). In an independent dilution series the reciprocal was tested. Extracted DNA from 2-KY0418: 3-KY0410 was diluted in dilution ratios: 0:1 (control for 3-KY0410), 1:1, 5:1, 10:1, 20:1, 100:1, 1000:1, 10000:1, 1:0 (control for 2-KY0418). A subsequent dilution series was then performed to more accurately determine the sensitivity of each primer set. This study assumes that the proportion of mtDNA in each total DNA extraction from each of the two experimental lines was the same.

After performing PCR amplification on the serial dilutions, visualization of PCR products were run on a 1.5% agarose gel stained with Sybrsafe (Invitrogen Australia Pty Ltd., Mulgrave, Australia). Gels were photographed with the Gene genius transilluminator (Syngene Pty Ltd., Maryland, USA). These gels determined the maximum detection power of each primer set to detect both mtDNA types and can be used to estimate the levels of heteroplasmy in experimental flies.

2.4 Determining frequency of paternal leakage Following determination of the sensitivity of each primer set flies from the experimental crosses were assayed. To avoid any bias in sampling small numbers of offspring from a specific cross, I focus on the 62 pairs that produced above 30

18

progeny in this thesis. A total of 33 offspring were collected from each of these 62 crosses (total of 2046). For clarity each fly is denoted by the nomenclature cross: individual. Thus 2:13 is cross number 2 and individual 13.

2.5 Confirmation of the suggested cases of mtDNA paternal leakage To confirm the occurrence of paternal leakage, all of the positive cases of heteroplasmy inferred from AS-PCR, along with their parents, were sequenced using the Dean common primer (5983-) and the Dean specific primer for 3-KY0410. There are 11 diagnostic nucleotide positions between 4726+ and 5983- in the mtDNA that can distinguish between siII and siIII mtDNA (see Table 2.1). The prediction is that the peaks will be intermediate in flies exhibiting heteroplasmy and show the pattern of from 3-KY0410 if paternal replacement occurs.

The DNA templates were purified using ExoSAP-IT® (USB Amersham, Buckinghanshire, UK) to remove un-incorporated dNTP's and excess primers from PCR products (Steuerwald et al. 2000, Mueller et al. 2009). ExoSAP reactions were carried out in 7 µL volume, containing 2 µL enzyme and 5 µL of PCR product. Reactions were incubated at 37 °C for 45 min, followed by inactivation at 80 °C for 15 min on a Bio-Rad® Dyad Peltier thermal cycler (Bio-Rad® Laboratories, Hercules, USA).

Sanger sequencing was applied to purified template in both directions using Big Dye® terminator chemistry (Applied Biosystems, CA, USA). Reactions were carried out in 10 µL containing 15-20 ng template and 3 pmol primer 5983-. Sequencing reactions were subjected to 96 ⁰C for 30 sec followed by 96 ⁰C for 10 sec, 50 ⁰C for 5 19

sec, and 60 ⁰C for 4 min for 25 cycles on a Bio-Rad® Dyad Peltier thermal cycler (Bio-Rad® Laboratories, Hercules, USA). Sequencing reactions were submitted to the Ramaciotti Centre (University of New South Wales, Australia) where they were run on an ABI 3730 DNA Sequencer (Applied Biosystems Inc., Foster City, USA). All DNA extractions suggesting possible cases of paternal leakage were aligned against their parental sequences and the reference sequences of 2-KY0418 and 3-KY0410 (Appendix 1) using Sequencher (Gene Code Corp., Ann Arbor, USA).

2.6 Calculating the frequency of paternal leakage The frequency of paternal leakage was calculated using basic maths. The total number of flies tested was A, and the total number of flies showed paternal leakage was B. The percentage of mtDNA paternal leakage in this study (X %) then was calculated using simple equation of X % = B/A × 100.

2.7 Testing for nonfunctional nuclear copies of mitochondrial genes (NUMTs) One alternate explanation for the detection of paternal mtDNA is that the primers could be amplifying nonfunctional nuclear copies of mitochondrial genes (NUMTs). To check whether the primers were amplifying mtDNA and not NUMTs, the paternal and maternal mtDNA representative sequences from intraspecific crosses were blasted against the D.simulans autosomal sequence. All sequences corresponded exactly to the mtDNA and none matched with any sequence in the nuclear genome.

20

3 Results

21

3.1 Allele Specific Polymerase Chain Reaction (AS-PCR) In this thesis, I aimed to test the frequency of paternal leakage of mtDNA from 3KY0410 males when mated to 2-KY0418 females. As such, I present the sensitivity test results when 3-KY0410 DNA is diluted by 2-KY0418 DNA. The reciprocal dilution is documented in Appendix 4.

For the Dean primer set, the 1/100 dilution of 3-KY0410 to 2-KY0418 was the maximum detection limit of primers (Fig. 3.1). A second intermediate dilution series was conducted to further investigate the sensitivity of this primer set: 1/100, 1/200, 1/400, 1/800 and 1/1000. These results show the maximum power limit of 1/100 for detecting 3-KY0410 mtDNA when it is diluted by 2-KY0418 (Fig. 3.2).

The maximum detection power of the 1432+/2314- & 1793- primer set was an order of magnitude greater as shown by a dilution series. The detection power of the 1432+/2314- & 1793- primer set was 1/1000 (paternal/ maternal) when 3-KY0410 mtDNA was diluted by 2-KY0418 (Fig. 3.3). A second intermediate dilution series confirmed these results (Fig. 3.4).

22

M

1

2

3

4

5

6

7

8

9

1257 bp 800 bp

Fig. 3.1 For the Dean primer set, the 1/100 dilution of 3-KY0410: 2-KY0418 was the maximum detection limit of experimentally mixed DNA. M: 100 bp ladder; Lane 1: 3-KY0410; Lane 2:

1/1, 3-KY0410: 2-KY0418; Lane 3: 1/10, 3-KY0410: 2-

KY0418; Lane 4: 1/100, 3-KY0410: 2-KY0418; Lane 5: 1/1000, 3-KY0410: 2KY0418; Lane 6: 1/10000, 3-KY0410: 2-KY0418; Lane 7: 1/ 100000, 3-KY0410: 2KY0418; Lane 8: 2-KY0418 only; Lane 9: negative control.

23

M

1

2

3

4

5

6

7

8

1257 bp 800 bp

Fig. 3.2 A second intermediate dilution series confirmed the 1/100 dilution of the Dean primer set was the maximum detection limit of experimentally mixed 3KY0410: 2-KY0418 DNA. M: 100 bp ladder; Lane 1: 3-KY0410 only; Lane 2: 1/100, 3-KY0410: 2-KY0418; Lane 3: 1/200, 3-KY0410: 2-KY0418; Lane 4: 1/400, 3-KY0410: 2-KY0418; Lane 5: 1/800, 3-KY0410: 2-KY0418; Lane 6: 1/1000, 3KY0410: 2-KY0418; Lane 7: 2-KY0418 only; Lane 8: negative control.

24

M

1

2

3

4

5

6

7

8

9

882 bp 361 bp

Fig. 3.3 For the 1432+/2314- &1793- primer set the 1/1000 dilution of 3-KY0410 to 2-KY0418 was the maximum detection limit of experimentally mixed DNA. M: 100 bp ladder: Lane 1: 3-KY0410 only; Lane 2: 1/1, 3-KY0410: 2-KY0418; Lane 3: 1/10, 3-KY0410: 2-KY0418; Lane 4: 1/100, 3-KY0410: 2-KY0418; Lane 5: 1/1000, 3KY0410: 2-KY0418; Lane 6: 1/10000, 3-KY0410: 2-KY0418; Lane 7: 1/ 100000, 3KY0410: 2-KY0418; Lane 8: 3-KY0410; Lane 9: negative control.

25

M

1

2

3

4

5

6

7

8

882 bp

361 bp

Fig. 3.4 A second dilution series using the 1432+/2314- &1793- primer set confirmed the 1/1000 dilution has the highest sensitivity of experimentally mixed 3-KY0410: 2KY0418 DNA. M: 100 bp ladder; Lane 1: 3-KY0410, only; Lane 2: 1/1000 3KY0410: 2-KY0418; Lane 3: 1/2000 3-KY0410: 2-KY0418; Lane 4: 1/4000 3KY0410: 2-KY0418; Lane 5: 1/8000 3-KY0410: 2-KY0418; Lane 6: 1/10000 3KY0410: 2-KY0418; Lane 7: 2-KY0418, only ; Lane 8: negative control.

26

3.2 Determining frequency of paternal leakage AS-PCR amplification of 2046 extracted DNA samples using the Dean primer set identified six possible cases suggestive of paternal leakage (Fig. 3.5). Surprisingly, three cases belong to cross 4 (4:1, 4:2, and 4:3) showed undetectable levels of maternal mtDNA as denoted by a single band size of 1257 bp. Further, one case from 21 (21:1) showed undetectable levels of maternal mtDNA. Two positive cases including one case belonging to cross 4 (4:4) and one belonging to cross 21 (21:2) showed two bands with amplicons 1257 bp and 800 bp indicating heteroplasmy (Fig. 3.5).

The second set of AS-PCR assays were performed using the 1432+/2314- &1793primer set that has an order of magnitude greater sensitivity. These results corroborated the previous results with six cases strongly suggestive of paternal leakage (Fig. 3.6). Again, four cases 4:1, 4:2, 4:3, and 21:1 presented a single band size of 361 bp. This result strongly suggests that the ratio of maternal mtDNA: paternal mtDNA was less than 1:1000. Two cases 4:4 and 21:2 showed double band sizes of 882 bp and 361 bp suggesting heteroplasmy (Fig. 3.6).

27

M 1 2 3

4

5 6 7

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1257 bp 800 bp

Fig. 3.5 Evidence for six cases of paternal leakage using the Dean primer set. M: 100 bp ladder; Lanes: 6, 11, 13 and 14, showed undetectable levels of 2-KY0418 mtDNA and are suggestive of mtDNA replacement by 3-KY0410; Lanes: 7 and 16, show a clear doublet suggest heteroplasmy by presenting two PCR product sizes of 1257 bp and 800 bp; Lanes: 1-5, 8-10, 12, 15 and 17-19 show no evidence of paternal leakage. Lane: 20, negative control; Lane: 21, 3-KY0410 mtDNA control; Lane: 22, 2KY0418 mtDNA control.

28

M 1 2

3

4

5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

882 bp 361 bp

Fig. 3.6 Evidence for six cases of paternal leakage using the 1432+/2314- &1793primer set. Lane: M: 100 bp ladder; Lanes: 6, 11, 13 and 14, showed undetectable levels of 2-KY0418 mtDNA and are suggestive of mtDNA replacement by 3KY0410; Lanes: 7 and 16 suggests heteroplasmy by presenting two PCR products; Lanes: 1-5, 8-9, 12, 15 and 17-19, 2-KY0418 mtDNA (negative cases); Lane: 20, negative control; Lane: 21, 3-KY0410 (control); Lane: 22, 2-KY0418 mtDNA (control).

29

3.3 Confirmation of mtDNA paternal leakage Paternal leakage was hypothesized to occur in two crosses (cross 4 and cross 21). To confirm paternal leakage, the mtDNA was amplified and sequenced from both sets of parents and individuals 4:1, 4:2, 4:3, 4:4, 21:1, 21:2. Sequencing was performed using the Dean common primer (5983- ) and the Dean specific primer for 3-KY0410.

Sequences were aligned against the 3-KY0410 and 2-KY0418 reference sequences (see Appendix 1) using Sequencher. The male parents of each cross were observed to be 3-KY0410 and the female parents 2-KY0418 (Table 3.1). As expected, the four cases (4:1, 4:2, 4:3, and 21:1) suggestive of paternal mtDNA replacement showed the same pattern of DNA changes as the males from 3-KY0410 (Table 3.1). In the two cases suggestive of heteroplasmy two positive cases 4:4 and 21:2 showed chromatograms with two peaks indicating heteroplasmy (Fig. 3.7). No unique singleton mutations were found in any of the flies harbouring paternally derived mtDNA (or their parents). The confirmation of paternal leakage using the common primer 1432+ from the primer set 1432+/2314- & 1793- is documented in Appendix 5.

3.4 Calculation the maximum frequency of the paternal leakage The rate of mtDNA paternal leakage in this study was approximately 0.3% with 6 positive cases from 2046 (6/ 2046 × 100). In total 2 of the 62 crosses showed signs of paternal leakage. Within these two crosses mtDNA paternal leakage rates were 4/33 (12%) and 2/33 (6%).

30

Table 3.1 Intraspecific variation in two positive cases of mtDNA paternal leakage in comparison with their parents and the reference sequences.

Nucleotide positions

5 1 0 4

5 5 5 5 5 2 2 2 2 3 1 5 6 7 0 1 2 9 6 3

5 3 4 3

5 3 8 5

5 5 4 6 1 5 9 2

5 7 0 9

2-KY0418 reference

A

A T C A T

A

A

T C

C

Female from cross 4

.

.

.

.

.

.

.

.

.

.

.

Female from cross 21

.

.

.

.

.

.

.

.

.

.

.

3-KY0410 reference

T

C

G T T

A

C

T C

T

G

Male from cross 4

T

C

G T T A

C

T C

T

G

Male from cross 21

T

C

G T T

A

C

T C

T

G

4:1

(cross 4, case 1)

T

C

G T T

A

C

T C

T

G

4:2

(cross 4, case 2)

T

C

G T T

A

C

T C

T

G

4:3

(cross 4, case 3)

T

C

G T T

A

C

T C

T

G

4:4

(cross 4, case 4)

W

.

Y

B

21:1

(cross 21, case 1)

T

C G T

T C

T

G

21:2

(cross 21, case 2)

.

M K

Y W Y M W Y

Y

.

K Y W W M W Y T

A

C

Sequences were aligned to the 2-KY0418 (siII) and 3-KY0410 (siIII) reference sequences (Appendix 1). Nucleotide positions are relative to reference sequences. Nucleotide codes: Y= C or T; M= A or C; W= A or T; B= C or G; K= G or T. (.): identical to consensus.

31

Fig. 3.7 Chromatograms belonging to the cases 4:1 and 4:4 at nucleotide position 5269 show one replacement case (4:1) and one heteroplasmic case (4:4) in comparison to their parents.

cross 4: Maternal mtDNA

cross 4: Paternal mtDNA

4:1, case of replacement mtDNA

4:4, case of heteroplasmy

32

4 Discussion

33

MtDNA is frequently assumed to be maternally inherited without testing this hypothesis. However, paternal leakage of mtDNA has been documented in at least 15 species spanning a wide range of taxa (Fontaine et al. 2007). Considering the importance of mtDNA in phylogeographic as well as phylogenetic studies it is vital to determine the frequency and mechanism of mtDNA paternal inheritance so that models of mtDNA evolution can be improved.

This study detected 0.3% paternal mtDNA in intraspecific crosses of siII (female) × siIII (male) in D.simulans. Further 3.23 % (2/62) of intraspecific crosses provided evidence of paternal leagake.

The most obvious question is why does paternal

leakage occur in D. simulans? One possible explanation is that increasing genetic differences within or between species might result in an inactivation (or decrease in efficiency) of any mechanism that is responsible for prevention of paternal mtDNA leakage (Fontaine et al. 2007). Species level mtDNA divergences tend to be less than 2% (Hebert et al. 2003) and the genetic differences between the siII and siIII mtDNA haplogroups is reported to be about 1.5% (Ballard 2000). The study by Magoulas and Zouros (1993) supports this hypothesis. Magoulas and Zouros (1993) studied the occurrence of paternal leakage in anchovy fish harbouring distinct mtDNA haplotgroups of AAA and BBC. They observed 3 heteroplasmic fishes (AAA/BBC) out of the 435 indicating 0.69% paternal leakage (Magoulas and Zouros 1993). The sequence divergence between these two haplotypes was reported as about 2.82% (Magoulas and Zouros 1993). If this is the case, the prediction is that paternal leakage would be higher in crosses between the siI and siIII mtDNA haplotypes of D. simulans because the genetic divergence is about 3% (Ballard 2000). This result is observed (Kondo et al. 1992, Sherengul et al. 2006). Currently, the mechanism by

34

which paternal mtDNA is excluded from the egg is not clear and the influence of genetic differences on mechanisms that prevent paternal leakage may be species specific (Pitnick and Karr 1998, Sutovsky 2003, Sakai et al. 2004).

The most surprising and unexpected result obtained in this thesis is that there was an apparent replacement of maternal mtDNA by paternal mtDNA in four cases. This does not exclude the possibility that mtDNA was present; however, it was below the detection limit of my assays. One plausible explanation for this result is that a subset of paternally derived protein: protein interactions must be maintained or the embryo dies. It is well established that four of the five complexes of the electron transport chain have both mitochondrial and nuclear encoded subunits (Scheffler 2002). If a small set of offspring express a specific nuclear encoded allele from the male parent the fly it may only mature and reach adulthood if the protein is interacting with proteins derived from siIII mtDNA. This is an intriguing hypothesis but no nuclear encoded subdivision has been found, to date, between individuals harbouring siII and siIII mtDNA (Ballard et al. 2007). Future studies should aim to rear through the offspring of heteroplasmic flies and flies experimentally demonstrated to have paternal mtDNA to investigate this result.

The experimental approach employed here was different from the three previous studies tested for paternal leakage in Drosophila (Kondo et al. 1990; Kondo et al. 1992, Sherengul et al. 2006) in four main aspects. First, I developed and implemented a highly sensitive and reliable molecular technique of AS-PCR. The protocol I developed had the same, or higher, sensitivity than the previous methods used for detection of paternal leakage (Satta et al. 1988, Kondo et al. 1990, Magoulas and

35

Zouros 1993, Schwartz and Vissing 2002, Kvist et al. 2003, Zhao et al. 2004, Gantenbein et al. 2005, Ciborowski et al. 2007, Guo et al. 2007, Ujvari et al. 2007, Wolff et al. 2008). The studies of paternal leakage in Drosophila have employed different sample sizes and unalike protocols with various detection sensitivities. In this study, 2046 flies were assayed and the detection sensitivity observed was 1% for the Dean primers and 0.1% for the 1432+/2314- &1793-. In Kondo et al. (1990), 3310 flies were tested and the sensitivity was reported to be 0.03%. In Kondo et al. (1992) 1600 flies were assayed and the sensitivity was 0.017%. In Sherengul et al. (2006), 500 flies were assayed and the sensitivity was between 10-3 and 10-6.

A second difference from previous studies was that putative cases of paternal leakage were confirmed with an entirely different set of primers. The two primer sets gave the same results and did not suggest any evidence for recombination among mtDNA types. The two primer sets did, however, have different efficiencies. Indeed, the primer set I designed was the more sensitive and enabled me to report that there was less than 0.1% of maternal mtDNA in four flies.

The third difference between this study and previous ones is that direct sequence comparison of both parents and putative cases suggested by AS-PCR confirmed all cases of paternal leakage. Further, this analysis did not suggest that additional mutations had accumulated in the mtDNA of the flies where paternal leakage was observed.

A fourth difference between this study and previous ones is that paternal leakage was quantified in the first generation intraspecific crosses of D. simulans harbouring

36

different mitochondrial genomes (siII and siIII). This reduced the potential for backcrossing to give biased estimates due to the loss of minor haplotypes by genetic drift (Kaneda et al. 1995, Sutovsky and Schatten 2000, Sherengul et al. 2006).

One additional feature of the experimental design employed in this thesis is that equal numbers of offspring from each cross were assayed. Assaying a large numbers of offspring minimises any potential sampling bias and enables accurate determination of the rates of paternal leakage (Milligan 1992). This further increases the robustness of the experimental design and allows accurate estimates of paternal leakage. In contrast, previous paternal leakage studies examined crosses in a less structured manner (Kondo et al. 1990, Gyllensten et al. 1991, Magoulas and Zouros 1993, Meusel and Moritz 1993, Shitara et al. 1998, Hoarau et al. 2002, Schwartz and Vissing 2002, Kvist et al. 2003, Zhao et al. 2004, Sherengul et al. 2006, Ciborowski et al. 2007, Fontaine et al. 2007, Guo et al. 2007, Theologidis et al. 2007). It is notable that paternal leakage was detected in multiple offspring from two separate crosses. This skewed distribution suggests that a mechanistic explanation is necessary to explain the observed pattern of paternal leakage in this study rather than a simple dilution of paternal mtDNA by maternal mtDNA (Shitara et al. 1998, Cummins 2000, Schwartz and Vissing 2003, White et al. 2008, Wolff and Gemmell 2008, Moriyama and Kawano 2010). However, additional paternal leakage data is required to test this hypothesis.

37

Comparison of mtDNA paternal leakage detected in this study with heteroplasmy levels in nature Clark (1988) constructed deterministic models of heteroplasmy. The models presented suggest stable heteroplasmy can be maintained in populations by the processes of paternal leakage, selection, mutation or any combination of these. When equilibria exist they are globally stable, and the conditions for the existence of equilibria are not stringent (Clark and Lyckegaard 1988).

In East Africa and Reunion heteroplasmy of D.simulans with siII and siIII in the range of 6-15% has been reported (Satta et al. 1988, Matsuura et al. 1991, Dean et al. 2003). MtDNA paternal leakage is one of the possible explanations for heteroplasmy (Magoulas and Zouros 1993, Bentley et al. 2010). I observed that that the average paternal contribution during paternal leakage is about 0.3% in the laboratory. It is not clear whether this same rate of paternal leakage occurs under field conditions where the environment is less stable and food resources more heterogeneous.

A second cause of heteroplasmy in the field is the maternal transmission of multiple mtDNA types. Loss of one mtDNA type is likely to be functionally determined by random genetic drift as well as selection. In the laboratory, heteroplasmy has been maintained in the laboratory for long periods suggesting that the rate of loss of one mtDNA type can be quite low (Solignac et al. 1984, Rand and Harrison 1986, Satta et al. 1988). Evidence for selective differences comes from a tendency for heteroplasmic types to generate an excess of one of the homoplasmic sub-lines that may be temperature dependent. Rand and Harrison (1986) observed that six out of seven of the offspring from a heteroplasmic female had a lower frequency of the larger

38

mtDNA. Matsuura et al. (1993) reported that mtDNA transmission in heteroplasmic Drosophila is temperature dependent. By fitting the data to sampling models, Solignac et al. (1984) estimate that that there are 400 segregating units per egg and that as many as 500 generations are need to get purely homoplastic lines from a heteroplasmic one. These estimates depend on the assumption of no mutation and no selection, since the appearance of mutations and/or selection favouring the heteroplasmy would lead to an overestimation of the number of segregating units.

Potential causes of bias and Limitations I suggest that it is unlikely that contamination biased the results of this thesis. Contamination may cause an artefact that appears to be paternal leakage (Bentley et al. 2010). Contamination may occur at two levels: biological and experimental. Transferring the parents of the flies individually to labelled vials before offspring hatched reduced biological contamination. Subsequently, offspring belonging to each cross were carefully transferred to a second labelled vial. Contamination may also occur in the PCR set-up. In this study positive and negative controls were always included. The presence of heteroplasmy in the female parent may also result in apparent paternal leakage to her offspring (McCauley et al. 2005). To rule this out, two independent regions of the mtDNA from both parents were amplified and sequenced to test for heteroplasmy. No parent showed double peaks in their sequence chromatograms at sites known to differ in the mtDNA genomes. These data suggest that if the flies were heteroplasmic it was below the detection limit.

39

The major limitation of this study is that the reciprocal cross has not been completed yet. The cross of siII (female) × siIII (male) showed 0.3% paternal leakage but the presence of paternal leakage in the reciprocal cross remains unknown. It is expected that the reciprocal crosses of siIII (female) × siII (male) would provide a higher level of mtDNA paternal leakage than current crosses, or at least the same percentage because micro-injection studies performed by Stordeur et al (1989) show the frequency of siII mtDNA tends to increase when the siII cytoplasm was injected into siIII eggs.

A second limitation is that the primers employed had limited sensitivity. The maximum sensitivity of the primers for detection of paternal mtDNA was 0.1% for the 1432+/2314- & 1793- primers and 1% for the Dean primers. The sensitivity of these primers may be increased in a variety of ways including the incorporation of the locked nucleic acids (LNAs) into oligonucleotide primers (Peleg et al. 2009). Incorporation of LNA causes (i) stronger binding strength for corresponding sequences, (ii) extensive mismatch discrimination, and (iii) intensification of duplex formation (Ballantyne et al. 2008).

Conclusion and future studies The frequency and extent of paternal leakage cases clearly indicates that it can no longer be defined as an exception to the general rule of mitochondrial inheritance but rather as an integrated aspect of mtDNA evolution (White et al. 2008). The presence of paternal leakage in some cases might only affect single individuals but in other cases replacement of mtDNA haplotypes may affect entire populations (Hale and Singh 1987, Zouros and al. 1992). Likely, the frequency of paternal leakage depends

40

on a multitude factors including genetic differences in both mtDNA and autosomal DNA within and between species (de Stordeur et al. 1989, Magoulas and Zouros 1993, James and Ballard 2003). D. simulans is an ideal model to investigate the mechanisms behind paternal leakage because the frequency of its occurrence in the laboratory is high. Further study regarding estimation of sperm/oocyte content and the mechanisms leading to elimination of paternal mtDNA such as ubiquitination of sperm mitochondria may lead to a better understanding of this phenomenon.

41

Reference

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Appendix 1

50

>Drosophila simulans, 2-KY0418, >14949 bases 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851

AATGAATTGC TTTCTGCATT ATTTAATAGA ATACACCAAA TTCATACCCC AATTCATCAA TACAGTTACA ATTTGTTATC ACAGAAGCTT TTTGTTATTT AAATTAATGA AAAAGCGGGG TTTAACATGA CTTTAATACT GTAATTTTAT TTTACGAAAA TAAGATCTTT TATTCATTTT ATTTCATTTA AATTTACATT TTAGGATTTT TCAATATTTT TTTTTTATTT AATAATTGAA TTTAATTATA TTTATTTTAT GCTATAAATA AATTGCAGTT TATTTGATTA TAAACTTCAG ATCATAAAGA ATAATCGGAA TGGAGCATTA ATGCTTTTAT TTTGGAAATA CCCGCGAATA TATTATTAGT GTTTACCCAC TTTAGCTATT CTGTAAATTT TTAGATCGAA ATTACTTTTA CAGATCGAAA CCTATTTTAT TATTTTAATT AATCAGGAAA GAAATTCCCG AGTCGGGATA TTATTGCAGT CATGGAACTC TGTATTTTTA CATCAGTAGA CATTATGTTT TCATTGATAT AAAGTCAATT CAACATTTTT AGATGCTTAC CATTATTAGG

CTGATAAAAA CATTGACTGA ATTAAACTAT ATATATTTAT ATTTATAAAG AAATTTTATT TCTAATTCTT TTTTATTCCC CTTTAAAATA TCTTCAATTT ATCTTTTACA CCGCTCCTTT ATAAATGCTT AATTACTTAT CAGTAATTAT CTAATAGCAT AATAATTAGA TATCATTTGT AATCAATTAT ATTTATAAAT TACCAAAATG TTATTATTAA ACGAATTTGT TCATAAAGAT ACTTTTTTTT ATTTTAAGGC AAGAAATTTC TGATATCATT GAATTAGGAC CCACTTAATC TATTGGAACT CATCATTAAG ATCGGAGATG TATAATTTTT AAAATGTGCC AATAATAGAG GAGAAGAATA CTTTATCTGC TTTTCTTTAC TATTACAACT TACCTTTATT TCATTACCAG TTTAAACACA ACCAACATTT TTACCTGGAT AAAAGAAACT CTAAAGGAGG GACGTTGATA TCCTACTGGT AACTTTCTTA TTTACAGTAG TATTATTTTA TATCAATAGG CCTTTATTTA TATTATTATA TAGGATTAGC ACAACATGAA AATTCTATTC

GGATTACCTT TTTATATGTT TTCTAAAAGT ATAAAAAGAT GTTATAGTCC TATTACAATT GGTTAGGAGC CTATTAAGAG TTTTTTAACC TATTAATATT TCCATAATTA CCATTTTTGA TGATATTAAT CTTAATATTA CGGAGCTATC TTTCTTCTAT GAATCAGTTT ATTAACATTT TTTCTTGATT TTTTTATCAT ACTTGTAATC TAATAATAAT TATTCTGCTT AAATATAATT CAATTTTTGG TTTAAGTTAA TTTAAGCCTT ATTGACTATA ATCCTGGAGC AGTGACCGCG TTATATTTTA AATTTTAATT ACCAAATTTA TTTATAGTTA TTTAATATTA TAACAACGTT GTTGAAAACG TGGAATTGCC ACTTAGCGGG GTAATTAATA TGTTTGATCA TATTAGCTGG TCATTTTTTG ATTTTGATTT TCGGAATAAT TTTGGTTCTT AAATTCCCGC CTCGAGCTTA ATTAAAATTT TTCCCCAGCT GGGGGTTAAC CATGATACTT AGCTGTATTT CTGGTTTAAC TTTATTGGAG TGGGATACCT ATGTTGTATC TTTTTTTATA

51

GATAGGGTAA ATTTATAAAG ATCAAAAACT AAGCTAATAA TTTTCTTTTT ATAATTATTG TTGAATAGGT ATAATAATAA CAAGCTTTAG AAAAAATAAT TTATATCAGC TTTCCTAATA AACTTGACAA AATATCTATT GGAGGGTTAA CAATCATTTA GGTTTATTTA ATATTTAATA TGTAAATAGA TGGGAGGATT CAACAATTAA ATCAACTTTA TTATAATAAA AGTAGTAATA ATTATTTATA TAAAACTAAT AGTAAAAATT AGACCTAATA ATGATTTACA ACAATGATTA TCTTTGGAGC CGAGCCGAAT TAATGTAATT TACCTATTAT GGTGCCCCTG TATACCATCT GAGCTGGGAC CACGGTGGAG AATTTCCTCA TACGGTCAAC GTAGTAATTA AGCTATTACT ACCCAGCTGG TTTGGTCACC CTCTCATATT TAGGAATAAT TAAGCTCATC TTTCACTTCA TCAGATGATT ATTTTATGAG AGGAGTTGTT ACTATGTAGT GCTATTATAG ATTAAATAAT TAAATTTAAC CGACGATATT TACTATTGGA TTATTTGAGA

ATCATGCAGT AAGGTTTTAT TTTGTGCATC AGCTACTGGG AATTTTTAAT GGACATTAAT TTAGAAATTA TTTAATATCT CTTCAACTGT TTAAATAATG TTTACTATTA TAATGGAAGG AAAATTGCAC ATTAATTAGA ACCAAACTTC GGGTGAATAC TTTTTTTTTT TTTTTAAATT AAAATTTTAA GCCCCCATTT CATTATGTAA ATTACATTAT TTATTTCGAA CTAATATATA ATTTCTTTAT AACCTTCAAA ACTCCTTCAA AAATTGTCCT ATCTATCGCC TTTTCTACAA TTGAGCTGGG TAGGACATCC GTAACTGCAC AATTGGTGGA ATATAGCATT GTGCTTTCTT AGGATGAACT CTTCAGTTGA ATTCTAGGAG AGGAATTTCA CTGCTTTATT ATATTATTAA AGGAGGAGAC CTGAAGTTTA ATTAGACAAG TTATATTGAG ACATATTTAC GCCACTATAA AGCTACTTTA CTTTAGGATT TTAGCTAATT AGCTCATTTT CAGGTTTTAT AAATGATTAA ATTTTTTCCT CAGATTATCC TCAACTATTT AAGTTTAGTA

2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 5901

TCACAACGAC GTATCAAAAT TAACAAATTA ATATATAAAG ATTTAGGTTT TTTCACGATC ATACTTAATA TACATGGACA TTATTATTTA AATTAATGAA GAAGCTACGA ATTCCAACAA TAACCGAGTA CTGCTGACGT GATGGAACAC AGGTTTATTT TTATACCAAT ATTTCTAGAA TTAAACCATT GTTAAATATA ATTTTTTAAT ATTTTTTCTA CATGCCAGAT CAATAAACTG CTATTTTTAA ATAATTCCAT AAATTCAATT CATCAGGTCA ATTTTATTTA AAGTCATTTA TTATATTATA GTCCCTCAAG AACTATTAGA CTAATATAAT CCTTCTATAT TTTATTAGTA CTTATTTCCT CAAATCACCC GCTATTGGAG ATATGATATG TATATCAATG CATACTTACG TTTATCAGAA GAAGTTTATC ATTATTTCAT ATTAGCTTCA ATAATCATTC GTATATTTTA TATCGCAGAT ATGGAATTCA CGACATTTAA AGCTGCATGA TCACAATTTA AATTAAACTA ATTTGACTTC TCTATTATTT AATTTTAGCT GATCTCCTTT TTTTCTTTAC AGAAATTGCA TTATAATTTG

AAGTAATTTA ACTCCACCAG ATTTCTAATA TATTTTACTT ACAAGATAGA ACGCATTATT TTTATATTAT ACTTATTGAA TTGCTCTTCC CCATCCGTAA ATATTCAGAT ATGAATTATC ATTTTACCTA TATTCATTCT CAGGACGATT TATGGTCAAT TGTAATTGAA ATAATTCTTC TTATAGTAAA TAACATTAGT CCCACAAATA TTACATTTAT TCACCTAAAT AAAATGATAA TTTATCACTT CAATTTACTG TTATTAACTC TAATGGATCT ATAATTTTAT ACTTTAACTT CGGTTGAATT GAACACCTGC AATATTATTC TGCTGGACAT CTTATTTATT TTAGAATCAG GTATTTAGAA TTTTCATTTA CTATAACAAC TCATTATTTT ATGACGAGAT CAGTAACTAT GTTTTATTTT CCCCGCCATT TTAATCCATT GGAGTTCAGG ACAAACTACT CAATACCTCC TCAGTTTATG TGTATTAATT ATAATCACTT TATTGACATT CTGGTGAGGA AATTTATTAA CAATCATAAG TTATTGCTTT TCAATTTTAT TGAATGCGGA GTTTTTTTTT TTAATTTTAC AACTATTACC

TCCAATTCAA CTGAACATAG TGGCAGATTA TTATTAGAAA GCCTCTCCTT AATTTTAGTA TTTTTAATAA ATAATTTGAA TTCTTTACGT CTTTAAAAAG TTTAATAATA AATTGATGGA TAAATTCTCA TGAACAATTC AAATCAAACT GTTCAGAAAT AGTGTTCCTG ATTAGATGAC TTAGCACTTA ATGTCAAACT GCACCAATTA TTTATTTTGT CTAATGAATT CAAATTTATT AATTGATTAA ATTAATACCT TTCATAAAGA ACTTTTATTT AGGATTATTT TATCTTTAGC AATCATACAC TATTTTAATA GACCTGGAAC TTATTATTAA AGTGACATTT CTGTAGCTAT CTTAATGAAG GTGGATTACA TGTATCAGGT TATTAGGTAA GTATCACGAG TGGTTTACGT TTGTAAGATT GAATTAGGAG CCAAATTCCC AGTTACTGTA CAAGGTTTAT AGCTTACGAA GATCAACATT GGAACAACTT TTCAAAAAAT TTGTTGATGT GGATAAGTTA TAATTAATTA GTCTATTAAT ATTAATTTTA CAAAAAAAGC TTTGACCCAA AATTACTATT CTATAATTAT TCTATTATTT

52

TTAAATTCAT ATATTCTGAA GTGCAATAGA ATAAATGTCT TAATGGAACA ATAATTACAG TTATGTAAAT CTATTTTACC TTACTTTACT AATTGGTCAT TTGAATATGA TTTCGATTAT AATTCGAATT CTGCTTTAGG AATTTTTTTA TTGTGGAGCT TAAATCACTT TGAAAGCAAG CTTCTAATGA AAAATTATTA GATGATTATT TCTATTAATT AAAAAACATT TTCTGTATTT GAACATTTTT TCTCGTTATA ATTCAAAACT TTATTTCTTT CCATATATTT TTTACCTTTA AACATATATT CCTTTTATAG ATTAGCTGTT CTCTTTTAGG TTATTAACAG AATTCAATCT TAAATTAATG GTCCATGACC ATAGTAAAAT TATTATTACT AAGGAACATA TGAGGAATAA TTTTTGAGCT CATCATGACC TTATTAAATA ACCTCATAGT TTTTCACAGT TATATTGAAG TTATATAGCA TTTTATTAGT CATCATTTTG AGTTTGATTA ATATTAATTT TCTATATAGT AAATAGTATA TTAATCACAA TTTAATCGAC AATCTTCATC ATTTTTTTAA TATTATAAAA TCATTTTAAT

CTATTGAATG TTACCACTAC TTTAAGCTCT ACATGAGCTA ATTAATTTTT TATTAGTGGG CGATTTCTTT AGCAATTATT TATTAGATGA CAATGATATT TTCATATATA TAGATGTTGA TTAGTGACAG AGTAAAAGTT TTAATCGACC AATCATAGAT TATTAAATGA TATTGGTCTC GAAAAAATTA AATAATTAAT ATTATTTATT ATTATTCTTA AATTTAAATT GACCCTTCAG AGGACTTTTA ATATTGTATG TTATTAGGAC ATTTTCATTA TTACAAGAAC TGATTATGTT TGCTCACTTA TATGTATTGA CGATTAACTG AAATACAGGA CTCAAATTGC TATGTATTTG TCTACACACT ATTAACAGGA GATTTCATCA ATTTTAACAG TCAAGGTTTA TCTTATTTAT TTTTTTCATA TCCTATGGGA CAGCTATTTT CTTATAGAAA TTTATTGGGA CTCCATTTAC ACAGGATTTC ATGTTTATTA GATTTGAAGC TTTTTATATA CATTAATATT ATAAAAGTAT GATAATTTTT CTATTGTTAT CGAGAAAAAA TCGATTACCA TTTTTGACGT TATTCTAATA TTTATTAATT

5951 6001 6051 6101 6151 6201 6251 6301 6351 6401 6451 6501 6551 6601 6651 6701 6751 6801 6851 6901 6951 7001 7051 7101 7151 7201 7251 7301 7351 7401 7451 7501 7551 7601 7651 7701 7751 7801 7851 7901 7951 8001 8051 8101 8151 8201 8251 8301 8351 8401 8451 8501 8551 8601 8651 8701 8751 8801 8851 8901 8951

GGTCTATATC TATATGAATA AAGTATTGAA TGCAGTTAGT AATTGAAGCC AAATTCCAAT TCTTTTAATG AAACCTTACA CTAAAATTAA CAATGTCAAA TTTATTAAAA AGAATTATTA GGTATAAATG TTTACAATTA TCTAATATAA ATATAAACAA TACCCAGATA ATAAATAGGT ATATTCTACC AGTATAATTC ATCTCCAGTT AACCTGTAGA ATTCTAACAA AGGTATCCCG AAGGTATATG TTTATATTAT TAAAGCATGA ATAAAATTCT ATTTTTTTTA AGTTAATCCA AAATAATATT GAAGAATGAA TAATCAAGAA TTATTAATCT AAATAAAAAA AAGAAGAAAA CAGCATTATA ACAAGTCCTA AATAATTAAT ATCGATTAAT ACTAAAGAAG ATCAAAAAGG CTCATTCAAT AAAAAACATA AATTGATAAA CCACAAATTA TGATTCTCTT TTAATAAATA AATAATTTTC TCTAAAAAAA AAACAATTCT GGAGCTGCTA AGGTATAAAA GACGTTCATA CCATGGGCAA AGTTAAAAGT CAATTAAAGC ACACCTCCTA TATTAATTGT TTAATATAAT TGAGCTTTAG

ATTATTCCTT AAGGGTTGTA TATTCAATCT TTCGACCTAA AAAAAGAGGC TAAGGAAGTA GTTAAATTCC TTTTCATTGT TTCATTATAT CTCTAAATAT TTAAAATTCA TGTATTAAAA CTGACCCCCA ATTGGCCATA GGTATAAATC AGATTTATTT AACCCCCCAC AAACAAATTA TCCAATAATT AACTTTCATC ATTGAATAAT AAAATAATAT TTTCTAAAAT CATAAAGCTA AATTCTTAAT GAATAATAGC GTTAATAAAT TATTATTAAA AATCAAATTC GATAATAATA AAATCGAATT CTAAAGCAGA GAAAAAGGAA ACCAATTACT TATAATTTCA GCTACATCTC AGATTTAATA ATCCATCTCA AATATTATTG ATTATTATCA CAATTATAAG AAGGTTATAA AAAATAAATT ATAAACTAAT TATTTCACGA GTATTTTTTT TTTAAAATTA CTCCCGAATT CATGTTGTCT GATAATAATG ATTTAATAAA TATTAGCTGA TTTAATAACC AGATACATTA TTATCAATGT CCTGATAAAA CTTTAAATCA CTAATCTAAT AAAAAAGCAA ACCTGCTAAA GTAATCATAA

GATTTCATTC GTTAATTATA ACCTTATTAA CCTTAGGTAA GTATCACTGT TGGTGATCAA ATTTATACTT AATAATAAAA CCAAAGATTT AAGCTATTTG AAATACAAAT ATAAAGTTTT AAATATTCTG ATTCAAAGGA ATATAGACCC AAAAAATATA AATACATACA TATAAGGAAA CTTATAATTA ATTTAATATA AAACTAATCG AAAAAAAATG TATATCCTTA AATTAGATAC CCCCCTATTA TCCAGCACAT GAAATATAGC CCTAATTGAC AAAATTAGCT ATAATAATTG AATAAATATA AACAGGTGTA TTTGAGCTCT AATATTTCAA TCTTCCATAA CAATTCGATT TTTTGAAAAT TCCTAATAAA ATAAAACAAA TTTCTTATAT AACAAAAGAT CAATTCTTGA ATATTATTTA AGAAATTAAA TCTAAAATGA TAAACTATTT ATAAATTTAA TTTCCTCTTC AAAAGAATAT ATAATATAAT GAAATTTCTC TCTTAATAAA CCTTATTAAT GCTAAACAAA GTAAGAACCA CAATTCCTAT GTTTGACGTA TCTAATTCAA TAACTCGTAA ATTATAGAAC ATGAACTAAA

53

ATGAATTGAT ACATTTGATT TTGAATATGA TTATTATACC TAATGATATA GTAAAAGCTG CTATTTATAT TATTATATTT AATAATCTCC GATATAAAAA AATATTAAAT AGAGTAAGTA ACCAACCTTG TAAAAAATTA TAAAAAAGTT AATTTCTTAA AATAATGTTA AGGAAAAATT ATAATCCTAT TTTAAACTAC AAATGAATAT AAAATATATT GAATAAAATC ATTAAAACAA AACGAATATC ATAAATAATA TAATTTAAAA TTAAAGTAGA CCTAACCCAG CCCTAACCAA CACCAGCTGT GGGGCAGCTA TTTAGTTATA ATTCATTTTG TTTAATATTC AGATAAGGCA AAATAACTAA ATTCTAATTA CATTAATACT ATTCCTTTCT ATAAACAATA AGAATTTAAA ATAAAAAATA TAAATGAATC ATTAATTCAT AAATATAATC AGGTAATCAA TAAATGAATA AAATATAAAG TATAGAAATT CTAATAAATT AATCATCATA TAATATTCTC ATAATCCAGA GATAAACCTC ATGAGCAACA AACAAACTAA ACAAATCTAT TATCCCATAA CAGAAACCGG AATATTGGTA

CAAATTAATT TGCATTCAAA AGCGATTAAT CTTATTCTTT ATTGAATTTT CTAACTTTTT AGTTTAAATA TTATAAATTA GTAACATCTT TAATAAAAAA AAATTTTTAA GATAATTTTT ATCAAAACTT TTCCATAAGT CTTAAATTAT CGAAATTAAA ATAATTTTAT AATCAATTTA TATTCCACGA CACAATTTAA CTAACAGTTA AACATTTCTA CAGCTAAAAA GCTGAAGTTA TTGAGAATTA ATGCTTTAAA AATCCTATAG TAAAGCAATA CTATAAATAT GAAGTTCTTA AACTAAAGTA TAGCTGCTGG GCAGCTAATA TATAATTTCT AAGCAATAGC GTTAATATAC ACAATAAGAA AATTTGGTCT AGTATAATAA ATAAAAAATT AACTTATTCA GAAACTACTT TAAACTTAAT TAATTCTACA ATCACTAACA ATAATATAAA TGTAAAAATA TACTCCAGAA TATAAGCAGC CAAGATCAAG CAATGTAGGA AAGTTATTGA CGACTTCCAA AGAACATAAA AGTAAGTTAT GACGAATAAG TCTAACTAAT ACTTCAAATT CCCCCTAATT AGCTTCAACA TTTTTACTAA

9001 9051 9101 9151 9201 9251 9301 9351 9401 9451 9501 9551 9601 9651 9701 9751 9801 9851 9901 9951 10001 10051 10101 10151 10201 10251 10301 10351 10401 10451 10501 10551 10601 10651 10701 10751 10801 10851 10901 10951 11001 11051 11101 11151 11201 11251 11301 11351 11401 11451 11501 11551 11601 11651 11701 11751 11801 11851 11901 11951 12001

AAAAGCACAT TATTTATTAA ATACCAATTA TACACCCGCC ATAATGTAGG CTTATTCTTG TAATAAAAAT CTAATAATAT GATAATATAT AAAATTATTT GTACCATTCA AAAAAAAAAA TCATTTCCAT TCCTTCACAT AATTTAATAT ATAAATTCTA AAAACAAAAT AAATTATAAT ATCAAAAATA TTTTCATTAA TATTCAATTA TAAATATAAT ATTTTTGTAT ATATATTTTA ATGTAACATC ACTGTATTTT TATTGACAAA CTATTATTAA AAATTATATA TTTATTAATT GACCTATTCG CACCCTTTAT AATTAATATC TAATTATTCA GATATTAATT TTATGGTTGA TTATTTGTAT ATATTCACTC AGGAACAGCT GAGGAGCTAC ATAGATCTAG TTTAACTCGA CTATAACTAT CCCATTGGAT TACATTTAAG CATTAGTATT CCAGCTAATC TTTATTTGCT TCATCGCATT AATTTAAGAA ATTTTGATCT GACCAGTTGA TATTTCTTAT TTTATTAAAT CATAAGATAG TAATAAAGAA TTAAAGAAAA TAACGAAATC AAAAGTTAAT AAATAACACA ATAAAAATTA

AATAAACAAA ATAAAAATTT ATATAGGTAA TGTAAACGTT AATTAATCTT AAAAAGTCAA AAATTTTTAT TAATGAACAA CACAACCTAA ATTAGTAAAA ATATATATTA TAATTTTTAA GAGTACGAAT ACTCTAAATG ATTTAAATAA AACTTAAAAG AATCCTAAAA CATTAGTTTT AGATTATTTC TCCCCAAAAT ATATTATACT TCATCCATTA GTATATTAAC TTTTTAATTT TTTAGCTTCT CTTCATTTAT ACTTCTTCTT TATAAATTCT ATTTTCCTAC ACTTTAATTG AATAATATCT TTAAAATTGC TCAAGATGAT AATTTTAACC TAGCTTTTTA TTATTACGAA TTATTTACAT CAACTTGATT TTTATAGGAT TGTAATTACT TTCAATGATT TTTTTTACAT AATTCATTTA TAAATTCTAA GATATTGTAG AATTAGACCA CTTTAGTTAC TATGCTATTT AGTTTTATCA AATTCCGAGG ATATTAGTTA AGAACCTTAT ATTATTTAGT TAAATAGTTA AATTTAATTT AATAATAAAA AGATAAAAAA GGGGTAAAGT TTTATATAAA AAATAATATT AAGCAAAACC

AATATAATAA ATAGAACCAA GGAAACTAAT CTGGTTGATA CTTTCAAAAA AATTAATAAT AATTATTATA ATTCATAAAC AAAATAAGAA AAATAAATCT TTAATAAAAC CATTATATAA TATAGAAACT TTAAAAATAT ATAAATAATA TATTGAAAGT TAAATAAAAT AATAGTTTAA TTTTAAAACT TAATATTTTA CATTAATTAT GCTTTAGGAT TGGATTAATA TTTTGGGAGG AATGAAATAT TTTGATTTTT CTTTATTTTT TATTTTATAG AAATTTTATT TTGTTGTAAA TAATTAATGA CAATAACGCT GAAATTTTGG GGATTATTTT TAGTGTTAAT CCTTACACGC GTAGGACGAG AATTGGAGTA ATGTATTACC AATTTATTAT ATGAGGTGGG TCCATTTCAT TTATTTCTTC TATTGATAAA GATTTATTGT AATTTATTAG ACCTGCCCAT TACGATCAAT ATCGCAATTC AATTCAATTT CAGTAATTTT GTATTAATTG AAACCCATTA ATGAGCTTGA TCTATTAACT TTTTAAATCC CATTTTCAAG ACCTCGAATT ATAATAAATT CTTATAAATA TCCTCTTCTA

54

ATCATAATTA TTTTATTTAT AAAGTATAAA ACCTCAACCT ATAAATAAAA AATAATAAAA TTTATTAATT TTAATAAAAT ATTTCTGATC AATAAAAAAT AAAGAGGACT TTCTAAAAGA AAAATTGATA TATTCTAAAA TAAAAAATAA AAATGTTTTC TATAGGTAAA TAAAAACATT TCAAGAGAAA AATAAACTAT TACTACTTCT TAACTTTATT ACTAAAAGTT AATACTTGTG TTAATTTATC ATATTAATTT AATAAACAAT AAAATTCTTT ACAATTTTAT AATTACAAAA ATAAACCTTT TTAGTAGATT ATCATTACTT TAGCTATACA CATATTTGTC TAACGGTGCA GAATTTATTA ATTATTTTAT TTGAGGACAA CAGCTATTCC TTTGCCGTTG TTTACCATTT ACCAAACAGG ATTCCTTTTC AATAATTTTT GAGACCCTGA ATTCAACCAG TCCAAATAAA TAATAATCTT TATCCTATTA ATTAACTTGA GACAAATTTT ATTACAAAAT ATAAGCGTAT TTACTAAAAA AATAAAAAAT CTAGATATAT CAAATAAAAA AAATACATCA AAATTCTTGC TATTCTACAT

AATATAAAAT AACATAAAAA ACAATAAATA AAAATCAAAA TATGAATAAT TAACAATATT CTTTCTCTAG TAATCCATAA AATAATTTAT ATTATAATTT TAAAAATACT TTGAAAATAA AACCTAAAGC TAATTTTCGT TATTAAAACA GATTAGATAC CTTCAGTATA GGTCTTGTAA AGAAATTTCT CTCTTGAAAT ATTATTTTTC AATTCAAACA TTTGATATTC TTATTTATTT AATTAAATTA TATCATTTAT GACATACAAT ATCTTTAAAT TAATAAATTA TTATTTAAAG ACGAAATTCT TACCAGCTCC GGGTTATGTT TTATACTGCT GAGATGTTAA TCATTTTTTT TGGTTCATAT TTTTAGTAAT ATATCATTTT TTATTTAGGT ATAACGCTAC ATTGTTCTTG TTCTAATAAC ATCCATATTT ATTTTAATTT TAATTTTATC AATGATATTT TTAGGAGGAG ACCTTTTTAT ATCAAGTAAT ATCGGAGCTC AACTGTTGTA GATGAGATAA GTTTTGAAAA AAATTCATTA AATAAATAAT TAATTTATCA CAAAAGAAAT CATCCTAAAA ATACTCAGCT TAAATCCTGA

12051 12101 12151 12201 12251 12301 12351 12401 12451 12501 12551 12601 12651 12701 12751 12801 12851 12901 12951 13001 13051 13101 13151 13201 13251 13301 13351 13401 13451 13501 13551 13601 13651 13701 13751 13801 13851 13901 13951 14001 14051 14101 14151 14201 14251 14301 14351 14401 14451 14501 14551 14601 14651 14701 14751 14801 14851 14901

AACTAATTCT CAGCTAATGA AAAAATCATA TCCAATTAAA AAGAAATAGT GAATTAGAAG ACAGCATAAA AAAAAGGTAT GGAGAAATAT TTTTGTAAAT AACCAACTTT CGTTCTAATA TAACAAACTT ATTTGTAATA GCCAAAATAG TAATATTTGG ACCAACCTGG GTCGAACAGA CCAACATCGA TTACGCTGTT TCATTTATTC TAATATCACC TATAATTAAA TTAAATAAAT TAAATGAAAC AAAAGACTAA TTAAAATTTT TGTTTTTGTT CTTTTCATAA TTGCTTAATT CAATAAATAA TGCTAATTCT TTATTTTATA AAATAAATAA AAAAACTAGA TTATAAATAA AATCGAGAAA AGGTACAATA TCAATTTTCT TTAAACAATA AAAAAATAAT TGCACAAAAA AAATTGTCAT ATCTTACCTT TAAAATCAAA AAATTTTTTC TTACTTAAAT TATTGAATAT ACTGATTACA AGGTTCCTCT ACATAACTAT ATCTAATCCT CATAATAATT ATTATTAATA GTGGTATAAC TGCTATTTCT TTTTATATTT TAAATCAAAT

GATTCTCCCT AATAGTTAAT TATAAACTTG AAAATAAAAG CTGAGCTACA ATCAACCAGC AAAAATAAAC ACATATTCAA AATATCTTAA AATTTAATTG ATTAGGACCT AAGTTAAAAA CCAATTAATG AAAATTACAT TTTTACATTA TCCTTTCGTA CTTACACCGG CTTAAAATTT GGTCGCAATC ATCCCTAAAG ATAAATTAAT CCAATAAAAT ATAAAAAAAA TTTAGCTTTT AGTTAATATC TGATTATGCT CAGTGGGCAG AAACAGGCGG AAATTAATTT TTATTAATTA TTTAATTTAT AAGCATATAT GCTTATCCCA TTAAGTAAAT TACCTTTAAA TTTTGTTACA AATAAATATT AATTAAATTT TTTACAATAC CTAAAACTAT AAAAATTAAT TCTTTTCAAT TCTAGATACA AATAATAAGA TTATTAATCT ATAATTTTAT ATAAGCTACA TATTATTCTT AACTCAAGTA AGATAGACTA TACTACTTTA AGTTTTTTAT TAAATATAAA AATTTAATTT CGCGACTGCT AAATTTCTTT ATTTTTAAAA TAATAACAAA

CAGCAAAATC CATACTAAAC ATAAAAAAAA ATAACAAAAT GCTCGCAAAC TACTATAACT CTCCTAAATT ACAAATAAAG ATAATTAGAT CATCACAAAA TTACGAATCT AGCTACACTC ATAAAATAAA AAATAAATTC ATAATATTCT CTAAAATATC TTTGAACTCA GAACGGCTAC TTTTTTATCG TAACTTAATT GTTTTTAAAA ATTTTTTTTT ATATAAAGAT TGACTAAAAA TCGTCCAACC ACCTTTGCAC GTTAGACTTT ATATTATTTT TAACATTATT AAATTAACAT AAAATAAATT TTATTAAATT TAAAATATTA TTATAATTTC AACGAATAAC TTAACTTAAA TATTTTTTAT TCTTTTTAAA TAATAAACTA AAATTTTATA AAATAAAAAC GTAAATGAAA CTTTCCAGTA GCGACGGGCG TTATAATTTT CCATATAAAT CCTTGATCTG ATAAAATATT AGGTCCATCG AAATACCGCC GCAATTTATT TAAAATTTTT ATTTCACTTA AATTATTACT GGCACCAATT AATTAATAAA TAAATATATA TTTTTAAGCC

55

AAAAGGAGTT CTATAGGAAA AAATAAATTA TAAAGCTAAA CTCCTAGTAA GTATAAACTC AAATGAATAT ATAAAAATAA AATAAAGGAT AGGTTGAGGA GAATATATCC ACTAATACAC TTCTATATAA TAAATTTATT TATAAAAAAT ACAATTTTTT GATCATGTAA ACCCAAAATT ATATGAACTC TTTTAATCAT ATTAAAAGTT ATTAAAATTT TTATAGGGTC ATAAAATTCT ATTCATTCCA AGTCAAAATA ATATATAATT TGCCGAATTC ATATACTAAT TTTAATAAAT ATAACATATT TATTTAATAT AAATTATAAA TAAATTAAAT ATTTCATTTC TATTATATTA TTAATAAACG TAAAAATTTT TTATTAAAAT GTTATTTCTA TAAATCAATT TACTTTACTT CATCTACTAT ATGTGTACAT ACTACTAAAT AAATTTATTG ATATAAATTT CTGATAACGA TGGATTATCG AAATTTTTTA TACATTTTAA TAACTTCAAT ATATATTTAA AAAAAAATTT TGGTCAATAC ATTAATTACT ATCATACAAA AAAATAAAAC

CGATTAGTTT TAAAATAATT TATTATAACT CTAACTTCAT AGCATAATTA CCAATCTAGT AATTTTACAA AGAAAAAATT AAGTTTGTTC ATTCCTATCA TAAAACTTTT AAATAATTAA AACAAGTACT GCACTAATCT ATAATTATTT AAAGATAGAA GAATTTAAAA ATATCTTAAT TCCAAAAAAA TATTAATGGA TTTTAAATTT AATTAATCTT TTCTCGTCTT ACAAAAATTT GCCTTCAATT CTGCGGCCAT CAAAAAGACA TTTATTTAAA TTTATCATTA AATTAAAATT TTTTAATAAT TTTTAAAAAT TTAATTAATT TTATTTCTTA TAATATAATA ACTCTTTTAA CTGATACACA TCAAATTATT TATTTTTTCT ATAATTAGAA TATATTGATT AATAAGCTTT GTTACGACTT ATTTTAGAGC CCACTTTCAA TAACCCATTA TTATTAAAAT CGGTATATAA ATTACAAAAC AGTTTCAAGA ATAATAGGGT TATATTTTTA TTTTATTATT ATTCGTATTA TTTTTAATAT GCGATTAAAT AATTTACATA TTTAA

>Drosophila simulans, 3-KY0410, >14949 bases 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851

AATGAATTGC TTTCTGCATT ATTTAATAGA ATACACCAAA TTCATACCCC AATTCATCAA TACAGTTACA ATTTATTATC ACAGAAGCTT TTTATTATTT AAATTAATGA AAAAGTGGGG TTTAACATGA CTTTAATACT GTAATTTTAT TCTACGAAAA TAAGATCTTT TATTCATTTT ATTTCATTTA AATTTACATT TTAGGATTTT TCAATATTTT TTTTTTATTT AATAATTGAA TTTAATTATA TTTATTTTAT GCTATAAATA AATTGCAGTT TTATTTGATT CTAAACTTCA AATCATAAAG AATAGTTGGA CTGGAGCATT CATGCTTTTA TTTTGGAATA TCCCACGAAT TTATTATTAG TGTTTACCCA ATTTAGCTAT GCTGTAAATT ATTAGATCGT TATTACTTTT ACAGATCGGA CCCTATTTTA ATATTTTAAT GAATCAGGGA CAAATTCCGG CAGTTGGAAT ATTATTGCAG ACATGGAACT TTGTATTTTT TCATCAGTAG TCATTATGTT TCCATTGATA AAAAGTCAAT TCAACATTTT CAGATGCTTA TCATTATTAG

CTGATAAAAA CATTGACTGA ATTAAACTAT ATATATTTAT ATTTATAAAG AAATTTTATT TCTAATTCTT TTTTATCCCC CTTTAAAATA TCTTCAATTT ATCTTTTACA CCGCTCCTTT ATAAATGCTT AATTTCTTAT CAGTAATTAT CTAATAGCAT AATAATTAGA TATCATTTGT AATCAATTAT ATTTATAAAT TACCTAAATG TTATTATTAT ACGAATCTGT TCATAAAGAT ACTTTTTTTT ATTTTAAGGC AAGAAATTTC TGATATCATT GAATTAGGAC GCCACTTAAT ATATTGGAAC ACATCATTAA AATTGGAGAT TTATAATTTT AGAATAGTAC AAATAATGAG TGAGAAGAAT CCCTTATCTG TTTTTCTTTA TTATTACAAC ATACCTTTAT ATCATTACCA ATTTAAATAC TACCAACATT TTTACCTGGG AAAAAGAAAC CTAATATTAG AGACGTTGAT TTCCTACTGG CAACTTTCTT ATTTACAGTA ATATTATTTT TTATCAATAG CCCTTTATTT TTATTATTAT TTAGGATTAG CACAACATGA GAATTCTATT

GGATTACCTT TTTATATATT TTCTAAAAGT ATAAAAAGAT GTTATAGTCC TATTACAATT GGTTAGGAGC CTATTAAGAG TTTTTTAACC TATTAATATT TCCATAATTA CCATTTTTGA TAGTATTAAT CTTAATATTA CGGAGCTATC TTTCTTCTAT GAATCAATTT ATTAACATTT TTTCTTGATT TTTTTATCAT ACTTGTAATT TAATAATAAT TATTCTGCTT AAATATAATT CAATTTTTGG TTTAAGTTAA TTTAAGCCTT ATTGACTATA ATCCTGGAGC CAGTAACCGC TTTATATTTT GAATTTTAAT GACCAAATTT TTTTATAGTT CTTTAATATT TATCAACGTC AGTTGAAAAT CTGGAATTGC CATCTAGCTG TGTAATTAAT TTGTTTGATC GTATTAGCAG ATCATTTTTT TATTTTGATT TTTGGAATAA TTTTGGTTCT GATTTATTGT ACTCGAGCTT TATTAAAATT ATTCCCCAGC GGAGGATTAA ACATGATACT GAGCTGTATT ACTGGATTAA ATTTATTGGA CTGGAATACC AATGTTGTAT CTTTTTTTAT

56

GATAGGGTAA ATTTATAAAG ATCAAAAACT AAGCTAATAA TTTTCTTTTT ATAATTATTG TTGAATAGGT ATAATAATAA CAAGCTTTAG AAAAAATAAT TTATATCAGC TTTCCTAATA AACTTGACAA AATATTTATT GGAGGATTAA CAATCATTTA GACTTATTTA ATATTTAATA TGTAAACAGA TAGGAGGATT CAACAATTAA ATCAACTTTA TTATAATAAA AGTAGTAATA ATTATTTATA TAAAACTAAT AGTAAAAATT AGACCTAATA ATAAATTTAC GACAATGATT ATTTTTGGAG TCGAGCCGAA ATAATGTAAT ATACCTATTA AGGTGCTCCT TATTCCAACT GGGGCTGGGA CCACGGTGGA GAATTTCTTC ATACGATCAA AGTAGTTATT GAGCTATTAC GACCCAGCTG TTTTGGTCAT TCTCTCATAT TTAGGAATAA ATGAGCTCAT ATTTCACTTC TTTAGATGAT TATTTTATGA CAGGAGTTGT TATTATGTAG TGCTATTATA CATTAAATAA GTAAATTTAA TCGACGATAT CTACTATTGG ATTATTTGAG

ATCATGCAGT AAGGTTTTAT TTTGTGCATC AGCTACTGGG AATTTTTAAT GGACATTAAT TTAGAAATTA TTTAATATCT CTTCAACTGT TTAAATAATG TTTATTATTA TAATGGAAGG AAAATTGCGC ATTAATTAGA ATCAAACTTC GGATGAATAT TTTTTTTTTT TTTTTAAATT AAAATTTTAA ACCTCCATTT CATTATGTAA ATTACATTAT TTATTTCGAA CTAATATATA ATTTCTTTAT AACCTTCAAA ACTCCTTCAA AAATTTGTCC AATCTATCGC ATTTTCTACA CTTGAGCTGG TTAGGACATC TGTAACTGCA TAATTGGTGG GATATAGCAT GTGTCTTTCT CAGGGTGAAC GCTTCAGTTG AATTCTAGGA CAGGAATTTC ACTGCTTTAT TATATTATTA GAGGAGGAGA CCTGAAGTTT TATTAGACAA TTTAATTCAG CATATATTCA AGCTACTATA TAGCTACTTT GCTTTAGGAT TTTAGCTAAT TAGCTCATTT GCAGGTTTTA TAAATGATTA CATTTTTTCC TCAGATTACC ATCAACTATT AAAGTTTAGT

2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 5901

ATCACAACGA GGTATCAAAA TTAACAAATT TATATATAAA AATTTAGGTT TTTTCACGAT GATACTTAAT TTACATGGAC TTTATTATTT AAATTAATGA TGAAGCTACG AATTCCAACA ATAACCGAGT GCTGCTGACG TGATGGAACA CAGGTTTATT TTTATGCCAA AATTTCTAGA CTTAAACCAT AGTTAAATAT TATTTTTTAA TATTTTTTCT ATATGCCAAA TCAATAAACT GCTATTTTTA AATAATTCCA GAAATTCAAT CCATCAGGTC AATTTTATTT CAAGTCACTT TTTATACTAT AGTACCCCAA AAACTATTAG GCTAATATAA ACCTTCTATA CTTTATTAGT GCTATTTGCT TCAAATCACC AGCTATTGGA AATATGATAT GTATATCAAT ACATACTTAT TTTTATCAGA AGAAGTTTAT AATTATTTCA TATTAGCTTC AATAATCATT AGTGTATTTT CTATTGCAGA CATGGAATTC ACGACATTTA CAGCTGCATG ATCACAATTT TAATTAAACT TATTTGACTT TTCTATTATT TAATTTTAGC AGATCTCCAT ATTTTCCTTA TAGAAATTGC ATTATAATTT

CAAGTAATTT TACTCCACCA AATTTCTAAT GTATTTTACT TACAAGATAG CATGCATTAT ATTTATATTA AACTTATTGA ATTGCTCTTC ACCATCCGTA AATATTCAGA AATGAATTAA AATTTTACCT TTATTCATTC CCGGGTCGAT TTATGGTCAA TTGTAATTGA AATAATTCTT TTTATAGTAA ATAACATTAG TCCCACAAAT ATTACATTTA TTCACCTAAA GAAAATGATA ATTTATCACT TCAATTTACT TTTATTGACT ATAACGGATC AATAATTTTA AACTTTAACT ATGGTTGAAT GGAACACCTG AAATATTATT TTGCTGGACA TCTTATTTAT ATTAGAATCA GTATTAAGAA CTTTTCATTT GCTATAACAA ATCATTATTT GATGACGAGA GCAGTAACTA GGTTTTATTT CACCCGCTAT TTTAATCCAT AGGATTCTGG CACAAACTAC ACAATACTTC TTCAGTTTAT ATGTATTAAT AATAATCACT ATATTGACAT ACTGGTGAGG AAATTTATTA CCAATCATAA TTTATTGCTT TTCAATTTTA TCGAATGCGG CGTTTTTTTT ATTAATTTTA GAACAATCAC

ATCCAATTCA GCTGAACATA ATGGCAGATT TTTATTAGAA AGCTTCTCCT TGATTTTAGT TTTTTTAATA AATAATTTGA CTTCTTTACG ACTTTAAAAA TTTTAATAAT CAACTGATGG ATAAACTCTC TTGAACAGTT TAAACCAAAC TGTTCAGAAA AAGTGTTCCT CATTAGATGA ATTAGCACTT TATGTCAAAC AGCACCAATT TTTTATTTTG TCTAATGAAT ACAAATTTAT TAATTGATTA GATTAATACC CTTCATAAAG TACTTTTATT TAGGATTATT TTATCTTTAG TAATCATACA CTATTTTAAT CGACCTGGAA TCTATTATTA TAGTAACATT GCTGTAGCTA CTTTATGAGA AGTGGATTAC CTGTATCAGG TTATTAGGTA TGTATCACGA TTGGTTTACG TTTGTAAGAT TGAATTAGGA TCCAAATTCC AGTTATTGTG TCAAGGTTTA AAGCTTATGA GGATCAACTT TGGAACAACT TTTCAAAAAA TTTGTTGATG AGGGTAAATT ATAATTAATT GGTCTATTAA TATTAATTTT TCAAAAAAAG ATTTGACCCA TAATTACTAT CCTATAATTA TTCTATTATT

57

ATTAAATTCA GATATTCTGA AGTGCAATAG AATAAATGTC TTAATGGAAC AATAATTACA ATTATGTAAA ACTATTTTAC TTTACTTTAT GAATTGGTCA ATTGAATTTG ATTTCGATTA AAATTCGAAT CCTGCTTTAG TAATTTTTTT TTTGTGGAGC GTAAATCACT CTGAAAGCAA ACTTCTAATG TAAAATTATT AGATGATTAT TTCTATTAAT TAAAAAACAT TTTCCGTATT AGAACATTTT TTCTCGTTAT AATTCAAAAC TTTATTTCTT TCCGTATATT CTTTACCTTT CAACATATAT ACCTTTTATA CATTAGCTGT ACTCTTTTAG TTTATTAACA TAATTCAATC GTAAATTAAT AGTCCATGAC TATAGTAAAA ATATTATTAC GAAGGAACAT TTGAGGAATA TTTTTTGAGC GCATCATGAC TTTATTAAAT ACCATCATAG TTTTTCACAG ATATATTGAA TTTATATAGC TTTTTACTAG TCATCATTTT TAGTTTGATT AATATTAATT ATCTATATAG TAAATAGTAT ATTAATCACA CTTTAATTGA AAATCTTCAT TATTTTTTTA TTATTATAAA TTCATTTTAA

TCTATTGAAT ATTACCACTA ATTTAAGCTC TACATGAGCT AATTAATTTT GTATTAGTAG TCGATTTCTT CAGCAATTAT TTATTAGATG TCAATGATAT ATTCATATAT TTAGATGTTG TTTAGTAACA GAGTAAAAGT ATTAATCGAC TAATCATAGA TTATTAAATG GTATTGGTCT ATAAAAAATT AAATAATTAA TACTATTTAT TATTATTCTT CAATTTAAAT CGACCCTTCA TAGGACTTTT AATATTGTAT TTTATTAGGT TATTTTCATT TTTACAAGAA ATGATTATGT TTGCTCACTT GTGTGTATTG TCGATTAACT GAAATACAGG GCTCAAATTG TTATGTATTT GTCTACACAC CATTAACAGG TGATTTCATC TATTTTAACA ATCAAGGTTT ATTTTATTTA TTTTTTTCAT CTCCTATAGG ACAGCTATTT TCTTATAGAA TTTTATTGGG GCTCCATTTA AACAGGATTT TATGTTTATT GGATTTGAAG ATTTTTATAT TCATTAATAT TATAAAAGTA AGATAATTTT ACTATTGTTA CCGAGAAAAA CTCGACTACC ATTTTTGATG ATATTCTAAT TTTTATTAAT

5951 6001 6051 6101 6151 6201 6251 6301 6351 6401 6451 6501 6551 6601 6651 6701 6751 6801 6851 6901 6951 7001 7051 7101 7151 7201 7251 7301 7351 7401 7451 7501 7551 7601 7651 7701 7751 7801 7851 7901 7951 8001 8051 8101 8151 8201 8251 8301 8351 8401 8451 8501 8551 8601 8651 8701 8751 8801 8851 8901 8951

TGGTCTATAT TTATATAAAT AAAGTATTGA TTGCAGTTAG TAATTGAAGC TAAATTCCAA TTCTTTTAAT AAAACCTTAC ACTAAAATTA TCAATGTCAA ATTTATTAAA AAGAATTATT TGATATAAAT TTTTACAATT TTCTAATATA TATATAAACA ATACCCAAAT TATAAATAGG AGTATTCTAC AAGTATAACT AATCCCCAGT AAACCTGTAG AATTCTAACA AAGGTATCCC AAAGGTATAT ATTTATATTA ATAAAGCATG GACAAAATTC AATTTTTTTT TAGTTAATCC AAAATAATAT AGAAGAGTGA GTAGTCAAGA ATTATTAATC TAAATAAAAA CAAGAAGAAA CCGGCATTAT AACAAGTCCT TAATAATTAA AATCGGTTAA TACTAAAGAA AATCAAAAAG TCCCATTCAA TAAAAAACAT AAATTGATAA ACCACAAATT ATGATTCTCT ATTAATAAAT AAACAATTTT CTCTAAAAAA GAAACAATTC AGGAGCTGCT AAGGTATAAA AGACGTTCAT ACCATGAGCA TAGTTAAAAG GCAATTAAAG TATACCTCCT TTATTAATTG TTTAATATAA ATGAGCCTTA

CATATTCCTT AAAGGGTTGT ATATTCAATC TTTCGACCTA CAAAAAGAGG TTAAGGAAGT GGTTAAATTC ATTTTCATTG ATTCATTATA ACTCTAAATA ATTAAAATTC ATGTATTAAA GCTGACCCCC AATTGACCAT AGGTATAAAT AAGATTTATT AAACCTCCTA TAAACAAATT CCCCAATAAT CAACTTTCAT TATTGAATAA AAAAATAATA ATTTCTAAAA ACATAAAGCT GAATTCTTAA TGAATAATAG AGTTAATAAA TTATTATTAA AAATCAAATT AGACAATAAT TAAATCGAAT ACTAAAGCAG AGAAAAAGGA TACCAATTAC ATATAATTTC AGCCACATCT AAGATTTAAT AATCCATCTC TAATATTATT TATTATAATC GCAATTATAA GAAAGTTATA TAAAATAAAT GACAAACTAA ATATTTCACG AGTATTTTTT TTTTAAAATT ACTCCCGAAT CCATGTTGAC AGATAATAAT TGTTTAATAA ATATTAGCTG ATTTAATAAC AAGATACATT ATTATTAATG TCCTGATAAA CCTTTAAATC ACTAATCTAA TAAAAAAGCA TACCTGCTAA GGTAATCATA

GATTTCATTC AGTTAATTAT TACCTTATTA ACCTTAGGTA CGTATCACTG ATGGTGATCA CATTTATACT TAATAATAAA TCCAAAGATT TAAGCTATTT AAAATACAAA AATAAAGTTT AAAATATTCT AATTCAAAGG CATATAGATC TAAAAAATAT CAATACATAC ATATAAGGAA TCTCATAATT CACTTAATAT TAAACTAATC TAAAAAAAAT TTATATCCTT AAATTAGATA TCCTCCTATT CCCCGGCACA TGAAATATAG ACCTAATTGA CAAAATTAGC AATAATAATT TAATAAATAA AAACAGGTGT ATTTGAGCTC TAATATTTCA ATCTTCCATA CCAATTCGAT ATTTTGAAAA ACCCCAATAA GATAAAACAA ATTTCTTATA GAACAAAAGA ACAATTCTTG TATATTATTT TAGAAATTAA ATCTAAAATG TTAAACTATT AATAAATTTA TTTTCCTCTT TAAAAGAATA GATAATATAA AGAAATTTCT ATCTTAATAA CCCTTATTGA AGCTAAACAA TATAAGAACC ACAATTCCTA AGTTTGACGT TTCTAATTCA ATAACTCGTA AATTATAGAA AATGAACTAA

58

ATGAAATTGA AACATTTGAT ATTGAATATG ATTATTATAC TTAATGATAT AGTAAAAGCT TCTATTTATA ATATTATATT TAATAATCTC GGATATAAAA TAATATTAAA TAGAATAAGT GATCAACCTT ATAAAAAATT CTAAAAAAGT AAATTTCTTA AAATAATGTT AAGGAAAAAT AATAATCCTA ATTTAAACTA GAAATGAATA GAAAATATAT AGAATAAAAT CATTAAAACA AAACGAATAT TATAAATAAT CTAATTTAAA CTTAAAGTAG TCCTAACCCA GTCCCAACCA ACACCAGCTG AGGAGCAGCC TTTTAGTTAT AATTCATTTT ATTTAATATT TAGATAAAGC TAAATAACTA AATTCTAATT ACATTAATAC TATTCTTTTC TATAAACAAT AAGAATTTAA AACAAAAAAT ATAAATGAAT AATTAATTCA TAAATATAAT AAGGTAATCA CTAAATGAAT TAAATATAGA TTATAGAAAT CCTAATAAAT AAATCATCAT TTAATATTCT AATAACCCAG AGATAAACCT TATGAGCAAC AAACAAACTA AACAAATCTA ATATTCCATA CCAGAAACTG AAATATCGGT

TCAAATTAAT TTGCATTCAA AAGCGATTGA CCTTATTCTT AATTGAATTT GCTAACTTTT TAGTTTAAAT TTTATAAATT CGTAACATCT ATAATAAAAA TAAATTTTTA AGATAATTTT GATCAAAACT ATTCCGTAAG TCTTATATTA AAGAAACTAT AATAATTTTA TAATCAATTT TTATTCCACG CCACAATTTA TCTAACAGTT TAACATTTCT CCAGCTAAAA GGCTGAAGTT CTTGAGAATT AATGCTTTAA AAATCCTATA ATAAAGCAAT GCTATAAATA AGAAGTTCTT TAACTAAAGT ATAGCTGCTG AGCAGCTAAT GTATAATTTC CAAGCAATAG AGTTAACATA AACAATAAGA AAATTTGGTC TAATATAATA TATAGAAAAT AAACTTATTC AGAAACTACT ATAAACTTAA CTAATTCTAC TATCACTAAC CATAACATAA ATGTAAAAAT ATACACCAGA GTATAAGCAG TCAAGATCAA TTAATGTAGG AAAGTTATCG TCGACTTCCA AAGAACATAA CAATAAGTCA AGACGAATAA ATCTAACTAA TACTTCATAT ACCCCCTAAT GAGCTTCAAC ATTTTTACTA

9001 9051 9101 9151 9201 9251 9301 9351 9401 9451 9501 9551 9601 9651 9701 9751 9801 9851 9901 9951 10001 10051 10101 10151 10201 10251 10301 10351 10401 10451 10501 10551 10601 10651 10701 10751 10801 10851 10901 10951 11001 11051 11101 11151 11201 11251 11301 11351 11401 11451 11501 11551 11601 11651 11701 11751 11801 11851 11901 11951 12001

AAAAAGCACA TTATTTATTA AATACCAATT ATACACCCGC AATAATGTAG TCTTATTCTT TTAATAAAAA GCTAATAATA AGATAATATA TAAAATTATT TGTACCATTC TAAAAAAAAA ATCATTTCCA CTCCTTCACA TAATTTAATA AATAAATTCT CAAAACAAAA AAAATTATAA AATCAAAAAT TTTTTCATTA TTATTCAATT CTAAATATAA AATTTTTGTA CATATATTTT TATGTAACAT AACTGTATTT TTATTGATAA TCTATTATTA TAAATTATAT ATTTATTAAT GGACCTATTC TCACCCTTTA CAATTAATAT TTAATTATTC TGATATTAAT ATTATGGTTG TTTATTTGTA TATATTTACT TAGGAACAGC TGAGGAGCTA TATAGATCTA CTTTAACTCG GCTATAACTA CCCAATTGGA TTACATTTAA TCATTAGTAT CCCAGCTAAC TTTTATTTGC GTTATCGCAT TAATTTAAGA TATTTTGATC CGACCAGTTG ATATTTCTTA ATTTATTAAA ACATAAGATA ATAATAAAGA TTTAAAGAAA ATAACGAAAC TAAAAGTTAA AAAATAACAC TATAAAAATT

TAATAAACAA AATAAAAATT AATATAGGTA TTGTAAACGT GAATTAATCT GAAAAAGTTA TAAATTTTTA TTAATGAACA TCACAACCTA TATTAGTAAA AATATATATT ATAATTTTTA TGAGTACGAA TACTCTAAAA TATTTAAATA AAACTTAAAA TAACCCTAAA TCATTAGTTT AAGATTATTT ATCCCCAAAA AATATTATAC TTCATCCACT TGTATATTAA ATTTTTAATT CTTTAGCTTC TCTTCATTTA AACTTCTTCT ATATAAATTC AATTTTCCTA TACTTTAATT GAATAATATC TTTAAAATTG CTCAAGATGA AAATTTTAAC TTAGCTTTTT ATTATTACGA TTTATTTACA CCAACTTGAT TTTTATAGGA CTGTAATTAC GTTCAATGAT ATTTTTTACA TAATTCATCT TTAAATTCTA GGATATTGTA TAATTAGACC CCTTTAGTTA CTATGCTATT TAGTTTTATC AAATTCCGAG TATATTAGTT AAGAACCTTA TATTATTTAG TTAAATAGTT GAATTTAATT AAATAATAAG AAGATAAAAA CGAGGTAAAG TTTTACATAA AAAATAATAT AAAGCAAAAC

AAATATAACA TATAGAACCA AAGAAACTAA TCTGGTTGAT TCTTTCAAAA AAATTAATAA TAATTATTAT AATCCATAAA AAAAATAAGA AAAATAAATC ATTAATAAAA ACATTATATA TTATAGAAAC GTTAAAAATA AATAAATAAT GTATTGAAAG ATAAATAAAA TAATAGTTTA CTTTTAAAAC TTAATATTTT TCATTAATCA AGCTTTAGGA CTGGATTAAT TTTTTAGGAG TAACGAAATA TTTTACTTTT TCTTTATTTT TTATTTTATA CAAATTTTAT GTTGTTGTAA TTAATTAATG CCAATAACGC TGAAATTTTG CGGATTATTT ATAGTGTTAA ACCTTACACG TGTAGGACGA TAATTGGGGT TATGTATTAC TAATTTATTA TATGAGGTGG TTCCATTTTA ATTATTTCTT ATATTGATAA GGATTTATTA AAATTTATTA CACCTGCTCA TTACGATCAA AATCGCAATT GAATTCAATT ACAGTAATTT TGTATTAATT TAAATCCATT AATGAGCTTG TTCTATTAAC ATTTTAAAAC ACATTTTCAA TACCTCGAGC AATAATAAAT TCTTATAAAC CTCCTCTTCT

59

AATCATAATT GTTTTATTTA TAAAGTATAA AACCTCAACC AATAAATAAA TAATAATAAA ATTTATTAAT CTTAATAAAA AATTTCTGAT TAATAAAAAA CAAAGAGGAC ATTCTAAAAG TAAAATTGAT TTATTCTAAA ATAAAAAATA TAAATGTTTT TTATAGGTAA ATAAAAACAT TTCAAGAGAA AAATAAACTA TTACTACTTC TTAACTTTAT AACTAAAAGT GAATACTTGT TTTAATTTAT AATATTAATT TAATAAATAA GAAAATTCTT TACAATTTTA AAATTACAAA AATAAACCTT TTTAGTAGAT GGTCATTACT TTAGCTATAC TCATATCTGT CTAACGGTGC GGAATTTATT AATTATTTTA CTTGAGGACA TCAGCTATTC ATTTGCTGTT TTTTACCATT CATCAAACAG AATTCCTTTT TAATAATTTT GGAGACCCTG TATTCAACCA TTCCAAATAA TTAATAATCT TTATCCTATT TATTAACTTG GGACAAATTC AATTACAAAA AACAAGCATA TTTACTAAAA CAATAAAAAA GCTAAATATA TCAAATAAAA TAAATACATC AAAATTCTTG ATATTCTACA

AAATATAAAA TAACATAAAA AACAATAAAT TAAAATCAAA ATATAAATAA ATAACAATAT TCTTTCTCTA TTAATCCATA CAATAATTTA TATTATAATT TTAAAAATAC ACTGAAAATA AAACCTAAAG ATAATTTTCA ATATTAAAAC CGATTAGATA ACTTCAATAT TGGTCTTGTA AAGAAATTTC CCTCTTGAAA TATTATTTTT TAATTCAAAC TTTTGATATT ATTATTTATT CAATTAAATT TTATCATTTA TGATATACAA TATCTTTAAA TTAATAAATT ATTATTTAAA TACGAAATTC TTACCAGCTC TGGATTATGT ATTATACTGC CGAGATGTTA ATCATTTTTT ATGGTTCATA TTTTTAGTAA AATATCATTT CTTATTTAGG GATAATGCTA TATTGTTCTT GTTCTAATAA CATCCATATT TATTTTAATT ATAATTTTAT GAATGATATT ATTAGGAGGA TACCTTTTTA AATCAAGTAA AATTGGAGCT TAACTGTTGT TGATGAGATA TGTTTTGAAA AAAATTCACT TAATAAATAA TTAATTTATC ACAAAAGAAA ACATCCTAAA CATATTCAGC TTAAACCCTG

12051 12101 12151 12201 12251 12301 12351 12401 12451 12501 12551 12601 12651 12701 12751 12801 12851 12901 12951 13001 13051 13101 13151 13201 13251 13301 13351 13401 13451 13501 13551 13601 13651 13701 13751 13801 13851 13901 13951 14001 14051 14101 14151 14201 14251 14301 14351 14401 14451 14501 14551 14601 14651 14701 14751 14801 14851 14901

AAACTAACTC TCAGCTAATG TAAAAATCAC TTCCAATTAA TAAGAAATAG AGAATTAGAA TACAACATAA AAAAAAGGTA TGGAGAAATA CTTTTGTAAA AAACCAACTT TCGCTCTAAT ATAACAAACT TATTTGTAAT TGCCAAAATA TTGATATTTG AACCAACCTG AGTCGAACAG TCCAACATCG ATTACGCTGT ATCATTTATT TTAATATCAC TTATAATTAA TTTAAATAAA TTAAATGAAA TAAAAGACTA TTTAAAATTT ATGTTTTTGT ACTTTTCATT ATTGCTTAAT TTAATAAATA TTGCTAATTC TTTATTTTAT TAAATAAATA AAAAAACTAG ATTATAAATA AAATCGAGAA AAGGTACAAT TTCAATTTTC TTTAAACAAT TAAAAAATAA TTGCACAAAA TAAATTGTCA TATCTTACCT CTAAAATCAA AAAATTTTTT ATTACTTAAA TTATTGAATA AACTGATTAC CAGGTTCCTC AACATAACTA TATCTAATCC ATATAATAAT TATTAATAAA GGTATAACCG CTATTTCTAA TTATATTTAT AATCAAATTA

TGATTCTCCT AAATAGTTAA ATATAAACTT AAAAATAAAA TTTGAGCTAC GATCAACCAG AAAAAATAAA TACATATCCA TAATATCTTA TAATTTAATT TATTAGGACC AAAGTTAAAA TCCAATTAAT AAAAATCACA GTTTTACATT GTCCTTTCGT GCTTACACCG ACTTAAAATT AGGTCGCAAT TATCCCTAAA CATAAATTAA CCCAATAAAA AATAAAAAAA TTTTAGCTTT CAGTTAATAT ATGATTATGC TCAGTGGGCA TAAACAGGCG AAAATTAATT TTTAATAATT ATTTAATTTA TAAGCATATA AGCTTATCCC ATTAAGTAAA ATACCTTTAA ATTTTGTTAC AAATAAATAT AAATTAAATT TTTTACAATA ACTAAAACTT TAAAAATTAA ATCTTTTCAA TTCTAGATAC TAATAATAAG ATTATTAATC CATAATTTTA TATAAGCTAC TTATTATTCT AAACTTAAGT TAGATAGACT TTACTACTTT TAGTTTTTTA TTAAATATAA TTTAATTTAA CGACTGCTGG ATTTCTTTAA TTTTAAAATA ATAACAAATT

TCAGCAAAAT TCATACTAAA GATAAAAAAA GATAATAAAA GGCTCGTAAA CCACTATAAC CCTCCTAAAT AACAAATAAA AATAATTAGA GCATCACAAA TTTACGAATC AAGCTACACT GATAAAATAA TAAATAAATT AATAATATTC ACTAAAATAT GTTTGAACTC TGAACGGCTA CTTTTTTATC GTAACTTAAT TGTTTTTAAA TATTTTTTTC AATATAAAGA TTAACTAAAA CTCGTCCAAC TACCTTTGCA GGTTAGACTT GATATTATTT TTAACATTAT AAAATTAACA TAAAATAAAT TTTATTAAAT ATAAAATATT TTTATAATTT AAACGAATAA ATTAACTTAA TTATTTTTTA TTCTTTTTAA CTAATAAACT TAAATTTTAT TAAATAAAAA TGTAAATGAA ACTTTCCAGT AGCGACGGGC TTTATAATTT TCCATATAAA ACCTTGATCT TATAAAATAT AAGGTCCATC AAAATACCGC AGCAATTAAT TTAAAATTTT AATTTCACTT TTATTACTAA CACCAATTTG TTAATAAAAT AATATATAAT TTTAAGCCAA

60

CAAAAGGAGT CCTATAGGAA AAAATAAATT TTAAAGCTAA CCCCCCAATA TGTATAAACT TAAATGAATA GACAAAAATA TAATAAAGGA AAGGTTGAGG TGAATATATC CACTAATACA ATTCTATATA CTAAATTTAT TTATAAAAAA CATAACTTTT AGATCATGTA CACCCAAAAT GATATGAACT TTTTTAATCA AATTAAAAGT TATTAAAATT TTTATAGGGT AATAAAATTC CATTCATTCC CAGTCAAAAT TATATATAAT TTGCCGAATT TATATACTAA TTTTAATAAA TATAACATAT TTATTTAATA AAAATTATAA CTAAATTAAA CATTTCATTT ATATTATATT TTTAATAAAC ATAAAATTTT ATTATTAAAA AGTTATTTCT CTAAATCAAT ATACTTTACT ACATCTACTA GATGTGTACA TACTACTAAA TAAATTTATT GATATAAATT TCTGATAACG GTGGATTATC CAAATTTTTT TTACATTTTA TCAACTTCAA AATATATTTA AAAAATTTAT GTCAATACTT TAATTACTGC CATACAAAAA AATAAAACTT

CCGATTAGTT ATAAAATAAT ATATTATAAC ACTAACTTCA AAGCATAATT CCCAATCTAG TAACTTTACA AAGAAAAAAT TAAGTTTGTT AATTCCTATT CTAAAACTTT CAAATAATTA AAACAAGTAC TGCACTAATC TATAATTATT TAAAGATAGA AGAATTTAAA TATATCTTAA CTCCAAAAAA TTACTAATGG TTTTTAAATT TAATTAATCT CTTCTCGTCT TACAAAAATT AGCCTTCAAT ACTGCGGCCA TCAAAAAGAC CTTTATTTAA TTTTATCATT TAATTAAAAT TTTTTAATAA TTTTTAAAAA ATTAATTAAT TTTATTTCTT CTAATATAAT AACTCTTTTA GCTGATACAC TTCAAATTAT TTATTCTTTC AATAATTAAA TTATATTGAT TAATAAGCTT TGTTACGACT TATTTTAGAG TCCACTTTCA GTAACCCATT TTTATTAAAA ACGGTATATA GATTACAAAA AAGTTTCAAG AATAATAGGG TTATATTTTT ATTTTATTAT TTGTATTAGT TTTAATATTG GATTAAATTT TTTACATATA TAA

Appendix 2

61

A. DNA extraction protocol Material: Cell lysis solution, protein precipitation solution, isopropanol, 70% ethanol, proteinase K.

Procedure 1. Prepare grinder (blue tip melted + 2 mL centrifuge tube) 2. Prepare 20 ml mixture of cell lysis buffer and 0.2 μl of proteinase K. 3. Grind single flies on to stage 2 solution using grinder (step 1) 4. Incubate at 55 ⁰C for 1.5 hours and vortex at 15 min, 30min and 45 min for 10 seconds. 5. Add 8ul protein precipitation solution. 6. Vertex and incubate on ice for 15 min. 7. Centrifuge in cold (4⁰C) for 15 min at 4,000 rpm. 8. Pipette out the supernatant and add it into a 2 Ml centrifuge tube with 20 μl of isopropanol inside. 9. Mix by inverting for 50 times. 10. Centrifuge in cold (4⁰C) for 15 min at 4,000 rpm. 11. Discard the supernatant and drain the tube on a clear piece of absorbent paper. 12. Add 20 μl of 70% Ethanol and centrifuge in cold (4⁰C) for 5 min at 4,000 rpm. 13. Discard Ethanol and let palled dry for 10-15 min. 14. Add 40 μl of TE buffer to dissolve the DNA.

62

B. PURP preparation for egg collection Material: Funnel, Molasses, Water, Agar, Plate, Yeast Procedure: 1. 50 ml of molasses 2. Add 450 ml of water 3. Add 20 g agar in cold water and mix throughfully 4. Microwave to boil 5. Pour to plates (cover of the vial) 6. Let it cold and cover the surface with some yeast Flies (around 20 pair) were allowed to mate for 48 hours and eggs were transferred into new bottle with Larva fluid. Eggs were washed with water and 3% of detergent prior to transfer into bottle.

63

Appendix 3

64

A. The Dean primer set optimization

In current study, I had to change the amount of dNTPs from that published in Dean et al. (2003). After implementation of dNTPs gradient including: 0.2-, 0.4- and 0.80 mM of dNTPs (on 50/50 mixture of 3-KY0410 / 2-KY0418 mtDNA templates, 3KY0410 mtDNA template and 2-KY0418 mtDNA template), result is only obtained from the PCR reaction containing 0.2 mM dNTPs (Fig. A3.1). The other ingredients used as written in Dean et al. (2003). The final PCR reactions were carried out in 25 μl reactions containing 30 ng DNA, 4.25 pmol of the primer 4726+, 4 pmol of 5983-, 2.25 pmol of 5183+, 3 mM MgCl2 (New England BioLabs Inc., MA, USA), 1x Crimson polymerase buffer (New England BioLabs Inc., MA, USA), 0.2 mM dNTPs (Bioline, London, UK), and 0.5 U Taq DNA polymerase (New England BioLabs Inc).

65

M

1

2

3

4

Fig. A3.1 Results from the Dean primer (after optimization of dNTPs). on 50/50 mixture 3-KY0410 and 2-KY0418 mtDNA template. M: 100 bp ladder; Lane 1: 50/50 mixture of 3-KY0410 / 2-KY0418 mtDNA templates; Lane 2: 3-KY0410 mtDNA template; Lane 3: 2-KY0418 mtDNA template; Lane 4: negative control.

66

B. The 1432+/2314- & 1793- primer set optimization First, I tested MgCl2 gradient (1.5 mM, 2.0 mM and 2.5 mM) on 3-KY0410 and 2KY0418 mtDNA templates, separately under the temperature gradient 55°C- 70°C (see Fig. A3.2 and A3.3). Under the 2.0 mM MgCl2 concentration, the 1432+/2314& 1793- primer set was specifically amplified the 3-KY0410 mtDNA (showed a single band in each temperature between 55°C and 63.9°C). However, this primer set was failed to specifically amplify the 2-KY0418 mtDNA template (multiband was presented). Thus, a primer gradient PCR including 3-, 5- and 10 pmol of 2314- (the specific primer for detection of 2-KY0418 mtDNA) was performed against the 2KY0418 mtDNA template (Fig A3.4). The amount of the other primers (1432+ & 1793- ) was set constant (10 pmol each).The final PCR reactions were carried out in 25 μl reactions containing 30 ng DNA contained 10 pmol of the primer 1432+ (common primer), 10 pmol of the primer 1793- (specific primer for 3-KY0410), and 3 pmol of the primer 2314- (specific primer for 2-KY0418), 2 mM MgCl2, 1x Crimson polymerase buffer, 0.2 mM dNTPs and 0.5 U Taq DNA polymerase.

67

M 1 2 3 4 5 6 7 8 9 10

A.

M 1 2 3 4 5 6 7 8 9

B.

M 1 2 3 4 5 6 7 8 9

C.

Fig. A3.2 Optimization of the 1432+/2314- & 1793- primer set using MgCl2 gradient (1.5 mM (A), 2.0 mM (B) and 2.5 mM (C)) in temperature gradient 55°C -70°C (55, 57.4, 59.2, 61.4, 63.9, 66.1, 68.9 and 70 °C ) on 3-KY0410 mtDNA template. A: M: 100 bp ladder; Lane 1: positive control; Lane 2: negative control; Lane 3-10: no bands (55°C -70°C). B: M: 100 bp ladder; Lane 1: specific band for 3-KY0410 at 55°C; Lane 2: specific band for 3-KY0410 at 57.4°C; Lane 3: specific band for 3-KY0410 at 59.2°C; Lane 4: specific band for 3-KY0410 at 61.4°C; Lane5: specific band for 3KY0410 at 63.9°C; Lane 6: no band at 66.1°C; Lane 7: no band at 68.9°C; Lane 8: no band at 70°C; Lane 9: negative control. C: Lane 1-4: multi-bands (unspecific reaction of 1432+/2314- & 1793- primer set on 3-KY0410 mtDNA template resulting in unspecific mtDNA amplification); Lane5: specific band for 3-KY0410 at 63.9°C; Lane 6: specific band for 3-KY0410 at 66.1°C. Lane 7: no band at 68.9°C; Lane 8: no band at 70°C; Lane 9: negative control.

68

M 1 2 3 4 5 6 7 8 9 10

A.

M 1 2 3 4 5 6 7 8 9

B.

M 1 2 3 4 5 6 7 8 9

C.

Fig. A3.3 Optimization of the 1432+/2314- & 1793- primer set using MgCl2 gradient (1.5 mM (A), 2.0 mM (B) and 2.5 mM (C)) in temperature gradient 55°C -70°C (55, 57.4, 59.2, 61.4, 63.9, 66.1, 68.9 and 70 °C ) on 2-KY0418 mtDNA template. A: M: 100 bp ladder; Lane 1-8: no bands (55°C -70°C); Lane 9: negative control; Lane 10: positive control. B: Lane 1-6: -bands (unspecific reaction of 1432+/2314- & 1793primer set on 2-KY0418 mtDNA template resulting in unspecific mtDNA amplification); Lane7: specific band for 2-KY0418 at 68.9°C; Lane 8: specific band for 2-KY0418 at 70°C; Lane 9: negative control. C: Lane 1-6: multi-bands (unspecific reaction of 1432+/2314- & 1793- primer set on 2-KY0418 mtDNA template resulting in unspecific mtDNA amplification); Lane7: specific band for 2-KY0418 at 68.9°C; Lane 8: no bands at 70°C; Lane 9: negative control.

69

M 1 2 3 4 5 6 7 8 9

A.

M 1 2 3 4 5 6 7 8 9

B.

M 1 2 3 4 5 6 7 8 9

C.

Fig. A3.4 Optimization of the 1432+/2314- & 1793- primer set using primer 2314gradient (3-(A), 5- (B), and 10 (C) pmol) in temperature gradient 55°C -70°C (55, 57.4, 59.2, 61.4, 63.9, 66.1, 68.9 and 70 °C ) on 2-KY0418 mtDNA template. A: M: 100 bp ladder; Lane 1: specific band for 2-KY0418 at 55°C; Lane 2: specific band for 2-KY0418 at 57.4°C; Lane 3: specific band for 2-KY0418 at 59.2°C; Lane 4: specific band for 2-KY0418 at 61.4°C; Lane 5: specific band for 2-KY0418 at 63.9; Lane 6-8: no bands (66.1, 68.9 and 70°C); Lane 9: negative control. B: M: 100 bp ladder; Lane 1-3: multi-bands (unspecific reaction of 1432+/2314- & 1793- primer set on 2KY0418 mtDNA template resulting in unspecific mtDNA amplification); Lane 4: specific band for 2-KY0418 at 61.4°C; Lane 5: specific band for 2-KY0418 at 63.9; Lane 6: specific band for 2-KY0418 at 68.9; Lane 7 -8: no bands (68.9 and 70°C); Lane 9: negative control. C: Lane 1-7: multi-bands (unspecific reaction of 1432+/2314- & 1793- primer set on 2-KY0418 mtDNA template resulting in unspecific mtDNA amplification); Lane 8: specific band for 2-KY0418 at 70°C; Lane 9: negative control.

70

Appendix 4

71

AS-PCR sensitivity test for -KY0418 diluted by 3-KY0418 (dilution series)

For the Dean primer set, the, 1/100 dilution of 2-KY0418 to 3-KY0410 was the maximum detection limit of primers (Fig. A4.1). To further investigate the sensitivity of this primer set a second intermediate dilution series was tested: 1/100, 1/200, 1/400, 1/800 and 1/1000. These results suggested the maximum power limit of 1/100 for detecting 2-KY0418 mtDNA when it is diluted by 3-KY0410 (Fig. A4. 2).

The maximum detection power of the 1432+, 2314- & 1793- primer set as shown by a dilution series was 1/10 when 2-KY0418 mtDNA was diluted by 3-KY0410 (Fig. A4.3). A second intermediate dilution series confirmed these results (Fig. A4. 4).

Combined, these data show that the Dean primers would be more robust than the 1432+, 2314- & 1793- primer set in detecting heteroplasmy levels in wild caught flies.

72

M

1

2

3

4

5

6

7

8

9

1257 bp 800 bp

Fig. A4.1 PCR dilution test with different ratios of experimentally mixed 2-KY0418: 3-KY0410 amplified with Dean primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe. M: 100 bp ladder; Lane1: 2-KY0418 DNA; Lane 2: 1/1, (2-KY0418/3-KY0410); Lane3: 1/10, (2-KY0418/3-KY0410); Lane 4: 1/100, (2-KY0418/3-KY0410); Lane 5: 1/1000, (2-KY0418/3-KY0410); Lane 6: 1/10000, (2-KY0418/3-KY0410); Lane 7: 1/ 100000, (2-KY0418/3-KY0410); Lane 8: 3-KY0410 DNA; Lane 9: negative control.

73

M

1

2

3

4

5

6

7

8

1257 bp 800 bp

Fig. A4.2 Complementary PCR dilution test with different ratios of of experimentally mixed 2-KY0418: 3-KY0410 amplified with Dean primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe. M: 100 bp ladder; Lane1: 2-KY0418 DNA; Lane 2: 1/100, (2-KY0418/3-KY0410); Lane3: 1/200, (2KY0418/3-KY0410); Lane 4: 1/400, (2-KY0418/3-KY0410); Lane 5: 1/800, (2KY0418/3-KY0410); Lane 6: 1/1000, (2-KY0418/3-KY0410); Lane 7: 3-KY0410 mtDNA; Lane 8: negative control.

74

M 1

2 3

4

5 6 7

8

9

882 bp 361 bp

Fig. A4.3 PCR dilution test with different ratios of experimentally mixed 2-KY0418: 3-KY0410 (maternal/paternal) amplified with 1432+/2314- &1793- primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe. M, 100 bp ladder; Lane1: 2-KY0418 DNA; Lane 2: 1/1 (2-KY0418/3-KY0410); Lane 3: 1/10 (2-KY0418/3-KY0410); Lane 4: 1/100 (2-KY0418/3-KY0410); Lane 5: 1/1000 (2-KY0418/3-KY0410); Lane 6: 1/10000 (2-KY0418/3-KY0410); Lane 7: 1/ 100000 (2-KY0418/3-KY0410); Lane 8: 3-KY0410 DNA; Lane 9: negative control.

75

M

1

2

3

4

5

6

7

8

882 bp 361 bp

Fig. A4.4 Complementary PCR dilution test with different volume ratios of 1/1000, 1/2000, 1/4000, 1/8000, and 1/10000, of experimentally mixed 2-KY0418: 3-KY0410 (maternal/paternal) with 1432+/2314- &1793- primer set and visualized after electrophoresis using a 1.5% agarose gel stained with Sybrsafe. M: 100 bp ladder; Lane1: 2-KY0418 DNA; Lane 2: 1/10 (2-KY0418/3-KY0410); Lane 3: 1/20 (2KY0418/3-KY0410); Lane 4: 1/40 (2-KY0418/3-KY0410); Lane 5: 1/80 (2KY0418/3-KY0410); Lane 6: 1/100 (2-KY0418/3-KY0410); Lane 7: 3-KY0410 DNA; Lane 8: negative control.

76

Appendix 5

77

Confirmation of the positive cases of mtDNA paternal leakage using 1432+/2314/1793- primer set

To confirm the sequencing results obtained by Dean primer sets the positive cases of mtDNA paternal leakage in crosses 4 and 21 (4:1, 4:2, 4:3, 4:4, 21:1, 21:2) along their parents were sequenced using common primer of 1432+ from the primer set of 1432+/2314-/1793-. The sequences were aligned against the 3-KY0410 and 2KY0418 reference sequences (see Appendix 1) using Sequencher® and the chromatograms were investigated. In the five diagnostic nucleotide positions of 1651, 1712, 1757, 1784, and 1895; four positive cases of 4:1, 4:2, 4:3, and 21:1 showed the same pattern as the males from siIII fly lines confirming the occurrence of mtDNA paternal leakage indicating replacement of siII by siIII (Table A5.1). Two positive cases of 4:4 and 21:2 showed individuals with both type of mtDNA (siII and siIII) indicating heteroplasmy (Table A5.1 and Fig. A5.1). Therefore, these results confirmed the sequencing results applied by Dean primer set.

78

Table A5.1 Intra-specific variation in six positive cases of mtDNA paternal leakage in comparison with their parents and reference sequences. Nucleotide positions 1 6 5 1

1 7 1 2

1 7 5 7

1 7 8 4

1 8 9 5

2-KY0418 reference

A

A

A

A

A

Female from cross 4

.

.

.

.

.

Female from cross 21

.

.

.

.

.

3-KY0410 reference

C

G

G

T

C

Male from cross 4

C

G

G

T

C

Male from cross 21

C

G

G

T

C

4:1

(cross 4, case 1)

C

G

G

T

C

4:2

(cross 4, case 2)

C

G

G

T

C

4:3

(cross 4, case 3)

C

G

G

T

C

4:4

(cross 4, case 4)

M

R

R

W

M

21:1

(cross 21, case 1)

C

G

G

T

M

21:2

(cross 21, case 2)

C

R

R

T

M

Sequences were aligned to the 2-KY0418 and 3-KY0410 reference sequences (Appendix 1). Nucleotide positions are relative to reference sequences. Nucleotide codes: M= A or C; W= A or T; R= A or G. (.): identical to consensus.

79

Fig. A5.1 Chromatogram comparison between a replacement case and a heteroplasmic case belong to 4:4 against their parents in nucleotide position 1712.

Nucleotide position 1 7 1 2 Maternal mtDNA, 4.female

Paternal mtDNA, 4.male

Replacement mtDNA,4:1

Heteroplasmy mtDNA, 4:4

80