UPTEC X09 012

Examensarbete 30 hpMars 2009

Construction of a linkage map of the zebra finch genome using SNP markers

Harriet Mellenius

 

Molecular Biotechnology Programme Uppsala University School of Engineering

UPTEC X 09 012 Date of issue 2009-03 Author

Harriet Mellenius Title (English)

Construction of a linkage map of the zebra finch genome using SNP markers

Title (Swedish) Abstract The zebra finch (Taeniopygia guttata) is one of the most used model organisms for studies of behaviour and neurology, especially pertaining to the learning process. Nevertheless, genetic information about the zebra finch has until recently been scarce. The aim of this project was to produce a linkage map over the zebra finch genome. A pedigree of 1,351 birds was investigated, and 1,080 were successfully genotyped for 1,424 single nucleotide polymorphisms (SNPs). The linkage analysis resulted in a framework map consisting of 423 markers, covering 32 chromosomes and 1,340.2 cM. The results reveal that physical and genetic distances show a non-linear relationship in the zebra finch. The map will be used to trace the genetic background of the phenotypic traits recorded in this zebra finch pedigree. Keywords Evolutionary genetics, genetic linkage analysis, linkage map, zebra finch, recombination, single nucleotide polymorphism Supervisors

Niclas Backström Uppsala universitet

Scientific reviewer

Mikael Thollesson Uppsala universitet

Project name

Sponsors

Language

English

Security

ISSN 1401-2138

Classification

Supplementary bibliographical information Pages 68

Biology Education Centre Biomedical Center Husargatan 3 Uppsala Box 592 S-75124 Uppsala Tel +46 (0)18 4710000 Fax +46 (0)18 555217

 

Construction of a linkage map of the zebra finch genome using SNP markers

Harriet Mellenius

Sammanfattning En genetisk karta informerar om både hur en arts arvsmassa är arrangerad och var man ska söka efter de gener som påverkar en viss egenskap. Kartan placerar de genetiska markörer man undersöker i grupper motsvarande artens kromosomer och i ordning längs kromosomerna. Avstånden mellan markörerna i en grupp mäts i förekomsten av överkorsning mellan dem; hur ofta två nästan likadana kromosomer utbytt information. Av de två nästan likadana kromosomerna kommer en från mamman och en från pappan. Därför är en genetisk markör en bit av arvsmassan som ofta skiljer sig åt mellan individer så att den kan påvisa överkorsningarna. I denna studie användes variation av enstaka byggstenar i den genetiska koden som markörer. Kartan byggs genom att man i ett släktträd observerar hur ofta markörerna tillsammans förs vidare till nästa generation. Om två markörer nästan alltid ärvs ihop betyder det att de tillhör samma kromosom, och när de ibland skiljs åt har överkorsning skett mellan dem. I detta projekt användes ett släktträd med 1351 zebrafinkar vars arvsmassa undersöktes i 1424 punkter, och den karta som byggdes inkluderade 423 markörer. Hos dessa zebrafinkar hade också många beteenderelaterade egenskaper studerats, vars genetiska bakgrund kanske kommer att kunna utforskas med hjälp av kartan.

Examensarbete 30hp

Civilingenjörsprogrammet Molekylär bioteknik

Uppsala universitet mars 2009

 

2

ABBREVIATIONS 3

INTRODUCTION 4

GENETIC MAPS, A HISTORY 4 GENETIC MAPPING AND SEQUENCING IN AVIAN GENOMES 4 RECOMBINATION IS NECESSARY FOR GENETIC MAPPING 5 GENETIC MARKERS 6 MAPPING METHODS 7 THE ZEBRA FINCH 8 THE POPULATION 9 AIMS AND STRATEGY 9

METHODS 10

SNP IDENTIFICATION 10 GENOTYPING 10 DATA PROCESSING 11 MAPPING 11 GRAPHICS 13 PHYSICAL DISTANCES 13

RESULTS 14

MARKER GENOTYPING 14 LINKAGE GROUPS 14 THE FRAMEWORK MAP 15 COMPARISONS 20 THE GENETIC MAPS OF THE ZEBRA FINCH 20 THE CHICKEN GENOME 21 GENETICAL VS. PHYSICAL DISTANCES 25

DISCUSSION 28

CONCLUSIONS 30 ACKNOWLEDGEMENTS 30

REFERENCES 32

LIST OF APPENDICES 34

3

Abbreviations SNP Single Nucleotide Polymorphism

cM centiMorgan

RFLP Restriction Fragment Length Polymorphism

QTL Quantitative Trait Loci

LOD Logarithm of Odds

BLAST Basic Local Alignment Search Tool

4

Introduction

Genetic maps, a history The aim of genetic mapping, unlike genetic sequencing, is not to determine every

nucleotide in the genetic sequence, but to construct a general representation of a

portion of the genome, consisting of the linear order of a set of selected markers.

The map, genetic or physical, can give information about the karyotype and

reveal the approximate position of the included markers. While a physical map

will give the distance between markers in physical units, that is, the number of

base pairs, the distances in a genetic map are based on the frequency of

recombination events over a distance.

Genetic mapping and sequencing in avian genomes The species that scientists have been most eager to genetically map are the ones

that have been used extensively in previous research, called model organisms, in

order to be able to compare previously obtained knowledge with new genetical

data. The very first genetic maps of chromosomes were developed for the

Drosophila melanogaster genome; one of the most commonly used model

organisms (Sturtevant 1913). Other species of interest have been organisms of

economical significance.

Those are the reasons why the chicken (Gallus gallus) was the first, and at

present the only, avian genome to be sequenced. Yet, the chicken is not

representative for all birds and yields only limited insight to wild avian

populations. The chicken belongs to the fowls, though the biggest of the avian

orders containing nearly half of all avian species is Passeriformes, the passerines

(Sibley and Ahlquist 1990, Barker et al. 2004), which have been the focus of many

ecological studies. At this point, the only passerine birds genetically mapped for

the whole genome are the zebra finch, for which a genetic map was published in

May 2008 (Stapley et al. 2008), and the collared flycatcher in July 2008

(Backström et al. 2008). Partial maps of passerine genomes worth mentioning

have been constructed for the great reed warbler (Hansson et al. 2005, Åkesson et

al. 2007) and the house sparrow (Hale et al. 2008).

5

Previous studies on chicken and passerine genomes have revealed an almost one-

to-one homology between chicken and passerine chromosomes. The exceptions

are chicken chromosomes 1 and 4, which are represented by two chromosomes

each in the passerine genomes (Derjusheva et al. 2004, Stapley et al. 2008).

However, even though the general chromosome organisation is preserved, there

is evidence of intrachromosomal rearrangements (Stapley et al. 2008, Backström

et al. 2008).

Recombination is necessary for genetic mapping Recombination is a process that occurs uniquely in meiosis, after DNA

replication. Homologous chromosomes, consisting of the two sister chromatids

with a common centromere, pair up forming tetrads, where chiasmata can be

formed along the chromosome arms. A chiasma is the complex where crossover,

or recombination, occurs. Since the homologous chromosomes originate from the

two parents, the recombined chromosome will be an assembly of genes from both

parents. When the newly formed gamete results in offspring, each chromosome

will be composed of genetic material from both grand-parents, either maternal or

paternal.

The unit of the genetic map is the centiMorgan, cM. The distance of one cM

between two linked markers implicates a 1% incidence that they are separated by

recombination. The cM is related to the physical distance measured in base pairs

in the sense that recombination is generally more likely to occur somewhere

along a long stretch of the chromosome than over a shorter one, but they are not

proportional as additional factors affect the rate of crossing-over. For instance,

the recombination rate varies along the chromosome, with reduced rate of

recombination close to the centromere. It has been suggested from studies on

chicken that this tendency is weaker in birds than in other vertebrates (Jensen-

Seaman et al. 2004, Schmid et al. 2005).

An interesting effect of recombination is that where only one of the two

chromosomes contains a variant, recombination can bring those variations

6

together onto the same chromosome, or, on the other hand, break up a collection

of genetic variants, called a haplotype. When genes are linked, grouped together

on the same chromosome, they are allowed to co-segregate through a pedigree. If

the genes of a haplotype interact to give a combined effect, this effect may be

observable in the pedigree, and may also be subject to selection. Thus, the

mapping of genetic linkage can be of use for gene annotation, and regions where

recombination seems to be underrepresented can be assumed to contain

informative haplotypes.

Genetic markers In order to distinguish the two chromosomes in a chromosome pair from each

other, the genetic marker must be a site where variation is common, so that a

large enough number of individuals in the investigated pedigree are heterozygous

for that site. Only where it is possible to tell the alleles apart, recombination

events can be detected. Throughout the history of genetic linkage analysis,

different types of markers have been used.

Among the first genetic markers to be used were the RFLPs, or restriction

fragment length polymorphisms (Grodzicker et al. 1974). Restriction enzymes are

used to cleave DNA strands at sites of short, specific sequences. Where there is

variation among the restriction sites between the chromosomes, the DNA

fragments resulting from restriction enzyme cleavage will be of distinctly

different lengths. The major benefit with RFLPs is the ease with which new

markers can be developed; the analysis is, on the other hand, slow and

inconvenient.

Sometimes, a nucleotide sequence can be repeated many times consecutively. The

number of times the sequence is repeated varies among individuals, and they can

thus be used as a genetic marker. If the sequence is very short, up to six base

pairs, the repeat is called a microsatellite (Ellegren 2004). Due to the presence of

multiple alleles, microsatellite polymorphisms are so variable in number of

repeats that they can even tell closely related individuals apart, and are

therefore the genetic marker used in DNA profiling. The variability is clearly a

7

big advantage when microsatellites are used as genetic markers, but also makes

comparisons between species troublesome.

The genetic markers used in this study were single nucleotide polymorphism, or

SNPs. A SNP is a substitution of a single nucleotide by mutation that makes a

common variation in the population. Typically, the SNP nucleotide alternates

between only two bases. The frequency of each of these in the population is called

allele frequency. For a SNP to be suitable as a genetic marker, the allele

frequency must be sufficiently high for it to be fairly common with biallelic

individuals in the population. An advantage using SNPs as markers, that was a

benefit to this study, is that the flanking sequences of the SNP is known, thus

enabling searches for homologues in other sequenced species.

Mapping methods Genetic linkage analysis is a statistical technique for building genetic maps from

linkage data. Linkage data is typically obtained through genetic analysis of a

pedigree where the segregation of markers can be observed, and recombination

events traced. The collected data will tell how often a pair or a set of markers are

inherited together. Allele combinations that are inherited together more often

than expected are assumed to pertain to the same chromosome or in the same

linkage group on a chromosome.

Subsequently, the markers within each linkage group can be ordered. The order

of the markers is based on how often recombination occurs between each pair of

markers, and to be able to determine where a recombination event has chanced,

the descent of the markers must be revealed. Only after deciding whether a

chromosome is paternal or maternal, and the parental markers are genotyped, it

is possible to detect recombination events.

Recombination events are detected by the sudden change from grand-maternal to

grand-paternal marker alleles along a chromosome, or vice versa. The fraction of

observed recombinations out of all informative meioses estimates the

recombination rate between a pair of markers, which is by definition the genetic

8

distance between them. However, there is always a risk that two or more

recombination events occur between two adjacent markers. On these occasions,

the total count of recombinations will be underestimated, as double

recombinations re-change the alleles. This problem is dealt with by the

recalculation of all recombination rates using a function that takes into account

the chance of double crossovers between adjacent markers. Moreover, the

probability of a recombination event is reduced close to other crossovers. This

phenomenon is called crossover interference (Sturtevant 1915), and the

mechanism behind it is not yet fully uncovered. The Kosambi mapping function

take this as well into account, and this function is hence used to make observed

recombination rates better represent the true recombination fractions along the

chromosome (Kosambi 1944, Zhao & McPeek 1996).

Using these data, the most likely marker order can be computed by addition of

the calculated genetic inter-distances between the markers. This information can

be ambiguous, so that there is seldom a single best order that includes all

markers, but more often many plausible marker orders of similar likelihood. The

concept of framework maps refers to that the final marker order has significantly

higher probability compared to the alternatives. This map unlikely includes all

markers as some can be placed at more than one locus with equal probability.

The Zebra Finch The zebra finch (Taeniopygia guttata) may not be one of the most well-known

model organisms, but is in fact the most used model organism for the study of

behaviour and neurology, especially pertaining to the learning process (Jin &

Clayton 1997, Bottjer et al. 1985). Every male bird learns to sing by imitating a

tutor, much like human babies learn to speak by listening to grown-ups

(Williams 2004). An interesting aspect of zebra finch singing is the social context,

such as the mating behaviour where the males sing in courtesy to the female.

Consequently, the singing has been widely studied due to an interest in

examining the genetic basis of learning. As a model organism, the zebra finch has

the advantages of being easy to breed in captivity, and it is just as popular among

pet owners as in scientific contexts.

9

A genetic map of the zebra finch genome will enable scientists to associate their

behavioural and neurological data to the regions in the genome that affect the

observed phenotype. Furthermore, the zebra finch genome is currently being

sequenced (http://genome.ucsc.edu/cgi-bin/hgGateway?db=taeGut1), which gives

the opportunity to compare physical and genetic distance to exhibit variations in

recombination rate over the chromosomes.

The population The population of zebra finches used in this study was bred at the Department of

Behavioural Ecology and Evolutionary Genetics at the Max Planck Institute for

Ornithology at Seewiesen by Dr Wolfgang Forstmeier. The original ancestors

were 63 males and 84 females, taken from the same population used in the zebra

finch linkage map constructed by Stapley et al. (2008). The ancestors, of unknown

kinship, gave rise to a pedigree of 1,204 individuals, all interrelated. The

pedigree consisted of 535 males, 524 females, and 145 offspring of unknown sex,

the youngest separated from the ancestors by at most four generations. The birds

were paired under controlled conditions. Maternity of offspring was determined

by observation, and paternity, when uncertain, by DNA analysis of ten

microsatellites. The entire pedigree contained 1,351 individuals.

The most interesting feature of the pedigree is how well-studied it is. Numerous

traits such as mass and tarsus length (relating to growth), beak colour and song

rate (relating to attractiveness of males), and aggressiveness and responsiveness

in mate choice situations (behavioural traits) have been monitored. A genetic

map covering this specific pedigree will hopefully enable the localisation of

approximate chromosome positions connected to these traits, which would be a

significant scientific progress.

Aims and Strategy The aim of the project was to produce a genetic map of the genome of the zebra

finch. Such a map would be beneficial for, inter alia, gene anchoring in the zebra

finch genome, in particular for genes involved in neurological functions, QTL

10

(quantitative trait loci) analysis and for comparative genomics of birds. This

genetic map would be obtained by linkage analysis of recombination data from a

pedigree of zebra finches, genotyped for some marker of preference. This project

used SNPs that were previously identified.

Methods The assignment of the degree project was the data analysis of the already

genotyped SNPs, but to put it in its correct context, the previous steps of the

mapping project need to be described.

SNP identification The original SNP panel consisted of 1,920 SNPs in total; 617 SNPs from the SNP

panel used by Stapley et al. (2008) in the first zebra finch map, 187 identified

from the sequencing of a number of pooled individuals from the pedigree in

Sheffield, 917 from the genome sequencing of a single zebra finch of American

origin, and 199 that had been identified in both of the latter reads. The newly

identified SNPs were chosen from sequences that had a single known chicken

homologue, as well as some of the Stapley SNPs. Hence 1,775 of the markers

were homologues to loci with known position in the chicken genome.

Genotyping The genotyping of the 1,920 SNPs was performed for 1,080 individuals from the

pedigree, using the Golden Gate Assay (Fan et al. 2003) from Illumina (San

Diego) at the SNP Technology Platform in Uppsala, Uppsala University

(http://www.genotyping.se). Prior to the genotyping, the DNA sequences flanking

the SNPs were examined for nucleotide composition, other polymorphisms etc. to

determine that they were appropriate as primers in the genotyping process. The

quality of the genotype data and the SNPs as genetic markers was controlled by a

set of quality control tests, including call rate, minor allele frequency, duplicate

tests for reproducibility and detection of inheritance conflicts. The call rate of

each SNP was calculated as the fraction of genotyping analyses that were

successful, including duplicates, for all individuals. The minor allele frequency

11

shows whether the SNP is polymorphic in this population; if the minor allele

frequency is zero, there is no variation to use for linkage analysis. An inheritance

conflict is the deviation from Mendelian inheritance of the genotype data of

parents and offspring. Inheritance conflicts in the data hence represent either

genotyping errors or errors in the pedigree of the population.

Data processing Prior to the linkage analysis, both the genotyping data and the pedigree had to be

revised to assure linkage data quality. Among the markers, 108 out of 1,920

SNPs failed completely in all genotyping attempts. In addition, 380 SNPs had a

minor allele frequency equal to zero and had to be discarded. Many inheritance

conflicts were reported, but most of them erroneously, as no respect were taken to

the fact that all markers on the Z chromosome were reported as homozygous. One

individual however had so many inheritance errors that its pedigree position was

considered wrong, and all its genotype data had to be removed. Fortunately, this

had only minor effect on the analysis as this individual had neither siblings nor

offspring. Eight SNPs had to be rejected due to excessive inheritance failures

that indicated genotyping difficulties.

Ultimately, 1,424 out of 1,920 markers persisted. The proportion of succeeded

markers for the SNPs of different origin was 587/617 from the Sheffield panel

(Stapley et al. 2008), 144/187 from the sequencing on pooled individuals, 506/917

from the sequencing of a single American zebra finch and 187/199 of the

combined. Obviously, the SNP identification in the reads from the single

American individual did not conform with the pedigree in question, perhaps due

to differences in allele frequencies between population as all other birds were

related to the Sheffield population, or due to individual variations being

mistaken for polymorphisms.

Mapping The linkage analysis was performed with CRI-MAP v. 2.4 (Green et al. 1990), but

with additions and modifications by Xuelu Liu (Monsanto) in order to be able to

manage the very complex pedigree in question. To start with, the TWOPOINT

12

program option was executed with LOD score > 3 (logarithm of odds,

corresponding to a p-value of 0.001). TWOPOINT makes twopoint linkage

analyses to establish linkage in between each pair of markers by calculating

whether two markers co-segregate through the pedigree. Henceforth, markers

were grouped by the command AUTOGROUP, an addition in the Monsanto

version. AUTOGROUP places markers that display linkage to each other in the

twopoint data output in linkage groups.

Twopoint analysis for 1,424 markers can make up in 1,013,176 different pairs in

theory. In practice, with a significant LOD score of at least 3 and linkage

presumably only within linkage groups, there were only 50,714 pairs with

significant linkage in the TWOPOINT analysis. However, with such an amount of

data points, a probability of one false positive in a thousand would make a

considerable amount of false linkages. A LOD score of 8 was consequently used

for the AUTOGROUP command as a threshold for inclusion in a linkage group,

producing only 1 expected false positive in each 108. This produced a set of

linkage groups that corresponded well with the expected chromosomes.

To include all significant linkages in the linkage groups, the AUTOGROUP

option was executed for each linkage group separately using LOD score 3 within

groups. In those executions, all markers suggested in first AUTOGROUP

analysis were included, as well as the markers suggested with reference to the

chicken chromosome organisation that had displayed significant linkage (of LOD

score > 3) in the original TWOPOINT execution. The linkage groups contained at

maximum 177 markers, which substantially reduced the risk of false positives.

Finally, all markers that had not been included in the AUTOGROUP executions

despite having significant linkage (but with unknown position in the chicken

genome) were assigned to linkage groups by their twopoint linkage data output.

Besides the LOD score, the AUTOGROUP command requires three additional

parameters to be specified to sort the markers into linkage groups; the minimum

number of informative meioses relative to the average number of informative

13

meioses, the maximum number of linkages to other groups, and minimum

fraction of linkages for each marker that are to the assigned group. These

parameters were set to 0, 20 and 0.3, respectively. Those constrains were all

relaxed so that the LOD score was hence the only restraining parameter.

When the linkage groups were established, the BUILD command that computes

the most likely marker order with LOD score > 3 was run in each linkage group

to produce a framework map. The BUILD command iteratively adds markers to

the set of ordered markers, if the maximum likelihood of the order is improved by

more than the threshold LOD score. Throughout the linkage analysis, distances

were sex-averaged and calculated in Kosambi cM, with exception for TgZ

(Taeniopygia guttata chromosome Z).

When building TgZ, individuals of unknown sex were excluded, using only 1,206

individuals of which 935 were genotyped. All females appeared as homozygous

for all loci due to females having only one Z chromosome, why one of the assumed

alleles for each locus was dismissed in females. Since recombination on sex

chromosomes only occurs in the homogametic sex, the distances calculated were

sex-specific for males.

Graphics Alignment of maps, as well as visualisation of maps, was performed using

MapChart (Voorips 2002).

Physical distances For the comparison of genetic and physical distances, the flanking regions of the

SNPs were used in BLAST searches in the zebra finch sequence assembly

(http://genome.ucsc.edu/cgi-bin/hgGateway?db=taeGut1) to obtain the physical

positions of the SNPs.

14

Results

Marker Genotyping The average success rate, i.e. sample call rate, among the residual markers was

95.04%. In this group, 18 out of 81,563 duplicates failed, giving a reproducibility

of 99.98%.

Linkage Groups The linkage group assignment resulted in 32 linkage groups, corresponding to

chromosome 1-28 plus chromosome Z, with double linkage groups for

chromosome 1, 24 and 25,

in the chicken genome and

named accordingly; where a

chicken chromosome

corresponded to more than

one linkage group in the

zebra finch linkage, those

were simply numbered

(Table 1). Using LOD score

> 3, 1,404 of 1,424 markers

displayed linkage to some

linkage group. All markers

included in the linkage

groups are listed in

Appendix A.

For chicken chromosome

22, there was only a single

marker with no linkage to

any other group. It is

included in the table of

linkage groups anyhow,

which thus contains 1,405

Linkage group

Number of

markers

Number of markers in

framework map

Distance covered in Kosambi cM

Tg1.1 150 36 118.3 Tg1.2 109 31 90.8 Tg2 177 28 75.7 Tg3 131 44 69.5 Tg4 125 17 44.4 Tg5 120 30 63.7 Tg6 76 22 60.2 Tg7 53 20 41.2 Tg8 55 23 47.1 Tg9 39 17 52.3 Tg10 31 13 55.0 Tg11 29 11 34.1 Tg12 38 16 34.8 Tg13 35 12 47.7 Tg14 20 7 46.1 Tg15 41 9 43.8 Tg16 3 2 27.5 Tg17 18 11 49.4 Tg18 10 5 30.7 Tg19 29 17 55.3 Tg20 24 14 52.4 Tg21 8 4 32.4 Tg22 1 1 0 Tg23 10 7 33.6

Tg24.1 3 2 4.2 Tg24.2 2 2 1.4 Tg25.1 4 2 14.9 Tg25.2 3 2 1.7 Tg26 7 5 64.7 Tg27 7 2 0.7 Tg28 8 2 0 TgZ 39 9 46.6

Total 1405 423 1340.2 Table 1. Number of markers by linkage group. LOD > 3. Linkage groups named according to chicken counterparts.

15

markers, owing to its presence in the former zebra finch map. Most of the

markers were assigned to the expected chromosome, with only three exceptions;

one marker located at chromosome 2 in chicken was found on chromosome 6 in

zebra finch, and another, located at chromosome 10 in chicken, appeared on

chromosome 14 in zebra finch, and lastly, a marker from chicken chromosome 4

was found in zebra finch chromosome 2.

Linkage group sizes correspond roughly with chicken chromosome lengths. As

expected, chicken chromosome 1 correspond to two big linkage groups. However,

chromosome 4, which was previously divided into two linkage groups as well, is

only represented by one linkage group in these results. This linkage group

contains markers from both chromosome 4 linkage groups in the previous zebra

finch map (Stapley et al. 2008).

The Framework Map The framework map provided by linkage analysis with LOD score > 3 contained

423 of the 1,404 linked markers (30.1%) and spanned 1,340.2 Kosambi cM. The

average genetic distance between adjacent markers was 3.43 cM, with a standard

deviation of 5.64 cM. Chromosome 22 was also included in the framework map

(Table 1, Figure 1) for further comparisons to other maps. Table 1 provides a

summary of the number of markers included in the framework map for each

linkage group. To have only two markers ordered is obviously equal to have no

markers ordered at all (as reading the linkage group from one end or the other

makes no difference). Linkage groups with only two ordered markers have

nevertheless been included in the framework map if the original linkage group

contained only very few markers (eight or less), where comparisons to other maps

are still informative.

16

TS1572

A16569A12272A13872TS0674A18344C00145A19836F09931A15078TS0017TS1455TS0649TS0262TS0168TS1188TS0016C00191C00198A14883A18836TS0832TS0444C00119TS1451A06985TS0816C00184TS0446A12605

TS0973

F10196F11969C00188C00149

TS0939

0

20

40

60

80

100

Tg1.1

TS1475

TS1538

C00269C00243TS0802TS0453TS0959C00281C00384TS0805A07057F27377TS0571TS1289C00130C00134C00059TS0924TS1660A04369TS1308TS1453A42430C00086TS0171TS1291

TS0898

A09491

TS0815TS0958

TS1393

0

20

40

60

80

Tg1.2

TS0789

TS1458

TS0744TS0403TS1243A33405A41865C00399TS0257TS0745C00113A42699F02848TS0724A07523TS0591TS0992A10115A13751F11064TS0894A13559F09532TS0025F13693TS0738C00205TS1304

0

20

40

60

Tg2

A19608

TS1441A16738F10496TS0558F05200TS0844F05987TS0180F06312F28042TS0796TS1211TS1069TS0365A41720TS1414TS0335A40085A41675A39343A40039C00369A34284A35359TS0422A37893A41302A37111A36468A25769A23077A29198A35582A17389A27742A30006A28961TS0121A35630C00381A35839A30742TS1178

0

20

40

60

Tg3

F15999

A29722TS0648

C00364

TS0810

TS1209A36052TS1220A33982TS1272TS0361F17130C00272C00212TS1505F14679

TS0011

0

20

40

Tg4

17

TS0368

TS0289

TS0391

TS1312TS0829TS0041TS1292TS0459C00041A03158A04695A43331A05544C00070TS0166A42516C00116TS0573C00456F23839TS1101TS1147TS1504TS1420A22924F01517TS0690A26084TS1091F17808

0

20

40

60

Tg5

A00955

TS1321

C00012TS0201

A01528TS0822A00201TS0491A25632TS1629A36826TS0023F21486TS0431A33993TS0987TS0786A31359TS0812A31051F18753

A28113

0

20

40

60

Tg6

TS0178A05508A43180A42625A05129TS0358TS0276TS1224A40829TS0486A28355A31013C00138C00240A27405F05452TS1066TS1113C00056

TS1463

0

20

40

Tg7

A27182TS1494A22212A21974A23550A23631A35525A37197TS0264A39248A38560A40409TS0191TS1382C00448A39167F25450TS1153A40627C00440TS0200TS1464

TS0823

0

20

40

Tg8

TS1419

TS1105

F23770C00338A30141TS0599C00284TS0903A25561A27076A22253A27226A21012C00214

TS0106

C00004

C00009

0

20

40

Tg9

TS0390

TS0095

F26842

A38288

A40455A35438C00376A28077F21558A32032A22173TS0934

TS0463

0

20

40

Tg10

18

TS0909

A27945

TS0797

C00331TS0946C00343F16314TS1467TS1431TS0971TS1444

0

20

Tg11

TS1476

A19389

A19735A17518A15050A12406TS1422TS1506A14062F11646F10615TS0911TS1344TS0635TS1454TS1383

0

20

Tg12

A00771

C00005TS0570C00228TS0870TS1346TS0477A20964C00215C00028TS1006

C00256

0

20

40

Tg13

A32839

TS0842

A02940TS0389TS0256

A01451

TS1550

0

20

40

Tg14

C00414

F25014

TS0585

TS1060

TS0333

A28843TS0341

C00350

A23726

0

20

40

Tg15

TS0725

TS1363

0

20

Tg16

TS0388

TS0732

A36774

A32346

TS0115

TS0369

TS0504TS0350

A28575TS0542TS1071

0

20

40

Tg17

A34181

TS1125

F26008

F22993F23695

0

20

Tg18

TS0864F22829A28744A18395

A32569TS0768TS0235

TS0997TS1253A27101

C00222

TS1158

F18227

F16699

TS0561

TS0590

TS1446

0

20

40

Tg19

A02020

TS0800

A22881

C00241F23558TS1590TS1119TS0920A31598

TS1325

TS1569TS0912TS1324

A37069

0

20

40

Tg20

19

F15517

A02955

F16527

A23214

0

20

Tg21

TS07870

Tg22

TS0627

TS0655

A02187TS1403TS1256

TS0149

C00007

0

20

Tg23

A01734

TS1263

0

Tg24.1

TS1315TS0948

0

Tg24.2

TS0675

TS0662

0

Tg25.1

C00218TS1376

0

Tg25.2

TS1117

TS1402

TS1565

TS1260

TS1371

0

20

40

60

Tg26

C00264TS1385

0

Tg27

TS0758TS1490

0

Tg28

A12246

A24644A08205

A34858A03084A37593

A34046A29010

A43515

0

20

40

TgZ

Figure 1. Linkage map over the zebra finch genome. The markers are ordered by LOD score > 3. Distances are measured in Kosambi cM.

20

The framework map (Figure 1) shows an obvious condensation of markers

somewhere in the middle of the chromosome for many linkage groups. This

condensation is assumed to represent chromosome areas close to the centromere,

where theory states that fewer recombination events occur and genetic distances

are thereby shorter.

Comparisons

The genetic maps of the zebra finch The genetic map of the zebra finch developed in this project had 587 markers in

common with the previous genetic map by Stapley et al. (2008). Out of these, 191

are included in the framework map. An alignment of the two zebra finch maps

exposes the good coherence between them, as is exemplified with linkage groups

Tg1.1-2, Tg2 and Tg3 in Figure 2.

Tg1.1 Tgu1B Tg1.2 Tgu1A Tg2 Tgu2 Tg3 Tgu3

Figure 2. Linkage groups Tg1.1-2, Tg2, and Tg3; a comparison between the framework map (to the left) and the map by Stapley et al. (2008) (to the right). All markers are included and common markers are connected. Tg1.1 and Tg3 have more than one homologous linkage group in the other map.

Tgu1

Tgu1C

Tgu3A

21

The chromosome assignment of the markers corresponds perfectly between the

two maps, and marker order is almost identical. Due to the higher power of the

linkage analysis of the Uppsala map, linkage was established to 7 linkage groups

(in Tg1.1, Tg3, Tg16, Tg20, Tg25 and Tg26) that were out-groups in the Sheffield

map (see Appendix B). The homologues to chicken chromosome 4, which was

represented by two linkage groups in the Sheffield map but only by one in this

study, would have been interesting to align, but unfortunately, all markers in

Tg4 in the framework map corresponded to the linkage group Tgu4A in the

Sheffield map. The whole alignment between the two zebra finch maps can be

found in Appendix B.

The chicken genome As the zebra finch SNPs were chosen in genes that were known to have chicken

orthologues, almost all of the markers in the framework map could be linked to

its chicken homologue. The linkage groups were, as mentioned above, almost

perfectly preserved between chicken and zebra finch with only a few exceptions.

There was, however, some disparity in gene order as is evinced in figure 3 that

aligns the framework map with a physical map over the chicken genome and

connects homologues.

22

Ggal1 Tg1.1 Ggal2 Tg2 Ggal3 Tg3 Ggal4 Tg4

Tg1.2

23

Ggal5 Tg5 Ggal6 Tg6 Ggal7 Tg7 Ggal8 Tg8 Ggal9 Tg9

Ggal10 Tg10 Ggal11 Tg11 Ggal12 Tg12 Ggal13 Tg13

Ggal14 Tg14 Ggal15 Tg15 Ggal16 Tg16 Ggal17 Tg17

24

Ggal18 Tg18 Ggal19 Tg19 Ggal20 Tg20 Ggal21 Tg21

Ggal22 Tg22 Ggal23 Tg23 Ggal24 Tg24.1 Ggal25 Tg25.1

Ggal26 Tg26 Ggal27 Tg27 Ggal28 Tg28 GgalZ TgZ

Figure 3. Alignment of zebra finch framework map (right) and the chicken homologues (left). The chicken markers are interspersed by physical distances. Chicken chromosome 1, 24 and 25 are represented by two linkage groups each in the zebra finch framework map. Homologue sequences are connected.

Tg25.2

Tg24.2

25

Genetical vs. physical distances In the BLAST search for physical positions of the SNPs, 1,408 out of the 1,424

markers (98.9%) were found. However, some of the sequences yielded duplicate

hits. Hits on other chromosomes than the assigned were dismissed. Duplicates

very close to each other, within ~20 kbp (much closer than any two markers),

were considered as one. Duplications that were further apart were not included

in the comparisons. The genetic distance was plotted against the physical

distance for all linkage groups containing more than 15 markers in the

framework map; linkage groups Tg1.1-2, Tg2-9, Tg12 and Tg19. Figure 4 displays

the plots for linkage groups Tg1.1-2, Tg2, Tg4, Tg12 and Tg19. The rest of the

plots can be found in Appendix C.

Tg1.1

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120 140

cM

Mbp

26

Tg1.2

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100

cM

Mbp

Tg2

0

20

40

60

80

100

120

140

160

180

0 10 20 30 40 50 60 70 80

cM

Mbp

27

Tg4

0

5

10

15

20

25

0 10 20 30 40 50

cM

Mbp

Tg12

0

5

10

15

20

25

0 5 10 15 20 25 30 35 40

cM

Mbp

28

Tg19

0

2

4

6

8

10

12

0 10 20 30 40 50 60

cM

Mbp

Figure 4. Physical distance plotted against genetic distance for linkage groups Tg1.1-2, Tg2, Tg4, Tg12 and Tg19.

Some linkage groups, such as Tg1.1-2 and Tg2-5, showed an apparent s-shaped

curve as expected if recombination rate is negatively correlated with the distance

to the centromere. For others, such as Tg19, the relation was a very evident

straight line, indicating no such correlation. Some of the plots, like Tg12, were

difficult to interpret, and, naturally, this concerned the plots with few data points

in particular.

Markers that appear outside the general curve or line are the ones for which the

genetical map and the zebra finch sequence are not in agreement. None of the

displayed plots contained any obvious out-groups, but in most plots, there were a

few data points that call for closer investigation in the future, such as the two

trios of markers that seem to have changed places at around 60 cM in Tg2.

Discussion The result of the linkage analysis was a linkage map containing 30% of the

markers, spanning 31 linkage groups. This map was compared with the previous

29

zebra finch linkage map (Stapley et al. 2008) and with physical positions of the

markers from the sequencing. The marker order was in general consentient. The

framework map was also aligned with a physical map of the chicken genome.

The most significant disparity between the previous zebra finch map and the

linkage groups from this study regarded the equivalent of chicken chromosome 4,

which had been divided into two linkage groups. This was not only proposed for

zebra finch by Stapley et al. (2008), but also by Derjusheva et al. (2004) for

domestic pigeon and the two passerines chaffinch and redwing, by Backström et

al. (2008) for the collared flycatcher, and others (Guttenbach et al. 2003). The fact

that chicken chromosome 4 was represented by only one linkage group in this

study does not rule out the possibility that it is also homologous to another

microchromosome. The only contradiction to previous results is the markers that

were included in both this study and the map by Stapley et al. (2008), which are

believed to belong to the same linkage group when a larger dataset provides more

power to the calculations.

As mentioned above, the chicken does not belong to the group of passerines

(species of the order Passeriformes), but to the fowls. This means that the chicken

and the zebra finch are separated by at least ~100 million years of evolution

(Sibley and Ahlquist 1990, Barker et al. 2004). The genetic map of the zebra finch

yields new insight in how the two lineages have evolved since the split between

them. Further studies might reveal how these changes are associated with the

phenotypic differences between the fowls and the passerines.

It has been suggested that the chromosome organisation of markers in avian

genomes are preserved, but not the synteny within chromosomes (Schmid et al.

2005). The first genetic map of the zebra finch was in support of this statement

(Stapley et al. 2008). The alignment of the Uppsala zebra finch framework map

and the chicken genome provides further support, as was expected from the

consensus of the two zebra finch maps.

30

In some chromosomes, such as chromosome 2, synteny seems to be remarkably

well-preserved despite the 100 million years that separates the two species. In

other, such as chromosome 15, there are rearrangements; however, the

rearrangements seem to be explainable by only a few major inversions, in this

example three.

The hypothesis that the in other vertebrates observed correlation between

recombination rate and distance to the centromere (Jensen-Seaman et al. 2004) is

not so strong in avian genomes (Schmid et al. 2005), based on the chicken

genome, was definitely contradicted by the physical-genetic distance plots of

zebra finch linkage groups Tg1.1-2 and Tg2-5. However, the tendency was not as

evident for all linkage groups, and not supported at all by Tg19. A possible

explanation is that microchromosomes are too small to demonstrate a

pronounced effect. It can nevertheless be concluded that the negative correlation

between recombination rate and distance to the centromere is applicable to at

least some avian genomes, if not chicken.

Conclusions The new framework map over the zebra finch genome has already provided new

insights in the passerine genome. The alignment against the chicken genome

reveals chromosomal rearrangements that have occurred since the divergence of

the orders of the fowls and the passerines. The physical-genetic distance plots in

the zebra finch linkage groups shed light on the correlation between physical and

genetic distances over the chromosome.

The natural progression of the project is to construct a best-order map including

all markers that were sorted into the linkage groups. These results will hopefully

be of use in exploring the genetic background to the phenotypic traits recorded in

this zebra finch pedigree.

Acknowledgements First, I want to thank my supervisor Niclas Backström for guidance and support,

and Professor Hans Ellegren, who initialised the project and provided valuable

31

advice. I thank Mikael Thollesson for accepting the task of the scientific reviewer,

and Maryam Montazerolghaem and Martin Dahlö for kindly accepting to be my

opponents. The whole research group of Professor Ellegren at the Department for

Evolutionary Biology was a support in creating a good scientific work

environment with many sensible ideas and opinions. I want to thank Axel

Künstner in particular for providing invaluable technical assistance. Mathieu

Authier’s help with the software was also appreciated. Dr Wolfgang Forstmeier

with colleagues at the Department of Behavioural Ecology and Evolutionary

Genetics at the Max Planck Institute for Ornithology at Seewiesen are gratefully

acknowledged for the breeding and the sampling of the pedigree. I thank Tomas

Axelsson and his colleagues at the SNP Technology Platform at Uppsala

University for the thorough SNP genotyping. Finally, Rickard Hedman is

thanked for all his considerate help during the writing of this report.

32

References Åkesson, M., B. Hansson, D. Hasselquist and S. Bensch, 2007. Linkage mapping of AFLP markers in a wild population of great reed warblers: importance of heterozygosity and number of genotyped individuals. Mol. Ecol. 16:2189–2202. Backström, N., N. Karaiskou, E. H. Leder, L. Gustafsson, C. R. Primmer, A. Qvarnström and H. Ellegren, 2008. A Gene-Based Genetic Linkage Map of the Collared Flycatcher (Ficedula albicollis) Reveals Extensive Synteny and Gene-Order Conservation During 100 Million Years of Avian Evolution. Genetics 179: 1479–1495. Barker, F. K., A. Cibois, P. Schikler, J. Feinstein and J. Cracraft, 2004. Phylogeny and diversification of the largest avian radiation. Proc. Natl. Acad. Sci. USA 101:11040–11045. Bottjer, S. W., S. L. Glaessner and A. P. Arnold, 1985. Ontogeny of brain nuclei controlling song learning and behavior in zebra finches. J. Neurosci. 5:1556-1562. Derjusheva, S., A. Kurganova, F. Habermann and E. Gaginskaya, 2004. High chromosome conservation detected by comparative chromosome painting in chicken, pigeon and passerine birds. Chromosome Res. 12:715–723. Ellegren, H., 2004. Microsatellites: simple sequences with complex evolution. Nat. Rev. Genet. 5:435-445. Fan, J. B., A. Oliphant, R. Shen, B. G. Kermani, F. Garcia et al., 2003. Highly parallel SNP genotyping. Cold Spring Harbor Symp. Quant. Biol. 68:69-78. Green, P., K. Falls and S. Crook, 1990. Documentation for CRIMAP, Version 2.4. Washington University School of Medicine, St. Louis. Grodzicker T., J. Williams, P. Sharp and J. Sambrook, 1974. Physical mapping of temperaturesensitive mutations of adenoviruses. Cold Spring Harbor Symp. Quant. Biol. 39:439- 446. Guttenbach M., I. Nanda, W. Feichtinger, J. S. Masabanda, D. K. Griffin and M. Schmid, 2003. Comparative chromosome painting of chicken autosomal paints 1–9 in nine different bird species. Cytogenet. Genome Res. 103:173–184. Hale M, H. Jensen, T. Birkhead, T. Burke and J. Slate, 2008. A comparison of synteny and gene order on the homologue of chicken chromosome 7 between two passerine species and between passerines and chicken. Cytogenet. Genome Res. 121:120–129.

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Hansson, B., M. Åkesson, J. Slate and J.M. Permberton, 2005. Linkage mapping reveals sex-dimorphic map distances in passerine bird. Proc. R. Soc. Lond. B. 272: 2289–2298. Jensen-Seaman M. I., T. S. Furey, B. A. Payseur, Y. Lu, K. M. Roskin, C. F. Chen, M. A. Thomas, D. Haussler and H. J. Jacob, 2004. Comparative recombination rates in the rat, mouse, and human genomes. Genome Res. 14:528-538. Jin, H. and D. F. Clayton, 1997. Localized changes in immediate-early gene regulation during sensory and motor learning in zebra finches, Neuron 19:1049–1059. Kosambi D. D., 1944. The estimation of the map distance from recombination values. Ann. Eugen. 12:172–175. Schmid, M., I. Nanda, H. Hoehn, M. Schartl, T. Haaf et al., 2005. Second report on chicken genes and chromosomes 2005. Cytogenet. Genome Res. 109:415–479. Sibley, C. and J. Ahlquist, 1990. Phylogeny and Classification of Birds: A Study in Molecular Evolution. Yale University Press, New Haven, CT. Stapley, J., T. R. Birkhead, T. Burke and J. Slate, 2008. A Linkage Map of the Zebra Finch Taeniopygia guttata Provides New Insights Into Avian Genome Evolution. Genetics 179:651–667. Sturtevant, A. H., 1913. The linear arrangement of six sexlinked factors in Drosophila, as shown by their mode of association. J. Exp. Zool. 14:43-59. Sturtevant, A. H., 1915. The behavior of the chromosomes as studied through linkage. Z. Indukt. Abstammungs. Vererbungsl. 13:234-287. Voorrips, R. E., 2002. MapChart: software for the graphical presentation of linkage maps and QTLs. J. Hered. 93:77–78. Williams, H., 2004. Birdsong and singing behavior, Ann. N. Y. Acad. Sci. 1016:1–30. Zhao H. and M. S. McPeek, 1996. On genetic map function. Genetics 142:1369–137.

34

List of appendices Appendix A. List of all markers included in the linkage groups (Appendices, p.1)

Appendix B. Complete comparison with Sheffield map (Appendices, p.28)

Appendix C. The remaining physical-genetic distance plots (Appendices, p.31)

35

Appendices Appendix A. List of all markers included in the linkage groups

SNP Chicken chromosome Zebra finch linkage group

In framework map?

A03701 chr1 Tg1.1 A06985 chr1 Tg1.1 yes A06992 chr1 Tg1.1 A07879 chr1 Tg1.1 A12263 chr1 Tg1.1 A12264 chr1 Tg1.1 A12272 chr1 Tg1.1 yes A12274 chr1 Tg1.1 A12281 chr1 Tg1.1 A12390 chr1 Tg1.1 A12605 chr1 Tg1.1 yes A12909 chr1 Tg1.1 A13002 chr1 Tg1.1 A13306 chr1 Tg1.1 A13603 chr1 Tg1.1 A13629 chr1 Tg1.1 A13669 chr1 Tg1.1 A13872 chr1 Tg1.1 yes A14092 chr1 Tg1.1 A14504 chr1 Tg1.1 A14657 chr1 Tg1.1 A14883 chr1 Tg1.1 yes A14891 chr1 Tg1.1 A14901 chr1 Tg1.1 A14941 chr1 Tg1.1 A15078 chr1 Tg1.1 yes A15165 chr1 Tg1.1 A15394 chr1 Tg1.1 A15865 chr1 Tg1.1 A16150 chr1 Tg1.1 A16569 chr1 Tg1.1 yes A16886 chr1 Tg1.1 A16982 chr1 Tg1.1 A17039 chr1 Tg1.1 A17590 chr1 Tg1.1 A17722 chr1 Tg1.1 A18161 chr1 Tg1.1 A18339 chr1 Tg1.1 A18344 chr1 Tg1.1 yes A18719 chr1 Tg1.1 A18836 chr1 Tg1.1 yes A19234 chr1 Tg1.1 A19836 chr1 Tg1.1 yes A20288 chr1 Tg1.1 A43644 chr1 Tg1.1 C00035 chr1 Tg1.1 C00119 chr1 Tg1.1 yes C00136 chr1 Tg1.1

36

C00139 chr1 Tg1.1 C00142 chr1 Tg1.1 C00143 chr1 Tg1.1 C00145 chr1 Tg1.1 yes C00147 chr1 Tg1.1 C00149 chr1 Tg1.1 yes C00152 chr1 Tg1.1 C00154 chr1 Tg1.1 C00155 chr1 Tg1.1 C00158 chr1 Tg1.1 C00171 chr1 Tg1.1 C00172 chr1 Tg1.1 C00174 chr1 Tg1.1 C00178 chr1 Tg1.1 C00180 chr1 Tg1.1 C00181 chr1 Tg1.1 C00183 chr1 Tg1.1 C00184 chr1 Tg1.1 yes C00188 chr1 Tg1.1 yes C00191 chr1 Tg1.1 yes C00198 chr1 Tg1.1 yes C00199 chr1 Tg1.1 C00202 chr1 Tg1.1 C00207 chr1 Tg1.1 F04399 chr1 Tg1.1 F06703 chr1 Tg1.1 F08918 chr1 Tg1.1 F09243 chr1 Tg1.1 F09317 chr1 Tg1.1 F09931 chr1 Tg1.1 yes F10196 chr1 Tg1.1 yes F11425 chr1 Tg1.1 F11429 chr1 Tg1.1 F11969 chr1 Tg1.1 yes F11984 chr1 Tg1.1 F12228 chr1 Tg1.1 F12778 chr1 Tg1.1 F13467 chr1 Tg1.1 TS0016 chr1 Tg1.1 yes TS0017 chr1 Tg1.1 yes TS0092 chr1 Tg1.1 TS0154 chr1 Tg1.1 TS0168 chr1 Tg1.1 yes TS0196 chr1 Tg1.1 TS0197 chr1 Tg1.1 TS0202 chr1 Tg1.1 TS0254 chr1 Tg1.1 TS0259 chr1 Tg1.1 TS0262 chr1 Tg1.1 yes TS0384 chr1 Tg1.1 TS0400 chr1 Tg1.1 TS0408 chr1 Tg1.1 TS0429 UN Tg1.1 TS0433 chr1 Tg1.1

37

TS0444 chr1 Tg1.1 yes TS0446 chr1 Tg1.1 yes TS0454 UN Tg1.1 TS0455 UN Tg1.1 TS0478 chr1 Tg1.1 TS0487 chr1 Tg1.1 TS0524 chr1 Tg1.1 TS0556 chr1 Tg1.1 TS0649 chr1 Tg1.1 yes TS0665 chr1 Tg1.1 TS0674 chr1 Tg1.1 yes TS0681 chr1 Tg1.1 TS0757 chr1 Tg1.1 TS0759 chr1 Tg1.1 TS0761 chr1 Tg1.1 TS0816 chr1 Tg1.1 yes TS0832 chr1 Tg1.1 yes TS0836 chr1 Tg1.1 TS0862 chr1 Tg1.1 TS0915 chr1 Tg1.1 TS0939 chr1 Tg1.1 yes TS0961 chr1 Tg1.1 TS0973 UN Tg1.1 yes TS0974 UN Tg1.1 TS1002 chr1 Tg1.1 TS1008 chr1 Tg1.1 TS1030 chr1 Tg1.1 TS1103 chr1 Tg1.1 TS1108 chr1 Tg1.1 TS1115 chr1 Tg1.1 TS1126 chr1 Tg1.1 TS1188 chr1 Tg1.1 yes TS1225 chr1 Tg1.1 TS1236 chr1 Tg1.1 TS1246 chr1 Tg1.1 TS1301 chr1 Tg1.1 TS1310 chr1 Tg1.1 TS1366 UN Tg1.1 TS1433 UN Tg1.1 TS1450 UN Tg1.1 TS1451 UN Tg1.1 yes TS1455 UN Tg1.1 yes TS1479 UN Tg1.1 TS1515 UN Tg1.1 TS1536 UN Tg1.1 TS1572 UN Tg1.1 yes TS1582 UN Tg1.1 TS1678 chr1 Tg1.1 A04369 chr1 Tg1.2 yes A04763 chr1 Tg1.2 A04902 chr1 Tg1.2 A05727 chr1 Tg1.2 A07057 chr1 Tg1.2 yes A07733 chr1 Tg1.2

38

A08195 chr1 Tg1.2 A09491 chr1 Tg1.2 yes A09775 chr1 Tg1.2 A11045 chr1 Tg1.2 A11361 chr1 Tg1.2 A22128 chr1 Tg1.2 A27553 chr1 Tg1.2 A27798 chr1 Tg1.2 A28548 chr1 Tg1.2 A28702 chr1 Tg1.2 A29647 chr1 Tg1.2 A31676 chr1 Tg1.2 A32293 chr1 Tg1.2 A32650 chr1 Tg1.2 A35146 chr1 Tg1.2 A37946 chr1 Tg1.2 A39183 chr1 Tg1.2 A39858 chr1 Tg1.2 A41768 chr1 Tg1.2 A42364 chr1 Tg1.2 A42367 chr1 Tg1.2 A42413 chr1 Tg1.2 A42430 chr1 Tg1.2 yes A43247 chr1 Tg1.2 C00037 chr1 Tg1.2 C00038 chr1 Tg1.2 C00048 chr1 Tg1.2 C00059 chr1 Tg1.2 yes C00072 chr1 Tg1.2 C00083 chr1 Tg1.2 C00086 chr1 Tg1.2 yes C00130 chr1 Tg1.2 yes C00134 chr1 Tg1.2 yes C00243 chr1 Tg1.2 yes C00269 chr1 Tg1.2 yes C00278 chr1 Tg1.2 C00279 chr1 Tg1.2 C00281 chr1 Tg1.2 yes C00384 chr1 Tg1.2 yes C00385 chr1 Tg1.2 C00393 chr1 Tg1.2 C00394 chr1 Tg1.2 C00436 chr1 Tg1.2 C00449 chr1 Tg1.2 F02396 chr1 Tg1.2 F04028 chr1 Tg1.2 F04095 chr1 Tg1.2 F05777 chr1 Tg1.2 F06395 chr1 Tg1.2 F07555 chr1 Tg1.2 F19832 chr1 Tg1.2 F20801 chr1 Tg1.2 F27252 chr1 Tg1.2 F27377 chr1 Tg1.2 yes

39

TS0091 chr1 Tg1.2 TS0171 chr1 Tg1.2 yes TS0199 chr1 Tg1.2 TS0207 chr1 Tg1.2 TS0223 chr1 Tg1.2 TS0227 chr1 Tg1.2 TS0239 chr1 Tg1.2 TS0261 chr1 Tg1.2 TS0326 chr1 Tg1.2 TS0364 chr1 Tg1.2 TS0373 chr1 Tg1.2 TS0407 chr1 Tg1.2 TS0418 chr1 Tg1.2 TS0453 chr1 Tg1.2 yes TS0468 chr1 Tg1.2 TS0472 chr1 Tg1.2 TS0529 chr1 Tg1.2 TS0553 chr1 Tg1.2 TS0571 chr1 Tg1.2 yes TS0576 chr1 Tg1.2 TS0683 chr1 Tg1.2 TS0692 chr1 Tg1.2 TS0766 chr1 Tg1.2 TS0802 chr1 Tg1.2 yes TS0805 chr1 Tg1.2 yes TS0815 chr1 Tg1.2 yes TS0849 chr1 Tg1.2 TS0878 chr1 Tg1.2 TS0898 chr1 Tg1.2 yes TS0924 chr1 Tg1.2 yes TS0958 chr1 Tg1.2 yes TS0959 chr1 Tg1.2 yes TS0982 chr1 Tg1.2 TS1086 chr1 Tg1.2 TS1136 chr1 Tg1.2 TS1181 chr1 Tg1.2 TS1265 chr1 Tg1.2 TS1286 chr1 Tg1.2 TS1289 chr1 Tg1.2 yes TS1291 chr1 Tg1.2 yes TS1308 chr1 Tg1.2 yes TS1333 UN Tg1.2 TS1386 UN Tg1.2 TS1393 UN Tg1.2 yes TS1418 UN Tg1.2 TS1453 UN Tg1.2 yes TS1475 UN Tg1.2 yes TS1538 UN Tg1.2 yes TS1660 chr1 Tg1.2 yes A03240 chr2 Tg2 A03277 chr2 Tg2 A03335 chr2 Tg2 A03790 chr2 Tg2 A03885 chr2 Tg2

40

A05152 chr2 Tg2 A05449 chr2 Tg2 A05759 chr2 Tg2 A06221 chr2 Tg2 A06365 chr2 Tg2 A06418 chr2 Tg2 A07256 chr2 Tg2 A07391 chr2 Tg2 A07523 chr2 Tg2 yes A07672 chr2 Tg2 A07779 chr2 Tg2 A08080 chr2 Tg2 A08187 chr2 Tg2 A08758 chr2 Tg2 A08971 chr2 Tg2 A08988 chr2 Tg2 A09151 chr2 Tg2 A09979 chr2 Tg2 A10115 chr2 Tg2 yes A10348 chr2 Tg2 A10361 chr2 Tg2 A11299 chr2 Tg2 A11463 chr2 Tg2 A11861 chr2 Tg2 A11985 chr2 Tg2 A12481 chr2 Tg2 A13559 chr2 Tg2 yes A13751 chr2 Tg2 yes A14702 chr2 Tg2 A15098 chr2 Tg2 A15310 chr2 Tg2 A15936 chr2 Tg2 A15969 chr2 Tg2 A16924 chr2 Tg2 A17365 chr2 Tg2 A17555 chr2 Tg2 A18705 chr2 Tg2 A18942 chr2 Tg2 A19061 chr2 Tg2 A19258 chr2 Tg2 A19271 chr2 Tg2 A19295 chr2 Tg2 A19759 chr2 Tg2 A19954 chr2 Tg2 A20726 chr2 Tg2 A25817 chr4 Tg2 A29441 chr2 Tg2 A29465 chr2 Tg2 A33405 chr2 Tg2 yes A36138 chr2 Tg2 A36166 chr2 Tg2 A37629 chr2 Tg2 A37782 chr2 Tg2 A39297 chr2 Tg2

41

A39631 chr2 Tg2 A40693 chr2 Tg2 A40713 chr2 Tg2 A40739 chr2 Tg2 A41837 chr2 Tg2 A41865 chr2 Tg2 yes A41914 chr2 Tg2 A42300 chr2 Tg2 A42699 chr2 Tg2 yes A42953 chr2 Tg2 A42964 chr2 Tg2 A42969 chr2 Tg2 A43011 chr2 Tg2 A43442 chr2 Tg2 A43475 chr2 Tg2 C00052 chr2 Tg2 C00053 chr2 Tg2 C00071 chr2 Tg2 C00074 chr2 Tg2 C00077 chr2 Tg2 C00103 chr2 Tg2 C00108 chr2 Tg2 C00110 chr2 Tg2 C00113 chr2 Tg2 yes C00120 chr2 Tg2 C00153 chr2 Tg2 C00161 chr2 Tg2 C00167 chr2 Tg2 C00173 chr2 Tg2 C00179 chr2 Tg2 C00186 chr2 Tg2 C00187 chr2 Tg2 C00189 chr2 Tg2 C00195 chr2 Tg2 C00201 chr2 Tg2 C00205 chr2 Tg2 yes C00399 chr2 Tg2 yes C00451 chr2 Tg2 C00459 chr2 Tg2 C00471 chr2 Tg2 F02848 chr2 Tg2 yes F06932 chr2 Tg2 F07545 chr2 Tg2 F07947 chr2 Tg2 F08517 chr2 Tg2 F08712 chr2 Tg2 F08999 chr2 Tg2 F09005 chr2 Tg2 F09532 chr2 Tg2 yes F11064 chr2 Tg2 yes F11067 chr2 Tg2 F11248 chr2 Tg2 F12277 chr2 Tg2 F13693 chr2 Tg2 yes

42

F25408 chr2 Tg2 F26022 chr2 Tg2 F27502 chr2 Tg2 F28095 chr2 Tg2 TS0025 chr2 Tg2 yes TS0030 chr2 Tg2 TS0064 chr2 Tg2 TS0096 chr2 Tg2 TS0131 chr2 Tg2 TS0141 chr2 Tg2 TS0205 chr2 Tg2 TS0220 chr2 Tg2 TS0234 chr2 Tg2 TS0244 chr2 Tg2 TS0257 chr2 Tg2 yes TS0260 chr2 Tg2 TS0306 chr2 Tg2 TS0315 chr2 Tg2 TS0403 chr2 Tg2 yes TS0482 chr2 Tg2 TS0535 chr2 Tg2 TS0591 chr2 Tg2 yes TS0601 chr2 Tg2 TS0611 chr2 Tg2 TS0620 chr2 Tg2 TS0702 chr2 Tg2 TS0724 chr2 Tg2 yes TS0738 chr2 Tg2 yes TS0744 chr2 Tg2 yes TS0745 chr2 Tg2 yes TS0771 chr2 Tg2 TS0789 chr2 Tg2 yes TS0818 chr2 Tg2 TS0853 chr2 Tg2 TS0894 chr2 Tg2 yes TS0944 chr2 Tg2 TS0955 chr2 Tg2 TS0976 chr2 Tg2 TS0992 chr2 Tg2 yes TS1029 chr2 Tg2 TS1042 chr2 Tg2 TS1043 chr2 Tg2 TS1096 chr2 Tg2 TS1150 chr2 Tg2 TS1170 chr2 Tg2 TS1184 chr2 Tg2 TS1186 chr2 Tg2 TS1192 chr2 Tg2 TS1240 chr2 Tg2 TS1243 chr2 Tg2 yes TS1274 chr2 Tg2 TS1294 chr2 Tg2 TS1304 chr2 Tg2 yes TS1305 chr2 Tg2

43

TS1313 chr2 Tg2 TS1338 UN Tg2 TS1341 UN Tg2 TS1370 UN Tg2 TS1390 UN Tg2 TS1458 UN Tg2 yes TS1466 UN Tg2 TS1498 UN Tg2 TS1500 UN Tg2 TS1524 UN Tg2 A12651 chr3 Tg3 A12658 chr3 Tg3 A16516 chr3 Tg3 A16518 chr3 Tg3 A16738 chr3 Tg3 yes A16739 chr3 Tg3 A16743 chr3 Tg3 A17266 chr3 Tg3 A17389 chr3 Tg3 yes A17466 chr3 Tg3 A19603 chr3 Tg3 A19608 chr3 Tg3 yes A19612 chr3 Tg3 A23063 chr3 Tg3 A23073 chr3 Tg3 A23077 chr3 Tg3 yes A23101 chr3 Tg3 A23112 chr3 Tg3 A23121 chr3 Tg3 A23530 chr3 Tg3 A25769 chr3 Tg3 yes A27740 chr3 Tg3 A27742 chr3 Tg3 yes A28961 chr3 Tg3 yes A29198 chr3 Tg3 yes A30006 chr3 Tg3 yes A30742 chr3 Tg3 yes A34263 chr3 Tg3 A34266 chr3 Tg3 A34284 chr3 Tg3 yes A35359 chr3 Tg3 yes A35582 chr3 Tg3 yes A35627 chr3 Tg3 A35630 chr3 Tg3 yes A35839 chr3 Tg3 yes A35847 chr3 Tg3 A35853 chr3 Tg3 A36465 chr3 Tg3 A36468 chr3 Tg3 yes A37111 chr3 Tg3 yes A37113 chr3 Tg3 A37116 chr3 Tg3 A37893 chr3 Tg3 yes A39343 chr3 Tg3 yes

44

A39383 chr3 Tg3 A39387 chr3 Tg3 A40006 chr3 Tg3 A40019 chr3 Tg3 A40020 chr3 Tg3 A40039 chr3 Tg3 yes A40085 chr3 Tg3 yes A40321 chr3 Tg3 A41286 chr3 Tg3 A41302 chr3 Tg3 yes A41604 chr3 Tg3 A41618 chr3 Tg3 A41675 chr3 Tg3 yes A41694 chr3 Tg3 A41696 chr3 Tg3 A41720 chr3 Tg3 yes A41723 chr3 Tg3 A42130 chr3 Tg3 C00369 chr3 Tg3 yes C00381 chr3 Tg3 yes C00425 chr3 Tg3 C00426 chr3 Tg3 C00435 chr3 Tg3 F02386 chr3 Tg3 F02771 chr3 Tg3 F03378 chr3 Tg3 F04948 chr3 Tg3 F05200 chr3 Tg3 yes F05987 chr3 Tg3 yes F06312 chr3 Tg3 yes F08508 chr3 Tg3 F10171 chr3 Tg3 F10496 chr3 Tg3 yes F10550 chr3 Tg3 F16718 chr3 Tg3 F19932 chr3 Tg3 F19936 chr3 Tg3 F26308 chr3 Tg3 F27664 chr3 Tg3 F27994 chr3 Tg3 F28042 chr3 Tg3 yes TS0121 chr3 Tg3 yes TS0139 chr3 Tg3 TS0140 chr3 Tg3 TS0162 chr3 Tg3 TS0180 chr3 Tg3 yes TS0190 chr3 Tg3 TS0228 chr3 Tg3 TS0295 chr3 Tg3 TS0299 chr3 Tg3 TS0316 chr3 Tg3 TS0335 chr3 Tg3 yes TS0365 chr3 Tg3 yes TS0422 chr3 Tg3 yes

45

TS0442 chr3 Tg3 TS0514 chr3 Tg3 TS0558 chr3 Tg3 yes TS0578 chr3 Tg3 TS0588 chr3 Tg3 TS0676 chr3 Tg3 TS0796 chr3 Tg3 yes TS0844 chr3 Tg3 yes TS0863 chr3 Tg3 TS0892 chr3 Tg3 TS0916 chr3 Tg3 TS0936 chr3 Tg3 TS0942 chr3 Tg3 TS0952 chr3 Tg3 TS1027 chr3 Tg3 TS1069 chr3 Tg3 yes TS1085 chr3 Tg3 TS1134 chr3 Tg3 TS1178 chr3 Tg3 yes TS1211 chr3 Tg3 yes TS1216 chr3 Tg3 TS1319 chr3 Tg3 TS1356 UN Tg3 TS1380 UN Tg3 TS1387 UN Tg3 TS1412 UN Tg3 TS1414 UN Tg3 yes TS1441 UN Tg3 yes TS1460 UN Tg3 TS1473 UN Tg3 TS1489 UN Tg3 TS1609 UN Tg3 TS1628 chr3 Tg3 A02995 chr4 Tg4 A03165 chr4 Tg4 A04383 chr4 Tg4 A04756 chr4 Tg4 A04769 chr4 Tg4 A05701 chr4 Tg4 A05990 chr4 Tg4 A08105 chr4 Tg4 A08543 chr4 Tg4 A09012 chr4 Tg4 A09735 chr4 Tg4 A09965 chr4 Tg4 A10296 chr4 Tg4 A10577 chr4 Tg4 A10656 chr4 Tg4 A10712 chr4 Tg4 A11324 chr4 Tg4 A23376 chr4 Tg4 A28739 chr4 Tg4 A29346 chr4 Tg4 A29722 chr4 Tg4 yes

46

A29731 chr4 Tg4 A30037 chr4 Tg4 A31294 chr4 Tg4 A31303 chr4 Tg4 A31386 chr4 Tg4 A32226 chr4 Tg4 A32910 chr4 Tg4 A33982 chr4 Tg4 yes A34614 chr4 Tg4 A34628 chr4 Tg4 A34637 chr4 Tg4 A34645 chr4 Tg4 A36052 chr4 Tg4 yes A37107 chr4 Tg4 A39437 chr4 Tg4 A39439 chr4 Tg4 A40155 chr4 Tg4 A40162 chr4 Tg4 A40952 chr4 Tg4 A42826 chr4 Tg4 A42843 chr4 Tg4 C00042 chr4 Tg4 C00043 chr4 Tg4 C00045 chr4 Tg4 C00046 chr4 Tg4 C00062 chr4 Tg4 C00063 chr4 Tg4 C00079 chr4 Tg4 C00080 chr4 Tg4 C00081 chr4 Tg4 C00087 chr4 Tg4 C00104 chr4 Tg4 C00112 chr4 Tg4 C00115 chr4 Tg4 C00127 chr4 Tg4 C00212 chr4 Tg4 yes C00272 chr4 Tg4 yes C00364 chr4 Tg4 yes C00367 chr4 Tg4 C00411 chr4 Tg4 C00430 chr4 Tg4 C00446 chr4 Tg4 C00450 chr4 Tg4 C00454 chr4 Tg4 C00470 chr4 Tg4 C00476 chr4 Tg4 C00478 chr4 Tg4 F03557 chr4 Tg4 F04077 chr4 Tg4 F05698 chr4 Tg4 F06458 chr4 Tg4 F07469 chr4 Tg4 F14285 chr4 Tg4 F14679 chr4 Tg4 yes

47

F15999 chr4 Tg4 yes F17130 chr4 Tg4 yes F17310 chr4 Tg4 F17445 chr4 Tg4 F19042 chr4 Tg4 F19182 chr4 Tg4 F20826 chr4 Tg4 F24095 chr4 Tg4 F26224 chr4 Tg4 F26679 chr4 Tg4 F27507 chr4 Tg4 TS0011 chr4 Tg4 yes TS0070 chr4 Tg4 TS0138 chr4 Tg4 TS0146 chr4 Tg4 TS0287 chr4 Tg4 TS0325 chr4 Tg4 TS0361 chr4 Tg4 yes TS0382 chr4 Tg4 TS0536 chr4 Tg4 TS0540 chr4 Tg4 TS0648 chr4 Tg4 yes TS0743 chr4 Tg4 TS0760 chr4 Tg4 TS0764 chr4 Tg4 TS0783 chr4 Tg4 TS0810 chr4 Tg4 yes TS0880 chr4 Tg4 TS0913 chr4 Tg4 TS0983 chr4 Tg4 TS1009 chr4 Tg4 TS1034 chr4 Tg4 TS1035 chr4 Tg4 TS1090 chr4 Tg4 TS1209 chr4 Tg4 yes TS1220 chr4 Tg4 yes TS1229 chr4 Tg4 TS1272 chr4 Tg4 yes TS1281 chr4 Tg4 TS1316 chr4 Tg4 TS1318 chr4 Tg4 TS1378 UN Tg4 TS1434 UN Tg4 TS1505 UN Tg4 yes TS1507 UN Tg4 TS1598 UN Tg4 TS1626 chr4 Tg4 TS1632 chr4 Tg4 TS1654 chr4 Tg4 TS1673 chr4 Tg4 A03158 chr5 Tg5 yes A03814 chr5 Tg5 A04285 chr5 Tg5 A04477 chr5 Tg5

48

A04505 chr5 Tg5 A04695 chr5 Tg5 yes A05287 chr5 Tg5 A05544 chr5 Tg5 yes A07012 chr5 Tg5 A07506 chr5 Tg5 A07530 chr5 Tg5 A07536 chr5 Tg5 A09857 chr5 Tg5 A12204 chr5 Tg5 A17306 chr5 Tg5 A18117 chr5 Tg5 A19204 chr5 Tg5 A22924 chr5 Tg5 yes A26084 chr5 Tg5 yes A26627 chr5 Tg5 A29591 chr5 Tg5 A29822 chr5 Tg5 A31668 chr5 Tg5 A32530 chr5 Tg5 A32730 chr5 Tg5 A34031 chr5 Tg5 A34675 chr5 Tg5 A38689 chr5 Tg5 A40568 chr5 Tg5 A42138 chr5 Tg5 A42516 chr5 Tg5 yes A42795 chr5 Tg5 A43328 chr5 Tg5 A43331 chr5 Tg5 yes C00041 chr5 Tg5 yes C00057 chr5 Tg5 C00065 chr5 Tg5 C00070 chr5 Tg5 yes C00076 chr5 Tg5 C00078 chr5 Tg5 C00100 chr5 Tg5 C00116 chr5 Tg5 yes C00122 chr5 Tg5 C00135 chr5 Tg5 C00335 chr5 Tg5 C00341 chr5 Tg5 C00344 chr5 Tg5 C00374 chr5 Tg5 C00396 chr5 Tg5 C00413 chr5 Tg5 C00427 chr5 Tg5 C00428 chr5 Tg5 C00447 chr5 Tg5 C00456 chr5 Tg5 yes C00458 chr5 Tg5 C00462 chr5 Tg5 F01517 chr5 Tg5 yes F03314 chr5 Tg5

49

F05366 chr5 Tg5 F05406 chr5 Tg5 F06187 chr5 Tg5 F06191 chr5 Tg5 F07531 chr5 Tg5 F07828 chr5 Tg5 F17808 chr5 Tg5 yes F22071 chr5 Tg5 F23668 chr5 Tg5 F23839 chr5 Tg5 yes TS0041 chr5 Tg5 yes TS0158 chr5 Tg5 TS0166 chr5 Tg5 yes TS0193 chr5 Tg5 TS0236 chr5 Tg5 TS0258 chr5 Tg5 TS0289 chr5 Tg5 yes TS0334 chr5 Tg5 TS0368 chr5 Tg5 yes TS0375 chr5 Tg5 TS0380 chr5 Tg5 TS0391 chr5 Tg5 yes TS0397 chr5 Tg5 TS0424 chr5 Tg5 TS0451 chr5 Tg5 TS0452 chr5 Tg5 TS0459 chr5 Tg5 yes TS0528 chr5 Tg5 TS0573 chr5 Tg5 yes TS0628 chr5 Tg5 TS0687 chr5 Tg5 TS0690 chr5 Tg5 yes TS0707 chr5 Tg5 TS0721 chr5 Tg5 TS0723 chr5 Tg5 TS0726 chr5 Tg5 TS0829 chr5 Tg5 yes TS0950 chr5 Tg5 TS0968 chr5 Tg5 TS0991 chr5 Tg5 TS0995 chr5 Tg5 TS0996 chr5 Tg5 TS1055 chr5 Tg5 TS1091 chr5 Tg5 yes TS1101 chr5 Tg5 yes TS1123 chr5 Tg5 TS1147 chr5 Tg5 yes TS1219 chr5 Tg5 TS1292 chr5 Tg5 yes TS1293 chr5 Tg5 TS1312 chr5 Tg5 yes TS1331 UN Tg5 TS1360 UN Tg5 TS1420 UN Tg5 yes

50

TS1465 UN Tg5 TS1504 UN Tg5 yes TS1527 UN Tg5 TS1556 UN Tg5 TS1557 UN Tg5 TS1603 UN Tg5 TS1610 UN Tg5 TS1639 chr5 Tg5 A00194 chr6 Tg6 A00201 chr6 Tg6 yes A00496 chr6 Tg6 A00955 chr6 Tg6 yes A01159 chr6 Tg6 A01170 chr6 Tg6 A01528 chr6 Tg6 yes A02457 chr6 Tg6 A02900 chr6 Tg6 A02916 chr6 Tg6 A02924 chr6 Tg6 A16704 chr6 Tg6 A21345 chr6 Tg6 A23291 chr6 Tg6 A25632 chr6 Tg6 yes A26317 chr6 Tg6 A26552 chr6 Tg6 A26560 chr6 Tg6 A26823 chr6 Tg6 A28113 chr6 Tg6 yes A30675 chr6 Tg6 A30768 chr6 Tg6 A31051 chr6 Tg6 yes A31359 chr6 Tg6 yes A32751 chr6 Tg6 A32826 chr6 Tg6 A32895 chr6 Tg6 A33747 chr6 Tg6 A33993 chr6 Tg6 yes A34343 chr6 Tg6 A36481 chr6 Tg6 A36826 chr6 Tg6 yes C00012 chr6 Tg6 yes C00022 chr6 Tg6 C00027 chr6 Tg6 C00245 chr6 Tg6 C00248 chr6 Tg6 C00268 chr6 Tg6 F04050 chr2 Tg6 F14277 chr6 Tg6 F14377 chr6 Tg6 F17356 chr6 Tg6 F18753 chr6 Tg6 yes F19332 chr6 Tg6 F21486 chr6 Tg6 yes F23331 chr6 Tg6

51

TS0023 chr6 Tg6 yes TS0039 chr6 Tg6 TS0123 chr6 Tg6 TS0201 chr6 Tg6 yes TS0281 chr6 Tg6 TS0304 chr6 Tg6 TS0415 chr6 Tg6 TS0428 chr6 Tg6 TS0431 chr6 Tg6 yes TS0491 chr6 Tg6 yes TS0492 chr6 Tg6 TS0493 chr6 Tg6 TS0494 chr6 Tg6 TS0503 chr6 Tg6 TS0515 chr6 Tg6 TS0534 chr6 Tg6 TS0700 chr6 Tg6 TS0752 chr6 Tg6 TS0786 chr6 Tg6 yes TS0812 chr6 Tg6 yes TS0822 chr6 Tg6 yes TS0835 chr6 Tg6 TS0987 chr6 Tg6 yes TS1016 chr6 Tg6 TS1321 chr6 Tg6 yes TS1381 UN Tg6 TS1447 UN Tg6 TS1553 UN Tg6 TS1554 UN Tg6 TS1629 chr6 Tg6 yes A04983 chr7 Tg7 A05129 chr7 Tg7 yes A05508 chr7 Tg7 yes A05883 chr7 Tg7 A09839 chr7 Tg7 A10260 chr7 Tg7 A27405 chr7 Tg7 yes A27408 chr7 Tg7 A28355 chr7 Tg7 yes A31007 chr7 Tg7 A31013 chr7 Tg7 yes A31033 chr7 Tg7 A32507 chr7 Tg7 A35682 chr7 Tg7 A40829 chr7 Tg7 yes A40909 chr7 Tg7 A41347 chr7 Tg7 A42625 chr7 Tg7 yes A43180 chr7 Tg7 yes C00056 chr7 Tg7 yes C00088 chr7 Tg7 C00138 chr7 Tg7 yes C00240 chr7 Tg7 yes C00327 chr7 Tg7

52

C00347 chr7 Tg7 C00389 chr7 Tg7 C00390 chr7 Tg7 C00445 chr7 Tg7 F03273 chr7 Tg7 F05452 chr7 Tg7 yes F16144 chr7 Tg7 F16426 chr7 Tg7 TS0026 chr7 Tg7 TS0161 chr7 Tg7 TS0178 chr7 Tg7 yes TS0276 chr7 Tg7 yes TS0297 chr7 Tg7 TS0348 chr7 Tg7 TS0349 chr7 Tg7 TS0358 chr7 Tg7 yes TS0398 chr7 Tg7 TS0486 chr7 Tg7 yes TS0701 chr7 Tg7 TS0703 chr7 Tg7 TS0748 chr7 Tg7 TS1066 chr7 Tg7 yes TS1113 chr7 Tg7 yes TS1146 chr7 Tg7 TS1224 chr7 Tg7 yes TS1232 chr7 Tg7 TS1298 chr7 Tg7 TS1463 UN Tg7 yes TS1568 UN Tg7 A03910 chr8 Tg8 A06503 chr8 Tg8 A21974 chr8 Tg8 yes A22212 chr8 Tg8 yes A23550 chr8 Tg8 yes A23622 chr8 Tg8 A23631 chr8 Tg8 yes A23639 chr8 Tg8 A27182 chr8 Tg8 yes A27875 chr8 Tg8 A28457 chr8 Tg8 A28459 chr8 Tg8 A28827 chr8 Tg8 A30147 chr8 Tg8 A34330 chr8 Tg8 A34852 chr8 Tg8 A35259 chr8 Tg8 A35525 chr8 Tg8 yes A37164 chr8 Tg8 A37197 chr8 Tg8 yes A38449 chr8 Tg8 A38560 chr8 Tg8 yes A38875 chr8 Tg8 A39167 chr8 Tg8 yes A39248 chr8 Tg8 yes

53

A40409 chr8 Tg8 yes A40627 chr8 Tg8 yes A42186 chr8 Tg8 C00275 chr8 Tg8 C00349 chr8 Tg8 C00382 chr8 Tg8 C00424 chr8 Tg8 C00440 chr8 Tg8 yes C00448 chr8 Tg8 yes F04004 chr8 Tg8 F25450 chr8 Tg8 yes F26562 chr8 Tg8 TS0191 chr8 Tg8 yes TS0200 chr8 Tg8 yes TS0209 chr8 Tg8 TS0264 chr8 Tg8 yes TS0354 chr8 Tg8 TS0479 chr8 Tg8 TS0617 chr8 Tg8 TS0823 chr8 Tg8 yes TS1001 chr8 Tg8 TS1095 chr8 Tg8 TS1153 chr8 Tg8 yes TS1208 chr8 Tg8 TS1271 chr8 Tg8 TS1382 UN Tg8 yes TS1464 UN Tg8 yes TS1493 UN Tg8 TS1494 UN Tg8 yes TS1665 chr8 Tg8 A00372 chr9 Tg9 A02205 chr9 Tg9 A02287 chr9 Tg9 A02645 chr9 Tg9 A20935 chr9 Tg9 A21012 chr9 Tg9 yes A22253 chr9 Tg9 yes A23237 chr9 Tg9 A24482 chr9 Tg9 A25561 chr9 Tg9 yes A27076 chr9 Tg9 yes A27226 chr9 Tg9 yes A30139 chr9 Tg9 A30141 chr9 Tg9 yes A32251 chr9 Tg9 A35641 chr9 Tg9 C00004 chr9 Tg9 yes C00009 chr9 Tg9 yes C00032 chr9 Tg9 C00214 chr9 Tg9 yes C00239 chr9 Tg9 C00284 chr9 Tg9 yes C00338 chr9 Tg9 yes F23770 chr9 Tg9 yes

54

TS0031 chr9 Tg9 TS0106 chr9 Tg9 yes TS0296 chr9 Tg9 TS0412 chr9 Tg9 TS0599 chr9 Tg9 yes TS0903 chr9 Tg9 yes TS0978 UN Tg9 TS0984 chr9 Tg9 TS1105 chr9 Tg9 yes TS1183 UN Tg9 TS1235 chr9 Tg9 TS1405 UN Tg9 TS1419 UN Tg9 yes TS1449 UN Tg9 TS1461 UN Tg9 A22173 chr10 Tg10 yes A28077 chr10 Tg10 yes A30204 chr10 Tg10 A30913 chr10 Tg10 A32032 chr10 Tg10 yes A34943 chr10 Tg10 A35438 chr10 Tg10 yes A36384 chr10 Tg10 A37263 chr10 Tg10 A38288 chr10 Tg10 yes A38294 chr10 Tg10 A38790 chr10 Tg10 A38897 chr10 Tg10 A40448 chr10 Tg10 A40455 chr10 Tg10 yes C00339 chr10 Tg10 C00375 chr10 Tg10 C00376 chr10 Tg10 yes F21558 chr10 Tg10 yes F26842 chr10 Tg10 yes TS0086 chr10 Tg10 TS0095 chr10 Tg10 yes TS0250 chr10 Tg10 TS0390 chr10 Tg10 yes TS0463 chr10 Tg10 yes TS0875 chr10 Tg10 TS0891 chr10 Tg10 TS0934 chr10 Tg10 yes TS1081 chr10 Tg10 TS1567 UN Tg10 TS1655 chr10 Tg10 A27945 chr11 Tg11 yes A30339 chr11 Tg11 A31145 chr11 Tg11 A31500 chr11 Tg11 A37540 chr11 Tg11 C00331 chr11 Tg11 yes C00343 chr11 Tg11 yes F16314 chr11 Tg11 yes

55

F17035 chr11 Tg11 F17942 chr11 Tg11 F18182 chr11 Tg11 F20181 chr11 Tg11 F20217 chr11 Tg11 F21501 chr11 Tg11 F22320 chr11 Tg11 TS0684 chr11 Tg11 TS0797 chr11 Tg11 yes TS0909 chr11 Tg11 yes TS0946 chr11 Tg11 yes TS0967 chr11 Tg11 TS0971 chr11 Tg11 yes TS1078 chr11 Tg11 TS1406 UN Tg11 TS1431 UN Tg11 yes TS1444 UN Tg11 yes TS1467 UN Tg11 yes TS1535 UN Tg11 TS1539 UN Tg11 TS1606 UN Tg11 A12406 chr12 Tg12 yes A13779 chr12 Tg12 A14062 chr12 Tg12 yes A14126 chr12 Tg12 A15050 chr12 Tg12 yes A16490 chr12 Tg12 A16650 chr12 Tg12 A16666 chr12 Tg12 A17518 chr12 Tg12 yes A17762 chr12 Tg12 A18191 chr12 Tg12 A19389 chr12 Tg12 yes A19502 chr12 Tg12 A19528 chr12 Tg12 A19735 chr12 Tg12 yes A20844 chr12 Tg12 F09327 chr12 Tg12 F10615 chr12 Tg12 yes F11205 chr12 Tg12 F11646 chr12 Tg12 yes F12549 chr12 Tg12 TS0049 chr12 Tg12 TS0527 chr12 Tg12 TS0635 chr12 Tg12 yes TS0785 chr12 Tg12 TS0911 chr12 Tg12 yes TS0960 chr12 Tg12 TS0981 chr12 Tg12 TS1223 chr12 Tg12 TS1344 UN Tg12 yes TS1348 UN Tg12 TS1369 UN Tg12 TS1383 UN Tg12 yes

56

TS1422 UN Tg12 yes TS1423 UN Tg12 TS1454 UN Tg12 yes TS1476 UN Tg12 yes TS1506 UN Tg12 yes A00771 chr13 Tg13 yes A01215 chr13 Tg13 A01472 chr13 Tg13 A01694 chr13 Tg13 A02604 chr13 Tg13 A13897 chr13 Tg13 A20964 chr13 Tg13 yes A22302 chr13 Tg13 A26252 chr13 Tg13 A26934 chr13 Tg13 C00005 chr13 Tg13 yes C00010 chr13 Tg13 C00026 chr13 Tg13 C00028 chr13 Tg13 yes C00029 chr13 Tg13 C00215 chr13 Tg13 yes C00225 chr13 Tg13 C00228 chr13 Tg13 yes C00242 chr13 Tg13 C00256 chr13 Tg13 yes F18408 chr13 Tg13 TS0024 UN Tg13 TS0029 UN Tg13 TS0035 chr13 Tg13 TS0078 chr13 Tg13 TS0255 chr13 Tg13 TS0362 chr13 Tg13 TS0477 chr13 Tg13 yes TS0513 chr13 Tg13 TS0570 chr13 Tg13 yes TS0870 chr13 Tg13 yes TS1006 chr13 Tg13 yes TS1257 chr13 Tg13 TS1346 UN Tg13 yes TS1430 UN Tg13 A01451 chr14 Tg14 yes A02940 chr14 Tg14 yes A26675 chr14 Tg14 A31376 chr14 Tg14 A31516 chr14 Tg14 A32839 chr14 Tg14 yes A34726 chr14 Tg14 F00674 chr14 Tg14 F00764 chr14 Tg14 TS0256 chr10 Tg14 yes TS0327 chr14 Tg14 TS0389 chr14 Tg14 yes TS0600 chr14 Tg14 TS0656 chr14 Tg14

57

TS0670 chr14 Tg14 TS0842 chr14 Tg14 yes TS1045 chr14 Tg14 TS1135 chr14 Tg14 TS1351 UN Tg14 TS1550 UN Tg14 yes A23726 chr15 Tg15 yes A23740 chr15 Tg15 A23915 chr15 Tg15 A24696 chr15 Tg15 A28843 chr15 Tg15 yes A33004 chr15 Tg15 A36057 chr15 Tg15 A38778 chr15 Tg15 C00262 chr15 Tg15 C00350 chr15 Tg15 yes C00352 chr15 Tg15 C00373 chr15 Tg15 C00387 chr15 Tg15 C00414 chr15 Tg15 yes C00441 chr15 Tg15 F19872 chr15 Tg15 F20400 chr15 Tg15 F25014 chr15 Tg15 yes F25075 chr15 Tg15 F26297 chr15 Tg15 F26717 chr15 Tg15 TS0004 chr15 Tg15 TS0145 chr15 Tg15 TS0225 chr15 Tg15 TS0246 chr15 Tg15 TS0324 chr15 Tg15 TS0333 chr15 Tg15 yes TS0341 chr15 Tg15 yes TS0462 chr15 Tg15 TS0585 chr15 Tg15 yes TS0659 chr15 Tg15 TS0672 chr15 Tg15 TS0933 chr15 Tg15 TS1022 chr15 Tg15 TS1040 chr15 Tg15 TS1060 chr15 Tg15 yes TS1337 UN Tg15 TS1417 UN Tg15 TS1503 UN Tg15 TS1534 UN Tg15 TS1576 UN Tg15 TS0725 chr16 Tg16 yes TS1363 UN Tg16 yes TS1365 UN Tg16 A22017 chr17 Tg17 A28575 chr17 Tg17 yes A32164 chr17 Tg17 A32346 chr17 Tg17 yes

58

A36774 chr17 Tg17 yes A37117 chr17 Tg17 C00383 chr17 Tg17 F20305 chr17 Tg17 TS0115 chr17 Tg17 yes TS0350 chr17 Tg17 yes TS0369 chr17 Tg17 yes TS0388 chr17 Tg17 yes TS0504 chr17 Tg17 yes TS0542 chr17 Tg17 yes TS0732 chr17 Tg17 yes TS1071 chr17 Tg17 yes TS1177 chr17 Tg17 TS1195 chr17 Tg17 A34181 chr18 Tg18 yes A34375 chr18 Tg18 F22993 chr18 Tg18 yes F23695 chr18 Tg18 yes F26008 chr18 Tg18 yes TS0679 chr18 Tg18 TS1070 chr18 Tg18 TS1125 chr18 Tg18 yes TS1347 UN Tg18 TS1588 UN Tg18 A18395 chr19 Tg19 yes A27101 chr19 Tg19 yes A28744 chr19 Tg19 yes A29186 chr19 Tg19 A32569 chr19 Tg19 yes A33929 chr19 Tg19 A35673 chr19 Tg19 C00222 chr19 Tg19 yes C00258 chr19 Tg19 F15660 chr19 Tg19 F16699 chr19 Tg19 yes F18227 chr19 Tg19 yes F22829 chr19 Tg19 yes TS0214 chr19 Tg19 TS0235 chr19 Tg19 yes TS0561 chr19 Tg19 yes TS0581 chr19 Tg19 TS0590 chr19 Tg19 yes TS0728 chr19 Tg19 TS0768 chr19 Tg19 yes TS0778 chr19 Tg19 TS0864 chr19 Tg19 yes TS0997 chr19 Tg19 yes TS1130 chr19 Tg19 TS1158 chr19 Tg19 yes TS1171 chr19 Tg19 TS1253 chr19 Tg19 yes TS1343 UN Tg19 TS1446 UN Tg19 yes A02020 chr20 Tg20 yes

59

A22881 chr20 Tg20 yes A23309 chr20 Tg20 A24986 chr20 Tg20 A31598 chr20 Tg20 yes A36958 chr20 Tg20 A37069 chr20 Tg20 yes C00241 chr20 Tg20 yes C00326 chr20 Tg20 F23558 chr20 Tg20 yes TS0248 chr20 Tg20 TS0800 chr20 Tg20 yes TS0912 chr20 Tg20 yes TS0920 chr20 Tg20 yes TS0957 chr20 Tg20 TS1119 chr20 Tg20 yes TS1162 chr20 Tg20 TS1324 chr20 Tg20 yes TS1325 chr20 Tg20 yes TS1569 UN Tg20 yes TS1570 UN Tg20 TS1590 UN Tg20 yes TS1591 UN Tg20 TS1597 UN Tg20 A02955 chr21 Tg21 yes A23214 chr21 Tg21 yes F15517 chr21 Tg21 yes F16527 chr21 Tg21 yes TS0718 chr21 Tg21 TS0919 chr21 Tg21 TS1185 chr21 Tg21 TS1602 UN Tg21 TS0787 chr22 Tg22 yes A02187 chr23 Tg23 yes C00007 chr23 Tg23 yes TS0149 chr23 Tg23 yes TS0627 chr23 Tg23 yes TS0655 chr23 Tg23 yes TS0663 chr23 Tg23 TS1167 chr23 Tg23 TS1238 chr23 Tg23 TS1256 chr23 Tg23 yes TS1403 UN Tg23 yes A01734 chr24 Tg24.1 yes C00013 chr24 Tg24.1 TS1263 chr24 Tg24.1 yes TS0948 chr24 Tg24.2 yes TS1315 chr24 Tg24.2 yes TS0662 chr25 Tg25.1 yes TS0675 UN Tg25.1 yes TS1436 UN Tg25.1 TS1522 UN Tg25.1 C00218 chr25 Tg25.2 yes TS1376 UN Tg25.2 yes TS1401 UN Tg25.2

60

TS0811 chr26 Tg26 TS1117 chr26 Tg26 yes TS1244 chr26 Tg26 TS1260 chr26 Tg26 yes TS1371 UN Tg26 yes TS1402 UN Tg26 yes TS1565 UN Tg26 yes C00261 chr27 Tg27 C00264 chr27 Tg27 yes TS0975 chr27 Tg27 TS1062 chr27 Tg27 TS1385 UN Tg27 yes TS1459 UN Tg27 TS1518 UN Tg27 A00633 chr28 Tg28 TS0010 chr28 Tg28 TS0089 chr28 Tg28 TS0758 chr28 Tg28 yes TS1490 UN Tg28 yes TS1508 UN Tg28 TS1595 UN Tg28 TS1601 UN Tg28 A03084 chrZ TgZ yes A04930 chrZ TgZ A05687 chrZ TgZ A05689 chrZ TgZ A07116 chrZ TgZ A07241 chrZ TgZ A08205 chrZ TgZ yes A08339 chrZ TgZ A09905 chrZ TgZ A11124 chrZ TgZ A11127 chrZ TgZ A12246 chrZ TgZ yes A24644 chrZ TgZ yes A29010 chrZ TgZ yes A29527 chrZ TgZ A33211 chrZ TgZ A34046 chrZ TgZ yes A34856 chrZ TgZ A34857 chrZ TgZ A34858 chrZ TgZ yes A35085 chrZ TgZ A37377 chrZ TgZ A37593 chrZ TgZ yes A37794 chrZ TgZ A38057 chrZ TgZ A38222 chrZ TgZ A43392 chrZ TgZ A43515 chrZ TgZ yes C00398 chrZ TgZ F28152 chrZ TgZ TS0183 chrZ TgZ TS0366 chrZ TgZ

61

TS0517 chrZ TgZ TS0582 chrZ TgZ TS0708 chrZ TgZ TS1007 chrZ TgZ TS1100 chrZ TgZ TS1309 chrZ TgZ TS1537 UN TgZ

62

Appendix B. Complete comparison with Sheffield map

Tg1.1 Tgu1B Tg1.2 Tgu1A Tg2 Tgu2 Tg3 Tgu3

Tg4 Tgu4A Tg5 Tgu5 Tg6 Tgu6 Tg7 Tgu7

Tgu1

Tgu1C

Tgu3A

Tgu4

63

Tg8 Tgu8 Tg9 Tgu9 Tg10 Tgu10 Tg11 Tgu11

Tg12 Tgu12 Tg13 Tgu13 Tg14 Tgu14 Tg15 Tgu15

Tg16 Tgu16 Tg17 Tgu17 Tg18 Tgu18 Tg19 Tgu19

Tgun4 Tgu18A

64

Tg24.1 Tgu24 Tg25.1 Tgun3 Tg26 Tgu26 Tg27 Tgu27

Tg28 Tgu28 TgZ TguZ

Tg24.2

Tg25.2

Tgu25

Tgun1

Tgu26A

65

Appendix C. The remaining physical-genetic distance plots, displaying Tg3 and

Tg5-9.

Tg3

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70 80

cM

Mbp

66

Tg5

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60

cM

Mbp

Tg6

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70

cM

Mbp

67

Tg7

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50

cM

Mbp

Tg8

0

5

10

15

20

25

30

0 10 20 30 40 50

cM

Mbp

68

Tg9

0

5

10

15

20

25

30

0 10 20 30 40 50 60

cM

Mbp