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Comparative genomics of arabidopsis thaliana

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Comparative genomics of arabidopsis thaliana

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Content COMPARATIVEGENOMICSOFARABIDOPSISTHALIANA
by
TinaT.Hu
ADissertationPresentedtothe
FACULTYOFTHEGRADUATESCHOOL
UNIVERSITYOFSOUTHERNCALIFORNIA
InPartialFulfillmentofthe
RequirementsfortheDegree
DOCTOROFPHILOSOPHY
(COMPUTATIONALBIOLOGYANDBIOINFORMATICS)
December2008
Copyright 2008 TinaT.Hu
Dedication
Tomyfamily...
ii
Acknowledgments
Firstandforemost,Iwouldliketoexpressmygreatappreciationtomyadvisor,Magnus
Nordborg, for all of his support, patience, and guidance throughout my time at USC. I
wouldalsoliketothankmycommitteemembers,bothpastandpresent: NormArnheim,
SteveFinkel,PaulMarjoram,SergeyNuzhdin,AntonioOrtega,andJeffWall. Iamalso
extremelygratefultohavebeenundertheformalandinformaltutelageofDetlefWeigel,
RichardClark,andChrisToomajian.
Of course, my time at USC would not have been as nearly as enjoyable had it not
been for my foosball teammates Sung Kim, Chris Toomajian, and Keyan Zhao as well
asmyfellowcompatriotsoftheextendedNordborglab: MuhammadAliAmer(Amer),
Mar´ ıa-Jos ´ eAranzana,SuziAtwell,PeteCalabrese,LizCooper,YuHuang,YokoIshino,
Badri Padhukasahasram, Vincent Plagnol, Alex Platt, Per Sj¨ odin, Aaron Tarone, Tom
Turner,andGlendaWillems.
iii
TableofContents
Dedication ii
Acknowledgments iii
ListofTables vi
ListofFigures vii
Abstract xii
Chapter1: Introduction 1
Chapter2: GenomeDifferencesBetweenA.thalianaandCloseRelatives 6
2.1 ChromosomalRearrangementsLeadingtoCurrentDayA.thalianaChro-
mosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 LossandGainofCentromeresandTelomeres . . . . . . . . . . . . . . 19
2.3 SegregatingInversions . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.4 ImpactofRearrangementsonPolymorphismLevels . . . . . . . . . . . 25
Chapter3: ShrinkageofA.thalianaGenome 28
3.1 Inter-ColinearGeneSpacingandTranscriptLengthComparisons . . . . 29
3.2 ImpactofRearrangements . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3 DeletionsUnderSelection . . . . . . . . . . . . . . . . . . . . . . . . 31
Chapter4: ComparisonofGenesBasedonColinearityStatus 38
4.1 AnalysisoftheImpactofGeneOrderonIndividualGenes . . . . . . . 40
4.1.1 LevelsofDivergence . . . . . . . . . . . . . . . . . . . . . . . 40
4.1.2 LevelsofPolymorphism . . . . . . . . . . . . . . . . . . . . . 43
4.2 AnalysisbyGeneFamily . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.3 TranspositionofFRIGIDA . . . . . . . . . . . . . . . . . . . . . . . . 54
4.3.1 TheFRILocus . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.3.2 ModeofTransposition . . . . . . . . . . . . . . . . . . . . . . 56
4.3.3 ComparisonofFRI
thal
andFRI
thal ancestral
. . . . . . . . . . . 57
iv
4.3.4 Helitrons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Chapter5: MaterialsandMethods 67
5.1 GenomeSequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
5.2 PolymorphismDatasetsinA.thaliana . . . . . . . . . . . . . . . . . . 68
5.3 IdentifyingColinearBlocks . . . . . . . . . . . . . . . . . . . . . . . . 68
5.4 PairwiseAlignmentsBetweenOrthologousGenePairs . . . . . . . . . 69
5.5 SequenceAnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
References 71
v
ListofTables
2.1 LocationsinA.thalianaofcentromeresandtelomeresinA.lyrata . . . 21
3.1 InsertionanddeletionpolymorphismsinA.thalianapolarizedbyA.lyrata 31
4.1 Categorizationofgenes . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.2 Kendall’srankcorrelationbetweencolinearityfractionandd
S
, d
N
,Θ
S
,
andΘ
A
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.3 Categorizationbycolinearitystatus . . . . . . . . . . . . . . . . . . . . 46
4.4 LevelsofnucleotidepolymorphisminFRI . . . . . . . . . . . . . . . . 56
5.1 Summaryofhits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
vi
ListofFigures
1.1 Phylogenyof27Brassicaceaemembers. Valuesalongbranchesaresup-
port values for the corresponding node with Bayesian posterior proba-
bilities above and parsimony bootstrap values below. Genome sizes in
pg (2C) and chromsome counts (2n) are given to the right of the taxa
(Fig. 1from[45]). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1 The events leading to chromosome number reduction in the course of
evolutionfromanancestorwithn=8chromosomestowardthekaryotype
of A. thaliana. Inversion (I), translocation (T), and fusion events (F) as
well as divergence time estimates are from Koch and Kiefer [32]. Only
consecutiveparacentricandpericentricinversions(Ipa/Ipe)withinAK4
are newly proposed. Events that require a distinct chronological order
arearrangedtogether. (Fig. 1Afrom[37]) . . . . . . . . . . . . . . . . 7
2.2 Idiogram of the A. thaliana karyotype indicating the composition of
chromosomes AT1 to 5 derived from the ancestral karyotype and the
events involved in chromosome number reduction in A. thaliana (Fig.
1Cfrom[37]). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.3 Orderoflarge-scalerearrangementeventsforAT1(inferredfrom[37]). 9
2.4 Orderoflarge-scalerearrangementeventsforAT2(inferredfrom[37]). 10
2.5 Orderoflarge-scalerearrangementeventsforAT3(inferredfrom[37]). 11
2.6 Orderoflarge-scalerearrangementeventsforAT4(inferredfrom[37]). 12
2.7 Orderoflarge-scalerearrangementeventsforAT5(inferredfrom[37]). 13
2.8 DotplotofhomologybetweenAT1toAL1andAL2. . . . . . . . . . . 14
2.9 DotplotofhomologybetweenAT2toAL3,AL4,andAL5. . . . . . . . 15
2.10 DotplotofhomologybetweenAT3toAL3andAL5. . . . . . . . . . . 16
vii
2.11 DotplotofhomologybetweenAT4toAL6andAL6. . . . . . . . . . . 17
2.12 DotplotofhomologybetweenAT5toAL6,AL7,andAL8. . . . . . . . 18
2.13 Patternofrepeatelement(redshading)andmethylation(blackline)den-
sity along the AT1-5 chromosomes. Chromosomal rearrangements as
compared to A. lyrata depicted below by arrows where color refers to
thecorrespondingAL. . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.14 List of Predicted Inversions in A. thaliana using the method described
inBansalet. al. [2]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.15 Crossover rates from a set of 17 F2 intercrosses genotyped at over 200
SNPs[57]. Resultsfromthedifferentcrossesarerepresentedascolored
linesandtheweightedaverageovereachcrossisshadedinblack. . . . 24
2.16 Polymorphism levels at fourfold degenerate sites (where all changes
remain synonymous) along the AT1-5 chromosomes (green shading).
Windowsthataresignificantlyspatiallyautocorrelatedaredenotedbeneath
by dark green ticks. Chromosomal rearrangements as compared to A.
lyratadepictedbelowbyarrowswherecolorreferstothecorresponding
AL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.17 Whole-genome tiling array recovery along the AT1-5 chromosomes for
each of the 20 A. thaliana accessions (Col-0 in black, Cvi-0 in salmon,
all other accessions in light blue, and repetitive probe density in gray
shading). ChromosomalrearrangementsascomparedtoA.lyratadepicted
belowbyarrowswherecolorreferstothecorrespondingAL. . . . . . . 27
3.1 Graphs comparing sizes of A. lyrata and B. stricta inserts from two-
hit clones in the filtered dataset to their homologous region in the A.
thaliana genome. The top panels show the direct comparison of the
sizes of the A. lyrata and B. stricta inserts from the genomic shotgun
libraries to the sizes of their A. thaliana homologs. The bottom panels
show the distribution of the size differences between the A. lyrata and
B.strictainsertstotheirA.thalianahomologs(Fig. 2from[45]).. . . . 33
3.2 Histogram of log
2
ratio of intergenic spacing between colinear pairs of
A.thalianaandA.lyratagenes. . . . . . . . . . . . . . . . . . . . . . . 34
3.3 Histogramoflog
2
ratiooftranscriptlengthsbetweenA.thalianaandA.
lyrataorthologousgenes. . . . . . . . . . . . . . . . . . . . . . . . . . 34
viii
3.4 Log
2
ratioofinter-colineargenespacingbetweencolineargenepairsof
A.thalianaandA.lyratagenes. Meanratioforallcolineargenepairsin
each100Kbwindowareshadedinbluewhereastheirindividualvalues
are show as light blue points. The ratio of the absolute distance based
on colinear gene pairs closest to the endpoints of each 100 Kb window
areplottedasdarkbluelines. . . . . . . . . . . . . . . . . . . . . . . . 35
3.5 Comparison of the log
2
ratio of 100 Kb windows and their orthologous
regionsinA.lyrata. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.6 Allelefrequencydistributionofinsertion/deletionpolymorphisms(polar- ized by A. lyrata) from the Nordborg et al. 2005 dataset [43]. Inser- tion/deletion polymorphisms in coding regions are plotted on top and
non-codingregionsonbottom. . . . . . . . . . . . . . . . . . . . . . . 37
4.1 Distributionofcolinearitystatusfor500Kbwindowsacrossthegenome.
Windows located in rearrangements are indicated in the first column in
red and windows where colinearity status could not be determined are
in yellow. Mean values of Ks andΘ
S
are computed for genes within
eachwindow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.2 Distributionofd
N
/d
S
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.3 Divergenceatnon-synonymoussites(d
N
). . . . . . . . . . . . . . . . . 47
4.4 Divergenceatsynonymoussites(d
S
). . . . . . . . . . . . . . . . . . . 47
4.5 DivergenceoftranscriptsasmeasuredbyKimura’s2-parameterdistance. 48
4.6 Comparison of transcript lengths with respect to their orthologs. Colin-
eargenesareinblueandtranspositionedgenesareinred. . . . . . . . . 48
4.7 Levelsofpolymorphism,asmeasuredbyπ,incodingregions. . . . . . 49
4.8 Distribution ofΘ
N
/Θ
S
. Colinear genes are in blue, transpositioned in
red, duplicated in green, partially homologous in purple, and lineage-
specificingray. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.9 Average recovery rates via whole-genome oligonucleotide tiling arrays
forcodingregionsacrossthe20accessionsqueriedinClarketal. 2007. 50
4.10 Distributionoftranscriptlengths. . . . . . . . . . . . . . . . . . . . . . 50
4.11 Codingsequencedivergence,asmeasuredbyKimura’s2-parameterdis-
tance,bygenefamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
ix
4.12 Divergence at synonymous (d
S
), non-synonymous (d
N
) and d
S
/d
N
by
genefamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.13 Distributionofcolinearitystatusbygenefamily. . . . . . . . . . . . . . 53
4.14 Gene model of FRI (AT4G00650.1) in the reference sequence (which
hasaloss-of-functionallele). . . . . . . . . . . . . . . . . . . . . . . . 55
4.15 MolecularstructureofdifferentFRIalleles. TheFRIgenecontainstwo
introns, 393 and 89 bp long, positioned 954 and 1520 bp downstream
of the Met residue of the likely translation start codon. The genomic
regionsofH51,Col,andLerallelesarerepresentedschematically,with
thechangesinnucleotideandaminoacidsequenceshownandtheposi-
tionsofthedeletionsindicated. (Fig2from[24]) . . . . . . . . . . . . 56
4.16 TheancestrallocationofFRIinA.thalianaisbetweenAT5G51080and
AT5G51090. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.17 VISTAviewofsequencesimilaritybetweenFRI
thal ancestral
andFRI
lyr
. 60
4.18 VISTA view of sequence similarity between FRI
thal ancestral
and the
orthologousregioncorrespondingtothegenesflankingFRIinA.thaliana. 61
4.19 DotplotofhomologybetweenFRI
thal
(AT4G00650.1)andFRI
lyr
(orthologous.scaffold 8.14317459.14382282). Focal gene models are
indicated in black, flanking gene models in gray, transposable elements
and repetitive elements in red, pseudogene predictions in purple, small
RNAsingreenandmethylationsitesinblue. . . . . . . . . . . . . . . 62
4.20 DotplotofhomologybetweenFRI
thal ancestral
(AT5G51090.1)andFRI
lyr
(orthologous.scaffold 8.14317459.14382282). Focal gene models are
indicated in black, flanking gene models in gray, transposable elements
and repetitive in red, pseudogene predictions in purple, small RNAs in
greenandmethylationsitesinblue. . . . . . . . . . . . . . . . . . . . 63
4.21 Dotplot of homology between FRI
thal
(AT4G00650) to FRI
thal ancestral
(AT5G51080,AT5G51090andAT5G51100). . . . . . . . . . . . . . . 64
4.22 GBROWSEviewofFRI
lyr
. TheorthologofFRIinA.lyrata
(scaffold 801542.1) is highlighted in blue and the Helitron elements
(HELITRON3,HELITRON1,andATREP15)arehighlightedingreen. . 65
x
4.23 StructureofHelitronelementsandtherollingcircletranspositionmech-
anism. (a)AgenericHelitronshowingsequencesandstructuralfeatures
that may be cis requirements for transposition. Helitrons from C. ele-
gans contain a single gene whereas Helitrons from A. thaliana and O.
sativa contain two or three. (b) A hypothetical mechanism for Helitron
transposition and gene acquisition based on the proposed rolling circle
mechanism for bacterial transposons. The element (in red) could be
either autonomous or nonautonomous. Two transposase molecules are
shown (blue ellipses) cleaving at the donor and target sites and binding
to the resulting 5’ ends. Replication at the cleaved donor site initiates
at the free 3’ OH and proceeds to displace one strand of Helitron. If
the palindrome and 3’ end of the element are recognized correctly, as
is shown on the Left, cleavage occurs after the CTRR sequence and the
one Helitron strand is transferred to the donor site where DNA replica-
tionresolvestheheteroduplex. TheillustrationontheRightdepictsone
way by which DNA flanking the 3’ end of the element (in green) could
betransferredalongwiththeelementtothedonorsite. Thismaybehow
Helitronshaveacquiredadditionalcodingsequences. (Fig. 1. of[16]) . 66
xi
Abstract
The highly selfing Arabidopsis thaliana differs from its closest relative, the obligate
outcrosser A. lyrata, both in genome size and organization. During approximately 5
million years of divergence, the A. thaliana genome has shrunk by approximately 40%
and chromosome number has decreased from 8 to 5. Comparative genomic analysis
between A. thaliana and A. lyrata suggests that genome rearrangements have had a
profoundeffectoncurrentpatternsofpolymorphismanddivergenceinA.thaliana.
xii
Chapter1
Introduction
Comparative genomic analysis is a powerful approach for understanding the evolution-
aryeventsthathaveshapedthemorphologicalandphysiologicalcharacteristicsofAra-
bidopsisthaliana. Throughthecomparisonwithitswell-studiedsisterspecies A.lyrata,
anunderstandingofhoweachspecieshasevolvedrelativetotheotherisrealized. Con-
served genomic regions that remain preserved in contemporary genome sequences typ-
ically reflect features common to the organisms being studied. These regions often
encode the elements responsible for the features that continue to be shared with their
last common ancestor. Conversely, divergent regions are generally responsible for gen-
erating differences between species. As such, genomic regions responsible for sim-
ilarities amongst species are generally maintained by purifying selection while those
regionsservingasthebasisfordifferencesbetweenspeciesaremaintainedthroughpos-
itive selection [22]. Another important aspect to note is the existence of neutral regions
thatareunconservedandpersistthroughrandomgeneticdrift. Theseregionswhichare
likely to be unimportant to the evolutionary success of the organism may resemble the
same selective forces leading to different evolutionary results in each of the lineages.
Identifying and understanding the impact of these conserved and divergent regions is
themajorprinciplebehindcomparativegenomics.
1
Even among close relatives, plant nuclear genomes vary tremendously in size and
arrangement. These differences are primarily the consequence of phenomenon ranging
fromthenumberandtimingofancestralpolyploidyevents(i.e.,doublingofentirechro-
mosome complements) to the variation in the degree of transposon amplification [4].
Despite such large variance in genome size, gene content remains relatively constant.
Thisparadoxbetweengenomesizeandorganismalcomplexityiscommonlyreferredto
asthe“Cvalueparadox”[53].
A. thaliana is a small flowering plant that is a member of the Brassicaceae family.
Membersofthisfamilyvaryconsiderablyfromlifehistoryandecologytogenomesize
and chromosome number. The wide ranging Brassicaceae family contains 338 genera
and 3,709 species distributed worldwide [1] and includes economically important crop
plants such as cabbage, horseradish, and broccoli. Although A. thaliana is of little
direct significance to agriculture, traits such as its short life cycle of six weeks, ability
to self-pollinate, small physical size, and small haploid genome size of ∼125 Mb have
madeitausefulmodelorganisminplantbiologyforunderstandingthegenetic,cellular,
and molecular biology of flowering plants. Among 27 members of the Brassicaceae
family surveyed, A. thaliana has the smallest genome as well as the fewest number of
chromosomes(Fig. 1.1).
A. thaliana and A. lyrata last shared a common ancestor approximately 5 million
years ago. During the last 5 million years, the A. thaliana plant and genome has under- goneseveraldrasticchanges. ProminentdifferencesbetweenA.thalianaanditsrecently
sequencedsisterspeciesA.lyratarangefromgenomesize(A.thalianaisapproximately
∼40% the size of A. lyrata), lifestyle (A. thaliana is a weedy annual and A. lyrata is
a perennial herb plant), to breeding structure (A. thaliana is predominantly inbreeding
whereas A. lyrata is outbreeding and self-incompatible). By analyzing the genomes of
A. thaliana and A. lyrata by means of a comparative framework, it will be possible to
2
futherourunderstandingofgenomeevolutioninA.thalianaandelucidatetheeventsthat
havecontributedtoitsspeciation. Specifically,theimpactofgenomerearrangementson
thecurrentpatternsofpolymorphismanddivergenceinA.thalianaareinvestigated.
In chapter 2, the large and small-scale chromosomal rearrangements between A.
thalianaandA.lyrataaredetailed. VariousstudiesanalyzingthecolinearitybetweenA.
thaliana and A. lyrata with a third species that predates the divergence of the two Ara-
bidopsisspeciesby∼10-14millionyears,havesuggestedtheancestralformtoresemble
A. lyrata, at least at the broad-scale [33, 62, 37]. Thus, most of the differences at the
genomic level are assumed to have occured in the A. thaliana lineage. To understand
theimpactoftheserearrangementsontheA.thalianagenome,variousgenomiccharac-
teristicsareanalyzedwithrespecttothepresenceofrearrangements.
In chapter 3, the shrinkage of the A. thaliana genome is investigated. This drastic
reduction in genome size may reflect directional selection towards faster transcription
for faster development, an important trait for a successful weedy lifestyle such as that
of A. thaliana [3, 7]. With respect to intron lengths, Wright et al. 2002 found that the
lengthsofintronsinA.thalianaareshorterthanthoseinA.lyrata[58]. Furthermore,in
concordance with the notion that shorter introns are selectively favored because of the
reduced costs to transcription, Seoighe et al. 2005 found that intron lengths of highly
expressed genes in A. thaliana were shorter in those genes specific to the gametophytic
(haploid) life stage, which are subject to stronger purifying selection, than those spe-
cific to the sporophytic (diploid) life stage [47]. Thus, the shrinkage of introns in A.
thalianaappeartobeconsistentwiththechangetowardsaweedylifestyle. Inthischap-
ter,theshrinkageoftheA.thalianagenomeisfurtherinvestigatedbycomparingrelative
lengths in non-coding regions based on distances between colinear gene pairs between
A.thalianaandA.lyrata.
3
In chapter 4, the impact of rearrangements on the evolution of individual genes is
investigated. The phenomenon of shared gene order between species suggests that the
orderingofgenesismaintainedthroughselection. Tounderstandtheextentofthispres-
sure exerted from selection at the level of individual genes, the divergence and poly-
morphism levels between colinear and non-colinear genes are explored and evaluated.
Furthermore, one of the previously identified non-colinear genes, FRIGIDA (FRI) [33],
isstudiedinfurtherdetail. InbothA.thalianaandA.lyrata,naturalvariationinflower- ingtimeisgovernedbytheFRIlocus[24,10,21,34]. However,FRIplaysalargerrole
in determining flowering time in A. thaliana and appears to have undergone a recent
selective sweep [56]. Following the duplication of a gene, it is believed that purify-
ing selection for the newly duplicated copy is relaxed thus allowing it to evolve a new
function [44]. Moreover, if the duplicate copy relocates to a new genomic context, it
is possible for that copy to escape from regulatory constraints at the ancestral location.
The mode of transposition of FRI, as well as the changes that have occured since its
transposition,arefutherinvestigated.
Together, these results suggest that genome rearrangements, both large and small,
haveplayedanotableroleinshapingthegenomeofA.thalianaandhavehadaprofound
effectoncurrentpatternsofpolymorphismanddivergenceinA.thaliana.
4
Figure1.1: Phylogenyof27Brassicaceaemembers. Valuesalongbranchesaresupport
values for the corresponding node with Bayesian posterior probabilities above and par- simony bootstrap values below. Genome sizes in pg (2C) and chromsome counts (2n)
aregiventotherightofthetaxa(Fig. 1from[45]).
5
Chapter2
GenomeDifferencesBetweenA.thalianaand
CloseRelatives
Even among close relatives, plant nuclear genomes vary tremendously in size and
arrangement. Such variation is primarily the consequence of phenomenona ranging
from polyploidization events to differing degrees of transposable element amplification
[4]. In the wide ranging Brassicaceae family, of which A. thaliana belongs to, mem-
bers vary considerably from life history and ecology to genome size and chromosome
number[1].
Chromosome number ranges widely from 4 to 128 within the Brassicaceae family
and between 5 and 8 within the Arabidopsis genus. Furthermore, among 27 members
of the Brassicaceae family surveyed, A. thaliana has the smallest genome as well as
the fewest number of chromosomes (Fig. 1.1). Based on previous comparative genetic
mapping and chromosome painting studies between A. thaliana with other members of
the Arabidopsis genus as well as various close relatives, the presumed ancestral kary-
otype(AK)stateis8chromosomes[33,62,37]. Thus,thechromosomalrearrangements
responsibleforthedifferenceinbothchromosomenumberandgenomearrangementare
presumedtohaveoccuredwithintheA.thalianalineage(Fig. 2.1).
6
2.1 ChromosomalRearrangementsLeadingtoCurrent
DayA.thalianaChromosomes
Previous studies estimate that at least 10 large-scale chromosomal rearrangements are
responsible for the current A. thaliana karyotype state of 5 chromosomes [33, 62, 37].
Thelarge-scalerearrangementeventsdetectedbythesemethodsinvolveatleast5inver- sion events, 2 reciprocal translocations, and 3 chromosomal fusions (Fig. 2.2, 2.3-2.7).
In addition to these rearrangements, chromosome features such as centromeres and
telomeres have been both lost and created in the process. With the genome sequence
ofA.lyratanearcompletion,fine-scalerearrangementscanfurtherresolvetheevolution
ofthesechromosomecomplements.
Figure2.1: Theeventsleadingtochromosomenumberreductioninthecourseofevolu-
tionfromanancestorwithn=8chromosomestowardthekaryotypeofA.thaliana. Inver- sion(I),translocation(T),andfusionevents(F)aswellasdivergencetimeestimatesare
from Koch and Kiefer [32]. Only consecutive paracentric and pericentric inversions
(Ipa/Ipe) within AK4 are newly proposed. Events that require a distinct chronological
orderarearrangedtogether. (Fig. 1Afrom[37])
7
Figure 2.2: Idiogram of the A. thaliana karyotype indicating the composition of chro-
mosomes AT1 to 5 derived from the ancestral karyotype and the events involved in
chromosomenumberreductioninA.thaliana(Fig. 1Cfrom[37]).
Based on the colinearity of similar genomic segments and orthologous protein-
codinggenesbetweenA.thaliana(AT)andA.lyrata(AL),∼30smaller-scalerearrange-
mentswerediscoveredinadditiontothepreviouslydeterminedlarge-scalegenomerear- rangements. These additional rearrangements support the theory that A. thaliana has
experiencedacomplexhistoryofgenomerearrangementssinceitlastsharedacommon
ancestor with A. lyrata. These fine-scale rearrangements include at least 23 inversion
and 7 translocation events. All detected chromosomal rearrangements are depicted as
breaks in colinearity in the homology dotplots between A. thaliana and A. lyrata chro-
mosomes(Fig. 2.8-2.12).
8
AK2
pericentric
inversion
AK1 & AK2
translocation
AT1 centromere
=
AK1 centromere
Figure2.3: Orderoflarge-scalerearrangementeventsforAT1(inferredfrom[37]).
9
AK3 & AK5
translocation
AK4
paracentric
inversion
AK4
pericentric
inversion
AK3/5 & AK4
translocation
AT2 centromere
=
AK3 centromere
Figure2.4: Orderoflarge-scalerearrangementeventsforAT2(inferredfrom[37]).
10
AK3 & AK5
translocation
AT3 centromere
=
AK5 centromere
goes on to form AT2
Figure2.5: Orderoflarge-scalerearrangementeventsforAT3(inferredfrom[37]).
11
AK6 & AK7
translocation
AK6/7
pericentric
inversion
AT4 centromere
=
AK6 centromere
goes on to form AT5
Figure2.6: Orderoflarge-scalerearrangementeventsforAT4(inferredfrom[37]).
12
AK6 & AK7
translocation
AK8
pericentric
inversion
AK6/7 & AK8
translocation
AT5 centromere
=
AK3 centromere
Figure2.7: Orderoflarge-scalerearrangementeventsforAT5(inferredfrom[37]).
13
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
AL 1
AL 2
AT 1 (position in MB)
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Figure2.9: DotplotofhomologybetweenAT2toAL3,AL4,andAL5.
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Figure2.12: DotplotofhomologybetweenAT5toAL6,AL7,andAL8.
18
2.2 LossandGainofCentromeresandTelomeres
Densemethylationandclusteringofrepetitiveelements,suchastransposableelements,
arefrequentlyfoundattranscriptionallyinactiveheterochromatin,suchascentromeres.
In a recent methylation map of A. thaliana, these heterochromatic regions indeed dis-
playedelevatedlevelsofmethylation,thoughpeakselsewhereinthegenomealsoappear
tobepresent[63].
During the transition from 8 to 5 chromosomes in A. thaliana, both centromeres
and telomeres were lost and/or gained (Table 2.1; Fig. 2.3-2.7). Depending on when
these heterochromatic regions were lost, remnants, appearing as slight peaks, of the
associated clustering of transposable elements and repeats appear to still be detectable
attheseformerlyheterochromaticregions(Fig. 2.13). However,morerecentrearrange-
ments causing heterochromatic regions to be introduced into euchromatic regions (and
viceversa)appeartoleaveastrongerfootprint. Thefootprintsofsuchrecentrearrange-
ments,likethepericentricinversiononAT4aswellasAT2,appearasseparateadjacent
peaks (Fig. 2.13) whereby the landscape of methlylation patterns and their associated
transposable elements and repeats are in their initial stages of readjusting to their new
locations.
2.3 SegregatingInversions
The 2.2 Mb pericentric inversion on AT 4 (between 1.6 and 2.8 Mb) corresponds to
the only known segregating rearrangement in A. thaliana [17]. Because the reference
orientation is identified, the reference allele is assumed to be the derived state. This
raises the possibility that the other identified rearrangements may not yet be fixed in
A. thaliana. To investigate whether any of these rearrangements are segregating, we
examinetheresultsoftwodifferentmethodsonalternatedatasets.
19
The first method attempts to identify large inversion polymorphisms using unusual
linkagedisequilibrium(LD)patternsfromhigh-densitySNPdata[2]. Specifically,posi-
tionswheretheorderingoftheSNPssuggestedbyLDpatternsisoppositetothatofthe
physical sequence correspond to regions where short-range LD patterns disagree with
long-rangeLDpatterns. Weapplythisalgorithmto262individualsgenotypedwiththe
250KSNPchip,designedtomaximizeLDwithallregionsofthegenome[30]. Results
of this algorithm detect the known segregating inversion and also predict 52 other seg-
regating inversions (Table 2.14). Most of these putative inversions are located within
centromeric regions and thus are difficult to verify further. Nonetheless, 5 putative
inversions appear to be highly likely as they coincide with inversions detected between
A.thalianaandA.lyrata(Fig. 2.8-2.12).
Thesecondmethodexaminestheestimatedrecombinationratesfromasetof17F2
intercrossesgenotypedatover200SNPs[57]. Tworegionswherethecrossoverratesare
strikingly different are strongly suggestive of inversions (Fig. 2.15). In particular, the
putativeinversiononchromosome5between15and19Mboverlapswithtwoinversions
as detected between A. thaliana and A. lyrata. Confirmation as to whether any of these
putativeinversionsareindeedsegregatingwillrequireexperimentalvalidationviaPCR.
20
Table2.1: LocationsinA.thalianaofcentromeresandtelomeresinA.lyrata
AT position(Mb) feature
1 0.4 telomereofAL1
1 21.4 telomereofAL1;bordersAL2centromere(lost)
1 24.1 telomereofAL2;bordersAL2centromere(lost)
1 30.0 telomereofAL2
2 9.0 telomereofAL3;telomereofAL4
2 11.3 borderscentromereofAL4(lost)
3 0.4 telomereofAL3
4 1.8 borderscentromereofAL6
4 7.9 borderscentromereofAL7
4 17.9 telomereofAL7
5 0.4 telomereofAL6
5 15.1 telomereofAL7
5 16.8 borderscentromereofAL8(lost)
5 19.4 borderscentromereofAL8(lost)
5 26.4 telomereofAL8
21
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
0.0
0.2
0.4
0.6
0.8
1.0
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
AT 1
AT 2
AT 3
AT 4
AT 5
transposable element, repeat & methylation density
Figure2.13: Patternofrepeatelement(redshading)andmethylation(blackline)density
alongtheAT1-5chromosomes. Chromosomalrearrangementsascomparedto A.lyrata
depictedbelowbyarrowswherecolorreferstothecorrespondingAL.
22
chr left bp. left bp. right bp. right bp. length best #samples
p-value with low p-value
1 11219348 11219890 11293988 11294794 74772 0.0010 7
1 14336223 14466028 15164469 15435474 898846 0.0000 7
1 14543157 14543196 15164444 15435474 756782 0.0000 9
1 15986528 15990879 16118186 16125257 133018 0.0000 7
1 16046371 16055234 16118186 16125257 70919 0.0010 10
1 17399322 17404301 17480046 17480305 78364 0.0010 7
1 23194778 23195014 23498602 23498686 303748 0.0010 8
2 3238982 3514923 3820731 3832859 449842 0.0000 7
2 3238982 3514923 4073405 4073603 696551 0.0000 7
2 3238982 3514923 3987805 3987956 610928 0.0000 7
2 3544429 3544525 3694794 3695355 150597 0.0000 8
2 3541304 3541458 3981932 3985161 442165 0.0020 8
2 3544138 3544394 3789553 3789665 245343 0.0000 7
2 3585801 3585835 3769283 3787588 192617 0.0000 9
2 3577735 3578352 3695471 3695816 117600 0.0030 10
2 3585835 3597873 4073603 4074668 482281 0.0010 9
2 3585801 3585835 3987345 3987601 401655 0.0010 7
2 3604024 3673052 3969066 3980572 336281 0.0000 7
2 3732048 3732612 3987956 3988364 255830 0.0020 7
2 3768285 3768434 3988431 3988484 220098 0.0000 8
2 3838860 3840181 4097215 4097343 257758 0.0010 8
2 3856480 3870508 4071108 4071473 207796 0.0000 7
2 3856480 3870570 3987805 3987956 124355 0.0010 10
2 4152820 4155755 4236648 4237191 82632 0.0000 10
2 4153223 4155755 4290970 4300566 141279 0.0000 10
2 4192068 4192139 4287087 4287410 95145 0.0000 8
2 4236648 4237191 4290970 4300566 58848 0.0030 10
2 4366249 4367425 4419114 4419321 52380 0.0000 9
2 4396099 4404343 4915895 4945834 530643 0.0000 9
2 5427772 5427926 5516470 5516556 88664 0.0130 7
2 5960614 5960809 6030046 6032925 70774 0.0010 7
2 9077746 9089494 9227581 9233185 146763 0.0000 9
2 9227223 9233185 11211306 11217225 1984061 0.0020 8
3 12263848 12264822 12352523 12352937 88395 0.0040 9
3 12461608 12463144 12529391 12529777 67208 0.0030 8
3 12953513 12953587 13156472 13165476 207424 0.0000 7
3 13156472 13165323 13325578 13325633 164708 0.0030 9
3 13293264 13315224 13473971 13474030 169756 0.0050 9
3 13390789 13393828 13456503 13471536 71711 0.0010 10
3 13531197 13534475 14136755 14168625 619854 0.0020 7
3 14001549 14014993 14076981 14080071 70255 0.0020 7
3 14001549 14015340 14170635 14171411 162578 0.0060 7
3 13999823 14000620 14250106 14250590 250126 0.0030 8
3 14073628 14073676 14169915 14170038 96324 0.0000 10
3 14071455 14073628 14330744 14335355 260508 0.0000 9
3 14076058 14076218 14244380 14244642 168373 0.0000 9
3 14176652 14177726 14326702 14328677 150500 0.0040 7
3 15035202 15035288 15190406 15190544 155230 0.0000 7
4 1613134 1613466 2782567 2783184 1169575 0.0000 9
5 11044762 11044956 11291854 11295980 249058 0.0080 7
5 11222303 11286140 11827109 11989704 654185 0.0000 9
5 13375947 13376600 13487760 13487914 111563 0.0050 7
Figure 2.14: List of Predicted Inversions in A. thaliana using the method described in
Bansalet. al. [2].
23
0e+00
1e−07
2e−07
3e−07
0e+00
1e−07
2e−07
3e−07
0e+00
1e−07
2e−07
3e−07
0e+00
1e−07
2e−07
3e−07
0e+00
1e−07
2e−07
3e−07
crossover rates
AT 1
AT 2
AT 3
AT 4
AT 5
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Figure 2.15: Crossover rates from a set of 17 F2 intercrosses genotyped at over 200
SNPs [57]. Results from the different crosses are represented as colored lines and the
weightedaverageovereachcrossisshadedinblack.
24
2.4 ImpactofRearrangementsonPolymorphismLevels
Inourprevioussurveyofpolymorphismin20A.thalianaaccessionsviawholegenome
tiling arrays [9], the analysis of polymorphism levels across the genome revealed that
this distribution varied dramatically along the chromosome in a strikingly nonrandom
fashion (Fig. 2.16). To understand the underlying forces responsible for these large-
scale fluctuations, we investigated several potential factors, such as selection on linked
sites and variation in recombination rates, though not all of the fluctuation could be
explained still. Based on the chromosomal rearrangements detected by comparing A.
thaliana with A. lyrata, it appears that the pattern of polymorphism in A. thaliana may
also be shaped by genome rearrangements, as some regions of spatial autocorrelation
coincidewiththeserearrangements.
Genome rearrangements may also be under selection. For instance, the transloca-
tion on AT1 (position 20.5 Mb) appears to delineate precisely the region with the most
extensive haplotype sharing detected in Clark et al. 2007. Furthermore, when plotting
therecoveryrateoftilingarraysacrossthegenome,itappearsthatsomeofthedipscor- respond to the endpoints of these rearrangements suggesting that conservation among
A. thaliana individuals in these regions is lower near rearrangement breakpoints (Fig.
2.17). Thus, the nonrandomness in polymorphism levels in A. thaliana may also be
partlyexplainedbychromosomalrearrangements.
25
0.000
0.005
0.010
0.015
| | || | | | | | | || || ||| || | | | | || | | || | | | | | | | | || | | | || | | | || | | | | | | | | ||| | | | | || | | | | | || | | || | | | | |
| ||||||||||||||||||||||||||||||||| ||||||||||||||||||||| | | ||| |||| ||||| | ||| ||| ||| | ||||||||| | ||||||||||||||||||||||||||||| |||| || | || |||| |||| |||||| ||||||||||| ||||| || ||| ||||||||||| |||||||||||||| ||||||||||||||||||| || || | || || || | ||||||||| | |||||||||||||||||||||||||| || | || || |||| |||||||| |||| | ||| | ||| ||| ||||||||| || | |||||||| |||||||||||| ||||||||| | || ||||
0.000
0.005
0.010
0.015
| | || | | | | | || | || || | || | | | | | | | | | | | | | | | | | | | | | | | || | | | | | | | | | |
| | |||||||||| | ||||||||||||||||||||||||| |||||| ||||| ||||||||| ||||||||||| ||| | |||||||||||||||||||||||||||||||||| |||||||| |||||||||||||||| | | ||| | |||| |||||| |||||| |||| |||| || ||||| ||||||||||| | | ||||||||||||||||||||||||||||||| |||| ||||||||||||||| | || || || | || || ||||||||||||||||| |||| |
0.000
0.005
0.010
0.015
| || | | ||| | | | | | | | | ||| | | | | | || || | | | | || | | | | | || | | | | ||| | | |
|| || |||| ||||| |||| |||||||||||||||| ||||| ||| || ||||||||||| ||||||||||||||||||| || ||||||||||||||||||||||| |||||||||||||||| |||||||| |||| |||| |||||| || |||| ||| | | |||| ||| |||| ||||||||| ||||| || || ||||||||| ||||||||||| | ||||||||||||||||||||| ||||||||| ||||| || || |||| | ||| || || | ||| || || ||||| ||||| ||||||||||||||||| ||||| || | |
0.000
0.005
0.010
0.015
|| | | | | | | || | | | | | || | | | | | | | | |
||||||| |||||||| |||||||||||||| || || | | |||||| ||||||| | | || | | || | |||||||||||||||||||||||||| || ||| | |||||| |||| | | ||| | |||||| | | | | || ||||||||||||||||||| | |||| | | |||| | |||| ||||
0.000
0.005
0.010
0.015
| | | || | | | | | || | || | | || | || | | | || | | | | | | | | | | | | | |||| | || ||| | || | | | | | | | || | | || | |||| | || | ||| | | | | | | | | | | |
| ||||||||||||||||||||||||||||| |||||||||| || | | | |||||| | ||||| |||||||||||| ||||||| || | | ||||| |||||| |||||||| ||||||||| ||||||||||| |||||||||||||||||| ||||||||||| || ||| |||||||||||||||| ||| |||||||||||| ||||||||| ||| ||| |||| | | |||||| |||| ||||||||||| |||| || | | ||| |||| ||||| ||||||| ||||||||| |||||||||||||| ||| |||||| || || | |||||| | |||| ||| || || ||| | |||||||||| | | | | | | | ||||| ||||| |||||| | |||| | | ||
AT 1
AT 2
AT 3
AT 4
AT 5
diversity at fourfold degenerate sites
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Figure2.16: Polymorphismlevelsatfourfolddegeneratesites(whereallchangesremain
synonymous) along the AT1-5 chromosomes (green shading). Windows that are signif-
icantly spatially autocorrelated are denoted beneath by dark green ticks. Chromosomal
rearrangements as compared to A. lyrata depicted below by arrows where color refers
tothecorrespondingAL.
26
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
tiling array recovery
AT 1
AT 2
AT 3
AT 4
AT 5
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Figure 2.17: Whole-genome tiling array recovery along the AT1-5 chromosomes for
each of the 20 A. thaliana accessions (Col-0 in black, Cvi-0 in salmon, all other acces-
sions in light blue, and repetitive probe density in gray shading). Chromosomal rear- rangementsascomparedtoA.lyratadepictedbelowbyarrowswherecolorreferstothe
correspondingAL.
27
Chapter3
ShrinkageofA.thalianaGenome
Primarily because of differences in the number and timing of ancestral polyploidy
eventstothevariationinthedegreeoftransposableelementamplification,plantnuclear
genomes vary tremendously in size and arrangement, even among close relatives [4].
Within A. thaliana, genome size has been found to vary by 10% among a sample of 21
Eurasianaccessions[46].
Inplants,variousmechanismsforgenomeexpansionandcontractionareresponsible
for the large variation in genome size in angiosperms [5]. The most prominent mecha-
nisms for genome expansion involve polyploidization and transposable element ampli-
fication. Mechanisms to counterbalance genome expansion are less known and range
from the loss of whole chromosomes, differential gene loss following polyploidization,
deletion-biasedmutationstoillegitimaterecombination. Itisalsoimportanttonotethat
additional factors such as cell size limitations and selection on cell division rates, par- ticularlyforselfingorganisms,caninfluenceareductioningenomesize[14].
It has been suggested that the change in lifestyle torwards a weedy annual may also
beresponsibleforthesmallergenomeofA.thaliana. Thisreductioningenomesizemay
reflectdirectionalselectiontowardsfastertranscription,animportanttraitforasuccess-
ful weedy lifestyle [3, 7]. With respect to intron lengths, Wright et al. 2002 found that
28
the lengths of introns are shorter in A. thaliana than in A. lyrata [58]. Furthermore, in
concordance with the notion that shorter introns are selectively favored because of the
reduced costs to transcription, Seoighe et al. 2005 found that intron lengths of highly
expressed genes in A. thaliana were shorter in those genes specific to the gametophytic
(haploid)lifestage,whicharesubjecttostrongerpurifyingselection,thanthosespecific
tothesporophytic(diploid)lifestage[47]. Thus,theshrinkageofintronsinA.thaliana
relative to A. lyrata appear to be consistent with the change towards a fast growing
weedylifestyle.
Withanestimatedgenomesizeof∼125Mb,A.thalianapossessesoneofthesmall-
est genomes of the angiosperms. Assuming an ancestral genome size similar to that
of A. lyrata, the A. thaliana genome has contracted by about 40%. Despite the drastic
difference in genome size, gene content is relatively comparable and most of the genes
in both species are orthologous. Together with results from the comparison of intron
lengths between A. thaliana and A. lyrata, the majority of the variation in genome size
appearstoresultingfromshrinkingnon-codingregions.
3.1 Inter-ColinearGeneSpacingandTranscriptLength
Comparisons
ToinvestigatethereductioningenomesizeinA.thalianarelativetoA.lyrata,theinter- colinear gene spacing and transcript lengths between colinear gene pairs are compared
(Fig. 3.2 and 3.3). Values of this ratio are greater than 0 if the spacing in A. thaliana
is longer and less than 0 if the spacing in A. lyrata is longer. Based on the histogram
in Figure 3.2, most of the spacing between inter-colinear gene pairs are shorter in A.
thaliana as compared to A. lyrata. Deletions resulting in these shorter distances in A.
thalianaarelikelyattributabletotheremovalofrepetitiveDNAsequencesand/orgenes.
29
Though the majority of these regions are just slightly shorter in A. thaliana, the peak at
the left tail of the histogram indicates that there exists an abundance of regions where
the spacing in A. lyrata is more than double that of A. thaliana. Conversely, transcript
lengthsbetweenA.thalianaandA.lyrataremainfairlyconsistent(Fig. 3.3),thoughthe
slightly longer length of A. thaliana transcripts may be an artifact from the inaccurate
annotationofuntranslatedregionsforA.lyratagenemodels.
Theseresultscomplementarecentstudycomparingtheinsertsizesof960genomic
shotgun clones between A. thaliana, A. lyrata, and a more distant relative Boechera
stricta which concluded that the majority of changes in A. thaliana’s genome size were
small but still included a considerable number of both large increases and decreases
(Fig. 3.1; [45]). Based on the results here and those of Oyama et al. 2008, it appears
thatthereductioningenomesizehasoccuredfairlyuniformlyinnon-codingintergenic
regionsthroughoutthegenome,withcertainregionsundergoingmoredrasticreductions
(Fig. 3.4).
3.2 ImpactofRearrangements
Whenviewedacrossthegenome,theregionswithmoredrasticreductions,correspond-
ing to the left tail of the histogram for inter-colinear gene spacing (Fig. 3.2), appear to
coincide with chromosomal rearrangements (Fig. 3.4). By comparing the endpoints of
100 Kb windows with the orthologous region in A. lyrata, the more drastically reduced
regions appear to be significantly associated with the presence of chromosomal rear- rangements (p-value =9.554e-10; Kolmogorov-Smirnov test; Fig. 3.5). Futhermore,
someofthesemoredrasticallyreducedregionscoincidewithregionsformerlyadjacent
to lost centromeres and telomeres (Table 2.1), thus likely reflecting the active removal
oftherepetitiveDNAsequencestypicalofheterochromaticregions.
30
3.3 DeletionsUnderSelection
Previous studies has suggested that the reduction in genome size may reflect the pref-
erence towards a smaller genome for weedy plants [3, 7]. With respect to the lengths
of introns in highly expressed genes in A. thaliana, Seoighe et al. 2005 found that
intron lengths were shorter in genes that were subject to stronger purifying selection
by comparing genes specifically expressed in the gametophytic to the sporophytic life
stage [47]. To investigate whether deletions are favored in the A. thaliana genome, the
frequency of insertion/deletion (indel) polymorphisms discovered among a set of 1213
PCRre-sequencedfragmentsdistributeduniformlyacrossthegenomein95individuals
[43] is examined. Among the 22,502 polymorphisms discovered, ∼10% (2,137) were
indel polymorphisms. Of these, only non-singleton indel polymorphisms which could
bepolarizedbyA.lyratawerefurtheranalyzed.
Consistent with mechanisms resulting in deletion-biased mutations, far fewer inser- tion than deletion polymorphisms are detected in both coding and non-coding regions
(Table 3.1). Futhermore, based on the allele frequency distribution of these indel poly-
morphisms, it appears that deletions are under positive selection in non-coding regions
while strongly selected against in coding regions (Fig. 3.6). That deletions may be
favored is reflected in the excess of high frequency deletion-derived alleles in non-
coding regions though the slight excess of low frequency deletion-derived alleles sug-
geststhatsomeofthesedeletionsmaybedeleterious. However,incodingregions,most
deletions are clearly deleterious as reflected in the excess of low frequency deletion-
derivedalleles.
Table3.1: InsertionanddeletionpolymorphismsinA.thalianapolarizedbyA.lyrata
annotation insertion deletion
coding 2 80
non-coding 17 594
31
Takentogether,thesefindingssuggestthatareductionmechanism,suchasdeletion-
biased mutations, in addition to directional selection for a smaller genome is operating
globallythroughouttheA.thalianagenome. Thoughthedeletionofgenescertainlycon-
tributestotheshrinkageofthegenomeinA.thaliana,themostprominentimpactcomes
fromtheshrinkageofintergenicregions. Also,becausegenecontentissimilarbetween
A. thaliana and A. lyrata and largely orthologous, the varying size of these intergenic
regions most likely reflects the different rates of gene transposition and, perhaps more
importantly, repetitive DNA insertion and deletion events. Nevertheless, the rate of this
reductioninA.thalianaappearstobemostsevere withinregionsofchromosomalrear- rangements. Furthermore,theanalysisofsegregatingindelsinA.thalianasuggeststhat
in addition to the higher occurrence of deletions, which may be enhanced in a selfing
organism, deletions appear to be under positive selection in A. thaliana. This supports
the previously suggested notion that the change to a weedy annual lifestyle, which is
characterizedbymorerapiddevelopment,mayselectforasmallergenome.
32
Figure 3.1: Graphs comparing sizes of A. lyrata and B. stricta inserts from two-hit
clonesinthefiltereddatasettotheirhomologousregionintheA.thalianagenome. The
toppanels showthedirect comparisonofthe sizesoftheA.lyrata andB.stricta inserts
from the genomic shotgun libraries to the sizes of their A. thaliana homologs. The
bottompanelsshowthedistributionofthesizedifferencesbetweentheA.lyrataandB.
strictainsertstotheirA.thalianahomologs(Fig. 2from[45]).
33
< −1
[−1,−0.8)
[−0.8,−0.6)
[−0.6,−0.4)
[−0.4,−0.2)
[−0.2,0)
[0,0.2)
[0.2,0.4)
[0.4,0.6)
[0.6,0.8)
[0.8,1)
>1
0
1000
2000
3000
4000
5000
6000
log2(thaliana/lyrata) intergenic regions
frequency
Figure 3.2: Histogram of log
2
ratio of intergenic spacing between colinear pairs of A.
thalianaandA.lyratagenes.
< −1
[−1,−0.8)
[−0.8,−0.6)
[−0.6,−0.4)
[−0.4,−0.2)
[−0.2,0)
[0,0.2)
[0.2,0.4)
[0.4,0.6)
[0.6,0.8)
[0.8,1)
>1
0
2000
4000
6000
8000
log2(thaliana/lyrata) transcript lengths
frequency
Figure 3.3: Histogram of log
2
ratio of transcript lengths between A. thaliana and A.
lyrataorthologousgenes.
34
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●
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●
−3
−2
−1
0
1
2
AT 1
AT 2
AT 3
AT 4
AT 5
log2(thaliana/lyrata)
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
c(0, 0)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Figure 3.4: Log
2
ratio of inter-colinear gene spacing between colinear gene pairs of A.
thaliana and A. lyrata genes. Mean ratio for all colinear gene pairs in each 100 Kb
windowareshadedinbluewhereastheirindividualvaluesareshowaslightbluepoints.
The ratio of the absolute distance based on colinear gene pairs closest to the endpoints
ofeach100Kbwindowareplottedasdarkbluelines.
35
< −1
[−1,−0.8)
[−0.8,−0.6)
[−0.6,−0.4)
[−0.4,−0.2)
[−0.2,0)
[0,0.2)
[0.2,0.4)
[0.4,0.6)
[0.6,0.8)
[0.8,1)
>1
colinear
rearrange
0.00
0.05
0.10
0.15
0.20
0.25
0.30
log2(thaliana/lyrata) for 100 Kb windows in thaliana
frequency
Figure 3.5: Comparison of the log
2
ratio of 100 Kb windows and their orthologous
regionsinA.lyrata.
36
0.125 0.25 0.375 0.5 0.625 0.75 0.875 1
coding
derived allele frequency
frequency
0.0 0.2 0.4 0.6 0.8
synonymous 596
nonsynonymous 807
deletion 80
insertion 2
0.125 0.25 0.375 0.5 0.625 0.75 0.875 1
non!coding
derived allele frequency
frequency
0.0 0.1 0.2 0.3 0.4
synonymous 596
nonsynonymous 807
deletion 594
insertion 17
Figure3.6: Allelefrequencydistributionofinsertion/deletionpolymorphisms(polarized
by A. lyrata) from the Nordborg et al. 2005 dataset [43]. Insertion/deletion polymor- phismsincodingregionsareplottedontopandnon-codingregionsonbottom.
37
Chapter4
ComparisonofGenesBasedonColinearity
Status
BasedonthesharedgeneorderoforthologousgenesbetweenA.thalianaandA.lyrata,
genes in each species can be classified into five major categories: colinear, transposi-
tioned, duplicated, partially homologous, and lineage-specific (Table 4.1). Orthologous
genes that are found in the same order between species are classified as colinear. As
can be expected of closely related species, the majority of genes in each of A. thaliana
and A. lyrata are categorized as colinear. Alternatively, orthologous genes not found
in their presumed ancestral context are considered as transpositioned. Movement of
genes from one genomic location to another can be generated by at least three molec-
ular mechanisms including excision and insertion mediated by mobile elements [55],
retrotransposition of a reverse-transcribed mRNA copy of a pre-existing gene [42], and
ectopic recombination between paralogous genes or gene-flanking repetitive sequences
[61]. The rate at which transpositions occur largely depends on the frequency of such
events. Perhaps because A. thaliana has fewer transposable elements from preliminary
estimates, it also has far fewer transpositioned genes relative to A. lyrata though the
geneorderofathirdspeciesisneededtoconfirmthisobservation.
38
The remaining categories encompass genes where orthology can not be confidently
established. Orthology relationships may be obscured from events such as gene dupli-
cations and gene conversion. Genes with multiple best hits are thus classified as dupli-
cated. These represent genes where multiple genes from one species map “best” to
the same gene in the other species. When only a portion of the transcript is found to be
homologouswiththeotherspecies,thegeneisclassifiedaspartiallyhomologous. When
similaritytoageneintheotherspeciescannotbefound, ageneisclassifiedaslineage-
specific. Lineage-specific genes can arise due to rapid divergence, pseudogenization or
geneloss. Thesegenesarepresumablyevolvingormutatingsorapidlythatorthologyis
nolongerpossible. Itisalsoimportanttonotethatbiasesmayexistintheannotationof
theA.lyratagenomewhichmayresultininaccuratecategorizationcounts. Forinstance,
the amount of lineage-specific genes detected in A. lyrata may be an underestimate as
thegenefindingalgorithmsusedinA.lyratarelyonA.thalianagenesastrainingsets.
Table4.1: Categorizationofgenes
Category A.thaliana A.lyrata
colinear 19649 21706
transpositioned 357 2379
duplicated 3223 1107
partiallyhomologous 1315 2346
lineage-specific 6700 2792
total 27235 32670
Anotherfactorthatmayinfluencethecolinearitystatusofageneisitsgenomiccon-
text. Regions which have experienced a rearrangement, such as inversions and translo-
cations,arelikelytolosetheircolinearitystatusduetothereducedefficacyofselection
in these regions as a result of suppressed recombination [41, 31]. In addition, the rate
at which colinearity status is lost further depends on the size of the rearrangement as
wellasthetimetofixation. Basedonthefractionofmembershiptothedifferentclassi-
ficationsofcolinearity,windowsthatoverlapwithrearrangementsappeartocontainthe
39
lowest fractions of colinear genes (Fig. 4.1). Correspondingly, the fraction of colinear
genes is significantly negatively correlated with d
S
, d
N
,Θ
S
, andΘ
A
(Table 4.2). The
weakercorrelationbetweend
S
andd
N
ascomparedtoΘ
S
andΘ
A
maybebecausethose
rearranged regions are still colinear in A. lyrata, while the stronger correlation between
Θ
S
andΘ
A
may be due to the effect of segregating rearrangements prior to fixation in
A.thaliana.
Table 4.2: Kendall’s rank correlation between colinearity fraction and d
S
, d
N
,Θ
S
, and
Θ
A
r p-value
d
S
-0.37250 <2.2e-16
d
N
-0.16288 1.332e-07
Θ
S
-0.29848 <2.2e-16
Θ
A
-0.36038 <2.2e-16
4.1 AnalysisoftheImpactofGeneOrderonIndividual
Genes
The prevalence of colinear genes across species spanning evolutionary time suggests
that selection may be operating to preserve the order of genes. Here, the impact of
this selection on gene order is evaluated by comparing the levels of divergence and
polymorphismbetweencolineargenesandtheothercategoriesofgenes.
4.1.1 LevelsofDivergence
With respect to levels of divergence, colinear genes are more conserved than transposi-
tioned genes. Comparison to the other categories are excluded as orthology is undeter- mined. Based on the comparison between colinear and transpositioned genes, transpo-
sitionedgeneshaveundergonemorerapiddivergencethangenesthatremainincolinear
40
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
colinear
transpositioned
duplicated
partially
homologous
lineage−specific
unknown
dS
thetaS
rearranged block
colinearity status
undetermined
Figure 4.1: Distribution of colinearity status for 500 Kb windows across the genome.
Windowslocatedinrearrangementsareindicatedinthefirstcolumninredandwindows
where colinearity status could not be determined are in yellow. Mean values of Ks and
Θ
S
arecomputedforgeneswithineachwindow.
41
order. This rapidity of divergence is reflected in the higher rates of protein evolution,
as measured by d
N
/d
S
, for transpositioned genes (Fig. 4.2). Nucleotide substitutions
in coding regions are either synonymous (does not change the amino acid) or non-
synonymous (changes the amino acid). Because non-synonymous changes are gener- ally detrimental, these types of substitutions are typically eliminated through purifying
selection. Thus, an indication of the amount of selection experienced by a gene can
be obtained by comparing the accumulation rates of synonymous and non-synonymous
substitutions(d
S
andd
N
,respectively).
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 >2
colinear
transpositioned
thaliana
dN/dS (Yang and Nielsen)
frequency
0.00 0.10 0.20 0.30
Figure4.2: Distributionofd
N
/d
S
.
Furthermore,therapidityofdivergencefortranspositionedgenesappearstobemore
pronounced and occuring throughout the transcript in full (Fig. 4.3, 4.4, and 4.5).
Because transpositioned genes can arise through mechanisms that involves duplication,
this pattern is consistent with the notion that purifying selection is relaxed immediately
followinggeneduplication[44].
42
The process of transposition involves the movement of a genomic region from one
locationtoanother. Toreiterate,thismovementcanbegeneratedbyatleastthreemolec-
ularmechanismsandtheamountofsequencetranspositionedisdependentonthemech-
anism at work. For instance, movement via retrotransposition creates an intron-less
and thus shorter copy of the progenitor gene whereas movement mediated by mobile
elements or ectopic recombination between paralogous genes or gene-flanking repeti-
tive sequences is dependent on the distance between the elements involved. Thus, the
lengths of transpositioned genes, as compared to their orthologs, are more variable pri-
marily because of the various mechanisms that generate transposition events. Whereas
the transcript length of colinear genes remains relatively constant, there is more vari-
ability for transpositioned genes (Fig. 4.6). The peaks for transpositioned genes corre-
spondingtothetailsofthisdistributionlikelyreflectsthesedifferentmodesoftransposi-
tionexperienced(i.e.,mRNA-mediatedtranspositionresultsinshorterintronlesscopies
while transpositions that may have involved flanking regions which have subsequently
fusedwiththetranspositionedtranscriptarelonger).
4.1.2 LevelsofPolymorphism
With respect to levels of polymorphism, colinear genes again appear to be the most
conserved relative to the other categories of genes. Within coding regions, colinear
genes appear to have the lowest levels of polymorphism (Fig. 4.7) and their proteins
are evolving much slower in general (Fig. 4.8). As expected for more conserved genes,
their recovery rates via the whole-genome oligonucleotide tiling array used in Clark et
al. 2007aremuchhigherdespitehavinglongertranscriptlengths(Fig. 4.9and4.10).
Theseresultsstronglysuggestthatinadditiontoretaininggeneorder,colineargenes
arealsoevolvingtheslowest,asreflectedbytheirlowlevelsofdivergenceandpolymor- phism. Thus,genomiccontextappearstoplayalargeroleintheevolutionofagene.
43
4.2 AnalysisbyGeneFamily
Gene families vary greatly in size and degree of similarity. The size of a gene family
over time depends on several factors, such as constraint due to physiological function
and susceptibility to duplications. Due to polyploidization events and both segmental
andtandemduplications,mostgenesinA.thalianaaremembersofgenefamilies. Based
on the previous annotation release of A. thaliana,∼71% of gene products are members
ofproteinfamilies[19]. Dependingonthefamilybuildingalgorithmemployed,thesize
of predicted protein families generally ranged between 2 to∼200 members, though the
highnumberofsmallfamiliesarelikelytoberemnantsofthemostrecentpolyploidiza-
tion event. Here, the degree of sequence conservation and physiological function for
severallargergenefamiliesinA.thalianaareinvestigated.
The analysis of divergence levels among gene families provides insight about the
degree of evolution and duplication experienced by the different families. Similar to
the observations from the analysis of polymorphism levels between gene families [9,
6], families involved in basic biological processes and those that belong to interaction
networks evolve more slowly whereas gene families involved in race-specific pathogen
defenseandsporophyticself-incompatibilityappeartoevolveatafasterpace(Fig. 4.11
and 4.12). The broader variance in levels of divergence for some gene families further
suggestthatnotallmembersinthesefamiliesareevolvingatthesamepace.
The analysis of colinearity status for gene family members provides an alternate
viewofcomparativechangesexperiencedbydifferentgenefamilies. Forinstance,rela-
tive rates of gene family expansion and contraction in one or both species will be over- represented for members classified as duplicated and partially homologous or lineage-
specific whereas members of more conserved gene families will mostly be classified
as colinear. Similar to the patterns of divergence, families involved in basic biolog-
ical processes and interaction networks are over-represented with colinear members
44
while gene families that benefit from variability such as those involved in race-specific
pathogen defense are over-represented with duplicated and partially homologous mem-
bers (Fig. 4.13 and Table 4.3). Also, further examination into cytoplasmic ribosomal
and cytochrome P450 gene families is needed as both these families appear to be heav-
ily over-represented with duplicated genes, suggesting that these gene families have
expandedwithintheA.thalianalineage,thoughitdoesnotprecludeanexpansioninthe
A.lyratalineage.
Interestingly,duplicatedmembersconstituteasignificantproportionofallgenefam-
ilies surveyed here, while there appears to be a severe under-representation of lineage-
specific members. The relatively constant fraction of duplicated members suggests that
either the surveyed gene families have all experienced expansions specific to the A.
thaliana lineage and/or the recurrent rate of gene conversion between family members
isfairly similarfor allfamilies whilemembership toa genefamily appearsto constrain
agenefromdevelopingnewfunctions.
45
Table4.3: Categorizationbycolinearitystatus
genefamily colinear transpositioned duplicated partially lineage-
homologous specific
reference 19649 357 3223 1315 6700
membrane 1037 14 137 16 8
transporter
ionchannel 44 5 4 3 3
families
proteinsynthesis 72 0 19 1 0
factors
metabolicpathway 1530 20 229 13 8
cytoplasmic 129 9 90 1 1
ribosomal
transcriptionfactor 1521 20 139 55 28
MYBtranscription 114 1 10 4 0
factor
bHLHtranscription 140 1 13 3 2
factor
glycosyltransferase 119 3 25 3 0
related
organicsolute 231 6 26 0 0
cotransporters
acyllipid 470 4 50 10 3
metabolism
glycosidehydrolase 239 0 46 7 0
cytochromeP450 129 3 69 4 1
RING 814 12 80 16 17
receptor-likekinase 329 2 31 15 3
F-box 462 30 139 182 34
diseaseresistance 144 10 87 46 12
S-locus 34 0 8 4 1
46
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! !! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! !! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !!! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! !! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! colinear
transpositioned
0.00 0.05 0.10 0.15 0.20 0.25
thaliana
dN
Figure4.3: Divergenceatnon-synonymoussites(d
N
).
! ! !! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! ! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! colinear
transpositioned
0.1 0.2 0.3 0.4
thaliana
dS
Figure4.4: Divergenceatsynonymoussites(d
S
).
47
! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! !! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! !! ! !! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! !! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! colinear
transpositioned
0.05 0.10 0.15 0.20
thaliana
divergence (entire transcript)
Figure4.5: DivergenceoftranscriptsasmeasuredbyKimura’s2-parameterdistance.
!0.8 !0.4 0 0.2 0.4 0.6 0.8 >1
thaliana
log2 ratio of transcript length
frequency
0.0 0.1 0.2 0.3 0.4
Figure 4.6: Comparison of transcript lengths with respect to their orthologs. Colinear
genesareinblueandtranspositionedgenesareinred.
48
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! !!!
! ! ! ! ! !! !!! !!
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! !! ! ! ! ! !! ! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! !
colinear
transpositioned
duplicated
partially
homologous
lineage!specific
0.000 0.002 0.004 0.006 0.008
pi (CDS)
Figure4.7: Levelsofpolymorphism,asmeasuredbyπ,incodingregions.
0.1 0.25 0.5 0.75 1 >1
theta_N/theta_S
frequency
0.0 0.1 0.2 0.3 0.4
Figure 4.8: Distribution ofΘ
N
/Θ
S
. Colinear genes are in blue, transpositioned in red,
duplicatedingreen,partiallyhomologousinpurple,andlineage-specificingray.
49
! !! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !!! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! !! ! !! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! !! ! ! ! ! !!! ! !! ! ! ! ! !! ! ! !! ! !! !! ! !! ! ! ! ! ! ! ! ! !! ! ! !! ! !! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! !! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! !!! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! !!! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! !
colinear
transpositioned
duplicated
partially
homologous
lineage!specific
0.0 0.2 0.4 0.6 0.8 1.0
average CDS recovery
Figure 4.9: Average recovery rates via whole-genome oligonucleotide tiling arrays for
codingregionsacrossthe20accessionsqueriedinClarketal. 2007.
! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! !! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! !! ! ! ! ! !! ! ! ! !! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! !! ! ! !! ! ! ! ! ! !! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! !! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! !!! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !
colinear
transpositioned
duplicated
partially
homologous
lineage!specific
0 2000 4000 6000
transcript length
Figure4.10: Distributionoftranscriptlengths.
50
! ! !
! ! ! ! !!! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! !
! ! ! ! ! ! ! ! ! !
! ! ! !! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! !
! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! !
! ! ! ! !
! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !
S!locus
disease resistance
F!box
receptor!like kinase
RING
cytochrome P450
glycoside hydrolase
acyl lipid metabolism
organic solute cotransporters
glycosyltransferase related
bHLH transcription factor
MYB transcription factor
transcription factor
cytoplasmic ribosomal
metabolic pathway
protein synthesis factors
ion channel families
membrane transporter
0.05 0.10 0.15 0.20 0.25
divergence (CDS)
Figure 4.11: Coding sequence divergence, as measured by Kimura’s 2-parameter dis-
tance,bygenefamily.
51
! !
! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !
! ! ! ! ! ! !!
! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!
! ! ! ! ! ! ! ! ! !
! ! !
! ! ! ! ! !
!
! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !
!
! ! !
! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !
S!locus
disease resistance
F!box
receptor!like kinase
RING
cytochrome P450
glycoside hydrolase
acyl lipid metabolism
organic solute cotransporters
glycosyltransferase related
bHLH transcription factor
MYB transcription factor
transcription factor
cytoplasmic ribosomal
metabolic pathway
protein synthesis factors
ion channel families
membrane transporter
0.1 0.2 0.3 0.4
dS
!
! ! !! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
!! ! ! !! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! !!
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! !
! ! ! ! ! ! ! !
! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! !! ! ! !
! ! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! !!! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !
! ! ! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! !!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
S!locus
disease resistance
F!box
receptor!like kinase
RING
cytochrome P450
glycoside hydrolase
acyl lipid metabolism
organic solute cotransporters
glycosyltransferase related
bHLH transcription factor
MYB transcription factor
transcription factor
cytoplasmic ribosomal
metabolic pathway
protein synthesis factors
ion channel families
membrane transporter
0.00 0.05 0.10 0.15 0.20 0.25
dN
!!
! ! !
! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! !
! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! !
! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! ! ! ! ! ! ! !
! ! ! ! !
! ! !
! ! ! ! ! ! ! !
! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
!! ! ! ! !
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !
! ! ! !
!
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! ! ! ! ! ! ! !! ! ! ! ! ! ! ! !! ! ! !
S!locus
disease resistance
F!box
receptor!like kinase
RING
cytochrome P450
glycoside hydrolase
acyl lipid metabolism
organic solute cotransporters
glycosyltransferase related
bHLH transcription factor
MYB transcription factor
transcription factor
cytoplasmic ribosomal
metabolic pathway
protein synthesis factors
ion channel families
membrane transporter
0.0 0.2 0.4 0.6 0.8 1.0
dN/dS
Figure4.12: Divergenceatsynonymous(d
S
),non-synonymous(d
N
)andd
S
/d
N
bygene
family.
52
S!locus
disease resistance
F!box
receptor!like kinase
RING
cytochrome P450
glycoside hydrolase
acyl lipid metabolism
organic solute cotransporters
glycosyltransferase related
bHLH transcription factor
MYB transcription factor
transcription factor
cytoplasmic ribosomal
metabolic pathway
protein synthesis factors
ion channel families
membrane transporter
reference
colinear
transpositioned
duplicated
partially
homologous
lineage!specific
unknown
Figure4.13: Distributionofcolinearitystatusbygenefamily.
53
4.3 TranspositionofFRIGIDA
In both A. thaliana and A. lyrata, natural variation in flowering time is governed by the
FRIGIDA (FRI) locus [24, 10, 21, 34]. In many plants, initiation of flowering requires
a long period of cold temperature (vernalization) [39]. In A. thaliana, flowering time is
highly variable. Flowering for winter-annual accessions requires vernalization whereas
summer-annual accessions are early-flowering. The elimination of the vernalization
requirementforsomesummer-annualaccessionsiscausedbyseveralindependentloss-
of-functionallelesin FRI[24]. ThoughfloweringtimeinA.lyrataisalsocontrolledby
FRI,variationinfloweringtimedoesnotappeartobepredominantlycontrolledbyFRI
asinA.thaliana. AllelicvariantsofFRIsampledamong295A.lyrataaccessionsappear
to have somewhat negligible effects on function [34]. Thus, allelic variation at the FRI
locussuggestsdifferentrolesinA.thalianaandA.lyrataandthatsomefunctionalityof
FRIinA.lyratamaybenecessarybutoptionalinA.thaliana.
This difference in the patterns of polymorphism may be attributed in part to differ- ences in life history as well as selfing vs. outcrossing modes of reproduction. Whereas
severalindependentloss-of-functionmutationsareunderstrongselectionforlocaladap-
tation in A. thaliana [56], mutations in A. lyrata are more subtle and possibly under
balancingselection[34].
Another important factor that may be responsible for the more drastic mutations
in A. thaliana are changes in genomic constraint. In a comparative linkage mapping
study between A. thaliana and A. lyrata, the ordering of various markers, including the
FRI locus, were investigated [33]. Though the marker order of A. lyrata was found to
be mostly similar to that of A. thaliana, the FRI locus was found to be out of order
and appears to have moved from chromosome 5 to chromosome 4 in A. thaliana. It
is thus tempting to speculate that in addition to the transition to selfing and an annual
lifestyle, the more prominent role FRI plays in A. thaliana may have been facilitated
54
by the transposition of FRI by separating it from possible regulatory elements and/or
disruptingpossiblycoordinatedgeneexpressionattheancestrallocation.
4.3.1 TheFRILocus
The normal and functional form of the FRI protein encodes 609 amino acids and is
predicted to contain two coiled-coil domains at two positions (between position 55-
100 and 405-450) (Fig. 4.14). In A. thaliana, two loss-of-function alleles found in the
Col and Ler accessions disrupt the FRI sequence and cause one of the two coiled-coil
domainstobelost(Fig. 4.15). InA.lyrata,a42bpindelpolymorphism,corresponding
to a 14 amino acid length difference, is situated at the end of the protein in exon 3. The
42 bp insertion is appears to be the ancestral form as A. thaliana contains the insertion.
Inaddition,thelog
2
ratiooftranscriptlengthbetweenA.thalianaandA.lyratais0.078
indicating that the transcript lengths are relatively similar and that A. thaliana has the
longertranscript.
269k 270k 271k
Chr4 Chr4
Annotation Units
F6N23 F6N23
Locus
AT4G00650
protein_coding_gene
Protein Coding Gene Models
AT4G00650.1
Pseudogenes
Noncoding RNAs
Figure 4.14: Gene model of FRI (AT4G00650.1) in the reference sequence (which has
aloss-of-functionallele).
The ratio of nonsynonymous to synonymous divergence (d
N
/d
S
) between A.
thalianaandA.lyratais0.37. Thissuggeststhatthegeneisunderfunctionalconstraint.
55
Figure 4.15: Molecular structure of different FRI alleles. The FRI gene contains two
introns,393and89bplong,positioned954and1520bpdownstreamoftheMetresidue
of the likely translation start codon. The genomic regions of H51, Col, and Ler alleles
are represented schematically, with the changes in nucleotide and amino acid sequence
shownandthepositionsofthedeletionsindicated. (Fig2from[24])
Consistent with patterns of a selective sweep in A. thaliana, overall within-species pol-
morphismlevelsarelowerinA.thalianathanin. A.lyrata(Table4.4). However,π
N
/π
S
islowerinA.lyrataindicatingthatFRIisevolvingmoreslowlyinA.lyrata.
Table4.4: LevelsofnucleotidepolymorphisminFRI
species π π
S
π
N
π
N
/π
S
reference
A.thaliana 0.00214 0.00275 0.00210 0.76 [10]
A.lyrata 0.00574 0.01283 0.00323 0.25 [34]
4.3.2 ModeofTransposition
WiththesequencingoftheA.lyratagenome,inferenceregardingthetranspositionmode
of FRI in A. thaliana is now possible. Based on the colinearity analysis of neighboring
genes of FRI (FRI
thal
), the ancestral copy of FRI in A. thaliana (FRI
thal ancestral
) is on
AT 5 between gene models AT5G51080 and AT5G51090 (Fig. 4.17 and 4.16). As for
FRI
thal
, the corresponding orthologous region in A. lyrata (FRI
lyr
) is located on AL 8
56
(Fig. 4.19) whereas the orthologous region to the genes flanking FRI in A. thaliana is
onAL6(Fig. 4.18and4.20).
20788k 20789k 20790k 20791k 20792k
Chr5 Chr5
Annotation Units
K3K7 K3K7
MWD22 MWD22
Locus
AT5G51080
protein_coding_gene
AT5G51100
protein_coding_gene
AT5G51090
protein_coding_gene
Protein Coding Gene Models
AT5G51080.1
AT5G51080.2
AT5G51080.3
AT5G51100.1 AT5G51090.1
Pseudogenes
Noncoding RNAs
Figure 4.16: The ancestral location of FRI in A. thaliana is between AT5G51080 and
AT5G51090.
4.3.3 ComparisonofFRI
thal
andFRI
thal ancestral
Pairwise sequence comparison between FRI
thal
and its ancestral location
FRI
thal ancestral
reveals that remnants of the transposition event of FRI still remain
(Fig 4.21). It appears that the transposition event involved first a duplication of
FRI
thal ancestral
followedbythedifferentialdeletionandrearrangementatboththenew
andancestrallocation.
Because the gene model of FRI
thal
is essentially unchanged from that of FRI
lyr
(Fig. 4.19),regionswithouthomologybetweenFRI
thal
andFRI
thal ancestral
areassumed
to have been lost in FRI
thal ancestral
. With respect to the gene model of FRI
thal
(Fig.
4.14), pairwise sequence comparison to the gene models in FRI
thal ancestral
(Fig. 4.16)
reveals that the first exon of FRI currently exists singly at FRI
thal
whereas the region
downstreamhasexperienceddifferentialretentioninFRI
thal ancestral
:
• AT5G51090istheproductofafusioneventbetweenthefirstintronandthelatter
portionofthethirdexonofFRI
thal
57
• theendofAT5G51080hasfusedontothethirdexonofFRI
thal
whiletheremain-
ingregionsofAT5G50180haspseudogenized
• the region in FRI
thal
that corresponds to AT5G51100 has become completely
pseudogenizedandhasalsotranspositioneddownstreamofFRI
thal
.
It is interesting to note that the longer transcript length of FRI
thal
, as compared to
FRI
lyr
, can be explained by the fusion of the 3’ UTR of AT5G51080 to the end of the
third exon in FRI
thal
. Because this fusion event involved an untranslated region and
occured at the 3’ end of the transcript past the termination signal, this mutation was
likelyneutral.
4.3.4 Helitrons
Based on the sequence comparison between FRI
thal
and FRI
thal ancestral
, the transpo-
sition of FRI does not appear to have occured by any of the three common mecha-
nisms of transposition. Because remnants of FRI still exist at the ancestral location on
AT 5, transposition via the excision and insertion mediated by mobile elements can be
excluded. Furthermore,thepresenceofbothintronsandtheconsistencyofgenemodels
with that of FRI
lyrata
suggests that the transposition of FRI was not mediated through
a reverse-transcribed mRNA copy of FRI. Lastly, the large transposition of FRI and
flanking sequence totaling∼4 Kb, in addition to the lack of homology in the flanking
regions, excludes transposition by ectopic recombination between paralogous genes or
gene-flanking repetitive sequences. However, upon closer inspection, the presence of
multipleHelitronelementsincloseproximity(within3Kb)ofFRI
thal lyr
(Fig. 4.22)in
addition to the duplication event from FRI
thal ancestral
to FRI
thal
strongly supports the
roleoftranspositionbyHelitrons.
58
Helitrons were first discovered “in silico” by (1) searching the genome sequences
of A. thaliana, rice, and nematode for DNA repeats, (2) grouping these repeats, and
then (3) generating consensus sequences for presumed autonomous elements without
the various insertion and deletions found in inactive or nonautonomous copies [28].
Helitrons are distinguished from other transposons in their structural features and their
proposed transposition mechanisms. Otherwise known as rolling-circle transposons,
Helitronshavebeenfoundtocapturehostgenefragmentsandarepresumedtotranspose
through rolling circle transposition involving replication and strand replacement rather
thanby“cut-and-paste”[29].
Since their discovery, Helitrons appear to be presently active in many organisms
[36, 11, 18, 65, 8, 59]. In maize, Helitrons have been found to be responsible for
intraspecific gene order between inbred lines [35] and mediate gene movement among
maize lines [36, 40]. In contrast, Helitrons do not appear to be as active in A. thaliana
buthavealsobeenfoundtocapturegenefragments[23].
DistinguishingfeaturesofatranspositioneventmediatedbyHelitronsincludeinser- tion between the host nucleotides A and T, 5’ TC and 3’ CTRR termini, a palindromic
sequence capable of forming a hairpin structure at the 3’ end of the element, and a lack
of both terminal repeats and duplication of the host sequence (Fig. 4.23). Though the
combination of these exact signatures have not been detected at either FRI
thal ancestral
or FRI
thal
, these signature sites may have been deleted as the A. thaliana genome has
shrunk by ∼40%. Thus, the transposition of FRI in A. thaliana by Helitrons is still
possiblebutwillneedfurtherinvestigation.
59
Figure4.17: VISTAviewofsequencesimilaritybetweenFRI
thal ancestral
andFRI
lyr
.
60
Figure4.18: VISTAviewofsequencesimilaritybetweenFRI
thal ancestral
andtheorthol-
ogousregioncorrespondingtothegenesflankingFRIinA.thaliana.
61
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ATHPOGON2
ATMU7
ATMU7
ATMU10
ATMU7
ATHPOGO
ATREP15
Helitron3
Helitron1
ATREP15
AT4G00650.1
orthologous.scaffold_8.14317459.14382282
268638 269138 269638 270138 270638 271138 271638 272138 272638
14337789 14338789 14339789 14340789 14341789 14342789 14343789 14344789 14345789 14346789 14347789 14348789 14349789
segregating 42 bp
indel in A. lyrata
where A. thaliana
has the insertion
Col-0 16 bp deletion
exon 1 3 2
AT4G00650
Figure4.19: DotplotofhomologybetweenFRI
thal
(AT4G00650.1)andFRI
lyr
(orthologous.scaffold 8.14317459.14382282). Focal gene models are indicated in
black, flanking gene models in gray, transposable elements and repetitive elements in
red, pseudogene predictions in purple, small RNAs in green and methylation sites in
blue.
62
most of FRI in lyrata
is deleted in the
ancestral location of FRI in thaliana
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ATMUN1
BRODYAGA2
RP1_AT
AtSB5
ATREPX1
Helitron4
ATHATN4
ATHAT8
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ATTIR16T3A
ATTIR16T3A
ATHPOGON2
ATMU7 ATMU7
ATMU10 ATMU7
ATHPOGO
ATREP15
Helitron3
Helitron1
ATREP15
Helitron4
AT5G51090.1
orthologous.scaffold_8.14317459.14382282
20776730 20781230 20785730 20790230 20794730 20799230 20803730 20808230 20812730 20817230
14317160 14322160 14327160 14332160 14337160 14342160 14347160 14352160 14357160 14362160 14367160 14372160 14377160
Figure 4.20: Dotplot of homology between FRI
thal ancestral
(AT5G51090.1) and FRI
lyr
(orthologous.scaffold 8.14317459.14382282). Focal gene models are indicated in
black, flanking gene models in gray, transposable elements and repetitive in red, pseu-
dogenepredictionsinpurple,smallRNAsingreenandmethylationsitesinblue.
63
| | | | | | | | | | |
| |
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|
ArnoldY1
! !
! !
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! ! !
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! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
!
! !
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! !
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! ! ! !
! !
! ! ! ! !
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!
AtSB5
AT4G00650.1
AT5G51090.1
269692 270192 270692 271192 271692 272192
20787272 20787772 20788272 20788772 20789272 20789772 20790272 20790772
AT5G51100
downstream of FRI
AT5G51090
maps to intron 1 and
the latter part of exon 3
AT5G51100 AT5G51090
exon 1 3 2
AT5G51080
AT5G51080
maps to the end of exon 3
AT4G00650
pseudogenes
Figure 4.21: Dotplot of homology between FRI
thal
(AT4G00650) to FRI
thal ancestral
(AT5G51080,AT5G51090andAT5G51100).
64
Figure4.22: GBROWSEviewofFRI
lyr
. TheorthologofFRIinA.lyrata
(scaffold 801542.1) is highlighted in blue and the Helitron elements (HELITRON3,
HELITRON1,andATREP15)arehighlightedingreen.
65
Figure 4.23: Structure of Helitron elements and the rolling circle transposition mecha-
nism. (a) A generic Helitron showing sequences and structural features that may be cis
requirementsfortransposition. HelitronsfromC.eleganscontainasinglegenewhereas
HelitronsfromA.thalianaandO.sativacontaintwoorthree. (b)Ahypotheticalmecha-
nismforHelitrontranspositionandgeneacquisitionbasedontheproposedrollingcircle
mechanism for bacterial transposons. The element (in red) could be either autonomous
ornonautonomous. Twotransposasemoleculesareshown(blueellipses)cleavingatthe
donor and target sites and binding to the resulting 5’ ends. Replication at the cleaved
donor site initiates at the free 3’ OH and proceeds to displace one strand of Helitron. If
the palindrome and 3’ end of the element are recognized correctly, as is shown on the
Left,cleavageoccursaftertheCTRRsequenceandtheoneHelitronstrandistransferred
to the donor site where DNA replication resolves the heteroduplex. The illustration on
the Right depicts one way by which DNA flanking the 3’ end of the element (in green)
couldbetransferredalongwiththeelementtothedonorsite. ThismaybehowHelitrons
haveacquiredadditionalcodingsequences. (Fig. 1. of[16])
66
Chapter5
MaterialsandMethods
5.1 GenomeSequences
The TAIR8 A. thaliana reference sequence and annotation were downloaded from
www.arabidopsis.org. The annotation of gene models in A. thaliana contained 27,235
protein coding genes, 4759 pseudogenes or transposable elements and 1288 ncRNAs
resultinginatotalof33,282genes(38,963genemodels). Theversion1.0releaseofthe
A.lyrata reference sequence containing 695 scaffolds and FilteredModels6 gene model
set were obtained from www.jgi.doe.gov/Alyrata. The preliminary annotation of gene
models in A. lyrata contained a total of 32,670 protein coding genes. For gene models
withmorethanoneisoform,thelongesttranscriptwasselected.
Gene family and superfamilies based on those analyzed in Clark et al. 2007 were
obtained from www.arabidopsis.org. In addition, lists of receptor-like kinase genes and
diseaseresistancegeneswerefurthersupplementedfrom[48]and[38],respectively.
Genome-wide methylation status for the reference Col-0 sequence was obtained
from www.arabidopsis.org [63]. Repeat masking of A. thaliana and A. lyrata refer- ence genomes was run with RepeatMasker version open-3.2.6 [49] on the 10-07-2008
RepBaserelease[25,26,27].
67
5.2 PolymorphismDatasetsinA.thaliana
Three polymorphism datasets in A. thaliana are utilized. The first dataset is a com-
prehensive survey of polymorphism via the re-sequencing by oligonucleotide tiling
arrays using a 1 base tile in a small sample of 20 individuals [9]. The pseu-
dochromosome sequences corresponding to these 20 accessions were obtained from
www.arabidopsis.org. The second dataset is somewhat of a subset of the first, but in
amuchlargersampleof95individuals[43]. The1213dideoxyre-sequencedfragments
in these 95 accessions were obtained from walnut.usc.edu. The third dataset is also a
subsetofthefirstandissimilartotheHapMapconceptofgenotyping250KTAGSNPs
in a large number of individuals. The genotyped calls for these 250K SNPs in 262
individualswereobtainedfromwalnut.usc.edu.
5.3 IdentifyingColinearBlocks
All 695 A. lyrata scaffolds were compared against each A. thaliana chromosome but
only results mapping to scaffolds 1 through 8 are analyzed in detail. To identify blocks
of colinearity, three complementary methods were employed for identifying pairwise
sequence similarity between A. thaliana and A. lyrata: 1) BLAST-ing between cod-
ing sequences [64], 2) MUMmer [13] and 3) Mauve [12]. BLASTN results with e-
values greater than 0.00001 were recorded. MUMmer and Mauve were run with the
default parameters. Although all three methods identified conserved coding regions
more readily, MUMmer and Mauve were able to additionally identify conserved non-
codingregions.
Tocombinehomologousmatchesintocolinearblocks,DAGchainer[20]wasrunon
theresultinghitsofsharedhomologyfromBLAST,MUMmerandMauve. DAGchainer
“chains” homology pairs between genomic regions by identifying paths through a
68
directed acyclic graph. Chained blocks of homology pairs are then assigned scores
based on the combination of the quality of the matches and their proximity to one
another (mismatches and gaps are allowed). As such, higher quality matches tended
to correspond to conserved coding regions and were given the most weight. After col-
inear blocks were identified by DAGchainer, manual inspection of the colinear blocks
wasrequiredtoeitherremove, partitionorjoinblocks. Thismanualstepwasnecessary
as blocks selected by DAGchainer were score-based such that a small inversion would
beoflittlecostforalargeblockofcolinearity. TheresultsofDAGchainercanbefound
inTable5.1andwaswiththefollowingparameters: GapUnitLen=3000,GapOpen=-10,
GapExtend=-20,MaxDist=1000000.
Table5.1: Summaryofhits
chromosome colinearblocks all coding used
1 22 14405 5438 5310
2 18 8229 3141 3122
3 11 10867 4266 4176
4 9 7484 3155 3075
5 19 12445 4925 4855
5.4 Pairwise Alignments Between Orthologous Gene
Pairs
Due to the degeneracy of the genetic code, alignments formed directly from the
nucleotide sequence may incorrectly align codons and/or generate false frameshifts.
Thus, to create accurate and reliable alignments between orthologous gene pairs, it is
preferabletocreatecodonalignmentsguidedbythealignmentofthecorrespondingpro-
teinsequences. Toaccomplishthis,orthologouspeptidesequenceswerefirstalignedby
MUSCLE[15]andthenreverse-translatedwithPAL2NAL[52]intocodonalignments.
69
5.5 SequenceAnalysis
Estimatesofnucleotidepolymorphismanddivergencewereperformedwiththepackage
’analysis’ provided by libsequence [54]. In addition, synonymous and nonsynonymous
substitution rates were computed with PAML [60]. Estimates of divergence were com-
puted via Kimura’s 2-parameter distance between all pairs of sequences in a file. All
BLAST output were parsed with the Bio::SearchIO module from the Bioperl project
[51, 50]. All other sequence manipulations and data analyses were accomplished with
customPerlscripts.
70
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Abstract (if available)

Abstract The highly selﬁng Arabidopsis thaliana differs from its closest relative, the obligate outcrosser A. lyrata, both in genome size and organization. During approximately 5 million years of divergence, the A. thaliana genome has shrunk by approximately 40% and chromosome number has decreased from 8 to 5. Comparative genomic analysis between A. thaliana and A. lyrata suggests that genome rearrangements have had a profound effect on current patterns of polymorphism and divergence in A. thaliana.

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Asset Metadata

Creator Hu, Tina T. (author)

Core Title Comparative genomics of arabidopsis thaliana

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Computational Biology

Publication Date 12/08/2009

Defense Date 10/29/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag arabidopsis,comparative genomics,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Nordborg, Magnus (committee chair), Marjoram, Paul (committee member), Nuzhdin, Sergey (committee member), Ortega, Antonio (committee member)

Creator Email tinahu@usc.edu,tinathu@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1890

Unique identifier UC1456184

Identifier etd-Hu-2459 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-141933 (legacy record id),usctheses-m1890 (legacy record id)

Legacy Identifier etd-Hu-2459.pdf

Dmrecord 141933

Document Type Dissertation

Rights Hu, Tina T.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu