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Development and Genetic Studies of a Solanum lycopersicoides Introgression Line
Library
By
Michael A. Canady
B.S. (Brigham Young University) 1994
M.S. (Brigham Young University) 1997
DISSERTATION
Submitted in partial satisfaction of the requirements of the degree of
DOCTOR OF PHILOSOPHY
in
Genetics
in the
OFFICE OF GRADUATE STUDIES
of the
UNIVERSITY OF CALIFORNIA
DAVIS
Approved:
fc-/ /
Committee in Charge
2002
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UMI Number: 3074553
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ACKNOWLEDGMENTS
I would first like to thank my major professor, Dr. Roger Chetelat, for his
willingness to mentor me during my time in his research group. Roger’s patient
demeanor and keen intellect helped guide me through the challenges o f my doctoral
program. Roger is the quintessential scientist/family man, and I’m fortunate to have
worked with him.
Next I would like to thank my dissertation committee, Drs. Jan Dvorak and David
Neale, for their helpful comments and willingness to help me improve the quality of my
manuscript. I truly appreciate the time they’ve spent consulting and advising me over the
past few years.
I am also grateful for my association with Dr. Charles Rick. Charley was a
constant source of wisdom, be it on tomatoes or any other subject, and is missed by all
who knew him.
Thanks also to the staff and students of the Tomato Genetics Resource Center,
including: Rachel Curtis, Samantha Smith, Jennifer Peterson, Boryana Stamova, Ricardo
Pertuze, Elaine Graham, Yuanfu Ji, Sompid Samipak, and Carl Jones. It has been a joy
working with all of you and you’ve each taught me much over the past five years.
I would also like to acknowledge my parents, Donald and Patricia Canady, for
always encouraging my curiosity and inspiring me to enjoy work. I’ll never be able to
repay you for what you’ve given me.
Lastly, I would never have made it this far in my educational career without the
love and constant support o f my beautiful wife Helen. And thanks to my children,
ii
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Clayton and Grace, who’ve given me my motivation to succeed. You’ve helped me
maintain perspective and balance in my life.
This dissertation is dedicated to the memory of my daughter, Grace Marie Canady.
iii
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TABLE OF CONTENTS
TITLE PAGE.......................................................................................................................... i
ACKNOWLEDGMENTS.....................................................................................................ii
TABLE OF CONTENTS.....................................................................................................iv
LIST OF TABLES............................................................................................................. vi
LIST OF FIGURES..........................................................................................................vii
ABSTRACT.............................................................................................................................1
INTRODUCTION................................................................................................................. 3
CHAPTER 1.......................................................................................................................... 9
ABSTRACT................................................................................................................ 9
INTRODUCTION..................................................................................................... 11
MATERIALS AND METHODS.............................................................................. 13
RESULTS AND DISCUSSION...............................................................................22
CHAPTER II........................................................................................................................ 32
ABSTRACT...............................................................................................................32
INTRODUCTION.....................................................................................................33
MATERIALS AND METHODS..............................................................................34
RESULTS.................................................................................................................. 36
DISCUSSION............................................................................................................49
CHAPTER H I...................................................................................................................... 53
ABSTRACT...............................................................................................................53
INTRODUCTION.....................................................................................................55
iv
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MATERIALS AND METHODS............................................................................. 57
RESULTS..................................................................................................................62
DISCUSSION............................................................................................................74
CONCLUSIONS AND FUTURE INVESTIGATIONS................................................ 78
REFERENCE LIST.............................................................................................................84
v
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LIST OF TABLES
Table 1.1 CAPS marker summaries
vi
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LIST OF FIGURES
Figure 1.1 Graphical genotypes...................................................... 15
Figure 1.2 ILm ap............................................................................ 18
Figure 1.3 Seed count per fruit histogram.................................... 25
Figure 2.1 Allele frequencies..........................................................37
Figure 2.2 Genotypic frequencies.................................................. 39
Figure 2.3 Recombination suppression..........................................44
Figure 2.4 Recombination rates..................................................... 46
Figure 2.5 Recombination rate vs. segment length.......................47
Figure 3.1 “Target/driver” crossing scheme..................................58
Figure 3.2 Segments used for “target/driver” study..................... 60
Figures 3.3-3.6 “Target/driver” results..........................................65
Figure 3.8 F2 IL x L. pennellii results............................................ 73
Figure 4.1 Hypothetical relative recombination rates................... 81
vii
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1
DEVELOPMENT AND GENETIC STUDIES OF A SOLANUM
LYCOPERSICOIDESINTROGRESSION LINE LIBRARY
ABSTRACT
The following three chapters describe a collection of Solanum lycopersicoides
Dun. nearly isogenic lines (NILs) in tomato (Lycopersicon esculentum Mill.), and their
use in the study o f segregation distortion and homeologous recombination. Despite
hybridization barriers that have impeded use of this wild species, we have identified a set
of introgression lines (ILs), representing the majority of the S. lycopersicoides genome,
which is directly accessible to breeding. Subnormal transmission rate of S.
lycopersicoides alleles and reduced recombination within individual introgressed
segments, due to lack of chromosome homology between tomato and S. lycopersicoides,
have impeded the completion of the IL library. The IL collection is divided into two
groups o f lines: the first set o f 63 lines was selected for maximum coverage and
minimum number of introgressed segments per line; the second group o f 41 lines
includes additional segment to improve mapping resolution. Overall, approximately 96%
of the S. lycopersicoides genome is represented in the IL collection, with 67% of the
primary and 12% of the secondary set homozygous for S. lycopersicoides. Consequently,
this genetic resource provides a unique opportunity to study segregation distortion and
homeologous recombination genome-wide. Recombination data were compiled from 45
different ILs representing most of the S. lycopersicoides genome. The length of
introgressed segments was positively correlated (1^ = 0.26) with recombination rate, and
recombination was least suppressed in terminal, paricentric segments. The IL library was
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also used to investigate the genetic basis for segregation distortion in tomato-5.
lycopersicoides hybrids. Fourteen segregation distortion loci associated with increased
transmission o f L. esculentum alleles and three loci correlated with increased
transmission o f 5. lycopersicoides alleles were identified. In addition, the feasibility of
using the 5. lycopersicoides IL collection to increase homologous and homeologous
recombination in tomato hybrids was also examined. The effect same-chromosome 5.
lycopersicoides segments have on homeologous recombination was also determined by
creating segregating populations with two combined segments. Juxtaposed terminal 5.
lycopersicoides segments enhanced recombination from zero to nearly 10% of the
control, thereby illustrating that combining two homeologous segments can offset
recombination suppression. Next, we crossed a 5. lycopersicoides IL with L. pennellii
and examined recombination in the region of homology (i.e. L. esculentum-L. pennellii)
adjacent to the region of homeology (i.e. L. pennellii-S. lycopersicoides). Recombination
in the homologous region was increased by 19%, demonstrating the potential to use
homeologous chromosome segments to increase recombination in neighboring regions.
Moreover, L. pennellii!S. lycopersicoides recombination increased 10-fold over that o f L.
esculentum/S. lycopersicoides, demonstrating the potential for using a third species as a
“bridge” to enhance recombination within introgressed regions. Therefore, the herein
described 5. lycopersicoides IL collection is a useful breeding resource with broad
applicability to both basic and applied plant genetics investigations.
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3
INTRODUCTION
Historically, agronomists and horticulturists have selectively bred traits that
generate the best yielding and highest quality crop. The selection process, in simple
terms, is two-fold: 1) “defective" traits are eliminated, and 2) yield is increased (Donald
1968). Using this process vast qualitative and quantitative improvements have been
made over the past 10,000 years of human agricultural history (Stalker 1980). However,
the domestication and modem breeding processes have reduced and restricted existing
crop genetic variability. For example, in several crops such as wheat (Triticum aestivum)
and oil-seed rape (Brassica napus), dormancy was eliminated early in the domestication
process. Unfortunately, elimination of dormancy and, therefore, delayed germination,
has caused grain pre-harvest sprouting problems (Holdsworth et al. 2001). Similar
compromises in fitness have been made in several crop species, as desirable traits have
been preferentially selected and fixed through successive generations o f breeding, while
less desirable characteristics have been removed. Other examples o f agricultural
“compromises” include: tomato (Lycopersicon esculentum) wall thickness vs. seed
production (Doganlar et al. 2000); sugar beet (Beta vulgaris) storage ability vs. sugar
content (Zeng et al. 1991); and alfalfa (Medicago sativa) cold tolerance vs. dormancy
period (Cunningham et al. 1995).
Plant breeders have typically relied on land races and their associated wild
relatives beyond primary gene pools as resources for increasing crop genetic variability,
and since the dawn of civilization, humans have attempted to improve performance
through such controlled matings. Over the centuries, thousands o f wild-relatives of crop
species have been used to introduce novel genetic and allelic variation into cultivated
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4
germplasm. The rice {Oryza sativa) cultivar ‘IR36’ is an excellent demonstration o f the
functionality of wild species for crop improvement (Plucknett et al. 1990). The wild rice
species O. nivara, as well as 13 rice cultivars from six different countries, were used in
the formation of this elite cultivar. ‘IR36’ is resistant to several insects and diseases, is
drought tolerant, and is immune to fluctuations in soil composition. Another example is
the potato (Solanum tuberosum) cultivar ‘Brodick,’ whose pedigree includes S.
tuberosum spp. andigena, S. demissum, S. phureja, S. simplicifolium, and S. vernei, as
well as other Solanum species, and is resistant to most fungal pathogens as well as several
potato viruses (Innes 1992).
Perhaps the best example of the successful use of wild germplasm for cultivar
enhancement is the tomato (L. esculentum). Prior to 1925, new cultivar development was
based solely on variability in existing tomato lines or on fortuitous heterogeneity
introduced by mutation or out-crossing. However, beginning in the 1930’s, various
Lycopersicon species were introduced into breeding programs and remarkable
improvements were made in cultivar fruit quality, yield and disease resistance. Nine
Lycopersicon species are all cross compatible with L. esculentum. Resistance to various
insect, fungal, bacterial and viral pathogens has been identified and incorporated into the
cultivated tomato using several Lycopersicon species (Alexander and Hoover 1955).
Gains in crop performance require continual improvements in accessibility to
sources o f genetic diversity where novel genes and alleles potentially reside. Sexually
compatible wild relatives are the most accessible source of genetic variation and they are
available for many crops. Often, breeders extend their search for novel genetic diversity
beyond immediate relatives and include more distantly related species. Wide-
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5
hybridization success is limited by several pre- and post-zygotic incompatibilities (De
Nettancourt 1977) and developmental barriers (Hogenboom 1972). Failures in the
physiological composition o f a would-be hybrid may include a malfunctioning style
and/or ovary (Wann 1962). Additional difficulties with hybrid seed development
(Alexander 1956) and eventual gamete formation and success (Khush and Rick 1963)
reinforce innate species barriers.
For some crops, use of related wild species in breeding involves incorporating
homeologous genomes whose chromosomes have diverged to the point where meiotic
recombination in the interspecific hybrid is drastically reduced. Homologous
chromosomes pair normally during meiosis, whereas homeologous chromosomes have
undergone sufficient sequence or structural change to the point that normal meiotic
pairing is restricted. Bread wheat (Triticum aestivum, 2n = 6x = 42), for example,
originated from 3 diploid species: T. urartu (A genome); T. speltoides (B genome); and
T. tauschii (D genome) (McFadden and Sears 1946; Sarkar and Stebbins 1956). In wheat
haploids, pairing between these homeologous chromosomes is virtually non-existent.
The gene P hi suppresses homeologous recombination between the A, B and D genomes
(as reviewed in Sears 1976). In barley (Hordeum vulgare), variability in recombination
levels between four breeding lines suggested genome-wide control of recombination,
possibly directed by several loci (Nilsson and Sail 1995). Hence, successful
hybridization between dissimilar genomes is affected not only by chromosome
homology, but also by specific loci that regulate recombination. In crops like wheat,
where access to wild species’ genetic diversity is inhibited by inter-genomic
recombination barriers, overcoming innate speciation mechanisms is often difficult.
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6
Obstacles to successful utilization of wild genomes can be overcome in some
cases through creative population structures. One such way to efficiently organize a
species’ genomic constitution is through an introgression line (IL) population.
Essentially, an IL collection contains a series of homozygous, single-segment,
overlapping chromosome segments representing the whole genome of one species, in the
genetic background o f another. Introgression lines (ILs) offer a convenient way of
maintaining interspecific hybrids indefinitely, as well as providing a stable genetic
environment in which individual locus effects can be measured. An important advantage
o f using ILs in quantitative trait loci (QTL) mapping is that once desirable traits are
identified, lines containing these traits can be used directly in breeding programs. In
addition, sterility present in interspecific hybrids is drastically reduced in IL populations.
Moreover, because all phenotypic variation between a line in the library and the recurrent
parent is due to the introgressed segment, epistatic effects are removed in IL populations.
Additionally, line homozygosity allows ILs to be readily reproduced (Zamir 2001).
Although all nine wild Lycopersicon species are cross compatible with L.
esculentum, few pre-bred lines containing the genetic material o f these taxa in cultivated
tomatoes exist. A recombinant inbred (RIL) population for L. cheesmanii was developed
(Paran et al. 1995) and backcross inbred lines (BILs) fo ri, hirsutum (Bemacchi et al.
1998b) and L. pimpinellifolium (Tanksley et al. 1996) have been created. Though
valuable as both a mapping resource and a method to permanently preserve exotic
genotypes, RIL assembly is not possible for all interspecific hybrids, specifically for
those hybrids where Fi sterility or self-incompatibility exists. Moreover, because RILs
potentially include large, multi-copied exotic chromosome segments, line sterility is
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8
parent (i.e. unilaterally incompatible), and tomato x S. lycopersicoides F, hybrids are
nearly male sterile (Chetelat et al. 1997). Consequently, exploiting S. lycopersicoides
through traditional backcross breeding is problematic. Transferring the S.
lycopersicoides genome into a tomato background via introgression lines (ILs) would be
an attractive method of utilizing this genetic resource. ILs are relatively fertile and
compatible, and are genetically stable, thus can be seed propagated indefinitely,
facilitating their maintenance and distribution.
The following three chapters describe development of a Solanum lycopersicoides
introgression line library, and its use to study homeologous recombination in tomato.
Chapter 1 describes the genetic constitution of each IL, overall composition of the
library, and various details pertinent to their maintenance. Chapter 2 describes
recombination and segregation trends within the S. lycopersicoides IL library. Chapter 3
examines strategies to increase recombination in breeding systems using the IL lines.
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9
CHAPTER I
A SOLANUM LYCOPERSICOIDES INTROGRESSION LINE LIBRARY IN TOMATO
A bstract
The majority of the Solanum lycopersicoides genome has been transferred into the
cultivated tomato (Lycopersicon esculentum) via a set of breeding lines containing
individual, overlapping chromosome segments in the variety ‘VF36’. Using RFLPs,
isozyme and morphological markers, we have screened thousands of inbred advanced
backcrossed progeny and have identified introgression lines (ILs) representing the
majority of the S. lycopersicoides genome. The IL collection consists of two groups of
lines: a primary set of 63 lines, which have been selected for maximum coverage o f the
Solanum lycopersicoides genome, a minimum number of introgressed segments per line
and homozygosity; a secondary set of 41 lines containing selected additional segments to
provide increased resolution for gene mapping. Three genomic regions from S.
lycopersicoides are missing from the IL collection: ~9 map units on chromosome 2, ~12
map units on chromosome 3, and ~29 map units on chromosome 4. Consequently, we
estimate that ~96% of the S. lycopersicoides genome is represented in the IL library.
Complete genome representation was impeded by subnormal transmission rates of S.
lycopersicoides segments and reduced recombination within individual introgressed
segments. Plants homozygous for long introgressed segments tended to be male-sterile,
and recovery o f shorter introgressed segments, though successful in many cases, was
limited by the lack of recombination. Consequently, only 67% o f the primary lines and
12% of the secondary set are homozygous for S. lycopersicoides introgressed segments.
For regions that resisted fixation of the S. lycopersicoides genotype, CAPS (cleaved
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10
amplified polymorphic sequence) markers were generated to facilitate identification of S.
lycopersicoides introgressions in segregating populations.
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Introduction
The use of wild germplasm in plant breeding programs is commonplace, and
perhaps no crop has benefited more from exotic genetic resources than the cultivated
tomato (Lycopersicon esculentum). Nine related wild Lycopersicon species, all cross
compatible with the cultigen, are available for breeding purposes. Detailed genetic maps
containing thousands of morphological, protein, and DNA-based markers have been
developed for the tomato genome (Fulton et al. 2002). Furthermore, several pre-bred
populations have been created which incorporate wild germplasm in the genome of
cultivated tomatoes. Examples include a recombinant inbred population for L.
cheesmanii (Paran et al. 1995), and backcross inbred lines representing L. hirsutum
(Bemacchi et al. 1998a), L. pimpinellifolium (Tanksley et al. 1996), L. peruvianum
(Fulton et al. 1997), and L. parvijlorum (Fulton et al. 1996) have also been created.
The use of introgression line (IL) populations is a viable alternative to
conventional backcross inbred lines, where long, multiple donor introgressions can
complicate gene and/or allele discovery. The principle objective of an introgression line
collection is to preserve and represent the entire genome o f one species in a genetic
background of another. In tomato, IL libraries exist for L. pennellii (Eshed and Zamir
1994a; Liu and Zamir 1999) and L. hirsutum (Monforte and Tanksley 2000).
The four tomato-like Solanum species, S. juglandifolium, S. ochranthum, S.
sitiens, and S. lycopersicoides, each possess unique traits not found in Lycopersicon.
Extending the circle of accessible germplasm to include these more distantly related
species requires overcoming additional reproductive barriers. Thus far, only S.
lycopersicoides and to a limited extent S. sitiens, have been successfully hybridized and
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12
hybrids backcrossed to tomato via sexual crosses. Solanum lycopersicoides is perhaps
more closely related to tomato and is more amenable to incorporation into tomato
breeding programs. This related Solanum species is native to high elevations (up to
3800m) in the Andes, and tolerates exposure to chilling and freezing temperatures, is
resistant to several insect pests and pathogens that impact production o f tomatoes
(Chetelat etal. 1997).
Solanum lycopersicoides is a diploid with the same chromosome number as
tomato (2n = 2x = 24). Crosses between them succeed only when tomato is used as the
female parent (unilateral compatibility). Resulting Fi hybrids are also unilaterally
incompatible with tomato, and pollen is essentially male sterile (Chetelat et al. 1997).
Meiotic chromosome pairing in L. esculentum x S. lycopersicoides hybrids is irregular,
with bivalents and a significant number of univalents in metaphase I (Rick 1951). In
addition, there are size differences in the two chromosome sets and a lack of
synchronization in condensation (Menzel 1962; Rick 1986). Comparative mapping has
demonstrated that the two genomes are collinear, with the exception o f a paracentric
inversion on chromosome 10L (Chetelat et al. 2000; Pertuze et al. 2002). The
chromosomes of tomato and S. lycopersicoides are therefore homeologous.
Consequently, S. lycopersicoides allele transmission and recombination are considerably
reduced in progeny o f intergeneric hybrids (Chetelat and Meglic 2000).
The most direct way of accessing the S. lycopersicoides genome for breeding
purposes is through the development of a comprehensive introgression line library. A
collection o f backcross-inbred families derived from a partially male-fertile intergeneric
hybrid provided the starting material for identifying ILs (Chetelat and Meglic 2000). A
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comprehensive IL library would ideally include both terminal and interstitial segments,
and would consist primarily of lines with single introgressed segments. The purpose of
the present study was to create such a S. lycopersicoides IL population through selection
for recombinant and homozygous segments, and elimination o f extraneous markers. The
ILs have been divided into a primary set of lines that provide maximal genome coverage
and an additional group o f lines that potentially provides greater genome mapping
resolution.
Materials and Methods
Population development
Production of the original BCi L. esculentum ’VF36’ x S. lycopersicoides LA2951
population, as well as the methods used to select BCi individuals for advanced backcross
generations, are described in Chetelat et al. (1997). Using restriction fragment length
polymorphisms (RFLPs), morphological, and isozyme markers to monitor S.
lycopersicoides introgression, Chetelat and Meglic (2000) advanced 32 BCi families to
various inbred-backcross generations. They identified 272 S. lycopersicoides-contoining
lines in a L. esculentum background.
In the present study, lines were selected based on the following criteria: (1)
homozygous lines were selected over heterozygotes; (2) overlap between adjacent
segments to maximize coverage; (3) presence of a single alien segment introgression per
line. Lines with multiple introgressed segments were backcrossed to the recurrent parent
(‘VF36’) and negative selection (i.e. progeny with multiple S. lycopersicoides segments
were eliminated) was performed using RFLP markers. Selection for the majority of the
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14
S. lycopersicoides introgressed segments was made at the BC 2 F2-7 generations, however,
selection for a few lines occurred at earlier (BC 1F6-7 ) or later (up to BC4F 6 ) stages.
Identification o f homozygotes and/or recombinants was performed in segregating
populations of approximately 100 plants per family. Recombinants of interest were fixed
by one more additional generation of selfing. Fertility of homozygotes was tested by
self- pollination, and those that failed to set seed were maintained in F2 or BC progeny as
heterozygotes. Pollinations, seed germination and seedling treatment were performed as
described in Chetelat and Meglic (2000).
Marker analysis
A combination of 116 RFLP, 17 isozyme, and 5 morphological markers were
used to identify S. lycopersicoides ILs (Figs. 1.1 a, I.lb and 1.2). DNA was isolated
according to Fulton et al. (1995) and restriction digestion and blotting were performed as
described in Chetelat and Meglic (2000), except an alkaline transfer buffer was used and
blots were stored wet (without vacuum drying) in Saran-Wrap at 4°C. Previously
mapped genomic (TG) and cDNA (CD and CT) markers were chosen from the tomato
RFLP map (Tanksley et al. 1992); probes were provided by Dr. Steve Tanksley (Cornell
University). Polymerase chain reaction (PCR) amplification of probes followed the
protocol described in Chetelat and DeVema (1991), and 32P-dCTP, dATP labeling and
detection as outlined in Feinberg and Vogelstein (1983) and Chetelat and Meglic (2000).
Isozyme analysis was performed as described in Chetelat et al. (1997).
For some introgressed segments, homozygotes were either nonviable or so sterile
that seed was not obtained from self-pollinations. However, in all cases, heterozygotes
were fertile and produced BC or F2 seed. To facilitate identification of these segments in
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15
Figure 1.1. Graphical genotype o f S. lycopersicoides introgression lines in L. esculentum.
The top row indicates chromosome with markers (not to scale), left column the line
numbers. Darkly shaded segments indicate homozygous introgression lines (LS), light
segments heterozygotes.
(A) primary IL set, (B) secondary set.
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Figure 1.2. Map o f the primary set of chromosome segments introgressed from S.
lycopersicoides into L. esculentum. The location and genotype of each segment is
indicated by vertical lines (solid lines homozygous, dashed lines heterozygous). Line
numbers (LS) are shown next to each segment and bins showing unique regions of
coverage are indicated by horizontal lines (I-A, 1-B, etc.). Markers and distances are
from the L. esculentum x L. pennellii F2 map (Tanksley et al. 1992) and the approximate
positions of centromeres are from Pillen et al. 1996. Shaded spaces between bins
indicate regions o f the S. lycopersicoides genome not represented in the ILs. Vertical
arrow on chr. 10 indicates paracentric inversion (Pertuze et al. 2002). Underlined RFLP
loci have been converted to CAPS markers. Isozyme markers are italicized.
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19
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Table 1.1. Summary of CAPS marker information, including primer sequences, reaction
conditions and expected results for each RFLP locus.
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22
segregating populations, we converted 16 RFLP loci to CAPS (cleaved amplified
polymorphic sequence) markers (Fig. 1.2, Table l.l). Other segregating segments are
identified by at least one isozyme or morphological marker. Sequence data for RFLP
probes were obtained from the Solanaceae Genomics Network
(http://www.sgn.comell.edu), and forward and reverse primer sequences were created
using Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). PCR
reaction annealing temperatures were first optimized using L. esculentum ‘VF36,’ S.
lycopersicoides LA2951, and the Fi (L. esculentum x S. lycopersicoides) using a
Stratagene RoboCycler Gradient 96 thermocycler. Optimized reactions were then
digested with the restriction enzymes HaeVSA, DdeI, Hinfi, Rsal, Msel, Hhal, Alul, Dpnl,
Mbol, and HapU according to manufacturer’s instructions and separated on a 1% agarose
gel. To confirm identity of CAPS markers, segregating IL populations were screened
using the optimized PCR conditions and restriction enzyme digestion for each primer set
and results were compared to genotypes obtained with corresponding RFLP probes on
Southern blots.
Results and Discussion
Generation of the S. lycopersicoides Introeression Line Library
A S. lycopersicoides backcross-inbred population in L. esculentum (Chetelat and
Meglic 2000) was used to develop a comprehensive IL library. The original 272 families
consisted primarily o f non-isogenic, terminal segments. Although homozygous S.
lycopersicoides segments covered the majority (66%) of the tomato genome, this
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23
population was an undesirable mapping and germplasm resource, because: 1) families
with longer alien segments were often sterile or had reduced fecundity, and were,
therefore, difficult to reproduce; 2) most lines (60%) contained more than one S.
lycopersicoides segment, which decreased their usefulness for mapping and QTL
discovery, and contributed to the sterility problems. For these reasons, lines were further
selected based on the following criteria: 1) maximum representation o f the 12 tomato
chromosomes with overlapping introgressions; 2) degree of isogenicity (single segments
preferred); 3) homozygosity. In order to reduce the size of longer introgressed segments,
recombinants were identified in F2 and/or BC populations. For example, line LSI5-2,
which has a S. lycopersicoides segment covering CT233 through TG465 (approximately
101 cM) on chromosome I, was self-pollinated and progeny genotypes were obtained
using RFLPs. Ten recombinant sub-lines were identified and included in the IL library
(Figs. 1.1, 1.2). Similarly, extraneous segments o f non-isogenic families were selected
against, and, occasionally, used to create new ILs. For instance, LSI 1-11, which
originally contained a heterozygous segment on chromosomes 1 and 6, was divided into 2
separate ILs. Each segment was fixed in sublines (LS11-11 A-chr. 1, LS11 -11 B-chr.6). In
addition, a recombinant sub-line LS 11-11BA, on chromosome 6, was identified. This
approach, which accumulates recombinant sub-ILs and selectively eliminates extraneous
IL segments, was used to create the currently described IL library.
Individual IL fecundity depends both on segment length and whether the IL is
hetero- or homozygous for the S. lycopersicoides introgression. Homozygous ILs with
longer segments were generally less fertile than those with shorter ones, and were nearly
always less fertile than heterozygotes (Fig. 1.3). Estimates of fertility based on seed yield
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24
for homozygotes and heterozygotes averaged 19 and 39 seed per fruit, respectively, as
compared to ~ 125 seed per fruit in ‘VF36.’ All heterozygous lines in the IL library were
self-fertile, indicating most sterility factors were recessive. Furthermore, seed yield of
heterozygous ILs was fairly consistent among the lines (data not shown). In addition,
homozygotes for several regions of the genome were either not obtained or were sterile.
Sterility o f the male gametes was more pronounced than that of the female gametes, since
backcross progeny were always possible to obtain.
The S. lycopersicoides IL collection is divided into two groups: a primary set of
63 lines, which have been selected for maximum genome coverage and homozygosity,
with a minimum number of segments per line; and a secondary set o f 41 lines, which
increase resolution by decreasing mapping bin sizes. Classifying ILs into primary and
secondary categories is an efficient method of prioritizing lines, allowing breeders
convenient access to all chromosomal regions with a minimal number of lines. Several
secondary ILs are recombinant sub-lines derived from primary lines. For instance, LSI 52 on chromosome I (not included in library because of sterility problems) yielded 6
additional recombinants: LS15-2A, 15-2B, 15-2C, 15-2D, 15-2F, and 15-2H. From
these 6 sublines, additional recombinant progeny were identified and were included in the
secondary line collection (Fig. 1.1B). Sublines on chromosomes 4 ,6 , and 11, included in
the secondary line collection, were derived from ILs in the primary line set. Conversely,
sublines on chromosomes 7, 8,9, 10 and 12 complemented the IL library better than their
parent lines, and were therefore included in the primary line library. Furthermore,
because segregation distortion towards the L. esculentum allele often facilitated the
elimination of extraneous segments in segregating generations, the sublines were often
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25
Figure 1.3. Histogram o f average seed count per fruit for homozygous S. lycopersicoides
introgression lines following self-pollination under greenhouse conditions. Horizontal
axis indicates chromosomes 1 through 12, including markers (to scale). Width of bars
symbolizes region covered by introgressed segment, vertical axis indicates average seed
per fruit for the line.
Sctdft
t-tf-l
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26
more nearly isogenic than the parental genotypes. In lines containing single alien
segment, the phenotypic effects o f each region are more readily determined than in lines
containing more than one introgression. Primary and secondary ILs were therefore
rigorously selected to eliminate extraneous S. lycopersicoides markers. The majority
o f the primary (68%) as well as secondary lines (90%) are nearly-isogenic. This
represents a significant improvement over the parent population, where only 40% of the
272 original lines had a single S. lycopersicoides segment. By way o f comparison, 100%
of the L. pennellii ILs and 65% of the L. hirsutum library contain single introgressions
(Eshed and Zamir 1994a; Liu and Zamir 1999; Monforte and Tanksley 2000). In this
respect, the S. lycopersicoides IL library is comparable to other tomato IL populations.
Genetic Characterization of the S. lycopersicoides IL Library
Ideally, an IL library would include primarily homozygous segments, which are
stable and relatively easy to maintain. However, homozygous ILs were not recovered for
several genomic regions from S. lycopersicoides. Sixty-seven percent of the primary
lines have been fixed, whereas only 12% of the secondary set are homozygous for the
targeted S. lycopersicoides segment. Overall, 45% of the lines in the complete library are
homozygous. Heterozygous ILs were self-pollinated and F2 progeny analyzed. Despite
these efforts, many ILs were resistant to fixation. In F2 populations o f 24 ILs, no S.
lycopersicoides homozygotes were recovered, indicating strong segregation distortion in
favor o f L. esculentum genotypes. Similarly, for 18 other lines where homozygous S.
lycopersicoides progeny were successfully obtained, pollen inviability in these individual
plants prevented recovery of self-fertilized seed (data not shown).
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27
As an alternative to RFLP markers, we developed CAPS markers to facilitate
genotyping o f segregating lines in IL population. Using RFLP probe sequence
information obtained from Solgenes (http://ukcrop.net/cgi-bin/WebAce/webace?db=
SolGenes), we developed 16 CAPS markers covering 9 L. esculentum chromosomes.
Primer sequences, optimal PCR annealing temperatures, polymorphism rates and
informative restriction enzymes for each converted marker are shown in Table 1.1.
Combined with existing isozyme and morphological loci, the CAPS markers provide at
least one high throughput marker per heterozygous IL.
An excess of terminal introgressions appears to be common to several IL
populations. Eshed and Zamir (1995) reported that 56% of the L. pennellii ILs were
terminal segments and 77% of the L. hirsutum IL population are terminal (Monforte and
Tanksley 2000). The majority (78%) of the original 272 S. lycopersicoides backcross
inbred lines contained terminal segments. Chetelat and Meglic (2000) used a simulated
tomato-like genome to predict segregation patterns, and determined that 62% of the
introgressed segments should be terminal. In the present IL population, primary-set IL
segments are 73% terminal, and overall, the entire population is comprised of 66%
terminal introgressions.
Additionally, recombination events involving terminal
segments rarely resulted in interstitial sub-ILs: when recombination events did occur,
they usually produced shorter terminal introgressions (Figs. 1.1, 1.2; see LS 11 -11B/11BA LS46-6/-6A, LS32-10/-10A, and LSI2-12/-12A). One notable exception to this
trend was LS15-2 on chromosome 1: several interstitial sub-lines were recovered when
this line was self-pollinated. The presence o f the centromere may have resulted in an
increase crossover frequency on chromosome IS, which would produce interstitial
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28
segments. Additionally, there is selection for S. lycopersicoides alleles around the Slocus (near TG51, bin 1-C). For all chromosomes, homozygous terminal segments were
recovered more frequently than interstitial homozygous segments. Despite these
obstacles, recovery and fixation o f interstitial segments was possible in many cases by
marker- assisted selection.
Chetelat et al. (2000) generated a BCi L. esculentum x S. lycopersicoides map,
and found the two genomes were largely collinear, despite genome-wide recombination
suppression that averaged approximately 27%. Little recombination was observed
between markers on the long arm of chromosome 10, suggesting the presence of a
genome rearrangement in this region. Inversions on chromosomes SS, 9S, 10L, 1 IS and
12S differentiate the potato and tomato genomes (Tanksley et al. 1992). Data from an F2
Solanum sitiens x S. lycopersicoides comparative map suggested an inversion for several
chromosome 10L markers distal to TG408 (Pertuze et al. 2002). The distribution of S.
lycopersicoides introgressed segments on chromosome 10 is consistent with the location
of this inversion: 1) a recombination “break-point” between TG596 and TG408
genetically bisects chromosome 10 into two non-overlapping sections, with several
segments on either side of the division; 2) smaller introgressed segments representing
portions of 10L are noticeably absent, consistent with a suppression of recombination.
For example, LSI2-12, which covers the entire putative inverted region, has a single subIL, which is terminal if the S. lycopersicoides arrangement was preserved.
Introgression Line Bin Mapping
The concept of bin mapping in tomato was first used to define individual RFLPdelimited introgressions in a L. pennellii IL library (Liu and Zamir 1999). Mapping bins
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29
represent unique genomic regions determined by the overlap between adjacent segments,
to which genes or QTLs can be assigned with an IL population. Using more than 1500
RFLP markers, an L. pennellii IL collection was divisible into 107 mapping bins,
averaging 12 cM per bin. The combined (primary and secondary) S. lycopersicoides IL
library reported here uses 142 markers to define 91 mapping bins, averaging 14 cM each
(Fig. 1.2). While increased marker saturation does not reduce bin-size, it does, however,
more precisely define the size of each introgression and bin, which will ultimately assist
QTL mapping efforts. The L. pennellii ILs have been successfully used to map a number
of genes and QTLs (Eshed and Zamir 1994b; Eshed and Zamir 1995; Monforte et al.
2001). In similar fashion, the S. lycopersicoides IL library is expected to be useful for
mapping traits or QTLs not expressed in Lycopersicon.
However, the S. lycopersicoides IL collection is deficient in 3 regions: 1) on
chromosome 2, a ~9 cM gap between TG308 and TG191; 2) on chromosome 3, a ~12 cM
gap between TG288 and TG42; and 3) a ~29 cM gap on chromosome 4 between Adh-l
and CT50. Non-overlapping ILs border each of these three regions, and attempts to
“bridge” the introgressions were unsuccessful (see shaded regions, Fig. 1.2).
Consequently, only 96% of the S. lycopersicoides genome (expressed in map units) is
represented in this IL library. Chetelat and Meglic (2000) reported 98% coverage in the
backcross inbred lines, but did not take into consideration regions absent on
chromosomes 2 and 3. Since these introgressed segments all trace to a limited number of
fertile BCi individuals, the three missing regions may not have been represented in the
original BCi population.
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30
Variation for bin-size among chromosomes helps convey the relative ease with
which introgressed segments were recovered on each chromosome: more ILs (per cM)
on a chromosome corresponds with smaller mapping bins. For simplicity, Fig. 1.2 shows
only the primary ILs, and thus does not include the additional mapping bins that define
the secondary lines. The combined population’s average bin-sizes range from 10 cM on
chromosomes 6 and 9, to 23 cM/bin on chromosome 3. There is no relationship between
bin size and chromosome length. Chromosome 1, which is the longest tomato
chromosome (136 cM; Tanksley et al. 1992), has an average bin-size of 11 cM thanks to
several interstitial (as well as terminal) recombinants identified. In contrast, chromosome
11, the shortest in the set (87 cM), is represented mainly by terminal S. lycopersicoides
segments, as a consequence of which the average bin-size is relatively large (IS cM).
Regional variation in the ratio of physical to genetic distance throughout the
tomato genome prevents precise evaluation of the actual alien segment sizes in physical
terms. Meiotic crossovers, and thus, recombination, are dramatically reduced in
heterochromatic sections of chromosomes (Tanksley et al. 1992). Molecular markers, if
situated in these regions, may be tightly linked genetically but far removed physically
(Sherman and Stack 1995). Approximately 77% of tomato nuclear DNA is packaged as
heterochromatin, mostly in the pericentric and subtelomeric regions (Peterson et al.
1996). Segments in these regions, especially those spanning the centromere, are
therefore likely to be quite long physically. Consequently, comparison of exact
introgression and bin sizes in different regions is limited by variation in the
recombination rates along the length of each chromosome.
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31
In conclusion, this IL library greatly improves access to the S. lycopersicoides
genome. Our results demonstrate the advantages as well as limitations of introgression
lines for transferring the genome of a distantly related wild species into diploid tomato.
We anticipate that similar genetic stocks could be synthesized for other tomato-like
nightshades, such as Solanum sitiens.
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32
CHAPTER H
TRANSMISSION AND RECOMBINATION IN S. LYCOPERSICOIDES
INTROGRESSION LINES OF TOMATO
Abstract
A S. lycopersicoides introgression line (IL) library in tomato provides a resource
to study segregation distortion and homeologous recombination genome-wide. Four
types of comparisons were made among lines with S. lycopersicoides introgressed
segments: 1) longer vs. shorter segments; 2) terminal vs. interstitial segments; 3)
pericentric vs. paracentric segments; and 4) segments on the long-arm vs. the short-arm.
Recombination data were compiled from progeny of 45 different ILs representing 88% of
the S. lycopersicoides genome. We found that introgression length is positively
correlated (r2 = 0.26) with recombination rate. At the 95% confidence level, no statistical
difference was detected between the recombination rates of terminal and interstitial
segments (t = 1.60, p = 0.12) and segments in chromosome long- and short-arms (t =
1.84, p = 0.087), whereas significant differences existed between peri- and paracentric
segments (t = 2.35, p = 0.025). We also used the IL library to investigate the genetic
basis for segregation distortion in tomato-5, lycopersicoides hybrids. We identified 14
segregation distortion loci associated with increased transmission of L. esculentum alleles
and three loci, sd l.l, sdl.3, and sd6.2 favoring 5. lycopersicoides transmission. The
mechanism of sdl.3 action appears to involve improper maturation of pollen containing
the L. esculentum allele borne on heterozygotes.
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33
Introduction
As plant breeders broaden their search for novel allelic and genotypic variability,
focus is placed on incorporating distantly related wild species into cultivation. Such
wide-crosses are frequently restricted by pre- and post-zygotic barriers, which prevent
successful exploitation of the genomes o f wild relatives (De Nettancourt 1977). Even if
these obstacles are overcome, segregation distortion may result in the selective exclusion
of the alien/species’ alleles in progeny of wide hybrids, thereby preventing complete
introgression. Moreover, meiotic recombination is often suppressed, which acts as
another barrier to gene transfer from a wild species to related crop plants (Rick 1971;
DeVicente and Tanksley 1991).
Nonetheless, many crop species form fully fertile hybrids with one or more o f
their immediate wild relatives. For example, comparative maps of various Lycopersicon
species and the cultivated tomato (L. esculentum) demonstrated interspecific chromosome
colinearity and homology (see for example Tanksley et al. 1992; Fulton et al. 1997).
Even so, hybrids between wild Lycopersicon species and tomato show varying degrees of
recombination suppression and/or allelic segregation distortion (towards the recurrent
parent) in a number of interspecific populations (Grandillo and Tanksley 1996; Paran et
al. 199S; Chetelat et al. 2000). In addition, reduced recombination has been observed in
male vs. female gametes (deVicente and Tanksley 1991) and in advanced vs. early
backcross generations (Rick 1969; Rick 1971; Fulton et al. 1997).
Four tomato-like Solanum species exist (S. juglandifolium, S. ochranthum, S.
sitiens, and S. lycopersicoides), each with distinct morphologies intermediate between the
potato and tomato. The potato (Solanum tuberosum) and tomato genomes differ by five
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34
paracentric (essentially whole-arm) inversions (Tanksley et al. 1992). Comparative
mapping of S. sitiens, S. lycopersicoides and tomato demonstrated that chromosomes of
these two Solanum species are colinear with tomato, except for an inversion on 10L
(Pertuze et al. 2002). L. esculentum x S. lycopersicoides Fi hybrids are male sterile due
to irregular chromosome pairing, presumably because of size differences in the two
chromosome sets and lack of chromosome sequence homology between the two species.
Therefore, tomato and S. lycopersicoides chromosomes are homeologous.
Recombination and transmission of S. lycopersicoides alleles are considerably reduced in
hybrid progeny (Chetelat and Meglic 2000; Chetelat et al. 2000).
A set of 104 S. lycopersicoides introgression lines, with 96% coverage o f the wild
genome has been developed (chapter 1). This introgression line library is divisible into
molecular marker-defined mapping bins, thus allowing specific phenotypes and
genotypes to be associated with a unique genomic region of S. lycopersicoides.
Additionally, this resource permits a detailed study of genomic trends influencing
homeologous recombination and single gene segregation distortion in backcross progeny
o f these two species.
Materials and Methods
Plant material, hybridization, and genotypic analysis
Chapter 1 describes the development of the S. lycopersicoides introgression line
(IL) library, including information relative to DNA isolation and RFLP, isozyme and
PCR analysis. For the majority o f the lines, segregation and recombination data were
compiled during the IL library selection process. The lines used in the current study were
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those advanced as part of the S. lycopersicoides IL library, which was a subset o f the
more than 400 unique recombinants identified in the backcross-inbred population
(Chetelat and Meglic 2000). Because emphasis was placed on this subset of
introgressions, several genomic regions are missing from the analysis: no S.
lycopersicoides introgressions were recovered for missing regions on chromosomes 2, 3
and 4, and S. lycopersicoides segments on chromosomes S, 9 and 11 were fixed early in
the selection process. Consequently, no segregating progeny were available for analysis
in these missing regions.
Pollen tube and pollen acetocarmine staining
Acetocarmine pollen stainability was performed as described in Tsuchiya (1971).
Statistical analysis
Conformity of allelic and genotypic frequencies with Mendelian values was tested
with the Chi-square goodness-of-fit statistic. Estimates of segment length were
calculated by the maximum likelihood method using Linkage-1 (Suiter et al. 1983). The
Kosambi mapping function was used to convert recombination fraction values to map
units (Kosambi 1944), and the Students-t test was used to for pair-wise comparisons
between peri- and paracentric, terminal and interstitial and long- and short-arm S.
lycopersicoides segments.
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36
Results
Distorted Segregation o f S. lycopersicoides Introgressions
The transmission rates o f individual S. lycopersicoides introgressions was
measured in F2 and BC progeny of heterozygotes (Figs. 2.1,2.2). All S. lycopersicoides
ILs in the library were genotyped during the process of identifying homozygotes. Since
several segments were still heterozygous when genotyping was performed, segregation
data was conveniently gathered for most ILs. Nearly all of the original 272 S.
lycopersicoides lines identified by Chetelat and Meglic (2000) were: 1) heterozygous and
in need of fixation; or 2) contained long introgressed segments with deleterious effects,
therefore needed to be sub-divided into smaller recombinant lines; or 3) contained
undesirable extraneous segments, thus requiring backcrossing to L. esculentum and
marker assisted selection to eliminate these segments and to fix the desired IL.
Preferential transmission of the *+’ (L. esculentum) allele was common in segregating S.
lycopersicoides IL populations. Segregation data based on alien segment genotypic
frequencies from several regions of the S. lycopersicoides genome showed especially
strong directional selection for the '+ ’ allele, including several overlapping segments that
cover entire chromosomes. All segregating S. lycopersicoides segments that were
genotyped on chromosomes 2 ,3 ,8 ,1 0 , 11 and 12 showed significant distortion towards
L. esculentum-, however, gaps in IL coverage on chromosomes 2 and 3 (see chapter 1)
prevent these two chromosomes from being completely classified as segregating in favor
o f L. esculentum. Segregation towards the *+’ allele was particularly severe in three
chromosome regions: chromosome 2, from TG48 to TG507 (approximately 44 cM);
chromosome 4S, from TG146 to the end of the chromosome
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37
Figure 2.1. Allele frequency in selected F2 and BC progeny of heterozygous S.
lycopersicoides ILs. Horizontal axis indicates chromosomes 1 through 12 of L.
esculentum, including markers (to scale). Width of bars indicates markers covered by
introgression and vertical position o f bar shows the frequency of 5. lycopersicoides
alleles (f(S)). Solid horizontal line indicates expected F2 allele frequency (0.5), dashed
line the expected BC frequency (0.25). Bars with circles are significantly different (p =
0.05) from expected values. BC data are indicated by arrows, all others are F2
populations.
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38
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39
Figure 2.2. Segregation distortion histogram for selected F2 and BC IL populations whose
genotypic frequencies deviate significantly from Mendelian values. Thin vertical lines
represent region covered by IL and 3 shaded vertical bars indicate the % deviation
(positive or negative) from Mendelian ratios of each genotypic class (+=£,. esculentum,
S=S. /ycopersicoides allele, where f(S)=S. lycopersicoides allele frequency). Putative
segregation distortion loci are indicated on chromosomes by arrows: dark shading,
segregation skewed towards S allele; light shading, region segregates towards +.
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40
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1 111
41
(approximately 35 cM); and chromosome 7S, including TG252 (approximately 43 cM).
Frequencies o f the *+’ allele in F2 families was ~0.8 for all three regions, due to an excess
of I. esculentum homozygotes. Segregation for adjacent ILs on chromosomes 4 (e.g.
LSI0-11) and 7 (e.g. LSI9-7) were closer to expected proportions. These segregation
distortion loci have been named sd2.2, sd4.1 and sd7.2, for regions o f chromosomes 2 ,4
and 7, respectively.
Conversely, S. lycopersicoides segments in three other regions of the genome
were transmitted in excess of the *+’ alleles: chromosome I, from the vicinity of the Slocus through TG59 (approximately 39 cM), and from TGI 7 to the end of the long arm
(approximately 36 cM); and chromosome 6L, including TG292 through CT206
(approximately 37 cM). These putative segregation distortion loci have been named
s d l.l, sd l.3 , and sd6.2, respectively. Introgressions favoring transmission o f L.
esculentum alleles border each of the three regions with an excess o f S. lycopersicoides
transmission, and thus delimit the location of these segregation distorter loci.
sdl.3 strongly promotes S. lycopersicoides allele transmission: the S.
lycopersicoides frequency in the F2 population was 0.70, which is significantly more than
the expected value o f 0.50. To further characterize segregation distortion at sdl.3,
heterozygous ILs covering sdl.2 (LSI0-2) were used in reciprocal backcrosses to L.
esculentum cv. ‘VF36.’ Allele frequencies in the progeny indicated that the segregation
distortion effect is expressed only on the male gametophyte: the cross L SI0-2 x L.
esculentum segregated 1:1, whereas the reciprocal produced 100% heterozygous progeny
(Figs. 2.1, 2.2). Pollen tube growth of LS 10-2 pollen was normal, suggesting selection
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42
for the S. lycopersicoides allele acts at other stages. Pollen viability, as measured by
acetocarmine-stainability, revealed a semi-sterility phenotype in heterozygotes: pollen
grains were either fully developed (67%) or malformed and apparently inviable (33%).
Thus, the mechanism of segregation distortion acts during meiosis or pollen development
and results in the abortion o f gametes containing the L. esculentum haplotype in LS10-2
heterozygotes.
sd6.2, which is bounded by two other sd factors which promote transmission of
the *+’ allele (sd6.I and sd6.3), includes the Beta (B) locus on 6L. Whether selection
operates on the S-allele producing gametophyte (pre-zygotic) or on the sporophyte (postzygotic) is unknown. Interestingly, other S. lycopersicoides introgressions that include B
(e.g. LS 32-14 and LS 4-17) don’t promote transmission o f the S. lycopersicoides allele
like sd6.2.
In total, 17 sd loci were identified within the 45 segregating S. lycopersicoides
introgressions on 11 different chromosomes. Three of the 17 segregation distortion
factors (18%) skew segregation towards S. lycopersicoides; the majority (82%),
therefore, are distorted towards L. esculentum. As mentioned earlier, the ILs used in the
present segregation and recombination experiments represent a portion (88%) of the S.
lycopersicoides genome. Consequently, the identified 17 sd loci likely represent a
fraction o f all existing segregation distortion factors in the S. lycopersicoides ILs.
Recombination in S. lycopersicoides Chromosome Segments
An IL library comprised of homeologous segments permits a detailed study of
genome-wide recombination trends. Estimation o f genetic distances in traditional
mapping populations (F2 , BC, recombinant inbred lines, etc.) can be affected by trans­
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
acting factors, which increase or decrease recombination in other regions. The ILs used
in the S. lycopersicoides library are mostly single-segment lines, thus recombination
effects can be associated with specific regions. In order to examine the effect of a
homeologous introgression on recombination rate, comparisons were made using four
contrasting types of S. lycopersicoides segments: 1) longer vs. shorter segments; 2)
terminal vs. interstitial segments; 3) peri- vs. paracentric segments; 4) long-arm vs.
short-arm terminal segments. Recombination data were compiled from F2 progeny of 45
different ILs representing 88% of the S. lycopersicoides genome (Figs. 2.3, 2.4).
To address the question of whether recombination rate is correlated with the size,
recombination rate (as a % of the reference map) was plotted as a function o f segment
length (Fig. 2.4). The size of introgressed segments was positively correlated (r2= 0.26)
with recombination rate. In other words, as S. lycopersicoides segment lengths increase,
homeologous recombination rates become more similar to control (homologous) values.
No considerations were made for interactions between chromosome segment size and
position; however, an examination of several sets of nested ILs demonstrates the positive
correlation between segment size and recombination rate within a chromosome. For
example, ILs on chromosome 1, that cover most o f the region between TG301 and
TG465 (approximately 100 cM), recombine at about 25% of control; in contrast, shorter
(~25 cM each), recombinant segments derived from longer ILs recombine at ~l-2%
control values. Another example is chromosome 7, where the substitution line
(introgressed whole chromosome) recombines at approximately 50% of the control value,
while other lines covering various portions o f the same chromosome recombine at —10%
control. Lastly, chromosome 9 contains two nested sub-lines, which are derived from
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44
Figure 2.3. Recombination suppression histogram for selected S. lycopersicoides IL F2
populations. Markers on each chromosome delimit region under examination and
vertical bars represents IL size as indicated on F2 L. esc. x L. pen. (Tanksley et al. 1992).
Horizontal boxes are proportional to the % recombination o f the indicated IL population
compared to control (i.e. F2 L. esc. x L. pen.).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
45
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Figure 2.4. Recombination rates in relation to chromosome position o f the S.
lycopersicoides introgression. For each category, values represent the average genetic
distance, expressed as percentage of the control distance, in the same marker intervals
from F2 L. esc. x L. pen. (Tanksley et al. 1992). Standard error bars indicate the 95%
confidence interval, where n is the number o f segments in each class. T-values and
significance (p) are given for pair-wise comparisons between categories 1 and 2, 3 and 4,
and 5 and 6. Asterisk indicates significance at 95% confidence level.
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47
Figure 2.5. Correlation between recombination rate and length o f introgressed S.
lycopersicoides chromosome segments in tomato. Recombination is expressed as a
percentage of the distance in the same marker intervals as the reference map (Tanksley et
al. 1992, based on F2 L. esc. x L. pen.). Length o f introgressed segments are also
estimated from the Tanksley map.
50
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100
48
the larger, terminal introgression. As the length o f these segments is increased,
recombination suppression becomes progressively more severe (Fig. 2.3). Three
different segment-positioning effects were examined, including: terminal peri- vs.
paracentric, interstitial peri- vs. paracentric, and long-arm vs. short-arm. Pair-wise
comparisons between each segment type were based on recombination values calculated
from the 45 F2 IL populations described earlier. Comparisons between the
aforementioned three S. lycopersicoides segment types (Fig. 2.4) showed significant
differences (at 95% confidence level) between peri- vs. paracentric segments (t = 2.35, p
= 0.025), but not long vs. short-arm segments (t = 1.84, p = 0.08) and terminal vs.
interstitial segments (t = 1.60, p = 0.12). Despite the lack o f statistical significance,
recombination in terminal S. lycopersicoides segments was generally higher than in
interstitial ones (Figs. 2.3,2.4). This point is demonstrated by two segments on
chromosome 12, where recombination in the shorter, paracentric terminal segment is
completely suppressed (LS 45-7sub; 0% control), whereas a second, longer segment
(LS45-7), which covers an additional chromosome region, including the centromere,
recombines at about 25% the control rate (Fig. 2.3). However, this difference may be due
to overall length (pericentric segments are longer) rather than position.
Some evidence of chromosome-specific differences in recombination levels was
detected, independent of introgression size and/or position. For example, the two
segments covering chromosome 3 are both terminally positioned and approximately 55
cM long. Surprisingly, recombination was completely suppressed in F2 populations
segregating for these segments. In contrast, segments on chromosomes 1, 7 and 9, of
similar size to those on chromosome 3, recombined at much higher levels (Fig. 2.3).
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49
Possible reasons for such reduced recombination on chromosome 3 include greater
sequence divergence or structural differences between L. esculentum and S.
lycopersicoides chromosomes. Rates of divergence between Lycopersicon species are no
greater on chromosome 3 than elsewhere (Baudry et al. 2001; Stephan and Langley
1998). Furthermore, the BCi L. esculentum x S. lycopersicoides map for this
chromosome shows a reduction in recombination of -40-50% o f control, somewhat lower
than average for the chromosomes (Chetelat et al. 2000). The most pronounced
reduction was in pericentromeric marker intervals, suggesting that differential
heterochromatin might contribute to recombination suppression.
The inversion on 10L of S. lycopersicoides suppressed recombination in segments
both within the inversion as well as in flanking segments. Ji and Chetelat (2002),
however, detected elevated recombination between markers adjacent to the inversion,
suggesting a compensatory response in these regions. Whole chromosomes that include
introgressed segments that recombine more regularly (e.g. chromosomes 1 and 7), on the
other hand, may have a high amount o f sequence conservation and/or have
recombination-enhancing loci. Regardless, regional heterogeneity in chromosome
structure and sequence possibly contribute to homeologous pairing and subsequent
recombination success.
Discussion
Segregation distortion towards the recurrent parent is common in wide crosses of
many crops, including the tomato (Zamir and Tadmor 1986). Though complicating the
recovery of desirable 5. lycopersicoides introgressions intended for the IL collection,
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50
higher than expected transmission o f L. esculentum alleles favored the elimination o f
segregating extraneous segments, which greatly assisted in the development of a nearly
isogenic population.
Harushima et al. (2001) used segregation analysis to map reproduction barriers in
rice by correlating deviations in allele frequencies with specific chromosomal regions. In
all, 33 barriers were identified, with some loci promoting the recurrent parent alleles,
others promoting the alien alleles. In similar fashion, the library o f segregating ILs has
highlighted specific regions o f the S. lycopersicoides genome influencing hybrid
fecundity. Epistatic effects, resulting from non-isogenic segments, which were
eliminated during the course o f this study, may have influenced previous IL segregation
data, so that segregation distortion loci identified in this study were not recognized in the
BCi generation (Chetelat et al. 1997; Chetelat et al. 2000).
Comparable recombination studies have been done in wheat (Triticum aestivum),
where it has been shown that recombination between chromosomes 3A and 5A and its
homeologues (3Am and 5Am; T. monococcum) is least suppressed in terminal segments
(Luo et al. 2000). In addition, Sherman and Stack (1995) demonstrated that
recombination nodules, and therefore, chiasmata and recombination, are more common in
euchromatin than in heterochromatin. Accordingly, chromosome genetic and physical
lengths do not directly correlate, as heterochromatic centromeric regions are typically
more recombinationally inert than euchromatic terminal regions. Terminal pericentric S.
lycopersicoides segments likely include euchromatic regions on both chromosome arms,
and are thus less likely to be recombinationally suppressed.
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51
Although no statistical difference was detected between terminal and interstitial S.
lycopersicoides segments, trends in the data suggest that terminal segments are less
suppressed than interstitial segments, which agrees with previous work done in wheat.
Luo et al. (2000) substituted chromosomes 3A and 5A of T. aestivum with their Triticum
monococcum homeologues (3Am and 5Am, respectively), and compared recombination
rates with those of the corresponding homologous chromosomes. Recombination in
proximal regions was suppressed much more severely than in distal areas. Devos et al.
(1995), and later Jones et al. (2002), compared recombination in normal and deletion
chromosomes in wheat and concluded that recombination frequency o f a chromosome
segment is mainly dependent on its spatial relationship to the telomere and centromere.
By crossing the same Triticum dicoccoides chromosome 1 segment into normal (cv.
Chinese Spring) and deletion stocks, the authors were able to transpose the identical
homeologous introgression proximally and distally along the long arm of wheat
chromosome IB. Homeologous recombination rates in the T. dicoccoides segment
increase in terminal deletion stocks (eg segment is moved closer to the telomere)
compared to the T. dicoccoides x cv. Chinese Spring control population. Unfortunately,
no such resource is available in tomato. However, with the S. lycopersicoides IL lines,
the same segment position manipulations are possible by using different introgressions
covering various genomic regions.
The present study demonstrates the effect of introgressed homeologous
chromosome segments on allele segregation and recombination in self-pollinated and
backcrossed progeny. As the size of an introgressed region o f homeology increases on
any given chromosome, the homologous portion simultaneously decreases, and,
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52
conceivably, recombination events are compelled to occur within unfavorable
homeologous chromosome segments. S. lycopersicoides introgressions that included
centromeric (heterochromatic) portions of the chromosome, especially those that extent
to the telomere (i.e. terminal), are physically longer than other chromosome segments.
Conceivably, these longer segments simply monopolize chromosome space so that
recombination is obliged to occur more frequently in longer segments.
Trends such as those described in this study not only affect the successful creation
of introgression line populations, but also can hinder the development of any mapping
population. Having a priori knowledge of recombination and segregation tendencies will
greatly assist in the planning of breeding experiments.
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53
CHAPTER III
STRATEGIES TO INCREASE HOMOLOGOUS AND HOMEOLOGOUS
RECOMBINATION IN TOMATO (L. ESCULENTUM) USING AN S.
LYCOPERSICOIDES INTROGRESSION LINE LIBRARY
Abstract
Reduced recombination in interspecific hybrids results in linkage drag in wide
crosses between most crop species and their wild relatives. To determine the feasibility
of using the S. lycopersicoides IL collection to increase homologous and homeologous
recombination in tomato introgressants, two populations were developed using different
IL combinations of segments. We first determined the effect two adjacent S.
lycopersicoides introgressions on the same chromosome have on homeologous
recombination within each segment. Each combination consisted o f a “target” and
“driver” (t/d) S. lycopersicoides segment in an adjacent region combined on the same
chromosome. Combinations (t/d) were created by crossing ELs on four different
chromosomes. Recombination rates were evaluated within each t/d segments in F2 and/or
BC progeny. Combinations o f terminal segments on chromosomes 2, 6 and 7 enhanced
recombination the most, with recombination increasing from zero to nearly 10% of
control populations, usually in both segments. These results demonstrate that a second
homeologous segment can partially offset recombination suppression in a neighboring
segment. Next, we crossed a S. lycopersicoides IL with L. pennellii (LA716) and
examined recombination in the region of homology (i.e. L. esculentum-L. pennellii) and
the region o f homeology (i.e. L. pennellii-S. lycopersicoides). Recombination in the
adjacent homologous region to the introgression was increased by 19%, while
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54
recombination between L. pennellii and S. lycopersicoides in the same cross increased
10-fold over!,, esculentum!S. lycopersicoides, demonstrating the potential for using a
third species (L. pennellii) as “bridge” to enhance recombination. These results
demonstrate the potential to use a homeologous chromosome segment to increase
recombination throughout the tomato genome.
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55
Introduction
The substructural theory of chromosome evolution predicts that accumulated
changes in DNA composition, including base pair substitutions, insertions, deletions and
rearrangements, are sufficient to effectively transform entire chromosome sets so that
meiotic pairing and recombination with the unmodified genome is severely impaired
(Dubcovsky et al. 1995). Sequence and structural divergence between such homeologous
chromosomes is an important speciation mechanism that prevents sexual hybridization
between evolutionarily isolated organisms. Wheat (Triticum aestivum) is a wellcharacterized allopolyploid, whose three non-homologous chromosome sets are
maintained independently both by virtue of intergenomic sequence differences (Dvorak
and Chen 1984), as well as by the action of specific anti-homeologous recombination
loci, such as P hi (Sears 1976). Though essential for preserving genomic autonomy,
excluding dissimilar chromosomes from meiosis simultaneously prevents potentially
beneficial wild genes and alleles from being incorporated.
Jones et al. (2002) were able modified homeologous recombination levels in a
wheat (T. aestivum) x T. dicoccoides cross by comparing the same T. dicoccoides
segment introgressed into normal, deletion and deficiency T. aestivum backgrounds.
Pairing in wheat chromosomes is initiated terminally (Lukaszewski 1997), implying that
recombination preferentially takes place in distal rather than proximal chromosome
intervals. As a result, recombination rate in the identical homeologous T. dicoccoides
segment increases as its position is “moved” further away from the centromere in wheat
whole-arm deletion and deficiency stocks.
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56
Few crops have as much accessible genetic diversity in wild relatives as the
cultivated tomato (Lycopersicon esculentum) with nine related Lycopersicon species, all
of which are cross compatible (Rick 1991). Consequently, efforts to increase
recombination in interspecific hybrids are potentially beneficial for tomato improvement.
Comparative maps o f all Lycopersicon species demonstrate that their chromosome sets
are fully homologous with those of cultivated tomato. Nonetheless, suppressed
recombination has been demonstrated in some interspecific hybrids and their derivatives
(Grandillo and Tanksley 1996; Paran et al. 1995), particularly in male gametes
(deVicente and Tanksley 1991) and in advanced backcross generations (Fulton et al.
1997). The tomato-like nightshade Solanum lycopersicoides is readily hybridized with
cultivated tomato, but Fi hybrids are male-sterile, and display abnormal meiosis, with
frequent univalents at metaphase I, differences in chromosome size, and lack of
synchronization of condensation. The chromosomes o f S. lycopersicoides are
homeologous with those of tomato. As a result of these obstacles, breeders have made
little use o f S. lycopersicoides, even though it possesses economic traits not found in
Lycopersicon. (Chetelat et al. 1997; Chetelat et al. 1998; Chetelat and Meglic 2000;
Chetelat et al. 2000; Menzel 1962; Pertuze and Chetelat 2002; Rick 1951; Rick et al.
1986; Rick 1988).
Recently, a population of introgression lines (ILs) was developed, representing
the majority of the S. lycopersicoides genome (chapter 1). These ILs are mostly single­
segment, overlapping homeologous introgressions distributed throughout the tomato
genome. Recombination within the ILs is extremely suppressed, with several as low as
0% of normal. In wheat hybrids, Dubcovsky et al. (1995) noted an increased crossover
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57
frequency in homologous chromosome stretches bordering homeologous segments.
Given this observation, there existed the potential for using the S. lycopersicoides
introgression lines (ILs) to enhance recombination in neighboring chromosome regions.
Unlike wheat, a collection of chromosome deletion stocks is not available in tomato.
Moreover, tomato is a diploid and has no known Ph-1-tike loci that restrict recombination
between homeologous chromosomes. Nonetheless, to determine the feasibility o f using
the S. lycopersicoides IL collection to increase homologous and homeologous
recombination in tomato hybrids, two populations were developed. We first determined
the influence of two adjacent S. lycopersicoides segments have on recombination in both
regions. Secondly, we crossed a S. lycopersicoides IL with L. pennellii (LA716) and
examined recombination both in the adjacent region of homology (e.g. L. esculentum-L.
pennellii) as well as the region of homeology (e.g. L. pennellii-S. lycopersicoides).
Materials and Methods
S. lycopersicoides introgression line library
Chapter 1 describes the development of the S. lycopersicoides introgression line
(IL) library, including DNA isolation, RFLP analysis, isozyme and PCR analysis, as well
as genome-ratio calculations.
‘Target/driver” populations and genotypic analysis
From the original 104 S. lycopersicoides ELs, five pairs of lines were combined
together, to create “target/driver” (t/d) combined genotypes. Each t/d genotype consisted
o f two adjacent S. lycopersicoides segments, oriented in repulsion phase on the same
chromosome (Fig. 3.1). The terms “target” and “driver” refer to the situation in which it
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58
Figure 3.1. Representation of a crossing scheme to produce paired S. lycopersicoides
introgressions or “target/driver” combinations, on one chromosome. The darkly shaded
and lightly shaded regions o f the chromosomes indicate the two S. lycop. segments, and
diagonal lines between homologues indicated expected crossover locations in the F2 and
BC generations.
Target
Driver
F,
L. esculentum
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BC
might be desirable to increase recombination in one introgressed region (target) by
crossing to another (driver). In the present experiments, these segments are arbitrarily
designated A, B and C. Using RFLP markers to identify each S. lycopersicoides
segment, paired ILs were combined together in individual plants (Fig. 3.2). To measure
recombination rates in each S. lycopersicoides segment in the t/d combinations,
segregating populations (F2 and BC) were created. Sex-based differences in
recombination were determined in some t/d crosses using the t/d Fi as both the male and
female parent in backcrosses with L. esculentum cv. ‘VF36’ (eg VF36 x t/d and t/d x
VF36). Recombination rates in F2 and BC populations were then assessed using RFLP
markers near the ends of each segment. Control measurements for t/d combinations were
obtained using F2 populations of each “target” and “driver” segment individually. For
segment 7B on chromosome 7, recombination data from a slightly longer (approximately
11 cM longer) S. lycopersicoides introgression was used as the control. Results from t/d
F2 populations were compared to control F2 populations by means of 9 x 2 contingency
X2 tests.
S. lycopersicoides IL x L. pennellii populations and genotypic analysis
To measure the effect a homeologous segment has on recombination in flanking
regions, we crossed a chromosome 2 S. lycopersicoides IL with L. pennellii (LA716).
After confirming the hybrid with RFLP markers, the Fi (S. lyc IL x LA716) was self­
pollinated to generate a segregating F2 population. Four RFLP markers were used to
measure recombination rates in the region of homology (between L. esculentum and L.
pennellii) and three markers for the region of homeology (between S. lycopersicoides and
L. pennellii). The F2 L. esculentum x L. pennellii (LA716) map (Tanksley et al. 1992)
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60
Figure 3.2. S. lycopersicoides segments used for “target/driver” study. The shaded
chromosome segments are shown adjacent to the markers they include from the tomato
RFLP map (Tanksley et al. 1992).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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62
was used as a control for the homologous region. For the homeologous region (S. lycop.
x L. penn.), data from the control cross for the same introgression (F 2 IL x L. esc.) were
not available. However, data from a related IL with a nearly identical introgression were
useful as a control.
Results
S. lycopersicoides segments combined in “target/driver” arrangement
Correlation between S. lycopersicoides segment length and recombination rate in
F2 and BC progeny (chapter 2) suggested that combining introgressed segments on the
same chromosome would increase each segment’s “effective total length,” thereby
increasing recombination rate in each. To this end, five pairs of S. lycopersicoides
segments on four different chromosomes were selected for the t/d study. The
chromosome segments on chromosome 1 (1A and IB) are both interstitial that cover
most of the long arm, whereas the chromosome 2 segments (2A and 2B) include 1
interstitial and one terminal. The paired segments on chromosomes 6 and 7 are each
terminal, on opposite arms (Fig. 3.2). Control F2 populations o f 6A and 6B detected no
recombination within the introgressed segments; consequently, it was o f interest to
determine if combining the segments in a double heterozygote would increase
recombination levels in either or both region(s). On chromosome 7, several nested
terminal introgressions were available (chapter 1), which provided a means to test the
effect homeologous segments o f different sizes would have on recombination in a
neighboring introgression. Consequently, we created two t/d populations that included
the long arm segment (7C), the first opposite a longer S. lycopersicoides segment (7A)
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63
that included most o f the short arm, and the second with a shorter segment that comprised
-h alf of 7S (e.g. 7B; Fig. 3.2).
Each individual t/d segment was compared to its corresponding single-segment
control via 9 x 2 contingency y} tests. Because only F2 control (single-segment)
populations were available for statistical comparisons, BC t/d populations were not
analyzed for y} significance. Nonetheless, backcross populations, in most cases,
supported trends observed in F2 recombination. At the 95% confidence level, only
recombination in t/d segment 7A (in combination with segment 7C) was significantly
increased over its corresponding, single-segment control. With the exception of t/d
segments on chromosome 1, recombination in all other t/d segments (both F2 and BC
populations) was measurably increased over control populations; however, x2 analysis
revealed no statistical significance.
The combination of segments on chromosome 1 showed no significant increase
(p=0.05) in recombination frequency over the single segment controls, although the BC
population for 1A recombined at a higher rate than the F2 (Fig. 3.3). Recombination in
segments 2A and 2B on chromosome two increased in t/d F2 and BC populations, with
increases 2B nearly reaching statistical significance (x2 = 12.56). Recombination in both
chromosome 6 t/d segments uniformly increased over control values, whereas
recombination in chromosome 7 t/d combinations increased varying amounts. The
7A+7C combination resulted in significant (p=0.05) elevation of recombination in
segments; however, only segment 7B in the 7B+7C combination was increased (x2 =
10.37).
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64
The juxtaposed S. lycopersicoides segments on chromosome 1 were both
positioned proximally: segments 1A and IB are 27 and 17 cM, respectively, from each
chromosome end. Recombination in each segment in combined (t/d) populations was
similar to single segment control rates. It is possible that chiasmata were localized to the
more preferable positions at the two terminal homologous regions, which reduced
crossover frequency between L. esculentum and S. lycopersicoides on 1A and IB (Fig.
3.3). In contrast, segments on chromosome 2 are positioned both terminally and
interstitially, with a 27 cM gap between the distal end of segment 2A and the
chromosome end. Recombination in the terminal segment (2B) is less affected by the t/d
arrangement than the other segment (2A). When combined with 2B, several 2A
recombinants were recovered (Figs. 3.4), representing a 6-18% decrease in recombination
suppression. These results provided further evidence that as crossovers are prevented in
terminal chromosome regions (via homeologous segments), recombination is
simultaneously increased in other portions on the same chromosome. The chromosome 2
t/d arrangement included one terminal S. lycopersicoides segment, and homeologous
recombination was enhanced. Both chromosome 6 segments are terminal, and, consistent
with our observations on chromosome 2, recombination rates are increased even more
dramatically (Fig. 3.5; approximately 5% reduction in recombination suppression in
segment 6A and 20% in segment 6B). Similarly, chromosome 7 t/d pairs, which are also
terminally positioned, show increased recombination over controls (see Fig. 3.6a-b).
Though recombination in only the longer 7A (vs. 7B) segment was significantly
enhanced, the data indicate that positioning t/d segments terminally has the greatest effect
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figures 3.3-3.6. Recombination in combined (target/driver) or single introgressed S.
lycopersicoides segments on chromosome 1, 2, 6 and 7 of tomato. Introgression segment
lengths are shown (vertical bars, far right) in reference to the F2 L. esculentum x L.
pennellii map for each chromosome (Tanksley et al. 1992). Control distances (cM),
population sizes (n) for individual S. lycop. segments are based on F2 populations. Inset
histograms show total recombination values for each segment alone or in combination,
with standard error, bars at 95% confidence level. Significant F2 x2 are indicated by
asterisk.
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66
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70
on homeologous recombination rates, facilitating the recovery of a number of
recombinants from recalcitrant S. lycopersicoides segments.
The proximity of target/drive segments to the distal end of each chromosome arm
appears to affect recombination rate more significantly than does increasing the total
genetic length of each chromosome that is heterozygous for homeologous segments. For
instance, the two t/d segment pairs on chromosomes 2 and 6 each represented -69% of
their chromosome's total genetic length. Non-significant recombination rates increased
in both chromosome 6 terminal segments (Fig. 3.5), whereas recombination in only one
of the chromosome 2 segments was positively affected (Fig. 3.4). Chromosome 1 t/d
populations included S. lycopersicoides segments that cover approximately 61% of
chromosome 1, but recombination in the combined lines was similar to control values
(Fig. 3.3). However, when both ends of the chromosome were covered by S.
lycopersicoides segments, recombination in a second segment was increased more
dramatically when paired with a longer segment. Segment 7C on chromosome 7 is
paired with two terminal segments, one longer (7 A, 53 cM) and the other shorter (7B, 25
cM). Recombination in 7C increased approximately 83% when combined with 7A,
whereas no significant increase in recombination rate was observed in the 7C/7B
combination. Similarly, when paired with 7C, recombination in the longer segment (7A)
is increased 267% (p = 0.05, x2== 17.86), yet recombination in 7B, when paired with 7C,
increases less dramatically (p=0.05, x2 ~ 10.37). These data indicate that increasing the
length o f homeology along a chromosome increases crossover frequency, at least when
one/both segments are terminal.
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71
Other factors affecting t/d recombination did not appear to influence rate
enhancement as significantly as segment position and size. Distance between S.
lycopersicoides t/d introgressions had no bearing on recombination in each segment;
rather, it was more important that the segment(s) be terminally positioned. For example,
only 20 cM separates segments 1A and IB, which showed little if any increase in
recombination, whereas 43 cM separates 6A and 6B, which did. However, because the
chromosome 6 segments are both terminal, recombination is enhanced in 6A and 6B,
while no increases are observed in 1A or IB (Figs. 3.3, 3.5).
Recombination in the homologous region between each t/d segment was also
observed. Most intervals experienced a 2 to 3-fold increase in recombination levels over
the reference L. esc. x L. penn. F2 population (Tanksley et al. 1992), regardless of
whether recombination in the individual t/d segments was enhanced (Figs. 3.3-6). For
instance, recombination in neither chromosome 1 t/d segment was significantly increased,
whereas the intervening region increased over 2-fold. Similar trends are observed in the
homologous gaps between the other t/d segments.
Recombination in male gametes of interspecific hybrids is typically reduced as
compared to female gametes (de Vicente and Tanksley 1991). Sex-based differences in
recombination can bias mapping results in backcross populations. For this reason, we
attempted to use each t/d line as both the male and female parent in backcross
experiments. Unfortunately, successful backcrosses in both directions were made for
only two of the five t/d combinations (2A/2B and 7B/7C). Low pollen fertility and
hybrid seed viability prevented the production o f progeny in the other three groups.
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72
Nonetheless, recombination in the chromosome 2 and 7 t/d backcross populations appears
to be equal in male and female gametes (Figs. 3.4, 3.6).
Recombination in IL x L. pennellii progeny
We measured homologous recombination adjacent to a homeologous region by
crossing a S. lycopersicoides IL with L. pennellii. Cultivated tomato (L. esculentum) and
L. pennellii are divergent enough to yield sufficient marker polymorphisms, yet similar
enough to have homologous chromosome sets. Unlike t/d populations, where x2
contingency analysis was used to compare control and t/d recombination values, control
genotypic values were not available, making such comparisons impossible in IL x L.
pennellii populations. Recombination between S. lycopersicoides and L. pennellii DNA
was reduced to ~69% o f normal in the distal segment of 2L, whereas recombination in
between L. esculentum and S. lycopersicoides in the same interval was only ~5% of
control. To determine the effect that IL the chromosome 2 IL has on the adjacent region,
we measured recombination between L. esculentum and L. pennellii in the proximal
portion o f chromosome 2L. Recombination in this region increased 19% over the
control, an effect that was most apparent in the intervals closest to the S. lycopersicoides
introgression. Similarly, recombination in the region bounded by the most proximal S.
lycopersicoides introgression marker (TG48) and the next L. esc. x L. penn. marker
(TG353) increased approximately 2.3 fold (Fig. 3.8).
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73
Figure 3.8. Recombination in homologous and homeologous regions o f chromosome 2 in
F2 IL x L. pennellii. Control distances are shown to the right o f F2 L. esc. x L.penn map
(Tanksley et al. 1992) for the regions o f homology (open) and homeology (lightly
shaded), as are results from an EL F2 population (dark). F2-population results for the
homologous (TG304-TG353, open) and homeologous (TG48-TG507, shaded regions are
shown at the left.
Ft
VLxL. penn
F2
L. esc. x L. penn
TG 304
TC 5S4
TC 308
TG 353
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TC 537T
41
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36
[]
L. esculentum x L. pennellii
|
L. esculentum x S. lycopersicoides
[]
L. pennellii x S. lycopersicoides
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74
Discussion
Sherman and Stack (199S) showed that recombination is not evenly distributed
along tomato chromosome arms, but that most crossovers occur in the distal euchromatic
regions. Additionally, evidence from wheat haploids demonstrates their preference to
pair homogenetically rather than with their homeologues (McGuire and Dvorak 1982).
In wheat chromosome pairs with mixtures o f homeologous and homologous segments,
there is no recombination in homeologous regions (Luo et al. 1996). When given the
opportunity, most crossovers would preferentially occur in non-proximal, homologous
regions. Moreover, recombination in homeologous chromosome segments is least
suppressed when they are terminally positioned (Luo et al. 2000; chapter 2). In the
current study, we have attempted to take advantage of these observations to increase
homeologous recombination in tomato-5, lycopersicoides hybrids. Near-isogenic 5.
lycopersicoides lines with chromosome introgressions on the same chromosome have
been hybridized and segregating F2 and BC populations created in so called
“target/driver” populations. By varying regions o f chromosome homeology via 5.
lycopersicoides segments selected for their chromosomal size and position (chapter 1),
rare crossover events have been “forced” to occur away from terminal and homologous
regions, thus increasing recombination in homeologous regions.
Chiasmata in wheat are most frequently distal; accordingly, recombination is
usually initiated in terminal chromosome regions (Lukaszewski and Curtis 1993), and
homeologous recombination in these regions is less suppressed than in proximal regions
(Luo et al. 2000; Jones et al. 2002; chapter 2). If at least one chiasma were required per
chromosome arm for proper chromosome segregation during meiosis, any modifications
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75
to chromosome homology in distal portions o f each arm would encourage alternative
chiasma positioning. Jones et al. (2002) were able to increase recombination rates in
Triticum dicoccoides segments in a cultivated wheat (cv. Chinese spring) background
using wheat deletion stocks. The authors adjusted the T. dicoccoides introgression’s
chromosome position by crossing it with wheat terminal deletion stocks, effectively
moving the alien segment closer to the chromosome’s telomere, and thus increasing its
recombination compared to control populations. It may be possible to use the S.
lycopersicoides IL library in the same way as the wheat deletion stocks, that is, to
increase recombination in adjacent chromosome regions. As with the wheat deletion
stocks, chiasma formation and recombination within S. lycopersicoides introgressions are
virtually absent, thus increasing the chances of recombination occurring elsewhere on the
chromosome. Positioning an alien Lycopersicon chromosome segment adjacent to a
single terminal S. lycopersicoides segment should, as with the wheat deletion stocks,
“force” compensatory recombination events to occur proximally (eg within the
introduced Lycopersicon segment). Positioning a second terminal S. lycopersicoides
introgression on the other arm of this same chromosome should, as demonstrated by t/d
results from chromosomes 2, 6 and 7, result in even greater increases in recombination in
the introgressed Lycopersicon segment (Figs. 3.4-6).
Using Triticum monococcum x L. aestivum hybrids to examine the effect a
homeologous segment has on recombination in adjacent chromosome regions, Luo et al.
(1996) observed significant increases in homologous recombination when the alien
segment was proximal to the region of homology. However, no enhancement in
recombination was found when the homeologous region was distal to the homologous
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76
segment. These data partially concur with the findings from the “target/driver” study,
where homeologous recombination was increased in juxtaposed S. lycopersicoides
segments when positioned terminally, but not proximally. In both populations, distal
homeologous S. lycopersicoides segments resulted in compensatory crossovers in
proximal homologous as well as homeologous regions, as demonstrated by localized
increases in recombination. Without additional S. lycopersicoides IL x L. pennellii
populations, including both proximal as well as additional distal S. lycopersicoides
segments in different genomic regions, definitive conclusions regarding the nature o f
compensatory crossovers are not possible. An increase in recombination was nonetheless
observed in the adjacent region of homology, thus demonstrating the potential to use
homeologous near isogenic lines, such as the S. lycopersicoides IL library, for increasing
homologous recombination.
Recombination between S. lycopersicoides and L. pennellii increased
approximately 10-times over the control IL F2 population (S. lycopersicoides x L.
esculentum) in distal portion of chromosome 2. Whether the increased recombination in
the region adjacent to the chromosome 2 S. lycopersicoides segment is due to c/s-effects
(factors localized to chromosome 2) or trans-effects (genome-wide factors) is uncertain.
Background effects were cited as the reason recombination in an advanced backcross L.
esculentum x L. pennellii population was more suppressed than in BCi population o f the
same species (Rick 1969; Rick 1971). Similarly, recombination in S. lycopersicoides
substitution lines o f L. esculentum was lower than in a BCi L. esculentum x S.
lycopersicoides population (Ji and Chetelat 2002). A set o f Lycopersicon pennellii
introgression lines with complete genomic coverage exist (Liu and Zamir 1999).
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77
Crossing the chromosome 2 S. lycopersicoides segment with L. pennellii ILs on
chromosome 2 (eg IL2-1, 2-2, 2-3 and 2-4) to determine if recombination is enhanced to
the same extent in these discrete segments as with L. pennellii would clarify if indeed the
observed increase were due to intra- or inter-chromosomal effects. Similarly, crosses
between other chromosomes’ L. pennellii and S. lycopersicoides segments will help
refine homologous/homeologous segment positioning, so that recombination can be
optimally enhanced.
Whether recombination enhancement in t/d populations actually works via a
recombination mechanism (i.e. increases in crossover frequency) or is the result of
gamete selection (i.e. a decrease in the frequency of parental, non-recombinant,
genotypes) is unknown. With two S. lycopersicoides segments, there is an increased
possibility that the chromosome will remain a univalent during prophase I o f meiosis and
aborted during gametogenesis. Genomic insitu hybridization (GISH) can be used to
examine the frequency of gametes carrying t/d S. lycopersicoides segments, in turn
identifying if these non-recombinant gametes are being selected against. Regardless,
recombination suppression is common in interspecific crosses and their derivatives and
thus any method to potentially increase the frequency o f recombinant gametes would be
beneficial to plant breeders. The observations presented in this study suggest one such
mechanism, that potentially increases rates of both homeologous and homologous
recombination using introgression lines containing marker-defined homeologous
segments. This technique could potentially benefit plant-breeding programs, where
undesirable linkage blocks frequently prevent complete expression o f a desired genotype.
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78
CONCLUSIONS AND FUTURE INVESTIGATIONS
Strong reproductive barriers have thwarted previous efforts to exploit the S.
lycopersicoides genome for tomato improvement. However, the herein described IL
collection, which includes a large share o f the nightshade’s genome, now enables
researchers to utilize this previously untapped resource of novel genetic diversity.
OTL identification
Other tomato IL populations have proved useful for breeding applications. For
example, using the L. pennellii introgression line population they developed, Eshed and
Zamir (1995) identified 23 QTL controlling total soluble solids and 18 for fruit mass.
They estimated this was twice the number o f loci identified than would have been
discovered using traditional mapping populations. Two chromosome 2 L. pennellii ILs
(IL2-5 and IL2-6) were fine-mapped to reveal three different QTL for fruit mass. Twelve
recombinant sub-lines were created from these two parent lines, which allowed more
precise genetic “dissection” of the L. pennellii genome in this region. It is doubtful that
this level of fine mapping is possible in the S. lycopersicoides IL population. Severe
recombination suppression, caused by reduced chromosome homology and an inversion,
would likely prevent the mapping resolution obtained in the L. pennellii ILs.
Nonetheless, results from mating strategies designed to enhance recombination within S.
lycopersicoides introgressions are encouraging (see chapter 3).
Introgression lines offer a convenient way of maintaining interspecific derivatives
indefinitely, as well as providing a stable genetic environment in which individual locus
effects can be measured. Once desirable traits are identified, lines containing these traits
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79
can be used directly in breeding programs. Most qualitative traits are inherited
quantitatively, e.g., a specific phenotype is controlled by several linked/unlinked loci
(QTLs). Individual ILs, clearly, would not likely carry all the loci required for
appropriate expression o f complex traits such as those involved in most fruit and
vegetative characters. However, once specific lines are correlated with the desired
phenotype, epistatic effects can be quantified through marker-assisted combining of ILs
into a single plant. Monforte et al. (2001) combined two ILs from L. peruvianum and L.
chmielewskii into a single tomato plant, and soluble solids, external and internal color
improved dramatically in the combination-genotype over the single-introgression
genotypes. The same study compared the effects of different alleles for several
agronomic and fruit traits on chromosome 4 of tomato. Segments covering most of 4L,
which originated from IL libraries for L. pennellii, L. hirsutum, L. peruvanium, were used
to identify several novel alleles o f known QTLs (Monforte et al. 2001). The S.
lycopersicoides XL population would potentially also be useful for QTL allele discovery.
Moreover, homozygous IL populations are ideal for replicated trials under different
environments. ILs with segregating introgressions can be pre-screened and advanced
using PCR-based or isozyme markers. However, several ILs have low fecundity and are,
therefore, potentially difficult to reproduce on a large scale. Typically, shorter
heterozygous introgressions are more fertile than longer, homozygous ones, and may,
therefore, be used instead.
It should be possible to further characterize segregation distortion (sd) through
fine mapping S. lycopersicoides introgressions carrying these loci, with the ultimate goal
to clone these genes. Using a Lycopersicon pennellii introgression line population, Eshed
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80
and Zamir (1995) identified a chromosome 2 IL that decreased fruit mass (FM) by 65%.
In order to further describe this trait, they identified overlapping recombinant sub-lines
for this introgression and again measured fruit mass. Three linked QTLs, within the
original chromosome 2 segment, were identified and the fruit mass gene Fm2-1 was
mapped to a 3.2 cM interval. Work with Fm2-l led to the identification and map-based
cloning of a second fruit mass gene (Fm2-2) (Frary et al. 2000). In the current study, the
putative segregation distortion locus sdl.3 was localized to a 17 cM region on IL. As
with Fm2-1 and Fm2-2, it should be possible to identify sdl. 2-recombinants and
determine which sub-lines affect pollen viability.
Applications to use S. lycopersicoides ILs to enhance recombination
For breeding applications, the recombination studies suggest a hierarchy of
chromosome segment arrangements that would be preferred: longer, terminal, pericentric
introgressions recombine at relatively higher rates. Accordingly, homeologous
recombination events will preferably take place in longer segments that include the
centromere (Fig. 4.1). The probability of obtaining a crossover in a given interval can be
maximized by judicious choice of ILs, allowing the unwanted linked DNA to be
separated from the target region with the least effort.
Initially, it might be worthwhile to combine both proximal and distal S.
lycopersicoides ILs with adjacent L. pennellii ILs to optimize homologous/homeologous
pairing. For instance, on chromosome 2, the same arrangement of S. lycopersicoides
chromosome segments (e.g. partial or complete cover of terminal chromosome regions)
could also be used to increase recombination in proximal S. lycopersicoides ILs, which
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81
Figure 4.1. Relative recombination rates for a hypothetical introgressed chromosome
segment occupying alternative positions on the chromosome.
centromere
region
of interest
unwanted
linkage Mock
terminal,
pericentric
terminal,
paracentric
interetitial,
paracentric
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82
are more recombinationally suppressed than terminal segments, thus increasing mapping
resolution within the S. lycopersicoides IL library.
Assuming that L. pennellii is o f intermediate chromosome homology between L.
esculentum and S. lycopersicoides, this Lycopersicon species may be useful as a
recombinational bridge between the cultivated tomato and S. lycopersicoides.
Potentially, L. pennellii ILs that correspond to the same chromosome locations as S.
lycopersicoides ILs could be used to mitigate recombination suppression in the latter
populations. Several S. lycopersicoides!L. pennellii IL pairings could be made
throughout the genome, which could generate many S. lycopersicoides sub-lines. L.
pennellii DNA, though not as readily eliminated in segregating progeny as S.
lycopersicoides, should not be too difficult to remove using medium to large-sized
segregating populations.
The “target/driver” approach to increasing recombination is a potentially useful
plant breeding tool. Strategically positioned terminal S. lycopersicoides segments,
arranged so as to be adjacent to the targeted gene block, could potentially promote
crossing over in the desired region of the chromosome. There exist terminal 5.
lycopersicoides segments for each of the 24 tomato chromosome arms, thus potentially
increasing recombination in all regions of the tomato genome. In fact, several
chromosomes have nested terminal introgressions, which would allow researchers to
modify the length of the homeologous segment to create the maximum number of
compensatory crossover events near the targeted region. The optimal scenario would be
to combine a whole-arm S. lycopersicoides introgression with a second terminal segment
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83
that extends proximally just to the distal border of the targeted chromosome block. Such
an arrangement would increase the frequency o f recombinants. After the desired
recombinants are identified, a generation o f backcrossing followed by selfing would
eliminate the S. lycopersicoides introgressions and fix the newly recombined segments.
Using homeologous segments to increase recombination in neighboring
chromosomal regions appears to be a practical way to promote recombination between
cultivated crops and their wild relatives. The S. lycopersicoides ILs provide nearly ideal
material for such efforts, since S. lycopersicoides chromosomes are more diverged than
Lycopersicon, and, consequently, compensatory recombination in neighboring
chromosome regions will likely be most encouraged with S. lycopersicoides ILs.
Furthermore, segregation distortion against S. lycopersicoides alleles will allow the
desired genotype to be recovered with minimal backcrossing generations.
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84
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