Transcript Genome Structure/Mapping - Plant Sciences Department

Genome
Structure/Mapping
Lisa Malm
05/April/2006
VCR 221
Genome
Structure/Mapping
 Characteristics of the tomato nuclear genome
as determined by sequencing undermethylated
EcoRI digested fragments
 Want et al. 2006
 Development of a set of PCR-based anchor
markers encompassing the tomato genome
and evaluation of their usefulness for genetics
and breeding experiments
 Frary et al. 2005
 Zooming in on a quantitative trait for tomato
yield using interspecific introgressions
 Fridman et al. 2004
Characterisitics of the tomato nuclear
genome as determined by sequencing
undermethylated EcorRI digested
fragments
 What is CpG and CpNpG
methylation?
 Methylcytosine
 Previous studies of unmethylated
DNA
 Focused on monocots
 Focus of Study
The Tomato Genome
 950 Mb of DNA
 25% in gene-rich
euchromatin @
distal ends of
chromosomes
 75% in genedeficient
heterochromatin
 One of the
lowest G+C
contents of any
plant species
 An estimated
23% of the
cytosine
residues are
methylated
Estimating the size of the
unmethylated portion of the tomato
genome based on EcoRI digested
fragments
 Detailed analysis of coding UGIs
 Undermethylated portion extends 676 bp
upstream and 766 bp downstream of coding
regions
 59% non-coding sequences, 12% transposons, and
1% organellar sequences
 Organellar sequences integrated into the
nuclear genome over the past 1 million years
 Accounts for majority of unmethylated genes in
the genome
 Estimated to constitute 61  15 Mb of DNA (~5%
of the entire genome)
 Indicates a significant portion of euchromatin is
methylated in the intergenic spacer regions
Implications for sequencing the
genome of tomato and other
solanaceous species
 310,000 sequence
reads estimated to
cover 95% of the
unmethylated
tomato gene space
 Solanaceous species
have same basic
chromosome # as
tomato (n=12)
 Similar chromosome
structure
 Similar gene content
 Assume methylation
patterns also similar
 Possible to apply
methylation filitration
sequencing to genomes
of other solanaceous
species
 Use order of tomato
sequence and synteny
maps to determine
derived order of UGI
genes
Development of a set of PCR-based
anchor markers encompassing the tomato
genome and evaluation of their
usefulness for genetics and breeding
experiments
 Genetic mapping of morphological traits
in tomato began in 1917
 Additional types of molecular markers
 Alternatives to RFLPs
 Cheaper, faster, less labor intensive
 Lack of PCR based map
 Map containing PCR-based markers would benefit
many studies
 Goals of this Study
PCR-based anchor
markers
 Consist of SSRs and
CAPs, based on
single-copy/coding
regions
 Encompass entire
genome, placed at
regular intervals,
anchored in linkage
map
 Priority given to
established
polymorphism
markers.
 Criteria:
 Detection of
polymorphism
 Visualization of
polymorphism
 Placement of
markers on map
 Additional SSR
markers
PCR Based Anchor Map of
Tomato
 76 SSRs placed on S. lycopersicum
x S. pennelli high density map
 76 CAP markers also mapped
 152 PCR-based anchor markers
 Uniformly distributed
 Encompass 95% of genome
 Locus specific
Applications
 Useful for mapping in other
interspecific populations
 Useful resource for:
 qualitative and quantitative trait
mapping
 Marker assisted seletion
 Germplasm identification
 Genetic diversity studies in tomato
Zooming In on a Quantitative Trait
for Tomato Yield Using Interspecific
Introgressions
 Previous QTL Projects
 Multiple Segregating vs Single Region
Segregating QTL
 Single region segregating QTL (ILs)
have higher genetic resolution
 Increased identification power for QTL
analysis
Exploring Natural Tomato
Biodiveristy
 Developed and examined a population
of 76 segmented introgression lines
 Utilized QTL database
 Examined total soluble content of
tomato fruit in “ketchup tomatoes”
measured in refractometer brix (B)
units
Characterizing the QTL
Brix9-2-5
 QTL improves B
w/out reducing total
yield
 Restricted to SNP
defined region of
484 bp of cell wall
invertase LIN5
 3 amino acid
differences, Asp366,
Val373, and Asp348,
are responsible for
QTL effects
 LIN5 exclusively
expressed in
conductive tissue
of flower
reproductive
tissues
 Supports role of
LIN5 as “sink
gene”
Characterizing the QTL
Brix9-2-5
 Maps to middle of
 Evaluated QTN
short arm of
SNP28378
chromosome arm  Responsible for
 But not present at
ASP348
this location in
substitution
any of the 5
 Role of ASP348
populations
and SNP28378
 All lines share 2
of 3 amino acids
Conclusions
 Example of the ability of a diverse
IL to provide detail information on
a QTL involved in increased sugar
yield in tomatoes