Using natural variation to understand diversity
Correlation of phenotype with environment (selective pressure?)Correlation of phenotype with phenotype
T. Mitchell-OldsWUE
Average yearly rainfall in collection siteMaloof et al, 2001: Correlation of latitude with light response andIdentification of responsible polymorphism in 2 ecotypesEl-Lithy et al, 2004: Correlation of seed size with early
but not late development rates
Using Natural Variation to Dissect Molecular Mechanisms Underlying Diversity
What are the genes responsible for morphological differencesamong closely related plants (ie. size, flower number, fruit size)?
What are the genes responsible for variation in environmentalresponses (fitness, resistance to pathogens and disease, resistance to stress, response to light)?
What kind of changes occur in evolution to allow plants to adaptUnder selective pressure (regulatory vs coding region changes)?
How do multiple genes interact to determine plant phenotype?
Can we develop better strategies for crop improvement?
4 5 6 7
Height
# ofpeople
Continuous Trait
ComplexOrQuantitativeTrait(under controlof multipleLoci)
SimpleTrait(1-2 geneticLoci control)
Two kinds of traits can create intra-species diversity
# ofpeople
Discontinuous Trait
Symptoms of Muscular Dystrophy
Examples of Complex Traits• Most human diseases
– Heart disease– Susceptibility to cancer– Asthma– Diabetes– Lifespan
Traits of Agricultural Importance– Yield– Stress Resistance– Growth Rate– Nutrient efficiency– lifespan
Elite Line #1 Elite Line #2
10 QTL that contribute to trait10 alleles that contribute positively to the trait210 possible combinations of QTL alleles
Markers for each QTL assist breeders in creating desired lines
Useful/cool things that QTL are good for
• Marker assisted breeding• Defining interactions between loci• Identifying unknown genes involved in traits
– Undetectable by forward genetics because of gene interactions, weak effect, redundancy, or null allele in starting accession
• Defining genes responsible for trait variation among lines of interest
• Asking questions about evolution and adaptation
COL LERF1
F2
InbredLines(F9)
1 2 3 4 5 6 7
In recombinant inbred lines, chromosomes are homozygous chimeras of parental chromosomes
x9
100502512.56.253.1251.6.8.4.2 = 99.8% homozygous
Population of RI lines
123456789…….Chromosome 1
L L S S S S S S L L S S S S L L L S S L S S S S S S S S S S L S S S S L S L S S S S L S L S S
Population of RI lines
123456789…….Chromosome 1
9 8 2 1 3 4 2 1 7 8 9 1 1 3 6 7 8 1 2 9 1 2 2 2 3 1 4 4 4 5 9 1 1 3 2 9 1 8 2 2 3 3 9 9 9 1 2
RED YELLOW
9878…
2134…
RED YELLOW82189
94217
Sample QTL map for chromosome 1
LODScore
Position on Chromosome 1 (cM)
0
5
10
SeedWeight(ng)
LOD
AdditiveFor redallele0
5
10
-5
1
Limitations on mapping with RI lines
2. Must be variation within population, preferably with transgression
Parents Parents
# ofRI lines
Seed weight Seed weight
# ofRI lines
3. Limited by # of RI line4. Limited by # and density of markers5. Limited by # of breakpoints in chromosomes
More RI populations, high-throughput marker identification and Lines with higher number of sib-crosses before inbreeding areIn progress.
1. Must be genotyped with high density of markers
Some examples of cool and innovative QTL mapping
Sergeeva et al., 2004Glc6-P (glycolysis) <----->Glc1-P (starch and cellulose)
PGM
1) Good distribution in RI lines from Cvi x Ler2) Tissue specificity of PGM activity is variable
Ler Cvi
• Most of the QTL for intensity of staining in distinct regions overlapped with QTL for activity in total extracts, with similar directions.
HoweverAdditional QTL found for individual tissues, and primary QTL for total extract activity doesn’t overlap with cot or root activity
This study reveals the presence and location of global regulators and organ specific regulators of inportant enzymatic activity
Steve Briggs, July 2004 Arabidopsis Conference
• Used level of gene expression in seedling as mapping trait• Identified QTL that regulate gene expression or are
upstream of gene in regulatory pathway• Compare QTL’s from many different mapping experiments
to find genes that are regulated by similar QTL’s and therefore may be co-regulated and/or function together.
• This kind of approach can lead to the development of transcriptional networks, or, if done with protein level, functional networks.
Cloning genes responsible for QTLs
MendelizeFine MapCandidate gene identification, if possibleProof of gene identity by allele swapping
Mendelizing a QTL
Create near isogenic line (NIL) to isolate a locus from one parent in theBackground of another parent.
Col Ler NIL
1-10 15-25 15-18
F2 from a NIL x Col cross
3: 1-101: 15-18
Fine MappingF2 from a NIL x Col cross
3: 1-101: 15-18
Gene in QTL region can now beFine-mapped using molecular markers by conventional methods
Candidate Gene identification (optional) based on genome annotation and knowledge of the genes affecting trait
Identification without a candidate gene and Confirmation
Swap alleles of the genes between parents by:
1) Introducing allele from one parent into null allele in other parentalbackground
2) Introducing dominant allele into parent carrying two copies ofthe recessive allele
IF NO CANDIDATE GENES KNOWN, THE ABOVE METHODSUSE LARGE REGIONS (BACs) TO CONFIRM PRESENCEOF GENE, AND THEN IDENTIFY GENE BY USING PROGRESSIVELY SMALLER PIECES.
Examples of cloning genes associated with QTL
QTL for flowering time assigned by candidate gene approach To CRY2 (blue light receptor), which was proved to be responsible for variation in 2 ecotypes.
QTL for insect herbivory assigned by fine-mapping and candidateGene approach to glucosinolate processing enzyme.
3 Heading time genes identified by map-based cloning ONLY in rice and found to correspond to known regulators of flowering in Arabidopsis (FT and constans)
Natural Variation in Arabidopsis ecotypes
Correlation of traits with environment and with eachotherIdentification of loci controlling complex traits
marker assisted breedingIdentification of novel genes and pathways that could not be foundby forward genetic screens due to interaction, small affect or null alleleDefining the molecular nature of intra-species diversity