Natural Variation in Arabidopsis ecotypes

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

Breeding for complex trait improvement

Elite Line #1 Elite Line #2

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