Classical genetics

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Classical genetics
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Arabidopsis thaliana- ideal model plant
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Arabidopsis thaliana L.
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Life cycle 6 weeks Arabidopsis thaliana
1 week 3 weeks
4 weeks 6 weeks
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Transformation of Arabidopsis via Agrobacteriem
tumefaciens
Dipping of inflorescence (with flower buds) to
Agrobacterial suspension
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Selection of transformed seedlings
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Organisms inherit traits from their parents
Gene ? protein ? trait
J. G. Mendel
?
??
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Terminology

• Gene
• segment of genomic information that specifies a
  trait
• basic unit of heridity in living organisms
• Genotypeenvironment?phenotype
• Interactions between genes (epistasis metabolic
  and signal pathways)

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• Allele form of a gene
• dominant vs. recesive
• genesis of new alleles by mutagenesis
• Locus position of a gene on chromosome
• Genetic linkage - genes are inherited jointly,
• reside on the same chromosome
• (gene distance cM of crossing over)
• Genetic (likage) maps x physical maps

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Comparison of genetic (likage) and physical maps
physical (bp)
genetic (cM)
Arabidopsis chromosom IV
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Genetic likage x crossing-over during meiosis
1.Cytologic event
2. Genetic result
Parental chromosomes
Parental Genotype (heterozygous Aa and Bb )
Locus A
Locus B
Meiosis
Without Crossing-over
Meiosis
Crossing-over
Gametes
Gametes
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3
2
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Not recombinant ( same as parental genotype )
Recombinant ( new )
Not recombinant
Recombinant
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Segregation in F2 generation
(PXXyy x xxYY, F1 XxYy frequency of gametes
depends on the linkage)
9331 (XYXyxYxy) x 4,75220,25
no linkage week genetic
linkage
different chromosoms
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• Genetic maps
• genes
• - markers ( any detectable feature with known
  position on chromosoms)

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• Aims of genetics
• understanding of gene function
• breeding (improvements of crops)

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Genetics classical (direct) x reverse
Direct from a trait (phenotype) to
identification of corresponding gene Reverse
from a gene to phenotype (study of gene function
by mutagenesis, modulated expression, ) -
both approaches need mutants
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Mutagenesis

• Classical
• chemical m. EMS (ethane metyl sulfonate
  point mutations)
• physical m. RTG, gama ... (usually short
  deletions)
• Advanced
• insertional m. T-DNA, transposons (allows
  simple determination of the site of insertion
  mutation)
• activation m., mutants with reporter genes (dr.
  Perry)

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Insertional mutagenesis by T-DNA
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Direct and reverse genetics in Arabidopsis
Determination of the site of insertion
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Selection of plants mutated in certain gene
(reverse genetics, in detail by Dr. Perry)

• - Publicly available Arabidopsis mutant
  collection (insertions in different positions in
  genom practically all genes)
• mutant selection in silico, order seeds

Gen1 Gen2
Gen3
1 2 3 4
5 6 7 8
line number
sites of T-DNA insertions in individual lines
(1-8)
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WWW interphase
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TILLING detection of mutants with point
mutations in certain gene

• Targeting induced local lesions in genomes
• Principle chemical mutagenesis (EMS)
• PCR- and heteroduplex analysis-based screen
• Point mutations! (changed regulation,
  interactions, )

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TILLING

• PCR of selected sequence from DNA stocks isolated
  from mutant population
• Reassociation with PCR fragment from wt plant
• Cleavage of ss sites of heteroduplex
  electrophoretic separation of end-labelled
  fragments

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TILLING strategy of screening
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Direct genetics - selection of mutants by altered
phenotype
shootmeristemless
agamous
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T-DNA mutated Arabidopsis

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• T-DNA mutated Arabidopsis

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The same phenotype can have different genetic
causesthere are numerous ways how to build up
house incorrectly - allelic mutations
mutation in the same gene (x different g.) How to
distinguish (recesive mutation)?
Crossing of homozygous mutants wt different
genes (complementation) - mutant allelic
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Identification of genes by specific expression
pattern

• - promotorless construct with a reporter gene

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GFP GUS - glucuronidase
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Identification of mutated gene

• insertional mutagenesis - simple (TAIL Thermal
  Asymmetric InterLaced PCR, iPCR, plasmid rescue)
• based on genetic map and segregation analysis
• (chemical, physical mutagenesis)

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Insertional mutagenesis
TAIL PCR
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Identification of mutated gene
Based on genetic map and segregation
analysis mapping determination of position of
the mutation in genetic map by cosegregation with
genetic markers (polymorphic between parental
genotypes) Identification of mutated sequence
chromosom walking, sequenation, comparison with
WT
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Markers

• Morphological
• Molecular
• DNA markers (differences in DNA sequence)
• e.g. isozymes (limited use)

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Genetic mapping

• classically indicator lines pr. NW-100

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Procedure

• crossing NW100 with homozygous mutant (y/y)
• F1 (heterozygous)
• Self pollination F2-segregation
• Counting frequency of phenotypic classes XY, Xy,
  xY a xy (for every X)
• Genetic linkage to markers (localization to
  chromosom arm)

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Segregation in F2 generation
(PXXyy x xxYY, F1 XxYy frequency of gametes
depends on the linkage)
9331 (XYXyxYxy) x 4,75220,25
no linkage week genetic
linkage
different chromosoms
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Molecular markers

• Looking for a band always cosegregating with the
  mutant or WT phenotype

r R
phenotype r
phenotype R
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Bulked segregant analysis

• Strong linkage

r
R
phenotype r
phenotype R
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After identification of two genetic markers
surrounding the mutation Chromosome walking
Big insert libraries YACs, BACs,
cosmids Identification of overlaps based on
hybridization
gen X
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Examples of DNA molecular markers

• Known sequence and position in the genome
• RFLP (Restriction fragment length polymorfism)
  Southern hybridization
• Unknown sequence and position (randomly
  visualized sequences), sequence and position
  determined only for those in genetic linkage with
  a trait (mutation)
• RAPD (Random amplified polymorphism detection)
• AFLP (Amplified fragment length polymorphism)

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RFLP
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RAPD
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AFLP
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• Marker assisted selection (MAS)
• Molecular marker in strong genetic linkage with
  certain trait can be used for screening of
  hybrids instead of the phenotypic
  characterization
• Advantages
• Not influenced by environmental conditions
• Screens of seedlings
• Often simple and cheaper
• Possibility to distinguish between homo- and
  heterozygots (using certain markers)

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Identification of genes by function (interaction)
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Yeast two-hybrid screen for protein interactors