genetics genetic mapping, classical approaches to study gene function
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GENETICSgenetic mapping, classical approaches to study gene
function
Basic aims:
• uncovering gene functionunderstand mechanisms of morphogenesis, development, metabolism, physiology etc. in connection with coordinated gene expression
• breedingproduction of plants (organisms) with improved characteristics or their combination
Terminology
Gene• segment of genomic information that specifies a trait• basic unit of heridity in living organisms
• Genotype + environment + ? = phenotype
• Interactions between genes/proteins (epistasis – metabolic and signal pathways)
Allele – form of a gene
• dominant vs. recesive
• genesis of new alleles by mutations
Locus – location of a gene on a chromosome
• Genetic linkage – inheriting of certain genes (their alleles) jointly,
because they reside on the same chromosome
(gene distance cM = % of recombinant gametes)
• Genetic (likage) maps x physical maps
- varying likelyhood of recombination – cM (0-50 cM)
What sequences are with lower recombination probability?
Genetic (linkage) and physical maps differ
Genetic likage x Genetic likage x crossing-overcrossing-over during meiosis during meiosis
1.Cytologic event
Parentalchromosomes
Meiosis WithoutCrossing-over Crossing-over
Gametes
1
2
3
4
Not recombinant Recombinant
2. Genetic result
ParentalGenotype(heterozygousAa and Bb )
Locus A
Locus B
Meiosis
Gametes
Not recombinant ( same as parental genotype )
Recombinant ( new )
Genetic maps - genes- markers (= any detectable feature with known position on chromosoms)
(identifiable)
Geneticsclassical (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
Mutagenesis
• Classical:– chemical m. – EMS (ethane metyl sulfonate;
point mutations)
– physical m. – RTG, gama ... (usually short deletions)
– wide spektrum of affects (regulation, interaction)– even dominant mutations, resamble natural
mutations, difficult/expensive identification of mutated gene
Direct – looking for certain phenotype in mutant populationReverse – targeted mutagenesis/modification of selected gene
Mutagenesis
• Advanced:– insertional mutagenesis – T-DNA, transposons – random insertions
– allows simple determination of the site of insertion = mutation attached to a tag (inserted sequence)
– various stratagies for gene isolation
Gene isolation based on phenotypic changecaused by insertion
Insertional inactivation- T-DNA tagging- transposon tagging
Activation mutagenesisinserted sequence contain promoter or enhancerthat can activate expression of adjacent otherwise inactive gene
Promotor, enhancer-trap- T-DNA with reporter gene without promoter (with minimal promoter)
original gene
selection based on reporter gene expression
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
Identification of mutated gene
Point mutations, short deletions
1) Based on genetic map and segregation analysis +
chromosom walking, sequencing(long, expensive)
2) Using NGS (quick, moderate expensive)- even in unknown genomes!!!- mixed samples (back crosses)- comparisons of frequencies of similar oligomers
Nordström et al. Nature Biotech.2013
Identification of mutated gene(responsible for the mutation)
Identification of mutated geneInsertional mutagenesis:
• sequencing of flanking region (low template concentration for direct sequencing!)
TAIL PCR (Thermal Asymmetric InterLaced PCR)adaptor PCR plasmid rescueiPCR
TAIL PCR:
1. three PCR (optimized Ta) with specific primer SP1-3 + certain AP2. product sequencing
SP1-3: complementary to inserted DNAAP: arbitrary (degenerated) primer
- several universal types, high P of anealing near insertion
SP1 SP2 SP3AP AP
SP1 AP
SP2 AP
SP3 AP
E E
Adaptor PCR:
E E
SP1 SP2 SP3 SAP
AP
1. cleavage (restriction endon., E)2. ligation of adaptors3. 2-3 PCR (spec. adapt. primer + spec. primers complementary
to inserted DNA)4. product sequencing
1. cleavage (E)2. circularization (ligation)3. transformation E.coli (ori, R)4. multiplication in bacteria 5. sequencing
ori bla/nptIII
E E
ori bla/nptIII
E
Plasmid rescue:
Inverse PCR:
E E
E1. cleavage (E)2. circularization (ligation)3. PCR4. sequencing
plasmid
E
Collections of insertion mutants
- publicly available (Arabidopsis, rice, …)- insertions in different positions in genome – practically all
genes (inactivation – 5’ exons, minimal promoter, confirmation by expression analysis necessary!)
– mutant selection in silico, ordering seeds
Gene1 Gene2 Gene3
= sites of T-DNA insertions in individual lines (1-8)
1 2 3 4 5 6 7 8 …line number
WWW interphase
http://signal.salk.edu/cgi-bin/tdnaexpress
Direct genetics - selection of mutants by altered phenotype
agamous
shootmeristemless
Mutant screens – phenotype, conditions, treatments, …
The same phenotypic change can result from different mutations
„there 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 F1 – wt = different genes (complementation)
- mutant = allelic
Direct and reverse genetics in Arabidopsis
Identification of mutation site + Tilling – „searching“ in non-characterizedcollection of lines by PCR and reasociation
reverse
direct
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, …)
TILLING1. PCR of selected
sequence from DNA stocks isolated from mutant population
2. Reassociation with PCR fragment from wt plant
3. Cleavage of ss sites of heteroduplex + electrophoretic separation of end-labelled fragments
TILLING – strategy of screening
Based on genetic map
1. mapping – genetic linkage with genetic markers
(necessity of dense polymorphic markers!)
2. identification of the gene
- chromosom walking- sequencing (sequence comparisons)
Identification/mapping of unknown (mutated) genes(„with phenotype“)
by cosegregation analysis
Genetic likage x Genetic likage x crossing-overcrossing-over during meiosis during meiosis
1.Cytologic event
Parentalchromosomes
Meiosis WithoutCrossing-over Crossing-over
Gametes
1
2
3
4
Not recombinant Recombinant
2. Genetic result
ParentalGenotype(heterozygousAa and Bb )
Locus A
Locus B
Meiosis
Gametes
Not recombinant ( same as parental genotype )
Recombinant ( new )
Basic set of genetic markers in
Arabidopsis thaliana
2-3 in every chromosomal arm
Ab c
aB CC
Ab c
aB
Abc
aBC
Ab caB C
AbC
Abc
aBC c
aB
P1 (homozygote)
P2 (homozygote)
F1 (heterozygote)
gametes gametes
gametes
F2 – full linkage:AB:Ab:aB:ab
2:1:1:0
Ab
aB
Ab
Ab
aBaB
F2 – without linkage: AC:Ac:aC:ac = 9:3:3:1
AC
A aC
AC
Ac
AAcc C C
AC
AcA
cc Ca a a
CCCc ccCaaa aa
A, B – full linkage!A, C – free recombination
Cosegragation analysis in F2 generation
Segregation in F2 generation
gamety XY (0.5) Xy xY xy (0.5)
XY (0.5) XXYY
XY (0.25)
XXYy
XY (0.5)
XxYY
XY (0.5)
XxYy
XY (0.25)
Xy XXYy
XY (0.5)
XXyy
Xy
XxYy
XY
Xxyy
Xy (0.5)
xY XxYY
XY (0.5)
XxYy
XY
xxYY
xY
xxYy
xY (0.5)
xy (0.5) XxYy
XY (0.25)
Xxyy
Xy (0. 5)
xxYy
xY (0. 5)
xxyy
xy (0.25)
(P=XXyy x xxYY, F1 = XxYy – frequency of gametes depends on the linkage)
9:3:3:1 (XY:Xy:xY:xy) x 4,75:2:2:0,25
= different chromosoms (arms)no linkage week genetic linkage
Looking for strong linkage!
Types of genetic markers = trait with known or identifiable position in genetic map with polymorphism between parental genotypes (e.g. different ecotypes)
• Morphological (limited number)
• Molecular– DNA markers – detectable differences in DNA
sequence
– isozymes
Natural morphological variability of Arabidopsis ecotypes
Morphological markersGene symbol
Name Phenotype Location (chr. - cM)
an-1 angustifolia narrow leaves, crinkled siliques 1-55.2
ap1-1 apetala no petals 1-99.3
py pyrimidine requiring
white leaves, restored by pyrimidine 2-49.1
er-1 erecta compact inflorescence, blunt siliques 2-43.5
hy2-1 long hypocotyl elongated hypocotyl, slender 3-11.5
gl1-1 glabra no trichomes 3-46.2
bp-1 brevipedicellus short pedicels, siliques bent downwards, short plant
4-15.0
cer2-2 eceriferum bright green stems, siliques bent downwards, short plant
4-51.9
ms1-1 male sterile no siliques 5-2.5
tt3-1 transparent testa
yellow seeds, no anthocyanin 5-57.4
Molecular markers in Arabidopsis
DNA molecular markers(= usually an electrophoretic band)
• RFLP (Restriction fragment length polymorfism) + Southern
• RAPD (Random amplified polymorphism detection)
• AFLP (Amplified fragment length polymorphism)
• SSR (Simple sequence repeats)
• SNP (Single nucleotide polymorphism)
Cosegregation analysis with molecular markers• Crossing of different genotypes with high polymorphism (multiple differences in markers)!!!• Possibility of analysis of high number of markers at ones
• Which marker A,B,C,D is linked with locus R?
fenotyp r fenotyp R
r RFenotyp:
Bulked segregant analysis• Strong linkage – possibility to analyze in bulk
phenotype r phenotype R
Rr
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 subsequently only for those in genetic linkage with a trait
• RAPD (Random amplified polymorphism detection)• AFLP (Amplified fragment length polymorphism)
RFLP
RAPD
AFLP
Finding of two markers surrounding mutated gene „Chromosome walking“
Mutovaný gen X Libraries of big
genomic fragments
YACs, BACs = yeast (bacterial) arteficial chromosome, ~ 300 (100) kbp
cosmids ( fág, 50 kbp)
Looking for overlaps using hybridization
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)
Identification of genes by function (interaction)
Yeast two-hybrid screen for protein interactors