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