GENETICS
genetic mapping, classical
approaches to study gene
function
Basic aims:
• uncovering gene function
understand mechanisms of morphogenesis,
development, metabolism, physiology etc. in
connection with coordinated gene expression
• breeding
production 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
Genetic (linkage) and physical maps differ
- varying likelyhood of recombination – cM (0-50 cM)
What sequences are with lower recombination probability?
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
1
3
2
4
Not recombinant
Recombinant
Not recombinant
( same as
parental genotype )
Recombinant
( new )
Genetic maps
- genes
(identifiable)
- markers (= any detectable feature with known position on chromosoms)
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
Mutagenesis
Direct – looking for certain phenotype in mutant population
Reverse – targeted mutagenesis/modification of selected gene
• 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
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 change
original gene
caused by insertion
Insertional inactivation
- T-DNA tagging
- transposon tagging
Activation mutagenesis
inserted sequence contain promoter or enhancer
that can activate expression of adjacent otherwise inactive gene
Promotor, enhancer-trap
- T-DNA with reporter gene without promoter
(with minimal promoter)
selection based on
reporter gene expression
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
Identification of mutated gene
(responsible for the mutation)
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
Insertional mutagenesis:
• sequencing of flanking region
(low template concentration for direct sequencing!)
TAIL PCR (Thermal Asymmetric InterLaced PCR)
adaptor PCR
plasmid rescue
iPCR
TAIL PCR:
SP1
SP2 SP3
AP
SP1
AP
SP2
AP
AP
AP
SP3
AP
1. three PCR (optimized Ta) with specific primer SP1-3 + certain AP
2. product sequencing
SP1-3: complementary to inserted DNA
AP: arbitrary (degenerated) primer
- several universal types, high P of anealing near insertion
Adaptor PCR:
E
E
E
E
SP1
SP2 SP3
SAP
1. cleavage (restriction endon., E)
2. ligation of adaptors
3. 2-3 PCR (spec. adapt. primer
+ spec. primers complementary
to inserted DNA)
4. product sequencing
Plasmid rescue:
ori
bla/nptIII
E
E
ori
bla/nptIII
E
plasmid
1.
2.
3.
4.
5.
cleavage (E)
circularization (ligation)
transformation E.coli (ori, R)
multiplication in bacteria
sequencing
Inverse PCR:
E
E
E
1.
2.
3.
4.
cleavage (E)
circularization (ligation)
PCR
sequencing
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
1
Gene2
23
4
Gene3
56
7
= sites of T-DNA insertions in individual lines (1-8)
8 …line number
WWW interphase
http://signal.salk.edu/cgi-bin/tdnaexpress
Direct genetics - selection of
mutants by altered phenotype
shootmeristemless
agamous
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
reverse
direct
Identification of mutation site
+ Tilling – „searching“ in non-characterized
collection of lines by PCR and reasociation
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, …)
TILLING
1.
2.
3.
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 endlabelled fragments
TILLING – strategy of
screening
Identification/mapping of unknown
(mutated) genes(„with phenotype“)
by cosegregation analysis
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)
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
1
3
2
4
Not recombinant
Recombinant
Not recombinant
( same as
parental genotype )
Recombinant
( new )
Basic set of
genetic
markers in
Arabidopsis
thaliana
2-3 in every
chromosomal
arm
A, B – full linkage!
A, C – free recombination
AA
bb cc
F1
(heterozygote)
A
bc
P1
(homozygote)
gametes
Aa
bB Cc
a
BC
gametes
aa
B B CC
P2
(homozygote)
gametes
A
bC
F2 – full linkage:
AB:Ab:aB:ab
2:1:1:0
AA a a a A
b b BB B b
A
bc
a
BC
a
Bc
Cosegragation analysis in F2 generation
F2 – without linkage: AC:Ac:aC:ac = 9:3:3:1
AA
Cc
AA
cc
AA
CC
Aa
Cc
Aa
cc
Aa
CC
aa
Cc
aa
CC
aa
cc
Segregation in F2 generation
(P=XXyy x xxYY, F1 = XxYy – frequency of gametes depends on the linkage)
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)
9:3:3:1 (XY:Xy:xY:xy) x 4,75:2:2:0,25
no linkage
= different chromosoms (arms)
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 markers
Gene
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
R
fenotyp r
fenotyp R
Bulked segregant analysis
• Strong linkage – possibility to analyze in bulk
r
phenotype r
R
phenotype R
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
Mutovaný
gen X
walking“
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