Transcript Forward Genetics

Maize Mutants and Their Analysis
Generating Mutants;
Forward Genetics

1. Generating your own mutants
– EMS
– Gamma/X-rays
– Transposon Tagging

2. Public Collections of Mutants
– Databases of EMS-generated mutants
– Databases of (likely) transposon-induced mutants
– The collection at Maize Genetics Cooperation Stock Center
EMS Mutagenesis

For creating a broad spectrum of new mutants

For creating new alleles of an existing mutant

For identifying suppressors and enhancers of a
particular mutant phenotype
EMS Mutagenesis of Pollen

Paraffin oil pollen treatment with EMS-- M.G. Neuffer-”Mutagenesis” in The Maize Handbook (M. Freeling and V.
Walbot, eds) 1993

Treated pollen is applied to silks

F1 (M1) seed is heterozygous for EMS-generated mutations
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Plant M1 seed, self each plant, collect and store M2 seed
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Plant 20-30 seeds from each M2, observe mutants segregating

A population of 3,000-5,000 M2 families should yield multiple
mutants for every locus (successful treatments yield frequencies
exceeding 1 recessive mutant per locus per 1000 M2 families
analyzed).
EMS Mutagenesis of ramosa1
ra1 is a mutation that strongly affects tassel and ear architecture
Two main purposes for EMS mutagenesis of ra1:
1. To find intragenic mutations in the RA1 locus (new alleles)
2. To find new loci involved in the RA1 pathway
ENHANCER/SUPPRESSOR SCREENING ON A WEAK ra1 ALLELE (ra1-RS)
ra1 weak phenotype
ra1-RS allele is due to a 2bp insertion at the very beginning of the coding sequence and encodes
for a slightly shorter protein
ra1-RS
2bp insertion
0
9 aa
0
166 aa
Erik Vollbrecht (unpublished)
EMS MUTAGENESIS AND SCREENING
ra1 weak
EMS treated pollen
ra1 weak
M0
M1
M2 families
~ 1100 M2 families screened so far for enhanced/suppressed phenotypes
(not a complete genome coverage)
Identified 16 families segregating new mutations affecting branching:
10 show an enhanced branched ear phenotype but no obvious change in tassel phenotype
2 show an enhanced branched ear and tassel phenotype
3 show a suppressed branched tassel phenotype
1 shows an enhanced tassel phenotype, but no effect on ear
M2 FAMILIES SEGREGATING FOR ENHANCED EAR BRANCHING
Branched ear phenotypes
00F20-120
00F20-247
00F20-284
00F20-318
M2 FAMILIESSEGREGATING FOR ENHANCED EAR AND TASSEL BRANCHING
Enhanced branched tassel and ear phenotypes
00F20-139
•Are the mutations intragenic or extragenic?
•Can sequence the ra1 locus and look for new mutations
00F20-392
SEQUENCING OF THE RA1 LOCUS FROM NEW MUTANTS
ra1-RS
00F20-120
2bp insertion
RA 21
-2,9 kb
•
•
-1 kb
RA 1
RA 44
0
RA 50
RA 13
176 aa
Sequenced ra1 gene from DNA of segregating mutant plants in M2 families
Have sequenced ra1 for 12 out of the 16 families
Results
•Found one new allele of ra1!
00F 20-120 family has a point mutation in ra1 locus in the coding
sequence causing a missense mutation (a G-C transvertion) resulting
in an amino acid change from Leu/Phe at position 160)
The mutation resides in a known repression domain
The mutation seems to affect ear branching only; no significant
enhancement in tassel branching phenotype!
00F20-120
What was obtained from the EMS mutagenesis of ra1-RS?
Potentially, several new loci involved in the branching pathways have been identified; more
are likely to be identified with continued screening.
One new interesting allele of ra1.
•Now the hard work begins
1. Are they allelic to each other?
Complementation tests
2. Do the enhancers increase branching in standard maize inbreds?
Back cross to different inbred lines.
3. Are these suppressors/enhancers known loci affecting branching?
Mapping and QTL
4. What is the developmental defect in the enhanced families?
Careful phenotypic analysis by SEM of immature ear and tassel
Generating Mutants;
Forward Genetics

1. Generating your own mutants
– EMS
– Gamma/X-rays
– Transposon Tagging
Gamma/X-rays for Mosaic and Clonal Analysis

Means of determining whether a gene acts in a cell autonomous
or non-autonomous manner.

To study the relationship of cell lineages during development of
the plant.

An example from a study of the control of leaf epidermal cell
morphology.
Frank, MJ et al. 2003 Development 130:753-762
Hake and Sinha1994 in The Maize Handbook
PLANT
brk1
CLONAL SECTOR
+
brk1
gamm a
lw2
+
brk2
vp5
+
irradiation
lw2
brk2
vp5
gamm a
irradiation
brk3
Oy700
+
+
brk3
gamm a
irradiation
Oy700
LEAF
Generating Mutants;
Forward Genetics

1. Generating your own mutants
– EMS
– X-rays
– Directed Transposon Tagging
Directed Transposon Tagging
A classic and (still current) means of gene cloning in maize!
SPM, AC and Mutator all proven sources for transposon tagging.
Mutator has higher mutation frequency than AC or SPM, so often
easier to find Mutator-induced mutations, but high copy number
often complicates identifying and cloning tagged gene.
AC and SPM-tagged alleles are often easier to clone due to lower
copy number in genome.
Lets look at an example of transposon tagging with Mutator.
Directed Transposon Tagging
An example---cloning the male sterile silky1 (si1) gene using Mutator.
si1 y1/si1 y1 X Si1 Y1/Si1 Y1, Mu-active
(white seed/silky tassel) X (Yellow seed/normal tassel)
all Yellow seed (y1/Y1)
si1 y1/Si1 Y1 (99.99%) Normal Phenotype
si1 y1/si1-mum Y1 (rare mutant 1/10000)
X Si1 y1/Si1 y1
Yellow seed have tagged si1 allele (Si1 y1/si1-mum Y1)
Grow plants from yellow seed and backcross to si1 y1/si1 y1.
Plant out seeds, score for silky vs normal tassel phenotype.
Prepare DNA from leaves of each type.
•Perform co-segregation analysis to identify
transposon linked to your mutant phenotype.
Southern Blot Hybridized with Mu1 Probe
(normal tassel)
(silky tassel (si1/si1-mum))
Generating Mutants;
Forward Genetics

1. Generating your own mutants
– EMS
– Gamma/X-rays
– Transposon Tagging

2. Public Collections of Mutants
– Databases of EMS-generated mutants
– Databases of (likely) transposon-induced mutants
– The collection at MGSC
Public Collections of Mutants

Maize GDB (http://www.maizegdb.org/)
(http://www.maizegdb.org/phenotype.php)
Has a vast collection of mutants and associated alleles, linked
to references containing background information about
locus.

Maize Inflorescence Architecture Project
(http://www.maizegdb.org/ems-phenotype.php)
A new project primarily cataloging EMS-generated
inflorescence (ear and tassel) mutants but also documenting
other adult and seedling mutant phenotypes.
•Generated in defined inbred backgrounds (B73 and A619) and
B73/Mo17 hybrid. Shooting for 3000-5000 M2 families.
Public Collections of Mutants

Databases of (likely) transposon-induced
mutants

Maize Gene Discovery Project (RescueMu
Phenotype Data In MaizeGDB)
(http://www.maizegdb.org/rescuemu-phenotype.php) A database
of over 31,000 mutants from screens of RescueMu-containing/Mutator
lines. Includes seed, seedling, and various stages of adult phenotypes.
F2 seeds for any phenotype can be ordered from MGCSC.

Functional Genomics of Maize Endosperm
(http://www.endosperm.org/) A database of seed mutants from
Mutator-containing lines in a W22 inbred background. All mutants
are in a Mu-off state. Order seed through web address.

Photosynthetic Mutant Library
(PML)(http://pml.uoregon.edu)
– Phenotype data of Mu-induced, non-photosynthetic maize mutants.
Public Collections of Mutants

The collection at the Maize Genetics Cooperation
Stock Center(http://w3.aces.uiuc.edu/maize-coop/)

A fantastic catalog of well maintained stocks of maize mutants of
all types, some dating back to the early 1900s. The source for
reference alleles.
Linkage groups often available (eg. o2 gl1 v5 ra1 on 7S)


Repository for mutant stocks from many genome projects,
including EMS mutants from the Maize Inflorescence
Architecture Project and Maize Gene Discovery Project.

Seed requests can be made on line, and orders are filled within
a couple of days!!
Maize Mutants and Their Analysis