Generating Mutants Reverse Genetics

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Generating MutantsReverse Genetics

• 1. Existing Reverse Genetic Resources
• TUSC
• MTM
• RescueMu
• UniformMu
• RNA silencing
• 2. Soon to Come Reverse Genetic Resources
• TILLING
• Putants, Problems and Pitfalls

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Trait Utility System for Corn (TUSC)

• A reverse genetics approach developed by Pioneer
  Hi-Bred (Dupont)
• Based on Mutator transposon mutagenesis
• PCR-based screen of DNAs from some 42,000
  individuals
• Available to the public at no charge (but
  licensing agreements must be made for each
  institution using this resource).

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Unique events (meiosis) are PCR positive in one
or few progeny
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P0.999
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Mu transpositions leaf cells contain S
parental germinal somatic
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Mu insertions 90 success rate true
nulls ??? (significantly lower)
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BARREN STALK1 MUTANT

• barren stalk1 mutant has defects in axillary
  meristem development (vegetative and
  reproductive)
• ba1 is required for axillary meristem
  initiation
• mapped on chromosome 3 L
• just one allele in mutant collection identified
  in 1928 (ba1-ref)

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LAX1 RICE MUTANT

• lax1 mutant in rice has a phenotype remarkably
  similar to ba1

• lax1 and ba1 mutants map in syntenic regions

Similarities between lax1 and ba1 mutants
phenotype, defect in inflorescence development,
map position
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2. Isolation of ZmLAX region in ba1-ref mutant
plants

• no differences in the coding sequence
• a 2.2 kb insertion is present at position -330
  in ba1-ref ZmLAX promoter

ZmLAX in ba1-ref plants
2.2 kb insertion
ATG
1.7 kb
- 306
- 2.2 kb
219 aa
3. Search for new ba1 alleles Screening of a
Mutator insertion pooled population (TUSC,
Pioneer) with gene specific primers 5
insertions were identified in proximal promoter,
5 UTR, coding sequence and 3 UTR
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PIONEER BA1 NEW ALLELES
ba1-ref
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PIONEER BA1 NEW ALLELES
Cosegregation analysis and the 3 new insertion
alleles indicate that ZmLAX is barren stalk1
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Generating MutantsReverse Genetics

• 1. Existing Reverse Genetic Resources
• TUSC
• MTM
• RescueMu
• UniformMu
• RNA silencing
• 2. Soon to Come Reverse Genetic Resources
• TILLING
• Putants, Problems and Pitfalls

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The MTM uses Mu-killer, a dominant inhibitor of
Mu, to reduce somatic sectors.
(Slotkin, Freeling and Lisch 2003)
Martienssen and Freeling
May et al. 2003
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Problem somatic transpositions of Mu may be
detected by PCR but not germinally transmitted.
hcf106Mu/ with active MuDR
hcf106Mu/hcf106Mu
/
hcf106Mu
somatic insertion
Hcf106
Light sectors are new, somatic Mu insertions in
Hcf106.
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Generating MutantsReverse Genetics

• 1. Existing Reverse Genetic Resources
• TUSC
• MTM
• RescueMu
• UniformMu
• RNA silencing
• 2. Soon to Come Reverse Genetic Resources
• TILLING
• Putants, Problems and Pitfalls

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Maize Gene Discovery Project (PI- Virginia
Walbot, Stanford University)
Lunde et al. 2003

• Has produced over 170,00O ESTs derived from a
  broad spectrum of organ types and developmental
  stages.
• Has cataloged over 31,000 mutants derived from
  Mutator-active lines.
• Has deposited over 178,000 sequence runs of
  genomic DNA flanking RescueMu insertion sites.
• Has created library plates of pooled rows and
  columns of RescueMu plasmids.
• Has generated cDNA microarrays for genome wide
  analyses of gene expression.

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RescueMu Tagging Grids

• Each tagging grid contains 48 rows and 48 columns
  of 2304 F1 progeny from RescueMu cross.

ROWS 1-48
X
COLUMNS 1-48

• Leaf samples pooled from a row yield an E. coli
  library that immortalizes the RescueMu insertion
  plasmids.
• Each grid generates 48 row 48 column gt 96 E.
  coli libraries.

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Grids Generate

• Selfed ears for phenotype evaluation
• Library plate of immortalized ROW and COLUMN
  RescueMu insertion events
• Grids with the Highest Forward Mutation Frequency
  enter the DNA sequencing queue.

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Grid Plants are Self-Pollinated

• Seeds are deposited at the Maize Coop at the
  University of Illinois and are freely available
• Seed requests are by ROW COLUMN address, based
  on DNA sequence information or PhenotypeDB

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Phenotype DB Finding Mutants on the Web

• Grid tagging plants are self-pollinated and
  evaluated for mutations at three life stages.
  Data are in a public database. Most mutants are
  caused by Mu elements.
• STAGE FAMILIES
• seed 35,000
• seedling 8,000
• adult 7,000

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DNA Sequencing Strategy

• Sequence all the rows of a grid to 95
  probability of identifying each heritable
  insertion at least once.
• Sequence 4 columns to cross-check that likely ROW
  germinal insertions are also present in the
  COLUMN libraries. Row Column samples are from
  different leaves.

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Sequencing Grid G

• 40 of row sequences are singlets and are
  likely somatic (non-heritable) insertions
• 60 are recovered multiple times and are
  likely germinal insertions. Cross-check with
  column sequences and progeny analysis confirms
  this.

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Likely Germinal Insertions

• Those identical plasmids found more than once per
  row or column during RescueMu sequencing
• Those identical plasmids found in both a row and
  a column by sequencing or by user PCR of a
  library plate

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RescueMu Genomic Survey Sequences (GSS)

• 65 match a maize or grass EST RescueMu
  targets RNA polymerase II transcription units
  (virtually no insertions into pol I and III
  genes)
• Almost no matches to retrotransposons
• Define maize genes when combined with ESTs

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1006126F03.y1 1006
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Community Uses of RescueMu

• Consult GenBank GSS to find maize genomic
  sequences. These are co-assembled with maize
  ESTs at ZmDB.
• Use PCR of 96-well LIBRARY PLATES containing the
  grid ROW COLUMN libraries to determine which
  plant has a particular insertion.
• Order plant seed for free from the Maize Coop at
  the University of Illinois.

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Generating MutantsReverse Genetics

• 1. Existing Reverse Genetic Resources
• TUSC
• MTM
• RescueMu
• UniformMu
• RNA silencing
• 2. Soon to Come Reverse Genetic Resources
• TILLING
• Putants, Problems and Pitfalls

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UniformMu
Mutator-induced mutants in a defined inbred
(W22)background.
Genomic sequence flanking Mu insertions is
obtained through MuTail PCR from MuDR-
backgrounds.
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UniformMu
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Search Mutants Search MuTAIL sequences Browse
Pedigrees
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Browse/search the seed mutant collection
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Browse MuTAIL libraries
Search MuTAIL database by BLAST
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Generating MutantsReverse Genetics

• 1. Existing Reverse Genetic Resources
• TUSC
• MTM
• RescueMu
• UniformMu
• RNA silencing
• 2. Soon to Come Reverse Genetic Resources
• TILLING
• Putants, Problems and Pitfalls

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RNA-induced Gene Silencing (RNA Interference)

• A transgenic approach to obtaining a mutant
  phenotype
• Express your targeted gene in an inverted repeat
  orientation (antisense-sense) under a strong
  constitutive promoter (Ubiquitin or 35S)
• Double stranded RNA induces a program of
  RNA-directed RNA degradation and RNA-directed DNA
  methylation of indogenous gene.

Wang and Waterhouse 2002 Waterhouse Helliwell
2003
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ZAP1 RNA Interference
Terminator
ADH Intron
3 ZAP1A
3 ZAP1A
35S
ZAP1 Silencing
Wild-type Tassel
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ZAP1A RNAi Transgenics
strong
intermediate
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transgenic
21-23 nuc siRNA
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Generating MutantsReverse Genetics

• 1. Existing Reverse Genetic Resources
• TUSC
• MTM
• RescueMu
• UniformMu
• RNA silencing
• 2. Soon to Come Reverse Genetic Resources
• TILLING
• Putants, Problems and Pitfalls

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TILLING Targeting Induced Limited Lesions IN
Genomes
Make mutant population
Maize Genetics Stock Center for distribution
EMS treat pollen
M2
c1 Sh1 Bz1
bz1
Bulk Seed
Grow-outs open to the general public
Minimum criteria 50 M2 seed with 60
germination
sh1
DNA
Forward screens for specific traits of interest
M3
C1 sh1 bz1
M1
Lyophilized leaf disks
Random intermating
M0
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DNA prepared and equal amounts of each sample
arrayed to microtiter wells, then these 8-fold
pooled
IR-labeled primers create double end-labeled
fragments
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Bands of complementary size are visible, one in
each image channel, corresponding to singly
end-labeled fragments cleaved by CELI at the
mutation site.
Mutant individuals identified from pools,
sequenced. Information and analysis returned to
User, along with Stock Center numbers for
ordering seed.
See Cliff Weil, Purdue
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Generating MutantsReverse Genetics

• 1. Existing Reverse Genetic Resources
• TUSC
• MTM
• RescueMu
• UniformMu
• RNA silencing
• 2. Soon to Come Reverse Genetic Resources
• TILLING
• Putants, Problems and Pitfalls

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Maize Mutants and Their Analysis
HAVE FUN!!!