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Genetics of tassel and ear developmentin maize
Erik VollbrechtDepartment of Genetics, Development and Cell BiologyIowa State University
Shoot ArchitecturePrincipal elements: Branch (shoot)
axesBranching variables:• Mode of branching
– Frequency– Placement
• Branch angle• Continuous, intermittent or
transient growth– Long or short shoots
• Persistence of branch
Ulmusamericana
Pinusponderosa
Architecture is an end product
SAM Properties• Iterative meristem functions
– Pattern formation – Organogenesis– Self renewal
Basic meristem structure(example: knotted1 gene in maize)
• kn1 encodes a homeoboxtranscription factor expressed in all shoot meristems
The knotted1 (kn1) gene maintains SAM function
• loss of function kn1 mutants display shoot meristem failure
wt kn1-E1 mutants
wt kn1-E1 mutant
kn1 mRNA expression pattern
Jackson et al. 1994 Vollbrecht et al. 2000
SAM Properties• Iterative meristem functions
– Pattern formation – Organogenesis– Self renewal
Basic meristem structure
• Meristem characteristics (meristem identity)– Primordium type– Determinacy: capacity for continued iterative growth
Contextual meristem behavior
• Maize inflorescence architecture – Inflorescence development– Developmental, molecular genetics of three ramosa
mutants• ramosa1 and inflorescence natural diversity
– A branch number QTL in maize– Maize domestication
• Genes that integrate whole shoot and inflorescence architecture
Outline
Maize inflorescence development
Ear and tassel spike, meristems:
1˚ = Inflorescence (IM)
2˚ = Spikelet pair (SPM)
3˚ = Spikelet (SM)
4˚ = Floral (FM)
Tassel base, meristems:
2˚ = Branch (BM)
3˚- 4˚- 5˚ = SPM - SM - FM
1˚2˚3˚
2˚
tassel ear
ra1-R
ramosa1 mutations alter branch architecture
Strong ra1 mutant Weak ra1 mutant
ra1-RS
Normal inbred
ra1-A619
The ra1 gene imposes determinacy on 2˚ meristems
ra1-R mutant
A
Bar = 250 um
**
C
*
D
G
*
Normal inbred
B
E
*
F
* *
H
*
*
Mutant ramosa tassels: novel 2˚ meristem determinacies
BM
SPM
Inbred A619
ID-SPM
mixed
~ID-SPM
BM
SPM
ra1-R
Novel 2˚ meristem determinacy
0%
20%
40%
60%
80%
100%
1 2 3 4
SPMIDSPMBM/IDSPMBM
B73 ra1-R ra2-Rra1-RSstrong allele
weak allele
Normal strong allele
Tass
el le
ngth
Mutations in 3 different ramosa genes
Strong ra3 mutant (chromo 7, bin 4)
Strong ra2 mutant (chromosome 3)
ra2-R ra3-R
Weak ra1 mutant (chromo 7, bin 2)
ra1-RS
ra1-RS weak
Normal
ramosa single mutants in B73 - ears
ra1-RS weak
Normal B73
ra2-Rstrong
ra3-R strong
ra1-R strong
X
The ramosa genes
LOB ra2 LOB domain(Bortiri et al. 2006)
EAR C2H2 ra1 EPF zinc finger(Vollbrecht et al. 2005)
ra3 trehalose 6-phosphatephosphatase homolog(Satoh et al. 2006)= phosphatase box
ra1 and ra2 are expressed only in inflorescences.
ra2 -RLOB*
ra2 regulates ra1EAR C2H2
EAR C1H2
EAR C2H2
ra1-B73
ra1-R
ra1-RS
ra2 -B73LOB
ra2 -mum4LOB
ra2 -dmLOB( )
ra1
ra2
ra1Vollbrecht et al. 2005, Bortiri et al. 2006
ramosa1 expression domain marks a boundary region, not the meristem
ra2-R mutantra1-Rpoint mutant
B73 inbred
ramosa2 expression: in the anlage and base, of all 2˚ and 3˚ meristems
CB
A
Transition inflorescence meristem
Developing inflorescence
ra1-R mutant
D
Bortiri et al. 2006
ra3 expression: discrete domains below all inflorescence axillary meristems
Satoh et al. 2006
2˚ meristems 3˚ meristems
Boundary domains and 2˚ meristems in inflorescence architecture
Geneba1 = barren stalk1
ra2 = ramosa2ra1 = ramosa1ra3 = ramosa3
bd1 = branched silkless1
Gene product (txn factor)(bHLH)
(LOB domain)(C2H2-EPF zinc finger)
phosphatase(ERF-AP2 domain)
overlayba1
2˚
3˚ 3˚
1˚
ra2
ra1 ra3
bd1
Gallavotti et al. 2004, Bortiri et al. 2006, Vollbrecht et al. 2005, Satoh et al. 2006, Chuck et al. 2002
A large-effect QTL for branch number in IHO maize
X
QTL populationBranch number QTL maps v. near ra1
IHO maize B73
Berke and Rocheford 1999, Upadyayula et al. 2006a, b
ra1-B73ra1-B73
ra1-IHOra1-IHO
Introgression lines for IHO geneticsXIHO maize B73
ra1-IHOra1-B73
x B73
ra1-R > B73 (BC6)ra1-T232 > B73 (BC6)
ra1-IHO > B73 (BC1)
ra1-IHO > B73 (BC6)
ra1-IHOra1-B73
x IHO
ra1-B73 > IHO (BC7)
ra1-R > IHO (BC7)ra1-T232 > IHO (BC7)
ra1-B73 > IHO (BC1)
ra1-IHO does not complement ra1-R
B73 inbred line introgressions:
ra1-IHOra1-B73
ra1-IHOra1-R
ra1-B73ra1-R
ra1-Rra1-R
mutant
The ra1-IHO allele is a common haplotype
retro
0 6080
ra1
2900 36501500
…CACTA retro
retro ra1…CACTA retro
e.g., ra1-IHO = ra1-T232:
ra1-T232ra1-R
ra1-IHOra1-R
ra1-Rra1-R
B73 introgressions:
Map-based clone the ra1-IHO modifier
retro
0 60802900 36501500
…CACTA retro??? ???
ra1-IHO
ra1-recombra1-R
ra1-IHOra1-R
ra1-Rra1-R
X
Frequency ?
retro …CACTA retro
ra1-R
A ramosa pathway
• ra1 quantitatively regulates endogenous, mobile indeterminacy signal
• Signal strength is modulated by ra1 activity level:
• RA1 protein function (weak alleles)
• level of wild type ra1 transcript (ra2 mutants)
• timing of expression
of wild type transcript (ra1-IHO)
ra1
ra2LOB
ra3
T6PP
C2H2
ra1 Limited outgrowth of 2˚ meristems (short branches)
DNA gel blotprobe = maize ra1
Sor
ghum
T. fl
orid
anum
T. fl
orid
anum
T. d
acty
loid
esZ.
per
enni
s
Z. d
iplo
pere
nnis
Z. lu
xuria
ns
Z. m
ays
hueh
ue...
Z. m
ays
mex
ican
aZ.
may
s pa
rvig
lum
is
B73
Z. m
ays
may
sOther teosintes
Tripsacum spp
Sorghum spp
Maize
Sister teosintes
ra1 in closely related grasses
Evolution
ra1 in closely related grasses
Crop domesticationCrop improvement
Sor
ghum
T. fl
orid
anum
T. fl
orid
anum
T. d
acty
loid
esZ.
per
enni
s
Z. d
iplo
pere
nnis
Z. lu
xuria
ns
Z. m
ays
hueh
ue...
Z. m
ays
mex
ican
aZ.
may
s pa
rvig
lum
is
B73
Z. m
ays
may
s
Domestication reduces genetic diversity
Yamasaki et al. 2005
Tripsacum
Mz1 Mz2 Mz3 Mz1 Mz2 Mz3 Mz1 Mz2 Mz3
Tripsacum
Tripsacum
Known Neutral Gene 1
Test gene 2: neutral
Test gene 3: selected
HKA test compares divergence:diversity among genes
Nucleotide diversity is low at ramosa1
0
0.01
0.02
0.03
0.04
75.5
151
226
301
376
451
526
601
676
751
826
901
976
1051
1126
1201
1276
1351
1426
1501
1576
1651
1726
1801
1876
1951
2026
2101
2176
2251
2326
2401
2476
2551
2626
2701
2776
2851
2926
3001
ATG TGARA1
1.1 kb 1.3 kb0.7 kb
HKA test region
0
0.01
0.02
0.03
0.04
75.5
151
226
301
376
451
526
601
676
751
826
901
976
1051
1126
1201
1276
1351
1426
1501
1576
1651
1726
1801
1876
1951
2026
2101
2176
2251
2326
2401
2476
2551
2626
2701
2776
2851
2926
3001
ra1, 12 teosintesAverage gene, parviglumis
ra1, 30 maize inbredsAverage gene, inbreds
Nuc
leot
ide
dive
rsity
/bp,
π
Inbreds vs. Tripsacum: p < 0.0001
Questions about selection
In HKA tests that use Tripsacum as an outgroup, the ramosa1 gene carries a signature of selection (Vollbrecht et al. 2005).
• Where in the Tripsacum-maize lineage, did selection occur?
• What is the genomic extent of the selected region?
• What was the selected character?
Teosintes:
Z. m. parviglumis: 12 haplotypes
Z. m. mexicana: 4 haplotypes
Maize Inbreds:
3 haplotypes
Survey of Nucleotide Diversity
Landraces:Colombia: Pira Naranja
Magdalena 399Huila 305Boyaca 476CariacoMagdalena 469Chococeno Negrito
Peru: Puno 19Puno 26Huanuco 63Lambayeque 5Cajamarca 15Cuzco 63
Argentina: Arg 538Arg 2334
Brazil: White Soft Corn, LenhaMexico: Veracruz 85
ConicoCelayaOnavenoMichoacan 286PepitillaJalaZapalote GrandeBenz 543Conico NortenoNal-tel
Cuba: Cuba 94U.S : Cherokee Flour Corn
OnavenoCooks Early Yellow DentSallu-yahBear Island Chippewa
Canada: Gehu
HKA test results
p=0.16593n/a0.00578ra1Teosinte
p=0.006160.004720.00318ra1;coding
Landrace
p=0.000130.000760.00028ra1; noncoding
Landrace
p=0.000170.001720.00099ra1Landrace
Not sign.n/a0.03774glb1Landrace
Not sign.0.016640.01664bz2Landrace
Not sign.0.020050.01978adh1LandraceNeutralgenes
T. dactyloideswas used as the outgroup
Population Gene pi theta p-value
Crop Domestication
Crop Improvement
teosinte (extant)
Modern Maize (inbred lines)
Primitive Maize (~extant landraces)
You get what you select for but … what was it?
Flowering time
Flowering time effects, lg2-
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9
day 3.55 lg2-R: day 3.56
Flowering time effects, ra1-
0
2
4
6
8
10
12
14
16
18
20
1 2 3 4 5 6 7 8 9 10 11 12
day 5.2 ra1-R: day 6.5
p < 0.005
Delayed flowering in ramosa mutantsFlowering time in segregating,
introgression lines
0
1
2
3
4
5
6
7
8
1 2 3 4 5 6
Day
of
flo
weri
ng
NormalMutant
lg2-R ra1-R ra2-R ra2-R ra1-RS ra3-R
ra3-R, B73 ra2-R, B73B73 GeneticBackground
SegregatingMutation***
tru1 mutants: gradient of change in successive axillaries
upper lower
= region of staminate (male) spikelets
Summary
• The ramosa pathway regulates meristem determinacy; ra1 is downstream of ra2 and ra3.
• For regulation, ra1 timing and activity (expression level or protein function) are important.– Tassel effects:
• branch number, branching complexity, flowering time– Ear effects:
• Branch suppression, row formation (straightness) & kernel packing
• Investigating the molecular basis of a QTL allele• Nucleotide diversity scans indicate selection: ra1 may be
a domestication gene.
http://www.public.iastate.edu/~vollbrec/index.html
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Iowa State UniversityBrandi SigmonXiang YangWei LiJustin ScharesBrad Hall
Becky WeeksJackie FarrellCory KnootKay-Marie Lamar
CollaboratorsEd Buckler CornellTom Brutnell BTI/Cornell
NSF Plant Genome - MIPSarah Hake USDA/UCBTorbert Rocheford UIUCBob Schmidt UCSDDavid Jackson CSHLVolker Brendel ISUToby Kellogg UMSL
Acknowledgments
Mechanism of ra1 action: Yeast 2-hybrid protein-protein interaction experiments# clones # expts confirm Interactor (similarity) Function
17 4 ¦ Ferritin Iron storage and binding11 5 ¦ Transthyretin-like (TTL) BRI1 substrate; signaling molecule?8 3 ¦ knotted1 homeobox gene Transcription factor, DNA binding 5 3 ¦ zinc finger POZ domain protein Transcription factor, protein binding2 2 ¦ Putative NAC 2 protein Transcription factor, DNA binding 1 1 ¦ NAM protein-like Transcription factor, DNA binding 2 2 ¦ Auxin response factor Hormone related transcription factor4 3 Mal d 1-associated protein Ribosome: transcription, translation3 2 60S ribosomal protein L10 Ribosome: transcription, translation4 1 Dynein binding Cytoskeleton3 2 Motor protein Cytoskeleton2 1 DNA directed RNA polymerase activity Transcription, DNA binding2 2 guanine nucleotide-binding protein Signal transduction12 6 Putative aldolase Lipopolysaccharide biosynthesis2 2 Phosphoribosylformylglycinamidine synthase Purine nucleotide biosynthesis2 2 peptidase/hydrolase Protein metabolism5 2 heat shock protein Protein metabolism
1 - 2 1 Many others
RA1 RA2 RA3RA1 yes - - RA2 yes - - RA3 no no no
Direct tests:
Reduced nucleotide diversity: causes
• Demographic: whole genome affected– Bottlenecks– Population founder events
• Directional selection (selective sweep): specific regions affected (selected and hitchhiking loci)
• Reduced recombination: all nucleotides in affected region show equivalent reduction