Transcript Diapositiva 1 - EPSO - European Plant Science Organisation

Maize genomic diversity and
productivity: is there a link?
Michele Morgante
Gent, September 8th, 2009
BREEDING MEETS GENES THROUGH GENOMICS:
LINKING GENE AND PHENOTYPE VARIATION
Bioinformatics
mRNA profiling
Protein profiling
Gene inactivation &
activation
Gene sequences (ESTs)
Genome sequence
Gene
Phenotype
Role of alleles
and haplotypes
Genetic
variation
DNA genotyping (SNPs)
Genetic diversity
Phenotypic
variation
Linkage maps
Association mapping
Physical/gene maps
Modified from Morgante and Salamini, 2003
Marker assisted
Selection (MAS)
+
Improved use of
germplasm
resources
Metabolic profiling
Phenotypic profiling
3-5 years gain
in new tobacco
crop
varieties to
market
RECONSTRUCTING DOMESTICATION:
MAIZE AND TEOSINTE
Selection at tb1 during domestication
Wang et al Nature 398 (1999) 236-239
Causative mutation lies 60-90 kb upstream of Tb1 (Clark et al. PNAS 2004)
RECONSTRUCTING MODERN BREEDING:
YELLOW MAIZE AND WHITE MAIZE
• A “natural” genetic modification
YELLOW MAIZE AND CAROTENOIDS
Geranylgeranyldiphosphate
Phytoene synthase (psy)
Phytoene
Phytoene desaturase (crtI)
-carotene desaturase
Lycopene
Increase
expression in (Y1)
endosperm
Lycopene -cyclase (lcy)
-carotene
-hydroxylase
Zeaxanthine
-carotene
lutein
Provitamin A
INTROGRESSION OF YELLOW ENDOSPERM
TRAIT THROUGH BACKCROSSING
X
Phytoene synthase (y1) gene variation in maize
YELLOW ENDOSPERM
WHITE ENDOSPERM
Palaisa et al. 2003 Plant Cell
EFFECTS OF INTROGRESSION
BREEDING
500000 DNA basepairs (and 10-20 genes) have been transferred into
modern maize together with the yellow allele
Palaisa, et al. (2004) Proc. Natl. Acad. Sci. USA 101, 9885-90
Copyright ©2004 by the National Academy of Sciences
GENOMICS AND
QUANTITATIVE VARIATION
• What type of quantitative genes?
– Micromutational view of quantitative variation
(R.A. Fisher, 1930)
• Infinite number of genes with v. small effects
– QTL view of quantitative variation
• Few genes of large effect plus other genes of small effect
• Breeding methods can be devised focused on
genotypes
– We can identify genes, look at the effects of
breeding on them, modify them, etc.
GENETIC DISSECTION OF TRAIT VARIATION:
A POSITIONAL CLONING APPROACH
Annotated genes
Mapped genetic
markers
Gene X
Prom.
5’UTR
Trait
CDS
Genetic Map
QTL mapping
Genome Sequence
or physical map
QTL mendelization
3’UTR
Clone 8
Clone 7
Clone 6
Clone 5
Clone 4
Clone 3
Clone 2
Clone 1
Candidate
genes
Diversity Map
positional cloning
POSITIONAL CLONING OF A FLOWERING TIME QTL
IN MAIZE (Zea mays): Vgt1
• vgt1 affects days to pollen shed (DPS)
and node number (ND)
• identified in cross between N28 (late)
and Gaspe’ flint (early)
• NIL with 6 cM flint introgression (C22-4)
• 4256 F2 progenies produced from N28 X
NIL cross
• 17 markers identified in a 6 cM interval
using ~40000 AFLP bands
• screening of 69 recombinant lines with
17 markers identified 2 AFLPs (13 and 14)
as flanking vgt1
• 16 recombinants left btw. AFLP13 and
AFLP14
STRUCTURE AND DIVERSITY OF
VGT1 REGION
AFLP13
Vgt1
Rap2.7
(Ap2-like)
0.1 cM
A
B
~2.6 kb
AFLP14
D
C
Mo17
Sequenced BAC clone
E
N28/B73
Sequenced BAC clone
P1,
late
P2,
early
C22-4
Sequenced cosmid clone
10 Kb
Salvi et al. 2007, PNAS
Vgt1 is a cis-regulatory mutation affecting
ZmRap2.7 expression
• Over-expressing maize ZmRAP2.7
– Late flowering observed
• Down-regulating maize ZmRAP2.7 by RNAi
– Early flowering observed
• Late allele of ZmRAP2.7 shows significantly
higher expression levels (2-3 fold) than early
allele
• Possible causative regulatory mutation: MITE
insertion
Salvi et al. 2007, PNAS
MANY QTL LOCI AFFECT A SINGLE
TRAIT
• 39 QTLs affect days to silking (flowering time)
• No large effect QTL
• Only one has been cloned: Vgt1
Buckler et al., Science, 2009
GENOMICS AND GENETIC
VARIATION
• Genetic variation: basis for natural
selection and artificial selection
• What is “natural” genetic variation?
– Differences in genomic DNA sequences
• Single nucleotide differences (SNPs)
• Differences in intergenic regions
• Differences in genic content
j: 0.47
THE HYPERVARIABLE MAIZE GENOME:
WIDESPREAD STRUCTURAL VARIATION
k:
0.17
O:
2.01
i:
4.15
n:
0.31
p:
0.99
30kb
Mo17
e:
2.83
SHARED SEQUENCE:
B73-specific:
f:
0.11
1
a:
b:
2.72 1.37
A
g:
2.18
3
2
B
U
C
m:
1.52
l:
0.70
c:
1.32
D
E
x:
0.22
4
Genes:
u:
1.32
t:
1.58
A) – T): geneA9002 – geneT9002
w:
0.54
s:
1.20
0.21
d:
2.54
F
0.43
NOPQ
M2
B73
r:
1.69
q:
0.61
z: 2.04
Mo17-specific:
v:
0.72
GH I J
y:
KLM
0.80
RS T 5
aa:
1.69
huck
ruda
giepum
opie
xilon
rire
non-LTR
1) Transposons:
jaws
zeon
shadowspawn
2) MITE:
prem
raider
dagaf
M2
0.63
3) Retrotransposons:
ji
Repetitive elements:
2.62
h:
1.39
locus9002 (bin 1.08; chromosome 1L)
Brunner et al., Plant Cell 2005
MAIZE GENOMIC DIVERSITY
• lack of colinearity is a general and frequent
phenomenon in maize
• more than 50% of the compared sequence
between B73 and Mo17 is non colinear
• large number of genes/gene fragments are
absent in one of the two inbred lines
– 34% of genic segments are not shared
• intergenic regions are largely non collinear
HYPER STRUCTURAL VARIATION (HSV)
THE PAN-GENOME CONCEPT
25%
50% 25%
core
Mo17
B73
genome
dispensable
genome
Morgante et al. Curr. Opin. Pl. Biol. 2007
THE MAIZE DISPENSABLE GENOME
25%
25%
Mo17
B73
1.67 Gbp of dispensable
genome
What is it made of?
How does it originate?
GENE TRANSDUPLICATION: FORMATION OF
NON-AUTONOMOUS HELITRON ELEMENTS
Gene A
Gene B
Gene C
A
B
C
TCT
CTAG
AT
AT
Morgante et al. Nature Genetics 2005
NON SHARED LTR-RETROTRANSPOSONS ARE
SIGNIFICANTLY YOUNGER THAN SHARED ONES
# of LTR-retrotransposons
30
25
20
15
10
5
.4
9
-4
4
3.
5
-3
.9
9
.4
9
-3
-2
2.
5
3
.9
9
.4
9
-2
2
1.
5
-1
.9
9
.4
9
1
-1
9
.9
-0
0.
5
0
-0
.4
9
0
Time of insertion in Myr
non-shared LTR-retrotransposons
shared LTR-retrotransposons
• consistent with recent movement of LTR-retrotransposons causing the
observed polymorphisms between the two inbred lines
Modified from Brunner et al., Plant Cell 2005
COMPOSITION AND ORIGIN OF THE
DISPENSABLE MAIZE GENOME
• DNA transposons
• Many carrying gene fragments
• LTR-retrotransposons
• Very recent movement of transposons of both
classes
THE MAIZE DISPENSABLE GENOME
25%
25%
Mo17
B73
dispensable
genome
Is it really dispensable?
Does it have a function?
DISPENSABLE GENOME:
NON SHARED GENE-CARRYING
TRANSPOSONS AND FUNCTION
GENE TRANSDUPLICATION: FORMATION OF
NON-AUTONOMOUS HELITRON ELEMENTS
Gene A
Gene B
Gene C
A
B
C
TCT
CTAG
mRNA
AT
AT
Morgante, Curr. Opin. Biotech., 2006
TRANSPOSONS AND GENE
TRANSDUPLICATION
•
•
•
•
Helitrons (maize)
Mu-like (rice, Arabidopsis)
CACTA (maize, soybean, sorghum)
Transposons as engines for new gene
formation?
– Most evidences point to pseudogenes rather than
genes
– Rare instances of complete genes observed
• Possible role in transcriptional regulation
through RNAi-like mechanisms to be
investigated
DISPENSABLE GENOME:
INTERGENIC REGION DIVERSITY
AND FUNCTION
SEQUENCE VARIATION AND
FUNCTIONAL POLYMORPHISMS
Coding variation
Cis-Regulatory variation
Humans, mouse
maize
Allele A
Allele A
Allele B
Allele C
Trans-acting
(regulator)
Cis-acting
(promoter, enhancer,
silencer, insulator)
INTERGENIC REGION DIVERSITY
AND CIS-REGULATORY VARIATION
Gene A
Retro B
Retro B
Retro A
Gene A
Retro C
Retro C
How is the different genomic environment
affecting expression pattern of gene A?
Transposons can provide:
• New promoter
• New enhancer / silencer / insulator
• Epigenetic changes / gene silencing /
chromatin modification
TRANSPOSON VARIATION AND REGULATORY VARIATION
How is the different genomic environment affecting expression pattern of gene A?
6
allelic ratio
5
4
3
2
1
1
B73
Mo17
The difference in allelic expression ratios between the Group 1 and
Group 2, 3, 4 genes is highly significant (P<0.001 Mann-Whitney U-test):
Group 1 genes show less regulatory variation than group 2, 3, 4
EXTENDING THE DISPENSABLE
GENOME CONCEPT
25%
25%
Mo17
B73
1.67 Gbp of dispensable
genome
What is it made of?
How does it originate?
FREQUENT LARGE STRUCTURAL
VARIANTS AMONG MAIZE INBREDS
Belo et al., TAG, in the press.
LARGE STRUCTURAL VARIANT
CONTAINING MULTIPLE GENES
Belo et al., TAG, in the press.
NEW GENETIC VARIATION
• Plant genomes: very dynamic and plastic
• Very recent movement of TEs of different
classes
• Our theory and methods have not yet fully
captured this
•
•
•
•
•
•
How much new vs. old variation
What types of variation
What mechanisms create it
At what rate
What is the functional role/significance
What is the relevance for breeding
CROP EVOLUTION AND THE CREATION OF
NEW GENETIC VARIATION
Doebley et al., Cell 2006
WILD
PROGENITOR
DOMESTICATION
DOMESTICATE
+ BREEDING
NGS technologies allow us to test this hypothesis
WHERE NEXT?
• High throughput identification of genes responsible
for traits of agronomic importance possible
– Biological resources, i.e. populations, and phenotyping
methods are key
• Need for complete resequencing of multiple maize
inbreds
– Reconstructing the dispensable portion of the genome may
be difficult
GENOME DIVERSITY AND
YIELD/HETEROSIS GENES
• Whole genome approaches
– Role of cis-regulatory variation in creating
(pseudo-)overdominance through expression
overdominance
– Role of large SVs in pseudo-overdominance
through a form of gene complementation
• Locus specific approaches
– Positional cloning of several yield/heterosis loci in
maize through QTL mendelization or association
mapping
– Use of newly created populations to improve trait
mapping and gene cloning efficiency
CONTRIBUTORS
Slobodanka Radovic
Federica Cattonaro
Daniele Trebbi
Gabriele Di Gaspero
Fabio Marroni
Sara Pinosio
Cristian Del Fabbro
Simone Scalabrin
Alberto Policriti
Alberto Stefan
Alberto Casagrande
• Kelly Palaisa
• Stefan Brunner
• Kevin Fengler
• Scott Tingey
• Antoni Rafalski
DuPont Crop Genetics
• Silvio Salvi
• Roberto Tuberosa
Universita’ di Bologna