Genomics-assisted breeding for maize improvement Roberto Tuberosa Dept. of Agroenvironmental Sciences & Technology University of Bologna, Italy 11 th Asian Maize Conference, 8-12 November 2011, Nanning, China
Genomics-assisted breeding for
maize improvement
Roberto Tuberosa
Dept. of Agroenvironmental Sciences & Technology
University of Bologna, Italy
11th Asian Maize Conference, 8-12 November 2011, Nanning, China
Outline
Setting the stage
Implementing genomics-assisted breeding (GAB)
Chasing genes and QTLs
Biparental (linkage) mapping
Association mapping
Nested associated mapping
Breeding applications
MAS, MABC and MARS
Genomewide selection
Conclusions and perspectives
Genomics-assisted breeding in maize
Setting the stage
Corn yield in the USA
© Chappatte - www.globecartoon.com
…more bad news…
Stocks of staples are low
Reduced funding for plant breeding and training
for several decades
Increase and sharp fluctuations in food prices
Decline in arable land
Higher protein consumption in China and India
7 billion people now and 9 billion people by 2050
Higher energy and water prices
Decrease in water available for irrigation
Q T L s
Los Angeles Times April 13, 2008
It follows that a given QTL can have positive,
null, or negative effects depending on the drought
scenario. This complication has slowed considerably
the utilization of QTL data for breeding.
Collins et al. (2008). Plant Physiol. 147: 469-486.
QTL
discovery
QTL characterization
- QTL x E x M
- Validation in different backgrounds
- Isogenization
Genomics-assisted breeding
- Cost-effectiveness
- High-throughput profiling
QTL
cloning
Perfect marker
TILLING, EcoTILLING
genetic engineering
Genomics-assisted breeding of quantitative traits
Genomics-assisted breeding in maize
Implementing GAB – Chasing genes and QTLs
QTL mapping and cloning strategies
Biparental
linkage mapping
(RIL, DH, BC, IL)
genome-wide
(high LD panel)
Association mapping
(> 200 unrelated
accessions)
candidate gene
(low LD panel)
QTL coarse
mapping
Near isogenic
lines (NIL)
Candidate gene validation
Positional
cloning
Genetic
resolution
10-20 cM
1-100 kb
To clone or not to clone QTLs?
Salvi & Tuberosa (2005). Trends in Plant Science
QTL cloning as an essential step to:
• understand the functional basis of quantitatve traits
• unlock the allelic richness of germplasm by
direct haplotyping and sequencing of target loci
• identify the perfect marker for selection
• genetically engineer quantitative traits.
QTL cloning:
A very tough nut
to crack!
QTL
Mapping and cloning QTLs for
drought tolerance at UNIBO
• flowering time (escape)
• root architecture (avoidance)
N28E N28
N28E N28
Vegetative to generative transition 1 (Vgt1)
Salvi et al., 2007. Proc. Nat. Acad. Sci. 104: 11376
Gaspé Flint
AFLP13 AFLP14 Vgt1
M8
M12
.34 .08 .08 cM
Physical mapping and cloning of Vgt1
.38 Genetic
map
N28
C22-4
* * * * * * * * * * * * * * * * * * * * * *
144-bp transposon
(mite) insertion Rec Rec
ca. 2.7 kb * = SNP
= INDEL
BAC
clone
ca. 70 kb
.42
Vgt1
M8 M12
Salvi et al., 2007. PNAS 104: 11376-11381.
B73 Gaspé Flint F1
20-d
ay d
iffe
ren
ce
7 days:
Vgt1
13 days:
loci?
Flowering time in B73 and Gaspé Flint
Intr
ogre
ssio
n lin
es
1 2 3 4 5 6 7 8 9 10
Intr
og
ress
ion
lin
e
Maize chromosomes
IL (BC5 B73 x Gaspe’) graphycal genotype
Root phenotypic difference between B73
and Gaspé Flint
1
2
3
1 2
3
abscence of seminal
roots
Root analysis
in pots
Root analysis
in paper rolls
1 2 3 4 5 6 7 8 9 10
Seminal
roots-roll
- +
Chromosomes IL
lin
es
NA
(Salvi et al., unpublished)
1 2 3 4 5 6 7 8 9 10
Seminal
roots-roll
Seminal
roots-
pots - +
Chromosomes IL
lin
es
Crown
roots-
pots (vs. B73)
NA NA
NA
- + - +
(Salvi et al., unpublished)
1 2 3 4 5 6 7 8 9 10
Seminal
roots-roll
Seminal
roots-
pots - +
Chromosomes IL
lin
es
Crown
roots-
pots (vs. B73)
NA NA
qSR1, bin 1.02
qSR2, bin 3.05-7
qSR3, bin 7.01-2
qSR4, bin 8.04-5
aroll = -1.30 (-45%)
apots = -1.11 (-39%)
aroll = -0.45 (-16%)
apots = -0.31 (-14%)
aroll = -0.75 (-16%)
apots = -0.32 (-14%)
aroll = -0.85 (-30%)
apots = -1.10 (-40%)
NA
- + - +
(Salvi et al., unpublished)
+ / +
ABA - / -
ABA
Lower yield Higher yield
Root-ABA1 (bin 2.04) (Landi et al. 2007, J. Exp. Bot. 58: 319)
Root-yield-1.06 (bin 1.06) (Landi et al, 2010, J. Exp. Bot. 61: 3553)
Lower yield Higher yield
QTL mapping and cloning strategies
Biparental
linkage mapping
(RIL, DH, BC, IL)
genome-wide
(high LD panel)
Association mapping
(> 200 unrelated
accessions)
candidate gene
(low LD panel)
QTL coarse
mapping
Near isogenic
lines (NIL)
Candidate gene validation
Positional
cloning
Genetic
resolution
10-20 cM
1-100 kb
QTL mapping/cloning by GWA
(Genome-Wide Association)
Belò et al. (2008) MGG
• 8,590 SNPs
• 553 maize
inbreds
• Phenotyped
for embryo
oleic acid
content
Fad2 (Fatty acid desaturase 2)
Science (2008), 319: 330-333
QTL mapping and cloning via linkage mapping and
GWAS
Krill et al. (2010). PLoS ONE 5, (4) e9958.
QTLs and candidate genes for Aluminum tolerance
Three F2s and a panel of 282 inbreds
Lu et al. (2010). PNAS 107: 19585–19590.
•QTLs and candidate genes for ASI and drought tolerance
•Three RIL populations + one panel of 305 inbreds
Li et al. (2011). Plos ONE 9, (6) e24699.
•QTL for palmitic acid (unsaturated/saturated ratio and oil content)
•One RIL + one BC population + one panel of 155 inbreds
CSA News, October 2011, 4-11.
What is NAM?
NAM is most powerful genetic resource for dissection of the
genetic bases of quantitative traits for any species.
Courtesy of Mike McMullen
Linkage Mapping Association Mapping
Recent recombination
High power
Low resolution
Analysis of 2 alleles
Moderate marker density
Genome scan
Historic recombination
Low power
High resolution
Analysis of many alleles
High marker density
Candidate gene testing
Nested Association Mapping
Recent and ancient recombination
High power
High resolution
Analysis of many alleles
Moderate genetic marker density
High projected marker density
Courtesy of Mike McMullen
Nested Association Analysis
Yu et al. (2008) Genetics 178: 539
25 DL
× B73
F1s
SSD
NAM
1
2
200
B97
CM
L10
3
CM
L22
8
CM
L24
7
CM
L27
7
CM
L32
2
CM
L33
3
CM
L52
CM
L69
Hp
30
1
Il1
4H
Ki1
1
Ki3
Ky2
1
M162W
M3
7W
Mo
18
W
MS
71
NC
350
NC
358
Oh
43
Oh
7B
P3
9
Tx
30
3
Tzi8
Courtesy of Mike McMullen
THE MAIZE DIVERSITY PROJECT
Courtesy of Jim Holland
Maize Phenomics:
Massively Parallel Phenotyping of the
Nested Association Mapping Population
Genomics-assisted breeding in maize
Implementing GAB – MAS, MABC and MARS
Selection for mapped loci
MAS: MARKER-ASSISTED SELECTION
Plants are selected for one or more (up to 8-10) alleles
MABC: MARKER-ASSISTED BACKCROSS
One or more (up to 6-8) donor alleles are transferred to an elite line
MARS: MARKER-ASSISTED RECURRENT SELECTION
Selection for several (up to 20-30) mapped QTLs relies on index
(genetic) values computed for each individual based on its haplotype
at target QTLs.
Development of markers for MAS
• Markers should be tightly-linked (< 5 cM) to target loci and
preferably within the sequences of interest
• Markers must be validated in different genetic backgrounds
• Original mapping markers should be converted to markers
more suitable for high-throughput profiling at the single locus
• Markers should preferably be codominant
• Success stories: QPM and pro-vitamin A, disease resistance
Marker-assisted backcrossing (MABC)
a) Select donor alleles at markers flanking target gene
b) Select recurrent parent alleles at other linked markers (to reduce
linkage drag around target gene)
c) Select for recurrent parent alleles in rest of genome (optional)
1 2 3 4
Target locus
1 2 3 4
‘RECOMBINANT’
SELECTION
1 2 3 4
„BACKGROUND’
SELECTION
‘TARGET
GENE/QTL’
SELECTION from: Collard and Mackill, 2006
a b c
Ribaut and Ragot (2007). J. Exp. Bot. 58: 351-360.
Under severe WS (ca. 60-80%
yield reduction), the best five
MABC-derived hybrids
outyielded by 50% the controls.
Under intermediate WS (< 50%
yield reduction), no difference
was observed between MABC-
derived hybrids and the controls.
No yield penalty of the MABC-
hybrids under WW conditions.
Outcome of MABC depends on:
• Number of genes/QTLs to transfer
• Genetic distance between genes and markers
• Nature of markers used
• Number of genotypes selected at each generation
• Genetic background
When much of the variation is controlled by minor QTLs, MABC has limited
applicability because estimates of QTL effects are inconsistent and
pyramiding becomes increasingly difficult as the number of QTLs increases.
A more effective strategy is to deploy MARS to increase the frequency of
favorable marker alleles in the population.
MARS involves (i) defining a selection index for F2 or F2-derived progenies
with desirable alleles at target QTLs, (ii) recombining selfed progenies of the
selected individuals and (iii) repeating the procedure for a number of cycles.
Marker-assisted recurrent selection (MARS)
Although the private sector has reported significant gains through MARS in
maize (Johnson, 2004; Eathington, 2005; Crosbie et al., 2006), fewer efforts
have been undertaken in the public sector.
Moreau et al. (2004) reported no advantage of MARS over phenotypic
selection for a multitrait performance index, probably due to the general high
heritability of traits and the limited (ca. 50%) σ2P accounted for by QTLs.
One shortcoming of MARS is caused by the inconsistency of QTL effects as
the genetic background changes during subsequent cycles of selection, a
problem which can be partially solved with the “Map as you go” (MAYGO)
approach suggested by Podlich et al. (2004).
Marker-assisted recurrent selection (MARS)
Genomics-assisted breeding in maize
Implementing GAB – Genomic selection
• Requires low-cost, high-density molecular markers (LD level)
• Unlike in MARS, GS considers the effects of all markers together and
captures most of the additive variation
• Marker effects are first estimated based on a so-called
“training population” that needs to be sufficiently large (> 300)
• Breeding value is then predicted for each genotype in the
“testing population” using the estimated marker effects
Genomic selection
• GS focuses on the genetic improvement of quantitative traits rather than
on understanding their genetic basis
• Simulation studies have shown that across different numbers of QTLs
(20, 40 and 100) and levels of H, responses to GS were 18 to 43%
larger than MARS (Bernardo and Yu, 2007)
• GS more effective with complex traits, low H and haplotypes rather than
single markers
• GS and QTL discovery are not mutually exclusive
• Application of GS as a function of objectives, resources of breeding
programs and the genetic architecture of traits
• Yield per se: difficult to identify major QTLs, particularly in elite x elite
Genomic selection
• Current maize inbreds have very little exotic germplasm
• Prebreeding via recurrent selection is usually required
• 10 cycles of testcross phenotypic selection require 20 years vs. 4 for GS
• The outcome of long-term (5-10 cycles) GS is unknown
Genomic selection for introgression of exotic germplasm
Response to 15 cycles of GS for
introgression of exotic germplasm
Bernardo, 2009
Crop Sci., 49: 419
F2 is preferable to BC1 and BC2
6-7 cycles of GS appear to be sufficient
After 7th cycle, reestimate of marker-
based selection index
Drought-tolerant corn by MAB; marketed by Pioneer in 2011
Accelerated Yield Technology (AYT™)
2009, 19, 10
Genomics-assisted breeding in maize
Perspectives and
conclusions
DROPS
EU-funded
Euro 8.7 M
15 Partners
5 companies
Plant Accelerator, ACPFG, Adelaide, Australia
Existence of a breeding program
Breeders familiar with molecular procedures, potential and shortcomings
Capacity to run 2-3 generations/year and produce DH
Capacity to automate DNA extraction
Access to high-throughput genotyping
Maintain a healthy pipeline between gene/QTL discovery and MAS
Access to an informatics platform to handle samples and data
Accurate and relevant phenotyping
Critical factors for the success of GAB
• Crop modeling will increasingly allow us to:
• Dissect complex traits into simpler components
• Help resolving G x E x M
• Support MAB with a breeding-by-design approach
Future opportunities for GAB
• Comparative genomics and other “omics” data will accelerate the
identification of candidate genes
• “Omics” platforms should be used in a very focused way
• Sequencing and novel bionformatic tools will facilitate collecting and
exploiting “omics” data
• Resequencing of target loci in mini-core collections for allele mining and
haplotype definition
Tying it all together
• On a case-by-case basis, develop appropriate breeding
strategies for the improvement of multiple traits and/or complex
traits.
• Delivering new cultivars via GAB will require a close collaboration
among molecular geneticists, breeders, physiologists, pathologists,
agronomists and other relevant stakeholders.
• Only an appropriate multi-disciplinary effort engagement will allow
us to effectively harness the potential of GAB while advancing our
quest to dissect the genetic make-up of agronomic traits.
G. Taramino et al., Pioneer Dupont, USA
M. Ouzunova et al., KWS, Germany
Many thanks to:
• Marco Maccaferri
• Silvio Salvi
• Maria C. Sanguineti
• Pierangelo Landi
• Silvia Giuliani
• Simona Corneti
• Sandra Stefanelli
• Marta Graziani
Funds: European Union, Pioneer-DuPont, KWS
INTERDROUGHT-IV 6-9 September 2013
Burswood Entertainment Complex
Perth, Western Australia
Congress Chair: Roberto Tuberosa, Italy Program Committee Chair: Graeme Hammer, Australia Local Organizing Committee Chair: Mehmet Cakir, Australia
www.interdrought4.com
Questionnaire on marker-assisted breeding
(sent to 5 seed companies)
What % of financial resources will be devoted to MAB in next 5 years?
Company A: 10-15%
Company B: MAB will be exploited in all our corn breeding projects
As to the resources devoted to MAB, what % is devoted to:
- MAS for simple traits
- MARS for complex traits
- GS for complex traits
Selection for complex traits is increasing, as is selection for both
simple and complex traits within the same breeding project
to a large extent
to a low extent
moderate with increasing importance
Questionnaire on marker-assisted breeding
Is GS fulfilling the potential expected from published simulations?
To a large extent Moderately
To what extent has AM allowed you to dissect complex traits?
Moderately Moderately
What are the 3 main factors limiting a more widespread use of MAB?
1: Cost; 2: Reluctance to change well-established breeding programs
3: Standardization
1: Experience; 2: Logistics; 3: Standardization
To what extent has GBS changed your perspective on MAB?
Moderately Moderately