Transcript Marker-assisted backcross breeding

Plant Breeding Approach
Abiotic and biotic resistance breeding
(disease/pest resistance, drought and salt tolerance)
Backcross breeding
BC1F1
P1
x
Classic Breeding
F1
P 1 x P2
F2
Main Street
F3
F4-5
F6-7
F8-10
Preliminary
Molecular Parent selection
breeding Predictive breeding
MAS
for simple traits
True/false,
self testing
Parent selection and progeny testing
Marker-assisted selection (MAS)
Genome-wide selection (GWS)
Marker-assisted backcross breeding (MABB)
QTL-based and genome-wide predictive breeding
Cultivar
variety
Release
Final Yield Test
MAS
for quantitative traits
Genotyping by sequencing (GBS)
RAD-seq and RNA-seq
SNP discovery and validation
QTL mapping and association analysis
Candidate gene identified and clone
Marker Development for Molecular Breeding
Donor Screening
Population Development
Phenotypic Data
Genotypic Data
Data Analysis
QTL Mapping
Association Analysis
Marker Identification
Marker Implementation
Molecular Breeding
Molecular Plant Breeding Approach
SNP is a single nucleotide (A, T, C or
G) mutation, and can be discovered
from PCR, Next generation sequencing
(NGS) such as RNA-Seq, RAD-Seq, GBS.
Tool: BioEdit, DNASTAR, SAMtools,
SOAPsnp, or GATK
Marker Discovery
(SNP, SSR)
SSR is repeating sequences of 2-5 (most of them)
base pairs of DNA such as (AT)n, (CTC)n, (GAGT)n,
(CTCGA)n
Tool: SSRLocator, BatchPrimer3, MEGA6, BioEdit
Genetic diversity
Genetic
Diversity
Genetic Map
Construction
Association
Analysis
Linkage/QTL
Mapping
Association analysis
MAS/GWS
QTL mapping
Genetic Map
Marker Identification
(SNP, SSR Markers)
Add
effect
Dom
effect
LOD
R^2
(%)
CoP930721_82 -0.123
-0.122
4.463
6.1
CoP930934_82 -0.076
0.274
2.807
3.9
SNP
Marker-assisted
Selection
Genome-wide
Selection
Molecular Breeding
SNP markers
Fall 2016 HORT6033
10/31/2016
Marker-assisted Selection
Marker-assisted Selection (MAS): using marker(s) to select trait of interest.
Marker type: SSR and SNP
QTL mapping :
Linkage analysis
Association Analysis
Marker: trait
Marker Identification
Marker Implementation
Parent selection and progeny testing
Early generation selection for simple trait
Marker-assisted backcrossing
Late generation selection for complex trait
Gene-pyramiding
Cultivar identity/assessment of ‘purity’
Jian-Long Xu, Institute of Crop Sciences, CAAS. Molecular Marker-assisted Breeding in Rice
Population Size for MAS
Jian-Long Xu, Institute of Crop Sciences, CAAS. Molecular Marker-assisted Breeding in Rice
Equation to Estimate Sample Size Required for QTL Detection
Progeny (Pedigree) Testing
Marker Implementation
• QTL/SNPs selection
Select 2-3 QTLs and 1-2 SNPs/QTL for each donor
• Epistasis
Select both QTLs if epistasis exists
• Low Linkage Disequilibrium (LD) blocks
Select QTLs located at low LD block region
• Yield info
Select QTLs independently to yield QTLs or no yield drag
Marker-assisted Backcrossing (MAB)
MAB has several advantages over conventional backcrossing:
• Effective selection of target loci
• Minimize linkage drag
• Accelerated recovery of recurrent parent
1
2
3
4
1
2
3
4
1
2
3
4
Target
locus
TARGET LOCUS
SELECTION
FOREGROUND
SELECTION
RECOMBINANT
SELECTION
BACKGROUND
SELECTION
BACKGROUND SELECTION
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
Backcrossing strategy
• Adopt backcrossing strategy for incorporating
genes/QTLs into ‘mega varieties’
• Utilize DNA markers for backcrossing for greater
efficiency – marker assisted backcrossing (MAB)
Bert Collard & David Mackill, Plant Breeding, Genetics and Biotechnology (PBGB) Division, IRRI. MARKER-ASSISTED BREEDING FOR RICE
IMPROVEMENT
Conventional backcrossing
• High yielding
• Susceptible for 1
trait
P1
Elite cultivar
x
P2
Donor
Desirable trait
e.g. disease resistance
P1 x F1
• Called recurrent
parent (RP)
P1 x BC1
P1 x BC2
Discard ~50% BC1
Visually select BC1 progeny that resemble RP
Repeat process until BC6
P1 x BC3
P1 x BC4
P1 x BC5
P1 x BC6
BC6F2
Recurrent parent genome recovered
Additional backcrosses may be required due to linkage drag
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
MAB: 1ST LEVEL OF SELECTION –
FOREGROUND SELECTION
• Selection for target gene or QTL
• Useful for traits that are difficult
to evaluate
• Also useful for recessive genes
1
2
3
4
Target locus
TARGET LOCUS
SELECTION
FOREGROUND SELECTION
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
Concept of ‘linkage drag’
TARGET
LOCUS
c
Donor/F1
BC1
BC3
TARGET
LOCUS
BC10
RECURRENT PARENT
CHROMOSOME
DONOR
CHROMOSOME
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
LINKED DONOR
GENES
• Large amounts of donor chromosome remain even after
many backcrosses
• Undesirable due to other donor genes that negatively
affect agronomic performance
• Markers can be used to greatly minimize the amount
of donor chromosome….but how?
Conventional backcrossing
TARGET
GENE
F1
BC1
c
c
BC2
BC3
BC10
BC20
Marker-assisted backcrossing
TARGET
GENE
c
F1
BC1
Ribaut, J.-M. & Hoisington, D. 1998 Marker-assisted selection:
new tools and strategies. Trends Plant Sci. 3, 236-239.
BC2
Bert Collard & David Mackill. MARKER-ASSISTED
BREEDING FOR RICE IMPROVEMENT
MAB: 2ND LEVEL OF SELECTION RECOMBINANT SELECTION
• Use flanking markers to
select recombinants between
the target locus and flanking
marker
• Linkage drag is minimized
• Require large population
sizes
• depends on distance of flanking
markers from target locus)
1
2
3
4
RECOMBINANT
SELECTION
• Important when donor is a
traditional variety
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
Step 1 – select target locus
BC1
Step 2 – select recombinant on either side of target locus
OR
Step 3 – select target locus again
BC2
Step 4 – select for other recombinant on either side of target locus
*
*
OR
* Marker locus is fixed for recurrent parent (i.e. homozygous) so does not need to be selected for in BC2
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
MAB: 3RD LEVEL OF SELECTION BACKGROUND SELECTION
• Use unlinked markers to select
against donor
• Accelerates the recovery of
the recurrent parent genome
• Savings of 2, 3 or even 4
backcross generations may be
possible
1
2
3
4
BACKGROUND
SELECTION
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
Background selection
Theoretical proportion of
the recurrent parent
genome is given by the
formula:
2n+1 - 1
2n+1
Where n = number of backcrosses,
assuming large population sizes
Percentage of RP genome after backcrossing
Important concept: although the average percentage of
the recurrent parent is 75% for BC1, some individual
plants possess more or less RP than others
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
CONVENTIONAL BACKCROSSING
P1 x
MARKER-ASSISTED BACKCROSSING
P2
P1 x
P1 x F1
P1 x F1
BC1
BC1
VISUAL SELECTION OF BC1 PLANTS THAT
MOST CLOSELY RESEMBLE RECURRENT
PARENT
P2
USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS
THAT HAVE MOST RP MARKERS AND SMALLEST %
OF DONOR GENOME
BC2
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
BC2
Advanced backcross QTL analysis
• Combine QTL mapping
and breeding together
• ‘Advanced backcross
QTL analysis’ by Tanksley
& Nelson (1996).
• Use backcross mapping
populations
• QTL analysis in BC2 or BC3
stage
• Further develop promising
lines based on QTL analysis
for breeding
P1 x
P2
P1 x F1
P1 x BC1
BC2
QTL MAPPING
Breeding program
References:
Tanksley & Nelson (1996). Advanced backcross QTL analysis: a method for the simultaneous discovery and
transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theor. Appl. Genet. 92: 191-203.
Toojinda et al. (1998) Introgression of quantitative trait loci (QTLs) determining stripe rust resistance in barley: an
example of marker-assisted line development. Theor. Appl. Genet. 96: 123-131.
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
QTL region based on physical and genetic maps
QTL_A1 on Chr 1
QTL_A2 on Chr 2
QTL_A3 on Chr 3
qtlA1
Linkage Disequilibrium
QTL_A3 is located on the region with high LD, it will linkage drag
Foreground and Background Selection (FBS)
for QTL Introgression in Molecular Breeding
Elite
×
QTL info
Genetic map
Donor
QTL position
Elite
×
F1
Foreground selection in BC1F1 and BC2F1: ~30 plants,
Elite
×
BC1F1
genotyping these plants using QTL markers from donor,
and select 0.5M * N with M number of QTL, < 4. Greater
sample size is need if M >= 4
Genotypes
BC2F1
BC2F2
BC2F3
Breeding Value
calculator
Validation of QTLs through
Phenotyping and SNP
marker genotyping and of
>105% yield of commercial
control through yield
testing
Cultivar
release
Germplasm
release
Background
marker position
Genetic tool for
foreground (QTL) and
background selection
Background selection in BC2F2: ~350
plants, genotyping with ~300 genomewide SNPs plus QTL markers and select 5
BC2F2 plants with fixed 3 QTLs and 90-92%
recurrent genome in BC2F2 increased to
>95% recurrent genome in BC2F3
Validation of QTLs through
phenotyping and SNP marker
genotyping and of >95% elite
genome through yield testing
MAB increases popcorn yield
For example
http://www.dnalandmarks.ca/services-and-technologies/genotyping/services/marker-assisted-backcrossing/
Gene Pyramiding
• Widely used for combining multiple disease
resistance genes for specific races of a pathogen
• Pyramiding is extremely difficult to achieve using
conventional methods
• Consider: phenotyping a single plant for multiple forms
of seedling resistance – almost impossible
• Important to develop ‘durable’ disease resistance
against different races
F0
F1
F2
F3
P1
P2
H1,2
P3
P4
P5
H3,4
P6
Founder
Parents
H5,6
H 1,2,3,4 (Node)
Pedigree
H 1,2,3,4,5,6 (Root genotype)
H (1,2,3,4,5,6)(1,2,3,4,5,6)
Gene Pyramiding Scheme
Ideotype
Gene Pyramiding for three genes of
rice blast resistance in rice
example
Jian-Long Xu, Institute of Crop Sciences, CAAS. Molecular Marker-assisted Breeding in Rice
Early generation MAS
• MAS conducted at F2 or F3 stage
• Plants with desirable genes/QTLs are selected
and alleles can be ‘fixed’ in the homozygous
state
• plants with undesirable gene combinations can be
discarded
• Advantage for later stages of breeding
program because resources can be used to
focus on fewer lines
References:
Ribaut & Betran (1999). Single large-scale marker assisted selection (SLS-MAS). Mol Breeding 5: 21-24.
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
Susceptible
P1
x
P2
Resistant
F1
F2
large populations (e.g. 2000 plants)
MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes
MAS for 2 QTLs – 94% elimination of (15/16) unwanted genotypes
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
SINGLE-LARGE SCALE MARKERASSISTED SELECTION (SLS-MAS)
PEDIGREE METHOD
P1
x
P2
P1
x
P2
F1
F1
F2
Phenotypic
screening
F2
F3
F4
Plants spaceplanted in rows for
individual plant
selection
Families grown in
progeny rows for
selection.
F5
F6
Preliminary yield
trials. Select single
plants.
F7
Further yield
trials
F8 – F12
Multi-location testing, licensing, seed increase
and cultivar release
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING
FOR RICE IMPROVEMENT
F3
MAS
Only desirable F3
lines planted in
field
F4
Families grown in
progeny rows for
selection.
F5
Pedigree selection
based on local
needs
F6
F7
F8 – F12
Multi-location testing, licensing, seed increase
and cultivar release
Benefits: breeding program can be efficiently
scaled down to focus on fewer lines
Cultivar identity/assessment of ‘purity’
‘purity’ testing example
Reading
• Collard, B.C.Y., and D.J. Mackill. 2008. Marker-assisted selection:
an approach for precision plant breeding in the twenty-first
century. Philosophical Transactions of the Royal Society B
363:557-572.
• Moose, S.P., and R.H. Mumm. 2008. Molecular plant breeding
as the foundation for 21st century crop improvement. Plant
Physiology 147:969-977.
• Tester, M., and P. Langridge. 2010. Breeding technologies to
increase crop production in a changing world. Science 327:818822.
• http://comp.uark.edu/~ashi/Ainong_UARK/presentation/prese
ntation/SMV_resistance_breeding.pdf