1. Plant Breeding Approach
Abiotic and biotic resistance breeding
(disease/pest resistance, drought and salt tolerance)
Backcross breeding P1
BC1F1 x
Cultivar
variety
Release
Classic Breeding
P1 x P2 F1 F2 F3
F4-5
F6-7
F8-10
Main Street
Visual selection
Preliminary
Intermediate
Molecular
breeding
Parent selection
Predictive breeding
MAS
for simple traits
Final Yield Test
True/false,
self testing
MAS
for quantitative traits
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
Genotyping by sequencing (GBS)
RAD-seq and RNA-seq
SNP discovery and validation
QTL mapping and association analysis
Candidate gene identified and clone

2. Population Development Marker Implementation
Marker Development for Molecular Breeding
Population Development
Phenotypic Data
Marker Implementation
Donor Screening
Data Analysis
Association Analysis
QTL Mapping
Genotypic Data
Marker Identification
Molecular Breeding

3. 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
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
Marker Discovery
(SNP, SSR)
Genetic diversity
QTL mapping
Genetic Diversity
Genetic Map Construction
Association Analysis
Linkage/QTL Mapping
Genetic Map
Association analysis
MAS/GWS
Marker Identification (SNP, SSR Markers)
SNP
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
Marker-assisted Selection
Genome-wide
Selection
SNP markers
Molecular Breeding

4. Marker-assisted Selection
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’

5. Jian-Long Xu, Institute of Crop Sciences, CAAS
Jian-Long Xu, Institute of Crop Sciences, CAAS. Molecular Marker-assisted Breeding in Rice

6. 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

7. Progeny (Pedigree) Testing

8. 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
Ainong: please explain 1) how did you interpretation your results and 2) how did you select (or rank) the QTLs for marker assisted selection

9. 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
Target locus
RECOMBINANT SELECTION
BACKGROUND SELECTION
TARGET LOCUS SELECTION
FOREGROUND SELECTION
BACKGROUND SELECTION
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT

10. 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

11. Conventional backcrossingP1 x P2
Desirable trait
e.g. disease resistance
High yielding
Susceptible for 1 trait
Called recurrent parent (RP)
Elite cultivar
Donor
P1 x F1
P1 x BC1
Discard ~50% BC1
Visually select BC1 progeny that resemble RP
P1 x BC2
Repeat process until BC6
P1 x BC3
P1 x BC4
P1 x BC5
Recurrent parent genome recovered
Additional backcrosses may be required due to linkage drag
P1 x BC6
BC6F2
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT

12. 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

13. Concept of ‘linkage drag’
Large amounts of donor chromosome remain even after many backcrosses
Undesirable due to other donor genes that negatively affect agronomic performance
TARGET LOCUS
LINKED DONOR GENES c
TARGET LOCUS
Donor/F1
BC1
BC3
BC10
RECURRENT PARENT CHROMOSOME
DONOR CHROMOSOME
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT

14. Markers can be used to greatly minimize the amount of donor chromosome….but how?
Conventional backcrossing F1 c c
TARGET GENE
BC1
BC2
BC3
BC10
BC20
Marker-assisted backcrossing F1 c
TARGET GENE
Ribaut, J.-M. & Hoisington, D Marker-assisted selection: new tools and strategies. Trends Plant Sci. 3,
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT
BC1
BC2

15. 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)
Important when donor is a traditional variety
RECOMBINANT SELECTION 1 2 3 4
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT

16. * BC1 OR BC2 OR Step 1 – select target locus
Step 2 – select recombinant on either side of target locus OR OR
BC2
Step 4 – select for other recombinant on either side of target locus
Step 3 – select target locus again *
* 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

17. 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

18. 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

19. BC2 P1 x F1 P1 x P2 BC1 P1 x P2 P1 x F1 BC1 BC2
CONVENTIONAL BACKCROSSING
BC2
MARKER-ASSISTED BACKCROSSING
P1 x F1
P1 x P2
BC1
USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE MOST RP MARKERS AND SMALLEST % OF DONOR GENOME
P1 x P2
P1 x F1
BC1
VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT PARENT
BC2
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT

20. 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 x P2 P1
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:
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:
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT

21. QTL region based on physical and genetic maps
QTL_A1 on Chr 1
QTL_A2 on Chr 2
QTL_A3 on Chr 3
Linkage Disequilibrium
QTL_A3 is located on the region with high LD, it will linkage drag
qtlA1

22. Breeding Value calculator
Foreground and Background Selection (FBS) for QTL Introgression in Molecular Breeding
Germplasm
release
Elite ×
Donor F1
BC1F1
Foreground selection in BC1F1 and BC2F1: ~30 plants, 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
BC2F1
BC2F2
Background selection in BC2F2: ~350 plants, genotyping with ~300 genome-wide 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
Genetic tool for foreground (QTL) and background selection
Genetic map
QTL info
QTL position
Background marker position
Genotypes
BC2F3
Breeding Value calculator
Validation of QTLs through Phenotyping and SNP marker genotyping and of >105% yield of commercial control through yield testing
Validation of QTLs through phenotyping and SNP marker genotyping and of >95% elite genome through yield testing
Cultivar
release

23. MAB increases popcorn yield
For example 

24. 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

25. Gene Pyramiding Scheme
Founder
Parents F0 F1 F2 F3
H 1,2,3,4,5,6 (Root genotype)
Pedigree
H1,2
H3,4
H5,6
H 1,2,3,4 (Node)
H (1,2,3,4,5,6)(1,2,3,4,5,6)
Ideotype
Gene Pyramiding Scheme

26. 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

27. 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:
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT

28. large populations (e.g. 2000 plants)x P2
Susceptible
Resistant F1 F2
large populations (e.g 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

29. SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS)
PEDIGREE METHOD
P1 x P2 F1 F2 F3
MAS
SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS) F4
Families grown in progeny rows for selection.
Pedigree selection based on local needs F6 F7 F5
F8 – F12
Multi-location testing, licensing, seed increase and cultivar release
Only desirable F3 lines planted in field
P1 x P2 F1
Phenotypic screening F2
Plants space-planted in rows for individual plant selection F3
Families grown in progeny rows for selection. F4 F5
Preliminary yield trials. Select single plants. F6
Further yield trials F7
Multi-location testing, licensing, seed increase and cultivar release
F8 – F12
Benefits: breeding program can be efficiently scaled down to focus on fewer lines
Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT

30. Cultivar identity/assessment of ‘purity’
‘purity’ testing example

31. Reading
Collard, B.C.Y., and D.J. Mackill Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Philosophical Transactions of the Royal Society B 363:
Moose, S.P., and R.H. Mumm Molecular plant breeding as the foundation for 21st century crop improvement. Plant Physiology 147:
Tester, M., and P. Langridge Breeding technologies to increase crop production in a changing world. Science 327: