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

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

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

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’

Jian-Long Xu, Institute of Crop Sciences, CAAS 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 Ainong: please explain 1) how did you interpretation your results and 2) how did you select (or rank) the QTLs for marker assisted selection

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

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 P1 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

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’ 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

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. 1998 Marker-assisted selection: new tools and strategies. Trends Plant Sci. 3, 236-239. Bert Collard & David Mackill. MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT BC1 BC2

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

* 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

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

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

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: 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 Linkage Disequilibrium QTL_A3 is located on the region with high LD, it will linkage drag qtlA1

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

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

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

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

large populations (e.g. 2000 plants) x P2 Susceptible 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 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

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 327:818-822. http://comp.uark.edu/~ashi/Ainong_UARK/presentation/presentation/SMV_resistance_breeding.pdf