1. PT Sampoerna Agro Tbk Sampoerna Strategic Square North Tower, 28th Floor Jl. Jend. Sudirman Kav. 45 Jakarta, Indonesia,12930 Development of Marker Assisted Selection through association mapping in Oil Palm Javier Herrero, PhD

2. Background  THE FACT: Nowadays African oil palm (Elaeis guineensis Jacq.) is the major source of vegetable oil. The application of genomic knowledge and tools in oil palm breeding is highly suitable due to the crop’s long generation cycle and large space requirement for field testing.  OBJECTIVE: As part of Sampoerna Agro breeding program improvement. AIMS to improve and accelerate its oil palm breeding process Molecular breeding laboratory (Palembang) Biotechnology Department (Vitoria, Spain) Research collaboration

3. Background  THE CHOICE: CANDIDATE-GENE ASSOCIATION MAPPING DEFINITION: “Association mapping is a statistical method to analyze the effects of existing alleles of candidate genes based on linkage disequilibrium (LD) and allows the discovery of strong associations between specific allele marker-trait expression in a set of genotypes, instead of in a designed mapping population” (Breseghello and Sorrells 2005; Ingvarsson 2005; Zhu et al., 2008). It involves finding marker-trait associations

4. Phenotypic data Interesting traits Candidate genes (CG) ASSOCIATION Phenotyping PLANT MATERIAL: Derived from Dura x Pisifera selected parents. Samples are selected based on 8 interesting traits. Extensively characterized (10 years). Every trait consists of average taken from 17 progenies with 2 GT which are segregated as best and worst performance. Set of 250 Genotypes (GT)

5. Genotypic data Interesting traits Candidate genes (CG) PhenotypingGenotyping Identification of trait-specific CG: Two contrasting bulks PER trait 1 1 BSA-cDNA-AFLP Quantitative trait loci VS Annotated sequences mEgCIR0059 linked to a QTL for MF in Lg 16 with high homology to a Transcription factor (Auxin Response factor) 2 2 Co-location analysis 3 3 Known genes DATABASES PUBLICATIONS With relevant biological meaning and related to the trait ASSOCIATION Set of 250 Genotypes (GT) Direct homology search

6. Genotypic data Interesting traits Candidate genes (CG) PhenotypingGenotyping Identification of trait-specific CG: ASSOCIATION Set of 250 Genotypes (GT) Total = 188 potential candidate genes 1 1 2 2 3 3 BSA-cDNA-AFLPCo-location analysisKnown genes *Example CG database

7. Genotyping Interesting traits Candidate genes (CG) Phenotyping Genotyping Identification of CG (3 aproaches): 1.BSA-cDNA-AFLP. 2.Co-location analysis. 3.Known genes. ASSOCIATION Amplicon sequencing in Set of GTs (ION Torrent) Set of 250 Genotypes (GT) filter and separate output amplicon sequencing Identification of CG’s allele patterns in the GTs ASSOCIATION MAPPING Statistical analysis ASPAM software 1 1 2 2 3 3 Pool of PCR products

8. …… Amplicon processing 1. Identification of allelic diversity for each CG. 2. Assignation of allele composition to each GT. 1 1 2 2 37 bp 187 bp Via Clustal: 2 SNPs. Pattern 1: 99 GTs Pattern 2: 60 GTs Sequence SELECTION PATTERN – GT association Short sequences and sequencing errors are discarded. Example: One batch consisted of 52 candidate genes in 250 bar-coded genotypes sent for sequencing using the ION Torrent system. It separates per CG and GT via several steps of local BLAST CG GT *OUTPUT = 5 million sequences. *Desired output product. *Example of CG sequence analysis. Alignment file of repeated patterns.

9. Association Pat S1 S2 S3 S4 Code Cultivar $ BN BW CPO HT FN FW MF OWM 2 2 1 1 1 1111 403/14 8.5 15.6 3.8 47 1001 9 92.9 49.5 3 2 2 2 2 1112 403/16 10.9 14.5 3.4 58 1230 7.8 71.2 45.2 2 2 1 1 1 1113 405/5 11 15.2 5.6 49 2515.0 4.5 76.6 61 3 2 2 2 2 1113 405/5 11 15.2 5.6 49 2515.0 4.5 76.6 61 2 2 1 1 1 1122 405/33 11.4 12.2 3 45 792.0 8.6 68.9 53.4 …………… *Example for a particular CG “Pattern Code Trait File”. ASPAM generates SAS files for Pattern and SNP analyses. Association Mapping STSTISTICAL ANALYSES mixed-model approach Stich et al. (2008) Pattern/SNPs GT/CODETrait/Phenotype 1. Identification of significant associations. 2. Effects of specific allel combination on traits (So far, 89 CG analyzed). 3 3 PATTERN – GT – TRAIT association

10. Results Table b): Pattern analysis.Table a): Example for Results of SNP analysis. b): CG INFLUENCES traits BN, CPO and OWM with indicates significances (0,0063, 0,0024 and 0,0058, respectively) with 5 patterns. For CPO Allele 1 (present in 55 GTs) yields the highest value with 4,5 (Ton/he), which is significantly better than that of pattern 2. a): SNP 1 of a specific CG has a significant effect on BW and HT. Level 2 of S1 (present in 11 GTs) leads to a significant higher bunch weight and Height increment than Level 1. 127 Significant effects for 12 CG alleles on trait expression for 7 different characters were identified. (So far, 89 potential CGs analyzed OUT OF 188). 127 Significant effects for 12 CG alleles on trait expression for 7 different characters were identified. (So far, 89 potential CGs analyzed OUT OF 188).

11. SUMMARY Interesting traits Candidate genes (CG) Phenotyping Genotyping Identification of CG (3 aproaches): 1.BSA-cDNA-AFLP. 2.Co-location analysis. 3.Known genes. Alleles of CG / Allele composition of GT Effects of specific allel combination on traits ASSOCIATION Amplicon sequencing in Set of GTs (ION Torrent) Set of 250 Genotypes (GT) Data Analysis (Association Mapping) Data Analysis (Association Mapping) Phenotypic values of 8 interesting oil palm traits: BN, BW, FN, FW, HI, MF, OWM & CPO

12. What’s next? Interesting traits Candidate genes (CG) Phenotyping Genotyping ASSOCIATION Association Mapping MORE TRAITs!! Sh gene Virescens Drought Tolerance Fertilizer efficiency MORE TRAITs!! Sh gene Virescens Drought Tolerance Fertilizer efficiency MORE CGs!! Involved in the desired trait performance MORE CGs!! Involved in the desired trait performance Obtained CGs WITH SIGNIFICANT EFFECTS Design of allel-specific primers GENOTYPING & VALIDATION (in independent set of Genotypes) Selection of parents with useful alleles GERMOPLASM Increment (New varieties) GERMOPLASM Increment (New varieties) MAS Model Building (New crosses) MAS Model Building (New crosses) Plant material selection:

13. Thanks for your attention Terima kasih