1. Identification of polymorphic microsatellite markers in oil palm By D.Ramajayam, P. Naveen Kumar, RK Mathur, G Ravichandran and G. Satyanarayana ICAR-Indian Institute of Oil Palm Research, Pedavegi-534450, West Godavari District, A.P., India Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR

2. Introduction African oil palm has the highest productivity amongst cultivated oleaginous crops with a global average yield of 3.5 tons of oil per ha (Potential yield is 11-18t/ha). Per hectare of crop land, oil palm plantations give 3-8 times more oil than any other oil crop. Alone is capable to fulfill the growing world demand for vegetable oils of 240 million tons by 2050 (Corley, 2009) Oil palm cultivation is one of the most profitable land use in the humid tropics (Sayer et al., 2012) In 2014, Palm oil was valued as a vegetable oil with the lowest production costs by the international commodities market, e.g. US$700 & US$850 per metric ton of oil palm and rapeseed oil, respectively (http://www.indexmundi.com) Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR

3. Commercial Assay kit for identification of three fruit farms in oil palm MPOB owns the intellectual property (IP) rights to the discovery through patents filed in Malaysia (PI 2013700413) and the US (61,847,853,USA) Rajinder Singh et al., 2014 Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR

4. Oil yield of tenera is up to 30% higher than dura Determination of exact fruit form is possible only after 2-3 years of field planting Dura contamination is possible in commercial seed garden which has to be tolerated at least for 25 years Oil palm breeder has to maintain unwanted fruit farms until the bunches are produced to distinguish them in the following crossing programmes – Production and evaluation of maternal lines (dura) In the tenera selfed or tenere x tenera crosses – Production and evaluation of paternal lines (pisifera) Teneras are either selfed, crossed (tenere x tenera) or crossed to pisifera (tenera x pisifera) Introduction Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR

5. World production of oil palm planting materials [DxP, Clones, Bi-/Semi-clones and Interspecific hybrids] World production of oil palm planting materials Type of planting materialsMillion Planting materials/year DxP315.0 Clones3.5 ( ~ 1%) Bi-clones/Semi-clones seeds1.0 Interspecific hybrids2.5 Grand Total321.5 Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR Clonal oil palm planting materials is expected to grow to 5 million by 2010 from 11 commercial tissue culture laboratories but the requirement is 40 million ramets by 2017. Kushairi et al (2010)

6. Mutert, E and Fairhurst, TH (1999) Oil palm clones: Productivity enhancement for the future, Better Crops International, 13(1): 45-47. Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR DxP without dura contamination

7. Objective To identify polymorphic microsatellite markers in oil palm with a special reference to different fruit forms Why microsatellites? – high distribution of loci within the genome – High PIC and reproducibility – Co-dominant and amenable for PCR Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR

8. Methodology Genomic DNA was extracted from spear leaves using kit (Mol Bio Himedia, HipurA TM Plant Genomic DNA Miniprep Purification Kit) DNA quantification and quality was ascertained by Nano Drop ND1000 spectrophotometer (NanoDrop Technologies Inc.) and gel electrophoresis on 0.8% agarose gel The PCR reaction was done in 25µl mixture using Ampli Taq Gold (R) 360 Master Mix (Applied Biosystems) The PCR was performed in the thermal cycler (Eppendorf Master Cycler Nexux Gradient) with initial denaturation for 5 min. at 95 °C; 35 cycles denaturation of 30s at 95 °C, annealing for 30 s at 50-55 °C and extending for 60s at 72 °C followed by final extension for 10 min at 72 °C and keep it α at 4 °C Gel documentation using Biorad Polymorphic DNA fragments were scored as present/absent in dura, tenera and pisifera samples Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR

9. Chromos ome No SSR Locus sequence (F)sequence (R)bp (F) bp (R)Ta Amplifi cation 1mEgCIR3639ACGTTTTGGCAACTCTCACTCCCCTCTTTGACAT17 52278 1mEgCIR3750GATGTTGCCGCTGTTTG CATCCCATTTCCCTCTT 17 52149 1mEgCIR0782CGTTCATCCCACCACCTTTCGCTGCGAGGCCACTGATAC201956187 1mEgCIR0905CACCACATGAAGCAAGCAGT CCTACCACAACCCCAGTCTC 20 52231 2mEgCIR2440TTCCAGAAGCTAAACGAATGACGCGGACAGTGCGAAGAGAGT222052182 2mEgCIR3732ATTTTATTTGGCTTGGTATAACTTTTCTATCTAATTCTTGAAGAT202552194 2mEgCIR3363CTTGACAATACCCTGAGTAGTAGGCTGTGCCTATCGGACTT231852195 2mEgCIR1773ATGACCTAAAAATAAAATCTCATACAGATCATGCTTGCTCACA232052322 2mEgCIR2422GCCCTCCCTCAACTCAAAAAATGGTGTCTGGGACTCTGAGTA202252248 3mEgCIR0783GAATGTGGCTGTAAATGCTGAGTGAAGCCGCATGGACAACTCTAGTAA24 58296 3mEgCIR3869CCAATGCAGGGGACATTGAAGCCAGTGGAAAGATAGT172052178 3mEgCIR3543GTTCCCTGACCATCTTTGAGGTCGGCGATTGATTAGATTC20 52232 3mEgCIR3358CCAAGGAACAACATAGAGTTCCCATCCTATTAGAC171852208 4mEgCIR3260AGGGCAAGTCATGTTTCTATAAGGGCGAGGTATT17 52221 4mEgCIR3301GCACTTGGTGGTTATGAAGCTGCTGATGGATATC17 52140 4mEgCIR3544AGCAGGGCAAGAGCAATACTTTCAGCAGCAGGAAACATC201952188 4mEgCIR0425AGCAAGAGCAAGAGCAGAACTCTTGGGGGCTTCGCTATC211858232 4mEgCIR3698AAGCCACCAGGATCATCGTCATTGCCACCTCTAACT171952182 Oil palm SSR primers isolated by CIRAD (http://tropgenedb.cirad.fr/oilpalm/publications.html) were used.http://tropgenedb.cirad.fr/oilpalm/publications.html (Billotte et al. 2015) Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR Methodology (Conti..)

10. Chromos ome No SSR Locus sequence (F)sequence (R)bp (F) bp (R)Ta Amplifi cation 5mEgCIR2423TCCAAGTAGCAAATGATGACTGCCCTGAAACCCTTGA201752342 5mEgCIR3535AGTTTGAGCTAGTGGTGTCACAGGGTTCTTTGTCTACT19 52167 6mEgCIR3439ACTTGTAAACCCTCTTCTCAGTTTCATTACTTGGCTTCTG20 52247 6mEgCIR3716GCAGACATGGCAGCAAAAAGGGGGATGTTCCTGGATATCA20 52200 7mEgCIR3886TTCTAGGGTCTATCAAAGTCATAAGAGCCACCACCACCATCTACT252052187 7mEgCIR2387TTGGTGAGCCATTTGCTACACCTCCTTCCACCCCTCTACT20 52243 8mEgCIR2575GGGACTTCGCAAACTGTAGCACGGTGGCGTATGGTGGATT211952269 8mEgCIR0408TTGCGGCCCATCGTAATCTCCCTGCAGTGTCCCTCTTT182058193 8mEgCIR3282GTAACAGCATCCACACTAACGCAGGACAGGAGTAATGAGT20 52245 9mEgCIR3788TTGTATGACCAAAGACAGCAGCGCAACATCAGACTA191752173 9mEgCIR1713GCTGAAGATGAAATTGATGTATTCAGGTCCACTTTCATTTA212052252 9mEgCIR0257GCAGCTAGTCACCTGAACGACGAGACTGGAAAGATG18 52286 10mEgCIR3672AAAGCCATTCCAGACTACCTCATAGCCTTTGTTGTGT181952159 10mEgCIR2427GAAGGGGCATTGGATTTTACCTATTACAGCGAGAGTG172052116 10mEgCIR0779AATGCAGACCAAGCTAATCATATACGTTCAGGTGATGGTGACTCAGATAG25 52238 11mEgCIR2380TTGGGATGCAAAATTCAACAGTAACCCATTGAGACTA172052177 11mEgCIR3402GGGCTTTCATTTTCCACTATGCTCAACCTCATCCACAC201852241 13mEgCIR3555CATCAGAGCCTTCAAACTACAGCCTGAATTGCCTCTC201752136 Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR Methodology (Conti..)

11. Chromos ome No SSR Locus sequence (F)sequence (R)bp (F) bp (R)Ta Amplifi cation 13mEgCIR3213GCTCTTTGTATTTCCTGGTTCAGCAGCAAACCCTACTAACT21205297 14mEgCIR3890GTGCAGATGCAGATTATATGCCTTTAGAATTGCCGTATC201952154 14mEgCIR2407TGCCGAGGTGCAAGAAGATTACTATGCCACCCTTCTGAAGTA202252187 14mEgCIR3711GTCTCATGTGGCTACCTCTCAGGCTCCCTGCTTTTAAGT201752175 14mEgCIR3622GCCAGTTAGGAATACAAGTCACGCATTTTTCTTG17 52142 14mEgCIR0555TACCATCACTGACCAATAACGTCTTTCTTGCTAACTACAC20 52226 15mEgCIR3641TGGGAAGGTGCTCTCATGGCTCCACATAATTTCTAC171952184 15mEgCIR3739CAGCCAGTAAGAGCCAAGTCCTCCAACATTGAGGAGACTAG202152207 15mEgCIR3400CAATTCCAGCGTCACTATAGAGTGGCAGTGGAAAAACAGT20 52148 15mEgCIR3362CCCATCATCTGCTCAGGATAGACACCCTCTCCTCTTGGGAAGA232052151 15mEgCIR3587TTGTTTCGTGCATGTGTAGGAAGAAGGCTGACAT17 52217 16mEgCIR2813GCTTTGTTGCAGTTTGACTAGTTTAGGATGTTGCGTGAT201952210 16mEgCIR0446CCCCTTCGAATCCACTATCAAATCCGACAAATCAAC18 52202 16mEgCIR0894TGCTTCTTGTCCTTGATACACCACGTCTACGAAATGATAA20 52186 Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR Methodology (Conti..)

12. P’ D’ D D D D P P P P T T EgSSR18 P’ D’ D D D D P P P P T T EgSSR19 EgSSR22 Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR Results

13. EgSSR29 EgSSR31 EgSSR53 EgSSR54 P’ D’ D D D D P P P P T T Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR Results (Conti..)

14. P’ D’ D D D D P P P P T T EgSSR3 P’ D’ D D D D P P P P T T EgSSR5 Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR Results (Conti..)

15. EgSSR67 EgSSR68 P’ D’ D D D D P P P P T T Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR Results (Conti..)

16. Chromos ome No SSR Locus sequence (F)sequence (R)bp (F) bp (R)Ta Amplifi cation 1mEgCIR3750GATGTTGCCGCTGTTTG CATCCCATTTCCCTCTT 17 52149 1mEgCIR0905CACCACATGAAGCAAGCAGT CCTACCACAACCCCAGTCTC 20 52231 4mEgCIR3260AGGGCAAGTCATGTTTCTATAAGGGCGAGGTATT17 52221 4mEgCIR3301GCACTTGGTGGTTATGAAGCTGCTGATGGATATC17 52140 4mEgCIR3698AAGCCACCAGGATCATCGTCATTGCCACCTCTAACT171952182 6mEgCIR3439ACTTGTAAACCCTCTTCTCAGTTTCATTACTTGGCTTCTG20 52247 6mEgCIR3716GCAGACATGGCAGCAAAAAGGGGGATGTTCCTGGATATCA20 52200 9mEgCIR3788TTGTATGACCAAAGACAGCAGCGCAACATCAGACTA191752173 9mEgCIR1713GCTGAAGATGAAATTGATGTATTCAGGTCCACTTTCATTTA212052252 11mEgCIR2380TTGGGATGCAAAATTCAACAGTAACCCATTGAGACTA172052177 11mEgCIR3402GGGCTTTCATTTTCCACTATGCTCAACCTCATCCACAC201852241 SSR loci with 100% similarity were 41 Polymorphic SSR loci which differentiated the tenera either from dura/pisifera were 11 Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR Results (Conti..)

17. Practical utility Useful in genetic diversity studies, linkage map and QTL analysis of oil palm. The technology developed will ensures supply of only hybrid tenera to the farmers by eliminating dura contamination in the nursery It reduces the cost of development of new cultivars and improve efficiency of breeding programme It is highly useful for the different enforcement agencies to check the quality of imported or indigenous planting materials In the long run, the developed technology will improve the productivity within the existing acreage. Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR