Dr D Ramajayam Senior Scientist Horti Fruit Science

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR 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 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 -18 t/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 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 Introduction • 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)

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR World production of oil palm planting materials [Dx. P, Clones, Bi-/Semi-clones and Interspecific hybrids] World production of oil palm planting materials Type of planting materials Million Planting materials/year Dx. P 315. 0 Clones 3. 5 (~ 1%) Bi-clones/Semi-clones seeds 1. 0 Interspecific hybrids 2. 5 Grand Total 321. 5 Kushairi et al (2010) 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.

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

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR 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 Methodology • Genomic DNA was extracted from spear leaves using kit (Mol Bio Himedia, Hipur. ATM Plant Genomic DNA Miniprep Purification Kit) • DNA quantification and quality was ascertained by Nano Drop ND 1000 spectrophotometer (Nano. Drop 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 thermal cycler (Eppendorf Master Cycler Nexux Gradient) with initial denaturation for 5 min. at 95 °C; 35 cycles denaturation of 30 s at 95 °C, annealing for 30 s at 50 -55 °C and extending for 60 s 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 Methodology (Conti. . ) Chromos ome No 1 1 2 2 2 3 3 4 4 4 SSR Locus m. Eg. CIR 3639 m. Eg. CIR 3750 m. Eg. CIR 0782 m. Eg. CIR 0905 m. Eg. CIR 2440 m. Eg. CIR 3732 m. Eg. CIR 3363 m. Eg. CIR 1773 m. Eg. CIR 2422 m. Eg. CIR 0783 m. Eg. CIR 3869 m. Eg. CIR 3543 m. Eg. CIR 3358 m. Eg. CIR 3260 m. Eg. CIR 3301 m. Eg. CIR 3544 m. Eg. CIR 0425 m. Eg. CIR 3698 sequence (F) ACGTTTTGGCAACTCTC GATGTTGCCGCTGTTTG CGTTCATCCCACCACCTTTC CACCACATGAAGCAGT TTCCAGAAGCTAAACGAATGAC ATTTTATTTGGCTTGGTATA CTTGACAATACCCTGAGTAGTAG ATGACCTAAAAATCTCAT GCCCTCAACTCAAAAA GAATGTGGCTGTAAATGCTGAGTG CCAATGCAGGGGACATT GTTCCCTGACCATCTTTGAG CCAAGGAACAACATAGA AGGGCAAGTCATGTTTC GCACTTGGTGGTTATGA AGCAGGGCAAGAGCAATACT AGCAAGAGCAGAACT AAGCCACCAGGATCATC sequence (R) ACTCCCCTCTTTGACAT CATCCCATTTCCCTCTT GCTGCGAGGCCACTGATAC CCTACCACAACCCCAGTCTC GCGGACAGTGCGAAGAGAGT ACTTTTCTAATTCTTGAAGAT GCTGTGCCTATCGGACTT ACAGATCATGCTCACA ATGGTGTCTGGGACTCTGAGTA AAGCCGCATGGACAACTCTAGTAA GAAGCCAGTGGAAAGATAGT GTCGGCGATTAGATTC GTTCCCATCCTATTAGAC TATAAGGGCGAGGTATT AGCTGCTGATGGATATC TTCAGCAGCAGGAAACATC CTTGGGGGCTTCGCTATC GTCATTGCCACCTCTAACT bp (F) 17 17 20 20 22 20 23 23 20 24 17 20 17 17 17 20 21 17 bp (R) 17 17 19 20 20 25 18 20 22 24 20 20 18 17 17 19 18 19 Ta 52 52 56 52 52 52 58 52 Amplifi cation 278 149 187 231 182 194 195 322 248 296 178 232 208 221 140 188 232 182 Oil palm SSR primers isolated by CIRAD (http: //tropgenedb. cirad. fr/oilpalm/publications. html) were used. (Billotte et al. 2015)

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR Methodology (Conti. . ) Chromos ome No 5 5 6 6 7 7 8 8 8 9 9 9 10 10 10 11 11 13 SSR Locus sequence (F) sequence (R) bp (F) m. Eg. CIR 2423 TCCAAGTAGCAAATGATGAC TGCCCTGAAACCCTTGA 20 m. Eg. CIR 3535 AGTTTGAGCTAGTGGTGTC ACAGGGTTCTTTGTCTACT 19 m. Eg. CIR 3439 ACTTGTAAACCCTCTTCTCA GTTTCATTACTTGGCTTCTG 20 m. Eg. CIR 3716 GCAGACATGGCAGCAAAAAG GGGGATGTTCCTGGATATCA 20 m. Eg. CIR 3886 TTCTAGGGTCTATCAAAGTCATAAG AGCCACCATCTACT 25 m. Eg. CIR 2387 TTGGTGAGCCATTTGCTACA CCTCCTTCCACCCCTCTACT 20 m. Eg. CIR 2575 GGGACTTCGCAAACTGTAGCA CGGTGGCGTATGGTGGATT 21 m. Eg. CIR 0408 TTGCGGCCCATCGTAATC TCCCTGCAGTGTCCCTCTTT 18 m. Eg. CIR 3282 GTAACAGCATCCACACTAAC GCAGGAGTAATGAGT 20 m. Eg. CIR 3788 TTGTATGACCAAAGACAGC AGCGCAACATCAGACTA 19 m. Eg. CIR 1713 GCTGAAGATGAAATTGATGTA TTCAGGTCCACTTTCATTTA 21 m. Eg. CIR 0257 GCAGCTAGTCACCTGAAC GACGAGACTGGAAAGATG 18 m. Eg. CIR 3672 AAAGCCATTCCAGACTAC CTCATAGCCTTTGTTGTGT 18 m. Eg. CIR 2427 GAAGGGGCATTGGATTT TACCTATTACAGCGAGAGTG 17 m. Eg. CIR 0779 AATGCAGACCAAGCTAATCATATAC GTTCAGGTGATGGTGACTCAGATAG 25 m. Eg. CIR 2380 TTGGGATGCAAAATTCA ACAGTAACCCATTGAGACTA 17 m. Eg. CIR 3402 GGGCTTTCATTTTCCACTAT GCTCAACCTCATCCACAC 20 m. Eg. CIR 3555 CATCAGAGCCTTCAAACTAC AGCCTGAATTGCCTCTC 20 bp (R) 17 19 20 20 17 20 18 19 20 25 20 18 17 Ta 52 52 58 52 52 52 Amplifi cation 342 167 247 200 187 243 269 193 245 173 252 286 159 116 238 177 241 136

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR Methodology (Conti. . ) Chromos ome No 13 14 14 14 15 15 15 16 16 16 SSR Locus m. Eg. CIR 3213 m. Eg. CIR 3890 m. Eg. CIR 2407 m. Eg. CIR 3711 m. Eg. CIR 3622 m. Eg. CIR 0555 m. Eg. CIR 3641 m. Eg. CIR 3739 m. Eg. CIR 3400 m. Eg. CIR 3362 m. Eg. CIR 3587 m. Eg. CIR 2813 m. Eg. CIR 0446 m. Eg. CIR 0894 sequence (F) GCTCTTTGTATTTCCTGGTTC GTGCAGATTATATG TGCCGAGGTGCAAGAAGATT GTCTCATGTGGCTACCTCTC GCCAGTTAGGAATACAA TACCATCACTGACCAATAAC TGGGAAGGTGCTCTCAT CAGCCAGTAAGAGCCAAGTC CAATTCCAGCGTCACTATAG CCCATCATCTGCTCAGGATAGAC TTGTTTCGTGCATGTGT GCTTTGTTGCAGTTTGACTA CCCCTTCGAATCCACTAT TGCTTCTTGTCCTTGATACA sequence (R) AGCAGCAAACCCTACTAACT CCTTTAGAATTGCCGTATC ACTATGCCACCCTTCTGAAGTA AGGCTCCCTGCTTTTAAGT GTCACGCATTTTTCTTG GTCTTGCTAACTACAC GGCTCCACATAATTTCTAC CTCCAACATTGAGGAGACTAG AGTGGCAGTGGAAAAACAGT ACCCTCTTGGGAAGA AGGAAGAAGGCTGACAT GTTTAGGATGTTGCGTGAT CAAATCCGACAAATCAAC CCACGTCTACGAAATGATAA bp (F) 21 20 20 20 17 20 20 23 17 20 18 20 bp (R) 20 19 22 17 17 20 19 21 20 20 17 19 18 20 Ta 52 52 52 52 Amplifi cation 97 154 187 175 142 226 184 207 148 151 217 210 202 186

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR Results P’ D’ D D P P T T Eg. SSR 18 P’ D’ D D P P T T Eg. SSR 19 P’ D’ D D P P T T Eg. SSR 22

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR Results (Conti. . ) P’ D’ D D P P T T Eg. SSR 29 P’ D’ D D P P T T Eg. SSR 53 P’ D’ D D P P T T Eg. SSR 31 P’ D’ D D P P T T Eg. SSR 54

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR Results (Conti. . ) P’ D’ D D P P T T Eg. SSR 3 P’ D’ D D P P T T Eg. SSR 5

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR Results (Conti. . ) P’ D’ D D P P T T Eg. SSR 67 P’ D’ D D P P T T Eg. SSR 68

Dr. D. Ramajayam, Senior Scientist (Horti. -Fruit Science), ICAR-IIOPR Results (Conti. . ) Chromos ome No 1 1 4 4 4 6 6 9 9 11 11 SSR Locus m. Eg. CIR 3750 m. Eg. CIR 0905 m. Eg. CIR 3260 m. Eg. CIR 3301 m. Eg. CIR 3698 m. Eg. CIR 3439 m. Eg. CIR 3716 m. Eg. CIR 3788 m. Eg. CIR 1713 m. Eg. CIR 2380 m. Eg. CIR 3402 sequence (F) GATGTTGCCGCTGTTTG CACCACATGAAGCAGT AGGGCAAGTCATGTTTC GCACTTGGTGGTTATGA AAGCCACCAGGATCATC ACTTGTAAACCCTCTTCTCA GCAGACATGGCAGCAAAAAG TTGTATGACCAAAGACAGC GCTGAAGATGAAATTGATGTA TTGGGATGCAAAATTCA GGGCTTTCATTTTCCACTAT sequence (R) CATCCCATTTCCCTCTT CCTACCACAACCCCAGTCTC TATAAGGGCGAGGTATT AGCTGCTGATGGATATC GTCATTGCCACCTCTAACT GTTTCATTACTTGGCTTCTG GGGGATGTTCCTGGATATCA AGCGCAACATCAGACTA TTCAGGTCCACTTTCATTTA ACAGTAACCCATTGAGACTA GCTCAACCTCATCCACAC bp (F) 17 20 17 17 17 20 20 19 21 17 20 bp (R) 17 20 17 17 19 20 20 17 20 20 18 Ta 52 52 52 Amplifi cation 149 231 221 140 182 247 200 173 252 177 241 • 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 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.