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 11-18t/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
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 [DxP,
Clones, Bi-/Semi-clones and Interspecific hybrids]
World production of oil palm planting materials
Type of planting materials
Million Planting materials/year
DxP
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.
DxP 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, HipurATM 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
Methodology (Conti..)
Chromos
ome No
1
1
1
1
2
2
2
2
2
3
3
3
3
4
4
4
4
4
SSR Locus
sequence (F)
sequence (R)
bp
(F)
mEgCIR3639 ACGTTTTGGCAACTCTC
ACTCCCCTCTTTGACAT
17
CATCCCATTTCCCTCTT
mEgCIR3750 GATGTTGCCGCTGTTTG
17
mEgCIR0782 CGTTCATCCCACCACCTTTC
GCTGCGAGGCCACTGATAC
20
CCTACCACAACCCCAGTCTC
mEgCIR0905 CACCACATGAAGCAAGCAGT
20
mEgCIR2440 TTCCAGAAGCTAAACGAATGAC
GCGGACAGTGCGAAGAGAGT
22
mEgCIR3732 ATTTTATTTGGCTTGGTATA
ACTTTTCTATCTAATTCTTGAAGAT 20
mEgCIR3363 CTTGACAATACCCTGAGTAGTAG GCTGTGCCTATCGGACTT
23
mEgCIR1773 ATGACCTAAAAATAAAATCTCAT ACAGATCATGCTTGCTCACA
23
mEgCIR2422 GCCCTCCCTCAACTCAAAAA
ATGGTGTCTGGGACTCTGAGTA
20
mEgCIR0783 GAATGTGGCTGTAAATGCTGAGTG AAGCCGCATGGACAACTCTAGTAA
24
mEgCIR3869 CCAATGCAGGGGACATT
GAAGCCAGTGGAAAGATAGT
17
mEgCIR3543 GTTCCCTGACCATCTTTGAG
GTCGGCGATTGATTAGATTC
20
mEgCIR3358 CCAAGGAACAACATAGA
GTTCCCATCCTATTAGAC
17
mEgCIR3260 AGGGCAAGTCATGTTTC
TATAAGGGCGAGGTATT
17
mEgCIR3301 GCACTTGGTGGTTATGA
AGCTGCTGATGGATATC
17
mEgCIR3544 AGCAGGGCAAGAGCAATACT
TTCAGCAGCAGGAAACATC
20
mEgCIR0425 AGCAAGAGCAAGAGCAGAACT
CTTGGGGGCTTCGCTATC
21
mEgCIR3698 AAGCCACCAGGATCATC
GTCATTGCCACCTCTAACT
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
52
52
52
58
52
52
52
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)
mEgCIR2423 TCCAAGTAGCAAATGATGAC
TGCCCTGAAACCCTTGA
20
mEgCIR3535 AGTTTGAGCTAGTGGTGTC
ACAGGGTTCTTTGTCTACT
19
mEgCIR3439 ACTTGTAAACCCTCTTCTCA
GTTTCATTACTTGGCTTCTG
20
mEgCIR3716 GCAGACATGGCAGCAAAAAG
GGGGATGTTCCTGGATATCA
20
mEgCIR3886 TTCTAGGGTCTATCAAAGTCATAAG AGCCACCACCACCATCTACT
25
mEgCIR2387 TTGGTGAGCCATTTGCTACA
CCTCCTTCCACCCCTCTACT
20
mEgCIR2575 GGGACTTCGCAAACTGTAGCA
CGGTGGCGTATGGTGGATT
21
mEgCIR0408 TTGCGGCCCATCGTAATC
TCCCTGCAGTGTCCCTCTTT
18
mEgCIR3282 GTAACAGCATCCACACTAAC
GCAGGACAGGAGTAATGAGT
20
mEgCIR3788 TTGTATGACCAAAGACAGC
AGCGCAACATCAGACTA
19
mEgCIR1713 GCTGAAGATGAAATTGATGTA
TTCAGGTCCACTTTCATTTA
21
mEgCIR0257 GCAGCTAGTCACCTGAAC
GACGAGACTGGAAAGATG
18
mEgCIR3672 AAAGCCATTCCAGACTAC
CTCATAGCCTTTGTTGTGT
18
mEgCIR2427 GAAGGGGCATTGGATTT
TACCTATTACAGCGAGAGTG
17
mEgCIR0779 AATGCAGACCAAGCTAATCATATAC GTTCAGGTGATGGTGACTCAGATAG 25
mEgCIR2380 TTGGGATGCAAAATTCA
ACAGTAACCCATTGAGACTA
17
mEgCIR3402 GGGCTTTCATTTTCCACTAT
GCTCAACCTCATCCACAC
20
mEgCIR3555 CATCAGAGCCTTCAAACTAC
AGCCTGAATTGCCTCTC
20
bp
(R)
17
19
20
20
20
20
19
20
20
17
20
18
19
20
25
20
18
17
Ta
52
52
52
52
52
52
52
58
52
52
52
52
52
52
52
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
14
14
15
15
15
15
15
16
16
16
SSR Locus
mEgCIR3213
mEgCIR3890
mEgCIR2407
mEgCIR3711
mEgCIR3622
mEgCIR0555
mEgCIR3641
mEgCIR3739
mEgCIR3400
mEgCIR3362
mEgCIR3587
mEgCIR2813
mEgCIR0446
mEgCIR0894
sequence (F)
GCTCTTTGTATTTCCTGGTTC
GTGCAGATGCAGATTATATG
TGCCGAGGTGCAAGAAGATT
GTCTCATGTGGCTACCTCTC
GCCAGTTAGGAATACAA
TACCATCACTGACCAATAAC
TGGGAAGGTGCTCTCAT
CAGCCAGTAAGAGCCAAGTC
CAATTCCAGCGTCACTATAG
CCCATCATCTGCTCAGGATAGAC
TTGTTTCGTGCATGTGT
GCTTTGTTGCAGTTTGACTA
CCCCTTCGAATCCACTAT
TGCTTCTTGTCCTTGATACA
sequence (R)
AGCAGCAAACCCTACTAACT
CCTTTAGAATTGCCGTATC
ACTATGCCACCCTTCTGAAGTA
AGGCTCCCTGCTTTTAAGT
GTCACGCATTTTTCTTG
GTCTTTCTTGCTAACTACAC
GGCTCCACATAATTTCTAC
CTCCAACATTGAGGAGACTAG
AGTGGCAGTGGAAAAACAGT
ACCCTCTCCTCTTGGGAAGA
AGGAAGAAGGCTGACAT
GTTTAGGATGTTGCGTGAT
CAAATCCGACAAATCAAC
CCACGTCTACGAAATGATAA
bp
(F)
21
20
20
20
17
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
52
52
52
52
52
52
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 D D P P P P T T
EgSSR18
P’ D’ D D D D P P P P T T
EgSSR19
P’ D’ D D D D P P P P T T
EgSSR22
Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR
Results (Conti..)
P’ D’ D D D D P P P P T T
EgSSR29
P’ D’ D D D D
P P P P T T
EgSSR53
P’ D’ D D D D P P P P T T
EgSSR31
P’ D’ D D D D P P P P T T
EgSSR54
Dr. D. Ramajayam, Senior Scientist (Horti.-Fruit Science),ICAR-IIOPR
Results (Conti..)
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..)
P’ D’ D D D D P P P P T T
EgSSR67
P’ D’ D D D D P P P P T T
EgSSR68
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
mEgCIR3750
mEgCIR0905
mEgCIR3260
mEgCIR3301
mEgCIR3698
mEgCIR3439
mEgCIR3716
mEgCIR3788
mEgCIR1713
mEgCIR2380
mEgCIR3402
sequence (F)
GATGTTGCCGCTGTTTG
CACCACATGAAGCAAGCAGT
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
52
52
52
52
52
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.