CONSTRUCTION OF A FUNCTIONAL GENETIC MAP IN OIL PALM (Elaeis guineensis Jacq.) J. Herrero1, M. Hernández1, A. Herrán1, E. Lopez de Armentia1, N. Billotte2 and E. Ritter1 1Department 2Centre
of Biotechnology (
[email protected]), NEIKER-Tecnalia. The Basque Institute of Agricultural Research and Development, P.O. Box 46, E01080 Vitoria, Spain. de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), UMR 1096 Plant Development and Molecular Improvemet. TA 40/03 Avenue Agropolis, 34398 Montpellier Cedex 5, France.
Introduction Genomic research can assist the improvement of perennial crop like Oil Palm and support the large efforts made by traditional techniques until now. The application of this knowledge in the major source of vegetable oil crop can save effort, time and space. Among other genomic tools, so far linkage maps have provided an important information about the palm genome and useful traits for breeding and selection. With the aim to link genes to trait, the increase of anchored molecular markers to the map will also provide more information. In this work we integrate additional cDNA-AFLP markers to the reference map published by Billotte et al. (2005). These Transcriptome Derived Fragment (TDF) markers represent coding sequences and are therefore particularly useful. Quantitative Trait Loci (QTLs), with agronomic interest, were also available and added to the map, allowing co-location analysis.
Material and Methods
Objective
PLANT MATERIAL: The progeny consisted of 116 full siblings derived from a cross between two heterozygous Elaeis guineensis Jacquin parents. Both parents were selected from a well known and high quality population. This cross transmits good oil yield and short trunk to its progeny.
i) to saturate the reference map with cDNA-AFLP markers and QTLs ii) to identify markers which are closely linked to QTL of interest
cDNA-AFLP assay (Bachem et al., 1998)
Results Total RNA extraction
Polymorphism analyses and mapping MAPRF V7.0 Table 1. PC: Primer Combination; PM: Polymorphic fragments; Dis: Nº of Displayed markers; Not displayed = AM: Associated markers; RF0: Recombination frequency of 0; NM: Not Mapped.
Markers PCs AFLP 132 SSR 278 cDNA-AFLP 164 QTLs TOTAL • •
PM Source 1120 Reference Map 398 TDFs 220 Billotte et al. 2010
Dis 545 279 193 191 1208
AM RF0
NM
170
218
200
mRNA isolation Digestion/Ligation
cDNA synthesis 200
• Taq I (frequent cutter) • Ase I (rare cutter) • Ligation of adaptors
Preamplification
27 170
From parents and progeny (spear leaves samples)
Ase+0/Taq+0
1578 245
Segregant fragments: Linkage test, recombination frequency and LOD score. QTLs from 27 traits of interest related with oil production, bunch components and vegetal growth.
Specific amplification Primer Combinations (PC) Ase+2 (*IRD)/Taq+2
Polyacrylamide GEL (LICOR System) MAPPING
Integrated MAP
Co-location analysis
Initially parental linkage maps were constructed firstly with dominant and co-dominant SSRs and then adding the rest of the markers. For integrating the parental maps a total of 114 codominant anchorpoints (mainly SSR alleles) were available.
•We obtained 19 loci (9 from male and 10 from female parent) where our TDFs were linked to QTLs within ± 1.1 centiMorgan. •In total, we could find 32 TDFs colocated with 25 QTLs. Legend: integrated linkage map of the cross. SSR markers are displayed in bold. Common fragments or common SSR alleles are underligned. AFLP frgaments are indicated with numbers corresponding to the PCs. New added cDNA-AFLP markers are indicated in red. In the integrated map markers descending from P2 are indicated in Italics.
AKNOWLEDGMENT: The research was supported by OPGP (Oil Palm Genome Project) Consortium.
Conclusions
REFERENCES: Corley, R.H.V. and Tinker, P.B. (2003) The oil palm, 4th edn. Blackwell Science, Oxford.
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Ritter E., Ruiz de Galarreta J.I., van Eck H.J. and Sánchez I. (2008) Construction of a potato transcriptome map based on the cDNA–AFLP technique. Theoretical and Applied Genetics ISSN: 0040-5752.
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Billotte N., Marseillac N., Risterucci AM., et al. (2005) Microsatellite-based high density linkage map in oil palm (Elaeis guineensis Jacq.). Theor Appl Genet 110: 754–765. Billotte N., Jourjon MF., Marseillac N., et al. (2010) QTL detection by multi-parent linkage mapping in oil palm (Elaeis guineensis Jacq.) Theor Appl Genet 120:1673–1687.
The anchoring of the Transcriptome map increased the genomic coverage of the Reference Map of this high-yielding commercial crop. cDNA-AFLP markers provide useful information since these markers are equivalent to expressed sequence tags.
OUTLOOK: We have isolated co-located TDFs from polyacrylamide gels, re-amplified and sent for sequencing in order to determine their biological meaning. cDNA-AFLP markers with relevant biological meaning could potentially explain these QTL.
Biospain 2012 (6th International Meeting on Biotechnology). Bilbao Exhibition Center, 19-21 Sept. 2012
