WO2010056107A2 - Method for identification of a molecular marker linked to the shell gene of oil palm - Google Patents

Abstract

The present invention relates to a method of genetic mapping of a tenera selfed palm in order to find a marker linked to the shell gene. The said method utilizes both co - dominant RFLP and SSR, SNP and dominant AFLP markers. Accordingly, a dense genetic map sufficient for establishing linkage relationships between the mapped markers and the traits of interest (shell gene) is achieved.

Description

METHOD FOR IDENTIFICATION OF A MOLECULAR MARKER LINKED TO

THE SHELL GENE OF OIL PALM

FIELD OF INVENTION

The present invention relates to the identification of a marker using restriction fragment technology, in particular Restriction Fragment Length Polymorphism (RFLP) marker linked to the shell gene for plant identification and breeding purposes, by way of generation or construction of the genetic map for tenera fruit type palm.

BACKGROUND OF INVENTION

It is generally agreed that scientific aspects play a major role in contributing to genetic improvement of various crop traits and in particular agriculture technology with respect to the cultivation of exceptional quality crop materials owing to sprouting technology advancements, which has become vital for the experts of the industry, and much has been written about them.

In most cases, the science of genetic manipulation in plants is focused on improvements of quality or high- value products, diversification, and in particular for assisting in improving or achieving increased total yield of crops or plants, in addition to obtaining desired vegetative characters and attributes.

Generally, in order to obtain value added products and complimentary fruit qualities of plants or crops, cross-breeding programmes and selection are highly favourable in this regard. In order to ensure effectiveness in providing methodological basis and tools for studies in improving the breeding efficiency of plants, a molecular linkage map is essential, whereby it can be considered as the first step towards developing markers linked to specific traits. It is known in the art that genetic mapping involves selection of markers, which are then placed evenly over the genome using certain experimental techniques, thus enhancing the probability of finding markers linked to qualitative, quantitative and complimentary traits. Linkage studies enable researchers to determine or at least identify markers which are consistently present under certain conditions. Therefore, in the event that a marker is found with the presence of certain condition, the marker and the subject gene are said to linked.

The most definitive heterologous and homologous markers used with respect to construction of genetic mapping include the genomic restriction fragment length polymorphism, complementary DNA (cDNA) restriction fragment length polymorphism (RFLP), (SSR) microsatellites, single nucleotide polymorphism (SNP), among others.

These genetic enhancement tools contribute significantly to increasing the accuracy and efficiency of a breeding programme, in particular acceleration of the breeding programme.

Proceeding from the above, one of the most important perennial crops amenable for such technological solutions with respect to the production of excellent quality products is the oil palm (E. guineensis), whereby the genetic efforts are typically focused on two main tissues of the oil palm, namely the mesocarp and kernel.

The oil palm (E. guineensis) are classified into three separate groups based on its fruit characteristics, whereby the said three groups are dura (thick shell), tenera (relatively thin shell) andpisifera (absence of shell).

According to studies and prior art, aided with genetic enhancement tools, oil palm species or groups as outlined above are capable of exchanging genes with one another, which thereby resulting to differentiations in fruit forms. For instance, tenera, which has been reported to have higher oil extraction efficiency, is a genetically formed hybrid between dura andpisifera.

As the essential embodiment of the breeding programmes in oil palm is to produce planting materials with increased oil yield, it is only natural that the tenera is the most preferred choice for commercial planting, and thus selected from crossing studies to achieve commercial seed production. Understandably, for this purpose, all offsprings should be of the tenera type. A person skilled in the art would comprehend that various control measures must be taken into account during the seed production, so as to avoid contamination in commercial seeds, which is rather a common occurrence in production of commercial seeds. In addition, a marker linked to the shell thickness can play a major role in breeding programmes, whereby markers can be used to efficiently separate the type of oil palms, as different type of palms, for instance relative to their shell thickness.

The prior art related to the tagging of shell thickness gene include an attempt by Mayes et al (1997), in which they have constructed a genetic map for population segregating for this trait. Accordingly, the map consists of 97 restriction fragment length polymorphism (RFLP) markers from genomic libraries in 24 groups. The shell gene was mapped in Group 8, about 8 cM away from the closest marker. However, this prior art has not been extended to providing the ability to distinguish fruit types in the different genotypes.

Another prior art comprise of the utilization of a combination of bulked segregation analysis, (BSA) and genetic mapping, reported a random amplified polymorphic DNA (RAPD) marker closely linked to the shell thickness locus (Moretzon et al., 2000). In contrast to the method disclosed earlier, the applicability of the marker in a wide range of breeding programmes is foreseen.

A relatively close prior art was reported recently (Billote et al., 2005) whereby there is provided a simple sequence repeat (SSR) -based high density linkage map for oil palm, which involved a cross between a thin shelled E. guineensis (tenera) palm and a thick shelled E. guineensis (dura) palm. The map consists of 255 SSR markers and 688 amplified fragment length polymorphism (AFLP) markers, and thus represents the first linkage map for oil palm to have 16 independent linkage groups which correspond to the haploid chromosome number of 16 in pil palm (Maria et. al., 1995). In addition, an AFLP marker was discovered to be mapped about 5 cM from the shell gene locus. Nevertheless, the prior art did not disclose or thus provide any ability of the said AFLP marker to distinguish the fruit forms. Consequently, the AFLP marker was therefore scored as dominant and thus it will not be able to distinguish the dura and the tenera fruit forms.

Following the above there has yet to a practical marker that can be used to separate dura, tenera and pisifera types in a typical nursery for the oil palm breeders, whereby in most cases plant breeders face difficulties with respect to the ability to distinguish closely related isolates with certainty.

It is therefore the main objective of the present invention to develop a dense genetic map for a selfed tenera palm, designated T 128 from the inventor's Nigerian germplasm collection.

It is yet another objective of the present invention to develop a dense genetic map for a selfed tenera type palm, using both co-dominant RPLP and SSR, SNP and dominant AFLP markers.

It is further an objective of the present invention to provide and thus establish the linkage relationships between the mapped markers and the traits of interest, in particular the shell gene of oil palm.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the combined AFLP, RFLP, SNP and SSR map in accordance with the present invention;

Figure 2 depicts the comparison of the closest marker identified to the shell gene in accordance to the present invention;

Figure 3 depicts the screening of probe SFB 83 on other four other genotypes, in accordance with an embodiment of the present invention.

SUMMARY OF INVENTION

In one embodiment the present invention relates to a method for identifying a genetic marker linked to a trait locus of an oil palm fruit using both co-dominant RFLP , SSR (micro satellite), SNP and dominant AFLP markers, said method comprising the steps of: a)preparing DNA sample based on the said oil palm; b)digesting the DNA fom step a)with a restriction enzyme; wherein the digesting step further comprising : i. preparing a first mixture of the said DNA from step a) , said mixture containing sterile water and a predetermined amount of DNA from step a); ii. preparing a second mixture containing a predetermined amount of restriction buffer and restriction enzyme; iii. adding the first mixture into the second mixture. conducting RFLP method, wherein the probe/restriction enzyme combinations having single/low copy of co-dominant profile are selected; conducting the AFLP method with pre-selective and selective amplification, wherein said AFLP method involved generating the AFLP markers with the usage of restriction enzymes; preparing microsatellite (SSR) primers from oil palm; and thus conducting the SSR method involving the said oil palm (SSR) microsatellite primers; preparing the oil palm single nucleotide polymorphisms (SNP) and genotyping based on said oil palm SNP primers; conducting data analysis based on RFLP, AFLP , SSR (microsatellite) and SNP markers; constructing the genetic map based on the results obtained in from said RFLP method, AFLP method SSR and SNP method.

In another embodiment, the present invention relates to a marker obtained and identified with the method as described herein.

In yet another embodiment of the present invention there is provided a selection marker obtained with the subject method comprising the DNA sequence of SEQ ID NO: 17 in the sequence listing.

DETAILED DESCRIPTION OF THE INVENTION

Further understanding of the object, construction, characteristics and functions of the invention, a detailed description with reference to the embodiments is given in the following.

It is therefore purpose of this invention is to present a genetic linkage map derived from the selling tenera palm.

It will be realized that the present invention utilizes SSR sequences isolated from the oil palm, therefore a person skilled in the art would comprehend that accordingly, said SSR probes prepared provides further advantages relative to the efficiency in the genetic mapping, as they will be more informative, apart from being more reliable and cost effective.

BEST MODE FOR CARRYING OUT THE INVENTION

The preparation of the present invention is described in detail by referring to the experimental examples. It should be understood that these experimental examples, while indicating preferred embodiments of the invention, are given by way better elucidation only. A person skilled in the art can ascertain the essential characteristics and embodiments of this invention, therefore various changes may be provided to adapt to various usages and conditions.

Materials and Methods

For the experimental examples, inventors used the mapping family derived from the selfing of the high iodine value (IV) tenera palm, T128, from Malaysian Palm Oil Board's (MPOB) Nigerian germplasm collection, whereby controlled self-pollination was preferred.

A total of 320 palms were planted at several locations in the country, whereby a total of 192 palms from selected locations, specifically MPOB-UKM, UIu Paka and United Plantations were used for the purpose of the present invention.

A number of related families were also used to confirm the marker linkages to the monogenic traits of shell thickness. The families used for this purpose were as follows:

Family 1: Tenera x Tenera (Trial 0.305 planted at MPOB-UKM Station, Bangi,

Selangor); Family 2: Tenera x Tenera (Trial 132 planted at United Plantations, Teluk Intan,

Perak);

Family 3: Dura x Tenera (Trial 35 planted at United Plantations, Teluk Intan, Perak); and

Family 4: Dura x Tenera (Trial 38 planted at United Plantations, Teluk Intan,

Perak). In accordance with the present invention, it should be noted that the palm variety on the left of the symbol 'x' denotes the female parent, while the palm on the right is the male parent.

From the families listed above, it will be realized that one of the parents of Family 1 is palm T 128, which was selfed to generate family used for the map construction for the present invention. The remaining tenera palms used for the crosses are all unrelated to palm Tl 28 and are unrelated to each other.

Further, Family 1 and Family 2 are segregating for all three fruit forms, while Family 3 and Family 4 are segregating for the dura and tenera fruit forms. The said families were used to confirm the marker associated with the shell gene locus of the present invention.

DNA Extraction This is considered as the first step of screening, whereby DNA is extracted and purified, to be used for the purpose of the present invention.

Unopened leaf samples, preferably spear leaf were collected from the individual palms and immediately frozen under liquid nitrogen and then stored at -8O⁰C until the initiation of DNA preparation step. DNA from the said leaf samples was extracted and subsequently purified using conventional method and means, for instance by Doyle and

Doyle (1990).

Leaf samples, approximately 3 grams, were accordingly ground to powder under liquid nitrogen using sterilized mortar and pestle. The said powder form tissue was then transferred to a 50 ml centrifuge tube, said tube was provided with 15 ml of modified CTAB buffer, preferably the amount of 2% CTAB w/v, 20 mM EDTA pH 8.0, 1.4 M NaCl, 100 mM tris-HCl pH 8.0, 5 mM ascorbic acid, 4 mM diethyldithiocarbamic acid sodium salt and 2% polyvinylpyrolidone-40) and 100 μl 2-mercaptoethanol.The mixture was placed at 6O⁰C for 30 minutes, after which an equal volume of chloroform : isoamyl alcohol (24:1) was added and mixed thoroughly.

The mixture was centrifuged at 10,000 rpm for 15 minutes at 2O⁰C and the upper aqueous phase was subsequently transferred to a 30 ml convex tube. The DNA in this aqueous phase was precipitated by adding 0.6 volume of ice-cold isopropanol. The solution was then stored at -20⁰C for 1 hour, and centrifuged at 12,000 rpm at 4°C for 15 minutes. The DNA pellet obtained was washed in 5 ml wash buffer which comprised of 76% ethanol, 10 mM ammonium acetate, and precipitated by centrifugation. The pellet was dried by using a speed- vac centrifuge, namely Savant Oligo Prep™ OP 120, for 1 hour and suspended in 4 ml TE buffer, said buffer comprising 10 mM tris-HCl pH 8.0 and 1 mM EDTA pH 8.0. The DNA solution was treated with 5 μl; RNase (10 mg/ml) at room temperature for 20 minutes. Subsequently, 0.5 volume of 7.5 M ammonium acetate, pH 7.7 was added and the mixture placed on ice for 20 minutes. After centrifugation at 12,000 rpm for 15 minutes at 4⁰C, the supernatant was transferred into a clean and sterile 30 ml corex tube. The DNA in the supernatant was precipitated by adding 2.5 volumes of ethanol and stored at -2O⁰C for 1 hour. The DNA was recovered by centrifuging the mixture at 12,000 rpm for 15 minutes at 4⁰C. The DNA pellet was washed with 5 ml 70% ethanol, dried and dissolved in 1 ml TE buffer.

The DNA concentration of the samples was determined by diluting an aliquot of the sample in 990μl sterile water (100 x dilutions) and optical density measurement at A₂₆₀, A_28O and A₃₅₀ was determined using spectrophotometer. The concentration and yield of DNA was determined using the following formula described by Sambrook et. al (1989):

DNA concentration (μg/μl) = A₂₆₀^A_350-X dilution factor

20 DNA yield (μg) = Concentration (μg/μl) x volume used in dissolving DNA (μl)

The integrity of the DNA was further examined by electrophoresing an approximate 5 μg of DNA in 0.9% agarose in Ix TAE buffer (0.04M tris base, 20 mM acetic acid, 2 mM

EDTA). The DNA was loaded onto the gel with 1/9 volume loading dye solution, FOG (0.1

M EDTA, 25% ficoll, 0.1% orange G and 50% glycerol). The digestibility of each of the

DNA sample extracted was also checked by restriction digestion of 3μg DNA with EcoRl

(six base pair cutter) and Haelll (four base pair cutter). The digestion was carried out as described below. The digested samples were run next to the undigested samples (for integrity testing). Also loaded was a 1 kb DNA ladder (New England Biolabs) and lOμl gel tracking dye (25% ficoll type 40, 1% orange G, 1% bromophenol blue, 1% xylene cyanol and 0.1 M

EDTA) in separate wells, and the gel was run at 70 V until the bromophenol blue reached the end of the gel tray. The gels were photographed under UV light (254 nm; Model Fotodyne Incorporated) using Polaroid camera.

Restriction enzyme (RE) digestion of DNA As known in the prior art, restriction enzymes are typically for assisting in digesting large pieces of DNA, from any source into smaller fragments of DNA relative to the characteristic of the enzyme.

For standard RFLP analysis, an aliquot of 20 μg genomic DNA which was obtained from the previous stage was transferred into a 1.5 ml microcentrifuge tube and sterile water was added to a final volume of 100 μl to form the first master mix. A second master mix consisting of the following was prepared: 20 μl of the appropriate 10x restriction buffer (supplied with the respective enzyme), 4 μl 0.1 M spermidine trihydrochloride (pH 7.0), 2 μl 10Ox Bovine Serum Albumin (BSA), the appropriate concentration of enzyme (varies according to the restriction enzyme used) and sterile water to a final volume of 100 μl. The second master mix was transferred to the microcentrifuge tube containing the first master mix to give a final volume of 200 μl of digestion mix. The digestion mix was incubated overnight at the optimum temperature, as recommended by the manufacturer of the respective enzymes. Subsequently 20 μl 3 M sodium acetate (pH 4.8) and 440 μl ice-cold absolute ethanol were added to the digested DNA and allowed to precipitate at -2O⁰C overnight. The DNA was recovered by centrifuging at 14,000 rpm at 4⁰C for 45 minutes using bench -top centrifuge (Eppendorf 5810R). The supernatant was aspirated out and the resulting DNA pellet washed with 70% ice cold ethanol by centrifuging at 14,000 rpm at 4°C for 15 minutes. The DNA pellet was then dissolved in 35 μl TE buffer, followed by addition of 5 μl of the loading dye, FOG. The samples were stored at 4°C (not longer than 72 hours) until required.

The restriction fragments produced from the above step are then separated, which will be described shortly herein, by way of electrophoresis by conventional means, for instance the agarose gel electrophoresis which was selected for this invention. Further, the standard blotting technique used with respect to the present invention is the Southern Blotting technique. Electrophoresis of genomic DNA for Southern analysis

Gel electrophoresis was carried out by using agarose gels. The percentage of the agarose gel used varied depending on the restriction enzymes used. A 0.9% agarose gel was used for enzymes having six base pair recognition sites (BamRl, Bell, BgIU, Dral, EcoRl, Hindi, Hinάlll, Seal, Ss /I, Xbal), while a 1.0% agarose gel was used for enzymes with four and five base pair recognition sites (Haelϊl, Rsal, Taql, BstNl). The agarose gel was prepared by weighing an appropriate amount of LE agarose powder (2.5 g for 1% and 2.25 g for 0.9%) in 250 ml of Ix TPE buffer (90 mM tris-phosphate buffer, 2 mM EDTA pH 8.0). The gel was boiled using a microwave oven with frequent stirring until the powder dissolved and the solution was left at room temperature for about 5 min until the temperature cooled down to about 6O C. The agarose gel was then cast in a 20 cm x 25 cm platform and allowed to solidify at room temperature.

The electrophoresis was carried out using a horizontal gel electrophoresis system (Horizon 20-25, Whatman Biometra), at 100 V for 5 min, followed by 25 V overnight.

The gel contained 5 ng 1 kb ladder (New England Biolabs) and 10 μl gel tracking dye in separate wells. Usually the gel was run until the bromophenol blue in the gel tracking dye reached the end of the gel tray. The gel was then stained with 20 μl ethidium bromide (10 mg/ml) in 200 ml electrophoresis buffer (Ix TPE) for 30 min. The stained gel was viewed under UV light and photographed using a Polaroid camera. Prior to blotting, the gel was briefly rinsed with distilled water.

Southern blotting was carried out using the VacuGene XL Vacuum Blotting System (GE Healthcare). The DNA was transferred onto Hybond™ N+ nylon membrane (GE Healthcare) under vacuum pressure of 50 mbar for 30 min using 0.4 M sodium hydroxide (NaOH) as the transfer buffer. The blotted membranes were washed with 2x SSC buffer (diluted 10 fold from 2Ox SSC stock solutions) before being fixed for 20 s at 254 run using a UV-crosslinker (Spectrolink XL-1000, Spectronics Corporation). The membranes were left to air dry and stored at 4 C until required.

RFLP probes

The RFLP probes used in this study were complementary DNA (cDNA) clones obtained from various cDNA libraries (young etiolated seedling, mesocarp, kernel and root) constructed previously as described by Cheah (1996). cDNA clones from a subtracted flower library (Cheah and Rajinder, 1998) were also used to screen the mapping population.

The cDNA clones were picked at random from the various libraries. Plasmid DNA was prepared from individual clones by using column purification, with Qiagen-tip 20 (Qiagen) as described by the manufacturer. The bacterial colonies containing the plasmid were initally grown overnight in 3 ml of LB broth (plus ampicillin), with shaking at 200 rpm at 37 C overnight. The bacterial pellet was recovered by spinning at 10,000 rpm for 10 min at 4⁰C and re-suspended in 0.3 ml of buffer Pl (resuspension buffer: 50 mM tris-Cl, pH 8.0; 10 mM EDTA; 100 μg/ml RNase A). Buffer P2 (200 mM NaOH, 1% SDS) was added, the solution mixed gently and incubated at room temperature for 5 minutes. Subsequently 0.3 ml of buffer P3 (neutralization buffer: 3.0 M potassium acetate, pH 5.5), pre-chilled on ice was added and the mixture incubated on ice for 5 min. The resulting suspension was spun at 10,000 rpm at 4 C for 5 min, and the supernatant immediately transferred to a new 1.5 ml microcentrifuge tube. The supernatant was passed through a Qiagen tip-20 column, pre- equilibrated with buffer QBT (equilibration buffer: 750 mM NaCl; 50 mM MOPS, pH 7.0; 15% isopropanol; 0.15% triton X-100). The solution was allowed to pass through the column under gravity flow. The column was washed with 4 x 1 ml of buffer QC (wash buffer; 1.0 M NaCl; 50 mM tris-Cl, pH 8.5; 15% isopropanol and the plasmid was eluted using 0.8 ml of buffer QF (elution buffer: 1.25 M NaCl; 50 mM tris-Cl, pH 8.5; 15% isopropanol). The plasmid DNA was precipitated by the addition of 0.56 ml isopropanol and recovered by spinning at 12,000 rpm for 30 min at room temperature. The plasmid DNA pellet was washed with 1 ml 70% ethanol, dried and dissolved in 12 μl TE buffer. The concentration of the prepared plasmids was determined by spotting an aliquot of the sample on ethidium bromide plates. (Sambrook et al. 1989).

The buffers used Pl, P2, P3, QBT and QC and QF were supplied by the manufacturer. The composition of the buffers was obtained from the Qiagen Plasmid Mini Handbook, page 28 (Qiagen).

The presence of DNA insert was examined by restriction digestion of 1 ug plasmid DNA with 10 U of the appropriate restriction enzyme for 3 hours, to release the insert DNA. One tenth (1/10) volume of loading buffer, FOG, was added to the digestion mix and the fragments were separated by electrophoresis on a 1.5% agarose gel in Ix TPE buffer. cDNA clones with insert size larger than 500 base-pairs (bp) were selected to screen for their ability to detect RFLP in the mapping population.

Probes for mapping were derived from the selected plasmids as polymerase chain reaction (PCR) amplified DNA fragments. Bacterial clones containing selected probes were maintained as frozen glycerol stocks at -80 C.

Labeling of selected DNA probes

It should be noted that the labeling of genetic probes may be achieved by various established procedures. The present invention has selected the Megaprime DNA Labelling System.

The DNA probe to be labeled was diluted to a concentration of 5 μg/μl in TE buffer. The DNA probe was then labeled using the Megaprime DNA Labelling system (GE Healthcare). A total of 50 ng of probe was placed into a clean and sterile microcentrifuge tube. To it was added 5 μl primer solution (supplied with the labeling kit) and sterile water to a final reaction volume of 33 μl. Denaturation of the solution was carried out by heating in a boiling water bath (lOO C) for 10 min, and then it was immediately chilled on ice. A brief spin was carried out to bring the contents to the bottom of the tube. Subsequently, 10 μl labeling buffer (provided in the Megaprime Labeling Kit), 5 μl α³²P dCTP (3000 Ci/mmol stock) and 2 μl Klenow enzyme (1 U/μl) were added to the mixture (to a final volume of 50 μl) and incubated at 37 C for 1 hour. The reaction was stopped by adding 25 μl blue dextran/orange G solution (1% w/v blue dextran, 1% w/v orange G in TE pH 8.0). The labeled probe was separated from the unincorporated nucleotides by purification through a Sephadex column as described in Sambrook et al. (1989).

Southern Hybridization

Initially DNA from a sample of 10 palms was each digested separately with 14 restriction enzymes (BamHl, BcR, BgRl, Dral, EcoRl, Hindi, Hindlll Seal, Sstl, Xbal, BstNl, Haelll, Rsal and Taql). The restricted DNA fragments were separated by electrophoresis in agarose gel and transferred onto nylon membranes (Hybond N+, GE

Healthcare) as described above.

The set of 140 samples were then hybridized in turn with each candidate probe to identify the probe/restriction enzyme combination that gave a segregation profile. In the case of more than one enzyme showing polymorphisms with a particular probe, the probe/enzyme combination that gave a single/low copy clear co-dominant profile was selected for screening the entire mapping family.

Pre-hybridization and hybridization were carried out in glass tubes in a rotiserrie oven at 65 ⁰C. The membranes were pre-hybridized for about 3 hours in a solution containing pre- hybridization buffer as follows : 5x SSPE solution (3 M NaCl, 0.2 M sodium phosphate, 20 mM EDTA pH 8.0), 0.5% SDS, 5x Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrolidone, 0.1% albumin bovine fraction V) and 100 μg/ml denatured herring sperm DNA. The pre-hybridization buffer was removed and replaced with hybridization solution containing 5x SSPE (3 M NaCl, 0.2 M sodium phosphate, 20 mM EDTA pH 8.0, 0.5% SDS and 100 μg/ml denatured herring sperm DNA. Labeled probes were denatured by heating in boiling water bath for 10 minutes and plunging into ice water before adding to the hybridization buffer. The probe was added to a concentration of about 1-3 x 10⁶ cpm/ml. Hybridization was carried out overnight at 65⁰C.

Hybridized membranes were washed twice in 2x SSC (0.3M NaCl, 30 mM trisodium citrate, pH 7.0) and 0.1% SDS at 65⁰C for 15 minutes each time, followed by once in Ix SSC (0.15 M NaCl, 15 mM trisodium citrate pH 7.0) and 0.1% SDS at 65°C for 10 minutes. The membranes were then autoradiographed at -8O⁰C using X-ray films with intensifying screens for seven to 10 days. X-ray films were developed using 0.22x Kodak's GBX developer for 5 minutes rinsed in distilled water, followed by a final wash with 0.2x Kodak's GBX fixer for 5 minutes.

The membranes hybridized with a particular probe could be re-used at least 3 more times with probe removal. The probes were removed by washing the membranes in 0.1 M sodium hydroxide (NaOH) at 42⁰C for 20 minutes followed by a solution containing 0.1 M tris-HCl pH 7.5, 0.1 x SSC (0.015 M NaCl, 1.5 mM tri-sodium citrate pH 7.0), 0.1% SDS at 42⁰C for 20 minutes and finally using a solution containing O.lx SSC, 0.1% SDS at room temperature for 30 minutes. The membranes were exposed to X-ray film as described above for 7 days, to make sure that probe removal was complete. AFLP analysis i. EcoBl/Msel Enzyme Pair

The AFLP assay with EcoRI/Msel enzyme pair in accordance with a preferred embodiment of the present invention was carried out using the GIBCO BRL AFLP Analysis System I essentially as described in the provided manual, with some minor modifications. 350 ng of genomic DNA was digested with 3.2 μl of EcoRI and Msel (1.25 U/ul each) at 37 C for 4 hours in a final volume of 25 μl. After heat inactivation of the enzymes at 7O C, the fragments (the whole 25 μl solution) were ligated to the EcoRI and Msel adapters (consisting of EcoRI/Msel adapters, 0.4 mM ATP, 10 mM tris-HCI pH 7.5, 10 mM Mg- acetate and 50 mM K-acetate) in the presence of T4 DNA ligase (1 U) at 20^°C for 3 hours. A pre-selective amplification was then carried out by amplifying a 10-fold dilution of the ligation mixture. The pre-selective amplification consisted of 5 μl of the diluted template DNA, 40 μl of pre-amp primer mix [supplied in the kit, consisting of EcoRI primer with one selective nucleotide (adenine) and Msel primer with one selective nucleotide (cytosine)], 5 μl 1 Ox PCR buffer (200 mM tris-HCI pH 8.4, 15 mM MgCl₂, 500 mM KCl) and Taq DNA polymerase (1 U) in a final volume of 51 μl. PCR was carried out for 20 cycles using a Perkin Elmer 9700 Thermocycler as follows: 94^°C for 30 s, 56^°C for 60 s and 72^°C for 60 s. A 50-fold dilution was performed on the pre-amplified PCR products for subsequent use in selective amplification.

For selective amplification, a selected EcoRI primer (with three selective nucleotides) was labeled with γ-³³P-ATP using T4 polynucleotide kinase. In the labeling reaction, 18 μl of the selected EcoRI primer (27.8 ng/μl), was mixed with 20 U of T4 polynucleotide kinase, 10 μl 5x kinase buffer (350 mM tris-HCI pH 7.6, 50 mM MgCl₂, 500 mM KCl, 5 mM 2-mercaptoethanol), and 10 μl of γ-³³P-ATP (2,000 Ci/mmol) in a final volume of 50 μl. The mixture was incubated at 37 C for 1 hour. The labeled EcoRI primer was mixed with a selected Msel primer (three selective nucleotides containing dNTPs) at a ratio of 1 :9 to form a primer master mix. The PCR contained 5 μl of a 30 μl 10-fold diluted preamplified DNA, 5 μl primer master mix, 0.5 U Taq DNA polymerase, 2 μl of a 10x PCR buffer in a final volume of 20 μl. PCR was performed as recommended by the manufacturer using a Perkin Elmer 9600 thermocycler as follows:

• One cycle at 94^°C for 30 s, 65^°C for 30 s and 72^°C for 60 s,

• The annealing temperature was lowered 0.7 C each cycle during the subsequent 12 cycles, giving a touch down phase of 13 cycles, • 23 cycles were then performed as follows; 94^°C for 30 s, 56^°C for 30 s and 72

^°C for 60s

Aliquots of the post PCR mixture were heated with an equal volume of formamide dye (98% (v/v) formamide, 10 mM EDTA, 0.2% (w/v) bromophenol blue, 0.2% (w/v) xylene cyanol) at 90 C for 3 min. A 5 μl sample was electrophoresed in a 6% (w/v) polyacrylamide sequencing gel with 7.5 M urea. The gel was dried and exposed to an X-ray film (Kodak XK- l) at -80^°C for 2 to 3 days.

Segregating AFLP markers were coded as EXXX/MYYY-N, where X and Y are the selective nucleotides used in the selective amplification, while N represents the fragment size in bp. E and M refer to the enzyme systems used EcoRl and Msel respectively.

The AFLP analysis using the TaqllHinάlll enzyme pairs was essentially performed as described by Rafalski et al. (1996), with slight modifications.

Digestion with Taql and Hindlll

1000 ng of DNA was digested with the enzyme Taql as follows:

DNA (100 ng/μl): 10 μl

10x One Phor All Buffer*: 4.0 μl

Taql (12U/μl): 0.6 μl

Sterile Water: 25.4 μl

The digestion mix was incubated at 65 C for 6 hours

[* 1Ox One Phor AU Buffer: 100 mM tris-acetate pH 7.5, 100 mM magnesium acetate, 100 mM potassium acetate, 50 mM DDT]

To the 40 μl digestion mix from above was added 1 μl 10x One Phor All Buffer, 0.6 μl

HmdIII enzyme (15 U/μl) and sterile water to a final volume of 50 μl. The reaction mixture was incubated at 37°C, overnight (at least 14 hours). Heat inactivation of the restriction enzymes was carried out at 85⁰C for 15 minutes.

Ligation to Taql and Hindlll Adapters

The Taql and Hindlll adapters were synthesized as follows: Hindlll -Forward adapter: 5' CTCGTAGACTGCGTACC- 3' Hindlll -Reverse adapter: 3'- CTG ACGC ATGGTCGA-5^*

Taql -Forward adapter: 5' GACGATGAGTCCTGAC- Y Taql -Reverse adapter:, ...» 3^*- T ACTC AGGACTGGC- 5'

The oligonucleotides above were purchased non-phosphorylated to avoid adapter to adapter ligation (Rafalski et al, 1996). The double stranded Taql adapter was produced by combining 5000 pmol of the forward and reverse primers in a final volume of 100 μl (to give a double stranded Taql adapter at 50 pmol/μl). The HmdIII double stranded adapter was produced by combining 500 pmol of each of the corresponding forward and reverse primers to a final volume of 100 μl (to give a double stranded Hindlll adapter of 5 pmol/μl). To generate double stranded molecules, each of the mixtures of the forward and reverse primers were incubated in sequentially decreasing temperatures of 65 C for 15 min, 37 C for 75 min, room temperature 15 min and followed by 4 C for about 10 min. The double stranded adapters were stored at -20 C until use.

Subsequently a ligation mix consisting of 1 μl 10x One Phor All Buffer, 1.2 μl Tαql adapter (50 pmol/μl), 1.2 μl HmdIII adapter (5 pmol/μl), 1.2 μl 10 mMATP, 1.2 μl T4 DNA

Ligase (1 U/μl), and sterile water to 10 μl was prepared. This ligation mix was added to the

50 μl heat inactivated digestion mix and incubated at 37 C for 4 hours. The ligated samples were diluted 10-fold with TE buffer.

Pre-selective and Selective Amplification

The pre-selective amplification consisted of 5 μl of the diluted template DNA, 2 μl of Taql pre-selective primer (50 ng/μl) with one selective nucleotide (adenine), 2 μl Hindlll primer (50 ng/μl) with one selective nucleotide (cytosine), 5 μl 10x PCR buffer (200 mM tris- ΗCI pΗ 8.4, 15 mM MgCl₂, 500 mM KCl), 1 μl of 5 mM dNTP mix, and Taq DNA polymerase (1 U) in a final volume of 50 μl. PCR was carried out using a Perkin Elmer 9700 Thermocycler as described above for pre-selective amplification using the EcoKl/Msel enzyme pair. A 50-fold dilution was performed on the pre-amplified PCR products for subsequent use in selective amplification. The pre-selective primers used were as follows: Hindlϊh S^r- GACTGCGTACCAGCTTC-3' Taql: 5' - GATGAGTCCTGACCGAA-3'

In the selective amplification reaction (20 μl volume), 2 μl of the diluted template DNA was combined with 1 μl of γ-³³P-ATP (2,000 Ci/mmol) labeled Taql primer (5 ng)

(with three selective nucleotides), 0.5 μl of the same unlabelled selective Taql primer (50 ng/μl stock), 0.6 μl of selective HmdIII primer (also consisting of three selective nucleotides)

(50ng/μl stock), 2 μl 10x PCR buffer, 0.8 μl 5 mM dNTP mix, 0.5 U Tag polymerase and

13.0 μl sterile water. The amplification reaction was carried out using a Perkin Elmer 9700 Thermocycler as described above for the selective amplification using the EcoRI/Msel enzyme pair.

The selective primers used for amplification were as follows:

Ϊ...

Taql : 5'-GATGAGTCCTGACCGAACT -3^*

5'- GATGAGTCCTGACCGAACC-3' 5' -GATGAGTCCTGACCGAAGC-S'

5 ^s- GATG AGTCCTG ACCG AAGG-3 '

tfmdIII; 5'- GACTGCGTACCAGCTTCAT-3'

5'- G ACTGCGTACC AGCTTCAA-3' 5'- GACTGCGTACCAGCTTCAGO'

5'- GACTGCGTACCAGCTTCTG-3'

The PCR amplification products were analyzed on a 6% (w/v) polyacrylamide sequencing gel with 7.5 M urea and subsequently visualized as described above. Segregating AFLP markers were coded as TXXX/ΗYYY-N, where X and Y are the selective nucleotides used in the selective amplification, while N represents the fragment size in bp. T and Η refer to the enzyme systems used Taql and HmdIII respectively. Microsatellites

A. Isolation of Microsatellites in Oil Palm

Degenerate primers (PCT4) (Fisher et al, 1996) and (PCTI) (Brachet et al, 1999) were used to isolate clones containing microsatellite sequences from oil palm. The primers contain microsatellite repeats, followed by a degenerate anchor at their 5' ends.

PCT4: KKVRVRV(CT)₆ and PCTI: KKYHYHY (GA)₁₅ (where K= G/T; V= G/C/A; R= G/A; Y=CAT; H=AAYC)

The PCR mixture was prepared essentially as described by Fisher et al. (1996) as follows:

30 ng genomic DNA, 0.2 mM each dNTPs, 1.5 mM MgCl₂, 10 mM tris-HCL (pH 8.3), 50 mM KCl, 3 U Taq DNA polymerase (INVITROGEN), 50 pMol degenerate primers (PCT4 or PCTl) and deionized water to 25 μl .

PCR was performed separately for the two parental DNA samples, T 128 (Nigerian guineensis) and UP 1026 (Colombian oleifera) using the protocol described by Fisher et al. (1996) as follows:

Ten μl of the post PCR samples was analyzed on a 1% agarose gel with Ix TAE buffer. Separately, 3 μL of the post PCR mix was cloned using the TOPO-TA cloning kit

(INVITROGEN, USA), essentially as recommended by the manufacturer. The cloning was carried out by using chemically competent E. coli cells, TOP 10, (provided in the kit) as follows:

Fresh PCR product : 3 μl

Salt solution (1.2 M NaCI, 0.06 Ml μl

TOPO Vector (PCR®2.1 -TOPO) 1 μl

The reaction was mixed gently and incubated at room temperature for 15 min. The reaction was then placed on ice. 2 μl of the above cloning reaction was then gently pipetted into TOP 10 chemically competent E.coli cells and incubated on ice for 30 min. Heat shock was then carried out at 42 C in a water bath for 42 s, after which the reaction mix was placed on ice. To this reaction mix was added 250 μl of room temperature SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 HiMMgCl₂, 10 mM MgSO₄, 20 mM glucose) and the tube was shaken horizontally (200 rpm) at 37^°C for 1 hour. 50 μl of the transformation mix spread on a LB plate containing 50 μg/ml ampicillin and X-gal and incubated overnight at 37 C. The resulting white colonies were chosen for subsequent analysis.

Plasmid DNA was obtained using the QIAGEN-tip 20 plasmid prep kit (QIAGEN) as described earlier. Presence of insert was checked with restriction digestion by EcόRI as follows:

Plasmid DNA: 500 ng

1 Ox Restriction buffer: 1.5 μl £coRI (10 U/μl): 1.2 μl

Sterile Water: To a final volume of 15μl.

Digestion was carried out at 37 C for 3 hours. The digested reaction was stopped by heating at 65^°C for 10 min. One tenth volume of loading buffer, FOG, was added to the digestion mix and the fragments separated by electrophoresis through a 1% agarose gel (1 g in 100 ml of Ix TAE buffer). The clones containing inserts larger than 150 bp were selected for sequencing and bacterial clones of the selected probes were stored as frozen glycerol stocks.

Sequencing was carried out on both strands using M13 forward and reverse primers

(INVITROGEN) using an ABI 377 sequencer (Applied Biosystems). Specific primers in the flanking regions of the microsatellites were designed using the PRIMER 3 software (Rozen and Skaletsky, 2000). For each primer pair, the annealing temperature was initially set at 4O C, and subsequently increased by 2^°C, until a single/low copy (not more than 4 bands) was observed on an agarose gel. Although the annealing temperature could have been estimated from the melting temperature generated by the primer design software, we used this information only as a guide in determining the most appropriate annealing temperature in order to eliminate or minimize nonspecific banding profile. B. Labeling of Microsatellite Primers and Analysis on Acrylamide Gel

One primer for each primer pair was 5' end labeled at 37 C for 30 min using T4 polynucleotide kinase (INVITROGEN). The labeling reactions contained 50 pmoles primer, 3 μl γ-³³p dATP (GE Healthcare Bioscience 3000 Ci/mmol), I U T4 polynucleotide kinase in a total volume of 25 μl.

Subsequently the PCR reaction was carried out in a 25 μl reaction containing I U Taq

Polymerase (INVITROGEN), 50 mM tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.2 μM of each primer, 0.2 mM dNTPs (INVITROGEN) and 50 ng of template DNA. PCR was performed in a Perkin Elmer 9700 thermocycler essentially as described by Billotte et al.

(2001), as follows:

94 C, 3 min (1 cycle);

93 ^OC/59^°C/72^OC, 30 s each (5 cycles);

93 ^°C/57 ^°C/72 ^°C, (35 cycles), 30 s each and

72 ^°C, 2 min (1 cycle).

After the PCR was completed, the reactions were stopped by the addition of 25 μl formamide buffer (0.3% bromophenol blue, 0.3% xylene cyanol; 10 mM EDTA pH 8.0; 97.5% deionized formamide). Each PCR reaction was subjected to electrophoresis on a 6% denaturing acrylamide gel containing 7 M urea using 0.5x TBE buffer at constant power of 40 W for 3 hours. The gels were then dried and exposed to X-ray film (Kodak) for 3-4 days at -80^°C. Sizing of each allele was done using AFLP molecular weight ladder (INVITROGEN, USA).

The nomenclature used to describe the SSR primers was as follows: PxNy; where x refers to "1 'and '4' if degenerate primer PCTl or PCT4 was used respectively to isolate the SSR loci; N is ^ΛT^P if palm T128 (E. guineensis) was used as the DNA source to isolate the SSR and O¹ if the palm UP 1026 (E. oleifera) was used instead; y refers to the clone number.

C. Application of Published Oil Palm Microsatellite Sequences

The 18 published microsatellite primer pairs (Billotte et al, 200 \) were also tested on the mapping populations. All the 18 primer pairs were synthesized based on the published sequences and tested on a small number of the mapping family as described above. The sequences of the informative primer pairs tested are provided in the Results Section. The nomenclature used for the SSR primers reported by Billotte et al (2001) was CIR X, where X refers to the clone number.

The second set of published SSR loci used was that reported by Chua et al. (2004). A total of five SSR primer pairs reported by Chua et al. (2004) were synthesized for testing on the mapping population. The SSRs described by Chua et al. (2004) were uncovered from oil palm expressed sequence tag (EST) collection of two different tissues (callus and embryoids) in the oil palm tissue culture process. The nomenclature given was EAP X and CNH X. EAP refers to SSR uncovered in an embryoid tissue EST collection while CNH refers to SSR obtained from a callus EST collection. X refers to the clone number.

Single nucleotide polymorphism (SNP) Array

Sequences for the entire collection of SNPs were analysed by bioinformaticians from Illumina to screen and identify suitable SNP sequences for primer design. These were ranked for their suitability for primer design. The first oil palm SNP array was then designed based on a random selection of 96 SNPs (ranking, gene identity were taken into consideration). The array was synthesized using the BeadArray Technology and deployed as a Sentrix 96 array matrix (SAM), hi this format, the first oil palm SNP array comprising of 96 SNPs (as 3- micron features) were assembled into the microwells of each Sentrix array. In this format, 96 samples can be genotyped simultaneously with each of the 96 SNPs. A total of 105 samples from the mapping population were genotyped in this experiment.

Data analysis

The AFLP loci were scored as dominant markers, and, as such, segregation was scored on the basis of presence or absence of the amplified band. The RFLP bands were scored as co-dominant markers and, for this selfed cross, loci segregating in the 1:2:1 ratio was scored. Some RFLP probes revealed complex patterns, for which the alleles were difficult to determine. For such cases, segregating bands were individually scored as being absent or present.

Table 1 provided below illustrates the 2 different types of segregation patterns observed in the mapping family from RFLP, SSR, SNP and AFLP markers.

Map construction

Map construction was carried out by using the Joinmap ver. 3.0 computer programme (Ooijen and Voorrips, 2001). The oil palm is an out-breeding species, and as such, a high degree of heterozygosity can be expected in its genome. The progeny palms from the selfed cross can thus be expected to behave like an F₂ population.

However, in map construction, it is important to first determine the "phases" of the markers, either in coupling or repulsion. For this reason, the selfed population was first analysed as a Cross Pollinator and type code CP was used in the analysis. For dominant markers, AFLP and some RPLP loci, the data were entered as "k-" for presence of a segregating band and "hh" for absence of a segregating band. The segregation type was entered as <hkxhk>. For co-dominant markers (RFLP and SSR) the data were scored as "hh": top band present; "hk": both bands present and "kk": bottom band present. Once the "phases" were determined the data was converted back for analysis as a "F2" population type. The data were converted back to avoid distortions in the goodness- of- fit to the expected Mendelian segregation ratios. The data were converted back as recommended by Ooijen (personal communication) as follows:

For markers showing phase (0,0) the dominant markers were converted as follows: Λk-' converted to ^Λc' 'hh¹ converted to V

For co-dominant markers, the conversion was as follows: 'hh' converted to 'a' 'kk' converted to 'b' 'hk' converted to 'h' For markers showing phase (1,1), the dominant markers were converted as follows: 'k-' converted to 'd' 'hh' converted to 'b'

For co-dominant markers, the conversion was as follows: 'hh' converted to 'b' 'kk' converted to 'a' 'hk' converted to 'h'

It is noted that two maps were constructed, one as a cross pollinator (CP) and the second as an "F2" population with the required marker conversion. Both analyses gave exactly the same outcome, indicating that marker conversion was carried out successfully.

Markers were first divided into linkage groups using a LOD score threshold of 4.6. After being divided into linkage groups stringent parameters were applied for map construction that is a recombination value of 0.32 and LOD score of 1.0. A ripple was performed after the addition of every three markers and map distances were calculated using the Kosambi map function. JoinMαp constructs maps in three cycles. In the first two cycles, the markers which exceeded the JUMP threshold are excluded. In the third cycle, markers excluded are inserted, with no restriction on the JUMP threshold. In this study, the ordering produced in the second cycle was taken. Markers which caused inconsistencies in the second cycle map were discarded.

Evaluation of monogenic traits The monogenic trait scored was shell thickness. The trait was evaluated on ripened bunches in the field. The criteria used to determine ripened bunches was described by Corley and Tinker (2003), one loose fruit per bunch (irrespective of palm height). Three fruits per bunch and three bunches per palm (for a total of nine fruits) were evaluated for the trait. All in the palms in the mapping family were examined for the trait.

In order to determine the shell thickness, the individual fruits were cut into equal halves using a sharp knife. A visual observation was made of the shell and classified as recommended by Corley and Tinker (2003). dura: thick shelled, 2-8 mm tenera: thin shelled, 0.5-4mm, fibre ring present pisifera: shell-less

In most cases, the presence of the fibre ring was sufficient to distinguish between dura and tenera fruit forms. Only in the very rare case where the fibre ring was not obvious, a physical measurement of the shell thickness was made using a vernier caliper, and designated as dura or tenera fruit form based on the above mentioned criterion.

Results

With the method described above, a novel selection marker SFB83 linked to shell gene comprising the DNA sequence SEQ ID NO: 17 in the sequence listing was determined.

Further in accordance with the method of the present invention, a total of 678 polymorphic loci (491 AFLP, 135 RFLP, 29 SSR and 23 SNP) were identified, 640 of which were found to be linked to at least one other marker at a LOD score of 4.6. A total of 31 markers were unlinked and not assigned to any linkage group. Among the unlinked markers, 26 were AFLP, four were SSR and one was RFLP.

The chosen software for the purpose of the present invention was JoinMap ver 3.0 software which carries out map construction in three cycles. The first two cycles are stringent, where markers meeting the set conditions are considered for map construction, while in the third cycle all available markers are mapped together in a particular linkage group. The method of the present invention uses only the markers linked in the second cycle.

A graphical summary of the genetic map obtained with the method of the present invention is as shown in Figure 1. A total of 515 markers (351 AFLP, 124 RFLP, 17 SSR and 23 SNP) mapped to 16 linkage groups, thirteen (13) of the groups had more than 20 markers each (Groups 1-13), two groups had between 16-18 markers (Groups 14-15) and one group had eight markers (Group 16). The oil palm has haploid chromosome number of 16, as shown by cytogenetic analysis (Maria et. al. 1995). The number of linkage groups observed in this study is 16, which is similar to the chromosome number of 16. In accordance with the present invention, the total length of the map constructed was 1,599.5 cM. The individual linkage groups varied between 32 cM and 172 cM, with a mean of about 100 cM. The average interval between two loci was 3 cM. In the present linkage map, no marker free regions of more than 20 cM were observed in any one linkage group.

The majority of the markers obtained or identified were dominant AFLP markers. The AFLP markers were mostly uniformly distributed over the linkage groups. Some of the groups (examples Group 1 and 5) had two or three AFLP markers clustering around a single locus but generally the clustering of the AFLP markers was minimal.

Further in accordance with the present invention, a total 124 RFLP loci were successfully mapped. The RFLP markers were also found to be well distributed over the linkage groups. All of the other linkage groups had at least one RFLP marker each (with nine groups having more than eight RFLP markers). In certain regions, for instance in Groups 1, 2, 7 and 8, a few of the RFLP markers were found to be tightly linked and mapped in close proximity to one another, without being interrupted by AFLP, SSR or SNP markers.

In addition, 17 out of 29 SSR loci were successfully mapped to eight of the linkage groups. All 23 SNP markers were also successfully mapped with six of the groups having at least one SNP marker.

At least four of the RFLP probes used with respect to the present invention revealed co-dominant duplicated loci — four alleles segregating. Duplicated loci for some of the probes, for instance SFB70, showed almost similar segregation profile, and as expected mapped in the same group (Group 1), and around the same region. Similar results were observed for SFB 56, where the locus for this marker was found to be duplicated around the same region in Group 2. The duplicated locus, G163, is mapped on separate linkage groups. Gl 63 detected loci on Groups 7 and 13 respectively. With respect to the other duplicated loci, SFB7, only one locus could be mapped.

A further 3 probes, namely MET25, ME51 and G39 also revealed more than one locus, each of which were scored as dominant markers (segregating in the 3:1 ratio as shown in Table 1), As such the 124 RFLP markers were generated from 118 independent cDNA probes, .The sequence listing of said 118 independent cDNA probes generating the RFLP markers is provided together with this specification.

Table 2: Analysis of palms for fruit type

Number of palms genotyped Fruit Type

Expected Ratio Observed Numbers Dura Tenera Pisifera Dura Tenera Pisifera Total^# χ²

105 1 2 1 21 48 30 99 1.7*

* Significant at P=0.05

" There was a discrepancy between the total number of palms genotyped and that observed for fruit type because some palms had not borne fruits at the time the observations were made.

It is noted that the shell gene trait in the mapping population used for the purpose of the present invention followed the expected Mendelian ratio (1 :2:1 for the dura, tenera and pisifera fruit forms, respectively). The shell gene locus was successfully mapped in Group 2, about 8 cM away from the closest marker, SFB83, which was an RFLP marker. The polymorphism for the RFLP marker was therefore revealed by digesting the DNA of the mapping family with the restriction enzyme Sstl.

Further in accordance with the present invention, the RFLP marker SFB83, was tested for its ability to distinguish among the three fruit types described in the preceding paragraphs. Figure 2 depicts autoradiograph of the segregation of the marker in a subset of the mapping family. The RFLP marker being co-dominant in nature was able to distinguish all three fruit forms (dura, tenera and pisifera) in the mapping family tested with about 85% accuracy. In Figure 2, it can be seen that the dura palms had the top band present, the tenera palms had both bands present while the pisifera palms mostly had the lower band present. The linkage of the probe SFB83 to the shell gene was tested in four other independent crosses. The probe was tested on DNA samples digested with the restriction enzyme Sstl. The results are shown in Figure 3. The marker could generally distinguish the different fruit fruits forms in all of the crosses with about 95% accuracy. The marker of the present invention could distinguish all three fruit forms in the tenera x tenera cross (Trial 0.305), with about 95% accuracy. The marker could further distinguish the dura and tenera fruit forms in the two independent dura and tenera (DxT) crosses with also about 90% accuracy. In DxT crosses only dura and tenera fruit forms are produced.

It is noted however from the obtained results that in the fourth independent cross involving a tenera x tenera cross (TTl 32), the probe SFB83 tested on DNA digested with the restriction enzyme Sstl, could not distinguish the three fruit forms. With another enzyme, namely Hindi, the probe SFB83 successfully could distinguish dura and tenera palms from the pisifera palms. Using this restriction enzyme, the probe displayed dominant profile whereby it was not able to distinguish the dura and tenera palms, both of which had the RPLP fragment present.

It is further noted that the pisifera palms could be largely distinguished from the dura and tenera palms, as all of the pisifera palms did not have any RFLP fragment present. The ability to distinguish pisifera palms alone provides advantages. In the production of commercial dura x pisifera (DxP) palms, pollen is usually collected from pisifera palms planted in different location or areas. An early selection marker will allow planting of pisifera palms in one geographical location, which can reduce time and effort in collecting pollen as well as making it easier to maintain the palms. Furthermore, a marker for pisifera could also facilitate the planting of pisifera palms at high density to encourage male inflorescence, and as such production of pollen for crossing programmes.

It will be realized that the distance of the RPLP marker from the shell gene loci obtained with the present invention is comparable to that of the genomic RPLP marker reported by another prior art, in particular by Mayes et. al, (1997) at 8 cM, but closer to that of the one reported by Moretzsohn et al, (2000), which is about 15 cM away from the locus.

The probe SFB83 of the present invention could distinguish all three fruit forms as it shows co-dominant profile. It is further discovered that the restriction enzyme Hindi may be utilized on the samples where the probe displayed a dominant profile and thus may be able to distinguish pisifera palms from that of the dura and tenera palms.

Conclusively, the selection marker of the present invention may allow at least 80 % enrichment of the desired genotype.

Although the present invention has been described with reference to the preferred embodiments and examples thereof, it is apparent to those skilled in the art that a variety of modifications and changes may be made without departing from the scope of the present invention which is intended to be defined by the appended claims.

Claims

1. A method for identifying a genetic marker linked to a trait locus of an oil palm fruit using co-dominant RFLP , SSR (microsatellite), SNP and dominant AFLP markers, said method comprising the steps of: a) preparing DNA sample based on the said oil palm; b) digesting the DNA from step a) with a restriction enzyme; wherein the digesting step further comprising :

1. preparing a first mixture of the said DNA from step a) , said mixture containing sterile water and a predetermined amount of DNA from step a); ii. preparing a second mixture containing a predetermined amount of restriction buffer and restriction enzyme; iii. adding the first mixture into the second mixture; conducting RFLP method, wherein the probe/restriction enzyme combinations having single/low copy of co-dominant profile are selected; conducting the AFLP method with pre-selective and selective amplification, wherein said

AFLP method involved generating the AFLP markers with the usage of restriction enzymes; preparing microsatellite (SSR) primers from oil palm; and thus conducting the SSR method involving the said oil palm (SSR) microsatellite primers; preparing the oil palm single nucleotide polymorphisms (SNP) and genotyping based on said oil palm SNP primers; conducting data analysis based on RFLP, AFLP , SSR (microsatellite) and SNP markers; and constructing the genetic map based on the results obtained in from said RFLP method,

AFLP method, SSR method and SNP method.

2. The method as claimed in Claim 1 wherein the method is used for construction of a dense genetic map for establishing the relationship between mapped markers with traits of interest.

3. The method as claimed in Claim 1 wherein the oil palm fruit is tenera.

4. The method as claimed in Claim 1 wherein the trait locus contributes to the shell thickness of said oil palm fruit.

5. The method as claimed in Claim 1 wherein a total of 515 markers, mapped to 16 linkage groups is obtained.

6. The method as claimed in Claim 1 wherein 351 AFLP markers are obtained.

7. The method as claimed in Claim 1 wherein 124 RFLP markers are obtained, whereby said 124 RFLP markers are generated from 118 independent cDNA probes as provided in the sequence listing.

8. The method as claimed in Claim 1 wherein 17 and 23 markers are obtained for SSR and SNP respectively.

9. The method as claimed in Claim 1 for use in plant identification and plant breeding purposes.

10. The method as claimed in Claim 1 wherein the genetic linkage map is constructed with the assistance of a suitable software.

11. A selection marker obtained and identified on a linkage group with the method as claimed in Claim 1.

12. The selection marker as claimed in Claim 12 wherein said marker is SFB83.

13. The selection marker as claimed in Claim 12 to 13 wherein said marker is able to distinguish dura, tenera, pisifera fruit forms with about 90% accuracy.

14. A selection marker comprising DNA described in SEQ ID NO: 17 in the sequence listing.