WO2011084047A2

WO2011084047A2 - Identification of molecular markers associated with fatty acid composition in plants

Info

Publication number: WO2011084047A2
Application number: PCT/MY2011/000003
Authority: WO
Inventors: Rajinder Singh Harminder Singh; Suan Choo Cheah; Rahimah Abd. Rahman; Cheng Li Leslie Ooi
Original assignee: Malaysian Palm Oil Board
Priority date: 2010-01-11
Filing date: 2011-01-07
Publication date: 2011-07-14
Also published as: CO6592066A2; WO2011084047A3

Abstract

A map was constructed from AFLP, RPLP and SSR analysis of the progeny derived from an interspecific cross involving a Colombian Elaeis oleifera (UP 1026) and a Nigerian E. guinneensis (T128). This interspecific cross was used to map genes associated with oil quality. A framework map was generated for the male parent, T128, using Joinmap ver. 3.0. In the paternal guineensis map, 389 markers (305 AFLP, 64 RFLP and 20 SSR) were ordered in 18 linkage groups (1571cM). The E. guineensis map was also used in scanning for quantitative trait loci (QTLs) controlling oil quality (measured in terms of iodine value and fatty acid composition). At a 99 % significant genomic wide threshold level, QTLs associated with iodine value (IV), C14:0, C16:0, C16:1, C18:0 and C18:1 were detected. The same genomic region appears to be influencing IV, C18:1 and C16:0 content. Considering the three traits are strongly correlated further supports the data observed. The QTLs also explain a significant proportion (about 35%) of the variation observed. Similarly significant QTLs for C16:1 and C14:0 were detected around the same locus in separate genomic region. This probably points to another major locus influencing fatty acid composition. Markers associated with IV and FAC (C14:0, C16:0, C16:1 and C18:1) were confirmed in a second mapping population or in palms of different genotype. The markers concerned are RFLP probes, namely CB75A (for IV, C16:0, C18:1) and RD56 (for C14:0 and C16:1) and microsatellite (SSR) marker P4T8 (C18:0 and C16:0).

Description

IDENTIFICATION OF MOLECULAR MARKERS ASSOCIATED WITH FATTY ACID COMPOSITION IN PLANTS

BACKGROUND OF THE INVENTION

. .

FIELD OF THE INVENTION

The present invention provides methods for selecting plants based on a desired characteristic. The subject invention relates particularly to methods of selecting plants based on the fatty acid content of the oil isolated from the plant.

DESCRIPTION OF THE PRIOR ART

The oil palm is perennial crop which belongs to the genus Elaeis and to the botanical · family Palmae. Palm oil is a form of edible vegetable oil obtained from the fruit of the oil palm tree. Palm oil has now surpassed soybean oil as the most widely produced vegetable oil in the world.

The palm fruit is the source of both palm oil (extracted from mesocarp of the fruit) and palm kernel oil (extracted from the fruit seeds).

Palm oil itself is reddish because it contains a high amount of betacarotene. It is used as cooking oil, to make margarine and is a component of many processed foods. Boiling it a few minutes destroys the carotenoids and the oil becomes white.

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Palm oil is one of the few vegetable oils relatively high in saturated fats (such as coconut oil) and thus semi-solid at room temperature.

The palm oil and palm kernel oil are composed of fatty acids esterified with glycerol just like any ordinary fat. Both are high in saturated fatty acids. The oil palm gives its name to the 16 carbon saturated fatty acid palmitic acid found in palm oil; monosaturated oleic acid is also a constituent of palm oil while palm kernel oil contains mainly lauric acid. Palm oil is the largest natural source of tocotrienol, part of the vitamin E family. Palm oil is also high in vitamin K and dietary magnesium.

The proximate concentration of fatty acids (FAs) in palm oil is as follows:

• Saturated (total: 49.9%)

o Palmitic CI 6:0 44.3%

o Stearic CI 8:0 4.6%

o Myristic C 1^*4:0 1.0%

· Monounsaturated

o Oleic CI 8:1 38.7%

• Polyunsaturated

o Linoleic C18:2 10.5% For palm kernel oil the fatty acid content is:

• Saturated (total : 82%)

■ Lauric C12:0 48.2%

■ Myristic C14:0 16.2%

■ Palmitic CI 6:0 8.4%

■ Capric C10:0 3.4%

■ Caprylic C8:0 3.3%

■ Stearic CI 8:0 2.5%

• Monounsaturated

■ Oleic C18:l 15.3%

• Polyunsaturated

■ Linoleic C18:2 2.3%

Within the genus Elaeis two species are distinguished, the economically important oil palm (Elaeis guineensis) originally native to Africa and a South American relative, Elaeis oleifera. Morphologically, the main feature of the E. oleifera palm which distinguishes it from the commercial species, E. guineensis, is its procumbent trunk (Corley and Tinker, The Oil Palm (4* edition), pp: 287-326, Blackwell Science, 2003). The individual fruits of E. oleifera are much smaller than those of E. guineensis, and the size of the bunch is about 15-20 kg, compared to about 25 kg on the average for the Deli E. guineensis (Latiff, Advances in Oil Palm Research, Volume 1, pp: 19-38, Malaysian Palm Oil Board (MPOB), 2000). In E. oleifera up to 90% of the fruits tend to be parthenocarphic in nature (Hartley, The oil palm (3^rd edition), pp: 47-94, Longman, London, 1988), which carries a much lower oil content (Meunier and Boulin, Oleagineux 30:5-8, 1975). As such, the oil yield of E. oleifera is much lower, with the oil to bunch ratio of 5%, as compared to the E. guineensis (tenera), which is more than 25% (Rajanaidu et al, In Advances in Oil Palm Research, Volume 1, pp:171-237, Malaysian Palm Oil Board, 2000). Nevertheless, E. oleifera does possess certain attributes that are of interest to oil palm breeders. The annual height increment is only 5-10 cm, less than one-fifth of that of E. guineensis (Corley and Tinker, The Oil Palm (4^th edition), pp: 287-326, Blackwell Science, 2003). The characteristic of its mesocarp oil is especially of interest. The iodine value (IV, which is a measure of oil unsaturation) of E. oleifera oil can reach up to more than 90, compared to E. guineensis oil, which on average has an rV of 53.3 (Rajanaidu et al, In Advances in Oil Palm Research, Volume 1, pp:171-237, Malaysian Palm Oil Board, 2000). The fatty acid composition is also unique, as E. oleifera oil has high levels of oleic and linoleic acid and lower levels of the palmitic acid and other saturated fatty acids, giving it a property quite similar to olive oil in composition. Interest in E. oleifera was also fuelled by the fact that it showed resistance to the bud rot disease in Colombia and its possible adaptation to a climate with a strong dry season (Hartley, The oil palm (3^rd edition), pp: 47-94, Longman, London, 1988). An interspecific E. oleifera x E. guineensis hybrid programme was proposed as a breeding method to introgress genes for slow height increment, high oil' unsaturation and disease tolerance from the E. oleifera into the high oil yielding E. guineensis (Meunier and Hardon, Oil Palm Research, pp:127-138, 1976). In Malaysia, one of the main objectives of the hybrid-breeding programme is to improve oil quality. This is a long term breeding strategy, with results obtained thus far showing that oil quality, taken as its unsaturated fatty acid content, being better in the hybrids and in their backcrosses than in the commercial E. guineensis (Suheimi and Lubis, Bulletin Pusat Penelitian Marihat 5:37-44, 1985; Chin et al, Palm Oil International Conference, Malaysian Palm Oil Board, pp: 36-50, 2003). Traditional breeding is playing an important role in achieving the aims of the hybrid breeding programme. However, it is impeded by the long selection cycle (10-12 years) (Oboh and Fakorede, Oleagineux 44:509-513, 1989) and the enormous resources (land, labour and field management) required for oil palm breeding programmes. The introduction of marker-assisted selection (MAS) techniques into the hybrid breeding programme can help to speed up the breeding objective of increasing the levels of unsaturated fatty acids. With MAS, selection in segregating generations of interspecific hybrids and their backcrosses based on the presence or absence of molecular markers linked to fatty acid composition is possible.

DNA based markers such as restriction fragement length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) markers, amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers have been applied to oil palm to investigate genetic diversity (Shah et al, Theoretical and Applied Genetics 89:713-718, 1994; Billotte et al, Genome 44:413-425, 2001), clone fingerprinting (Mayes et al, Molecular Breeding 2:175-180, 1996) and at attempts in identifying markers for somaclonal variation (Rival et al, Plant Breeding 117:73-76, 1998; Matthes et al, Theoretical and Applied Genetics 102:971-979, 2001). A number of these marker systems have also been applied to genetic mapping in oil palm. RFLP markers from genomic libraries have been applied to oil palm linkage mapping (Mayes et al, Genome 40:116-122, 1997). The map harbors 97 RFLP markers in 24 groups of two or more and was generated using a selfed guineensis cross. Moretzsohn et al, Theoretical and Applied Genetics 100:63-70 (2000) reported genetic linkage mapping for a single controlled cross of oil palm using RAPD markers and the pseudo-testcross mapping strategy. More recently Billotte et al, Theoretical and Applied Genetics 110:754-765 (2005) reported a SSR-based high density linkage map for oil palm, involving a cross between a thin shelled E. guineensis (tenera) palm and a thick shelled E. guineensis (dura) palm. The map consisting of 255 microsatellite markers and 688 AFLPs, represents the first linkage map for oil palm to have 16 independent linkage groups which correspond to the haploid chromosome number of 16 in oil palm (Maria et al, Elaeis 7:122-134, 1995). Despite the advances being made and the progress achieved in genetic mapping of oil palm, only a limited number of economically important traits have been tagged to date. Mayes et at, Genome 40:116-122 (1997), Moretzsohn et at, Theoretical and Applied Genetics 100:63-70 (2000) and Billotte et at, Theoretical and Applied Genetics 110:754-765 (2005) reported the identification of RFLP, RAPD and AFLP markers respectively, linked to the shell thickness locus. Shell thickness is an important economic trait which exhibits monofactorial inheritance, and is the basis for the classification of the E. guineensis species into three fruit forms, dura (thick shelled), tenera (thin shelled and pisfera (shelless). However, most of the traits of economic interest in oil palm exhibit quantitative inheritance. In this area, Ranee et at, Theoretical and Applied Genetics 103:1302-1310 (2001), expanding on the genetic map developed by Mayes et at, Genome 40:116-122 (1997), reported the detection of QTLs associated with vegetative and yield components of oil palm. The work reported above represents important developments in the application of MAS in oil palm breeding programmes.

In the present invention complementary DNA (cDNA) probes were exploited as RFLP markers. The cDNA clones occur in the expressed regions of the genome and represent gene fragments. Their identity can be determined via sequencing and such sequences are known as expressed sequence tags (ESTs). The usefulness of ESTs as markers has been demonstrated in several plant species (Matthews et at, Crop Science 41 : 516-521, 2001; Pfaff and Kahl, Molecular Genetics and Genomics 269:243-251, 2003). The use of EST probes help to map known genes as well as provide anchor probes for comparative mapping. Furthermore, by mapping ESTs closely linked to or co-segregating with a trait allows the gene for that trait to be identified by the. candidate gene approach. Apart from RFLP markers, AFLP and SSR markers were also used in the study. Bibliographic details of the publications referred to in this specification are also collected at the end of the description.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID Nos: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:l), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table A. A sequence listing is provided before the claims. Primer sequences of microsatellite primer sets are provided in Attachment 1.

The present invention is predicated in part on the determination of genetic markers which enable plants to be selected based on the fatty acid content (FAC) of oil isolated from the plant.

Accordingly, one aspect of the present invention provides a method for selecting a plant based on fatty acid content (FAC) of oil isolated from the plant, the method comprising screening for the presence or absence of a genetic marker in the plant, the genetic marker corresponding to a nucleotide sequence having at least 90% identity to at least 20 contiguous nucleotides of SEQ ID NO:3, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of the marker is indicative of a plant having oil with a high unsaturated FAC.

Another aspect of the present invention is directed to a method of selecting a plant based on the FAC of oil, wherein the genetic marker is CB75A. In another aspect the present invention is directed to a method of selecting a plant based on the FAC of oil, wherein the saturated fatty acid is selected from the group consisting of: myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0). In a further aspect, the present invention is directed to a method of selecting a plant based on the FAC of oil, wherein the unsaturated fatty acid is selected from the group consisting of oleic acid (CI 8:1), linoleic acid (CI 8:2) and alpha-linoleic acid (C18:3).

The present invention further provides, a method of selecting a plant based on the FAC of oil, wherein the absence of the genetic marker is further indicative of a plant having a high iodine value (IV). In another aspect, the present invention is directed to a method of selecting a plant based on fatty acid content (FAC) of oil isolated from the plant, the method comprising screening for the presence or absence of a genetic marker in the plant, the genetic marker corresponding to a nucleotide sequence having at least 90% identity to at least 20 contiguous nucleotides of SEQ ID NO:56, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of the marker is indicative of a plant having oil with a high unsaturated FAC.

Another aspect of the present invention is directed to a method of selecting a plant based on the FAC of oil, wherein the genetic marker is RD56.

In a related aspect, the present invention is directed to a method of selecting a plant based on fatty acid content (FAC) of oil, wherein the saturated fatty acid is myristic acid.

In another aspect, the present invention is directed to a method of selecting a plant based on fatty acid content (FAC) of oil isolated from the plant, the method comprising screening for the presence or absence of a genetic marker in the plant, the genetic marker corresponding to a nucleotide sequence having at least 90% identity to a contiguous nucleotides of SEQ ID 65 which is the primer sequence for microsatellite primer set P4T8, as shown in Table A and Attachment 1, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of the marker is indicative of a plant having oil with a high unsaturated FAC.

Another aspect of the present invention is directed to a method of selecting a plant based on the FAC of oil, wherein the genetic marker is P4T8.

In a related aspect, the present invention is directed to a method of selecting a plant based on fatty acid content (FAC) of oil, wherein the saturated fatty acid is stearic acid (C18:0) and wherein the unsaturated fatty acid is palmitoleic (C16:l) acid.

In a further aspect, the present invention is directed to a DNA molecule associated with FAC homologous or complementary to the DNA molecule disclosed in SEQ ID NO:3 located on linkage group 1 of the mapping population of T128, an oil palm of the species Elaeis guineensis or progeny thereof and is of a sufficient length to be useful as a DNA marker for an allele of a quantitative trait locus, wherein the allele determines the FAC content of a plant.

In another aspect, the present invention is directed to a DNA molecule associated with FAC homologous or complementary to the DNA molecule disclosed in SEQ ID NO:56 located on linkage group 12 of the mapping population of T128 or progeny thereof and is of a sufficient length to be useful as a DNA marker for an allele of a quantitative trait locus, wherein the allele determines the FAC content of a plant.

In another aspect, the present invention is directed to a DNA molecule associated with FAC homologous or complementary to the DNA molecule disclosed in SEQ ID NO:P4T8 located on linkage group 12 of the mapping population of T128 or progeny thereof and is of a sufficient length to be useful as a DNA marker for an allele of a quantitative trait locus, wherein the allele determines the FAC content of a plant. A summary of sequence identifiers used throughout the subject specification is provided in Table A and Attachment 1.

TABLE A

Summary of Sequence Identifiers

65 , Forward and Reverse primer sequence of clone P4T8

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a photographical representation of autoradiograms showing the segregating loci revealed by RFLP markers KT19 (A), FDA39 (B). UP 1026 and T128 are the female parent, Colombian oleifera and male parent, Nigerian guineensis, respectively. (<— ) indicates segregating bands.

Figure 2 is a schematic representation of a combined AFP, RFLP and SSR map of interspecific hybrid (Palm T128).

Figure 3 is a graphical representation of QTL graphs for IV and fatty acid composition (C14:0, C16:0, C16:l, C18:0). Figure 4 is a photographical representation of the validation of Probe CB75A in independent palms.

Figure 5 is . a graphical representation of QTL graphs for fatty acid composition (C14:0, and CI 6:1) in the second mapping population. The same markers RD56 and P4T8, appear to be linked to C14:0 and C16:l content.

DETAILED DESCRIPTION OF SOME PREFERRED EMBODIMENTS

OF INVENTION

The present invention provides methods for selecting a plant based on the fatty acid content (FAC) of the oil isolated' from said plant. The invention particularly relates to the use of molecular markers particularly genetic markers in the identification of fatty acid content particularly the content of saturated and unsaturated fatty acid in the oil isolated from said plant. The said saturated fatty acids include myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (CI 8:0). The unsaturated fatty acids include palmitoleic acid (C16:l), oleic acid (C18:l), linoleic acid (C18:2) and alpha- linoleic acid (C18:3).

In describing or claiming the present invention, the following terminology are used in accordance with the definitions set forth below. Before describing the present invention detail, it is to be understood that unless otherwise indicated, the subject invention is not limited to specific formulations or components, manufacturing methods, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly indicates otherwise. Thus, for example, reference to a "compound" includes a single compound, as well as two or more compounds; references to "an active agent" includes a single active agent, as well as two or more active agents; "a holocyclotoxin" includes a single holocyclotoxin or two or more holocyclotoxins and so forth.

The term "oil palm", as used herein should be understood to refer oil palm plants which include species such as Elaeis oleifera and Elaeis guinneensis including modified varieties or genetic variants thereof.

The term "fruit" as used herein is intended to encompass any object or objects that are produced by a plant in response to a fertilization event, whether a self- or non- self fertilization, and whether or not the resulting fruit is sterile or non-sterile. For example, both an apple and the seeds of the apple should be viewed as "fruit" herein. As another example, seedles fruits such as grapes and tangerines are fruit. "Fruit" are not limited to edible objects. For example, the poisonous berries of the yew are "fruits" as are any fruits and nuts produced by palms. Other exemplary "fruit" include peanuts, tomatoes, corn, bananas, wheat berries, pears, etc.

The term "oil" used interchangeably herein to refer to any material or mixture of materials that is primarily composed of one or more highly hydrophobic substances such as fatty acids or true fats (e.g. esters of fatty acids and glycerol). The term "molecular marker" includes any gene fragment which can be used to select for a phenotype or which when expressed facilitates the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof. The terms such as "hybridization", "hybridizing" and the like are used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA, U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.

In a preferred embodiment of the present invention, an interspecific mapping population derived from the cross of the Colombian female parent, UP 1026 {Elaeis oleifera) x the male Nigerian parent, T128 {Elaeis guinneensis) was analyzed. In order to determine markers associated with iodine value (IV), restriction fragment length polymorphism (RFLP) markers (designated CB75A and RD56) which are able to distinguish palms having high and low unsaturated oil (measured in terms of iodine value, IV) were developed. These restriction fragment length polymorphism (RFLP) markers, CB75A and RD56 are able to distinguish palms having high and low unsaturated oil (measured in terms of iodine value, IV) as well as palms having high and low amounts of fatty acids, namely myristic acid (CI 4:0), palmitic acid (C16:0), palmitoleic acid (C16:l), stearic acid (C18:0) and oleic acid (C18:l). The RFLP probes used to screen the interspecific mapping family were cDNA clones obtained from various cDNA libraries (young etiolated seedlings, mesocarp, kernel and root) wherein the marker, CB75A, is a complementary DNA (cDNA) probe obtained from a callus library, while marker, RD56, is a cDNA probe obtained from a root library. For the screening procedure, Southern blotting, pre-hybridization and hybridization were carried out. Alternatively, the present invention also involves the application of amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers for further analysis on the fatty acid content (FAC) from the oil palm plant. A map was constructed from AFLP, RFLP and SSR analysis of the progeny derived from the above mentioned interspecific cross involving the Colombian female parent, UP1026 {Elaeis oleifera) and the male Nigerian parent, T128 {Elaeis guinneensis) wherein the said interspecific cross was used to map genes associated with oil quality. A framework map was generated for the male Nigerian parent, T128 {Elaeis guinneensis), using Joinmap ver. 3.0. In the paternal guinneensis map, 389 markers (305 AFLP, 64 RFLP and 20 SSR) were ordered in 18 linkage groups (1571cM). The E. guinneensis map was also used in scanning for quantitative trait loci (QTLs) controlling oil quality which is measured in terms of iodine value, IV and fatty acid composition, FAC. RFLP, AFLP and SSR were used to study marker screening, segregation analyses of AFLP, RFLP and SSR markers in the mapping population, linkage analysis, segregation of markers associated with quantitative trait loci (QTLs) and validation of probes on independent palms and second mapping population. The markers were uncovered in the genetic mapping experiment using the above interspecific cross involving the Colombian species Elaeis oleifera (UP 1026) and Nigerian species Elaeis guinneensis (T128). The E. guinneensis map was used in scanning for quantitative trait loci (QTLs) controlling oil quality (measured in terms of iodine value and fatty acid composition) as the quantitative traits associated with oil quality were of interest of this study. At a 99% significant genomic wide threshold level, QTLs associated with iodine value (IV), C14:0, C16:0, C16:l, C18:0 and CI 8:1 were detected. The QTLs also explain a significant proportion (about 35%) of the variation observed. Markers associated with TV and FAC (C14:0, C16:0, CI 6:1 and CI 8:1) were confirmed in a second mapping population or in palms of different genotype. The markers concerned are RFLP probes, namely CB75A (for IV, CI 6:0, CI 8:1) and RD56 (for C14:0) and microsatellite (SSR) marker P4T8 (for CI 8:0 and C16:l). It has been postulated that identification of these markers associated with IV and FAC, will make it easier for breeders to select desirable palms for planting at the nursery stage and/or select the appropriate palms for subsequent crossing to help alter palm oil saturation levels or fatty acid composition. The markers can be immediately applied to selection for genotypes with different levels of unsaturation, as well as fatty acid composition wherein with the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of said marker is indicative of a plant having oil with a high unsaturated FAC.

Some major aspects of the present invention have been described in the preceding section titled "SUMMARY OF THE INVENTION".

Further aspects or embodiments of the present invention will now be described in the following paragraphs. Further aspects of the invention provide a method for selecting a plant based on fatty acid content (FAC) of oil isolated from said plant, said method comprising screening for the presence or absence of a genetic marker in the plant, said genetic marker corresponding to a nucleotide sequence having at least 90% identity to at least 20 contiguous nucleotides of SEQ ID NO:3, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of said marker is indicative of a plant having oil with a high unsaturated FAC or wherein the genetic marker is CB75A and/or wherein the saturated fatty acid is selected from the group consisting of: myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0). In the aforesaid further aspects or embodiments of the. invention, the level of saturated FAC may be greater than each of the following percentages of total FAC, namely greater than 50%, 60%, 70%, 80%, 90%, 95% or 98% of total FAC.

Yet further aspects or embodiments of the present invention provide a method for selecting a plant based on fatty acid content (FAC) of oil isolated from said plant, said method comprising screening for the presence or absence of a genetic marker in the plant, said genetic marker corresponding to a nucleotide sequence having at least 90% identity to at least 20 contiguous nucleotides of SEQ ED NO:3, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of said marker is indicative of a plant having oil with a high unsaturated FAC or wherein the genetic marker is CB75A and/or wherein the unsaturated fatty acid is selected from the group consisting of oleic acid (CI 8:1), linoleic acid (CI 8:2) and alpha-linoleic acid (CI 8:3). In the aforesaid further aspects or embodiments of the invention, the level of unsaturated FAC may be greater than each of the following percentages of total FAC, namely greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of total FAC. In the aforesaid further aspects/embodiments of the invention, the level of oleic acid may be 60%. Where the level of oleic acid is 60%, the level of palmitic acid may be 25%.

Further aspects of the invention provide a method for selecting a plant based on fatty acid content (FAC) of oil isolated from said plant, said method comprising screening for the presence or absence of a genetic marker in the plant, said genetic marker corresponding to a nucleotide sequence having at least 90% identity to at least 20 contiguous nucleotides of SEQ ID NO:3, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of said marker is indic tive of a plant having oil with a high unsaturated FAC or wherein the genetic marker is CB75A and/or wherein the absence of the genetic marker is further indicative of a plant having a high iodine value (TV). In the aforesaid further aspects of the invention, the iodine value (IV) may be greater than each of the following IV values, namely greater than 60, 70, 80, 90, 95 or 98.

In yet further aspects of the invention, there are provided a method of selecting a plant based on fatty acid content (FAC) of oil isolated from said plant, said method comprising screening for the presence or absence of a genetic marker in the plant, said genetic marker corresponding to a nucleotide sequence having at least 90% identity to at least 20 contiguous nucleotides of SEQ ID NO:56, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of said marker is indicative of a plant having oil with a high unsaturated FAC or wherein the genetic marker is RD56 and/or wherein the saturated fatty acid (saturated FAC) is myristic acid . In the aforesaid further aspects of the invention, the level of saturated FAC may be greater than each of the following percentages of total FAC, namely greater than 50%, 60%, 70%, 80%, 90%, 95% or 98% of total FAC. In the aforesaid aspects of the invention, the unsaturated FAC may be palmitoleic (CI 6:1) acid. In the aforesaid further aspects of the invention, the unsaturated FAC may be greater than each of the following percentages of total FAC, namely greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% of total FAC. Further aspects of the invention also provide a method of selecting a plant based on fatty acid content (FAC) of oil isolated from said plant, said method comprising screening for the presence or absence of a genetic marker in the plant, said genetic marker corresponding to a nucleotide sequence having at least 90% identity to at least 20 contiguous nucleotides of SEQ ID NO:65, which is the primer sequence for microsatellite primer set P4T8, as shown in Attachment 1, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of said marker is indicative of a plant having oil with a high unsaturated FAC, or wherein the genetic marker is P4T8 and/or wherein the saturated fatty acid (saturated FAC) is stearic acid. In the aforesaid further aspects of the invention, the level of saturated FAC may be greater than each of the following percentages of total FAC, namely greater than 50%, 60%, 70%, 80%, 90%, 95% or 98% of total FAC. In the aforesaid aspects of the invention, the unsaturated FAC may be palmitoleic (CI 6: 1) acid. In the aforesaid further aspects of the invention, the unsaturated FAC may be greater than each of the following percentages of total FAC, namely greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% of total FAC.

In all the aspects/embodiments of the invention described in the preceding graphs of this section as well as the section titled "SUMMARY OF THE INVENTION", the plant may be an oil palm plant.

The present invention is further described by the following non-limiting Examples.

EXAMPLES

Example 1

Plant materials

An interspecific mapping population derived from the cross UP1026 (E. Oleifera) x T128 (E. guineensis) was analyzed. The female parent, UP1026, is a Colombian oleifera and the male parent, T128, is a Nigerian guineensis which produces oil with a high iodine value (IV). Controlled self-pollination was used to generate the mapping population.

A total of 117 palms were planted and evaluated at one location at United Plantations, Teluk Intan, Perak, Malaysia. Unopened leaf samples (spear leaf) were collected from all 117 individual palms and immediately frozen under liquid nitrogen and then stored at - 80°C until DNA preparation.

In order to determine markers associated with iodine value (IV), palmitic acid (C16:0) and oleic acid (C18:l), leaf samples were harvested from 13 palms of a different interspecific cross. The fatty acid composition (FAC) for each of the palms had been determined previously.

A second mapping population was also used to verify the markers associated with myristic acid content (C14:0) and palmitoleic acid content (C16:l). The mapping population used was derived from the selfing^'of the high iodine value (IV) tenera palm, T128, from MPOB's Nigerian germplasm collection (Rajanaidu, Major developments in oil palm (Elaeis guineensis) breeding, Proceedings of thel2th Plenary Meeting of Association for the Taxonomic Study of the Flora of Tropical Africa (AETFAT), pp 39-52, Mitteilungen Inst. Allg. Bot. Hamburg, Germany, 1990; Cheah and Rajinder, Project Completion Report No. 0057/98, 16th July 1999, Malaysian Palm Oil Board (MPOB), 1999). Controlled self-pollination was used to generate the mapping family. A total of 320 palms were planted at several locations in Malaysia, including the MPOB-UKM Research Station at Bangi, Selangor, Ulu Paka Research Station at Terengganu, Keratong Research Station at Kluang, Johore, Lahad Datu Research Station at Sabah and United Plantations, Teluk Intan, Perak. In this study, a total of 192 palms from MPOB-UKM, Ulu Paka and United Plantations were used for the purpose of genetic linkage mapping.

Preparation of genomic DNA

DNA was prepared from young spear leaves based on the method of Doyle and Doyle, FOCUS 12:13-15 (1990). Amplified fragment length polymorphism (AFLP) procedure

AFLP analysis was carried out by using the EcoRVMsel and Tagil Hindlll enzyme pairs. The EcoRVMsel assay was carried out by using the GIBCO BRL AFLP Analysis System 1 (INVITROGEN, USA), essentially as described in the manufacturer's manual. The AFLP analysis using the TagVHindlll enzyme pairs was essentially performed as described by Rafalski et al, Non-mammalian Genomic Analysis-A Practical Guide, ed. B. Birren, and E. Lai, San Diego, Academic Press (1996).

Example 2

Restriction fragment length polymorphism (RFLP) analysis i. RFLP Probes

The RFLP probes used to screen the interspecific hybrid mapping family were cDNA clones obtained from various cDNA libraries (young etiolated seedlings, mesocarp, kernel and root) constructed previously as described by Cheah, Project Completion Report No. 0011/95, 4^th My 1996, Malaysian Palm Oil Board (MPOB) (1996). cDNA clones from a subtracted flower library (Cheah and Rajinder, Project Completion Report No. 0057/98, 16th July 1999, Malaysian Palm Oil Board (MPOB), 1999) were also used to screen the mapping population. The sequences for these clones are disclosed in SEQ ID NOs: 1 to 64.

Plasmid DNA was prepared from individual clones by using column purification (Qiagen, USA). The presence of the DNA insert was examined for by restriction digestion (EcoRl) and electrophoresing on a 1.5% agarose gel. cDNA clones with insert sizes larger than 500 base-pairs (bp) were selected to screen for their ability to detect RFLP in the mapping population.

The DNA probes were diluted to a concentration of 5 ng/μΐ in TE buffer. The DNA probe was then labeled with a?²P-dCTP (3000 Ci/mmol stock) b using the Megaprime DNA Labeling system (GE Healthcare), as recommended by the manufacturer. The labeled probe was separated from the unincorporated nucleotides by purification through a Sephadex column as described in Sambrook et al, A Laboratory manual, (2^nd edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). ii. Southern Blotting and Hybridization

For the screening procedure, DNA samples (20 μg) from 10 palms (including the parental palms) were digested with 14 restriction enzymes (BamHl, Bell, BgUl, Dral, EcoBI, Hindi, Hindlll, Seal, Sstl, Xbal, BsMl, Haelll, Rsal and TaqT). The restricted DNA fragments were separated by electrophoresis in 0.8% agarose gel in 1 x TAE (0.04 M Tris-acetate, pH 7.9, 1 mM EDTA) buffer and then transferred onto nylon membranes (Hybond N+, GE Healthcare Biosciences, UK) by vaccum blotting.

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 polymorphism with a particular probe, the probe/enzyme combination that gave a single/low copy clear profile was selected for screening the entire mapping population, cleaved with the. appropriate restriction enzyme.

Pre-hybridization and hybridization were carried out in glass tubes in a rotisserie oven at 65 °C. Membranes were pre-hybridized for about 6 hours in a solution containing pre-hybridization buffer containing: 5 x SSPE solution (3 M NaCl, 0.2 M sodium phosphate, 20 mM EDTA pH 8.0), 0.5% SDS, 5 x 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 of 5 x 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 a boiling water bath for 10 min and plunging into ice water, before addition 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 65 °C. Hybridized membranes were washed twice in 2 x SSC (0.3 M NaCl, 30 mM trisodium citrate, pH 7), 0.1% SDS at 65 °C for 15 min each time, followed by once in 1 x SSC (0.15 M NaCl, 15 mM tri sodium citrate pH 7), 0.1% SDS at 65 °C for 15 min. The membranes were then autoradiographed at -80 °C using X-ray films with intensifying screens for 7 to 10 days, X-ray films were developed using 0.22 x Kodak's GBX developer for 5 min, rinsed in distilled water, followed by a final wash with 0.2 x Kodak's GBX fixer for 5 min. EXAMPLE 3

Microsatellites

A. Isolation of Microsatellites in Oil Palm

Degenerate primers described by Fisher et al, Nucleic Acids Research 24:4369- 4371 (1996) and Brachet et al, Molecular Ecology 8:160-163 (1999) were used to isolate clones containing microsatellite sequences from oil palm. PCR was performed separately for the two parental DNA samples, T128 (Nigerian guineensis) and UP 1026 (Colombian oleifera) using the protocol described by Fisher et al, Nucleic Acids Research 24:4369-4371 (1996). The post PCR mix was cloned using the TOPO-TA cloning kit (INVITROGEN, USA), essentially as recommended by the manufacturer. Sequencing was carried out on both strands using Ml 3 forward and reverse primers (INVITROGEN, USA) using a ABI 377 . sequencer (Applied Biosystems). Specific primers in the flanking regions of the microsatellites were designed using the PRIMER 3 software (Rozen and Skaletsky, Bioinformatics Methods and Protocols: Methods in Molecular Biology, pp:365- 386, 2000).

One primer for each primer pair was 5' end labeled at 37 °C for 30 min using T4 polynucleotide kinase (INVITROGEN, USA). The labeling reactions contained 50 pmoles of primer, 3 μΐ of γ-³³ρ dATP (Amersham 3000 Ci/mmol), 1 U of T4 polynucleotide kinase in a total volume of 25 μΐ. Subsequently the PCR reaction was carried out essentially as described by Billotte et al, Genome 44:413-425 (2001). After the PCR was completed, the reactions were stopped by the addition of 25 μΐ of 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 0.5 x 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 five EST-SSRs described by Chua et al, Scientific Meeting, ppl27, Melaka, Malaysia 19th -21st July 2004 (2004) and developed in the lab at MPOB were also tested on the mapping population.

B. Application of Published Oil Palm Microsatellite Sequences

The 18 published microsatellite primer pairs (Billotte et al, Genome 44:413-425, 2001) were also tested on the mapping population. All the primer pairs were synthesized based on the published sequences and tested on a small number of mapping population as described above. The informative primer pairs were than used to screen the entire mapping population.

Data analysis

The molecular results were analyzed according to a double pseudo testcross model described by Grattapaglia and Sederoff, Genetics 137:1121-1137 (1994). Data from RFLP, AFLP and SSR were scored and coded according to Lespinasse et al, Theoretical and Applied Genetics 100:127-138 (2000) and Billotte et al, Theoretical and Applied Genetics 110:754-765 (2005). The segregating bands were evaluated by using chi-square tests for goodness of fit to the expected segregation ratios (P=0.05). The segregation profiles observed and scored are summarized in Table 1.

Map construction

Map construction was carried out by using the JoinMap version 3.0 computer programme (Ooijen and Voorrips, JoinMap 3.0: Software for calculation of genetic linkage maps. Wageningen, 2001). The interspecific cross was analyzed as a population resulting from a cross between two heterozygous diploid parents. The population type code Cross Pollinator (CP) was used in the analysis. The data set from the female parent and the male parent were analyzed separately.

The markers were first divided into linkage groups by using a LOD score threshold of 5.0. The LOD score calculated in JoinMap version 3.0 is based on the chi-square test for independence of segregation. After being divided into- linkage groups, stringent parameters were applied for map construction that is a recombination value of 0.32 and a LOD score of 1.0. A ripple was performed after the addition of every three markers and the map distances were calculated by using the Kosambi map function. The ordering produced in the second cycle was used for map construction.

In a few cases a marker was discarded during the mapping stage if its presence caused inconsistencies in the map. Such markers were identified by inspection of the chi-square value after each extension on the map. A jump in the goodness-of-fit value indicates that the marker just added to the map causes 'internal friction' (Stam, The Plant Journal 3 : 739-744, 1993).

Quantitative data analysis

Quantitative traits associated with oil quality were of interest in this study. The criteria used to determine ripened bunches was as described by Corley and Tinker, The Oil Palm (4^th edition), pp: 287-326, Blackwell Science (2003), one loose fruit per bunch (irrespective of palm height).

Bunches after harvesting were carefully tagged and brought back to the laboratory. Oil was harvested from the mesocarp of the bunch as described by Rao et al, PORIM Occasional Paper 9: 1-28 (1983). The oil samples were stored in dark vials at -20 °C prior to being sent to MPOB's analytical laboratory for the following analysis:

i) Iodine value (IV)

ϋ) Fatty acid composition (FAC), namely:

C14:0 : myristic acid

CI 6:0 : palmitic acid CI 6:1 : palmitoleic acid

CI 8:0 : stearic acid

C18:l : oleic acid

CI 8:2 : linoleic acid

Sampling was performed on all 117 palms in the mapping population. In total, 81 palms were successfully sampled, oil extracted from their bunches and the appropriate analysis carried out. The data were obtained over a 2 year period QTL mapping analysis was performed by using interval mapping as implemented in MapQTL version 4.0 (Ooijen, MapQTL 4.0: Software for the calculation of QTLs position on genetic map. Wageningen, 2002). The empirical thresholds for QTL detection were calculated using the permutation test, also implemented via MapQTL version 4.0 (Ooijen, MapQTL 4.0: Software for the calculation of QTLs position on genetic map. Wageningen, 2002).

Example 4

Marker Screening

A total of 3,993 AFLP loci were scored in the progeny by using the 67 AFLP primer pairs (Table 2). The number of detectable fragments obtained for each combination ranged from 25 to 125, with band sizes ranging from 50 to 500bp. Generally a lower number of bands were obtained with primers which had CG dinucleotides. Primers with AT content usually gave higher numbers of bands (data not shown). This also confirms the low frequency of CG dinucleotides in interspecific hybrids of oil palm, similar to that reported for other plant genomes (Moore et al, Bio/Technology 11:584-589, 1993). Of the 3993 AFLP bands observed, 418 (10.5%) were polymorphic and segregating in the progeny (Table 2). Generally, majority of the segregating markers scored, 410 (98%) were in the pseudo-testcross configuration where either, the male parent was heterozygous and the fragment was absent in the female parent (type b profile) or vice versa (type a profile) (Table- 1).

A total 287 cDNA probes from various cDNA libraries were tested for their ability to detect segregation in the progeny using the RFLP approach. Of the 287 clones screened, 69 (24%), showed polymorphisms with at least one restriction enzyme, 167 (58%) were monomorphic and 51 (18%) gave no clear banding pattern. Figure 1 shows_. the banding profile produced by low copy probes segregating in the mapping population. The percentage of polymorphic probes identified (24%) was similar to the 25% rate of polymorphic RFLP probes (from genomic library) reported by Mayes et al., Genome 40:116-122 (1997) for oil palm previously. Of the 69 RFLP probes showing polymorphisms, 64 (93%) were inherited from the male E. guineensis parent (Table 2). This appeared to indicate that the RFLP probes used in this study mainly scanned the homozygous regions of the E. oleifera parental palm which were not segregating in the mapping progeny, thus reducing the number of polymorphic probes revealed. Four of these 69 probes, revealed two polymorphic loci, giving a total of 73 polymorphic loci. Among the 33 SSR primer pairs developed in the course of this study, nine were informative and segregating in the mapping population. The SSR loci developed by Billotte et al, Genome 44:413-425 (2001) were also tested on the mapping population. Of the 18 primer pairs tested, seven segregated in the mapping population. Six segregated in the male E. guineensis parental gametes only, while one segregated in the female E. oleifera gametes only. Three of five EST-SSRs tested, (C H0887, CNH1537 and EAP3339) showed polymorphism in the mapping population. All three informative primer pairs segregated only in the male parent, E. guineensis gametes. Information on the informative SSR primer pairs is provided in Table 3 and Attachment 2. Three of the informative SSR primers revealed two loci each.

Example 5

Segregation analyses of AFLP, RFLP and SSR markers in the mapping

population

The majority of the segregating bands were found to be segregating in the gametes of the male parent (palm T128, E. guineensis). Of the 513 (418 AFLP, 73 RFLP and 22 SSR) markers identified segregating in the mapping population, 454 (365 AFLP, 68 RFLP and 21 SSR) were segregating in the gametes of the male parent, Nigerian E. guineensis and 51 (9.9%) were segregating in the gametes of the female parent, the Colombian oleifera (Table 2). This indicates that the male parent is more heterozygous than the female parent, E. oleifera. A high proportion of polymorphic markers in the female parent did not segregate, most probably because they were homozygous at those loci. As such, sufficient markers could only be generated under the present experimental procedure to enable development of a genetic linkage map for the male parent. It is therefore concluded that it would be more appropriate to analyze this cross as a "one-way pseudo-testcross" in which the male, E. guineensis is considered to be the heterozygous parent and the Colombian E. oleifera, the homozygous parent. The low level of heterozygosity detected in the Colombian oleifera could be explained by the fact that E. oleifera is found in scattered areas in the South American country (Rajanaidu, Elaeis oleifera collection in Central and South America. Proceedings of International Workshop on Oil Palm Germplasm and Utilisation, 26-27 March 1985, Selangor, Malaysia, 1985). This could have encouraged inbreeding, resulting in a relatively high homozygous genome.

Although some of the RFLP markers showed the presence of- duplicated loci, pointing to a polyploidy ancestry, almost all the markers evaluated showed clear disomic inheritance. Majority of the markers showed either a 1 :1 segregation ratio (type a, b, d & e), where the band was present only in one parent or 3:1 segregation ratio (type c), where the band was present in both parents, which is the expected case in disomy (Lespinasse et al, Theoretical and Applied Genetics 100:127-138, 2000). The limited number of markers that showed type "f " & "g" profiles also showed clear disomy inheritance profiles, in which the parents and the resultant progeny had a maximum of two alleles, which were never absent at the same time.

Example 6

Linkage analysis

A total of 365 AFLP, 68 RFLP and 21 SSR markers (totaling 454 markers) were used to generate a linkage map for the male T128 parent. Only markers segregating in the pseudotestcross configuration (type b, e, f & g profiles, Table 1) were used for linkage analysis. Markers showing "Type c" profile with a 3:1 segregation ratio (Table 1), were not employed as the recombination frequencies obtained with such markers are typically inaccurate (Maliepaard et al, Theoretical and Applied Genetics 97: 60-73, 1998). Generally 447 of the markers analyzed could be linked to at least one other marker, indicating a good genome coverage. Seven of the markers (four AFLP, two RFLP and one SSR) remained unlinked at LOD 5.0. These unlinked markers could be sampling parts of the genome where there are few other markers, in which case they would be very valuable in the future (Marques et al, Theoretical and Applied Genetics 96:72' '-737, 1998).

A graphical representation of the genetic linkage map obtained is shown in Figure 2. In total, 389 markers (305 AFLP, 64 RFLP and 20 SSR) mapped in 18 linkage groups. The mean number of markers per linkage group was 21. The total genetic distance covered by the markers was 1571 cM, with an average interval of 4.1 cM between adjacent markers. The map distance of the tenera, T128 parental palm was similar to the tenera map distance of 1,597 cM reported by Billotte et al, Theoretical and Applied Genetics 110:754-765 (2005). Excluding the two smallest groups (17 and 18) which have only two markers each, the length of individual linkage groups varied from 28 cM to 177 cM, with an average of 96 cM. The average length of the linkage groups is close to the expected size of 100-150 cM found in most agricultural crops (Maliepaard et al, Theoretical and Applied Genetics 97: 60-73, 1998).

An excess of linkage groups in relation to the haploid chromosome number was obtained despite the relatively high number of markers used. Failure to obtain the basic chromosome number, despite high numbers of markers applied, has been reported for other species as well (Grattapaglia and Sederoff, Genetics 137:1121- 1137, 1994; Simone et al, Molecular Breeding 3:415-425, 1997). The reason for this perhaps could be due to the relatively small sample size of the F_\ progeny used in this study. Another possible explanation could be the lack of polymorphic markers in particular chromosomal regions, which could be due to the marker systems being employed and/or presence of large homozygous regions in the genome of the female E. oleifera parental palm used to create the interspecific hybrid population used in this study.

The markers were found to be well distributed over all the 18 linkage groups. There were no gaps larger than 20 cM, with the largest gap being 20 cM at Group 3 and 18 cM at Group 1. This indicates that the map offers a relative homogeneity of marker distribution and will be useful for tagging traits of economic interest for the purpose of marker-assisted selection. We also expect the existing gaps to disappear with the addition of more markers to the map. The backbone of this linkage map is composed of AFLPs that appear to cover the genome uniformly. However there were some regions of the map with (e.g. Groups 1 and 3), where some clustering of AFLP markers around the same locus was observed. The clustering of markers is commonly observed for many crop species and may have a biological basis as they may correspond to regions of reduced recombination frequencies such as centromeres and or telomeric regions (Tanksley et al, Genetics 132: 1141-1160, 1992; Keim et al, Crop Science 37:537-543, 1997). The limited clustering of the AFLP markers observed in this study was not related to the primers combinations used for generating these markers, but is probably due to the limited resolution of the mapping population as pointed out by Grando et al, Theoretical and Applied Genetics 106:1213-1224 (2003).

Of the 68 RFLP loci used for linkage analysis, 64 (94%) were successfully mapped.

The RFLP markers were generally well distributed throughout the linkage groups.

There were certain instances (e.g. Groups 4 and 11), where the RFLP markers are seldom interrupted by AFLP loci, which in fact tended to flank the RFLP markers.

However, there were many regions where both markers systems intermingled and as such probably do not at this stage of the study represent distinct regions.

Nevertheless since the RFLP markers were well distributed across the linkage groups, this augurs well for them to be used as potential anchor markers for integration or comparison of maps between several populations. The RFLP markers used are listed in Table A. In order to improve the coverage of the genome, mapping of SSR loci was also attempted. Of the 21 SSR loci segregating in the male parent gametes, 18 mapped at the LOD score of 5.0. Admittedly due to the low number of SSR markers employed, only nine of the 18 groups had at least one SSR marker each. Nevertheless, the presence of SSR markers in these groups together with the RFLP markers makes it more convenient for genetic map integration or comparison. Development of additional SSR from the existing ESTs collection is in progress, and it is anticipated that the EST-SSRs will assist with map saturation in the future. The proportion of segregation ratio distorted markers in this study was about 22% (Table 2). This was slightly higher than that reported for oil palm previously (less than 10%) (BiUotte et al, Theoretical and Applied Genetics 110:754-765, 2005) and other species, such as Eucalyptus (15%) (Marques et al, Theoretical and Applied Genetics 96:727-737, 1998) and apricot (17% for AFLP markers) (Vilanova et al, Theoretical and Applied Genetics 107: 239-247, 2003). However, the segregation distortion was much lower than those observed for roses (27%) (Rajapakse et al, Theoretical and Applied Genetics 103:575-583, 2001) and coffee (30%) (Ky et al, Theoretical and Applied Genetics 101:669-676, 2000). Nevertheless 78% of the markers segregated in the expected ratios, indicating that a majority of the markers were inherited in a stable Mendelian manner.

Loci showing skewed distortions were mostly located in Group 4, 7 and 11. In these groups markers showing distorted ratios mapped close to one another forming clusters of distorted markers. The clustering of distorted markers in a particular region of the linkage group might suggest a biological basis, reflecting possibly a high rate of hetroduplex formation and mismatch during meiosis, which gives rise to segregation distortions (Ky et al, Theoretical and Applied Genetics 101 :669-676, 2000). Hetroduplex formation and meiosis mispairing are expected to occur in interspecific hybrids due to differences in DNA content among parents used in the cross (Barre et al, Cytometry 24:32-38, 1996). Some of the distorted fragments were dispersed in different linkage groups and were not clustering, suggesting that in these groups, statistical reasons (relatively small mapping population) rather than a biological basis was responsible for the distortion (Marques et al, Theoretical and Applied Genetics 96:727-737, 1998).

Example 7

Quantitative traits

QTLs associated with iodine value (IV) and fatty acid composition (FAC) in oil palm were mapped. IV is a measure of the un-saturation of fats and oils.

All of the traits showed a pattern of continuous distribution around the mean, although some of the traits did not follow a perfect normal distribution (data not shown). The frequency distribution of IV, C16:0, C18:0, C18:l and C18:2 did not differ significantly from normality as assessed by the Shapiro-Wilk statistic calculated using the SPSS statistical programme. However the frequency distribution of C14:0 and C16:l showed deviation from normality. Deviation of a trait from a perfect normal distribution has been observed in QTL analysis experiments (Septiningsih et al, Theoretical and Applied Genetics 107:1433-1441, 2003).

The correlation coefficients between the various traits and their values have also been computed and are provided in Table 4. The IV content as expected is positively correlated with unsaturated fatty acids, CI 8:1 and CI 8:2. The results also indicate that the saturated fatty acids, CI 4:0 and CI 6:0 are negatively correlated with IV, CI 8:1 and CI 8:2. The results obtained here are as expected and similar to those reported previously (Rajanaidu et al, Palm Oil Research Institute (PORIM) Bulletin 7:9-20, 1983; Sambanthamurthi et al, Journal of Oil Palm Research 24-33, 1999). The expected correlation observed is a good indication of the accuracy of the fatty acids and IV measurements carried out in this study, which is an extremely important criterion for accurate QTL detection.

QTL analysis

QTL analysis was performed for each of the measured traits using the interval mapping method implemented by MapQTL version. 4.0 (Ooijen, MapQTL 4.0: Software for the calculation of QTLs position on genetic map. Wageningen, 2002). At a genomic wide significant threshold of PO.01 and PO.05, significant QTLs were detected for IV, C14:0, C16:0, C16:l, C18:0 and C18:l (Table 5). Significant QTLs were not detected for CI 8:2. The significant genomic wide threshold for IV content was determined to be 4.7 for PO.01 and 3.8 for PO.05. At this threshold, a significant QTL for IV was mapped on Group 1 (Figure 3A). The QTLs for C16:0 and CI 8:1 were mapped on the same group (Table 5, Figure 3D and 3H). All three QTLs showed similar shaped likelihood profiles, suggesting that the same QTL may be influencing the three traits. The fact that the traits are significantly correlated (Table 4) further supports this assumption. IV is a measure of unsaturation of oils and fats, C18:l is the most abundant un-saturated fatty acid while CI 6:0 is the most abundant saturated fatty acid in palm oil. As such, it is of no surprise that the same locus could be influencing the three traits. The QTLs mapped on Group 1 explains a significant portion of the variation observed for traits that is 35.6% for IV, 28.3% for C18:l and 36.8% for C 16:0 (Table 5).

QTLs were also detected at the PO.01 significance threshold levels for C14:0 (in Groups 4 and 12), CI 6:1 (Group 12) and CI 8:0 (Groups 3 and 12). Significant QTLs for C16:l and C18:0 were detected around the same region in Group 12. This probably points to another major locus influencing fatty acid composition. In the fatty acid synthesis pathway, C18:0 is converted to CI 8:1, by the actions of a desaturase enzyme known as stearoyl ACP desaturase (Sambanthamurthi et al, Journal of Oil Palm Research:24-33, 1999). This enzyme although highly specific to the conversion of C18:0 to C18:l, is also known to sometimes act on C16:0 as poor substrate and convert it to CI 6:1 (Sambanthamurthi et al, Journal of Oil Palm Research 24-33, 1999). This probably explains the strong correlation (r=-0.734) between C18:0 (stearic acid) and C16:l (palmitoleic acid). This also probably explains why the same QTL may be influencing both traits. The likelihood profile for the QTLs affecting both traits in Group 12 (Figure 3E and 3G) is also very similar adding further strength to the argument that the same locus is influencing both traits. For C18:0, an additional QTL, explaining a further 18.9% of the total phenotypic variation was also detected in Group 3 (Table 5). The trait C14:0 was found not correlated with CI 8:0 (at a significance level of PO.Q5) in the interspecific hybrid mapping population used in this study, but the QTL for the trait overlaps with CI 8:0 in Group 12. Overlapping of map positions of QTLs for different and unrelated traits has been reported previously, and can be caused by a single gene with pleitropic effects (Schneider et al, Theoretical and Applied Genetics 104:1107-1113, 2002). For CI 4:0, the QTL detected at Grou 12 accounted for 21.7% of the total variation, while the additional QTL detected at Group 4 explained another 20.9% of the total variation.

Apart from Interval Mapping, the rank sum test of Kruskal-Wallis was also used to detect markers with linkages to specific QTLs. This was to establish if there was an agreement between both test (Interval Mapping and Kruskal-Wallis). The Kruskal- Wallis test is regarded as the non-parametric equivalent to the one-way analysis of variance (Ooijen and Maliepaard, MapQTL™ version 3.0: Software for the calculation of QTL positions on genetic maps. CPRO-DLO, Wageningen, 1996), and was also carried out using the MapQTL software. The results are also summarized in Table 5. Generally the results from both analyses were in agreement. For all traits, the markers with the largest LOD score on the linkage group (which are close to the estimated position of the QTL), also showed very high significance for the presence of a segregating QTL in the Kruskal-Wallis test. The two tests appear to point to a common locus influencing QTLs associated with fatty acid composition.

Eight (8) QTLs were detected for IV and the five components of the fatty acid composition (C14:0, C16:0, C16:l, C18:0 and C18:l) in three different linkage groups. Since similar work has not been carried out for oil palm prior to this, a direct comparison with findings from other research groups could not be made. However, a comparison with other crops (mainly annual crops) is possible. For example in maize, Alrefai et al, Genome 38:894-901 (1995) detected 15 QTLs (in eight groups) associated with C16:0 only. Similarly Mangolin et al, Euphytica 137:251-259 (2004) detected 13 QTLs, distributed in eight chromosomes, for kernel oil content in maize. The low number of QTLs detected, in. this, study, were however ia agreement with the work by Somers et al, Theoretical and Applied Genetics 96:897-903 (1998) and Jourdren et al, Euphytica 90:351-357 (1996), who found that a few QTL loci could explain a big proportion of the phenotypic variation associated with one of the fatty acid components, CI 8:3 (linolenic acid) in Brassica napus. Nevertheless, it is important to note that the differences in QTLs mapped in this research cannot be directly compared to those reported above because of the different crop, type of markers, mapping population structure and the density of the genetic maps used in the analysis.

Example 8

Segregation of markers associated with QTLs

This study also correlated the actual segregation of the markers (which were pointing to the position of the QTLs) and the traits of interest in the mapping population. Since the pseudo-testcross strategy was used in map construction, we separated palms in the mapping population to those that either had the band present ("ab") or band absent ("aa") for a particular marker pointing to a QTL. The trait values were averaged and compared between palms with the "aa" and "ab" genotypes. The results obtained are summarized in Table 6. As is shown for the RFLP marker CB75A (SEQ ID NO:3) [This corresponds to sequence No. 3 in the list of RFLP probes] there was a significant difference for IV between palms having the "aa" and "ab" genotypes. The absence of the CB75A band (aa) resulted in high levels of IV in other words, high levels of unsaturation of the oil. Similar results were observed for C18:l .

The RFLP probe, CB75A was also associated with the QTL for C16:0 (palmitic acid). Carrying out a similar analysis, there was significant difference in C16:0 content between palms having the "ab" and "aa" genotypes. In this case, the presence of the RFLP band resulted in higher levels of the saturated fatty acid C16:0. The results indicate that the presence of the CB75A band points to a higher level of saturated fatty acid (C16:0), lower levels of un-saturation (lower IV reading). In a similar way, the traits CI 6:1 and C18:0 were negatively correlated and the QTLs overlapped in the same position on Group 12. The presence of the SSR allele, P4T8 (which points towards the estimated position of the QTL) resulted in high levels of C 18 :0, but reduced levels of C 16 : 1.

Example 9

Validation of probe CB75A in independent palms The probe CB75A was tested on 13 independent palms, for which the C16:0, C18:l and IV content had already been determined- previously. The results are^' shown in Figure 4.

The palms were arranged in order of increasing CI 6:0 content which also indicates that the palms are in order for decreasing amounts of CI 8:1 and IV content. The probe was able to distinguish palms contaimng high and low CI 6:0, CI 8:1 and IV content.

Validation of Probes RD56 and SSR marker P4T8 on a second mapping population

A second mapping population (selfing of the E. guineensis palm T128), was also used to detect QTLs associated with C14:0 and C16:l . The results for the QTL analysis are presented in Figure 5. Generally the same markers, RD56 (SEQ ID NO:56) [ΝΘΤΕ: this corresponds to sequence No. 56 in the list of RFLP probes] and P4T8 [ Note: the primer sequence is provided in Attachment 2], were associated with C14. and C16:l content in the second mapping population.

The association of the markers with C14:0 and C16:l content is further illustrated in Table 7 and 8.

The results here point to the verification of the markers associated with CI 4:0 and CI 6:1, content, where the markers are able to distinguish palms with high and low C14:0 and C16:l content.

Example 10

Molecular Breeding for oil qualit

The present invention, for the first time, has revealed QTLs associated with IV and FAC in oil palm. Markers which explain a large proportion of the phenotypic variation (up to 45% for C14:0, C16:0, C16:l, C18:0 and C18:l) were identified. The traits were largely controlled by a limited number of genomic regions with large effects.

The identification of QTLs provides important tools to breeders to manipulate the FAC- content in oil palm. For example, absence of the RFLP probe, CB75A, isindicative of palms having oil with higher unsaturation levels. The absence of the RFLP marker resulted in an increase of about 2.6% above the family mean for TV (level of un-saturation), and a decrease of about 6.5% below the family mean for CI 6:0 content (saturated fatty acid).

Although the efforts in Malaysia are largely directed towards decreasing levels of unsaturation, increasing levels of certain saturated fatty acids can also have some economic benefits. In this respect, there is interest in increasing the stearic acid content (CI 8:0), which can give rise to new applications such as, cocoa butter substitution and personal care products such as lotions, shaving creams and rubbing oils (Parveez, AgBiotechNet, 5: July, ABN 113, 2003). This is also partly motivated by the substantial price differential between cocoa butter and commodity oils (Cheah, Palm Oil Developments 20:28-34, 1994). The oil palm like most plant oils, have low stearate content of about 2% (Sambanthamurthi et al, Journal of Oil Palm Research:24-33, 1999). The SSR marker, P4T8, could play an important role in MAS for high stearate palms. The presence of the SSR alleles resulted in an increase of 6.8% above the family mean for C18:0 content.

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Claims

1. A method for selecting a plant based on fatty acid content (FAC) of oil isolated from said plant, said method comprising screening for the presence or absence of a genetic marker in the plant, said genetic marker corresponding to a nucleotide sequence having at least 90% identity to at least 20 contiguous nucleotides of SEQ ID NO:3, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of said marker is indicative of a plant having oil with a high unsaturated FAC.

2. The method of Claim 1 , wherein the genetic marker is CB75 A.

3. The method of any one of Claims 1 and 2, wherein the saturated fatty acid is selected from the group consisting of: myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C 18:0).

4. The method of any one of Claims 1 and 2, wherein the level of saturated FAC is greater than 50% of total FAC.

5. The method of any one of Claims 1 and 2, wherein the level of saturated FAC is greater than 60% of total FAC.

6. The method of any one of Claims 1 and 2, wherein the level of saturated FAC is greater than 70% of total FAC.

7. The method of any one of Claims 1 and 2, wherein the level of saturated FAC is greater than 80% of total FAC.

8. The method of any one of Claims 1 and 2, wherein the level of saturated FAC is greater than 90% of total FAC.

9. The method of any one of Claims 1 and 2, wherein the level of saturated FAC is greater than 95% of total FAC.

10. The method of any one of Claims 1 and 2, wherein the level of saturated FAC is greater than 98% of total FAC.

11. The method of any one of Claims 1 and 2, wherein the unsaturated fatty acid is selected from the group consisting of oleic acid (C18:l), linoleic acid (C18:2) and alphalinoleic acid (CI 8:3).

12. The method of any one of Claims 1 and 11, wherein unsaturated FAC is greater than 20% of total FAC.

13. The method of any one of Claims 1 and 11 , wherein unsaturated FAC is greater than 30% of total FAC.

14. The method of any one of Claims 1 and 11 , wherein unsaturated FAC is greater than 40% of total FAC.

15. The method of any one of Claims 1 and 11 , wherein unsaturated FAC is greater than 50% of total FAC.

16. The method of any one of Claims 1 and 11, wherein unsaturated FAC is greater than 60% of total FAC.

17. The method of any one of Claims 1 and 11, wherein unsaturated FAC is greater than 70% of total FAC.

18. The method of any one of Claims 1 and 11 , wherein unsaturated FAC is greater than 80% of total FAC.

19. The method of any one of Claims 1 and 11, wherein unsaturated FAC is greater than 90% of total FAC.

20. The method of any one of Claims 1 and 1 1, wherein unsaturated FAC is greater than 95% of total FAC.

21. The method of any one of Claims 1 and 1 1, wherein unsaturated FAC is greater than 98% of total FAC.

22. The method of any one of Claims 1 and 11, wherein the level of oleic acid is 60%.

23. The method of Claim 22, wherein the level of palmitic acid is 25%.

24. The method of any one of Claims 1 and 2, wherein the absence of the genetic marker is further indicative of a plant having a high iodine value (IV).

25. The method of Claim 24, wherein the IV is greater than 60.

26. The method of Claim 24, wherein the IV is greater than 70.

27. The method of Claim 24, wherein the TV is greater than 80.

28. The method of Claim 24, wherein the IV is greater than 90.

29. The method of Claim 24, wherein the IV is greater than 95.

30. The method of Claim 24, wherein the IV is greater than 98.

31. A method of selecting a plant based on fatty acid content (FAC) of oil isolated from said plant, said method comprising screening for the presence or absence of a genetic marker in the plant, said genetic marker corresponding to a nucleotide sequence having at least 90% identity to at least 20 contiguous nucleotides of SEQ ID NO:56, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of said marker is indicative of a plant having oil with a high unsaturated FAC.

32. The method of Claim 31 , wherein the genetic marker is RD56.

33. The method of any one of Claims 31 and 32, wherein the saturated fatty acid is myristic acid.

34. The method of any one of Claims 31 and 32, wherein the level of saturated FAC is greater than 50% of total FAC.

35. The method of any one of Claims 31 and 32, wherein the level of saturated FAC is greater than 60% of total FAC.

36. The method of any one of Claims 31 and 32, wherein the level of saturated FAC is greater than 70% of total FAC .

37. The method of any one of Claims 31 and 32, wherein the level of saturated FAC is greater than 80% of total FAC.

38. The method of any one of Claims 31 and 32, wherein the level of saturated FAC is greater than 90% of total FAC.

39. The method of any one of Claims 31 and 32, wherein the level of saturated FAC is greater than 95% of total FAC.

40. The method of any one of Claims 31 and 32, wherein the level of saturated FAC is greater than 98% of total FAC.

41. A method of selecting a plant based on fatty acid content (FAC) of oil isolated from said plant, said method comprising screening for the presence or absence of a genetic marker in the plant, said genetic marker corresponding to a nucleotide sequence having at least 90% identity to the contiguous nucleotides of SEQ ID NO:65, or a complementary sequence thereto, wherein the presence of the genetic marker is indicative of a plant having oil with a high saturated FAC and the absence of said marker is indicative of a plant having oil with a high unsaturated FAC.

42. The method of Claim 41 , where the genetic marker is P4T8.

43. The method of any one of Claims 41 and 42, wherein the saturated fatty acid is stearic acid.

44. The method of any one of Claims 41 and 42, wherein the level of saturated FAC is greater than 50% of total FAC.

45. The method of any one of Claims 41 and 42, wherein the level of saturated FAC is greater than 60% of total FAC.

46. The method of any one of Claims 41 and 42, wherein the level of saturated FAC is greater than 70% of total FAC.

47. The method of any one of Claims 41 and 42, wherein the level of saturated ^' FAC is greater than 80% of total FAC.

48. The method of any one of Claims 41 and 42, wherein the level of saturated FAC is greater than 90% of total FAC.

49. The method of any one of Claims 41 and 42, wherein the level of saturated FAC is greater than 95% of total FAC.

50. The method of any one of Claims 41 and 42, wherein the level of saturated FAC is greater than 98% of total FAC.

51. The method of any one of Claims 41 and 42, wherein the unsaturated fatty acid is palmitoleic (C16:l) acid.

52. The method of any one of Claims 41 and 42, wherein the unsaturated FAC is greater than 20% of total FAC.

53. The method of any one of Claims 41 and 42, wherein the unsaturated FAC is greater than 30% of total FAC.

54. The method of any one of Claims 41 and 42, wherein the unsaturated FAC is greater than 40% of total FAC.

55. The method of any one of Claims 41 and 42, wherein the unsaturated FAC is greater than 50% of total FAC.

56. The method of any one of Claims 41 and 42, wherein the unsaturated FAC is greater than 60% of total FAC.

57. The method of any one of Claims 41 and 42, wherein the unsaturated FAC is greater than 70% of total FAC.

58. The method of any one of Claims 41 and 42, wherein the unsaturated FAC is greater than 80% of total FAC.

59. The method of any one of Claims 41 and 42, wherein the unsaturated FAC is greater than 90% of total FAC.

60. The method of any one of Claims 41 and 42, wherein the unsaturated FAC is greater than 95% of total FAC.

61. The method of any one of Claims.41 and 42, wherein the unsaturated FAC is greater than 98% of total FAC.

62. A DNA molecule associated with FAC homologous or complementary to the DNA molecule disclosed in SEQ ID NO:3 located on linkage group 1 of the mapping population of T128, an oil palm of the species Elaeis guineensis or progeny thereof and is of a sufficient length to be useful as a DNA marker for an allele of a quantitative trait locus, wherein the allele determines the FAC content of a plant.

63. A DNA molecule associated with FAC homologous or complementary to the DNA molecule disclosed in SEQ ED NO:56 located on linkage group 12 of the mapping population of T128, an oil palm of the species Elaeis guineensis or progeny thereof and is of a sufficient length to be useful as a DNA marker for an allele of a quantitative trait locus, wherein the allele determines the FAC content of a plant.

64. A DNA molecule associated with FAC homologous or complementary to the DNA molecule disclosed in SEQ ID NO:65 located on linkage group 12 of the mapping population of T128, an oil palm of the species Elaeis guineensis or progeny thereof and is of a sufficient length to be useful as a DNA marker for an allele of a quantitative trait locus, wherein the allele determines the FAC content of a plant.

65. The method of any of Claims 1 to 61, wherein the plant is an oil palm plant.