BR112017027201B1 - METHOD FOR PREDICTING THE BARK PHENOTYPE OF A PALM TREE OR A PALM SEED AND METHOD FOR SEGREGATING A PLURALITY OF PALM TREES INTO DIFFERENT CATEGORIES BASED ON A PREDICTED BARK PHENOTYPE - Google Patents

Abstract

MADS-BOX DOMAIN ALLELES FOR CONTROLLING BARK PHENOTYPE IN PALM. Nucleic acid and polypeptide sequences for predicting and controlling bark phenotype in palm.

Description

CROSS-REFERENCE TO RELATED ORDERS

[001] This application claims priority from U.S. Provisional Application No. 62/180,042, filed June 15, 2015, the contents of which are incorporated herein by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

[002] Oil palms (E. guineensis and E. oleifera) can be classified into separate groups based on their fruiting characteristics, and have three naturally occurring fruit types that vary in shell thickness and oil yield. Dura palms are homozygous for a wild-type allele of the shell gene (sh+/sh+), have a thick seed coat (2 to 8 mm), and produce approximately 5.3 tonnes of oil per hectare per year. Tenera palms are heterozygous for a wild-type and mutant allele of the shell gene (sh+/sh-), have a relatively thin shell surrounded by a distinct fiber ring, and produce approximately 7.4 tonnes of oil per hectare per year. Finally, pisifera palms are homozygous for a mutant allele of the shell gene (sh-/sh-), have no shell or seed coat, and are generally female sterile (Hartley, 1988) (Table 1). Therefore, inheritance of the single gene controlling the shell phenotype is a major contributor to palm oil yield.

[003] Tenera palms are hybrids between the dura and pisifera palms. Whitmore (1973) described the various fruit forms as different varieties of oil palm. However, Latiff (2000) agreed with Purseglove (1972) that varieties or cultivars as proposed by Whitmore (1973) do not occur in the strict sense in this species. Therefore, Latiff (2000) proposed the term “race” to differentiate dura, pisifera and tenera. Race was considered an appropriate term as the term reflects a permanent microspecies, in which the different races have the capacity to exchange genes with each other, which has been adequately demonstrated in the different fruit forms observed in oil palm (Latiff, 2000). In fact, the characteristics of the three different races are controlled simply by the inheritance of a single gene. Genetic studies have revealed that the shell gene shows codominant monogenic inheritance, which is exploitable in breeding programs (Beirnaert and Vanderweyen, 1941).

[004] The shell gene responsible for this phenotype was first reported in the Belgian Congo in the 1940s (Beirnaert and Venderweyan, 1941). However, tenera fruit forms were recognized and extensively exploited in Africa before then (Devuyst, 1953; Godding, 1930; Sousa et al., 2011). Given the central role played by the shell gene, oil palm breeding utilizes reciprocal recurrent selection of maternal (dura) and paternal (pisifera) seed banks using the North Carolina Model 1 maize breeding project (Rajanaidu et al., 2000). The Deli dura population, direct descendants of the four original African palms planted in the Bogor Botanical Garden, Indonesia (1848), has excellent combinatory ability with AVROS (Algemene Vereniging van Rubberplanters ter Oostkust van Sumatra) and other pisifera palm parents. The AVROS pisifera palms were derived from the famous “Djongo” palm of Congo, but more recently several different accessions of dura and pisifera have also originated from Africa (Rajanaidu et al., 2000).

[005] Tenera fruit types have a higher mesocarp to fruit ratio, which directly translates to significantly higher oil yield than both the duro palm and pisifera palm (as illustrated in Table 1). TABLE 1: Comparison of duro, tenera, and pisifera fruit forms * usually sterile female clusters rot prematurely ** fiber ring is present in the mesocarp and is often used as a diagnostic tool to differentiate dura and tenera palms. (Source: Hardon et al., 1985; Hartley, 1988)

[006] Since the core of oil palm breeding programs is to reproduce plant material with higher oil yields, the tenera palm is the preferred choice for commercial planting. It is for this reason that substantial resources are invested by commercial seed producers to cross selected dura and pisifera palms to produce hybrid seed. And despite the many advances that have been made in the production of hybrid oil palm seed, two significant problems remain in the seed production process. First, tenera seed lots that will produce the high-oil-yielding tenera palm type are frequently contaminated with dura seed (Donough and Law, 1995). Dura contamination of tenera seed is currently estimated to be as high as 5% (reduced from as high as 20 to 30% in the early 1990s as a result of improved quality control practices). Seed contamination is due in part to the difficulties of producing pure tenera seeds under open plantation conditions, where workers use ladders to hand pollinate tall trees, and where the palm is allowed to flower for a given cluster to mature over a period of time, making it difficult to pollinate all the flowers in a cluster with a single hand pollination event. Some flowers in the cluster may have matured prior to hand pollination and therefore may have had the opportunity to be wind pollinated from an unknown tree, thus producing contaminating seeds in the cluster. Alternatively, premature flowers may exist in the cluster at the time of hand pollination, and may mature after pollination has occurred allowing them to be wind pollinated from an unknown tree, thus producing contaminating seeds in the cluster. Prior to the invention described herein, it was not possible to identify the type of fruit from a given seed or a given plant that appears from a seed until the plant matures sufficiently to produce a first batch of fruit, which typically takes approximately six years after germination. Notably, in the four to five year interval from germination to fruit production, significant land, labor, financial, and energy resources are invested in what are believed to be tenera trees, some of which will ultimately be the undesirable low-yielding contaminant fruit types. By the time these suboptimal trees are identified, it is impractical to remove them from the field and replace them with tenera trees, and producers thus achieve lower oil palm yields over the 25 to 30 year production cycle of the contaminant trees. Therefore, the problem of contamination of tenera seed lots with dura or pisifera seeds is a problem for oil palm breeding, highlighting the need for a method to predict the fruit type of nursery seeds and seedlings with high accuracy.

[007] A second problem in the seed production process is the investment that investment seed producers make in maintaining dura and pisifera lines, and in other expenses incurred in the hybrid seed production process. Traditionally, there has been no known way to produce a tree with an ideal bark phenotype that, when crossed with itself or another tree with an ideal bark phenotype, would produce seed that would generate only ideal bark phenotypes. Therefore, there has been a need to manipulate trees to breed true from one generation to the next for the ideal bark phenotype. There is also a need to separate predicted tenera plants (e.g., seeds or seedlings) from any contaminating dura and/or pisifera plants produced during the hybrid production process. Similarly, there is a need to separate predicted dura plants from pisifera and/or tenera plants and predicted pisifera plants from dura and/or tenera plants to maintain breeding stocks for hybrid production.

[008] Genetic mapping of the SHELL gene was initially attempted by Mayes et al. (1997). A second group in Brazil, using a combination of bulk segregation analysis (BSA) and genetic mapping, reported two random amplified polymorphic DNA (RAPD) markers flanking the shell locus (Moretzsohn et al., 2000). More recently, Billotte et al., (2005) reported a high-density simple sequence repeat (SSR)-based linkage map for oil palm involving a cross between a plain-shelled palm E. guineensis (tenera) and a thick-shelled palm E. guineensis (dura). A patent application filed by the Malaysian Palm Oil Board (MPOB) describes the identification of a marker using restriction fragment length technology, in particular, a Restriction Fragment Length Polymorphism (RFLP) marker linked to the shell gene for plant identification and breeding purposes (RAJINDER SINGH, LESLIE OOI CHENG-LI, RAHIMAH A. RAHMAN and LESLIE LOW ENG TI. 2008. Method for identification of a molecular marker linked to the shell gene of oil palm. Patent Application No. PI 20084563. Patent filed on 13 November 2008). The RFLP marker (SFB 83) was identified by generating or constructing a genetic map for a tenera fruit type palm. Patent application publications U.S. 2013/024729 and U.S. 2015/0037793, filed by MPOB, describe the identification of the SHELL gene, two pisifera alleles (shAVROS and shMPOB), and methods for predicting fruit shape phenotype by detecting wild-type and pisifera alleles of the SHELL gene.

BRIEF SUMMARY OF THE INVENTION

[009] Described herein is the identification of novel alleles of the SHELL gene responsible for different fruit shape phenotypes and methods for predicting or determining the shell phenotype of a palm tree (including, but not limited to, an entire palm tree or palm seed). The SHELL gene is an oil palm MADS-box gene substantially similar to Arabidopsis SEEDSTICK (STK), also referred to as AGAMOUS-like 11 (AGL11), as well as Arabidopsis SHATTERPROOF (SHP1), also referred to as AGAMOUS-like 1 (AGL1).

[010] Two SHELL alleles, shMPOB and shAVROS, have previously been identified, either of which results in the preferred tenera fruit form when present in an oil palm that has one copy of a mutant allele and one wild-type allele. For example, heterozygous oil palms that include the wild-type SHELL allele, ShDeliDura, on one chromosome and either of the two mutant SHELL alleles on the other chromosome exhibit a tenera phenotype.

[011] Nine additional mutations in exon one of the SHELL gene are described herein, referred to as SHELL alleles three (3), four (4), five (5), six (6), seven (7), eight (8), nine (9), ten (10), and eleven (11). The amino acid sequences of the SHELL gene product resulting from alleles 311 are depicted in SEQ ID NOs: 3 through 11 respectively. The nucleotide sequences for exon 1 of the SHELL gene for alleles 3-11 are depicted in SEQ ID Nos: 13 through 21 respectively. As with the shMPOB and shAVROS alleles, the presence of these SHELL alleles can result in a tenera phenotype when heterozygous with a wild-type allele or a pisifera phenotype when either homozygous or heterozygous with another non-functional SHELL allele.

[012] Referring to the wild-type SHELL gene (ShDeliDura), allele 3 polymorphism is an adenosine to cytosine (A→C) mutation at nucleotide position 67 of exon 1 of the SHELL gene. Allele 3 results in a lysine to glutamine substitution within the conserved MADS-box domain of SHELL. As diagrammed in Figure 1, the entire MADS-box domain of SHELL is encoded by exon 1 of the SHELL gene. The variant amino acid occurs at the 6 amino acid N-terminus for the recurring amino acid substitution of the shMPOB allele, at the 8 amino acid N-terminus for the resulting amino acid substitution of the shAVROS allele, and at position 23 of the translated open reading frame of exon 1 (Figures 2 and 3).

[013] Similarly, the allele 4 polymorphism is a cytosine to adenosine (C→A) mutation at nucleotide position 122 of exon 1 of the SHELL gene. Allele 4 results in an alanine to aspartate substitution within the conserved MADS-box domain of SHELL. The variant amino acid occurs at position 41 of the translated open reading frame of exon 1 (Figures 2 and 3).

[014] Allele 5 polymorphism is an adenosine to thymine (A→T) mutation at nucleotide position 69 of exon 1 of the SHELL gene. Allele 5 results in a lysine to asparagine mutation at position 23 of the translated open reading frame of exon 1 (Figures 2 and 3). Allele 6 polymorphism is a guanosine to cytosine (G→C) mutation at position 34 of exon 1 of the SHELL gene. Allele 6 results in a glutamate to glutamine mutation at position 12 of the translated open reading frame of exon 1 (Figures 2 and 3). The allele 7 polymorphism is a fifteen-nucleotide deletion at positions 23 to 37 of exon 1 of the SHELL gene (or nucleotides 22 to 36 because the alignment of the range is ambiguous). The allele 7 results in an in-frame deletion of five amino acids at positions 8 to 12 of the translated open reading frame of exon 1 (Figures 2 and 3). Amino acid positions 8 to 12 of the SHELL gene are encoded by nucleotides 22 to 36. The allele 8 polymorphism is a guanosine-to-adenosine (G→A) mutation at position 71 of exon 1 of the SHELL gene. The allele 8 results in an arginine-to-histidine mutation at position 24 of the translated open reading frame of exon 1 (Figures 2 and 3).

[015] Allele 9 polymorphism is a cytosine to guanosine (C→G) mutation at position 70 of exon 1 of the SHELL gene. Allele 9 results in an arginine to glycine mutation at position 24 of the translated open reading frame of exon 1. Allele 10 polymorphism is a thymine to adenosine (T→A) mutation at position 110 of exon 1 of the SHELL gene. Allele 10 results in a valine to aspartate mutation at position 37 of the translated open reading frame of exon 1 (Figures 2 and 3).

[016] Allele 11 polymorphism is a thymine to cytosine (T→C) mutation at position 114 of exon 1 of the SHELL gene. Allele 11 is a silent mutation in that it does not affect the resulting amino acid sequence of the SHELL gene product (Figures 2 and 3). This mutation can be detected to confirm or predict the presence or absence of a wild-type SHELL gene product and therefore predict a dura phenotype when homozygous or heterozygous with another wild-type allele in a palm tree and a tenera phenotype when heterozygous with an inactive SHELL allele. Alternatively, in some embodiments, this mutation can affect gene expression and/or transcriptional or translational regulation of the SHELL gene. Consequently, in such embodiments, the mutation may correlate with a pisifera when homozygous or heterozygous with an inactive SHELL allele in a palm or tenera when heterozygous with a wild-type allele.

[017] Also described herein is a mutation in intron 1 of the SHELL gene that was discovered in a subset of oil palm plants that have the allele 3 mutation. This mutation is referred to herein as allele 12 and depicted in SEQ ID NO:12. The mutation results in the deletion of four nucleotides at positions 43 to 46 of intron 1 of the wild-type SHELL gene (ShDeliDura). The mutation may be silent in that it may not by itself contribute to the presence or absence of a fruit-shaped SHELL phenotype (e.g., dura, tenera, or pisifera). However, because of the close physical distance (i.e., genetic linkage) between the intron 1 and exon 1 mutation, the contribution of parental germplasm known to have a particular SHELL allele (wild-type or mutant) within exon 1 and the intron 1 marker can be traced with a high degree of confidence in progeny by detecting the allele 12 mutation rather than a mutation in exon 1. Furthermore, in some cases, the intron 1 mutation may be in linkage disequilibrium with exon 1 or a portion thereof. Alternatively, allele 12 may alter transcriptional regulation or splicing and thus exhibit a pisifera SHELL phenotype when homozygous or a tenera phenotype when heterozygous with a wild-type SHELL allele.

[018] Nuclear proteins, such as transcription factors, must be actively transported and retained within the nucleus to be functional. The mechanism of nuclear localization involves the binding of nuclear localization protein signals on the nuclear protein to importin and importin subunits in the cytoplasm. Importin binds to the nuclear localization signal (NLS), while importin interacts with importin as well as the nuclear pore. In plant MADS-box proteins, the prominent NLS amino acid motif is KR[K or R]X4KK (SEQ ID NO:29), where X can be any amino acid (Gramzow and Theissen, 2010). The MADS-box domain of SHELL includes this motif (KRRNGLLKK; SEQ ID NO:30) at amino acids 23 to 31. MADS-box proteins may also have a bipartite NLS that involves additional upstream amino acids. An example is the bipartite NLS of petunia FLORAL Splicing PROTEIN 11 (FBP11) that includes the sequence MGRGKIEIKRIENNTNRQVTFCKRRNGLLKK (SEQ ID NO:31). The bipartite NLS is composed of NLS amino acids (underlined) as well as conserved basic amino acids (in italics), all of which contribute to the nuclear localization mechanism (Immink et al., 2002).

[019] The MADS-box domain of SHELL includes a very similar bipartite NLS that includes amino acids 3, 5, 9, and 10, and 21 to 31 (MGRGKIEIKRIENTTSRQVTFCKRRNGLLKK; SEQ ID NO:32) (Figures 2 and 3). It is notable that, of the ten sequence changes resulting in amino acid substitutions or deletions reported here (shAVROS, shMPOB, and alleles 3 to 10), six change one or more of these highly conserved NLS amino acids (shAVROS, shMPOB, allele 3, allele 5, allele 7, allele 8, and allele 9), and a 7th (shMPOB) introduces a proline substitution at a variable position within the prominent NLS that would be expected to significantly alter the secondary structure of the protein within the NLS domain (Figures 2 and 3). These findings suggest that a common mechanism conferring the pisifera (when homozygous or heterozygous with another nonfunctional SHELL allele) or tenera (when heterozygous with a wild-type SHELL allele) phenotype may be the reduction or prevention of nuclear localization of nonfunctional SHELL proteins or SHELL protein dimers with other MADS-box transcription factors. Therefore, it is likely that mutation of any of the conserved NLS amino acids (boxed in Figures 2 and 3), or any mutation that disrupts SHELL NLS function, may be associated with the pisifera or tenera phenotype.

[020] Accordingly, in one aspect, methods for determining or predicting the phenotype of bark of a palm tree (e.g., oil palm) (including, but not limited to, a whole palm tree or palm seed) are provided. In some embodiments, the method comprises providing a sample of the plant or seed; and determine, from the sample, the genotype of a polymorphic marker at a position in exon 1 of the SHELL gene selected from the group consisting of nucleotides: (i) 7, 8, 9, 13, 14, 15, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 109, 110, 111, 114, 121, 122 and 123; (ii) 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 67, 69, 70, 71, 110, 114 and 122; or (iii) 7 to 9, 13 to 15, 25 to 30, 61 to 75 and 88 to 92.

[021] In some cases, heterozygosity at one or more of the polymorphic markers for a pisifera allele and a dura allele predicts the presence of the tenera bark phenotype. In some cases, homozygosity for a pisifera allele genotype predicted at one or more of the polymorphic markers predicts the presence of the pisifera bark phenotype. In some cases, the polymorphic marker genotype may comprise one or more of the predicted pisifera allele genotypes depicted in SEQ ID NOs: 13 through 21.

[022] In some cases, a mutation relative to the wild-type SHELL gene (ShDeliDura) at one or more of the nucleotide positions that results in an amino acid substitution (e.g., non-conservative substitution), deletion, insertion, or frameshift may predict a pisifera phenotype when homozygous or heterozygous with a different mutation relative to the wild-type SHELL gene (ShDeliDura), or a tenera phenotype when heterozygous for the wild-type allele. For example, a mutation relative to the wild-type SHELL gene (ShDeliDura) at one or more of the nucleotide positions that results in an amino acid substitution (e.g., non-conservative substitution), deletion, insertion, or frameshift may predict a pisifera phenotype when heterozygous with a different mutation that results in a non-functional SHELL gene, such as a mutation that results in a different substitution (e.g., non-conservative substitution), deletion, insertion, or frameshift.

[023] In some embodiments, the genotype of the polymorphic marker comprises a deletion or mutation of one or more nucleotides selected from the group consisting of nucleotides: (i) 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, and 37; (ii) 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36; or (iii) 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, and 37, of exon 1 of the SHELL gene. In some embodiments, the genotype of the polymorphic marker comprises a deletion of one or more, or all, of nucleotides 23 to 37 (or 22 to 36) of exon 1 of the SHELL gene. In some embodiments, the genotype of the polymorphic marker comprises a mutation of nucleotide 34 of exon 1 of the SHELL gene (e.g., a mutation relative to ShDeliDura). In some embodiments, the mutation comprises a missense (e.g., non-conservative substitution), nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the genotype of the polymorphic marker comprises a cytosine (C) at nucleotide 34 of exon 1 of the SHELL gene.

[024] In some embodiments, the genotype of the polymorphic marker comprises a mutation of nucleotide 67 of exon 1 of the SHELL gene (e.g., a mutation relative to ShDeliDura). In some embodiments, the mutation comprises a missense mutation (e.g., non-conservative substitution), nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the genotype of the polymorphic marker comprises a cytosine (C) at nucleotide 67 of exon 1 of the SHELL gene. In some embodiments, the genotype of the polymorphic marker comprises a mutation of nucleotide 69 of exon 1 of the SHELL gene (e.g., a mutation relative to ShDeliDura). In some embodiments, the mutation comprises a missense mutation (e.g., non-conservative substitution), nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the genotype of the polymorphic marker comprises a thymine (T) at nucleotide 69 of exon 1 of the SHELL gene.

[025] In some embodiments, the genotype of the polymorphic marker comprises a mutation of nucleotide 70 of exon 1 of the SHELL gene (e.g., a mutation relative to ShDeliDura). In some embodiments, the mutation comprises a missense mutation (e.g., non-conservative substitution), nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the genotype of the polymorphic marker comprises a guanosine (G) at nucleotide 70 of exon 1 of the SHELL gene. In some embodiments, the genotype of the polymorphic marker comprises a mutation of nucleotide 71 of exon 1 of the SHELL gene (e.g., a mutation relative to ShDeliDura). In some embodiments, the mutation comprises a missense mutation (e.g., non-conservative substitution), nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the genotype of the polymorphic marker comprises an adenosine (A) at nucleotide 71 of exon 1 of the SHELL gene. In some embodiments, the genotype of the polymorphic marker comprises a mutation at nucleotide 110 of exon 1 of the SHELL gene (e.g., a mutation related to ShDeliDura). In some embodiments, the mutation comprises a missense (e.g., non-conservative substitution), nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the genotype of the polymorphic marker comprises an adenosine (A) at nucleotide 110 of exon 1 of the SHELL gene.

[026] In some embodiments, the genotype of the polymorphic marker comprises a mutation of nucleotide 114 of exon 1 of the SHELL gene (e.g., a mutation relative to ShDeliDura). In some embodiments, the mutation comprises a missense mutation (e.g., non-conservative substitution), nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the genotype of the polymorphic marker comprises a cytosine (C) at nucleotide 114 of exon 1 of the SHELL gene. In some embodiments, the genotype of the polymorphic marker comprises a mutation of nucleotide 122 of exon 1 of the SHELL gene (e.g., a mutation relative to ShDeliDura). In some embodiments, the mutation comprises a missense mutation (e.g., non-conservative substitution), nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the polymorphic marker genotype comprises an adenosine (A) at nucleotide 122 of exon 1 of the SHELL gene.

[027] In any of the preceding embodiments, the method may comprise providing a sample of the plant or seed; and determining, from the sample, the genotype of a polymorphic marker at a position in exon 1 of the SHELL gene selected from the group consisting of nucleotides: (i) 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 110, 114, and 122; (ii) 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 67, 69, 70, 71, 110, 114 and 122; or (iii) 67, 69, 70 and 71.

[028] In some cases, heterozygosity at one or more of the polymorphic markers for a pisifera allele and a dura allele predicts the presence of the tenera bark phenotype. In some cases, homozygosity for a genotype of a pisifera allele predicted at one or more of the polymorphic markers predicts the presence of the pisifera bark phenotype. In some cases, heterozygosity for a genotype of a first pisifera allele predicted at one or more of the polymorphic markers and a second pisifera allele predicted at one or more of the polymorphic markers predicts the presence of the pisifera bark phenotype. In some cases, the genotype of the polymorphic marker may comprise one or more of the predicted pisifera allele genotypes depicted in SEQ ID NOs: 13, 15, 17, 18, and 19.

[029] In some embodiments, the method comprises providing a sample of the plant or seed; and determining, from the sample, the genotype of a polymorphic marker at a position in intron 1 of the SHELL gene selected from the group consisting of nucleotides 43, 44, 45, and 46. In some cases, heterozygosity at one or more of the polymorphic markers for a pisifera allele and a dura allele predicts the presence of the tenera shell phenotype. In some cases, homozygosity for a genotype of a pisifera allele predicted at one or more of the polymorphic markers predicts the presence of the pisifera shell phenotype. In some cases, heterozygosity for a genotype of a first pisifera allele predicted at one or more of the polymorphic markers and a genotype of a second pisifera allele predicted at one or more of the polymorphic markers predicts the presence of the pisifera shell phenotype. In some cases, the genotype of the polymorphic marker may comprise one or more, or all, of the deleted nucleotides of intron 1 depicted in SEQ ID NO:12.

[030] In some embodiments, the method comprises providing a sample of the plant or seed; and detecting in the sample a genotype of a polymorphic marker encoding a mutation in the SHELL gene product at one or more amino acid positions selected from the group consisting of amino acid positions 3, 5, 8, 9, 10, 11, 12, 21, 22, 23, 24, 25, 26, 27, 28, 30, 37, and 41, selected from the group consisting of amino acid positions 8, 9, 10, 11, 12, 23, 24, 37, and 41 or selected from the group consisting of amino acid positions 8, 9, 10, 11, 12, 23, 24, 31, 37, and 41. In some cases, the genotype of the polymorphic marker comprises a deletion of one or more, or all, of the amino acids at positions 8 through 12 of the wild-type SHELL gene product. In some cases, heterozygosity at one or more of the polymorphic markers for a pisifera allele and a dura allele predicts the presence of the tenera shell phenotype. In some cases, homozygosity for a genotype of a pisifera allele predicted at one or more of the polymorphic markers predicts the presence of the pisifera shell phenotype. In some cases, heterozygosity for a genotype of a first pisifera allele predicted at one or more of the polymorphic markers and a second pisifera allele predicted at one or more of the polymorphic markers predicts the presence of the pisifera shell phenotype. In some cases, the polymorphic marker genotype may comprise one or more of the predicted pisifera allele SHELL gene products depicted in SEQ ID NOs: 3 through 10, or one or more of the predicted pisifera allele SHELL gene products depicted in SEQ ID NOs: 3, 5, 7, 8, and 9.

[031] In some embodiments, the genotype of the polymorphic marker comprises a mutation at amino acid position 23 as compared to the wild-type SHELL gene product. In some cases, the mutation comprises a lysine to glutamine mutation or a lysine to asparagine mutation at amino acid position 23. In some embodiments, the genotype of the polymorphic marker comprises a mutation at amino acid position 24 as compared to the wild-type SHELL gene product. In some cases, the mutation comprises an arginine to histidine mutation or an arginine to glycine mutation at amino acid position 24. In some embodiments, the genotype of the polymorphic marker comprises a mutation at amino acid position 37 of the wild-type SHELL gene product. In some cases, the mutation comprises a valine to aspartate mutation at amino acid 37. In some embodiments, the genotype of the polymorphic marker comprises a mutation at amino acid position 41 of the wild-type SHELL gene product. In some cases, the mutation comprises an alanine to aspartate mutation at amino acid 41.

[032] In some embodiments, the method comprises providing a sample of the plant or seed; and detecting in the sample a genotype of a polymorphic marker encoding a mutation in the SHELL gene product at a position in the nuclear localization signal (NLS) of the SHELL gene product, wherein the mutation at the position in the NLS comprises a mutation at an amino acid position selected from the group consisting of amino acid position 3, 5, 9, 10, 21, 22, 23, 24, 25, 26, 27, 28, and 30; or amino acid position 23, 24, 25, 26, 27, 28, and 30 of the SHELL gene product. In some cases, the mutation is at an amino acid position selected from the group consisting of amino acid position 23 and 24 of the SHELL gene product. In some cases, the mutation at amino acid position 23 comprises a mutation from lysine to glutamine. In some cases, the mutation at amino acid position 23 comprises a mutation from lysine to asparagine. In some cases, the mutation at amino acid position 24 comprises a mutation from arginine to histidine. In some cases, the mutation at amino acid position 24 comprises a mutation from arginine to glycine.

[033] In some embodiments, the plant or seed is generated from i) a cross between a plant having the hard bark phenotype and a plant having the pisiferous bark phenotype, ii) the self-pollination of a tenera palm, iii) a cross between two plants having the tenera bark phenotype, iv) a cross between a plant having the hard bark phenotype and a plant having the tenera bark phenotype, or v) a cross between a plant having the tenera bark phenotype and a plant having the pisiferous bark phenotype. In some embodiments, the plant is less than 5 years old. In some embodiments, the plant is less than one year old. In some embodiments, the polymorphic marker is, or is at least, 86, 88, 90, 92, 94, 96, 97, 98, or 99% predictive of the tenera phenotype.

[034] In some embodiments, the method further comprises selecting the seed or plant for cultivation if the plant is heterozygous for the polymorphic marker (e.g., heterozygous for a dura marker and a pisifera marker predicting a tenera phenotype). In some embodiments, the method further comprises selecting the seed or plant for cultivation if the plant is homozygous for a polymorphic marker (e.g., indicating a dura phenotype or a pisifera phenotype). In some embodiments, plants or seeds are discarded, stored (e.g., stored separately from tenera plants or seeds), or cultivated (e.g., cultivated separately from tenera plants or seeds) if the plants or seeds do not have a genotype predictive of the tenera bark phenotype, such as if the plants or seeds have a genotype predictive of a pisifera phenotype or have a genotype predictive of a dura phenotype.

[035] Also provided is a method for segregating a plurality of palm plants (e.g., oil palm) into different categories based on predicted bark phenotype. In some embodiments, the method comprises providing a sample of each plant in the plurality of plants; determine, from the samples, the genotype of at least one polymorphic marker at a position in exon 1 of the SHELL gene selected from the group consisting of: (i) nucleotides 7, 8, 9, 13, 14, 15, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 109, 110, 111, 114, 121, 122 and 123; (ii) nucleotides 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37; (iii) nucleotide 34; (iv) nucleotides 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91 and 92; (v) nucleotides 67, 69, 70, and 71; (vi) nucleotide 67; (vii) nucleotide 69; (viii) nucleotide 70; (ix) nucleotide 71; (x) nucleotide 110; (xi) nucleotide 114; or (xii) nucleotide 122; and segregate the plants into groups based on the genotype of the polymorphic marker, where the groups correspond to plants predicted to have the softshell phenotype, plants predicted to have the hardshell phenotype, and plants predicted to have the softshell phenotype.

[036] Kits for determining the husk phenotype of a palm seed or plant are also provided. In some embodiments, the kit comprises one or more oligonucleotide primers or probes that independently comprise: a sequence of at least, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 (or 20, 22, 24, 30, or more) consecutive nucleotides of SEQ ID NO:27; or; a sequence 100% complementary to at least, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 (or 20, 22, 24, 30, or more) consecutive nucleotides of SEQ ID NO:27, wherein the one or more primers or probes independently hybridize to a sequence that is within, or within about, 5,000; 2,500; 1,000; 750; 500; 250; 200; 150; 100; 75; 25, or 1 bp from a position in exon 1 of the SHELL gene selected from the group consisting of: (i) nucleotides 7, 8, 9, 13, 14, 15, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 109, 110, 111, 114, 121, 122 and 123; (ii) nucleotides 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and 37; (iii) nucleotide 34; (iv) nucleotides 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91 and 92 (v) nucleotides 67, 69, 70 and 71; (vi) nucleotide 67; (vii) nucleotide 69; (viii) nucleotide 70; (ix) nucleotide 71; (x) nucleotide 110; (xi) nucleotide 114; or (xii) nucleotide 122.

[037] In some embodiments, the one or more primers or probes independently hybridize to a sequence that is adjacent to, or contains, a position in exon 1 of the SHELL gene selected from the group consisting of one or more of the preceding groups of nucleotides (i) through (xii).

[038] In some embodiments, the one or more primers or probes specifically hybridize to palm DNA or RNA.

[039] In some embodiments, a detectable identifier is linked (e.g., covalently linked) to the oligonucleotide. In some embodiments, the detectable identifier is fluorescent.

[040] In some embodiments, the kit further comprises a polynucleotide encoding a polypeptide comprising a sequence substantially (e.g., at least 80, 85, 90, 95, 97, 98, 99%) identical or identical to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, or 21, wherein the polynucleotide comprises a mutation depicted in SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, or 21 relative to wild-type SHELL shAVROS, or shMPOB.

[041] Also provided is an isolated nucleic acid comprising a polynucleotide encoding a polypeptide comprising a sequence substantially (e.g., at least 80, 85, 90, 95, 97, 98, 99%) identical or identical to at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive amino acids of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, or 11, wherein the polynucleotide comprises a mutation depicted in SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, or 21 relative to wild-type SHELL, shAVROS, or shMPOB.

[042] Also provided is a cell or seed or plant comprising a heterologous expression cassette, the expression cassette comprising a heterologous promoter operably linked to a polynucleotide encoding a polypeptide comprising a sequence substantially (e.g., at least 80, 85, 90, 95, 97, 98, 99%) identical to or identical to SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, or 11, for example, wherein the polynucleotide comprises a mutation depicted in SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, or 21 relative to wild-type SHELL shAVROS, or shMPOB. In some embodiments, the seed or plant is a palm (e.g., oil palm) seed or palm (e.g., oil palm) fruit. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the heterologous promoter results in the level of expression of an RNA encoding the polypeptide in the seed or plant that is less than, equal to, or greater than the expression of an endogenous SHELL RNA in the seed or plant. In some embodiments, the seed or plant comprises two dura alleles of an endogenous SHELL gene. In some embodiments, the seed or plant produces fruit that have mature shells that are, on average, less than 2 mm thick, less than 3 mm thick, or are between 0.5 and 3 mm thick.

[043] Also provided is a cell or seed or plant comprising a heterologous expression cassette, the expression cassette comprising a promoter operably linked to a polynucleotide having at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, or 21, or a complement thereof, which polynucleotide, when expressed in the seed or plant, reduces the expression of an endogenous SHELL polypeptide in the seed or plant (compared to a control plant lacking the expression cassette), wherein reduced expression of the SHELL polypeptide results in reduced shell thickness of future seeds produced by the plant. In some embodiments, the polynucleotide encodes a siRNA, antisense polynucleotide, a microRNA, or a sense suppression nucleic acid, thereby suppressing the expression of an endogenous SHELL gene. In some embodiments, the seed or plant produces mature shells that are, on average, less than about 2 mm thick, less than about 3 mm thick, or are between 0.5 and 3 mm thick.

[044] Also provided is a method of producing a plant as described above or elsewhere herein, comprising introducing the expression cassette into a plant.

[045] A method of cultivating the plants described herein is also provided.

[046] Other modalities will become evident from reading the rest of the revelation.

DEFINITIONS

[047] A "shell phenotype" refers to the three fruit forms of E. guineensis - hard, tenera, and pisifera. The hard (wild-type) fruit form is exemplified by the presence of a shell that has an average thickness of at least 2 to 8 mm and is typically found in palms that have a homozygous wild-type SHELL genotype. The pisifera fruit form is exemplified by the absence of a shell and is typically found in palms that lack a functional SHELL gene. For example, a pisifera palm may have two nonfunctional SHELL genes (e.g., homozygous for one nonfunctional SHELL genotype or heterozygous for two different nonfunctional SHELL genotypes). The tenera fruit form is exemplified by the presence of a thin shell that has an average thickness of less than about 3 mm (e.g., approximately 0.5 to 3 mm) and is typically found in palms that are heterozygous for one functional SHELL gene and one nonfunctional SHELL gene. Heterologous palms that overexpress or underexpress the SHELL gene or gene product or that partially or completely interfere with the activity of an endogenous SHELL gene product may also exhibit a hard, tenera, or pisifera fruit form phenotype.

[048] A "polymorphic marker" refers to a genetic marker that distinguishes between two alleles. The polymorphic marker may be a nucleotide substitution, insertion, deletion, or rearrangement, or a combination thereof.

[049] As used herein, “detecting a genotype” refers to: (i) analyzing a nucleic acid to determine a genotype by performing a sequence-specific sequencing, hybridization, polymerization, or alignment digestion reaction, or by detecting the bulk of the nucleic acid, or a portion thereof; or (ii) analyzing a polypeptide, or portion thereof, encoded by the nucleic acid by performing a sequence-specific sequencing, detection (e.g., ELISA), or proteolytic digestion reaction, or by detecting the bulk of the polypeptide, or a portion thereof.

[050] As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” refer to nucleic acid regions, nucleic acid segments, primers, probes, amplicons, and oligomer fragments. The terms are not limited in length and are generic to linear polymers of polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other purine or pyrimidine base N-glycoside, or modified purine or pyrimidine bases. These terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.

[051] A nucleic acid, polynucleotide or oligonucleotide may comprise, for example, phosphodiester linkages or modified linkages including, but not limited to, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages.

[052] A nucleic acid, polynucleotide or oligonucleotide may comprise the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil) and/or bases other than the five biologically occurring bases.

[053] Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.

[054] “Percent sequence identity” is determined by comparing two ideally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for ideal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage sequence identity.

[055] The term "substantial identity" of polypeptide sequences means that a polypeptide comprises a sequence that has at least 75% sequence identity. Alternatively, percent identity may be any integer from 75% to 100%. Exemplary embodiments include at least: 75%, 80%, 85%, 90%, 95%, or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard or default parameters as described below. One of skill will recognize that these values may be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Polypeptides that are "substantially similar" share sequences as noted above except that residual positions that are not identical may diverge by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues that have similar side chains. For example, a group of amino acids that have aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; a group of amino acids that have aliphatic hydroxyl side chains are serine and threonine; a group of amino acids that have amide-containing side chains are asparagine and glutamine; a group of amino acids that have aromatic side chains are phenylalanine, tyrosine, and tryptophan; a group of amino acids that have basic side chains are lysine, arginine, and histidine; a group of amino acids that have acidic side chains are aspartic acid and glutamic acid; and a group of amino acids that have sulfur-containing side chains are cysteine and methionine. The preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

[056] One indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other, or to a third nucleic acid, under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. In general, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those at which the salt concentration is about 0.02 molar at pH 7, and the temperature is at least about 60 °C.

[057] The term "promoter" or "regulatory element" refers to a determinant region or sequence located upstream or downstream of the initiation of transcription and that is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of plant origin, for example, promoters derived from plant viruses, such as the CaMV35S promoter, may be used.

[058] The term "plant" includes whole plants, shoots, plant organs/structures (e.g. daughters, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryos, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, seed tissue, ground tissue and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny thereof. The class of plants that can be used in the method of the present invention is, in general, as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous. In one exemplary embodiment, the plant is an oil palm (E. guineensis or E. oleifera, or a hybrid thereof). In some cases, the plant is E. guineensis.

[059] An “expression cassette” refers to a nucleic acid construct, which, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. Antisense constructs or sense constructs that are not or cannot be translated are expressly included by this definition. The expression cassette may contain a heterologous promoter.

[060] The term "operatively linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or transcription factor binding site array) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[061] A polynucleotide sequence or amino acid sequence is “heterologous to” an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a heterologous promoter operably linked to a coding sequence refers to a promoter from a species other than that from which the coding sequence was derived, or, if from the same species, a promoter that is different from any naturally occurring allelic variants, or a promoter that is not naturally found to be operably linked to the specified coding sequence in the specified plant.

[062] As used herein, the term “nucleotide position” and the like, in the context of a nucleotide position of exon 1 of the SHELL gene refers to the position of a nucleotide relative to the adenosine of the wild-type SHELL gene initiator methionine triplet (“ATG”) codon (i.e., amino terminus). Thus, for example, nucleotide position 1 refers to the adenosine of the wild-type SHELL gene initiator methionine triplet ATG codon; and, position 2 refers to the next nucleotide (e.g., “T” of the wild-type SHELL gene initiator methionine triplet ATG codon), and so on. Similarly, in the context of a nucleotide position of intron 1 of the SHELL gene, the term “nucleotide position” and similar terms refer to the position of a nucleotide relative to the first nucleotide of intron 1 of the wild-type SHELL gene. Thus, the first nucleotide of intron 1 of the SHELL gene is at position 1, the second at position 2, and so on.

[063] Similarly, the term "amino acid position" in the context of a particular amino acid, or group of amino acids, of the SHELL gene refers to an amino acid position relative to the initiator (i.e., amino terminus) methionine of the SHELL gene. Thus, for example, amino acid position 1 refers to the amino terminal methionine, amino acid position 2 refers to the adjacent glycine of the wild-type SHELL or an alternative amino acid or deletion found in a mutant SHELL allele at the same position. It will be appreciated that these positions are independent of any N-terminal degradation or conjugation or other post-translational processing. For example, in a SHELL polypeptide in which the N-terminal methionine amino acid is post-translationally removed, position 2 still refers to the previously adjacent glycine amino acid and position 3 refers to the adjacent arginine amino acid of the wild-type SHELL or an alternative amino acid or deletion found in a mutant SHELL allele at the same position.

BRIEF DESCRIPTION OF THE DRAWINGS

[064] Figure 1. SHELL gene model. Exons (boxes) and introns (horizontal lines) were validated by RNA-seq. A diagram of protein domains encoded by the indicated exons is provided below the gene diagram. MADS-box, I, K and C domains of the SHELL protein are indicated.

[065] Figure 2. Nucleotide variants in the SHELL gene. The DNA sequence of wild-type exon 1 (ShDeliDura) encoding the MADS-box domain of SHELL (SEQ ID NO: 25) is shown in the top row of the DNA sequence alignment. Sequences of the alleles AVROS, MPOB, allele 3, allele 4, allele 5, allele 6, allele 7, allele 8, allele 9, allele 10, and allele 11 (SEQ ID NO: 22, 23, 13 to 21, respectively) are shown aligned to the dura sequence. Single nucleotide variants are indicated by boxes. Deleted bases (allele 7) are indicated by dashes.

[066] Figure 3. Amino acid variants in the SHELL gene. The peptide sequence of the wild-type MADS-box domain (ShDeliDura) domain (SEQ ID NO: 24) is shown in the top row of the peptide alignment. The peptide sequences of AVROS, MPOB, allele 3, allele 4, allele 5, allele 6, allele 7, allele 8, allele 9, allele 10, and allele 11 (SEQ ID NO: 1-11, respectively) are shown aligned to the dura peptide sequence. Variant amino acids caused by missense single nucleotide variants are indicated by the appropriate single-letter amino acid code. Deleted amino acids (allele 7) are indicated by asterisks. Amino acids that are unchanged from the dura peptide sequence are indicated by dashes.

DETAILED DESCRIPTION OF THE INVENTION I. INTRODUCTION

[067] The present disclosure describes the discovery of alleles 3-10 of the SHELL gene that are predicted to modulate the fruit shape phenotype of palm trees (e.g., oil palm). Similarly, alleles 11 (depicted in SEQ ID NOs: 11 and 21) and 12 (depicted in SEQ ID NO:12) are both predicted to modulate the fruit shape phenotype directly and can be used to infer the genotype of the SHELL gene due to their close physical linkage to allele polymorphisms 3 through 10. A polymorphic marker closely linked to the SHELL gene, or the identification of the presence, absence, or copy number of alleles 3 through 11 in an oil palm can be used by seed producers as a quality control tool to i) reduce or eliminate dura or pisifera contamination of tenera seed or seedlings, ii) reduce or eliminate dura or tenera contamination of pisifera seed or seedlings, iii) reduce or eliminate pisifera or tenera contamination of dura seed or seedlings, iv) positively identify seed or tenera seedlings which are then selected as suitable planting material for commercial palm oil production, v) positively identify dura seeds or seedlings which can then be selected as suitable planting material for commercial dura germplasm production, or vi) positively identify pisifera seeds or seedlings which can then be selected as suitable planting material for commercial pisifera germplasm production.

[068] Identification of the SHELL gene or a marker genetically linked to the bark tract is also of importance in breeding programs. The marker or gene alleles responsible for the tract can be used to separate dura, tenera and pisifera plants in the nursery; the advantage here is that they could be planted separately based on shell fruit shape phenotype. This is of interest as pisifera palms generally show very vigorous vegetative growth, so in a test consisting of all three types, bias in results could occur due to inbreeding competition. Furthermore, separating pisifera palms and planting them in high density encourages male inflorescence and this facilitates the production of pollen that is used in breeding programs (Jack et al., 1998). Consequently, following detection of the presence or absence of a SHELL genotype predicted to result in a dura, pisifera, or tenera phenotype, or a linked marker as described below, an additional step of: (1) reducing and eliminating dura or pisifera contamination from tenera seed or seedlings, (2) positively identifying tenera seeds or seedlings that are then selected as suitable planting material for commercial palm oil production, or (3) separating dura, tenera, and pisifera plants into two or more groups (e.g., plants predicted to be tenera in one group and plants predicted to be dura or pisifera in a second group; plants predicted to be dura in one group and plants predicted to be tenera or pisifera in a second group; plants predicted to be pisifera in one group and plants predicted to be dura or tenera in a second group, or separation into three groups: dura, pisifera, and tenera) may be achieved.

[069] Any marker that exists that is polymorphic between the parent dura and pisifera trees in a cross and is linked to the shell locus has the potential to serve as a molecular signal to identify tenera trees in a cross. For example, if a dura tree that is homozygous for “T” (i.e., T/T) at a given SNP position near the shell locus is crossed with a pisifera tree that is homozygous for “A” (i.e., A/A) at the same SNP position, then one could genotype seeds from the cross, or one could genotype seedlings that arise from seeds from the cross, at the SNP position to screen and identify contaminating seeds or seedlings. Seeds that are determined to be heterozygous at the SNP position, (i.e., A/T) are very likely to be tenera, unless recombination between the marker and the SHELL gene has occurred in the individual that is genotyped. Similarly, seeds that are homozygous at the SNP position for “A” or “T” (i.e., A/A or T/T) are contaminant trees of pisifera or dura, respectively, and when these trees become sexually mature in several years, they will produce suboptimal fruit types. Additionally, seeds or seedlings that have a “C” or “G” at the SNP position, neither of which is present in the parental palm of the cross, are likely trees that originated from a pollen donor other than the one intended in the cross and can therefore be ruled out as contaminant seeds or seedlings. Markers that are in closer proximity to the SHEL locus would have greater predictive accuracy than markers that are further from the shell locus, because the closer the marker is to the SHELL gene, the less likely that recombination would occur that would break the linkage between the marker and the SHELL gene. Consequently, polymorphic markers within the SHELL gene itself are expected to have the strongest predictive power, and analysis of multiple markers closely linked to or within the SHELL gene may be advantageous.

II. DETERMINATION OF SHELL PHENOTYPE BASED ON NUCLEIC ACID DETECTION

[070] In view of the finding that the SHELL genotype segregates with the tenera/pisifera/hard bark phenotype, genotyping of a plant or seed at the SHELL locus or adjacent genomic regions can be used to predict the bark phenotype of a palm.

[071] SEQ ID NO:24 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the protein expressed in hard-fruited oil palm (ShDeliDura). The endogenous protein includes additional C-terminal amino acids not included in SEQ ID NO:24. In hard-fruited oil palm, proteins derived from both alleles of the gene include: (i) an isoleucine (I), lysine (K), arginine (R), isoleucine (I), and glutamate (E) at positions 8 to 12 respectively, which are deleted in the predicted pisifera allele 7; (ii) a glutamate (E) at position 12, which is mutated to a glutamine (Q) in predicted pisifera allele 6; (iii) a lysine (K) at position 23 that is mutated to a glutamine (Q) in predicted pisifera allele 3 and an asparagine (N) in predicted pisifera allele 5; (iv) an arginine (R) that is mutated to a histidine (H) in predicted pisifera allele 8 and a glycine (G) in predicted pisifera allele 9; (v) a leucine (L) at position 29 that is mutated to a proline in the pisifera shMPOB allele; (vi) a lysine (K) at position 31 that is mutated to an asparagine in the pisifera shAVROS allele; (vii) a valine (V) at position 37 that is mutated to an aspartate (D) in predicted pisifera allele 10; and (viii) an alanine (A) at position 41, which is mutated to an aspartate (D) in the predicted pisifera allele 4.

[072] SEQ ID NO:1 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the protein expressed in oil palm of the pisifera fruit type that is derived from the Zaire line (shAVROS). The endogenous protein includes additional C-terminal amino acids not included in SEQ ID NO:1. This polypeptide includes an asparagine (N) amino acid at the 31st amino acid position. A nucleotide sequence encoding exon 1 of the shAVROS allele is provided in SEQ ID NO:22.

[073] SEQ ID NO:2 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the protein expressed in oil palm of the pisifera fruit type that is derived from the Nigerian line (shMPOB). The endogenous protein includes additional C-terminal amino acids not included herein. This polypeptide includes a proline (P) amino acid at the 29th amino acid position. A nucleotide sequence encoding exon 1 of the shMPOB allele is provided in SEQ ID NO:23.

[074] SEQ ID NO:3 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the oil palm expressed protein of the predicted pisifera fruit type 3 SHELL allele. The endogenous protein includes additional C-terminal amino acids not included herein. This polypeptide includes a glutamine (Q) amino acid at the 23rd amino acid position. A nucleotide sequence encoding exon 1 of allele 3 is provided in SEQ ID NO:13.

[075] SEQ ID NO:4 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the oil palm expressed protein of the predicted pisifera fruit type SHELL allele 4. The endogenous protein includes additional C-terminal amino acids not included herein. This polypeptide includes an aspartate (D) amino acid at the 41st amino acid position. A nucleotide sequence encoding exon 1 of allele 4 is provided in SEQ ID NO:14.

[076] SEQ ID NO:5 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the oil palm expressed protein of the predicted pisifera fruit type SHELL allele 5. The endogenous protein includes additional C-terminal amino acids not included herein. This polypeptide includes an asparagine (N) amino acid at the 23rd amino acid position. A nucleotide sequence encoding exon 1 of allele 5 is provided in SEQ ID NO:15.

[077] SEQ ID NO:6 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the oil palm expressed protein of the predicted pisifera fruit type SHELL allele 6. The endogenous protein includes additional C-terminal amino acids not included herein. This polypeptide includes a glutamine (E) amino acid at the 12th amino acid position. A nucleotide sequence encoding exon 1 of allele 6 is provided in SEQ ID NO:16.

[078] SEQ ID NO:7 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the oil palm expressed protein of the predicted pisifera fruit type SHELL allele 7. The endogenous protein includes additional C-terminal amino acids not included here. This polypeptide has a deletion of the amino acids lysine (K), arginine (R), isoleucine (I), and glutamate (E), at positions 8 to 12, respectively, compared to the wild-type ShDeliDura allele. A nucleotide sequence encoding exon 1 of allele 7 is provided in SEQ ID NO:17.

[079] SEQ ID NO:8 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the oil palm expressed protein of the predicted pisifera fruit type SHELL allele 8. The endogenous protein includes additional C-terminal amino acids not included herein. This polypeptide includes a histidine (H) amino acid at the 24th amino acid position. A nucleotide sequence encoding exon 1 of allele 8 is provided in SEQ ID NO:18.

[080] SEQ ID NO:9 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the oil palm expressed protein of the predicted pisifera fruit type SHELL allele 9. The endogenous protein includes additional C-terminal amino acids not included herein. This polypeptide includes a glycine (G) amino acid at the 24th amino acid position. A nucleotide sequence encoding exon 1 of allele 9 is provided in SEQ ID NO:19.

[081] SEQ ID NO:10 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the oil palm expressed protein of the predicted pisifera fruit type SHELL allele 10. The endogenous protein includes additional C-terminal amino acids not included herein. This polypeptide includes an aspartate (D) amino acid at the 37th amino acid position. A nucleotide sequence encoding exon 1 of allele 10 is provided in SEQ ID NO:20.

[082] SEQ ID NO:11 represents the predicted amino acid sequence of the N-terminal 181 amino acids of the oil palm expressed protein of the predicted pisifera fruit type SHELL allele 11. The endogenous protein includes additional C-terminal amino acids not included herein. The allele encodes a silent mutation relative to a wild-type SHELL gene (ShDeliDura). A nucleotide sequence encoding exon 1 of allele 11 is provided in SEQ ID NO:21. As described herein, this silent mutation may affect transcriptional or translational regulation and therefore provide a pisifera phenotype despite encoding a wild-type protein sequence. Alternatively, the nucleotide sequence encoding such a silent mutation can be used to infer the presence or absence of a genotype at one or more of the preceding polymorphic nucleotide (e.g., one or more of the wild-type relative polymorphic markers exemplified in SEQ ID NOs: 13-20, 13-20 and 23, 13-20 and 22, or 13-20 and 22-23) or amino acid markers (e.g., one or more of the wild-type relative polymorphic markers exemplified in SEQ ID NOs: 3-10, 2-10, 1 and 3-10, or 1-10).

[083] SEQ ID NO:12 represents the nucleotide sequence of the first 56 nucleotides of intron 1 of SHELL allele 12 in which nucleotides 43, 44, 45, and 46 are deleted relative to intron 1 of a wild-type SHELL allele (ShDeliDura). Since this polymorphism is within a non-coding region of the SHELL gene, it is a silent mutation. As described herein, this silent mutation may affect transcriptional or translational regulation or splicing and therefore provide a pisifera phenotype. Alternatively, the presence or absence of allele 12 can be used to infer the presence or absence of a genotype at one or more of the preceding polymorphic nucleotide (e.g., one or more of the wild-type relative polymorphic markers exemplified in SEQ ID NOs: 13-20, 13-21, 13-20 and 22, 13-20 and 23, or 13-23) or amino acid markers (e.g., one or more of the wild-type relative polymorphic markers exemplified in SEQ ID NOs: 3-10, 3-11, 2-10, 2-11, 1-10, 1 and 3-10, 1 and 3-11, or 1-11).

[084] Oil palm trees of the pisifera fruit type are the result of one of at least four possibilities: i) two homozygous SHELL alleles having a nucleotide sequence encoding one of the following protein sequences: SEQ ID NOs: 3 to 10; ii) two heterozygous SHELL alleles having two different nucleotide sequences that independently encode one of the following protein sequences: SEQ ID NOs: 3 to 10, or iii) one SHELL allele encoding the ShAVROS or ShMPOB protein sequence and the other allele encoding a mutation relative to the wild type represented in one or more of the following protein sequences: SEQ ID NOs: 3 to 10. In some cases, the nucleotide sequences comprising SEQ ID NO: 12 and/or 21 are similarly predicted pisifera alleles. In such cases, a pisifera fruit type may result in plants that are homozygous for SEQ ID NO: 12 or 21 or heterozygous for SEQ ID NO: 12 or 21 and a different allele selected from the group consisting of any of SEQ ID Nos:13 through 23 (e.g., any of SEQ ID Nos:13 through 20) or encode any of SEQ ID Nos:1 through 10 (e.g., any of SEQ ID NOs:3 through 10).

[085] Oil palm trees of the tenera fruit type are the result of an allele encoding one or more of the pisifera alleles described herein and an allele encoding a wild-type SHELL protein (ShDeliDura). It will be appreciated that SEQ ID Nos:1 through 11 and 24 are representative sequences and that different individual palms may have an amino acid sequence that has one, two, three, four, or more amino acid changes relative to SEQ ID Nos:1 through 11 and 24, due to, for example, natural variation. Similarly, SEQ ID Nos:12 through 23 and 25 are representative sequences and different individual palms may have a nucleotide sequence that has one, two, three, four, or more nucleotide changes relative to SEQ ID Nos:12 through 23 and 25 due to, for example, natural variation.

[086] One or more polymorphisms between SHELL pisifera and dura alleles can be used to determine the bark phenotype of a palm or other plant. For example, when the polymorphism is codominant (detectable independently of the other allele) then: the presence of only one SHELL dura allele indicates that the plant has or will have a hard bark phenotype; the presence of only one SHELL pisifera allele indicates that the plant has or will have a pisifera bark phenotype; and the presence of one SHELL pisifera allele and one SHELL dura allele indicates that the plant has or will have a soft bark phenotype.

[087] However, genomic regions adjacent to the SHELL gene are also useful in determining the likelihood that a palm will manifest a particular bark phenotype. Due to genetic linkage to the SHELL gene, polymorphisms adjacent to the SHELL locus are predictive of bark phenotype, albeit with reduced accuracy as a function of increasing distance from the SHELL locus. SEQ ID NO:27 provides an approximately 3.4 Mb genomic region of the palm genome comprising the SHELL gene. Table A of U.S. Patent Application Publication No. 2013/0247249 discloses 8217 SNPs identified within SEQ ID NO:27. A selection of these SNPs have been genetically mapped to the SHELL locus. The estimated predictive values of these SNPs are also described in Table A of U.S. Patent Application Publication No. 2013/0247249. Thus, as an example, the SNP listed in row 1 of U.S. Patent No. 2013/0247249, Table A as having an estimated prediction success of 83, represents a SNP that is accurate in predicting the bark phenotype 83% of the time. Stated another way, using this SNP as a genetic marker, one can correctly predict the bark phenotype of palm trees 83 out of 100 times. Thus, even at a significant physical distance from the SHELL locus on the palm chromosome, polymorphic markers allow for relatively accurate prediction of plant bark phenotype. In some embodiments, the polymorphic marker is within 1, 10, 20, 50, 100, 200, 500, 1,000 kb of the SHELL gene (e.g., the gene corresponding to SEQ ID NO:28).

[088] Accordingly, methods of detecting one or more polymorphic markers within a region of the palm genome corresponding to SEQ ID NO:27 are provided. Such methods are useful in predicting the bark phenotype of palm trees, for example. Although over 8,200 specific polymorphisms are provided in U.S. Patent No. 2013/0247249, it should be noted that the polymorphisms represented are merely an example of polymorphisms within the genomic region corresponding to SEQ ID NO:27. Additional polymorphisms may be identified as desired and also be used to predict the bark phenotype of a palm tree. Such additional polymorphisms are intended to be encompassed in the methods described herein. Furthermore, it will be appreciated that SEQ ID NO:27 is a representative sequence and that different individual palms may have a corresponding genomic region that has one or more nucleotide changes relative to SEQ ID NO:27 due to, for example, natural variation. As noted elsewhere herein, however, identifying the region of a genome corresponding to SEQ ID NO:27 can be readily determined using alignment programs, etc.

[089] The nucleic acid sequences provided herein were generated by nucleotide sequencing and, on occasion, include one or more stretches of "N's". These stretches of N's represent gaps in the assembly of sequences of an estimated size. The precise number of N's in a sequence is an estimate (e.g., 100 N's may only represent 30 bases). N's can be any base, and are likely repetitive sequences in the genome.

[090] Detecting specific polymorphic markers can be accomplished by methods known in the art for detecting sequences at polymorphic sites. For example, standard techniques for genotyping the presence of SNPs and/or microsatellite markers can be used, such as fluorescence-based techniques (Chen, X. et al., Genome Res. 9(5): 492-498 (1999)), using PCR, LCR, nested PCR, and other techniques for nucleic acid amplification. Specific commercial methodologies available for SNP genotyping include, but are not limited to, TaqMan™ genotyping assays and SNPlex platforms (Applied Biosystems), gel electrophoresis (Applied Biosystems), gel spectrometry (e.g., Sequenom MassARRAY system), minisequencing methods, real-time PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), array hybridization technology (e.g., Affymetrix GeneChip; Perlegen), BeadArray technologies (e.g., Illumina GoldenGate and Infinium assays), array labeling technology (e.g., Parallele), and endonuclease-based fluorescence hybridization technology (Invader; Third Wave). Some of the available array platforms, including Affymetrix SNP Array 6.0 and Illumina CNV370-Duo and 1M BeadChips, include SNPs that tag specific copy number variants.

[091] In certain embodiments, polymorphic markers are detected by sequencing technologies. Obtaining sequence information about an individual plant identifies particular nucleotides in the context of a sequence. For SNPs, sequence information about a single unique sequence site is sufficient to identify alleles at that particular SNP. For markers comprising more than one nucleotide, sequence information about the nucleotides in the individual that contain the polymorphic site identifies the individuals alleles for the particular site.

[092] Various methods for obtaining nucleic acid sequence are known to one of skill in the art, and all such methods are useful for practicing the invention. Sanger sequencing is a known method for generating nucleic acid sequence information. Recent methods for obtaining large amounts of sequence data have been developed, and such methods are also contemplated as useful for obtaining sequence information from a plant, if desired. These include, but are not limited to, pyrosequencing technology (Ronaghi, M. et al. Anal Biochem 267:65-71 (1999); Ronaghi, et al., Biotechniques 25:876-878 (1998)), e.g., 454 pyrosequencing (Nyren, P., et al. Anal Biochem 208:171-175 (1993)), Illumina/Solexa sequencing technology (www.illumina.com; see also Strausberg, R L, et al Drug Disc Today 13:569-577 (2008)), Supported Oligonucleotide Ligation and Detection (SOLiD) Platform technology (Applied Biosystems, www.appliedbiosystems.com); Strausberg, R L, et al., Drug Disc Today 13:569-577 (2008), single-molecule real-time sequencing (Pacific Biosciences) and IonTorrent technology (ThermoFisher).

[093] Polymorphism detection methods can be performed on any type of plant biological sample that contains nucleic acids (e.g., DNA, RNA). As a particular advantage of the methods is to predict the bark phenotype of young plants prior to field cultivation, in some embodiments, the samples are obtained from a plant that has been germinated less than 1, 2, 4, 6, months or less than 1, 2, 3, 4, or 5 years. In some embodiments, the plants are generated from i) a cross between dura and pisifera palms, ii) the self-pollination of a tenera palm, iii) a cross between two plants having the tenera bark phenotype, iv) a cross between dura and tenera palms, and v) a cross between tenera and pisifera palms. Because such crosses are not 100% efficient, such crosses result in some percentage of seeds or plants that will not produce seeds or plants with the tenera shell phenotype in the future (in the case of i), and the observed number of tenera palms observed does not follow the expected Mendelian segregation (ii, iii & iv). By testing seeds or plants resulting from attempted crosses, one can reduce or eliminate contaminating seeds or plants other than tenera from material planted for cultivation (optionally discarding those plants that are impaired for being dura and/or pisifera). Alternatively, one can identify and segregate plants based on their predicted SHELL genotype, allowing the selection and cultivation of fields of pure pisifera and dura trees if desired, for example, for further breeding purposes.

III. TRANSGENIC PLANTS

[094] As discussed above, the palm SHELL gene has been found to control bark phenotype. Accordingly, in some embodiments, plants that have modulated the expression of a SHELL polypeptide are provided. The most desirable bark phenotype (tenera, which has bark less than 2 mm thick) occurs naturally as a heterozygote between the dura and pisifera alleles.

[095] Alleles of SHELL pisifera have been found to contain missense mutations in portions of the gene encoding the MADS-box domain of the protein, which plays a role in transition regulation. Thus, it is hypothesized that the tenera phenotype may result from a mechanism involving protein:protein interaction of pisifera types of non-DNA binding SHELL proteins with fully functional types of SHELL (homodimers) or other MADS-box family members (heterodimers). Thus, in some embodiments, plants heterologously expressing a SHELL polypeptide having a functional M, I, and K domain and a functional C-(MADsbox) domain are provided. The M, I, K, and C domains are described in, e.g., Gramzow and Theissen, 2010 Genome Biology 11:214-224 and the corresponding domains can be identified in the palm sequences described herein. By expressing such a protein that has active protein:protein interaction domains but a non-functional DNA binding domain, proteins that interact with the modified SHELL protein will be removed from biological action, thus resulting in reduced shell thickness. Thus, for example, one could express any of the pisifera alleles described herein under the control of a heterologous promoter in the plant (e.g., a palm tree, for example, a dura tree), thus resulting in reduced shell thickness.

[096] Similarly, many pisifera SHELL alleles have been found to contain mutations in a nuclear localization signal (NLS) within a MADS-box domain of the SHELL protein. Thus, it is hypothesized that the tenera phenotype may result from a mechanism involving protein:protein interaction between one or more pisifera SHELL allele proteins that lack a functional NLS and one or more fully functional SHELL types (homodimers) or other MADS-box family members (heterodimers). By expressing such a protein that has active protein:protein interaction domains but a non-functional NLS, proteins that interact with the modified SHELL protein may be inhibited (e.g., prevented) from entering the nucleus or removed from the nucleus, thereby reducing the amount of biologically active SHELL protein and its interacting protein (e.g., splicing partner) in the nucleus and resulting in reduced shell thickness. Thus, for example, one can express any of the pisifera alleles containing an NLS mutation described herein, or a SHELL gene encoding a SHELL protein mutated in any of the NLS amino acids (e.g., conserved) under the control of a heterologous promoter in the plant (e.g., a palm tree, e.g., a dura tree), thus resulting in reduced shell thickness.

B. USE OF NUCLEIC ACIDS OF THE INVENTION TO ENHANCE GENE EXPRESSION

[097] Nucleic acid sequences encoding all or an active portion of a SHELL polypeptide (including, but not limited to, polypeptides substantially identical to any one or more of SEQ ID NOs: 1 to 10 (e.g., any one of SEQ ID NOs: 3 to 10), SHELL polypeptides that have a functional M, I, and K domain and a non-functional C domain, or SHELL polypeptides that have a non-functional NLS, which, when expressed, controls shell thickness) can be used to prepare expression cassettes that enhance, or increase, SHELL gene expression. Where overexpression of a gene is desired, the desired SHELL gene from a different species can be used to decrease potential sense suppression effects.

[098] Any of a number of means known in the art may be used to increase SHELL activity in plants. Any organ may be targeted, such as shoot plant organs/structures (e.g., leaves, stem, and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers, and ovules), seed (including embryos, endosperm, and seed coat), and fruit. Alternatively, a SHELL gene may be expressed constitutively (e.g., using the CaMV 35S promoter).

[099] One of skill will recognize that the polypeptides encoded by the genes of the invention, like other proteins, have different domains that perform different functions. Thus, the gene sequences need not be full length, as long as the desired functional domain of the protein is expressed.

III. PREPARATION OF RECOMBINANT VECTORS

[100] In some embodiments, to use isolated sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of larger plant species are known and described in the technical and scientific literature. See, e.g., Bardin et al. Ann. Rev. Genet. 22, 421-2622 (1988). A DNA sequence encoding the desired polypeptide, e.g., a cDNA sequence encoding a full-length protein, will preferably be combined with transcriptional and translational initiation regulatory sequences that will direct transcription of the gene sequence in the intended tissues of the transformed plant.

[101] For example, for overexpression, a plant promoter fragment can be employed which will direct expression of the gene in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of cellular development or differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transition initiation region, the 1'- or 2'-T-DNA-derived promoter of Agrobacterium tumefaciens, and other transition initiation regions of various known plant genes to the elements of interest.

[102] Alternatively, the plant promoter may direct expression of the polynucleotide of the invention in a specific tissue (tissue-specific promoters) or may otherwise be under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, or flowers. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light.

[103] If proper polypeptide expression is desired, a polyadenylation region at the 3' end of the coding region must be included. The polyadenylation region may be derived from the natural gene, from a variety of other plant genes, or from T-DNA.

[104] The vector comprising the sequences (e.g., promoters or coding regions) of genes of the invention may optionally comprise a marker gene that confers a selectable phenotype in plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluphoron or Basta.

[105] SHELL nucleic acid operatively linked to a promoter is provided that, in some embodiments, is capable of driving transcription of the SHELL coding sequence in plants. The promoter can be, for example, derived from plant or viral sources. The promoter can be, for example, constitutively active, inducible, or tissue-specific. In constructing recombinant expression cassettes, vectors, transgenics, of the invention, different promoters can be chosen and employed to differentially direct gene expression, for example, in some or all tissues of a plant or animal. In some embodiments, as discussed above, desired promoters are identified by analyzing the 5' sequences of a genomic clone corresponding to a SHELL gene as described herein.

V. PRODUCTION OF TRANSGENIC PLANTS

[106] DNA constructs of the invention may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs may be introduced directly into plant tissue using ballistic methods such as DNA particle bombardment. Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria.

[107] Several methods of palm transformation have been described. See, for example, Masani and Parveez, Electronic Journal of Biotechnology Vol. 11 No. 3, 15 July 2008; Chowdury et al., Plant Cell Reports, Volume 16, Number 5, 277–281 (1997).

[108] Microinjection techniques are known in the art and described in the scientific and patent literature. Introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. EMBO J. 3:2717–2722 (1984). Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. U.S.A. 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70–73 (1987).

[109] Agrobacterium tumefaciens-mediated transformation techniques, including disarmament and use of binary vectors, are described in the scientific literature. See, for example, Horsch et al. Science 233:496–498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. U.S.A. 80:4803 (1983)

[110] Transformed plant cells that are derived from any transformation technique can be cultured to regenerate an entire plant that has the transformed genotype and thus the desired phenotype. Such regeneration techniques are based on the manipulation of certain phytohormones in a tissue culture growth medium, which optionally contains a biocide and/or herbicide marker that has been introduced along with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124–176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21–73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from callus, explants, plant organs, or parts thereof. Such regeneration techniques are described in general in Klee et al. Ann. Rev. of Plant Phys. 38:467–486 (1987).

[111] The nucleic acids of the invention can be used to confer desired traits on essentially any plant. Thus, the invention has use on a wide range of plants including species of the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis, Vigna and Zea. Plants having a bark, and thus those of use in the present invention, include, but are not limited to, dicotyledons and monocotyledons including, but not limited to, palm trees.

EXAMPLES

[112] The following examples are offered to illustrate, but not limit, the claimed invention.

EXAMPLE 1. IDENTIFICATION OF ALLELES 3 TO 11, CORRESPONDING TO SEQ ID NOS: 12 TO 21 OF NUCLEOTIDES AND TO SEQ ID NOS: 3 TO 11 OF POLYPEPTIDES

[113] It has been previously reported that the AVROS and MPOB mutations in exon 1 of the SHELL gene are responsible for the pisifera oil palm fruit shape phenotype when homozygous (e.g., AVROS/AVROS, MPOB/MPOB or AVROS/MPOB) and the tenera oil palm fruit shape phenotype when heterozygous together with a wild-type dura allele (e.g., AVROS/dura or MPOB/dura). Although the tenera oil palm is the preferred phenotype for commercial oil palm production, it is difficult to completely avoid the occurrence of the wild-type dura palm in commercial populations. To estimate the degree of dura contamination within commercial oil palm populations and to search for novel alleles of the SHELL gene that are causative of the pisifera/tenera phenotype, 5,158 oil palm trees from 6 different smallholder plantations across Malaysia were tested for the presence of AVROS and/or MPOB alleles using an allele-specific PCR assay for each allele (dura, shAVROS and shMPOB). As expected, the majority of palms were heterozygous for both the shAVROS and shMPOB alleles (Table 2).

[114] However, 504 palms were predicted to be homozygous for the ShDeliDura allele at both SNP positions. Exon 1 of the SHELL gene, which encodes the entire MADS-box domain, was sequenced in each of these 504 palms. Sequencing was performed on PCR-amplified amplicons using primers flanking exon 1. PCR was performed under standard conditions. Amplicons were purified and Sanger sequenced in one direction using an internal primer to the PCR amplification primers. Sequencing reads were individually analyzed within CONSED to determine the sequence and zygosity of each nucleotide position within exon 1. As shown in Table 2, 13 palms were determined to be heterozygous for the shAVROS allele (2 from site 1, 4 from site 2, 2 from site 3, 3 from site 4, and 2 from site 5), indicating that these palms were indeed genotypically tenera palms. Three palms were determined to be heterozygous for the shMPOB allele (1 from site 2, 1 from site 4, and 1 from site 5), indicating that they were also genotypically tenera palms. However, the remaining 488 palms were homozygous for the ShDeliDura allele at both SNP positions, suggesting that these trees were either genotypically dura or that they carried previously unidentified mutant alleles of the SHELL gene.

[115] Allele 3 (SEQ ID NO: 3 and 13) was found to be heterozygous in 68 palms (Table 2), allele 4 (SEQ ID NO: 4 and 14) was found to be heterozygous in 66 palms, and allele 5 was found to be heterozygous in 1 palm. Each of these 135 palms was independent of the others. The amino acids encoded by alleles 3 and 5 occur within the NLS of SHELL, as do the AVROS and MPOB mutations (Figures 3). The amino acid encoded by allele 4 is C-terminal with 10 amino acids to the amino acid mutated by the shAVROS allele. TABLE 2. Determination of SHELL exon 1 genotypes in palms sampled from the smallholder plantations aNumber of independent palms tested for MPOB by allele-specific PCR assays for AVROS mutations and bNumber of independent palms additionally analyzed by DNA sequencing of exon 1 of the SHELL gene

[116] Next, 3,952 palms from seven oil palm nursery sites across Malaysia were genotyped (Table 3). Again, the majority were heterozygous for both the shAVROS and shMPOB alleles, indicating that they were genotypically tenera palms. However, 536 palms were predicted to be homozygous for the ShDeliDura allele at both SNP positions. Exon 1 of the SHELL gene, which encodes the entire MADS-box domain, was sequenced in each of these 536 palms as described above.

[117] As shown in Table 3, 6 palms were determined to be heterozygous for the shAVROS allele, indicating that these palms were indeed genotypically tenera palms. One palm was determined to be heterozygous for the shMPOB allele. However, the remaining 529 palms were determined to be homozygous for the ShDeliDura allele at both SNP positions, suggesting that these trees were either genotypically dura or that they carried previously unidentified mutant alleles of the SHELL gene. Allele 3 (SEQ ID NO: 3 and 13) was found to be heterozygous in 36 palms (Table 3), and allele 4 (SEQ ID NO: 4 and 14) was found to be heterozygous in 2 palms. Each of these 38 palms was independent of the others. TABLE 3. Determination of SHELL exon 1 genotypes in palms sampled from oil palm nurseries aNumber of independent palms tested for AVROS and MPOB mutations by allele-specific PCR assays bNumber of independent palms additionally analyzed by DNA sequencing of exon 1 of the SHELL gene

[118] To further identify SHELL exon 1 variants, exon 1 of the SHELL gene was sequenced in 148 palms from germplasm collections collected from various geographic regions (64 from Angola, 28 from Ghana, 27 from Nigeria, 27 from Tanzania, and 2 from Guinea). The shAVROS allele was the most common among populations expected to be of the tenera phenotype (50 palms from Angola, 10 palms from Ghana, 6 palms from Nigeria, and 22 palms from Nigeria), while the shMPOB allele was detected in 5 palms from Angola, 5 palms from Ghana, and 19 palms from Nigeria (Table 4). Allele 4 (also detected in smallholder plantations and nurseries) was detected in 1 palm from Ghana and 2 palms from Guinea.

[119] Additionally, six novel SHELL alleles were detected. Allele 6 was detected in 4 palms from Tanzania and encodes an aspartate to glutamine amino acid change relative to dura at amino acid position 12 (Figures 2 and 3). Allele 7, an in-frame deletion that removes 5 amino acids relative to dura, was detected in 1 palm from Nigeria. Alleles 8 and 9 change the same amino acid relative to dura. The conserved arginine at amino acid position 24 is changed to histidine in allele 8 and to glycine in allele 9 (Figures 2 and 3). Allele 10 was detected in a palm from Ghana, and it encodes a valine to glutamate amino acid substitution relative to dura at amino acid position 37. Finally, allele 11 was detected in two palms from Angola, and it encodes a synonymous single nucleotide polymorphism (Figures 2). It is worth noting that, like the shAVROS and shMPOB mutations, alleles 3, 5, 7, 8, and 9 affect amino acids that are part of the highly conserved NLS of the SHELL protein. TABLE 4. Determination of SHELL exon 1 genotypes in germplasm collections.

[120] Of the palms sequenced as part of the germplasm collection, 32 were visually phenotyped for oil palm fruit type (dura, tenera, or pisifera). Of the three Angola palms phenotyped as tenera, two were heterozygous for the shAVROS allele and were wild-type (dura) at all other exon 1 nucleotide positions within exon 1. Of the two Angola palms phenotyped as dura, both were heterozygous for the variant Allele 11 (Allele 11/ShDeliDura) and were wild-type (dura) at all other exon 1 nucleotide positions, consistent with the expectation that the synonymous switch is not directly involved in the tenera/pisifera phenotype. One Angola palm phenotyped as tenera was wild-type (dura) at all nucleotide positions of exon 1. None of the Angola palms in this study were phenotyped as pisifera.

[121] Of the 10 Ghanaian palms phenotyped as tenera, 1 was heterozygous for Allele 4 (Allele 4/ShDeliDura) and was wild-type (dura) at all other nucleotide positions of exon 1, 4 were heterozygous for Allele 8 (Allele 8/ShDeliDura) and were wild-type (dura) at all other nucleotide positions of exon 1, 4 were heterozygous for Allele 9 (Allele 9/ShDeliDura) and were wild-type (dura) at all other nucleotide positions of exon 1, and 1 was heterozygous for Allele 10 (Allele 10/ShDeliDura) and was wild-type (dura) at all other nucleotide positions of exon 1. None of the Ghanaian palms in this study were phenotyped as dura.

[122] Of the 8 Nigerian palms phenotyped as tenera, 3 were heterozygous for the shAVROS allele (shAVROS/ShDeliDura) and wild-type (dura) at all other nucleotide positions, 3 were heterozygous for the shMPOB allele (shMPOB/ShDeliDura) and wild-type (dura) at all other nucleotide positions of exon 1, and 1 was heterozygous for the 7 allele (7/ShDeliDura allele) and wild-type (dura) at all other nucleotide positions of exon 1. One Nigerian palm phenotyped as tenera was wild-type (dura) at all nucleotide positions of exon 1. Of the two Nigerian palms phenotyped as pisifera, 1 was homozygous for the shAVROS allele and was wild-type (dura) at all other nucleotide positions of exon 1. exon 1 nucleotide, while the other was heterozygous for the shAVROS allele (shAVROS/ShDeliDura) and was wild-type (dura) at all other exon 1 nucleotide positions. None of the Nigerian palms in this study were phenotyped as dura.

[123] Of the 2 Tanzanian palms phenotyped as tenera, 1 was heterozygous for the shAVROS allele (shAVROS/ShDeliDura) and was wild-type (dura) at all other nucleotide positions of exon 1, while the other was a compound heterozygote with the shAVROS allele on one chromosome and Allele 6 on the other chromosome (shAVROS/Allele 6) and was wild-type (dura) at all other nucleotide positions of exon 1. Furthermore, 3 of the 3 Tanzanian palms phenotyped as dura were heterozygous for Allele 6. This suggests that although Allele 6 may not contribute to the tenera phenotype, it may be a marker for closely linked alleles that contribute to the phenotype. No Tanzanian palms in this study were phenotyped as pisifera.

[124] Of the 2 Guinea palms phenotyped as tenera, 1 was heterozygous for Allele 4 (Allele 4/ShDeliDura) and was wild type (dura) at all other nucleotide positions of exon 1. The other was homozygous for Allele 4 and was wild type (dura) at all other nucleotide positions of exon 1. None of the Guinea palms in this study were phenotyped as dura or pisifera. REFERENCES Beirnaert, A. and Vanderweyen, R. 1941. Contribution to l’etude genetic et biometricque des varieties d’Elaeis guineensis Jacq. Publs. INEAC, Series Ser. Sci. (27).101. Bhasker, S. & Mohankumar, C. Association of lignifying enzymes in shell synthesis of oil palm fruit (Elaeis guineensis--dura variety). 2001. Indian J Exp Biol 39:160-4 S.C., Rohde, W and Charrier, A. 2005. Microsatellite-based high density linkage map in oil palm (Elaeis guineensis Jacq.). Theoretical and Applied Genetics 110: 754 to 765 Birchler, J. A., Auger, D. L. & Riddle, N. C. 2003. In search of the molecular basis of heterosis. Plant Cell 15: 2236-9 Cheah, S. C. 1996. Restriction Fragment Length Polymorphism (RFLP) in Oil Palm. Project Completion Report No. 0011/95, 4 July 1996, Malaysian Palm Oil Board (MPOB), Bangi, Malaysia. Cheah, S. C. and Rajinder, S. 1998. Gene expression during flower development in the oil palm. Project Completion Report No. 0057/98, 16 July 1999. Palm Oil Research Institute of Malaysia (PORIM), Bangi, Malaysia. Corley, R. H. V. and Tinker, P. B. 2003. Care and maintenance of oil palms. In The Oil Palm (4th Edition), pages:287 to 326. Oxford: Blackwell Science. Danielsen, F. et al. 2009. Biofuel plantations on forested lands: double jeopardy for biodiversity and climate. Conserv Biol 23: 348 to 58 Devuyst, A. 1953. Selection of the oil palm (Elaeis guineensis) in Africa. Nature 172: 685 to 686 Dinneny, J. R. and Yanofsky, M. F. 2005. Drawing lines and borders: how the dehiscent fruit of Arabidopsis is patterned. Bioessays 27: 42 to 9 Donough, C. R. and Law, I. H. 1995. Breeding and selection for seed production at Pamol Plantations Sdn Bhd and early performance of Pamol D x P. Planter 71:513 to 530. Doyle, J. J. and Doyle, J. L. 1990. Isolation of plant DNA from fresh tissue. FOCUS 12:13 to 15. Ferrandiz, C., Liljegren, S. J. & Yanofsky, M. F. 2000. Negative regulation of the SHATTERPROOF genes by FRUITFULL during Arabidopsis fruit development. Science 289: 436 a 8 Godding, R. 1930. Observation de la production de palmiers selectionnes a Mongana (Equateur). Bull Arig. Congo belge 21: 1263. Gschwend, M. et al. 1996. A locus for Fanconi anemia on 16q determined by homozygosity mapping. Am J Hum Genet 59: 377 to 84 Gu, Q., Ferrandiz, C., Yanofsky, M. F. & Martienssen, R. 1998. The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development. Development 125: 1509 to 17 Gramzow, L. and Theissen, G. 2010. A hitchhiker’s guide to the MADS world of plants. Genome Biology 11: 214 to 225 Hardon, J.J., Rao, V., and Rajanaidu, N. 1985. A review of oil palm breeding. In Progress in Plant Breeding, ed G.E. Rusell, pages 139 to 163, Butterworths, R. U. Hartley, C. W. S. 1988. The botany of oil palm. In The oil palm (3rd Edition), pages:47 to 94, Longman, London. Huang, H., Tudor, M., Su, T., Zhang, Y., Hu, Y., and Ma, H. 1996. DNA binding properties of two Arabidopsis MADS domain proteins: Binding Consensus and Dimer Formation. The Plant Cell 8: 81 to 94 Immink, R. G., Gadella, T. W. J., Ferrario, S., Busscher, M., and Angenent, G. 2002. Proc. Natl. Academic. Sci. 99: 2416 to 2421 Immink, R. G., Kaufmann, K. & Angenent, G. C. 2010. The 'ABC' of MADS domain protein behavior and interactions. Semin Cell Dev Biol 21: 87 to 93 Jack, P. L., James, C., Price, Z., Rance, K., Groves, L., CorleY, R. H. V., Nelson, S and Rao, V. 1998. Application of DNA markers in oil palm breeding. In: 1998 International Oil Palm Congress- Commodity of the past, today and future, 23-25 September 1998, Bali, Indonesia. Krieger, U., Lippman, Z. B. & Zamir, D. 2010. The flowering gene SINGLE FLOWER TRUSS drives heterosis for yield in tomato. Nat Genet 42: 459 to 463 Lander, E. S. and Botstein, D. 1987. Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children. Science 236: 1567 to 1570 Latiff, A. 2000. The Biology of the Genus Elaeis. In: Advances in Oil Palm Research, Volume 1, ed. Y. Basiron, B. S. Jalani, and K. W. Chan, pages:19 to 38, Malaysian Palm Oil Board (MPOB). Liljegren, S. J., Ditta, G. S., Eshed, Y., Savidge, B., Bowman, J. L., Yanofsky, M. F. 2000. SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature 404: 766 to 770 Maria, M., Clyde, M. M. and Cheah, S. C. 1995. Cytological analysis of Elaeis guineensis (tenera) chromosomes. Elaeis 7:122 to 134. Mayes, S., Jack, P. L., Marshall, D. F., and Corley, R. H. V. 1997. Construction of an RFLP genetic linkage map for oil palm (Elaeis guineensis Jacq.). Genome 40:116 to 122. Moretzsohn, M. C., Nunes, C. D. M., Ferreira, M. E. and Grattapaglia, D. 2000. RAPD linkage mapping of the shell thickness locus in oil palm (Elaeis guineensis Jacq.). Theoretical and Applied Genetics 100:63 to 70. Ooijen, J. W. V. 2006. JoinMap 4.0: Software for calculation of genetic linkage maps. In experimental populations. Kyazma B.V., Wageningen, Netherlands Pinyopich, A. et al. 2003. Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature 424: 85 to 88 Purseglove, J. W. 1972. Tropical Crops. Monocotyledons. Longman, London. pages: 607. Rajanaidu, N., Rao, V., Abdul Halim, H. & A.S.H., O. 1989. Genetic resources: New developments in Oil Palm breeding. Elaeis 1: 1 to 10 Rajanaidu, N. 1990. Major developments in oil palm (Elaeis guineensis) breeding. In Proceedings of the 12th Plenary Meeting of AETFAT, pages: 39 to 52 Hamburg, Germany. Rajanaidu, N. et al. 2000. in Advances in Oil Palm Research (eds. Basiron, Y., Jalani, B. S. & Chan, K. W.) 171-237 (Malaysian Palm Oil Board (MPOB), Bangi, Selangor). Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular cloning: A Laboratory manual, (2nd Edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Singh R, Tan SG, Panandam JM, Rahman RA, Ooi LCL, Low ETL, Sharma M, Jansen J, and Cheah SC. 2009. Mapping quantitative trait loci (QTLs) for fatty acid composition in an interspecific cross of oil palm. BMC Plant Biology 9: 114. Sousa, J., Barata, A. V., Sousa, C., Casanova, C. C. and Vicente, L. 2011. Chimpanzee oil-palm use in southern Cantanhez National Park, Guinea-Bissau. Am J Primatol 73: 485 to 497 Tani, E., Polidoros, A. N. & Tsaftaris, A. S. Characterization and expression analysis of FRUITFULL- and SHATTERPROOF-like genes from peach (Prunus persica) and their role in split-pit formation. 2007. Tree Physiol 27: 649 to 459 Vrebalov, J., Pan, I.L., Arroyo, A.J.M., McQuinn, R., Chung, M., Poole, M., Rose, J., Seymour, G., Grandillo, S., Giovannoni, J., and Irish, V.F. 2009. Fleshy fruit expansion and ripening are regulated by the tomato SHATTERPROOF gene TAGL1. The Plant Cell 21: 3041 to 3062 Whitmore, T. C. 1973. The Palms of Malaya. Longmans, Malaysia, pages:56 to 58. The term “a” or “an” is intended to mean “one (or more). The term “comprise” and variations thereof, such as “comprise” and “comprising”, when preceding the citation of a step or element, are intended to mean that the addition of additional steps or elements is optional and not excluded. All patents, patent applications, and other published reference materials cited in this application are hereby incorporated by reference in their entirety. INFORMAL LISTING OF EXEMPLARY SEQUENCES SEQ ID NO:1 Predicted protein sequence of SHELL, mutation underlined, italicized and in bold [pisifera, Zaire allele; shAVROS] MGRGKIEIKRIENTTSRQVTFCKRRNGLLKNAYELSVLCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:2 Predicted protein sequence of SHELL, mutation underlined, italicized and in bold [pisifera, Nigeria allele; shMPOB] MGRGKIEIKRIENTTSRQVTFCKRRNGL P KKAYELSVLCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:3 Predicted protein sequence of SHELL, mutation underlined, italicized and in bold [predicted pisifera, Allele 3] MGRGKIEIKRIENTTSRQVTFC Q RRNGLLKKAYELSVLCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:4 Predicted protein sequence of SHELL, mutation underlined, italicized and in bold [predicted pisifera, Allele 4] MGRGKIEIKRIENTTSRQVTFCKRRNGLLKKAYELSVLCD D EVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:5 Predicted protein sequence of SHELL, mutation underlined, italicized and in bold [predicted pisifera, Allele 5] MGRGKIEIKRIENTTSRQVTFC N RRNGLLKKAYELSVLCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:6 Predicted protein sequence of SHELL, mutation underlined, italicized and in bold [predicted pisifera, Allele 6] MGRGKIEIKRIQ NTTSRQVTFCKRRNGLLKKAYELSVLCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:7 Predicted protein sequence of SHELL deleted amino acids indicated by a dash (“-“) [predicted pisifera, Allele 7] MGRGKIE NTTSRQVTFCKRRNGLLKKAYELSVLCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:8 Predicted protein sequence of SHELL, mutation underlined, italicized and in bold [predicted pisifera, Allele 8] MGRGKIEIKRIENTTSRQVTFCKH RNGLLKKAYELSVLCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:9 Predicted protein sequence of SHELL, mutation underlined, italicized and in bold [predicted pisifera, Allele 9] MGRGKIEIKRIENTTSRQVTFCKG RNGLLKKAYELSVLCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:10 Predicted protein sequence of SHELL, mutation underlined, italicized and in bold [predicted pisifera, Allele 10] MGRGKIEIKRIENTTSRQVTFCKRRNGLLKKAYELS D LCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:11 Predicted protein sequence of SHELL, silent mutation results in wild-type amino acid sequence [predicted pisifera, Allele 11] MGRGKIEIKRIENTTSRQVTFCKRRNGLLKKAYELSVLCDAEVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:12 Deletion in intron 1 of SHELL gene, nucleotides deleted in reference to wild type indicated with a dash (“-“) GTATGCTTTGATGACGCCTTCTCTTCCTTCGCTCATATCAAG - - - - TTTTATGGCTTCA T SEQ ID NO:13 SHELL exon 1 allele 3 sequence, mutation underlined, italicized and in bold ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGC CAACGCCGAAATGGACTGCTGAAGAAAGCTTATG AGTTGTCTGTCCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTCTCCAGCC GGGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:14 SHELL exon 1 allele 4 sequence, mutation underlined, italicized, and bold ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGCAAACGCCGAAATGGACTGCTGAAGAAAGCTTATG AGTTGTCTGTCCTTTGTGATG A TGAGGTTGCCCTTATTGTCTTCTCCAGCC GGGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:15 SHELL exon 1 allele 5 sequence, mutation underlined, italicized, and bold ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGCAA TCGCCGAAATGGACTGCTGAAGAAAGCTTATGA GTTGTCTGTCCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTCTCCAGCCG GGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:16 Sequence from SHELL exon 1 allele 6, mutation underlined in bold italics ATGGGTAGAGGAAAGATTGAGATCAAGAGGATC CAGAACACCACAAGCCG GCAGGTCACTTTCTGCAAACGCCGAAATGGACTGCTGAAGAAAGCTTATG AGTTGTCTGTCCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTCTCCAGCC GGGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:17 SHELL exon 1 allele 7 sequence, nucleotides deleted in reference to wild type indicated by a dash (“-“) ATGGGTAGAGGAAAGATTGAGA - - - - - - - - - - - - - - - ACACCACAAGCCGGCAGG TCACTTTCTGCAAACGCCGAAATGGACTGCTGAAGAAAGCTTATGAGTTGT CTGTCCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTCTCCAGCCGGGGCC GCCTCTATGAGTACGCCAATAACAG SEQ ID NO:18 SHELL exon 1 allele 8 sequence, mutation underlined, italicized, and in bold ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGCAAAC A CCGAAATGGACTGCTGAAGAAAGCTTATGA GTTGTCTGTCCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTTCTCCAGCCG GGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:19 SHELL exon 1 allele 9 sequence, mutation underlined, italicized, and in bold ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGCAAAG GCCGAAATGGACTGCTGAAGAAAGCTTATG AGTTGTCTGTCCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTCTCCAGCC GGGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:20 SHELL exon 1 allele 10 sequence, mutation underlined, italicized, and in bold ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGCAAACGCCGAAATGGACTGCTGAAGAAAGCTTATG AGTTGTCTG A CCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTCTCCAGCC GGGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:21 SHELL exon 1 allele 11 sequence, mutation underlined, italicized, and in bold ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGCAAACGCCGAAATGGACTGCTGAAGAAAGCTTATG AGTTGTCTGTCCT CTGTGATGCTGAGGTTGCCCTTATTGTCTTCTCCAGCC GGGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:22 SHELL exon 1 sequence of shAVROS allele, mutation underlined, italicized and in bold ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGCAAACGCCGAAATGGACTGCTGAAGAATGCTTATGA GTTGTCTGTCCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTTCTCCAGCCG GGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:23 SHELL exon 1 sequence of shAVROS allele shMPOB, mutation underlined, in bold italics ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGCAAACGCCGAAATGGACTGC C GAAGAAAGCTTATG AGTTGTCTGTCCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTCTCCAGCC GGGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:24 Predicted protein sequence of wild-type SHELL (ShDeliDura). Amino acids mutated in shAVROS, shMPOB, and alleles 3 to 6 and 8 to 10 are underlined, in bold italics. Amino acids mutated in allele 7 are underlined. [dura, ShDeliDura] MGRGKIEIKRIE NTTSRQVTFC KR RNGL L KKAYELS VL CD A EVALIVFSS RGRLYEYANNSIRSTIDRYKKACANSSNSGATIEINSQYYQQESAKLRHQ IQILQNANRHLMGEALSTLTVKELKQLENRLERGITRIRSKKHELLFAEI EYMQKREVELQNDNMYLRAKIAENERAQQAA SEQ ID NO:25 Wild-type SHELL (ShDeliDura) exon 1 sequence, nucleotides mutated in shAVROS, shMPOB, and alleles 3 to 6 and 8 to 11 are underlined, italicized, and bold. Nucleotides deleted in allele 7 are underlined ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAA CACCACAAGCCG GCAGGTCACTTTCTGC AAACG CCGAAATGGACTGC TGAAGAAA GCTTATG AGTTGTCTG TCCTTTGTGATG CTGAGGTTGCCCTTATTGTCTTCTCCAGCC GGGGCCGCCTCTATGAGTACGCCAATAACAG SEQ ID NO:26 Wild-type SHELL (ShDeliDura) exon 1 merged with nucleotides 1 to 119 of intron 1 ATGGGTAGAGGAAAGATTGAGATCAAGAGGATCGAGAACACCACAAGCCG GCAGGTCACTTTCTGCAAACGCCGAAATGGACTGCTGAAGAAAGCTTATG AGTTGTCTGTCCTTTGTGATGCTGAGGTTGCCCTTATTGTCTTCTCCAGCC GGGGCCGCCTCTATGAGTACGCCAATAACAGGTATGCTTTGATGACGCCT TCTCTTCCTTCGCTCATATCAAGTTAATTTTATGGCTTCATTTGTTCTATGGC CAAGCCAAATTCTTTTTAAAGTTCTAGAATGTTAATGATGGTAGTTT

Claims (5)

1. METHOD FOR PREDICTING THE PHENOTYPE OF A PALM TREE BARK OR A PALM TREE SEED, the method being characterized by the fact that it comprises: (a) determining, from a nucleic acid sample of the palm tree or palm tree seed, the presence, absence or number of pisifera or dura alleles, (i) detecting the genotype of the polymorphic marker corresponding to nucleotide 67 of exon 1 of the SHELL gene, and in which the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, in which the detection of an A → C mutation of the polymorphic marker corresponding to nucleotide 67 of exon 1 of the SHELL gene, as shown in SEQ ID NO.13 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 69 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of an A→T mutation of the polymorphic marker corresponding to nucleotide 69 of exon 1 of the SHELL gene as shown in SEQ ID NO.15 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 70 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a C→G mutation of the polymorphic marker corresponding to nucleotide 70 of exon 1 of the SHELL gene as shown in SEQ ID NO.19 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 71 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker 23 indicates the presence of a pisifera allele, wherein the detection of a G → A mutation of the polymorphic marker corresponding to nucleotide 71 of exon 1 of the SHELL gene as shown in SEQ ID NO.18 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 110 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a T → A mutation of the polymorphic marker corresponding to nucleotide 110 of exon 1 of the SHELL gene as shown in SEQ ID NO.20 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 122 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a C → A mutation of the polymorphic marker corresponding to nucleotide 122 of exon 1 of the SHELL gene as shown in SEQ ID NO.14 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 34 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a G → C mutation of the polymorphic marker corresponding to nucleotide 34 of exon 1 of the SHELL gene indicates the presence, as shown in SEQ ID NO.16, of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 114 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a T → C mutation of the polymorphic marker corresponding to nucleotide 114 of exon 1 of the SHELL gene as shown in SEQ ID NO.21 indicates the presence of a pisifera allele; or (ii) detecting a genotype of a polymorphic marker within a region of the palm genome corresponding to SEQ ID NO:27 to thereby predict the genotype of at least one polymorphic marker corresponding to nucleotide 67, 69, 70, 71, 110, 122 or 34, of exon 1 of a SHELL gene; and (b) predict the phenotype of palm bark or palm seed based on the presence, absence, or number of pisifera or dura alleles. 2. A METHOD FOR SEGREGATING A PLURALITY OF PALM TREES INTO DIFFERENT CATEGORIES BASED ON A PREDICTED BARK PHENOTYPE, comprising: (i) predicting the palm bark or palm seed phenotype based on the presence, absence or number of pisifera or dura alleles, (ii) detecting the genotype of the polymorphic marker corresponding to nucleotide 67 of exon 1 of a SHELL gene and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of an A → C mutation of the polymorphic marker corresponding to nucleotide 67 of exon 1 of the SHELL gene as shown in SEQ ID NO. 13 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 69 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of an A→T mutation of the polymorphic marker corresponding to nucleotide 69 of exon 1 of the SHELL gene as shown in SEQ ID NO.15 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 70 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a C→G mutation of the polymorphic marker corresponding to nucleotide 70 of exon 1 of the SHELL gene as shown in SEQ ID NO.19 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 71 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker 23 indicates the presence of a pisifera allele, wherein the detection of a G → A mutation of the polymorphic marker corresponding to nucleotide 71 of exon 1 of the SHELL gene as shown in SEQ ID NO.18 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 110 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a T → A mutation of the polymorphic marker corresponding to nucleotide 110 of exon 1 of the SHELL gene as shown in SEQ ID NO.20 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 122 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a C → A mutation of the polymorphic marker corresponding to nucleotide 122 of exon 1 of the SHELL gene as shown in SEQ ID NO.14 indicates the presence of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 34 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a G → C mutation of the polymorphic marker corresponding to nucleotide 34 of exon 1 of the SHELL gene indicates the presence, as shown in SEQ ID NO.16, of a pisifera allele; or detecting the genotype of the polymorphic marker corresponding to nucleotide 114 of exon 1 of the SHELL gene, and wherein the detection of a mutation relative to the wild type of the polymorphic marker indicates the presence of a pisifera allele, wherein the detection of a T → C mutation of the polymorphic marker corresponding to nucleotide 114 of exon 1 of the SHELL gene as shown in SEQ ID NO.21 indicates the presence of a pisifera allele; or (iii) detecting a genotype of a linked marker within a region of the palm genome corresponding to SEQ ID NO:27 to thereby predict the genotype of at least one polymorphic marker corresponding to nucleotide 67, 69, 70, 71, 110, 122 or 34 of exon 1 of a SHELL gene; and (c) segregating the palms or palm seeds into groups based on the genotype of the polymorphic marker, wherein at least one group contains only one of (i) palms or palm seeds predicted to have the soft bark phenotype, (ii) palms or palm seeds predicted to have the hard bark phenotype, or (iii) palms or palm seeds predicted to have the pisiferous bark phenotype. 3. METHOD according to any one of claims 1 to 2, characterized in that the palm trees are less than 5 years old. 4. METHOD, according to any one of claims 1 to 2, characterized by the fact that it additionally comprises selecting the palm trees or palm seeds for cultivation if the palm trees or palm seeds are heterozygous for the polymorphic marker. 5. METHOD according to any one of claims 1 to 2, characterized in that the palms or palm seeds are discarded if the palms or palm seeds do not have a genotype predictive of the tenera bark phenotype.