MANTLE PHENOTYPE DETECTION IN PALM
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] The present application claims the benefit of priority to U.S. Provisional Patent
Application No. 61/988,132, filed on May 2, 2014, and U.S. Provisional Patent Application
No. 62/091,471, filed on December 12, 2014, the contents of each of which are hereby
incorporated by reference in the entirety and for all purposes.
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
[0002] The Sequence Listing written in file SEQ 96380-94121 l_ST25.txt, created on
April 30, 2015, 420,872 bytes, machine format IBM-PC, MS-Windows operating system, is
hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] The oil palm belongs to the genus Elaeis which contains two species, E. guineensis
and E. oleifera. It is regarded as the most efficient oil bearing crop in the world out yielding
all other crops of the same genre, e.g., soybean, rapeseed and sunflower. The ability to
produce oil at an average yield of 3.74 tonne/ha/year, on land 10 times smaller than the
requirement for soybean (Oil World, 2007) and with a productive life cycle of 25-30 years,
makes the oil palm a lucrative agricultural crop. However, of late the oil yield has reached
stagnation. Nevertheless, demand for edible oils is predicted to escalate to feed the growing
world population.
[0004] The oil palm has gone through at least two known cycles of yield improvements
since its introduction as an oil crop in Malaysia, the first wave being the introduction of the
hybrid tenera (DxP), which replaced the dura as commercial planting material. This
demonstrated an increase in oil yield of up to 30% by merely manipulating a single gene
(Kushairi et al., 2006; Singh et al, 2013). However, the average oil yield in Malaysia has
hovered between 3.5 and 3.9 t/ha/yr for the last two decades. Having dropped to the number
two spot in palm oil production, Malaysia—and all other palm oil producing countries—is in
need of yield improvement. This is further compounded by the fact that agricultural land is
becoming a rarity. Therefore increased production by planting larger areas is no longer seen
as an alternative.
[0005] Through years of breeding and selection, the palm oil industry has already produced
palms yielding as high as 13.6 t/ha/yr (Sharma and Tan, 1999) which are close to the
theoretical yield of 18.2 t/ha/yr (Corley, 1998). The best experimental plot has produced an
average of 9.8 t/ha/yr of palm oil (Musa and Gurmit, 2008) with selected progenies able to
achieve up to 12.2 t/ha/yr (Rajanaidu et al., 1990). Cloning these super palms may provide
the industry with the much-needed high-yielding planting materials to get it out of the
stagnation rut. Hence, clones for commercial use are touted as the second wave of crop
improvement for the oil palm.
[0006] Due to its biological structure, the oil palm has no natural means of vegetative
propagation and conventional hybrid breeding methodology would require at least three
generations, or over 20 years, to realize such superior yields (Soh et al., 2005). Successful
vegetative propagation of oil palm was first described in the 1970s (Jones, 1974; Rabechault
and Martin, 1976). Jones (1995) gave a rather comprehensive and personal account of its
development. These successful reports of oil palm cloning prompted the development of
tissue culture laboratories to provide clonal oil palm planting material. Encouraging results
from early field trials set the pace for more laboratories to follow suit. By the mid- 1980's,
there were already 10 clonal oil palm laboratories in Malaysia (Wooi, 1990) and others
elsewhere (Le Guen et al., 1991).
[0007] However, when Corley et al. (1986) reported the mantling phenomenon for the first
time, the whole clonal industry led by the pioneering Bakasawit/ Unifield and Tropiclone
commercial laboratories decided to cut back on production and reverted to research and
development. The then, Palm Oil Research Institute of Malaysia (PORIM), now known as
Malaysian Palm Oil Board (MPOB), as the custodian of the palm oil industry, was assigned
the task of spearheading research in clonal abnormalities.
[0008] Through a concerted effort, by the early 1990's, the results obtained suggested that
better tissue culture protocols needed to be established, which included subculturing
procedures and the use of less devastating types of growth regulators. Alternative methods
were also proposed such as suspension and protoplast cultures as a means to avoid
subculturing. Cloning of dura and pisifera parents, followed by conventional crossing to
circumvent the potential occurrence of somaclonal variants from clonal teneras, was amongst
the different methods discussed (Ong-Abdullah, Viva 562/201 1). Interestingly, up to 10% of
abnormal palms spontaneously reverted to normal and remained normal for some time
(Durand-Gasselin et al, 1990). Seedlings developed from Mantled fruits e.g., clone 115E,
were normal; refuting the possibility that abnormality is due to a dominant gene effect or to
maternally transmitted factors. Through conventional genetic crossings conducted by Rao
and Donough (1990), this trait was also shown to behave in a non-Mendelian manner.
[0009] Earlier attempts that employed techniques such as flow cytometry, random amplified
polymorphic DNA (RAPD) or the classical amplified fragment length polymorphisms
(AFLP) analysis failed to yield any detectable differences between Mantled and normal
palms (Rival et al. 1997, 1998; Matthes et al. 2001). However, when methylation sensitive
or related technologies were utilized, the methylation level of the Mantled genome appeared
to be altered (Jaligot et al. 2002, Matthes et al. 2001, Jaligot et al. 2004).
[0010] Subsequently, further research concentrated on understanding the underlying
molecular cause(s) and epigenetic regulation of mantling. It was also known that in Mantled
oil palms, staminodes and stamens of pistillate and functional flowers develop respectively as
pseudocarpels (Morcillo et al, 2006). In severe cases, the flowers are sterile with abortive
fruits leading to lower yields. It was postulated that since homeotic modifications had taken
place, it was highly likely that the B-function homeotic MADS box genes of the ABCDE
model for flower organ identity (Murai, 2013) are involved.
[0011] Following the MADS box hypothesis, MADS-box containing genes from the oil
palm were isolated (Alwee et al., 2006; Auyong, 2006) using the MADS box-directed
profiling technique (van der Linden et al. 2002). This method allows the visualization of
DNA polymorphisms in restriction sites at the MADS box vicinity among normal, abnormal
and reverted oil palms. Two markers, namely MM77 and MM78 (EP Patent Appl. No.
13 162130.2) were identified and the latter was widely used for further validation although it
was found not to fall in the class of MADS box genes. In the course of validating MM78 and
from past experiences with other unrelated markers, it was confirmed that the functional use
of these markers is genotype dependent. Therefore, they have little or no use when tested on
clones from other genetic backgrounds. This has been the main point of contention in
biomarker development for clonal fidelity of the oil palm.
[0012] Previous studies have found an overall decrease in DNA methylation in mantled
palms relative to ortets and normal ramets (Jaligot et al. 2000; Matthes et al. 2001; Jaligot et
al. 2002; Jaligot et al. 2004). These results are similar to observations in Arabidopsis and
other plant cell cultures, in which transposable elements (TEs) are hypomethylated and
expressed (Tanurdzic et al. 2008; Miguel et al. 201 1; Castilho et al. 2000; Kubis et al. 2003).
In addition to TEs, somaclonal regenerants in rice and maize undergo extensive gene and
promoter hypomethylation (Stroud et al. 2013; Stelpflug et al. 2014), which might also
contribute to somaclonal variation in oil palm and other crops. The homeotic transformations
observed in mantled palms resemble defects in B-function MADS box genes, suggesting that
retroelements within one or more MADS box genes, or the MADS box genes themselves are
candidates for epigenetic modification (Adam et al. 2005). However, decades of research
into DNA methylation changes in candidate retroelements (Castilho et al. 2000; Kubis et al.
2003; Jaligot et al. 2014) and candidate homeotic genes (Syed Alwee et al. 2006; Adam et al.
2007; Jaligot et al. 2014) have yet to identify epigenetic changes that are consistently found
in somaclonal mantled palms. And indeed, recent studies of rice and Arabidopsis plants
regenerated from tissue culture implicate genetic rather than epigenetic mechanisms as being
responsible for somaclonal variation (Jiang et al. 201 l;Miyao et al. 2012.
BRIEF SUMMARY OF THE INVENTION
[0013] Described herein are methods, compositions, and kits for predicting the presence or
absence of a somaclonal abnormality (e.g. , Mantled) in an oil palm plant, plant cell, or plant
tissue. In some embodiments, the present invention provides a method for segregating an oil
palm plant comprising: a) obtaining a biological sample from the plant; b) determining the
methylation status of at least one cytosine within a differential methylation region (DMR) in
the sample from the plant, wherein the DMR is within a sequence of DNA at least 70%, 80%,
90%, 95%, or 99% identical, or identical, to SEQ ID NO:l; c) correlating the methylation
status of the at least one cytosine to the presence or absence of a somaclonal abnormality in
the plant, wherein the correlation comprises predicting the presence or absence of somaclonal
abnormality in the plant; and d) physically separating a plant predicted to have a somaclonal
abnormality from one or more plants predicted to lack a somaclonal abnormality.
[0014] In some aspects, the DMR is within a DNA meta-region in the sample from the
plant, where the DNA meta-region is at least 90%>, 95%>, or 99%> identical, or identical, to a
sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and
70. In some aspects, the DMR is within a DNA region in the sample from the plant, where
the DNA region is at least 70%, 80%, 90%, 95%, or 99% identical, or identical, to a sequence
selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In some cases,
the determining step comprises determining the methylation status of at least one cytosine in
a biomarker, wherein the biomarker is at least 90%, 95%, or 99% identical, or identical, to a
sequence selected from the group consisting of SEQ ID NO: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, 71, and 72.
[0015] In some aspects the method comprises predicting the presence of a somaclonal
abnormality when the methylation status of the at least one cytosine is reduced relative to a
control locus. In some cases, the presence of a somaclonal abnormality is predicted when the
methylation status of the at least one cytosine in the DNA meta-region at least 70%, 80%,
90%, 95%, or 99% identical, or identical, to the sequence selected from the group consisting
of SEQ ID NO:63, 64, 65, 66, 67, 69, and 70 (or selected from the group consisting of SEQ
ID NO:63, 64, 65, 66, 67, 68, 69, and 70) is reduced relative to a control locus. In some
cases, the presence of a somaclonal abnormality is predicted when the methylation status of
the at least one cytosine in the DNA region is at least 70%, 80%, 90%, 95%, or 99%
identical, or identical, to the sequence selected from the group consisting of SEQ ID NO:35,
36, 39, 40, 42, 43, 44, 45, 46, 48, 49, 51, 52, 57, 58, 59, 60, 61, and 73 is reduced relative to a
control locus. In some cases, the presence of a somaclonal abnormality is predicted when the
methylation status of the at least one cytosine in the biomarker at least 90%, 95%, or 99%
identical, or identical to the sequence selected from the group consisting of SEQ ID NO:7, 8,
11, 12, 14, 15, 16, 17, 18, 20, 21, 23, 24, 29, 30, 31, 32, 33, and 7 1 is reduced relative to a
control locus.
[0016] In some aspects, the method comprises predicting the presence of a somaclonal
abnormality when the methylation status of the at least one cytosine is increased relative to a
control locus. In some cases, the presence of a somaclonal abnormality is predicted when the
methylation status of the at least one cytosine in the DNA meta-region at least 70%, 80%,
90%, 95%, or 99% identical, or identical, to the sequence selected from the group consisting
of SEQ ID NO:63, 64, 65, 66, 67, 68, and 69 (or selected from the group consisting of SEQ
ID NO:63, 64, 65, 66, 67, 68, 69, and 70) is increased relative to a control locus. In some
cases, the presence of a somaclonal abnormality is predicted when the methylation status of
the at least one cytosine in the DNA region at least 70%, 80%, 90%, 95%, or 99% identical,
or identical, to the sequence selected from the group consisting of SEQ ID NO:35, 36, 37, 38,
41, 42, 47, 50, 52, 53, 54, 55, 56, 57, 62, and 74 is increased relative to a control locus. In
some cases, the presence of a somaclonal abnormality is predicted when the methylation
status of the at least one cytosine in the biomarker at least 90%, 95%, or 99% identical, or
identical to the sequence selected from the group consisting of SEQ ID NO:7, 8, 9, 10, 13,
14, 19, 22, 24, 25, 26, 27, 28, 29, 34 and 72 is increased relative to a control locus.
[0017] In some aspects, the method comprises predicting the presence of a somaclonal
abnormality when the methylation status of the at least one cytosine is either increased or
decreased relative to a control locus. In some cases,the control locus is an endogenous
control locus. In some cases, the control locus is an exogenous control locus.
[0018] In some aspects, the determining step comprises determining the methylation status
of at least one cytosine in at least two, three or four different differential methylation regions
(DMRs), wherein each DMR is independently within a sequence of DNA at least 70%>, 80%>,
or 90%, 95%, or 99% identical, or identical, to SEQ ID NO: 1. In some cases, each DMR is
within a DNA meta-region in the sample from the plant, where each DNA meta-region is at
least 70%, 80%>, 90%>, 95%, or 99% identical, or identical, to a sequence independently
selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and 70. In some
cases, each DMR is within a DNA region in the sample from the plant, where the DNA
region is at least 70%>, 80%>, 90%>, 95%, or 99% identical, or identical, to a sequence
independently selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In
some cases, the determining step comprises determining the methylation status of at least one
cytosine in a biomarker in each DMR, wherein each biomarker is at least 90%, 95%, or 99%
identical, or identical, to a sequence independently selected from the group consisting of SEQ
ID NO: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, 71, and 72.
[0019] In any of the foregoing embodiments, aspects, or cases, the somaclonal abnormality
can comprise a reduction in fruit yield, oil yield, growth, or reproduction of the plant relative
to a control plant. In some cases, the control plant is a parental plant. In some cases, the
control plant is a wild-type plant of the same fruit form phenotype {dura, tenera, or pisifera)
as the plant predicted to have a somaclonal abnormality. In some cases, the somaclonal
abnormality exhibits a Mantled phenotype.
[0020] In any of the foregoing embodiments, aspects, or cases, the determining the
methylation status can comprise bisulfite conversion; and/or the determining the methylation
status can comprise digesting genomic DNA with a methylation-dependent endonuclease;
and/or the determining the methylation status can comprise digesting genomic DNA with a
methylation-sensitive endonuclease; and/or the determining of the methylation status can
comprise measuring rates of methylated base incorporation during sequencing; and/or the
determining of the methylation status can comprise measuring current as molecules including
methylated bases pass through a nanopore. In any of the foregoing embodiments, aspects, or
cases, the determining the methylation status can comprise methylated DNA
immunoprecipitation, methylated DNA capture by affinity purification, or reduced
representation bisulfite sequencing. In any of the foregoing embodiments, aspects, or cases,
the determining the methylation status can comprise nucleic acid hybridization, e.g.,
microarray or bead array hybridization.
[0021] In any of the foregoing embodiments, aspects, or cases, the physically separating
can comprise selecting plants predicted to have a somaclonal abnormality for destruction;
and/or selecting plants predicted to lack a somaclonal abnormality for cultivation. In some
cases, the plants selected for cultivation are germinated, planted, or transplanted. In some
cases, the plants not selected for cultivation are discarded or destroyed.
[0022] In some embodiments, the present invention provides a computer program product
for determining the presence or absence of a somaclonal abnormality in an oil palm plant, the
computer program product comprising: a computer readable medium encoded with program
code, the program code including: program code for receiving a methylation value
representing a methylation status of at least one cytosine within a differential methylation
region (DMR) in a sample from the oil palm plant, wherein the DMR is within a sequence of
DNA at least 70%, 80%, 90%, 95%, or 99% identical, or identical, to SEQ ID NO: 1; and
program code for comparing the methylation value to a control value, wherein the control
value distinguishes between plants with and without a somaclonal abnormality, wherein the
comparison of the methylation value to the control value is predictive of the presence or
absence of a somaclonal abnormality in the plant.
[0023] In some aspects, the DMR is within a DNA meta-region in the sample from the
plant, where the DNA meta-region is at least 70%, 80%, 90%, 95%, or 99% identical, or
identical, to a sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67,
68, 69, and 70. In some aspects, the DMR is within a DNA region in the sample from the
plant, where the DNA region is at least 70%, 80%, 90%, 95%, or 99% identical, or identical,
to a sequence selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In
some aspects, the at least one cytosine is in a biomarker, wherein the biomarker is at least
90%o, 95%o, or 99% identical, or identical, to a sequence selected from the group consisting of
SEQ ID NO: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, 71, and 72.
[0024] In some aspects, the control value is a methylation value for a control locus
exogenous to the plant. In some aspects, the control value is a methylation value for a control
locus endogenous to the plant.
[0025] In some aspects, wherein the program code comprises program code for receiving
the methylation status of at least one cytosine in at least two, three or four different DMRs,
wherein each DMR is independently within a sequence of DNA at least 70%>, 80%>, 90%>,
95%o, or 99%o identical, or identical, to SEQ ID NO: 1. In some cases, each DMR is within a
DNA meta-region in the sample from the plant, where each DNA meta-region is at least 70%,
80%, 90%, 95%, or 99%> identical, or identical, to a sequence independently selected from the
group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and 70. In some cases, each DMR
is within a DNA region in the sample from the plant, where each DNA region is at least 70%,
80%, 90%, 95%, or 99%> identical, or identical, to a sequence independently selected from the
group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In some cases, each DMR is within
a biomarker, wherein each biomarker is at least 90%, 95%, or 99% identical, or identical, to a
sequence independently selected from the group consisting of SEQ ID NO: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, 7 1 and 72.
[0026] In any of the foregoing computer program products, the computer program product
can, in some cases, predict the presence or absence of a somaclonal abnormality in the plant.
In some cases, the somaclonal abnormality exhibits a Mantled phenotype.
[0027] In some embodiments, the present invention provides a kit for determining the
methylation status of at least one DMR in a biological sample from an oil palm plant, the kit
comprising: (1) a polynucleotide (e.g., detectably labeled polynucleotide), or a pair of
polynucleotides (e.g., wherein one or both polynucleotides of the pair are detectably labeled),
capable of specifically amplifying at least a portion of a DMR, wherein the DMR is within a
sequence of DNA at least 70%, 80%, 90%, 95%, or 99% identical, or identical, to SEQ ID
NO:l; and a methylation-dependent, a methylation sensitive restriction enzyme, and/or
sodium bisulfite; or (2) sodium bisulfite, primers, and adapters for whole genome
amplification, and at least one polynucleotide to quantify the presence of the converted
methylated and/or the converted unmethylated sequence of at least one cytosine from a DMR,
wherein the DMR is within a sequence of DNA at least 70%, 80%, 90%, 95%, or 99%
identical, or identical, to SEQ ID NO:l; or (3) methylation sensing restriction enzymes,
primers and adapters for whole genome amplification, and at least one polynucleotide to
quantify the number of copies of at least a portion of a DMR, wherein the DMR is within a
sequence of DNA at least 70%, 80%, 90%, 95%, or 99% identical, or identical, to SEQ ID
NO:l; or (4) a methylation sensing binding moiety and at least one polynucleotide to quantify
the number of copies of at least a portion of a DMR, wherein the DMR is within a sequence
of DNA at least 70%, 80%, 90%, 95%, or 99% identical, or identical, to SEQ ID NO:l.
[0028] In some aspects, the DMR is within a DNA meta-region in the sample from the
plant, where the DNA meta-region is at least 70%, 80%, 90%, 95%, or 99% identical, or
identical, to a sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67,
68, 69, and 70. In some aspects, the DMR is within a DNA region in the sample from the
plant, where the DNA region is at least 70%, 80%, 90%, 95%, or 99% identical, or identical,
to a sequence selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In
some cases, the DMR is within a biomarker, wherein the biomarker is at least 90%, 95%, or
99% identical, or identical, to a sequence selected from the group consisting of SEQ ID
NO: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, 71, and 72.
[0029] In some aspects, the kit comprises at least two, three, or four polynucleotides-or
two, three, or four pairs of polynucleotides-capable of specifically amplifying at least a
portion of two, three, or four different DMRs, wherein each DMR is independently within a
sequence of DNA at least 70%, 80%, 90%, 95%, or 99% identical, or identical, to SEQ ID
NO: 1. In some cases, each DMR is within a DNA meta-region, where the DNA meta-region
is at least 70%>, 80%>, 90%>, 95%>, or 99%> identical, or identical, to a sequence independently
selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and 70. In some
cases, each DMR is within a sequence of DNA at least 70%, 80%, 90%, 95%, or 99%
identical, or identical, to a sequence independently selected from the group consisting of SEQ
ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 73 and 74. In some cases, each DMR is within a biomarker, wherein each
biomarker is at least 90%, 95%, or 99%> identical, or identical, to a sequence independently
selected from the group consisting of SEQ ID NO: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, 7 1 and 72.
[0030] In some aspects, the kit further comprises a detectably labeled polynucleotide probe
that specifically detects an amplified DMR, or portion thereof. In some cases, the
polynucleotide probe specifically detects an amplified DMR, or portion thereof, in a real-time
amplification reaction.
[0031] In some embodiments, the present invention provides a method of predicting the
presence or absence of somaclonal abnormality in an oil palm plant comprising: a) obtaining
a biological sample from the plant; b) determining the methylation status of at least one
cytosine within a differential methylation region (DMR) in the sample from the plant,
wherein the DMR is within a sequence of DNA at least 70%, 80%, 90%, 95%, or 99%
identical, or identical, to SEQ ID NO:l; and c) correlating the methylation status of the at
least one cytosine to the presence or absence of a somaclonal abnormality in the plant,
wherein the correlation comprises predicting the presence or absence of somaclonal
abnormality in the plant.
[0032] In some aspects, the DMR is within a DNA meta-region in the sample from the
plant, where the DNA meta-region is at least 70%, 80%, 90%, 95%, or 99% identical, or
identical, to a sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67,
68, 69, and 70. In some aspects, the DMR is within a DNA region in the sample from the
plant, where the DNA region is at least 70%, 80%, 90%, 95%, or 99% identical, or identical,
to a sequence selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73 and 74. In
some cases, the determining step comprises determining the methylation status of at least one
cytosine in a biomarker, wherein the biomarker is at least 90%, 95%, or 99% identical, or
identical, to a sequence selected from the group consisting of SEQ ID NO: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, 7 1 and 72.
[0033] In some aspects, the method comprises predicting the presence of a somaclonal
abnormality when the methylation status of the at least one cytosine is reduced relative to a
control locus. In some cases, the presence of a somaclonal abnormality is predicted when the
methylation status of the at least one cytosine in the DNA meta-region at least 70%, 80%,
90%, 95%o, or 99% identical, or identical, to the sequence selected from the group consisting
of SEQ ID NO:63, 64, 65, 66, 67, 69, and 70 (or selected from the group consisting of SEQ
ID NO: 63, 64, 65, 66, 67, 68, 69, and 70) is reduced relative to a control locus. In some
cases, the presence of a somaclonal abnormality is predicted when the methylation status of
the at least one cytosine in the DNA region at least 70%, 80%, 90%, 95%, or 99% identical,
or identical, to the sequence selected from the group consisting of SEQ ID NO:35, 36, 39, 40,
42, 43, 44, 45, 46, 48, 49, 51, 52, 57, 58, 59, 60, 61, and 73 is reduced relative to a control
locus. In some cases, the presence of a somaclonal abnormality is predicted when the
methylation status of the at least one cytosine in the biomarker at least 90%, 95%, or 99%
identical, or identical to the sequence selected from the group consisting of SEQ ID NO:7, 8,
11, 12, 14, 15, 16, 17, 18, 20, 21, 23, 24, 29, 30, 31, 32, 33, and 7 1 is reduced relative to a
control locus.
[0034] In some aspects, the method comprises predicting the presence of a somaclonal
abnormality when the methylation status of the at least one cytosine is increased relative to a
control locus. In some cases, the presence of a somaclonal abnormality is predicted when the
methylation status of the at least one cytosine in the DNA meta-region at least 70%, 80%,
90%, 95%, or 99% identical, or identical, to the sequence selected from the group consisting
of SEQ ID NO:63, 64, 65, 66, 67, 68, and 69 (or selected from the group consisting of SEQ
ID NO:63, 64, 65, 66, 67, 68, 69, and 70) is increased relative to a control locus. In some
cases, the presence of a somaclonal abnormality is predicted when the methylation status of
the at least one cytosine in the DNA region at least 70%, 80%, 90%, 95%, or 99% identical,
or identical, to the sequence selected from the group consisting of SEQ ID NO:35, 36, 37, 38,
41, 42, 47, 50, 52, 53, 54, 55, 56, 57, 62, and 74 is increased relative to a control locus. In
some cases, the presence of a somaclonal abnormality is predicted when the methylation
status of the at least one cytosine in the biomarker at least 90%, 95%, or 99% identical, or
identical to the sequence selected from the group consisting of SEQ ID NO:7, 8, 9, 10, 13,
14, 19, 22, 24, 25, 26, 27, 28, 29, 34, and 72 is increased relative to a control locus.
[0035] In some aspects, the method comprises predicting the presence of a somaclonal
abnormality when the methylation status of the at least one cytosine is either increased or
decreased relative to a control locus. In some cases, the control locus is an endogenous
control locus. In some cases, the control locus is an exogenous control locus.
[0036] In some aspects, the determining step comprises determining the methylation status
of at least one cytosine in at least two, three or four different differential methylation regions
(DMRs), wherein each DMR is independently within a sequence of DNA at least 70%, 80%>,
90%, 95%, or 99% identical, or identical, to SEQ ID NO:l. In some cases, each DMR is
within a DNA meta-region in the sample from the plant, where each DNA meta-region is at
least 70%, 80%>, 90%>, 95%, or 99% identical, or identical, to a sequence independently
selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and 70. In some
cases, each DMR is within a DNA region in the sample from the plant, where each DNA
region is at least 70%>, 80%>, 90%>, 95%, or 99% identical, or identical, to a sequence
independently selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In
some cases, the determining step comprises determining the methylation status of at least one
cytosine in a biomarker in each DMR, wherein each biomarker is at least 90%, 90%, 95%, or
99% identical, or identical, to a sequence independently selected from the group consisting of
SEQ ID NO: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, 71, and 72.
[0037] In some aspects, the somaclonal abnormality comprises a reduction in fruit yield, oil
yield, growth, or reproduction of the plant relative to a control plant. In some cases, the
control plant is a parental plant. In some cases, the control plant is a wild-type plant of the
same fruit form phenotype {dura, tenera, or pisifera) as the plant predicted to have a
somaclonal abnormality.
[0038] In some aspects, the somaclonal abnormality exhibits a Mantled phenotype.
[0039] In some aspects, the determining the methylation status comprises bisulfite
conversion; and/or digesting genomic DNA with a methylation-dependent endonuclease;
and/or digesting genomic DNA with a methylation-sensitive endonuclease.
[0040] In some embodiments, the present invention provides a method comprising:
providing a prediction of a presence or absence of a somaclonal abnormality in a plurality of
plants, wherein the presence or absence of a somaclonal abnormality is determined by a
methylation status of at least one cytosine within a differential methylation region (DMR) in
a sample from each plant, wherein the DMR is within a sequence of DNA at least 70%, 80%,
90%, 95%, or 99% identical, or identical, to SEQ ID NO:l; and physically separating a plant
predicted to have a somaclonal abnormality from a plant predicted to lack a somaclonal
abnormality.
[0041] In some aspects, the DMR is within a DNA meta-region in the sample from the
plant, where the DNA region is at least 70%, 80%, 90%, 95%, or 99% identical, or identical,
to a sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69,
and 70. In some aspects, the DMR is within a DNA region in the sample from the plant,
where the DNA region is at least 70%, 80%, 90%, 95%, or 99% identical, or identical, to a
sequence selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In some
cases, the determining step comprises determining the methylation status of at least one
cytosine in a biomarker, wherein the biomarker is at least 90%, 95%, or 99% identical, or
identical, to a sequence selected from the group consisting of SEQ ID NO: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, 71, and 72.
[0042] In some aspects, the present invention provides a method for detecting or predicting
a somaclonal abnormality for an oil palm plant, the method comprising: a) obtaining a
biological sample from the plant; b) determining the methylation status of at least one
cytosine within a differential methylation region (DMR) in the sample from the plant,
wherein the DMR is within a sequence of DNA at least 90%>, 95%>, or 99%> identical, or
identical, to SEQ ID NO:l; and c) correlating the methylation status of the at least one
cytosine to the presence or absence of the somaclonal abnormality in the plant. In some
embodiments, the method further comprises physically separating a plant predicted to have
the somaclonal abnormality from one or more plants predicted to lack a somaclonal
abnormality. In some cases, the physically separating comprises selecting plants predicted to
have a somaclonal abnormality for destruction.
[0043] In some cases, the physically separating comprises selecting plants predicted to lack
a somaclonal abnormality for cultivation. In some cases, the plants selected for cultivation
are germinated, planted, or transplanted. In some cases, the plants not selected for cultivation
are discarded or destroyed. In some cases, the plants not selected for cultivation are treated to
reduce the likelihood of a somaclonal abnormality. In some embodiments, the at least one
cytosine is a first cytosine in a CHG sequence, wherein H is C, A, or T.
[0044] In some embodiments, the DMR is within a DNA meta-region in the sample from
the plant, where the DNA meta-region is at least 90%>, 95%>, or 99%> identical, or identical, to
a sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and
70. In some embodiments, the DMR is within a DNA region in the sample from the plant,
where the DNA region is at least 90%, 95%, or 99% identical, or identical, to a sequence
selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74.
[0045] In some cases, the determining step comprises determining the methylation status of
at least one cytosine in a biomarker, wherein the biomarker is at least 90%, 90%, 95%, or
99% identical, or identical, to a sequence selected from the group consisting of SEQ ID
NO: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, 71, and 72. In some cases, the DMR is within a DNA region in the sample from
the plant, where the DNA region is at least 90%>, 95%>, or 99%> identical, or identical, to SEQ
ID NO:84, 87, or 90.
[0046] In some cases the at least cytosine is in an AlwNl, Bbvl, ScrFl, or Rsal restriction
endonuclease recognition site. In some cases, the method comprises determining the
methylation status of a first and a second cytosine, wherein the first cytosine is within a DMR
within a DNA region in the sample from the plant, where the DNA region is at least 90%,
95%, or 99% identical, or identical, to SEQ ID NO:87, and wherein the second cytosine is
within a DMR within a DNA region in the sample from the plant, where the DNA region is at
least 90%, 95%, or 99% identical, or identical, to SEQ ID NO: 90. In some cases, the first
cytosine is in a Bbvl restriction endonuclease site, and the second cytosine is in a Rsal
restriction endonuclease site.
[0047] In some cases, the method comprises predicting the presence of a somaclonal
abnormality when the methylation status of the at least one cytosine is reduced relative to a
control locus. In some cases, the method comprises predicting the presence of a somaclonal
abnormality when the methylation status of the at least one cytosine is increased relative to a
control locus. In some cases, the method comprises predicting the presence of a somaclonal
abnormality when the methylation status of the at least one cytosine is either increased or
decreased relative to a control locus. In some cases, the control locus is an endogenous
control locus. In some cases, the control locus is an exogenous control locus.
[0048] In some cases, the determining step comprises determining the methylation status of
at least one cytosine in at least two, three or four different differential methylation regions
(DMRs), wherein each DMR is independently within a sequence of DNA at least 90%, 95%,
or 99% identical, or identical, to SEQ ID NO:l.
[0049] In some cases, the somaclonal abnormality comprises a reduction in fruit yield, oil
yield, growth, or reproduction of the plant relative to a control plant. In some cases, the
control plant is a parental plant. In some cases, the control plant is a wild-type plant of the
same fruit form phenotype {dura, tenera, or pisifera) as the plant predicted to have a
somaclonal abnormality.
[0050] In some cases, the somaclonal abnormality is predicted to exhibit a Mantled
phenotype.
[0051] In some cases, the determining the methylation status comprises bisulfite
conversion. In some cases, the determining the methylation status comprises digesting
genomic DNA with a methylation-dependent endonuclease. In some cases, the determining
the methylation status comprises digesting genomic DNA with a methylation-sensitive
endonuclease. In some cases, the genomic DNA is amplified after digesting.
[0052] In some cases, the determining the methylation status comprises bisulfite
conversion; and/or the determining the methylation status comprises digesting genomic DNA
with a methylation-dependent endonuclease; and/or the determining the methylation status
comprises digesting genomic DNA with a methylation-sensitive endonuclease; and/or the
determining of the methylation status comprising measuring rates of methylated base
incorporation during sequencing; and/or the determining of the methylation status comprising
measuring current as molecules including methylated bases pass through a nanopore. In
some cases, the determining the methylation status can comprise methylated DNA
immunoprecipitation, methylated DNA capture by affinity purification, or reduced
representation bisulfite sequencing. In some cases, the determining the methylation status
can comprise nucleic acid hybridization, e.g. , microarray or bead array hybridization.
[0053] In some aspects, the present invention provides a method for detecting or predicting
a somaclonal abnormality for an oil palm plant, the method comprising: a) obtaining a
biological sample from the plant; b) determining the expression level of at least one small
RNA in the sample from the plant, wherein the at least one small RNA is encoded by a
sequence comprising a polynucleotide at least 90%>, 95%, or 99% identical or identical to
SEQ ID NO:91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 130, 131,
132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,
150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161; and c) correlating the
expression level of the at least one small RNA to the presence or absence of the somaclonal
abnormality in the plant. In some embodiments, the expression level of the at least one small
RNA is at least 2-fold increased or decreased relative to expression of the at least one small
RNA in a normal control plant.
[0054] In some cases, the at least one small RNA in the sample from the plant is encoded
by a sequence comprising a polynucleotide at least 90% (e.g., 91%, 92%, 93%>, 94%>, 95%>,
96%, 97%, 98%, 99%, 99.5%, or 100%) identical to any one of SEQ ID NOs: 144-161. In
some cases, the expression level of the at least one small RNA that is at least 90% identical to
any one of SEQ ID NOs: 144-161 in a sample from a plant predicted to have a somaclonal
abnormality is less than 50% of the expression level of the at least one small RNA in a
normal control plant. In some cases, the at least one small RNA in the sample from the plant
is encoded by a sequence comprising a polynucleotide at least 90%> (e.g., 9 1 >, 92%>, 93%>,
94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identical to SEQ ID NO:91 . In some
cases, the expression level of the at least one small RNA that is at least 90% identical to SEQ
ID NO:91 in a sample from a plant predicted to have a somaclonal abnormality is less than
50%, 40%, 30%, or 10% of the expression level of the at least one small RNA in a normal
control plant.
[0055] In some cases, the biological sample is derived from shoot apex tissue of the plant.
In some cases, the biological sample is derived from < 2 cm stage inflorescens tissue of the
plant. In some cases, the biological sample is derived from at least 2 cm stage inflorescens
tissue of the plant. In some cases, the biological sample is derived from an in vitro tissue
cultured plant cell, a seed, or a seedling.
[0056] In some embodiments, the method further comprises physically separating a plant
predicted to have the somaclonal abnormality from one or more plants predicted to lack a
somaclonal abnormality. In some embodiments, the physically separating comprises
selecting plants predicted to have a somaclonal abnormality for destruction. In some cases,
the physically separating comprises selecting plants predicted to lack a somaclonal
abnormality for cultivation. In some cases, the plants selected for cultivation are germinated,
planted, or transplanted. In some cases, plants not selected for cultivation are discarded or
destroyed. In some cases, the plants not selected for cultivation are treated to reduce the
likelihood of a somaclonal abnormality. In some cases, the somaclonal abnormality is
predicted to exhibit a Mantled phenotype.
[0057] In some aspects, the present invention provides, a method for detecting or predicting
a somaclonal abnormality for an oil palm plant, the method comprising: a) obtaining a
biological sample from the plant; b) determining the expression level of a transcript encoded
by SEQ ID NO:5, 75, 78, or 80; and c) correlating the expression level to the presence or
absence of the somaclonal abnormality in the plant. In some embodiments, the plant is
predicted to have a somaclonal abnormality when the expression level of SEQ ID NO:5 is
decreased relative to a wildtype control plant, or when the expression level of SEQ ID
NO:75, or 78, or 80 is increased relative to a wildtype control plant. In some embodiments,
the plant is predicted to have a somaclonal abnormality when the expression level of SEQ ID
NO:75 or 78 or 80 is increased relative to an expression level of SEQ ID NO:5.
[0058] In some embodiments, the method further comprises physically separating a plant
predicted to have the somaclonal abnormality from one or more plants predicted to lack a
somaclonal abnormality. In some cases, the physically separating comprises selecting plants
predicted to have a somaclonal abnormality for destruction. In some cases, the physically
separating comprises selecting plants predicted to lack a somaclonal abnormality for
cultivation. In some cases, the plants selected for cultivation are germinated, planted, or
transplanted. In some cases, the plants not selected for cultivation are discarded or destroyed.
In some cases, the plants not selected for cultivation are treated to reduce the likelihood of a
somaclonal abnormality.
[0059] In some embodiments, the somaclonal abnormality is predicted to exhibit the
Mantled phenotype.
[0060] In some aspects, the present invention provides a computer program product for
predicting the presence or absence of a somaclonal abnormality in an oil palm plant, the
computer program product comprising: a computer readable medium encoded with program
code, the program code including: program code for receiving a methylation value
representing the methylation status of at least one cytosine within a differential methylation
region (DMR) in the sample from the oil palm plant, wherein the DMR is within a sequence
of DNA at least 70%, 80%, 90%, 95%, or 99% identical, or identical, to SEQ ID NO:l; and
program code for comparing the methylation value to a control value, wherein the control
value distinguishes between plants with and without a somaclonal abnormality, wherein the
comparison of the methylation value to the control value is predictive of the presence or
absence of a somaclonal abnormality in the plant.
[0061] In some embodiments, the DMR is within a DNA meta-region in the sample from
the plant, where the DNA meta-region is at least 90%, 95%, or 99% identical, or identical, to
a sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and
70. In some cases, the DMR is within a DNA region in the sample from the plant, where the
DNA region is at least 90%, 95%, or 99% identical, or identical, to a sequence selected from
the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In some cases, the at least one
cytosine is in a biomarker, wherein the biomarker is at least 90% 95%, or 99% identical, or
identical, to a sequence selected from the group consisting of SEQ ID NO: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, 71, and 72.
[0062] In some cases, the control value is a methylation value for a control locus
exogenous to the plant. In some cases, the control value is a methylation value for a control
locus endogenous to the plant. In some cases, the program code comprises program code for
receiving the methylation status of at least one cytosine in at least two, three or four different
DMRs, wherein each DMR is independently within a sequence of DNA at least 90%, 95%, or
99% identical, or identical, to SEQ ID NO: 1. In some cases, each DMR is within a DNA
meta-region in the sample from the plant, where each DNA meta-region is at least 90%, 95%,
or 99% identical, or identical, to a sequence independently selected from the group consisting
of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and 70.
[0063] In some cases, each DMR is within a DNA region in the sample from the plant,
wherein each DNA region is at least 90%, 95%, or 99% identical, or identical, to a sequence
independently selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In
some cases, each DMR is within a biomarker, wherein each biomarker is at least 90%, 95%,
or 99% identical, or identical, to a sequence independently selected from the group consisting
of SEQ ID NO: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, 7 1 and 72. In some cases, the somaclonal abnormality is predicted to
exhibit a Mantled phenotype.
[0064] In some aspects, the present invention provides a computer program product for
determining the presence or absence of a somaclonal abnormality in an oil palm plant, the
computer program product comprising: a computer readable medium encoded with program
code, the program code including: program code for receiving a value representing i). an
expression level of a small RNA (e.g. , an expression level of a small RNA in a sample from a
plant), wherein the small RNA is encoded by a sequence comprising a polynucleotide at least
90%, 95%, or 99% identical, or identical, to SEQ ID NO:91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 121, 122, 123, 124, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,
141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
159, 160 or 161; or ii). an expression level of a transcript at least 90%>, 95%>, or 99%>
identical, or identical, to SEQ ID NO:5, 75, 78, or 80; and program code for comparing the
expression level value to a control value, wherein the control value distinguishes between
plants with and without a somaclonal abnormality, wherein the comparison of the expression
level value to the control value is predictive of the presence or absence of a somaclonal
abnormality in the plant.
[0065] In some cases, the at least one small RNA in the sample from the plant is encoded
by a sequence comprising a polynucleotide at least 90%> (e.g., 91%, 92%>, 93%>, 94%>, 95%>,
96%, 97%, 98%, 99%, 99.5%, or 100%) identical to any one of SEQ ID NOs: 144-161. In
some cases, the expression level of the at least one small RNA that is at least 90%>, 95%>, or
99% identical to any one of SEQ ID NOs: 144-161 in a sample from a plant predicted to have
a somaclonal abnormality is less than 50% of the expression level of the at least one small
RNA in a normal control plant. In some cases, the at least one small RNA in the sample from
the plant is encoded by a sequence comprising a polynucleotide at least 90%> (e.g., 91%>, 92%>,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identical to SEQ ID NO:91. In
some cases, the expression level of the at least one small RNA that is at least 90%>, 95%>, or
99% identical to SEQ ID NO:91 in a sample from a plant predicted to have a somaclonal
abnormality is less than 50%>, 40%>, 30%>, or 10%> of the expression level of the at least one
small RNA in a normal control plant.
[0066] The computer program product can, in some cases, predict the presence or absence
of a somaclonal abnormality in the plant. In some cases, the somaclonal abnormality exhibits
a Mantled phenotype. In some cases, a plant predicted to have a somaclonal abnormality by
application of the computer program product is physically separated from one or more plants
predicted to lack a somaclonal abnormality.
[0067] In some aspects, the present invention provides a kit for determining the
methylation status of at least one DMR in a biological sample from an oil palm plant,
wherein the DMR is within a sequence of DNA at least 90%, 95%, or 99% identical, or
identical, to SEQ ID NO:l, the kit comprising: (1) sodium bisulfite, oligonucleotide
amplification primers, and at least one polynucleotide to quantify the presence of the
unconverted methylated or the converted unmethylated sequence of at least one cytosine from
the DMR; (2) a methylation-sensitive or dependent restriction enzyme, oligonucleotide
amplification primers, and at least one polynucleotide to quantify the number of copies of at
least a portion of the DMR; (3) a methylation sensing binding moiety and at least one
polynucleotide to quantify the number of copies of at least a portion of the DMR, wherein the
methylation status of the at least one cytosine is predictive of a somaclonal abnormality of the
oil palm plant.
[0068] In some embodiments, the methylation-sensitive or dependent restriction enzyme is
heterologous to the oil palm plant. In some embodiments, the methylation-sensitive or
dependent restriction enzyme is selected from the group consisting of AlwNl, Bbvl, Rsal, and
ScrFl. In some embodiments, the kit comprises Bbvl, and Rsal. In some embodiments, the
at least one polynucleotide to quantify the presence of the unconverted methylated or the
converted unmethylated sequence of at least one cytosine from the DMR comprises a
sequence that specifically hybridizes to a sequence from the DMR containing a bisulfite
converted cytosine. In some embodiments, the at least one polynucleotide to quantify the
number of copies of at least a portion of the DMR comprises a sequence that specifically
hybridizes to a sequence from the DMR containing a bisulfite converted cytosine.
[0069] In some embodiments, the methylation sensitive binding moiety is an antibody. In
some embodiments, the DMR is within a DNA meta-region in the sample from the plant,
where the DNA meta-region is at least 90%, 95%, or 99% identical, or identical, to a
sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and
70. In some embodiments, the DMR is within a DNA region in the sample from the plant,
where the DNA region is at least 90%, 95%, or 99% identical, or identical, to a sequence
selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74. In some cases,
the DMR is within a biomarker, wherein the biomarker is at least 90%, 95%, or 99%
identical, or identical, to a sequence selected from the group consisting of SEQ ID NO: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, 71, and 72.
[0070] In some embodiments, the kit comprises at least two, three, or four polynucleotidesor
two, three, or four pairs of polynucleotides-capable of specifically amplifying at least a
portion of two, three, or four different DMRs, wherein each DMR is independently within a
sequence of DNA at least 90%, 95%, or 99% identical, or identical, to SEQ ID NO:l. In
some cases, each DMR is within a DNA meta-region, where the DNA meta-region is at least
90%, 95%o, or 99%o identical, or identical, to a sequence independently selected from the
group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, and 70.
[0071] In some cases, each DMR is within a sequence of DNA at least 90%, 95%, or 99%
identical, or identical, to a sequence independently selected from the group consisting of SEQ
ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 73 and 74. In some cases, each DMR is within a biomarker, wherein each
biomarker is at least 90%>, 95%>, or 99%> identical, or identical, to a sequence independently
selected from the group consisting of SEQ ID NO: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, 7 1 and 72. In some cases, the kit
further comprises a detectably labeled polynucleotide probe that specifically detects an
amplified DMR, or portion thereof. In some cases, the polynucleotide probe specifically
detects an amplified DMR, or portion thereof, in a real-time amplification reaction.
[0072] In some aspects, the present invention provides a kit for detecting the expression
level of an RNA in an oil palm plant, the kit comprising: a) an oligonucleotide primer capable
of specifically hybridizing to a small RNA encoded by a sequence comprising a
polynucleotide at least 90%, 95%, or 99% identical, or identical, to SEQ ID NO:91, 92, 93,
94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,
156, 157, 158, 159, 160 or 161; or b) an oligonucleotide primer capable of specifically
hybridizing to a transcript encoded by SEQ ID NO:5, 75, 78, or 80, wherein the detected
expression level is predictive of a somaclonal abnormality of the oil palm plant. In some
cases, the kit further comprises a detectably labeled oligonucleotide probe; or wherein the
oligonucleotide primer is detectably labeled. In some cases, the oligonucleotide primer of b)
comprises SEQ ID NO: 125, 126, 127, 128, or 129. In some cases, the oligonucleotide primer
of a) is capable of is capable of specifically hybridizing to a small RNA encoded by a
sequence comprising a polynucleotide at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 99.5%, or 100%) identical to one of SEQ ID NOs: 144-161.
[0073] In some aspects, the present invention provides a method of reducing somaclonal
abnormalities an oil palm plant propagated by in vitro tissue culture comprising: exogenously
applying to the plant an mRNA encoded by SEQ ID NO:5 or a sequence at least 90%>, 95%,
or 99% identical to SEQ ID NO:5; or exogenously applying to the plant a small RNA
encoded by a sequence comprising a polynucleotide at least 90%>, 95%, or 99% identical, or
identical, to SEQ ID NO:91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 112, 113, 114, 116, 117, 123, 124, 130, 131, 132, 133, 134, 136,
137, 138, 139, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
157, 158, 159, 160 or 161. In some embodiments, the exogenously applying the mRNA or
small RNA comprises contacting a cytoplasm or nucleus of the plant with the mRNA or
small RNA. In some embodiments, the exogenously applying the mRNA or small RNA
comprises contacting the plant with an expression cassette comprising a heterologous
promoter operably linked to a polynucleotide at least 90%, 95%, or 99% identical, or
identical, to SEQ ID NO:5.
[0074] In some embodiments, the exogenously applying the mRNA or small RNA
comprises contacting the plant with an expression cassette comprising a heterologous
promoter operably linked to a polynucleotide encoding a small RNA, wherein the
polynucleotide comprises a sequence at least 90%, 95%, or 99% identical, or identical, to
SEQ ID NO:91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 116, 117, 123, 124, 130, 131, 132, 133, 134, 136, 137, 138,
139, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
159, 160 or 161. In some embodiments, the exogenously applying the mRNA or small RNA
comprises contacting an in vitro tissue cultured plant cell with the mRNA or small RNA.
[0075] In some asepcts, the present inventino provides an expression cassette comprising a
heterologous promoter operably linked to: i) a polynucleotide encoding a small RNA,
wherein the polynucleotide comprises a sequence at least 90%, 95%, or 99% identical, or
identical, to SEQ ID NO:91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 112, 113, 114, 116, 117, 123, 124, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,
154, 155, 156, 157, 158, 159, 160 or 161; or ii) a polynucleotide encoding an mR A,
wherein the polynucleotide comprises a sequence at least 90%, 95%, or 99% identical, or
identical, to SEQ ID NO:5. The expression cassette can be a heterologous expression
cassette. In some aspects, the present invention provides a recombinant plant comprising any
one of the foregoing expression cassettes.
[0076] In some embodiments, the present invention provides a method of predicting the
presence or absence of somaclonal abnormality in an oil palm plant comprising: a) obtaining
a biological sample from the plant; b) determining a methylation density of a differential
methylation region (DMR), or sub-region, in the sample from the plant, wherein the DMR is
within a sequence of DNA at least 70%, 80%, 90%, 95%, or 99% identical, or identical, to
SEQ ID NO:l; and c) correlating the methylation density to the presence or absence of a
somaclonal abnormality in the plant, wherein the correlation comprises predicting the
presence or absence of somaclonal abnormality in the plant.
[0077] In some aspects, the DMR is within a DNA meta-region in the sample from the
plant, where the DNA meta-region is at least 70%, 80%, 90%, 95%, or 99% identical, or
identical, to a sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67,
68, 69, and 70. In some aspects, the DMR is within a DNA region in the sample from the
plant, where the DNA region is at least 70%, 80%, 90%, 95%, or 99% identical, or identical,
to a sequence selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73 and 74. In
some cases, the determining step comprises determining the methylation density in a
biomarker, wherein the biomarker is at least 90%, 95%, or 99% identical, or identical, to a
sequence selected from the group consisting of SEQ ID NO: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, 7 1 and 72.

WHAT IS CLAIMED IS:
1. A method for detecting or predicting a somaclonal abnormality for an
oil palm plant, the method comprising:
a) obtaining a biological sample from the plant;
b) determining the methylation status of at least one cytosine within 5 a
differential methylation region (DMR) in the sample from the plant, wherein the DMR is
within a sequence of DNA at least 90% identical, or identical, to SEQ ID NO:1; and
c) correlating the methylation status of the at least one cytosine to the presence
or absence of the somaclonal abnormality in the plant.
10 2. The method of claim 1, wherein the method further comprises
physically separating a plant predicted to have the somaclonal abnormality from one or more
plants predicted to lack a somaclonal abnormality.
3. The method of any one of the preceding claims, wherein the at least
one cytosine is a first cytosine in a CHG sequence, wherein H is C, A, or T.
15 4. The method of any one of the preceding claims, wherein the DMR is
within a DNA meta-region in the sample from the plant, where the DNA meta-region is at
least 90% identical, or identical, to a sequence selected from the group consisting of SEQ ID
NO:63, 64, 65, 66, 67, 68, 69, and 70.
5. The method of any one of claims 1-3, wherein the DMR is within a
20 DNA region in the sample from the plant, where the DNA region is at least 90% identical, or
identical, to a sequence selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and
74.
6. The method of claim 5, wherein the determining step comprises
25 determining the methylation status of at least one cytosine in a biomarker, wherein the
biomarker is at least 90% identical, or identical, to a sequence selected from the group
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consisting of SEQ ID NO: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, 71, and 72.
7. The method of any one of claims 1-3, wherein the DMR is within a
DNA region in the sample from the plant, where the DNA region is at least 90% identical, or
identical, to SEQ ID NO:84, 87, or 905 .
8. The method of any one of claims 1-3, wherein the method comprises
determining the methylation status of a first and a second cytosine, wherein the first cytosine
is within a DMR within a DNA region in the sample from the plant, where the DNA region is
at least 90% identical, or identical, to SEQ ID NO:87, and wherein the second cytosine is
10 within a DMR within a DNA region in the sample from the plant, where the DNA region is at
least 90% identical, or identical, to SEQ ID NO: 90.
9. The method of any one of claims 1 - 8, wherein the method comprises
predicting the presence of a somaclonal abnormality when the methylation status of the at
least one cytosine is reduced relative to a control locus.
15 10. The method of any one of claims 1 -8 , wherein the method comprises
predicting the presence of a somaclonal abnormality when the methylation status of the at
least one cytosine is increased relative to a control locus.
11. The method of any one of claims 9-10 - , wherein the control locus is
an endogenous control locus.
20 12. The method of any one of claims 9-10 - , wherein the control locus is
an exogenous control locus.
13. The method of any one of the preceding claims, wherein the
somaclonal abnormality comprises a reduction in fruit yield, oil yield, growth, or
reproduction of the plant relative to a control plant.
25 14. The method of claim 13, wherein the control plant is a parental plant.
15. The method of claim 13, wherein the control plant is a wild-type plant
of the same fruit form phenotype (dura, tenera, or pisifera) as the plant predicted to have a
somaclonal abnormality.
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16. The method of any one of the preceding claims, wherein the
somaclonal abnormality is predicted to exhibit a Mantled phenotype.
17. The method of any one of the preceding claims, wherein the
determining the methylation status comprises bisulfite conversion.
18. The method of any one of the preceding claims, wherein th5 e
determining the methylation status comprises digesting genomic DNA with a methylationdependent
endonuclease.
19. The method of any one of the preceding claims, wherein the
determining the methylation status comprises digesting genomic DNA with a methylation10
sensitive endonuclease.
20. The method of claim 18 or 19, wherein the genomic DNA is amplified
after digesting.
21. A method for detecting or predicting a somaclonal abnormality for an
oil palm plant, the method comprising:
15 a) obtaining a biological sample from the plant;
b) determining the expression level of at least one small RNA in the sample
from the plant, wherein the at least one small RNA is encoded by a sequence comprising a
polynucleotide at least 90% identical or identical to SEQ ID NO:91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
20 117, 118, 119, 120, 121, 122, 123, 124, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,
140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,
158, 159, 160 or 161; and
c) correlating the expression level of the at least one small RNA to the
presence or absence of the somaclonal abnormality in the plant.
25 22. The method of claim 21, wherein the expression level of the at least
one small RNA is at least 2-fold increased or decreased relative to expression of the at least
one small RNA in a normal control plant.
23. The method of claim 21 or 22, wherein the biological sample is
derived from shoot apex tissue of the plant.
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24. The method of claim 21 or 22, wherein the biological sample is
derived from < 2 cm stage inflorescens tissue of the plant.
25. The method of claim 21 or 22, wherein the biological sample is
derived from at least 2 cm stage inflorescens tissue of the plant.
26. The method of claim 21 or 22, wherein the biological sample i5 s
derived from an in vitro tissue cultured plant cell, a seed, or a seedling.
27. The method of any one of claims 21-25, wherein the method further
comprises physically separating a plant predicted to have the somaclonal abnormality from
one or more plants predicted to lack a somaclonal abnormality.
10 28. The method of any one of claims 21-27-, wherein the somaclonal
abnormality is predicted to exhibit a Mantled phenotype.
29. A method for detecting or predicting a somaclonal abnormality for an
oil palm plant, the method comprising:
a) obtaining a biological sample from the plant;
15 b) determining the expression level of a transcript encoded by SEQ ID NO:5,
75, 78, or 80; and
c) correlating the expression level to the presence or absence of the
somaclonal abnormality in the plant.
30. The method of claim 29, wherein the plant is predicted to have a
20 somaclonal abnormality when the expression level of SEQ ID NO:5 is decreased relative to a
wildtype control plant, or when the expression level of SEQ ID NO:75, or 78, or 80 is
increased relative to a wildtype control plant.
31. The method of claim 29, wherein the plant is predicted to have a
somaclonal abnormality when the expression level of SEQ ID NO:75 or 78 or 80 is increased
25 relative to an expression level of SEQ ID NO:5.
32. The method of any one of claims 29-31, wherein the method further
comprises physically separating a plant predicted to have the somaclonal abnormality from
one or more plants predicted to lack a somaclonal abnormality.
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33. The method of any one of claims 29-32-, wherein the somaclonal
abnormality is predicted to exhibit the Mantled phenotype.
34. A kit for determining the methylation status of at least one DMR in a
biological sample from an oil palm plant, wherein the DMR is within a sequence of DNA at
least 90% identical, or identical, to SEQ ID NO:1, the kit comprising5 :
(1) sodium bisulfite, oligonucleotide amplification primers, and at least one
polynucleotide to quantify the presence of the unconverted methylated or the converted
unmethylated sequence of at least one cytosine from the DMR;
(2) a methylation-sensitive or dependent restriction enzyme, oligonucleotide
10 amplification primers, and at least one polynucleotide to quantify the number of copies of at
least a portion of the DMR;
(3) a methylation sensing binding moiety and at least one polynucleotide to
quantify the number of copies of at least a portion of the DMR,
wherein the methylation status of the at least one cytosine is predictive of a somaclonal
15 abnormality of the oil palm plant.
36. The kit of claim 34, wherein the methylation-sensitive or dependent
restriction enzyme is selected from the group consisting of AlwNI, BbvI, RsaI, and ScrFI.
37. The kit of claim 34, wherein the kit comprises BbvI, and RsaI.
38. The kit of claim 34, wherein the at least one polynucleotide to quantify
20 the presence of the unconverted methylated or the converted unmethylated sequence of at
least one cytosine from the DMR comprises a sequence that specifically hybridizes to a
sequence from the DMR containing a bisulfite converted cytosine.
39. The kit of claim 34, wherein the at least one polynucleotide to quantify
the number of copies of at least a portion of the DMR comprises a sequence that specifically
25 hybridizes to a sequence from the DMR containing a bisulfite converted cytosine.
40. The kit of claim 34, wherein the methylation sensitive binding moiety
is an antibody.
41. The kit of claim 34, wherein the DMR is within a DNA meta-region in
the sample from the plant, where the DNA meta-region is at least 90% identical, or identical,
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to a sequence selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69,
and 70.
42. The kit of claim 34, wherein the DMR is within a DNA region in the
sample from the plant, where the DNA region is at least 90% identical, or identical, to a
sequence selected from the group consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42, 435 ,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73, and 74.
43. The kit of claim 42, wherein the DMR is within a biomarker, wherein
the biomarker is at least 90% identical, or identical, to a sequence selected from the group
consisting of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
10 26, 27, 28, 29, 30, 31, 32, 33, 34, 71, and 72.
44. The kit of claim 34, wherein the kit comprises at least two, three, or
four polynucleotides-or two, three, or four pairs of polynucleotides-capable of amplifying at
least a portion of two, three, or four different DMRs, wherein each DMR is independently
within a sequence of DNA at least 90% identical, or identical, to SEQ ID NO:1.
15 45. The kit of claim 44, wherein each DMR is within a DNA meta-region,
where the DNA meta-region is at least 90% identical, or identical, to a sequence
independently selected from the group consisting of SEQ ID NO:63, 64, 65, 66, 67, 68, 69,
and 70.
46. The kit of claim 44, wherein each DMR is within a sequence of DNA
20 at least 90% identical, or identical, to a sequence independently selected from the group
consisting of SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 73 and 74.
47. The kit of claim 44, wherein each DMR is within a biomarker, wherein
each biomarker is at least 90% identical, or identical, to a sequence independently selected
25 from the group consisting of SEQ ID NO: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, 71 and 72.
48. The kit of claim 34, wherein the kit further comprises a detectably
labeled polynucleotide probe that detects an amplified DMR, or portion thereof.
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49. The kit of claim 48, wherein the polynucleotide probe detects an
amplified DMR, or portion thereof, in a real-time amplification reaction.
50. A kit for detecting the expression level of an RNA in an oil palm plant,
the kit comprising:
a) an oligonucleotide primer capable of specifically hybridizing to a smal5 l
RNA encoded by a sequence comprising a polynucleotide at least 90% identical, or identical,
to SEQ ID NO:91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
10 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161; or
b) an oligonucleotide primer capable of specifically hybridizing to a transcript
encoded by SEQ ID NO:5, 75, 78, or 80,
wherein the detected expression level is predictive of a somaclonal abnormality of the oil
palm plant.
15 51. The kit of claim 50, wherein the kit further comprises a detectably
labeled oligonucleotide probe; or wherein the oligonucleotide primer is detectably labeled.
52. The kit of claim 50, wherein the oligonucleotide primer of b)
comprises SEQ ID NO:125, 126, 127, 128, or