ABSTRACT
Cycads, be it extinct or extant are
unique among gymnosperms and plants in general due to their absolute dioecious
nature. Apart from the living cycads, no bisexual cycad cones or individuals
have ever been found in the fossil record. This dioecy can be correlated with
occurrence of sex chromosome having unequal length but occasional sex change as
encountered in cycads compounds the problem of explaining the sexuality.
Whatever factor behind the sex expression, distinguishing the maleness or
femaleness at an early vegetative stage is virtually impossible for cycads.
This poses serious problems in raising a population prior undertaking a proper
conservation strategy either through in situ or ex situ means.
The present review, hence aims to address the classical and state-of-art
molecular methods of sex determination in cycads at pre sporangial stage. Since
genome information of cycads is scanty, most of the PCR (Polymerase Chain
Reaction) based approaches adopt anonymous markers, which later on are usually
converted to more informative SCAR (Sequence Characterized Amplified Region)
markers. Even more reliable marker for sex determination in cycads at pre
sporangial stage will plausibly emanate from a technique like
Methylation—Sensitive Amplification Polymorphism (MSAP), which considers the
epigenetic mechanism, especially DNA methylation since perturbation of
environmental cue leads to occasional sex change.
Introduction
Gymnosperms include the earliest living
lineages with innovations of a greatly reduced male gametophyte (pollen),
pollination as well as seeds, but with extremely large genome. Recent molecular
phylogenetic studies indicate that the
five major lineages of extant gymnosperms (cycads, Ginkgo, Gnetales, Pinaceae, and all other conifers)
form a monophyletic group that is sister
to angiosperms, although an alternate placement of Gnetales as sister to the angiosperms cannot be unambiguously
rejected. Cycads represent the likely sister lineage to all other extant
gymnosperms and are consistently included in comparative developmental and
molecular systematic studies. As a basal lineage, cycads provide exemplars to
help ascertain the generalized gymnosperm reproductive features from which flowering
plant morphology and genetic controls were likely modified (http://www. greenbac.org/tree.html: The green plant BAC
library project).
Charles Darwin hypothesized that
ancestral lineages were hermaphroditic (each individual produces both
functional female and male gametes) and that dioecy evolved as a derived
condition (Darwin 1873). In plants and animals, evolution from hermaphroditism
to strict dioecy almost certainly occurred via an intermediate stage that
involved individuals who were both hermaphroditic and either functional females
or males (Darwin 1873; Charlesworth and Guttman 1999; Gorelick 2003). These
intermediate stages are referred to as gynodioecy (functional females) or
androdioecy (functional males). However, in spite of being an ancient lineage
(existing for 275 to 300 million years in near modern form), hermaphrodite
sexual morph was never found in cycads and dioecy appears to be quite a
primitive trait among cycads. No bisexual cycad cones or individuals have ever
been found in the fossil record (Gorelick and Osborne 2007) though occurrence of
bisporangiate cones has been reported in conifers (Flores-Renteria et al.
2011). The only instances of anything other than strict dioecy that have ever
been seen in cycads are those examples of sex change (Osborne and Gorelick
2007).
The living cycads can be divided into
three families; Cycadaceae, Stangeriaceae and Zamiaceae which consisted of 11
genera, 297 species and sub-species. Since all cycads are dioecious, they
appear to possess little if any sexual dimorphism as far as vegetative
structures is concerned. Therefore, one can hardly distinguish the sexes of a
species by visual inspection unless their reproductive organs are present. This
poses serious problems in raising a population prior undertaking a proper
conservation strategy either through in situ or ex situ means
since many of the cycads, including Zamia, have already been placed in
the endangered red list category (Roy et al. 2012). Attempt towards artificial
pollination leading to recovery of large number of viable seeds is, hence, the
only way to date, for scaling up the already diminishing population of these
threatened plants, for which substantial number of donor plants of both sexes
is a prerequisite.
In the backdrop of this, the present
review is aimed to address the classical and state-of-art methods of sex
determination in cycads, which are a popular area of research among experts in
various fields, including plant molecular biology, agriculture, horticulture,
ecology and environmental protection.
Ecological Approach: Sex and Population Differences
Four natural populations of Cycas
micronesica growing under differing ecological conditions were surveyed
over a 4 year period to assess the response of juveniles before and after the
introduction of the cycad aulacaspis scale insect (Aulacaspis yasumatsui Takagi)
and to test the hypothesis that monopodial ovulate plants are taller than
pollen-producing plants with equivalent diameter (D). Height (H), D, and leaf
and stem tip numbers were recorded for 297 ovulate and 186 pollen-producing
(“male” and “female”, respectively) plants and a total of 493 juveniles
(n=976). Among the 483 adults, mean female plant H and D did not differ significantly
from those of male plants. However, population-specific differences in mean
plant size were observed; i.e., female plants achieve greater H and have
significantly smaller leaf and stem tip numbers than do their male counterparts
with equivalent increases in D. Population differences, however, were not statistically
significant for juvenile plants (Niklas and Marler 2008).
Cytological Techniques towards Identification of Sex Chromosome
In many animals (but only in a handful of
plants), sex chromosome of different lengths can be identified. For example, in
humans, the Y chromosome is much shorter than the X chromosome. Evolution of sex
chromosomes is usually explained by a population genetic model known as
Muller’s ratchet (Griffin et al. 2002; Gorelick 2003). One of the prerequisites
for Muller’s ratchet for sexual organisms is that the haploid stage of the life
cycle should be largely secluded from selection. This is the case in most
animals, which generally have small and short-lived gametes. Plants, on the
other hand, have large multi-celled and long-lived haploid stages
(gametophytes), with cycads being extreme in this regard. Cycad gametophytes
are enormous; occupying much of the volume of what becomes a seed following
fertilization. Female cycad gametophytes are also long-lived; sometimes more
than a year passes between pollination and fertilization (Norstog and Nicholls
1997). Male gametophytes are also large and complex compared with other plants.
Although there are no data concerning whether cycad haploid stages express most
of the genes expressed by their diploid stages, the structural complexity, size
and age of cycad gametophytes indicate that they should be largely immune from
the Muller’s ratchet. Therefore, the chance of cycads (and most other plants) developing
unequal length of sex chromosome is remote (Gorelick 2005). Another argument
for cycads not having unequal length sex chromosome is that occasional sex
change does occur in cycads and it could only be possible if large chromosomal
rearrangements had taken place, which is virtually impossible (Gorelick and
Osborne 2007).
However, contrary to the aforesaid
rationale, the study of Sangduen et al. (2007), encompassing three species of Cycas
and Zamia of Nong Nooch Tropical Botanical Garden, Thailand,
revealed that all the three species of both Cycas and Zamia have
an equal chromosome number in each species, namely 2n = 2x = 22 in Cycas and
2n = 2x = 16 in Zamia. The karyotype formulae of Cycas varied
into 3 groups: 12 M + 8 SM + 2 A, 10 M + 8 SM + 2 A and 12 M + 8 SM + 2 T and
of Zamia were 12 M + 4 SM. Nevertheless, of all the three Cycas species
studied, the karyotype of female and male plants could be distinguished. The Zamia
evidence, however, was further complicated as all three species had the
same chromosome number and karyotype pattern as well. Only Z. pumila could
be differentiated between male and female plants (Sangduen et al. 2007). Hence,
concepts and/or reports on identification of sex at pre-sporangial stage
through cytology or karyomorphological study seems to be plausibly conflicting.
The recent elegant review on sex
chromosomes in land plants (Ming et al. 2011); however, is a proponent of
unequal length of sex chromosomes in cycads. The hypothesis of six stages of
plant sex chromosome evolution as proposed by them have placed the candidature
of Cycas revoluta in the fifth stage where severe degeneration of the Y
chromosome has caused the loss of function for most genes, and loss of
nonfunctioning Y chromosome sequences resulting in a shrinking of the Y
chromosome. They have hypothesized that some sex chromosome systems might not have
undergone this phase of shrinking but instead kept expanding and degenerating
until a complete loss of the Y chromosome had taken place. In either case, a
small portion of the Y chromosome has continued to meiotically pair with the X
chromosome allowing proper disjunction. Since there were no known angiosperm
sex chromosomes at this stage, they (Ming et al. 2011) are of opinion that the
gymnosperm species Cycas revoluta having heteromorphic sex chromosomes
with a reduced Y chromosome has played the intermediary stage (Fig. 1).
More refined and state-of-art technique
like FISH (Fluorescence in situ Hybridization) as attempted by Tagashira
and Kondo (2001), who have studied the chromosome phylogeny of Zamia and
Ceratozamia by rDNA analysis through FISH may throw some light in this
direction. Theoretically, at least, it
can be postulated that there must have an epigenetic control behind this
conflict regarding the existence of sex chromosome having unequal length.
Cycads appear to have retained an ancestral form of dioecy, with the sex of an
individual being determined by cytosine methylation down regulating genes
responsible for production of gametes or sex chromosomes. Cycads have thereby
retained the phenotypic plasticity to change sex via removing methylation in
the face of large environmental perturbations. It is not obvious how many other
plants have retained this plasticity in sex determination. It does not appear
that any genetic assimilation of the epigenetic mechanism of sex determination
has occurred in cycads. However, such canalization of dioecy may have been unnecessary
because cycads cannot revert to a hermaphrodite condition via allopolyploidy. Finally,
it appears that cycads are immune from Muller’s ratchet because they have
haploid stages that express most of the genes expressed in their diploid
stages. Micro array studies could be used to test this inference of immunity
from Muller’s ratchet on the large size, complexity and longevity of cycad
gametophytes (Gorelick and Osborne 2007).
Molecular Marker Techniques for Sex Determination in Pre Sporangial
Stage
If the epigenetic basis of sex expression
is conceived in case of cycads, then it is the outcome of environmental
perturbation mediated phenotypic plasticity, which is defined as the ability of
an organism to change its phenotype in response to changes in the environment.
However, while looking for an ideal marker for sex determination in pre
sporangial stage, the markers should be completely independent of environmental
conditions and should be detected at virtually any stage of plant development.
The DNA based molecular markers per se probably satisfy all the
essential criteria of ‘true and full proof’ markers. These markers are based on
naturally occurring polymorphisms in DNA sequences (i.e., base pair deletions, substitutions,
additions or patterns). There are various methods to detect and amplify these
polymorphisms so that they can be used for genetic analysis. The DNA based
molecular markers are superior to other forms of markers because they are
relatively simple to detect, abundant throughout the genome, completely
independent of environmental conditions and can be detected at virtually any
stage of plant development. There are five conditions that characterize a
suitable molecular marker (Gupta and Varshney 1999): 1) Must be polymorphic 2)
Co-dominant inheritance 3) Randomly and frequent distribution throughout the
genome 4) Easy and cheap to detect 5) Reproducible. Consequently, these markers
have been used for several different applications including: germplasms
characterization, characterization of transformants, study of genome
organization, phylogenic analysis and genetic vis-à-vis sex diagnostics. Almost
all of the molecular markers are either based on DNA-DNA hybridization or
polymerase chain reaction principle or sometimes a combination of both. Some of
these important markers, which have been used in sex determination, particularly
in case of gymnosperms are as follows.
RAPD
The discovery of RAPD or Random Amplified
Polymorphic DNA (Williams et al. 1990) based genetic marker in the beginning of
1990s probably revolutionized the application of till then sophisticated Polymerase
Chain Reaction technique. The instant popularity of this technique was probably
due to its simplicity since RAPD analysis does not involve hybridization/ autoradiography
or high technical expertise. It uses one or sometimes two short arbitrary
primers (usually 8–10 bases) to amplify anonymous stretches of DNA which are
then separated and visualized usually by simple agarose gel electrophoresis.
Many different fragments are normally amplified using each single primer; the
technique has, therefore, proved to be a fast method for detecting
polymorphisms. Furthermore, the requirement of only tiny quantities of target
DNA and relatively easy procurement of the different series of random primers
from the commercial manufacturer made the unit costs per assay quite low.
Suddenly all the field of Biology jumped to it and to the Population
Geneticists it was initially probably the perfect technique they were looking
for so long. However, the theoretical and practical limitations of this
technique soon became quite evident.
From the practical point of view it was
observed that RAPD does suffer from sensitivity to changes in PCR conditions
resulting in changes to some of the amplified fragments. Considering
theoretically, it was understood that RAPD has problems of co-migration: and
often it was found that result interpretation became problematic since it was
difficult to predict whether same RAPD derived band is same DNA fragment and
whether one band is solely one fragment. This is because the type of gel electrophoresis
used, while able to separate DNA quantitatively (i.e., according to size), cannot
separate equal-sized fragments qualitatively (i.e., according to base sequence).
Finally, the dominant nature of the RAPD markers was found to be another
bottleneck for distinguishing homozygotes from heterozygotes thus proving this
system quite unsuitable in Marker Assisted Selection particularly at the stage
of F1 hybrids.
In spite of the above shortcomings, the
most popular markers for sex determination in plants surprisingly include RAPD
(Milewicz and Sawicki 2013) and gymnosperms vis-à-vis cycads are no exception
to that. The reason probably is simple since in the absence of any specific genome
information in most of the non-model plants and particularly the gymnosperms,
the workers have to adopt this fast yet random technique to look for
polymorphism between plants of expressed sex and later try to correlate their
findings in population of unknown or yet to be expressed sex (i.e., at pre
sporangial stage).
The group of the present reviewers has
successfully used RAPD technique for detecting sex related marker in Cycas
circinalis and observed readily distinguishable sexual dimorphism of this
Gymnosperm in members of contrasting sex within population (Gangopadhyay et al.
2007). Of the RAPD fingerprints generated from a number of random primers, the
profiles of primers OPB 01 and OPB 05 were noteworthy, since they represented
one male-specific (686 bp) and another female specific (2048 bp) band respectively
(Fig. 2) (Gangopadhyay et al. 2007). Sequencing of these two cloned DNA
fragments, followed by BLASTX searching, revealed maximum homology with reverse
transcriptase of Ginkgo biloba (score 69.3 bits; NCBI accession no.
AAY87195) in case of male-specific DNA fragment (NCBI accession DQ386640, dated
22.02.2006), while the female-specific DNA fragment did not result in any
significant match.
Unique RAPD derived polymorphism was also
detected by the present group between male and female Zamia fischeri plants
(Roy et al. 2012). The RAPD profiles of both OPB03 and OPB04 primers showed one
male and one female-specific DNA fragments in each of the cases (Fig. 2). The
molecular mass of male and female specific fragments in case of OPB03 was 294
and 534 kb, while those were 1320 and 1015 kb respectively in case of OPB04. The
specific DNA fragments of both male and female samples of OPB03 were eluted out
from gel, cloned and subsequently sequenced. Sequencing of these two cloned DNA
fragments, followed by BLASTN searching, revealed homology with Araucaria
angustifolia (a conifer) clone AAng27 micro satellite sequence (maximum
identity 83%; GenBank accession no. AY865591) in case of male specific DNA
fragment (GQ141708), while the female specific DNA fragment (GQ141709) did not
result in any relevant homology with the available database.
Similar RAPD based experimental approach
was undertaken for sex identification in Encephalartos natalensis,
another cycad (Prakash and Staden 2006). Initially, the workers used 140
primers to amplify the bulk DNA of five individuals each of known male and
female sexuality. While a high degree of polymorphism was observed in the
amplification profiles of male and female plants, only primer OPD-20 generated
a specific band (~850 bp) in female DNA (Fig. 2). To validate this observation,
this primer was re-tested with 69 individuals of E. natalensis. The ~850
bp DNA band was present in all 38 female individuals tested and it was
consistently absent in all 31 male plants tested.
An even greater endeavor was undertaken
by Ling et al. (2003) while searching for a sex-associated RAPD marker in the
living fossil, Ginkgo biloba. The workers screened one thousand and two
hundred random decamers and landed up to a single 682 bp RAPD marker, which
appeared to be maleness associated after scoring a staggering figure of 8,372
amplicons.
SCAR
Though RAPD is the first choice to look
for sex related polymorphism in many plants including gymnosperms but due to
some inherent shortcomings of this technique, adoption of next level of marker
is often recommended (Milewicz and Sawicki 2013). Conversion of RAPD polymorphic
band into SCAR or Sequence Characterized Amplified Region marker though technically
demanding but greatly enhances the reliability of the marker. Longer (20–30 bp)
and more specific primers for these markers facilitate amplification of the
desirable sequence, and they would guarantee repeatability of measurement
results. Furthermore, development of sex-linked SCAR markers support sex
differentiation even of a single individual—the sex-specific band appears or
does not. For other marker systems, which generate the whole band patterns, it
would be difficult to identify a gender without the need of comparing the band
patterns for both sexes.
Endeavor in this direction has resulted
in conversion of RAPD marker into female sex specific co dominant SCAR marker,
which has provided a possibility of identifying the sex of Cycas tanqingii before
sexual maturation, which is very important for in situ or ex situ conservation
(Jing et al. 2007). There are further
issues in SCAR marker development. Ideally, a researcher should use one or two
different SCAR markers which create products of different length in males and
females in the same amplification. Sex-linked markers for Ginkgo biloba were
determined in line with the above method. SCAR markers generated products with
the length of 571 bp for males and 688 bp for females (Fig. 2) (Liao et al.
2009). Annealing temperature differed for both primer pairs. Situations such as
those encountered in the study of Ginkgo biloba happen rarely. Even if
the makers of both sexes are found in the same species, they are rarely
discovered by the same research team, and their identification is a laborious
process, leaving aside the luck or chance factor (Milewicz and Sawicki 2013).
AFLP
Described as the most full-proof of all
molecular marker techniques, AFLP or Amplified Fragment Length Polymorphism
(Vos et al. 1995) involves the following steps: Digestion of DNA with two
specific restriction enzymes, one frequent cutter and the other rare cutter;
ligation of oligonucleotide ‘adapters’ to the ends of each fragment ensuring
amplification of only the fragments, which have been cut by both frequent and
rare cutters; designing of primers from the known sequences of the adapters
plus 1–3 selective nucleotides, which extend into the fragment sequences; PCR
followed by visualization of fragments in gel after autoradiography. The high
number of bands eases analysis by providing better chance of polymorphism.
Though theoretically sound but this time, skill and cost intensive technique is
hardly suitable for evaluating large number of individuals of a population effectively.
Hence, report of use of AFLP in determining sex of gymnosperm is relatively
scarce. Only one report of identify cation of three female and one male specific
AFLP polymorphism is available in Ginkgo biloba (Wang et al. 2001).
Micro satellites (SSR)
Micro satellite loci or simple sequence
repeats (SSR) exhibit high levels of variability because of differences in the
number of repeated units. The high allelic diversity and abundance of micro
satellites in the eukaryotic genome make these co dominant molecular markers
popular for detailed genetic studies as genetic diversity and genetic structure
(Chase et al. 1996). Unfortunately, plausibly due to complexity of most of the
gymnosperm genome as well as paucity of research materials in case of
endangered ones, research is yet to be flourished in this direction unlike
angiosperm crop plants. Only one report of development of twenty-nine
species-specific and highly polymorphic micro satellite loci is available in Araucaria
angustifolia from a genomic library enriched for AG/TC repeats (Schmidt et
al. 2007). Future endeavor to link those SSRs with sex loci may throw some
light in early detection of sex in gymnosperms.
Functional Genomics in Sex Determination
A promising functional genomics approach
was undertaken by Zhang et al. (2002) to understand the molecular mechanisms
controlling development of sexual characters in Cycas edentate. Their
attempt towards cloning genes expressing differentially in male or female
reproductive organs culminated in a novel gene, named Fortune-1 (Ft-1), with
enhanced expression in male reproductive organs. The 593-base-pair Ft-1 cDNA
was predicted to encode a 77-amino-acid protein, and exists as a single copy
gene in the C. edentata genome. Ft-1 expression was enhanced in male
cones, including the cone axis, microsporophylls and microsporangia, but was
reduced in ovules and undetectable in megasporophylls. The secondary structure
prediction and homology search of Ft-1 protein showed that it has a
helix–loop–helix motif, and predictably it was without any homologue in the
database indicating paucity of basic research work in non-model, rare plant
materials.
Conclusion
Since sex is the queen of problems in
evolutionary biology (Bell 1982), understanding the molecular factor(s) behind
sex expression has immense importance both in basic and applied research.
Despite the growing body of research, the mechanism of sex determination in
many plant species remains unexplained, and this is even truer in case of
gymnosperms and cycads to be precise. Though the search for molecular
sex-linked markers paves the way for future scientific discoveries, on the
other hand, sex-linked markers alone do not explain the molecular mechanism of
sex determination in dioecious plants, but the number of markers, their
sequence structure and homology between sequences characteristic of males and
females provide a certain venture point for studies into sex determination
mechanisms. Still, it appears that there remains a large gap between the
theoretical understanding and the technique/marker of choice for early sex
determination (in pre sporangial condition) that will be helpful for breeding
program prior undertaking a proper conservation strategy. If an epigenetic control
behind the conflict regarding the existence of sex chromosome having unequal
length is finally envisaged on the backdrop of occasional sex change in cycads,
then a proper technique, which considers the epigenetic mechanism, especially
DNA methylation has to be adopted for future research towards sex determination
in cycads. In this regard adoption of technique like Methylation-Sensitive Amplification
Polymorphism (MSAP) seems to be promising, which has been first tried for sex
determination in some cycads (Kanchanaketu et al 2007). The attempt of their
work using modified AFLP technique using isochizomer enzyme (MspI and HpaII) or
MSAP was carried out to assess the pattern of cytosine methylation in both
sexes of Cycas and Zamia. Using seven pairs of primers, a total
of 364 bands, some of which showing sex-specific were produced and classified
into three groups. The first group was non-polymorphic markers, whereas the
second group was chosen from the results of differentiation ability of MspI and
HpaII to cut the methylated sequences, but sex-different markers were still not
obtained. Markers in the third group were methylation-sensitive and they also
showed some polymorphic patterns between the two sexes. Their end suggestion
that sex in cycads may be associated with DNA methylation is probably justified,
which however, warrants further studies in this direction with state-of-art techniques
to reach a final conclusion.
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