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.
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.
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.
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).
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.
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.