Young organs (Figure 2A and 2D) were collected during the spring from the opening buds of short shoots immediately after their emergence. At this stage, the megasporangium consists of an axis typically bearing two ovules (Figure 2A–C). The ovule is composed of a single integument surrounding a developing nucellus (Figure 2C). The male structure consists of a main axis bearing two or more microsporangia (Figure 2D–F). RNA was extracted from the following organs: megagasporangia, microsporangia and two sets of leaves collected from either male or female trees. mRNA isolated from all four tissues was used to construct four separate cDNA libraries. (Both male and female leaf sequences were pooled during subsequent bioinformatic analysis). Size fractionation was used to enrich for full-length cDNAs during library construction. From this cDNA library, 6,434 sequence reads (Expressed Sequence Tags, ESTs) were generated. All Ginkgo biloba EST reads have been deposited in GenBank. It was determined that 3,739 (58%) of the cDNA clones were over 500 bp long. 3618 of the reads were generated from the 5' end of the cDNA, and 2816 were sequenced from the 3' end. Cluster analysis on the EST sequence produced a unigene set of 3,830 contigs consisting of 2,851 singletons and 979 assemblies. Of the clusteredESTs, the longest contig was 2,172 bp. The entire unigene set or complete Ginkgo BLAST files can be downloaded at the following website 23. Each G. biloba contig is given a numeric identifier. The constituent ESTs for each contig can be obtained at this website. Additional bioinformatic analysis of the Ginkgo biloba dataset can be accessed at the open Sputnik Comparative Genomics Platform at 24. This site features sequence annotations, peptide sequence predictions, protein domain architectures and putative molecular markers (ISSRs) for the ginkgo EST derived unigenes. The sequence can be downloaded either as a fasta file, a clustered fasta file or as the derived peptide fasta file. In addition, BLAST analysis can be performed with the clustered ESTs from a given ginkgo organ against all genes in Arabidopsis thaliana or distinct plant clustered EST datasets using the ViCoGenTa program available at the New York Plant Genomics Consortium website 23.

TBLASTX (expect < 1eX10-5) was used to compare the G. biloba unigenes against all available plant ESTs from TIGR (The Institute for Genomic Research) 25, and the Plant Genome DataBase (Plant GDB) 26. ESTs from these databases were downloaded and clustered into unigenes, which were used in the comparison. Next the Ginkgo unigene set was compared against the Arabidopsis and rice genome annotated protein sequences downloaded from TIGR. All genes used in this comparison against Ginkgo were divided into one of three taxonomically relevant categories: 1. angiosperms, 2. gymnosperms, and 3. non-seed plants. The angiosperm category encompasses all annotated rice and Arabidopsis genes identified from their respective genomic sequences, as well as all higher plant ESTs. The majority of the gymnosperm ESTs came from the conifer groups pine and spruce but also include ESTs generated from the Plant Genomics Consortium containing ESTs from the two other gymnosperm clades, Cycadales and Gnetales. The non-seed plant category consisted of genes from all remaining plant ESTs including ferns, fern allies, bryophytes and algae available with the majority of the sequences originating from Physcomitrella patens and Chlamydamonas reinhardtii.

A Venn diagram shown in Figure 3A displays the number of Ginkgo contigs, which are shared between one or more of the plant EST datasets at low BLAST stringency value (expect < 1eX10-5). From the Venn diagram, it can be seen that a majority of Ginkgo unigenes (2749/3830) match genes in other plants, and 1081 have no match to other plant genes. Of those 2749 Ginkgo biloba unigenes with matches to other plant genes, a subgroup of 256 unigenes had no corresponding match to genes in the angiosperm dataset. Of these 256 Ginkgo genes that do not match angiosperms, 4 also match genes in non-seed plants.

The 252 Ginkgo biloba unigenes that only match gymnosperm genes were next partitioned into matches between the three other gymnosperm orders: Cycadales, Coniferales, and Gnetales (Figure 3B). Since there are significantly more conifer unigenes (>60,000) than cycad unigenes (5459) that were used in this comparison, one would expect that the number of matches between Ginkgo and conifers would be significantly greater than matches between Ginkgo and cycads. However the actual number of matches between Ginkgo and conifers (215) is only slightly more then for Ginkgo and cycads (163). In other words despite the fact that there is over 10 times the number of conifer genes then cycad genes used in this comparison, Ginkgo matches to conifers are only 1.3 times greater then matches between Ginkgo and cycads. Of the matches between Ginkgo and gymnosperms, 31 match only cycads, (22 match Cycas rumphii, 6 match only unigenes from the cycad species, Zamia furfuracea, (753 contigs deposited in Genbank from Brenner et al, unpublished data); and 3 Ginkgo contigs match unigenes from both Cycas rumphii and Zamia furfuracea).

As one might expect, unigenes with matches to other plants are somewhat longer then those that have no match to other plants. Of all Ginkgo unigenes with matches to other plants, 89% are greater than 300 bp, whereas 72% are greater than 300 bp then those Ginkgo unigenes with no matches to other plants.

Our comparative analysis of the Ginkgo EST dataset builds upon our results from a previous genomic study on the cycad, Cycas rumphii 18. In our current analysis, three (CB090673:GinkgoA3816, CycadCB089620:GingkoA3730, and CycadCB089926:GinkgoA1532) of the fourteen unigenes from cycads previously found only among gymnosperms (after the full-length clone was sequenced), were also homologues to Ginkgo genes found only in gymnosperms. Considering the relatively small number of unigenes from Ginkgo (3,830) and cycads (4706) available for our comparative studies, the detection of the same gene match in Ginkgo and cycads with homology to only gymnosperms strengthens the argument that these genes are gymnosperms specific.

An additional four Ginkgo unigenes that are not found in angiosperms were detected in non-seed plants. Three of these Ginkgo unigenes (GinkgoA2411, GinkgoA3214 and GinkgoA325) match non-seed plants and other gymnosperm genes whereas the forth Ginkgo gene (GinkgoA2273) only matches non-seed plants with similarity to a gene in Chlamydomonas.

Each contig from the Ginkgo dataset was automatically assigned to a functional category (FunCat) based on its top match against the MIPS FunCat list of functionally annotated gene sequences from S. cerevisiae and A. thaliana databases using BLASTP. A non-stringent expect value (E-value) of < e-10 was chosen as the threshold. Table 1 below illustrates the relative fraction that each functional category comprises within the entire unigene set compared to our previous study in Cycas rumphii 18. The four largest categories of Ginkgo ESTs according to this functional categorization are: "cellular organization" (19%), "metabolism" (11%), "unclassified proteins" (14%), and "protein synthesis" (8%). In general, these same categories are also the highest in the cycad EST library from our previous work, except for "protein synthesis", which appears increased in Ginkgo, whereas interestingly, the category of cell growth, cell division and DNA synthesis is reduced in Ginkgo compared to cycads.

Analysis of the Ginkgo biloba dataset revealed a number of ESTs with highest BLAST similarity to genes with known roles in higher plant developmental processes. A sampling of some of these genes is shown below (Table 2). These genes included the Polycomb gene CURLY LEAF 2728 as well as LATERAL ORGAN BOUNDARIES (LOB) 29, EARLY FLOWERING 5 (ELF5) 30, FLOWERING LOCUS T (FT) 31, and CONSTANS (for review see 32 as well as five ESTs that match MADS box genes, some of which appear identical to previously cloned fragments of MADS genes from Ginkgo biloba including the G. biloba ortholog of AGAMOUS 19. Other genes in our EST library have homologies to proteins that regulate development through protein turnover including SPA-1 33COP1 34 and COP9 3536. This sampling reveals that the EST dataset of Ginkgo biloba is a rich source of genes encoding proteins with known roles in development at the transcriptional as well as post-transcriptional stage.

As the sole remaining species of an ancient genus of plants which has survived nearly 170 million years from the Jurassic 5, Ginkgo biloba is a taxonomic and geographic relict that may be even older because fossils displaying a "ginkgophyte" vegetative morphology have been found as early as the Permian 4. Ginkgo has a number of plesiomorphic (unspecialized) as well as apomorphic (derived) traits that make it a valuable tool to study the evolution of seed plants. Here we used a genomic approach to investigate the genes involved in regulating development in Ginkgo by creating an EST library from both reproductive and vegetative tissues.

Similar to our previous analysis in Cycas rumphii, our Ginkgo EST study has found significant BLAST homology between Ginkgo ESTs with plant genes in gymnosperms and non-seed plants but not in angiosperms. Since ESTs, even when clustered in contiguous genes, may not represent the complete gene 37, often one will find homology to angiosperm genes when the remaining Ginkgo gene sequence is revealed. For example in the gymnosperm, Pinus taeda EST collection, contigs of increasing length have a higher likelihood then shorter contigs matching a known gene in the Arabidopsis genome 38. However, in this same study a significant subcategory of very long contigs (>1900 bp) have no homology to Arabidopsis 38. It is likely that at least some of these long contigs with no match to angiosperm genes represent full length genes that are specific to the gymnosperm and/or seed-less plant clades. Our strategy to address this question involves screening for these same genes in additional taxa of gymnosperms, in the case of this study, Ginkgo biloba. In our analysis three Ginkgo genes that were only found in gymnosperms also matched the fourteen ESTs from our previous study of gymnosperm common cycad genes.

Along these same lines, our results suggest the presence of genes common to non-seed plants and gymnosperms that are not present in angiosperms. This non-seed plant/gymnosperm grouping is not surprising considering the fact that gymnosperms have morphologically common characters that are not found in the angiosperms – particularly in their reproductive structures. For example, the megagametophyte is highly reduced both in cell number and in structural organization in angiosperms when compared to gymnosperms. Although these results cannot say for certain that these genes are specific to non-seed plants and gymnosperms, or more specifically that these genes are found in gymnosperm structures that are not found in seed plant, it nonetheless represents an important starting point to correlate the presence or absence of gymnosperm genes in angiosperms and/or lower plants.

One result from our study found that the number of Ginkgo contig matches to conifers are only 1.3 times greater then matches between Ginkgo and cycads despite the fact that there is over 10 times the number of conifer genes then cycad genes used in this comparison,. Taken together these results might indicate a closer evolutionary association between Ginkgo and cycads then between Ginkgo and conifers. This bias towards cycad/Ginkgo similarity correlates with the fact that the majority of molecular phylogenetic studies place as the cycads sister group to Ginkgo. Hopefully, this preliminary data will encourage further phylogenomic studies to fully resolve the hierarchy among extant gymnosperm orders. Until the full genome sequence becomes available for key gymnosperm taxa, EST sampling provides an important initial step for large scale identification of molecular markers to generate robust phylogenetic trees.

In Ginkgo biloba we note here a variety of genes with similarity to developmental regulators in angiosperms. We also note below that homologues to some of these developmental regulators are also present in our Cycas rumphii library as either orthologs to those found in higher plants or at least, belonging to the same gene family. An EST from Ginkgo biloba that was detected in the megagametophyte library has high similarity to the Arabidopsis CURLY LEAF (CLC) gene, which belongs to the Polycomb-group proteins (PcGs). PcGs epigenetically regulate downstream target genes 28. PcGs modify chromatin-protein complexes that repress homeotic gene transcription and influence cell proliferation. In Arabidopsis PcG genes have been shown to regulate MADS box genes 39. The CLC protein product regulates the expression of AGAMOUS 27, a gene controlling floral organ identity 40. Interestingly, an ortholog for angiosperm AGAMOUS was also detected in the Ginkgo megagametophyte library (Table 2). Ginkgo AGAMOUS, (previously named GBM5) was identified in a study where the MADS domains were examined in Ginkgo 19. In this work Ginkgo AGAMOUS was shown via RT-PCR to be expressed in not only female but also in male and vegetative tissue. In our analysis, five total MADS box homologues were also detected in the Ginkgo EST dataset. Three of the Ginkgo ESTs from our library, GinkgoA2340, GinkgoA2730 and GinkgoA2850, are perfectly identical to the MADS domain gene fragments previously cloned by 19 as degenerate PCR products. The other two unigenes from our dataset have homologies to MADS genes (GinkgoA629 and GinkgoA352), but do not specifically match any of the PCR fragments isolated in their study. These two MADS box unigenes either do not include the small region amplified in their degenerate PCR screen or could alternative be unique MADS genes not isolated in their study. Unlike the degenerate primer approach used to isolate MADS genes, our EST approach offers the additional advantage of cloning entire genes or at least substantially large gene fragments. Among the few developmental genes examined in gymnosperms, most attention has focused on the expression of MADS homologs 4142.

Other developmental genes found in the Ginkgo EST library include those with homology to regulators of flowering such as EARLY FLOWERING 5 (ELF5), which controls the levels of the gene FLC, which itself is a central regulator of flowering 30. Another Ginkgo EST includes FLOWERING LOCUS T (FT), which belongs to a small family of genes (FT/TFL1) that act to promote flowering as a downstream component from CONSTANS 43. CONSTANS is a transcription factor that has a critical role integrating circadian rhythms and light signals (for review see 32). As one would expect an EST homolog for the CONSTANS gene family was found in Ginkgo. CONSTANS belongs to a large gene family, which may have redundant roles in plants 44. Not surprisingly, we also found homologs to CONSTANS in our previous study on cycad leaf ESTs 18. In that flowering plants are believed to have evolved from gymnosperms, a survey of CONSTANS, ELF, and FT in gymnosperms, particularly in very young reproductive tissue might help define the origins of reproductive induction in non-flowering plants. Among the other genes related to developmental regulators includes a homologue to LATEROL ORGAN BOUNDARIES (LOB) domain gene family which in Arabidopsis has over 40 members 29. The molecular mechanism of LOB domain containing genes is unknown, but one gene in Arabidopsis, ASYMMETRIC LEAVES2, is required for normal leaf development, by potentially acting as a regulatory repressor of KNOX genes 45. A KNOX homolog is also present in our EST library and was found in male reproductive tissues and HOX genes were also detected in our previous analysis in C. rumphii

Another important component regulating development occurs at the level of protein degradation. A gene recognized in our EST library includes COP1. COP1, serves as an E3 ubiquitin targeting photomorphogenic factors such as HY5 for degradation 33. Another Ginkgo EST from the library has highest similarity to COP9. In our previous EST analysis in Cycas rumphii an EST was also isolated with similarity to COP9 18. COP9 is a subunit of the COP9 signalosome complex that controls multiple signaling pathways that regulate development in all eukaryotes 3536. In Arabidopsis, the cop9 and cop1 mutants are constitutively photomorphogenic in dark grown seedlings 46. Unlike angiosperms, seedlings from conifers are constitutively photomorphogenic when grown in the dark 4748. In Ginkgo, chlorophyll and chloroplast development is completely dependent on light, however this process proceeds at a markedly slower pace then in flowering plants. That is, photomorphogenic development in Ginkgo seedlings is strongly delayed after transfer from dark grown conditions to light grown conditions when compared to seed plants 2021. The dark grown phenotype of cycads is unreported. Considering this variability in photomorphic development among and between the gymnosperms and the angiosperms, the discovery of genes encoding photomorphogenic regulators in gymnosperms will help understand the evolution of photomorphogenesis in seed plants.

Taken together, our genomics analysis of Ginkgo biloba is an important additional step to analyze the role of molecular development of early seed plants. Thus the stage is set to further determine the role of these genes during the development of ancillary structures found between Ginkgo, cycads and other gymnosperms with higher plants as well as the role of those in structures that are unique to gymnosperms and/or the non-seed plants as a step to understand the evolution of the seed plant habit.

Newly emerged microsporangia from accession 76163B, megasporangia from accession 76163D, and immature leaves from both accessions were collected from newly opened buds of Ginkgo biloba growing in the New York Botanical Garden outdoor collection on April 12, 2002. Organs were snap frozen in liquid nitrogen. RNA was collected from each organ and a cDNA library was constructed from fractionated cDNA according to 49.

Ginkgo apices were collected on April 19. Bract tissues were removed from the apex leaving the leaves and reproductive tissue, which was fixed in FAA (50% ethanol, 5% glacial acetic Acid, 3.7% formaldehyde) under vacuum (20 In. Hg) at room temperature. Fresh FAA was vacuum infiltrated two additional times. Tissue was stored in 70% ethanol at 4°C.

For histology, tissue was prepared by sequential (overnight 4 C incubation at each alcohol grade) dehydration in 80, 90, 95 and finally 100% ethanol plus Eosin Y (National Medicinal Products) followed by two treatments in 100% ethanol for 2 hours at room temperature. The tissue was next placed in a 1:1 solution of ethanol and toluene, then twice in toluene alone, each time for 2 hours at room temperature. The tissue was then placed in toluene with a quarter-volume of paraffin (PARAPLAST X-TRA^® (Fisher)) chips at 60°C overnight. The tissue was then embedded in melted paraffin with six wax changes over the course of three days at 55°C. Apices were sectioned on a MICROM HM 355 microtome. 8 μm thick sections were taken using a blade angle of 9°. The tissue was stained with Astra Blue and Safranin. After mounting on slides, sections were imaged using a Nikon DXM1200F digital microscope camera.

For scanning electron microscopy, fixed materials were dissected, dehydrated in ethanol and critical point dried in a Denton critical point dryer. Dried materials were affixed to aluminium E. M. stubs and coated with between 80–240 A of palladium in a Hummer II Sputter Coater. Coated materials were then observed using a Jeol scanning electron microscope at 15 or 20 kV. Images were digitally recorded and evaluated using Adobe Photoshop 9.0.

Plasmid DNA was collected as described in the manual (Stratagene), catalogue number 200450 in the in vivo mass excision section. Sequence analysis was performed at CSHL using an ABI 3700 Capillary sequencer for separation and nucleotide detection. Reactions were performed using a 1/16 Big Dye Terminator. Sequencing was performed with either the -21 M13 forward and/or reverse primer. ESTs were assembled using Phrap 5051 and clustered into contigs using the CAP3 program 52

Peptide sequences were derived for all unigenes using the ESTScan application 53 run with the default parameters. Prior to the ESTScan predictions, a Ginkgo species-specific ESTScan model was created. ESTScan was trained with Ginkgo ORFs identified from the best match of BLASTX analyses performed on the unigene sequence against the Swissprot protein database. All BLASTX matches were filtered using the arbitrary expectation value of 1e-10.

Sequence annotation on each of the Ginkgo cluster consensus sequences and derived peptides were performed within the openSputnik application 54. Results were assessed for possible contamination by searching for homology to the E. coli and human genomes and were scored for homology to a wide range of non-coding RNAs and plant chloroplast and mitochondrial genomes. Homology searches were performed using the BLAST application 55 and results were filtered using the expectation value < 1e-10. Functional assignment was performed on both cluster consensus sequence and the peptide sequence. Assignments were made using BLASTX and BLASTP respectively against the MIPS catalogue of functionally assigned proteins (funcat) 5051, tentative functional assignments were filtered using the expectation value < 1e-10.

All Ginkgo contigs sequences were aligned against a PlantEST database using TBLASTX 55 and BLASTX against the NR (aa) database. The PlantEST database was created by downloading all plant ESTs in GenBank and assembling them using Phrap 5051. Todd Wood from Clemson University provided the PERL script that creates the PlantEST databases as described above. The NR (aa) database is a non-redundant database of protein sequences from GenBank.

All available plant ESTs were downloaded from GenBank and separated into three datasets consisting of angiosperms (monocots and dicots), gymnosperms, or non-seed plants (ferns, mosses and algae). Downloaded ESTs were assembled using Phrap 5051. All matches with an expect value < 1e 10-5 are considered significant.