Gibberellins regulate masculinization through the SpGAI-SpSTM module in dioecious spinach.

The sex of dioecious plants is mainly determined by genetic factors, but it can also be converted by environmental cues such as exogenous phytohormones. Gibberellic acids (GAs) are well-known inducers of flowering and sexual development, yet the pathway of gibberellin-induced sex conversion in dioecious spinach (Spinacia oleracea L.) remains elusive. Based on sex detection before and after GA3 application using T11A and SSR19 molecular markers, we confirmed and elevated the masculinization effect of GA on a single female plant through exogenous applications of GA3, showing complete conversion and functional stamens. Silencing of GIBBERELLIC ACID INSENSITIVE (SpGAI), a single DELLA family protein that is a central GA signaling repressor, results in similar masculinization. We also show that SpGAI can physically interact with the spinach KNOX transcription factor SHOOT MERISTEMLESS (SpSTM), which is a homolog of the flower meristem identity regulator STM in Arabidopsis. The silencing of SpSTM also masculinized female flowers in spinach. Furthermore, SpSTM could directly bind the intron of SpPI to repress SpPI expression in developing female flowers. Overall, our results suggest that GA induces a female masculinization process through the SpGAI-SpSTM-SpPI regulatory module in spinach. These insights may help to clarify the molecular mechanism underlying the sex conversion system in dioecious plants while also elucidating the physiological basis for the generation of unisexual flowers so as to establish dioecy in plants.

Genome-wide analysis of transposable elements and satellite DNA in Humulus scandens, a dioecious plant with XX/XY1Y2 chromosomes.

Transposable elements (TEs) and satellite DNAs, two major categories of repetitive sequences, are expected to accumulate in non-recombining genome regions, including sex-linked regions, and contribute to sex chromosome evolution. The dioecious plant, Humulus scandens, can be used for studying the evolution of the XX/XY1Y2 sex chromosomes. In this study, we thoroughly examined the repetitive components of male and female H. scandens using next-generation sequencing data followed by bioinformatics analysis and florescence in situ hybridization (FISH). The H. scandens genome has a high overall repetitive sequence composition, 68.30% in the female and 66.78% in the male genome, with abundant long terminal repeat (LTR) retrotransposons (RTs), including more Ty3/Gypsy than Ty1/Copia elements, particularly two Ty3/Gypsy lineages, Tekay and Retand. Most LTR-RT lineages were found dispersed across the chromosomes, though CRM and Athila elements were predominately found within the centromeres and the pericentromeric regions. The Athila elements also showed clearly higher FISH signal intensities in the Y1 and Y2 chromosomes than in the X or autosomes. Three novel satellite DNAs were specifically distributed in the centromeric and/or telomeric regions, with markedly different distributions on the X, Y1, and Y2 chromosomes. Combined with FISH using satellite DNAs to stain chromosomes during meiotic diakinesis, we determined the synapsis pattern and distinguish pseudoautosomal regions (PARs). The results indicate that the XY1Y2 sex chromosomes of H. scandens might have originated from a centric fission event. This study improves our understanding of the repetitive sequence organization of H. scandens genome and provides a basis for further analysis of their chromosome evolution process.

Lineage-specific amplification and epigenetic regulation of LTR-retrotransposons contribute to the structure, evolution, and function of Fabaceae species.

BACKGROUND: Long terminal repeat (LTR)-retrotransposons (LTR-RTs) are ubiquitous and make up the majority of nearly all sequenced plant genomes, whereas their pivotal roles in genome evolution, gene expression regulation as well as their epigenetic regulation are still not well understood, especially in a large number of closely related species. RESULTS: Here, we analyzed the abundance and dynamic evolution of LTR-RTs in 54 species from an economically and agronomically important family, Fabaceae, and also selected two representative species for further analysis in expression of associated genes, transcriptional activity and DNA methylation patterns of LTR-RTs. Annotation results revealed highly varied proportions of LTR-RTs in these genomes (5.1%~68.4%) and their correlation with genome size was highly positive, and they were significantly contributed to the variance in genome size through species-specific unique amplifications. Almost all of the intact LTR-RTs were inserted into the genomes 4 Mya (million years ago), and more than 50% of them were inserted in the last 0.5 million years, suggesting that recent amplifications of LTR-RTs were an important force driving genome evolution. In addition, expression levels of genes with intronic, promoter, and downstream LTR-RT insertions of Glycine max and Vigna radiata, two agronomically important crops in Fabaceae, showed that the LTR-RTs located in promoter or downstream regions suppressed associated gene expression. However, the LTR-RTs within introns promoted gene expression or had no contribution to gene expression. Additionally, shorter and younger LTR-RTs maintained higher mobility and transpositional potential. Compared with the transcriptionally silent LTR-RTs, the active elements showed significantly lower DNA methylation levels in all three contexts. The distributions of transcriptionally active and silent LTR-RT methylation varied across different lineages due to the position of LTR-RTs located or potentially epigenetic regulation. CONCLUSION: Lineage-specific amplification patterns were observed and higher methylation level may repress the activity of LTR-RTs, further influence evolution in Fabaceae species. This study offers valuable clues into the evolution, function, transcriptional activity and epigenetic regulation of LTR-RTs in Fabaceae genomes.

Impact of LTR-Retrotransposons on Genome Structure, Evolution, and Function in Curcurbitaceae Species.

Long terminal repeat (LTR)-retrotransposons (LTR-RTs) comprise a major portion of many plant genomes and may exert a profound impact on genome structure, function, and evolution. Although many studies have focused on these elements in an individual species, their dynamics on a family level remains elusive. Here, we investigated the abundance, evolutionary dynamics, and impact on associated genes of LTR-RTs in 16 species in an economically important plant family, Cucurbitaceae. Results showed that full-length LTR-RT numbers and LTR-RT content varied greatly among different species, and they were highly correlated with genome size. Most of the full-length LTR-RTs were amplified after the speciation event, reflecting the ongoing rapid evolution of these genomes. LTR-RTs highly contributed to genome size variation via species-specific distinct proliferations. The Angela and Tekay lineages with a greater evolutionary age were amplified in Trichosanthes anguina, whereas a recent activity burst of Reina and another ancient round of Tekay activity burst were examined in Sechium edule. In addition, Tekay and Retand lineages belonging to the Gypsy superfamily underwent a recent burst in Gynostemma pentaphyllum. Detailed investigation of genes with intronic and promoter LTR-RT insertion showed diverse functions, but the term of metabolism was enriched in most species. Further gene expression analysis in G.pentaphyllum revealed that the LTR-RTs within introns suppress the corresponding gene expression, whereas the LTR-RTs within promoters exert a complex influence on the downstream gene expression, with the main function of promoting gene expression. This study provides novel insights into the organization, evolution, and function of LTR-RTs in Cucurbitaceae genomes.

DNA methylation is involved in sexual differentiation and sex chromosome evolution in the dioecious plant garden asparagus.

DNA methylation is a crucial regulatory mechanism in many biological processes. However, limited studies have dissected the contribution of DNA methylation to sexual differentiation in dioecious plants. In this study, we investigated the variances in methylation and transcriptional patterns of male and female flowers of garden asparagus. Compared with male flowers, female flowers at the same stages showed higher levels of DNA methylation. Both male and female flowers gained DNA methylation globally from the premeiotic to meiotic stages. Detailed analysis revealed that the increased DNA methylation was largely due to increased CHH methylation. Correlation analysis of differentially expressed genes and differentially methylated regions suggested that DNA methylation might not have contributed to the expression variation of the sex-determining genes SOFF and TDF1 but probably played important roles in sexual differentiation and flower development of garden asparagus. The upregulated genes AoMS1, AoLAP3, AoAMS, and AoLAP5 with varied methylated CHH regions might have been involved in sexual differentiation and flower development of garden asparagus. Plant hormone signaling genes and transcription factor genes also participated in sexual differentiation and flower development with potential epigenetic regulation. In addition, the CG and CHG methylation levels in the Y chromosome were notably higher than those in the X chromosome, implying that DNA methylation might have been involved in Y chromosome evolution. These data provide insights into the epigenetic modification of sexual differentiation and flower development and improve our understanding of sex chromosome evolution in garden asparagus.

Chromosome-level genome assembly, annotation and evolutionary analysis of the ornamental plant Asparagus setaceus.

Asparagus setaceus is a popular ornamental plant cultivated in tropical and subtropical regions globally. Here, we constructed a chromosome-scale reference genome of A. setaceus to facilitate the investigation of its genome characteristics and evolution. Using a combination of Nanopore long reads, Illumina short reads, 10× Genomics linked reads, and Hi-C data, we generated a high-quality genome assembly of A. setaceus covering 710.15 Mb, accounting for 98.63% of the estimated genome size. A total of 96.85% of the sequences were anchored to ten superscaffolds corresponding to the ten chromosomes. The genome of A. setaceus was predicted to contain 28,410 genes, 25,649 (90.28%) of which were functionally annotated. A total of 65.59% of the genome was occupied by repetitive sequences, among which long terminal repeats were predominant (42.51% of the whole genome). Evolutionary analysis revealed an estimated divergence time of A. setaceus from its close relative A. officinalis of ~9.66 million years ago, and A. setaceus underwent two rounds of whole-genome duplication. In addition, 762 specific gene families, 96 positively selected genes, and 76 resistance (R) genes were detected and functionally predicted in A. setaceus. These findings provide new knowledge about the characteristics and evolution of the A. setaceus genome, and will facilitate comparative genetic and genomic research on the genus Asparagus.

Nuclear Integrants of Organellar DNA Contribute to Genome Structure and Evolution in Plants.

The transfer of genetic material from the mitochondria and plastid to the nucleus gives rise to nuclear integrants of mitochondrial DNA (NUMTs) and nuclear integrants of plastid DNA (NUPTs). This frequently occurring DNA transfer is ongoing and has important evolutionary implications. In this review, based on previous studies and the analysis of NUMT/NUPT insertions of more than 200 sequenced plant genomes, we analyzed and summarized the general features of NUMTs/NUPTs and highlighted the genetic consequence of organellar DNA insertions. The statistics of organellar DNA integrants among various plant genomes revealed that organellar DNA-derived sequence content is positively correlated with the nuclear genome size. After integration, the nuclear organellar DNA could undergo different fates, including elimination, mutation, rearrangement, fragmentation, and proliferation. The integrated organellar DNAs play important roles in increasing genetic diversity, promoting gene and genome evolution, and are involved in sex chromosome evolution in dioecious plants. The integrating mechanisms, involving non-homologous end joining at double-strand breaks were also discussed.

Cytogenetic and genomic organization analyses of chloroplast DNA invasions in the nuclear genome of Asparagus officinalis L. provides signatures of evolutionary complexity and informativity in sex chromosome evolution.

BACKGROUND: The transfer of chloroplast DNA into nuclear genome is a common process in plants. These transfers form nuclear integrants of plastid DNAs (NUPTs), which are thought to be driving forces in genome evolution, including sex chromosome evolution. In this study, NUPTs in the genome of a dioecious plant Asparagus officinalis L. were systematically analyzed, in order to investigate the characteristics of NUPTs in the nuclear genome and the relationship between NUPTs and sex chromosome evolution in this species. RESULTS: A total of 3155 NUPT insertions were detected, and they represented approximated 0.06% of the nuclear genome. About 45% of the NUPTs were organized in clusters. These clusters were derived from various evolutionary events. The Y chromosome contained the highest number and largest proportion of NUPTs, suggesting more accumulation of NUPTs on sex chromosomes. NUPTs were distributed widely in all of the chromosomes, and some regions preferred these insertions. The highest density of NUPTs was found in a 47 kb region in the Y chromosome; more than 75% of this region was occupied by NUPTs. Further cytogenetic and sequence alignment analysis revealed that this region was likely the centromeric region of the sex chromosomes. On the other hand, the male-specific region of the Y chromosome (MSY) and the adjacent regions did not have NUPT insertions. CONCLUSIONS: These results indicated that NUPTs were involved in shaping the genome of A. officinalis through complicated process. NUPTs may play important roles in the centromere shaping of the sex chromosomes of A. officinalis, but were not implicated in MSY formation.

The landscape of transposable elements and satellite DNAs in the genome of a dioecious plant spinach (Spinacia oleracea L.).

BACKGROUND: Repetitive sequences, including transposable elements (TEs) and satellite DNAs, occupy a considerable portion of plant genomes. Analysis of the repeat fraction benefits the understanding of genome structure and evolution. Spinach (Spinacia oleracea L.), an important vegetable crop, is also a model dioecious plant species for studying sex determination and sex chromosome evolution. However, the repetitive sequences of the spinach genome have not been fully investigated. RESULTS: We extensively analyzed the repetitive components of draft spinach genome, especially TEs and satellites, by different strategies. A total of 16,002 full-length TEs were identified. Among the most abundant long terminal repeat (LTR) retrotransposons (REs), Copia elements were overrepresented compared with Gypsy ones. Angela was the most dominating Copia lineage; Ogre/Tat was the most abundant Gypsy lineage. The mean insertion age of LTR-REs was 1.42 million years; approximately 83.7% of these elements were retrotransposed during the last two million years. RepeatMasker totally masked about 64.05% of the spinach genome, with LTR-REs, non-LTR-REs, and DNA transposons occupying 49.2, 2.4, and 5.6%, respectively. Fluorescence in situ hybridization (FISH) analysis showed that most LTR-REs dispersed all over the chromosomes, by contrast, elements of CRM lineage were distributed at the centromeric region of all chromosomes. In addition, Ogre/Tat lineage mainly accumulated on sex chromosomes, and satellites Spsat2 and Spsat3 were exclusively located at the telomeric region of the short arm of sex chromosomes. CONCLUSIONS: We reliably annotated the TE fraction of the draft genome of spinach. FISH analysis indicates that Ogre/Tat lineage and the sex chromosome-specific satellites DNAs might participate in sex chromosome formation and evolution. Based on FISH signals of microsatellites, together with 45S rDNA, a fine karyotype of spinach was established. This study improves our knowledge of repetitive sequence organization in spinach genome and aids in accurate spinach karyotype construction.

Characterization of the complete chloroplast genome of Asparagus setaceus.

Asparagus setaceus is an important ornamental plant and is important to study the sex chromosome evolution in genus Asparagus. Here the complete chloroplast (cp) genome sequence of A. setaceus was determined. The cpDNA is 156,978 bp in length, contains a large single copy region of 85,311 bp and a small single copy region of 18,641 bp, which was separated by a pair of inverted repeat regions of 26,513 bp each. The cp genome contains 132 genes, including 88 protein-coding genes, 6 ribosomal RNA genes, and 38 transfer RNA genes. Phylogenetic analysis indicates that A. setaceus is closely related to Asparagus officinalis and Asparagus schoberioides.

Genome-wide characterization of microsatellites and genetic diversity assessment of spinach in the Chinese germplasm collection.

Spinach is a nutritional leafy green vegetable, and it also serves as a model species for studying sex chromosome evolution. Genetic marker development and genome structure analysis are important in breeding practice and theoretical evolution studies of spinach. In this study, the frequency and distribution of different microsatellites in the recently released draft spinach genome were characterized. A total of 261,002 perfect microsatellites were identified (estimated frequency: ~262.1 loci/Mbp). The most abundant microsatellites were tetranucleotide and trinucleotide, accounting for 33.2% and 27.7% of the total number of microsatellites, respectively. A total of 105 primer pairs were designed and screened, and 34 were polymorphic among the detected spinach cultivars. Combined with seven primer sets developed previously, 41 primer pairs were used to investigate genetic diversity among 43 spinach cultivars in China. The average polymorphism information content value of the 41 markers was 0.43, representing an intermediate level. The spinach cultivars had a low genetic diversity, and no detectable common factors were shared by each group in the UPGMA dendrogram. This study's findings facilitate further investigations on the organization of the microsatellites in spinach genome and provide clues for future breeding applications of spinach in China.

The application of MutMap in forward genetic studies based on whole-genome sequencing.

Classical forward genetic analysis relies on construction of complicated progeny populations and development of many molecular markers for linkage analysis in genetic mapping, which is both time- and cost-consuming. The recently developed MutMap is a new forward genetic approach based on high-throughput next-generation sequencing technologies. It is more efficient and affordable than traditional methods. Moreover, new extended methods based on MutMap have been developed: MutMap+, which is based on self-crossing; MutMap-Gap, which is used to recognize the causative variations occurring in genome gap regions; QTL-seq, a method similar to MutMap for mapping quantitative trait loci. These methods are free from constructing complicated mapping population, genetic hybridization and linkage information. They have greatly accelerated the identification of genetic elements associated with interested phenotypic variation. Here, we review the basic principles of MutMap, and discuss their future applications in next generation sequencing-based forward genetic mapping and crop improvement.

Chromosome Evolution in Connection with Repetitive Sequences and Epigenetics in Plants.

Chromosome evolution is a fundamental aspect of evolutionary biology. The evolution of chromosome size, structure and shape, number, and the change in DNA composition suggest the high plasticity of nuclear genomes at the chromosomal level. Repetitive DNA sequences, which represent a conspicuous fraction of every eukaryotic genome, particularly in plants, are found to be tightly linked with plant chromosome evolution. Different classes of repetitive sequences have distinct distribution patterns on the chromosomes. Mounting evidence shows that repetitive sequences may play multiple generative roles in shaping the chromosome karyotypes in plants. Furthermore, recent development in our understanding of the repetitive sequences and plant chromosome evolution has elucidated the involvement of a spectrum of epigenetic modification. In this review, we focused on the recent evidence relating to the distribution pattern of repetitive sequences in plant chromosomes and highlighted their potential relevance to chromosome evolution in plants. We also discussed the possible connections between evolution and epigenetic alterations in chromosome structure and repatterning, such as heterochromatin formation, centromere function, and epigenetic-associated transposable element inactivation.

Comparative transcriptome analysis reveals differentially expressed genes associated with sex expression in garden asparagus (Asparagus officinalis).

BACKGROUND: Garden asparagus (Asparagus officinalis) is a highly valuable vegetable crop of commercial and nutritional interest. It is also commonly used to investigate the mechanisms of sex determination and differentiation in plants. However, the sex expression mechanisms in asparagus remain poorly understood. RESULTS: De novo transcriptome sequencing via Illumina paired-end sequencing revealed more than 26 billion bases of high-quality sequence data from male and female asparagus flower buds. A total of 72,626 unigenes with an average length of 979 bp were assembled. In comparative transcriptome analysis, 4876 differentially expressed genes (DEGs) were identified in the possible sex-determining stage of female and male/supermale flower buds. Of these DEGs, 433, including 285 male/supermale-biased and 149 female-biased genes, were annotated as flower related. Of the male/supermale-biased flower-related genes, 102 were probably involved in anther development. In addition, 43 DEGs implicated in hormone response and biosynthesis putatively associated with sex expression and reproduction were discovered. Moreover, 128 transcription factor (TF)-related genes belonging to various families were found to be differentially expressed, and this finding implied the essential roles of TF in sex determination or differentiation in asparagus. Correlation analysis indicated that miRNA-DEG pairs were also implicated in asparagus sexual development. CONCLUSIONS: Our study identified a large number of DEGs involved in the sex expression and reproduction of asparagus, including known genes participating in plant reproduction, plant hormone signaling, TF encoding, and genes with unclear functions. We also found that miRNAs might be involved in the sex differentiation process. Our study could provide a valuable basis for further investigations on the regulatory networks of sex determination and differentiation in asparagus and facilitate further genetic and genomic studies on this dioecious species.

Identification of male-specific AFLP and SCAR markers in the dioecious plant Humulus scandens.

In this study, 17 male-specific amplified fragment length polymorphism (AFLP) markers were identified between male and female Humulus scandens plants. BLAST analysis revealed that 7 of the 17 sex-linked sequences were highly similar to retrotransposons. Two stable male-specific sequence-characterized amplified regions (SCAR) markers were developed. These AFLP and SCAR markers are novel molecular probes that can be used efficiently to identify the genetic gender of H. scandens and may provide a basis for further investigations on the evolution of sex chromosomes.

Genes encoding Δ(8)-sphingolipid desaturase from various plants: identification, biochemical functions, and evolution.

∆(8)-sphingolipid desaturase catalyzes the C8 desaturation of a long chain base, which is the characteristic structure of various complex sphingolipids. The genes of 20 ∆(8)-sphingolipid desaturases from 12 plants were identified and functionally detected by using Saccharomyces cerevisiae system to elucidate the relationship between the biochemical function and evolution of this enzyme. Results showed that the 20 genes all can encode a functional ∆(8)-sphingolipid desaturase, which catalyzes different ratios of two products, namely, 8(Z) and 8(E)-C18-phytosphingenine. The coded enzymes could be divided into two groups on the basis of biochemical functions: ∆(8)-sphingolipid desaturase with a preference for an E-isomer product and ∆(8)-sphingolipid desaturase with a preference for a Z-isomer product. The conversion rate of the latter was generally lower than that of the former. Phylogenetic analysis revealed that the 20 desaturases could also be clustered into two groups, and this grouping is consistent with that of the biochemical functions. Thus, the biochemical function of ∆(8)-sphingolipid desaturase is correlated with its evolution. The two groups of ∆(8)-sphingolipid desaturases could arise from distinct ancestors in higher plants. However, they might have initially evolved from ∆(8)-sphingolipid desaturases in lower organisms, such as yeasts, which can produce E-isomer products only. Furthermore, almost all of the transgenic yeasts harboring ∆(8)-sphingolipid desaturase genes exhibit an improvement in aluminum tolerance. Our study provided new insights into the biochemical function and evolution of ∆(8)-sphingolipid desaturases in plants.

DPTEdb, an integrative database of transposable elements in dioecious plants.

Dioecious plants usually harbor 'young' sex chromosomes, providing an opportunity to study the early stages of sex chromosome evolution. Transposable elements (TEs) are mobile DNA elements frequently found in plants and are suggested to play important roles in plant sex chromosome evolution. The genomes of several dioecious plants have been sequenced, offering an opportunity to annotate and mine the TE data. However, comprehensive and unified annotation of TEs in these dioecious plants is still lacking. In this study, we constructed a dioecious plant transposable element database (DPTEdb). DPTEdb is a specific, comprehensive and unified relational database and web interface. We used a combination of de novo, structure-based and homology-based approaches to identify TEs from the genome assemblies of previously published data, as well as our own. The database currently integrates eight dioecious plant species and a total of 31 340 TEs along with classification information. DPTEdb provides user-friendly web interfaces to browse, search and download the TE sequences in the database. Users can also use tools, including BLAST, GetORF, HMMER, Cut sequence and JBrowse, to analyze TE data. Given the role of TEs in plant sex chromosome evolution, the database will contribute to the investigation of TEs in structural, functional and evolutionary dynamics of the genome of dioecious plants. In addition, the database will supplement the research of sex diversification and sex chromosome evolution of dioecious plants.Database URL: http://genedenovoweb.ticp.net:81/DPTEdb/index.php.

Repetitive sequences and epigenetic modification: inseparable partners play important roles in the evolution of plant sex chromosomes.

MAIN CONCLUSION: The present review discusses the roles of repetitive sequences played in plant sex chromosome evolution, and highlights epigenetic modification as potential mechanism of repetitive sequences involved in sex chromosome evolution. Sex determination in plants is mostly based on sex chromosomes. Classic theory proposes that sex chromosomes evolve from a specific pair of autosomes with emergence of a sex-determining gene(s). Subsequently, the newly formed sex chromosomes stop recombination in a small region around the sex-determining locus, and over time, the non-recombining region expands to almost all parts of the sex chromosomes. Accumulation of repetitive sequences, mostly transposable elements and tandem repeats, is a conspicuous feature of the non-recombining region of the Y chromosome, even in primitive one. Repetitive sequences may play multiple roles in sex chromosome evolution, such as triggering heterochromatization and causing recombination suppression, leading to structural and morphological differentiation of sex chromosomes, and promoting Y chromosome degeneration and X chromosome dosage compensation. In this article, we review the current status of this field, and based on preliminary evidence, we posit that repetitive sequences are involved in sex chromosome evolution probably via epigenetic modification, such as DNA and histone methylation, with small interfering RNAs as the mediator.

Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences.

The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related X,X(m) and Y. Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However,there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.

Isolation of differentially expressed sex genes in garden asparagus using suppression subtractive hybridization.

Garden asparagus (Asparagus officinalis L.) is a dioecious species whose male and female flowers are found in separate unisexual individuals. A region called the M-locus, located on a pair of homomorphic sex chromosomes, controls sexual dimorphism in asparagus. To date, no sex determining gene has been isolated from asparagus. To identify more genes involved in flower development in asparagus, subtractive hybridization library of male flowers in asparagus was constructed by suppression subtraction hybridization. A total of 107 expressed sequence tags (ESTs) were identified. BLASTX analysis showed that the library contained several genes that could be related to flower development. The expression patterns of seven selected genes believed to be involved in the development of asparagus male flower were further analyzed by semi-quantitative or real-time reverse-transcription polymerase chain reaction (RT-PCR). Results showed that AOEST4-5, AOEST12-40, and AOEST13-38 were strongly expressed in the male flower stage, whereas no transcript level of AOEST13-38 was detected in the female flower stage. The expression levels of AOEST13-87, AOEST13-92, AOEST13-40, and AOEST18-87 in the male flower stage were also higher than those in the female flower stage, although these transcripts were also expressed in other tissues. The identified genes can provide a strong starting point for further studies on the underlying molecular differences between the male and female flowers of asparagus.