Long-Dou Lu

Karyotype of asparagus by physical mapping of 45S and 5S rDNA by FISH

Garden asparagus (Asparagus officinalis L.) is an economically important plant with 2n = 2x = 20 chromosomes and a haploid genome size of 1323 Mb (Bennett and Leitch 2003). The karyotype of asparagus consists of five long (L), one medium (M) and four small (S) chromosomes (Löptien 1976). Plants of this dioecious species differ in producing either male or female flowers. The sexual dimorphism in asparagus is controlled by a region located on a pair of homomorphic sex chromosomes (chromosome L5) (Löptien 1979) termed the M locus (Flory 1932; Uno et al. 2002). Maleness is dominant with males normally heterogametic (Mm) and females homogametic (mm). Supermales (MM) can be produced from anther culture or from rarely occurring andromonoecious plants. The identification of chromosome L5 is important for studying sex chromosome evolution in asparagus. However, it was observed that the sex chromosome pair is homomorphic: X and Y chromosomes do not differ in morphology. Telgmann-Rauber et al. (2007) tried to clone the region determining sex in asparagus from its position in the genome. Molecular cytogenetic and sequence analyses of bacterial artificial chromosomes (BACs) flanking M locus indicate that the BAC contain highly repetitive sequences that localize to centromeric and pericentromeric locations on all asparagus chromosomes. However, the L5 chromosome could not be distinguished by BAC-FISH (Telgmann-Rauber et al. 2007). Recently, FISH has become a powerful and useful tool for the direct detection of specific DNA in genome. In this technique, ribosomal DNA genes (45S and 5S rDNA) are commonly used as markers for the physical mapping of plant chromosomes to analyse genomic organization (Nakayama et al. 2001; Lan et al. 2006; Grabowska-Joachimiak et al.

Analysis of transposable elements in the genome of Asparagus officinalis from high coverage sequence data.

Asparagus officinalis is an economically and nutritionally important vegetable crop that is widely cultivated and is used as a model dioecious species to study plant sex determination and sex chromosome evolution. To improve our understanding of its genome composition, especially with respect to transposable elements (TEs), which make up the majority of the genome, we performed Illumina HiSeq2000 sequencing of both male and female asparagus genomes followed by bioinformatics analysis. We generated 17 Gb of sequence (12×coverage) and assembled them into 163,406 scaffolds with a total cumulated length of 400 Mbp, which represent about 30% of asparagus genome. Overall, TEs masked about 53% of the A. officinalis assembly. Majority of the identified TEs belonged to LTR retrotransposons, which constitute about 28% of genomic DNA, with Ty1/copia elements being more diverse and accumulated to higher copy numbers than Ty3/gypsy. Compared with LTR retrotransposons, non-LTR retrotransposons and DNA transposons were relatively rare. In addition, comparison of the abundance of the TE groups between male and female genomes showed that the overall TE composition was highly similar, with only slight differences in the abundance of several TE groups, which is consistent with the relatively recent origin of asparagus sex chromosomes. This study greatly improves our knowledge of the repetitive sequence construction of asparagus, which facilitates the identification of TEs responsible for the early evolution of plant sex chromosomes and is helpful for further studies on this dioecious plant.

Microdissection and painting of the Y chromosome in spinach ( Spinacia oleracea )

Spinach has long been used as a model for genetic and physiological studies of sex determination and expression. Although trisomic analysis from a cross between diploid and triploid plants identified the XY chromosome as the largest chromosome, no direct evidence has been provided to support this at the molecular level. In this study, the largest chromosomes of spinach from mitotic metaphase spreads were microdissected using glass needles. Degenerate oligonucleotide primed polymerase chain reaction was used to amplify the dissected chromosomes. The amplified products from the Y chromosome were identified using the male-specific marker T11A. For the first time, the largest spinach chromosome was confirmed to be a sex chromosome at the molecular level. PCR products from the isolated chromosomes were used in an in situ probe mixture for painting the Y chromosome. The fluorescence signals were mainly distributed on all chromosomes and four pair of weaker punctate fluorescence signal sites were observed on the terminal region of two pair of autosomes. These findings provide a foundation for the study of sex chromosome evolution in spinach.

Assessment of genetic diversity of Lycoris longituba (Amaryllidaceae) detected by RAPDs.

Lycoris longituba Y. Hsu et Q. J. Fan is a perennial, bulbiferous, herbaceous plant of the family Amaryllidaceae. It is endemic in China, with a very limited distribution, being found only at Langyashan in Anhui province, and Baohuashan and Xuyi city in Jiangsu province, all located about 50 km from one another (see figure 1 and table 1 in electronic supplementary material at http://www.ias.ac.in/jgenet/). A unique biological characteristic of this plant is that its leaves come out in spring and die in early summer, followed by flowering. Thus, its vegetative growth and reproduction are discrete, which is rare in angiosperms. With a vigorous and long scape, its flowers are diverse in both colour and floral form, and fragrant in smell, and have high ornamental value. Also called Chinese tulip, this species is ideal for ikebana, and its bulb has medicinal properties. We have collected more than 70 variants of this species in our laboratory with different floral colours and forms. Population-genetic structure is considered as spatiotemporal distribution of genetic variation (Ge 1997), and reflects evolutionary processes in populations (Hamrick and Loveless 1989). To take right measures to protect wild resources of L. longituba, it is important to study the range of genetic variation, genetic structure, diversification trend and other factors affecting genetic structure of the populations of this important and sparsely distributed species. Presently, many biochemical and DNA-based markers are used in studies of population structure. The random amplified polymorphic DNA (RAPD) method (Williams et al. 1990) is frequently applied to reveal population-genetic variation, divergence and biogeography (Schaal and Leverich 2001). A large number of studies have successfully applied this method to study genetic variation in plants owing to

Effect of 5-azaC on the growth, flowering time and sexual phenotype of spinach

Spinach (Spinacia oleracea L.) is a diploid dioecious plant with a pair of homomorphic sex chromosomes X and Y. Plant DNA methylation, a known process for genome epigenetic modification, regulates gene expression in plants. To explore the effects of DNA methylation on spinach growth and sexual development, spinach seeds were treated with the demethylating reagent 5-azaC. The resulting phenotypes were then investigated, including germination percentage, root length, plant height, flowering time, and sexual phenotype. Results showed that 5-azaC at a low concentration (30 µM) only slightly influenced spinach development but promoted seed germination. The germination percentage, root length, and plant height negatively correlated with 5-azaC at 100–1000 µM. The flowering time significantly reduced at all four treatments with 5-azaC. In addition, 5-azaC influenced the sexual phenotype of spinach and remarkably increased the percentage of monoecious individuals. These results may suggest that vegetative and reproductive growth are both epigenetically regulated by DNA methylation.

Detection of genome DNA methylation change in spinach induced by 5-azaC

DNA methylation has been implicated in the regulation of gene expression, genome imprinting, and chromatin remodeling in eukaryotes. In this study, we analyzed possible alterations in levels and patterns of cytosine methylation in male and female spinach plants after treatment with demethylation agent 5-azacytidine (5-azaC) using two methods: (1) direct determination of 5-methylcytidine (5 mC) amounts in genomic DNA by high-performance liquid chromatography (HPLC) separation and quantification of nucleosides and (2) methylation-sensitive inter-simple sequence repeat (MS-ISSR) technique. HPLC analysis revealed that the DNA methylation events in male and female spinach leaves markedly decreased upon 30 μM 5-azaC treatment, and the methylation level gradually decreased with the increase in 5-azaC concentration. To study the altered DNA methylation patterns in spinach after 5-azaC treatment, untreated and 500 μM 5-azaC-treated samples were analyzed by MS-ISSR assay. A total of 385 informative profiles were resolved using 35 ISSR primer sets. MS-ISSR analysis showed various altered methylation patterns between untreated and 5-azaC-treated spinach plants. These alterations were mainly demethylation events, which were largely consistent with the HPLC results. Both HPLC and MS-ISSR analyses showed that the changes in DNA methylation levels and patterns were similar in male and female spinach leaves, which implies that sex was not the main factor influencing DNA methylation levels and patterns in the vegetative organs of spinach. This study could provide a molecular basis of the altered DNA methylation induced by 5-azaC, and lay a foundation for further investigation of the relationship between methylation and sex determination and development in this dioecious plant spinach.

Identification of sex chromosome of spinach by physical mapping of 45s rDNAs by FISH

Male and female spinach (Spinacia oleracea L, 2n = 12) were karyotyped by chromosome measurements and subjected to fluorescence in situ hybridization with 45s rDNA The chromosome complement comprised two long nearly subtelocentrics, two short subtelocentrics, two acrocentrics, and six submetacentrics. The X and Y chromosomes were not distinguished by morphological analysis; thus, six obvious 45s rDNA sites were labeled with Texas red and clearly observed as red signals on three pairs of chromosomes. Among the three pairs of signal sites, a pair of the largest one was located on the short arms of chromosome 5, which is a satellite chromosome. Another prominent signal site was found on the short arms of chromosomes 2 and 6. However, from single images with 45s rDNA (red) of male or female spinach, weak 45s rDNA sites labeled with Texas red were observed on chromosomes 1 and 3. The weak 45s rDNA signals site were located on the large arm of chromosome 3 of both male and female spinach. Interestingly, different fluorescence signals were found on chromosome 1. In male spinach, weak signals were located on the short arm of one chromosome of the largest chromosome pairs, but signals on another chromosome were not observed. In female spinach, weak signals were located on the entire short arm of largest chromosome pairs. For the first time, the largest chromosome pairs were directly identified as the sex chromosomes. The identification of these sex chromosomes is very helpful for cloning and manually locating the sex determination region of spinach.

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.

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, Xm 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.

Three homologous genes encoding functional ∆8-sphingolipid desaturase in Populus tomentosa

Abstract∆8-sphingolipid desaturase is characterized by its ability to catalyze desaturation at the C8 position of the long-chain base of sphingolipids in plants. No previous studies have been conducted on genes encoding Δ8-sphingolipid desaturases in the woody plant Populus tomentosa. In this study, three genes that encode Δ8-sphingolipid desaturase were isolated from P. tomentosa. Among these genes, PtD8A and PtD8B showed high sequence similarity; whereas PtD8C exhibited large sequence divergence. RT-PCR results showed that PtD8A and PtD8B were expressed in all tissues detected, whereas PtD8C was not expressed in roots. Heterologous expression in yeast revealed that PtD8A/B/C were functional Δ8-sphingolipid desaturases, and can catalyze the C18-phytosphingenine desaturation to produce 8(Z)- and 8(E)-C18-phytosphingenine. However, the conversion rate and ratios of the two products differed. Compared with control cells, transgenic yeasts expressing PtD8A/B/C exhibited enhanced aluminum tolerance. Our findings further elucidated the biochemical functions and evolutionary history of Δ8-sphingolipid desaturases in plants. Candidate genes for breeding new poplar germplasm resources with enhanced tolerance ability to aluminium were also provided.