W. Q. Song

DNA Methylation in Genomes of Several Annual Herbaceous and Woody Perennial Plants of Varying Ploidy as Detected by MSAP.

Polyploidization is known to accompany altered DNA methylation in higher plants, which plays an important role in gene expression regulation and maintaining genome stability. While the characteristics of DNA methylation in different polyploid plants are still to be elucidated; here, status of genomic DNA methylation in a series of diploid, triploid, and tetraploid annual herbaceous plants (watermelon and Salvia) and woody perennials (pear, Poplar, and loquat) were explored by methylation-specific amplified polymorphism analysis. The results indicated that levels of DNA methylation in triploid watermelon and Salvia were lower than their diploid parents. In triploid Poplar and pear, higher levels of DNA methylation were detected, and no significant difference was observed between triploid and tetraploid in all tested materials. Further data analysis suggested that about half of the total detected sites underwent changes of DNA methylation patterns in triploid watermelons and Salvia, as well as an obvious trend towards demethylation. However, the changes of DNA methylation patterns in three triploid woody perennials were only 17.54–33.40%. This implied that the characteristics of DNA methylation are significantly different during the polyploidization of different plant species. Furthermore, the results suggested that the level of DNA methylation was nonlinearly related to the ploidy level, and triploid plants displayed more interesting DNA methylation status. The characteristics and possible functions of DNA methylation in different ploidy series are further discussed.

Differentiating sex chromosomes of the dioecious Spinacia oleracea L. (spinach) by FISH of 45S rDNA

Spinacia oleracea L. (spinach) is a dioecious species with both male and female plants having 2n = 2x = 12 chromosomes, consisting of two large metacentrics, two long subtelocentrics, two short subtelocentrics, two acrocentrics, and four submetacentrics. The location of 45S rDNA was investigated on metaphase chromosomes using fluorescence in situ hybridization (FISH). The numbers of 45S rDNA foci in diploid sets of chromosomes from females was six and from males was five. All the fluorescent foci lay in secondary constrictions and the satellites. Our results indicate that an XY-type sex chromosome system could be present in spinach where the Y chromosome lacks a 45S RNA focus.

A Report of Triploid Populus of the Section Aigeiros

Abstract This screening study analyzed ploidy levels by counting the chromosome number of 61 Aigeiros cultivars grown in China. Triploid Aigeiros has been found in four of these cultivars: Populus x euramericana (Dode) Guiner cv. Wuhei-1, P. × Liaohenica, P. Langfangensis-3 Wang (P. deltodide Barry cv. “Shanhaiguan” × P. simonii × P. pyramidalis-12 +Ulmus pumila Linn.), and P. × euramericana (Dode) Guinier. cv. “Zhonglin-46”. The karyotype analysis indicates that triploid Aigeiros might be derived from original allotriploid. Because growth of the triploid trees was faster than their respective diploid hybrids or clones in the plantations where we collected the materials, we expect that they will play a significant role in breeding, reforestation and fiber production in China.

Application of ISSR Markers to Fingerprinting of Elite Cultivars (Varieties/Clones) From Different Sections of the Genus Populus L.

Abstract The Inter-Simple Sequence Repeat (ISSR) was used in this study for genetic fingerprinting and identification of 28 important Populus L. (poplar) cultivars (varieties/ clones), and determination of the genetic relationships among these cultivars. These 28 cultivars belonged to sections Aigeiros, Tacahamaca, Leuce, Turanga, and hybrids between sections Aigeiros and Tacahamaca. Out of 27 ISSR primers tested, eight primers generated clear multiplex profiles. The best three primers produced 154 easily detectable fragments, 129 (84%) of which were polymorphic among the cultivars. Each of these 3 primers produced fingerprint profiles unique to each of the accessions studied, and thus could be solely used for their identification. Twenty-five markers, unique to 10 of the cultivars studied, were detected. These markers may be converted into cultivar-specific probes for identification purposes. Genetic relationships among the cultivars were evaluated by generating a similarity matrix based on the simple matching coefficient and the unweighted pair group method with arithmetic average (UPGMA) dendrogram. The results showed a clear-cut separation of cultivars among different sections of poplar, and were in agreement with the genealogy of the sampled cultivars. The present study shows that ISSR markers could generate abundant polymorphism, are reproducible, and are quick for characterization of poplar cultivars. In the future, the markers used in this study, in combination with other molecular techniques, could provide a useful panel of ISSR markers for largescale DNA fingerprinting of poplar cultivars and determination of the genetic relationships among these cultivars.

Construction of poplar (Populus tremula) chromosome 1-specific DNA library by using a microdissection technique

A method for single-chromosome microdissection and microcloning was established in forest plants using poplar (Populus tremula) as a model. By use of meristematic cell division in root tip and the wall degradation hypotonic method, well-spread poplar metaphase chromosome spreads showing low contamination were quickly prepared and fitted for chromosome microdissection. An individual chromosome 1 was microdissected from the metaphase spreads of poplar root-tip cells with a fine glass needle controlled by a micromanipulator. The dissected chromosome was amplified in vitro by theSau3A linker adaptor-mediated PCR technique, by which 200- to 3000-bp smear DNA fragments were obtained. Southern hybridization results showed that the PCR products from the single poplar chromosome were homogeneous with poplar genomic DNA, indicating that DNA from the single chromosome has been successfully amplified. Next, the second-round PCR products from the single chromosome 1 were cloned into T-easy vectors to generate a DNA library of the chromosome 1. About 3×105 recombinant clones were obtained. Evaluation based on 160 randomly selected clones showed that the sizes of the cloned inserts varied from 230–2200 bp, with an average of 800 bp. Therefore, this research suggests that microdissection and microcloning of single small chromosomes in forest plants is feasible.

Cloning of differential expression fragments in cauliflower after Xanthomonas campestris inoculation

A near isogenic line (NIL) of Brassica oleracea var. botrytis with resistant and susceptible lines C712 and C731, was used in this study. More than 100 differentially expressed cDNA fragments were obtained from black rot resistant cauliflower plants obtained using cDNA-amplified fragment length polymorphism (AFLP) after infection with the pathogen. Thirteen of these fragments were cloned and subjected to reverse Northern blot analysis using both infected and control cDNA pools. Two positive clones, M2 and M6, were isolated. Northern dot blot and Northern blot analyses showed that M2 was constitutively expressed, whereas M6 contained a gene that was differentially expressed during pathogen infection. Moreover, M6 cDNA fragment was also highly expressed 16–24 h after H2O2 treatment. Southern blots showed that M6 is a single copy gene in the cauliflower genome, and encodes a protein with 84 % homology to gene on Arabidopsis chromosome 1. The deduced M6 protein has 91 % positive homology with the Arabidopsis 2A6 protein, which regulates ethylene synthesis; 76 % homology with a 1-aminocyclopropane-1-carboxylate oxidase (ACO), the last enzyme in ethylene synthesis; and 70 % homology with an ethylene induced DNA binding factor. These results suggest that M6 gene fragment is a new H2O2 downstream defense related gene fragment and can be induced by Xanthomonas campestris pv. campestris and H2O2.

Identification and sequence analysis of cDNA fragments relative to ovary development of Cymbidium hybridium after pollination

In this study, seven cDNA fragments of genes, differentially expressed in ovaries after pollination in Cymbidium hybridium, were identified and characterized by mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR). Four (CDD-313, CDD-272, CDD-265, CDD-243) among these seven cDNA fragments showed no significant homology with ESTs or genes in the databases of NCBI; another three (CDD-193, CDD-218, CDD-470) showed significant homologies with sequences encoding components of an ABC-type transporter, a GTPase and 40S ribosomal S3 proteins (RPS3), respectively. The differential expression patterns of them were confirmed by reverse Northern dot blot analysis. More interestingly, CDD-470 appeared to be present and highly expressed in the pollinated ovaries and encoded a new factor of RPS3 participating in cell growth and proliferation. We deduced that this 40s ribosomal S3 like protein was involved in ovary development of orchids.

Physical mapping of 45S rDNA to metaphase chromosomes in 30 taxonomically diverse plant species

Summary The genomic distribution of ribosomal RNA genes (rDNA) has been determined by fluorescence in situ hybridisation (FISH) in 30 diverse plant species, using 45S rDNA as a probe. In most species, physical mapping of 45S rDNA to metaphase chromosomes is reported for the first time. These species could be divided into four groups based on differences in the number and positions of their rDNA loci. Fluorescent signals lay in secondary constrictions and satellites of satellite-chromosomes (SAT-chromosomes) in 14 of the 30 species, including Berberis thunbergii, Plantago major, Sanicula lamelligera, Ficus carica and Robinia pseudoacacia. In another seven species, including Robinia hispida, Kolkwitzia amabilis, Litchi chinensis, Catalpa speciosa and Acer buergerianum, FISH markers were also detected in the terminal and/or peri-centromeric regions of some chromosomes beside secondary constrictions and satellites. There was only one species (Viburnum sargentii) in which four pairs of SAT-chromosomes were found, only one pair of which gave FISH signals. In the other eight species, including Kerria japonica f. pleniflora, Salvia miltiorrhiza, Cucumis sativus and Ulmus pumila, no visible satellites existed and the positions of 45S rDNA loci on the chromosomes were terminal or peri-centromeric. Our results indicate that 45S rDNA can be used to identify individual chromosomes. The polymorphism of 45S rDNA loci is also discussed in this paper.

Cytosine methylation at CG and CNG sites is differential during the development of triploid black poplar

It has been widely shown that polyploidization can result in changes in cytosine methylation. However, little is known regarding how cytosine methylation changes in polyploids development, especially in polyploid trees. In this study, we investigated drifting changes of DNA methylation status at 5′-CCGG sites in the apical bud, young and mature leaf tissues of triploid black poplar (Populus. euramericana) with methylation-sensitive amplification polymorphism (MSAP) and assessed the expression of multiple DNA methyltransferases (MTases) and DNA demethylase during different developmental stages. MSAP analysis detected methylation levels at CG and CNG sites of diploid tissues reduced during development from bud to leaves, while for the triploid, methylation at CNG sites increased during development, but levels of methylation at CG sites first decreased in young leaves before increasing in mature leaves. MTase genes related to CG or CNG methylation were respectively preferential in different triploid tissues with high CG or CNG methylation levels. High expression of DNA demethylase was observed in tissue with high demethylation trends. These finding suggest CG and CNG methylation and their related enzymes are involved with different biological functions and networks of gene regulation in different developmental stages of triploid.

Development of a sequence characterized amplified region (SCAR) marker associated with high rooting ability in Larix

In this study, bulked segregant analysis (BSA) was used on Larix leptolepis × Larix olgensis hybrids to identify a random amplified polymorphic DNA (RAPD) marker associated with high rooting ability in larch. Two DNA bulks: H (high rooting ability) bulk and L (low rooting ability) bulk were constructed according to the rooting percentages of the stock plants. Among the 328 primers, only S356 could amplify a specific band, named S356445, which only existed in the H bulk and was further confirmed following selective genotyping of individual hybrids. Grounded on the border sequences, S356445 was converted to a sequence characterized amplified region (SCAR) marker, HRL445, which can be useful in marker-assisted selection (MAS) to screen for larch with high rooting ability. All the results strongly indicated that S356445 and HRL445 were closely associated with high rooting ability in larch.