Xiulan Li

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.

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.

Characters of DNA constitution in the rye B chromosome.

We have used chromosome microdissection and microcloning to construct a DNA library of the entire B chromosome (B) of rye. New rye B-specific sequences have been screened from this pool, blasted with other sequences and analyzed to elucidate the characters of DNA constitution and the possible pathway of the origin of the rye B chromosome. We report the discovery of a new sequence that is specific to the rye B centromere.

Chromosome Microdissection, Cloning and Painting of the Chromosome 1 in Poplar (Populus tremula)

Abstract The chromosome microdissection, cloning and painting technology has evolved into an efficient tool for genomic research. Application of these techniques has rarely been applied for forest plants, largely due to the difficulty of chromosome preparation. The present study was performed to establish a method for single chromosome microdissection, cloning and painting in forest plants using poplar (Populus tremula) as a model. An individual chromosome 1 was microdissected from the metaphase spreads of poplar root-tip cells with fine glass needle controlled by a micromanipulator. The dissected chromosome was amplified in vitro by the Sau3A linker adaptor mediated PCR (LA-PCR) technique, by which 200bp to 3,000bp smear DNA fragments were obtained. Then, 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. Approximately 3 x 105 recombinant clones were obtained. The second round PCR products were used as a complex probe mixture for fluorescent in situ hybridization (FISH) on the metaphase spreads of poplar. Hybridization signals were observed, mainly, along the entire chromosome 1, at the same time, signals were also present on telomeric and centromeric regions of other chromosomes. Therefore, this research suggests that chromosome microdissection, cloning and painting of the single small chromosome in forest plants are feasible.

Cloning and Characterization of Disease Resistance Gene Analogs from Poplar (Populus tremula) Chromosome 1

The majority of verified plant disease resistance genes (R‐genes) isolated to date are of the NBS‐LRR class, encoding proteins with a predicted nucleotide‐binding site (NBS) and a leucine‐rich repeat (LRR) region. The conservation between different NBS‐LRR R‐genes opens the avenue for the use of PCR‐based strategies in isolating and cloning other R‐gene family members or analogs using degenerate primers for these conserved regions in plants. However, the screening of target sequences among the numerous amplified resistance gene analogs (RGAs) can be very laborious and time‐consuming. The amplification of RGAs from specific chromosomes could greatly reduce the number of RGAs to be screened and, consequently, speed up the identification of target RGAs. We have developed a technique that combines chromosome microdissection and homologous sequence amplification in order to acquire RGAs from single chromosomes in forest tree poplars (Populus tremula). A number of RGAs were amplified with the DNA fragments of P. tremula chromosome 1 as template, using consensus‐degenerate hybrid oligonucleotide primers based on conserved motifs of the NBS domain. Here, we report the sequence characterization and diversity analysis of these RGAs and their relationships with the NBS sequences of known R‐genes from other plant species. These data permit insights into the origin, diversification, and evolution of NBS‐LRR R‐genes in perennial species such as poplar.

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.

Homologous analysis on centromeric region sequences of A and B chromosomes from rye by FISH

Four centromeric segments from 6 chromosomes (Bs) of rye have been microdissected and amplified by linker adapter PCR (LA-PCR). The PCR products ranged from 100 to 2 000 bp. Fluorescencein situ hybridization (FISH) experiment has been carried out using PCR products as the probe, which was labeled by DIG-11-dUTP. The result confirms that these PCR products from Bs centromeric region are homologous with that of A chromosomes (As) in rye. It also proves that Bs are originated from As.

Chromosome G-banding in plants by inducing with trypsin and urea *

Clear G-bands were revealed by applying the TUG method on chromosomes of 5 species of higher plants (Lilium davidii, Viciafaba, Hordeum vulgare, Ginkgo biloba and Triticum monococcum). Some details of the TUG method which consisted of treating chromosomes with both trypsin and urea were also studied. The mechanisms that might account for the presence of G-bands in plants were discussed.