Nuclear genome size and karyotype analysis in Papaver for ВАС library construction

The objective of the research carried out is study of the pathway of alkaloid production in Papaver species and cell lines, and integration data on physical mapping of newly developed marker DNA sequences with existing difference in expression of genes for key enzymes of alkaloid biosynthesis. This research requires the knowledge on genome structure and organization, and development of genomic resources for detailed characterization of opium poppy genome. The work is focused on the investigation of some features of genome organization of Papaver somniferum and related species, Papaver bracteatum and Papaver rhoeas. These characteristics are necessary for the construction of a BAC library which would be used as appropriate genomic resource for characterization of karyotype changes in poppy stocks with altered alkaloid biosynthesis pathway. Some of these stocks (cell lines) were generated and differed in the types of alkaloids they accumulated.

Introduction.The data from several different alka loid-producing plants suggest that their biosynthesis and accumulation involve a highly regulated process that includes cell-, tissue-, development and envi ronment-specific controls [1,2].The evolution of alkaloid pathways together with their cellular compartmentation appears to be closely associated with the primary reactions from which they have evolved.Opium poppy, Papaver somniferum, is cultivated for its alkaloid-rich latex.Tyrosine decarboxylase (TyDC) is the first enzyme in poppy alkaloid bio synthesis and is encoded by a small gene family.Members of this family are differentially expressed in organs of the plant and cultivated cells [3 ].With the availability of an increasing number of genes involved in alkaloid biosynthesis, increasing efforts would be made to identify the regulators [4] associated with the development of specialized cell types which relate to alkaloid biosynthesis and accumulation.Gene loca-lization and isolation require a detailed knowledge of genome structure.While genetic maps provide infor mation on relative order of molecular markers and genes along the chromosomes, physical mapping pro vides data on the physical position of DNA sequence within a genome.P. somniferum, P. bracteatum and P. rhoeas belong to one taxonomic section within genus Papaver and produce morphinan and benthophenanthridine type alkaloids which belong to dif ferent biosynthetic pathways.
Large-insert DNA libraries are one of the key resources that facilitate gene isolation by positional cloning and the analysis of genome organization, structure and evolution.The easy handling and propagation of the clones make BACs (bacterial artificial chromosomes) an invaluable tool in genomic research, used for a variety of applications, including physical mapping and genome sequencing [5].The production of full-length cDNA molecules from geno mic DNA libraries, representing genes of interest, is of paramount importance in basic plant biology re-search as well as plant biotechnology [6].Estimation of genome size, karyotype parameters, testing restric tion endonucleases patterns are the necessary charac teristics for BAC library construction.
This study was undertaken to determine nuclear genome size and genomic distribution of ribosomal DNA loci and two families of repetitive DNA sequen ces in Papaver species representing one taxonomic section within genus Papaver, with the aim to expand the number of species where these characteristics are known, and aid in the construction of a BAC library wich would be used as appropriate genomic resource for characterization of karyotype changes in poppy stocks with altered alkaloid biosynthesis pathway.
Material and Methods.Plant material.For DNA extraction and chromosome slide preparations we used seedlings and roots of poppy species, P. somniferum, P. bracteatum and P. rhoeas (accessions from Kyiv Central Botanic Garden collection).DNA was extracted from etiolated seedlings following Ausubel et al. [7].
Determination of genome size.According to Dolezel et al. [8 ], approximately 50 mg of midrib was cut from a poppy, P. somniferum, young leaf and transferred to a glass Petri dish.About 10 mg of a young leaf of maize (Zea mays cv.CE-777) with 2C = = 5.43 pg DNA was added and served as an internal reference standard.The tissues were chopped simul taneously in 1 ml of Otto I buffer (0.1 M citric acid, 0.5 % v/v Tween 20).Crude suspension of isolated nuclei was filtered through a 50 pm nylon mesh.Nuclei were then pelleted (300 g, 5 min), resuspended in 200 fil Otto I and incubated for 1 h at room temperature.Finally, 600 fil Otto II buffer (0.4 M Na 2 HP0 4 ), supplemented with 50 [tg/ml RNAse and 50 iKg/ml propidium iodide (PI), was added.Samples were analysed using Partec PAS flow cytometer («Partec GmbH», «Munster», Germany) equipped with 488-nm argon laser.The gain of the instrument was adjusted so that peak representing maize Gl nuclei appeared approximately on channel 100 on histogram of relative fluorescence intensity when using 512-channel scale.About 5,000 nuclei were analysed at rate 10-25 nuclei/s.Three plants were measured per accession.Analysis of each plant was repeated three times on different days.Nuclear DNA content was calculated from individual measurements.
Fluorescence in situ hybridization (FISH).FISH probe for ribosomal DNA was obtained by labelling a pTa71 DNA clone containing 18S, 5.8S and 26S rRNA genes [9] with biotin-16-dUTP («Roche», Germany) by Nick Translation.Along with rDNA probe two new clones were used.These were made by sonicating total genomic DNA from P. somniferum (Danish flag accession) to an average length of 200 to 600 bp.The fragments were denatured at 100 °C in 0.1 M sodium phosphate buffer, pH 7.5, for 10 min and incubated at 60 °C to C 0 t of 0.02.The samples were treated with SI nuclease.The resulting highly repeated double-stranded DNA sequences were clo ned into pUCJ8 at the PstI site.From several thousand clones, 200 were chosen randomly for dot hybridization with 32 P-labelled total DNA from the representatives of four Papaver taxonomic sections (Meconella, Pilosa, MacranthalPapaver, Argemonidium).Two clones, pPs21 and pPs41 gave very strong signals with P. somniferum DNA and no visible signals with three other sections members.These clones were selected for further studies on in situ and southern hybridizations.Digoxigenin-labelled probes for these repeats were prepared using PCR with M13 direct and reverse primers and pPs21 and pPs41 clones as a templates.
Metaphase spreads were prepared according to Alkhimova et al. [10].The slides were treated with 100 mg/ml RNAse in a 2 x SSC solution at 37 °C for 1 h in a humid chamber, washed 3x5 min in 2 x SSC at room temperature.After two washes in 2 x SSC the slides were treated in 4 % paraformaldehyde for 10 min at room temperature, washed in 2 x SSC, dehydrated in ethanol series, and air dried.Prior to hybridization, the probes were mixed in a solution containing 50 % formamide, 10 % dextran sulphate, 0.12 % SDS in 2 x SSC and 50 ng/,ul salmon sperm DNA. 1 fil of probe in 30 fil hybridization mixture per slide was used.The hybridization mixture was dena tured at 70 °C for 10 min and incubated on ice for 10-15 min before being added to the preparations.The chromosomes together with the probes were denatured at 70 °C for 5 min and the hybridization was performed overnight at 37 °C in a humid cham ber.The slides were then washed in 2 x SSC at 42 °C and rinsed in a stringent washing solution of 20 % formamide in 0.1 x SSC at 42 °C for 10 min, followed by several washes in 2 x SSC and 4 x SSC (0.2 % Tween).The sites of digoxigenin-and biotin-labelled probe hybridization were detected using anti-digoxigenin fluorescein («Roche») and streptavidin conju gated to Cy3 («Sigma», USA), respectively.Finally, the preparations were counterstained with DAPI (0.2 The preparations were evaluated using Olympus BX60 microscope equipped with optical filter sets appropriate for DAPI, fluorescein and Cy3 fluo rescence.The images of DAPI, fluorescein and Cy3 fluorescence were acquired separately with a b/w CCD camera, which was interfaced to a PC running the ISIS software («Metasystems», «Altlussheim», Germany).The images were superimposed after con trast and background optimization.

Results and Discussion. Estimation of genome size of Papaver somniferum.
Flow cytometry is a rapid technique that allows accurate estimation of nuclear DNA content [11].To determine nuclear DNA con tent in absolute units, fluorescence intensity of nuclei is compared with that of nuclei isolated from a species with known nuclear genome size.Fluorescence of Pl-stained nuclei were analysed using a Partec PAS II flow cytometer.Nuclei isolated from Z. mays cv.CE-777 with known nuclear genome size (2C = = 5.43 pg DNA) were used as internal standard to estimate nuclear DNA content of P. somniferum in absolute units.Small amounts of leaf tissues (stan dard and sample) were simultaneously chopped in buffer, supplemented with PI and RNAse.Suspension of nuclei was filtered through 50 p.m nylon and stored on ice prior to analysis.2C DNA content of P. somniferum was calculated according to formula: The results of the study showed (Fig. 1) that the size of P. somniferum nuclear genome equal 6.46 pg is smaller than previously estimated [12].
Karyotype of P. somniferum and related species, P. bracteatum and P. rhoeas.A karyotype, which is characterized by the number and morphology of chromosomes, is an important characteristic of a species.Methods for chromosome preparation and in situ hybridization essentially followed Heslop-Harrison et al. [13].Root tips were fixed, partially digested with enzymes and cells were spread on slides.At least ten well-spread metaphase plates with similar degree of chromatin condensation were used to make chromosomal measurements.For constructing the karyotype, the chromosomes were arranged in order of decreasing size and increasing asymmetry (Fig. 2).
Localization of rDNA loci and repetitive DNA clones.Fluorescence in situ hybridization on P. som niferum chromosomes showed that pPs21 and pPs41 DNA sequences were distributed mostly uniformly along all chromosomes with gaps near centromeres and nucleolar organizer regions (data not shown).FISH with P. bracteatum, the representative of the same taxonomic group Macranthal Papaver, is shown in Fig. 3. DNA sequences have been shown to be dispersed over the P. bracteatum genome.It had substantially more copies of pPs41 dispersed along most chromosomes (Fig. 3, b) than pPs21 which gave less number of dots.
Preparation of BAC library construction.BAC library is a source of DNA clones, which will be used as landmarks for chromosome identification, and also for the search of clones consisted of single copy sequences.An important step toward the structural analysis of a functional DNA domains is the const ruction of a large-insert libraries.Their inserts repre sent large DNA fragments that can be easily localized on mitotic chromosomes using FISH and allow selec- Fluorescence in situ hybridization has been per formed to investigate the physical distribution of repetitive clones along the chromosomes of P. somni ferum and P. bracteatum species.Two new clones have been used, pPs21 and pPs41.In situ hybridi zation on P. somniferum chromosomes has shown that pPs21 and pPs41 DNA sequences are distributed mostly uniformly along all chromosomes with gaps near centromeres and nucleolar organizer regions (data not shown).These repetitive sequences have been shown to be dispersed over the P. bracteatum genome distinguishing by the copy numbers of their repeats (Fig. 3).For the first time, the localization of the ribosomal DNA on P. bracteatum chromosomes is shown.
BAC library is a source of new DNA clones, which will be used as landmarks for the chromosome identification, and also for searching the clones con sisted of single copy sequences.An important step toward the structural analysis of functional DNA domains is the construction of large-insert libraries.

NUCLEAR GENOME SIZE AND KARYOTYPE ANALYSIS IN PAPAVER
The construction of the representative ВАС library requires the determination of the genome size to obtain the appropriate amount of clones.The results of the study show that the size of the P. somniferum nuclear genome equal to 6.46 pg is smaller than previously estimated.The genome size of P. somni ferum (In = 22) which represents one copy of nuclear DNA equal to 1C, has been determined as 3100 Mb.Based on this haploid genome size and average insert size of fragments around 150 kb that can be cloned efficiently in the ВАС vector [5], the library repre senting five genome equivalents would consist of 100000 clones.Using cDNA probes will allow the verification of the coverage.The results obtained will be used for the construction of the large insert size genomic library as a new molecular resource.
NUCLEAR GENOME SIZE AND KARYOTYPE ANALYSIS IN PAPAVER ml) and mounted in Vectashield antifade solution («Vector Laboratories*, USA).

Fig. 2 .Fig. 3 .Fig. 4 .
Fig. 2. Karyoidiogram of Papaver somniferum (a), P. bracteatum (b) and P. rhoeas (c) showing the length of individual chromosomes and centromere position.Distribution of rDNA loci revealed by in situ hybridization with pTa71 probe tion of BAC clones with deficiency in repetitive DNA sequences.This approach requires determination of restriction endonucleases suitable for library const ruction, vector selection, appropriate ratio of high molecular weight DNA and chosen vector, optimal ligation and transformation conditions.Testing conditions for different restriction en zymes.EcoRI, BamHI, or Hindlll, the restriction enzymes most frequently used in BAC cloning [5].High molecular weight DNA of P. somniferum, obtai ned by purification of protoplasts from the seedlings in enzyme solution followed by embedding on lowmelting-point agarose, was digested with the five restriction enzymes, which can be ranged according to