Plant Systems Biology Group

The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis -type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequence assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species. Copyright © 2009 S. Karger AG, Basel Accepted after revision: November 25, 2008 by B. Friebe Nurul Faridi Forest Science Laboratory, Texas A&M University 2585-TAMU College Station , TX, 77843-2585 (USA) Tel. +1 979 862 3908, Fax +1 979 845 3272, E-Mail [email protected] © 2009 S. Karger AG, Basel 1424–8581/09/1251–0074$26.00/0 Accessible online at: www.karger.com/cgr Fluorescent in situ Hybridization in Populus trichocarpa Cytogenet Genome Res 2009;125:74–80 75 and reported for poplars [e.g., Bradshaw et al., 1994; Grattapaglia and Sederoff, 1994; Bradshaw, 1996; Cervera et al., 1996, 2001; Yin et al., 2004]. However, little work has been completed at the cytogenetic level, especially when compared to other plant models such as Arabidopsis [Jackson et al., 1998; de Jong et al., 1999; Fransz et al., 2000, 2002], rice [Cheng et al., 2001, 2002; Zhao et al., 2002; Cheng et al., 2005; Tang et al., 2007], and sorghum [Islam-Faridi et al., 2002; Kim et al., 2005a, b]. Cytogenetic analysis in these plant species has been useful in aiding genome assembly by facilitating integration of linkage maps with physical maps and in determining the order of sequence scaffolds and their respective contigs [Zhao et al., 2002; Cheng et al., 2005; Kim et al., 2005a]. This has provided a more complete understanding of the structural and functional properties of these genomes. Detailed cytogenetic analysis of poplar chromosomes should help improve the genome assembly, which currently consists of 2,447 major scaffolds 1 5 kb [Tuskan et al., 2006]. In this paper, we utilized fluorescent in situ hybridization (FISH) to study and localize the 18S-28S and 5S rDNA and the Arabidopsis -type telomere repeat sequence (ATRS) sites in P. trichocarpa . In addition, we used FISH to localize 6 marker-selected BAC clones that represent 2 linkage groups of P. trichocarpa . Our specific objectives were to 1) determine the number and location of the rDNA sites, 2) determine the distribution of the ATRS sites, and 3) study the feasibility of utilizing BACFISH in Populus for chromosome localization. The 3rd objective is important for furthering our work in establishing a cytomolecular map for each poplar chromosome and to better understand the structural details of the Populus genome. Materials and Methods Chromosome Preparation Chromosome spreads were prepared for P. trichocarpa clone Nisqually-1 (383–2499), the same genotype that was used for whole genome sequencing [Tuskan et al., 2006]. Actively growing root tips about 1.5 cm long were harvested from rooted cuttings growing in potting soil in a greenhouse. Harvested root tips were immediately pre-treated with an aqueous solution of -monobromonaphthalene (0.8%, Sigma) for 1.5 to 1.75 h at room temperature (RT) in the dark and then fixed in 4: 1 ethanol:glacial acetic acid to arrest cell division at metaphase. Fixed root tips were processed enzymatically (5% cellulase Onozuka R-10, Yakult Honsha Co. Ltd., and 1.25% pectolyase Y-23, Kyowa Chemical Products Co. Ltd.) in 0.01 M citrate buffer, and the chromosome spreads were prepared as described elsewhere [Jewell and IslamFaridi, 1994]. The chromosome spreads were checked with a phase contrast microscope (Axioskop, Carl Zeiss, Inc.), and slides containing good chromosome spreads were selected and stored at –80 ° C for use in FISH. Probe DNA The following probes were used in the current experiments: 18S-28S rDNA of maize [Zimmer et al., 1988], 5S rDNA including a spacer region of sugar beet [Schmidt et al., 1994], Arabidopsis type telomere repeat sequence (TTTAGGG) n (kindly provided by Dr. T. McKnight, Department of Biology, Texas A&M University), and BAC clones from 2 P. trichocarpa linkage groups LG-I and LG-VI. The BAC clones were derived from a library prepared from Nisqually-1 [Stirling et al., 2001]. Positions of the BACs were determined by alignment of end sequences to the genetic map-anchored whole genome sequence assembly. The repeat content of each BAC clone was inferred based on the frequency of constituent 16mers in the full set of 7.5 million sequence reads from the Populus genome sequencing project and the abundance of protein coding sequences contained in the BAC. The selected BACs were 66B19, 75P22, and 87F21 from LG-I and 78O18, 88A10, and 93N12 from LG-VI (http://www.bcgsc.ca/platform/mapping/ data/?searchterm=poplar). BAC DNA was isolated from overnight liquid cultures in selective media by alkaline lysis, digested with Eco RI, and followed by further purification using Plant DNeasy spin columns (QIAGEN, Valencia, CA) as described elsewhere [Childs et al., 2001]. Probe DNAs with and without whole plasmids were labeled with biotin-16-dUTP (Biotin-Nick Translation Mix, Roche Diagnostics) and/or digoxigenein-11-dUTP (dig) (Dig-Nick Translation Mix, Roche Diagnostics) by nick translation as recommended by the manufacturer. Labeled probes were dot-blotted to verify incorporation of label. Fluorescent in situ Hybridization (FISH) The hybridization mixture consisted of deionized formamide (50%, Fisher Chemical), dextran sulfate (10%, Fisher Chemical), 2 ! SSC, labeled probe DNA (30 ng/slide), carrier DNA ( E. coli DNA, 7.5 g/slide), and blocking DNA (poplar Nisqually-1 Cot-1 DNA, 300 to 600 ng/slide, depending on singleor dualcolor FISH, respectively). The poplar Cot-1 DNA was prepared as described by Zwick et al. [1997] and was used only for FISH with BAC probes (BAC-FISH). The BAC-FISH hybridization mixture was denatured in a boiling water bath for 10 min, immediately placed on ice for 5 to 10 min, and then incubated at 37 ° C for 30 min to allow the Cot-1 DNA to hybridize with the repetitive DNA sequences of the BAC probes. Chromosome spreads were denatured in 70% deionized formamide at 72 ° C for 1.5 min in an oven on a metal block followed by dehydration through a series of ethanol (70, 85, 95, and 100%) washes at –20 ° C for 3 min each. The slides were dried with forced-air and on the bench at RT for 25 to 30 min prior to hybridization with probe DNA. The hybridizations were accomplished by loading 25 l of hybridization mixture on the chromosome spread and placing and sealing a 22 ! 30 mm glass coverslip over the mixture. The cover slips were sealed with rubber cement, and the slides were placed in a humidity chamber and incubated over night at 37 ° C. Following hybridization, the coverslips were washed off with 2 !