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Tomoya Baba1), Satoshi Katagiri1), Hiroshi Tanoue1), Chiden1), Shoko Saji1), Masao Hamada1), Marina Nakashima1), Masako Okamoto1), Mika Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries Kamiyokoba, Tsukuba, Ibaraki, 305-0854, Japan1) National Institute of Agrobiological Resources Kannnondai, Tsukuba, Ibaraki, 305-8602, Japan2) A P1-derived artificial chromosome (PAC) library and a bacterial artificial chromosome (BAC) library have been established for Oryza sativa ssp. japonica are referred to as the RGP (Rice Genome Research Program) Nipponbare PAC library and the RGP Kasalath BAC library. The PAC library has a coverage of about 16 genome equivalents and consists of 69276 recombinant clones carrying inserts (generated by partial Sau3AI digestion) with an average size of about 112 kb in a PAC vector, pCYPAC2. Hybridization with organellar DNA revealed the presence of 11.8 % clones with chloroplast DNA and 0.9 % clones with mitochondrial DNA. On the other hand, the BAC library has a coverage of about 14 genome equivalents and consists of 47194 recombinant clones carrying inserts (generated by partial MboI digestion) of an average size of about 133 kb in a BAC vector, pBeloBAC11. Hybridization with organellar DNA revealed the presence of 2.5 % clones with chloroplast DNA and 0.6 % clones with mitochondrial DNA. With extensive genome coverage, these libraries provide excellent of the two major subspecies of rice.
Rice has a genome size of 430 Mb which is the smallest among the major cereal crops such as maize, barley and wheat (Arumuganathan and Earle 1991). It is also considered to be a model cereal (Moore et al. 1993) because of its synteny with other cereal genomes (Ahn and Tanksley 1993, Kurata et al. 1994, Gale and Devos 1998). Oryza sativa genome, particularly toward its entire sequencing project started from 1998 (Sasaki 1998), created a strong demand for high-quality PAC or BAC libraries. Two Oryza sativa BAC libraries from , Shimokita (Nakamura et al. 1997) have been established for gene cloning experiments. Based on the insert size and number of clones, each BAC library has genome coverage of about 3 and 7 genome equivalents, respectively. At the sequencing project of Arabidopsis thaliana, the total genomic libraries have more than 10 genome equivalents (Mozo et al. 1998, 1999). Here we report the construction of two genomic libraries of rice: a PAC library for japonica bout 2−4 µg of partially digested 180-230 kb DNA fragments from 150 g leaves of Nipponbare or 4x105 recombinants. The transformation efficiencies of 105-106 transformants per µg of vector DNA were obtained in constructing each library. Although the restriction enzymes, Sau3AI and MboI, used for partial digestion of rice HMW nuclear DNA recognize the same 4 bp target sequence, GATC, the efficiency of digestion may differ depending on the degree of DNA methylation. To obtain partially digested DNA fragments from 180 to 230 kb, the required restriction enzyme units of Sau3AI for was 250 times greater than the units of MboI for DNA, this may reflect the difference in digestion effciency observed here. The pUC linker, which is replaced by insert DNA, is a vector self-ligated without the pUC linker or insert DNA, then the host E. coli cell became lethal due to the expression of sacB gene. Therefore only recombinants carrying insert DNA would survive and this could facilitate selection of recombinant clones. The obtained by blue or white colony color selection system using lacZ gene. Approximately 300 and 150 transformations were performed, and a total of 71040 PAC clones and 47232 BAC clones were picked and transferred into 740 and 492 96-well microtiter plates, respectively. During the replication and further analysis of these libraries however, 1764 PAC clones and 35 BAC clones did not grow in the microtiter plates. Thus at present, the To determine the average insert DNA size, 192 clones were randomly selected from each library, digested with NotI and separated by pulsed-field gel electrophoresis (Fig. 3). Figure 4 illustrates the size distribution of the analyzed clones of both libraries. Nipponbare PAC library clones and 7.1 % of the RGP BAC library clones had insert sizes below 60 kb, the majority of the clones, 61.5 % of Nipponbare PAC library and 71.4 % of size range of approximately 100 to 200 kb. The presence of insert DNA clones of less than 100 kb is probably due to the entrapment of smaller DNA restriction fragments in the compression band even after the second size selection. Recently, Osoegawa et al. (1998) reported that small DNA fragments can be removed efficiently by inverting the migration direction of the DNA fragments to the nearest gel edge in a first separation step. The average insert DNA size of the RGP Nipponbare PAC library is 112 kb. On the other corrected number of recombinants and the average insert size, the genome coverage of the Nipponbare PAC and Kasalath BAC libraries of RGP was calculated at 18.0 and 14.6 In order to estimate mitochondrial or chloroplast DNA contamination in each library, a part of PAC or BAC library was gridded at high density on Hybond-N+ nylon filters with 6 x 6 dots. These were used for hybridization with probes containing cloned DNA specific to the Oryza sativa organellar genomes (Fig. 5). Each colony hybridization filter BAC library consisted of 7296 PAC clones and 6912 BAC clones, corresponding to 10.3 % and 14.6 % of the libraries, respectively. Three chloroplast DNAs, ndhA, psbA and rbcL are spaced evenly across the chloroplast genome (Fig. 6) and are thought to be suitable for estimating the chloroplast DNA content in the library. For the same purpose, five mitochondrial DNAs, atpA, coxI, cob, atp6 and atp9, distributed across the genome were used as probes (Fig. 6). A total of 862 PAC clones and 170 BAC clones were screened using chloroplast DNA as probes, whereas 68 PAC clones and 40 BAC clones were screened using mitochondrial DNA as probes. As a result, chloroplast and mitochondrial DNA contamination for the PAC library were estimated at 11.8 % and 0.9 %, respectively. In the case of the BAC library, chloroplast DNA contamination was 2.5 % and mitochondrial DNA contamination was 0.6 %. Wang et al. (1995) reported that 0.3 % of the clones in their rice BAC library contained chloroplast or mitochondrial DNA based on two probes, rbcL for chloroplast and coxI for mitochondrial DNA in hybridization analysis. Using three chloroplast DNA, ndhA, psbA and rbcL, Nakamura et al. (1997) detected 7 % of chloroplast DNA contamination in their rice BAC library. We made a detailed analysis of both mitochondrial and chloroplast DNA contamination in order to obtain high quality libraries. Although both of our PAC and BAC libraries contained small amount of mitochondorial DNA, there was a large amount of chloroplast DNA contamination because the HMW nuclear DNA was We would like to express our appreciation to Dr. Atsushi Hirai, Professor of University of Tokyo for providing the plasmid and lambda clones carrying rice chloroplast and mitochondrial DNAs. We also thank Dr. Kosuke Tashiro of Kyushu University for kind technical advice.
The digested DNA vector was dephosphorylated with 2.4 U of calf intestine alkaline phosphatase (CIAP, Boehringer mannheim) per microgram DNA at 37 for 30 min. After CIAP inactivation by incubation . The short BamHI/ScaI linker fragments were removed by spin dialysis through Microcon 100 cartridges (Amicon) at 3 weeks the plants were transferred into the dark condition for 3 days. Young leaves were collected and stored at -80 (HMW) DNA was isolated from the nuclei according to Zhang embedded in agarose plugs and treated with proteinase K. Then the plugs were cut in half, washed 3 times with 10 mM Tris-HCl pH 7.5 at 30 min and equilibrated with 100 µl of reaction solution (MgCl2 free) containing 1.5 U of Sau3AI (TaKaRa) at 4 overnight. Digestion reaction was started by addition of MgCl2 to be the final concentration of 15 mM and incubated at 37 performed for MboI partial digestion using 0.006 U of the enzyme. Partial digests of HMW DNA were loaded in 1 % NA agarose (Pharmacia) gel and size selection was performed by pulsed-field gel electrophresis (PFGE) using CHEF Mapper (BioRad) with agarose strip containing 180-230 kb fragments was excised and transferred in 1 % low melting point agarose, SeaPlaque GTG (TaKaRa) gel for second size selection. Electrophoresis was carried out in 0.5X TBE at 14 for 18 hours, and the strip carrying 180 - 230 kb was again excised. The for 10 min and treated with 1 U of β-agarase (NEB) per 100 µl of melted gel for 2 hours at 42 The size-selected 10 ng of genomic DNA were ligated to 10 ng of vector DNA in a 40 µl reaction volume using 1 U of T4 DNA ligase (MBI) for 16 hours at 14 , followed by incubation for 6 hours at 4 . After ligation, the reaction was drop-dialysed against 0.1x TE buffer for 1 hour using 0.025 µm type VS membrane (Millipore). A 10 µl of the ligated DNA was added to 50 µl of ElectroMAX DH10B competent cells (GibcoBRL) and electroporated by Gene Pulser (BioRad) at a setting of 1.8 kV, 25 µF capacitance, resistance. PAC recombinants were selected on LB plates containing 40 µg/ml kanamycin and 5 % sucrose. BAC recombinants were selected on LB agar plates Recombinant PACs or BACs were isolated by standard alkaline lysis procedure from 2 ml overnight cultures in LB medium containing kanamycin Plasmid DNA inserts were analyzed with NotI (TaKaRa) digestion and separated by pulsed-field gel electrophoresis with 1 % agarose gel in a 0.5X , 6 V/cm with a 5-40 s pulse time of 14 hours.
Rice chloroplast DNAs (psbA, ndhA, rbcL) cloned in pUC18 and mitochondrial DNAs (atpA, coxI, cob, atp6, atp9) cloned in lambda dash II were kindly provided by Dr. Atsushi Hirai (Univ. of Tokyo, Japan). These organellar DNAs were amplified by polymerase chain reaction (PCR) with primer sets shown in Table 1.
Amplified organellar DNAs were nonradioactively labeled with the ECL direct nucleic acid labelling and detection system (Amersham) according to the instructions of Each library was gridded at high density on Hybond-N+ nylon filters (Amersham) using BioGrid (BioRobotics). The dotted filters were incubated the instructions of the ECL direct nucleic acid labelling and detection In order to provide genomic resources for rice genome analysis such as P1-derived artificial chromosome (PAC) library for Oryza sativa ssp. japonica Nipponbare and bacterial artificial chromosome (BAC) library for Oryza sativa ssp.
consisted of 69276 recombinant clones carrying inserts generated by partial Sau3AI digestion of an average size of about 112 kb in a PAC vector, pCYPAC2. Hybridization with organellar DNA revealed the presence of 11.8 % clones with chloroplast DNA and 0.9 % clones with mitochondrial DNA. This library, called the RGP Nipponbare PAC library, corresponds to about 16 genome equivalents of rice. The BAC library consisted of 47194 recombinant clones carrying inserts generated by partial MboI digestion of an average size of about 133 kb in a BAC vector, pBeloBAC11. Hybridization with organellar DNA revealed the presence of 2.5 % clones with chloroplast DNA and 0.6 % clones with mitochondrial DNA. This library, called the BAC library, corresponds to about 14 genome equivalents of rice. Both Structure of PAC (pCYPAC2) and BAC (pBeloBAC11) vectors SacB positive selection system of PAC (pCYPAC2) vector DNA was digested with NotI and separated by pulsed-field gel electrophoresis.
The PAC (pCYPAC2) vector band and sizes of lambda HindIII fragments or lambda Insert DNA size distribution of the Nipponbare PAC library and the Kasalath BAC library.
A total of 192 clones were analyzed from each libraries.
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pUC linker
sacB
promoter

sacB
pBeloBAC11
Kamr
F1 plasmid ori
P1 plasmid replicon
pUC linker
chloroplast
mitochondrial
Table 1. Oligonucleotide sequence of primer sets for amplification of organellar DNA 5'-AGAAGATCGAAAGAGGGGGG-3' 5'-CAATGGCACCGAAGATGAAA-3' 5'-AGCTCATGGGAGTGTCTGCC-3' 5'-TGCAATCCCATCTCCAACTG-3' 5'-TGAGAGATGTTGAAGGGGGC-3' 5'-TTGTCCACGGAGAAACCACC-3' 5'-GGCATTGAATAGGCAGGCTC-3' 5'-GTCGACCCAACGGAAAACAT-3' 5'-TTATGGGACTGCAATCGTGC-3' 5'-GCAGCTCTTTCCTGGGTTTTT-3' 5'-CGAAAGTACAAGCCTGTGGG-3' 5'-GATGCAGCTTCCCAAATTGG-3' 5'-CCCCTGGGACTGCTTCAAGC-3' 5'-CTAATAGATTGAGCGGCAGC-3' 5'-GCGTTGGAGGGACCGTTTTG-3' 5'-TCCTGAGTGAAAAAGATACC-3' a) mitochondrial DNA : coxI, atpA, cob, atp6, atp9 (Iwahashi et al . 1992) chloroplast DNA : psbA, ndhA, rbcL (Hiratsuka etal . 1989)

Source: http://rgp.dna.affrc.go.jp/E/rgp/PAC-library.pdf

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