RAPD-based screening of genomic libraries for positional cloning
RAPD-based screening of genomic libraries for positional cloningWaly Dioh, Didier Tharreau1 and Marc-Henri Lebrun*
Génétique Moléculaire des Champignons Phytopathogènes, Institut de Génétique et Microbiologie, CNRS-URA 2255, Bâtiment 400, Université Paris-Sud, 91405 Orsay, France and 1UR-PHYMA, CIRAD-CA, BP 5035, 34032 Montpellier, France
Received July 4, 1997;Revised and Accepted October 31, 1997
ABSTRACT
RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea. Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.
RAPD markers (Random Amplified Polymorphic DNA; 1 ) are widely used as starting molecular markers for positional cloning of a gene of interest (2 ). However RAPD markers frequently contain repeated sequences (3 ,6 ) that prevent the screening of genomic libraries by hybridisation. To identify single copy sequences within such RAPD markers, a large number of subclones must be analysed individually by Southern hybridisation using genomic DNA. Such a strategy might be time consuming or unsuccessful. RAPD markers can also be cloned and sequenced. Oligonucleotides (20-24 bp) complementary to ends of the RAPD marker are designed to specifically amplify a fragment of this locus (SCAR; 4 ). Pools of cosmids, BACs or YACs from genomic libraries are screened by PCR using SCAR oligonucleotides (5 ). Although this strategy can be successful, the molecular characterisation of RAPD markers is time consuming.
We present here a rapid and efficient strategy to screen a genomic library for clones bearing a RAPD marker. First, we performed a RAPD analysis of cosmid pools from a genomic library. Second, the RAPD marker of interest was used as a probe on colony filters of cosmid pools amplifying the RAPD marker. Third, each cosmid clone hybridising to the RAPD marker was analysed for amplification of the marker using a RAPD protocol. The efficiency of this strategy was evaluated with RAPD markers composed of single copy or repeated sequences.
Four RAPD markers linked to avirulence genes of the rice blast fungus Magnaporthe grisea were identified bybulk segregant analysis (6 ). OPE-Y13 (1.3 kb) was linked to avirulence gene AvrIrat7-1. OPE-D16 (0.3 kb) and OPE-M18 (1.2 kb) were linked to avirulence gene AvrMednoï-1. OPE-S9 (0.9 kb) was linked to avirulence gene AvrKu86-1. OPE-S9 and OPE-M18 were shown to contain repeated sequences, while OPE-D16 and OPE-Y13 contained only single copy sequences. We constructed two M.grisea genomic libraries, one with 4100 clones (four times the genome size) using isolate 96/0/76 and cosmid vector pMOcosX and the other with 2880 clones using isolate Guy11 and vector pHC79. The 96 cosmids from each microtitration plate were pooled for growth and their DNA extracted (7 ). RAPD analysis of cosmid pools and clones were performed with a PTC100 MJ-Research PCR apparatus with standard conditions: one cycle of 2 min 30 s at 95°C and 45 cycles of 1 min at 95°C, 1 min 30 s at 37°C, 1 min 30 s at 72°C followed by one cycle of 15 min at 72°C. Four Operon primers were used: S9 (CCTGGTCCCC), M18 (CACCATCCGT), D16 (AGGGCGTAAG) and Y13 (GGGTCTCGGT) at 1 µM with 50 ng of DNA, 2.5 µl of 10* buffer (Appligene: MgCl2 at 1.5 mM final), 0.2 mM dNTP (Eurobio), 1 U Taq polymerase (Appligene) and autoclaved water up to 25 µl. Amplification products were separated by gel electrophoresis on 1% agarose gel in TAE at 5 V/cm for 3 h. For hybridisation, RAPD markers were separated by gel electrophoresis, extracted from the gel by glassmilk purification (Jetsorb, Bioprobe) and labelled with [alpha]-32P (Amersham) by random priming (Pharmacia). Hybridisation was performed at 65°C overnight in buffer 1 (6* SSC, 0.5% SDS, 5* Denhardt's) with two washes at 65°C with buffer 2 (0.1* SSC, 0.2% SDS) for 1 h.
RAPD analysis of cosmid pools from 96/0/76 genomic library with Operon primer Y13 showed that two pools (D31 and D42, Table 1 ) amplified a marker similar in sizeto OPE-Y13 (1.3 kb). Colony filters corresponding to these positive pools were probed with OPE-Y13. We did not detect hybridisation signals in pool D42, while a strong signal was detected in pool D31 (cosmid D31C12, Table 1 ). A marker similar in sizeto OPE-Y13 was amplified from cosmid D31C12. The screening of the whole 96/0/76 cosmid library by hybridisation to the single copy marker OPE-Y13 led to the detection of the same cosmid. RAPD analysis of cosmid pools from 96/0/76 library with Operon primer D16 revealed six pools (D11, D12, D27, D28, D35 and D37; Table 1 ) amplifying a marker similar in sizeto OPE-D16 (0.3 kb). Colony filters corresponding to these positive pools were probed with OPE-D16. One cosmid from each pool strongly hybridised to OPE-D16 (Table 1 ). A RAPD marker
aThe first letter refers to the cosmid library: D for 96/0/76 and G for Guy 11. The first number refers to the microtitration plate number. The second letter and the second number refer to the row and column numbers of the microtitration plate.
similar in sizeto OPE-D16 was amplified from each of these cosmids. When the whole cosmid library was hybridised to the single copy RAPD marker OPE-D16, we only detected the six cosmids already identified by our RAPD-based screening.
6 Dioh,W., Tharreau,D., Gomez,R., Roumen,E., Orbach,M., Notteghem,J.-L. and Lebrun,M.-H. (1996) In Kush,S. (ed.) Rice Genetics III. IRRI press, Los Banos, Philippines, pp. 916-920.
7 Bowring,F.J. and Catcheside,D.E.A. (1995) Fungal Genet. Newslett., 42, 18-20.
*To whom correspondence should be addressed at present address: Physiologie Cellulaire Végétale, UMR 41 CNRS-RPA, Rhône-Poulenc Agrochimie, 14 rue Pierre Baizet, 69009 Lyon, France. Tel: +33 472 85 24 81; Fax: +33 472 85 22 97; Email: marc-henri.lebrun@rhone-poulenc.com
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