6 T-DNA PCR TAIL-PCR T-DNA. 69% 30% T-DNA left border LB right border RB T-DNA A T-DNA

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1 Research Paper Acta Microbiologica Sinica February 2011 ISSN CN / Q http / / journals. im. ac. cn / actamicrocn Botrytis cinerea T-DNA * Botrytis cinerea T-DNA Agrobactirium tumfacience T-DNA PCR TAIL-PCR T-DNA T-DNA 69% 30% T-DNA left border LB right border RB T-DNA T-DNA T-DNA T-DNA TAIL-PCR Q933 A Botrytis cinerea 200 T-DNA 1 T-DNA 3-6 T-DNA PCR thermal asymmetric interlaced polymerase chain 2 reaction TAIL-PCR 7 T-DNA Magnaporthe oryzae T-DNA 4 8 T-DNA Agrobactirium tumfacience mediated-transformant T-DNA AMT 2 AMT T-DNA T-DNA 9 T-DNA T-DNA 2009R10030 * Tel thzhu@ zjut. edu. cn

2 204 Jia Liu et al. / Acta Microbiologica Sinica TAIL-PCR T-DNA DNA 23 PDA DNA PCR 1 hptf TTCGATGTAGGAGGGCGTGGA-3 / hptr 5 -CGCGT B. cinerea CTGCTGCTCCATACAAG-3 B Paul Tudzynski 1. 4 T-DNA TAIL-PCR A. tumfacience AGL1 TAIL-PCR TAIL-PCR 1 Taq pcambiai1390 T-DNA DNA 9 Biopsin patmt T-DNA 1. 2 pcambiai1390 RSP3 5 -CCCGCCTTCAGTTTAAACTAT-3 pcambiai1300 CaMV35S 7 TAIL-PCR 3 TrpC patmt ng DNA T-DNA TAIL- 20 μl 2 dntp 2. 5 mmol / L 1. 5 μl PCR 1 1 RSP1 10 μmol / L 4 μl 10 μmol / L μl Taq 5U / μl ddh2o 20 1 μl 3 RSP2 5 -GGCGAATGCTAGAGCAGCTT-3 DNA RSP μl DNA RSP3 PCR 9 Fig. 1 1 T-DNA Illustration of T-DNA region in binary vector. LAD arbitrary degenerate primer LB left border RB right border LSP1-3 left specific primer RSP1-3 right specific primer. RSP1 5 -CCAACAGTTGCGCAGCCTGAAT T-DNA PCR pmd19-t TaKaRa TAIL-PCR TAIL- Broad Institute PCR T-DNA http / / www. broad. mit. edu / T-DNA annotation / genome / botrytis_cinerea Blast DNA PCR DNAStar TAIL-PCR 2 PCR 2. 1 T-DNA 465 T-DNA 1. 6 PCR EZ- 10 Spin Column DNA Gel Extraction Kit BIO BASIC INC Canada DNA 3 PCR

Botrytis cinerea T-DNA. / 2011 51 2 205 pcambiai1390 15 T-DNA 5 patmt 100 μg / ml DNA PCR 2 T-DNA T-DNA 5 T-DNA left border LB LB 59% - 90% T-DNA transformants of Botrytis cinerea.

3 Botrytis cinerea T-DNA. / pcambiai T-DNA 5 patmt 100 μg / ml DNA PCR 2 T-DNA T-DNA 5 T-DNA left border LB LB 59% - 90% T-DNA transformants of Botrytis cinerea. M Standard DNA molecular weight marker WT Wild type B. cinerea 1-6 Transformants % RSP3 LSP3 7 RSP3 LSP3 right border RB T-DNA RB RB 5 -TTTG- -A-3 T-DNA T-DNA 3-A 3-B 3 2 T-DNA T-DNA PCR illegitimate recombination Fig. 2 PCR amplification of hygromycin resistance gene from some non-homologous T-DNA 3 microhomology 11 T-DNA T-DNA 12 T-DNA Magnaporthe oryzae T-DNA 3 6 TAIL-PCR T-DNA LB 36 T-DNA truncated LB 12 RB small Blast deletions T-DNA LB RB T-DNA 35 bp 93% - 96% RB 98% T-DNA % T-DNA 30 69% T-DNA % T-DNA 69% TAIL-PCR 30% 6. 9% T- T-DNA DNA PCR T-DNA TAIL-PCR 100% TAIL-PCR T-DNA 2. 3 T-DNA 2 T- 43 T-DNA DNA T-DNA

4 206 Jia Liu et al. / Acta Microbiologica Sinica T-DNA A B Fig. 3 The alignment of junctions between T-DNA left A and right B border with B. cinerea genome DNA. A L1-L12 LB regions for different T-DNA inserts LB region of T-DNA from vector and the dashed line indicates the standard LB sequence The black shaded are T-DNA regions and the unshaded are flanking sequences. B R1-R21 RB regions for different T-DNA inserts RB region of T-DNA from vector and the dashed line indicates the standard RB sequence The black shaded are T- DNA regions and the unshaded are flanking sequences. T-DNA 2 Michielse CB Hooykaas PJJ van den Hondel CAMJJ Ram AFJ. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Current Genetics Mayerhofer R Konczkalman Z Nawrath C Bakkeren G 1 Williamson B Tudzynsk B Tudzynski P Van Kan JAL. Botrytis cinerea the cause of grey mould disease. Molecular Plant Pathology Crameri A Angelis K Redei GP Schell J Hohn B Koncz C. T-DNA Integration - a mode of illegitimate recombination in plants. Embo Journal Choi J Park S Ji M Park J Jun J Goh J Yoo S Kim B Han S Kim S Lee Y. Integration of Agrobacterium tumefaciens T-DNA into the chromosomes of Magnaporthe

5 Botrytis cinerea T-DNA. / grisea. Phytopathology S24-S24. 5 Zhu CF Wu JH He CZ. Induction of chromosomal inversion by integration of T-DNA in the rice genome. Journal of Genetics Genomics Kim SI Veena Gelvin SB. Genome-wide analysis of Agrobacterium T-DNA integration sites in the Arabidopsis genome generated under non-selective conditions. Plant Journal Liu YG Chen Y. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques Choi J Park J Jeon J Chi MH Goh J Yoo SY Park J Jung K Kim H Park SY Rho HS Kim S Kim BR Han SS Kang S Lee YH. Genome-wide analysis of T-DNA integration into the chromosomes of Magnaporthe oryzae. Molecular Microbiology T- systematic analysis of T-DNA insertion events in DNA. Magnaporthe oryzae. Fungal Genetics Biology Acta Microbiologica Sinica Chinese Journal of Rice Science Muller AE Atkinson RG Sandoval RB Jorgensen RA. Microhomologies between T-DNA ends and target sites often occur in inverted orientation and may be responsible for the high frequency of T-DNA-associated inversions. Plant Cell Reports Li GH Zhou ZZ Liu GF Zheng FC He CZ. Characterization of T-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae. Current Genetics Meng Y Patel G Heist M Betts MF Tucker SL Galadima N Donofrio NM Brown D Mitchell TK Li L Xu JR Orbach M Thon M Dean RA Farman ML. A Analysis of T-DNA integration pattern in Botrytis cinerea genome Jia Liu Jianyun Zhang Tingheng Zhu * Kun Wang Zhifeng Cui College of Biological & Environmental Engineering Zhejiang University of Technology Hangzhou China Abstract Objective To analyze the T-DNA integration pattern in the genome of grey mold Botrytis cinerea. Methods T-DNA Transfer DNA inserted mutant library of Botrytis cinerea was created by Agrobactirium tumfacience mediated transformation. By using TAIL-PCR Thermal asymmetric interlaced polymerase chain reaction we amplified and cloned the chromosomal regions flanking T-DNA insertions. The obtained T-DNA flanking sequences were subjected to alignment with standard T-DNA border sequence for identification and analysis of integration. Results Up to 69% T-DNA inserted at noncoding regions and 30% inserted at exons. Recombination including deletion or addition of bases in T-DNA region was observed. The right borders of the T-DNA were frequently truncated and by contrast the left borders were less prone to degradation and appeared to have been inserted in a relatively integrated manner. Extra sequence additions also occurred in T-DNA integration sites. Conclusion Analysis of T-DNA integration pattern in B. cinerea genome will stimulate the functional genomics study of this fungus. Keywords Botrytis cinerea T-DNA TAIL-PCR Supported by the Qianjiang Excellence Project of Zhejiang Province 2009R10030 * Corresponding author. Tel thzhu@ zjut. edu. cn Received 8 Octember 2010 / Revised 17 November 2010

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