| To clone an avirulence gene cluster containing Avr-Pi1, Avr-Pi2 and Avr-Pi4a in Magnaporthe grisea, we previously screened BAC library of 81278 by using molecular marker SC1088, and obtained four positive clones (805L15, 807P11, 809L6 and 811C13). In this study, we subcloned three of them, and sequenced some of subclones. After blasting the sequences in Magnaporthe grisea databases, we found the sequence of subclones of 805L15 were homologous to different supercontig, and the same in the 807P11 and 809L6. According to the sequence of subclones, we developed a polymorphic marker P807-11. P807P11 is linked to the Avr gene cluster and the genetic distance between P807P11 and Avr-Pi1, Avr-Pi2, Avr-Pi4a is 43.2 cM, 50.0 cM and 33.8 cM respectively.According to the genetic map of the Avr cluster and the molecular markers P807-11, BAC809L6, the BAC clone 805L15 was selected to sequence. According to the sequence data, we developed five polymorphic markers (L230, L891-2, L469-1, L469-3, L213-1) and an SSR marker S85-1, which were all linked to the Avr cluster. Among these markers, L469-1, BAC809L6 and L213-1 were located on one side of the Avr cluster (Avr-Pi2), and L230, L469-3, S85-1 and P807-11 were on another side of the Avr cluster (Avr-Pi4a). The closest markers were L213-1 and L230 on both side of the Avr cluster. The genetic distance between L213-1 and Avr cluster, L230 and Avr cluster was 10.2 cM and 15.4cM respectively. Marker L891-2 was located on the Avr cluster, and the genetic distance between L891-2 and Avr-Pi1, Avr-Pi2, Avr-Pi4a was 9.5 cM, 9.5 cM and 11.4 cM respectively. Thus, the linkage group, which was composed of the Avr cluster and markers L213-1, L230, covered a map distance of 46.2 cM. The results further confirmed that the Avr cluster was located on BAC clone 805L15, and the Avr cluster was in between the 805L15-t7 BAC end and the marker L230.Based on analysis, 48 genes were annoated from the sequence of BAC clone 805L15, among which eight are predicted as extracellular secreted protein, and one is as GPI-anchor protein.The predicted secreted protein and GPI anchor protein were then cloned into pCX62 (with native promotor) or pDL1 (only ORF) by using yeast homologous recombination. In total, 11 constructs named SP1-pCX62, SP4-pCX62, SP5-pCX62, SP6-pCX62, SCP9-pCX62, SP1-pDL1, SP4-pDL1, SP5-pDL1, SP6-pDL1, SP7-pDL1 and SP8-pDL1 were obtained. The BAC clone 805L15 were then subcloned into 9 cosmid clones Swaâ… -2, Swaâ… -4, Swaâ… -7, Swaâ… -8, Swaâ… -10, Notâ… -1, SgrAâ… -2, Fseâ… -2 and Fseâ… -3. All these constructs were transformed into protoplasts of virulent strain GUY11. Independent hygromycin-resistant transformants of different constructs were obtained, except for Swaâ… -2 construct. All hygromycin-resistance transformants and GUY11 were used for pathogenicity test on CO39 near-isogenic lines of rice. However, the pathogenicity of all transformants were the same to GUY11, suggesting that the avirulence genes may not be present or could not express normal fuction in tested transformants. |
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