Cloning, sequencing and clone library construction

Our cloned region was part of the SSU rDNA gene (length ~550 bp). Surface soil samples (0–20 cm depth) were used for AMF T-RFLP profile database construction. Primer pair (without fluorescence labeling) and PCR cycling parameters of the clone-sequencing procedure were similar to those described above. Twelve soil samples (3 P treatments, 4 replicates) were amplified with NS31/AM1 and the 4 replicated PCR products of each P treatment were pooled together to form one sample for clone library construction. In this study three soil sample clone libraries were built to distinguish the identification of T-RFs.

Cloning was conducted by the method of Liu et al.²⁴ and Lee et al.⁵⁶. In each clone library, clones containing the converted DNA fragments were selected by blue/white screening and 96 white clones were randomly picked up. All clones were sequenced by ZhongKeXiLin Biotechnology Company (ABI 3730XL, Beijing, China). After sequencing, each positive clone was used as a template for PCR amplification (NS31-HEX/AM1-FAM) and was digested and T-RFLP analysis was performed. AMF sequences obtained from the clone library were also used in simulated restriction endonuclease enzyme digestion using ChromasPro software using Hinfl and Hin1II. Unknown T-RFLP profiles were matched with profiles of sequenced clones and all T-RFs within 1.5 base pairs were required to be detected for a positive match. T-RFLP detected all or almost all terminal restriction fragments predicted from the cloned sequences. This step allowed us to conclude with confidence that all peaks taken into account were true T-RFs even when they were of low intensity.