DNA extraction, PCR, nucleotide sequencing and microsatellite genotyping
Genomic DNA was extracted from the powdered leaf tissue following a CTAB procedure (Doyle, 1991). The DNA was dissolved in sterile, deionized water and stored at -20 ̵̊C waiting for further process. We screened three noncoding spacers of cpDNA: atp B-rbc L (Sang, Crawford, & Stuessy, 1997), psb A-trn H (T. Y. Chiang & Peng, 1998) andpsb B-psb H (Shaw et al., 2005) and five low copy nuclear genes (Yi Qing Gong & Gong, 2016): PHYP (phytochrome P),AC 5 (actin 5), PPRC (pentatricopeptide repeat-containing protein), AAT (aspartate aminotransferase) and SAMS(S-adenosylmethionine Synthetase) for further amplification and sequencing. The primer, reaction mixture and program of PCR for cpDNA were the same with the previous study of C. multipinnata (Y. Q. Gong et al., 2015), and the information of the primers for nuclear genes was the same as a previous study (Table S1) of C. debaoensis (Yi Qing Gong & Gong, 2016). PCR products were checked by electrophoresis in 1% agarose gel using 1 × TAE buffer. Then the purified amplified products were initially directly sequenced using the amplification primers, using an ABI 3730 automated sequencer at Shanghai Majorbio Bio-pharm Technology Co., Ltd. All sequences were deposited in GenBank with the accession numbers: OM451238-OM451426.
Individuals were genotyped at 16 microsatellite loci, which were selected from 65 primers published for Cycas . One primer of each pair was end-labeled with a fluorescent tag (FAM, TAM or HEX). The PCR mixture and conditions were the same to that of C. multipinnata(Y. Q. Gong et al., 2015). PCR products were mixed with a fluorescent size standard, ROX 500, and run on an ABI 3730xl Capillary DNA Analyzer by Sangon Biotech (Shanghai) Co., Ltd. Fragment length sizes were assessed using GeneMapper version 4.0 (Applied Biosystems, Foster City, CA).