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).