Edta na2

Fresh leaf tissues were collected from Cremastra appendiculata, Calanthe davidii, Epipactis mairei, and Platanthera japonica in the Qinling Mountains, Shaanxi Province, China. The leaves were cleaned and preserved in a −80 °C refrigerator at Northwest University. The voucher specimens of the four species materials were deposited into the Northwest University Herbarium (NUH). The total genomic DNA was isolated using the modified Cetyltrimethyl Ammonium Bromide (CTAB) method [69 ], which added the EDTA buffer (Amresco, Washington, DC, USA) (1.0 mol/L Tris-HCl (Amresco, Washington, DC, USA) (pH 8.0), 0.5 mol/L EDTA-Na₂ (Amresco, Washington, DC, USA), 5.0 mol/L NaCl) solution before isolating the high-quality DNA with the CTAB solution (1.0 mol/L Tris-HCl (pH 8.0), 0.5 mol/L EDTA-Na₂, 2% CTAB). Following this, we constructed a pair-end (PE) library with 350 bp insert size fragments using TruSeq DNA sample preparation kits (Sangon, Shanghai, China). Subsequently, we sequenced at least 4.5 GB of clean data for each orchid species. The detailed next-generation sequencing was conducted on the Illumina Hiseq 2500 platform by Sangon Biotech (Shanghai, China).

Following overnight fasting, a 5 ml venous blood sample was collected from the elbow, anticoagulated with EDTA Na₂ (Amresco, LLC, Solon, OH, USA) and stored at −80°C for the subsequent experiments. Genomic DNA was extracted from the blood using a TIANamp genomic DNA kit (Tiangen Biotech, Beijing, China), according to the manufacturer’s instructions, and the concentration was determined using ultraviolet spectrophotometry (UV3100; Hitachi, Ltd., Tokyo, Japan). PCR amplification of the GDF9, BMP15, FSHR and INHBB genes was performed using the primers shown in Table I, in a 25 μl reaction volume containing 1 μl DNA template, 12.5 μl PCR mix, 0.75 μl forward and reverse primers, 0.25 μl Taq DNA polymerase and 10.5 μl ddH₂O, under the PCR amplification conditions shown in Table I. The PCR was conducted on an ABI 3100 sequencer (Applied Biosystems, Foster City, CA, USA). The PCR products were confirmed using gel electrophoresis on a 2% agarose gel (Sangon Biotech Co., Ltd., Shanghai, China). All PCR reagents, primers and kits were purchased from Sangon Biotech Co., Ltd. Subsequently, the PCR products were sequenced for GDF9, the BMP15 gene protein coding region, INHBB gene exon 2 and the FSHR Ala307Thr and Ser680Asn variants at the Beijing Genomics Institute (Beijing, China) using a dideoxy chain termination method (27 (link)).