*2.3. DNA Extraction and Marker Analysis*

Booting stage plant leaves were gathered for use in extracting genomic DNA, and extraction was performed based on the method of Causse et al. (1994) with some minor modifications [30]. The Cetyl trimethylammonium bromide (CTAB) method was used to extract plant DNA. Leaf fragments from each plant were placed in a 2.0-mL microcentrifuge tube with aluminum beads (4 mm) and 500 μL of CTAB extraction bu ffer added. The tubes were incubated at 55 ◦C in a dry oven in CTAB bu ffer (2% CTAB, 100 mM pH 8.0 Tris-HCI, 20 mM EDTA, 1.4 M NaCl, 0.2% β-mercaptoethanol) for 30 min right after grinding. An equal volume of chloroform was added and the solution was mixed well. The tubes were centrifuged at 4 ◦C for 15 min at 13,000 rpm. The 200-μL upper supernatant layer was transferred into a new 1.5-mL tube and mixed with 2/3 volume of isopropanol, and after gently mixing, incubated at 4 ◦C for 15 min at 12,000 rpm. The supernatant was discarded very carefully, and the DNA pellets were washed with 70% ethanol once. After washing the DNA pellets, they were kept at room temperature to dry out the rest of ethanol and then diluted in 100 μL TE bu ffer (10 mM Tris pH 8, 1 mM EDTA). The concentration of the DNA was checked with a NanoDropTM 2000 spectrophotometer (Thermo Scientific Inc., Wilmington, DE, USA).

A 15-μL of PCR reaction mixture containing 5 μL DNA template (2~5 ng/μL), 1 μL of forward and reverse primer (10 pmol each), 1.5 μL of 10× PCR bu ffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.1% Gelatin), 1 μL of dNTP (2.5 mM each), 0.1 μL of *Taq* polymerase (5 unit/μL), and 6.4 μL triple distilled water was used. The PCR was performed as described in Panaud et al. (1996) with minor modifications: 94 ◦C for 5 min, followed by 33–35 cycles of 94 ◦C for 30–40 s, 52–60 ◦C (based on annealing temperature of each marker) for 30 s, and 72 ◦C for 30 s, and a final extension at 5 min at 72 ◦C [31]. Separation of PCR products was conducted on 3% of metaphor agarose gel stained with Staining Safe Nucleic Acid Gel Stain (RBC, Taiwan). A total of 18 markers, including 15 Simple Sequence Repeat (SSR) and 3 Insertion-Deletion (InDel) markers were used to map QTLs (Table S1). Three InDel markers (qLTG3-1\_18D, qLTG10\_InDel3, and qLTG10\_InDel4) and one SSR marker (CRM22) were designed and used for mapping [27,28].
