*2.2. Phenotyping*

The phenotypic data were recorded under natural conditions in the experimental area of Guangxi University (Guangxi, China; 22◦38 N, 108◦13 E) in spring (from March to July) and fall (from July to November). A randomized complete block design (RCBD) was used with three replications for CSSLs, CSSL combinations and their parents. The parent plants and 20 CSSL plants were selected from each replication for data collection. The data for 17 traits were recorded, including leaf sheath color (LSC), leaf margin color (LMC), tiller angle (TA), heading date (HD), plant height (PH), grain shattering (SH), apiculus color (AC), stigma color (SC), glume color (GC), number of grains per panicle (NGPP), 1000-grain weight (GWT), grain length (GL), grain width (GW), grain length to width ratio (GLWR), awn length (AL), seed coat color (SCC) and cold tolerance (CT). The data for TA was recorded on a scale from 1 to 9 representing the angles of 0–10◦, 11–20◦, 21–30◦, 31–40◦, 41–50◦, 51–60◦, 61–70◦, 71–80◦ and 81–90◦, respectively [17–20]. The HD was recorded when the first panicle to emerge reached about 2-cm-long and the number of days from sowing to heading were scored for each plant. PH was measured for each plant at the mature stage from the base of the stem to the tip of the higher panicle. NGPP, GL and GW were recorded according to the previously established methods [19,20]. The GLWR and 100-GWT of the filled grains were investigated after the rice was harvested at the mature and naturally dried stage [10].

### *2.3. Evaluation of Cold Tolerance at Seedling Stage*

To preliminarily evaluate the cold tolerance variation of CSSLs, the experiment was conducted in controlled conditions at seedling stage. Thirty seedlings of each CSSL were planted in soil. Plants were grown in a controlled environment at a day temperature of 25 ◦C and night temperature of 19 ◦C till three leaf stage. At three leaf stage, seedlings were exposed to cold stress according to a previously established method [21]. The depth of the water was about 5 cm measured from the surface of the soil in the trey. The cold stress treatment lasted 5 days and the conditions alternated between 10 ◦C for 10 h during the day and 8 ◦C for 14 h at night. After the cold treatment, the seedlings were subjected to natural standard growth conditions at 26 ◦C and the survival rate was investigated. After 5 days of treatment, cold tolerance was evaluated on the basis survival rate and injury level. The experiment was repeated three times under the same cold stress treatment. The average data of three replications were used. The data for CT were recorded on a scale from 0 to 9, representing the survival rates of 0–9%, 10–19%, 20–29%, 30–39%, 40–49%, 50–59%, 60–69%, 70–79%, 80–89% and 90–100%, respectively [21]. The secondary mapping population used to fine-map the major QTL for cold tolerance was developed by backcrossing a CSSL(RZ34) with the recipient parent (93-11). Three hundred and eleven F2 plants of that cross were genotyped using five InDel markers and fifty-seven cold tolerant and twenty-one cold sensitive plants selected for phenotypic evaluation under cold stress treatment.

### *2.4. Construction of CSSLs and Genome Sequencing and Development of InDel Markers*

CSSLs of common wild rice were constructed by hybridization, backcrossing and marker-assisted selection (MAS) according to the previously described method [22,23]. The genomic DNA of DP30 and 93-11 was prepared and whole-genome re-sequencing (WGRS) was performed on an Illumina HiSeq2500™ by Novo Generation Company, Beijing, China. The standard Illumina protocol was followed for sample preparation and sequencing. The quality trimming (phred quality score, <Q20) was carried out by using FastQC [24] and the Cutadapt software was used for adapter trimming with the parameters of −O 5 and −m 32 [25]. The Burrows–Wheeler Aligner (BWA) software was used to map clean reads to the 93-11-reference genome [26,27]. InDel polymorphisms were detected by the GATK tool software with the defined length of insertions and deletions between 1 bp and 10 bp [28]. The larger sized (≥2 bp) InDel regions and high sequencing depths (DP, ≥5-fold) were extracted to design the InDel markers. The primer pairs were designed based on parental sequence differences by the DNAMAN v6.0 software and screened using the NCBI (https://www.ncbi.nlm.nih.gov/) database.
