**3. Results**

### *3.1. Phenotypic Variation of Plant Architecture, Agronomic Traits and Cold Tolerance in DP30-CSSLs*

We evaluated the variation in plant architecture, agronomic and cold tolerance traits (DP30, DP30-CSSLs and 93-11) and calculated the phenotypic values of these traits during two seasons (fall and spring). The DP30-CSSLs showed significant variation in all of these traits. Furthermore, this variation in HD, GWT, GL, GW, GLWR and AL were normally distributed, while TA, PH, NGPP, SH and CT followed biased distribution (Figure S1). First, we speculated that the biased distribution was due to continuous backcrossing, that the CSSLs without related trait substitution segments were similar to 93-11. Second, we counted 132 lines, so an insufficient population may have led to biased distribution. Third, these distributions were frequently skewed because of interactions between the alleles, nonallelic genes or other environmental factors.

### *3.2. Genome Re-Sequencing and Selection of InDel Markers*

The DP30 genome was sequenced by Illumina high-throughput sequencing technology, and the resulting sequence was assembled using IRGSP-1.0, with the 93-11 genome as a reference (Figure 1A). The DP30 genome is displayed according to the input reads (Table S1). The sequencing results reveal that 1,894,103 bp across all 12 chromosomes were different in DP30 and 93-11, representing 6.1% of the genome (Figure 1B). InDel molecular markers were designed such that the base mutation exceeded 20 bp. Based on the lengths of the PCR products (150–200 bp) and the results of gel electrophoresis, we selected 285 InDel markers with an average distance of 1.2 Mb (Table 1). The sequences of the 285 InDel markers (Table S2).

**Figure 1.** Whole-genome sequencing of DP30 and genome-wide sequence difference. (**A**) Circos plot for SNPs and InDels detected in 93-11 and DP30. The outermost circle depicts the ideogram of the 12 chromosomes in the rice genome. The innermost lines represent the frequency SNPs and InDel markers identified between DP30 (blue) and 93-11 (red); (**B**) genome-wide distribution of sequence differences between 93-11 and DP30. Red and blue colors represent the degree of variation between DP30 and 93-11. Depth of the color indicates the level of variation frequency.


**Table 1.** Number of single-nucleotide polymorphism (SNPs) detected in wild rice (DP30).

SNP-No—number of SNPs detected in DP30; Chr.-length (bp)—chromosome length; Length (Mb)—average length between adjacent markers in each chromosome; SNP Percentage—percentage of SNP; Marker No.—the number of molecular markers on each chromosome; Mb—million bp.

### *3.3. Development and Substitution Segment Analysis of DP30-CSSLs*
