3.3.2. Substitution Segments of DP30-CSSLs

In total, we identified 80 BC4 F2 plants from 171 CSSLs and named as DP30-CSSLs. Then, 24 BC4 F2 plants were selected and 44 BC5 F2 plants were obtained after continuous backcrossing. Finally, 18 BC6 F2 were obtained with a relatively complex genetic background to continue backcrossing. The 132 CSSLs formed a DP30-CSSLs population, and the target replacement segments of these CSSLs accumulated a total length of 536.4 Mb, covering 91.42% of the DP30 genome. The CSSLs were arranged according to the position of their target substitution segments. The 99 substitution lines contained only one substitution segmen<sup>t</sup> from DP30. The total length of these CSSLs substitution segments was 359.66 Mb and the coverage rate was 61.36% (Figure 3).

**Figure 3.** Graphic genotypes of the 132 DP30-CSSLs developed in this study. Red bars indicate homozygous substituted segments derived from DP30. Blue bars indicate heterozygous substituted segments derived from DP30. Gray bars indicate the genetic background of the recipient parent 93-11, "Chr."—chromosome.

Chromosomes with more than 90% coverage of the DP30 genome by chromosome substitution segments include chromosomes 1, 2, 3, 5, 7, 11 and 12. Among them, chromosome 5 had the highest coverage rate (97%), while the lowest coverage rate belonged to chromosome 8 (Table 2).

The average length of DP30-CSSLs population substitution target segmen<sup>t</sup> was 4.06 Mb, which ranges from 0.5–22 Mb. The length of the substitution segmen<sup>t</sup> of 25 CSSLs was less than 2 Mb; The length range of substitution segments of 55 substitution lines was 2–4 Mb and 30 CSSLs were 4–6 Mb and the length of the substitution segmen<sup>t</sup> of 6 CSSLs was larger than 8 Mb.


**Table 2.** Substitution segmen<sup>t</sup> analysis in DP30-CSSL.

CSSLs No.—number of substitution lines on chromosomes; Chr.-Length (Mb)—chromosome length; Coverage length (Mb)—covering length of the substitution segmen<sup>t</sup> to the chromosome; Percentage (%)—coverage of the substitution segment.

### *3.4. Wild rice QTLs in the DP30-CSSLs*

We detected the genotypes of all CSSLs in DP30-CSSLs (Table S3). In total, 36 QTLs were identified in the CSSLs.

### 3.4.1. Tiller Angle (TA) and Heading Date (HD)

Four QTLs (*qTA1.1, qTA7.1, qTA9.1* and *qTA2.1*) related to TA were detected in the CSSLs. Seven CSSLs, (RZ18, RZ20, RZ38, RZ104, RZ129, RZ130 and RZ168) had di fferent TAs those of 93-11 (Figure S2A,B). *qTA1.1* was located in the overlapping segmen<sup>t</sup> of RZ18 and RZ20, which belong to C1-26–C1-27 in chromosome 1. *qTA7.1* was detected in an overlapping segmen<sup>t</sup> of RZ104 and RZ168 that located in C7-2 on chromosome 7, the *PROG1* was also in the same position of chromosome [17]. *qTA9.1* and *TAC1* were detected in overlapping segments of RZ129 and RZ130, which was in the C9-14–C9-15 region of chromosome 9 [20] (Table 3). The TAs of the RZ38 plants were between 0◦ to 10◦, while 93-11 plants had TAs of 30◦ to 15◦, in both seasons (Figure S1A,B). The QTL *qTA2.1* in the segmen<sup>t</sup> of RZ38 was located near the molecular marker C2-25 on chromosome 2 (Table 3; Figure S6).

Three CSSLs (RZ142, RZ8 and RZ13) showed significant di fferences in HD when compared with 93-11 (Figure S2C). These three lines had an overlapping segmen<sup>t</sup> near C11-4 on chromosome 11, so we identified a new QTL (*qHD11.1*) in this region that has negative additive e ffects on HD (Table 3; Figure S6).


**Table 3.** List of 36 QTLs identified from DP30-CSSLs.


**Table 3.** *Cont.*

List of wild rice QTLs identified in this study. Position—physical position of molecular markers on chromosomes (Mb); length—length of the overlapping part of the lines; a—additive effect; Gene— cloned genes in the overlapping segments. TA— tiller angle; HD—heading date; PH—plant height; NGPP—number of grains per panicle; GWT—1000 grain weight; GL—grain length; GW—grain width; GLWR—grain length to width ratio; AL—awn length; SH—grain shattering; and CT—cold tolerance.
