**4. Discussion**

### *4.1. Constructing CSSLs in Guangxi Wild Rice (*O. rufipogon *Gri*ff*.) DP30*

Since QTL detection is based on the natural allelic differences between parental cultivars, it is important to select parental cultivars that show large phenotypic variation in the target traits [49]. There is rich polymorphism between Guangxi wild rice DP30 (*O. rufipogon* Griff.) and cultivated rice 93-11 (*O. sativa* L.) due to their distant genetic bases [10]. In this study, we performed MAS using Guangxi wild rice DP30 (*O. rufipogon* Griff) as a donor parent to develop 132 DP30-CSSLs. The coverage rate of the DP30 wild rice was 91.55%. The average length of each replacement segmen<sup>t</sup> was 0.5–22 Mb. Compared with the previously reported CSSLs in wild rice, the DP30-CSSLs had a higher substitution segmen<sup>t</sup> coverage rate and more polymorphic primers for MAS [50,51]. Clearly, the genomic constitution quality of the CSSLs population is important. The chromosome position and genetic effect of QTLs locating on dp30-CSSLs will be more accurately assessed.

### *4.2. Identification of Quantitative Trait Loci and Measurement of Various Traits in Fall and Spring*

Previous reports have shown that the CSSLs of wild rice are effective in the mining and transferring of wild alleles into cultivated rice [10]. Furthermore, several studies have reported the development of CSSLs and the identification of several agronomic and plant architecture traits [17,35–41]. Ma et al. (2019) detected eighteen QTLs were two known grain length- and width-related genes and four novel QTLs. In addition, two QTLs were verified, and two novel QTLs were identified, for panicle neck length, a domestication-related trait [49]. Tan et al. (2004) identified quantitative trait loci (QTLs) associated with plant height and the days to heading in the BC3 F2 population. Putative QTLs derived from O. *rufipogon* were detected for plant height on chromosome 1 and identified 6 QTLs for days to heading on chromosomes 1, 3, 7, 8 and 11 [50].

In the present work, we compared the phenotypes of DP30-CSSLs to the phenotypes of known genes and used related molecular markers to confirm whether any of these genes/allele genes were present in the segments. We identified five QTLs related to TA, *qTA7.1* and *PROG1* were in the same position on the chromosome; *qTA9.1* may be the allelic form of *TAC1* (Table 3). *PROG1* (LOC\_Os07g05900) controls the creeping growth habits of common wild rice [17]. *TAC1* (LOC\_Os09g35980) is a recently discovered gene that controls the TA of rice corresponding to a major QTL [20]. We found that these five QTLs had positive additive effects on TA (Table 3). A QTL named *qHD11.1* had negative additive effects on HD. Three of the QTLs identified in this study (Table 3) had positive additive effects for phenotypic variations in PH in both fall and spring. *qPH1.1* and *Sd1* were in the same position on the chromosome (Table 3). The *Sd1* (LOC\_Os01g66100) gene, which controls gibberellin biosynthesis, was among these QTLs [36]. Three QTLs showed negative additive effects on NGPP and *qGWT1.1* may be the allele of *OsAGPL2* (Table 3). *OsAGPL2* (LOC\_Os01g44220) is a member of the *OsPYL* gene family that regulates the filling rate of grains, leading to lower final grain weight and yields [37]. Except for the QTLs locus near the *OsAGPL2* gene, we also identified eight other QTLs. All of them showed negative additive effects on 100GWT in both seasons (Table 3). The *SG1* (LOC\_Os09g28520) gene has shorter grains than the wild type and a dwarf phenotype [39]. Similar to the *SG1 gene,* one new QTL we identified, which showed negative additive effects on GW (Table 3). We identified five AL-related QTL, *qAL4.1* and *qAL4.2* as alleles of *An-1* and *An-2*, respectively (Table 3). *An-1* (LOC\_Os04g28280) regulates the formation of awn primordia, promoting awn elongation and increasing the length of grains [40]. *An-2* (LOC\_Os04g43840) also increases the length of awns and makes them spinier (Figure S4) [41]. The QTL *qSH4.2*, which is related to grain shattering, the *SH4* (LOC\_Os04g57530) was in the same chromosome position [42] (Table 3). Cold tolerance in seedlings is one of the important traits for the stable production of rice [43–45]. Here, we identified seven QTLs related to cold tolerance including these loci in a similar region with these three previously cloned genes. *qCT1.1*, *qCT5.1* and *qCT6.1* may be the allelic form of *OsRAN1*, *OsiSAP8* and *OsPYL9*, respectively (Table 3). *OsRAN1* (LOC\_Os010611100) participates in cell division and the cell cycle and promotes the formation of intact nuclear membranes, thus improving the cold tolerance of rice [43]. *OsiSAP8* (LOC\_Os06g41010) is a zinc finger protein gene that enhances salt, drought and cold stress tolerance in rice [44]. *OsPYL9* (LOC\_Os06g33690) is a member of the *OsPYL* gene family and is a possible abscisic acid (ABA) receptor [45]. In addition, there are six quality trait loci, which contain three alleles [46–48]. The alleles of eleven cloned genes showed the reliability of DP30-CSSLs. No cloned genes were found in other QTL, which may contain new genes.

### *4.3. Construction of Secondary Population and Mapping of qCT2.1*

Map-based cloning and mapping of cold tolerance genes in rice have always been a classical method for cold tolerance research in rice.

Previous studies used di fferent populations to obtain some cold tolerance genes of rice [51,52]. According to the published data, more than 250 QTL of low-temperature tolerance has been found on 12 chromosomes of rice. In DP30-CSSLs, *qSCT-3-1* was identified in the RM15031-RM3400 region of the long arm of chromosome three near to the centromere, and the genetic distance between the linkage markers was found to be 1.8 cM [53]. In this study, F2 populations were constructed by Guangxi common wild rice seedling cold-tolerant segmen<sup>t</sup> replacement line RZ34 and cold-sensitive recurrent parent 93-11. Through map-based cloning, it was found that the main cold tolerance QTL *qCT2.1* of rice at the seedling stage was located on chromosome 2 and located in the range of 1.7 Mb between molecular marker dxw-4 and dxw-9. To date, there is no cloned cold tolerance gene at the seedling stage in this interval. *qCT2.1* could enhance cold tolerance at the seedling stage, which has a strong dominant e ffect, so it is expected to be used in rice breeding.

Rice breeding entered to a new era with the utilization of MAS and whole-genome sequencing to link genotypes with phenotypes. The introduction of wild rice CSSLs promoted gene QTLs mapping and genomic research. This study also suggests using Guangxi common wild rice accessions will provide a broad platform for genomic research and may lead to the discovery of new QTLs that will benefit rice breeding.
