**1. Introduction**

Wild rice (*Oryza rufipogon* Griff.) contains many novel and useful alleles that control tiller number, shattering, dormancy, pericarp color, mating type, panicle architecture and grain size and number [1]. Therefore, the potentially beneficial genes in wild rice are an important goal to improve cultivated rice (*Oryza sativa* L.) [2]. Although many quantitative trait loci (QTLs), for plant architecture, agronomic traits and cold tolerance (CT) have been identified in rice [3–5], however, there are few reports on those traits that were discovered in same chromosome segmen<sup>t</sup> substitution lines (CSSLs). The hybridization between *Oryza sativa* and wild rice and use of marker-assisted selection (MAS) of

individuals retaining a part of the wild chromosome in the background revealed the transfer desirable genes to cultivated rice [6]. QTL identification through CSSLs is advantageous because it completely removes the genetic background interference and provides QTL visualization as a single Mendelian factor [7]. The development of CSSLs is laborious and time-consuming, but it is useful to scientists and plant breeders [8–10]. To date, several CSSLs in rice have been developed and many QTLs for traits of biologic and economic interest have been mapped [8]. The potential of the rice progenitor as a genetic resource has been explored for improving O. *sativa* with 33 chromosome segmen<sup>t</sup> substitution lines (CSSLs) of O. *rufipogon* (W0106) [9]. Single segmen<sup>t</sup> substitution lines (SSSLs) libraries also has been utilized to detect several QTLs related to plant height, heading date, seed setting rate and 1000-grain weight [10]. These achievements have undoubtedly enhanced the understanding of complex traits and encouraged plant genomic studies.

Some QTLs and genes for plant architecture traits and agronomic traits have also been detected from CSSLs [11,12]. *GL4* can control the grain length and seed shattering ability of African cultivated rice by regulating the longitudinal cell elongation of the outer glume and inner glume [11]. *RLS3* plays an important role in regulating chloroplast degradation and the normal growth of rice [12]. The additive effect, main effect and epistatic effect of QTLs were also studied based on CSSLs. In a previous study, eight SSSLs as experimental material were utilized to estimate the additive and dominant effects of six QTLs (*Hd1, Ehd1, Hd3a or RFT1, EH3, OsMADS50* and *DTH8*) and their epistatic effects among dual QTLs [13].

Cold injuries have been observed at many stages of growth and may result in the failure of germination, retarded seedling growth, stunting, discoloration, panicle tip degeneration, a prolonged duration of cultivation, sterility and irregular maturity [14]. Previous studies identified strong interactions between the cold tolerant QTLs and their environments [15,16]. Therefore, the screening of cold-tolerant germplasm and identifying the QTLs related to cold tolerance will help reduce the losses caused by low temperatures and improve rice production in marginal lands.

In this study, a broad population of DP30-CSSLs was constructed via the backcrossing, selfing and masker-assisted selection (MAS) of cultivated rice (93-11) and Guangxi wild rice (*Oryza rufipogon* Griff. DP30). By performing high-throughput whole genome sequencing, we designed 285 InDel molecular markers that were evenly distributed across the twelve chromosomes of rice. These molecular markers were utilized to select 132 substitution lines through MAS. The rate of coverage of the substitution segments to the whole genome of DP30 wild rice was 91.55%. Meanwhile, we investigated eighteen traits found in the CSSLs. thirty-six QTLs, some of which have been found in previous studies while others are new. Furthermore, we selected a cold-tolerant line (RZ34) to construct a secondary population and *qCT2.1* was located in a 1.7 Mb region on chromosome 2.
