*Article* **Genetic Dissection of Germinability under Low Temperature by Building a Resequencing Linkage Map in** *japonica* **Rice**

**Shukun Jiang 1,\*,**†**, Chao Yang 2,**†**, Quan Xu 3, Lizhi Wang 1, Xianli Yang 1, Xianwei Song 2, Jiayu Wang 3, Xijuan Zhang 1, Bo Li 1, Hongyu Li 4, Zhugang Li 1,\* and Wenhua Li 1,\***


Received: 25 January 2020; Accepted: 13 February 2020; Published: 14 February 2020

**Abstract:** Among all cereals, rice is highly sensitive to cold stress, especially at the germination stage, which adversely impacts its germination ability, seed vigor, crop stand establishment, and, ultimately, grain yield. The dissection of novel quantitative trait loci (QTLs) or genes conferring a low-temperature germination (LTG) ability can significantly accelerate cold-tolerant rice breeding to ensure the wide application of rice cultivation through the direct seeding method. In this study, we identified 11 QTLs for LTG using 144 recombinant inbred lines (RILs) derived from a cross between a cold-tolerant variety, Lijiangxintuanheigu (LTH), and a cold-sensitive variety, Shennong265 (SN265). By resequencing two parents and RIL lines, a high-density bin map, including 2,828 bin markers, was constructed using 123,859 single-nucleotide polymorphisms (SNPs) between two parents. The total genetic distance corresponding to all 12 chromosome linkage maps was 2,840.12 cm. Adjacent markers were marked by an average genetic distance of 1.01 cm, corresponding to a 128.80 kb physical distance. Eight and three QTL alleles had positive effects inherited from LTH and SN265, respectively. Moreover, a pleiotropic QTL was identified for a higher number of erected panicles and a higher grain number on Chr-9 near the previously cloned *DEP1* gene. Among the LTG QTLs, *qLTG3* and *qLTG7b* were also located at relatively small genetic intervals that define two known LTG genes, *qLTG3-1* and *OsSAP16*. Sequencing comparisons between the two parents demonstrated that LTH possesses *qLTG3-1* and *OsSAP16* genes, and SN-265 owns the *DEP1* gene. These comparison results strengthen the accuracy and mapping resolution power of the bin map and population. Later, fine mapping was done for *qLTG6* at 45.80 kb through four key homozygous recombinant lines derived from a population with 1569 segregating plants. Finally, *LOC\_Os06g01320* was identified as the most possible candidate gene for *qLTG6*, which contains a missense mutation and a 32-bp deletion/insertion at the promoter between the two parents. LTH was observed to have lower expression levels in comparison with SN265 and was commonly detected at low temperatures. In conclusion, these results strengthen our understanding of the impacts of cold temperature stress on seed vigor and germination abilities and help improve the mechanisms of rice breeding programs to breed cold-tolerant varieties.

**Keywords:** *japonica* rice; cold stress; germinability; high-density linkage map; QTLs

#### **1. Introduction**

Rice (*Oryza sativa* L.), which is a staple food and nutritional source for many countries, fulfills the nutritional requirements for over half of the world's population and is cultivated across the globe, except in a few areas where icy conditions prevail during most of the year [1]. Since rice originated in tropical and sub-tropical climates, it is one of the most sensitive cereals to cold stress [2], which limits its growth, development, and yield formation, especially when cold stress prevails at the germination stage [3]. Cold stress impacts all growth stages of rice, including tillering, booting, flowering, and grain-filling, but if stress dominates at the germination stage, it proves adverse for rice development at later growth stages. Low-temperature stress during the germination stages of rice affects seedling vigor and produces poor seedling emergence and an uneven stand establishment with a lower growth rate, which delays panicle development and enhances spikelet sterility [4]. Across China's mainland, most of the rice cultivation areas are affected by frequent cold stresses. The Chinese agricultural sector suffers from an average loss of rice of about 3–5 million tons of rice every year due to these frequent cold stresses [5]. Two kinds of cold stresses occur in Chinese rice-growing regions. The (1) "cold spring" and (2) "cold autumn wind" often cause severe yield losses in double-cropping rice regions across the Yangtze River in China. In Northeast China (NEC), commonly considered a rice region at a high latitude, and the Yunnan-Guizhou Plateau, considered a rice cultivation region at a low latitude, severe cold summer damage was observed, with an average of three to four years of cold stress. These areas are expected to encounter more severe damage in the near future due to low temperature stress [6].

Traditional genetic and molecular analyses on Arabidopsis, rice, and other model plants have revealed that C-repeat binding factors (CBFs) are mainly involved in the cold signaling pathways. Recent studies have further revealed that the protein kinases and transcription factors are also involved in cold signaling in plants [7]. Additionally, genetic research on rice has detected numerous quantitative trait loci that control cold tolerance on nearly all 12 chromosomes [8]. Among these loci, only a few quantitative trait loci (QTLs) have been thoroughly researched and cloned, while the functional mechanisms of most are still largely unknown. Among all the QTLs for low-temperature germination in rice, only *qLTG3-1* and *OsSAP16* were cloned. *qLTG3-1* encodes a protein with glycine-rich and lipid trans-protein domain structures [9], and *OsSAP16* encodes a zinc-finger protein that positively regulates germination under low temperatures [10]. The QTLs *qCTS7*, *LTG1*, *COLD1*, *qCTS9*, *bZIP73*, *qPSR10*, and *HAN1* control the pathways for cold tolerance in rice. *qCTS7* increases cold tolerance at the seedling stage due to its overexpression [11]. *LTG1* encodes a casein kinase that plays a role in regulating the rice growth rate under cold stress [4]. A regulator of Ca2<sup>+</sup> signaling in the plasma membrane and endoplasmic reticulum is encoded by *COLD1* [12], whereas a novel protein that interacts with Brassinosteroid Insensitive-1 is encoded by *qCTS9* [13]. There is a functional interaction between *bZIP73* and *bZIP71* that makes rice seedlings tolerant to greater cold [14]. A single-nucleotide polymorphism (SNP), SNP2G, at position 343 in *qPSR10*, is responsible for conferring cold tolerance during the seedling stage [15]. *HAN1* encodes an oxidase that provides functional contributions to the Jasmonic acid mediated cold response in temperate *japonica* rice [16]. The other three QTLs, *Ctb1*, *CTB4a*, and *bZIP73*, control cold tolerance at the booting stage. The first encodes an F-box protein [17], the second encodes a leucine rich repeat kinase that enhances seed setting through increased ATP-synthase activity under low temperature stress [3], and the third increases the cold tolerance rate by enhancing the soluble sugar transport from anthers to pollens at the booting stage [18].

Advances in genome-wide sequencing technology have provided an effective method to detect DNA sequencing differences among closely related rice materials and to ensure the presence of sufficient markers for a genetic mapping analysis. A genotype calling method for RILs that utilizes resequencing data was developed [19], which determined the construction of resequencing bin maps and accelerated genetics-based studies for many crops, including cereals [19–27]. Based on the above discussions, many important advances have been achieved in the study of rice cold stress, but we still need to use high-throughput sequencing technology to mine further cold-tolerance genes from *japonica* rice, especially cold tolerance genes at the germination stage for breeding practice.

The current study was arranged with the following objectives: (1) constructing a high density bin map by re-sequencing a set of 144 RILs with large differences in germination abilities under cold stress; (2) identifying QTLs for LTG in RIL populations by using the built linkage map; and (3) creating an accurate map of *qLTG6*, with a high low-temperature germinability (LOD) score and relatively small genetic intervals.
