*Article* **Salt Tolerance Improvement in Rice through E**ffi**cient SNP Marker-Assisted Selection Coupled with Speed-Breeding**

**Md Masud Rana 1,2, Takeshi Takamatsu 1,3, Marouane Baslam <sup>3</sup> , Kentaro Kaneko <sup>1</sup> , Kimiko Itoh 1,3, Naoki Harada 1,3, Toshie Sugiyama 1,3, Takayuki Ohnishi <sup>4</sup> , Tetsu Kinoshita <sup>5</sup> , Hiroki Takagi <sup>6</sup> and Toshiaki Mitsui 1,3,\***


Received: 19 April 2019; Accepted: 22 May 2019; Published: 26 May 2019

**Abstract:** Salinity critically limits rice metabolism, growth, and productivity worldwide. Improvement of the salt resistance of locally grown high-yielding cultivars is a slow process. The objective of this study was to develop a new salt-tolerant rice germplasm using speed-breeding. Here, we precisely introgressed the *hst1* gene, transferring salinity tolerance from "Kaijin" into high-yielding "Yukinko-mai" (WT) rice through single nucleotide polymorphism (SNP) marker-assisted selection. Using a biotron speed-breeding technique, we developed a BC3F<sup>3</sup> population, named "YNU31-2-4", in six generations and 17 months. High-resolution genotyping by whole-genome sequencing revealed that the BC3F<sup>2</sup> genome had 93.5% similarity to the WT and fixed only 2.7% of donor parent alleles. Functional annotation of BC3F<sup>2</sup> variants along with field assessment data indicated that "YNU31-2-4" plants carrying the *hst1* gene had similar agronomic traits to the WT under normal growth condition. "YNU31-2-4" seedlings subjected to salt stress (125 mM NaCl) had a significantly higher survival rate and increased shoot and root biomasses than the WT. At the tissue level, quantitative and electron probe microanalyzer studies indicated that "YNU31-2-4" seedlings avoided Na<sup>+</sup> accumulation in shoots under salt stress. The "YNU31-2-4" plants showed an improved phenotype with significantly higher net CO<sup>2</sup> assimilation and lower yield decline than WT under salt stress at the reproductive stage. "YNU31-2-4" is a potential candidate for a new rice cultivar that is highly tolerant to salt stress at the seedling and reproductive stages, and which might maintain yields under a changing global climate.

**Keywords:** *hst1*; Na<sup>+</sup> accumulation; SNP; rapid generation advance; salt tolerant; variant annotation; whole-genome sequencing

### **1. Introduction**

Projected climate change will aggravate a variety of abiotic stresses of rice plants, including salinity, heat, drought, and submergence, thus reducing world rice production [1–4]. At the same time, we must increase global rice production by at least 70% to feed the anticipated 9.6 <sup>×</sup> <sup>10</sup><sup>9</sup> people by 2050 [5,6]. Under these conditions, the improvement of the salinity tolerance of locally grown high-yielding rice cultivars is one of the most promising breeding objectives by which to meet global food demand.

Rice is considered the most salt-sensitive cereal crop [7], with a threshold EC<sup>e</sup> (electrical conductivity of saturated extract) of 3 dSm−<sup>1</sup> , above which yield starts to decline [8–10]. Salinity imposes osmotic effects, ion toxicity, and nutritional imbalance and substantially affects almost all phases of growth [7,11]. Possible salt tolerance mechanisms in rice involve ion homeostasis and compartmentalization, ion transport and uptake, biosynthesis and accumulation of osmoprotectants, osmolytes, and compatible solutes, activation of antioxidant enzymes for ROS detoxification, and hormone modulation [12–17]. The *Saltol* [18,19] and *SHOOT K*<sup>+</sup> *CONCENTRATION 1* [20,21] genes have been identified from major quantitative trait loci (QTLs) of salt-tolerant landraces Pakkali and Nona Bokra, respectively. These QTLs have been introgressed into some widely grown, high-yielding rice cultivars to improve salt tolerance [22–26], but the rate of improvement is slow.

Rice biotechnology has made advances in identifying single nucleotide polymorphisms (SNPs) controlling salinity tolerance [27–31]. Mutant lines of "Hitomebore" were generated by treatment with ethyl methanesulfonate (EMS), an inducer of nucleotide substitutions, and isolated a salt-tolerant line carrying the *hitomebore salt tolerant 1* (*hst1*) gene. The causative SNP conferring the high salinity tolerance of the *hst1* mutant line corresponded to the third exon of the *Os06g0183100* gene, which is predicted to encode a B-type response regulator designated *OsRR22*. We backcrossed the *hst1* line with "Hitomebore" to breed the salt-tolerant cultivar "Kaijin", with a yield ability of 5.88 t ha−<sup>1</sup> [32,33]. "Yukinko-mai" is an early-maturing standard cultivar derived from a cross between "Yukino-sei" and "Domannaka" at the Niigata Agricultural Research Institute's Crop Research Center; it has a high yield potential of 6.84 t ha−<sup>1</sup> [34] and is tolerant to high temperatures during grain filling [35].

To combat earthquake- and tsunami-induced soil salinity in Japan, it is crucial to improve the salt resistance of locally grown popular rice cultivars, most of which are salt sensitive [32,36–38]. In addition, developing Japanese cultivars for international appeal and fine-tuning their yield performance under various ecosystems around the world are time-demanding tasks. To generate new rice cultivars quickly, in response to evolving consumer preferences and crises, we crossed the salt-tolerant "Kaijin" with "Yukinko-mai" to develop a salt-tolerant line with elite agronomic traits through the use of marker-assisted selection (MAS). MAS is the most advanced tool yet developed for the precise introgression of genes of interest into elite rice cultivars [39,40], and allows breeders to recover most of the recurrent parent genome in only two or three generations [41]. Salt, heat, and drought stress-responsive genes or QTLs revealed by recent advances in genomics and biotechnology are being used for MAS of rice all over the world [42–44].

In recent years, rapid generation-advance technology called "speed-breeding" has been used to shorten the generation cycle, accelerating the progress of genomics and breeding studies in multiple crops [45–48]. This technique has been used for the genetical improvement of rice, such as recombinant inbred lines, backcrossed inbred lines, and isogenic cultivars [46,47]. The speed breeding method has been reported for six major crops such as spring wheat (*Triticum aestivum*), durum wheat (*Triticum durum*), barley (*Hordeum vulgare*), chickpea (*Cicer arietinum*), pea (*Pisum sativum*), and canola (*Brassica napus*), that uses a prolonged photoperiod to reduce the generation time [48]. Nagatoshi and Fujita [49] developed a breeding technique for short-day soybean plant applying supplemental CO<sup>2</sup> in combination with long-day and appropriate temperature cycles. Using speed-breeding, which combines temperature, light duration, and humidity control, tiller removal, and embryo rescue, breeders can obtain four to five advanced generations in a year [45,46].

In this study, we precisely transferred the *hst1* (*OsRR22*) gene, which confers salinity tolerance, from "Kaijin" into high-yielding "Yukinko-mai" (WT) rice through SNP MAS coupled with speed-breeding. We sequenced the whole genome of a BC3F<sup>2</sup> *hst1* homozygous line and determined the genome recovery rate. We also examined important physiological and biochemical parameters of the BC3F<sup>3</sup> generation that confer salt tolerance and evaluated the phenotype under salt stress and normal field conditions.
