Identification and fine mapping of qGR6.2, a novel locus controlling rice seed germination under salt stress

Background Rice growth is frequently affected by salinity. When exposed to high salinity, rice seed germination and seedling establishment are significantly inhibited. With the promotion of direct-seeding in Asia, improving rice seed germination under salt stress is crucial for breeding. Results In this study, an indica landrace Wujiaozhan (WJZ) was identified with high germinability under salt stress. A BC1F2 population derived from the crossing WJZ/Nip (japonica, Nipponbare)//Nip, was used to quantitative trait loci (QTL) mapping for the seed germination rate (GR) and germination index (GI) under H2O and 300 mM NaCl conditions. A total of 13 QTLs were identified, i.e. ten QTLs under H2O conditions and nine QTLs under salt conditions. Six QTLs, qGR6.1, qGR8.1, qGR8.2, qGR10.1, qGR10.2 and qGI10.1 were simultaneously identified under two conditions. Under salt conditions, three QTLs, qGR6.2, qGR10.1 and qGR10.2 for GR were identified at different time points during seed germination, which shared the same chromosomal region with qGI6.2, qGI10.1 and qGI10.2 for GI respectively. The qGR6.2 accounted for more than 20% of phenotypic variation under salt stress, as the major effective QTL. Furthermore, qGR6.2 was verified via the BC2F2 population and narrowed to a 65.9-kb region with eleven candidate genes predicted. Based on the microarray database, five candidate genes were found with high transcript abundances at the seed germination stage, of which LOC_Os06g10650 and LOC_Os06g10710 were differentially expressed after seed imbibition. RT-qPCR results showed the expression of LOC_Os06g10650 was significantly up-regulated in two parents with higher levels in WJZ than Nip during seed germination under salt conditions. Taken together, it suggests that LOC_Os06g10650, encoding tyrosine phosphatase family protein, might be the causal candidate gene for qGR6.2. Conclusions In this study, we identified 13 QTLs from a landrace WJZ that confer seed germination traits under H2O and salt conditions. A major salt-tolerance-specific QTL qGR6.2 was fine mapped to a 65.9-kb region. Our results provide information on the genetic basis of improving rice seed germination under salt stress by marker-assisted selection (MAS). Supplementary Information The online version contains supplementary material available at 10.1186/s12870-020-02820-7.


Background
Soil salinity is the primary abiotic stress affecting crop growth and productivity worldwide [1]. It is estimated that 6% of the Earth's landmass and 20% of irrigated land are affected by salinity [2]. Rice is the most important staple food, feeding more than half of the world's population. Compared to wheat, rice is more sensitive to salt stress, and approximately 30% of the rice-growing area in the world is affected by salinity [3]. According to previous reports, high salinity inhibits seed germination and seedling establishment, reduces plant growth and diminishes rice yield [4,5]. Although saline soil could be improved by large-scale irrigation, drainage schemes, and chemical treatment, all these solutions are overly costly [6]. Hence, genetic improvement of salt tolerance has been an important and feasible objective for rice breeding in coastal areas [7].
Salt tolerance is a polygenic trait and highly influenced by the environment [8][9][10]. To date, hundreds of saltresponse QTLs have been reported at different developmental growth stages in rice [11]. Lin et al. [12] detected two major QTLs (qSKC-1 and qSNC-7) for Na + /K + content in the seedling shoot on chromosomes 1 and 7. Based on the results of QTL mapping, the major QTL qSKC-1 has been cloned, which encodes an HKT-type transporter protein regulating K + content in the shoot [13]. Another major QTL, Saltol, was identified and used to breed new salt-tolerant varieties by MAS, such as Pusa44, CR1009, and PR114, which enhances salt tolerance at the seedling stage [14,15].
Salt tolerance at the seed germination stage is not consistently related to other stages, such as the seedling and reproductive stages [9,16]. Seed germination is a key parameter of prime significance, and fundamental to total biomass and yield in a plant's life cycle, starting with the uptake of water followed by the protrusion of the radicle through the seed envelopes [17]. During seed germination, salinity results in many disorders and metabolic changes such as changed the enzymes activity causing high solute leakage [18], decreased K + efflux and impeding α-amylase activity [19]. Few studies have aimed at genetically dissecting seed germination under salt stress in rice. Wang et al. [4] detected 16 QTLs of rice seed germination ability at 100 mM NaCl from the japonica variety Jiucaiqing. Approximately 50 loci have been identified for seed germination under salt stress by genome-wide association studies (GWAS) [20][21][22]. Fujino et al. [23] reported that qLTG3-1 was associated with germination under low temperature through tissue vacuolation and weakening, and also with good seed germinability under high salinity. Recently, a QTL qSE3 promoting seed germination and seedling establishment was identified from a japonica landrace Jiucaiqing at 300 mM NaCl, which encodes a potassium transport OsHAK21 and mediates seed germination under salt stress through abscisic acid (ABA) metabolism [24]. With the increasing promotion of rice direct-seeding methods, it is of considerable importance to explore more loci or genes for seed germination under salt stress and develop cultivars with a high capacity for seed germination under salt stress by MAS in coastal areas.
In this study, an indica landrace WJZ from Yunan province in China [21] was identified with a high capacity for seed germination under high salt stress. To identify QTL for seed germination, the germination rate (GR) and germination index (GI) of the BC 1 F 2 population derived from a cross between WJZ and Nip were evaluated under H 2 O and 300 mM NaCl conditions. A major QTL qGR6.2 on the short arm of chromosome 6 was specifically identified under 300 mM NaCl conditions. Additionally, qGR6.2 was verified among the BC 2 F 2 population and fine mapped within a 65.9-kb region between Z654 and Z619 markers. This work could be valuable in elucidating the genetic and molecular basis of seed germination under salt stress.

Characteristics of seed germination for two parents under salt stress
The germination rate (GR), seedling percentage (SP) and germination index (GI) for indica WJZ and japonica Nip seeds were evaluated after 10 days ( (Table 1), indicating that rice seed germination was inhibited and delayed by salt stress. When exposed to 350 mM NaCl, WJZ seeds displayed 80.03% of GR, in contrast to 12.22% of GR for Nip ( Table 1), suggesting that WJZ was considerably more salt-tolerant than Nip during seed germination. Considering the greatest variation in GR, SP and GI between the two rice parents, seed germination under salt stress was assessed with 300 mM NaCl in later experiments.
To better understand the characteristics of the high seed germination ability and salt tolerance of WJZ, the differences in GR and SP between the two parents were further analyzed under H 2 O and 300 mM NaCl conditions. Under H 2 O conditions, although all seeds of both parents germinated and established seedlings after 120 h (5 d) of imbibition (Fig. 1a, b), WJZ germinated faster and had higher values of GR and SP than Nip at the beginning of seed germination. Under 300 mM NaCl conditions, significant differences in GR and SP between WJZ and Nip were observed from 3 to 14 d during seed germination ( Fig. 1c-e). The WJZ began to germinate after 2 d of imbibition, and its GR reached 90% after 7 d of imbibition (Fig. 1c), with a strong seedling establishment capacity being observed (Fig. 1d, e). However, Nip started to germinate after 7 d of imbibition and showed only 58.89% GR after 14 d of imbibition (Fig. 1c). Quantification and statistical analysis of germination rate (a) and seedling percentage (b) of two-parent varieties from 36 h to 120 h under H 2 O conditions. c-d Quantification of germination rate (c) and seedling percentage (d) of two-parent varieties from 1 to 14 d under 300 mM NaCl conditions. Each point represents the mean ± standard deviation. e Morphology of two varieties during seed germination after 3, 5, 7 and 9 d imbibition under 300 mM NaCl conditions. Scale bar = 1 cm  (Fig. 2a, b), and a symmetrical distribution of GI (Fig. 2c). Under 300 mM NaCl conditions, GR showed a left-skewed distribution at 5 d, a right-skewed distribution at 9, 11 and 13 d during seed germination (Fig. 2d, f-h), and a symmetrical distribution at 7 d (Fig. 2e). The GI under 300 mM NaCl conditions showed a right-skewed distribution (Fig. 2i)  Under 300 mM NaCl conditions, six QTLs for GR and three for GI of seed germination were identified on chromosomes 6, 8, and 10, respectively ( Table 2). All these QTLs showed a negative additive effect, indicating that the positive alleles originated from WJZ. Among the six QTLs for GR, qGR6.2 and qGR10.2 were continuously identified at 7, 9, 11, and 13 d of seed imbibition, qGR10.1 at 5, 7, 9, 11 and 13 d (Fig. 3 (Table 2). By comparison, qGR6.2 was co-localized with qGI6.2 between Z604 and RM276 on chromosome 6, and qGR10.1 shared the same region with qGI10.1 in the interval of W13~W20 on chromosome 10, qGR10.2 was co-localized with qGI10.2 between W20 and RM6824 on chromosome 10. Among those QTLs, the major QTL qGR6.2 or qGI6.2 with a high LOD value (> 8) could specifically enhance GR or GI of seeds under salt conditions (Table 2). To validate the major qGR6.2 controlling seed germination under salt stress, we further structured a BC 2 F 2 population consisting of 70 individuals. There was a significant peak between markers Z604 and Z605 based on GR at 13 d under 300 mM NaCl conditions, and its phenotypic variation and LOD values were 19.50% and 9.31, respectively (Fig. 4). This result indicated that qGR6.2 could improve rice seed germination under salt stress.
A large BC 2 F 3 population consisting of 1205 individuals was developed to narrow the region of qGR6.2. Eighty-six recombinants were identified between Z604 and RM276 markers (Fig. 5). Eighteen recombinant events were between Z604 and Z616, 57 recombinant events were between Z617 and Z619, and eleven recombinant events were between Z605 and RM276 (Fig. 5). Based on the genotypes, these 86 recombinants were classified into four groups (A-D). For each group, we selected the homozygous individuals as the heterozygous region from the recombinants' progeny by selfing. The homozygous individuals were further divided into two kinds of genotypes, one genotype is from WJZ, and the other is from Nip. Seed germination under salt stress was assessed by the average GR values of the two different genotypes. In groups B or D, the average value of GR at 10 d for homozygous WJZ alleles was significantly higher than that for Nip alleles, while there was no difference in groups A or C. qGR6.2 was delimited between the Z617 and Z619 markers (Fig. 5). Similarly, the larger BC 2 F 4 population derived from heterozygous BC 2 F 3 plants in markers Z617 and Z619 was developed, containing 2318 individuals. Three types of recombination were obtained (E, F and G), consisting of 17 recombinants (Fig. 5), and the assay of each homozygous individual (BC 2 F 5 ) from the recombinant group was conducted. Finally, the qGR6.2 locus was narrowed down to a 65.9-kb region between markers Z654 and Z619 (Fig. 5).
Based on RNA-Seq data and array database deposited in GENEVESTIGATOR, the expression profiles of 10 ORFs in various developmental stages and seed imbibition were obtained, except for ORF10 encoding transposon protein (Fig. 6). The results showed that at the seed germination stage, there were higher transcript abundances for five genes, LOC_Os06g10650, LOC_Os06g10660, LOC_Os06g10690, ORF7 (LOC_Os06g10710) and ORF9 (LOC_Os06g10730), low transcript abundances for two genes, LOC_Os06g10670 and LOC_Os06g10750, and almost no expression for three genes, LOC_Os06g10680, LOC_Os06g10700 and  (Fig. 6a). During seed germination, the expression of LOC_Os06g10650 in the whole seeds or the isolated embryo was significantly upregulated, and LOC_Os06g10710 was down-regulated obviously (Fig. 6b).
With the quantitative real-time PCR (RT-qPCR) approach, we subsequently detected the expression of 5 ORFs (LOC_Os06g10650, LOC_Os06g10660, LOC_Os06g10690, LOC_Os06g10710 and LOC_Os06g10730) in WJZ and Nip during seed germination under 300 mM NaCl conditions, which showed high transcript abundances at the seed germination stage based on GENEVESTIGATOR database. The expression of LOC_Os06g10650 was up-regulated dramatically in both parents during seed germination under 300 mM NaCl conditions (Fig. 7a). The expression of LOC_ Os06g10660 and LOC_Os06g10690 were smooth over time (Fig. 7b, c). The expression of LOC_Os06g10710 and LOC_ Os06g10730 was slightly down-regulated during seed germination under salt stress (Fig. 7d, e). Compared to Nip, the significant higher expression of LOC_Os06g10650 in WJZ seeds was observed after imbibition for 24 h and 36 h. Taken together with gene function annotation and expression profiles, it indicates that ORF1 (LOC_Os06g10650), encoding a tyrosine phosphatase family protein might be the causal candidate gene for seed germination under salt stress in the qGR6.2 locus.

Discussion
Salinity seriously affects rice seed germination and seedling establishment, especially in the direct-seeding area, leading to rice reduction in yields [10,11,20]. In this study, the indica landrace WJZ from Yunnan Province in China showed a strong capability of seed germination and seedling establishment under high salinity. When exposed to 300 mM NaCl, the seeds of WJZ could start to germinate after 2 days of imbibition and established normal seedlings after 5 days of imbibition. This finding suggests that WJZ is important germplasm with a strong capability of seed germination under high salinity, similar to other rice accessions N22-C-334-3 [25], Italica Livorno [23] and Jiucaiqing [24]. WJZ was considerably taller than Nip with 167.62 cm vs. 84.18 cm of plant height and fell down easily in field planting. Hence, it is of great importance to explore elite genes controlling seed germination under salt stress from WJZ, which will be beneficial for improving rice seed germination under salt stress in direct-sowing areas. As reported in previous studies, rice suffered from salinity stress during the whole growth stage, and salt tolerance at one developmental stage might not be correlated with salt tolerance at other stages [20,26,27]. WJZ has a very strong tolerance to salt stress at seed germination stage, however, it needs to be studied at other growth stages. Through pyramiding various salt-tolerance loci, or those loci expressing at germination, seedling, tillering or booting stages, it may be possible to develop new rice varieties with salt tolerance across all growth stages, and so improve production in rice direct-seeding areas or saline soils.
Evaluating the phenotype of salt tolerance comprehensively and accurately is the most crucial step for QTL mapping [26]. Previous studies showed that it's a good combination of final germination rate with germination index as germination parameters [4,28]. Both parameters showed correlation and provided reliable information on germination levels likewise temporal aspects of germination [28]. In this study, we evaluated seed germination using the parameters of GR and GI under H 2 O and NaCl conditions for the BC 1 F 2 population that was derived from a BC 1 F 1 individual containing approximately 37.76% genetic region of Nip (Fig. S1b). The continuous distribution and wide range of genetic variations in the BC 1 F 2 population were found during seed germination under H 2 O and Fig. 5 Fine mapping of qGR6.2. The qGR6.2 region was narrowed down to an approximately 65.9-kb region flanked by the markers Z654 and Z619 on the short arm of chromosome 6. On the left, numbers between two markers represent recombinant individuals. A-G represents seven groups according to genotype. The physical positions of the map are based on RAP-DB (http://rapdb.lab.nig.ac.jp/index.html). On the right, the phenotype is the average GR after 10 d of seed imbibition under 300 mM NaCl conditions. All individuals for assessing GR were derived from the left corresponding recombinant progeny by selfing. Orange bars, light blue bars, and light green bars represent the WJZ, Nip, and heterozygous genotypes or corresponding phenotypes, respectively. Arrows indicate eleven ORFs in this region according to the Rice Genome Annotation Project (RGAP) NaCl conditions, suggesting seed germination was regulated by various genes. Additionally, GR at 7 d under 300 mM NaCl conditions showed symmetrical distribution, suggesting that there was great genetic variation at this time point and might be a crucial period for breaking through the seed coat to germinate under salt stress (Fig. 2e).
In this study, a total of 13 QTLs controlling seed germination were identified via QTL mapping under H 2 O and 300 mM NaCl conditions. All these QTLs could be used to improve rice seed germination and salt tolerance through gene pyramiding by MAS in the future. By comparison, qGR6.1, qGR8.1, qGR8.2, qGR10.1, qGR10.2 and qGI10.1 were consistently identified under both H 2 O and 300 mM NaCl conditions, suggesting they may simultaneously regulate seed germination and salt stress. qGR6.2, qGR10.1 and qGR10.2 were identified at different time points of seed germination and shared the same region with qGI6.2, qGI10.1 and qGI10.2, suggesting they are curial loci for seed germination under salt stress. By comparing chromosomal locations of reported QTLs, qGR3.3, qGR6.2, qGR10.1, qGI6.2 and qGI10.1 in the BC 1 F 2 population were located in the same or adjacent regions as previously reported QTLs. qGR3.3 was near the region of qLTG-3-2 for low-temperature germination ability reported by Fujino et al. in 2004 [29] and qGR3-1 for germination rate reported by Cui et al. in 2002 [30]. qGR6.2 and qGI6.2 were close to the qIR-6 position for seed germination under salt stress reported by Wang et al. in 2011 [4], and one gene, OsRR22 was located within this region and involved salt tolerance at the seedling stage reported by Takagi et al. in 2015 [31]. The regions of qGR10.1 and qGI10.1 were similar to qSKC10 and qRKC10 identified for the shoot and root potassium content under salt stress at the seedling stage [26]. These results indicated that the co-localized QTLs at the different developmental stages were the weightily genomic regions for salt tolerance in rice.
Here, we focused on the major QTL qGR6.2, which was associated with both GR and GI under salt conditions. At last, qGR6.2 was mapped in a region of 65.9 kb with eleven candidate genes. Among these eleven candidate genes, only ORF2 named LYP6 has been reported to play dual roles in peptidoglycan and chitin perception in rice innate immunity [32]. As reported previously, PTP family proteins have been reported to regulate signal transduction and control plant growth and development [33], and the PHD finger has been identified as one of the major families of histone reader domains, being involved in recognition of methylated H3K4 [34], so we speculated ORF1 (LOC_Os06g10650) and ORF5 (LOC_Os06g10690) might play similar functions in rice. Based on the GENEVESTIGATOR database and RT-qPCR, the expression of LOC_Os06g10650 was significantly upregulated, and there was a significant higher level in WJZ than Nip during seed germination under salt conditions, suggesting important roles of LOC_Os06g10650 in seed germination under salt stress. According to previous studies, AtPTP1, the first PTP family gene in the plant, was upregulated by high salt stress [35]. Another PTP family gene At5g23720 was reported to play import role in ABA signaling, of which mutant phs1-3 exhibited a strong ABAinduced inhibition of seed germination in Arabidopsis [36]. In rice genome, there were 132-protein phosphatase-coding genes in silico investigation [37], and they were categorized into PP2A, PP2C, PTP, DSP and LMWP classes according to domain analysis and phylogenetic analysis [37,38]. Phylogenetic relationship results revealed that LOC_Os06g10650 (OsPP68) belongs to PP2C class [37]. Expression profiles showed that there were 46 genes of phosphatase family to be differentially expressed under three abiotic stress conditions (salt, cold and drought) [37]. All these imply that LOC_Os06g10650 might be the causal candidate gene of qGR6.2. The function of it will be validated by genetic transformation using CRISPR/Cas9 or other method.

Conclusions
In this study, we identified 13 QTLs for seed germination traits under H 2 O and salt conditions, which provide information on the genetic basis of improving salt tolerance during seed germination by MAS. Of these loci, the major QTL qGR6.2, specifically for seed germination under salt stress, was fine mapped within a region of 65.9 kb with one more likely causal gene, LOC_Os06g10650. Sequence analysis and genetic transformation will be carried out in the future to validate the function of the candidate gene and elucidate the molecular mechanism underlying seed germination under salt stress. The major QTL qGR6.2 could be highly useful for improving seed germination under salt stress by the MAS strategy.

Plant materials
The the radicle (2 mm) through the surrounding tissue, and the seedling establishment was considered when the root length reached the seed length and the shoot length reached half of the seed length [21]. Germination ability was observed every day to calculate the germination rate (GR) and seedling percentage (SP). The germination index (GI) was calculated as GI = ∑ (Gt/t), where Gt is the number of germinated seeds on day t [4]. Three replications of each line were performed.

DNA extraction and PCR analysis
Total genomic DNA was extracted from the young leaves of each plant using the cetyltrimethylammonium bromide (CTAB) method. PCR was performed as described by Chen et al. [39]. The PCR products were separated by electrophoresis through 8% nondenaturing polyacrylamide gels and visualized by silver staining [40].

QTL mapping
According to the International Rice Microsatellite Initiative (IRMI, http://www.gramene.org) [41], a total of 157 SSR or InDel markers were polymorphic between WJZ and Nip and scattered on 12 chromosomes (Table S1). The BC 1 F 2 population with 181 individuals was used to construct a genetic map by Mapmaker/Exp 3.0 [42]. GR at 2 and 3 d under H 2 O conditions and GI, GR at 5, 7, 9, 11 and 13 d and GI under 300 mM NaCl conditions were used for QTL mapping. QTL analysis was carried out by Inclusive Composite Interval Mapping (ICIM) [43] with a threshold of LOD > 3 operating 1000 permutations. The phenotypic variation and additive and dominance effects of each QTL were estimated.
Validation and fine mapping of qGR6.2 Six polymorphic InDel markers were developed and used for validation and fine mapping of qGR6.2 (Table S2). A linkage map of 70 individuals from the BC 2 F 2 population was analyzed with 9 SSR markers (Fig. 4) on chromosome 6 to ensure the presence of the major QTL qGR6.2. A total of 1205 BC 2 F 3 individuals were used to screen recombinants between the Z604 and RM276 markers, and 2318 BC 2 F 4 individuals were used to screen recombinants between the Z617 and Z619 markers. In total, seven types of recombinants were identified. Twenty progenies of each recombinant were planted and screened for homozygous plants from each group. These homozygous plants (BC 2 F 4 , BC 2 F 5 ) were tested for seed germination under 300 mM NaCl conditions. The average GR value of seed in each group after 10 d of imbibition was used for fine mapping.

Prediction and expression analysis of candidate genes
Open reading frames (ORFs) in the region of markers Z654 and Z619 were predicted by the Rice Annotation Project Database (http://rice.plantbiology.msu.edu/). GENEVE STIGATOR (https://genevestigator.com/gv/) was employed to analyze the expression patterns of eleven candidate genes based on 2836 Affymetrix microarray datasets and 565 RNA-Seq data in seed imbibition with a significance level of P < 0.05. Seeds of two parents were sampled after 0 h, 6 h, 12 h, 24 h and 36 h imbibition at 300 mM NaCl, frozen quickly in liquid nitrogen and stored at − 80°C for RNA extraction. Total RNA was isolated from approximately 80∼100 mg powder with a total RNA Kit (BioTeke, http://www.bioteke.com). The first-strand cDNA was synthesized with random oligonucleotides using the HiScript II Reverse kit (Vazyme Biotech, http://www. vazyme.com/) according to the manufacturer's protocol. To measure the mRNA levels of genes, RT-qPCR was conducted using a CFX96 Real-time System (Bio-Rad, USA) with SYBR Green Mix (Vazyme). The rice housekeeping gene OsActin (LOC_Os03g50885) was used as an internal control [44]. The PCR conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. A final melt curve stage of 65-95°C was performed to confirm the specificity of the primers. Relative quantification of transcript levels was obtained based on the 2 −ΔΔCT method [45]. The amount of the transcripts in the WJZ after imbibing for 0 h was set at 1.0. All of the primers used for RT-qPCR (Table  S3) were designed according to http://quantprime. mpimp-golm.mpg.de/. Three biological replicates were conducted.

Data analysis
The experimental data were analyzed using Statistical Analysis System (SAS) software (Cary, NC, USA) and compared with Student's t-test at the 5 and 1% levels of probability.