Seed Dormancy

The CSSL and BIL populations were used to dissect the genetic basis of seed dormancy in rice. These two populations were derived using different approaches from the same cross between the two genome-sequenced rice cultivars, NIP and 9311. NIP is classified as subspecies japonica and exhibits seed dormancy, whereas 9311, an elite restorer line is classified as subspecies indica and lacks seed dormancy. The first population of the CSSLs consisted of 122 lines and was developed from a cross between NIP (as the donor) and 9311 (as the recurrent parent) using a backcross scheme with a marker-assisted selection approach (Additional file 1: Fig. S1). In the CSSLs, each line contained one or a few introduced NIP segments within the 9311 background (Tan et al. 2011 ). The other population (BILs) comprising 437 lines derived from a NIP × 9311 F₁ backcrossed to 9311 and advanced to the F₈ by single-seed descent (Additional file 1: Fig. S1). The genomic DNA from each line of BILs was extracted and subjected to genotyping as described previously (Yuan et al. 2019 ). To validate the effect of qSD3.2, one CSSL (NY38) that carried a 1.6-Mb introduced NIP segment surrounding qSD3.2 on chromosome 3 and a background NIP segment on chromosome 10 was crossed with 9311 to generate an F₂ population (hereafter referred to as the NY38-derived population). The other CSSL (NY61) harboring two introduced NIP segments surrounding qSD3.1 and qSD3.2, was used to generate an additional F₂ population (referred to as the NY61-derived population). Two near-isogenic lines (NILs) containing either qSD3.1 or qSD3.2 were obtained during the development of these mapping populations. The NILs and the mapping populations along with the parental lines were grown at the Wuhan Experimental Station of Huazhong Agricultural University, China. Each line was planted in four rows with ten individuals per row with spacing of 16.7 × 26.6 cm. Field management was carried out according to the local standard practices (Tan et al. 2011 ).

The diploid Ae. tauschii ssp. tauschii accession T093 was originally derived from Henan province, which is resistant to PHS with long seed dormancy time after harvest. Zhoumai 18, a typical white-grain wheat with high susceptibility to PHS, was applied as recurrent parent in this work. Hybrid F₁ plants were obtained through hybridization of Ae. tauschii accession T093 as female parents with Zhoumai 18, which were then treated with colchicine to generate synthetic octaploid wheat (AABBDDDD, 2n = 8x = 56). The next year, emasculated florets of Zhoumai 18 were pollinated by synthetic octaploid wheat to generate BC₁F₁ seeds. Afterwards, the BC₁F₁ plants, as female parents, were successively backcrossed two times by Zhoumai 18 and then selfed four generations to produce advanced backcross population (BC₃F₄ population) (Figure 1). Phenotypic traits of strains within the group were stabilized after several generations of backcross and selfing, demonstrating consistent ripening rates with the recurrent parent Zhoumai 18. The mapping population and Zhoumai 18 were cultivated on the 2014–2015 crop season in the wheat breeding farm of Plant Germplasm Resources and Genetic Engineering Laboratory, Henan University. Seeds were sown with 10 cm distance between plants and 30 cm row gap, which were grown under consistent field conditions.

We selected 478 accessions from 46 countries and areas with an appropriate growth period and without distinct unfavorable traits in southern China (Additional file 2: Table S2) in the 3 K RGP [13 (link)] to evaluate salt tolerance at the seed germination stage. In total, there were 305 indica accessions, 85 japonica accessions (9 japonica, 34 temperate japonica, and 43 tropical japonica), 65 aus accessions, 16 basmati accessions, and 7 intermediate accessions. All seeds used in your study were from the International Rice Genebank Collection at the International Rice Research Institute.
A total of 120 yellow-ripe seeds of each accession were dried at 50 °C for 3 days to break seed dormancy. The seeds were surface-sterilized with 15% sodium hypochlorite solution for 20 min and then rinsed three times with sterile distilled water before the germination experiment. Two replications for each treatment, each consisting of 30 seeds from each accession, were placed in 9-cm-diameter Petri dishes on two layers of filter paper, to which 10 mL of NaCl solution was added to simulate salt-stress conditions. In the control, the filter paper was soaked with 10 mL water. The seeds were incubated in a growth chamber at 30 °C under a 12-h light/12-h dark photoperiod with 80% relative humidity for 10 days. To determine the most suitable salt concentration, we selected 35 accessions for treatment with NaCl at three different concentrations (60, 80, and 100 mM). More genetic variation was observed in the 60 mM NaCl treatment than in the other treatments. Therefore, the seed GRs were evaluated among all the accessions under a 60 mM NaCl treatment and control (water) conditions. The solution was replaced every day to maintain the NaCl concentration and the distilled water volume.
Dry seeds, and seeds incubated for 24 and 48 h, were weighed independently to calculate seed IR (mg/g) using the method of Wang et al. [6 (link)], as follows: IR = (W₂ – W₁)/W₁ × 1000, where W₁ (g) represents the dry seed weight, and W₂ (g) represents the total seed weight after imbibition for 24 h or 48 h. The seeds that germinated were observed each day to calculate the GR and GI. We considered a seed germinated when its shoot length was greater than half of the seed length and the root length was greater than the seed length. The GR was the germination percentage at a certain day (GR = N_t/N₀ × 100%, where N_t represents the number of germinated seeds at day t and N₀ represents the total number of experimental seeds). The GI was calculated by the method of Wang et al. [6 (link)] as follows: GI = ∑(G_t/T_t), where G_t is the accumulated number of germinated seeds at day t and T_t is the time corresponding to G_t in days. The MGT was calculated for the GR using the following formula: MGT = ∑T_iN_i/∑N_i, where N_i is the number of newly germinated seeds at day t [34 ]. We chose 10 germinated seeds to measure the average shoot length after 10 days, and the seed VI was calculated using the formula [6 (link)]: VI = GI × average shoot length. The responses of the genotypes to salt stress were expressed according to the SSIs calculated using the methods of Fischer and Maurer [35 (link)] as follows: SSI = (1 − Ys/Yp)/D, where Ys = mean performance of a genotype under stress; Yp = mean performance of the same genotype without stress; D (stress intensity) = 1 − (mean Ys of all of the genotypes/mean Yp of all of the genotypes).

The freshly mature seeds of Amomum tsaoko were collected from 10-year-old plants from a plantation in Napo County, Guangxi province, China (101 N, 113E). After mixing with moist perlite (volume ratio 1:3), seeds were stored in temperature-controlled incubators at 15 ℃ as a warm stratification [2 ]. To test seed germination, perlite was used as medium and the incubator was set to 20/30 ℃ (night/day) with a 12 h light/12 h dark cycle as reported in our previous study [2 ]. Germination capacity was recorded after 28 days of the experiment and the seed germination rate was calculated as the ratio of the number of seeds with the radicle protruding from the seed coat to the total number of seeds tested. There were three replicates per treatment with 100 seeds per germination box. Seeds were collected for transcriptome and proteome analysis after warm stratification of 30d (S30, early seed dormancy release period), 60d (S60, middle seed dormancy release period) and 90d (S90, late seed dormancy release period), respectively. The fresh seeds without stratification treatment were considered as the control (CK). Embryos were stripped with sterilized blades, frozen in liquid nitrogen immediately and stored at -80 ℃.

Since 30d, 60d and 90d of warm stratification represented critical time points of seed dormancy release of A. tsaoko as reported in our previous study [18 ], the embryos of CK, S30, S60 and S90 were used in RNA extraction and transcriptome profiling analysis. To prepare Illumina RNA-seq libraries, the total RNA of 12 embryo samples from CK, S30, S60, and S90 (three replicates per dormancy release stage) were extracted using Takara MiniBEST Universal RNA Extraction Kit (Beijing, China) by the manufacturer’s instructions. The purity, concentration and integrity of each RNA sample were validated using 1% agarose electrophoresis, Nanodrop 2000 microspectrophotometer and Agilent 2100 Bioanalyzer, respectively. For RNA-seq libraries construction, RNA samples (RIN>7.5) were enriched by Oligo-dT magnetic beads. The purified RNA was fragmented into short pieces by fragment buffer and then the reverse transcription was carried out with N6 primer. The double-strand cDNA was end-repaired, adaptor-ligated, and followed by PCR amplification. The amplified PCR product was heat-denatured into single-stranded DNA, single-stranded DNA was then circularized to make a single-stranded circular DNA library. Finally, paired-end sequencing of the cDNA library was performed on the BGISEQ-500 platform.

Dormant seeds of uniform texture with an average weight of 2.04 mg, an average moisture content of 5.79%, and an average viability of 88.89% were selected, soaked in distilled water at 20 ± 5 °C for 48 h and 0.2% (v/v) NaClO solution for 10–15 min, and finally rinsed with water. The coat of these disinfected seeds was stripped on ice (to avoid enzyme inactivation), and the peeled embryos were used for the subsequent experiments [14 (link)]. The embryo obtained after pre-treatment is shown in Figure 7.