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 (d) of imbibition under H2O and various salt concentration conditions (150, 200, 250, 300 and 350 mM NaCl). Both WJZ and Nip germinated readily, with approximately 100% of the GR and SP for WJZ and Nip under H2O conditions (Table 1). However, WJZ had a significantly higher GI (13.93) than Nip (10.51), indicating that WJZ germinated faster than Nip under H2O conditions. There was a significant decrease in GR, SP, or GI of both WJZ and Nip under various NaCl concentrations (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. Since the greatest variation in GR, SP and GI between the two rice parents was under 300 mM NaCl, seed germination was assessed with 300 mM NaCl in our later experiments.
To better understand the characteristics of the high seed germination ability and salt tolerance of WJZ, the dynamic traits of GR and SP between the two parents were further analyzed under H2O and 300 mM NaCl conditions. Under H2O conditions, although all seeds of both parents germinated and established seedlings after 6 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, d). The WJZ began to germinate after 3 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). However, Nip started to germinate after 7 d of imbibition and showed only 58.89% GR after 14 d of imbibition.
Variation in seed germination among the BC1F2 populations under salt stress
A BC1F2 population consisting of 181 individuals was derived from the selfing of one salt-tolerance BC1F1 single plant, which was produced by the backcross of one salt-tolerance F3 individual (Nip × WJZ) with Nip (Fig. S1a). The variations in GR and GI among this BC1F2 population under H2O and 300 mM NaCl conditions were analyzed. All the traits observed, including GR from 2 to 3 d and GI under H2O conditions, GR from 5 to 13 d and GI under 300 mM NaCl conditions, showed a continuous distribution and had a wide range of genetic variations (Fig. 2). Under H2O conditions, there was a left-skewed distribution of GR after 2 d of imbibition, a right-skewed distribution at 3 d (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). These results indicated that the traits of GR and GI are polygenic characteristics and might be regulated by various genes at the early and later stages of germination under either H2O or NaCl conditions.
QTL mapping of seed germination traits under H2O and salt conditions
A molecular linkage map consisting of 70 simple sequence repeat (SSR) or InDel (Insertion/Deletion) markers was constructed with the above BC1F2 population consisting of 181 individuals for QTL mapping of seed germination traits, GR and GI under H2O and salt conditions. Under H2O conditions, eight QTLs of GR during seed germination were identified on chromosomes 3, 6, 8 and 10, and two QTLs of GI were identified on chromosomes 6 and 10 (Table 2). GR for 2 d was regulated by three QTLs (qGR8.1, qGR8.2 and qGR10.1), and GR for 3 d was regulated by six QTLs (qGR3.1, qGR3.2, qGR3.3, qGR6.1, qGR10.1 and qGR10.2). The phenotypic variance of GR explained by a single QTL ranged from 7.32 to 23.99%. One major QTL, qGR6.1explained 23.99% of the phenotypic variation. The qGI6.1 for the GI, identified within the interval of RM190~Z602 on chromosome 6, and qGI10.1 within the interval of W13~W20 on chromosome 10, accounted for 10.39 and 8.86% of phenotypic variation, respectively. By comparison, qGR6.1 and qGI6.1 shared the same interval of RM190~Z602 on chromosome 6, and qGR10.1 and qGI0.1 shared the same interval of W13~W20 on chromosome 10 (Table 2). The additive effects of all these QTLs detected under H2O conditions were negative, ranging from -0.36 to -9.95 (Table 2), suggesting that the positive alleles were derived from WJZ.
Under 300 mM NaCl conditions, six QTLs of GR and three of GI during 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 of GR, qGR6.2 and qGR10.2 were continuously identified after 7, 9, 11, and 13 d of imbibition, qGR10.1 at 5, 7,9,11, and 13 d (Fig. 3), and qGR8.1 and qGR8.2 only at 5 d and qGR6.1 only at 13 d. It suggested that qGR6.2, qGR10.2 and qGR10.1 might be key QTLs of salt tolerance for seed germination(Table 2). The qGR6.2 flanked by Z604 and RM276, explaining more than 20.0% of phenotypic variation, could be a major-effective QTL. Three QTLs of GI, qGI6.2, qGI10.1, and qGI10.2, accounted for 24.39, 17.41 and 13.18% of phenotypic variation, respectively (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, another chromosomal region (W20~RM6824) on chromosome 10 was identified to control GR and GI at the same time. Among those loci, a major QTL qGR6.2 with a high LOD value (>8) could specifically enhance GR and GI under salt conditions (Table 2).
Validation and fine mapping of qGR6.2
To validate the major qGR6.2 identified for GR under 300 mM NaCl conditions, we further structured a BC2F2 population consisting of 70 individuals. There was a significant peak between markers Z604 and Z605 based on GR at 13 d under salt stress, and its phenotypic variation and LOD values were 19.50% and 9.31, respectively (Fig. 4; Table S2). This result indicated that qGR6.2 could obviously improve rice seed germination under salt stress.
A large BC2F3 population consisting of 1,205 individuals was developed to narrow the region of qGR6.2. Eighty-six recombinants were identified between Z604 and RM276 markers (Fig. 5a). Four new polymorphic markers (Z616, Z617, Z619 and Z605) between Z604 and RM276 were further developed. Based on the genotypes, the 86 recombinants were classified into four groups. Eighteen recombinant events were between Z604 and Z616, 57 recombinant events were between Z617 and Z619, and 11 recombinant events were between Z605 and RM276 (Fig. 5b). The progeny of these recombinants were tested to identify homozygous individuals in the heterozygous region of each group. Salt tolerance during seed germination was determined by the average values of these homozygous individuals derived due to segregation in the recombinant heterozygous region. 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. 5b). Similarly, the larger BC2F4 population derived from heterozygous BC2F3 plants in markers Z617 and Z619 was developed, containing 2,318 individuals. Seventeen recombinants consisting of three types of recombination were obtained (E, F and G) (Fig. 5b), and the progeny assay of each homozygous individual 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. 5b).
Prediction and expression analysis of candidate genes in the qGR6.2 locus
According to the MSU Rice Genome Annotation Project Database (http://rice.plantbiology.msu.edu), eleven opening reading frames (ORFs) were annotated within the 65.9-kb region located in the qGR6.2 locus, including five functional proteins, one transposon protein and five expressed proteins without annotation (Table 3). Five genes with functional annotation showed that ORF1 (LOC_Os06g10650) encodes a tyrosine phosphatase (PTP) family protein, ORF2 (LOC_Os06g10660) encodes a lysM domain-containing GPI-anchored protein 1 precursor, ORF3 (LOC_Os06g10670) encodes an aspartic proteinase nepenthesin-1 precursor, ORF5 (LOC_Os06g10690) encodes a PHD-finger domain-containing protein, and ORF11 (LOC_Os06g10750) encodes an integral membrane protein DUF6-containing protein.
The expression profiles of 10 ORFs, including five functional proteins and five expressed proteins without annotation in various developmental stages and incubation of salt stress based on mRNASeq data and array database deposited in GENEVESTIGATOR (Fig. 6), were determined. The results showed that there were higher transcript abundances of seven genes, including ORF1 (LOC_Os06g10650), ORF2 (LOC_Os06g10660), ORF3 (LOC_Os06g10670), ORF5 (LOC_Os06g10690), ORF7 (LOC_Os06g10710), ORF9 (LOC_Os06g10730) and ORF11 (LOC_Os06g10750), at all developmental stages, while almost no expression was observed for the other three genes, ORF4 (LOC_Os06g10680), ORF6 (LOC_Os06g10700) and ORF8 (LOC_Os06g10720) (Fig. 6a). When the root and seedling samples were exposed to salt stress, ORF11 (LOC_Os06g10750) showed significantly down-regulated expression patterns after salt incubation (Fig. 6b).
With the quantitative real-time PCR (RT-qPCR) approach, we subsequently detected the expression of all 11 ORFs in WJZ and Nip during seed germination under 300 mM NaCl conditions. The expression of ORF1 (LOC_Os06g10650) was dramatically induced by salt stress after imbibition for 24 h with a nearly 20-fold change compared with 0 h in WJZ and 14-fold change compared with 0 h in Nip (Fig. 7a). ORF2 (LOC_Os06g10660) and ORF5 (LOC_Os06g10690) showed smooth expression (Fig. 7b, e). Eight genes, ORF3 (LOC_Os06g10670), ORF4 (LOC_Os06g10680), ORF6 (LOC_Os06g10700), ORF7 (LOC_Os06g10710), ORF8 (LOC_Os06g10720), ORF9 (LOC_Os06g10730), ORF10 (LOC_Os06g10740) and ORF11 (LOC_Os06g10750), showed down-regulated expression patterns during seed germination under salt stress (Fig. 7). Comparing the expression level between the two parents, we found that ORF11 (LOC_Os06g10750) in WJZ was nearly 10-fold lower than that in Nip (Fig. 7k), indicating the different role of ORF11 (LOC_Os06g10750) in the two parents. Taken together with gene function annotation and expression profiles, these results indicated that ORF1 (LOC_Os06g10650), encoding a tyrosine phosphatase (PTP) family protein, and ORF11 (LOC_Os06g10750), encoding an integral membrane protein DUF6-containing protein, might be the causal candidate gene for salt tolerance in the qGR6.2 locus.