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

Abstract

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).

Keywords: Fine mapping; Marker-assisted selection (MAS); Quantitative trait loci (QTLs); Rice; Salt stress; Seed germination.

Fig. 1

Evaluation of seed germination and seedling establishment between WJZ and Nip under H₂O and 300 mM NaCl conditions. a-b Quantification and statistical analysis of germination rate (a) and seedling percentage (b) of two-parent varieties from 36 h to 120 h under H₂O 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. 2

Performance of seed germination traits in the BC₁F₂ population on different days under H₂O and 300 mM NaCl conditions. Performance of GR at 2 d (a) and 3 d (b) imbibition, and GI (c) under H₂O conditions, GR at 5 d (d), 7 d (e), 9 d (f), 11 d (g), 13 d (h) imbibition and GI (i) under 300 mM NaCl conditions. The mean, range and CV (coefficient of variation) of GR and GI were listed in figure

Fig. 3

QTLs associated with GR in the BC₁F₂ population under 300 mM NaCl conditions from 5 to 13 d. PVE, phenotypic variation explained by QTL. Dashed lines show the trends of PVE for GR

Fig. 4

QTL mapping and validation of qGR6.2. On the left, the red line represents the region of QTLs among the BC₁F₂ population, on the right, validation of qGR6.2 among the BC₂F₂ population under 300 mM NaCl conditions. The LOD curve indicates the strength of evidence for the presence of qGR6.2 within Z604~Z605 on chromosome 6. Dashed lines show LOD thresholds of 3.0. Marker names and genetic distances are shown on the right of the chromosome. Chr., chromosome; cM, centimorgan; LOD, the likelihood of odds; PVE, phenotypic variation explained by QTL; ADD, the additive effect of substituting a WJZ allele for a Nip allele, which its negative value indicates that WJZ contains the positive allele; DOM, dominance effect

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)

Fig. 6

Expression patterns of candidate genes in various developmental stages (a) and response to seed imbibition (b) based on mRNA-Seq data and Affymetrix microarray datasets from GENEVESTIGATOR (http://www.genevestigator.com). The expression potential of a gene is a robust indicator for the maximal expression level of this gene. The ‘OS-nnnnn’ refers to the experiments ID

Fig. 7

Expression levels of candidate genes between WJZ and Nip during seed germination under 300 mM NaCl conditions. Expression analysis of five ORFs (a-e) after seed imbibition for 0 h, 6 h, 12 h, 24 h and 36 h under 300 mM NaCl conditions between WJZ and Nip. Data represent the mean ± SD (n=3). * and ** indicate significant differences between WJZ and Nip at the 5 and 1% levels, respectively. n.s. indicates no significant difference