| Soil salinity is one of the major abiotic stress affecting crop growth and productivity worldwide.When plants were exposed to salinity,seed germination and seedling establishment were significantly inhibited.Rice(Oryza sativa L.)is one of the most important food crops in the world,which is sensitive to saline stress.The salinization of available arable land seriously restricts rice production.In recent years,with the rapid development of economy and the advancement of rural urbanization,light-cultivated rice models such as direct-seeding rice have become more and more common,which requires high seed vigor and stress-tolerance for varieties during seed germination.It is of great importance to identify salt tolerance genes during seed germination and elucidate its mechanism,which could provide a basis for cultivating and promoting salt-tolerant rice varieties,and improving rice productivity in saline-alkali land.Main research findings are as follows:1.Map-based cloning and functional analysis of salt tolerance related genes gss1 during seed germination in Rice.(1)We performed a genetic screening of a population mutation from Nipponbare(Oryza sativa L.ssp.japonica,Nip)by Ethyl methanesulfonate(EMS),and isolated a dominant mutant gss1,named germination salt sensitive,which showed specific salt sensitive at the seed germination stage.Under 200 mM NaCl condition,the seed germination rate of mutant gss1 was significantly lower than that of wild-type Nip.The salt sensitivity of mutant gss1 was not significantly different from that of wild-type Nip at the bud and seedling stages.There were no differences in agronomic traits of between mutant gssl and wild-type Nip,such as heading date,plant height,panicle length,effective number of tillers,grain length,grain width and grain thickness and so on.(2)A F2 population consisting of 116 individuals was derived from crossing mutant gss1 with Nip.Forty extreme phenotypes individuals based on germination rate(GR)after seven days imbibition and germination index(GI)were selected for Mutmap.Three SNP index greater than 0.75 were obtained,which located in the exon regions of three candidate genes(LOC_Os05g47790,LOC_Os07g48130 and LOC_Os07g48610),resulting the substitution of coding amino acids.Among them,LOC_Os07g48130 encodes a potassium transporter OsHAK9 with the highest SNP index-0.97;LOC_Os05g47790 encodes a conserved hypothetical protein with SNP index-0.82;LOC_Os07g48610 encodes an alpha/beta fold family domain containing protein with SNP index-0.77.qRT-PCR analysis revealed that LOC_Os07g48130 and LOC_Os07g48610 were significantly up-regulated during seed germination at 200 mM NaCl,and LOC_Os05g47790 showed smooth expression pattern.Furthermore,the single candidate gene with SNP mutation was selected from the F3～4 population to identify the salt tolerance at 200 mM NaCl.The results revealed that GR of mutant LOC_Os07g48130 was significantly lower than that of wild-type(Nip),mutant of LOC_Os05g47790 and LOC_Os07g48610 were not significantly different from wild-type(Nip).These results indicated that the salt-sensitive phenotype of mutant gss1 during germination was caused by the mutation of OsHAK9(LOC_Ds07g48130).(3)In this study,T-DNA insertion mutant oshak9-pro,CRISPR/Cas9 mutant CC-HAK9,over-expression transgenic lines OE-HAK9 and complementation transgenic lines COMHAK9 were further obtained for function complementation of OsHAK9.Both T-DNA insertion mutant and CRISPR/Cas9 mutant lines showed salt sensitivity during seed germination compared with wild-type at 200 mM NaCl.Over-expression transgenic lines showed higher salt tolerance germination capacity than that of wild-type,and complementation transgenic lines significantly rescued the salt-hypersensitive phenotype of mutant gss1.All these results demonstrated that OsHAK9 is the candidate gene for gss1,which plays a vital role in salt tolerance during seed germination.(4)Bioinformatics analysis showed that OsHAK9 is a potassium transporter with a typical transmembrane domain,belonging to the second branch of the KUP/KT/HAK family.Subcellular localization analysis revealed that OsHAK9 was localized on the plasma membrane.Electronic expression data showed that OsHAK9 was significantly up-regulated by salt and drought induction.qRT-PCR analysis showed that OsHAK9 was expressed in various tissues and organs,mainly in roots and panicle and low transcripts in leaves and stems.The results of function complementation in yeast system showed that OsHAK9 confers no or minimal K+-transporting activity in the potassum absorption-deficient yeast CY162.Physiological and biochemical analysis showed that OsHAK9 can significantly reduce potassium ion efflux,change the gene expression of GA metabolic pathway,promote the accumulation of GA and promote seed germination under salt stress.2.Identification and fine mapping of QTLs related to salt tolerance during seed germination from an indica landrace Wujiaozhan(WJZ)(1)In previous study,we identified an indica landrace Wujiaozhan(WJZ)with high capability of seed germination.The seed germination traits were analyzed by germination rate(GR),seedling percentage(SP)and germination index(GI).Under water conditions,WJZ germinated faster than Nip.Under salt stress conditions,the values of GR,SP and GI were significantly higher than that of Nip,especially under high salt stress.These data indicated that WJZ had strong salt tolerance during seed germination.(2)A BC1F2 population consisting of 181 individuals was derived from the backcross of F3(Nip × WJZ)with Nip.A genetic map was constructed using 167 polymorphic SSR/InDel molecular markers among this BC1F2 population.Salt tolerance during seed germination was evaluated by GR,SP and GI under water conditions(0 mM)and salt stress(300 mM NaCl)for 14 days.A total of 19 QTLs related to seed vigor were detected under water conditions,and 16 QTLs were detected under salt stress conditions.The phenotypic variation ranged from 7.07 to 61.45,and the additive effects were all negative,indicating the effect originated from the donor parent WJZ.By comparison,6 QTLs(qGR6.2,qSP1,qSP8.1,qSP6.4,qG16.2,qGI10.2)were salt specific,and 6 QTLs(qGR6.2,qGR10.1,qGR10.2,qSP6.1,qSP10.1 and qSP10.2)were continuously expressed during germination under salt stress.A major QTL qGR6.2 was colocalized with qSP6.4 and qGI6.2 between Z604 and RM276 on chromosome 6,accounting for more than 20%of phenotypic variation.(3)The large BC2F3 and BC2F4 population were used to screen recombinant plants.To fine mapping qGR6.2 locus,new polymorphic molecular markers were developed.The progeny of recombinants was tested to identify homozygous individuals in the heterozygous region.Salt tolerance during seed germination was determined by the average values of these homozygous individuals.Finally,qGR6.2 was narrowed down to a 65.9 kb region between the Z654 and Z619 markers,with eleven open reading coding frames(ORFs)predicted.Based on the microarray database,six genes(LOC_Os06g10650,LOC_Os06g10660,LOC_Os06g10690,LOC_Os06g10710,LOC_Os06g10730,and LOC_os06g10750)were differentially expressed by seed imbibition or salt incubation.qRT-PCR showed that two genes(LOC_Os06g10650 and LOC_Os06g10750)were significantly induced by salt stress and had different expression patterns in two parents during seed germination,which could be casual genes for qGR6.2.In this study,the EMS mutants generated by EMS and the high seed vigor indica landrace variety WJZ were used to identify salt tolerance locus during seed germination.A novel gene OsHAK9 responsible for salt tolerance during seed germination was successfully mapped and cloned,the its mechanism was analyzed.Besides,a major salt-tolerance-specific QTL qGR6.2 was identified and fine mapped with prediction candidate genes at 300 mM NaCl during seed germination.Our study provides new information to better understand the mechanism of salt tolerance during seed germination,which could help breeders to use marker-assisted selection for cultivating new salt-tolerant rice varieties during seed germination and promoting the direct seeding rice technology. |
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