Identification Of Key Loci For Seed Germination Under Salt And Drought Stress In Rice And Prediction Of QSG3 Candidate Genes

Salt and drought stress become the important abiotic stresses for rice production with the increases in soil salinization and global climate changes.Seed germination is an critical process during crop growth and development,which is greatly affected by salt stress and drought stress.Therefore,it is important to identify the genes related to salt and drought tolerances at seed germination stage in rice,and to reveal their regulatory mechanisms of rapid seed germination and seedling growth under salt and drought stress for varieties breeding.In this study,168 rice germplasm resources（natural populations）that have been sequenced were used as research materials to detect the loci for seed germination under normal condition,salt stress and drought stress,and to explore their candidate genes.The main results are as follows:1、The traits of seed germination,including germination rate（GR）,the time required for the germination rate to reach 50%（T50）,germination index（GI）and germination level（GL）,were evaluated using the 168 rice germplasm seeds planted in the field in autumn 2017 under normal（H2O）,salt stress（200 mM NaCl）and simulated drought stress（20%PEG 6000）.Under normal condition,the average values of GR,T50,GI,and GL of the natural rice population were 98.49%,2.05 d,12.35,and 9.43,respectively,and their coefficients of variation were 2.14%,23.50%,15.96%,and 6.46%,respectively.Under salt stress,the average values of GR,T50,GI and GL of the natural rice population were 93.84%,4.20 d,6.96 and 7.35,and the coefficients of variation were 10.90%,34.64%,27.25%and 14.96%,respectively;under simulated drought stress,the average values of GR,T50,GI and GL were 88.15%,4.51 d,6.29 and 6.97,and the coefficients of variation were 19.84%,43.51%,31.86%and 21.58%,respectively.The results showed that the seed germination rate（GR）,the time required for the germination rate to reach 50%（T50）,germination index（GI）and seed germination level（GL）have extensive genetic variation under normal condition,salt stress and simulated drought stress,salt stress and simulated drought stress significantly inhibited the four seed germination indexes of rice.2、Based on the GR,T50,GI,and GL values of 168 rice germplasm seeds measured under three germination conditions,Efficient mixed model association eXpedited（EMMAX）was used for genome-wide association analysis（GWAS）to detect the loci significantly associated with seed germination traits,A total of 17,41 and 35 loci were detected under normal,salt and drought conditions respectively.Among them,under normal condition,4,1,8,and 4 loci controlling GR,T50,GI,and GL traits were detected,and 4 loci were detected at least two indexes;under salt stress,6,4,8,and 23 loci controlling GR,T50,GI,and GL traits were detected,and 8 loci were detected at least two indexes;under simulated drought stress conditions,5,5,4,and 21 loci controlling GR,T50,GI,and GL traits were detected,and 7 loci were detected at least two indexes.Under the three germination conditions,the 19 loci detected by more than two seed germination indexes were named L1--L19 respectively.These loci were compared with the results obtained in 2015 in our laboratory,it was found that 4 loci were stably expressed in 2015 and 2017.Interestingly,one stablely expressed loci was detected simultaneously under normal condition,salt stress and drought stress,and thus it might be a key loci for seed germination in rice,named as qSG3.3、The candidate genes for the above key loci qSG3 were analyzed and verified in rice.Results showed that a total of 20 candidate genes were predicted in the region of qSG3.In which,two most likely candidate genes LOC_Os03g2 7280（OsSAPK1）and LOC_Os03g27590（OsSCP18）were predicted based on the function annotation,and homologous gene and haplotype analyses.Every two haplotypes were identified for OsSAPK1 and OsSCP18 in rice natural population.The rice accessions containing hap1 of OsSAPK1 and OsSCP18 had significantly higher seed germination compared with that of accessions containing hap2 under salt and drought stresses.Furthermore,the homozygous mutants of ossapkl and osscp 18 were constructed using the CRISPR/Cas9 approach in rice.The significantly lower seed germination was observed in ossapkl and osscpl8 compared with that of wild-type（WT）under normal condition,and thus the possible qSG3 candidate gene OsSAPKl and OsSCP 18 were further verificated.4、The salt-tolerant and drought-resistant rice varieties were identified using the index of germination level（GL≥8）under stress and the elite haplotypes（hapl）of candidate genes.Eight elite accessions that were both salt-tolerant and drought-resistant were identified,including EMATA A 16-34,SLO 17,Honduras,KPF-16,Saber,Berenj,Saturn and Chiem Chanh.These results will provide the basis for functional analysis of qSG3 in rice,and for breeding new stress-tolerant varieties in the future.