Fine-mapping And Candidate Gene Prediction Of QSTS8,a Quantitative Trait Locus Associated With Salt Tolerance At Seedling Stage In Rice

Soil salinization is one of the important abiotic stresses harmful to rice production.Recently,the degree of salinization of agricultural land in China has been deepening with the influence of climate warming and agricultural measures such as,irrational irrigation and excessive application of chemical fertilizers.Rice is one of the main grain crops with salt-sensitive in China,and soil salinization has a great impact on the development of rice production.The use of molecular ways to map and clone salt-tolerant genes in rice seedlings,and that be used in the breeding of salt-tolerant rice varieties has become an important way to improve the salt tolerance of rice,and make full use of saline-alkali cultivated land.In this study,we used the chromosomal fragment substitution mapping method to carry out the fine mapping of the salt-tolerant gene qSTS8 at seedling stage and the prediction analysis of the genome sequence in corresponding interval.The specific results were as follows:1.A salt-tolerant F3 line derived from a cross between Nipponbare and Faguodao was used as the female parent,and the salt-sensitive parent Nipponbare was continuously backcrossed to obtain a BC2F2 population which was used as the materials in the present study.The qSTS8 was narrowed to a interval of 366Kb on the long arm of chromosome 8 in rice,and two pairs of upstream and downstream flanking markers RM515 and RM223 closely linked to the target gene were obtained.With P<10-6 as the threshold,single-point analysis was performed.A total of seven single-point sites controlling STS were detected,and the contribution rate ranged from 22%to 30%.The additive effects of these seven sites were positive and the distribution range was from 0.62 to 0.74.The dominant effects were also positive with the distribution range were 0.31 to 0.46.The sites with the highest contribution rate were RM515 and RM223.Compared with the initial mapping results,the contribution rate was further increased by 8%,and the additive effect was increased by 0.21.2.We used the mixed population of BC2F2 and BC3F2 as materials.The molecular marker-assisted selection was used to obtain the individuals with heterozygous segment in the target gene and the Nipponbare genetic background,which constituted a fine-mapping group composed 4355 individual plants.The flanking markers closely linked to the gene RM515 and RM223,and 7 pairs of polymorphic markers developed in the interval were used to select the recombinant individuals.189 individuals were obtained within the interval,and 105 of them were used for fine mapping.The chromosomal fragment substitution mapping method was used and finally we located the salt-tolerant QTL qSTS8 between the markers RM8265 and RM223,the interval was 167Kb.3.Combined with the qSTS8 fine mapping interval and the physical location of the marker was found on the rice genome annotation site(http://rice.plantbiology.msu.edu/),and 18 predicted genes in the interval were annotated and screened.Represented by LOC_Os08g33150,LOC_Os08g33154,LOC_Os08g33160 and LOC_Os08g33170,bioinformatics analysis on these 18 candidate genes were performed.We found that the gene LOC_Os08g33]50 contains HTH_MYB and other related protein elements.There was a transmembrane_regions in the LOC Os08g33154 gene.The gene LOC_Os08g33160 was a member of TIFY family has a ZIM motif which may be involved in the regulation of stress resistance in plants.4.We used 590 BC3F2 plants obtained from the salt-tolerant parent Faguodao and salt-sensitive parent Nipponbare backcross as test materials,genotypes of each individuals were detected using 32 SSR markers covering introgressed segments of Faguodao in the backcross population.Genomic marker-assisted selection was carried out in genome foreground and background,the target gene segment was selected as homozygous Faguodao fragment,and other genetic backgrounds were as close as possible to the recurrent parent Nipponbare.There were 120 individuals with qSTS8 segment as the Faguodao homozygous genotype,accounting for 20.3%of the total,which could be used as an alternative population of qSTS8 salt-tolerant near-isogenic lines.In this group,the distribution range of Faguodao introgressed segments was from 2 to 16,the average number of introgressed segments was 6.4,the number of homozygous segments was 2.2,and the number of heterozygous segments was 4.3.The distribution of segments was consistent with normal distribution.Among them,16668-122 has the highest salt tolerance level of 6.4,and can be used as a salt-tolerant introgression line to construct a salt-tolerant near-isogenic line for functional verification of the target genes.