| As a moderately salt-sensitive plant,the growth and development process of cultivated tomato(Solanum lycopersicum)can be relatively easier affected by high salt environment.Studies have shown that wild tomato Solanum Pennellii LA0716(S.pennellii)has higher salt tolerance than cultivated tomatoes.Wild tomato Solanum galapagense(S.galapagense)originates from Galapagos Island and has a higher salt tolerance than Solanum pennellii.It can grow in the environment of 70% concentration of seawater.Wild tomatoes were shown to have higher salt tolerance.In this study,circ RNA,mi RNA and m RNA in response to salt stress in cultivated tomato M82 and wild tomato S.pennellii were identified for the first time by small RNA sequencing and full transcriptome sequencing.Based on the competitive endogenous theory,several competitive endogenous regulatory networks of circ RNA-mi RNA-m RNA in response to salt stress were established.The regulatory elements and signal pathways that may be related to salt tolerance were screened.We also established an F2 segregating population stemmed from a cross between S.galapagense and M82 and located the SNP sites and candidate regions that were significantly associated with the salt target phenotype through the bulked segregant analysis(BSA)analysis.Provide theoretical foundation for the salt tolerance mechanism of wild tomatoes and present a strategy to study the salt tolerance mechanism of other materails.1.The roots of cultivated tomato M82 and wild tomato S.pennellii were treated with salt stress for 0h and 12 h and sampled to be materials.The small RNA sequencing was carried out after the RNA was extracted.The results showed that a total of 135 known mi RNA were identified and 109 novel mi RNAs were predicted in M82 and S.pennellii.The length of known mi RNAs was mainly 21 nt,while the length of novel mi RNAs was mainly 24 nt.In order to find out the difference of mi RNAs between two varieties,it was found that 117 known mi RNA were expressed in both varieties,10 known mi RNA were specifically expressed in M82 and 8 were specifically expressed in S.pennellii.Combined with previous studies,we found that the homologues of some salt-responsive mi RNAs in other species had been identified to be salt stress-related mi RNA,indicating that these mi RNA may be a more conservative part of the salt-responsive mechanisms evolved by different species.At the same time,34 novel mi RNAs were identified in S.pennellii specifically.Through the analysis of the function of target genes,it was found that six mi RNAs(sly-mi R164a-3p,sly-mi R171 e,sly-mi R171 f,sly-mi R319 a,sly-mi R394-5p and sly-mi R477-3p)were involved in response to salt stress in S.pennellii.2.By whole-genome sequencing,circ RNAs and m RNAs in response to salt stress in two tomato varieties were analyzed and compared.There were 4324 circ RNAs were identified,consisted of 2495 circ RNAs that were only expressed in M82,and 1509 circ RNAs that were only expressed in S.pennellii.The circ RNAs were located on 12 chromosomes.Among them,chromosome 1 had the most circ RNAs(579)and chromosome 5 had the lowest circ RNAs(249).By differentially expressed(DE)analysis,27up-regulated circ RNAs and 20 down-regulated circ RNAs were found in M82.There were 33up-regulated circ RNAs and 30 down-regulated circ RNAs in S.pennellii.sly?circ?3614,sly?circ?3635and sly?circ?3485 were differentially expressed in both two materails.Among them,sly?circ?3635 and sly?circ?3614 were significantly down-regulated in S.pennellii.Circ RNAs act as competitive endogenous RNAs that can adsorb mi RNAs to regulate the expression of target genes.By predicting the regulation relationship between circ RNAs and mi RNAs in tomatoes,there were 18 and 30DE-circ RNAs identified in M82 and S.pennellii that could specifically bind to the DE-mi RNAs that in response to salt stress.Some circ RNAs contained several mi RNA binding sites,and multiple circ RNAs could also share the same mi RNA binding site.Several circ RNA-mi RNA-m RNA regulatory networks in tomatoes were established.We found that suberin biosynthetic process was significantly enriched only in S.pennellii,including 9 DE-circ RNAs in S.pennellii,5 DE-mi RNAs and 7 salt-related genes under salt stress.The regulatory network of suberin biosynthetic process was based on the targeting relationship.Among them,CYP86 A had been confirmed to be a positive regulatory factor in Arabidopsis under salt stress.These results indicated that the circ RNAs of S.pennellii could control the suberin biosynthetic process by bestowing salt tolerance onto the plants.3.By whole-genome resequencing,4,187,929 high?quality SNP markers and 566,269 high?quality In Dels were detected between the parents,5,202,108 high?quality SNP markers and 666,647high?quality In Dels were between two mixed pools.The 904 kb region that was significantly associated with the trait of salt tolerance were identified through association analysis,which contained 140 genes.The enrichment analysis result of 140 genes showed that there were 6 genes in the ethylene signaling pathway enriched in the plant hormone signaling transduction pathway.There were also 5 genes enriched in purine and pyrimidine metabolism pathway.In conjunction with RNA-seq data,only EIN4 was significantly up-regulated in M82.The combined analysis results showed that sly?circ?309 and sly?circ?154 might be adsorbed by novel?mir?007,in which the inhibition of EIN4 was relieved and showed reduced tolerance to Na Cl.In summary,this study indentified circ RNAs,mi RNAs and m RNAs in two tomato materails based on the high-throughput sequencing technology and biological analysis,and the basic characteristics of circ RNAs and mi RNAs in cultivated and wild tomatoes were also revealed.The similarities and differences of circ RNAs and mi RNAs between two materails were also compared.Based on RNA-based interactions the ce RNA regulation networks were constructed under salt stress.It was confirmed that the circ RNAs and mi RNAs in tomatoes were widely involved in response to salt stress,and the specific regulatory elements associated with salt stress in wild tomato were also analyzed.Finally,it was found that sly?circ?309 and sly?circ?154 could control the expression of ethylene receptor EIN4 by adsorbing novel?mir?007 by combining the candidate genes that were identified by BSA analysis with RNA-seq analysis results under salt stress. |
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