QTL Mapping Of Drought-tolerance And Identification Of Drought-responsive MicroRNA In Tomato Introgression Lines

As sessile organisms,plants suffer from various environmental stresses.Among them,drought is one of the most frequent environmental stresses,which limits crop growth,development,yield,and product quality.Plant drought tolerance is fine-tuned by a complex gene regulatory network,which is genetically controlled by multiple genes.Using introgression lines can reduce the interference of complex genetic background,and they have been proved to be the classic materials for genetic mapping of complex quantitative traits.As a class of endogenous non-coding RNA and functions at the transcriptional and post-transcriptional level,micro RNA plays important roles in plant growth,development and stress response.Tomato(Solanum lycopersicum)is one of the most important vegetable species grown worldwide.With abundant nutritions,it has become an important part of our daily diet.However,abiotic stress,such as drought,is a major limit for the sustainable production of tomato.Stress not only reduces tomato yield,but it also affects the fruit quality.Therefore,it would be very important for tomato genetic improvement to identify drought-tolerant genes and to elucidate their mechanisms.Modern tomato cultivars have very narrow genetic bases after the long-term and high-pressure of selection,and the vast majority of them are sensitive to drought stress.However,elite genes for stress tolerance are well reserved in some wild tomato species.For instance,S.pennellii grows in Andean regions in South America where is extremely drought,and accessions from this species show high level of drought tolerance.Using the processing tomato cultivar M82 as the recurrent parent,previous researchers have developed an introgression line population which covers the whole genome of S.pennellii LA0716,and it serves as a classic population for addressing complex quantitative trait loci(QTLs),including drought tolerant QTLs.Previously,we have identified two important drought-tolerant introgression lines(IL2-5,IL9-1)from the LA0716 introgression population.Based on the progresses,this study aims at(1)analysis of the drought-tolerant mechanisms of IL2-5 and IL9-1using physiological and biochemical methods;(2)construction of sub-introgression lines of IL2-5 and unitization of them in the mapping of drought-tolerant QTL,and then selection of drought-tolerant candidate genes by analyzing the differentially-expressed genes under drought stress and sequence variation within the introgressed chromosomal segments between the introgression lines and their recurrent parent;(3)profiling of drought-responsive micro RNAs in IL9-1,IL2-5 and their recurrent parent,M82.The main results are as follows:1.Elucidation of drought-tolerant mechanisms underlying the introgression lines at the physiological and biochemical level.The leaf wax contents of IL2-5,IL9-1 and M82 were measured.It was found that there was no difference in wax composition among the three genotypes,however,the wax content of IL2-5 was significantly higher than those of IL9-1 and M82.The root parameters(total root length,area and volume),photosynthetic parameters,leaf soluble sugar and H₂O₂content were investigated for LA0716,IL2-5,IL9-1 and M82 under normal and drought stress conditions.The results indicated that the root system is not a contributor to the drought tolerance of IL2-5 and IL9-1.Although all the four genotypes accumulated soluble sugars under drought stress,IL2-5 accumulated a much higher level than the other three lines.The contents of H₂O₂ in the three drought-tolerant genotypes were significantly lower than that in the sensitive line M82.Regarding photosynthesis,the actual photosynthetic efficiency in the four tomato genotypes increased under a mild drought stress,suggesting that mild drought could promote photosynthesis.However,with the increase of drought stress,the maximum and actual photosynthetic efficiency decreased in all the lines,but IL2-5and IL9-1 could maintain at a significantly higher level relative to M82,indicating that drought stress had less influence on the photosynthesis of the drought-tolerant genotypes.Thus,it was speculated that higher wax content,higher capability of H₂O₂elimination,and more accumulation of soluble sugar under drought stress were involved in the drought tolerance of IL2-5,while H₂O₂ scavenging ability also contributed to the drought tolerance of IL9-1.2.QTL mapping and identification of drought-tolerant candidate genes from IL2-5.Forty polymorphic DNA markers were developed,which were distributed evenly in the introgressed region of IL2-5.A sub-introgression population,constituting 23 lines,was developed,and the genotypes of the sub-lines were analyzed.By using survival rate under drought stress and recovery as the indicator,two major QTLs were mapped into a 1.69 M and a 1.79 M region,which were designated region cd and ef,respectively.It was speculated that the two major QTLs in these two regions had additive effects.A negative QTL of drought tolerance was also mapped to a 2.24 M region(region ab).Drought-responsive genes were identified in IL2-5 and M82 using RNA-Seq,and 3028 and 1432 differentially-expressed genes were identified.Based on gene annotation,GO and pathway analysis,it was found that the expression of many genes involved in stress-tolerance related pathways was significantly changed.Some GO terms were specifically enriched in IL2-5,such as response to oxidative stress.In addition,the genomic variations in the QTL regions between S.pennellii and M82 were analyzed using Snp Eff software,a large number of SNPs,In Dels were detected.Candidate genes were selected based on the integrated analysis of different expression and sequence variation.3.Characterization of drought-responsive miRNAs in the introgression lines.miRNA-Seq was used to profile drought-responsive miRNAs in the introgression lines.In the comparison of M82 and IL9-1,105 conserved and 179 novel miRNAs were identified in the two genotypes,among them,54 and 98 were differentially expressed upon drought stress,respectively.Twelve differentially-expressed miRNAs with similar expression trend were shared by the two genotypes,however,more miRNAs showed different expression between them.In the comparison of M82 and IL2-5,a total of 108 conserved and 208 novel miRNAs were identified,among them,32 and 68 were significantly changed in expression after stress.Further,1936 putative target genes were predicted for those differentially-expressed miRNAs.GO and pathway analyses showed that many of the target genes were involved in stress resistance.Overall,our results suggested that miRNAs play essential roles in tomato drought tolerance.This work will help to further characterize specific miRNAs functioning in drought tolerance.