| Tomato(Solanum lycopersicum L.or Lycopersicon esculentum Mill.)from Solanaceae,originating in the Andes Mountains of South America,is one of the most economically important vegetables widely grown in the world.Intensive selection of a few economic traits during domestication has led to the loss of genetic diversity among the commercial tomatoes.A lot of valuable genes have been lost during the domestication of tomatoes.Wild tomatoes with many valuable genes are very useful in cultivars improvement.It is of great importance to have a clear understanding of the genetic diversity and relationship between cultivated tomatoes and their wild species for the conservation and further utilization of tomato germplasm resources.As a consequence of global warming,high temperature has become an urgent issue of global agriculture.High temperature is a common abiotic stress when tomatoes were cultivated through summer.When the environmental temperature is higher than suitable temperature,the growth of tomato plants could be inhibited and the yield of tomato fruit could be decreased together with deteriorate quality.Therefore,evaluation and identification of the heat tolerance in tomato germplasm resources and uncover the mechanisms of heat tolerance in physiological and molecular levels are very important.This is not only the critical scientific problem in terms of tomato research,but also the urgent demand in tomato production.Our aim is to evaluate and screen the heat tolerance of tomato germplasm resources and uncover the physiological and molecular mechanisms for heat tolerance in tomato.Firstly,the genetic diversity of some cultivated and wild tomatoes with enlarged samples were analysed.Secondly,the heat tolerance of tomato germplasm resources were evaluated and screened.Then,with the tomatoes differing in heat tolerance as materials,the physiological responses of tomatoes at vegetative and reproductive phase to high temperature were studied.Finally,miRNAs and their target genes in heat-tolerant wild tomato under high temperatures were identified.The main contents and results are as follows:1.With 50 cultivated and wild tomato genotypes as plant materials,morphological traits and molecular markers were used to assess their genetic diversity.The three components of principal component analysis(PCA)explained 78.54%of the total morphological variation.Three dimensional PCA plot based on the morphological descriptor separated the 50 tomatoes genotypes into distinct section.Morphological cluster divided the 50 genotypes into six clusters.In addition,15 polymorphic genomic simple sequence repeat(genomic-SSR)and 13 polymorphic expressed sequence tag-derived SSR(EST-SSR)amplified 1115 and 780 clear fragments,respectively.Genomic-SSR detected a total of 64 alleles and,while EST-SSR detected 52 alleles.The polymorphism information content(PIC)was slightly higher in genomic-SSR(0.49)than the PIC in EST-SSR(0.45).The average similarity coefficient among the wild tomatoes and wild relative is lower than the average similarity coefficient among the cultivated tomatoes.The dendrogram based on the genetic distance divided the 50 tomato genotypes into eight clusters.The Mantel test between genomic-SSR and EST-SSR matrices revealed a good correlation coefficient,whereas the morphological matrices and the molecular matrices correlated weakly.High variability of the 50 tomato genotypes,especially for the wild tomatoes,was observed at the morphological and molecular levels.2.Initially,67 tomato genotypes were ranked according to maximum quantum efficiency of photosystem ? photochemistry(Fv/Fm)in three-step heat stress.Two genotypes with higher Fv/Fm(heat-tolerant group)and two genotypes with lower Fv/Fm(heat-sensitive group)were acquired.Physiological responses of the four genotypes to seven days of heat stress were validated.Pollen germination of the four genotypes was investigated at high temperature.The results showed that the heat-tolerant group maintained higher leaf pigment contents and higher total phenolic content than the heat-sensitive group under heat stress.The heat-tolerant group maintained unaltered stomata and pore area and net photosynthetic rate,but increased stomatal conductance under heat stress compared with the control.Chloroplasts in the heat-tolerant group maintained normal shape,whereas the chloroplasts in the heat-sensitive group became swollen with decomposed starch grain after heat stress.The heat-tolerant group showed higher pollen germination rate,longer pollen tube length compared with the heat-sensitive group at high temperatures.In conclusion,Fv/Fm is an early indicator when tomato plants are exposed to heat stress and could be used for heat tolerance identification.The stay-green trait,improved ability of stomata regulation and higher contents of reactive oxygen species scavengers are protective mechanisms in heat-tolerant tomatoes.3.With 'LA1994'(heat-tolerant)and 'Aromata'(heat-sensitive)at the seedling and anthesis stages as plant materials,photosynthetic parameters,maximum quantum efficiency of photosystem?photochemistry(Fv/Fm),chlorophyll and carbohydrate content in the leaves of plants grown under control and high temperature was analysed.The net photosynthetic rate(PN)of 'Aromata' decreased significantly during high temperature at both stages,whereas the PN of 'LA1994' remained the same.The Fv/Fm and chlorophyll content were significantly lower in 'Aromata' than in 'LA1994' under HS at both stages.High temperature induced a similar change in carbohydrate content in the young leaves of both cultivars at anthesis.Fructose and sucrose content was unaffected in the mature leaves of 'Aromata' with decreased starch content but significantly increased in 'LA1994' with unaltered starch content under high temperature at anthesis.High temperature inhibited fruit set due to flower wilting and abnormal abscission only in 'Aromata'.The results indicate that physiological responses to high temperature at seedling stage correspond to those at anthesis.A decrease in the PN in the mature leaf(source)of tomatoes induced an insufficient carbohydrate translocation to the fruit(sink),indicating a suppression of flower and fruit set under high temperature.4.Three small RNAs(sRNAs)libraries and three degradome libraries were constructed by leaves of 'LA2093'(Solanum pimpinellifolium L.)at normal temperature as control(26/18?,NT),moderately elevated temperature(33/33?,MET)and acutely elevated temperature(40/40?,AET).Following high throughput sequencing and filtering,662 conserved miRNAs and 97 novel miRNAs were identified in the three sRNAs libraries,which enriched the amount of miRNAs in tomato.Of these miRNAs,39 conserved and four novel miRNAs were significantly up-regulated;38 conserved and 15 novel miRNAs were significantly down-regulated in the MET library as compared to the NT library.There were 62 conserved and 20 novel miRNAs with significant up-regulated expression levels and 57 conserved and 11 novel miRNAs with significant down-regulated expression levels in the AET library as compared to the NT library.By degradome sequencing,349 sequences were targeted by 138 conserved miRNAs and 13 sequences were targeted by eight novel miRNAs.The functional annotation showed that the target genes of the miRNAs in the wild tomato plants at the elevated temperatures were mainly involved in regulation of transcription,sequence-specific DNA-binding transcription-factor activity,DNA binding and catalysis.The expression levels of four miRNAs and five target genes were validated by quantitative real-time PCR(qRT-PCR),which were largely consistent with the sequencing results.Moreover,the network between the miRNAs with significantly different expression levels and their target genes indicated that the regulation of the target genes mediated by tomato miRNAs played positive roles in response to high temperatures.This study enriched the amount of the heat-responsive miRNAs and laid a foundation for clarifying the miRNAs response and the regulatory mechanism of the target genes mediated by miRNAs in plants at high temperatures. |
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