Exploiting Key Genes Regulated To Malate Accumulation By Genome-wide Association Analysis In Tomato Fruit

Tomato（Solanum lycopersicum）is one of most popular fruit and vegetable worldwide due to the suitable sugar-acid ratio and abundant of sugar.Tomato also serves as an important source of fiber and antioxidant nutrients in the human diet and play as a key model for the study of fruit biology.High-yielding,disease resistant,storage and transportation were the main objective for tomato breeding because of the limitations of our basic national conditions in the past years.Naturally,the flavor and nutritional quality was ignored.It is contradictory with the consumption concept of "delicious,nutritious,safe and healthy".In order to address this global concern,quality improvement will become urgent.Content of soluble sugar and organic acids and their composition ratio is an important basis for the formation of fruit flavor.Additionally,Al-activated release of organic acids including malate from root was the best documented physiological mechanism for plant A1 tolerance in acid soil.Therefore,studying the mechanism of malate accumulation in different tomato genotypes provides large knowledge about tomato quality evaluation and improvement.With the development of sequencing technologies,the cost of sequencing has been greatly reduced.The completion of the whole genome sequencing of different species provides a huge amount of genomic information for our further exploiting of gene function.In this current study,metabolic genome-wide association study based on-5.5 million SNPs obtained from 360 diverse accessions of tomato was carried out.Moreover,BSA was employed for the linkage mapping of fruit malate.The main results are as follows:1.We determined the levels of 40 metabolic traits in the red fruit of 272 diverse global collections of tomato accessions using gas chromatography-mass spectrometry（GC-MS）at two locations.On the basis of the levels of metabolites detected in our study,cluster analysis grouped the varieties into three distinct clusters:PIM,CER and BIG.2.Using the SNP-based genotype and fruit malate concentrations of 272 accessions we performed a GWAS using the compressed mixed linear model（CMLM）.In this genome-wide scan we observed a single region on chromosome 6 that contained multiple SNPs highly associated with fruit malate concentrations.An aluminum activated malate transporter protein named SIALMT9 in this region was supposed to be good candidates for the causal gene underlying the observed malate QTL.3.To further genetically characterize the fruit malate QTL on chromosome 6 identified using GWA analysis,we generated an experimental F2 population in which the alternate alleles of most malate associated SNPs on chromosome 6 were segregating.The F1 generation of this cross showed a fruit malate concentration significantly lower than the TS-40 parent,and slightly higher than the TS-66 parent.We sequenced 2 bulk populations（HM-pool and LM-pool）with extreme concentration of fruit malate,each consisting of 40 progenies from an F2 population of 350 individuals,to a depth of 40×.Our linkage mapping confirmed the results we obtained from our GWA analysis,and further supported SIALMT9 on chromosome 6 as candidate genes driving the natural variation in fruit malate accumulation we observed in our global toamto population sample.4.SlALMT9 contained six exons and five introns,encoding a protein of 559 aa in length,which is predicted to contain 5 transmembrane domains with molecular mass of 62.54 kDa.SlALMT9 exhibits highest amino acid sequence similarity to Ma（58%）of apple.5.Using the 360 re-sequenced accessions,we examined the predicted protein coding haplotypes of SlALMT9.A clear association between haplotype and malate concentration is observed,i.e.,accessions in haplotype Ⅰ and haplotypes Ⅱ showed dramatic difference in malate concentration.Since there is only one H/R307 difference between haplotype Ⅰ and haplotype Ⅱ,we supposed that H/R307 in the C-terminus hydrophobic region of SlALMT9 was likely the determinant of malate accumulation in tomato fruit.6.To obtain the expression pattern of SlALMT9,the expression of SlALMT9 was investigated using the GUS reporter gene driven by the presumed SlALMT9 promoter in transgenic tomato plants（ProSlALMT9::GUS）.The SlALMT9 promoter had no transcription activities in either main roots or lateral roots under normal conditions,but showed activity upon Al3+ treatment under low pH.7.Transient expression of CaMV35S::SlALMT9-GFP fusion construct in Nicotiana benthamiana leaves showed that SlALMT9 localized in the tonoplast.8.Among the sequenced SIALMT9 genes in 17 accessions,seven SNPs were found and all significantly associated with malate concentration.These only non-synonymous polymorphisms of SNP6（A→G）lead to the substitution of H/R30.The geographic and evolutionary analysis indicated that SNP ch06₄1343002 were selected in the process of tomato domestication and breeding.9.Over expressed SlALMT9-G in TS-66 leads to a significantly increase of malate content in both fruit and root.In addition,the functionality of the SIALMT9 gene was investigated via ectopic expression in yeast.The yeast strain with SlALMT9-G showed stronger A1 resistance than that of SIALMT9-A yeast strain.10.Compared with the TS-66 wild-type,some sugar and organic acids such as sucrose and citric acid in red fruit were significantly changed.11.RNA-seq analysis of wild-type（Ts-66）and overexpressing line（OX1）was employed to investigate potential regulation network of SIALMT9.Among 1,887 differentially expressed genes,1,231 were up-regulated（65.2%）and 656 were down-regulated.Compared with the wild type,some organic acid transporter protein,sugar transporter protein,vacuolar membrane transporter protein,structural genes involved in sugar and organic acid and disease resistance protein showed different expressed.These expression differences partially coincide with changes of sugar and organic acid concentration.12.Two PCR-based markers were developed based on polymorphism of ALMT9SNP6-A and ALMT9SNP6-G.Among the 68 Fi hybrids tested,the marker showed a co-segregation with malate content that ALMT9SNP6-A and ALMT9SNP6-A/G cultivars had lower malate accumulation,but ALMT9SNP6-G cultivars contained higher malate content.13.We carried out a genome-wide analysis of ALMT gene family in tomato and indentified 16 SlALMTs which can be divided into 4 groups（Cladel,2,3 and 4）in reference to Arabidopsis,rice and potato.Gene structure analysis showed that all SlALMTs contained six exons and five introns and the N-domain contained 5 or 6 transmembrane domains.Expression abundance of most SlALMTs,especially for SlALMT9,can be induced by A1 treatment in root,stem and leaf,suggesting that SIALMT9 may play an important role in response to A1 stress in tomato.In addition,RNA-seq combined with metabolism analysis was used to investigate global dynamic changes in gene expression and metabolite accumulation during the development and ripening of fruit from two tomato cultivars,Ailsa Craig（AC）and HG6-61.The main results are as follows:1.The metabolically dynamic analysis of fruit at seven developmental stages（7,14,21,28,35,42 and 49 days after flowering）from two tomato cultivars（AC and HG6-61）was evaluated using LC-MS and HPLC.The results showed that the dynamic changes of majority metabolites in both genotypes are consistent,but some metabolites such as lycopene and naringenin chalcone accumulated earlier in the AC than in HG6-61.2.The transcriptomes of fruit at seven developmental stages（7,14,21,28,35,42 and 49 days after flowering）from two tomato cultivars（Ailsa Craig and HG6-61）were evaluated using the Illumina sequencing platform.A total of 26,397 genes,which were expressed in at least one developmental stage,were detected in the two cultivars,and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis.3.A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid,carotenoid and flavonoid biosynthesis（20,34 and 37 respectively）.Moreover,some metabolic related transcription factors were validated by two sequenced varieties cultivar Heinz and the wild relative Solanum pimpinellifolium.4.To further validate the transcriptional association between the transcription factors and structural genes,we performed agroinfiltration to verify the putative regulatory activity of the selected transcription factors[MYB（Solyc09g010840.1）,NAC（Solycl2g013620.1）and ZIF（Solyc06g065440.1）]on genes associated;with ascorbic acid biosynthesis.The expression levels of most selected structural genes involved in ascorbic acid metabolism were up-regulated.