Melon Genome Assembly And Mining Of Major Genes Associated With Fruit Sugar Accumulation

Melon(Cucumis melo L.),originating in Africa,is cultivated across the globe.Two subspecies of melon,C.melo ssp.melo and C.melo ssp.agrestis,were domesticated independently.Accurate reference genome and abundant genetic resources make it possible to study evolution and map genes for important agronomic traits at the genome level.Agrestis melon has many special traits that can be used for germplasm improvement,especially in terms of disease resistance,fruit flavor and environmental adaptability.However,the high-quality genomic information of melon(ssp.agrestis)has not been resolved.Sugar content is the most important quality trait of melon fruit.However,the sugar accumulation in the fruit is very complicated,and the key factor for controlling the sugar accumulation is unclear yet.In this study,we assembled the chromosome-level genome of the agresits subspecies by combination of third generation sequencing(Pac Bio)and Hi-C tecnologies:Through comparative genomics analysis,positive selection genes for phloem assimilate transport in cucurbit crops were identified,and the mechanism of oligosaccharide transport in Cucurbiteace was revealed from an evolutionary perspective;RNA-seq during fruit development and whole genome DNA methylation sequencing were used to discover the regulatory relationship of DNA methylation and gene expression during the development period of melon fruit;recombinant inbred lines were used to map 5 QTLs related to fruit sugar accumulation.The main results of this study are as follows:1.The assembly of chromosome-level melon(ssp.agrestis)‘HS' genomeWe used single-molecule real-time sequencing SMRT,and high-throughput chromatin capture technology Hi-C combined with third generation pair-end reads sequencing and ultra-high-density genetic map assistance and other multi-tools for assembly,and finally assembled 359.4 Mb HS genome in size,contig_N50 length is3.45 Mb,contig_N90 length is 926.7 Kb,super-scaffold_N50 length is 29.76 Mb,and98.15% of the sequences are anchored on 12 pseudochromosomes.The BUSCO complete assessment was 91.0%,and 28,898 protein-coding genes were annotated.Compared with the published melon genomes,it has higher integrity,continuity and accuracy.By comparing the ‘HS',DHL92 and ‘Payzawat' genomes,there are chromosomal structural variations(SVs)caused by intrachromosomal inversions and translocations on Chr₀1,Chr₀4,Chr₀8 and Chr₁1.2.Evolutionary analysis of phloem oligosaccharide transport in cucurbitaceous cropsAccording to the estimation of the phylogeny and divergence time base on single copy genes,it was found that the differentiation time of melo and agrestis was about2.6-4.9 million years ago.It has been identified that some gene families related to carbohydrate metabolism and carbohydrate binding are significantly expanded in the genome of Cucurbitaceae,which may be related to the transport of carbohydrates.Evolutionary analysis found that the key enzyme uridine diphosphate pyrophosphorylase(UDP-sugar pyrophosphorylase,MELO05057)responsible for the frist step in the synthesis of raffinose oligosaccharide family(RFOs),and the initial enzyme ?-galactosidase(MELO06810),which is hydrolyzed before being transported into the fruit,were subject to positive selection among Cucurbiteace,and were deduced to be related to the oligosaccharides transport of cucurbit crops.3.DNA methylation and regulation of gene expression during melon fruit developmentTranscriptome sequencing and whole-genome DNA methylation sequencing were carried out on the flesh samples from the fruit development stage of ‘HS' and DHL92 and combined analysis after determination of sugar content that DHL92 accumulated sharply after DAP20 while ‘HS' kept rather low throughout all stages.The codifferentially expressed and methylated genes of the fruit at 20 days after pollination were mainly enriched in genes which are focused on the metabolic pathways related to fruit quality,in which 127 of all 164 genes related to sugar metabolism are both differentially expressed at the transcriptional level and modified by methylation.DNA methylation can apparently regulate the expression of sugar accumulation-related genes and is associated with the accumulation of sugar in melon fruit.4.Mapping major genes for sugar accumulation in melon fruit and functional analysis of candidate genesUsing recombinant inbred line populations(RILs),phenotype of sugar accumulation in both spring and fall were analysed,which in general showed a normal distribution of sucrose content in the RILs population,then we mapped three QTLs related to fruit sugar accumulation via genotyping-by-sequencing in spring and fall,respectively.Three loci are located in Chr₇,Chr₁0 and Chr₁1 in spring while another three located in Chr₄,Chr₅ and Chr₁0 in fall.In these intervals,there are 5genes that may be related to the sugar accumulation and metabolism of melon fruit through gene function annotation,gene expression,and genotyping analysis.They are:MELO21653(alkaline/neutral invertase 1,CINV1),MELO21692(sucrosephosphatase 2,SPP2),MELO16006(?-galactosidase,?-Gal),MELO16058(sugar phosphate/phosphate transporter,SPPT),MELO09061(?-galactosidase,?-Gal).