Comparative Genomic Research On The Organellar Genome Of Watermelon And Melon

Watermelon（Citrullus lanatus L.）and melon（Cucumis melo L.）are all the worldwide famous horticultural crops,due to their significant commercial worth.Organelle genome is an important component of a species whole genome.Studying organelles（chloroplast and mitochondria）genome is an indispensable way to understand the complete genetic information of watermelon and melon after nuclear genome project and achieve the molecular design breeding in watermelon and melon.At present,there are few studies on the organellar genome of watermelon and melon,which could hamper the development of researches based on the organellar genome.Hence,there is a substantial need to study on the organellar genomes of watermelon and melon.In this existing study,we first sequenced,assembled and published the chloroplast genome sequence of cultivated watermelon and whole mitochondrial genome of cultivated melon,and then combined them with genomic data published in the GenBank database,a comprehensive comparative genomic analysis on organellar genomes of watermelon and melon was conducted.The main research contents and results are as follows:1.The first chloroplast genome of cultivated watermelon was completed and published.The chloroplast genome of cultivated watermelon is composed of a single-ring double-stranded DNA molecule,and its total length is 156,906bp,showing a typical four segment structure.Large single copy（LSC）length is 86,844bp,small single copy（SSC）length is 17,896bp,and two inverted repeats（IRa and IRb）regions have same length（26,083bp）.The whole genome encodes 131 genes,including 86 protein-encoding genes,37 tRNA and 8 rRNA.The structure,GC content,gene arrangement and codon preference of the genome are similar to the chloroplast genomes of other higher plants.In addition,there were 19 tandem repeats（TR）,26 pairs of dispersed repeat sequences（DR）and 53 simple sequence repeats（SSR）in the watermelon chloroplast genome,polyA and poly T are the most of SSR types,mainly distributed in non-coding regions.2.A new efficient strategy for de novo chloroplast genome based on high-throughput sequencing data is proposed.This study proposed a new strategy for de novo chloroplast genome based on the sequencing data from Illumina Hiseq.The chloroplast genome sequences can be obtained efficiently and economically without any separation and purification of chloroplast genome DNA from total DNA by using this strategy.We successfully completed and published the first chloroplast genome of cultivated watermelon by using this method.For validating the good universality and practicality of method,we successfully use this method to assemble and published the first complete chloroplast genome of Citrullus colocynthis,Citrullus rehmii,Citrullus amarus,Cucurbita moschata,Cucurbita maxima,Momordica charantia,Lagenaria siceraria.Through a comparative genomic analysis of chloroplast whole genome sequences of 15 cucurbit plants,we found the chloroplast genomes of Cucurbitaceae are single-ring double-stranded DNA molecule,and GC content ranged in 36.7³7.2%,which is similar to watermelon,whole genome length is about 155¹59kb,its average length is about 157kb,encoding gene number is in 130¹33,and the average number is 131;chloroplast genome collinearity among 15 cucurbit plants are fine,no rearrangement phenomenon was found.Additionally,we developed 7 DNA barcoding and 38 SSR markers for species identification,phylogenetic and population genetic research in Cucurbitaceae based on the alignment of chloroplast genome sequences of 15 cucurbit plants.Phylogenetic relationship analysis based on chloroplast whole genome sequence,the results similar with the nuclear genome analysis can be obtained.3.The first complete genome sequence of mitochondrial genome of cultivated melon was completed and published.In this study,the PacBio Sequel sequencing technology with long-reads advantage was applied to the mitochondrial genomic research of cultivated melon,successfully completed and published the complete mitochondrial genome sequence of cultivated melon with longest sequence in Cucurbitaceae.The genome consists of 3 circular DNA molecules.The main loop length is 2,709,526bp,the lengths of remaining two small loops were 149,555bp and47,592bp,respectively A total length of genome is about 2.9Mb（2,906,673bp）,and the GC content is 44.77%,higher than the GC content of melon nucleus genome and chloroplast genome.The genome is encoding 88 genes,including 40 protein-encoding genes,8 rRNAs and 40 tRNAs,in which all protein-encoding genes are located in the main loop,only 5 tRNAs distributed in second loop,no encoding gene is found on the third loop.The non-coded sequence accounts for the vast majority of the mitochondrial genome of cultivated melon,with a percentage of 98.32%.The chloroplast-derived and nuclear-derived sequence in the melon mitochondrial genome sequence were 2.73%and 46.47%,respectively.In addition,there were 4,861 pairs of forward repeats（FR）,439 pairs of inverted repeats（IR）,653 TRs and 218 SSRs in the melon mitochondrial genome sequence.The total length of repeat sequences was about 44.2%of the total genome.A comparative genomic analysis of mitochondrial genome structure features of melon,watermelon,cucumber and zucchini,found that large content of repeat sequences and nuclear-derived sequences is the main reason for increasing and variation of melon mitochondrial genome,which could provide an insight into the genetic variation of cucurbit plant mitochondrial genomes.4.The chloroplast genome was first applied to the population genetics of watermelon and melon.We identified 82 SNP loci on the chloroplast genome of watermelon natural population contained 313 watermelon materials,and 192 SNP loci on the chloroplast genome of melon natural population contains 348 melon material,these SNP loci mainly distributed in the LSC and SSC of chloroplast genome,the two IRs（IRa and IRb）almost does not contain SNP loci.Then the population structure of watermelon and melon population,and fruit-related traits and genetic diversity within each group were analysed based on these SNP loci.The results showed that 82SNP loci can effectively divided the watermelon natural populations are into 4 groups:wild group,semi-wild group,America ecotype cultivated group and Asia ecotype cultivated group,their genetic diversity decreased sequentially,respectively is 6.6×10^-5,2.4×10^-5,9.8×10^-66 and 5.41×10^-6,the wild watermelon group have the significant difference with the other three groups in single fruit weight（SFW）,soluble solid content（SSC）,fruit color value（FCV）and 1000 seed weight（1000SW）;192 SNP can effectively divided melon populations into 3 groups:wild group,thick-grind type cultivated melon group and thin-grind type cultivated melon group,its genetic diversity were decreased,1.12×10^-3,4.71×10^-44 and 2.85×10^-4,respectively,there is a remarkable difference in flesh thickness（FT）among three groups.These results suggest that chloroplast genome can be used to study the population genetics of watermelon and melon,and also contribute to the classification and identification of core germplasm resources.In this study,the first cultivated watermelon chloroplast genome and first mitochondrial reference genome of melon were published.A new efficient strategy for de novo chloroplast genome was constructed,and DNA barcoding and SSR were developed for plant research in Cucurbitaceae,which would provide a theoretical foundation for the studies based on chloroplast and mitochondrial genomes of watermelon and melon in the future,such as phylogenetic relationship,species identification,population genetics,nucleo-cytoplasmic interaction,and RNA editing.