Map-based Cloning Of A Dwarfness Related Gene Cldf(Citrullus Lanatus Dwarfism) In Watermelon

Watermelon is an important cucurbit crop worldwide,Plant height in watermelon is a vital agronomic architecture trait that can increase fruit yield and reduce labor costs in crop cultivation and pruning.Therefore,it is of great significance to map and clone the genes that related to watermelon dwarf for breeding new watermelon cultivars with a dwarfism phenotype.In this study,for inheritance analysis and causal gene identification,the parents of dwarf line"N21"and normal line"M08"were used to produce F₂ segregating populations,the genetic analysis showed that watermelon dwarf was a recessive trait controlled by a single gene,named Cldf?Citrullus lanatus dwarfism?.At the same time,Genome resequencing technology was used to complete the precise localization of Cldf gene.The candidate gene Cla015407 was determined.The main results are as follows:1. Compared with the normal line M08,the dwarf inbred line N21 with smaller leaves showed compact plant architecture.The objective phenotype can be visibly distinguished at the seedling stage and obviously classified as dwarfism or normal throughout the whole development stage.To compare the internode length and plant height among two parents and their F₁ progenies,five individuals for each line were randomly selected.The internode length of N21?4.0±0.8 cm,22 internodes?was much shorter than that of M08?9.6±1.7 cm,22 internodes?,and the plant height of the former?88.0±6.1 cm?was also significantly less than that of the latter?211.6±8.9 cm?.The plant height of reciprocally crossed F₁ plants,as well as internode length,was also significantly higher than the N21 dwarf line,but significantly less than the ordinary line M08,indicating that the normal vine phenotype is dominant to dwarfism.2. To analyze the inheritance of the dwarfism phenotype,we collected phenotypic data from two reciprocal F₂ segregation populations.There were 315 normal and 106 dwarf plants in the M08×N21 F₂ population?total 421 individuals?,fitting a 3:1 Mendelian ratio??²=0.0008,p=0.93?.Moreover,364 N21×M08 F₂ individuals contained 266 normal and 98 dwarf vine plants,which was also consistent with the Mendelian ratio of 3:1??²=0.62,p=0.40?.Taken together,these data suggest that the dwarfism phenotype in N21 is controlled by a single recessive gene and is hereafter designated Cldf.3. In this study,to obtain enough SNPs and indels for developing polymorphic marks,genomes of two parental lines were re-sequenced.152,894 high-confidence SNPs and 4018indels were obtained and utilized to develop CAPS markers in the mapping strategy.To locate the Cldf gene on the chromosome,polymorphic markers were designed for the other eleven chromosomes.As a result,the marker W12181817 on chromosome 9 was confirmed to be linked with the dwarfism locus,populations were subjected to genotype with new markers.The dwarfism trait was delimited within a 235.67 kb region between markers W1222182 and W1221186,with 1?1.04 c M?and 3?3.13 c M?recombinants,respectively.4. To further narrow down the initial mapping interval,the remaining larger segregating populations?1996 strains?were subjected to genotype with the primary flanking markers W1222182 and W1221186 and the recombinant was identified.Four new polymorphic markers were developed to genotype the recombinants.As a result,the Cldf locus was finally narrowed down to a 32.88 kb region between markers W1222183 and W1222185.One cosegregated marker,W1221184,was obtained,which can be used for marker selection breeding programs.5. According to the annotated version of the reference genome,only three genes were annotated in the fine mapping region.Gene annotation of the corresponding region revealed that the Cla015407 gene encoding a gibberellin 3?-hydroxylase functions as the best possible candidate gene for Cldf.Sequence analysis showed that the fourth polymorphism site?a G to A point mutation?at the 3?AG splice receptor site of the intron leads to a 13 bp deletion in the coding sequence of Cldf in dwarf line N21 and thus results in a truncated protein lacking the conserved domain for binding 2-oxoglutarate.6. Real-time fluorescence quantitative PCR was used to analysis the expression levels of the candidate gene Cla015407 in tissue-specific of the parents,and the results showed that similar expression patterns were observed in the five organs between two parental lines,except for tissue tendrils.Real-time fluorescence quantitative PCR was used to analysis the expression levels of of pathway-related genes in apical shoots of two parental lines.And the results showed that the transcription levels of both CPS and KAO homologs,as well as three GA20ox members,were upregulated in dwarf line N21 compared to that in M08,suggesting possible feedback regulation,the two Cl GID1s exhibited distinct expression patterns between two parental lines,suggesting their possible distinct functions,five DELLA homolog genes were identified in watermelon,of which only one gene,Cla019759,was repressed in dwarf mutant Cldf.7. We investigated the phenotypes of homozygous recessive individuals from the F₂population,which were treated with exogenous GA₃ application.The results showed that,plant heights could be rescued by the application of GA₃.8. To further recover the evolution of the GA3ox family in plants,a phylogenetic tree was constructed with 28 GA3oxs from Cucurbitaceae and 19 homologs from other families.It seems that there are three ancient lineages in the common ancestor of Cucurbitaceae,and one of them?subgroup IIa?is specific to cucurbit crops.