Genetic And Molecular Mechanisms Of Delayed Green Leaf Color And Short Internode Length In Watermelon(citrullus Lanatus)

Cultivated watermelon?Citrullus lanatus?is one of the most important food crops in the Cucurbitaceae family.Its diversification after domestication has lead cultivated watermelon to display diverse fruit quality and agronomic traits?leaf color and plant architecture?.Recently,there has been increase interest in cloning and identification of genes conferring important agronomic traits,which plays a significant role in the final productivity of the crop.Currently,our understanding on these important agronomic traits in watermelon is very rare,because only few research works have been reported on molecular genetics and their regulation mechanisms.Here,we conducted a research on delayed green leaf color and short internode length,cloning and identification of candidate genes controlling both traits through molecular biology to investigate their molecular genetics and regulation mechanisms.In order to clone and identify candidate genes controlling delayed green leaf color and short internode length in watermelon,research studies were carried out using watermelon inbred lines having extreme phenotypes:Charleston gray?green leaf?and Houlv?delayed green leaf?,and Zhengzhouzigua?long internode length?and Duan125?short internode length?as mapping parents.For each trait,the two parental lines were crossed to generate F₁ progenies and then F₁ plants were self-pollinated to produce F₂ population.Besides,F₁ plants backcrossed with both parental lines to generate BC₁P₁ and BC₁P₂.Thus,this research work comes up with the following outcomes:All F₁ plants derived from the cross between Charleston gray?green leaf?and Houlv?delayed green leaf?showed green leaf color,suggesting that the green leaf was dominant over the delayed green leaf.The inheritance analysis of F₂ and BC₁ progenies were consistent with the expected Mendelian segregation ratio of 3:1 and 1:1,respectively.Through whole genome resequencing using bulk segregant analysis approach,the delayed green?dg?locus was localized within 16884692-16938229 bp?7.48 Mb?mapping region on chromosome 3.Then,using 765 F₂ individuals the region was fine mapped to 53.54 kb region between SNP130 and SNP135 markers,having three putative genes.Sequence alignment of the three putative genes indicated that there was a single SNP mutation?G/A?in the coding region of ClCG03G010030 in Houlv parent,which resulted in an amino acid change of Arginine to Lysine.The gene functional annotation indicated that ClCG03G010030 encodes FtsH extracellular protease family,involved in early chloroplast development.The expression analysis of the candidate gene showed that higher expression was observed in the green leaf plants than that in delayed green leaf plants at the early leaf development.Likewise,in the delayed green plants,reduced amount of pigment contents,chlorophyll precursors,photosynthetic and chlorophyll fluorescence parameters and abnormal chloroplast development was observed.However,as the leaf development progresses these characteristics were showed an increasing trend and had no significant difference compared with the green leaf plants.Taken together,these results indicated that ClCG03G010030 might be the candidate gene controlling the delayed green leaf phenotype in watermelon.This finding provides useful information for delayed green leaf color variation,chloroplast development and marker-assisted selection in watermelon and for the study of delayed green leaf color in other plants.The transcriptomic analysis identified 196 differentially expressed genes between green leaf and delayed green leaf plants.The expression level of chlorophyll biosynthesis genes encoding glutamyl-tRNA synthetase?GTS?,protoporphyrin IX Mg-chelatase subunit H?CHLH?and protoporphyrin IX Mg-chelatase subunit I?CHLI?,protoporphyrin IX Mg-methyltransferase?CHLM?,and chlorophyll a oxygenase?CAO?were down-regulated.While,protoporphyrin IX Mg-chelatase subunit D?CHLD?was up-regulated.Concurrently,phytoene synthase?PSY?,15-cis-zeta-carotene isomerase?Z-ISO?and Lycopene epsilon cyclase?LCYE?encoding genes were down regulated in the delayed green leaf plants.Furthermore,genes associated with photosystem II?PSII?such as ClCG00G002330?D1 subunit,PsbA?and ClCG08G005640?PsbS?were down-regulated;whereas,ClCG07G005950?CP47 subunit,PsbB?and ClCG02G011250?PsbL?were up-regulated.Likewise,the expression level of ClCG02G010780?PsaA?and ClCG01G009360?PsaB?were also down-regulated in the delayed green plants than that in green leaf plants.Most of the genes encoding light harvesting protein complexes I and II?LCHI and LHCII?were down-regulated in the delayed green leaf plants.These results provide new insight into the molecular mechanisms of delayed green leaf color and expression of genes involved in pigments biosynthesis,chloroplast cell division,chloroplast protein components,photosynthesis,which are potentially applicable for further molecular selection breeding in watermelon.We identified a locus responsible for short internode length in watermelon using Zhengzhouzigua?long internode length?and Duan125?short internode length?as mapping parents.Genetic analysis indicated that F₁ plants were in accordance with long internode length plants,suggesting that the long internode length was dominant over the short internode length.The observed F₂ and BC₁ progenies were fitted to the expected phenotypic segregation ratios of 3:1 and 1:1,respectively.Through whole genome resequencing?WGR?using bulked segregant analysis?BSA-seq?approach,the locus was mapped on chromosome 9.Furthermore,the region was narrowed down to 8.525 kb having only one putative gene?Cla015407?flanking by CAPS90 and CAPS91 markers,which encodes gibberellin 3?-hydroxylase?GA3?-hydroxylase?.The sequence alignment of the candidate gene between Zhengzhouzigua and Duan125indicated that a 13 bp deletion was detected in the Duan125 parent,which resulted in a truncated protein.In the short internode length plants,the expression level of the Cla015407 was threefold lower than that of the long internode length plants in the stem tissue.All together,these results suggested that Cla015407 might be the candidate gene controlling the short internode length in watermelon.