| Watermelon(Citrullus lanatus,2 n = 22)belong to the Cucurbitaceae family,an annual vining plant.The quality traits and plant architecture of watermelon are always the breeding goal of breeders.The stripe pattern of watermelon is an essential commodity trait,which has a higher diversity and determines the choice of consumer.Hypocotyl is the component of plant architecture in watermelon seeding,and the length of hypocotyl is associate with the lodging resistance,seeding transportation,survival rate and the eventually watermelon production.However,the molecular mechanisms of watermelon stripe appearance and hypocotyl elongation remain unclear.In this study,a dark-green stripe and normal hypocotyl line WT2,a netted stripe and short hypocotyl line WM204 were used to construct F1 and F2 populations,which were used for genetic analysis and fine-mapping.And the major results are as follows:1.WM204,light green rind and has netted stripe.WT2,light green rind and has darkgreen stripe.Then,there was an obvious difference for chloroplast number and size between WT2 and WM204 through analysis the cellar structure of fruit rind between parental lines by using Transmission electron microscopy.Furthermore,we developed F1 and F2 population by crossed WT2 and WM204.The segregation data for stripe in the F2 population showed a dominant gene Cl GS controlling the formation of dark-green stripe.2.A total of 1256 SSR markers were used for polymorphism screening between WT2 and WM204,242 SSR markers showed clear bands and polymorphism.Among of the 242 good polymorphism SSR markers by using a BSA method,12 were further identified to have polymorphism between stripe bulk and netted bulk,and all of which were located on the end of watermelon chromosome 6.Based on the data of two parental lines re-sequencing,we identified many SNPs and Indels in the candidate region and developed new molecular markers for mapping Cl GS.The locus of Cl GS was located in a 107 Kb region between the d CAPS3 and d CAPS8 on chromosome 6 finally.There were 11 genes(Cla019202-Cla019212)in candidate region,we identified 64 SNPs in the 11 genes,and 25 of them changed the sequence of amnio acid.Moreover,ten of them showed different expression level between ovary of parental lines.3.41 natural lines showed netted and 33 natural lines showed dark-green stripe were selected and analysized in 414 natural lines resequencing data.Then,we found a Indels located in the 8th exon of Cla019205 showed higher conservative between two bulks.We further developed the Indel marker based on the indels and showed co-separation in another 25 natural accessions.Furthermore,the 597 th base upstream of Cla019205 start carbon in 41 netted lines were deletion.Therefore,these evidences suggested Cla019205 is associate with dark-green stripe formation of watermelon.4.The length of WT2 hypocotyl is 6.17 ± 0.43 cm and the length of WM204 hypocotyl is 2.08 ± 0.28 cm.Genetic analysis indicated the normal hypocotyl was dominant over the short hypocotyl,and the observed F2 plants fitted the expected phenotypic segregation ratios of 3:1.Which suggested a recessive gene Clsh controlling the short hypocotyl.242 polymorphism SSR markers between parental lines were used for bulker-segregant analysis to primary mapping Clsh.3 SSR markers located on the chromosome 9 showed the clear polymorphism between normal hypocotyl plants and short hypocotyl plants in F2 population and linkage with Clsh.The new SSR markers were developed in the candidate region.Using a F2 population with 1408 individuals screened the SSR markers and constructed a linkage map,and we found Clsh was located on a 50 Kb region of chromosome 9.According to the annotation of 97103 V1 genome,there were 3 genes(Cla015408,Cla015407,Cla015406)in candidate region.Among of them,Cla015406 encodes a 2-oxoglutarate-dependent dioxygenase.Cla015408 and Cla015407 encode Gibberellin 3-beta-hydroxylase.5.By analysis the sequence difference in candidate gene between parental lines,2 SNPs were identified on the exon of Cla015408 and 1 SNP was identified on the exon of Cla015406 respectively.Then,2 SNPs markers were developed in Cla015408 and Cla015406 respectively.Which were used screened recombinant individual.The result showed the Clsh is Cla015407.We also found 2 SNPs in the intron of Cl015407 between parental lines.In order to the find the key mutant,we analyzed the sequence of Cla015407 in 100 natural accessions.And the mutant of 626 th base in Cla015407 has more conservative than the other one.By spraying the parental lines with GA3 and PAC respectively,we found the hypocotyl length of WM204 was partially restored.These results indicated the short hypocotyl of WM204 caused by the mutant of Cla015407,which affected the bio-activate GA content in vivo and changed the phenotype of hypocotyl. |
PDF Full Text Request