Fine Mapping Of Gene Cmsf1(Cucumis Melo Sutured Fruit 1)in CUCUMIS MELO L.and The Germplasm Selection Of Melon Regeneration System

Melon fruit suture is one of the important appearance qualities of melon fruit.Fine mapping and cloning the melon fruit suture functional gene will contributue to application of molecular marker in breeding program and uncover the underlying mechanism of this important agricultural trait.In this study,the melon inbred line'Y101'without fruit suture and the melon inbred line'0426'with fruit suture were used as experimental materials to observe the morphological and cytological characteristics of fruit suture,and the six generation population was constructed with the two materials as parents.The genetic law of fruit suture was completed by statistical analysis of population phenotype.Furthermore,BSA and molecular markers were used to complete the fine mapping of melon fruit surface regulatory genes.Finally,through gene function annotation and sequence analysis,candidate genes were screened.In order to verify the gene function by genetic transformation technology,the induction rate of adventitious buds of 7 melon germplasm was also preliminarily screened in this study,which laid a foundation for the establishment of regeneration system system.The results of this study are as follows:1. In the experiment,the fruits of the inbred lines'Y101'without suture and'0426'with fruit suture were used as the experimental materials.The morphological observation showed that the fruit of the parent'0426'had striated pits on the surface of the peel at the embryo stage?1 d before flowering?,which was the fruit suture phenotype;The fruit suture character can be observed in the whole fruit growth period until the fruit matures.With the fruit expanding?15 d after pollination?,the fruit suture gradually deepens,which is more conducive to observation.The results of paraffin section showed that compared with melon without fruit suture,the cell volume of peel tissue in melon increased and the cell arrangement was loose.2. Using germplasm‘Y101'and‘0426'as parents,the six generation population was constructed and the genetic analysis of fruit suture was completed by phenotypic statistical analysis:P₂?Y101?,F₁ and BC₁P₂ had no fruit suture;P₁?0426?had fruit surface ditch;the ratio of number of fruit suture to number of fruit suture in BC₁P₁ population was 1:1 population was 3:1?c²=0.617,P=0.432?.The results showed that the character of melon fruit suture was a quality character controlled by a single gene,and the character with fruit suture was recessive for that without fruit suture;the gene controlling melon fruit suture was named Cmsf1.3. In order to locate the Cmsf1 gene,249 pairs of SSR primers were synthesized and screened to obtain 132 pairs of polymorphic markers. Furthermore,BSA method was used to construct fruit suture gene pool and non fruit suture gene pool.Linkage analysis showed that the marker Chr11-15 located on chromosome 11 was linked to fruit suture character.After pool removal,Chr11-15 was used to mark two wings polymorphic markers to screen recombinant single The Cmsf1 gene was initially located between the markers Chr11-13 and Chr11-16 with a physical distance of 7.29 Mb.4. In order to precisely locate the Cmsf1 gene,125 recombinants?2300 individuals?were obtained by using the initial mapping markers Chr11-13 and Chr11-16.38 pairs of high-density molecular markers?28 pairs of CAPS primers,2 pairs of In Del Markers and 8pairs of SSR primers?were developed by using the parent re sequencing to scan the recombinants.Finally,the Cmsf1 gene was located between ZC11-85 and ZC11-84.The two markers and the Cmsf1 base were located The genetic distance between them was 0.01 and0.02 c M,and the location interval was 3.67 kb.5. Using the reference genome DHL92?V3.6.1?of melon to analyze the location region,we found that the location region sequence includes a 1000 bp Gap and a gene with ID of MELO3C001616.2.1.The functional annotation of the gene is U-box domain containing protein 7,and there is no difference in the sequence between the two parents.Using FGENESH,an ORF2 was found in the location region,which had no difference in the middle sequence of the two parents.In order to amplify Gap,four pairs of primers were synthesized on both sides of Gap.672 bp and 1430 bp fragments were amplified by nested PCR,which were not the target sequence.It is concluded that there is a difference between the length of gap sequence in genome and the actual length.6. In this study,the adventitious bud induction rates of 7 different melon germplasm cotyledon nodes were analyzed.It was found that the adventitious bud induction rates of'0426'and'0544'cotyledon nodes were 78.3%and 75%respectively,which were significantly higher than other germplasm,indicating that'0426'and'0544'were suitable for regeneration system.