| Flower is an important ornamental part of garden plants.It has great economic benefits for the study of variation of flower shape,flower color and other traits.Meanwhile it has always been a hot spot in flower development research.The double petal flower is a kind of mutant flower types,that has large ornamental value.The research on the molecular mechanism of this formation is of great significance for improving the ornamental value of plants.Petunia is not only a widely used bed flower of garden plants,but also a model plant for flower development research,like Arabidopsis thaliana and Antirrhinum majus.Therefore,it is an ideal material for studying double traits.This study combines the results of previous research,and start from the perspective of forward genetics to conduct the experiment.In this study,a high-density genetic linkage map was constructed by whole genome resequencing.At the same time,molecular markers and near-isogenic lines were used to locate the double gene.The results of mapping were combined to delineate the interval region of the double gene to obtain the candidate gene.The main findings obtained so far are as follows:1.Using the original markers,we aligned to 9 Scaffolds of double petunia genome.According to the single petunia and double petunia resequencing and transcriptome data,459 genes were annotated on 9 Scaffolds.By using IGV to screen differencial genes,there were obvious differences in the cds of 52 genes between single and double petunia.Differential sequences amplifications were performed preferentially on genes that were completely missing in single petunia showing in either resequencing data or transcriptome.28 primers were designed in total,and 4 pairs showed polymorphism in single and double petunia.They had a stable difference in amplification in single and double petunia when expanded the population.At the same time,37 genes with significant differences in the promoter sequence in single and double petunia which were up-regulated in double petunia were obtained.2.Two combinations of single and double petunia from NIL were selected to hybrid for obtaining two new screening populations.There were 720 and 384 individual plants respectively in the two segregation populations.Based on the markers developed in the previous experiments and the newly screened markers in this experiment,there were totally 19 markers.Referring to the scaffold sequence of the micro-collinearity analysis of the double petunia genome and the potato and tomato genome,markers on the scaffoldthat near the ends are preferentially selected to detect.Scf4-1 on the Scaffold676 and Scf43 on the Scaffold1027 are used for the new round of testing.No exchange of individual plants was detected on the current populations.3.Petunia axillaris which was single petal flower and double petal petunia were used as parents to cross.Then a single petunia and a double petunia in F1 progeny were selected for hybridization to get the next generation.And the hybrid population from F1 progeny was used to construct a genetic linkage map by whole genome resequencing.Two kinds of software were used to carry out linkage group division and map construction.The map constructed by JoinMap4.1 software contained 4474 markers with a total genetic distance of 2022.786 cM.An average genetic distance was 0.758 cM.The map constructed by using LepMap3 spanned 453.39 cM and consisted of 8030 markers.The average genetic distance was 1.00 cM.Then the double flower trait was mapped to LG5 by genetic map. |
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