Fine Mapping,Candidate Gene Cloning And Application In Pumpkin (Cucurbita Maxima Duch.) Breeding Of The Strong Female Characteristics

Cucurbita maxima Duch.is an annual trailing herb.As an important breeding target in pumpkin crops,the strong female characteristic of C.maxima can greatly increase the field yield,effectively improve seed production efficiency and seed purity,reduce labor costs and expand economic benefits..Therefore,fine mapping and candidate gene analysis of the strong female characteristic are not only important for improving the efficiency of pumpkin breeding,but also necessaryfor analyzing the molecular mechanism of strong female.Till now,there are only two reports on the mapping of strong female trait in pumpkin.There are some problems such as large location interval,insufficient linkages between molecular markers and target genes.In addition,the candidate gene has not been cloned.Therefore,on the basis of previous research,we further conducted fine mapping of strong female characteristic in pumpkin using re-sequencing.Then we conducted prediction and expression analysis of the candidate genes.At the same time,the gene mapping results were applied to the screen the strong female individuals and create excellent inbred lines in pumpkin breeding.The main results of this paper are as follows:1 We obtained 8 Gb reads for each parent of the mapping population by using re-sequencingBy using the C.maxima genome sequence as reference,we re-sequenced the parents of the mapping population,and obtained 8.6 Gb and 8.4 Gb reads,respectively.Based on the previous mapping result of this trait obtained by our laboratory,the initial location area?3 Mb?was spliced.By analyzing the sequence differences between the two parents,1,025 InDels and 3,258 SNPs were obtained.2 The strong female gene of C.maxima was finely mapped to an interval of 48 kb on LG 2Using the obtained In Del markers to analyze the 177 individuals of F₂ population and the 97 individuals of BC₁ population,we ampped the strong female candidate gene to an interval of 48 kb on LG 2.3 The 48 kb interval contains 29 coding genes,among which one gene may be a candidate controlling the strong female trait of pumpkinWe conducted identification of candidate genes by using the genome-wide annotation of C.maxima and found 29 coding genes in the interval.Among them,Cma3₀000021 was associated with gibberellin signal transduction and may be a candidate for controlling strong female trait.The sequence of this gene has a 2,958bp long sequence difference between the strong female and the normal plant,resulting in early termination of protein encoding.4 Expression analysis of candidate gene Cma3₀000021The expression pattern of Cma3₀000021 was analyzed.The results showed that Cma3₀000021 had the highest abundance in the female flower of the plant,with little expression in the stems and leaves and no expression in the root.5 Marker-assisted selection was carried out using the molecular marker Cma02-08co-segregating with the strong female traitUsing marker Cma02-08,which was co-segregated with the strong female trait,the seedling screening of the strong female trait was carried out to assist the breeding of strong female inbred lines.After screening and planting of nearly 1500 individuals,the field observation of strong female trait was consistent with the results of molecular marker screening..This method of identification of strong female trait is fast and reliable,and greatly saves the breeding workload.