Construction Of A High-density Genetic Map And The X/Y Sex-determining Gene Fine Mapping In Spinach

Cultivated spinach(Spinacia loeracea L.)belongs to the family Chenopodiaceae,which is a diploid with 2n = 12 chromosomes.Spinach is generally considered a dioecious species with separate male and female plants and occasional monoecious plants with both male and female flowers.The sex of spinach is determined by X/Y gene.Spinach is an ideal dioecious plant model for investigating the mechanism of sex determination.However,researches about the construction of genetic map and the sex are few,the clone of the sex-determining gene in spinach has not been reported and the molecular mechanism is unclear.In this study,a high density genetic map based on the combination of SLAF-seq and High Map was constructed and the sex-determining gene was also finely mapped by the SRAP-BSA and super-BSA technologies,moreover,some candidate gene were obtained through the gene annotation.The main results are as follows: 1.A high-density genetic map of spinach based on SLAF-seq technology was ultimately constructed.After linkage analysis,4,080 SLAF markers were mapped onto the genetic map.It was divided into 6 linkage groups corresponding to the number of spinach chromosomes.The map spanned 1,125,97 c M with an average genetic distance of 0.31 c M between the adjacent marker locis.It is the highest density genetic map of spinach reported at present.2.A total of 1280 different SRAP primer combinations were employed to screen the male and female gene bulks.Eventually,20 primer combinations were closely linked with the spinach sex gene.These 20 markers were chosen to genotype the 148 BC1 mapping population.Eventually,the markers were mapped to a 15.9 c M region encompassing the sex locus,with an average distance of 0.72 c M between adjacent markers.The sex-determining gene was located in a 0.4 c M region.3.The sex-determining gene was also finely mapped by the super-BSA technologies.According to the sex phenotype survey results,the sequencing data of 50 individual male plants and 50 individual female plants in the BC1 population were extracted as the male pool and female pool sequencing results respectively to identify regions with target trait.Finally,323 associated SLAF markers were identified.Combined with the high density genetic map of spinach,the sex-determining regions were located in two regions 66.98 c M-69.72 c M and 75.48 c M-92.96 c M of LG4.4.Using spinach high density genetic map to assist spinach genome assembly,scaffolds were anchored to the corresponding sex linkage markers.Through the analysis of scaffolds information with linkage markers,166 candidate genes were obtained.According to the gene annotations,there were 32 genes associated with hormonal regulation and flowering.5.The SRAP 27.47 and SRAP 30.39 were convert to high stability and good specificity SCAR markers.One d CAPs marker D4.3 was converted to a KASP marker for rapid genotyping of the large population.Both of the two typical molecular markers could fast distinguish male and female plants before bolting.This may highly improve the progress in marker-assisted selection(MAS).