Mapping a Partial Andromonoecy Locus in Citrullus lanatus Using BSA-Seq and GWAS Approaches

Abstract

The sexual expression of watermelon plants is the result of the distribution and occurrence of male, female, bisexual and hermaphrodite flowers on the main and secondary stems. Plants can be monoecious (producing male and female flowers), andromonoecious (producing male and hermaphrodite flowers), or partially andromonoecious (producing male, female, bisexual, and hermaphrodite flowers) within the same plant. Sex determination of individual floral buds and the distribution of the different flower types on the plant, are both controlled by ethylene. A single missense mutation in the ethylene biosynthesis gene <i>CitACS4</i>, is able to promote the conversion of female into hermaphrodite flowers, and therefore of monoecy (genotype <i>MM</i>) into partial andromonoecy (genotype <i>Mm</i>) or andromonoecy (genotype <i>mm</i>). We phenotyped and genotyped, for the <i>M/m</i> locus, a panel of 207 <i>C. lanatus</i> accessions, including five inbreds and hybrids, and found several accessions that were repeatedly phenotyped as PA (partially andromonoecious) in several locations and different years, despite being <i>MM</i>. A cosegregation analysis between a SNV in <i>CitACS4</i> and the PA phenotype, demonstrated that the occurrence of bisexual and hermaphrodite flowers in a PA line is not dependent on <i>CitACS4</i>, but conferred by an unlinked recessive gene which we called <i>pa</i>. Two different approaches were performed to map the <i>pa</i> gene in the genome of <i>C. lanatus</i>: bulk segregant analysis sequencing (BSA-seq) and genome wide association analysis studies (GWAS). The BSA-seq study was performed using two contrasting bulks, the monoecious M-bulk and the partially andromonoecious PA-bulk, each one generated by pooling DNA from 20 F2 plants. For GWAS, 122 accessions from USDA gene bank, already re-sequenced by genotyping by sequencing (GBS), were used. The combination of the two approaches indicates that <i>pa</i> maps onto a genomic region expanding across 32.24-36.44 Mb in chromosome 1 of watermelon. Fine mapping narrowed down the <i>pa</i> locus to a 867 Kb genomic region containing 101 genes. A number of candidate genes were selected, not only for their function in ethylene biosynthesis and signalling as well as their role in flower development and sex determination, but also by the impact of the SNPs and indels differentially detected in the two sequenced bulks.