Genetic and molecular control of monoecy stability, fruit set and fruit quality in watermelon

  1. Aguado Donaire, Encarnación
Supervised by:
  1. Manuel Jamilena Quesada Director
  2. Susana Manzano Medina Co-director

Defence university: Universidad de Almería

Fecha de defensa: 23 July 2020

  1. Dolores Garrido Garrido Chair
  2. María del Mar Rebolloso Fuentes Secretary
  3. Luis Armando Rivera Burgos Committee member

Type: Thesis

Teseo: 631593 DIALNET lock_openriUAL editor


As in other species of the Cucurbitaceae family, the different sex morphotypes of Citrullus lanatus can be differentiated according to the distribution and occurrence of male, female, bisexual and hermaphrodite flowers on the main and secondary shoots. Depending on the distribution of the four types of flowers, the 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. The main objective of this thesis is to analyze the genetic and molecular control of monoecy, andromonoecy and partial andromonoecy in watermelon. To study the genetic control of andromonoecy, a segregation analysis of three independent F2 populations derived from the crosses between three monoecious lines and one andromonoecious line was performed. It has been demonstrated that the andromonoecious trait is controlled by a single gene with two alleles, the monoecious M allele, and the andromonoecious m allele. The M allele is semidominant respect to m, in such a way that the heterozygous Mm genotype is partially andromonoecious. In other cucurbit species andromonoecy is conferred by mutations in the ethylene biosynthesis genes CmACS7, CsACS2 and CpACS27A in melon, cucumber, and squash, respectively. The gene with the highest homology in watermelon is CitACS4, which encodes for an ACS type III enzyme that is predominantly expressed in pistillate flowers. In the andromonoecious line, we detected a missense mutation in a very conserved residue of CitACS4 (C364W) that cosegregated with the andromonoecious phenotype in three independent F2 populations. These data indicated that CitACS4 is likely to be involved in the ethylene biosynthesis required for stamen arrest during the development of female flowers. The C364W mutation would reduce the production of ethylene in pistillate floral buds, promoting the conversion of female into hermaphrodite flowers, and therefore of monoecy (genotype MM) into partial andromonoecy (genotype Mm) and andromonoecy (genotype mm). In parallel we have studied whether the CitACS4 gene could be also involved in other ethylene-regulated traits during flower and fruit development, including pistillate flowering transition and the number of female flowers per plant, the development of floral organs other than stamens, as well as fruit and seed set, and fruit development. A linkage analysis approach was performed in three independent F2 populations segregating for the two alleles of the gene (M, monoecious; m, andromonoecious), and the different traits under study. The CitACS4 m allele not only co-segregated with andromonoecy, but also with earlier pistillate transition, an increased number of pistillate flowers per plant, and a slower growth and maturation of petals and carpels, which delayed anthesis time in hermaphrodite flowers. The m allele was also found to be linked to a reduced fruit set, which was not caused by a deficiency in pollination or fertilization. The gene also affected the longitudinal and transverse growth rates of the ovary and fruit, which means that fruits from andromonoecious plants (mm) were rounder than those from monoecious (MM) ones. Taken together, these data indicate that the locus defined by the ethylene biosynthesis and sex determining gene CitACS4 acts as a pleiotropic regulator of the complete development of the pistillate flower and the earlier development of the fruit. In the last part of this thesis we have performed genetic and molecular analysis of the partially andromonoecious phenotypes of watermelon. We phenotyped and genotyped, for the M/m locus, a panel of 207 Citrullus lanatus accessions from all parts of the world, including 5 inbreds and hybrids, and found several accessions that were repeatedly phenotyped as partially andromonoecious in several locations and different years, despite being MM. A cosegregation analysis between a SNV in CitACS4 and the partially andromonoecious phenotype, demonstrated that the occurrence of bisexual and hermaphrodite flowers in a partially andromonoecious line is not dependent on CitACS4, but conferred by an unlinked recessive gene which we called pa. Two different strategies were performed to map the pa gene in the genome of Citrullus lanatus: bulk segregant analysis sequencing (BSA-seq) and genome wide association analysis studies (GWAS). The BSAseq 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 possessing the most contrasting phenotypes for monoecy and PA, respectively. For GWAS, 122 accessions from USDA gene bank, already re-sequenced through genotyping by sequencing (GBS), were used. The combination of the two strategies indicates that pa maps onto a genomic region expanding across 32.24-36.44 Mb in chromosome 1 of watermelon. Fine mapping narrowed down the pa locus to a 867 Kb genomic region containing 101 genes. Finally, several candidate genes were selected, for their function in ethylene biosynthesis and signaling, as well as in flower development and sex determination, but also by the impact of the SNPs and indels differentially detected in the two sequenced bulks.