Fig 1.
Sexual expression of watermelon lines P86, P86 and P87 and F1 hybrids derived from crosses P85xP87 and P86xP87.
(A) Phenotype of watermelon hermaphrodite, bisexual, female and male flowers. (B) Distribution of staminate and pistillate flowers in the 20 first nodes of the main shoot. In each node, white, black, green and red bars represent the percentages of male, female, bisexual and hermaphrodite flowers in the total number of plants analysed (n ≥ 10 for each genotype). The lack of bar in a node indicates the absence of flower in that node for some of the analysed plants.
Table 1.
Segregation ratio of monoecious, partially andromonoecious and andromonoecious plants in F2 populations derived from two crosses between monoecious and andromonoecious inbred lines.
Fig 2.
Molecular characterisation of CitACS4 gene and protein.
(A) Gene structure of CitACS4, CpACS27A, CmACS7 and CsACS2 in watermelon, squash, melon and cucumber, respectively. The numbers indicate the size of the three exons (filled boxes) and the two introns (black lines). The identified polymorphisms between DNA sequences in the monoecious and andromonoecious inbred lines are shown in above CitACS4. The missense mutation (C1477G) producing the amino acid substitution C364W in the protein is highlighted in red. (B) Alignment of watermelon CitACS4 with CpACS27A, CmACS7 and CsACS2 in squash, melon and cucumber. The amino acid changes between monoecious and andromonoecious lines in the different species are highlighted in red, blue, yellow and green, respectively.
Fig 3.
Phylogenetic analysis of CitACS4 protein.
Evolutionary tree performed for 37 ACS proteins from different plants: Arabidopsis thaliana (AtACS1, AAM91649.1; AtACS2, AAG50097.1; AtACS4, Q43309.1; AtACS5, Q37001.1; AtACS6, Q9SAR0.2; AtACS7, AEE85169.1; AtACS8, Q9T065.1; AtACS9, Q9M2Y8.1; AtACS11, AEE82593.1), Cucurbita máxima (CmaxACS3, BAB47124.1; CmaxACS4, BAB47123.1; CmaxACS11, CBAA00839.1), Cucurbita pepo (CpACC1A, AAA33111.1; CpACC1B, AAA33112.1; CpACS27A, KF113530), Cucumis melo (CmACS1, BAA83618.1; CmACS2, BAB18464.1; CmACS3, ACO83163.1; CmACS7, ACG70849.1; CmACS11, XP_008445556.1), Cucumis sativus (CsACS1, BAA93714.1; CsACS1G, ABI33818.1; CsACS2, ACG70849.1; CsACS3-like, XP_004142909.2), Citrullus lanatus (CitACS1, AFI49625.1; CitACS3, ABO76787.1; CitACS4, EF154458.1), Malus x domestica (MdACS3a-2, AEP82201.1; MdACS3c, BAE94692.1) and Solanum lycopersicon (SlACS1A, AAF97614.1; SlACS1B, AAF97615.1; SlACS2, P18485.2; SlACS3, NP_001234026.1; SlACS5, NP_001234156.1; SlACS6, NP_001234164.1; SlACS7, AAK72432.1; SlACS8, AAK72431.1). The tree was inferred using the UPGMA method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) is shown next to the branches.
Fig 4.
Relative expression of CitACS4 in different tissues of watermelon cv. Premium.
The values are the average and standard deviation of three biological replicates. St stem, Le leaves, Fe female flowers, Ma male flowers, Bi bisexual flowers. The utilized flowers were at early stages of development (S0).
Fig 5.
Expression of CitACS4 and ethylene production during the development of pistillate flowers in monoecious and andromonoecious lines of watermelon.
(A) Stages of development studied. (B) Relative expression of the gene in female flowers of monoecious (P85 and P86) and in the hermaphrodite flowers of andromonoecious (P87) lines. At S0, the expression corresponds to complete flowers, but in the other stages (S1 to S3), the expression in the ovary was separated to that in the rest of the floral organs (petals, style and stigma, and stamens). (C) Ethylene production in female, hermaphrodite and male flowers of monoecious and andromonoecious lines was measured at earlier developmental stages (S0-S1). Each value is the average from at least three biological replicates. Error bars indicate standard deviation.
Table 2.
Segregation of the M and A alleles of CitACS4 with sex monoecy/andromonoecy phenotype in the two F2 populations derived from crosses monoecious x andromonoeicous.
Table 3.
Evaluation of sex expression (transition to pistillate flowering and % pistillate flowers per plant) in F1 and F2 populations derived from crosses monoecious x andromonoecious.