Genotype related to sex expression in ‘Dokanari-sennnari’ cucumber plants
Analysis of the genotype related to sex expression in ‘Dokanari-sennari’ cucumber plants revealed significant information. Sequence analysis of the full length of the M (CS-ACS2) genome in ‘Dokanari-sennari’ cucumber plants revealed no mutations in the genome containing exons 1 (Fig. 3a, asterisk), 2, and 3 (Data not shown). PCR analysis using both dominant and co-dominant markers for the F gene revealed that ‘Dokanari-sennari’ cucumber plants possess FF gene (Fig. 3b, c). Thus, the genotype related to sex expression in ‘Dokanari-sennari’ cucumber plants was confirmed to be MMFF.
Sex expression of the F 1 hybrid cucumber plants resulting from the cross between ‘Lemon’ (mmff) pollens and ‘Dokanari-sennari’ (MMFF) pistils
To investigate the heredity of the female flowers with short ovaries produced in ‘Lemon’ (mmff) cucumber plants, using the pollens of ‘Lemon’ (mmff) cucumber plants to pollinate the pistils of female flowers bearing normal ovaries and then analyzing the expression of female flowers with short ovaries in the progeny plants is considered useful. Based on this idea, F1 hybrid plants were created by using the pollens of ‘Lemon’ (mmff) cucumber plants to pollinate the pistils of ‘Dokanari-sennari’ (MMFF) cucumber plants (Fig. 1a, b). In the 8 F1 hybrid plants (MmFf), only male flowers were produced up to node 6, and male flowers and female flowers with normal ovaries were produced from nodes 7 to 25. Theoretically, cucumber plants carrying the MmFf genotype should exhibit a gynoecious sex phenotype; however, the F1 hybrid plants (MmFf) displayed a monoecious sex phenotype. Thus, female flowers with normal ovaries were produced but female flowers with short ovaries were not produced in the F1 hybrid plants (MmFf) resulting from the cross between ‘Lemon’ (mmff) and ‘Dokanari-sennari’ (MMFF) cucumber plants.
Approximate sex expression of the F 2 segregating plants generated through self-fertilization of the F1 hybrid plants (MmFf)
To investigate the heredity of the female flowers with short ovaries produced in ‘Lemon’ (mmff) cucumber plants, the approximate sex expression of the 45 F2 segregants generated through self-fertilization of the F1 hybrid plants (MmFf) was analyzed. Among the 45 F2 segregants, 29 produced female flowers with normal ovaries, whereas 16 produced bisexual flowers with short ovaries. A Chi-squared test indicated that the segregation of plants producing female flowers with normal ovaries (MM, Mm) and that of those producing bisexual flowers with short ovaries (mm) followed the theoretical ratio of 3:1 (p-value = 0.10). Thus, female flowers with short ovaries were absent in the 29 plants (MM, Mm) but present in 3 of the 16 plants (mm). Because AgNO3 was applied to the shoot apices of the cucumber plants carrying FF gene, i.e., MMFF and MmFF, to induce male flowers for self-fertilization, analyzing the exact sex expression in the 45 F2 segregants was not possible. In summary, among the 45 F2 segregants, 29 were gynoecious or monoecious, 13 plants were andromonoecious, and 3 plants were trimonoecious.
Genotype related to sex expression in individuals 4 and 45 selected from the 45 F 2 segregants
To investigate the heredity of the female flowers with short ovaries produced in ‘Lemon’ (mmff) cucumber plants, individuals 4 and 45, which exhibited trimonoecious sex phenotype and produced both bisexual and female flowers with short ovaries, were selected from the 45 F2 segregants (Fig. 1a, b).
The genotype related to sex expression was analyzed in both F2 individuals 4 and 45. Sequence analysis of the full length of the M (CS-ACS2) genome revealed that a base substitution from G to T (c.97G > T) resulted in a single amino acid mutation (G33C) in exon 1 in the genome of both F2 individuals 4 and 45, similar to ‘Lemon’ (mmff) cucumber plants (Fig. 3a, asterisk). No mutations were found in exons 2 and 3 in the M (CS-ACS2) genome of either F2 individual (Data not shown). PCR analysis using both dominant and co-dominant markers for the F gene revealed that the F2 individuals 4 and 45 possess ff and FF gene, respectively (Fig. 3b, c). Thus, the genotype related to sex expression in the F2 individuals 4 and 45 was confirmed to be mmff and mmFF, respectively.
Morphology of flowers in F 5 plants generated from the F2 individual 4 and F4 plants generated from the F2 individual 45 through continuous self-fertilization
Morphological analysis of flowers with short ovaries in the F5 plants (mmff) generated through continuous self-fertilization of the F2 individual 4 (mmff) revealed important characteristics (Fig. 1a). Dissection of flowers with short ovaries (Fig. 4a, asterisk) and visible stamens (Fig. 4b) revealed developed stamens containing filled pollen grains (Fig. 4c, d, e) and developed pistils (Fig. 4f), indicating bisexual flowers with short ovaries. Dissection of flowers with short ovaries (Fig. 4i, asterisk) and without visible stamens (Fig. 4j) revealed stamens that had ceased development midway (Fig. 4k, l, arrowheads) and lacked pollen grains (Fig. 4m) along with developed pistils (Fig. 4n), signifying female flowers with short ovaries. Dissection of flowers with short ovaries (Fig. 4q, asterisk) and without visible stamens (Fig. 4r) revealed arrested stamens lacking pollen grains (Fig. 4s, t, u) and developed pistils (Fig. 4v), indicating female flowers with short ovaries. Additionally, male flowers were produced, akin to those observed in ‘Lemon’ (mmff) cucumber plants (Fig. 2a–f). Thus, some F5 plants (mmff) generated through continuous self-fertilization of the F2 individual 4 (mmff) produced three kinds of flowers: male flowers, bisexual flowers with short ovaries, and female flowers with short ovaries.
Morphological analysis of flowers with short ovaries in the F4 plants (mmFF) generated through continuous self-fertilization of the F2 individual 45 (mmFF) revealed significant information (Fig. 1b). Dissection of flowers with short ovaries (Fig. 5a, asterisk) and visible stamens (Fig. 5b) revealed developed stamens containing filled pollen grains (Fig. 5c, d, e) and developed pistils (Fig. 5f), indicating bisexual flowers with short ovaries. Dissection of flowers with short ovaries (Fig. 5i, asterisk) and three slightly visible stamens (Fig. 5j, arrowheads) revealed stamens that had ceased development midway (Fig. 5k, l, arrowheads) and lacked pollen grains (Fig. 5m) along with developed pistils (Fig. 5n), signifying female flowers with short ovaries. Dissection of flowers with short ovaries (Fig. 5p, asterisk) and without visible stamens (Fig. 5q) revealed arrested stamens lacking pollen grains (Fig. 5r, s, t) and developed pistils (Fig. 5u), indicating female flowers with short ovaries. Additionally, male flowers were produced, akin to those observed in ‘Lemon’ (mmff) cucumber plants (Fig. 2a–f). Thus, some F4 plants (mmFF) generated through continuous self-fertilization of the F2 individual 45 (mmFF) produced three kinds of flowers: male flowers, bisexual flowers with short ovaries, and female flowers with short ovaries.
Flower and fruit traits and sex phenotype in the F 5 cucumber plants (mmff) generated through continuous self-fertilization of the F2 individual 4 (mmff)
To clarify the number of both bisexual and female flowers with short ovaries produced on both the main stem and lateral branches throughout the growth period in the F5 cucumber plants (mmff) generated through continuous self-fertilization of the F2 individual 4 (mmff), 10 individuals (from 4-2-6-1 to 4-2-6-10) were investigated. On the main stem, 94 bisexual flowers with short ovaries and 11 female flowers with short ovaries were produced (Table 2). Notably, male flowers were produced at all nodes on the main stem in all 10 plants. On the lateral branches, 61 bisexual flowers with short ovaries and 16 female flowers with short ovaries were produced (Table 2). The lateral branches elongated between nodes 3 and 35 on the main stem. Furthermore, bisexual flowers with short ovaries were produced in all 10 individuals, whereas female flowers with short ovaries were produced in 8 of the 10 F5 plants (mmff) generated through continuous self-fertilization of the F2 individual 4 (mmff) (Table 2). Overall, the order of flower differentiation was male flowers, bisexual flowers with short ovaries, and female flowers with short ovaries. Thus, the sex phenotype of the 8 individuals was determined to be trimonoecious, whereas that of the remaining 2 individuals was determined to be andromonoecious (Table 2).
To clarify the number of fruits derived from both bisexual and female flowers with short ovaries produced on both the main stem and lateral branches throughout the growth period in the F5 cucumber plants (mmff) generated through continuous self-fertilization of the F2 individual 4 (mmff), 10 individuals were investigated. On the main stem, 19 fruits derived from bisexual flowers with short ovaries (Fig. 4g) and 3 fruits derived from female flowers with short ovaries (Fig. 4o, w) were observed (Table 2). On the lateral branches, 9 fruits derived from bisexual flowers with short ovaries (Fig. 4h) and 4 fruits derived from female flowers with short ovaries (Fig. 4p, x) were observed (Table 2). All fruits were white in color (Fig. 4g, h, o, p, w, x). Notably, on the main stem, the shape of fruits derived from female flowers with short ovaries (Fig. 4o, w) was spherical, akin to that of fruits derived from bisexual flowers with short ovaries (Fig. 4g). Likewise, on the lateral branches, the shape of fruits derived from female flowers with short ovaries (Fig. 4p, x) was spheroidal, akin to that of the fruits derived from bisexual flowers with short ovaries (Fig. 4h). Thus, the shape of the fruits derived from female flowers with short ovaries closely resembled that of the fruits derived from bisexual flowers in the 10 F5 cucumber plants (mmff) generated through continuous self-fertilization of the F2 individual 4 (mmff). Compared to ‘Lemon’ (mmff) cucumber plants, the 10 F5 cucumber plants (mmff) generated through continuous self-fertilization of the F2 individual 4 (mmff) exhibited early differentiation of female flowers with short ovaries on both the main stem and lateral branches, facilitating cross-pollination and fruit set.
Flower and fruit traits and sex phenotype in the F 4 cucumber plants (mmFF) generated through continuous self-fertilization of the F2 individual 45 (mmFF)
To clarify the number of bisexual and female flowers with short ovaries produced on both the main stem and lateral branches throughout the growth period in the F4 cucumber plants (mmFF) generated through continuous self-fertilization of the F2 individual 45 (mmFF), 10 individuals (from 45-1-1 to 45-1-10) were investigated. On the main stem, 65 bisexual flowers with short ovaries and 3 female flowers with short ovaries were produced (Table 3). Additionally, male flowers were produced at all nodes on the main stem. On the lateral branches, 43 bisexual flowers with short ovaries and 53 female flowers with short ovaries were produced (Table 3). The lateral branches elongated between nodes 12 and 44 on the main stem. Bisexual flowers with short ovaries were produced in all 10 individuals, whereas female flowers with short ovaries were produced in 9 of the 10 individuals (Table 3). Overall, the order of flower differentiation was male flowers, bisexual flowers with short ovaries, and female flowers with short ovaries. Thus, the sex phenotype of the 9 individuals was determined to be trimonoecious, whereas that of the remaining 1 individual was determined to be andromonoecious (Table 3).
To clarify the number of fruits derived from bisexual and female flowers with short ovaries produced on both the main stem and lateral branches throughout the growth period in the F4 cucumber plants (mmFF) generated through continuous self-fertilization of the F2 individual 45 (mmFF), 10 individuals were investigated. The main stem exhibited 15 fruits derived from bisexual flowers with short ovaries (Fig. 5g), whereas no fruits derived from female flowers with short ovaries were observed (Table 3). On the lateral branches, 8 fruits were derived from bisexual flowers with short ovaries (Fig. 5h), whereas 10 fruits were derived from female flowers with short ovaries (Fig. 5o, v) (Table 3). All fruits exhibited a light yellow color (Fig. 5g, h, o, v). On the main stem, the shape of the fruits derived from bisexual flowers with short ovaries (Fig. 5g) was spherical. Likewise, the shape of the fruits derived from female flowers with short ovaries (Fig. 5o, v) was spheroidal, akin to that of the fruits derived from bisexual flowers with short ovaries (Fig. 5h) on the lateral branches. Thus, the shape of the fruits derived from female flowers with short ovaries closely resembled that of the fruits derived from bisexual flowers on the lateral branches in the 10 F4 plants (mmFF) generated through continuous self-fertilization of the F2 individual 45 (mmFF). Compared to ‘Lemon’ (mmff) cucumber plants, the 10 F4 cucumber plants (mmFF) generated through continuous self-fertilization of the F2 individual 45 (mmFF) exhibited early differentiation of female flowers with short ovaries, particularly on the lateral branches, facilitating cross-pollination and fruit set.
Analysis of the sex phenotype of F 5 cucumber plants (mmFF) generated through continuous self-fertilization of the F2 individual 45 (mmFF)
To investigate the heredity of the sex phenotype in the F4 cucumber plants (mmFF) generated through continuous self-fertilization of the F2 individual 45 (mmFF), the sex phenotype of the F5 cucumber plants (mmFF) generated through self-fertilization of the F4 plants (mmFF) was analyzed. The sex phenotype of all 6 F5 plants (mmFF) derived from the trimonoecious F4 individual 45-1-2 (mmFF) was trimonoecious (Table 3). The sex phenotype of the 6 F5 plants (mmFF) derived from the andromonoecious F4 individual 45-1-3 (mmFF) was as follows: 5 F5 plants (mmFF) were trimonoecious and 1 F5 plant (mmFF) was andromonoecious (Table 3). The sex phenotype of the 7 F5 plants (mmFF) derived from the trimonoecious F4 individual 45-1-7 (mmFF) was as follows: 4 F5 plants (mmFF) were trimonoecious and 3 F5 plants (mmFF) were andromonoecious (Table 3). The sex phenotype of all 6 F5 plants (mmFF) derived from the trimonoecious F4 individual 45-1-8 (mmFF) was trimonoecious (Table 3). Thus, there were no clear genetic rules governing the determination of sex phenotypes (trimonoecious or andromonoecious), specifically regarding the production of female flowers with short ovaries in cucumber plants carrying the mm gene (CS-ACS2 gene with c.97G > T mutation).