l. ’— _—-—— ———— __———-—-— _———-— _—_—— —.—— —_——— ._————-' __—————— __—_——— _———— _———— ————— __—_———— 333333330333 SE OF SEX EX?R;533033 ‘3’ ’L' 133$ 33:33:30 32.5333” .3333'333'353 23:3. 3.) 333 3323331333.:ee “3 3-33 8. 35.303 333.333 3... "3TE 1.3335231831‘.‘ 333333 ”33333532 3003‘? 3.934 ’ _ . —L —- -— - _ — \ — ‘ - ., ' ‘ W w d . - 9 ~ .— - _ c---—~- _—.—..'—.__ -~ 9 -pM.). ‘ “A ,h .1»; -i—Wfi_ ~——— ——‘ -—.- —— ~_————‘f’—.‘ o . I 3 . s 7 i. , v "-dé-b-“~] . ,- -../. 7rfiflam —— .8. — I - 1" ABSTRACT INHERITANCE OF SEX EXPRESSION IN DIOECIOUS CUCUMBER (CUCUMIS SATIVUS L.) By John Warner Scott The hybrids and segregating populations of H gynoecious lines crossed with M androecious lines were analyzed to determine the inheritance of sex expression in dioecious cucumber (Cucumis sativus L.). Sex expression of the hybrids was characterized by gynoecious and predominantly female phenotypes. Both phenotypes are characterized by a continuous pistillate stage of flowering on the main stem. No reciprocal cross differences were observed. Backcrosses to the gynoecious parents produced plants with a continuous female stage. Backcrosses to the androecious parent pro- duced plants with a continuous pistillate, monoecious (with- out a continuous pistillate stage), and androecious pheno— types in a 2:1:1 ratio, respectively. The F2 generation segregated 12:3:1 continuous pistillate, monoecious, and androecious phenotypes, respectively. Two major loci were proposed to control sex expression in the populations studied. The a locus permits male (aa) versus female (5:) John Warner Scott flower expression. The agr_locus conditions the intensity of femaleness where £333 is epistatic to aa_and results in a continuous pistillate stage. Accordingly, gynoecious and predominantly female geno- types are homozygous or heterozygous for a933, while monoecious and androecious phenotypes are agr:_homozygotes. With an acr+acr+ genotype A:_conditions monoecism and a3 conditions androecism. INHERITANCE OF SEX EXPRESSION IN THE DIOECIOUS CUCUMBER (CUCUMIS SATIVUS L.) By John Warner Scott A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 197A Guidance Committee: This thesis is condensed into a format suited and intended for publication in the Journal of the American Society for Horticultural Science. ii ACKNOWLEDGMENTS The author expresses thanks to his professor, Dr. L. R. Baker for his valuable assistance with this work and to committee members Drs. K. C. Sink and M. W. Adams for their input. I'm grateful to Dr. Jehoshua Rudich for his unselfish data contribution and suggestions. Thanks also to Dr. J. H. Gill for his statistical advice and to H. M. Slatis for his help with the genetic interpretation. Finally, thanks to Drs. E. T. Mescherov and M. Yordanov who made the whole study possible by supplying the androecious seed to our breeding program. iii TABLE OF CONTENTS LIST OF TABLES . INTRODUCTION . . . MATERIALS AND METHODS RESULTS AND DISCUSSION SUMMARY AND CONCLUSIONS LITERATURE CITED iv FJH \OCOUTI'UH Table 1. LIST OF TABLES Page Sex expression of S plants from gynoecious and androecious lin s of cucumber . . . . . 6 Sex expression in the cross of gynoecious x androecious (MSU 1A1) cucumber . . . . . . 7 Sex expression in the cross of gynoecious x androecious (MSU 1A2) cucumber . . . . . . 8 Sex expression in the cross of gynoecious x androecious (MSU 1A3) cucumber . . . . . . 9 Sex expression in the cross of gynoecious x androecious (MSU 2A) cucumber . . . . . . 10 Proposed genetic model for sex expression from the cross of gynoecious x androecious cucumber . . . . . . . . . . . . . l3 INTRODUCTION Kubicki (9) reported that androecious (all—male) expression of cucumber (Cucumis sativus L.) was controlled by a single recessive gene a and was also influenced by the agg locus. The influence of the agr locus on sex expression had been reported earlier (16,17). The agr_locus is proba- bly analogous to the st locus (A), which in still earlier work was called f (20). Kubicki (9) obtained entirely gynoecious (all-female) F1 plants from some crosses of gynoecious x androecious. This result stimulated interest in the use of an androecious parent for hybrid seed produc- tion. Use of a vigorous, androecious pollinator might be advantageous over current monoecious (11) or proposed hermaphroditic (5,12) pollinators. It might also be useful as a pollinator for 3—way hybrid seed production (1“). The purpose of this study was to determine the inherit- ance of sex expression in crosses of gynoecious and androecious cucumber. This information is essential to determine the feasibility of using androecious lines as pollinators for hybrid seed production. MATERIALS AND METHODS In August 1972 crosses involving A gynoecious and A androecious lines of cucumbers were planted in the green- house to obtain F1 and S1 seed. The A gynoecious inbred parents were: 1) GylA, a white spined pickling line developed by Clemson University; 2) MSU 713-5, a black spined pickling line develOped by Michigan State University; 3) Tablegreen 68G, a white spined slicer line developed by Cornell University; and A) MSU 39AG an experimental white spined pickling line developed by Michigan State University. The androecious parents consisted of 3 lines of black spined slicing cucumbers designated MSU 1A1, MSU 1A2, and MSU 1A3. The fourth androecious line, designated MSU 2A, was a white spined slicer line with prolific growth and late flowering.1 A second planting of parental and F1 seed was made in November 1972 to obtain reciprocal Fl (RFl), 8C1, reciprocal BC (RBCl), BC2, reciprocal BC (RBCZ), and F2 seed. The 1 2 sex expression of these Fl plants under greenhouse condi- tions was recorded. Gynoecious parents were sprayed 3x at A day intervals with 50 ppm GAA/7 beginning at the 1-leaf stage to induce male flowers (13) for selfing and reciprocal crosses. The androecious parents were sprayed with 50 ppm ethephon at 1Seed of MSU 1A1, MSU 1A2, and MSU 1A3 was supplied by Dr. E. T. Mescherov, All-Union Institute of Plant Industry, Leningrad, USSR. Seed of MSU 2A was supplied by Dr. M. Yordanov, Plovdiv, Bulgaria. the 3—leaf stage to induce female flowers (1) for selfing and reciprocal crosses. To obtain staminate flowers for F2 and BC seed the gynoecious F plants were sprayed 3x with l 50 ppm GAA/7’ whereas PF F plants were sprayed 2x after 1 classification. Seed obtained from the various crosses was planted at 2 field locations in the summer of 1973. The 81’ F1, RFl, BC RBC BC RBC , and F generations were planted near 1’ l’ 2’ 2 2 East Lansing, Michigan on June 15 and 26. On July 12, a second planting was made near Sodus, Michigan, approximately 120 miles southwest of East Lansing. A completely random- ized design was used at both locations with 3 replications. Plants were thinned to 25 plants per 9.1A meter (30 foot) plot to avoid excessive crowding. Twenty—five plants were desired yet not always attained due to variable plant stands. Plots were fertilized with 22.59 kg (A9.8 lb) N, 11.7 kg (25.8 lb) P, and 22.59 kg (A9.8 lb) K by using 336 kg/ha (300 lb/acre) 20—20-20 before planting and side- dressed with 8.16 kg (18 lb) N using 56 kg/ha (50 1b) NH“ NC at the 6-leaf stage. 3 For East Lansing, daylength ranged from 15 l/A hr to 13 hrs. Average maximum temperature for East Lansing was 26.0°C (78.8°F), average minimum temperature was l3.83°C (56.9°F), and average mean temperature was 20.5°C (68.9°F). At Sodus, daylength ranged from 15 hr to 13 hrs. Sodus average maximum temperature was 26.6°C (79.9°F), average minimum temperature was 15.8900 (60.6°F), and the average mean temperature was 21.28°C (70.3°F). All plants were classified for sex over the entire growing season (June through September) and placed into A categories: 1) gynoecious, all female flowers; 2) predominantly female (PF), some early male flowers followed by a continuous pistillage stage; 3) monoecious, many male with some female flowers, but no continuous female stage; and A) androecious, only male flowers with no female flowers or in some cases with very late female flowers formed on third order laterals. Each plot was coded for replicate number, F2 sister (if an F2), pedigree, generation, and location, together with the frequencies of the observed sex phenotypes. Genetic analysis consisted of testing for homogeneity with X2 contingency tables (18) in order to pool and simplify the data. Homogeneity was tested in the following order: replicates of same plot and location, F2 pOpulations of the same pedigree and location, reciprocal crosses within gen- eration within location, plots of the same pedigree (plots derived from sister plants - this includes Sl plants) and location, pedigrees within generation within location, and location within pedigree within generation. RESULTS AND DISCUSSION Replicates within plots and locations, F2 sisters (3 to 5) within pedigree and location, reciprocal crosses of F1’ BCl’ 2 same pedigree, generation, and location) were homogeneous and BC within location, and sister plots (of the (p > .05) and were pooled. Progenies from 2 selfed plants (81) for each parental line were homogeneous (p > .05) with each other and between locations (Table l). Pedigrees within generation within location proved heterogeneous and are reported separately. When pedigrees within a genera— tion were compared between locations, most proved to be homogeneous. Location differences were not significant (p > .05) within crosses, excluding those involving Table- green 68G and a single F population involving MSU 39AG x 2 MSU 1A2. Thus all other data are reported with locations pooled (Tables 2 to 5). No definite location effect could be determined for heterogeneous crosses except that loca- tions may have influenced sex expression in different ways. For all crosses (Tables 2 to 5), the F generation 1 segregated gynoecious and PF plants with exceptional monoecious segregates resulting from 3 crosses. Hence, the heterozygote resulting from the cross of gynoecious x androecious exhibited a low percentage of gynoecious with a relatively high percentage of PF plants. Therefore, no genetic basis for differences between these 2 classes could be proposed. Table 1. Sex expression of S androecious parent lines of cucumber. plants from gynoecious and Variety G Pgex A Total plants GylA 93 12 O 105 MSU 713-5 126 10 O 136 MSU 39AG 130 8 O 138 may 75 12 o 87 MSU 1A1 O O 63 63 MSU 1A2 O O 72 72 MSU 1A3 O 0 68 68 MSU 2A 0 O 79 79 ZG Gynoecious, PF = Predominantly Female, M = Monoecious, A Androecious. yTG = Tablegreen 68G .mSOHomopoc< u ¢ mmonooozoz .COHpmoOH msoom n .mz .COHmeOH mcchmq ummm u .H.mx .omm cmmsmenme u 09% z mmHmEmm mecmcHEoompm n mm mmsoHomocmo n 0N mm.A mmmo.o NumHumw moH n mH mm m: .m I H .95 for 12 goodness of fit to this ratio (Tables 2 to 5). Based on the ratios observed in the BC2 and F2 generations, an independently inherited digenic system is proposed. The significant number of androecious segregates in both the BC and F generations seems to discount a more complex 2 2 system of inheritance for androecious expression. The 2 loci involved are designated as a after Kubicki (9) and agr_as originally designated by Shiffriss (16,17) and then by Kubicki (6,7,9). These designations will be used here to avoid confusion with nomenclature. The A allele as the female flower allele is dominant to a the male flower allele. The agr_locus controls female inten- sity with ag§3_homozygotes being of high female intensity while £33: homozygotes exhibit a low female intensity. The achacr+ heterozygote is intermediate between the homo- zygotes, but tends toward the ag§E_homozygote phenotypically (6). An agri_complement exhibits epistasis with aa. The proposed model is outlined in Table 6. An agfii complement results in a continuous pistillate stage, i.e. gynoecious or PF; whereas agr: homozygotes do not. The difference between gynoecious and PF may be due to different alleles at the agg locus (7) and/or minor modifier genes (7,8,16) and/or environment (2,3,A,10,16,19). In common between monoecious and androecious genotypes is acr+acr+. The difference between monoecious and androecious is that monoecious phenotypes require an A: genotype whereas 13 Table 6. Proposed genetic model for sex expression from the cross of gynoecious x androecious cucumber. Generation Ratio Genotype Phenotype Gynoecious F F Parent 1 AA acr acr Gynoecious Androecious + + Parent 1 gg acr acr Androecious F + 2 Fl 1 Ag acr acr Gynoecious/PF F F 3/8 A: acr acr Gynoecious 1/8 gg achach Gynoecious BCPl F + 3/8 A: acr acr Gynoecious/PF 1/8 gg achacr+ Gynoecious/PF l/A Agachacr+ Gynoecious/PF l/A gg_achacr+ Gynoecious/PF BCP2 + + l/A Ag acr acr Monoecious l/A gg acr+acr+ Androecious 3/16 A:_achach Gynoecious 1/16 gg achach Gynoecious 3/8 A: achacr+ Gynoecious/PF F2 F + 1/8 gg acr acr Gynoecious/PF 3/16 A: acr+acr+ Monoecious 1/16 gg acr+aq£: Androecious 2 PF = Predominantly Female. 1A androecious phenotypes require gg. Except for the differ- ence between monoecious and androecious, it is beyond the scope of the data to show that A: adds to the femaleness of other sex phenotypes. For example, gg achach and A: gggi: ach are assumed of equal female intensity for this study and the proposed model. The phenotypic difference between these 2 genotypes is likely small. A major deviation from the prOposed genetic model occurs with crosses involving MSU 2A (Table 5). Greenhouse experiments at Michigan State University in the Fall of 1973, demonstrated that the androecious expression of MSU 2A was unstable under low temperature and/or short day condi- tions. Only under high temperature and long day conditions (as with 1973 field experiments) is MSU 2A stable for androecious expression. Under short day (10 to 11 hr) and/ or low night temperature (10 to 12°C) conditions MSU 2A exhibits monoecious expression (15). Environmental influences on sex expression have been observed previously (2,3,A,10,l6,19). It is generally accepted that stronger femaleness is observed with short days and low temperature conditions. However, some lines are environmentally stable, such as MSU 713-5 (3) and MSU 1A1 (15). Such a genetic system for sex expression which causes certain varieties to be environmentally sensitive while others are stable has not been reported. Thus, the genotype of unstable MSU 2A might be ggacr+acr+ with a gene complement which causes 15 femaleness under short days and/or cold temperatures or the genotype A: acr+acr+ with a gene complement which causes maleness under long day and high temperature condi- tions. The cross of MSU 2A and MSU 1A1 might provide an answer to this question. For this data, MSU 2A does not fit a 2:1:1 BC1 or a 12:3:1 F2 ratio so the androecious and monoecious classes were combined and 1:1 BCP2 and 3:1 F2 ratios, typical of monoecious inheritance (7,16) were tested and found to be acceptable fits (Table 5). This suggested that the geno- type of MSU 2A is A: acr+acr+ with modifier genes for unstable androecious expression resulting in maleness under long day, high temperature conditions. Other significant deviations (p < .05) from expected F ratios occurred with Tablegreen 680 x MSU 1A1 (East 2 Lansing location), Tablegreen 680 x MSU 1A2 (Sodus location), 39AG x MSU 1A2 (East Lansing location) and MSU 39AG x MSU 1A1. In the case of Tablegreen 680 x MSU lAl (East Lansing), the significant deviations are due to a higher than expected female tendency, that is more gynoecious and PF phenotypes. But, the monoecious and androecious classes are observed to be high in the Tablegreen 680 x 1A2 (Sodus). In the first case, the greater female intensity is not too surprising based on higher percentage of gynoecious segregates in other generations as compared to the other pedigrees. Varying intensities of femaleness among "gynoecious" 16 varieties has been reported previously (7,8,16). Table- green 680 is observed to express a strong female intensity such that it is extremely difficult to induce male flowers after treatment with GAA/7 (unpublished data). This study lends no evidence for a genetic basis for such a strong female tendency. Its occurrence could lend support to: 1) multiple alleles at the g23_1ocus (7), in this case having a very strong gggi expression, or 2) another locus controlling female intensity (8), or 3) it may be due to an accumulation of highly female polygenes (7,8,16), or A) combinations of the above. Yet certain Tablegreen 680 crosses segregated monoecious phenotypes in the F1 and BCPl generations which is incongruous with its strong female expression (Tables 2, 3 A). Also the S1 segregates the highest percentage of PF's of all the parents (Table 1). These incidences of greater maleness are likely related to the Tablegreen 680 x MSU 1A2 F2 (Sodus) which expressed a greater than expected frequency of monoecious and androecious segregates. Further work must be done with Tablegreen 680 to explain this apparent disparity. In the F of MSU 39AG X MSU 1A1 and MSU 39AG X MSU 1A2 2 (East Lansing location), the significant deviations result from a higher than expected frequency of gynoecious and PF plants. High female intensity is evident in other crosses with MSU 39AG (Tables 2 to 5). The S1 of MSU 39AG 17 segregated the lowest percentage of PF plants indicating a stronger female tendency than the other gynoecious lines (Table 1). This strong female intensity might be analogous to the high female intensity of Tablegreen 680. The possi- bility of a unique environmental effect in the F2 popula- tions of MSU 39AG x 1A2 (East Lansing) and Tablegreen 68G x MSU 1A1 (East Lansing) is apparent since the same crosses at Sodus were consistent with the expected. Additional experiments would be necessary to determine any modifier genes or multiple ggg alleles which might cause the significant deviations in sex expression. If actual numbers of male and female flowers were counted, this would provide quantitative data which might elucidate the modifier genes affecting sex expression. 18 SUMMARY AND CONCLUSIONS The inheritance of sex expression in the cross of gynoecious and androecious phenotypes appears to be con— trolled by 2 major, independently inherited loci; viz., g and ggg. A flower sex allele A conditions pistillate flowers and is dominant to g which allows staminate flower development. The sex phenotype is controlled by alleles at a female intensity locus ggg. The achach or achacr+ genotypes condition a continuous pistillate stage on the main runner of the plant as opposed to the acr+acr+ geno- type which is not associated with a continuous pistillate stage. The gggi allele exhibits epistasis to the g allele (9). The only major genetic difference between monoecious and androecious phenotypes is that androecious phenotypes require gg whereas monoecious require A;. Other modifier genes and environment also influence phenotypic sex expression. For hybrid seed production an androecious pollinator would be used in the same way as monoecious pollinators (11, 1A) in producing highly female Fl varieties. Hermaphrodites seem more suitable for production of seed of all-gynoecious F1 (5,12) varieties which are necessary for parthenocarpic cucumber production (12). l) 2) 3) A) 5) 6) 7) 8) 9) 10) 19 LITERATURE CITED Augustine, J. J., L. R. Baker, and H. M. Sell. 1973. Female flower induction on androecious cucumber, Cucumis sativus L. Jour. Amer. Soc. Hort. Sci. 98: 197-199. Atsmon, D. and E. Galun. 1962. Physiology of sex in Cucumis sativus (L.). Leaf age patterns and sexual differentiation of floral buds. Ann. Botany 26:137- 1H6. Fukushima, Eiji, Eisuke Matsuo, and Junimitsu Fujieda. 1968. Studies on the growth behavior of cucumber Cucumis sativus L. J. Fac. Agr. Kyushu Univ. lA:3A9- 366. Galun, E. 1961. 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