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Sex determination in cucumber. Anandkumar Surendrarao VC221: Vegetable crop breeding May 10, 2006. Perfect flowers or Hermaphroditic flowers Both male and female reproductive parts are present on the same flower. Perfect flowers or Hermaphroditic flowers

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Sex determination in cucumber

Sex determination in cucumber

Anandkumar Surendrarao

VC221: Vegetable crop breeding

May 10, 2006


Perfect flowers or Hermaphroditic flowers

Both male and female reproductive parts are present on the same flower


Perfect flowers or Hermaphroditic flowers

Both male and female reproductive parts are present on the same flower


Monoecious plants – Imperfect flowers

Separate male and female flowers are present on the same plant


Dioecious plants – Imperfect flowers

Male and female flowers are present on individual separate plants


Dioecious plants – Imperfect flowers

Male and female flowers are present on individual separate plants


Sex determination in cucumber1
Sex determination in cucumber

♀flower

♂flower




Developmental arrest of whorl 4 in male and whorl 3 in female flowers
Developmental arrest of whorl 4 in male and whorl 3 in female flowers


Class C

Class B

Cucumber floral MADS box gene expression and sequenceProbing female cDNA library with petunia MADS box gene


Amino acid conservation amongst mads box genes
amino-acid conservation amongst MADS box genes

CUM1 = Ath AGAMOUS (69%)

CUM1 = Antirhinum PLENA (71%)

CUM26 = Ath PISTLLATA (69%)

CUM26 = Antirhinum GLOBOSA (70%)

CUM26 = Petunia FLORAL BINDING PROTEIN 1 (71%)


In situ hybridization analyses of CUM1 and CUM26 expression in wild type male and female flowerswith antisense probe to divergent 3’ UTR sequence

Wild type

Expression of homeotic genes is observed even in arrested primordia

CUM1 – class C, whorls 3 and 4; CUM26 – class B, whorls 2 and 3


Gp mutant flower phenotypes at 22 c a d sepal sepal flower x sepal sepal x carpel
gp mutant flower phenotypes at 22°CA-D♂ sepal-sepal-flower-X♀ sepal-sepal-X-carpel

gp mutant flower phenotypes at ≥ 30°CE-J♂ sepal-sepal-carpel-X ♀ sepal-sepal-X-carpel


In situ hybridization analyses of cum1 and cum26 expression in gp mutant male flowers
In situ hybridization analyses of CUM1 and CUM26 expression in gp mutant male flowers

35°C

35°C

22°C

22°C

gp mutant

CUM26 = GP = class B mutant

CUM1 – class C, whorls 3 and 4; CUM26 – class B, whorls 2 and 3


CUM1 hypermorph (over-expression)

Unisexual to bisexual

floral conversion

CUM1 hypomorph

(co-suppression)



Selective repression of male or female reproductive organs depends on floral whorl position rather than organ identity


Paper 2
Paper #2 depends on floral whorl position rather than organ identity


Genetic and environmental control of cucumber sex determination
Genetic and environmental control of depends on floral whorl position rather than organ identitycucumber sex determination

Genotypes

Gynoecious - F-M- - ♀

Andromonoecious - ffmm - ♂ and ♀

Monoecious - ffM- - ♂ and ♀

Hermaphrodite - F-mm - ♀

Ethylene and ethephon – induction of ♀ flowers

AVG and AgNO3 – induction of ♂ flowers


Sex of different cultivars used in this study
Sex of different cultivars used in this study depends on floral whorl position rather than organ identity


Development of flower buds in gynoecious cucumber plants
Development of flower buds in depends on floral whorl position rather than organ identitygynoecious cucumber plants


Development of flower buds in gynoecious cucumber plants1
Development of flower buds in depends on floral whorl position rather than organ identitygynoecious cucumber plants


Development of flower buds in depends on floral whorl position rather than organ identitymonoecious cucumber plants


Development of flower buds in depends on floral whorl position rather than organ identitymonoecious cucumber plants


Avg masculinizes between node 8 and 13 ethephon feminizes between nodes 10 and 14
AVG masculinizes between node 8 and 13, Ethephon feminizes between nodes 10 and 14

Floral stages immediately before and after differentiation of stamen primordia

are responsive to both AVG and ethephon treatments


Monoecious Gynoecious Andromonoecious Monoecious

Antisense CS ACS2

Antisense CS ERS

Antisense CS ETR1

Antisense CS ETR2

Sense CS ACS2

Sense CS ERS

Sense CS ETR1

Sense CS ETR2


In situ hybridization results
In situ Monoecious hybridization results


The expression patterns for MonoeciousCS-ACS2, CS-ERS, CS-ETR1, and CS-ETR2 are all different among monoecious, gynoecious and andromonoecious plants.

CS-ACS2 and CS-ETR2 are expressed in identical domains in monoecious plants and overlapping domains in gynoecious plants.

In andromonoecious plants, none of the ethylene receptors transcripts accumulated in the stamen primordia.

Atleast one ethylene receptor transcript is expressed in the stamen and pistil primordia of monoecious and gynoecious flowers, and pistil primordium of andromonoecious flowers.


Cells producing and sensing ethylene are identical. Eg. Overlapping CS-ACS2 and CS-ETR2 mRNA expression in monoecious and gynoecious plants, direct determination of female flowers by inducing pistil development.

Cells producing and sensing ethylene are adjacent. Eg. mRNA expression of CS-ACS2 in adaxial side of petals but all the receptors in stamen primordia in monoecious plants. (diffusion?)

Cells producing and sensing ethylene are distant. Eg. mRNA expression of CS-ACS2 in pistil primordia but that of receptors in the stamen primordia. (diffusion?)


Paper 3
Paper #3 Overlapping CS-ACS2 and CS-ETR2 mRNA expression in monoecious and gynoecious plants, direct determination of female flowers by inducing pistil development.


What are the downstream targets of the sex determination machinery that allow the selective arrest of stamen and pistil primordia development?

Use suppression subtractive hybridization on NILs of gynoecious (FFMMaa), hermaphrodite (FFmmaa), androecious (ffMMaa) and monoecious (ffMMA-) genotypes.

AgNO3 induced male flowers in gynoecious plants, and ethephon induced female flowers in hermaphrodite plants used for SSH.

Controls for SSH were female and male flowers from gynoecious and androecious plants respectively.


Results from ssh
Results from SSH machinery that allow the selective arrest of stamen and pistil primordia development?


Selection of 21 178 clones by dot blot analyses
Selection of 21/178 clones by dot blot analyses machinery that allow the selective arrest of stamen and pistil primordia development?

11/21 differentially expressed in hermaphrodite buds –

Clone #38 is putative CS nt sugar epimerase

10/21 differentially expressed in gynoecious plants


Putative sugar nt epimerase expressed lower in gynoecious than in hermaphrodite plants
Putative sugar nt epimerase expressed lower in gynoecious than in hermaphrodite plants

Leaves

Floral buds


Putative sugat nt epimerase expressed higher in natural/induced male flowers compared to natural/induced female flowers

Monoecious + no treatment ♂ plants + ethephon ♀ plants + AgNO3


No detectable polymorphisms at gdna level between gynoecious and hermaphrodite plants
No detectable polymorphisms at gDNA level between gynoecious and hermaphrodite plants

Southern blot hybridization with 19 different restriction enzymes


Mechanistic role for sugar nt epimerase in stamen primordia outgrowth and arrest
Mechanistic role for sugar nt epimerase in stamen primordia outgrowth and arrest

UDP glucose-4-epimerase converts UDP-glucose to UDP-galactose.

These are required for the synthesis of AGPs (Arabino-Galactan proteins) and cell wall polysaccharides that are necessary for cell wall expansion and therefore primordial outgrowth.


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