Download 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 cucumber
♀flower
♂flower
ABC model of floral development
Paper #1
Developmental arrest of whorl 4 in
male and whorl 3 in female flowers
Cucumber floral MADS box gene expression and sequence
Probing female cDNA library with petunia MADS box gene
Class C
Class B
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 flowers
with 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 ≥ 30°C
E-J
♂ sepal-sepal-carpel-X
♀ sepal-sepal-X-carpel
In situ hybridization analyses of CUM1 and CUM26
expression in gp mutant male flowers
22°C
35°C
22°C
gp mutant
CUM26 = GP = class B mutant
CUM1 – class C, whorls 3 and 4; CUM26 – class B, whorls 2 and 3
35°C
CUM1
hypermorph
(over-expression)
Unisexual to bisexual
floral conversion
CUM1 hypomorph
(co-suppression)
ABC model of floral development
Selective repression of male or female reproductive organs
depends on floral whorl position rather than organ identity
Paper #2
Genetic and environmental control of
cucumber sex determination
Gynoecious
Andromonoecious
Monoecious
Hermaphrodite
-
Genotypes
F-Mffmm
ffMF-mm
-
♀
♂ and ♀
♂ and ♀
♀
Ethylene and ethephon
– induction of ♀ flowers
AVG and AgNO3
– induction of ♂ flowers
Sex of different cultivars used in this study
Development of flower buds in
gynoecious cucumber plants
Development of flower buds in
gynoecious cucumber plants
Development of flower buds in
monoecious cucumber plants
Development of flower buds in
monoecious cucumber plants
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
Sense CS ACS2
Antisense CS ERS
Sense CS ERS
Antisense CS ETR1
Sense CS ETR1
Antisense CS ETR2
Sense CS ETR2
In situ hybridization results
The expression patterns for CS-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
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
Selection of 21/178 clones by dot blot analyses
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
Floral buds
Leaves
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
Southern blot hybridization with 19 different restriction enzymes
Mechanistic role for sugar nt epimerase in stamen
primordia outgrowth and arrest
UDP glucose-4-epimerase converts UDP-glucose to UDPgalactose.
These are required for the synthesis of AGPs (ArabinoGalactan proteins) and cell wall polysaccharides that are
necessary for cell wall expansion and therefore
primordial outgrowth.