Unreduced Gametes in Brassica:
Effects of Genotype, Temperature
and Interspecific Hybridization
Annaliese S. Mason, The University of
Queensland, Australia
[email protected]
Guijun Yan, The University of Western Australia
Wallace A. Cowling, Canola Breeders Western Australia
Matthew N. Nelson, The University of Western Australia
Summary
•
•
•
•
What is an unreduced gamete?
Why are unreduced gametes interesting?
Experimental material
Hypotheses: hybridisation, genotype and
temperature effects
• Results from pollen measurements
• Results from sporad observations
• Results from hybridisation experiment
What is a reduced gamete?
Maternal chromosome
Paternal chromosome
Reduced
gametes
Pre-meiosis DNA
content doubles;
Normal
meiosis
two identical
chromatids formed
“2n” DNA
content
“n” DNA
content
What is an unreduced gamete?
Maternal chromosome
Paternal chromosome
Unreduced
gametes
Pre-meiosis DNA
content doubles;
Abnormal
meiosis
two identical
chromatids formed
“2n” DNA
content
“2n” DNA
content
Evolution of polyploid plants through union
of unreduced gametes
Species A
AA
A
A
A
2n = AA
New polyploid
species
pollen
A
A
A
2n = AB
Species B
2n = BB
B
B
B
B
B
BB
B
B
B
B
2n = AABB
ovules
U’s Triangle
of Brassica
B. rapa
B. juncea
B. napus
AA
AABB
BB
B. nigra
AACC
BBCC
B. carinata
CC
B. oleracea
U’s Triangle
of Brassica
B. rapa
B. juncea
B. napus
AA
AABB
x
BB
B. nigra
x
AACC
x
BBCC
B. carinata
CC
B. oleracea
AABB
B. carinata
B. napus
B. juncea
x
AACC
BBCC
x
x
~2400 bud
pollinations
Interspecific
hybrids
1500
13
genotypes
seeds
AABC
BBAC
CCAB
total
Unreduced gametes: hypotheses
• Do interspecific hybrid Brassica produce more
unreduced gametes than their natural parents?
• Are there differences between Brassica
genotypes in unreduced gamete production?
• Will unreduced gametes contribute to form
higher ploidy progeny in interspecific crosses?
• Does temperature influence unreduced gamete
production?
Estimating unreduced gamete production
Method 1: Pollen measurements
“Normal”
viable pollen
(reduced)
“Giant” viable
pollen
(unreduced)
Parent pollen
Number of pollen grains
100
300
500
700
Hybrids produce
Pollenhigh
sizefrequencies
results of viable
unreduced pollen grains
Hybrid pollen
0
“Giant” pollen
20
25
30
35
40
45
50
Pollen minimum diameter in micrometres
55
Normal Meiosis
Meiosis I
Meiosis II
Normal tetrad
Parallel Spindles (a common means of 2n gamete formation)
Meiosis I
Dicotyledonous plants
Meiosis II
FDR-type dyad
Estimating unreduced gamete production
Method 2: Sporad observation
Tetrad
(4 nuclei)
reduced
Dyads
(2 nuclei)
unreduced
Dyads observed in sporad population (%)
Sporad results: interspecific hybrids produce more
unreduced gametes than their parent species
8%
7%
6%
0.02%
1.32%
4 dyads out
of ~10 000
sporads
680 dyads
out of ~27500
sporads
5%
4%
3%
2%
1%
0%
Natural species
Hybrid
AABCA
Hybrid
BBACB
Hybrid
CCABC
Brassica genotype
Mason et al (2011) BMC Plant Biology 11:103
Genotypic differences
2n gametes in viable pollen
2n gametes in sporads
50%
45%
40%
c
35%
c
30%
25%
abc
20%
b
15%
10%
b
b
b
5%
a
a
B1
B2
0%
A1
A2
A3
A4
A5
C1
C2
C3
C4
C5
C6
Hybrid genotype
Mason et al (2011) BMC Plant Biology 11:103
Does temperature affect unreduced
gamete production?
Temperature treatments – four controlled
environment rooms, night/day temperature
•
•
•
•
“cold”
“cool”
“warm”
“hot”
–
–
–
–
5°C / 10°C
13°C / 18°C
15°C / 25°C
25°C / 30°C
• Five genotypes (B. napus, B. juncea, B.
carinata) and five of their interspecific hybrids
• Two plants x 10 genotypes in each room
• 2 x 300 sporad counts per plant
Mason et al (2011) BMC Plant Biology 11:103
The effect of temperature on unreduced gamete production
% Male unreduced gametes
30%
cold
cool
warm
hot
25%
20%
*
15%
*
10%
5%
0%
C1
C2
J1
N1
N2
J1N1 J1N2 J1C1 N1C2 N2C2
Genotype
Mason et al (2011) BMC Plant Biology 11:103
Allohexaploid Brassica
B. rapa
AA
B. nigra
B. oleracea
BB
CC
B. juncea
B. napus
B. carinata
AABB
AACC
BBCC
AABBCC?
Diploid
2n = 2x
Tetraploid
2n = 4x
Hexaploid
2n = 6x
Can we use unreduced gametes to produce this
hexaploid from the tetraploid species?
Mason et al (2012) Euphytica In Press
Experimental crossing
design
Hybrid
AABC
AB
B. juncea
AABB
AC
AABC
B. napus
2n
AACC
AB
AC
AABBCC
AB
AC
ABCC
2n
ABBC
Hybrid
ABBC
BC
BC
AA
BB
CC
Indicate the three
basic genomes with
A=10, B=8 & C=9
chromosomes
Hybrid
ABCC
2n
B. carinata
BC
XX
BBCC
XXXX
2n
reduced (n) gamete
unreduced (2n) gamete
30
20
10
5.5x 6.5x
0
of individuals
of individuals
Number Number
40
DNA content (estimated by flow cytometry) for second cross
hybrid population
0.0
0.5
3.0
1.0
6.0
1.5
9.0
DNA content (proportional
to expected value
for an AABBCC hexaploid)
Relative
DNA content:
~AABBCC
= 6.0x
Approximate ploidy level of progeny
Cross
AABC x
BBCC
BBAC x
AACC
CCAB x
AABB
Plants
46
64
All ~4x
All ~4x
1 plant ~5.5x
2
Mason et al (2012) Euphytica In Press
1 plant ~6.5x
Phenotype of “~6.5x” plant
Pollen viability: 0-22%
Self seed set: 0
OP seed set: 13
“~6.5x” plant chromosome spreads
2n ~ 58
Expected AABBCC = 54 chromosomes
Phenotype of “5.5x” plant
Pollen viability: 25%
Self seed set: 166
OP seed set: 458
“5x” plant chromosome spreads
2n ~ 50
Expected AABBCC = 54 chromosomes
Molecular marker results
Normal tetrad
6.5x plant
AjBj from B. juncea + 2(0.5A + 0.5B + Cn or c)
from CnCcAnBc hybrid
SDR-type dyad
Explanation: Meiosis II failed to separate
sister chromatids
5.5x plant
AjBj from B. juncea + Cn/cCn/cAnBc from
CnCcAnBc hybrid (minus a few chromosomes)
= AjAnBjBcCnCc
Explanation: Meiosis I failed to separate
homologous chromosomes
FDR-type dyad
Unreduced gametes: conclusions
• Interspecific hybrid Brassica produce more
unreduced gametes than their natural parents
• Genotype influences unreduced gamete
production
• Unreduced gametes can be used to produce
higher ploidy progeny in interspecific crosses,
depending on genotype
• Cold ambient temperature increases unreduced
gamete production in some genotypes
Thanks and acknowledgements
Supervision
• Prof. Wallace Cowling
• Dr. Matt Nelson
• Assoc. Prof. Guijun Yan
Funding
• Australian Postgraduate Award (Industry) – Australian
Research Council with industry partners NPZ Lembke
(Germany) and COGGO Ltd (Australia)
Help and technical assistance
• Kathy Heel Miller and Tracy Lee Pullen – flow
cytometry (CMCA UWA Perth)