Locating Brassica A and C genome
centromeres by half-tetrad analysis
Matthew Nelson
Annaliese Mason, Mathieu Rousseau-Gueutin, Philipp Bayer, Aneeta
Pradhan, Naghmeh Besharat, Anouska Cousin, Jérôme Morice, AnneMarie Chèvre, Isobel Parkin, Jacqui Batley
Presentation overview
• Centromeres and cell division
• Locating centromeres
• What is half-tetrad analysis?
• Our experimental approach
• Brassica centromere positions
• Utility of this approach for other
species
Centromeres
• Comprise a core functional
domain where spindle
fibres attached via the
kinetochore
• ‘Point’ centromeres are tiny
(125bp in yeast)
• ‘Regional’ centromeres are
larger (0.5-1.8 Mb in
Arabidopsis)
Sullivan et al. (2001) Nature Genetics 2:584-596
• Brassicaceae centromeres
comprise satellite repeats
Centromeres and cell division
Tachibana-Konwalski (2015) Nature 517:441-442
Why locate centromeres?
• Basic understanding of chromosome
evolution
• Managing recombination in breeding
programs
– Recombination is generally suppressed
at centromeres
How to locate centromeres?
• Cytogenetically using FISH (hard)
• Genetically through suppressed
recombination (imprecise)
• Motif analysis in genome sequences
(imprecise)
• Tetrad analysis (restricted to fungi,
Arabidopsis)
• Half-tetrad analysis
What is half-tetrad analysis?
• Uses widespread phenomenon of unreduced
(2n) gamete formation
• First division restitution (FDR) yields 2n gametes
comprising non-sister chromatids
– Maximum heterozygosity at centromeres
• Second division restitution (SDR) yields 2n
gametes comprising sister chromatids
– Minimum heterozygosity at centromeres
Contrasting heterozygosity in FDR and SDR
Park et al. (2007) Genetics 176:85–94
FDR unreduced gametes in Brassica
Visible as dyads at the
sporad stage of microspore
development
Visible as giant mature
pollen
Gamete formation (male, dicot):
Reduced versus FDR unreduced
Meiosis II
Normal
spindles
Premeiotic
doubling
Reduced
gametes
Meiosis I
Parallel
spindles
Unreduced
gametes
Our experimental approach
• Intercrossing Brassica allotetraploids
– Elevated levels of unreduced gametes
– Highly heterozygous
– High gamete viability
• Microspore culture and testcrosses
• Genotyping by 60K Infinium SNP chip
B. rapa
U’s Triangle
n = 10
AA
AABC
hybrids
B. napus
B. juncea
n = 18
AABB
n = 19
AACC
X
X
B. nigra
B. carinata
n=8
BB
n = 17
BBCC
B. oleracea
CCAB
hybrids
n=9
CC
Nelson et al. (2009) TAG
X
B. napus
B. carinata
CCAB
hybrids
microspores
embryos
SNP genotyping
plants
Derivation of experimental progeny through microspore culture
Results: AABC progeny
• 86 microspore-derived progeny from
4 AABC hybrids
• 7,168 polymorphic A-genome SNPs
• 77 progeny (90%) were FDR-derived
• Two pairs of identical twins
• 75 unreduced progeny used for
centromere mapping
Results: CCAB progeny
• 124 microspore-derived progeny
from 4 CCAB hybrids
• 13,819 polymorphic C-genome SNPs
• 92 progeny (74%) were FDR-derived
• 15 pairs of identical twins
• Of 65 testcross progeny, 35 (54%)
were FDR-derived
• 112 unreduced progeny used for
centromere mapping
Results: Centromere mapping
Mb position
40
A02
30
20
“Trace”
10
Centromere
0
50
60
70
80
90
100
% heterozygosity
Cheng et al. (2013) 25:1541–1554
Summary of HTA results
• Centromeres defined to 40 kbp to
6.7 Mbp (average 1.7 Mbp) intervals
• A-genome positions matched
previous centromere motif-defined
positions except for A02 and A09
(Cheng et al. 2013)
• C-genome positions refined from our
earlier analysis (Parkin et al. 2014)
• Congruence with gene and
transposable element densities
Half-tetrad analysis in other species
• Unreduced gametes occur in most
plant species at low levels
– Enriched in interspecific and
interploid crosses
– Stimulated by cold
• Flow cytometric sorting combined
with single molecule genome
sequencing could allow this method
in any plant species
Acknowledgements
UWA, Perth
Anouska Cousin
Naghmeh Besharat
Aneeta Pradhan
Philipp Bayer
Jacqui Batley
Shyam Sundar Dey
Linda Thomson
Marie-Claire Castello
Guijun Yan
Wallace Cowling
JLU, Giessen
Annaliese Mason
INRA Le Rheu, Rennes
Mathieu Rousseau-Gueutin
Jérôme Morice
Anne-Marie Chèvre
AAFC, Saskatoon
Isobel Parkin
UQ, Brisbane
Rowan Bunch