How many beans make five?
Genetic diversity analysis and
taxonomy in the legume Ononis
Jane Kloda
Dr D MacDonald, Dr PGD Dean
and Dr S Mayes
Morphology
O. repens
O. spinosa
Morphology
Ononis repens subsp maritima
Morphology
Ononis spinosa
Geography
Ononis repens
Ononis spinosa
Preston, CD, Pearman, DA and Dines, TD (2002) New At
of the British and Irish Flora
Cytology
Scale bar = 2μm
O. repens
2n=60
O. spinosa
2n=30
Morisset (1967) Watsonia 12:145-153
Flow Cytometry
O. repens
Mode =59
O. spinosa
Mode = 30
Taxonomy
Linnaeus
(1753),
Morisset
(1967)
Greuter
(1986)
Jarvis,
Cannon and
Ivimey Cook
(1983)
Sirjaev
(1932)
Stace (1997)
Rouy and
Foucaud
(1897)
O. spinosa L.
O. repens L.
O. spinosa
subsp.
spinosa
O. spinosa
subsp.
intermedia
(Rouy) P.
Fourn.
O. campestris G. Koch
O. spinosa
subsp.
procurrens
(Wallr.) Briq.
O. spinosa L. (divided into
four subspecies plus 11
varieties)
O. spinosa L. O. x
pseudohircina
Schur.
O. campestris O. intermedia
Koch et Ziz
Mey
O. repens L. (divided into 8
varieties)
O. spinosa
subsp.
maritima
(Dumort.) P.
Fourn.
O. repens L.
O. repens L.
O. repens
subsp.
maritima
O. procurrens O. repens L.
Wallr
Aims
• How many groups are British
Restharrows divided into genetically?
• Are Ononis repens and Ononis spinosa
interbreeding?
• How does this compare with samples
from continental Europe?
• Do present levels of genetic diversity
give cause for concern?
Tools
• Ten microsatellite markers
• DNA sequence
– chloroplast
– nuclear: coding and non-coding DNA
• 700 plant samples from 40 populations
Collection
sites
Key:
O. spinosa
O. repens
O. natrix
Other
Ten highly polymorphic
microsatellites
No of
Locus alleles
1
3
11
16
17
21
22
24
26
28
47
33
21
25
24
46
39
42
47
46
Mean alleles per
population
8.5
4.6
4.3
6.3
3.7
5.6
8.4
7.1
8.7
6.2
The trouble with tetraploids
10 ACCCTCGCATTACACACACACACACACACACAAAGGTCGACCGTTCAC
10 ACCCTCGCATTACACACACACACACACACACAAAGGTCGACCGTTCAC
10 ACCCTCGCATTACACACACACACACACACACAAAGGTCGACCGTTCAC
08 ACCCTCGCATTACACACACACACACACAAAGGTCGACCGTTCAC
Is indistinguishable from:
10 ACCCTCGCATTACACACACACACACACACACAAAGGTCGACCGTTCAC
10 ACCCTCGCATTACACACACACACACACACACAAAGGTCGACCGTTCAC
08 ACCCTCGCATTACACACACACACACACAAAGGTCGACCGTTCAC
08 ACCCTCGCATTACACACACACACACACAAAGGTCGACCGTTCAC
Quantitative scoring:
Qualitative scoring:
10,10,10,08 and 10,10,08,08
10,08,00,00 and 10,08,00,00
Principal Coordinates Analysis
• Similarities between cases
• Euclidian distance
 p
2
dij   ( xik  x jk )
 k 1
– xik variable Xk individual i
– xjk same variable individual j
PCO British individuals
PCO case scores (Euclidean)
2.4
1.9
repens
1.4
1.0
Intermedia
Axis 2
0.5
-2.4
-1.9
-1.4
-1.0
-0.5
0.5
1.0
1.4
1.9
2.4
-0.5
maritima
-1.0
-1.4
-1.9
Axis 1 = 10.3 %
Axis 2 = 4.2 %
-2.4
Axis 1
spinosa
PCO British populations
PCO case scores (Euclidean)
1.3
1.0
repens
0.8
0.5
Axis 2
0.3
-1.3
-1.0
-0.8
-0.5
-0.3
0.3
0.5
0.8
1.0
1.3
-0.3
-0.5
-0.8
-1.0
Axis 1 = 27 %
Axis 2 = 11.5 %
-1.3
Axis 1
spinosa
PCO all populations
PCO case scores (Euclidean)
1.7
Spanish O. spinosa
1.3
British O. repens
1.0
German O. repens
0.7
Axis 2
British O. spinosa
0.3
Spanish O. natrix
Spanish O. diffusa
-1.0
-0.7
-0.3
0.3
0.7
1.0
1.3
1.7
French O. repens
-0.3
French O. spinosa
-0.7
Spanish O. viscosa
Axis 1 = 19.5 %
Axis 2 = 14.8 %
-1.0
Axis 1
Nei’s genetic distance
Ds   log e ( J XY / J X J Y )
xi, yi frequencies of ith allele in populations X and Y
probability that two randomly chosen genes in population X are
identical is jx=Σxi2,
in population Y it is jy=Σyi2
probability of identity for both populations is jxy=Σxiyi.
Jx, Jy and Jxy arithmetic means of jx, jy and jxy, over all loci.
Nei’s genetic distance
O.spinosa 14
O.spinosa 21
O.spinosa 7
O.spinosa 15
O.spinosa 3
O.spinosa 9
O.repens 18
O.repens 17
O.spinosa 16
O.repens 6
O.repens 19
O.spinosa 12s
O.repens 8
O.repens 12r
O.repens 1
O.spinosa 13
O.repens 10
O.repens 2
1
O.repens 22
O.repens 4
O.repens 5
DNA sequencing
• Chloroplast DNA – trnL spacer
• Nuclear – β-amyrin
sequence:intron/exon/protein
• Nuclear – microsatellite locus 1 flanking
region
DNA sequencing
• For O. repens and O. spinosa:
– No variation in chloroplast DNA sequence
(except German)
– Low variation in β-amyrin sequence, no
species differentiation
– High variation in microsatellite flanking
regions, no species differentiation
• For other Ononis species, consistent
relationships were revealed
Conclusions
• The ten microsatellites are effective
– at differentiating O. spinosa and O. repens
in Britain
– for studying genetic diversity in populations
of continental Ononis species
• The DNA sequences are effective
– for cross-species comparisons
Conclusions
• Ononis spinosa and O. repens are not freely
interbreeding in Britain
• The same pattern appears in France
• There is no further genetic differentiation
between ecotypes
• O. spinosa and O. repens are similar in the
DNA sequences studied
• Levels of genetic diversity are high and do
not give cause for concern
Thanks!
It is my pleasure to acknowledge:
Dr Sean Mayes
Dr Don MacDonald
Dr Peter Dean
Chris Maddren
Dr. François Balloux
Dr. Johannes Vogel
Cambio and BBSRC