Selection tests of Nod factor receptors (NFRs) in

Glycine subgenus Glycine

Craig Richard
Doyle lab
Mentor: Adrian Powell

Outline
Nodulation
Processes
Potential applications

Polyploidy
Significance to nodulation

Hypothesis
Methods
Data and results
Conclusion

Legumes and Nodulation

Nodulation

Skorupska et al., 2010

Nodulation

Skorupska et al., 2010

Nodulation

Skorupska et al., 2010

Nodulation

Skorupska et al., 2010

Nod Factor Receptors

Ryu et al.

Potential applications
Engineering crops to nodulate
Reduction in spending on fertilizers
Reduction in the use of fertilizers

http://commons.wikimedia.org

Polyploidy
What is it?
How does it occur?
Why it is important?

Hufton & Panapoulou, 2009

Study system
G. max, G. soja

2n = 40
~5 MYA

subg. Glycine

2n = 38, 40, 78, 80

ycine subgenus Glycine Polyploid Comple

G. hirticaulis
Craven and
Tindale

T6
G. tomentella
2n=78

D5B
G. tomentella
2n=40

D1
G. tomentella
2n=38

2n=40
T1
G. tomentella
2n=78

T4
G. tomentella
Hayata
2n=80

G. hirticaulis
2n=80

H genome

T5
G. tomentella
2n=78

D3
G. tomentella
2n=40

T3
G. tomentella
2n=80

D5A
G. tomentella
2n=40

T2
G. dolichocarpa
Tateishi &
H. Ohashi
2n=80

Based on Doyle et al.

2003

BBBB
G. tabacina
(Labill.) Benth.
2n=80

BB
G. stenophita
2n=40
G. clandestina
J.C. Wendl.

2n=40

B genome

D4
G. syndetika

AABB
G. pescadrensis
Hayata
2n=80

Pfeil and Craven

Glycine tomentella complex

BB
B genome
complex
2n=40

2n=40

A genome Glycine tabacina

complex

Hypotheses
If NFRs mediate a critical relationship
between specific Nod factor and receptor,
genes should be subject to strong purifying
selection
Positive or directional selection would
indicate diversification
If constraints are less stringent, genes may
be selectively neutral

Approaches
Selection

Tests

Purifying

Tajimas D and variants

Positive

McDonald-Kreitman (MK)

Neutrality

Absence of significant deviations in

above tests

G. max NFRs
NFR5

NFR1

NFR1

NFR5

G. max NFRs

NFR5

NFR5
lysM Domain

Region 1

Transmembra
ne Domain
Kinase Domain

Region 2

Image from Phytozyme.

Methods and Procedures

Primers were designed to NFR5 in G. max
Polymerase Chain Reaction conditions were optimized for the primer
pairs
PCR was used to amplify extracted DNA from D1, D3, G. syndetika
and G. clandestina
Products were examined via gel electrophoresis
Sequences were obtained from accessions of D1, D3, G. syndetika and
G. clandestina using the Sanger method
Tools used

Sequences aligned using MUSCLE in DNASTAR software package

DnaSP (Rozas et al. 2003)

Results: Summary Statistics

NFR5 Region 1 (357 bp)

NFR5 Region 2 (282 bp)

Results: Selection Tests

NFR5 Region 1

NFR5 Region 2

*Bold values indicate P < 0.05

McDonald-Kreitman (MK)
Test

G. max used as outgroup for

estimating divergence

In some cases, contingency table

could not be computed
In all cases where test could be
conducted, no statistically significant
deviations from neutrality

Discussion
Data indicates neutrality, purifying
selection
De Mita et al. (2007): Selective
neutrality of nodulation receptorencoding genes in Medicago
truncatula

Future Work
Increased sample size is necessary
for diploid species
Complete sequencing of NFR5
Develop homeologue-specific
primers for allopolyploids in the
Glycine subgenus Glycine polyploid
complex
Continue with study of NFR5 and
NFR1

Acknowledgments
I would like to thank:
NSF PGRP
The Doyle Lab
The Boyce Thompson Institute for
Plant Research
Cornell University