Amplified Fragment Length Polymorphism

171 Pages·2013·8.82 MB·English

Stem Rust Resistance tn Avena strigosa Schreb.:
lnheritance, Gene Transfer, and ldentification of an
Amplified Fragment Length Polymorphism
(AFLP) Marker
by
Taye Zegeye
A Thesis submitted to the Faculty of Graduate Studies of
The University of Manitoba
in partial fulfilment of the requirements of the degree of
Doctor of Philosophy
Department of Plant Science
University of Manitoba
Winnipeg, Manitoba
Copyright O 2008 by Taye Zegeye
THE UNIVERSITY OF MANITOBA
FACULTY OF GRADUATE STUDIES
COPYRIGHT PBRMISSION
Stem Rust Resistance in Avena strigosa Schreb.: lnheritance, Gene Transfer, and
ldentification of an Amplified Fragment Length Polymorphism (AFLp) Marker
BY
Taye Zegeye
A ThesislPracticum submittetl to the Faculty of Graduate Sturlies of The University of
Manitoba in partial fulfillment of the requirement of the degree
of
Doctor of Philosophy
Taye Zegeye O 2008
Permission has been granted to the University of Manitoba Libraries to lentl â copy of this
thesis/practicum, to Library and Archives Canada (LAC) to lend a copy of this thesis/þracticum,
and to LAC's agent (UMlÆroQuest) to microfilm, sell copies and to puUi.ft an abstract of this
thesis/practicum.
This reproduction or copy of this thesis has been made availabte by authority of the copyright
owner solely for the purpose of private study and research, and may ónly be reirotluced ana c"opieO
as permitted by copyright laws or with express written authorizaiion f.o- thà copyright ownér.
ACKNOWLEDGEMENTS
Throughout the duration of the study, many people helped me out in one form or
another. Some offered their time and provided me with their valuable inputs while others
gave me the moral support that I needed and encouraged me all the way. It will be
difficult to list the names of all the people who have contributed to the success of this
project. I appreciate all the help that was offered to me and thank you all for the same.
However, there are few people who went extra miles to see me complete my studies and
they deserve a special thank you.
First of all I like to thank my advisor Dr. Thomas Fetch Jr. for taking me as his
student. Dr. Fetch was very willing to take me as his first Ph.D student when I was
looking for an advisor after my first advisor informed me that he was not in a position to
be my advisor due to health reasons. I appreciate the advice Dr. Fetch offered, the time
he devoted to me, and also the patience he had with me when the project was going at a
slow pace. His support helped me to get going and complete the project. Thank you Dr.
Fetch.
I like to thank professor Lakhdar Lamari for agreeing to be my co-advisor and for
all the helpful inputs he provided me throughout the study. The much relaxed and
friendly environment whenever I approached Dr. Lamari has indeed helped me to boost
the much needed energy to bring the project back on track.
Dr. P.B.E. McVetty, Dr. Daryl Somers, and Dr. Brent McCallum agreed to be
members of my advisory committee at the first request and I thank them all for that. The
', l
ii
valuable inputs and constructive suggestions they provided me whenever I approached
them and during advisory committee meetings are highly appreciated.
Dr. Jim Bole, the former director of the Cereal Research Centre (CRC), provided
me a huge help when the project was initiated. It was Dr. Bole who facilitated my
smooth transition from a technician position to being a full time student. He also played
a major role in finding me a supervisor. Thank you Dr. Bole for ali the support.
Dr. James Chong deserves a special thank you for motivating me and agreeing to
take me as his student until he began having health problems. I am also very grateful for
the positive suggestions he offered me whenever I approached him.
I also like to thank the staff at the CRC in Winnipeg, particularly the rust
laboratory technicians, Ken Dunsmore, Courtney Leclerc, Rebecca Dueck, Wayne
Mayert, Janet Gruenke, Pat Seto-Goh, and Laura Trump for their moral support and
technical inputs. The patience and support of technicians in the molecular biology
laboratory Leslie Bezte, Yanfen Zheng, Mira Popovic, Zlatko Popovic, Monica Eng, and
Debbie Miranda is greatly appreciated. I am very grateful to them for accommodating
me in the laboratory and teaching me the techniques while I was doing the molecular
work. Technicians in the doubled haploid laboratory, Debbie Jones, Elaine Martineau,
and Tobi Malasiuk, deserve a special thank you. Had it not been for their help and
willingness to accommodate me in the doubled haploid laboratory I wouldn't have been
able to produce the octaploid oat plants. I also like to thank technicians in the molecular
cytogenetics laboratory at the CRC, particularly Jadwiga Budzinski, for the help she
offered in the cytogenetics work.
111
I thank Dr. Axel Diedrichsen, Plant Gene Resource Canada (PGRC) in Saskatoon,
for providing the original Avena strigosa seeds that were used in this study.
I take this opportunity to thank graduate students in the Department of Plant
Science, particularly those at the CRC, for all the support they gave me. A special thank
you goes to Colin Hiebert, a graduate student in the Department of Botany, who was very
helpful whenever I needed his help in cytogenetics.
It was a great honor and privilege to be offered the opportunity to undertake this
study by being granted a leave of absence with a financial support. The support of
Agriculture and Agri-Food Canada (AAFC) in this regard is much appreciated. The
financial support of Vy'estern Grains Research Foundation (WGRF) that partially funded
the study is also gratefully acknowledged. It was a great pleasure and privilege to have
worked with two great institutions, the Department of Plant Science at the University of
Manitoba and the Cereal Research Centre of Agriculture and Agri-Food Canada.
Last but not least, I take this opportunity to thank my family, my wife Aikaterini,
our daughter Hagere Selam, and our son Theodoros for being so patient and
understanding while I was working on this project at the expense of the family time. Had
it not been for the understanding and full support of my family, the project wouldn't have
come to its conclusion. I owe my family a lot.
1V
TABLE OF CONTENTS
rNTRODUCTION..............
1.0 GENERAL .............1
2.0 LITERATURE REVTEW ..................6
0AT..
2.1 ............6
cultivation
2.1.1 Origin and ...............7
Dßtribution
2.1.2 .............8
CløssiJícøtion....
2.1.3 .......9
2.1.4 Oat crossing .............;.... . . ..10
production
2.1.5 Octt ......12
oat........
2.1.6 ases of ..... 13
Avenø...
2.1.7 Cytogenetics of .........,15
strigosø..
2.1.8 Avena .....16
RUST.......
2.2 T}JE ............17
rust..
2.2.1 Oøt stem .......17
Classfficationofoøtstemrust..
2.2.2 ................18
2.2.3 Life cycle of Puccinia gramínis øvenae...... ...............19
Environmentalføctors..........
2.2.4 ....................20
damøge................
2.2.5 Stem rust ......................21
2.2.6 Oat stem rust resístance genes in North Americø .....21
resistance
2.2.7 Adult plant .............22
hypothesß
2.2.8 Gene-for-gene .......23
2.2.9 Differential infection in oat........ ..............24
methods....
2.2.10 Control ................25
practices.........
2.2.10.1 Cultural ....................2s
control......
2.2.10.2 Chemical ......26
...........
2.2.10.3 Genetic host resßtance .........27
POLYMORPISM
2.3 AMPLIFIED FRAGMENT LENGTH ..............29
AFLP...
2.3.1 Application of ............31
ANALYSIS...............
2.4 BULKED SEGREGANT ..............32
3.0 INHERITANCE OF OAT STEM RUST RESISTANCE IN SEVEN AVENA
ACCESSIONS.
STRTGOSA ....................33
ABSTRACT......
3.1 ...........33
INTRODUCTION
3.2 .......35
M8THODS...............
3.3 MATERIALS AND .....38
study....
3.3.1 Genetìc .....38
greenhouse..........
3.3.2 Stem rust inoculatíon in the ...... ..40
ønalyses
3.3.3 Dßease røting and statisticøl .....42
inoculøtion
3.3.4 Field .....................42
study ....i.............
3.3.5 Temperature ..................43
3.3.6 Muki-pøtltotype øllelism test............ . ..44
RESULTS.........
3.4 ...........46
F3families...........
3.4.1 F2populations ønd ......47
Backcrosses......
3.4.2 .....50
øccessions
3.4.3 Intercrosses umong the seven ..................51
P96.......
3.4.4 Allelßmto .................51
study.........
3.4.5 Temperature .........54
............
3.4.6 Multi-pathotype allelism test ........ 55
DISCUSSION
3.5 ..............57
Fjfamilies...........
3.5.1 F2populations ønd ......57
.........
3.5.2 Temperøture study .........59
test............
3.5.3 Muki-pathotype allelism ........61
CONCLUSIONS
3.6
........62
4.0 TRANSFER OF RESISTANCE GENE TO RACE TJJ (NA67) FROM,4 VENA
II')............
STRIGOSA SCHREB. "îO AWNA SATIVA L. (,SUN ............64
4.1
4.2
V1
M8THODS...............
4.3 MATERIALS AND .....69
crosses..
4.3.1 Avena strigosa by hexøploid .....69
Stemrustinoculum-raceTJJNA6T)
4.3.2 ....................70
Cross¿.Í...............
4.3.3
......................71
4.3.4 Avenø longiglumis by 'Sun ff' cross.... ......................74
technique..........
4.3.5 The doubled høploid @H) ..............74
counting...
4.3.6 Chromosome . .....77
RESULTS.........
4.s
...........79
crosses..
4.5.1 Avenø strigosa by hexøploid ......79
4.5,2 Avenø longiglumis (Cw57) by 'Sun II' cross.... .........87
DTSCUSSION
4.6 ..............88
crosses..
4.6.1 Avena strigosø by hexøploid .....88
cross..
4.6.2 Avenø longiglumis by hexaploid .....................94
CONCLUSION
4.7 ...........97
5.0 IDENTIFICATION OF AN AFLP MOLECULAR MARKER FOR OAT
STRIGOSA
STEM RUST RESISTANCE IN AVENA ..............99
5.1
5.2
M8THODS...............
5.3 MATERIALS AND ...105
extrøction.
5.3.1 Plant materials and DNA .....105
ampliJicøtion................
5.3.2 Digestion, Iigation, ønd ......I06
analysis....
5.3.3 Bulked SegregøntAnalysis (BSA) and gel ..............108
RESULTS.........
s.4
.........110
Analysis
5.4.1 Bulked Segregant ...110
DISCUSSION
5.5 ............116
5.5.1 BulkedSegregøntAnølysis (BSA). ...... . ........ . ......119
CONCLUSION
5.6 .........122
DISCUSSION..
6.0 GENERAL ...........123
CONCLUSIONS...............
7.0 GENERAL
...........129
8.0 SIGNIFICANCE OF THIS STUDY AND FURTURE WORK.... ...I32
CITED
9.0 LITERATURE
..................134
APPENDICES..........
............151
vlt
LIST OF TABLES
Table 1. Effective/ineffective Pg genes for 23 oat stem rust races, Puccinia graminis
Pers. f. sp. avenae Eriks. and E. Henn. .....45
Table 2. Stem rust reactions of Avena strigosa accessions and 'AC Assiniboia' in
TJJ.
greenhouse and field experiments using race .....47
Table 3. Chi-square analyses of F2populations tested with race TJJ at the seedling stage.
47
Table 4. Chi-square analyses of F3 families tested with race TJJ at the seedlingstage.49
Table 5. Chi-square analyses of backcross populations tested with race TJJ at the
stage.
seedling ....... 50
Table 6. F2 populations of intercrosses between seven A. strigosa accessions tested with
stage.........
race TJJ at the seedling ........... 52
Table 7. Allelism test of Fz populations from the seven A. strigosa accessions crossed to
stage.
Pgó (CN56818) and tested with race TJJ at the seedling ..................... 52
Table 8. F: seedlings from four F2 plants originally rated as susceptible when tested with
TJJ.
race ................ 53
Table 9. Rust reaction of nine A. strigosa accessions and 'AC Assiniboia' to race TJJ at
regimes.
three temperature .....54
Table 10. Seedling reactions of eight A. strigosa accessions, 12 single gene differential
lines, and fhe Pg-a complex fo 23 races of Puccinia graminis f. sp. avenae. ..................56
Table 11. Seedling reactions of diploid A. strigosa and hexaploid oat cultivars to oat
TJJ.
stem rust race ..................73
Table 12. Ft and colchicine doubled F1 plants producing seeds from interploidy crosses
..........
between diploid (A. strigosa) and hexaploid (1. sativa) oat. ...............79
Table 13. Seeds produced from colchicine-treated and non-treated F¡ plants ftom A.
crosses.
strigosaby A. sativd ....................80
vll1
Table 14. BCIFI seeds from octaploids backcrossed to 'Sun II' as well as from
crosses.
reciprocal ...................83
............
Table 15. PslI and Msel primers' and adapters' sequences. ...........107
Table 16. Selective three base-pair PslI and Mselprimers used for screening. ...........108