AN ABSTRACT OF THE DISSERTATION OF
Harish Tulshiramii Gandhi for the degree of Doctor of Philosophy in Crop
Science presented on June 16, 2005
Title: Jointed Goatgrass (Aeqilops cylindrica Host) Genetic Diversity and
Hybridization with Wheat (riticum aestivum L.)
Abstract
approv
Redacted for privacy
car Riera-Lizarazu
Carol A. MaIlory-Sm7'
Jointed goatgrass (Aegilops cylindrica Host; 2n=4x28; CCDD) is an
agriculturally important species both as a weed and as a genetic resource for
wheat (Triticum aestivum L.; 2n=6x=42; AABBDD) improvement. In order to
better understand the evolution of this species, the diversity of Ac. cylindrica
was evaluated along with its progenitors, Ae. markgrafii (Greuter) Hammer
(2n=2x=14; CC) and Ac. tauschii Coss. (2n=2x=14; DD), using chloroplast and
nuclear microsatellite markers. Ac. cylindrica had lower levels of plastome and
nuclear diversity than its progenitors. The plastome diversity of Ac. cylindrica
was lower than its nuclear diversity. Ac. cylindrica was found to have either Cor D-type plastomes, derived from Ac. markgrafll or Ac. tauschll, respectively,
where the C-type plastome was found to occur at a lower frequency than the
D-type plastome. The nuclear genomes of Ac. cylindrica accessions with C-or
D-type plastome were found to be very closely related, suggesting a
monotypic origin. Furthermore, analyses suggests that Ac. tauschii ssp.
tauschii contributed its D genome and D-type plastome to Ac. cylindrica. Ac.
cylindrica accessions collected near Van Lake in southeastern Turkey, an area
where Ac. tauschii ssp. tauschii and Ac. markgrafll overlap, showed high
allelic diversity and may represent the site where Ae. cylindrica formed.
Population structure analyses suggested a lack of regional genetic structure in
Ae. cylindrica and evidence of migration of Ae. cylindrica among various
regions. Finally, Ae. cylindrica accessions in the USA were found to be closely
related to accessions from at least three regions of its native range central
Anatolia, central East Turkey and western Armenia, and Caucasia.
Wheat and jointed goatgrass are closely related and both have the Dgenome. These two species can hybridize and produce backcross derivatives
under natural conditions, a situation that may allow gene flow between these
two species. In order to better understand mating patterns between these two
species, a total of 413 first-generation backcross (BC1) seeds obtained from
127 wheat-jointed goatgrass F1 hybrids, produced under natural conditions,
were evaluated for their parentage using chloroplast and nuclear microsatellite
markers. Of the 127 F1 hybrids evaluated, 109 had jointed goatgrass as the
female parent, while the remaining 18 F1 plants had wheat as the female
parent. Of the 413 BC1 plants analyzed, 358 had wheat and 24 had jointed
goatgrass as the recurrent male parent. The male parentage of 31 BC1 plants
could not be determined. Although the majority of hybrids were pollinated by
wheat, backcrossing of hybrids to jointed goatgrass would enable gene flow
from wheat to jointed goatgrass. Though the observed frequency of jointed
goatgrass-backcrossed hybrids (F1 X jointed goatgrass) was low under field
conditions, their absolute number is dependent on frequency of hybrids, which
in turn, depends on the density of jointed goatgrass in wheat fields. Therefore,
the recommendations to control jointed goatgrass in wheat fields and adjacent
areas and to plant jointed goatgrass free wheat seed should be followed in
order to avoid gene flow from wheat to jointed goatgrass.
© Copyright by Harish Tulshiramji Gandhi
June 16, 2005
All Rights Reserved
Jointed Goatgrass (Aegilops cylindrica Host) Genetic Diversity and
Hybridization with Wheat (Triticum aestivum L.)
by
Harish Tulshiramji Gandhi
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented June 16, 2005
Commencement June 2006
Doctor of Philosophy dissertation of Harish Tuishiramli Gandhi presented
on June 16, 2005
APPROVED:
Redacted for privacy
r Professor- representing Crop Science
Redacted for privacy
Co-Major Professor,
resenting Crop Science
Redacted for privacy
Head of the Department of Crop and Soil Science
Redacted for privacy
Dean of th,éAraduate School
I understand that my dissertation will become part of the permanent
collection of Oregon State University libraries. My signature below
authorizes release of my dissertation to any reader upon request.
Redacted for privacy
Harish Tulshiramji Gandhi, Author
ACKNOWLEDGMENTS
The success I achieved in my academic and research career is a result
of support, inspiration and love I received from the various individuals who
came to my life either as teachers, friends, colleagues or family members.
Although it is impossible for me to express gratitude for everything I have
received and to recognize everybody who has contributed, following is an
attempt.
I express my sincere thanks to Dr. Oscar Riera-Lizarazu for serving as
my co-advisor, for providing critical suggestions and motivating words during
the course of my doctoral studies. His suggestions and ideas were primers for
many of the studies conducted in my dissertation research. Similarly, I would
like to sincerely express my appreciation to Dr. Carol Mallory-Smith, a coadvisor for my doctoral studies. I was grateful to have her constant help,
guidance and input during conduction of my doctoral research.
I would like to thank Dr. M. Isabel Vales for serving as a committee
member, and for providing constructive inputs and suggestions during the
course of my research. I also would like to sincerely appreciate Christy J.
Watson for her multifaceted help during various stages of research.
I thank Dr. Thomas Wolpert and Dr. Mina McDaniel for serving on my
graduate committee. I sincerely appreciate Dr. Thomas Wolpert for agreeing to
serve on my graduate committee during the later stages of my doctoral
studies. I also would like thank Dr. Steve Knapp for serving as a member on
my graduate committee during part of my doctoral studies.
I would like to recognize the excellent and courteous service of Caprice
Rosato, Alex Krupkin and Naoki at the central service lab. I also thank my
laboratory colleagues Pradeep Tempalli, Jason Nunes, Vamsi Nalam, Dr. Jeff
Leonard, Robin Truer and Shannon Bell for their help and camaraderie. I
thank the staff of the Crop Science Department, especially Peggy Mullet,
Barbara Reed, and Susan Wheeler for guiding me through administrative
steps for various purposes.
Thanks to all of my fellow graduate students of Crop Science, including
Felix Schuppert, Guillermo Pizarro, Matt Faletti and Jan Von Zitzewitz with
whom I enjoyed the days of graduate studies. I sincerely thank Balaji
Megarajan, Sivasai Yerubandi and many other friends who I met in Corvallis
for their constant support and friendship. I also would like to thank Ramesh
Pudake, Kishore Ambekar and others friends for expressing their care and
concern from thousands of miles away.
I also thank my numerous family friends in Corvallis, who made
Corvallis a home-away-from-home for me and my wife. I express special
thanks to the Satsang team, Kishore, Sonali Gandhi, Srinivas Rao, Sankar
Chakraborty, Ravi Inampudi, Anjan Bacchu and others, for providing healthy
philosophical discussions, which served as an enduring energy during testing
times. I also express special thanks to Anjan Bacchu for writing software,
Binary (Beta version), on request.
Words can not express the contribution of my mother, father, brothers
(Kamal bhau and Kunju
bhaiya),
sisters (Usha and Jyotij(/i), sister-in-laws
(Maya and Rupa
bhabhi),
and brother-in-laws (Pradeep and Jayprakashjijaji).
Each of these individuals has always been a source of encouragement and
moral support for all of my aspirations. I also recognize encouragement and
compliments provided by members of my in-laws family. I express my special
thanks and appreciation to my wife, Gayatri, whose friendship, help, support,
understanding and love were persistent during the final, but most elusive, days
of my doctoral studies. Last but not least, thanks to all others who have
knowingly or unknowingly contributed to the advancement of my career though
their services.
CONTRIBUTION OF AUTHORS
Dr. Oscar Riera-Lizarazu guided and proposed the research conducted.
He was also involved in the preparation of manuscripts and provided the
necessary feedback during various stages of the research. Dr. Carol MallorySmith extended her guidance in various aspects of the research projects and
also contributed to the preparation of the manuscripts. Dr. M. Isabel Vales
helped in various laboratory and greenhouse related activities and provided
constructive inputs at various stages of research and manuscripts preparation.
Dr. Robert Zemetra was a collaborator for the proposed research and
reviewed manuscripts. Dr. Laura Morrison, helped in procuring plant material
and performed part of the diversity assays. Dr. Naoki Mon contributed
important discussion to the preparation of a manuscript. He also provided
primers for the wheat chloroplast microsatellite markers. Christy J.W. Watson
helped in laboratory and greenhouse related activities and in preparation of
manuscripts. Maqsood Rehman helped with cytological analyses.
TABLE OF CONTENTS
Page
INTRODUCTION
.1
CHLOROPLAST AND NUCLEAR MICROSATELLITE ANALYSIS OF
Aegilopscylindrica ........................................................................ 6
ABSTRACT ................................................................ 7
INTRODUCTION ......................................................... 8
MATERIALS AND METHODS ....................................... 11
RESULTS ................................................................. 20
DiSCUSSION ............................................................ 35
ACKNOWLEDGMENTS .............................................. 42
LITERATURE CITED .................................................. 43
GENETIC STRUCTURE ANALYSIS OF Aegilops cylindrica.................. 49
ABSTRACT ............................................................... 50
INTRODUCTION ....................................................... 51
MATERIALS AND METHODS ....................................... 54
RESULTS ................................................................. 60
DISCUSSION ............................................................ 89
ACKNOWLEDGMENTS ............................................. 104
LITERATURE CITED ................................................ 105
PATTERNS OF MATING BETWEEN Triticum aestivum AND Aegiiops
cylindrica UNDER FIELD CONDITIONS .............................................. 111
ABSTRACT ............................................................. 112
INTRODUCTION ...................................................... 113
TABLE OF CONTENTS (Continued)
Page
MATERIALS AND METHODS
.115
RESULTS ............................................................... 120
DISCUSSION .......................................................... 128
ACKNOWLEDGMENTS ............................................. 132
REFERENCES ........................................................ 133
CONCLUSION ........................................................................... 136
BIBLIOGRAPHY ........................................................................ 143
APPENDICES ........................................................................... 153
LIST OF FIGURES
Figure
Page
2.1 Neighbor-joining tree showing chioroplast genetic relatedness
between Ae. cylindrica and its relatives ........................................ 26
2.2 Neighbor-joining tree showing the nuclear genetic relatedness
between Ae. cylindrica and its relatives ........................................ 28
2.3 Apical portions of spikes from Ae. markgrafii, Ae. tauscliii,
andAe. cylindrica .................................................................. 32
2.4 Mitotic metaphase chromosome spreads and genomic in situ
hybridization (GISH) of the Ae. cylindrica accession TK 116
(P1 486249) ................................................................................ 34
3.1 Map showing the distribution of Ae. cylindrica collections from
various regions ........................................................................ 55
3.2 Neighbor-joining tree showing chloroplast genetic relatedness
between Ae. cylindrica and its relatives ....................................... 76
3.3 Model-based clustering plots for chloroplast microsatellite data ......... 78
3.4 Neighbor-joining tree showing nuclear genetic relatedness
between Ae. cylindrica and its relatives ........................................ 80
3.5 Plot of model-based clustering of 173 Ae. cylindrica accessions
in six subpopulations .................................................................85
4.1 Distribution and male parentage of BC1 plants across sites ............ 127
LIST OF TABLES
Table
Page
2.1 List of accessions along with their region of origin and the
geographical coordinates of their collection sites ........................... 12
2.2 Heterozygosity indices, number of alleles, and allele size range
for Ae. markgrafli, Ae. tauschii, and Ae. cylindrica using
chloroplast microsatellite markers ................................................ 21
2.3Heterozygosity indices, alleles, and allele size range for
Ae. markgrafll, Ae. tauschll, and Ae. cylindrica using nuclear
microsatellite markers ............................................................... 23
3.IA Allele frequency and diversity indices of C-type Ae. cylindrica for
selected ch loroplast microsatellite markers ................................... 61
3.1B Allele frequency and diversity indices of D-type Ae. cylindrica for
chloroplast microsatellite markers ................................................. 63
3.2 Allele frequency and diversity indices for nuclear microsatellite
makers .................................................................................. 67
3.3 Model-based clustering of 173 Ae. cylindrica accessions in six
subpopulations using nuclear microsatellite markers ....................... 84
3.4 The pair-wise Rst (above diagonal) and Fst (below diagonal)
estimates for genotypes from various regions ................................ 88
4.1 Accessions of jointed goatgrass and wheat varieties used for
marker characterization ........................................................... 118
4.2 Microsatellite marker allele sizes for jointed goatgrass accessions
and wheat varieties from F1 hybrid and BC1 plant collection sites...... 121
LIST OF TABLES (Continued)
Table
Page
4.3 Parentage of F1 and BC1 plants based on chloroplast and nuclear
microsatellite analyses ............................................................ 125
4.4 Parentage and collection information of wheat-jointed goatgrass
hybrids and BC1 plants by collection site ..................................... 126
Jointed Goatgrass (Aegilops cylindrica Host) Genetic Diversity and
Hybridization with Wheat (Triticum aestivum L.)
CHAPTER 1
INTRODUCTION
Jointed goatgrass (Aegilops cylindrica Host, 2n=4x= 28, CCDD) is an
allotetraploid species of the tribe Triticeae (Poaceae family). This species is an
important weed of wheat (Triticum aestivum L., 2n=4x=42, AABBDD) in
various parts of the world, including the USA (van Slageran 1994). In the USA,
Ae. cylindrica infests millions of hectares of cultivable and non-cultivable land
and is spreading at the rate of 20,000 hectares per year. Annual yield and
quality losses of wheat due to infestation of Ae. cylindrica in the USA are
estimated to be $145 million (White, 2003). Besides being an important weed,
Ae. cylindrica a close relative of wheat, is also a source of biotic and abiotic
stress resistance genes for wheat improvement. This species may be a useful
source of salt tolerance (Farooq et al. 1992), Hessian fly resistance (El
Bouhssini et al. 1998), and freezing tolerance (Iriki et al. 2001). Moreover,
some breeding programs have obtained disease resistant wheat lines through
introgression of Ae. cylindrica chromatin (Galaev et al. 2003).
Jointed goatgrass is a product of amphidiploidization of hybrids
between Ae. tauschii (2n=2x=14; DD) and Ae. markgrafii (2n=2x=14; CC)
(Kihara and Matsumura 1941; Kimber and Zhao 1983; Dubcovsky and Dvorak
1994). Studies on phenotypic (Maan 1976; Tsunewaki 1996) and organellar
2
DNA variation among alloplasmic lines of wheat (Ogihara and Tsunewaki
1988; Wang et al. 1997; Wang et al. 2000a) suggested cytoplasmic homology
between Ae. cylindrica and Ae. tauschii (D-type cytoplasm). Because the
cytoplasm is inherited uniparentally through the female in the tribe Triticeae,
these studies indicated that Ae. tauschii was the maternal parent in the
formation of Ae. cylindrica. However, cytoplasmic homology between Ae.
tauschll and Ae. cylindrica was established using only one or two accessions
of each species. Therefore, additional studies with a larger sample are
necessary to evaluate cytoplasmic variation in Ae. cylindrica.
The native distribution of jointed goatgrass is believed to encompass
parts of the Mediterranean, the Near East, the Caucasian region, areas
around the Black Sea and Central Asia. This species is adventive in the USA,
parts of Europe and China (van Slageren 1994). The genetic variation in
jointed goatgrass has been studied using allozyme (Watanabe et al. 1994), Cbanding (Badaeva et al. 2002), RAPD (Okuno et al. 1998; Goryunova et al.
2004), a combination of RAPD and AFLP (Pester et al. 2003), and DNA
sequence polymorphisms (CaIdwell et al. 2004). By comparing the C-banding
patterns of chromosomes of Ae. cylindrica and Ae. tauschii Badaeva et al.
(2002) speculated that the origin of Ae. cylindrica is more recent compared to
other D-genome polyploid species of the Triticeae tribe. Furthermore, owing to
the presence of two haplotypes of the Gss (granule-bound starch synthase)
locus from Ae. tauschii in Ae. cylindrica, Caidwell et al. (2004) suggested that
Ac. cylindrica was formed recurrently. Overall this and other studies noted a
3
low level of genetic diversity in Ae. cylindrica compared to its progenitors and
related genera. However, these studies had better representation of samples
either from the native (e.g. CaIdwell et al. 2004) or the non-native distribution
range (e.g. Pester et al. 2003). An evaluation of a larger set of Ae. cylindrica
accessions encompassing both its native and non-native areas of distribution
is necessary to provide a better picture of nuclear genetic variation in Ae.
cylindrica and to address questions related to Ae. cylindrica's origin, formation,
and population genetic structure.
Jointed goatgrass is a close relative of wheat and both have the Dgenome derived from Ae. tauschll (Kimber and Zhao 1983). Moreover, natural
and partially female fertile hybrids between wheat and jointed goatgrass have
been reported in Europe, Eurasia and the USA (van Slageren 1994; MallorySmith et al. 1996; Seefeldt et al., 1998; Zemetra et al. 1998; Guadagnuolo et
al., 2001). These hybrids can backcross with either parent under field
conditions, which suggests that gene flow between wheat and jointed
goatgrass is feasible (Zemetra et al. 1998; Wang et al. 2000b, 2001; Snyder et
al. 2000). Thus, research on hybridization dynamics between wheat and
jointed goatgrass can be used to ascertain the potential of gene flow between
these species. Information on hybridization patterns will also aid in designing
better management strategies in herbicide resistant wheat fields to avoid the
transfer of herbicide resistance from wheat to jointed goatgrass.
In order to characterize the hybridization dynamics between wheat and
jointed goatgrass, the hybridization rate between these species, the parentage
4
of the hybrids and the male parentage of backcross generations (wheat or
jointed goatgrass) needs to be measured and characterized. Earlier research
suggested that jointed goatgrass and wheat hybridize at an average rate of
1.8% under field conditions (Morrison et al. 2002a). Depending on various
conditions, the hybridization rate can range from 0 to 8% (Guadagnuolo et al.
2001; Morrison et al. 2002a). The evaluation of naturally produced hybrids and
the first-generation backcross (BC1) individuals with respect to parentage
suggested that jointed goatgrass was the predominant female parent (70%) in
the formation of
F1
hybrids and wheat was the predominant backcross parent
(91%). These studies relied on root ball analysis to deduce the female
parentage of the hybrid plants. The seed remnant on a root ball suggested
wheat was the female parent, while the presence of spikelet indicated jointed
goatgrass was the female parent of a hybrid. Therefore, the determination of
parentage of hybrids using root ball analysis was possible only in the cases
where the seed or spikelet could be found attached to the roots. To determine
male parentage, high molecular weight (HMW) glutenin markers were used.
The HMW glutenin markers provided limited nuclear genome coverage, which
led to determination of parentage for only 51% of the BC1 plants. Thus, these
methods allowed parentage analyses on a subset of plant material collected
(Morrison et al. 2002a). The use of DNA-based markers can overcome these
limitations and their efficiency allows screening of a larger collection. The
screening of a larger sample set is necessary to obtain a clearer picture of
hybridization dynamics between wheat and jointed goatgrass under field
conditions.
Ae. cylindrica is an agriculturally important species because of its
occurrence as a weed, its role in potential crop-to-weed gene flow, and its
utility as a source of genetic variation for wheat improvement. Therefore, it is
critical to understand various aspects of Ae. cylindrica biology for both its
better management and beneficial use. In this regard, the aims of this
research were: to increase our knowledge base with respect to cytoplasmic
and nuclear diversity in jointed goatgrass; and to use this information to better
understand hybridization dynamics between wheat and jointed goatgrass
under field conditions. The specific objectives of this dissertation research
were to:
1.
Assess nuclear and cytoplasmic DNA variation in Aegilops cylindrica
2.
Assess the population genetic structure of jointed goatgrass across its
geographic distribution
3.
Determine patterns of mating between wheat and jointed goatgrass
under field conditions using both nuclear and cytoplasmic DNA-based
markers.
These objectives are addressed in Chapters 2, 3, and 4, respectively, while a
summary of conclusions from these studies is presented in Chapter 5 of this
dissertation.
CHAPTER 2
CHLOROPLAST AND NUCLEAR MICROSATELLITE ANALYSIS OF
Aegilops cylindrica
Harish T. Gandhi, M. Isabel Vales, Christy J. W. Watson, Carol A. MallorySmith, Naoki Mori, Maqsood Rehman, Robert S. Zemetra and Oscar RieraLizarazu
Harish T. Gandhi, M. Isabel Vales, Christy J. W. Watson, Carol A. Mallory-
Smith and Oscar RieraLizarazut
Department of Crop and Soil Science, 107 Crop Science Building, Oregon
State University, Corvallis, Oregon 97331-3002, USA.
Phone 541-737-5879
Fax 541-737-1334
E-mail: oscar.rieraoregonstate.edu
tCorresponding author
Naoki Mon
Laboratory of Plant Genetics, Faculty of Agriculture, Kobe University, 1
Rokkodai-cho, Nadu-ku, Kobe 657-8501, Japan.
Maqsood Rehman, and Robert S. Zemetra
Department of Plant, Soil and Entomological Sciences, University of Idaho,
Moscow, Idaho 83844-2339, USA.
Theoretical and Applied Genetics (in press)
Publisher: Springer-Verlag GmbH
7
ABSTRACT
Aegilops cylindrica Host (2n=4x=28; genome CCDD) is an allotetraploid
formed by hybridization between the diploid species Ae. tauschii Coss.
(2n=2x=14; genome DD) and Ae. markgrafii (Greuter) Hammer (2n2x14;
genome CC). Previous research has shown that Ae. tauschii contributed its
cytoplasm to Ae. cylindrica. However, our analysis with chforoplast
microsatellite markers showed that one of the 36 Ae. cylindrica accessions
studied, TK 116 (P1 486249), had a plastome derived from Ae. markgrafii
rather than Ae. tauschii. Thus, Ae. markgrafii has also contributed its
cytoplasm to Ae. cyllndrica. Our analysis of chioroplast and nuclear
microsatellite markers also suggests that D-type plastome and the D genome
in Ae. cylindrica were closely related to and were probably derived from the
tauschii gene pool of Ae. tauschii. A determination of the likely source of the C
genome and the C-type plastome in Ae. cylindrica was not possible.
INTRODUCTION
Jointed goatgrass (Aegilops cylindrica Host; 2n=4x=28, genome CCDD) is an
allotetraploid of the Triticeae tribe (Poaceae family). Ae. cylindrica is a close
relative of wheat (Triticum aestivum L.; 2n=6x=42, genome AABBDD) and the
two share the D genome (Riley and Law 1965; Kimber and Zhao 1983). This
species is of worldwide economic importance for various reasons. First, jointed
goatgrass is a widespread weed of bread wheat, chronically infesting fields in
the Midwestern and Western United States as well as fields in the Middle East
and parts of Europe (Dewey 1996; Ogg and Seefeldt 1999; Guadagnuolo et al.
2001). Hybridization between jointed goatgrass and wheat and partial female
fertility of the resulting naturally-produced hybrids suggest the possibility of
crop-to-weed gene movement (Zemetra et al. 1998; Morrison et al. 2002).
Jointed goatgrass a'so has been identified as a source of useful genetic
variation for wheat improvement (Farooq et al. 1992; El Bouhssini et al. 1998;
Iriki et al. 2001). Therefore, there is considerable interest in understanding
various aspects of the evolution of Ac. cylindrica for its better management
and use.
Jointed goatgrass formed through amphidiploidization of a hybrid
between Ac. tauschii Coss. (2n=2x=14, genome DD) and Ae. markgrafii
(Greuter) Hammer (syn. Ae. caudata L.; 2n=2x=14; genome CC). This
determination is based on data from a variety of sources including
chromosome pairing studies in interspecific hybrids (Kihara and Matsumura
1941, Kimber and Zhao 1983), karyotype analysis (Chennaveeraiah 1960),
and analyses of protein and nuclear DNA variation (Jaaska 1981, Nakai 1981,
Masci et al. 1992, Dubcovsky and Dvorak 1994). Furthermore, studies on
phenotypic (Maan 1976; Tsunewaki, 1996) and organellar DNA variation
among alloplasmic lines of wheat (Ogihara and Tsunewaki 1988; Wang et al.
1997; Wang et al. 2000a) established cytoplasmic homology between Ae.
cylindrica and Ae. tauschii (D-type cytoplasm). These analyses suggested that
Ac. tauschii was the maternal parent in the formation of Ae. cylindrica.
However, studies on cytoplasmic variation in Ac. cylindrica have not been
undertaken.
The nuclear genetic diversity of jointed goatgrass has been studied
using allozyme (Watanabe et al 1994), C-banding (Badaeva et al. 2002),
RAPD (Okuno et al. 1998; Goryunova et al. 2004), a combination RAPD and
AFLP (Pester et al. 2003), and DNA sequence polymorphisms (Caldwell et al.
2004). These studies suggested that Ac. cylindrica had very low levels of
genetic diversity and that this allotetraploid originated recurrently. Although
some studies indicated that the D genomes of wheat and Ac. cylindrica were
apparently contributed by genetically distinct biotypes of Ac. tauschii (Badaeva
et al. 2002; Caldwell et al. 2004), the relationship between Ac. cylindrica with
if,]
subspecies of Ae.
between
tauschii is
Ae. cylindrica
not well defined. Similarly, the relationship
and genetically differentiated populations of Ae.
markgrafii(Ohta 2000; 2001) is unknown.
In this study, nuclear and chioroplast microsatellite markers were
employed to investigate the relationships between Ae. cylindrica and its
progenitors,
Ae. tauschll,
and Ae. markgrafii. This analysis and the new
insights that it provides with respect to the evolution of Ae.
discussed.
cylindrica is
11
MATERIALS AND METHODS
Plant material
Chloroplast and nuclear microsatellite analyses were performed on 36
accessions of Ae. cylindrica, 17 accessions of Ae. tauschll, seven accessions
of Ae. markgrafii, two accessions of T. aestivum, and two accessions of T.
turgidum. The list of accessions along with their region of origin, the
geographical coordinates of their collection sites, and seed sources are
presented in Table 2.1.
DNA isolation and molecular marker assays
DNA was extracted from 35 mg of leaf tissue following the protocol described
by Riera-Lizarazu et al. (2000). Twenty wheat chloroplast (WCt) microsatellite
markers (lshii et al. 2001; Table 2.2) were used to characterize the chloroplast
genome and nineteen Gaterslaben wheat microsatellite (gwm) markers (Roder
et al. 1998; Table 2.3) were used to evaluate the nuclear genome. For
microsatellite marker assays, one primer was labeled with a fluorescent dye
16-Carboxyfluorescein (FAM), or 4,7,2',4',5',7'-Hexachloro-6-carboxyfluroscein
(HEX), or 4,7,2', 7'-tetrachloro-6-carboxyflu roscein (TET)1. Polymerase chain
reactions (PCR) were carried out in 10 p1 reactions comprising 0.03 units Taq
Table 2.1 List of accessions along with their region of origin and the geographical coordinates of their collection sites
Geographical
coordinatesc
Region of
Speciesa
Accessions
Germplasm 1Db
Latitude
Longitude
Oriq in
Ae. markgrafiivar.
KU0006(A)
KU0006-2(A)
Syria
37.13
36.12
GR GB89
G591
Turkey
37.06
37.33
KU5472
KU5472
Iraq
35.54
44.84
Ae. markgrafii var. polyathera
KU5852(B)
KU5852(B)
Turkey
40.65
35.83
Ae. markgrafii var. markgrafii
KU5864 (C)
KU5864 (C)
Turkey
40.266
28.357
Ac. markgrafiivar.
markgrafii
KU5871(D)
KU5871(D)
Greece
NA
NA
Ac.
markgrafii
TK GB9O
84TK159-036
Turkey
38.033
28.917
Ac. tauschii ssp. tauschii
AE1039/95
AE1039195
Tadjikistan
NA
NA
Ae. tauschii ssp. strangulata
AE145196
AE145/96
Azerbaijan
NA
NA
Ac. tauschii ssp. strangulata
AE1 84/78
AE1 84/78
Iran
NA
NA
Ac.
markgrafii
Ac.
markgrafiivar.
markgrafii
var. polyathera
markgrafiivar.
markgrafli
-
Ae. tauschii ssp. strangulata
AE246/76
AE246/76
Uzbekistan
NA
NA
Ae. tauschii ssp. tauschii
AE257/76
AE257176
Kyrgyzstan
NA
NA
Ae. tauschii ssp. tauschiI
AE276/00
AE276/00
Afghanistan
NA
NA
Ae. tauschii ssp. strangulata
AE457/94
AE457194
Georgia
41.69
44. 80
Ae. tauschii ssp. strangulata
AE498/79
AE498179
Dagestan
NA
NA
Ae. tauschii ssp. tauschii
AE499/81
AE499181
Turkmenistan
NA
NA
Ae. tauschii ssp. tauschii
G5792
G5792
China
NA
NA
Ae. tauschii
IRGB93
G1279
Iran
NA
NA
Ae. tauschii ssp. tauschii
TA10143
TA10143
Syria
35.31
38.45
Ae. tauschii ssp. tauschii
TA10144
TA10144
Syria
35.37
38.45
Ae. tauschii ssp. tauschii
TA10145
TA10145
Syria
35.37
38.45
Ae. tauschii ssp. tauschii
TA10146
TA10146
Syria
36.53
38.14
Ae. tauschii ssp. tauschii
TA1588
TA1588
Turkey
38.5
43.3
Ae. tauschii ssp. tauschii
TA2460
TA2460
Iran
NA
NA
Ae. cylindrica
AF 26
P129889 1
Afghanistan
35.72
64.90
()
Ac. cylindrica
AR 147
1G48754
Armenia
39.83
44.83
Ac. cylindrica
AZ 133
1G48031
Azerbaijan
39.28
47.05
Ac. cylindrica
BG 137
1G48325
Bulgaria
42.02
23.65
Ac. cylindrica
DG 135
1G48260
Dagestan
41.93
48.37
Ac. cylindrica
GE 29
P1314406
Georgia
41.72
44.78
Ae. cylindrica
GR 159
PC
Greece
NA
NA
Ac. cylindrica
IQ 34
P1254864
Iraq
37.12
42.68
Ac. cylindrica
IR 149
1G48914
Iran
37.47
57.33
Ac. cylindrica
JO 146
1G48584
Jordan
31.78
36.80
Ac. cylindrica
LB 148
1G48789
Lebanon
34.47
36.33
Ac. cylindrica
SY119
1G44621
Syria
33.92
36.70
Ac. cylindrica
TJ 142
1G48558
Tadjikistan
39.45
68.33
Ac. cylindrica
TK 1
P1172357
Turkey
40.27
40.25
Ae. cylindrica
TK 107
P1407639
Turkey
39.48
32.34
Ac. cylindrica
TK115
P1554230
Turkey
37.13
44.52
-
Ac.
cylindrica
TK 116
P1486249
Turkey
40.18
42.63
Ae.
cylindrica
TK 120
1G47699
Turkey
40.23
28.20
Ae.
cylindrica
TK 127
1G47906
Turkey
38.83
32.08
Ae.
cylindrica
TK 129
1G47927
Turkey
38.97
35.60
Ac.
cylindrica
TK 131
1G47959
Turkey
38.42
39.33
Ae.
cylindrica
TK 14
P1542179
Turkey
39.35
26.75
Ac.
cylindrica
TK 15
P1554201
Turkey
38.37
37.77
Ac.
cylindrica
TK 16
P1486236
Turkey
37.30
44.57
Ae. cylindrica
TK 17
P1554206
Turkey
37.23
44.65
Ae.
cylindrica
TK 19
P1554225
Turkey
38.40
42.60
Ac.
cylindrica
TK2
P1172358
Turkey
40.05
42.18
Ae.
cylindrica
TK 39
G404
Turkey
36.85
40.05
Ac.
cylindrica
TK 5
P1554203
Turkey
38.30
43.17
Ae.
cylindrica
TM 139
1G48529
Turkmenistan
38.25
56.33
Ac.
cylindrica
US/co 61
PW27
USA
NA
NA
Ae. cylindrica
US/NE 45
PW6
USA
NA
NA
Ae.cylindrica
US/OR 13
FC13
USA
NA
NA
Ae. cylindrica
US/UT 21
FC21
USA
NA
NA
Aecylindrica
UZ35
P1314185
Uzbekistan
41.37
69.55
Ae. cylindrica
YU 37
P1344778
Yugoslavia (Serbia)
44.02
20.92
T. turgidum ssp. Durum
394
P194705
Palestine
32.00
35.00
T. turgidum ssp. Durum
Langdon
Cltr 13165
USA
NA
NA
T. aestivum ssp. aestivum
Alcedo
TA 2933
Germany
NA
NA
T. aestivum
Chinese Sorina
Cltr 14108
China
NA
NA
SSD.
aestivum
a
The variety (Ae. markgrafii) and subspecies (Ae. tauschii) designations are based on passport data, Pestova et al.
(2000), Ohta (2000, 2001), and our own observations.
blhe first letter(s) of the germplasm ID makes reference to the sources of the germplasm. Accessions starting with "G"
were obtained from Dr. J. Giles Waines, University of California, Riverside, CA, U.S.A.; "KU" accessions were obtained
from Dr. Shoji Ohta, Fukui Prefectural University, Japan; "AE" accessions were obtained from Institute of Plant Genetics
and Crop Plant Research (IPK), Germany; "TA" accessions were obtained from Wheat Genetic Resource Center, Kansas
State University, KS, U.S.A.; "IG" accessions were obtained from the International Center for Agricultural Research in the
Dry Areas (ICARDA), Aleppo, Syria; "Cltr" and "P1" accessions were obtained from U.S. Department of Agriculture,
National Small Grains Collection, Aberdeen, ID, U.S.A.; FC, PW and PC (personal collections) accessions are maintained
at Oregon State University, USA.
C
Longitude and latitude co-ordinates are in the decimal system. NA indicates that the co-ordinates were not available.
0)
17
polymerase with IX PCR buffer containing 1.5 mM MgCl2 (Qiagen, Valencia,
CA, USA), 2% sucrose in 0.04% cresol red, 0.2 mM of each dNTP, and 0.2pM
of each primer. The PCR consisted of denaturation at 95 ° for 5 mm, followed
by 40 cycles of 95 ° for 1 mm, 50-60 ° (depending on primers) for I mm, and
72 ° for I mm, with final extension at 72 ° for 10 mm. Fragment analysis was
carried out using an ABI Prism® 377 DNA Sequencer and ABI Prism® 3100
Genetic Analyzer. ABI GeneScan® 2.1 and Genotyper® 2.0 software (Applied
Biosystems, Foster City, CA, USA) were used to size fragments based on an
internal lane standard [n,n,n',n'-tetramethyl-6-carboxyrhodamine (TAMRA) or
6-carboxy-x-rhodamine (ROX)].
Spike morphology assessments
Spike and apical spikelet morphology can be used to distinguish Ae. cylindrica
from its progenitors (Kimber and Feldman 1987; van Slageren 1994). Thus,
spike morphology and the presence or absence of awns on apical glumes and
lemmas were evaluated in some Ae. cylindrica, Ac. tauschll, and Ae.
markgrafii accessions to verify their identities.
iE]
Cytological analyses
Root-tip collection, pre-treatment, and chromosome spread preparations for
chromosome counting and karyotypic observations were carried out as
described in Riera-Lizarazu et al. (1996). Slides were analyzed with a Zeiss
Axiokop 2 (Carl Zeiss AG, Germany) microscope. Images were photographed
with black and white Agfapan APX 100 film (Agfa-Gevaert N.V., Mortsel,
Belgium). Sample collection, treatments, and slide preparations for genomic
in situ hybridization (GISH) performed on root-tip mitotic chromosome spreads
of TK 116 were performed as described by Wang et al. (2002). Ae. markgrafii
genomic DNA was used as the C-genome probe (biotinylated) and unlabeled
Ae. tauschii genomic DNA was used as the D-genome hybridization
competitor. Biotinylated DNA was detected with fluorescein conjugated Avidin
followed by signal amplification with biotinylated anti-avidin-D coupled with
another layer of fluorescein-labeled Avidin. Unlabeled chromatin was
counterstained with propidium iodide. Slides were analyzed with a microscope
(Nikon Eclipse E1000) equipped with an epifluorescence attachment (with
FITC, TRITC and a dual band FITC/Pl filters, Chroma Technology Corp,
Brattleboro, VT). Images were taken with a built-in digital camera and were
later processed using Adobe ® Photoshop
San Jose, CA).
7.0 (Adobe Systems Incorporated,
19
Statistical analyses
The number and frequency of alleles for each microsatellite marker were
determined and used for the calculation of expected heterozyosity (Botstein et
al. 1980). For both chloroplast and nuclear microsatellite markers, MICROSAT
2.0 (Minch et al. 1997) was used to generate a genetic distance (dissimilarity)
matrix based on the proportion of shared alleles (Bowcock et al. 1994). The
genetic distance matrix was then subjected to MEGA 2.0 for tree formation
(Kumar et al. 2001) using the neighbor-joining method (Saitou and Nei 1987).
Tree View 1.6.6 (Page 2001) and MEGA 2.0 were used to produce graphical
outputs.
20
RESULTS
For Ae. cylindrica, Ae. tauschll, and Ae. markgrafii, the average expected
heterozygosity and number of alleles per marker were greater for nuclear than
for chioroplast microsatellite markers (Tables 2.2 and 2.3). Since there were
only two genotypes each from T. aestivum and T. turgidum, their
heterozygosity values were not calculated. The average expected
heterozygosity for Ae. cylindrica, for both chloroplast and nuclear
microsatellites, was lower than its progenitors, Ae. markgrafii and Ae. tauschll.
For both chloroplast and nuclear microsatellite markers, Ae. tauschll showed
the highest level of variation expressed as average expected heterozygosity
and allele number per marker. Chloroplast markers with the highest average
expected heterozygosity values were WCt 3 in Ae. markgrafii (0.69), WCt 11 in
Ae. tauschii (0.78), and WCt 5 in Ae. cylindrica (0.56) (Table 2.2). The nuclear
marker gwm458 showed the highest heterozygosity in Ae. markgrafii (0.84),
while nuclear marker gwm3l4 showed the highest heterozygosity in Ae.
tauschii and Ae. cylindrica (0.94 and 0.83, respectively) (Table 2.3).
Based on genetic similarity analysis with 20 chloroplast microsatellite
markers, genetic distance between any two accessions ranged from 0 (most
similar) to 0.9 (most dissimilar). This analysis also allowed the distinction of
species with respect to various plastome types. Seven markers (WCt 1, WCt 2
Table 2.2 Heterozygosity indices, number of alleles, and allele size range for Ae. markgrafii, Ae. tauschii, and Ae.
cylindrica using chioroplast microsatellite markers
Marker
WCt1
Aegilops markgrafii
Allele size
No. of
range
H
alleles (base pairs)
113-114
0.24
2
Aegilops tauschii
Allele size
range
No. of
H
alleles (base pairs)
111-112
0.21
2
Aegilops cyIindrica'
Allele size
range
No. of
alleles (base pairs)
TK 116
Ha
Allele size
(base pairs)
2
110-111
0.11
112
WCt2
2
124-125
0.49
5
128-131
0.65
3
128-130
0.36
124
WCt3
4
151-159
0.69
4
147-154
0.53
2
146-147
0.45
156
WCt4
3
193-198
0.61
2
193-197
0.57
1
196
0.00
197
WCt 5
2
81-82
0.49
4
81-84
0.63
3
82-84
0.56
83
WCt6
1
187
0.00
4
184-188
0.66
2
186-187
0.16
187
WCt8
1
148
0.00
2
148-149
0.11
3
147-149
0.21
147
WCt9
1
120
0.00
1
120
0.00
1
120
0.00
120
WCtIO
2
194-195
0.49
3
192-194
0.46
2
192-193
0.16
195
WCt1I
3
167-169
0.61
5
166-170
0.78
2
166-167
0.24
166
1.)
WCt12
2
146-147
0.49
4
148-151
0.67
2
149-150
0.06
146
WCt13
1
104
0.00
3
105-107
0.55
2
104-106
0.16
104
WCt15
2
103-104
0.41
3
98-110
0.49
2
98-99
028
104
WCt 16
2
97-98
0.24
4
97-101
0.31
1
98
0.00
97
WCt17
1
147
0.00
2
145-146
0.50
3
145-147
0.16
145
WCt18
2
198-199
0.24
3
197-199
0.21
2
198-199
0.06
198
WCt19
2
152-153
0.49
3
151-154
0.55
2
151-152
0.11
152
WCt22
1
188
0.00
4
196-198
0.70
2
196-197
0.24
188
WCt23
1
106
0.00
1
106
0.00
1
106
0.00
106
WCt24
1
178
0.00
4
179-186
0.46
1
184
0.00
178
Average
1.8
0.28
3.15
0.45
1.95
a
b
0.17
The expected heterozygosity was calculated as described by Botstein et al. (1980).
Calculations did not include data from TK 116.
r\)
Table 2.3 Heterozygosity indices, alleles, and allele size range for Ae. markgrafii, Ae. tauschii, and Ae. cylindrica using
nuclear microsatellite markers
Marker
gwm232
Aegilops markgrafii
Allele size
range
No. of
H
alleles (base pairs)
null, 139-310 0.61
4
Aegilops tauschii
Allele size
range
No. of
H
(base pairs)
alleles
127-310
0.85
10
Aegilops cylindrica
Allele size
range
No. of
H
(base pairs)
alleles
137-310
0.19
5
gwm337
2
null, 166
0.41
12
152-213
0.89
6
164-193
0.41
gwm458
7
94-129
0.84
11
96-133
0.88
6
101-132
0.66
gwm642
8
169-191
0.83
18
108-200
0.88
5
170-187
0.70
gwm3Ol
3
159-225
0.58
12
161-222
0.87
8
159-197
0.76
gwm455
4
120-133
0.61
9
128-188
0.76
3
127-187
0.10
gwm484
5
null, 112-154
0.72
12
114-164
0.90
5
111-115
0.56
gwm6O8
7
110-134
0.84
3
101-110
0.54
1
110
0.00
gwm3
5
64-95
0.68
9
59-76
0.79
1
64
0.00
gwm3l4
2
Null, 99
0.24
17
null, 99-268
0.94
10
171-183
0.83
rJ
gwm383
7
null, 132-229
0.82
12
180-228
0.89
5
203-233
0.63
gwml86
5
null, 95-147
0.78
4
null, 96-169
0.56
3
null, 98-99
0.35
gwml9O
6
229-246
0.82
10
null, 184-231
0.87
6
192-235
0.40
gwm2O5
3
133-136
0.57
5
127-310
0.70
4
129-147
0.28
gwm272
3
124-126
0.61
10
118-150
0.80
2
125-126
0.39
gwm325
3
null, 114-127
0.65
7
114-142
0.81
2
113-114
0.05
gwm469
4
84-88
0.66
10
140-176
0.83
5
156-162
0.50
gwm437
3
null, 159-165
0.53
11
83-129
0.86
5
null, 87-99
0.59
gwm44
4
null, 156-278
0.66
3
116-178
0.21
5
276-283
0.24
Average
4.47
0.66
9.74
0.78
4.58
The expected heterozygosity was calculated as described by Botstein et al. (1980)
0.40
25
WCt 12, WCt 13, WCt 17, WCt 22, and WCt 24) permitted the differentiation of
64 accessions from five species into plasmon types B, C and D (Figure 2.1).
An unanticipated finding was that one accession of Ae. cylindrica, TK 116 (P1
486249), exhibited some microsatellite alleles that were neither present in Ae.
cylindrica nor in Ae. tauschii accessions but matched the allelic constitution of
some Ae. markgrafll accessions (Table 2.2).
Thirty-five Ae. cylindrica and 17 Ae. tauschii accessions formed a single
major cluster (D-type plastome). Of the 17 Ae. tauschii accessions studied, 14
formed a dispersed group while three accessions (TA 1588, TA 10143, and TA
10145) intermingled with Ae. cylindrica. The Ae. cylindrica accession TK 2 (P1
172358), which had been previously used to determine that Ae. cylindrica had
plasmon type D (Maan 1976) fell in this major cluster with other Ae. cylindrica
accessions. The wheat lines Chinese Spring, Alcedo, Langdon, and 394, and
seven Ae. markgra f/i accessions were part of a cluster with two distinct groups
(B- and C-type plastomes). One Ae. cylindrica accession, TK 116, grouped
with Ae. markgrafii. This was consistent with our observation that the allelic
constitution of this accession was more similar to Ae. markgrafii than Ae.
tauschii.
Nineteen nuclear microsatellites were also used to study the genetic
relatedness of Ac. tauschii, Ae. markgrafll, and Ae. cylindrica accessions. The
genetic distances ranged from 0.05 (most similar) to 0.98 (most dissimilar).
26
Figure 2.1 Neighbor-joining tree showing chloroplast genetic relatedness
between Ae. cylindrica and its relatives. TK 116 and TK 2 (an accession
used in alloplasmic interaction studies) are underlined. Ae. tauschii
accessions interfused with Ae. cylindrica are italicized. The prefixes used
before the name of each accession stand for the following: AE= T.
aestivum, CL= Ae. cylindrica, DU= T. turgidum, MK= Ae. markgra f/i, and
TU= Ae. tauschll. Clusters of accessions designated as B, C, and D
correspond to individuals with plasmon types B, C, and D, respectively.
27
CL-TK5
15
CL-TK1
CL-TK1 6
-AZ133
CL-US/NE45
CL-TK1 07
TU-TA 10145
CL-TK19
CL-TK1 5
CL-TK2
TU-TA 10143
CL-TM 139
CL-TK1 20
CL-TK1 4
CL-GR1 59
CL-BG1 37
CL-TK129
CL-tQ34
CL-AF26
CL-US/C061
CL-TK131
CL-TK1 7
CL-GE29
TU-TA 1588
CL-1R149
CL-US/UT21
CL-US/OR1 3
CL-JO 146
CL-SY1 19
CL-DG135
CL-TK39
CL-TJ142
CL-UZ35
CL-TK127
-f
CL-AR 147
TU-AE257
TU-TA10144
TU-AE1 039
I
I
I)
I
TU-AE276
I
-TU-AE145
I
TU-AE246
I
TU-TA2460
TU-AE495
TU-1R0B93
TU-AE184
I
I
Cl)
I
C)
I
-TU-AE499
I
TU-G5792
I
TU-AE457
I
TU-TA10146
CL-TKII6
MK-KU0006
MK-KU5472
MK-TKGB9O
MK-KU5852
MK-KU5864
MK-GRGB89
MK-KU5871
AE-394
AE-Alcedo
AE-Langdon
AE-Chinese Spring
0.1
C
Figure 2.2 Neighbor-joining tree showing the nuclear genetic relatedness
between Ae. cylindrica and its relatives. TK 116 is underlined and in bold.
The two major clusters are labeled as I and II. Based on membership,
major clusters were subdivided into groups labeled CM (Ae. cylindrica and
Ae. markgrafiil), TU1 (Ae. tauschii), TU2 (Ae. tauschii and T. aestivum) and
DU (T. turgidum). The CM sub-cluster was further split into the CL (Ae.
cylindrica) and MK (Ae. markgrafii) groups. Ae. tauschii accessions in the
TU1 grouping belong to the tauschii gene pooi while Ae. tauschii in the
TU2 group belong to the strangulata gene pool. The meaning of prefixes
used before the name of each accession is same as figure 2.1.
29
CL-1Q34
CL-PF26
CL-US/OR1 3
CL-TJ 142
CL-IJS/UT2I
CL-SY119
f8
TId'4 I
- CL-TK19
CL-BG1 37
CL-TK1 4
CL-GR1 59
CL-Th1
CL-TK1 07
CL-Th1 7
CL-JO1 46
--LiiUS/NE45
CL-US/C061
CL-TK120
CL
CL-TK129
CL-TK15
CM
CL-LB1 48
CL-TK2
CL-AZ1 33
CL-UZ35
I
CL-TK5
1
CL-TK1 6
CL-TK1 31
CL-TK115
CL-IR1 49
L______ CL-TM139
CL-TK1 27
CL-TK39
CL-GE29
CL-YU37
CL-PR147
CL-DG1 35
FK-KU5472
K-ThGB90
?vl(-KU5864
M<-KU0006
MK
M(-KU5871
rvK-GRGB89
- Iv1c-KU5852
T!J-TA1 588
TU-TA10144
TIJ-TA10145
TU-TA1 0143
TU 1
Th-PE499
Th-AE257
11JAE1 039
TU-PiE276
TU-G5792
TU-TA10146
TUAE457
TU-.4E498
Th-AE1 45
TU-AE246
TU-TA2460
TU-IRGB93
Th-AE1 84
TU2
II
A4E-PJcedo
i4E-Chinese Spring
DU-Langdon
DU-394
0.1
I DU
I
30
The 65 accessions studied grouped into two major clusters (Figure 2.2).
Ae. cylindrica, Ae. markgrafll, and nine Ae. tauschll accessions grouped in
cluster I while tetraploid and hexaploid wheat and eight Ae. tauschii
accessions grouped in cluster II (Figure 2.2, Table 2.3). Cluster I was
subdivided into a group with Ae. cylindrica and Ae. markgrafii accessions
(group CM) and a group of nine Ae. tauschii accessions (group TU1). The CM
group was composed of Ae. cylindrica (group CL), five Ae. markgrafIi
accessions (group MK), and two other Ae. markgrafii accessions that grouped
between Ae. markgrafii and Ae cylindrica (Figure 2.2). TK 116 was present in
the CL group. The Ae. markgrafii accessions KU 5472 and TK GB9O were
most closely related to Ae. cylindrica (Figure 2.2). Cluster II was subdivided
into a group represented by tetraploid wheat (group DU), a group with
hexaploid wheat and six Ae. tauschii accessions (group TU2), and two other
Ae. tauschll accessions (Figure 2.2).
Spike morphology and cytological analyses were also conducted to
investigate the identity of TK 116. The apical spikelets of Ae. cylindrica have
four prominent awns with one pair originating from glumes and one pair from
lemmas of the apical spikelet (van Slageren 1994). On the other hand, apical
spikelets of Ae. markgrafii have two prominent awns coming from the apical
glumes, while apical spikelets of Ae. tauschii have two awns originating from
two
lemmas. In the present study, similar characteristics were noted for Ae.
31
markgrafii and Ac. tauschii (Figure 2.3). The spikes of TK 116 and anotherAe.
cylindrica accession (USA/OR 13) have a cylindrical structure and bear four
prominent awns on glumes and lemmas from apical spikelets. The overall
similarity of TK 116 with other Ac. cylindrica accessions with respect to spike
morphology and the number of awns in apical spikelets supports its
classification as an Ac. cyllndrica accession. Based on chromosome counting
and GISH analysis, TK 116 was found to be a 28-chromosome allotetraploid
with both C- and D-genome chromosomes (Figure 2.4a and 4b).
32
Figure 2.3 Apical portions of spikes from Ae. markgrafii, Ae. tauschii, and
Ae. cylindrica. a. Apical spikelet of the Ae. markgrafii accession GR GB89
showing two awns originating from the apical glumes. b. Apical spikelet of
the Ae. tausci-ill accession AE 276 showing two awns originating from two
apical lemmas. c. and d. Apical spikelets of the Ae. cylindrica accessions
US/OR 13 and TK 116, respectively. Apical spikelets in picture C and D
show four awns originating from both lemmas and glumes.
33
34
a
.14,;
Figure 2.4 Mitotic metaphase chromosome spreads and genomic in situ
hybridization (GISH)oftheAe. cylindrica accession TK 116 (Fl 486249). a.
Chromosome spread of TK1 16 showing 28 chromosomes with a combination
of chromosomes with terminal, sub-l:erminal, sub-median, and median
centromeres. b. GISH of a mitotic chromosome spread of TK1 16. Fourteen
fluorescein-labeled chromosomes (yellow-green) correspond to C genome
chromosomes while 14 red-orange (propidium iodide) colored chromosomes
correspond to D-genome chromosomes.
35
DISCUSSION
The evaluation of both chloroplast and nuclear microsatellite variation
revealed various patterns (Tables 2.1 and 2.2). First, the level of
chloroplast variation compared to nuclear variation was lower for all
species studied. The lower levels of variation in chloroplast compared to
nuclear microsatellites reflect the uniparental inheritance of chloroplast
genomes and their slower rate of evolution relative to nuclear genomes
(Wolfe et al. 1987; Provan et al. 1999; Provan et al. 2004). Second, Ae.
cylindrica was less diverse than either of its diploid progenitors (Ae.
markgrafll and Ac. tauschii) whether chloroplast or nuclear markers were
used. Since allopolyploids are formed from one or few relatively recent
hybridization events, these contain only a subset of the genetic variation
present in their progenitors. Thus, allopolyploids like Ac. cylindrica are
commonly less diverse than their progenitors. Third, Ac. tauschll was more
diverse than Ac. markgrafii. Goryunova et al. (2004) also made this
observation and suggested that this reflected a more ancient origin for Ac.
tauschll relative to Ae. markgrafll. Although our observations are consistent
with those of Goryunova et al. (2004), a larger sampling of Ae. markgrafii
accessions will be needed to fully address this difference in genetic
diversity. Finally, Ac. cylindrica was more closely related to Ac. markgrafli
than Ac. tauschii when nuclear microsatellites were analyzed. The close
36
relationship between Ae. cylindrica and Ae. markgrafll was also observed
using repetitive DNA markers (Dubcovsky and Dvorak 1994), RAPD
markers (Goryunova et al 2004), and analysis of the internal transcribed
spacers (ITS) of ribosomal RNA genes (Wang et al. 2000b). These
observations demonstrate that the C genome in Ae. cylindrica is less
divergent from the C genome of Ae. markgrafll than its D genome is from
the D genome of Ae. tauschii.
Plasmon analysis using wheat alloplasmic lines indicated that Ae.
tauschii ( D-type cytoplasm) was the maternal parent in the formation of
Ae. cylindrica (Tsunewaki 1996; Wang et al. 1997; Wang et al. 2000a).
However, our current investigation showed that one accession of Ae.
cylindrica, TK 116 (P1 486249), had chloroplast microsatellite alleles that
were neither present in Ae. cylindrica nor in Ae. tauschii accessions but
matched the allelic constitution of some Ae. markgrafii accessions (Table
2.2, Figure 2.1). This finding suggested that the chloroplastgenome of TK
116 was derived from Ae. markgrafii (C-type cytoplasm). Since our nuclear
microsatellite markers analysis (Figure 2.2), spike morphology
assessments (Figure 2.3), and karyotype evaluations (Figure 2.4) showed
that TK 116 was a bonafide Ae. cylindrica accession, we conclude that C
and D types of cytoplasm derived from Ae. markgrafii and Ae. tauschii,
respectively, are present in Ae. cylindrica.
37
We contemplated the possibility that our results with respect to TK
116 could be explained by chioroplast microsatellite allele size homoplasy
(Doyle et al. 1998; Hale et al. 2004). However, we reasoned that this was
unlikely since we evaluated a sizeable number of accessions with 20
chioroplast microsatellite markers. Other researchers also have found that
homoplasy was unlikely for chloroplast markers when evaluating closely
related genera, including species of the Triticeae, due to their relatively
slow rate of evolution compared to nuclear loci (Provan et al. 2004).
The occurrence of two types of cytoplasm in Ae. cylindrica may be
simply explained by reciprocal hybridization between Ae. markgrafii and
Ae. tauschii during the formation of Ae. cylindrica. Since reciprocal hybrids
between Ae. tauschll and Ae. markgrafll have been produced
experimentally (Sears 1941; Knobloch 1968), it is plausible that reciprocal
hybridization in nature led to the formation Ae. cylindrica with both C- and
D-type cytoplasm. Interestingly, reciprocal hybridization between Ae.
markgrafii and Ae. umbellulata Zhuk. (2n=2x=14; UU) has also been
proposed in the origin of the allotetraploid species Ae. triuncialis L.
(2n=4x=28; genome CCUU) (Murai and Tsunewaki 1986; Wang et al.
1997; and Vanichonon et al. 2003). Since evidence for multiple
hybridization events in the formation of Ae. cylindrica has been recently
presented by Caldwell et al. (2004), reciprocal hybridization is an attractive
mechanism to explain the presence of C- and D-type plastomes in this
38
species. However, cytoplasmic introgression or substitution should also be
considered (Rieseberg and Soltis, 1991; Brubaker et al. 1993; van
Raamsdonck et al. 1997). In this scenario, hybridization between Ac.
markgra f/i (female parent) and Ae. cylindrica (male parent) followed by
backcrossing with Ac. cylindrica (male parent) would also result in Ac.
cylindrica with C-type cytoplasm (Kihara and Matsumura 1941).
Based on a comprehensive survey of Ac. tauschiigermplasm with
nuclear DNA markers, Dvorak et al. (1998) suggested that the distribution
of present-day Ac. tauschii originated by expansion of two geographically
isolated subspecies - Ae. tauschii ssp. strangulata in coastal areas of
eastern Caspian Iran and ssp. tauschll in an inland area of northwestern
Iran. According to Dvorak et al. (1998), expansion of the distribution of ssp.
tauschii preceded that of ssp. strangulata leading to the spread of ssp.
tauschii westward to Turkey and eastward to Afghanistan, Turkmenistan,
Pakistan, Tadjikistan, and China. Subsequently, expansion of the
distribution of ssp. strangulata and gene flow between the subspecies in
the Caspian region and north-central Iran was argued to have resulted in
the observed discontinuity in the distribution of ssp. tauschii in Iran
(Lubbers et al. 1991; Dvorak et al. 1998). Furthermore, Dvorak et al. (1998)
suggested that Ac. tauschii germ plasm should be viewed as being
composed of two gene pools, strangulata and tauschii, rather than two
subspecies based on morphology. Nonetheless, this and other studies
39
have shown that the D genome in hexaploid wheat is more closely related
to the D genome of the strangulata gene pool of Ae. tauschii (Lubbers et al.
1991; Dvorak et al 1998; Pestsova et al. 2000).
Based on our analysis of nuclear microsatellite markers, Ae. tauschii
clustered in two distinct groups (TUI and TU2) (Figure 2.2). The TU2
group was composed of Ae. tauschii and hexaploid wheat (Alcedo and
Chinese Spring). Ae. tauschii accessions in the TU2 group belong to the
strangulata gene pool while the Ae. tauschii accessions in the TUI group
that are more closely related to Ae. cylindrica belong to the tauschll gene
pool (Table 2.1) (Dvorak et al. 1998; Petsova et al. 2000). Furthermore,
three accessions of the TU1 group (TA 1588, TA 10143 and TA 10145)
were interspersed with Ae. cylindrica in the dendrogram based on
chloroplast microsatellite data (Figure 2.1). Overall, this suggests that the
D genome and D-type plastome in Ae. cylindrica are closely related to and
were probably derived from the tauschii gene pool of Ae. tauschii. This
conclusion is consistent with molecular cytogenetic analyses showing that
D-genome chromosomes in Ae. cylindrica and common wheat were
derived from different Ae. tauschii biotypes (Badaeva et al. 2002).
Based on spike morphology, two taxonomic varieties of Ae.
markgrafii have been described (Eig 1929; Hammer 1980). Variety typica
(syn. Ae. markgrafii var. markgrafii) is characterized by well-developed
awns on apical glumes and awnless glumes of lateral spikelets while var.
40
polyathera (syn. Ae. markgrafll var. polyathera) has awned apical and
lateral spikelets. Irrespective of this varietal differentiation, studies on
intraspecific hybrid sterility and the genetic variation for the development of
awns on lateral spikelets suggested that Ae. markgrafii is divided into two
genetically differentiated populations (Ohta 2000, 2001). One population is
present in the western region encompassing Greece and West Anatolia
while the other population is present in the eastern region consisting of
central, southern, and eastern Anatolia, Syria, and northern Iraq.
In our analysis with chioroplast and nuclear markers, the genetic
differentiation of Ae. markgrafii accessions from the west and east was not
evident. The Ae. markgarfii accessions KU 0006 (typica from northwestern
Syria), KU 5852 (polyathera from north-central Turkey), KU 5864 (typica
from northwestern Turkey), and KU 5871 (typica from mainland Greece)
formed a single group (MK) in our dendrogram based on nuclear markers
(Figure 2.2). On the dendogram based on chloroplast markers, KU 5852,
KU 5864, and KU5871 formed a sub-group while KU 0006 associated with
otherAe. markgrafii accessions (Figure 2.2). Thus, KU 0006 and KU 5852
that correspond to Ohta's (2000) A and B testers of the eastern region and
KU 5864 and KU 5871 that correspond to the C and D testers of the
western region, respectively, were all closely related. This inability to
differentiate Ae. markgrafll genotypes from the west from those of the east
did not allow the identification of a probable source for the C genome or C-
41
type plastome in Ae.
cylindrica.
The two Ac. markgrafii accessions most
closely related to Ac.
cylindrica
based on nuclear markers were a typica
form the east, KU 5472 (from northern Iraq), and typica from the west, TK
GB9O (from western Turkey) (Figure 2.2).
Maps with collection sites of Ac. markgrafii and Ae. tauschii suggest
that the geographic distribution of these species overlap in southeastern
Turkey, northeastern Syria, northern Iraq, and northwestern Iran (van
Slageren 1994; Ohta 2000; Dvorak et al. 1998). Assuming that the
distributions of these species were not significantly different in the past,
then the central part of the Fertile Crescent is likely to be where Ae.
cylindrica
formed. Our observation that Ac. tauschll of their western region
of distribution (tauschii gene pool) are most closely related to Ac. cylindrica
is consistent with this hypothesis. However, this pattern was not evident
with the sample of Ae. markgraflithatwe used. An analysis of a more
comprehensive sample of Ac. markgra f/i accessions and an assessment of
the population structure of this species may be necessary before a
connection to Ac.
sample of Ae.
cylindrica
cylindrica
is possible. Similarly, a study with a larger
and its progenitors may be necessary to obtain a
more precise picture of these genetic and geographical connections.
42
ACKNOWLEDGEMENTS
We would like to gratefully acknowledge funding from the United States
Department of Agriculture Initiative for Future Agriculture and Food
Systems (IFAFS) and National Research Initiative (NRI) Competitive
Grants Programs.
43
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49
CHAPTER 3
GENETIC STRUCTURE ANALYSIS OF Aegilops cylindrica
Harish T. Gandhi*, Laura Morrison*, Christy J. W. Watson*, M. Isabel Vales*,
Carol MaIlorySmith*, Robert Zemetrat and Oscar RieraLizarazu*
*Department of Crop and Soil Science, Oregon State University, Corvallis,
Oregon 97331-3002, USA. tDepartment of Plant, Soil and Entomological
Sciences, University of Idaho, Moscow, Idaho 83844-2339, USA.
50
ABSTRACT
Jointed goatgrass (Ae. cylindrica; 2n=4x=28, genome CCDD) formed
through amphidiploidization of a hybrid between Ae. tauschii Coss. (2n2x14;
genome DD) and Ae. markgrafii (Greuter) Hammer (2n2x=14; genome CC).
In the present study, chloroplast and nuclear microsatellite markers were used
to gain a better understanding of the diversity, formation, origin, spread, and
population genetic structure of Ae. cylindrica. These analyses suggested low
chioroplast and nuclear genetic diversity in Ae. cylindrica. Both, C- and D-type
plastomes were found to occur in Ae. cylindrica from its native and non-native
(USA) distribution ranges, however the frequency of C-type plastome was
lower (13%) than the D-type plastome (87%). The nuclear genomes of the Ae.
cylindrica with C- and D-type plastome were found to be very closely related,
suggesting a monotypic origin. The Ae. cylindrica accessions from a region
near Van Lake in eastern Turkey, where the distribution of Ae. markgrafii and
Ae. tauschii overlap, showed the greatest level of nuclear allelic diversity. This
study suggests a lack of regional population genetic structure in Ae. cylindrica.
Analyses indicated that genetically distinct genotypes! populations of Ae.
cylindrica have migrated between regions. Finally, these analyses also
suggested that Ae. cylindrica in the USA originated from at least three regions
of its native range - central Anatolia, central East Turkey and western Armenia,
and Caucasia.
51
INTRODUCTION
Jointed goatgrass (Aegilops cylindrica Host; 2n=4x=28; genome CCDD)
is an autogamous allotetraploid of the Triticeae tribe (Poaceae family). It
formed through amphidiploidization of a hybrid between Ae. tauschii Coss.
(2n=2x=14; genome DD) and Ae. markgrafii (Greuter) Hammer (syn. Ae.
caudata L.; 2n=2x=14; genome CC). The progenitors of Ae. cylindrIca were
identified using data from a variety of sources including chromosome pairing
studies in interspecific hybrids (KHARA and MATSuMURA 1941, KIMBER and
ZHAO 1983), karyotype analysis (CHENNAVEERAIAH 1960), and analyses of
protein and nuclear DNA variation (JAASAKA 1981, NAKAI 1981, MAsCI et al.
1992, DUBCOVSKY and DVORAK 1994). Furthermore, studies on phenotypic
(MAAN 1976; TsuNEwAKI1996) and organellar DNA variation among
alloplasmic lines of wheat (OGHIHARA and TSUNEWAKI, 1988; WANG et al. 1997;
WANG et al. 2000) established cytoplasmic homology between Ae. cylindrica
and Ae. tauschii (D-type cytoplasm). Thus, indicating that Ae. tauschii was the
cytoplasmic donor of Ae. cylindrica. However, an analysis with chloroplast
microsatellite markers (presented in Chapter 2) showed that Ae. markgrafii (Ctype cytoplasm) has also contributed its plastome to Ae. cylindrica. Therefore,
genotypes with either C-or D-type plastome are present in Ae. cylindrica.
However, the relative frequency of C- or D-type plastome containing Ae.
cylindrica and the mechanism for their occurrence are presently unknown.
52
The native distribution of Ac. cylindrica involves parts of the
Mediterranean, the Near East and central Asia. Ae. cylindrica has spread
westward to Greece, Bulgaria, Romania, Kosovo, Montenegro, Serbia and
Hungary. To the east, Ae. cylindrica is found in central Asia. Northwards, it is
present in the Caucasus region and along the Black Sea coast. Though rare,
this species is also present in the western arc of the Fertile Crescent involving
Lebanon, Jordan, and Syria. Ac. cylindrica is adventive in parts of Europe and
the USA (VAN SLAGEREN, 1994). Present understanding about the genetic
relatedness in Ac. cylindrica collected from some of these regions is derived
from genetic diversity studies (WATANABE et al. 1994; OKuNO et al. 1998;
PESTER et al. 2003; GORYUNOVA et al. 2004; CALDWELL et al. 2004). These
studies suggested a lower level of diversity in Ac. cylindrica compared to other
Aegilops and Triticum species. However, studies of the population genetic
structure in Ac. cylindrica across its distribution have not been undertaken.
Throughout its range of distribution, Ac. cylindrica is considered a
weedy species, except where it is rare (VAN SLAGEREN, 1994). Since its
introduction into the USA in the late 1800s or early 1900s, Ac. cylindrica has
spread to many states and various hypotheses about its mode of introduction
have been proposed. It is generally accepted that Ac. cylindrica was brought
to the USA with the importation of hard red winter wheat by immigrants from
southern Russia, researchers from the United States Department of
Agriculture (USDA) and/ or private millers. There is also speculation that Ac.
cylindrica escaped to fields from grass gardens in Pullman, Washington
53
(MAYFIELD 1927; JoHNsToN and PARKER 1929). However, exact information
about the sources of Ae. cylindrica in the USA is unavailable. The information
about the sources of Ae. cylindrica in the USA might prove helpful for better
management of Ae. cylindrica (PESTER et al. 2003).
Therefore, studies are required to gain knowledge about the frequency
and formation of C- and D-type plastomes, the population genetic structure in
Ae. cylindrica and also to ascertain the likely source for accessions in the USA.
54
MATERIALS AND METHODS
Plant Material: One hundred and seventy-three Ae. cylindrica
accessions were analyzed using nuclear and chloroplast microsatellite
markers. These accessions were collected from 18 countries covering parts of
the native and non-native distribution of Ae. cylindrica (Appendix 1). Five Ae.
tauschll, three Ae. markgrafll, and three T. aestivum accessions also were
included in the assays. For chioroplast microsatellite marker analyses, an
additional 15 Ae. tauschii and six Ae. markgrafii accessions were used. Based
on topological and agro-climatic features, the Ae. cylindrica collection sites
from the native and non-native distribution were divided into 12 geographic
regions (henceforth referred to as region! regions; RI- R9, US1-US3). The RI
to R9 regions had accessions from Eastern Europe, the Near East, Caucasia,
central Asia and the Levant (Figure 3.1A), while the US1 to US3 regions had
accessions from the Great Plains, and western states of the USA (Figure
3.1 B). The list of accessions along with their area of origin, the geographical
coordinates of the collection sites, their regional assigment (Ri to US3), and
seed source information are provided in the Appendix 1. Eight seeds per
accession were planted and leaf tissue was harvested from each of the
germinated plants and bulked for DNA extraction.
DNA Isolation and molecular marker analysis: DNA was extracted
from 35 mg of leaf tissue following the protocol described by
FIGURE 3.1 --Map showing the distribution of Ae. cylindrica collections from various regions. Grouping of accessions into
regions was based on topological and geographical information. Regions are labeled and demarcated. Accessions of
each reagion are located by unique symbols. A. Locations of accessions belonging to native distribution of Ae. cylindrica.
Regions are labeled as Ri to R9. R4 and R5 are sympatric for Ae. tauschii spp. tauschii and Ae. markgrafii. B.
Approximate locations of accessions belonging to Ae. cylindrica in the USA. Regions in the USA are labeled as US1 to
US3.
A
*
*
sack Sea
*
Fu
*
L
;
t._
**
,
E
*
*
a
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R2'..
a
S
S
S
R6
#?F
LS
qp
?
+
S
)
E
c-fl
C)
57
RIERA-LIZARAZU et al. (2000). Twenty wheat chioroplast (WCt) microsatellite
markers (IsHli et al. 2001; Table 3.IB) were used to characterize the
chloroplast genome and 24 Gaterslaben wheat microsatellite (gwm) markers
(RODER et al. 1998; Table 3) were used to evaluate the nuclear genome. Of
the 24 nuclear markers analyzed, marker gwm 165 and gwm 205 consistently
detected two loci, increasing the total number of markers for analysis to 26.
For microsatellite marker assays, one primer was labeled with a
fluorescent dye [6-carboxyfluorescein (FAM), or 4,7,2',4',5',7'-hexachloro-6carboxyflu roscein (HEX), or 4,7 ,2',T-tetrachloro-6-carboxyfluroscein (TET)1.
Polymerase chain reactions (PCR) were carried out in lO-pI reactions
containing 0.03 units Taq polymerase with IX PCR buffer containing 1.5 mM
MgCl2 (Qiagen, Valencia, CA, USA), 2% sucrose in 0.04% cresol red, 0.2 mM
of each dNTP, and 0.2 pM of each primer. The PCR consisted of an initial
DNA-denaturing step at 95 ° for 5 mm, followed by 40 cycles of denaturation at
95 ° for I mm, annealing at 50-60 ° (depending on primers) for I mm, and
extension at 72 ° for 1 mm, with a final step of extension at 72 ° for 10 mm.
Fragment analysis was carried out using either ABI Prism® 377 DNA
Sequencer or ABI Prism® 3100 Genetic Analyzer. ABI GeneScan® 2.1 and
Genotyper® 2.0 software (Applied Biosystems, Foster City, CA, USA) were
used to size fragments based on an internal lane standard [n,n,n',n'tetramethyl-6-carboxyrhodamine (TAM RA) or 6-carboxy-x-rhodamine (ROX)].
Statistical analyses:
Descriptive statistics such as the number and
frequency of alleles, unbiased heterozygosity, and frequency (%) of
polymorphic loci were calculated for each microsatellite marker and region
using TFPGA 1.3 (MILLER 1997). For chioroplast microsatellites, indices of
diversity for regions and markers were obtained separately for Ae.
cylindrica
accessions with C- and D-plastome types. The calculation of unbiased
expected heterozyosity was based on NEI (1978). In this chapter, unbiased
expected heterozygosity is always referred to as heterozygosity. The plot of
genotypic diversity vs. number of loci was obtained for nuclear microsatellites
markers using Multilocus 1.2 (AGAPOW and BURT, 2001). Indices of population
differentiation, Fst and Rst were calculated as per the methods described by
WEIR and COCKERHAM (1984) and ROUSETT (1996), respectively. Estimates of
Fst and Rst statistics were obtained for each pair of regions using Arlequin 2.0
(SCHNEIDER et al. 2000). The statistical significance of pair-wise Fst and Rst
estimates was tested by performing 10,000 iterations of re-sampling using
Arlequin.
The program MICROSAT 2.0 (MINCH et al. 1997) was used to generate
a genetic distance (dissimilarity) matrix based on the proportion of shared
alleles (BowcocK et al. 1994). The genetic distance matrices were then
subjected to the neighbor-joining method (SAITOU and NEI 1987) of tree
formation using MEGA 2.0 (KUMAR et at. 2001). MEGA 2.0 was also used to
produce graphical trees.
59
Population structure analyses: Population structure analysis was
performed using a model-based method implemented in Structure 2.1
(PRITCHARD et at. 2000; FALUSH et at. 2003). Structure estimates the number
of subpopulations (K) in samples by performing simulations according to a
particular model with multilocus genotypic data. Each simulation, while
estimating for the value of K, provides a Bayesian log-likelihood probability. In
the present analysis, we used a model which assumes no admixture and
independent allele frequency between K populations. For each value of K,
simulations involving five iterations of 40,000 steps after 20,000 steps of burnin were performed. Simulations, for chioroplast data, were performed by
separating accessions into two groups and for K values one to five. The first
group was comprised of data from genotypes of Ae. cylindrica with C-type
plastome and Ae. markgrafii, while the second group consisted of data from
accessions of Ae. cylindrica with D-type plastome and Ao. tauschii. For both of
these groups, log-likelihood estimates reached a plateau at values of K
2. In
the case of nuclear microsatellite data, simulations were performed only with
data from Ae. cylindrica and for K values ranging from one to 20. A value of
K= 6 was found to best describe the relationship among the Ae. cylindrica
accessions. The results obtained using Structure were graphically depicted by
using the Distruct program (ROSENBERG, 2002).
RESULTS
Chloroplast microsatellite diversity: Of the 173 Ae. cylindrica
accessions analyzed, 12 were found to have more than one allele at some
chloroplast microsatellite loci. These heterogeneous samples were de-bulked
and re-analyzed with chloroplast microsatellite markers. Thus, the total
number of Ae. cylindrica samples analyzed with chloroplast markers increased
to 185.
Of the 20 chloroplast microsatellite markers used, markers WCt 2, WCt
3, WCt 10, WCt 15, WCt 19, WCt 22, and WCt 24 showed distinct alleles for
C- and D-type plastomes. For Ae. cylindrica with C-type plastome (C-type Ae.
cylindrica), the number of alleles per marker ranged from one to three, with an
average of 1.5 alleles per marker (Table 3.1A). A total of 30 alleles were
observed for 20 markers. Of the 20 markers analyzed, 12 (60%) were
monomorphic and three markers (15%) had heterozygosity values lower than
the average of 0.09 over all loci and regions. Markers WCt 5 (HE = 0.51) and
WCt 11 (HE = 0.50) showed the highest values of heterozygosity for C-type Ae.
cylindrica.
In the case of Ae. cylindrica with D-type plastome (D-type Ae.
cylindrica), a total of 39 alleles were observed for 20 markers. The number of
alleles per marker ranged from one to four, with an average of 1.95 alleles
(Table 3.IB). Of the 39 total alleles observed, six were unique to a specific
61
TABLE 3.IA
Allele frequency and diversity indices of C-type Ae. cylindrica for
selected chloroplast microsatellite markers
Region (number of accessions)
and allelic frequency
Marker
WCt 5
0.50
0.44
1.00
-
0.54
-
0.50
0.56
-
1.00
0.46
0.00
0.67
0.51
0.00
0.00
0.51
-
0.04
0.75
1.00
0.92
1
-
-
0.06
2
1.00
1.00
0.94
3
-
-
0.25
-
0.04
0.00
0.00
0.12
0.43
0.00
0.16
1
1.00
1.00
0.94
1.00
1.00
0.96
2
-
-
0.06
0.00
0.00
0.12
0.00
0.00
0.08
1
-
0.50
0.69
0.58
1.00
0.50
0.31
0.25
0.75
1.00
2
-
0.41
0.00
0.67
0.44
0.43
0.00
0.50
0.06
0.75
1.00
0.92
1
1.00
1.00
2
-
-
3
-
HE
WCt 13
HE
a
-
0.25
-
0.04
0.94
-
-
0.04
0.00
0.12
0.43
0.00
0.16
1
1.00
1.00
0.94
1.00
1.00
0.96
2
-
-
0.06
-
0.00
0.00
0.12
0.00
0.00
0.06
1
1.00
1.00
0.94
1.00
1.00
0.92
2
-
-
0.06
-
0.08
0.00
0.00
0.12
0.00
0.00
0.08
1
1.00
1.00
1.00
0.50
1.00
0.92
2
-
-
0.50
-
0.08
0.00
0.00
0.57
0.00
0.16
0.09
0.00
0.09
HE
WCt 18
0.04
0.00
HE
WCt 16
Marker
diversity
2
HE
WCt 12
US3
(1)
1.00
HE
WCt 11
(16)
US2
(4)
1
HE
WCt 9
USI
Alleles
HEa
WCt 6
R9
(2)
R6
(1)
Total
HE
0.00
0.07
Unbiased expected heterozygosity as per
0.00
0.08
NEI
(1972)
0.04
62
region. The accessions from the US1 region showed two unique alleles, while
those from the Ri, R2, R7, and US3 regions showed a single unique allele
each. Accessions from other regions did not exhibit any unique alleles.
Interestingly, the lines from the Ri, R7, R8 and R9 regions shared the alleles
occurring at low frequency only with the accessions from USI and US3
regions of the USA. Of the 20 markers used on D-type Ae. cyllndrica, 10
markers were monomorphic or nearly monomorphic (HE
0.02), while four
markers showed estimates of heterozygosity lower than the average over all
loci (HE < 0.08). Marker WCt 6 (HE = 0.44) and WCt 5 (HE = 0.34) had the
highest values of heterozygosity. Among the regions, accessions from USI
(HE = 0.12) had the highest values of heterozygosity followed by accessions
from R7(HE
0.11).
Nuclear microsatellite diversity: The nuclear microsatellite markers
were more diverse compared to the chloroplast markers. A total of 122 alleles
were observed with 25 markers, with an average of 4.9 alleles per marker. The
number of alleles per marker ranged from one for gwm 3 and gwm 205.1 to 16
for gwm 314. The average heterozygosity over 25 loci or 12 regions was 0.27.
Markers gwm 314 (HE= 0.86) and gwm 165.2 (HE= 0.85) showed the highest
values of heterozygosity, followed by gwm 301 (HE = 0.68) (Table 3.2).
In a plot of genotypic diversity vs. number of nuclear markers, genotypic
diversity of accessions reached a plateau after the addition of 17 of the 26
marker loci. Thus, suggesting that addition of more markers to this array of
TABLE 3.IB
Allele frequency and diversity indices of D-type Ae. cylindrica for chloroplast microsatellite markers
Region (number of accessions) and allelic frequency
Ri
Marker
WCt 1
R9
(13)
US1
US2
(9)
US3
(29)
Marker
diversity
-
0.02
1.00
0.98
-
0.006
0.05
2
0.85
0.89
1.00
3
-
0.05
-
0.27
0.2
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.05
1.00
1.00
1.00
0.89
1.00
1.00
0.88
1.00
1.00
1.00
1.00
1.00
0.99
-
0.12
-
0.01
0.00
0.23
0.00
0.00
0.00
0.00
0.01
-
0.88
0.25
-
-
0.03
0.02
0.00
(28)
-
1.00
1.00
0.00
1.00
1.00
1.00
1.00
1.00
1.00
-
0.12
-
0.00
0.23
0.00
0.00
-
2
1.00
1.00
1.00
1.00
1.00
1.00
0.12
0.75
1.00
1.00
1.00
0.97
0.98
0.00
0.00
0.00
0.00
0.00
0.00
0.23
0.43
0.00
0.00
0.00
0.07
0.04
1
0.92
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.79
1
0.97
0.95
2
0.08
-
-
-
0.21
-
0.93
0.05
0.15
0.00
0.00
0.00
0.34
0.00
0.13
0.10
-
-
-
0.04
-
0.006
HE
1
-
-
1
HE
WCt 5
R8
(4)
0.15
HE
WCt4
R7
(8)
1
2
WCt 3
R6
(6)
(13)
1
R4
(9)
R5
(18)
Alleles
HEa
WCt 2
R3
(4)
R2
(20)
-
0.00
-
0.00
0.00
-
0.00
0.00
0)
()
2
0.93
0.79
0.43
0.00
0.13
0.34
0.62
0.46
0.56
0.76
0.68
0.25
0.38
0.54
0.44
0.24
0.32
0.40
0.43
0.49
0.51
0.52
0.37
0.44
0.83
0.88
0.75
0.85
0.96
0.78
0.90
0.88
0.22
0.17
.0.13
0.25
0.15
0.04
0.22
0.10
0.12
0.21
0.35
0.30
0.23
0.43
0.27
0.07
0.37
0.19
0.21
1.00
1.00
1.00
1.00
1.00
0.92
0.89
0.89
1.00
0.97
-
0.01
.0.11
.0.11
-
0.03
0.00
0.15
0.19
0.21
0.00
0.06
0.11
0.10
0.07
0.39
0.17
0.25
1.00
0.95
0.50
0.22
0.61
0.83
0.75
1.00
0.00
0.10
0.57
0.37
0.49
0.30
0.40
0.00
1
0.77
0.79
0.50
0.78
0.72
0.67
0.75
0.75
2
0.23
0.21
0.50
0.22
0.28
0.33
0.25
0.37
0.34
0.57
0.37
0.41
0.48
1
0.92
0.89
1.00
0.89
0.78
2
0.08
.0.11
.0.11
0.15
0.19
0.00
1.00
1.00
1.00
HE
HE
wct ii
1.00
0.78
HE
wctg
0.21
0.50
3
WCt6
0.07
0.05
1
2
0.00
HE
WCt 12
1
0.00
0.00
0.00
0.00
-
2
3
1.00
1.00
4
0.00
HE
WCt 13
1
0.00
0.75
0.89
0.25
0.11
0.43
0.21
1.00
0.00
0.31
0.25
0.69
0.71
0.00
0.00
-
0.13
0.23
-
-
0.04
1.00
0.75
1.00
0.92
0.13
-
0.08
0.43
0.00
0.15
0.00
-
0.73
0.42
0.06
0.89
0.21
0.83
0.89
0.07
0.04
0.30
0.21
0.03
0.01
2
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.97
0.99
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.07
0.01
-
-
-
-
-
-
-
-
0.18
0.11
0.03
0.04
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.82
0.89
0.97
0.96
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.30
0.21
0.07
0.08
1
-
-
-
-
-
-
-
-
0.08
0.04
-
0.02
2
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.92
0.96
1.00
1.00
0.98
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.15
0.07
0.00
0.00
0.04
0.92
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.99
-
-
-
-
-
-
-
-
0.01
HE
WCt 16
1
2
HE
WCt 17
HE
WCt 18
HE
1
0.08
2
0.15
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.01
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.75
0.92
1.00
1.00
1.00
0.99
1
-
-
-
-
-
-
0.25
0.08
2
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.43
0.15
0.00
0.00
0.00
0.02
1.00
1.00
1.00
1.00
1.00
1.00
0.88
1.00
1.00
1.00
1.00
1.00
0.99
1
-
-
-
-
-
-
0.12
-
-
-
2
0.00
0.00
0.00
0.00
0.00
0.00
0.23
0.00
0.00
0.00
0.00
0.00
0.01
0.06
0.05
0.11
0.09
0.09
0.12
0.08
0.06
0.08
WCt22
HE
WCt23
HE
a
0.05
0.05
0.08
0.07
Total
HE
Unbiased expected heterozygosity as per NEI (1972)
0.01
0.01
C)
01
Ae. cylindrica
accessions would not provide additional information about the
genotypic diversity of the samples studied (Appendix 2).
In order to evaluate the levels of diversity across geographic regions,
along with the values of heterozygosity, accessions from each region were
assessed for other indices including the composition of unique and rare alleles.
Of the 122 total alleles, 41(33%) were unique to one of the 12 regions. The
alleles which were present in two to three regions in the native distribution (Ri
to R9) were considered rare. A total of 30 rare alleles were found in regions
Ri through R9. When indices of diversity were compared across the regions,
R5 had the highest value of heterozygosity
(HE
= 0.28), the highest number of
total alleles (69), the highest number of rare alleles (14), and the highest
number of unique alleles (11) (Table 3.2). These results indicated that
accessions from R5 have retained maximum allelic diversity. Using these
measures, accessions from R4 and R8 were found to have the least allelic
diversity.
Interestingly, six unique alleles were observed in accessions from the
USA. Accessions from the USI and US3 regions had greater numbers of total
and unique alleles compared to U52 (Table 3.2). Moreover, of the 30 rare
alleles found in the native distribution, 18 were present in US1, 12 in U52 and
ii in US3. It was important to note that, accessions from the Ri, R2, R5, R6,
and R7 regions shared five to seven of their rare alleles with the accessions in
the USA. In summary, accessions from the USI, US2 and US3 regions had
TABLE 3.2
Allele frequency and diversity indices for nuclear microsatellite makers
Regions (number of accessions) and allele frequency
Ri
Marker
gwm 3
R8
(4)
R9
(13)
USI
(28)
US2
(9)
US3
(29)
Marker
diversity
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1
-
-
-
-
0.03
-
-
-
-
-
2
1.00
1.00
1.00
1.00
0.94
1.00
1.00
1.00
1.00
1.00
1.00
0.99
-
-
-
-
-
-
-
-
0.002
0.00
0.00
0.00
0.00
0.11
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.01
1
1.00
1.00
1.00
1.00
1.00
0.93
1.00
1.00
1.00
1.00
1.00
1.00
0.99
2
-
-
-
-
-
0.07
-
-
-
-
-
0.00
0.00
0.00
0.00
0.00
0.14
0.00
0.00
0.00
0.00
0.00
0.00
0.006
1.00
1.00
1.00
1.00
1.00
1.00
0.88
1.00
1.00
0.97
0.58
0.88
0.94
-
-
-
-
0.03
0.42
0.13
0.06
0.00
0.00
0.00
0.00
0.00
0.00
0.23
0.00
0.00
0.06
0.51
0.22
0.12
1.00
0.95
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.99
0.05
-
-
-
-
-
-
-
-
-
-
0.01
0.10
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.01
1
2
HE
1
2
HE
R7
(8)
1.00
HE
gwm 161
R6
(6)
1
HE
gwm 157
R5
(18)
(13)
3
gwm 44
R4
(9)
Alleles
HEa
gwm 16
R3
(4)
R2
(20)
0.00
1.00
0.03
0.12
0.002
0.003
C)
gwm 165.1
1
2
0.92
3
0.08
0.00
1
0.04
-
2
-
0.05
0.05
3
4
-
5
0.08
6
0.13
7
0.25
8
0.00
0.00
-
1.00
0.87
1.00
-
-
0.21
0.23
0.00
0.00
1.00
0.00
-
-
-
0.005
1.00
1.00
1.00
0.98
-
0.02
0.00
0.02
-
0.002
0.00
0.00
-
0.13
-
0.29
0.13
-
0.29
-
-
0.06
0.16
0.13
0.06
0.47
0.5
0.65
0.11
-
0.33
0.11
-
0.44
10
0.17
0.05
0.25
0.22
11
0.08
-
12
0.08
-
-
-
-
0.04
0.08
0.5
0.11
-
0.13
0.33
0.21
0.11
-
0.01
-
0.02
-
0.06
-
0.14
0.14
0.13
-
0.13
0.1
0.08
0.02
0.09
0.5
0.25
0.07
0.47
0.42
0.26
0.29
0.12
-
0.13
0.06
0.02
0.07
0.75
0.43
-
0.13
-
-
-
0.07
0.07
0.13
0.08
-
0.05
-
0.01
-
0.18
-
0.14
0.12
0.09
0.17
0.88
0.74
0.75
0.68
0.56
0.84
0.73
0.43
0.77
0.73
0.72
0.65
0.85
1.00
0.89
1.00
1.00
0.94
1.00
1.00
1.00
1.00
0.97
0.92
0.90
0.95
-
0.06
-
0.03
0.08
0.10
0.04
2
3
0.88
0.13
0.12
0.17
1
HE
1.00
9
HE
gwm 186
1.00
-
0.16
HE
gwm 165.2
1.00
-
0.05
-
0.06
0.00
0.20
-
0.005
0.00
0.00
0.11
0.00
0.00
0.00
0.00
0.06
0.16
0.19
0.09
gwm 190
-
2
-
-
-
-
0.14
3
0.08
0.11
-
0.06
0.29
4
0.62
0.84
1.00
0.69
0.57
5
0.30
0.05
-
6
-
-
-
-
0.06
-
-
-
0.54
0.28
0.00
0.00
0.50
0.62
0.61
0.00
0.13
-
-
0.06
-
0.25
1.00
-
1
1.00
-
-
-
0.13
-
-
0.06
0.56
1.00
0.93
0.94
-
0.07
0.06
0.11
0.75
0.77
0.42
0.69
0.38
3
0.08
0.09
-
-
0.11
-
0.19
4
0.15
-
-
5
-
-
-
-
6
0.19
0.29
0.25
0.23
0.36
0.21
0.19
-
0.11
0.12
0.62
0.54
0.43
0.37
0.70
0.36
0.78
0.00
0.20
0.21
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1
0.92
1.00
1.00
1.00
0.81
1.00
0.81
1.00
2
0.08
-
-
3
-
-
1
4
-
-
0.89
0.88
0.11
0.08
-
0.005
-
0.05
1.00
0.97
0.86
-
0.03
0.04
-
0.01
0.00
0.07
0.25
0.08
0.11
0.04
0.92
0.79
0.74
-
-
-
-
-
-
-
-
0.19
-
0.04
-
0.02
0.02
0.003
0.09
0.17
0.16
0.37
0.43
1.00
1.00
1.00
1.00
0.00
0.00
0.00
0.00
0.00
1.00
1.00
0.92
0.89
0.94
0.08
0.04
0.02
-
0.07
0.02
-
0.02
-
-
-
-
0.62
0.06
0.03
-
0.58
HE
gwm 205.2
-
2
HE
gwm 205.1
0.31
-
HE
gwm 194
0.14
1
-
HE
gwm 210
1
0.15
0.00
0.00
0.00
0.34
0.00
0.33
0.00
0.00
0.00
0.16
0.20
0.11
0.96
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.75
1.00
1.00
1.00
0.98
-
-
-
-
-
-
-
0.25
-
-
-
-
-
-
-
-
-
-
-
-
0.003
0.00
0.39
0.00
0.00
0.00
0.05
-
0.002
2
3
0.08
0.00
0.00
0.00
0.00
0.00
0.00
1
-
-
-
-
-
-
-
2
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.97
1.00
1.00
1.00
0.99
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.07
0.00
0.00
0.00
0.05
1
-
-
-
-
-
-
-
-
-
-
0.04
0.006
2
0.06
-
-
-
-
-
-
-
0.006
3
-
-
-
-
-
-
-
-
0.04
0.003
4
0.06
-
-
-
-
-
-
-
0.41
0.33
0.22
0.07
-
-
-
-
-
-
0.13
0.55
0.61
0.33
0.38
0.25
0.27
-
0.5
0.38
-
-
HE
gwm 272
HE
gwm 314
0.04
0.02
5
-
6
7
0.11
-
0.33
8
-
0.17
-
-
0.25
-
0.17
0.11
0.33
9
0.08
0.06
0.25
10
-
0.11
0.25
-
11
0.25
0.39
0.25
0.11
12
0.17
0.05
-
-
13
0.08
-
-
0.03
0.25
0.33
0.06
0.16
0.16
0.02
0.06
-
0.01
-
-
0.20
0.07
0.16
-
0.04
0.06
0.17
0.13
0.21
0.23
0.16
0.07
0.24
0.27
0.13
-
0.63
0.03
0.03
0.13
0.52
0.21
-
-
-
0.03
-
0.01
-
0
14
-
-
-
-
-
-
15
-
-
-
-
-
0.13
16
-
-
-
-
-
-
0.81
0.86
0.58
0.79
0.63
0.82
0.05
0.13
0.06
-
-
HE
gwm 301
0.08
0.81
1
0.006
-
0.003
0.03
-
-
0.006
0.83
0.74
0.57
0.66
0.86
0.07
0.07
0.17
0.04
0.05
0.17
0.61
0.50
0.18
0.20
0.68
0.50
0.44
0.28
0.71
0.50
1.00
0.67
0.23
3
-
0.21
0.12
0.44
0.50
0.14
0.25
-
0.27
0.10
4
0.08
0.05
0.25
0.12
0.17
0.25
-
-
0.31
0.42
0.18
0.17
-
-
0.27
0.25
-
0.08
HE
-
0.16
2
0.50
HE
1
1.00
1.00
-
0.63
-
-
0.66
0.48
-
-
0.97
1.00
1.00
-
-
0.88
1.00
-
-
0.12
-
0.03
0.67
-
-
0.01
0.00
0.50
0.77
0.03
-
0.97
1.00
1.00
-
-
0.002
0.74
0.58
0.68
0.003
1.00
1.00
0.99
-
0.003
-
0.003
0.00
0.00
0.25
0.00
0.06
0.00
0.00
0.00
0.07
0.00
0.00
0.00
0.02
0.92
0.95
0.88
1.00
0.97
1.00
1.00
1.00
1.00
0.97
1.00
1.00
0.98
-
-
0.03
-
-
-
-
-
-
2
3
0.75
-
3
4
0.14
-
1
0.08
4
HE
-
0.92
6
gwm 337
-
2
5
gwm 325
0.60
-
0.12
0.05
0.15
0.10
0.25
0.03
-
0.00
-
0.06
0.00
0.002
0.00
0.01
0.005
-
0.00
0.00
0.06
0.00
0.00
0.05
gwm 383
1
-
-
0.14
-
-
0.27
0.03
-
0.33
0.17
0.43
0.63
0.50
0.60
0.97
1.00
0.66
0.83
0.29
0.37
0.50
0.13
0.18
0.25
3
0.69
0.76
0.75
4
0.15
0.06
0.49
0.39
0.43
0.47
0.29
0.75
0.50
0.57
0.57
0.06
1
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
2
-
-
-
0.00
0.00
0.00
-
0.00
0.00
1
-
2
-
0.05
0.25
3
0.85
0.26
0.25
4
0.15
0.68
0.50
-
0.00
-
1.00
5
HE
gwm 458
0.00
0.00
0.00
0.17
0.13
0.005
-
0.15
HE
gwm 437
0.14
2
HE
gwm 455
-
-
1.00
0.73
-
0.19
0.00
0.00
0.43
1.00
0.97
0.99
-
0.03
0.005
0.00
0.00
0.07
0.01
-
0.10
0.17
0.61
0.14
0.07
0.10
0.08
0.05
0.06
0.34
0.64
0.06
0.17
-
-
0.75
0.67
0.75
0.75
0.93
0.71
0.67
0.11
-
0.12
0.25
-
0.03
0.08
-
-
0.43
0.43
0.13
-
-
0.08
0.07
0.13
0.06
0.02
0.27
0.47
0.71
0.00
0.43
0.55
1
-
0.05
-
-
-
-
2
0.04
0.05
0.11
-
3
0.38
0.53
0.75
0.55
0.64
0.29
-
0.75
0.27
0.3
0.29
0.81
0.47
4
0.27
0.26
0.25
0.22
0.11
0.29
0.79
0.25
0.73
0.33
0.08
0.07
0.28
5
0.15
0.11
0.11
0.19
0.29
0.14
-
0.31
0.58
0.12
0.19
6
0.08
-
0.07
0.04
0.04
-
0.02
-
0.47
0.54
0.52
0.55
-
0.005
0.01
-
-
-
F\)
7
0.08
-
0.06
8
0.77
gwm 469
0.65
2
-
-
-
3
-
-
-
4
0.08
-
-
5
0.92
0.92
1.00
6
7
8
HE
1
0.65
0.03
1
gwm 484
0.43
-
0.05
0.15
0.15
-
-
0.81
-
0.005
-
0.55
0.79
0.38
0.03
-
-
-
-
8
-
0.62
0.05
0.12
0.25
0.30
0.42
0.50
0.18
-
0.01
0.25
0.25
0.79
0.26
0.38
0.16
0.24
-
-
-
-
0.005
0.26
-
-
-
-
-
0.40
0.43
0.35
0.71
0.67
-
-
-
-
0.11
0.22
0.71
0.50
0.50
0.30
0.84
0.55
0.27
0.14
0.50
0.25
0.57
0.16
0.06
-
0.33
-
0.60
0.77
0.005
-
0.005
0.83
0.28
0.47
0.17
0.72
0.42
0.005
-
0.25
0.13
0.08
0.005
-
0.48
0.61
0.005
-
0.33
0.005
0.62
-
-
0.06
0.54
0.01
0.14
0.06
6
0.04
-
0.55
0.14
7
-
0.34
-
0.88
-
0.06
0.21
0.56
0.58
0.002
0.37
0.33
0.42
0.61
0.21
0.00
5
0.33
0.75
0.06
-
0.59
0.75
-
0.37
0.71
0.008
0.86
-
0.46
0.40
-
0.58
0.33
4
0.01
0.01
0.19
0.08
-
-
-
3
0.43
-
2
HE
0.005
0.14
9
HE
-
0.53
0.71
0.59
0.27
0.29
0.41
0.60
-.1
gwm 608
1.00
1.00
1.00
0.94
0.92
1.00
0.94
-
-
0.06
0.08
-
0.06
0.00
0.00
0.00
0.11
0.16
0.00
0.13
0.00
1
0.04
-
-
-
-
-
-
2
0.50
0.50
0.50
0.50
0.47
0.50
-
-
0.06
0.03
0.50
0.50
0.44
0.44
-
1
2
HE
gwm 642
3
HE
Unique
alleles
Rare allelesc
Rare alleles
contributed to
4
0.46
0.50
5
-
-
0.56
0.51
0.57
0.58
0.59
7
6
1
0
10
7
5
6
5
61
0.06
0.95
0.97
0.04
1.00
0.05
0.03
0.00
0.08
0.00
0.10
0.06
-
-
-
-
-
0.002
0.50
0.50
0.46
0.31
0.38
0.52
0.45
0.50
-
0.04
0.16
0.13
0.50
0.50
0.53
0.50
0.48
0.49
-
-
-
0.005
1.00
1.00
0.96
0.04
-
-
0.54
0.53
0.52
0.55
0.61
0.62
0.51
0.56
10
5
1
1
4
2
1
3
41
3
14
5
8
1
3
18
11
10
4
2
7
5
6
1
2
-
-
62
43
42
70
50
50
36
52
63
50
55
122
57.7
53.8
42.3
38.5
69.2
46.2
47.6
30.8
53.9
57.7
46.2
57.7
92.31
0.25
0.23
0.22
0.19
0.28
0.25
0.27
0.17
0.21
0.22
0.22
0.21
0.27
USAd
Total allelese
0/
0
'ol"-
morphic loci
Total
HE
8Unbiased expected heterozygosity as per NEI (1972)
b
Allele present in only one of the regions
C
Allele present in three or less regions of native distribution (RI to R9)
d
Number of rare alleles contributed by a region to USA
e
Number of total alleles across all markers
75
levels of allelic diversity that were greater than accessions from R8 and R4,
but were less than the R5 region.
Relative frequency of C- and D-type plastomes in Ae. cylindrica: In
the chioroplast microsatellite marker tree, the accessions clustered into three
major groups, B, C, and D (Figure 3.2). These groupings corresponded to
known plasmon types defined by Tsunewaki (1996). Group B corresponded to
plasmon B with two wheat genotypes, group C corresponded to plasmon C
with Ae. markgrafii and 24 Ae. cylindrica accessions (C-type Ae. cylindrica)
and group D corresponded to plasmon D with Ae. tauschll and the remaining
161 accessions of Ae. cylindrica (D-typeAe. cylindrica). Thus, itwas found
that the C-type Ae. cylindrica was present at the frequency of 13% compared
to 87% of Ae. cylindrica with the D-type plastome. The frequency of Ae.
cylindrica accessions with C-type plastome in the USA (24.3%) was greater
than in its native area of distribution (3%). The majority of the Ae. cylindrica
with C-type plastome were collected in US1 (16) and US2 (4) regions, and
most of these accessions were closely related (Figure 3.2). The R6 and US3
regions contributed a single accession each, while R9 had two Ac. cylindrica
accessions with the C-type plastome.
Genetic relationship between Ae. cylindrica and its progenitors
based on chioroplast microsatellites: Within the C-type cluster, Ac.
markgrafll var. polyathera genotype MK-3 was more closely related to the
accessions of Ae. cylindrica than to other Ac. markgrafii accessions
76
FIGURE 3.2---Neighbor-joining tree showing chloroplast genetic relatedness
between Ae. cylindrica and its relatives. The prefixes used before the name of
each accession stand for the following: R= Ae. cylindrica; AE= T. aestivum,
MK= Ac. markgrafll, and TU= Ac. tauschll. Prefix for Ae. cylindrica accessions
includes information about their region of origin, for e.g. Ri-CL, would indicate
Ac. cylindrica from RI. Clusters of accessions are designated as B, C and D
corresponding to individuals with plasmon types B, C and D, respectively.
A
Ae. cylindnca
(161)
Ae. fauschii
(20)
B
Ae. cylindrica
(24)
Ae. markgrafii
(8)
FIGURE 3.3---Model-based clustering plots for chloroplast microsatellite data. The horizontal axis corresponds to
accessions which are separated by black lines, while the vertical axis of the plots indicate proportion of the genome
belonging to a given subpopulation A. D-type Ae. cylindrica and Ae. tauschii accessions. The two species are labeled
along with number of accessions in parenthesis. Two subpopulations (K=2) were observed in Ae. tauschii - TC-K1
(yellow), TC-K2 (orange). All of the D-type Ae. cylindrica accessions belonged to subpopulation TC-K2. B. C-type Ae.
cylindrica and Ae. markgrafii accessions. The two species are labeled along with number of accessions in parenthesis.
Two subpopulations (K=2) were observed in Ae. markgra f/i - MC-K1 (blue), MC-K2 (green). The majority of C-type Ae.
cylindrica accessions belonged to subpopulation MC-K1.
79
(Figure 3.2). Similarly, one oftheAe. cylindrica accessions (US1-CL1I1) did
not group with other C-type Ae. cylindrica and was more closely related to Ae.
markgrafii accessions MK-2, MK-4 and MK-5. In cluster D, Ae. tauschii ssp.
tauschii accessions la-i, lu-i 5, TU-i 7 and TU-1 9 were the most closely
related to Ae. cylindrica with the D-type plastome.
A similar pattern was observed when model-based clustering was used
to study the relationship between the plastomes of Ae. tauschii or Ae.
markgrafii and Ae. cylindrica. This analysis suggested that the chioroplast
genomes of Ae. markgrafii and Ae. tauschii could be differentiated into two
subpopulations each (Figure 3.3). Of the two plastome populations identified in
Ae. markgrafii, accessions MK-i and MK-3 had the greatest coefficient of
ancestry (membership) to subpopulation MC-K1, while the rest of the
accessions had complete membership to the other subpopulation labeled MCK2. Of the 24 C-type Ae. cylindrica accessions, 21 had membership to the
MC-K1 subpopulation, while the remaining three accessions had membership
to both subpopulations. For Ae. tauschii, 16 accessions belonged to the
subpopulation named TC-K1, two belonged to the subpopulation TC-K2, and
two accessions had membership to both subpopulations. All of the D-type Ae.
cylindrica accessions had membership to the TC-K2 subpopulation. The Ae.
tauschii accessions which had membership to the IC-K2 subpopulation
belonged to ssp. tauschii and were closely related to Ae. cylindrica in the
chloroplast phylogenetic tree (Figure 3.2; la-i, TU-1 5, Tu-i 7 and TU-i 9).
FIGURE 3.4---Neighbor-joining tree showing nuclear genetic relatedness
between Ae. cylindrica and its relatives. The prefixes used before the name of
each accession stand for the following: R= Ae. cylindrica; AE= T. aestivum,
MK= Ae. markgrafll, and TU= Ae. tauschii. Prefix for Ae. cylindrica accessions
includes information about their region of origin, for e.g. RI-CL, would indicate
Ae. cylindrica from Ri. The major clusters (A, B, C and D) and sub-clusters (I
to IX) of Ae.cylindrica accessions are labeled.
Ix
IjIIi
VII
VI
IV
III
Nuclear genetic relationship in Ae. cylindrica: In the nuclear
microsatellite marker tree, accessions were grouped according to their known
taxonomic classifications (Figure 3.4). All Ae. cylindrica accessions grouped in
a single cluster. Accessions of Ac. markgrafll were more closely related to Ac.
cylindrica than to Ae. tauschii. The Ae. tauschll accessions that were most
closely related to Ac. cylindrica were Ae. tauschii ssp. tauschii ( Figure 3.4;
Appendix 1). The Ae. tauschll ssp. strangulata accessions were found to
cluster with T. aestivum. Four major clusters (A, B, C, and D) included all of
the Ac. cylindrica accessions except single accession each from the R5 and
R6 regions. These accessions (R5-CL45, and R6-CL63) were at the base of
the Ac. cylindrica cluster.
The four major clusters (A, B, C, and D) of Ac. cylindrica were
subdivided into nine sub-clusters. Among the nine sub-clusters there were five
groups with the accessions from the USA (I, II, IV, VI, IX) and four groups with
the accessions from the RI to R9 regions of Ac. cylindrica's native range (Ill,
V, VII, VIII). Four of the five groups with USA accessions had at least one or
two accessions from the R2, R3, R6, and R7 regions and a single group had
one accession from the Rl region. Each of the major four clusters (A, B, C,
and D) of Ac. cylindrica had accessions with C-type cytoplasm. Seventeen of
24 Ac. cylindrica accessions with C-type plastome were closely related and
grouped in sub-cluster VI. The remaining seven C-type accessions were
present in sub-clusters I, II, IV and V (Figure 3.4).
The Structure program was used to evaluate the level of genetic
relatedness and population genetic structure in the Ae. cylindrica accessions.
In the simulations to estimate K, values of log-likelihood probability increased
with K values of one to 20. However, for K > 6 a change in log-likelihood
estimates approached a plateau. Thus, a value of K=6 or the existence of six
subpopulation among 173 accessions of Ae. cylindrica best described the data
set. However, the value of K= 6 is an approximation and may not reflect the
actual number of subpopulations in Ae. cylindrica.
The six subpopulations of Ae. cylindrica predicted using the Structure
program, were labeled CL-K1 to CL-K6 (Table 3.3; Figure 3.5). The majority of
accessions had membership to subpopulations CL-K1 and CL-K4.
Membership to the subpopulation CL-K5 was the lowest. Accessions from the
native range had membership to all six subpopulations (Table 3.3). However,
accessions with membership to the subpopulation CL-K5 in the native range
always shared their ancestry with other subpopulations (Figure 3.5). The
majority of genotypes collected in the USA had membership to four
subpopulations (CL-K2, CL-K3, CL-K5, and CL-K6; Figure 3.5). An accession
from the US3 region also had membership to subpopulation CL-K1 (Table 3.3).
Interestingly, accessions with complete membership to the subpopulation CLK5 had C-type plastomes. However, C-type Ae. cylindrica accessions also had
membership to subpopulations CL-K1, CL-K2, and CL-K3 (Figure 3.5).
TABLE 3.3
Model-based clustering of 173 Ae.
cylindrica
accessions in six subpopulations using nuclear microsatellite
markers
Coefficients of subpopulation membership
Source
Native
USA
Total
CL-K2
Ri
CL-K1
0.58
R2
No. of
accessions
0.01
CL-K3
0.00
CL-K4
0.38
CL-K5
0.03
CL-K6
0.00
0.66
0.20
0.00
0.08
0.01
0.05
19
R3
0.46
0.07
0.21
0.00
0.00
0.26
4
R4
0.11
0.03
0.00
0.85
0.00
0.00
9
R5
0.02
0.07
0.05
0.85
0.00
0.01
18
R6
0.38
0.09
0.07
0.30
0.00
0.16
7
R7
0.28
0.58
0.01
0.13
0.00
0.00
8
R8
0.41
0.08
0.00
0.51
0.00
0.00
4
R9
0.66
0.00
0.00
0.34
0.00
0.00
15
Native
0.40
0.13
0.04
0.38
0.004
0.05
97
US1
0.00
0.24
0.28
0.00
0.28
0.20
36
US2
0.00
0.26
0.28
0.00
0.28
0.18
11
US3
0.01
0.26
0.14
0.00
0.00
0.59
29
USA
0.005
0.26
0.23
0.00
0.19
0.32
76
0.33
0.17
0.09
0.32
0.05
0.12
173
Region
13
FIGURE 3.5 ----- Plot of model-based clustering of 173 Ae. cylindrica
accessions in six subpopulations. Vertical axis of plot corresponds to
accessions which are separated by black lines. Horizontal axis of plot
indicates coefficient of ancestry of an accession to subpopulation(s).
Subpopulations are labeled and color coded (on right) as CL-K1, brown; CLK2 blue; CL-K3, yellow; CL-K4, pink; CL-K5, green; and CL-K6, purple. Labels
of subpopulations which had membership from the USA accessions are
underlined and boldfaced. Accessions from the native source in
subpopulations with membership from the USA genotypes are marked by
colored arrow head with a label indicating region of origin (on left). Accessions
with C-type plastome are marked by empty black (USA) or color (native)
headed arrows (on left).
CL-K5
CL-K4
CL-K3
CL-K2
CL-K1
The genotypes of each region had membership to two to five
subpopuations. Accessions from the R2, R3, R5, R6, US1, and US2 regions
had membership to four or more subpopulations (Table 3.3). The remaining
regions had genotypes with membership to three or less subpopulations.
Moreover, genotypes from neighboring regions had comparable proportion of
subpopulation membership. This was especially noticeable for genotypes in
regions R8 and R9 and USI and US2 (Figures 3.1A, 3.1B; Table 3.3). It is
important to note that subpopulations (CL-K2, CL-K3, and CL-K6), which had
the majority of accessions from the USA also had four genotypes from the R2
region, one genotype from the R3 region, two genotypes from the R5 region,
three genotypes from the R6 region, and five genotypes from the R7 region.
Thus, suggesting that accessions from the R2, R3, R5, R6, and R7 regions
were closely related to genotypes in the USA.
Pair-wise comparisons between the regions for Fst and Rst estimates
were made to further investigate the relationship between the accessions from
these regions (Table 3.4). The pair-wise estimates of Fst and Rst between the
groups of accessions from regions were found to be comparable. The regions
with the similar population membership patterns (Table 3.3) had lower and
non-significant Fst and Rst estimates, while the regions with dissimilar
population membership patterns had higher and significant Fst and Rst
estimates (Table 3.4). The group of genotypes from regions in the USA
showed statistically significant Rst and Fst estimates with the group of
genotypes from most of the regions in the native range.
TABLE 3.4
The pair-wise Rst (above diagonal) and Fst (below diagonal) estimates for genotypes from various regions
Region
RI
Ri
R2
R3
R4
0 05
003
R5
R7
R8
R9
0 12
0.13**
0 12
0.14**
005
-0.02
0 14** 0 13***
0.17*** 0.16***
0.09
0 10** 0 13
0.16*** 0.13***
0 23*** 0 31***
0.22*** 0.29***
-0.01
-
0.18**
0.09
0.08
0.13*
0.12
0 18*
0 03
0.09
0 26**
0 11
0 13*
0 21
-
0 19**
0 20
0 10*
0.25*
0.20
0 32*** 0 41***
0 08
0 14***
0 21*** 0 27***
0 10
0 16
0 23***
0 1 2**
0 13*
0 1 8**
0 14
0 1 7***
0 1 5***
0 1 9***
0 30***
0 09
0 1 8**
0 29**
0 36***
0 1 9***
0 29***
0 38***
0 00
0 24***
-
0 21***
R2
0.07
R3
R4
0.09
0 12**
R5
0 14***
0 17
0 19***
R6
0 04
0 11 **
0 13
0 03
0 14*
R7
0 06
0 11 **
0 11
0 1 5**
0 1 3***
0 07
R8
0 06
0 11
0 14
0 09
0 10
0 09
0 13
R9
0 1 3***
0 1 9***
0 17
0 17**
0 1 3**
0 1 7***
0 1 5**
0 1 3**
0 22**
0 29*** 0 28***
0.29*** 0.27***
USI
0 15*** 0 18***
US2
0 22*** 0 24***
0.16***
0.19k
US3
US2
R6
0 18**
0.11
0.12
0 1 3**
0 23*** 0 25***
* Pair-wise differences were significant at p-value <0.05
** Pair-wise difference significant at p-value <0.01
Pair-wise differences significant at p-value <0.001
0 11
0 15*** 0 18**
0 13
0 19*** 0 20*** 0 23**
0.21*** 0.21*** 0.20
US1
0 18***
0 24***
0 04
O.23
0.17
US3
0 38***
0.18-
DISCUSSION
Genetic diversity in Aegilops cylindrica: This study was the first to
extensively evaluate genetic diversity in a large collection of Ac. cylindrica.
The level of variation observed for nuclear microsatellites was greater
compared to chloroplast markers (Tables 3.IA, 1B, and 3.2; Figures 3.2, 3.3,
3.4, and 3.5). This result was expected since chloroplast genomes have
uniparental inheritance and a slower rate of evolution relative to nuclear
genomes (WOLFE et al. 1987; PROVAN et al. 1999; PROVAN et al. 2004).
Average expected heterozygosity obtained for nuclear
chloroplast microsatellites
(HE
(HE
=0.27) and
=0.08, D-type) were lower than our earlier
analysis, in which 36 Ac. cylindrica accessions were evaluated with 19 nuclear
(HE
=0.40) and 20 chloroplast
(HE
=0.17) microsatellite markers (Chapter 2).
However, the majority of the markers used in both of these studies were the
same. In the earlier analyses, heterozygosity was estimated using the method
described by BOTSTEIN et al. (1980), while in the present study Nei's unbiased
estimate of heterozygosity (NEI 1972) was used to avoid sample size bias.
With an increase in sample size, the heterozygosity estimates obtained from
these two methods are expected to be comparable. However, if a species has
low genetic diversity, it is reasonable to observe a decline in heterozygosity
values with an increase in sample size. Therefore, the decrease of
heterozygosity observed in the present study reflects low genetic diversity in
Ac. cylindrica. This is consistent with other studies which have reported a
lower genetic diversity in Ae. cylindrica compared to its progenitors and other
relatives (OKUN0 et al. 1998; PESTER et al. 2003; GORYUNOVA et al. 2004).
Formation and origin of Ae. cylindrica: Ae. tauschii is one of the
progenitors of Ae. cylindrica. Ae. tauschii is composed of four morphological
varieties, which are grouped in two subspecies - Ae. tauscliii spp. strangulata
(var. strangulata) and Ae. tauschii spp. tauschll (var. typica, var. meyeri, and
var. anathera) (EIG, 1929; KIHARA and TANAKA, 1958; TANAKA, 1983). It has
been suggested that the D genomes of Ac. cylindrica and T. aestivum were
contributed by different biotypes of Ae. tauschii (BADAEVA 2002; CALDWELL et
al. 2004). In various studies, the D genome of hexaploid wheat has been
shown to be more closely related to the D genome of Ac. tauschii ssp.
strangulata than to Ae. tauschii ssp. tauschii (LUBBERS et al. 1991; DVORAK et
al 1998; PESTSOVA et al. 2000; Chapter 2). We have also determined that the
D-type plastome and the D genome of Ae. cylindrica are more closely related
to Ae. tauschii ssp. tauschii than to Ac. tauschii ssp. strangulata (Chapter 2).
Furthermore, it has been observed that between the two subspecies of Ac.
tauschii only Ac. tauschii spp. tauschii shares its distribution with Ae.
markgrafii, the other progenitor of Ac. cylindrica (VAN SLAGERAN 1994;
DVORAK et al.1 998). Therefore, it is very likely that Ac. tauschii ssp. tauschii
donated the D-type cytoplasm and D genome to Ac. cylindrica.
In the present study, model-based clustering of chioroplast marker data
suggested the presence of two plastome subpopulations, TC-K1 and TC-K2,
in Ac. tauschii (Figure 3.3A). Ac. tauschii ssp. strangulata and some Ae.
91
tauscliii spp. tauschii accessions had membership in the TC-K1
subpopulation, while the remaining Ac. tauschll ssp. tauschll accessions had
membership to subpopulation TC-K2. All of the accessions of Ae. cylindrica
with the D-type plastome had membership to the subpopulation TC-K2. In the
genetic distance trees based on chioroplast and nuclear markers (Figures 3.2
and 3.4), Ac. tauschii spp. tauschii accessions were found to be closely
related to Ae. cylindrica. Thus, these studies confirmed the observations made
in our earlier analyses (Chapter 2) and indicate that Ae. tauschii spp. tauschii
contributed one of its plastome type to Ac. cylindrica.
In a previous study with chloroplast and nuclear microsatellite markers
(Chapter 2), the reported genetic differentiation of Ac. markgrafii (OHTA 2000,
2001) was not observed. In the present study, model-based clustering of
chloroplast marker data suggested the presence of two plastome
subpopulations, MC-K1 and MC-K2, in Ac. markgra f/i (Figure 3.3B). All C-type
Ae. cylindrica accessions studied belonged to the MC-K1 subpopulation but
three of these accessions also had some membership to subpopulation
MC-K2. Interestingly, two Ac. markgrafii accessions (MK-1 and MK-3) with the
greatest membership to subpopulation MC-K1 were of the polyathera variety
that is commonly present in the eastern region of Ac. markgrafiis distribution
(OHTA 2000, 2001). Thus, it seems likely that the plastome in C-type Ac.
cylindrica might have originated from genotypes of this region. Still, an
analysis of a larger number of Ac. markgrafll accessions will be needed to
more clearly elucidate the relationship between its cytoplasm and that of Ctype Ae. cylindrica.
Of the 173 genotypes studied in nuclear marker analyses, accessions
from the R4, R5 and R6 regions had some distinctive features which might
help elucidate the region of origin of Ae. cylindrica. The R5 and R6 regions
encompass an area (Figure 3.IA) with geographical features characterized by
mountains, table-lands and plateaus; though the R4 region also had similar
geographical features it is separated from the R5 and R6 regions by high
mountains (3000-4000m). R4 and R5 are the only regions, where both of the
progenitors of Ae. cylindrica, Ae. tauschll and Ae. markgrafii, have been
collected (Figure 3.IA; VAN SLAGERAN 1994; DVORAKet al. 1998; OHATA
2000). Accessions of the R4 region had the least nuclear allelic diversity
among the regions studied, whereas accessions of Ae. cylindrica from R5
were found to have the greatest allelic diversity (Table 3.2). Genotypes
belonging to the R6 region were present in multiple clusters (Figure 3.4) and
had membership to five of the six subpopulations of Ae. cylindrica (Table 3.3).
Furthermore, an accession each from R5 and R6 were basal to the Ae.
cylindrica cluster (Figure 3.4), suggesting that these regions have Ae.
cylindrica accessions with an ancestral state. Thus, accessions from the R5
and R6 regions represent a substantial amount of genetic variation of Ae.
cyllndrica and are likely to be areas of origin of Ae. cylindrica. However, the
distributions of Ae. tauschii and Ae. markgrafii based on plant collection
information, overlaps only in the R5 region (VAN SLAGERAN 1994; DVORAK et
93
al. 1998). Therefore, we suggest that Ae. cylindrica probably formed in the R5
region and might have later migrated to the neighboring R6 region and
elsewhere. Interestingly, the R5 region is in the Fertile Crescent, where
various founder crops were domesticated (reviewed in YADUN et al. 2000;
DIAMOND, 2002).
Since the R5 region is the probable area of origin of Ae. cylindrica, we
expected its accessions to have the greater levels of population variation
compared to other regions. The greater population variation in the R5 region
would be reflected in membership of its accessions to various subpopulations
or phylogenetic clusters as observed for the genotypes from the R2, R3 and
R6 regions (Table 3.3; Figure 3.4). In our analysis the majority of accessions
from the R5 region belonged to a single major cluster (Figure 3.4) and had
membership to the subpopulation CL-K4 (Table 3.3). Though the accessions
of R5 had membership to other subpopulation (CL-K1, CL-K2, CL-K3, and CLK6; Table 3.3), membership to these subpopulations were less frequent. The
lack of population genetic variation in R5, thus, was unexpected. However, we
reasoned that under an adaptive evolution model, a species passes through
bottlenecks and a population(s) of species with better adaptation takes over
the distribution. Depending on the selection pressure, adaptive evolution leads
to dramatic changes in the original allele frequencies and population
membership patterns of a region. Therefore, we think that the genotypes with
membership to subpopulation CL-K4 might have displaced other Ac. cylindrica
and dominated the R5 region. Accessions from all regions of the native range
94
except the R3 region had membership to CL-K4 subpopulation, indicating that
accessions with membership to CL-K4 were widely distributed, adaptable and
competitive (Table 3.3). Thus, it is possible that low frequencies of unique and
rare alleles in the R5 region (Table 3.2) and the less frequent membership of
accessions of R5 to other subpopulations are, in fact, signatures of adaptive
spread of some of the accessions of Ae. cylindrica after its formation in the R5
region.
Population genetic structure in Ae. cylindrica: Analyses with
Structure using nuclear microsatellite data suggest that each region is
composed of two or more subpopulations in varied proportions. Furthermore,
genotypes in regions with geographical proximity shared their membership for
at least one subpopulation. In a phylogenetic tree based on nuclear genetic
distances, no distinct clusters were observed according to regions. Instead
accessions from a particular region were interspersed in the tree, suggesting
that Ae. cylindrica is not geographically structured.
Indices of population genetic differentiation, Fst and Rst, measure the
extent of genetic variability within or among the regions which can be allotted
to genetic differentiation. This genetic differentiation may occur due to genetic
drift, migration (also referred to as gene flow), or mutation. The parameter Fst
assumes migration-drift equilibrium among the regions and either excludes or
assumes a very low rate of mutation as the cause of genetic differentiation.
Since microsatellites have been suggested to mutate at higher rates, the Rst
parameter is recommended when microsatellites are used to measure genetic
95
variation (SLATKIN 1995). Interestingly, if genetic differentiation is caused
either by equal rates of mutation and migration, or higher rates of migration
than mutation, Fst and Rst statistics are comparable (BALLOUX and GAUDET
2002). However, Rst is expected to be larger than Fst if the mutation rate is
higher than the rate of migration (BALLOUX and GAUDET 2002; HARDY et al.
2003). Therefore, comparisons of the Fst and Rst estimates can help elucidate
causes of genetic differentiation.
In the present analysis, for most of the pair-wise comparisons the Fst
and Rst estimates were comparable (Table 3.4). Furthermore, when the Fst
and Rst estimates between the various regions were tested for statistical
significance (p-value
0.05), both provided a similar pattern of genetic
differentiation (Table 3.4). This result suggests that the observed genetic
differentiation between these regions may be due to either a higher rate of
migration than mutation or equal rates of migration and mutation. If migrations
were the cause of genetic differentiation, we should expect the presence of the
genetically similar accessions in various regions. In model-based clustering,
accessions with membership to any single subpopulation were found
distributed in multiple regions (Table 3.3). This was also observed in the
nuclear genetic distance based tree, where genetically similar accessions
originated from various regions (Figure 3.4). This result indicated that
accessions with membership to distinct subpopulations or clusters have
migrated among various regions. Furthermore, we found that the pair-wise
comparisons of regions with a similar population membership pattern (Table
3.3) have low and non-significant Fst and Rst scores (Table 3.4). Therefore,
we made two main inferences from population structure analyses: i) migration
has shaped the genetic make up of each region, and ii) variation in level of
genetic differentiation between any pair of regions was due to their similar or
dissimilar genotypic make up. We further suggest that the absence of
geographic genetic structuring in Ae.
cylindrica is
probably due to migration of
genetically distinct accessions between the regions.
Introduction and population genetic structure of Ae. cylindrica in
the USA: In our analyses, no single region of the native range could have
contributed all of the genetic variability observed in accessions from the USA.
Therefore, accessions in the USA were introduced from more than one source.
Of the nine regions of the native range, accessions from the R7 region shared
low-frequency chloroplast alleles (Table 3.IB) and six rare nuclear alleles
(Table 3.2) with the samples in the USA. Furthermore, in both the genetic
distance- and the model-based clustering five of the eight accessions of the
R7 region were genetically very similar to genotypes from the USA (Figures
3.3 and 3.5). Therefore, the R7 region, which is comprised of accessions from
parts of Caucasia, seemed to be among the most likely sources for the USA
accessions. Beside the R7 region, the samples from the R2 and R6 regions,
which are comprised of accessions from central Anatolia, and central East
Turkey and western Armenia, also shared rare nuclear alleles with the
accessions from the USA (Table 3.2). In addition, the R2 and R6 regions,
contributed the second and third largest number of accessions, respectively
97
after the R7 region which were all closely related to genotypes from the USA
(Figure 3.3 and 3.5). Therefore, accessions from the R2 and R6 regions must
also be among the sources of the Ae. cylindrica in the USA. Furthermore,
accessions from the R2, R6, and R7 regions together contain most of the
allelic variability and population variation observed in accessions from the USA
(Tables 3.1A and B, and 3.2). Accessions from the RI, R3, and R5 regions
also shared some degree of similarity to the genotypes from the USA,
however only in a few of the analyses conducted (Tables 3.1 B and 3.2;
Figures 3.4 and 3.5). Therefore, we think that the R2, R6, and R7 regions
were the primary sources of Ae. cylindrica that was introduced into the USA
and that the Rl, R3, and R5 regions may be secondary sources.
Though R2, R6 and R7 seemed to be the primary sources for the USA
accessions, a few unique and rare alleles of chloroplast and nuclear
microsatellites markers in accessions from the USA were not present in any
accession of the native range (Tables 3.1B and 3.2). If the occurrence of
unique and rare alleles in the accessions from the USA is not due to sampling,
it is possible that accessions in the USA may have originated from region(s)
other than those studied in the present analysis. The wheat imports, which
might have led to Ae. cylindrica introduction in the USA, were from southern
Russia (present day eastern Europe) (MAYFIELD 1927; QUISENBERRY and
REITZ 1974; SAUL 1989). Thus, it is possible that the region of Ac. cylindrica's
native range, which have been suggested to be the source of Ac. cylindrica in
the USA in the present analysis, did not directly contribute accessions to the
USA but they were the source of accessions for a site or sites from where
wheat imports to the USA had occurred. A survey of accessions from sites in
eastern Europe should clarify this issue.
Ae. cylindrica in the USA was first reported in Kansas (MAYFIELD 1927;
JOHNSTON and PARKER 1929). In the present study, the USI region, had
accessions from Kansas and other neighboring states including Colorado,
Oklahoma, and Wyoming. If US1 is a region where Ae. cylindrica was first
introduced, we would expect its accessions to have greater genetic variability
than accessions from other regions in the USA. Indeed, we found that the
accessions from the USI region were the most diverse. Moreover, the allelic
diversity within the USA was less for the accessions from the US2 and US3
regions (Tables 3.IB and
3.2).
Thus, these results support the suggestion that
Ae. cylindrica in the USA may have first arrived in parts of the USI region.
Since its introduction, Ae. cylindrica has moved considerably within the
USA. The close genetic relationship between accessions suggests that Ae.
cylindrica in the USA spread through a few founder genotypes (Table 3.3;
Figure
3.4).
Spread of Ae. cylindrica through founder effect was further evident
from the close genetic relationship observed between some of the C-type Ae.
cylindrica collected in the USA (Figure
3.4).
If the US1 region is the source for
accessions to other regions in the USA, then the similar population
membership pattern observed for the accessions from the US1 and US2
regions (Table
3.3)
and low non-significant Rst and Fst scores between the
US1 and US2 regions suggest that accessions from the US1 region moved to
the US2 region (Table 3.4).
In the case of the US3 region, most of its accessions had subpopulation
and cluster membership similar to accessions from the US1 and US2 regions
(Figures 3.4, and 3.5; Table 3.3). Therefore, statistically significant pair-wise
Fst and Rst estimates observed between the USI and US3 regions, and the
US2 and US3 regions were most probably due to variation in the proportion of
genotypes with a given subpopulation membership (Tables 3.3 and 3.4). We
found that 28% of accessions from the US1 and US2 regions had membership
to subpopulation CL-K5; however, accessions with membership to CL-K5 were
absent in the US3 region. Moreover, accessions with membership to CL-K6 in
the US3 region were present at a higher proportion than in the USI and US2
regions. Thus, it can be inferred that the significant differentiation observed
between US1 and US3 or US2 and US3 were mostly due to the differences in
overall genotypic makeup in these regions and not due to genetic differences
of their accessions. Therefore, despite significant genetic differentiation
between US1 and US3 or US2 and US3 we believe that most of the
accessions of US3 originated from the US1 or US2 regions. However, an
accession from the US3 region had membership to the subpopulation CL-K1
to which accessions in the US1 and US2 regions had no membership (Table
3.3). If this finding is not simply due to sampling, the US3 region may have
received a few of its accessions from a source other than the US1 or US2
regions.
100
C- and D-type plastomes in Aegilops cylindrica: Analyses with
chloroplast microsatellite markers confirmed the presence of C- and D-type
plastomes in Ae. cylindrica (Figure 3.2; Figure 3.3A and B). However, the
frequency of the C-type plastome in Ae. cylindrica was lower than the D-type
plastome. The difference in the frequency of plastome types in the native
distribution (Ri to R9) was dramatic, where D-type Ae. cylindrica was present
at the rate of 97%. This result suggests that the D-type Ae. cylindrica was
preferred to Ae. cylindrica with the C-type plastome. Preference for
cytoplasmic or plastome types is not unknown in the tribe Triticeae. For
example, five of the seven D genome allopolyploids have D-type cytoplasm or
its closest variant (D2-type) (TSUNEWAKI et al. 2002). This preference for
cytoplasmic or plastome types in allopolyploids may be attributed to
interactions between the nuclear and cytoplasmic genomes. Earlier studies
with alloplasmic lines showed that plasmon types can have significant effects
on phenotypes, including traits related to reproduction (TSUNEwAKI 1996;
TSUNEWAKI et al. 2002). Thus, it is possible that preference for the D-type
plastome in Ae. cylindrica is due to the favorable nucleo-cytoplasmic
interactions.
In the present analysis, no C-type Ae. cylindrica was identified in the
present distribution range of Ae. markgrafii (RI, R2, R4, and R5 regions), the
donor of the C-type plastome (Figure 3.2; Appendix 1). Although, three C-type
accessions were observed in the native range (R6 and R9 regions) these were
collected at sites beyond the distribution range of Ae. markgrafii (OHATA 2000
101
and 2001) and probably represent genotypes that had migrated to these
regions. The absence of C-type Ae. cylindrica within Ao. markgrafiis
distribution range is perplexing; however, this result might be due to the very
low frequency (3%) of Ae. cylindrica with C-type plastome in the native range
(RI to R9) and insufficient sampling. Thus, a larger sample of Ae. cylindrica
from the RI, R2, R4 and R5 regions might yield C-type Ae. cylindrica
accessions.
The occurrence of C-type Ae. cylindrica accessions in the USA was
significantly higher than the native distribution (Figure 3.2). Therefore, it is
possible that conditions in the USA are more favourable for C-type
Ae.cylindrica than in the native range. Many of the C-type accessions
collected in the USA were genetically very similar to each other (Figure 3.2;
Figure 3.4), which probably represents the classic case of spread of Ae.
cylindrica through a few founder accessions. Since a similar observevation
was made in our analyses of nuclear markers, we think that founder effects
have led to an increase in the frequency of a particular group of Ae. cylindrica
(for e.g. C-type) in the USA compared to the native range (Table 3.3).
In a nuclear phylogenetic tree, each of the four major clusters (A, B, C,
D) had accessions with C- and D-type plastomes. Similarly, C-and D-type Ae.
cylindrica accessions had membership to three common subpopulations (CL-
Ki, CL-K2, and CL-K3; Figure 3.5). The C-type Ae. cylindrica accessions also
had exclusive membership to subpopulation CL-K5. However, C-type
accessions belonging to CL-K5 were grouped in sub-cluster VI of the nuclear
102
genetic distance tree and were closely related to other D-type Ae. cylindrica
accessions (Figure 3.4). Therefore, the results from the genetic distance- and
model-based clustering indicate that the nuclear genomes of C- and D-type Ae.
cylindrica are closely related.
Recently, CALDWELL et al. (2004) suggested the recurrent origin for the
D-genome of Ae. cylindrica through multiple hybridization events. It is possible
that the individual or group of clusters! populations observed in Ae. cylindrica
might be associated with an individual hybridization event (Figure 3.3; Figure
3.5). However, as the C-type Ae. cyllndrica accessions did not associate with
any specific cluster or a subpopulation type, the formation or existence of Cand D-type plastomes does not appear to correspond to a unique hybridization
event in the formation of Ae. cylindrica. The absence of a distinct cluster for Ctype accessions in a nuclear tree suggests that genomes of C- and D-type Ae.
cylindrica have a monotypic origin. The monotypic origin with the two
cytoplasmic types in Ae. cylindrica can be the result of cytoplasmic
introgression from Ae. markgrafii after the formation of Ae. cylindrica. Though
it is tempting to suggest that cytoplasmic introgression could be mechanism
for the occurrence of the C-type plastome in Ae. cylindrica, it is necessary to
establish that the observed close nuclear genetic relationship between C- and
D-type accessions is not due to reticulate evolution or reciprocal hybridization
between progenitors with very narrow genetic bases.
In summary, our analyses provide a view of Ae. cylindrica diversity,
origin, formation, and population genetic structure. In addition, we also provide
103
insights on the origin and spread of Ae. cylindrica in the USA and the
mechanisms for the occurrence of C- and D-type plastomes in Ae. cylindrica.
We believe that low genetic diversity and no geographic genetic structuring in
Ae. cylindrica are probably associated with its recent origin (BADAEVA et al.
2002) and weedy and invasive nature. Still, additional analyses are necessary
to test the various hypotheses posited in this study. As the suggested area of
origin of Ae. cylindrica near Van Lake, Turkey (R5 region; Figure 3.1A) is a
part of the Fertile Crescent, the movement of Ae. cylindrica from its area of
origin, may be associated with the spread of agriculture. This is, in fact,
exemplified by the introduction of Ae. cylindrica to the USA with wheat.
Therefore, besides increasing our knowledge base, research on population
genetics of Ae. cylindrica is germane to understanding the history and spread
of agriculture from the Fertile Crescent.
104
ACKNOWLEDGMENTS
We would like to acknowledge support from United States Department
of Agriculture-National Research Initiative (Grant# 2001-35320-09918). We
would like to thank Gayatri Gandhi for her help in preparation of tables and
other parts of manuscript. We greatly acknowledge the germplasm we
received from United States Department of Agriculture-National Small Grains
Collection (USDA-NSGC), Dr. Waines, University of California, Riverside, USA,
Dr. P. Westra, Colorado State University, Fortcollins, USA, International
Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria
and Institute of Plant Genetics and Crop Plant Research (IPK), Gaterslaben,
Germany.
105
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Ill
CHAPTER 4
PATTERNS OF MATING BETWEEN Triticum aestivum AND Aegilops
cylindrica UNDER FIELD CONDITIONS
Harish T. Gandhi, Carol A. Mallory-Smith, Laura A. Morrison, Robert S.
Zemetra, Christy J. W. Watson, M. Isabel Vales and Oscar Riera-Lizarazu
Harish T. Gandhi, Carol A. Mallory-Smith, Laura A. Morrison, Christy J. W.
Watson, M. Isabel Vales and Oscar Riera-Lizarazu, Department of Crop
and Soil Science, Oregon State University, Corvallis, OR 97331; Robert S.
Zemetra, Department of Plant, Soil, and Entomological Sciences,
University of Idaho, Moscow, ID 83844-2339.
112
ABSTRACT
Jointed goatgrass (Aegilops cylindrica Host) is an important weed of
wheat (Triticum aestivum L.) in the United States and other parts of the world.
Wheat and jointed goatgrass can hybridize and produce backcross derivatives
under natural conditions, a situation that may allow gene flow between these
two species. In order to gain a better understanding of the factors governing
gene flow, 413 first-generation backcross (BC1) seeds obtained from 127
wheat-jointed goatgrass
F1
hybrids, produced naturally under field conditions,
were evaluated for their parentage using chloroplast and nuclear microsatellite
markers. Of the 127 hybrids evaluated, 109 (85.8%) had jointed goatgrass as
the female parent, while the remaining 18
F1
plants (14.2%) had wheat as the
fema'e parent. Of the 413 BC1 p'ants ana'yzed, 358(86.7%) had wheat and
24 (5.8%) had jointed goatgrass as the recurrent male parent. The male
parentage of 31 BC1 (7.5%) plants could not be determined. This study
suggests that under natural conditions wheat is the prevalent pollen donor for
the production of hybrids and first-generation backcross derivatives but
hybrids and backcrosses where jointed goatgrass was the male parent were
also observed.
113
INTRODUCTION
Jointed goatgrass (Aegilops cylindrica Host; 2n=4x=28, genome CCDD)
is a close relative and an important weed of wheat (Triticum aestivum L.;
2n=6x=42, genome AABBDD). It infests wheat in the Great Plains and
Western United States, the Near East, and parts of Europe. Wheat and jointed
goatgrass can hybridize and naturally produced hybrids have been reported in
Eurasia, Europe and the USA (van Slageren 1994; Guadagnuolo et al. 2001;
Seefeldt et al. 1998; Zemetra et al. 1998). Wheat-jointed goatgrass hybrids are
male sterile but have partial fema'e fertiUty (Mal'ory-Smith et al. 1996; Zemetra
et al. 1998; Wang et al. 2001). Backcrossing of these hybrids by wheat or
jointed goatgrass under natural conditions can result into the production of
backcross derivatives (Snyder et al. 2000). Moreover, self-fertility is gradually
restored in advanced backcross generations (Snyder et al. 2000; Wang et al.
2000, 2001). The co-existence of wheat and jointed goatgrass, their
successful hybridization, the ability of hybrids to backcross, and restoration of
self-fertility of advanced backcross generations suggest a potential for gene
flow between wheat and jointed goatgrass.
Gene flow and its direction depend on factors such as hybridization
rates between jointed goatgrass and wheat and backcross frequencies by
each of the parent species (wheat or jointed goatgrass). In earlier studies,
hybridization rates between jointed goatgrass and wheat under field conditions
ranged from 0 to 8% with an average of 1.8% (Guadagnuolo et al. 2001;
Morrison et al. 2002a). Using material collected in northeastern Oregon, USA,
114
Morrison et al. (2002a) evaluated the parentage of hybrids and their firstgeneration backcross (BC1) derivatives by performing root ball (identification of
seed or spikelet remnant) and high molecular weight (HMW) glutenin marker
analyses. These analyses suggested that jointed goatgrass was the
predominant female parent (69%) in the formation of F1 hybrids and that wheat
was the predominant backcross male parent (91%) in the formation of BC1
seeds. However, root ball analysis was possible only in the cases where the
seed/spikelet was still attached to root system of the F1 plant. Hence, female
parentage for only a subset of 55 F1 hybrids could be deduced. Similarly,
HMW glutenins markers provided limited nuclear genome coverage. Thus, the
male parentage for only 51% of the collected BC1 plants was determined
(Morrison et al. 2002a)
Since wheat-jointed goatgrass hybrids are male sterile, seeds produced
on F1 hybrids must be the result of cross pollination. Therefore, the female
parentage of the F1 generation can be deduced from their BC1 progeny using
maternally inherited chioroplast DNA-based markers. Similarly, the male
parentage of BC1 plants can be deduced using co-dominant, nuclear DNAbased markers that distinguish between wheat and jointed goatgrass. In an
attempt to extend earlier analyses (Morrison et al. 2002a), we used chloroplast
and nuclear DNA-based molecular markers to evaluate parentage of 413 BC1
individuals that originated from 127 wheat-jointed goatgrass F1 plants. The
results obtained from this analysis are presented in this chapter.
115
MATERIALS AND METHODS
Plant Material
Seven accessions of jointed goatgrass and 13 wheat cultivars were
used to identify and characterize diagnostic molecular markers (Table 4.1).
With the exception of one jointed goatgrass accession, TK 116, all other
genotypes used were obtained from the site or county where wheat-jointed
goatgrass hybrid plants (and BC1 seeds) had been collected. The wheat
genotypes analyzed were commonly grown cultivars in Oregon (Anonymous,
1995-1999). These cultivars may have either B- or D2-type cytoplasm
(Edwards, 2002) (Table 4.1).
Seeds for 413 BC1 plants were harvested from 127 F1 hybrids. The F1
hybrids were collected from 11 field sites. These sites represent the seven
major wheat growing counties of Oregon. A maximum number of 41 F1 plants
were collected from Site 26a, followed by 17 F1 plants from Site 13b and 15
from Site 27a. The greatest numbers of BC1 plants originating from F1 hybrids
were collected at Sites 13b (94) and 13d (93) (Figure 4.1; Table 4.4). A
detailed description of collections and the location of these sites were reported
by Morrison et al. (2002a).
DNA isolation and Molecular Marker Analysis
DNA was extracted from 20 to 50 mg of leaf tissue as described by
Riera-Lizarazu et al. (2000). Three wheat chloroplast microsatellite markers
116
(Table 4.1; lshii et al. 2001) were used to determine the cytoplasmic donor of
each BC1 plant, while seven D-genome wheat nuclear microsatellite markers
(Table 4.1; Röder et al. 1998; Pestova et al. 2000) were used to identify the
male parent of each individual BC1 plant. Polymerase chain reaction (PCR)
assays were performed in 10 p1 reactions containing lx PCR buffer with 1.5
mM of MgCl2, 0.2 mM of each dNTP, 0.3 U Taq polymerase (Qiagen, Valencia,
CA. USA), 0.2 pM to 0.5pM of each primer and 2% sucrose in 0.04% cresol
red. PCR cycles consisted of an initial denaturation step at 95 °C for 5 minutes,
followed by 40 cycles of 95 °C for 1 minute, 50-60 °C (depending on primers)
for 1 minute, and 72 °C for 1 minute, with final extension at 72 °C for 10
minutes. In the PCR assays, one primer of the pair was labeled with a
fluorescent dye [6-carboxyfluorescein (FAM), or 4,7,2' ,4' , 5', 7'-hexachloro-6-
carboxyflu roscein (HEX), or 4,7,2' ,7'-tetrachloro-6-carboxyfluroscein (TEl)] to
facilitate the genotyping using an automated fragment analyzer, ABI Prism®
3100 Genetic Analyzer or ABI Prism® 377 DNA Sequencer. Software ABI
GeneScan® 2.1 and Genotyper® 2.0 (Applied Biosystems, Foster City, CA.
USA) were used to size PCR amplified fragments based on internal lane
standards [n, n n', n'-tetramethyl-6-carboxyrhodam me (TAM RA) or 6-carboxy,
rhodamine (ROX)J.
Parentage Analysis
Since previous studies have shown maternal inheritance of plastomes
in Triticum and Aegilops (Ogihara and Tsunewaki 1982; Wang et al. 1997), the
117
maternal parent of an F1 plant was deduced from the plastome of its progeny
(BC1). Thus, the species (Ae.
cylindrica
or T. aestivum) which contributed
plastome alleles to the BC1 plant was designated as the cytoplasmic donor or
female parent. To determine the male parent of a given BC1 individual, an
exclusion test was used. If a BC1 plant did not have a nuclear marker allele
originating from T. aestivum then T. aestivum was excluded as the recurrent
male parent in the production of a BC1 plant. Similarly, if a BC1 plant did not
have an allele originating from Ae.
cylindrica
then Ae.
cylindrica
was excluded
as the male parent in the production of a given BC1 plant. If a BC1 plant
showed alleles at all loci from both wheat and jointed goatgrass, the male
parent of a given BC1 plant could not be determined. For each BC1 plant, the
combined results obtained from the exclusion test of all seven D-genome
nuclear markers were used to determine its parentage. The distinctions
between wheat and jointed goatgrass alleles were based on a survey of
accessions from both species (Tables 4.1 and 4.2).
Table 4.1. Accessions of jointed goatgrass and wheat cultivars used for marker characterization
Species
Aegilops cylindrica
Triticum aestivum
Accession/Cultivar
Collection site/ Pedigree
Plasmon types
TK 116 (P1 486249)t
Kars (Turkey)
C
USA/OR 170
05b (Wasco County, Oregon, USA)
D
USA/OR 171
23a (Wasco County, Oregon, USA)
D
USA/OR 173
26a (Sherman County, Oregon, USA)
D
USA/OR 180
20 (Umatilla County, Oregon, USA)
D
USA/OR 181
33a (Umatilla County, Oregon, USA)
D
USA/OR 182
12a (Union County, Oregon, USA)
D
Gene
Cleo/ Pichon//Zenzontli
B
Hill-81
Yamhill I Hyslop
B
MacVicar
Yamhill I McDermid II T.spelta var. Alba 131 Suwon92 IRoedel
/4/NB68513 /Hyslop //Backa
B
o:
Malcolm
Ste phensll63-1 89/Bezostaja
B
Nugaines
(Norini 0/Brevor,Cl 1 3253,Sel. 14)16/(Se13,Cl I 2692,Orfed
/5/(Hybrid 50 Turkey Red/
Florence//Fortyfold/Federation/4/Oro//Turkey
Red/Florencel3lOrollFortyfoldlFederation))/7IBu rt
B
Penewawa
Potam70/Fielder
B
Rely
Tres/Tyee
B
Rod
Luke/Daws//Hil 181
B
Rohde
Paha/Selection72//Daws
B
Stephens
Nord DesprezlPullman Sel. 101,CI 13438
B
Yamhill
HeinesVll/Alba (Redmond)
B
Madsen
VPM1/Moissong5l//2*H ill 81
D2
Hyak
VPM1/Moisson42l//2*Tyee
D2
tThe label in parenthesis indicates germplasm ID for TK 116. Other Ae. cylindrica accessions and wheat varieties were part of
personal collections at Oregon State University.
Plasmon types are based on Tsunewaki (1996), Edwards (2002), and Chapters 2 and 3.
(0
120
RESULTS
Characterization of Microsatellite Markers
The chloroplast microsatellite marker WCt 3 allowed the distinction
of the possible plastome type (B, C, D, and D2) combinations. The
chloroplast markers, WCt 11 and WCt 24 confirmed results obtained with
WCt 3 (Table 4.2). Therefore, these markers were found to be useful in the
parentage analysis of wheat-jointed goatgrass hybrids and their derivatives.
Compared to other jointed goatgrass accessions, TK 116 showed distinct
allele sizes for markers WCt 3 and WCt 24 (Table 4.2). TK 116 was shown
to have a C-type plastome, while other jointed goatgrass accessions have
D-type plastomes (Chapters 2 and 3). Similarly among all wheat cultivars
analyzed, Madsen and Hyak showed unique allele sizes for the chloroplast
microsatellite markers WCt 3 and WCt 11 (Table 4.2). Pedigree information
suggests that the cytoplasm of the cultivars Madsen and Hyak were
derived from the wheat breeding line VPMI. In turn, the breeding line
VPMI has its cytoplasm apparently derived from Ae. ventricosa (2n4X28;
NNDD; D2-type plasmon) (Doussinault et al. 1981). Thus, Madsen and
Hyak have D2-type cytoplasm, while other wheat cultivars used in our
analysis have B-type cytoplasm (Edwards, 2002).
Ae. cylindrica accessions did not show variation for any of the
nuclear markers analyzed, except gwm 437. On the other hand, wheat
cultivars showed variation for all the markers used, except gwm 157.
Table 4.2 Microsatellite marker allele sizes for jointed goatgrass accessions and wheat cultivars
F1
hybrid and BC1 plant collection
sites
Allele size (base pairs)
Chloroplast marker
Accession/
Cultivar
Nuclear marker (wheat chromosome)
gdml26
gwml57
gdml29
gwml9O
gwm325
gwm437
(4D)
(5D)
(6D)
(7D)
WCt 3
WCt 11
WCt 24
(1 D)
(2D)
gwm3
(3D)
TK116
156
167
179
183
97
67
116
229
110
93
USA/OR 170
146
166
184
183
97
67
116
229
110
82
USA/OR 171
146
166
184
183
97
67
116
229
110
91
USA/OR 173
146
166
184
183
97
67
116
229
110
93
USAIOR 180
146
166
184
183
97
67
116
229
110
93
USA/OR 181
146
166
184
183
97
67
116
229
110
93
USA/OR 182
146
166
184
183
97
67
116
229
110
82
Gene
150
172
189
195
103
77
101, 124
197
132
105
HiIl-81
150
171
189
195
103
85
101, 124
199, 207
138
113
MacVicar
150
172
189
195
103
79
101, 124
207
138
115
Malcolm
150
172
189
195
103
79
101, 124
209
138
113
Nugaines
150
172
189
195
103
85
101, 126
207
138
105
Penawawa
150
172
189
195
103
85
101, 124
210
131
101
Rely
150
172
189
195
103
85
101, 124
207
145
105
Rod
150
172
189
195
103
83
101, 124
207
138
97
Rohde
150
172
189
197
103
79
101, 124
204
134
105
Stephens
150
172
189
195
103
77
101, 126
205
138
113
Yamhill
150
172
189
195
103
79
101, 124
198
138
105
Madsen
157
167
189
195
103
85
101, 124
197
134
105
Hyak
157
167
189
195
103
77,85
101, 124
218
148
105
1\)
N)
123
Marker gwm 437 was variable within
Ae. cylindrica
and T. aestivum, while
gwm 157 had only a single allele each in these two species. As all of the
seven nuclear markers used had distinct alleles between wheat and jointed
goatgrass and were co-dominant in nature, these markers were considered
to be useful for the parentage analyses (Table 4.2).
Female Parentage
Of the 413 BC1 plants tested, 67 had a plastome that originated from
wheat (B- or D2- type) and 346 BC1 plants had a plastome that originated
from
Ae. cylindrica
plastome of Ae.
(D-type). None of the BC1 plants had the C-type
cylindrica.
were produced on 16
F1
Of the 67 BC1 plants with wheat cytoplasm, 63
plants with D2-type plasmon, while four were
produced on two F1 plants with B-type plasmon. Thus, a total of 18 F1
plants (14.2%) had wheat as the maternal parent (F1W). On the other hand,
109
F1
plants (85.8%) had D-type cytoplasm, indicating that jointed
goatgrass was their maternal parent (F1J). This analysis suggests that
under field conditions the jointed goatgrass X wheat type
F1
hybrid (F1J) is
more frequent than wheat X jointed goatgrass-type F1 hybrid (F1W; Table
4.3).
F1
plants collected at 8 out of 11 sites had only jointed goatgrass as
the maternal parent, whereas all 15
F1
plants collected from Site 27a and
the single F1 collected from Site 20a had wheat as the female parent. Site
29a was the only location where both wheat and jointed goatgrass served
as the female parent in the production of F1 plants (Figure 4.1, Table 4.4).
124
Male Parentage of BC1 Plants
Of the 413 BC1 plants analyzed, 358 (86.7%) had wheat and 24
(5.8%) had jointed goatgrass as the male parent. Thirty-one BC1 plants
(7.5%) showed nuclear microsatellite alleles originating from both wheat
and jointed goatgrass and their male parent could not be determined (Table
4.3). Based on the female parentage of each hybrid, BC1 plants were
c'assified into two groups consisting of 346 that formed on F1J type hybrids
and 67 that formed on F1W type hybrids. These groupings were used to
determine the relative proportion of four types of backcrosses. A backcross
with the constitution of F1J X wheat occurred at the greatest rate (72.2%),
followed by F1W X wheat (145%), F1J X jointed goatgrass (4.8%), and F1W
Xjointed goatgrass (1%)
Table 4.3. Parentage of
F1
and BC1 plants based on chloroplast and nuclear microsatellite analyses
Type of original F1 based on
chloroplast microsatellite analysist
Jointed goatgrass XWheat (F1J)
No.of
%of
No.of
F1
total
BC1
plants
109
F1
analyzed
346
WheatX Jointed goatgrass (F1W)
l8
85.8
14.2
67
%of subtotal
No. of
(F1J or F1W)
% of total
BC1
BC1
BC1
20
5.8
4.8
Wheat
298
86.1
72.1
Indeterminable
28
8.1
6.8
Subtotal(F1J)
346
100.0
83.8
Jointed Goatgrass
4
6.0
1.0
Wheat
60
89.6
14.5
3
4.5
0.7
67
100.0
16.7
Recurrent male
parent
Jointed Goatgrass
Indeterminable
Subtotal
Total
t Deduced from BC1 progeny of a given
127
F1
100.0
413
(
F1W)
413
100.0
plant
t 16 F1 plants (12.6 %) had D2-type cytoplasm
t\)
Table 4.4. Parentage and collection information of wheat-jointed goatgrass hybrids and BC1 plants by collection site
No. of BC1 plants with male parentage from-
No. of F1
plants
collected
Type of original
F1 plants
(Number)t
No. of BC1
plants
analyzed
Fj(8)
Jointed goatgrass
Wheat
Non-determinant
8
12
0
12
0
Site
05b
County
Wasco
Year of
collection
1998
13b
Wallowa
1998
17
Fj(17)
94
2
83
9
18a
Morrow
1998
5
23
4
13
6
20a
Umatilla
1998
1
Fij(5)
F1j(1)
3
0
3
0
13d
Wallowa
1999
12
Fj(12)
93
1
85
7
23a
Wasco
1999
12
Fij(12)
26
3
23
0
26a
Sherman
1999
41
Fj(41)
80
4
71
5
27a
Sherman
1999
15
Fiw(14+1)
54
4
47
3
29a
Gilliam
1999
3
F(1), Fiw(2)
11
0
11
0
33a
Umatilla
1999
11
Fj (11)
15
5
9
1
39a
Union
1999
2
Fj(2)
2
1
1
0
413
24
358
31
Total
127
t Female parent determination were based on chloroplast microsatellite analysis
stands for jointed goatgrass X wheat F1 hybrid
§ F1w stands for wheat X jointed goatgrass F1 hybrid
hybrids were produced with D2-type cytoplasm containing wheat cultivars
11 These F1
0)
128
DISCUSSION
The evaluation of 127
F1
hybrids and their 413 BC1 derivatives to
characterize patterns of mating between wheat and jointed goatgrass
represents the largest set of wheat-jointed goatgrass hybridization material
analyzed to date. The plant material was collected from the major wheat
growing areas of Oregon. Since the plant material collected was distributed
across sites, results obtained should provide a clearer picture of the wheatjointed goatgrass hybridization dynamics at these locations.
The F1J type hybrids (jointed goatgrass X wheat) occurred at a rate
of 85.8%. The greater proportion of F1J compared to F1W hybrids was also
observed by Morrison et al. (2002a), but at the rate of 70% of total BC1
plants. Because the material used by Morrison et al. (2002a) was a subset
of the material used in the present study, the difference in estimated
frequency of F1J is probably due to differences in the method used to
deduce female parentage and the number of individuals studied. Of the 413
BC1 plants analyzed, 86.7% had wheat as their male parent (BC1W), while
jointed goatgrass was the male parent for 5.8% of BC1 plants (BC1J).
Results with respect to male parentage of BC1 plants, were similar to those
reported by Morrison et al. (2002a) and Crèmieux (2000), where they
observed wheat as the major male parent for 91% and 90% of BC1 plants,
respectively.
The greater occurrence of F1J type hybrids and F1J X wheat or F1W
X wheat plants may have various explanations. The plant material was
129
collected from field sites where the wheat plant density was much greater
than that of jointed goatgrass. The unequal proportion of plant densities
would result in a higher proportion of wheat pollen, increasing the chances
for wheat to pollinate jointed goatgrass and F1 hybrids. However, because
the site of collections of the
F1
plants were within fields, it is possible that
the majority of F1W type hybrids were harvested with wheat in the previous
generation.
All of the F1J hybrids had D-type plastome, while F1W type hybrids
had either D2-or B-type plastomes (Table 4.3). The involvement of only Dtype jointed goatgrass in the production of F1J hybrid was possibly due to
the lower frequency of C-type plastome in Ae.
cylindrica
populations
(Chapter 2 and 3). F1W hybrids with D2-type plastome were observed at a
greater frequency than those with B-type plastome. The majority of the
wheat cultivars grown in Oregon have B-type cytoplasm, so the relative
frequency of D2-type F1W is disproportionate to the relative frequency of
D2-type wheat cultivars grown in the area (Anonymous, 1995-1 999). The
F1W hybrids with D2-type cytoplasm were observed at two sites, 27a and
29a (Table 4.3). At Site 29a where F1W hybrids with only D2-type plastome
were found, the wheat cultivar Hyak (D2-type plastome) was grown a year
before the
F1
hybrids were collected. At Site 27a both B- and D2- type
plastome F1W hybrids were found. However, the majority of F1W hybrids at
Site 27a had D2-type plastome. This biased occurrence of F1W hybrids
with D2-type plastome at Site 27a may be due to preferential formation or
survival of F1W hybrids with D2-type plasmon. The preferential presence of
130
F1W plants with D2-type cytoplasm may be due to the involvement of
nucleo-cytoplasmic interactions during the formation or survival of F1W
hybrids. Nucleo-cytoplasmic interactions have been previously observed in
interspecific hybridization involving wheat (Tsunewaki 1996). Thus, the
reason for prevalence of F1W hybrids with D2-type cytoplasm compared to
F1W hybrids with B-type cytoplasm needs to be further evaluated.
The determination of male parentage in the BC1 plants was based
on unique alleles of seven nuclear microsatellite markers for wheat and
jointed goatgrass. However, the male parentage of 7.5% or 31 BC1 plants
could not be determined because they contained alleles of wheat and
jointed goatgrass for all seven nuclear markers. Since the probability for
heterozygosity for all seven markers is low (0.5), we believe that these
BC1 plants represent cases, where an unreduced gamete from a hybrid
combined with a gamete either from wheat or jointed goatgrass. Unreduced
gametes can form in interspecific hybrids through meiotic non-reduction or
restitution (Kihara and Lilienfeld 1949; Maan and Sasakuma 1977). In the
cases where complete meiotic restitution has occurred, gametes of the
hybrids carry the entire genetic complement of both parents. Of the 37
wheat-jointed goatgrass BC1 plants studied by Crèmieux (2000), five plants
(13.5%) were reported to have been produced from completely restituted
hybrid gametes. Similarly, Wang et al. (2002) reported two of the 20 BC1
plants (10%) were the product of complete meiotic restitution. Therefore,
our failure to establish the male parentage of some BC1 plants may be due
131
to complete meiotic restitution in the wheat-jointed goatgrass hybrids from
which they originated.
This study provides a comprehensive view of hybridization
dynamics between wheat and jointed goatgrass under field conditions.
Production of BC1J (F1W X jointed goatgrass or F1J X jointed goatgrass)
plants under field conditions would enable gene flow from wheat to jointed
goatgrass. Therefore, the results obtained from this study could be
important in designing better management strategies in herbicide-resistant
wheat fields (Lazar et at. 2003; Haley et al. 2003) to avoid transfer of the
resistance gene from wheat to jointed goatgrass. Even though the
frequency of backcross offspring of the BC1J type is low (5.8%) under field
conditions, the absolute number of these events is dependent on the
frequency of hybrids. Wheat and jointed goatgrass hybridize at an average
rate of 1.8% so if jointed goatgrass in the wheat fields goes unchecked,
there will be an increase in the number of hybrids produced per unit area
(Guadagnuolo et al. 2001; Morrison et at. 2002a; Morrison et al 2002b).
Therefore, we emphasize that in order to avoid gene flow from herbicideresistant wheat to jointed goatgrass, guidelines for growing herbicideresistant wheat cultivars should be followed. These guidelines include
planting jointed goatgrass free wheat seed and control of jointed goatgrass
plants present in the wheat field and adjacent areas.
132
ACKNOWLEDGEMENTS
We acknowledge funding support to conduct this research from the
National Research Initiative Competitive Grants Program, USDA (Grant#
2001-35320-09918). We also would like to thank wheat growers who
kindly participated in this research.
133
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136
CHAPTER 5
CONCLUSIONS
Jointed goatgrass (Acgilops cylindrica; 2n=4x=28; CCDD) is an
agriculturally important species because of its invasion of crop fields as a
weed, its utility as a source of biotic and abiotic stress resistance genes in
wheat improvement, and its role in crop-to-weed gene movement. The
aims of this dissertation research were to increase our knowledge with
respect to cytoplasm and nuclear diversity in jointed goatgrass as well as
its population structure, and to use this information to better understand
hybridization dynamics between wheat and jointed goatgrass under field
conditions.
The first objective of this research was to evaluate the nature of
cytoplasmic and nuclear variation in Ac. cylindrica. In the present analyses,
the nature of cytoplasmic and nuclear variation in Ac. cylindrica was
evaluated along with its progenitors, Ac. markgrafii and Ac. tauschii, using
chloroplast and nuclear microsatellite markers (Chapters 2 and 3). In these
studies, Ac. cylindrica was found to have lower plastome and nuclear
variability compared to its progenitors. Furthermore, variability for nuclear
microsatellite markers within Ac. cylindrica was found to be greater than for
chloroplast microsatellite markers (Chapters 2 and 3).
137
When Ac. cylindrica was evaluated for cytoplasmic variation, it was
found to have either C- or D-type plastomes (Chapter 2). This was
unexpected as Ac. cylindrica has been suggested to only have D-type
cytoplasm (Maan 1976; Tsunewaki 1996; Wang et al. 1997; Wang et al.
2000a). Ac. cylindrica with the C-type plastome (C-type Ae. cylindrica) was
found to occur at a lower frequency than Ae. cylindrica with the D-type
plastome (D-type Ae. cylindrica) (Chapter 3). Furthermore, the nuclear
genomes of C-and D-type Ac. cylindrica were found to be very closely
related (Chapter 3). This led to the suggestion that the presence of C- type
plastome in Ac. cylindrica was probably not due to the reciprocal
hybridization between Ae. markgrafii and Ae. tauschii during the formation
of Ac. cylindrica. Instead, it is suggested that the C-type plastome in Ac.
cylindrica was introgressed after Ac. cylindrica had formed.
In earlier studies, the D genomes of Ac. cylindrica and T. aestivum
were speculated to be contributed from different biotypes of Ac. tauschii
(Badaeva et al. 2002; CaIdwell et al. 2004). Researchers have further
suggested that the D genome of T. aestivum was closely related to Ac.
tauschiissp. strangulata (Lubbers et al. 1991; Dvorak et al. 1998; Pestsova
et al. 2000). In this study, the D genome and D-type plastome of Ac.
cylindrica were found to be more closely related to Ac. tauschii ssp.
tauschii than to Ac. tauschii ssp. strangulata (Chapter 2). The close
relationship observed between Ac. cylindrica and Ac. tauschii ssp. tauschii
138
was confirmed using model- and genetic distance-based clustering
methods (Chapter 3). These results were consistent with the work of
Badaeva et al. (2002) and CaIdwell et al. (2004) and the observation that
among the two subspecies of Ae. tauschii only Ae. tauschii ssp. tauschii
shares its distribution range with Ae. markgrafii (van Slageran 1994;
Dvorak et al. 1998; Ohta 2000). Therefore, it can be concluded that the D
genome and D-type plastome of Ae. cylindrica were derived from Ae.
tauschii ssp. tauschii (Chapters 2 and 3).
The second objective of this research was to evaluate the
population genetic structure in Ac. cylindrica. For this purpose, 173 Ac.
cylindrica accessions collected from 12 geographic regions were analyzed
with nuclear and chloroplast microsatellite markers (Chapter 3). Of the 12
regions, nine were located in Ae. cylindrica's native area of distribution (Ri
to R9), while three regions were from the USA (US1 to US3). Data
generated in the population genetic structure assessment were also
analyzed for regional genetic diversity. Ac. cylindrica accessions collected
from a region near Van Lake, Turkey (R5), where the distribution of Ac.
tauschii ssp. tauschii and Ae. markgrafii overlap, showed a substantial
amount of Ae. cylindrica allelic diversity. Thus, the area near Van Lake,
Turkey is proposed as the area where Ac. cylindrica originated.
In order to evaluate the population genetic structure, nuclear
microsatellite marker data were analyzed using the model- and genetic
139
distance-based clustering methods (Chapter 3). In these analyses,
accessions from each of the 12 regions were found to have membership to
multiple subpopulations or clusters, suggesting a lack of regional genetic
structure in Ae. cylindrica. Furthermore, comparisons of the Fst and Rst
estimates of population differentiation for pair-wise comparisons among
regions, together with the results obtained from model- and genetic
distance-based clustering, suggested that migration of Ae. cylindrica has
occurred among regions. The migration of Ae. cylindrica accessions
among regions has probably shaped the observed subpopulation
membership patterns of a given region and has possibly led to a lack of
regional genetic structure in Ae. cylindrica (Chapter 3).
Results presented in Chapter 3 also provide information on the
sources and diversity of Ae. cylindrica accessions from the USA. Ae.
cylindrica accessions in the USA showed an intermediate level of diversity
compared to accessions from regions of the native range. These results
further suggest that Ae. cylindrica accessions in the USA probably
originated from more than a single area. In the present study, close
similarity was observed between the accessions from the USA and
accessions from three geographic regions
central Anatolia (R2), central
East Turkey and western Armenia (R6), and Caucasia (R7). Therefore, the
R2, R6, and R7 regions were suggested to be the primary sources for
accessions collected in the USA (Chapter 3).
140
Ac. cylindrica may have been introduced in the USA through the
importations of hard red winter wheat by immigrants, researchers at the
United States of Department of Agriculture, and/ or private millers from
southern Russia (present day eastern Europe) into the Great Plains
(Mayfield 1927; Johnston and Parker 1929). However, some chioroplast
and nuclear alleles observed in the accessions from the USA were unique
(Chapter 3). Thus, it is possible that the region of Ac. cylindrica's native
range, which has been suggested to be the source of Ac. cylindrica in the
USA, did not directly contribute accessions to the USA but they were the
source of accessions for site(s) from where wheat imports to the USA
occurred. A survey of accessions from adventive sites in eastern Europe
might clarify this issue.
In the present analysis, accessions from the Great Plains (US1) had
higher allelic diversity than other regions of the USA. The results also
suggest that the Great Plains was a source of Ae. cylindrica for other
regions in the USA. Furthermore, the close nuclear genetic relationship
observed between most of the C-type Ae. cylindrica accessions from the
USA and comparisons of population membership patterns suggested that
the spread of Ae. cylindrica in the USA has experienced founder effects
(Chapter 3).
The last objective of this research was to understand hybridization
dynamics between wheat and jointed goatgrass under field conditions. In
141
order to achieve this objective, the molecular marker (chloroplast and
nuclear) information generated in Chapters 2 and 3 was used to identify
markers to perform the parentage analysis on wheat-jointed goatgrass firstgeneration backcross (BC1) plants (Chapter 4). The uniparental maternal
inheritance of plastomes in Triticum and Aegilops genera allowed the
female parentage determination of F1 hybrids from the BC1 plants using
chioroplast markers. A total of 413 BC1 plants obtained from 127 F1
hybrids, naturally formed in Northeast Oregon, were analyzed (Chapter 4).
In this study, jointed goatgrass was found to be the female parent
for the production of 85.8% wheat-jointed goatgrass hybrids (F1J).
However, when jointed goatgrass was the pollinator for 14.2% of the
hybrids (F1W), the wheat cultivars with the B-type cytoplasm (B-type
cultivars) were the female parents for 1 .6% of the hybrids, while cultivars
with the D2-type cytoplasm (D2-type cultivars) were the female parents for
12.6% of F1 hybrids. Since B-type cultivars are grown at a higher rate in
Northeast Oregon, the occurrence of D2-type cultivars as a major wheat
female parent in the production of hybrids was unexpected and suggested
a preference for D2-type over B-type wheat in the formation of hybrids
where wheat was the female parent (Chapter 4).
Of the 413 BC1 plants evaluated, 358 (86.7%) had wheat as the
recurrent male parent, while 24 BC1 plants (5.8%) were produced by
backcrossing from jointed goatgrass (Chapter 4). The bias for wheat pollen
142
load in a field probably accounts for wheat being the major pollinator for the
production of hybrids and BC1 plants under field conditions. The male
parentage for 31 BC1 plants (7.5%) could not be resolved. Thus, this study
provides a comprehensive view of hybridization dynamics between wheat
and jointed goatgrass under field conditions. The production of BC1J type
backcross derivatives (F1W X jointed goatgrass or F1J X jointed goatgrass)
under field conditions would enable gene flow form wheat to jointed
goatgrass. Even though the observed frequency of BC1J type backcross
derivatives was low (5.8%) under field conditions, the absolute number is
dependent on frequency of wheat-jointed goatgrass hybrids, which in turn,
depends on the density of jointed goatgrass in wheat fields. Therefore,
control of jointed goatgrass present in the wheat and adjacent areas and
planting of jointed goatgrass free wheat seed, as advised in the guidelines
for growing herbicide resistant wheat cultivars, should be thoroughly
followed in order to avoid gene flow from wheat to jointed goatgrass.
143
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153
APPENDICES
Appendix I
List of accessions along with their area of origin and geographical coordinates of collection sites.
Geographical
coordinatesc
Species8
Region
assigned
Accession 1Db
New ID assigned
Area of origin
Ri
P1176853
Ri-CL1
P1 542179
P1 551082
P1 344778
P1 374378
R1-CL2
Latitude
Longitude
R1-CL12
R1-CL13
Turkey
Turkey
Greece
Serbia
Serbia
Turkey
Turkey
Turkey
Turkey
Bulgaria
Bulgaria
Greece
Greece
40.95
39.35
39.95
44.02
42.48
40.95
40.23
41.83
41.67
43.20
42.02
39.95
40.52
28.82
26.75
21.37
20.92
21.50
28.82
28.20
27.28
27.05
27.88
23.65
21.37
21.27
R2-CL14
R2-CL15
R2-CL16
R2-CL17
Turkey
Turkey
Turkey
Turkey
40.27
38.37
39.58
40.48
40.25
37.70
30.93
33.67
Ae. cylindrica
G 404
IG 47699
1G47753
1G47754
IG 47816
1G48325
1G107273
PC2
R2
P1172357
P1 554201
Pt 573363
Pt 573366
Ri-CL3
R1-CL4
R1-CL5
R1-CL6
Ri-CL7
R1-CL8
R1-CL9
R1-CL1O
Ri-CL11
(-n
R2-CL32
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
40.03
39.48
39.85
40.82
40.48
40.33
39.92
39.13
38.83
38.67
38.97
39.17
38.42
39.93
39.45
32.92
32.57
32.82
32.98
33.68
32.67
33.25
33.33
32.08
36.25
35.60
37.50
39.33
32.93
32.50
P1 573367
P1 407639
R2-CL18
R2-CL19
Pt 554219
R2-CL2O
R2-CL21
P1 573364
Pt 573365
IG 47857
IG 47870
IG 47882
IG 47906
IG 47922
IG 47927
IG 47938
IG 47959
P1 573368
P1 573369
R2-CL22
R2-CL23
R2-CL24
R2-CL25
R2-CL26
R2-CL27
R2-CL28
R2-CL29
R2-CL3O
R2-CL31
R3
IG 46621
1G48584
IG 48789
1G110842
R3-CL33
R3-CL34
R3-CL35
R3-CL36
Syria
Jordan
Lebnon
Lebnon
33.92
31.78
34.47
34.20
36.70
36.80
36.33
36.08
R4
P1 486236
P1 486237
P1 486238
P1 554206
P1 554209
P1 254864
R4-CL37
R4-CL38
R4-CL39
R4-CL4O
R4-CL41
Turkey
Turkey
Turkey
Turkey
Turkey
R4-CL42
Iraq
37.30
37.20
37.33
37.23
37.78
37.12
44.57
44.62
44.53
44.65
44.33
42.68
01
01
R5
P1 554230
P1 486239
1G49132
R4-CL43
R4-CL44
R4-CL82
Turkey
Turkey
P1172683
Fl 486241
Pt 554225
R5-CL45
R5-CL46
R5-CL47
R5-CL48
R5-CL49
P1 574461
P1 486243
R5-CL5O
R5-CL51
IG 48032
Unknown
R5-CL52
R5-CL53
R5-CL54
R5-CL55
R5-CL56
R5-CL57
R5-CL58
R5-CL59
P1 486244
P1 486245
R5-CL6O
R5-CL61
IC 48754
P1172358
P1 486242
P1 554203
P1 486235
P1 486240
P1 554212
P1 554213
Pt 554226
P1 554232
R6
P1 486246
P1 486248
P1 486250
Iran
37.13
37.78
38.20
44.52
44.33
46.58
R5-CL62
Turkey
Turkey
Turkey
Turkey
Turkey
Azerbaijan
Turkey
Armenia
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Armenia
38.93
38.58
38.83
38.30
38.40
39.25
38.92
39.40
38.30
38.58
38.82
38.92
38.42
38.53
38.00
39.62
39.70
39.83
44.03
43.93
43.43
43.17
42.60
45.50
43.60
45.52
43.17
43.55
43.42
43.60
43.30
43.33
43.00
44.18
44.08
44.83
R6-CL63
R6-CL64
R6-CL65
R6-CL66
Turkey
Turkey
Turkey
Turkey
40.05
40.15
40.12
39.83
42.18
43.37
42.67
41.80
0)
P1 349035
P1 486247
P1 486249
R7
P1 574462
P1 314406
P1 428560
P1 428561
IG 48030
IG 48031
IG 48260
IG 48277
R8
Pt 276976
G 406
IG 49083
IG107058
R9
P1 392331
P1 298891
P1 298893
P1 314185
P1
IG
IG
IG
IG
568162
48495
48529
48541
48549
R6-CL67
R6-CL68
R6-CL69
Armenia
Turkey
Turkey
R7-CL7O
R7-CL71
R7-CL72
R7-CL73
R7-CL74
R7-CL75
R7-CL76
R7-CL77
Azerbaijan
Georgia
Georgia
Georgia
Georgia
Azerbaijan
Daghestan
Daghestan
R8-CL78
R8-CL79
R8-CL81
R8-CL83
R9-CL8O
R9-CL84
R9-CL85
R9-CL87
R9-CL88
R9-CL89
R9-CL9O
R9-CL91
R9-CL92
40.50
40.13
40.18
45.00
43.07
42.63
40.50
47.00
41.72
44.78
42.00
43.50
43.50
44.15
47.05
42.00
41.42
39.28
41.93
42.20
48.37
47.92
Iran
Iran
Iran
Iran
36.27
50.00
35.63
47.15
36.17
50.33
33.95
50.08
Uzbekistan
Afghanistan
Afghanistan
Uzbekistan
Uzbekistan
Turkmenistan
Turkmenistan
Uzbekistan
Uzbekistan
41.00
64.00
35.72
64.90
35.85
64.52
41.47
69.55
41.70
38.17
70.10
58.37
38.25
56.33
40.83
68.50
39.97
67.50
-
IG 48558
IG 48562
IG 48569
Unknown
IG 48914
P1 499259
USI
CO-Ol
CO-02
CO-04
CO-05
CO-06
CO-07
CO-09
CO-lO
CO-Il
CO-12
CO-13
CO-42
CO-17
CO-18
CO-19
CO-21
CO-26
CO-27
CO-41
CO-44
R9-CL93
R9-CL94
R9-CL95
R9-CL96
R9-CL97
R9-CL86
Tadjikistan
Tadjikistan
Uzbekistan
Uzbekistan
US1-CL98
US1-CL99
US1-CL100
Nebraska
Nebraska
Nebraska
Nebraska
Nebraska
Nebraska
Nebraska
Oklahoma
Oklahoma
Oklahoma
Oklahoma
Oklahoma
Colorado
Colorado
Colorado
Colorado
Colorado
Colorado
Colorado
Colorado
US1-CL1O1
US1-CL1O2
US1-CL1O3
US1-CLIO4
US1-CL1O5
US1-CL1O6
USI-CL1O7
USI-CL1O8
US1-CLIO9
US1-CL11O
USI-CL111
US1-CL112
USI-CL113
US1-CLII4
US1-CL115
USI-CL116
USI-CLII7
Iran
China
39.45
40.08
40.77
40.93
37.47
35.00
68.13
69.12
70.67
70.03
57.33
105.00
41.11
-102.33
-102.34
-102.99
-103.66
-103.66
-103.66
-103.00
NA
NA
NA
NA
-97.41
-105.07
-102.74
-102.63
-103.07
-104.09
-102.35
NA
-102.96
41.62
41.22
41.23
41.23
41.87
42.83
NA
NA
NA
NA
35.55
40.56
38.43
40.64
40.16
38.84
38.82
NA
40.15
(n
03
US2
US1-CL132
US1-CL133
Colorado
Colorado
Kansas
Kansas
Kansas
Kansas
Wyoming
Wyoming
Wyoming
Wyoming
South Dakota
Nebraska
Colorado
Nebraska
Colorado
NA
NA
37.65
39.66
38.53
38.88
41.15
41.15
41.18
41.76
43.36
41.22
40.56
42.83
40.64
US2-CL128
US2-CL134
US2-CL135
US2-CL136
US2-CL137
US2-CL138
US2-CL139
US2-CL14O
US2-CL14I
US2-CL142
US2-CL143
Montana
Idaho
Idaho
Idaho
Utah
Utah
Utah
Utaft
Utah
Idaho
Utah
46.10
43.19
42.56
CO-52
CO-56
CO-24
CO-25
CO-49
CO-50
CO-35
CO-36
CO-37
CO-38
CO-51
CO-03
CO-16
CO-08
CO-20
US1-CL118
US1-CL119
US1-CL12O
US1-CL12I
US1-CL122
US1-CL123
US1-CL124
US1-CL125
US1-CL126
US1-CL127
CO-40
CO-14
CO-15
CO-43
CO-29
CO-30
CO-32
CO-47
CO-48
PC 1
CO-31
USI-CL129
USI-CL13O
US1-CL13I
42.01
41.51
41.51
41.69
41.30
40.33
NA
41.51
NA
NA
-98.11
-99.57
-99.31
-98.70
-104.66
-104.66
-104.07
-104.82
-103.14
-102.99
-105.07
-103.00
-102.63
-108.88
-112.34
-114.46
-111.81
-112.02
-112.02
-111.75
-111.92
-111.16
NA
-112.02
(C
US3
FC 121
US2-CL144
Utah
CO-22
CO-23
CO-34
CO-45
CO-46
CO-28
CO-53
CO-54
PC 3
FC 105a
FC 105b
US3-CL145
US3-CL146
US3-CL147
US3-CL148
US3-CL149
US3-CL15O
US3-CLI51
US3-CL152
US3-CL153
US3-CL154
US3-CL155
US3-CL156
US3-CL157
US3-CL158
US3-CL159
US3-CL16O
US3-CL161
US3-CL162
US3-CL163
US3-CL164
US3-CL165
US3-CL166
US3-CL167
US3-CL168
US3-CL169
Washington
Washington
Washington
Washington
Washington
Oregon
Oregon
Oregon
Washington
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
Oregon
FC223a
FC 107a
FC 226
FC 227
FC 109
FC 229
FC118a
FC231
FC 246
FC 120
FC 233a
FC112
FC239a
FC 242a
NA
NA
46.73
-117.16
-117.88
-118.38
-117.05
-117.53
-119.82
-119.82
-120.69
NA
-121.18
-121.18
-121.18
-120.72
-120.70
-120.73
-120.18
-120.18
-119.82
-119.56
-118.79
-118.79
-118.79
-117.92
-117.92
-117.92
46.81
47.13
46.34
46.43
45.50
45.50
45.41
NA
45.60
45.60
45.60
44.91
45.59
45.48
45.23
45.23
45.50
45.35
45.67
45.67
45.67
45.57
45.57
45.57
C)
C
FC 113b
US3-CL17O
FC114
FC237
FC 350
Ae. tauschii
ssp. tauschii
ssp. strangulata
ssp. strangulata
ssp. tauschii
ssp. tauschii
ssp. tauschii
ssp. strangulata
ssp. strangulata
ssp. strangulata
ssp. tauschii
ssp. tauschii
ssp. strangulata
ssp. strangulata
ssp. tauschii
ssp. tauschii
ssp. tauschii
ssp. tauschii
ssp. tauschii
ssp. tauschii
ssp. tauschii
84TK154-043
G 1278
G 1279
G 435
G 5792
AE1039/95
AE145/96
AE1 84/78
AE246/76
AE257/76
AE276/00
AE457/94
AE498/79
AE499/81
TA10143
TA10144
TA10145
TA10146
TA1 588
TA2460
US3-CL17I
US3-CL172
US3-CL173
Oregon
Oregon
Oregon
Washington
45.90
45.57
45.93
NA
TU-1
Turkey
TU-2
TU-3
TU-4
TU-5
TU-6
TU-7
TU-8
TU-9
Iran
Iran
NA
37.38
NA
35.70
NA
NA
NA
NA
NA
NA
NA
41.69
NA
NA
35.31
35.37
35.37
36.53
38.5
NA
TU-1 0
TU-1 1
TU-12
TU-1 3
TU-14
TU-15
TU-16
TU-17
TU-18
TU-19
TU-20
Afghanistan
China
Tadjikistan
Azerbaijan
Iran
Uzbekistan
Kyrgyzstan
Afghanistan
Georgia
Dagestan
Turkmenistan
Syria
Syria
Syria
Syria
Turkey
Iran
-117.31
-117.53
-118.39
NA
NA
49.28
NA
64.25
NA
NA
NA
NA
NA
NA
NA
44. 80
NA
NA
38.45
38.45
38.45
38.14
43.3
NA
0)
Ae. markgrafii
var. polyathera
var. markgrafii
var. polyathera
var. markgrafii
var. markgrafii
var. polyathera
var. markgrafii
var. markgrafii
G 591
MK-1
84TK159-036
G 758
KU0006-2(A)
KU5472
KU5852(B)
KU5864 (C)
KU5871(D)
MK-2
MK-3
MK-4
MK-5
MK-6
MK-7
MK-8
Turkey
Turkey
Unknown
Syria
Iraq
Turkey
Turkey
Greece
37.06
38.03
NA
37.13
35.54
40.65
40.266
NA
37.33
28.92
NA
36.12
44.84
35.83
28.357
NA
T. aestivum
Chinese Spring
AE-1
China
NA
NA
84TK593
AE-2
Turkey
43.83
39.05
Madsen
AE-3
USA
NA
NA
a
Ae. markgrafii varieties (var.) and Ae. tauschll subspecies (ssp.) designations were based on passport data, PESTOVA et
al. (2000), OHTA (2000, 2001), and our own observations.
b
The first letter(s) of the germplasm ID makes reference to the sources of the germplasm. Accessions starting with "G"
were obtained from Dr. J. Giles Waines, University of California, Riverside, CA, U.S.A.; "KU" accessions were obtained
from Dr. Shoji Ohta, Fukui Prefectural University, Japan; "AE" accessions were obtained from Institute of Plant Genetics
and Crop Plant Research (IPK), Germany; "TA" accessions were obtained from Wheat Genetic Resource Center, Kansas
State University, KS, U.S.A.; "IG" accessions were obtained from the International Center for Agricultural Research in the
Dry Areas (ICARDA), Aleppo, Syria; "P1" and "84" accessions were obtained from U.S. Department of Agriculture,
National Small Grains Collection, Aberdeen, ID, U.S.A.; FC, PW and PC (personal collections) accessions are maintained
at Oregon State University, USA.; "CO" accessions were obtained from Dr. Phillip Westra, Colorado State University, Co,
USA. Chinese Spring and Madsen wheat cultivars were obtained from Wheat Breeding Program, Oregon State University,
USA.
C
Longitude and latitude coordinates are in the decimal system. Geographical coordinates should be considered
approximate for the accessions collected in USA. NA indicates that the coordinates were not available.
C)
N.)
163
Appendix 2
Plot of genotypic diversity vs. number of loci. X-axis corresponds to
number of loci, while Y-axis indicates amount genotypic diversity additively
explained by n number of loci.
I
1
I
I
3
I
I
5
I
7
I
9
I
11
I
13
I
I
15
I
17
Number of loci
19
21
23
25