Open Journal of Genomics
Open Journal of Neuroscience, 2011, 1-3
6-AN disrupts embryonic blood-CSF barrier
OPEN ACCESS
Research Article
Gene Sequences Reveal Heterokaryotic Variations and
Evolutionary Mechanisms in Puccinia striiformis, the
Stripe Rust Pathogen
Bo Liu1,2, Xianming Chen2,3, Zhensheng Kang1
1
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University,
Yangling, Shaanxi 712100, PR China
2
Department of Plant Pathology, Washington State University, Pullman, WA 99164- 6430, USA
3
United States Department of Agriculture, Agricultural Research Service, Wheat Genetics, Quality, Physiology and
Disease Research Unit, Pullman, WA 99164-6430, USA
Corresponding Author & Address:
Xianming Chen
361 Johnson Hall, Washington State University, P. O. Box 646430, Pullman, WA 99164-6430, USA; Email:
[email protected]; (Tel): 509-335-8086; (Fax): 509-335-9581
Published: 5th January, 2012
Received: 13th November, 2011
Accepted:
Revised:
5th January, 2012
20th December, 2011
Open Journal of Genomics, 2012, 1-1
© Chen et al.; licensee Ross Science Publishers
ROSS Open Access articles will be distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided that the original work will always be cited properly.
Keywords: Puccinia striiformis, stripe rust, yellow rust, gene sequencing, genetic recombination,
heterokaryosis, phylogenetic relationship
ABSTRACT
Puccinia striiformis (Ps), the causal agent of stripe rust, is an obligate biotrophic fungus with two nuclei in its
uredinial stage. Heterokaryosis has been postulated to be involved in the pathogen variation. To determine the
mechanisms and importance of heterokaryosis in the pathogen evolution, sequences of three genes, betatubulin (BT), elongation factor (EF) and mitogen-activated protein kinase (MAPK), were compared among
different Ps races from the US and China. Of 101 polymorphic base pair sites detected in the three genes, 64
(63%) had heterokaryotic variations, indicating that heterokaryosis is very common in the population of the
stripe rust pathogen. Using the polymorphic base pair sites, a total of 14 genotypes were identified from 21
tested isolates, which were grouped into four sequence lineages. The phylogenetic relationships for the races
revealed that mutation is the major evolutionary mechanism to create genetic variations including
heterokaryotic variation. This is the first report of high heterokaryotic variation at the gene sequence level for
the Ps fungus.
INTRODUCTION
Puccinia striiformis Westend. (Ps) is a fungal
species causing stripe rust (yellow rust) on cereal
crops and various grass species. Puccinia
striiformis f. sp. tritici (Pst) causes wheat stripe
rust, which is one of the most important wheat
diseases worldwide [2, 31, 33]. Stripe rust on
barley, caused by P. striiformis f. sp. hordei (Psh),
Open Journal of Genomics, 2012, 1-1
is also a serious problem of barley production in
many parts of the world [6, 10]. Puccinia
striiformis was commonly assumed to have a
macrocyclic lifecycle but with missing pycnial and
aecial stages until very recently it was shown to be
able to infect some Berberis species [16].
However, the role of sexual reproduction in the
evolution of the pathogen under natural
conditions in the US Pacific Northwest may be
limited [36]. Because the stripe rust fungus is
biotrophic and a transformation system is not
available, mutation and somatic recombination,
which have been considered as the major
mechanisms of genetic variations for Ps [18, 19,
23, 30, 37, 39], have not been demonstrated at
the molecular level.
The
one-celled,
dikaryotic
(n+n)
urediniospores [22, 33] are able to cause large
scale epidemics that can result in significant yield
reduction and poor grain quality. Urediniospores
of the pathogen can be spread by wind for
hundreds of miles and new virulent races
(pathotypes), which are subgroups or biotypes of
the stripe rust pathogen distinguished by their
virulence and avirulence patterns on wheat or
barley cultivars possessing different resistance
genes, can be spread by wind or human activities
between continents [2, 12, 14, 37]. More than 140
Pst races and 80 Psh races have been identified in
the US [1-3, 8, 23]. Numerous races of Pst and Psh
were also reported in other countries [2, 33, 35,
37]. The large number of races shows the rapid
evolution of the pathogen virulence and selections
by host crop cultivars with various resistance
genes.
The genetic diversity of Pst has been
investigated since the 1990s using various
molecular techniques. For the US Pst population, a
high genetic diversity was found among 115
single-spore isolates using the random amplified
polymorphic DNA (RAPD) technique [4]. Later,
using the amplified fragment length polymorphic
(AFLP) technique, recent isolates (collected since
2000) were found to be genetically distinct from
older isolates (collected before 2000) from the
south central US [28]. Extensive diversity was also
found in the Chinese Pst population using DNA
fingerprinting with genome-specific repetitive
sequences [32, 41]. More recently, remarkable
phenotypic and genotypic diversities were
reported in relatively small or large stripe rust
Stripe Rust Heterokaryotic Variations
epidemic regions in Northwest China, using simple
sequence repeat (SSR) markers [26, 29]. Their
results suggest extensive genetic recombination in
the Chinese population. In contrast, relatively low
genetic diversities have been reported using AFLP
markers for the Australian and European Pst
populations as the pathogen appears to be clonal
[11-13].
The mechanisms by which new races and
genotypes are created in Ps are not fully
understood. Mutation has been considered as the
major mechanism for changes from avirulent to
virulent pathotypes [23, 37]. In Australia and New
Zealand, constant monitoring of races shows that
newly detected ones appear to differ from preexisting ones only at single virulence loci,
suggesting a sequential pattern of single gene
mutations for virulence [37]. Re-assortment of
whole nuclei was suggested as a mechanism of
genetic recombination [38, 40]. Such somatic
recombination of whole nuclei during germ-tube
fusion can result in new races, demonstrated by
producing a novel race of Pst through inoculation
of the same plant with mixing urediniospores of
two races [25, 39]. If genetic recombination,
either somatic, sexual or both, is common, the
population should be composed of races having
combinations of virulences from coexisting races.
In North America, races with new virulence factors
often appeared as ones with a narrow virulence
spectrum, but soon recombine presumably with
previously existing races to form more complex
ones [22, 23]. Using a statistical approach,
approximately 30% of North America Pst races
identified thus far was found to be evolved
through recombination [5]. Using co-dominant
SSR markers, a recent study identified most Pst
isolates tested as lineage A, Psh isolates as lineage
B, and many isolates from grasses as lineage C
that appears to be a hybrid of genotypes A and B
[9]. Interestingly, isolates of genotype C were able
to infect some genotypes of both wheat and
barley sets for differentiating Pst and Psh races,
respectively. The results provide strong evidence
that the two closely related formae speciales are
able to recombine. Genetic recombination was
identified between SSR markers, suggesting the
existence of a sexual or parasexual cycle in the Pst
population in Tianshui, China [29]. However, it is
not clear if the genetic recombination is through
sexual or somatic hybridization.
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Open Journal of Genomics, 2012, 1-1
All of the previous studies on Ps
polymorphism using various molecular markers
are based on DNA fragment size. Markers based
on fragment size may not provide accurate
estimates as fragments of the same or similar
sizes may have different origins. Although the
recent studies with SSR markers have provided
some evidence for genetic recombination, little is
known about how Ps has evolved into different
formae speciales and virulence races. None of the
earlier findings were based on individual genes
and sequences. A full-length cDNA library was
constructed using Pst urediniospores [24]. This
Stripe Rust Heterokaryotic Variations
library and the genes with identified functions
have provided opportunities to study how the
fungus has evolved into the complex structures of
populations in both phenotypic and genetic
variations. The objectives of this study were to
identify polymorphic genes for determining the
mechanisms of the pathogen variation, determine
the commonness of heterokaryosis, and
determine evolutionary relationships among
major virulence races of the stripe rust pathogen
in China and the United States through comparing
their sequences of the polymorphic and
heterokaryotic genes.
Table 1. Races of Puccinia striiformis f. sp. tritici (PST) and P. striiformis f. sp. hordei (PSH) used in this study and the
year collected, number and rate of heterogeneous base pairs, polymorphic genotypes, and sequence lineages
Race
Isolate
Virulence on differential cultivarsa
Year
PST-1
PST-45
PST-6
PST-8
PST-11
PST-15
PST-16
PST-25
PSH-19
PSH-12
PST-21
CYR32
CYR27
CYR29
PST-3
PST-78
PST-100
PST-127
CYR31
CYR8
PSH-53
CDL-1-5
CDL-45-1
CDL-6-2
CDL-8-1
CDL-11
CDL-15-3
CDL-16-1
CDL-25-1
PSH94ID-1
PSH93AZ-5
CDL-21-5
CYR32-1B
CYR27
CYR29-S3
CDL-3-1
2K041-Yr9
03-202-10-sp1
07-211-13-sp1
CYR31-3
CYR8
01-248
1,2
1,3,12,13,15
1,6,8,12
1,3,9
1
1,3,6,8,10
1,3,9,11
1,3,6,8,9,10,12
1,3,5,6,7,8
1,2,3,4,5,8
2
1,2,3,5,6,8,9,10,11,12,13,14,15,16,17,18,19,20
1,8,10,11,12,16,17,20
1,2,3,5,6,8,9,10,11,12,13,14,15,16,18,19
1,3
1,3,11,12,16,17,18,19,20
1,3,8,9,10,11,12,16,17,18,19,20
1,2,3,5,6,8,9,10,11,12,13,15,16,17,18,19,20
1,5,8,10,11,12,14,15,16,17,20
1,3,5,8,10,11,12,13,14,15,16,17,20
1,8,9
1963
1990
1974
1975
1976
1977
1977
1982
1995
1993
1980
1994
1980
1985
1964
2000
2003
2007
1993
1960
2001
No. and (%) of
heterogeneous
base pairsb
54 (53.5)
54 (53.5)
57 (56.4)
57 (56.4)
57 (56.4)
57 (56.4)
57 (56.4)
57 (56.4)
57 (56.4)
30 (29.7)
26 (25.7)
26 (25.7)
30 (29.7)
26 (25.7)
21 (20.8)
15 (14.9)
15 (14.9)
0 (0.0)
4 (4.0)
0 (0.0)
1 (1.0)
Polymorphic Heterogeneous Sequence
genotype
genotype
lineage
1-1-1
1-1-1
1-2-1
1-2-1
1-2-1
1-2-1
1-2-1
1-2-1
1-2-1
1-3-2
1-3-3
1-4-4
1-5-2
1-5-5
2-6-6
3-6-7
3-6-8
4-5-9
4-5-2
4-7-10
5-8-11
1-1-1
1-1-1
1-2-1
1-2-1
1-2-1
1-2-1
1-2-1
1-2-1
1-2-1
1-3-2
1-3-3
1-3-4
1-4-2
1-4-4
2-5-4
3-5-3
3-5-4
4-4-4
4-4-2
4-3-4
4-6-5
1
1
1
1
1
1
1
1
1
2
2
2
2
2
3
3
3
4
4
4
4
a
For the PST and CYR races, virulence were tested on US differential cultivars (1 = Lemhi, 2 = Chinese 166, 3 = Heines VII, 4 = Moro,
5 = Paha, 6 = Druchamp, 7 = AvSYr5NIL, 8 = Produra, 9 = Yamhill, 10 = Stephens, 11 = Lee, 12 = Fielder, 13 = Tyee, 14 = Tres, 15 =
Hyak, 16 = Express, 17 = AvSYr8NIL, 18 = AvSYr9NIL, 19 = Clement, and 20 = Compair. PSH races on US barley differential cultivars
(1 = Topper, 2 = Heils Franken, 3 = Emir, 4 = Astrix, 5 = Hiproly, 6 = Varunda, 7 = Abed Binder 12, 8 = Trumpf, 9 = Mazurka, 10 = Bigo,
11 = I 5, and 12 = Bancroft); CYR races were also tested on Chinese differential cultivars (1 = Trigo Eureka, 2 = Fulhard, 3 = Lutescens
128, 4 = Mentana, 5 = Virgilio, 6 = Abbondanza, 7 = Early Premium, 8 = Funo, 9 = Danish 1, 10 = Jubilejina 2, 11 = Fengchan 3, 12 =
Lovrin 13, 13 = Kangyin 655, 14 = Suwon 11, 15 = Zhong 4, 16 = Lovrin 10 and 17 = Hybrid 46 with additions 18 = AvSYr1NIL, 19 =
AvSYr6NIL and 20 =AvSYr7NIL). The Chinese differential virulence formula for CYR 8 is 1, 2, 3, 4, 6, 7, 8, 10 and 14; for CYR 27 is 2, 3,
4, 5, 6, 7, 10, 19 and 20; for CYR 29 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 16, 18 and 19; for CYR 31 is 1, 2, 4, 6, 7, 8, 10, 11, 14, 19 and 20;
and for CYR 32 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19 and 20.
b
The number of heterogeneous base pairs for the isolates were determined with genes (EF, BT and MAPK) and as that of a total
101 polymorphic base pairs.
MATERIALS AND METHODOLOGY
Fungal isolates and genes
A total of 21 Ps isolates representing 21
different races of Pst and Psh (Table 1) were
selected for this study to represent a wide range
of race groups based on previous virulence studies
in the US and China [1-4, 6-8, 22, 34]. For the
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Open Journal of Genomics, 2012, 1-1
Stripe Rust Heterokaryotic Variations
initial experiment, 7 Pst single-spore isolates [4]
and 1 Psh isolate from the US and 2 Chinese Pst
isolates were used for analyzing sequences of 7
genes including elongation factor (EF, clone
80N15), beta-tubulin (BT, clone 58H22), TATA-box
binding protein (TBP, clone 58E6), serinethreonine kinase receptor-associated protein
(STKRAP, clone 70E5), conidiation protein (CP,
clone 10I12), mitogen-activated protein kinase
(MAPK, clone 55B10), and cell wall glucanase
(CWG, clone 70I2). Based on the polymorphic data
of the initial experiment, genes BT, EF and MAPK
(Table 2) were selected for sequencing analysis
with all 21 Ps isolates.
Table 2. Genes and their primers and annealing temperatures used to sequence the full-length of the genes in various
races of Puccinia striiformis
Clone
b
no.
GenBank
accession
BT
58H22
EG374306
EF
80N15
EG374397
MAPK
55B10
EG374277
Gene
a
Primer
c
58H22-F
58H22-R
58H22-WF1
58H22-WF2
58H22-WR1
80N15-F
80N15-R
80N15-WF
55B10-F
55B10-R
55B10-WF
55B10-WR
Sequence
5’-GAAATCGTTCATCTCCAA A-3’
5’-TCGTAACCCTCTTCAACTTC-3’
5’-CTCTTCCGTCCCGACAACTTT G-3’
5’-AAGACTTGTTCAAGCGGGTGG-3’
5’-AGTCCATAGTTCCGGGCTCCA-3’
5’-ACTTCTACAATGGGTAAAGA-3’
5’-ACTACTTCTTGGCACCG-3’
5’-CGTCAAGAAGGTCGGATA-3’
5’-ATGGTCGGCCCTAGCTTT-3’
5’-TTAAAAGTCACGAGTGACGAG-3’
5’-CGTTGGTATCGCGCTCCTGAA A-3’
5’-ATCCGTCTCAAACCCTCTA-3’
a
BT = beta-tubulin, EF = elongation factor and MAPK = mitogen-activated protein kinase/
b
Clone numbers are from the full-length cDNA library of P. striiformis f. sp. tritici [24].
o
Tm ( C)
52
52
55
52
57
51
54
53
56
53
58
56
c
Primer pairs with “F” and “R” were used to amplify the full-length sequence and those with “WF” and “WR” were used to amplify
the fragments for filling the sequences not obtained from sequencing the full length.
DNA extraction
DNA samples of races PST-1, 3, 6, 8, 11, 15,
16, 21, 25 and 45 of Pst and PSH-12 and 19 of Psh
were from the previous studies by Chen et al. [4,
6]. DNA samples of the other isolates (Table 1)
were extracted directly from urediniospores using
a modified cetyltrimethylammonium bromide
(CTAB) procedure as previously described [4]. For
each isolate, 20 mg urediniospores were ground
with sterile sand into a fine powder. The powder
was transferred into a 1.5 mL Eppendorf tube and
500 μL extraction buffer (50 mM Tris-HCl, pH 8.0,
150 mM NaCl and 100 mM EDTA) was added.
After adding 30 μL 20% SDS, 75 μL 5 M NaCl and
65 μL CTAB/NaCl, and mixing thoroughly, the tube
was incubated at 65°C for 60 min. Then the
mixture was extracted with equal volume
saturated phenol (phenol/ chloroform/ isoamyl
alcohol 25:24:1) and 0.1 volume 3 M sodium
acetate (pH 5.3), and centrifuged for 10 min at
13,000 rpm. The top aqueous phase was
transferred to a clean tube. After adding an equal
volume of chloroform, the tube was inverted
gently, and centrifuged for 10 min at 13,000 rpm.
DNA was precipitated by adding equal volume of
isopropyl alcohol and 0.1 volume 3 M sodium
acetate (pH 5.3), and keeping at -20°C for 120 min.
After centrifuging for 30 min at 4°C, the pellet was
rinsed twice with cold 70% ethanol and 100%
ethanol separately, dried and dissolved in 500 μL
TE buffer. The DNA solution was treated with
RNase (final concentration 20 μg/mL) and kept at
37°C for 60 min to completely digest RNA. The
DNA was re-precipitated, rinsed with ethanol,
dried and dissolved in 30 μL of TE buffer. DNA
concentrations were diluted to 20 ng/μL with TE
buffer before storing at -20°C in small aliquots.
PCR and sequencing
Primers for PCR amplification were designed
to sequence the full-length and partial sequences
of the seven selected genes (Table 2). Each 50 μL
amplification reaction consisted of 1× GoTaq Flexi
Buffer, 2 mM MgCl2, 0.2 mM each of dNTP, 0.2
μM of each primer, 2 unit of GoTaq Flexi DNA
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Open Journal of Genomics, 2012, 1-1
Polymerase (Promega, Madison, WI, USA) and 50
ng of template DNA. PCR amplification was
performed under the following conditions: 94°C
for 5 min; 94°C for 1 min, 42 - 60°C for 1 min
depending upon primers, 72°C for 1 - 2 min, 35
cycles; and 72°C for 10 - 15 min. PCR products
were purified using ExoSAP-IT PCR Product Cleanup (Affymetrix, Santa Clara, CA, USA), then directly
applied for sequencing using corresponding PCR
primers. The sequencing reactions were done with
the cycling program; 10 s at 96°C, 15 s at 50°C and
4 min at 60°C for 25 cycles. Dye-labeled fragments
were cleaned by Performa DTR Gel Filtration
Cartridge (Edge BioSystems, Gaithersburg, MD,
USA), following the manufacturer’s instructions.
Sequencing was done by the Sequencing Core
Facility of Washington State University. All the
fragments were sequenced twice by two
directions to avoid misreading.
To verify if the heterogeneous base pairs
were not due to spore contaminations or
sequencing errors, 3 PCR fragments, 1 for each of
the 3 genes that showed polymorphic base pairs,
were purified and ligated into pGEM-T Easy Vector
(Promega, Madison, WI, USA). Twenty positive
clones for each fragment were sequenced. The reamplification, cloning and sequencing were
repeated twice. The chi-squared test was used to
determine two different sequences from a single
isolate fit an expected 1:1 ratio among 20
sequenced clones. Probability (P) values of the chisquared tests were obtained using the “chitest”
formula of the Data Analysis Tool in the Excel of
Microsoft Office (Microsoft, Redmond, WA, USA).
Sequence analyses
The sequence homologies and structures of
the Ps genes were compared with the Pg
homologous genes using the Pg database
(http://www.broadinstitute.org/annotation/geno
me/puccinia_group.1/MultiHome.html).
For each gene, ChromasPro, version 1.33
(Technelysium Pty Ltd, Tewantin, QLD, Australia)
was used for assembling overlapping sequences
from both directions into a consensus gene
genomic sequence, then the split sites were
analyzed by comparing with the cDNA sequences.
CLUSTALX, version 2.0.9 (21) was used for
alignment of the sequences. All final alignments
were edited visually by inspecting all polymorphic
positions and using the chromatograms generated
Stripe Rust Heterokaryotic Variations
during DNA sequencing to confirm the
polymorphism at each site. For phylogenetic
analysis, the maximum parsimony (MP) trees of
each gene were constructed using the MEGA
Version 4.0.2 software package (The Biodesign
Institute, Tempe, AZ, USA) [20]. The consensus
network was done using SplitsTree (version 4.1)
[15, 34].
For easily visualizing genetic relationships
among the 21 races, a three-dimensional diagram
was generated with the DNA sequence
polymorphism values of BT, EF and MAPK as the
three axes. PST-1, which was the earliest race
detected in the US, was used as the starting point
and the genetic distance of each of the remaining
races was calculated for each of the three genes. If
the different base pair site was homogeneous in
both PST-1 and the other race in a pair-wise
comparison, the polymorphic site was treated as
1, while if one of the races had heterogeneous
base pair at the site, the polymorphic site was
treated as 0.5 because one of the heterogeneous
nucleotide was the same as the homogeneous
nucleotide in the other race. This method is
similar to a modified three-principal approach
used in a previous study to generate threedimensional diagrams [6].
RESULTS AND OBSERVATIONS
Length polymorphisms and structures of the
genes
As shown in Table 3, the full-length genomic
DNA sequences of BT, EF and MAPK were 1843,
1952 and 2327 bp and the cDNA sequences were
1347, 1383 and 1443, respectively. Interestingly,
the lengths of genomic DNA sequences of these
genes were all shorter than the Pg homologues
and their cDNA sequences were also shorter
except that the BT cDNAs had the same length.
The lengths of the genes were the same among
the 21 races except the following deletions: 1)
CYR8 and CYR32 had a two-bp deletion in an
intron of the EF gene at base pair positions 107
and 108. 2) PST-21 and PSH-53 had a two-bp
deletion in an intron of the MAPK gene at base
pair positions 190 and 191. 3) Compared to other
isolates, PST-127, CYR31, CYR8, and PSH-53 had a
base pair deletion in an intron at position 385 of
the BT gene. In contrast, 4) PST-3, PST-78, PST100, PST-127, CYR8, CYR31, and PSH-53 had a
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Open Journal of Genomics, 2012, 1-1
nucleotide G insertion in an intron at position 389
Stripe Rust Heterokaryotic Variations
of the BT gene (Supplement Table 1).
Table 3. Lengths and percentages of identical sequences of Puccinia striiformis (Ps) genes and their percentages of
identical sequences to the homologues of P. graminis (Pg) at the DNA, cDNA, and predicted protein levels
Gene a
BT
EF
MAPK
Total or (average)
a
Full-length (bp)
Ps isolates
Pg isolates
DNA cDNA
DNA cDNA
1952 1347
2046 1347
1843 1383
2124 1425
2327 1443
2396 1482
6122 3173
6566 4254
Identical sequences (%)
Among Ps isolates
DNA cDNA Protein
98.05 99.89 100.00
98.75 99.56 99.90
98.15 99.89 99.90
98.32 99.78 99.93
Between Ps and Pg
DNA cDNA
Protein
75.16 91.24
98.88
68.99 87.37
94.95
65.07 78.65
84.18
69.74 85.75
92.67
BT = beta-tubulin, EF = elongation factor and MAPK = mitogen-activated protein kinase.
Fig. 1) Genomic structures and protein domains of Puccinia striiformis genes used in this study. On the left, the exons and introns
are depicted as black boxes and straight horizontal lines, respectively. The numbers above indicates their positions and the arrows
mark approximate sites of primers (Table 2). On the right, arrows indicate major protein domains and the length of amino acid
sequence is shown above. A: mitogen-activated protein kinase (MAPK, 55B10); B: beta-tublin (BT, 58H22); and C: elongation factor
(EF, 80N15).
Splice sites were analyzed corresponding to
the cDNA sequences of the three genes as shown
in Fig. 1A-1C. MAPK (Fig. 1A) had 12 and both BT
(Fig. 1B) and EF (Fig. 1C) had 8 exons. MAPK had
the same intron-exon numbers as those of the Pg
homologue. Furthermore, the position and size of
each intron and exon were similar to those in the
Pg homologue. The BT and EF genes lacked the
last intron of the Pg homologues. The sequence
homologies between Ps and Pg were presented in
Table 3. The results showed that Ps and Pg are
closely related, but substantially different.
Sequence homology
Genes ET, EF and MAPK were all
polymorphic in sequence among the tested Ps
isolates (Table 3, Supplement Table 1). Each of the
genes had a higher similarity between any two Ps
isolates than between Ps and Pg. When compared
among the Ps isolates, the genes had identical
base pairs all above 98.00% at the genomic DNA
level, 99.00% at the cDNA level and 99.90% at the
amino acid level. In contrast, each of the Ps genes
had identical sequences ranging from 65.07%
(MAPK) to 75.16% (BT) at the genomic DNA level,
78.65% (MAPK) to 91.24% (BT) at the cDNA level
and 84.18% (MAPK) to 98.88% (BT) at the amino
acid level with the Pg homologues (Table 3).
A total of 101 DNA base pairs that were
polymorphic among the Ps isolates were detected
in the sequences of the three genes (BT, EF and
MAPK) (Table 4). Detailed information on
positions and nucleotides of polymorphic base
pairs for these genes among Ps isolates can be
found in Supplement Table 1. The 101 base pairs
were mostly discontinuously distributed in each
gene sequence. Continuous base pairs consisted
of only 2 bp or 3 bp with two (one 3 bp and one 2
bp) in BT, three (all 2 bp) in EF and two (both 2 bp)
in MAPK. These results indicated that the
polymorphic base pairs likely originated from
point mutations.
Genes MAPK, BT and EF had 30, 35 and 36
polymorphic base pairs, respectively, among the
tested Ps isolates. Of the 101 base pairs, only 32
(0.5% of the total of 6122 bp) were found in
exons.
The
polymorphisms
resulted
in
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Open Journal of Genomics, 2012, 1-1
Stripe Rust Heterokaryotic Variations
substitutions of six amino acids (Table 4). The
polymorphism rate was 0.01% of the total number
of base pairs, 6122 bp (Table 3), for the three
genes. For individual genes, rates of
polymorphism were 1.29%, 1.79% and 1.95% at
the genomic DNA level and 0.83%, 0.82%, and
0.65% in MAPK, BT, and EF at the cDNA level,
respectively (Table 4). Based on the cDNA
polymorphism rates, MAPK had a higher variation
and a faster evolution speed than BT and EF.
Heterogeneous base pairs within individual
isolates
Of the 101 polymorphic base pairs, 37 were
homogeneous base pairs within isolates but
polymorphic among isolates and 64 were
heterogeneous base pairs within isolates and
polymorphic among Ps isolates (Table 4).
Heterogeneous base pairs were identified with all
three genes in all isolates, except CYR8
(Supplement Table 1). In the sequencing
verification experiments, two distinct, but
expected sequences were obtained from different
clones for each of the three fragments. Moreover,
the two sequences were detected in the expected
1:1 ratio (P = 0.37, 1.00 and 0.65 for BT, EF and
MAPK, respectively). These results clearly showed
that the isolates were truly dikaryotic with
different sequences.
The number of heterogeneous base pairs
varied greatly among the isolates, from 0 in CYR8
to 57 in seven isolates (PST-6, PST-8, PST-11, PST15, PST-16, PST-25 and PSH-19) among the 101
total polymorphic base pairs in the three genes
(BT, EF and MAPK). The number and percentage of
heterogeneous base pairs for the 21 tested
isolates are summarized in Table 1. The
heterogeneous base pair rates also varied greatly
among the three genes from 0.17% in MAPK to
1.63% in EF (Table 4). The heterogeneous base
pairs occurred with a higher frequency (70%) in
introns than in exons. However, those that
occurred in exons were synonymous substitutions
that did not change amino acids. Therefore, these
heterogeneous base pairs would not alter the
gene functions. The patterns and percentages of
heterogeneous base pairs indicated similar
evolutionary relationships among the 21 tested
races based on all detected polymorphic base
pairs as presented below.
Table 4. The numbers of polymorphic base pairs and the rates of heterogeneous DNA base pairs of five genes among
Puccinia striiformis isolates
Genea
BT
EF
MAPK
Total or average
Polymorphic base pairs
Number of bp or aab
Total In exon Amino acids
35
11
0
36
9
1
30
12
5
101 32
6
Rate (%)
DNA level
1.79
1.95
1.29
1.68
cDNA level
0.82
0.65
0.83
0.77
Heterogeneous base pairs
Number of bpb
Rate (%)
Total In exon
DNA level
29
10
1.49
31
8
1.63
4
1
0.17
64
19
1.10
a
BT = beta-tubulin, EF = elongation factor and MAPK = mitogen-activated protein kinase.
b
bp = base pairs for DNA and aa = amino acids.
Genetic relationships among Ps isolates
revealed by polymorphic base pairs
Genes BT, EF, and MAPK separated the 21
tested isolates into 5, 8 and 11 genotypes,
respectively. The evolutionary relationships of the
genotypes identified by BT, EF and MAPK are
shown in Fig. 2A, 2B and 2C, respectively.
When all of the 101 polymorphic base pairs
including the 64 heterogeneous base pairs were
used to determine relationships of the isolates, a
total of 14 genotypes were obtained (Table 1).
PST-1 and PST-45 had a same genotype 1-1-1 (for
cDNA level
0.74
0.58
0.07
0.46
genotypes of BT-EF-MAPK) and seven isolates
(PST-6, PST-8, PST-11, PST-15, PST-16, PST-25, and
PSH-19) belonged to genotype 1-2-1. The
remaining 12 isolates each had a single genotype,
but with various levels of genetic relationships.
When the polymorphic base pairs of the two
house keeping genes (BT and EF) were used to
group all 21 isolates, six sequence groups (Fig. 2D,
Table 1) were obtained. When the MAPK gene
was included with BT and EF, the 21 isolates were
classified into 14 genotypes and more detailed
genetic relationships among the races were
obtained (Fig. 2E).
When only the 64 heterogeneous base pairs
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Open Journal of Genomics, 2012, 1-1
of the three genes were used, the BT gene
classified the 21 isolates into 4 genotypes; EF 6
genotypes, and MAPK 5 genotypes, which were
fewer than the genotypes separated by the total
Stripe Rust Heterokaryotic Variations
of 101 polymorphic base pairs by 1, 2, and 6
genotypes, respectively. However, when these
genes were used together, also 14 different
genotypes were obtained (Table 1).
Fig. 2) Evolutionary trees for 21 selected isolates of Puccinia striiformis. The trees were generated with each of the three genes
using the maximum parsimony method of the MEGA software package version 4.0.2. A: beta-tublin (BT, clone 58H22); B:
elongation factor (EF, clone 80N15); C: mitogen-activated protein kinase (MAPK, clone 55B10); D: house-keeping genes, BT and EF
together; and E: BT, EF, and MAPK together. The numbers before isolates indicate lineage groups and those at branches indicate
the boostrap values in percentage from 1,000 replications.
When the heterogeneous base pairs were
excluded from the 101 polymorphic base pairs
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Open Journal of Genomics, 2012, 1-1
Stripe Rust Heterokaryotic Variations
(using only 37 homogeneous polymorphic base
pair sites), the BT gene classified the 21 isolates
into 4 groups, EF 5 groups, and MAPK 9 groups.
Overall, the 21 isolates formed 12 genotypes, two
genotypes fewer as classified by only the
heterogeneous base pair sites (Table 1). The
relationships of the races determined only by the
heterogeneous base pairs (dendrograms not
shown) were similar to those determined with the
101 base pair sites as shown above.
PSH-53
CYR8
SL 4
PST-127
CYR31
BT
SL 3
20
PST-100
PST-3
19
PST-78
18
17
16
15
14
13
12
CYR32
PST-21
11
SL 2
10
9
CYR29
8
7
CYR27
PSH-12
10
6
17
9
5
8
MA
PK
7
14
4
12
6
SL 1
3
5
4
9
2
6
3
1
2
1
PST-6**
PST-1*
1
2
3
4
7
8
10
15
18
16
13
11
EF
5
Fig. 3) Three-dimensional diagram showing genetic relationships among 21 races of Puccinia striiformis. The diagram was generated
using the DNA sequence polymorphisms of genes encoding beta-tublin (BT), elongation factor (EF) and mitogen-activated protein
kinase (MAPK). The scale of the axes is based on polymorphic base pairs, in which a base pair site different between two
homokaryotic races was treated as 1 while that different between a homokaryotic race and a heterokaryotic race was treated 0.5
as one of the heterokaryotic nucleotides was the same as the nucleotide in the homokaryotic race. * PST-1 and PST-45 and ** PST6, PST-8, PST-11, PST-15, PST-16, PST-25 and PSH-19 had identical sequences of the three genes. Races in each of the sequence
lineages are included in a dash cycle.
The genetic relationships among the stripe
rust races were best visualized in a three-
dimensional diagram (Fig. 3). In this analysis, PST-1
that was the earliest detected Pst race in the US
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Open Journal of Genomics, 2012, 1-1
was used as the starting point and the other 20
races were compared to it with sequence
polymorphism values determined with BT, EF and
MAPK. The four sequence lineages (SLs) have
different levels of polymorphism among races
within each SL. SL 1 consisted of two genotypes
(PST-1 and PST-45 as one genotype and PST-6,
PST-8, PST-11, PST-15, PST-16, PST-25 and PSH-19
as another genotype), which was separated by
only 1.5 polymorphism points by EF. The races in
SL 1 were all collected from the US before 2000.
SL 2 consisted of five races (PSH-12, CYR27,
CYR29, CYR32 and PST-21), each as a different
genotype. These races had identical sequences of
BT, but separated mainly by polymorphic
sequences of MAPK. SL 3 consisted of three races,
PST-3, PST-78 and PST-100 collected from the US
in 1964, 2000 and 2003, respectively. These races
shared unique heterogeneous sequences Y (C+T),
R (A+G), and R at base pair sites 1078, 1276 and
1826 of BT, respectively, and Y at base pair
position 976 of EF. Races CYR8, CYR31, PST-127
and PSH-53 in SL 4 were more diverse. They did
not have any heterogeneous base pairs and
shared unique T, C, T and T at the base pair sites
25, 26, 27, and 29, respectively in BT. SL 1 and SL 2
had identical BT sequences, but separated by
polymorphic sequences in EF. SL 2, SL 3 and SL 4
were separated from each other mainly by
polymorphic BT sequences.
DISCUSSION
Polymorphic sequences of genes are useful in
determinations of genetic variations among
Ps isolates
The primary objective of this study was to
identify polymorphic genes for determining
genetic variations of Ps at the race level with the
emphasis on Pst. Therefore, the isolates used here
could represent the maximum genetic diversity
observed in the US within the wheat stripe rust
pathogen based on previous virulence and
molecular studies [2-4, 6-8, 23, 35]. The US Pst
isolates that were analyzed cover from the earliest
races such as PST-1 and PST-3 [2, 23] to the latest
race such as PST-127 [8], and from the races with
the narrowest virulence spectrum such as PST-11
and PST-21 [2, 22] to those with the widest
virulence spectrum such as PST-127 [8]. The
Chinese races were also selected considering the
year of collection and virulence factors. Use of
Stripe Rust Heterokaryotic Variations
isolates from the two countries could further
increase the pathogen diversity in this study. The
genes selected for this study have three general
functional categories including protein translation
(EF), cell structure and growth (BT) and cell
signaling (MAPK). The three genes produced 101
polymorphic base pair sites, which are suitable for
determining different levels of phylogenetic
relationships among Ps isolates. Based on
polymorphism rates of the three genes, BT and EF
are more conserved than MAPK, and should be
more suitable for classifying races or isolates into
basic phylogenetic lineages while MAPK is more
suitable for separating more closely related
isolates as demonstrated by the different numbers
of genotypes separated by the different genes.
Heterogeneous base pairs are common in Ps
isolates
One of the most important observations of
this study was the high proportion of
heterogeneous base pairs within single isolates. Of
the 101 polymorphic base pair sites, about 63%
were heterogeneous. The percentage of
heterogeneous base pairs of the polymorphic base
pairs was 83% in BT, 86% in EF, and only 13% in
MAPK. The heterogeneous base pairs in this study
were found not to change amino acids and
therefore, should not alter the gene functions.
Such neutral nature could be the reason that the
large number of mutants had been maintained
during the rust evolutionary process over the past
more than 40 years. More importantly, the
heterogeneous base pairs in general revealed
similar genetic relationships among isolates as did
the homogeneous polymorphic base pairs.
Heterokaryosis has long been postulated as
one of the mechanisms for variation in dikaryotic
fungi like rusts. However, direct evidence of the
effect of heterokaryosis on pathogenicity and
virulence of fungal plant pathogens has been
limited. Several papers were published in the
1990s that suggested heterokaryosis as a possible
mechanism of variation after obtaining strains
with new virulence combinations from coinoculation of an albino isolate and eight Chinese
races of Pst [17-19, 27]. Scientists have often
attempted to connect heterokaryosis with somatic
recombination [30, 39] with limited success. Based
on the results of the present study, heterokaryosis
should be considered a general rule for the stripe
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Open Journal of Genomics, 2012, 1-1
rust fungus. Probably, every isolate of the stripe
rust fungus could be heterokaryotic, as we
observed for 20 of the 21 tested isolates.
Point mutation as a major evolutionary
mechanism
The data of this study suggest that point
mutation is the major mechanism for the
sequence variation, including the heterokaryotic
variation within isolates and homokaryotic
variation among isolates. Except for few cases of
two or three continuous base pair sites, the most
of the polymorphic base pairs are not continuous.
If introgenic recombination had occurred, we
would have detected differences of sequence
blocks. It is also worthy to note that only two
nucleotides were found for every base pair site,
indicating that the directions of mutation are not
random toward all of the four nucleotides.
Moreover, the two nucleotides in the most of the
polymorphic base pair sites were not in an equal
1:1 ratio, suggesting clonal reproduction, although
the 21 isolates were arbitrarily selected. However,
our results could not completely rule out the
possibility of somatic or sexual recombination if
exchanges of chromosomes and/or crossovers
occur between the genes. In fact, some of the
polymorphic patterns may suggest recombination
between different genes. For example, if we
assume that genotypes 1-1 (for BT-EF) in races
PST-1 and PST-45 and genotypes 4-5 in PST-127
and CYR31 as two genetically distant genotypes,
then genotype 1-5 (CRY27 and CYR 29) appear to
be a recombination of BT-1 and EF-5. A clearer
example of recombination between genes among
Chinese races will be discussed below. Grouping of
some of isolates into different genotype groups
using different individual genes and gene
combinations may also support the recombination
(either somatic or sexual) hypothesis. Whole
genome sequencing of multiple isolates, which is
currently being undertaken, should provide
clearer evidence with sequences of more genes.
Evolutionary relationships of Ps races
The results of this study provide some
insights for the evolutionary relationships for Pst
races in the US and China. The 13 US races (PST-1,
3, 6, 8, 11, 15, 16, 21, 25, 78, 100 and 127), which
represent major races or race groups over the last
45 years [2, 3, 8, 23], were separated into two
Stripe Rust Heterokaryotic Variations
major groups by polymorphic base pairs of EF (Fig.
3). The first group consisted of eight Pst races
(PST-1, 45, 6, 8, 11, 15, 16 and 25) in SL 1. PST-1
and PST-45 had identical sequences for the three
genes and were different from each other by only
one base pair in another five genes (data not
shown). The other races differed from PST-1 and
PST-45 by only three base pair sites, 713, 714 and
738, of the EF gene. Races in this group had the
highest number of base pairs of being
heterogeneous. The cluster results clearly show
that the races in SL 1 with various virulence
factors (Table 1) were evolved through mutation
from the presumably earliest race, PST-1 (virulent
to differential genotypes 1 and 2), rather than
PST-3 with virulences to differential genotypes 1
(Lemhi) and 3 (Heines VII) which are in many of
other races. The second group, consisting of US
races PST-21; PST-3, 78 and 100; and PST-127 in
SLs 2; 3; and 4, respectively (Fig. 3), was more
diverse. PST-21 (virulent to only Chinese 166 of
the differential genotypes) is related to SL 1
because of the identical BT sequences, but does
not have any heterogeneous base pairs in the EF
sequence.
PST-78 was postulated to be introduced to
the US based on virulence [8] and molecular
markers [28]. PST-100 was postulated to be
evolved from PST-78 [2, 3, 8]. In the present study,
the two races had identical sequences of BT and
EF and CWG (data not shown), and just differed in
six base pairs of MAPK and two base pairs of
STKRAP sequences (data not shown). These two
races were the most closely related to PST-3, one
of few old races frequently detected in the southcentral US before the year 2000 [23, 28]. Grouping
PST-3, PST-78 and PST-100 together was also
supported by their identical CWG sequences (data
not shown). Based on this result, PST-78 was
possibly evolved from PST-3 or a PST-3 like lineage
outside of the US. The two races are very different
in their virulence spectra as shown in Table 1. The
separation of the US races into the two major
groups agreed with the early report of two races,
especially PST-1 and PST-3, which was postulated
to have different origins [22].
The five Chinese Pst races were grouped
into two sequence lineages, SL2 and SL4. CYR27
and CYR29 are most closely related as they have
identical sequences of BT and EF, and only have
four different base pars in MAPK. As discussed
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Open Journal of Genomics, 2012, 1-1
above, CYR29 might have evolved from CRY27 by
homogenizing the four heterogeneous base pairs
through a single event of nucleus or chromosome
re-assortment. CYR32 might have evolved from
CYR29 as it is more closely related to CYR29 than
to CYR27.
PST-127 has the widest virulence spectrum
(virulent to 17 of the 20 differential genotypes) [8]
and is the most recent US isolate used in this
study. It has identical sequences of BT and EF with
a Chinese race, CYR31. The two races were also
grouped with CYR8 in SL 4 (Fig. 3), which may
indicate that these races may have a common
origin. Grouping some US and Chinese races into
same sequence lineages agrees with the
hypothesis that the US Pst population might have
come from Asia, rather than Europe [22].
However, we cannot rule out the possibility of the
origin of Europe and other regions as we did not
include isolates from these regions. It is also
possible that the grouping of some US and
Chinese races together is caused by homoplasy
effect as previously reported [14]. However, the
numerous base pair polymorphisms used in this
study should reduce the possibility of homoplasy
effect. A study in the global scale is currently
underway, which may shield more lights on this
issue.
CONCLUSION
The results of the present study show that
polymorphic sequences of genes are useful in
determination of genetic variations among
isolates of the stripe rust pathogen.
Heterogeneous base pairs are very common and
contribute greatly to the genetic variation among
isolates of the pathogen. Point mutation revealed
by the polymorphic sequences is a major
mechanism for the pathogen evolution. Based on
the results and discussion, we can make the
following
conclusions
on
evolutionary
relationships of Ps races: 1) The early US Pst races,
except for PST-3, have the same origin and the
other races have diverse origins, suggesting
separate evolutionary and migration events. 2)
Races from the US and China are not clearly
separated into distinct groups, indicating that the
Stripe Rust Heterokaryotic Variations
earliest introduced or spread population was a
mixture, as reported for Psh [6], and has
continued mutating and spreading. 3) Barley
stripe rust may not be genetically very different
from wheat stripe rust. Further studies with more
genes and more isolates may provide more
insights to the evolutionary mechanisms of the
stripe rust fungus. The polymorphic base pairs can
be used to develop single nucleotide
polymorphism (SNP) markers and those at the
restriction sites (data not shown) can be used to
develop cleaved amplified polymorphic sequence
(CAPS) markers, all of which can be more useful in
characterizing a large number of samples for
routinely monitoring the rust populations.
DISCLOSURES
None of the authors have any conflicts of
interest.
ACKNOWLEDGMENTS
This research was supported by the US
Department of Agriculture, Agricultural Research
Service (Project No. 5348-22000-014-00D) and
Washington Wheat Commission (Project No. 13C3061-3923). PPNS No. 0543, Department of Plant
Pathology, College of Agricultural, Human, and
Natural Resource Sciences, Agricultural Research
Center, Project Number WNP00823, Washington
State University, Pullman, WA 99164-6430, USA.
The scholarship from China Scholarship Council to
Bo Liu is appreciated. The research is also part of
the Northwest A&F University Plant Pathology
“111” Project. We thank Peng Cheng for technical
assistance and Drs. Lee Hadwiger and Scot Hulbert
for critical reviewing the manuscript.
SUPPLEMENTARY MATERIAL
Supplementary material associated with this
article can be found, in the online version, at URL:
http://www.rossscience.org/ojgen/articles/20759061-1-1_ST01.xls
Supplement Table 1: Polymorphic nucleotide sites
in sequences of genes beta-tubulin (BT, 58H22),
elongation factor (EF, 80N15) and mitogenactivated protein kinase (MAPK, 55B10) among
races of Puccinia striiformis.
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Stripe Rust Heterokaryotic Variations
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