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Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis
f u n g a l b i o l o g y 1 1 6 ( 2 0 1 2 ) 5 2 2 e5 2 8
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Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis Yanchun WANGa,b, Baojun HAOa, Qiang ZHANGa, Enlai TUOa, Guangyu SUNa,*, Rong ZHANGa, Shelin JINc, Mingqi ZHUa, Yang WANGa, Tom HSIANGd a
State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, PR China b Chifeng Agriculture and Animal Husbandry Science Academy, Chifeng 024031, Inner Mongolia, PR China c Institute of Plant Protection, Gansu Agricultural Science Academy, Lanzhou, Gansu, 730070, PR China d Environmental Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
article info
abstract
Article history:
In a search for specific molecular markers for population analysis of Puccinia striiformis f. sp.
Received 24 January 2011
tritici, the ribosomal DNA (rDNA) intergenic spacer (IGS) 1 region (rDNAeIGS1, between the
Received in revised form
28S and the 5S rDNA genes) was amplified, cloned, and sequenced. It was found to exhibit
2 February 2012
multiple bands and length polymorphism. Surprisingly, single isolates were found to pos-
Accepted 5 February 2012
sess between three to five different IGS1 haplotypes. Bands were cloned and sequenced,
Available online 17 February 2012
and two highly variable regions (a and b) were found between conserved regions, with re-
Corresponding Editor:
peat units interspersed in both types of regions. There were 14 different repeat units, and
Kentaro Hosaka
these were sometimes grouped further into four combinations of repeat units, with a few individual nucleotides (A or C) inserted between the repeats. Among three geographically
Keywords:
dispersed isolates, the variable region a was divided into eight types, and the variable re-
Concerted evolution
gion b was divided into two types based on repeat units. Most of the 14 repeat units
Intergenic spacer
were shared by the variable and the conserved regions. Among the three isolates, there
Polymorphism
were a total of 12 IGS1 haplotypes, but some of these were shared between isolates such
Ribosomal DNA
that there were only eight unique haplotypes. The occurrence of multiple haplotypes
Stripe rust
within single isolates may be useful for analyzing the population structure, tracking the origin of annual epidemics and providing insights into evolutionary biology of this pathogen. ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Stripe rust of wheat, also known as yellow rust, is caused by Puccinia striiformis Westend. f. sp. tritici Erikss., and is one of the most devastating diseases of wheat throughout the world. The dikaryotic uredial stage plays the major role in infection and epidemics on wheat. Barberry (Berberis spp.) was recently found to be the aecial host of P. striiformis (Jin et al. 2010); however the importance of the aecial host on the disease cycle of
stripe rust is not known. This fungus is separated into physiological races according to virulence interactions on differential wheat cultivars (Stubbs 1985). For a deeper understanding of the epidemiology, population structure, and genetic diversity of fungal pathogens, it is necessary to find appropriate genetic markers. The use of double-stranded RNA (dsRNA) was reported by Newton et al. (1985) to study the relationships of races of stripe rust in England, but no differences were found. Similarly, limited
* Corresponding author. Tel./fax: þ86 29 87092075. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2012.02.002
Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis
variations using dsRNA markers were observed in Australia (Dickinson et al. 1990). Randomly amplified polymorphic DNA (RAPD) markers were used to reveal low genetic variation in a North American collection of stripe rust (Chen et al. 1993), and no polymorphisms were found in Australian isolates (Steele et al. 2001). Low levels of polymorphism between 160 isolates from China were found using restriction fragment length polymorphism (RFLP) markers (Shan et al. 1998). Amplified restriction fragment length polymorphism (AFLP) can reveal high levels of polymorphism and is considered a robust method to study genetic diversity of wheat yelal et al. 2002; Becerra et al. 2007; Liu et al. low rust (Villare 2011), to understand the evolution of pathotypes (Zheng et al. 2001), and to trace back aerial dispersal of this pathogen to other geographical regions (Justesen et al. 2002; Enjalbert et al. 2005). AFLP markers have revealed a clonal population in Northwestern Europe (HovmØller et al. 2002), and no amplified fragment length polymorphisms were found among Australian and New Zealand isolates (Steele et al. 2001). Liu et al. (2011) found 41 AFLP genotypes among 140 isolates from Yunnan province, China, and 22 genotypes out of 40 isolates from Gansu province, China. Ribosomal DNA (rDNA) is one of the best-studied genomic regions in Eukaryotes (Weider et al. 2005). The 18S, 5.8S, 28S, and 5S rDNA genes are found as multiple copies arranged as tandem repeats separated by noncoding spacers: the internal transcribed spacers (ITS) between the 18S, 5.8S, and 28S RNA sequences, and the intergenic spacers (IGSs) between transcription units. These tandem repeats are usually located in the nuclear organizer region of one or several chromosomes (Mateos & Markow 2005; Rooney & Ward 2005). rDNA sequences have been used for the identification of many fungal species (Takamatsu 1998; Simon & Weiß 2008), and the IGS is commonly considered the most variable part of the rDNA unit, both in sequence and in length, and has been found to be polymorphic between and sometimes even within species (Gerbi 1985; Jennings et al. 1997; James et al. 2009). Two or three IGS haplotypes were found in Laccaria bicolor strains (Martin et al. 1999). For 16 isolates of Lentinula edodes, the sizes of the DNA bands from IGS2 were 1e3 kbp, and several different bands were detected for each isolate (Saito et al. 2002). The objective of this work was to sequence the IGS1 (region between the 28S and 5S rDNA gene) to attempt to reveal more molecular markers for stripe rust. This region was examined in three isolates differing in geographical origin (one from the U.S.A. and two from China) and in virulence spectrum. Surprisingly, more than two IGS1 haplotypes were found for each isolate. The occurrence of multiple haplotypes within single isolates may be useful for analyzing the population structure, tracking the origin of annual epidemics and providing insights into evolutionary biology of stripe rust of wheat.
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the presence of at least three different-sized bands in each of the isolates. Among these isolates, three were chosen for further study: Baihua, Shui4 and Shui5. Isolate Baihua was kindly provided by Dr E.L. Sharp of Montana State University in the 1980’s and has been stored in Laboratory of Plant Immunology, Northwest A&F University. Its virulence spectrum based on testing on Chinese differential sets was found to differ from any race found in China. Isolates Shui4 (race SY 11-4) and Shui5 (race SY 11-5) were sampled from Gangu County, Gansu Province, China in May 2006. The virulence spectra of these latter two isolates were determined using Chinese differential sets of 17 differential cultivars (Wan et al. 2004). Each isolate was purified and bulked up by a series of monouredial transfers on seedlings of wheat (Triticum aestivum L.) cv. Mingxian 169 at seedlings (Li & Zeng 2002), so each is considered to be a single lineage. PCR reactions were conducted at least three times for each experiment.
DNA preparation Total DNA was extracted according to Roose-Amsaleg et al. (2002) with slight modifications. Approximately 1 mg nongerminated uredospores was crushed in a 1.5 ml microcentrifuge tube with 50 ml of extraction buffer [100 mM TriseHCl, pH 9.0; 20 mM EDTA, pH 8.0; 1.4 mM NaCl; 2 % cetyltrimethylammonium bromide (CTAB)] using plastic pestles (Bio Basic Inc. Markham, Ontario, Canada). And then 550 ml of extraction buffer were added to each tube, and the solution incubated for 2 h at 65 C. Proteins were denatured and removed by repeated extractions with 600 ml Tris saturated phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v). Phases were separated by centrifugation, the aqueous phase removed and DNA precipitated with 700 ml of isopropanol. DNA was pelleted by centrifugation, dried and resuspended in 40 ml of TriseEDTA buffer (10 mM TriseHCl, pH 7.6; 1 mM EDTA).
PCR amplification of IGS1
Materials and methods
The IGS1 region was amplified with the primers L318 50 -GCTACGATCCACTGAGGTTC-30 (Kim et al. 1992) and 5SK 50 -CTTCGCAGATCGGACGGGAT-30 (Wolters & Erdmann 1988), which were synthesized by Sangon Biotech, Shanghai, China. Each PCR reaction was set up in a 25 ml volume containing 2 ml of DNA extract, 2 mM MgCl2, 1 U of DNA polymerase, 1 mM of each of the primer pair, 200 mM of deoxy-ribonucleoside triphosphate (dNTP) mixture and 2.5 ml of 10 reaction buffer (TriseHCl 100 mmol ml1，KCl 500 mmol ml1, Takara Biotechnology Co. Ltd, Dalian, China). The cycling conditions followed Roose-Amsaleg et al. (2002) with 30 cycles of 1 min at 95 C, 1 min at 55 C, and 3 min 30 s at 72 C followed by 10 min at 72 C, and holding 4 C. The PCR products were analyzed by electrophoresis through 6 % polyacrylamide gels (SigmaeAldrich Inc., St. Louis, USA from Wolsen, China) to visualize the products for 10 h at a constant power of 20 W in a Protean II xi cell (BIO-RAD, Beijing).
Fungal isolates
IGS1 sequence analysis
Eleven isolates of Puccinia striiformis f. sp. tritici were initially used this study. Preliminary PCR analysis of the IGS1 revealed
The IGS1 PCR products were purified using the HQ&Q Gel Extraction Kit (U-gene Biotechnology, Anhui, China), and cloned
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into pMD19-T vector (Takara Biotechnology Co. Ltd, Dalian, China) as described by the manufacturer’s protocols. From each of the three isolates, at least 60 clones were obtained, and the distribution of PCR band sizes assessed for each isolate. At least one representative of each band size was subjected to sequencing by the dideoxy sequencing method (Sangon Biotech, Shanghai, China) with M13 forward and reverse primers. The sequence data were viewed using Chromas software (Technelysium, Queensland, Australia), aligned with Clustal X, compared and adjusted manually by BioEdit version 5.0.9.1, and subjected to BLASTN (Altschul et al. 1990) analysis against the GenBank NR database. Dot-plot matrix analyses were performed using the program Dotter (Sonnhammer & Durbin 1995). This program plots the regions of identity between two DNA sequences. The length of the lines formed by a series of dots gives an indication of the length of the common motif. Therefore, when a sequence is plotted against itself, the matrix is perfectly symmetrical, with a main diagonal from bottom left to top right as expected. Shorter secondary diagonals reflect the presence of repeated motifs.
Verification of IGS1 polymorphisms To confirm the presence of multiple IGS haplotypes, a primer pair was designed. Because there were large numbers of repeats or combinations of repeats in both variable regions and in conserved regions, we cloned and compared all five sequences from isolate Baihua. We chose regions without repeat units on either side of variable region a to design the specific primer pair, RI94 and RIn, with the software Primer Premier 5.0 (Premier Biosoft International, Palo Alto, California, USA). The PCR reactions were set up in a 25 ml volume containing 2 ml of DNA extract from isolate Baihua, 2 mM MgCl2, 1 U of DNA polymerase, 1 mM of each primer, 200 mM of dNTP mixture and 2.5 ml of 10 reaction buffer. The cycling conditions were as follows: 94 C for 5 min, followed by 20 cycles of 94 C for 30 s, 55 C for 30 s (with a decrease by 1 C of the annealing temperature every cycle), and 72 C for 30 s, followed by 20 cycles of 94 C for 30 s, 45 C for 30 s, and 72 C for 30 s, a final extension of 72 C for 10 min. The PCR products were visualized after electrophoresis through 1 % agarose gels.
Results PCR amplification of IGS1 The rDNAeIGS region from three isolates of Puccinia striiformis f. sp. tritici was successfully amplified using conserved primers in the 28S and 5S regions. Surprisingly, the dikaryons yielded three to five bands per isolate: three bands in Shui5, four bands in Shui4, and five bands in Baihua (Fig. 1). To further verify IGS1 polymorphism, a pair of primers was designed to amplify a variable region. The primer pair sequences were as follows: RI94 50 -AGGGCTAGTTA CTGTTGTT-30 , and RIn 50 -ATGGTCCTTGGCTATTGT-30 . The multiple amplification products of RI94 and RIn from isolate Baihua ranging from 500 to 900 bp confirmed that the IGS1 of P. striiformis showed length polymorphisms.
Y. Wang et al.
Fig. 1 e PCR amplification of the IGS1 in isolates of Puccinia striiformis. Lanes 1, 2, and 3 show amplification of a fragment of the IGS1 between the 28S and the 5S rDNA genes from isolates Shui5, Baihua and Shui4, respectively, using primer pair L318 (in 28S) and 5SK (in 5S). M [ molecular size marker Marker-F.
IGS1 sequence analysis The amplification products of IGS1 were purified and directly cloned into vector pMD19-T and sequenced with the M13 forward and reverse primers. The IGS1 haplotypes and their size and frequency in each of the three isolates are presented in Table 1. The overall structure of the IGS1 was analyzed by the program Dotter, and shown as dot-matrix plots (Fig. 2) in which sequence B-1 (the shortest sequence in all sequences) was compared with itself (Fig. 2A) and sequence S4e4 (the longest sequence in all sequences) (Fig. 2B). This plot produced
Table 1 e Isolate name, band code, band size, number of clones, and GenBank accession number for IGS1 bands amplified with conserved 28S and 5S primers (L318 and 5SK) from three isolates of Puccinia striiformis. Isolate
Band code
Band size (bp)
Clones
GenBank accession
Baihua
B-1 B-2 B-3 B-4 B-5 S4-1 S4-2 S4-3 S4-4 S5-1 S5-2 S5-3
1113 1125 1193 1275 1343 1125 1218 1312 1501 1125 1218 1343
16 8 3 14 19 6 24 28 2 9 15 36
FJ914254 FJ914255 FJ914256 FJ914257 FJ914258 FJ914259 FJ914260 FJ914261 FJ914262 FJ914263 FJ914264 FJ914265
Shui4
Shui5
Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis
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Fig. 2 e Dot-plot matrix of IGS1 sequences generated with the program Dotter. Diagonal lines indicate sequence similarity (A) Sequence B-1 from isolate Baihua compared against itself. (B) Sequence S4-4 from Shui4 compared with B-1. The grey zones below the plots correspond to the regions of mostly unique sequences between the two compared sequences, while the white boxes correspond to insertion/deletion sites. The short diagonals represent repeat sequences.
many secondary diagonals, indicating the presence of repeated motifs. It appeared that the IGS1 region was composed of two variable regions between unique conserved regions (Fig. 3), and pairwise comparisons between all sequences from different bands of the three isolates showed that the length of the insertion/deletion regions were highly variable (data not shown). Based on the dot-matrix plot results, all sequences were aligned with Clustal X, and then compared and edited manually in BioEdit (Hall 1999). This showed that both the variable regions and the unique conserved regions were composed of repeat units and combinations of repeat units (Table 2, Fig. 3). There were 14 repeat types found, respectively labelled as b, d, e, f, h, j, k, m, n, p, q, r, s, and y. The repeat units were found to be further grouped into combinations, designated by the capital letters E, F, H, and I, which represent respectively, drhd, qhdn, msA/CjAbh, and msCjbh (Table 2). A few nucleotides, such as A or C were inserted into the repeats The detailed structure of IGS1 region, the variable region a and the variable region b is shown in Fig. 3. Lowercase letters represent repeat units while uppercase letters (except for the
nucleotides A, T, C, and G) represent combinations of repeat units. Among the three isolates, the variable region a was divided into eight types as follows: dp in type i (16 bp), dpp in type ii (28 bp), dpHdp in type iii (85 bp), dpIFHdp in type iv (178 bp), dpIFHEhdp in type v (230 bp), FHdp in type vi (110 bp), FHFHdp in type vii (204 bp), FHFHFHFHdp in type viii (239 bp) (Fig. 3). Only type i and type v had no repeats, while the other types all had combinations or tandem repeats. The variable region b was divided into two types: k in type x (10 bp), and kGCk in type xi (22 bp) (Fig. 3). Most of the 14 repeat types were shared by the variable and conserved regions. The repeat types, b, d, h, j, m, n, p, q, r, and s appeared in variable region a and the conserved regions. Repeat type k appeared in variable region b and the conserved regions. Among the three isolates, there were a total of 12 IGS size haplotypes (Fig. 1), but some of these were shared between isolates such that there were only eight unique size haplotypes, named I to VIII. IGS1 size haplotype II was shared by all three isolates, but it actually had differing variable regions which coincidentally were of the same length, and hence would be detected as the same haplotype (based on size) if
Fig. 3 e Diagram showing the detailed structure of IGS1 genes in Puccinia striiformis. Eight types of variable region a and two types of variable region b are shown, as well conserved regions. As detailed in Table 2, lowercase letters represent repeat units, while uppercase letters are combinations of repeat units, and A, T, C, G represent nucleotides.
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Table 2 e Basic repeat units of IGS1 sequences, with nucleotide arrays arranged in subrepeat units in Puccinia striiformis. Namea
Length (bp)
Repeat unit
Sequence
b d e f h j k m n p q
4 4 7 7 7 8 10 10 12 12 18
Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides
r
20
Oligonucleotides
s
22
Oligonucleotides
y
29
Oligonucleotides
E F H I
35 41 53 52
Combinations Combinations Combinations Combinations
CAAG CTGG CTCCAAG CTTCAAG TTCAGGG CGTTTGGC ATATATATTT TATAACGTTT AGATTTTTATTA CATATTTTGGTC CCGGTGGTGATCT CTGAC GTCACTTTGAGCCT CAAGTT CGATTCTTTACCTAT GAGATTT CAATCGTTTACCTAT GAGATTTACGTGAG drhd qhdn m s A/C j A b h msCjbh
a A lowercase letter represents a series of nucleotides as a repeat unit. Capital letters (except for the nucleotides A, T, G, C) are combinations of repeat units sometimes with additions such as nucleotides A or C. A/C indicates either A or C.
screened by the conserved IGS1 primers (from 28S to 5S). Isolate Baihua contained IGS1 size haplotypes I, II, III, V, and VII; isolate Shui4 had IGS1 haplotypes III, IV, VI, and VIII; and isolate Shui5 had IGS1 size haplotypes II, IV, and VIII. When considering each isolate individually, the combination of haplotypes gave each isolate a unique set of patterns (Table 3).
Table 3 e Composition of various IGS1 size haplotypes of Puccinia striiformis. The lowercase roman numerals i, ii, iii, iv, v, vi, vii, and viii indicate type of variable region for a, and x and xi for b. The uppercase roman numerals I, II, III, IV, V, VI, VII, and VIII refer to the IGS1 size haplotype. IGS1 haplotype (size, bp) I (1113) II (1125)a III (1193) IV (1218) V (1275) VI (1312) VII (1343) VIII (1501)
Variable region a
Variable region b
Band code
i i ii iii vi iv vii v viii
x xi x xi xi xi xi xi xi
B-1 S4-1, S5-1 B-2 B-3 S4-2, S5-2 B-4 S4-3 B-5, S5-3 S4-4
a IGS1 size haplotype II was shared by all three isolates, but it actually had differing variable regions which coincidentally were of the same length, and hence would be detected as the same haplotype (based on size) when screened with conserved 28S and 5S primers (L318 and 5SK).
Discussion The three isolates tested were of divergent virulence spectra and different geographical origins. The PCR amplification products of the three isolates gave a ladder-like pattern on a gel, with different numbers of rungs. The PCR products were cloned, revealing that the IGS1 of these three isolates of Puccinia striiformis f. sp. tritici was composed of multiple haplotypes that varied within and between isolates, with three to five variants per isolate. In a previous study with the same pathogen, P. striiformis f. sp. tritici, Roose-Amsaleg et al. (2002) found two type length variants (IGS1-1 and IGS1-2) for three European isolates belonging to three races with different geographic origins and virulence spectra. Their IGS1 sequences were similar and highly conserved between the three isolates, and had no significant sequence similarity with other fungi or other organisms. In our study, extensive length variations were observed in all three isolates tested, with these differences based on two variable regions composed of different repeat units. Compared to Roose-Amsaleg et al. (2002), a greater number and more highly variable IGS1 haplotypes were observed. One possible reason for the difference might be in the gel electrophoresis for the IGS1 amplification products. Agarose gel was used by Roose-Amsaleg et al. (2002), but polyacrylamide gels were used here to give finer resolution. We had attempted agarose gel electrophoresis at the beginning of the experiment with the three isolates, and only two or three bands could be visualized per isolate (data not shown). Another possible source of the greater variation observed might be the geographic diversity of the isolates examined. Puccinia striiformis has a strict clonal behaviour and a low genetic diversity in European populations (HovmØller et al. 2002; Enjalbert et al. 2005). But Duan et al. (2010) revealed much more polymorphisms using the same primers as HovmØller et al. (2002) with the local Gansu population. Our isolates Shui4 and Shui5 all sampled from Gansu, so these may be actually much more variable than the European population sampled by Roose-Amsaleg et al. (2002). rDNA is a repetitive gene family presented in tandem array in all eukaryotes. Each repeat unit contains three highly conserved rRNA genes: the 28S large subunit (LSU), 18S small subunit (SSU), and 5.8S rRNA genes. The ITS1 region lies between the 18S and 5.8S genes; the ITS2 region lies between the 5.8S and 28S genes (White et al. 1990; Simon et al. 1992). The large IGS is between transcription units (Long & Dawid 1980). rDNA copy number varies in range of 30 to 30 000 in most eukaryotes (Prokopowich et al. 2003). Due to the high copy number of rDNA and extensive datasets available, these regions provide a means for analyzing phylogenetic relationships over a wide range of taxonomic levels. The SSU and LSU genes evolve relatively slowly, and are useful for studying distantly related organisms, generally in high levels, such as phylum, class, family and genus. The ITS and IGS regions evolve faster and may vary among species within a genus. rDNA plays a key role in ribosome biogenesis and its sequences are extremely conservative throughout evolution. One of the most remarkable characteristics of rDNA tandem repeats is the homogeneity in sequence among transcription units within a genome, and this is achieved primarily by concerted evolution (Elder & Turner 1995).
Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis
Although the most of studies have shown that rRNA multigene families evolve in the strictly concerted evolution pattern, some exceptional variations have been found in plants, animals, fungi, and human beings (Carranza et al. 1996; Gonzalez & Sylvester 2001; Roose-Amsaleg et al. 2002; Saito et al. 2002; Kauserud & Schumacher 2003; Rooney 2004; Rooney & Ward 2005; James et al. 2009; Lindner & Banik 2011). Due to the large number of polymorphisms found between ribosomal genes of single individuals, Simon & Weiß (2008) assumed that concerted evolution did not always work strictly with nuclear ribosomal genes. In our study, surprisingly, highly variable length variations were observed in all isolates tested (11 isolates were examined, and only data of three isolates are shown). Eight unique and shared IGS1 size haplotypes were found. Such high variation shows that P. striiformis f. sp. tritici may be evolving faster than compensations by concerted evolution, as has been observed in other living organisms such as the grasshopper Podisma pedestris (Keller et al. 2006). Variation in IGS within single isolates might also result from rDNA differences among chromosomes. In fungi, tandem arrays may contain 45e200 copies of the ribosomal region (Maleszka & Clark-Walker 1990; Ganley & Kobayashi 2007), and these copies may be distributed across one or more chromosomal locations (Pasero & Marilley 1993), thus allowing for significant variation within the genome of one isolate. Cytological studies using light and electron microscope techniques showed that P. striiformis has six chromosomes (Goddard 1976). Another explanation for the occurrence of variation in the IGS regions may be the presence of distinct genotypes in different nuclei in the same spore. Some researchers have suggested that heterokaryosis as a possible mechanism of variation for obtaining strains with new virulence combinations from coinoculation of different races of stripe rust (Little & Manners 1967; Kang et al. 1993; Ma et al. 1993). Microscopic observations support these results (Ma et al. 1993), while Wang et al. (2004) reported spores with three nuclei (0.42 %) and four nuclei (0.55 %) among naturally collected urediospores. Recently, Jin et al. (2010) found that Berberis spp. are aecial hosts infected by P. striiformis, and hypothesized that sexual recombination has likely played an active role in contributing to the variability of P. striiformis (Jin 2011). Sexual reproduction may allow for increased variation in IGS haplotype combinations, and the finding of multiple IGS haplotypes hints that sexual reproduction may be occurring in local populations, but more investigation is needed to verify this hypothesis. Many molecular markers have been used in the study of wheat stripe rust, such as dsRNA, RAPD, RFLP, and AFLP, but few or no polymorphisms have been found, and these general ally show no relationship to different pathotypes (Villare et al. 2002). Roose-Amsaleg et al. (2002) were the first to find IGS1 length polymorphism in P. striiformis f. sp. tritici, with two types of IGS1 in uredospores of each isolate. Our results confirmed the occurrence of IGS1 polymorphism of stripe rust both within and between isolates, and at higher levels than previously observed. All three tested isolates showed unique sets of IGS1 haplotype combinations. If a large number of isolates were investigated for such combinations, these might be useful as genetics markers for different stripe rust
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populations. These results open up greater avenues for molecular marker analyses of this fungus, and may be useful for strain identification, providing insights into the epidemiology, population structure and dynamics, and genetic diversity of wheat stripe rust.
Acknowledgements This work was supported by National Natural Science Foundation of China (31170015, 31171797), the 111 Project from Education Ministry of China (B07049), Top Talent Project of Northwest A&F University and the Natural Sciences and Engineering Research Council of Canada. We also thank two anonymous referees for comments and corrections that improved the manuscript.
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journal homepage: www.elsevier.com/locate/funbio
Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis Yanchun WANGa,b, Baojun HAOa, Qiang ZHANGa, Enlai TUOa, Guangyu SUNa,*, Rong ZHANGa, Shelin JINc, Mingqi ZHUa, Yang WANGa, Tom HSIANGd a
State Key Laboratory of Crop Stress Biology in Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, PR China b Chifeng Agriculture and Animal Husbandry Science Academy, Chifeng 024031, Inner Mongolia, PR China c Institute of Plant Protection, Gansu Agricultural Science Academy, Lanzhou, Gansu, 730070, PR China d Environmental Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
article info
abstract
Article history:
In a search for specific molecular markers for population analysis of Puccinia striiformis f. sp.
Received 24 January 2011
tritici, the ribosomal DNA (rDNA) intergenic spacer (IGS) 1 region (rDNAeIGS1, between the
Received in revised form
28S and the 5S rDNA genes) was amplified, cloned, and sequenced. It was found to exhibit
2 February 2012
multiple bands and length polymorphism. Surprisingly, single isolates were found to pos-
Accepted 5 February 2012
sess between three to five different IGS1 haplotypes. Bands were cloned and sequenced,
Available online 17 February 2012
and two highly variable regions (a and b) were found between conserved regions, with re-
Corresponding Editor:
peat units interspersed in both types of regions. There were 14 different repeat units, and
Kentaro Hosaka
these were sometimes grouped further into four combinations of repeat units, with a few individual nucleotides (A or C) inserted between the repeats. Among three geographically
Keywords:
dispersed isolates, the variable region a was divided into eight types, and the variable re-
Concerted evolution
gion b was divided into two types based on repeat units. Most of the 14 repeat units
Intergenic spacer
were shared by the variable and the conserved regions. Among the three isolates, there
Polymorphism
were a total of 12 IGS1 haplotypes, but some of these were shared between isolates such
Ribosomal DNA
that there were only eight unique haplotypes. The occurrence of multiple haplotypes
Stripe rust
within single isolates may be useful for analyzing the population structure, tracking the origin of annual epidemics and providing insights into evolutionary biology of this pathogen. ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Stripe rust of wheat, also known as yellow rust, is caused by Puccinia striiformis Westend. f. sp. tritici Erikss., and is one of the most devastating diseases of wheat throughout the world. The dikaryotic uredial stage plays the major role in infection and epidemics on wheat. Barberry (Berberis spp.) was recently found to be the aecial host of P. striiformis (Jin et al. 2010); however the importance of the aecial host on the disease cycle of
stripe rust is not known. This fungus is separated into physiological races according to virulence interactions on differential wheat cultivars (Stubbs 1985). For a deeper understanding of the epidemiology, population structure, and genetic diversity of fungal pathogens, it is necessary to find appropriate genetic markers. The use of double-stranded RNA (dsRNA) was reported by Newton et al. (1985) to study the relationships of races of stripe rust in England, but no differences were found. Similarly, limited
* Corresponding author. Tel./fax: þ86 29 87092075. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2012.02.002
Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis
variations using dsRNA markers were observed in Australia (Dickinson et al. 1990). Randomly amplified polymorphic DNA (RAPD) markers were used to reveal low genetic variation in a North American collection of stripe rust (Chen et al. 1993), and no polymorphisms were found in Australian isolates (Steele et al. 2001). Low levels of polymorphism between 160 isolates from China were found using restriction fragment length polymorphism (RFLP) markers (Shan et al. 1998). Amplified restriction fragment length polymorphism (AFLP) can reveal high levels of polymorphism and is considered a robust method to study genetic diversity of wheat yelal et al. 2002; Becerra et al. 2007; Liu et al. low rust (Villare 2011), to understand the evolution of pathotypes (Zheng et al. 2001), and to trace back aerial dispersal of this pathogen to other geographical regions (Justesen et al. 2002; Enjalbert et al. 2005). AFLP markers have revealed a clonal population in Northwestern Europe (HovmØller et al. 2002), and no amplified fragment length polymorphisms were found among Australian and New Zealand isolates (Steele et al. 2001). Liu et al. (2011) found 41 AFLP genotypes among 140 isolates from Yunnan province, China, and 22 genotypes out of 40 isolates from Gansu province, China. Ribosomal DNA (rDNA) is one of the best-studied genomic regions in Eukaryotes (Weider et al. 2005). The 18S, 5.8S, 28S, and 5S rDNA genes are found as multiple copies arranged as tandem repeats separated by noncoding spacers: the internal transcribed spacers (ITS) between the 18S, 5.8S, and 28S RNA sequences, and the intergenic spacers (IGSs) between transcription units. These tandem repeats are usually located in the nuclear organizer region of one or several chromosomes (Mateos & Markow 2005; Rooney & Ward 2005). rDNA sequences have been used for the identification of many fungal species (Takamatsu 1998; Simon & Weiß 2008), and the IGS is commonly considered the most variable part of the rDNA unit, both in sequence and in length, and has been found to be polymorphic between and sometimes even within species (Gerbi 1985; Jennings et al. 1997; James et al. 2009). Two or three IGS haplotypes were found in Laccaria bicolor strains (Martin et al. 1999). For 16 isolates of Lentinula edodes, the sizes of the DNA bands from IGS2 were 1e3 kbp, and several different bands were detected for each isolate (Saito et al. 2002). The objective of this work was to sequence the IGS1 (region between the 28S and 5S rDNA gene) to attempt to reveal more molecular markers for stripe rust. This region was examined in three isolates differing in geographical origin (one from the U.S.A. and two from China) and in virulence spectrum. Surprisingly, more than two IGS1 haplotypes were found for each isolate. The occurrence of multiple haplotypes within single isolates may be useful for analyzing the population structure, tracking the origin of annual epidemics and providing insights into evolutionary biology of stripe rust of wheat.
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the presence of at least three different-sized bands in each of the isolates. Among these isolates, three were chosen for further study: Baihua, Shui4 and Shui5. Isolate Baihua was kindly provided by Dr E.L. Sharp of Montana State University in the 1980’s and has been stored in Laboratory of Plant Immunology, Northwest A&F University. Its virulence spectrum based on testing on Chinese differential sets was found to differ from any race found in China. Isolates Shui4 (race SY 11-4) and Shui5 (race SY 11-5) were sampled from Gangu County, Gansu Province, China in May 2006. The virulence spectra of these latter two isolates were determined using Chinese differential sets of 17 differential cultivars (Wan et al. 2004). Each isolate was purified and bulked up by a series of monouredial transfers on seedlings of wheat (Triticum aestivum L.) cv. Mingxian 169 at seedlings (Li & Zeng 2002), so each is considered to be a single lineage. PCR reactions were conducted at least three times for each experiment.
DNA preparation Total DNA was extracted according to Roose-Amsaleg et al. (2002) with slight modifications. Approximately 1 mg nongerminated uredospores was crushed in a 1.5 ml microcentrifuge tube with 50 ml of extraction buffer [100 mM TriseHCl, pH 9.0; 20 mM EDTA, pH 8.0; 1.4 mM NaCl; 2 % cetyltrimethylammonium bromide (CTAB)] using plastic pestles (Bio Basic Inc. Markham, Ontario, Canada). And then 550 ml of extraction buffer were added to each tube, and the solution incubated for 2 h at 65 C. Proteins were denatured and removed by repeated extractions with 600 ml Tris saturated phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v). Phases were separated by centrifugation, the aqueous phase removed and DNA precipitated with 700 ml of isopropanol. DNA was pelleted by centrifugation, dried and resuspended in 40 ml of TriseEDTA buffer (10 mM TriseHCl, pH 7.6; 1 mM EDTA).
PCR amplification of IGS1
Materials and methods
The IGS1 region was amplified with the primers L318 50 -GCTACGATCCACTGAGGTTC-30 (Kim et al. 1992) and 5SK 50 -CTTCGCAGATCGGACGGGAT-30 (Wolters & Erdmann 1988), which were synthesized by Sangon Biotech, Shanghai, China. Each PCR reaction was set up in a 25 ml volume containing 2 ml of DNA extract, 2 mM MgCl2, 1 U of DNA polymerase, 1 mM of each of the primer pair, 200 mM of deoxy-ribonucleoside triphosphate (dNTP) mixture and 2.5 ml of 10 reaction buffer (TriseHCl 100 mmol ml1，KCl 500 mmol ml1, Takara Biotechnology Co. Ltd, Dalian, China). The cycling conditions followed Roose-Amsaleg et al. (2002) with 30 cycles of 1 min at 95 C, 1 min at 55 C, and 3 min 30 s at 72 C followed by 10 min at 72 C, and holding 4 C. The PCR products were analyzed by electrophoresis through 6 % polyacrylamide gels (SigmaeAldrich Inc., St. Louis, USA from Wolsen, China) to visualize the products for 10 h at a constant power of 20 W in a Protean II xi cell (BIO-RAD, Beijing).
Fungal isolates
IGS1 sequence analysis
Eleven isolates of Puccinia striiformis f. sp. tritici were initially used this study. Preliminary PCR analysis of the IGS1 revealed
The IGS1 PCR products were purified using the HQ&Q Gel Extraction Kit (U-gene Biotechnology, Anhui, China), and cloned
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into pMD19-T vector (Takara Biotechnology Co. Ltd, Dalian, China) as described by the manufacturer’s protocols. From each of the three isolates, at least 60 clones were obtained, and the distribution of PCR band sizes assessed for each isolate. At least one representative of each band size was subjected to sequencing by the dideoxy sequencing method (Sangon Biotech, Shanghai, China) with M13 forward and reverse primers. The sequence data were viewed using Chromas software (Technelysium, Queensland, Australia), aligned with Clustal X, compared and adjusted manually by BioEdit version 5.0.9.1, and subjected to BLASTN (Altschul et al. 1990) analysis against the GenBank NR database. Dot-plot matrix analyses were performed using the program Dotter (Sonnhammer & Durbin 1995). This program plots the regions of identity between two DNA sequences. The length of the lines formed by a series of dots gives an indication of the length of the common motif. Therefore, when a sequence is plotted against itself, the matrix is perfectly symmetrical, with a main diagonal from bottom left to top right as expected. Shorter secondary diagonals reflect the presence of repeated motifs.
Verification of IGS1 polymorphisms To confirm the presence of multiple IGS haplotypes, a primer pair was designed. Because there were large numbers of repeats or combinations of repeats in both variable regions and in conserved regions, we cloned and compared all five sequences from isolate Baihua. We chose regions without repeat units on either side of variable region a to design the specific primer pair, RI94 and RIn, with the software Primer Premier 5.0 (Premier Biosoft International, Palo Alto, California, USA). The PCR reactions were set up in a 25 ml volume containing 2 ml of DNA extract from isolate Baihua, 2 mM MgCl2, 1 U of DNA polymerase, 1 mM of each primer, 200 mM of dNTP mixture and 2.5 ml of 10 reaction buffer. The cycling conditions were as follows: 94 C for 5 min, followed by 20 cycles of 94 C for 30 s, 55 C for 30 s (with a decrease by 1 C of the annealing temperature every cycle), and 72 C for 30 s, followed by 20 cycles of 94 C for 30 s, 45 C for 30 s, and 72 C for 30 s, a final extension of 72 C for 10 min. The PCR products were visualized after electrophoresis through 1 % agarose gels.
Results PCR amplification of IGS1 The rDNAeIGS region from three isolates of Puccinia striiformis f. sp. tritici was successfully amplified using conserved primers in the 28S and 5S regions. Surprisingly, the dikaryons yielded three to five bands per isolate: three bands in Shui5, four bands in Shui4, and five bands in Baihua (Fig. 1). To further verify IGS1 polymorphism, a pair of primers was designed to amplify a variable region. The primer pair sequences were as follows: RI94 50 -AGGGCTAGTTA CTGTTGTT-30 , and RIn 50 -ATGGTCCTTGGCTATTGT-30 . The multiple amplification products of RI94 and RIn from isolate Baihua ranging from 500 to 900 bp confirmed that the IGS1 of P. striiformis showed length polymorphisms.
Y. Wang et al.
Fig. 1 e PCR amplification of the IGS1 in isolates of Puccinia striiformis. Lanes 1, 2, and 3 show amplification of a fragment of the IGS1 between the 28S and the 5S rDNA genes from isolates Shui5, Baihua and Shui4, respectively, using primer pair L318 (in 28S) and 5SK (in 5S). M [ molecular size marker Marker-F.
IGS1 sequence analysis The amplification products of IGS1 were purified and directly cloned into vector pMD19-T and sequenced with the M13 forward and reverse primers. The IGS1 haplotypes and their size and frequency in each of the three isolates are presented in Table 1. The overall structure of the IGS1 was analyzed by the program Dotter, and shown as dot-matrix plots (Fig. 2) in which sequence B-1 (the shortest sequence in all sequences) was compared with itself (Fig. 2A) and sequence S4e4 (the longest sequence in all sequences) (Fig. 2B). This plot produced
Table 1 e Isolate name, band code, band size, number of clones, and GenBank accession number for IGS1 bands amplified with conserved 28S and 5S primers (L318 and 5SK) from three isolates of Puccinia striiformis. Isolate
Band code
Band size (bp)
Clones
GenBank accession
Baihua
B-1 B-2 B-3 B-4 B-5 S4-1 S4-2 S4-3 S4-4 S5-1 S5-2 S5-3
1113 1125 1193 1275 1343 1125 1218 1312 1501 1125 1218 1343
16 8 3 14 19 6 24 28 2 9 15 36
FJ914254 FJ914255 FJ914256 FJ914257 FJ914258 FJ914259 FJ914260 FJ914261 FJ914262 FJ914263 FJ914264 FJ914265
Shui4
Shui5
Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis
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Fig. 2 e Dot-plot matrix of IGS1 sequences generated with the program Dotter. Diagonal lines indicate sequence similarity (A) Sequence B-1 from isolate Baihua compared against itself. (B) Sequence S4-4 from Shui4 compared with B-1. The grey zones below the plots correspond to the regions of mostly unique sequences between the two compared sequences, while the white boxes correspond to insertion/deletion sites. The short diagonals represent repeat sequences.
many secondary diagonals, indicating the presence of repeated motifs. It appeared that the IGS1 region was composed of two variable regions between unique conserved regions (Fig. 3), and pairwise comparisons between all sequences from different bands of the three isolates showed that the length of the insertion/deletion regions were highly variable (data not shown). Based on the dot-matrix plot results, all sequences were aligned with Clustal X, and then compared and edited manually in BioEdit (Hall 1999). This showed that both the variable regions and the unique conserved regions were composed of repeat units and combinations of repeat units (Table 2, Fig. 3). There were 14 repeat types found, respectively labelled as b, d, e, f, h, j, k, m, n, p, q, r, s, and y. The repeat units were found to be further grouped into combinations, designated by the capital letters E, F, H, and I, which represent respectively, drhd, qhdn, msA/CjAbh, and msCjbh (Table 2). A few nucleotides, such as A or C were inserted into the repeats The detailed structure of IGS1 region, the variable region a and the variable region b is shown in Fig. 3. Lowercase letters represent repeat units while uppercase letters (except for the
nucleotides A, T, C, and G) represent combinations of repeat units. Among the three isolates, the variable region a was divided into eight types as follows: dp in type i (16 bp), dpp in type ii (28 bp), dpHdp in type iii (85 bp), dpIFHdp in type iv (178 bp), dpIFHEhdp in type v (230 bp), FHdp in type vi (110 bp), FHFHdp in type vii (204 bp), FHFHFHFHdp in type viii (239 bp) (Fig. 3). Only type i and type v had no repeats, while the other types all had combinations or tandem repeats. The variable region b was divided into two types: k in type x (10 bp), and kGCk in type xi (22 bp) (Fig. 3). Most of the 14 repeat types were shared by the variable and conserved regions. The repeat types, b, d, h, j, m, n, p, q, r, and s appeared in variable region a and the conserved regions. Repeat type k appeared in variable region b and the conserved regions. Among the three isolates, there were a total of 12 IGS size haplotypes (Fig. 1), but some of these were shared between isolates such that there were only eight unique size haplotypes, named I to VIII. IGS1 size haplotype II was shared by all three isolates, but it actually had differing variable regions which coincidentally were of the same length, and hence would be detected as the same haplotype (based on size) if
Fig. 3 e Diagram showing the detailed structure of IGS1 genes in Puccinia striiformis. Eight types of variable region a and two types of variable region b are shown, as well conserved regions. As detailed in Table 2, lowercase letters represent repeat units, while uppercase letters are combinations of repeat units, and A, T, C, G represent nucleotides.
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Table 2 e Basic repeat units of IGS1 sequences, with nucleotide arrays arranged in subrepeat units in Puccinia striiformis. Namea
Length (bp)
Repeat unit
Sequence
b d e f h j k m n p q
4 4 7 7 7 8 10 10 12 12 18
Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides Oligonucleotides
r
20
Oligonucleotides
s
22
Oligonucleotides
y
29
Oligonucleotides
E F H I
35 41 53 52
Combinations Combinations Combinations Combinations
CAAG CTGG CTCCAAG CTTCAAG TTCAGGG CGTTTGGC ATATATATTT TATAACGTTT AGATTTTTATTA CATATTTTGGTC CCGGTGGTGATCT CTGAC GTCACTTTGAGCCT CAAGTT CGATTCTTTACCTAT GAGATTT CAATCGTTTACCTAT GAGATTTACGTGAG drhd qhdn m s A/C j A b h msCjbh
a A lowercase letter represents a series of nucleotides as a repeat unit. Capital letters (except for the nucleotides A, T, G, C) are combinations of repeat units sometimes with additions such as nucleotides A or C. A/C indicates either A or C.
screened by the conserved IGS1 primers (from 28S to 5S). Isolate Baihua contained IGS1 size haplotypes I, II, III, V, and VII; isolate Shui4 had IGS1 haplotypes III, IV, VI, and VIII; and isolate Shui5 had IGS1 size haplotypes II, IV, and VIII. When considering each isolate individually, the combination of haplotypes gave each isolate a unique set of patterns (Table 3).
Table 3 e Composition of various IGS1 size haplotypes of Puccinia striiformis. The lowercase roman numerals i, ii, iii, iv, v, vi, vii, and viii indicate type of variable region for a, and x and xi for b. The uppercase roman numerals I, II, III, IV, V, VI, VII, and VIII refer to the IGS1 size haplotype. IGS1 haplotype (size, bp) I (1113) II (1125)a III (1193) IV (1218) V (1275) VI (1312) VII (1343) VIII (1501)
Variable region a
Variable region b
Band code
i i ii iii vi iv vii v viii
x xi x xi xi xi xi xi xi
B-1 S4-1, S5-1 B-2 B-3 S4-2, S5-2 B-4 S4-3 B-5, S5-3 S4-4
a IGS1 size haplotype II was shared by all three isolates, but it actually had differing variable regions which coincidentally were of the same length, and hence would be detected as the same haplotype (based on size) when screened with conserved 28S and 5S primers (L318 and 5SK).
Discussion The three isolates tested were of divergent virulence spectra and different geographical origins. The PCR amplification products of the three isolates gave a ladder-like pattern on a gel, with different numbers of rungs. The PCR products were cloned, revealing that the IGS1 of these three isolates of Puccinia striiformis f. sp. tritici was composed of multiple haplotypes that varied within and between isolates, with three to five variants per isolate. In a previous study with the same pathogen, P. striiformis f. sp. tritici, Roose-Amsaleg et al. (2002) found two type length variants (IGS1-1 and IGS1-2) for three European isolates belonging to three races with different geographic origins and virulence spectra. Their IGS1 sequences were similar and highly conserved between the three isolates, and had no significant sequence similarity with other fungi or other organisms. In our study, extensive length variations were observed in all three isolates tested, with these differences based on two variable regions composed of different repeat units. Compared to Roose-Amsaleg et al. (2002), a greater number and more highly variable IGS1 haplotypes were observed. One possible reason for the difference might be in the gel electrophoresis for the IGS1 amplification products. Agarose gel was used by Roose-Amsaleg et al. (2002), but polyacrylamide gels were used here to give finer resolution. We had attempted agarose gel electrophoresis at the beginning of the experiment with the three isolates, and only two or three bands could be visualized per isolate (data not shown). Another possible source of the greater variation observed might be the geographic diversity of the isolates examined. Puccinia striiformis has a strict clonal behaviour and a low genetic diversity in European populations (HovmØller et al. 2002; Enjalbert et al. 2005). But Duan et al. (2010) revealed much more polymorphisms using the same primers as HovmØller et al. (2002) with the local Gansu population. Our isolates Shui4 and Shui5 all sampled from Gansu, so these may be actually much more variable than the European population sampled by Roose-Amsaleg et al. (2002). rDNA is a repetitive gene family presented in tandem array in all eukaryotes. Each repeat unit contains three highly conserved rRNA genes: the 28S large subunit (LSU), 18S small subunit (SSU), and 5.8S rRNA genes. The ITS1 region lies between the 18S and 5.8S genes; the ITS2 region lies between the 5.8S and 28S genes (White et al. 1990; Simon et al. 1992). The large IGS is between transcription units (Long & Dawid 1980). rDNA copy number varies in range of 30 to 30 000 in most eukaryotes (Prokopowich et al. 2003). Due to the high copy number of rDNA and extensive datasets available, these regions provide a means for analyzing phylogenetic relationships over a wide range of taxonomic levels. The SSU and LSU genes evolve relatively slowly, and are useful for studying distantly related organisms, generally in high levels, such as phylum, class, family and genus. The ITS and IGS regions evolve faster and may vary among species within a genus. rDNA plays a key role in ribosome biogenesis and its sequences are extremely conservative throughout evolution. One of the most remarkable characteristics of rDNA tandem repeats is the homogeneity in sequence among transcription units within a genome, and this is achieved primarily by concerted evolution (Elder & Turner 1995).
Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis
Although the most of studies have shown that rRNA multigene families evolve in the strictly concerted evolution pattern, some exceptional variations have been found in plants, animals, fungi, and human beings (Carranza et al. 1996; Gonzalez & Sylvester 2001; Roose-Amsaleg et al. 2002; Saito et al. 2002; Kauserud & Schumacher 2003; Rooney 2004; Rooney & Ward 2005; James et al. 2009; Lindner & Banik 2011). Due to the large number of polymorphisms found between ribosomal genes of single individuals, Simon & Weiß (2008) assumed that concerted evolution did not always work strictly with nuclear ribosomal genes. In our study, surprisingly, highly variable length variations were observed in all isolates tested (11 isolates were examined, and only data of three isolates are shown). Eight unique and shared IGS1 size haplotypes were found. Such high variation shows that P. striiformis f. sp. tritici may be evolving faster than compensations by concerted evolution, as has been observed in other living organisms such as the grasshopper Podisma pedestris (Keller et al. 2006). Variation in IGS within single isolates might also result from rDNA differences among chromosomes. In fungi, tandem arrays may contain 45e200 copies of the ribosomal region (Maleszka & Clark-Walker 1990; Ganley & Kobayashi 2007), and these copies may be distributed across one or more chromosomal locations (Pasero & Marilley 1993), thus allowing for significant variation within the genome of one isolate. Cytological studies using light and electron microscope techniques showed that P. striiformis has six chromosomes (Goddard 1976). Another explanation for the occurrence of variation in the IGS regions may be the presence of distinct genotypes in different nuclei in the same spore. Some researchers have suggested that heterokaryosis as a possible mechanism of variation for obtaining strains with new virulence combinations from coinoculation of different races of stripe rust (Little & Manners 1967; Kang et al. 1993; Ma et al. 1993). Microscopic observations support these results (Ma et al. 1993), while Wang et al. (2004) reported spores with three nuclei (0.42 %) and four nuclei (0.55 %) among naturally collected urediospores. Recently, Jin et al. (2010) found that Berberis spp. are aecial hosts infected by P. striiformis, and hypothesized that sexual recombination has likely played an active role in contributing to the variability of P. striiformis (Jin 2011). Sexual reproduction may allow for increased variation in IGS haplotype combinations, and the finding of multiple IGS haplotypes hints that sexual reproduction may be occurring in local populations, but more investigation is needed to verify this hypothesis. Many molecular markers have been used in the study of wheat stripe rust, such as dsRNA, RAPD, RFLP, and AFLP, but few or no polymorphisms have been found, and these general ally show no relationship to different pathotypes (Villare et al. 2002). Roose-Amsaleg et al. (2002) were the first to find IGS1 length polymorphism in P. striiformis f. sp. tritici, with two types of IGS1 in uredospores of each isolate. Our results confirmed the occurrence of IGS1 polymorphism of stripe rust both within and between isolates, and at higher levels than previously observed. All three tested isolates showed unique sets of IGS1 haplotype combinations. If a large number of isolates were investigated for such combinations, these might be useful as genetics markers for different stripe rust
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populations. These results open up greater avenues for molecular marker analyses of this fungus, and may be useful for strain identification, providing insights into the epidemiology, population structure and dynamics, and genetic diversity of wheat stripe rust.
Acknowledgements This work was supported by National Natural Science Foundation of China (31170015, 31171797), the 111 Project from Education Ministry of China (B07049), Top Talent Project of Northwest A&F University and the Natural Sciences and Engineering Research Council of Canada. We also thank two anonymous referees for comments and corrections that improved the manuscript.
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