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Available online at www.sciencedirect.com
ScienceDirect Journal of Genetics and Genomics xx (2014) 1e9
JGG ORIGINAL RESEARCH
Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatLThinopyrum ponticum Partial Amphiploids Qi Zheng a, Zhenling Lv a, Zhixia Niu b, Bin Li a, Hongwei Li a, Steven S. Xu b, Fangpu Han a, Zhensheng Li a,* a
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China b United States Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, Fargo, ND 58102-2765, USA Received 24 January 2014; revised 27 May 2014; accepted 9 June 2014 Available online - - -
ABSTRACT Partial amphiploids created by crossing common wheat (Triticum aestivum L.) and Thinopyrum ponticum (Podp.) Barkworth & D. R. Dewey are important intermediates in wheat breeding because of their resistance to major wheat diseases. In this study, we examined the chromosome compositions of five Xiaoyan-series wheatTh. ponticum partial amphiploids (Xiaoyan 68, Xiaoyan 693, Xiaoyan 784, Xiaoyan 7430, and Xiaoyan 7631) using GISH, multicolor-GISH, and multicolor-FISH. We found several chromosome changes in these lines. For example, wheat chromosomes 1B and 2B were added in Xiaoyan 68 and Xiaoyan 7430, respectively, while wheat chromosome 6B was eliminated from Xiaoyan 693 and Xiaoyan 7631. Chromosome rearrangements were also detected in these amphiploids, including an interspecific translocation involving chromosome 4D and some intergenomic translocations, such as AeB and AeD translocations, among wheat genomes. Analysis of the Th. ponticum chromosomes in the amphiploids showed that some lines shared the same alien chromosomes. We also evaluated these partial amphiploids for resistance to nine races of stem rust, including TTKSK (commonly known as Ug99). Three lines, Xiaoyan 68, Xiaoyan 784, and Xiaoyan 7430, exhibited excellent resistance to all nine races, and could therefore be valuable sources of stem rust resistance in wheat breeding. KEYWORDS: Thinopyrum ponticum; Triticum aestivum; Partial amphiploid; Multicolor-genomic in situ hybridization (mc-GISH); Multicolor-fluorescent in situ hybridization (mc-FISH); Stem rust
INTRODUCTION Wild relatives of common wheat (Triticum aestivum L.) have been used extensively to transfer beneficial alien genes into wheat (Friebe et al., 1996; Bommineni and Jauhar, 1997; Sepsi et al., 2008). Tall wheatgrass, Thinopyrum ponticum (Podp.) Barkworth and D. R. Dewey [ ¼ Agropyron elongatum ssp. ruthenicum Beldie; Elytrigia pontica (Podp.) ´ Holub; Lophopyrum ponticum (Podp.) A Lo¨ve] (2n ¼ 10x ¼ 70) is a perennial species in the tribe Triticeae * Corresponding author. Tel: þ86 10 6480 6607, fax: þ86 10 6480 6605. E-mail address: [email protected] (Z. Li).
that is important for wheat improvement because of its resistance to a number of wheat diseases, as well as its stress tolerance and high crossability with various Triticum species (Shannon, 1978; Sharma et al., 1989; Cox, 1991; McIntosh, 1991). To transfer resistance genes from Th. ponticum to wheat, creating partial amphiploids from crosses between common wheat and tall wheatgrass is an important intermediate step. In the past several decades, a number of stable wheateTh. ponticum partial amphiploids have been developed that carry useful agronomic traits, such as resistance to wheat streak mosaic virus, barley yellow dwarf virus, common root rot, Fusarium head blight, tan spot, and Stagonospora nodorum blotch (Chen et al., 1998a, 1998b; Thomas
http://dx.doi.org/10.1016/j.jgg.2014.06.003 1673-8527/Copyright Ó 2014, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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et al., 1998; Fedak et al., 2000; Li et al., 2004; Oliver et al., 2006). Understanding of the compositions and structures of the introgressed Th. ponticum chromosomes in partial amphiploids is useful for further transferring desirable genes into common wheat. Therefore, the chromosome compositions of many wheateTh. ponticum partial amphiploids have been characterized. Chen et al. (1998a, 1998b) reported two octoploid Agrotriticum partial amphiploids, Agrotana and OK7211542, which had eight Js-genome chromosomes and eight E-or J-genome chromosomes. Oliver et al. (2006) described four partial wheateTh. ponticum amphiploids with different amounts of alien chromatin. Based on their genomic in situ hybridization (GISH) results, Fedak et al. (2000) revealed that each of six wheateTh. ponticum partial amphiploids contained a synthetic alien genome composed of different combinations of St-, J-, or Js-genome chromosomes. Li et al. (1985) reported the production of partial amphiploids, and designated them the Xiaoyan series, from hybrids between common wheat and Th. ponticum. Zhang et al. (1996) investigated the chromosome complements of this series with an Stgenome probe and A-, B-, D- and E-genome blocks. Xiaoyan 693 and Xiaoyan 7631 had identical St-genomes, while Xiaoyan 784, Xiaoyan 68, and Xiaoyan 7430 had a synthetic alien genome, including six pairs of St-genome chromosomes and one pair of Ee- (or Eb-) genome chromosomes. However, the specific wheat genome compositions in these partial amphiploids, including the missing, substituted, and translocated chromosomes, remain unclear. To more extensively explore these partial amphiploids for wheat improvement, detailed information on their genomic constitutions is needed. In the present study, we used a combination of multicolorgenomic in situ hybridization (mc-GISH) and multicolorfluorescent in situ hybridization (mc-FISH) to examine the cytogenetic compositions of five partial amphiploids in the Xiaoyan series (Xiaoyan 68, Xiaoyan 693, Xiaoyan 784, Xiaoyan 7430, and Xiaoyan 7631). In addition, because stem rust Ug99-lineage races are currently a major threat to global wheat production (Singh et al., 2011) and some wheateTh. ponticum partial amphiploids have excellent resistance to Ug99 (Xu et al., 2009; Turner et al., 2013), we evaluated seedlings of these five lines for resistance to stem rust Ug99 and eight other major races of stem rust from Africa and North America and confirmed that three partial amphiploids are good breeding material for stem rust resistance. RESULTS mc-GISH analysis revealed intergenomic rearrangements in the partial amphiploids In the mc-GISH analysis of the five partial amphiploids, the mitotic chromosomes were probed by D-genomic DNA (Aegilops tauschii) with Texas-red-5-dCTP (red), and total genomic DNA of Th. ponticum and Triticum urartu was probed with Fluorescein-12-dUTP (green) and blocked by Sgenome (Aegilops speltoides) DNA. Based on the distribution
of hybridization signals on chromosomes, the A-, B-, and Dgenome from common wheat and the genomes from Th. ponticum could be distinguished at the same time. The A-, B-, and D-genome chromosomes were labeled with yellowegreen, gray, and red florescences, respectively, while the Th. ponticum chromosomes were labeled with green florescences. A number of intergenomic rearrangements were detected in the wheat genome by mc-GISH analysis. As illustrated in Fig. 1A, Xiaoyan 68 had 12 green-fluorescing chromosomes that originated from tall wheatgrass and two interspecific translocation chromosomes with green terminal fragments on red short arms. Among the remaining 42 wheat chromosomes, 14 fluoresced yellowegreen, 16 gray, and 12 red. Among the 12 red-fluorescing chromosomes, one pair carried a terminal yellowegreen signal, suggesting that an intergenomic rearrangement involving the A and D genomes had taken place in the wheat genome. Therefore, in addition to 12 alien chromosomes and 2 wheateTh. ponticum translocation chromosomes, Xiaoyan 68 also had 14 A-genome, 16 B-genome, 10 D-genome, and 2 AeD translocation chromosomes. Similarly, Xiaoyan 784 possessed 14 Th. ponticum chromosomes and 42 wheat chromosomes composed of 10 Agenome, 14 B-genome, and 12 D-genome chromosomes plus two pairs of AeD translocation chromosomes and one pair of AeB translocation chromosomes (Fig. 1C). Xiaoyan 7430 contained 12 alien chromosomes plus 44 wheat chromosomes comprising 8 A-genome, 16 B-genome, and 10 D-genome chromosomes, 4 pairs of AeD translocation chromosomes, and one pair of AeB translocation chromosomes (Fig. 1D). Among the four pairs of AeD translocation chromosomes in Xiaoyan 7430, three were mainly D genome with a small Agenome segment on the distal region of the long arm, and one comprised a large part of the A genome with a small Dgenome fraction on its long arm. Both Xiaoyan 693 and Xiaoyan 7631 carried 16 Th. ponticum chromosomes plus 40 wheat chromosomes consisting of 12 A-genome, 12 Bgenome, 14 D-genome, and two AeB translocation chromosomes (Fig. 1B and E). Sequential GISH and mc-FISH analyses revealed the chromosome compositions of the partial amphiploids Although each of the five lines consistently had 56 chromosomes, the number of alien chromosomes ranged from 12 to 16, suggesting that chromosome deletion and substitution occurred from the wheat genome. Chromosome elimination and addition can be detected by sequential GISH and mc-FISH methodology (Fig. 2AeJ). The results of mc-GISH analysis revealed that a pair of B-genome chromosomes was absent from Xiaoyan 693 and Xiaoyan 7631 (Fig. 2D and J). Comparing their mc-FISH results using probes pAs1 and pSc119.2 with that of common wheat (Mukai et al., 1993), the missing chromosome was determined to be 6B, because of faint pAs1 bands on the satellite and short arm and a terminal and an interstitial pSc119.2 band on the long arm. In both Xiaoyan 68 and Xiaoyan 7430 a pair of B-genome chromosomes was added (Fig. 2B and H). The added
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Fig. 1. Multicolor (mc)-GISH patterns of the five partial amphiploids. The different florescence signals show A-genome chromosomes (yellow-green), B-genome chromosomes (gray), D-genome chromosomes (red) and the chromosomes of Th. ponticum (green). Arrows indicate AeD translocation chromosomes in Xiaoyan 68 (A), Xiaoyan 784 (C) and Xiaoyan 7430 (D); arrow heads indicate AeB translocation chromosomes in Xiaoyan 693 (B), Xiaoyan 7430 (D) and Xiaoyan 7631 (E); and asterisks indicate a wheateTh. ponticum translocation chromosomes.
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Fig. 2. Sequential GISH and mc-FISH analyses of the five partial amphiploids. GISH analysis of the Thinopyrum ponticum chromosomes in Xiaoyan 68 (A), Xiaoyan 693 (C), Xiaoyan 784 (E), Xiaoyan 7430 (G) and Xiaoyan 7631 (I). Bright yellow signals marked the Th. ponticum chromosomes or chromosomal segments. The Arabic figures refer to the alien chromosomes according to the size from big to small. The wheat chromosomes were counterstained with PI (red). Mc-FISH on the same metaphase chromosome spreads in Xiaoyan 68 (B), Xiaoyan 693 (D), Xiaoyan 784 (F), Xiaoyan 7430 (H) and Xiaoyan 7631 (J) by pAs1 (green) and pSc119.2 (red) simultaneously. Arrows refer to the AeD translocation chromosomes in H.
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Fig. 2. continued.
chromosomes in Xiaoyan 7430 showed a distinctive 2B pattern, with strong pSc119.2 bands in the terminal region of the short arm and in an intercalary region of the long arm. In contrast, the extra chromosomes in Xiaoyan 68 had strong hybridization sites with pSc119.2 on the distal end of the long arm, indicating that they were chromosome 1B. In addition, mc-FISH results revealed that the wheateTh. ponticum translocation chromosomes in Xiaoyan 68 were wheat chromosome 4D with a terminal alien segment on the short arm. The alien fragment was very small, amounting to 20% of the translocated short arm. Although many intergenomic rearrangements in the wheat genomes occurred in these five wheat lines, only two pairs of AeD translocated chromosomes were identified in Xiaoyan 7430 using mc-FISH analysis because of a lack of distinctive bands. Moreover, the fluorescence signals of repetitive DNA sequences pAs1 and pSc119.2 were observed on the Th. ponticum-derived chromosomes (Fig. 3AeE). The pAs1 bands were located on almost all of the alien chromosomes. They were more frequent in the terminal regions than in the interstitial
ones but were absent in the centromeric and most of the pericentromeric regions. The pSc119.2 sites were observed in terminal regions of a few wheatgrass chromosomes, such as three alien chromosomes in Xiaoyan 7430, two in Xiaoyan 693, one in Xiaoyan 7631, and one in Xiaoyan 784, but were missing from alien chromosomes in Xiaoyan 68. Based on the diagram of both sequence signals, three perennial grass chromosomes were identical in Xiaoyan 693 (Fig. 3B, chromosomes 1, 3, and 5) and in Xiaoyan 7631 (Fig. 3E, chromosomes 1, 3 and 8). The fifth-ranked chromosomes in Xiaoyan 68 and Xiaoyan 784 originated from one decaploid wheatgrass chromosome, because they had duplicate fluorescence bands. In addition, the third alien chromosomes of Xiaoyan 784 and Xiaoyan 7430 possessed the same pAs1 signals, indicating that they were identical. Reactions of five partial amphiploids to stem rust The infection types of the partial amphiploids to the seven North American (RTQQC, TCMJC, TPMKC, QFCSC,
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Fig. 3. The fluorescence signal of repetitive DNA sequences on the Th. ponticum-derived chromosomes. Idiogram of Thinopyrum ponticum chromosomes in Xiaoyan 68 (A), Xiaoyan 693 (B), Xiaoyan 784 (C), Xiaoyan 7430 (D) and Xiaoyan 7631 (E), showing the locations of pAs1 (green) and pSc119.2 (red) on drawn chromosomes side by side with FISH photographs of the same chromosomes.
MCCFC, TMLKC, and TTTTF) and two African (TTKSK and TRTTF) races of the stem rust pathogen are listed in Table 1. As expected, the susceptible control cultivar, Chinese Spring (CS), had infection types (ITs) 3þ or 4 to all races. The five Xiaoyan-series partial amphiploids exhibited various levels of resistance. Xiaoyan 693 was highly or moderately resistant to four races, RTQQC (23), TCMJC (12), TPMKC (123), and TMLKC (23), but it was susceptible to QFCSC (34) and MCCFC (32). Xiaoyan 7631 was highly resistant to RTQQC (12), TCMJC (11), MCCFC (12), and TMLKC (12) and moderately resistant to TPMKC (23), but it was susceptible to QFCSC (32). Other three partial amphiploids were highly resistant to all nine races. Among them, Xiaoyan 68 and Xiaoyan 7430 were near-immune (IT ;) to all nine races and Xiaoyan 784 expressed variable levels
of resistance from near-immunity (IT ;) to moderate resistance (2;), suggesting that the three partial amphiploids are valuable source with resistance to Ug99 and other races of stem rust. DISCUSSION Zhang et al. (1996) proposed that Th. ponticum has two basic genomes, E and St, and suggested the formula StStEeEbEx for this decaploid. In Th. ponticum, Ee and Eb are two sub-genome types of the E genome and originated from T. elongatum and T. bessarabicum, respectively. Ex indicates either Ee or Eb, depending on the accession, and the St genome is donated by the mono-genome genus Pseudoroegneria. The GISH results showed that both Xiaoyan 693 and Xiaoyan 7631 had identical
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Table 1 Infection types produced by five wheateTh. ponticum partial amphiploids to nine races of Puccinia graminis f. sp. tritici Infection type to racea
Line RTQQC
QFCSC
TCMJC
MCCFC
TPMKC
TMLKC
TTKSK
TRTTF
TTTTF
Xiaoyan 68
;
;
;
;
;
;
;
0;
0;
Xiaoyan 693
23
34
12
32
123
23
Xiaoyan 784
;
12
1
12
1
1
2;
;
2;
Xiaoyan 7430
;
;
;
;
;
;
;
;
;
Xiaoyan 7631
12
32
11
12
23
12
Chinese Spring
4
4
4
4
4
4
3þ
3þ
3þ
Infection types (ITs) were followed Stakman et al. (1962), where 0 = immune, ; ¼ necrotic flecks, 1 ¼ small necrotic pustules, 2 ¼ small to medium sized chlorotic pustules with green island, 3 ¼ medium sized chlorotic pustules, and 4 ¼ large pustules without chlorosis. ITs 0, ;, 1, 2, or any combination of these indicated resistance, and ITs 3 or 4 indicated susceptibility. Hence 123 is predominantly IT 1, with decreasing amounts of IT 2 and IT 3. 1 indicates small IT 1. 2 indicates small IT 2. 3þ indicates large IT 3. a
St-genome chromosomes, but Xiaoyan 784, Xiaoyan 68, and Xiaoyan 7430 had a synthetic alien genome that included six pairs of St-genome chromosomes and one pair of Ee- (or Eb-) genome chromosomes. Zhang and Dong (1994) found that many univalents were present in test crosses among Xiaoyan 784, Xiaoyan 68, and Xiaoyan 7430, suggesting that many modifications exist to the St genomes in Th. ponticum. In this study, although Xiaoyan 693 and Xiaoyan 7631 shared some Th. ponticum chromosomes, they also have five different chromosomes. Thus, the cytogenetical evidence in our study indicated that Xiaoyan 7631 and Xiaoyan 693 are two different lines. In the previous study (Zhang and Dong, 1994), seven pairs of tall wheatgrass chromosomes were found in both Xiaoyan 68 and Xiaoyan 7430. However, Xiaoyan 7430 had six pairs of alien chromosomes, and Xiaoyan 68 possessed six pairs of Th. ponticum chromosomes as well as a pair of Th. ponticume wheat translocation chromosomes. These findings suggested that some octoploid Trititrigia are not cytogenetically stable, and the genome compositions of these partial amphiploids can change without altering the chromosome number. The pAs1 probe was previously reported to be located in the terminal regions of both arms and dispersed throughout all 70 chromosomes, except in the centromeric and most of the pericentromeric regions, while that pSc119.2 labeling was distributed on the short arms of 17 chromosomes and the long arms of five others (Brasileiro-Vidal et al., 2003). In this study, however, some alien chromosome arms completely lacked this tandem repeat, which could be explained by recombination and modification within Th. ponticum chromosome segments during formation of the synthetic species from the perennial grass. Based on the ITs, the stem-rust resistance of five Xiaoyanseries partial amphiploids could be divided into levels I (Xiaoyan 68 and Xiaoyan 7430), II (Xiaoyan 784), and III (Xiaoyan 7631 and Xiaoyan 693). The partial amphiploids with levels I and II were clearly resistant to all races. In particular, Xiaoyan 68 and Xiaoyan 7430 were most immune and should be valuable sources with stem rust resistance, while resistance in Xiaoyan 784 varied from high to moderate. The
partial amphiploids in level III had distinctly less resistance to most of rust races tested, and they may have less utility in wheat breeding for resistance to stem rust. MATERIALS AND METHODS Plant materials Five partial amphiploids Xiaoyan 68, Xiaoyan 693, Xiaoyan 784, Xiaoyan 7430, Xiaoyan 7631 were produced from wheat e Th. ponticum hybrids by the wheat distant hybridization group in Northwest Institute of Botany, Chinese Academy of Sciences (Yangling, Shaanxi, China). Wheat cv. “Chinese Spring” and Th. ponticum accessions R431 were also used in this study. All the above materials were collected in our laboratory. Chromosome preparation Chromosome spread preparation was carried out following the procedures of Kato et al. (2006) and Han and Lv (2013) with some minor modifications. Seeds were germinated on moist filter paper at 23 C for 48 h. Root tips of 1e2 cm length were collected and pretreated in N2O at 10 atm pressure for 2 h to accumulate metaphases, and then fixed in 90% acetic acid. Multicolor-genomic in situ hybridization (mc-GISH) Mc-GISH was performed using published protocols with some modification (Han et al., 2012). Total genomic DNA of Th. ponticum, T. urartu, A. speltoides and A. tauschii was isolated from the young fresh leaves (Kidwell and Osborn, 1992). Fluorescein-12-dUTP (green) labeled Th. ponticum and T. urartu genomic DNA and Texas-red-5-dCTP (red) labeled A. tauschii genomic DNA served as probes. Sheared A. speltoides genomic DNA was used as a block. After hybridization, the slides were washed in 2 SSC and mounted in Vectashield mounting medium (containing DAPI; Vector Laboratories, USA). Detection and visualization were carried out as described by Han et al. (2009).
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Sequential genomic and multicolor-fluorescent in situ hybridization (sequential GISH and mc-FISH) Procedures for sequential GISH and mc-FISH were followed according to Zheng et al. (2006). Briefly, Th. ponticum genomic DNA was labeled with digoxigenin-11-dUTP and served as a probe, and wheat DNA was used as a block. The ratio between probe and block was 1:250. The hybridized probes were detected with anti-digoxigenin conjugated by fluorescence isothiocyanate (FITC). The slides were counterstained with propidium iodide (PI, 0.25 mg/ mL) in the Vectashield mounting medium (Vector Laboratories, USA). Mc-FISH was carried out after GISH analysis using two probes, pAs1 labeled with digoxigenin-11-dUTP and pSc119.2 labeled with biotin-11-dUTP. Two probes were mixed 1:1 before the hybridization. After the hybridization, anti-digoxigenin-FITC and avidin-rhodamine were used for detection of the two probes simultaneously. The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI). All cells with good hybridization signals were captured by DVC CCD camera attached to an Olympus BX50 epifluorescence microscope. The images captured for each color channel were merged using program Image-Pro Plus 4.0 (Digital Video Camera, USA). Measurements were made on the digital images of the signals. A minimum of five translocation chromosomes were measured for each line. The identification of the alien chromosomes and their idiograms were determined based on the chromosome length, arm ratio, and mc-FISH patterns instead of the homology to wheat. Stem rust resistance evaluation The six major North American races RTQQC, TCMJC, TPMKC, QFCSC, MCCFC, and TMLKC were firstly used for the stem rust testing of the five partial amphiploids at USDAARS, Northern Crop Science Laboratory, Fargo, ND. The seedlings were grown in the greenhouse at 20 Ce23 C with 16/8 (day/night) photoperiod. Seven-day-old seedlings were inoculated as described by Williams et al. (1992). Inoculated plants were then moved to a greenhouse at 20 C23 C. Based on the availability of the seed samples, three lines, Xiaoyan 68, Xiaoyan 784, and Xiaoyan 7430, were further evaluated for reactions to two African races, TTKSK (Ug99) and TRTTF, and the North American race TTTTF, all with broad virulence spectra at the seedling stage. The inoculation with the three races was performed using the procedure described by Jin et al. (2007) at USDA-ARS, Cereal Disease Laboratory, St. Paul, MN, USA. In both evaluation trials, infection types (ITs) were scored 14 days after inoculation using the system of Stakman et al. (1962), where 0 ¼ immune, ; ¼ necrotic flecks, 1 ¼ small necrotic pustules, 2 ¼ small to medium sized chlorotic pustules with green island, 3 ¼ medium sized chlorotic pustules, and 4 ¼ large pustules without chlorosis. ITs 0, ;, 1, 2, or any combination of these indicated resistance, and ITs 3 or 4 indicated susceptibility.
ACKNOWLEDGEMENTS We thank Dr. Yue Jin for his critical reading the manuscript. This work was supported by the grants from the National Natural Science Foundation of China (No. 31171539), the National High-Tech Research and Development Program of China (No. 2011AA1001) and the National Key Technology R&D Program of China (No. 2013BAD05B01). REFERENCES Bommineni, V.R., Jauhar, P.P., 1997. Wide hybridization and genome relationships in cereals: an assessment of molecular approaches. Maydica 42, 81e105. Brasileiro-Vidal, A.C., Cuadrado, A., Brammer, S.P., Zanatta, A.C.A., Prestes, A.M., Moraes-Fernandes, M.I.B., Guerra, M., 2003. Chromosome characterization in Thinopyrum ponticum (Triticeae, Poaceae) using in situ hybridization with different DNA sequences. Genet. Mol. Biol. 26, 505e510. Chen, Q., Ahmad, F., Collin, J., Comeau, A., Fedak, G., St-Pierre, C.A., 1998a. Genomic constitution of a partial amphiploid OK7211542 used as a source of immunity to barley yellow dwarf virus for bread wheat. Plant Breeding 117, 1e6. Chen, Q., Conner, R.L., Ahmad, F., Laroche, A., Feda, G., Thomas, J.B., 1998b. Molecular characterization of the genome composition of partial amphiploids derived from Triticum aestivum Th. intermedium as sources of resistance to wheat streak mosaic virus and its vector, Aceriella tosichella. Theor. Appl. Genet. 97, 1e8. Cox, T.S., 1991. The contribution of introduced germplasm to the development of U.S. wheat cultivars. Special Publication No. 17. In: Shands, H.L., Wiesner, L.E. (Eds.), Use of Plant Introductions in Cultivar Development: Part 1. Crop Science Society of America, Madison, Wisconsin, pp. 25e47. Fedak, G., Chen, Q., Conner, R.L., Laroche, A., Petroski, R., Armstrong, K.W., 2000. Characterization of wheat-Thinopyrum partial amphiploids by meiotic analysis and genomic in situ hybrization. Genome 43, 712e719. Friebe, B., Jiang, J., Raupp, W.J., McIntosh, R.A., Gill, B.S., 1996. Characterization of wheat-alien translocations conferring resistance to diseases and pests: current status. Euphytica 91, 59e87. Han, F., Gao, Z., Birchler, J., 2009. Reactivation of an inactive centromere reveals epigenetic and structural components for centromere specification in maize. Plant Cell 21, 1929e1939. Han, F., Liu, B., Li, J., Guo, X., Qi, B., Lv, Z., Fu, S., 2012. Molecular cytogenetic characterization of wheateThinopyrum elongatum addition, substitution and translocation lines with a novel source of resistance to wheat fusarium head blight. J. Genet. Genomics 39, 103e110. Han, F., Lv, Z., 2013. Multicolor Fluorescence In Situ Hybridization (MFISH) Method for Quickly Analyzing and Identifying Alien Chromosome of Wheat. Patent Publication Number: CN 103205500 A. http://www.google. com/patents/CN103205500A?cl¼zh. Accessed 5 July, 2014. Jin, Y., Singh, R.P., Ward, R.W., Wanyera, R., Kinyua, M., Njau, P., Fetch, T., Pretorius, Z.A., Yahyaoui, A., 2007. Characterization of seedling infection types and adult plant infection responses of monogenic Sr gene lines to race TTKS of Puccinia graminis f. sp. tritici. Plant Dis. 91, 1096e1099. Kato, A., Albert, P.S., Vega, J.M., Birchler, J.A., 2006. Sensitive FISH signal detection using directly labeled probes produced by high concentration DNA polymerase nick translation in maize. Biotech. Histochem. 81, 71e78. Kidwell, K.K., Osborn, T.C., 1992. Simple plant DNA isolation procedures. In: Bechmann, J.S., Osborn, T.C. (Eds.), Plant Genomes: Methods for Genetic and Physical Mapping. Kluwer Academic Publishers, London, pp. 1e13. Li, H., Conne, R.L., Chen, Q., Li, H., Laroche, A., Graf, R.J., Kuzyk, A.D., 2004. The transfer and characterization of resistance to common root from Thinopyrum ponticum to wheat. Genome 47, 215e223. Li, Z., Rong, S., Chen, S., Zhong, G., Mu, S., 1985. Wheat Wide Hybridization. Chinese Scientific Publishing Co., Beijing.
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Q. Zheng et al. / Journal of Genetics and Genomics xx (2014) 1e9 McIntosh, R., 1991. Alien sources of disease resistance in bread wheat. In: Sasakuma, T., Kinoshita, T. (Eds.), Proc. of Dr. H. Kihara Memorial Int. Symp. on Cytoplasmic Engineering in Wheat: Nuclear Organ Genomes Wheat Species. Kihara Institute for Biological Research, Yokohama City University, Yokohama, pp. 320e332. Mukai, Y., Nakahara, Y., Yamamoto, M., 1993. Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic highly repeated DNA probes. Genome 36, 489e494. Oliver, R.E., Xu, S.S., Stack, R.W., Friesen, T.L., Jin, Y., Cai, X., 2006. Molecular cytogenetic characterization of four partial wheat-Thinopyrum ponticum amphiploids and their reactions to Fusarium head blight, tan spot, and Stagonospora nodorum blotch. Theor. Appl. Genet. 112, 1473e1479. Sepsi, A., Molnar, I., Szalay, D., Molnar-Lang, M., 2008. Characterization of a leaf rust-resistant wheateThinopyrum ponticum partial amphiploid BE-1, using sequential multicolor GISH and FISH. Theor. Appl. Genet. 116, 825e834. Singh, R.P., Hodson, D.P., Huerta-Espino, J., Jin, Y., Bhavani, S., Njau, P., Herrera-Foessel, S., Singh, P.K., Singh, S., Govindan, V., 2011. The emergence of Ug99 races of the stem rust fungus is a threat to world wheat production. Annu. Rev. Phytopathol. 49, 465e481. Shannon, M.C., 1978. Testing salt tolerance variability among tall wheatgrass lines. Agron. J. 70, 719e722. Sharma, H.C., Ohm, H.W., Lister, R.M., Foster, J.E., Shukle, R.H., 1989. Response of wheatgrass and wheat wheatgrass hybrids to barley yellow dwarf virus. Theor. Appl. Genet. 77, 369e374.
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Stakman, E.C., Steward, D.M., Loegering, W.Q., 1962. Identification of Physiologic Races of Puccinia graminis var. tiritici. U.S. Gov. Print. Off, Washington, DC. Thomas, J., Chen, Q., Talbert, L., 1998. Genetic segregation and the detection of spontaneous wheat-alien translocation. Euphytica 100, 261e267. Turner, M.K., DeHaan, L.R., Jin, Y., Anderson, J.A., 2013. Wheatgrass-wheat partial amphiploids as a novel source of stem rust and Fusarium head blight resistance. Crop Sci. 53, 1994e2005. Williams, N.D., Miller, J.D., Klindworth, D.L., 1992. Induced mutations of a genetic suppressor of resistance to wheat stem rust. Crop Sci. 32, 612e616. Xu, S.S., Jin, Y., Klindworth, D.L., Wang, R.R.C., Cai, X., 2009. Evaluation and characterization of seedling resistances to stem rust Ug99 races in wheat-alien species derivatives. Crop Sci. 49, 2167e2175. Zhang, X., Dong, Y., 1994. Cytogenetic research on a hybrid of Triticum with Thinopyrum ponticum as well as its derivatives: II. Comparative research of eleven partial amphiploids derived from hybrid offspring of T. aestivum with both Th. ponticum and Th. intermedium. Acta Genet. Sin. 21, 287e296 (in Chinese with an English abstract). Zhang, X., Dong, Y., Wang, R.R., 1996. Characterization of genomes and chromosomes in partial amphiploids of the hybrid Triticum aestivum Thinopyrum ponticum by in situ hybridization, isozyme analysis and RAPD. Genome 39, 1062e1071. Zheng, Q., Li, B., Zhang, X., Mu, S., Zhou, H., Li, Z., 2006. Molecular cytogenetic characterization of wheat-Thinopyrum ponticum translocations bearing blue-grained gene(s) induced by g-ray. Euphytica 152, 51e60.
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Available online at www.sciencedirect.com
ScienceDirect Journal of Genetics and Genomics xx (2014) 1e9
JGG ORIGINAL RESEARCH
Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatLThinopyrum ponticum Partial Amphiploids Qi Zheng a, Zhenling Lv a, Zhixia Niu b, Bin Li a, Hongwei Li a, Steven S. Xu b, Fangpu Han a, Zhensheng Li a,* a
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China b United States Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, Fargo, ND 58102-2765, USA Received 24 January 2014; revised 27 May 2014; accepted 9 June 2014 Available online - - -
ABSTRACT Partial amphiploids created by crossing common wheat (Triticum aestivum L.) and Thinopyrum ponticum (Podp.) Barkworth & D. R. Dewey are important intermediates in wheat breeding because of their resistance to major wheat diseases. In this study, we examined the chromosome compositions of five Xiaoyan-series wheatTh. ponticum partial amphiploids (Xiaoyan 68, Xiaoyan 693, Xiaoyan 784, Xiaoyan 7430, and Xiaoyan 7631) using GISH, multicolor-GISH, and multicolor-FISH. We found several chromosome changes in these lines. For example, wheat chromosomes 1B and 2B were added in Xiaoyan 68 and Xiaoyan 7430, respectively, while wheat chromosome 6B was eliminated from Xiaoyan 693 and Xiaoyan 7631. Chromosome rearrangements were also detected in these amphiploids, including an interspecific translocation involving chromosome 4D and some intergenomic translocations, such as AeB and AeD translocations, among wheat genomes. Analysis of the Th. ponticum chromosomes in the amphiploids showed that some lines shared the same alien chromosomes. We also evaluated these partial amphiploids for resistance to nine races of stem rust, including TTKSK (commonly known as Ug99). Three lines, Xiaoyan 68, Xiaoyan 784, and Xiaoyan 7430, exhibited excellent resistance to all nine races, and could therefore be valuable sources of stem rust resistance in wheat breeding. KEYWORDS: Thinopyrum ponticum; Triticum aestivum; Partial amphiploid; Multicolor-genomic in situ hybridization (mc-GISH); Multicolor-fluorescent in situ hybridization (mc-FISH); Stem rust
INTRODUCTION Wild relatives of common wheat (Triticum aestivum L.) have been used extensively to transfer beneficial alien genes into wheat (Friebe et al., 1996; Bommineni and Jauhar, 1997; Sepsi et al., 2008). Tall wheatgrass, Thinopyrum ponticum (Podp.) Barkworth and D. R. Dewey [ ¼ Agropyron elongatum ssp. ruthenicum Beldie; Elytrigia pontica (Podp.) ´ Holub; Lophopyrum ponticum (Podp.) A Lo¨ve] (2n ¼ 10x ¼ 70) is a perennial species in the tribe Triticeae * Corresponding author. Tel: þ86 10 6480 6607, fax: þ86 10 6480 6605. E-mail address: [email protected] (Z. Li).
that is important for wheat improvement because of its resistance to a number of wheat diseases, as well as its stress tolerance and high crossability with various Triticum species (Shannon, 1978; Sharma et al., 1989; Cox, 1991; McIntosh, 1991). To transfer resistance genes from Th. ponticum to wheat, creating partial amphiploids from crosses between common wheat and tall wheatgrass is an important intermediate step. In the past several decades, a number of stable wheateTh. ponticum partial amphiploids have been developed that carry useful agronomic traits, such as resistance to wheat streak mosaic virus, barley yellow dwarf virus, common root rot, Fusarium head blight, tan spot, and Stagonospora nodorum blotch (Chen et al., 1998a, 1998b; Thomas
http://dx.doi.org/10.1016/j.jgg.2014.06.003 1673-8527/Copyright Ó 2014, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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et al., 1998; Fedak et al., 2000; Li et al., 2004; Oliver et al., 2006). Understanding of the compositions and structures of the introgressed Th. ponticum chromosomes in partial amphiploids is useful for further transferring desirable genes into common wheat. Therefore, the chromosome compositions of many wheateTh. ponticum partial amphiploids have been characterized. Chen et al. (1998a, 1998b) reported two octoploid Agrotriticum partial amphiploids, Agrotana and OK7211542, which had eight Js-genome chromosomes and eight E-or J-genome chromosomes. Oliver et al. (2006) described four partial wheateTh. ponticum amphiploids with different amounts of alien chromatin. Based on their genomic in situ hybridization (GISH) results, Fedak et al. (2000) revealed that each of six wheateTh. ponticum partial amphiploids contained a synthetic alien genome composed of different combinations of St-, J-, or Js-genome chromosomes. Li et al. (1985) reported the production of partial amphiploids, and designated them the Xiaoyan series, from hybrids between common wheat and Th. ponticum. Zhang et al. (1996) investigated the chromosome complements of this series with an Stgenome probe and A-, B-, D- and E-genome blocks. Xiaoyan 693 and Xiaoyan 7631 had identical St-genomes, while Xiaoyan 784, Xiaoyan 68, and Xiaoyan 7430 had a synthetic alien genome, including six pairs of St-genome chromosomes and one pair of Ee- (or Eb-) genome chromosomes. However, the specific wheat genome compositions in these partial amphiploids, including the missing, substituted, and translocated chromosomes, remain unclear. To more extensively explore these partial amphiploids for wheat improvement, detailed information on their genomic constitutions is needed. In the present study, we used a combination of multicolorgenomic in situ hybridization (mc-GISH) and multicolorfluorescent in situ hybridization (mc-FISH) to examine the cytogenetic compositions of five partial amphiploids in the Xiaoyan series (Xiaoyan 68, Xiaoyan 693, Xiaoyan 784, Xiaoyan 7430, and Xiaoyan 7631). In addition, because stem rust Ug99-lineage races are currently a major threat to global wheat production (Singh et al., 2011) and some wheateTh. ponticum partial amphiploids have excellent resistance to Ug99 (Xu et al., 2009; Turner et al., 2013), we evaluated seedlings of these five lines for resistance to stem rust Ug99 and eight other major races of stem rust from Africa and North America and confirmed that three partial amphiploids are good breeding material for stem rust resistance. RESULTS mc-GISH analysis revealed intergenomic rearrangements in the partial amphiploids In the mc-GISH analysis of the five partial amphiploids, the mitotic chromosomes were probed by D-genomic DNA (Aegilops tauschii) with Texas-red-5-dCTP (red), and total genomic DNA of Th. ponticum and Triticum urartu was probed with Fluorescein-12-dUTP (green) and blocked by Sgenome (Aegilops speltoides) DNA. Based on the distribution
of hybridization signals on chromosomes, the A-, B-, and Dgenome from common wheat and the genomes from Th. ponticum could be distinguished at the same time. The A-, B-, and D-genome chromosomes were labeled with yellowegreen, gray, and red florescences, respectively, while the Th. ponticum chromosomes were labeled with green florescences. A number of intergenomic rearrangements were detected in the wheat genome by mc-GISH analysis. As illustrated in Fig. 1A, Xiaoyan 68 had 12 green-fluorescing chromosomes that originated from tall wheatgrass and two interspecific translocation chromosomes with green terminal fragments on red short arms. Among the remaining 42 wheat chromosomes, 14 fluoresced yellowegreen, 16 gray, and 12 red. Among the 12 red-fluorescing chromosomes, one pair carried a terminal yellowegreen signal, suggesting that an intergenomic rearrangement involving the A and D genomes had taken place in the wheat genome. Therefore, in addition to 12 alien chromosomes and 2 wheateTh. ponticum translocation chromosomes, Xiaoyan 68 also had 14 A-genome, 16 B-genome, 10 D-genome, and 2 AeD translocation chromosomes. Similarly, Xiaoyan 784 possessed 14 Th. ponticum chromosomes and 42 wheat chromosomes composed of 10 Agenome, 14 B-genome, and 12 D-genome chromosomes plus two pairs of AeD translocation chromosomes and one pair of AeB translocation chromosomes (Fig. 1C). Xiaoyan 7430 contained 12 alien chromosomes plus 44 wheat chromosomes comprising 8 A-genome, 16 B-genome, and 10 D-genome chromosomes, 4 pairs of AeD translocation chromosomes, and one pair of AeB translocation chromosomes (Fig. 1D). Among the four pairs of AeD translocation chromosomes in Xiaoyan 7430, three were mainly D genome with a small Agenome segment on the distal region of the long arm, and one comprised a large part of the A genome with a small Dgenome fraction on its long arm. Both Xiaoyan 693 and Xiaoyan 7631 carried 16 Th. ponticum chromosomes plus 40 wheat chromosomes consisting of 12 A-genome, 12 Bgenome, 14 D-genome, and two AeB translocation chromosomes (Fig. 1B and E). Sequential GISH and mc-FISH analyses revealed the chromosome compositions of the partial amphiploids Although each of the five lines consistently had 56 chromosomes, the number of alien chromosomes ranged from 12 to 16, suggesting that chromosome deletion and substitution occurred from the wheat genome. Chromosome elimination and addition can be detected by sequential GISH and mc-FISH methodology (Fig. 2AeJ). The results of mc-GISH analysis revealed that a pair of B-genome chromosomes was absent from Xiaoyan 693 and Xiaoyan 7631 (Fig. 2D and J). Comparing their mc-FISH results using probes pAs1 and pSc119.2 with that of common wheat (Mukai et al., 1993), the missing chromosome was determined to be 6B, because of faint pAs1 bands on the satellite and short arm and a terminal and an interstitial pSc119.2 band on the long arm. In both Xiaoyan 68 and Xiaoyan 7430 a pair of B-genome chromosomes was added (Fig. 2B and H). The added
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Fig. 1. Multicolor (mc)-GISH patterns of the five partial amphiploids. The different florescence signals show A-genome chromosomes (yellow-green), B-genome chromosomes (gray), D-genome chromosomes (red) and the chromosomes of Th. ponticum (green). Arrows indicate AeD translocation chromosomes in Xiaoyan 68 (A), Xiaoyan 784 (C) and Xiaoyan 7430 (D); arrow heads indicate AeB translocation chromosomes in Xiaoyan 693 (B), Xiaoyan 7430 (D) and Xiaoyan 7631 (E); and asterisks indicate a wheateTh. ponticum translocation chromosomes.
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Fig. 2. Sequential GISH and mc-FISH analyses of the five partial amphiploids. GISH analysis of the Thinopyrum ponticum chromosomes in Xiaoyan 68 (A), Xiaoyan 693 (C), Xiaoyan 784 (E), Xiaoyan 7430 (G) and Xiaoyan 7631 (I). Bright yellow signals marked the Th. ponticum chromosomes or chromosomal segments. The Arabic figures refer to the alien chromosomes according to the size from big to small. The wheat chromosomes were counterstained with PI (red). Mc-FISH on the same metaphase chromosome spreads in Xiaoyan 68 (B), Xiaoyan 693 (D), Xiaoyan 784 (F), Xiaoyan 7430 (H) and Xiaoyan 7631 (J) by pAs1 (green) and pSc119.2 (red) simultaneously. Arrows refer to the AeD translocation chromosomes in H.
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Fig. 2. continued.
chromosomes in Xiaoyan 7430 showed a distinctive 2B pattern, with strong pSc119.2 bands in the terminal region of the short arm and in an intercalary region of the long arm. In contrast, the extra chromosomes in Xiaoyan 68 had strong hybridization sites with pSc119.2 on the distal end of the long arm, indicating that they were chromosome 1B. In addition, mc-FISH results revealed that the wheateTh. ponticum translocation chromosomes in Xiaoyan 68 were wheat chromosome 4D with a terminal alien segment on the short arm. The alien fragment was very small, amounting to 20% of the translocated short arm. Although many intergenomic rearrangements in the wheat genomes occurred in these five wheat lines, only two pairs of AeD translocated chromosomes were identified in Xiaoyan 7430 using mc-FISH analysis because of a lack of distinctive bands. Moreover, the fluorescence signals of repetitive DNA sequences pAs1 and pSc119.2 were observed on the Th. ponticum-derived chromosomes (Fig. 3AeE). The pAs1 bands were located on almost all of the alien chromosomes. They were more frequent in the terminal regions than in the interstitial
ones but were absent in the centromeric and most of the pericentromeric regions. The pSc119.2 sites were observed in terminal regions of a few wheatgrass chromosomes, such as three alien chromosomes in Xiaoyan 7430, two in Xiaoyan 693, one in Xiaoyan 7631, and one in Xiaoyan 784, but were missing from alien chromosomes in Xiaoyan 68. Based on the diagram of both sequence signals, three perennial grass chromosomes were identical in Xiaoyan 693 (Fig. 3B, chromosomes 1, 3, and 5) and in Xiaoyan 7631 (Fig. 3E, chromosomes 1, 3 and 8). The fifth-ranked chromosomes in Xiaoyan 68 and Xiaoyan 784 originated from one decaploid wheatgrass chromosome, because they had duplicate fluorescence bands. In addition, the third alien chromosomes of Xiaoyan 784 and Xiaoyan 7430 possessed the same pAs1 signals, indicating that they were identical. Reactions of five partial amphiploids to stem rust The infection types of the partial amphiploids to the seven North American (RTQQC, TCMJC, TPMKC, QFCSC,
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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A
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Fig. 3. The fluorescence signal of repetitive DNA sequences on the Th. ponticum-derived chromosomes. Idiogram of Thinopyrum ponticum chromosomes in Xiaoyan 68 (A), Xiaoyan 693 (B), Xiaoyan 784 (C), Xiaoyan 7430 (D) and Xiaoyan 7631 (E), showing the locations of pAs1 (green) and pSc119.2 (red) on drawn chromosomes side by side with FISH photographs of the same chromosomes.
MCCFC, TMLKC, and TTTTF) and two African (TTKSK and TRTTF) races of the stem rust pathogen are listed in Table 1. As expected, the susceptible control cultivar, Chinese Spring (CS), had infection types (ITs) 3þ or 4 to all races. The five Xiaoyan-series partial amphiploids exhibited various levels of resistance. Xiaoyan 693 was highly or moderately resistant to four races, RTQQC (23), TCMJC (12), TPMKC (123), and TMLKC (23), but it was susceptible to QFCSC (34) and MCCFC (32). Xiaoyan 7631 was highly resistant to RTQQC (12), TCMJC (11), MCCFC (12), and TMLKC (12) and moderately resistant to TPMKC (23), but it was susceptible to QFCSC (32). Other three partial amphiploids were highly resistant to all nine races. Among them, Xiaoyan 68 and Xiaoyan 7430 were near-immune (IT ;) to all nine races and Xiaoyan 784 expressed variable levels
of resistance from near-immunity (IT ;) to moderate resistance (2;), suggesting that the three partial amphiploids are valuable source with resistance to Ug99 and other races of stem rust. DISCUSSION Zhang et al. (1996) proposed that Th. ponticum has two basic genomes, E and St, and suggested the formula StStEeEbEx for this decaploid. In Th. ponticum, Ee and Eb are two sub-genome types of the E genome and originated from T. elongatum and T. bessarabicum, respectively. Ex indicates either Ee or Eb, depending on the accession, and the St genome is donated by the mono-genome genus Pseudoroegneria. The GISH results showed that both Xiaoyan 693 and Xiaoyan 7631 had identical
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Table 1 Infection types produced by five wheateTh. ponticum partial amphiploids to nine races of Puccinia graminis f. sp. tritici Infection type to racea
Line RTQQC
QFCSC
TCMJC
MCCFC
TPMKC
TMLKC
TTKSK
TRTTF
TTTTF
Xiaoyan 68
;
;
;
;
;
;
;
0;
0;
Xiaoyan 693
23
34
12
32
123
23
Xiaoyan 784
;
12
1
12
1
1
2;
;
2;
Xiaoyan 7430
;
;
;
;
;
;
;
;
;
Xiaoyan 7631
12
32
11
12
23
12
Chinese Spring
4
4
4
4
4
4
3þ
3þ
3þ
Infection types (ITs) were followed Stakman et al. (1962), where 0 = immune, ; ¼ necrotic flecks, 1 ¼ small necrotic pustules, 2 ¼ small to medium sized chlorotic pustules with green island, 3 ¼ medium sized chlorotic pustules, and 4 ¼ large pustules without chlorosis. ITs 0, ;, 1, 2, or any combination of these indicated resistance, and ITs 3 or 4 indicated susceptibility. Hence 123 is predominantly IT 1, with decreasing amounts of IT 2 and IT 3. 1 indicates small IT 1. 2 indicates small IT 2. 3þ indicates large IT 3. a
St-genome chromosomes, but Xiaoyan 784, Xiaoyan 68, and Xiaoyan 7430 had a synthetic alien genome that included six pairs of St-genome chromosomes and one pair of Ee- (or Eb-) genome chromosomes. Zhang and Dong (1994) found that many univalents were present in test crosses among Xiaoyan 784, Xiaoyan 68, and Xiaoyan 7430, suggesting that many modifications exist to the St genomes in Th. ponticum. In this study, although Xiaoyan 693 and Xiaoyan 7631 shared some Th. ponticum chromosomes, they also have five different chromosomes. Thus, the cytogenetical evidence in our study indicated that Xiaoyan 7631 and Xiaoyan 693 are two different lines. In the previous study (Zhang and Dong, 1994), seven pairs of tall wheatgrass chromosomes were found in both Xiaoyan 68 and Xiaoyan 7430. However, Xiaoyan 7430 had six pairs of alien chromosomes, and Xiaoyan 68 possessed six pairs of Th. ponticum chromosomes as well as a pair of Th. ponticume wheat translocation chromosomes. These findings suggested that some octoploid Trititrigia are not cytogenetically stable, and the genome compositions of these partial amphiploids can change without altering the chromosome number. The pAs1 probe was previously reported to be located in the terminal regions of both arms and dispersed throughout all 70 chromosomes, except in the centromeric and most of the pericentromeric regions, while that pSc119.2 labeling was distributed on the short arms of 17 chromosomes and the long arms of five others (Brasileiro-Vidal et al., 2003). In this study, however, some alien chromosome arms completely lacked this tandem repeat, which could be explained by recombination and modification within Th. ponticum chromosome segments during formation of the synthetic species from the perennial grass. Based on the ITs, the stem-rust resistance of five Xiaoyanseries partial amphiploids could be divided into levels I (Xiaoyan 68 and Xiaoyan 7430), II (Xiaoyan 784), and III (Xiaoyan 7631 and Xiaoyan 693). The partial amphiploids with levels I and II were clearly resistant to all races. In particular, Xiaoyan 68 and Xiaoyan 7430 were most immune and should be valuable sources with stem rust resistance, while resistance in Xiaoyan 784 varied from high to moderate. The
partial amphiploids in level III had distinctly less resistance to most of rust races tested, and they may have less utility in wheat breeding for resistance to stem rust. MATERIALS AND METHODS Plant materials Five partial amphiploids Xiaoyan 68, Xiaoyan 693, Xiaoyan 784, Xiaoyan 7430, Xiaoyan 7631 were produced from wheat e Th. ponticum hybrids by the wheat distant hybridization group in Northwest Institute of Botany, Chinese Academy of Sciences (Yangling, Shaanxi, China). Wheat cv. “Chinese Spring” and Th. ponticum accessions R431 were also used in this study. All the above materials were collected in our laboratory. Chromosome preparation Chromosome spread preparation was carried out following the procedures of Kato et al. (2006) and Han and Lv (2013) with some minor modifications. Seeds were germinated on moist filter paper at 23 C for 48 h. Root tips of 1e2 cm length were collected and pretreated in N2O at 10 atm pressure for 2 h to accumulate metaphases, and then fixed in 90% acetic acid. Multicolor-genomic in situ hybridization (mc-GISH) Mc-GISH was performed using published protocols with some modification (Han et al., 2012). Total genomic DNA of Th. ponticum, T. urartu, A. speltoides and A. tauschii was isolated from the young fresh leaves (Kidwell and Osborn, 1992). Fluorescein-12-dUTP (green) labeled Th. ponticum and T. urartu genomic DNA and Texas-red-5-dCTP (red) labeled A. tauschii genomic DNA served as probes. Sheared A. speltoides genomic DNA was used as a block. After hybridization, the slides were washed in 2 SSC and mounted in Vectashield mounting medium (containing DAPI; Vector Laboratories, USA). Detection and visualization were carried out as described by Han et al. (2009).
Please cite this article in press as: Zheng, Q., et al., Molecular Cytogenetic Characterization and Stem Rust Resistance of Five WheatThinopyrum ponticum Partial Amphiploids, Journal of Genetics and Genomics (2014), http://dx.doi.org/10.1016/j.jgg.2014.06.003
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Sequential genomic and multicolor-fluorescent in situ hybridization (sequential GISH and mc-FISH) Procedures for sequential GISH and mc-FISH were followed according to Zheng et al. (2006). Briefly, Th. ponticum genomic DNA was labeled with digoxigenin-11-dUTP and served as a probe, and wheat DNA was used as a block. The ratio between probe and block was 1:250. The hybridized probes were detected with anti-digoxigenin conjugated by fluorescence isothiocyanate (FITC). The slides were counterstained with propidium iodide (PI, 0.25 mg/ mL) in the Vectashield mounting medium (Vector Laboratories, USA). Mc-FISH was carried out after GISH analysis using two probes, pAs1 labeled with digoxigenin-11-dUTP and pSc119.2 labeled with biotin-11-dUTP. Two probes were mixed 1:1 before the hybridization. After the hybridization, anti-digoxigenin-FITC and avidin-rhodamine were used for detection of the two probes simultaneously. The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI). All cells with good hybridization signals were captured by DVC CCD camera attached to an Olympus BX50 epifluorescence microscope. The images captured for each color channel were merged using program Image-Pro Plus 4.0 (Digital Video Camera, USA). Measurements were made on the digital images of the signals. A minimum of five translocation chromosomes were measured for each line. The identification of the alien chromosomes and their idiograms were determined based on the chromosome length, arm ratio, and mc-FISH patterns instead of the homology to wheat. Stem rust resistance evaluation The six major North American races RTQQC, TCMJC, TPMKC, QFCSC, MCCFC, and TMLKC were firstly used for the stem rust testing of the five partial amphiploids at USDAARS, Northern Crop Science Laboratory, Fargo, ND. The seedlings were grown in the greenhouse at 20 Ce23 C with 16/8 (day/night) photoperiod. Seven-day-old seedlings were inoculated as described by Williams et al. (1992). Inoculated plants were then moved to a greenhouse at 20 C23 C. Based on the availability of the seed samples, three lines, Xiaoyan 68, Xiaoyan 784, and Xiaoyan 7430, were further evaluated for reactions to two African races, TTKSK (Ug99) and TRTTF, and the North American race TTTTF, all with broad virulence spectra at the seedling stage. The inoculation with the three races was performed using the procedure described by Jin et al. (2007) at USDA-ARS, Cereal Disease Laboratory, St. Paul, MN, USA. In both evaluation trials, infection types (ITs) were scored 14 days after inoculation using the system of Stakman et al. (1962), where 0 ¼ immune, ; ¼ necrotic flecks, 1 ¼ small necrotic pustules, 2 ¼ small to medium sized chlorotic pustules with green island, 3 ¼ medium sized chlorotic pustules, and 4 ¼ large pustules without chlorosis. ITs 0, ;, 1, 2, or any combination of these indicated resistance, and ITs 3 or 4 indicated susceptibility.
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