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Pm67, a new powdery mildew resistance gene transferred from Dasypyrum villosum chromosome 1 V to common wheat (Triticum aestivum L.)
Journal Pre-proofs Pm67, a new powdery mildew resistance gene transferred from Dasypyrum villosum chromosome 1V to common wheat (Triticum aestivum L.) Ruiqi Zhang, Chuanxi Xiong, Huanqing Mu, Ruonan Yao, Xiangru Meng, Lingna Kong, Liping Xing, Jizhong Wu, Yigao Feng, Aizhong Cao PII: DOI: Reference:
S2214-5141(20)30176-8 https://doi.org/10.1016/j.cj.2020.09.012 CJ 550
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The Crop Journal
Received Date: Revised Date: Accepted Date:
30 July 2020 3 September 2020 3 November 2020
Please cite this article as: R. Zhang, C. Xiong, H. Mu, R. Yao, X. Meng, L. Kong, L. Xing, J. Wu, Y. Feng, A. Cao, Pm67, a new powdery mildew resistance gene transferred from Dasypyrum villosum chromosome 1V to common wheat (Triticum aestivum L.), The Crop Journal (2020), doi: https://doi.org/10.1016/j.cj.2020.09.012
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Pm67, a new powdery mildew resistance gene transferred from Dasypyrum villosum chromosome 1V to common wheat (Triticum aestivum L.) Ruiqi Zhang a, 1, *, Chuanxi Xiong a, 1, Huanqing Mu a, Ruonan Yao a, Xiangru Meng a, Lingna Kong a, Liping Xing a, Jizhong Wu b, Yigao Feng a, Aizhong Cao a a
College of Agronomy/JCIC-MCP/National Key Lab of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural
University, Nanjing 210095, Jiangsu, China b
Institute of Germplasm Resources and Biotechnology/Provincial Key Lab of Agrobiology, Jiangsu Academy of Agricultural
Sciences, Nanjing 210014, Jiangsu, China
Abstract: Powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), is a global disease that poses a serious threat to wheat production. To explore additional resistance gene, a wheat-Dasypyrum villosum 1V#5 (1D) disomic substitution line NAU1813 (2n = 42) with high level of seedling resistance to powdery mildew was used to generate the recombination between chromosomes 1V#5 and 1D. Four introgression lines, including t1VS#5 ditelosomic addition line NAU1815, t1VL#5 ditelosomic addition line NAU1816, homozygous T1DL·1VS#5 translocation line NAU1817, and homozygous T1DS·1VL#5 translocation line NAU1818 were developed from the selfing progenies of 1V#5 and 1D double monosomic line that derived from F1 hybrids of NAU1813/NAU0686. All of them were characterized by fluorescence in situ hybridization, genomic in situ hybridization, 1V-specific markers analysis, and powdery mildew tests at different developmental stages. A new powdery mildew resistance gene named Pm67 was physically located in the terminal bin (FL 0.70–1.00) of 1VS#5. Lines with Pm67 exhibited seedling stage immunity and tissue-differentiated reactions at adult plant stage. The sheaths, stems, and spikes of the Pm67 line were still immune, but the leaves showed a low degree of susceptibility. Microscopic observation showed that most penetration attempts were stopped in association with papillae on the sheath, and colonies cannot form conidia on the susceptible leaf of Pm67 line at adult plant stage, suggesting that the defence layers of the Pm67 line is tissue-differentiated. Thus, the T1DL·1VS#5 translocation line NAU1817 provides a new germplasm in wheat breeding for improvement of powdery mildew resistance.
Keywords: Wheat; Dasypyrum villosum; Powdery mildew; Pm67 1. Introduction Wheat (Triticum aestivum L.) is a widely produced grain crop that contributes to global food security. Existing and new races of fungal pathogens constantly threaten the high and stable yields of wheat. Among them, powdery *
Corresponding author: Ruiqi Zhang, E-mail address: [email protected].
1
These authors contributed equally to this work.
Received: 2020-07-30; Revised: 2020-09-03; Accepted: 2020-11-03. 1
mildew, caused by Blumeria graminis f. sp. tritici (Bgt), occurs in many wheat-growing regions, especially in highly productive areas where semi-dwarf cultivars are planted under high input conditions [1]. Powdery mildew interferes with photosynthesis, thereby reducing plant growth, heading, grain filling, and end-use quality. When weather is favorable and the mycelium occurs at flag leaf emergence or during heading, grain yield losses of up to 40% can occur [2]. Utilization of resistant cultivars is the most practical and cost-effective means to control this disease. The traditional powdery mildew resistance breeding strategy involves the use of a single major resistance gene in the major wheat production where powdery mildew usually causes significant yield losses. Genotypes produced by this strategy provide strong selection pressure for pathogen variants with an increased ability to reproduce upon them. Consequently, the effectiveness of most deployed resistance genes is rapidly lost as the pathogens acquire virulence. Although Pm2, Pm4, Pm6, Pm8, Pm13, Pm21, and Pm30 genes are frequently detected in winter wheat cultivars in China [3–6], only Pm21 provides sufficient protection from powdery mildew in the field currently. Varieties with the T6AL·6VS translocation containing Pm21 are planted on more than 4 million hectares annually [6,7]. Moreover, more than 40% of elite lines carrying Pm21 were detected in the main wheat production region Yangze River, China, where powdery mildew can cause significant yield losses and most of the known Pm genes have lost their effectiveness. So far due to good protection of Pm21 the use of this gene continues, which will increase the selection pressure for virulent variants of the pathogen. Thus, the discovery and utilization of additional sources of powdery mildew resistance to address this vulnerability is necessary for wheat improvement programs in China and elsewhere. Wild relatives of wheat are an important source of genetic variation for variety improvement [8]. Most species belonging to the tertiary gene pool are not common hosts for Bgt and their resistance appears to be broad-spectrum and effective over long periods of time [9,10]. Among them Dasypyrum villosum (2n = 14, VV) is recognized as an excellent species of new resistance genes because it is a near-nonhost to many wheat pathogens and has widespread immunity to multiple diseases, such as stripe rust, leaf rust and powdery mildew [11]. Two D. villosum accessions, 91C43 (#4, originally introduced from the Cambridge University Botanical Garden, UK), and 01I140 (#5, collected in the Pisa region of Italy), have been used to develop wheat-D. villosum introgression lines at the Cytogenetics Institute of Nanjing Agricultural University (CINAU) [12,13]. Three formally designated powdery mildew resistance genes, Pm21, Pm55, and Pm62, have been transferred from D. villosum to wheat by means of compensating Robertsonian translocations (RobTs), confirming that D. villosum is indeed an important source of powdery mildew resistance genes for wheat [13–15].
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A set of 1V#5 to 7V#5 disomic addition/substitution lines have been developed in wheat variety NAU0686 background from the progenies of NAU0686/NAU1802 (AABBVV (#5)) at CINAU. Their responses to stripe rust, leaf rust, and powdery mildew were assessed in field disease nurseries and the greenhouse. Results indicated that wheat-D. villosum 1V#5 (1D) disomic substitution line NAU1813 conferred effective resistance against Bgt at both seedling and adult stages. The objectives of this study were to: (1) characterize the response of NAU1813 and its chromosome 1V#5 to powdery mildew, (2) introgress the chromosome 1V#5 powdery mildew resistance gene from NAU1813 into the wheat genome through the breakage-fusion mechanism, and (3) physically map a new powdery mildew resistance gene on chromosome 1V#5.
2. Materials and methods 2.1. Plant materials Durum variety ZY1286 introduced from CIMMYT was initially crossed with D. villosum accession 01I140 (#5) to generate the amphiploid NAU1802 (2n = 42, AABBVV). The 1V#5 (1D) disomic substitution line NAU1813 and
1V#5
deletion
line
NAU1814
were
obtained
from
BC4F2
progenies
of
the
cross
NAU0686/NAU1802//4*NAU0686. The T1DL·1VS#4 translocation line NAU1811 and T1DS·1VL#4 translocation line NAU1812 derived from D. villosum accession 91C43 (#4) [16] were used as positive controls in molecular marker analyses. An elite T5DL·5VS#4 translocation line TF5V-1 (pedigree: NAU0686/NAU415 (T5DL·5VS#4)//4*NAU0686) harboring Pm55 was used to pyramid powdery mildew resistance genes. All the genetic stocks developed and maintained at CINAU were summarized in Table S1. All the 1V#5 introgression lines were planted in the field powdery mildew nursery at the Jiangpu Experiment Station of Nanjing Agricultural University for examination of powdery mildew response at different developmental stages. Following inoculation, the materials were covered with plastic sheeting for protection and enhancing disease development during the winter season. Ten plants were grown in each 1.0 m row with 25 cm spacing between rows. Each line with the exception of F2 segregating individuals was grown in four rows with three replications. The recurrent parent NAU0686 planted on both sides of each experimental row served as an inoculum spreader of Bgt and susceptible control. 2.2. Cytogenetic analysis Root tips from the germinating wheat seedlings were soaked in 0.2 μmol L−1 amiprophos-methyl (APM) solution for 2 h at 24 °C and then treated with nitrous oxide for 1.5 h and 90% acetic acid for 10 min following Komuro et al. [17]. Slides were prepared for genomic in situ hybridization (GISH) and fluorescence in situ hybridization 3
(FISH) as described by Du et al. [18]. Total genomic DNA of D. villosum was labeled with fluorescein-12-dUTP (green) as the GISH probe. FISH was performed using mixed oligo-pAs1-2 and oligo-pAs1-5 probes that preferentially paint tandem repeats on D genome chromosomes of common wheat. The oligo-probes labeled by 6-carboxytetramethylrhodamine (TAM) producing red signals were synthesized by the Tsingke Biological Technology Company (Nanjing, China) following the procedures described by Du et al. [18]. After hybridization, chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and colored blue (Invitrogen Life Science, Carlsbad, CA, USA), and slides were examined under an Olympus BX60 microscope (Olympus Co.). Images were captured with a SPOT Cooled Color Digital Camera (Diagnostic Instruments, Sterling Heights, MI, USA) and processed using Adobe Photoshop CS 7.0 (Adobe Systems, San Jose, CA, USA). 2.3. Molecular marker analysis Genomic DNA of the genetic stocks were isolated from seedling leaves using a DNA Secure Plant Kit (Tiangen Biotech Co., Ltd, Nanjing), according to the manufacturer’s instructions. Four chromosome 1V-specific markers, including 1V-207, 1V-253, 1EST-493, and P1/P5 developed previously [19,20] were used to screen the progenies of NAU0686/NAU1802//4*NAU0686 for lines carrying chromosome 1V#5 or parts thereof. Markers 1V-207 and 1V-253 were specific for chromosome arm 1VS, and 1EST-493 and P1/P5 were specific for 1VL (Table S2). PCR amplification was conducted using a T100TM Thermal Cycler (Bio-Rad Laboratories, Hercules, USA) in 10 μL reaction volumes containing 40 ng of genomic DNA, 2.5 mmol L−1 of each dNTP, 2 μmol L−1 of each primer, 2.5 mmol L−1 MgCl2, 1×PCR buffer (10 mmol L−1 Tris-HCl, 50 mmol L−1 KCl, pH 8.5), and 0.2 U Taq DNA polymerase. Amplification of DNA samples was programmed at 95 °C for 4 min followed by 32 cycles at 94 °C, 58 °C for 45 s, and 72 s of elongation at 72 °C, with a final extension at 72 °C for 10 min. PCR products were separated on 8% non-denaturing polyacrylamide gels (Acr:Bis = 39:1) and visualized by silver staining. 2.4. Evaluation of powdery mildew response Seedlings of chromosome 1V#5 introgression lines and recurrent parent NAU0686 were planted in a greenhouse at 18/22 °C (night/day) with 80% relative humidity and photoperiod of 16 h light/8 h darkness. Bgt isolates E26 and E31, and a mixed field sample collected from Yangze River region were used in separate tests under controlled greenhouse conditions. Infection types (ITs) were recorded on a 0–4 scale at 10 days post inoculation when susceptible NAU0686 control plants were heavily diseased [3]. In addition, a mixture of Bgt isolates (collected from Yangze River region) was used to infect NAU0686 and 1V#5 introgression lines at the three-leaf growth stage in the field powdery mildew nursery. Reactions of 1V#5 4
introgression lines evaluated at the tillering, stem elongation, booting, heading, and grain-filling stages were recorded on a 0–9 IT scale [21]. 2.5. Microscopic evaluation For evaluation of the difference in mycelium development, the leaf and sheath segments of the resistant line NAU1813 and susceptible line NAU1814 at the grain-filling stage were examined by an endogenous peroxidase-dependent in situ histochemical staining procedure. About 5 cm segments of leaf and sheath were transferred to tubes to distain by a solution of acetic acid-ethanol (1:3, v/v) for 24 h. After that, about 1.5 cm segments were cut and immersed into a solution containing 0.6% Coomassie Brilliant Blue, 15% trichloroacetic acid in 99% methanol (1:1 v/v) for 25 min [10]. Microscope slides were prepared by embedding the stained segments of leaf and sheath in 100% glycerol, with the adaxial side up. Slides were screened using an Olympus BX60 microscope (Olympus Co.) under a white light. 2.6. Evaluation of agronomic traits The plants with and without T1DL·1VS#5 translocated chromosome were evaluated for plant height (PH, cm), main spike length (SL, cm), grains per spike (GPS) and 1000-kernel weight (TKW, g). Differences in main agronomic traits between the genotypes were evaluated by means of Tukey’s post hoc test (SPSS 16.0) at the P <0.05 significance level.
3. Results 3.1. Development and identification of the 1V#5 introgression lines Two wheat-D. villosum 1V#5 introgression lines in the BC4F2 progeny of NAU0686/NAU1802//4*NAU0686 were firstly identified by GISH/FISH and molecular markers. NAU1813 had 40 wheat chromosomes and two D. villosum chromosomes with one strong hybridization band in the proximal centromere region, and one strong band each in the subtelomeric and telomeric regions of the short arm (Fig. 1a). FISH patterns produced with the oligo-pAs1-2 and oligo-pAs1-5 probes and four 1V-specific marker analysis confirmed that the wheat chromosome 1D pair had been substituted by a pair of D. villosum chromosomes 1V#5 (Figs. 1a and 2). NAU1814 had 42 wheat chromosomes and two D. villosum chromosomes with one strong hybridization band in the proximal centromere region but missing the bands in the subtelomeric and telomeric regions of the short arm (Fig. 1b). The specific bands for molecular markers 1V-207, 1EST-493, and P1/P5 were present but the 1VS-specific band of 1V-253 was missing (Fig. 2a and b), suggesting that NAU1814 had a deletion of the distal 5
region of the short arm of chromosome 1V#5 with the breakpoint at FL 0.70. Seedling stage powdery mildew tests showed that NAU1813 was immune (IT 0) whereas NAU1814 was susceptible (IT 4), indicating that a powdery mildew resistance gene(s) was located in the terminal region of chromosome arm 1VS.
Fig. 1 – Identification of the six wheat-D. villosum 1V#5 introgression lines through genomic in situ (GISH) and fluorescent in situ (FISH) hybridization. D. villosum genomic DNA labeled with fluorescein-12-dUTP (green) as probes was used for GISH. Probes for FISH were Oligo-pAs1-2 and Oligo-pAs1-5 labeled with TAM (red). Wheat chromosomes were counterstained with DAPI (blue). (a) GISH/FISH patterns of NAU1813 (1V#5 (1D), 2n = 42); Chromosome 1V#5 shows one strong hybridization band at the proximal centromere region, and one strong band each in the subtelomeric and telomeric regions of the short arm. (b) GISH/FISH patterns of NAU1814 (2n = 44), which has a spontaneous deletion of distal part of the short arm of chromosome 1V#5 with the breakpoint at FL0.70. (c) GISH/FISH patterns of NAU1815 (2n = 44), which was a t1VS#5 ditelosomic addition line. (d) GISH/FISH patterns of NAU1816 (2n = 44), a t1VL#5 ditelosomic addition line. (e) GISH/FISH patterns of NAU1817 (2n = 42), a homozygous T1DL·1VS#5 translocation line. (f) GISH/FISH patterns of NAU1818 (2n = 42), a homozygous T1DS·1VL#5 translocation line. Bars, 10 μm
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Fig. 2 – PCR amplification patterns of representative 1VS- and 1VL-specific markers in the wheat-D. villosum introgression lines. 1VS-specific markers 1V-207 (a) and 1V-253 (b); 1VL-specific markers 1EST-493 (c) and P1/P5 (d). Marker, DL2000. The 1DL·1VS lines NAU1811 and NAU1817 have 1VS- and 1DL-specific bands but lack the 1DS-specific band. The 1DS·1VL lines NAU1812 and NAU1818 have 1VL- and 1DS-specific bands but lack the 1DL-specific band.
In order to identify Robertsonian whole-arm translocations of chromosome 1V and a wheat chromosome, the progeny of double monosomic 1V#5 and 1D hybrids of NAU1813 crossed with NAU0686 were screened. Among 250 selfing progenies of 1V#5 and 1D double monosomic plant Bx-4b-13 screened with four 1V-specific markers, 15 plants (6%) had only the 1VS-specific markers 1V-207 and 1V-253. GISH/FISH analysis showed that 14 of these plants had a 1VS#5 telosome and one plant (YL-2b-11) was heterozygous with a normal 1D chromosome and a T1DL·1VS#5 translocation chromosome. Other 13 plants (5.2%) had only the 1VL-specific markers 1EST-493 and P1/P5; 12 had a 1VL#5 telosome and one (YL-2b-3) was heterozygous for a T1DS·1VL#5 Robertsonian translocation. Lines derived from these various plants included t1VS#5 ditelosomic addition line 7
NAU1815 (Fig. 1c), t1VL#5 ditelosomic addition line NAU1816 (Fig. 1d), homozygous T1DL·1VS#5 translocation line NAU1817 (Fig. 1e), and homozygous T1DS·1VL#5 translocation line NAU1818 (Fig. 1f). In addition, 100 seedlings among the selfing progenies of YL-2b-11 (heterozygous T1DL·1VS#5) were examined by GISH; 20 were disomic for a T1DL·1VS#5 recombinant chromosome pair, 58 were heterozygous and 22 lacked alien chromatin indicating normal gametic transmission of the translocated chromosome relative to its intact 1D homologue (χ21:2:1 = 2.56, P >0.05). Measures of agronomic traits shown in Table S3 revealed no obvious differences in plant height, 1000-kernel weight, main spike length, and seeds per main spike between plants with and without the T1DL·1VS#5 recombinant chromosome. 3.2. Powdery mildew reactions Seedlings of above YL-2b-11 selfing-plants were tested for reaction to the field isolates Bgt. All 78 plants harboring 1VS#5 chromosome arm were immune (IT 0–0;), whereas the remaining 22 plants lacking 1VS#5 chromatin were susceptible (IT 3–4) (Table S3). Thus, the powdery mildew resistance gene(s) located on chromosome arm 1VS#5 was dominant and formally designated Pm67. For characterization of Pm67 resistance at different growth stages NAU1813, NAU1814, NAU1815, NAU1816, NAU1817, and NAU1818 were tested separately for reactions to Bgt isolates E26 and E31 and a field collection at two- to three-leaf stage. NAU1813, NAU1815, and NAU1817 had IT of 0 (Fig. 3a), whereas NAU1814, NAU1816, and, NAU1818 were susceptible (IT 4) as was the recurrent parent NAU0686. In field nursery evaluations, NAU1813, NAU1815, and NAU1817 were highly resistant (IT 0) at the tillering stage (Fig. 3b), but showed differences in leaf and leaf sheath reactions from the booting stage. The sheaths of these lines continued to be near immune at all growth stages but the leaves showed a low degree of susceptibility (IT 1–2) (Fig. 3c and d). The other three lines were highly susceptible on all tissues similar to those of NAU0686. As the terminal deletion in chromosome 1VS in line NAU1814 led to a highly susceptible response at all growth stages (Fig. 4a), Pm67 was therefore physically located in the distal region (FL 0.70–1.00) of chromosome 1VS#5.
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Fig. 3 – Powdery mildew reactions of NAU1817 (T1DL·1VS#5) and its recurrent parent NAU0686 at different developmental stages. (a) Seedling (three-leaf) stage, NAU1817 shows immune response to Bgt. (b) Tillering stage, NAU1817 still shows immune response to Bgt. (c) Stem elongation stage, the sheath of NAU1817 shows immune but leaf shows mild necrotic reaction phenotype. (d) Heading stage, the sheath of NAU1817 shows immune but leaf shows a low degree susceptibility.
Fig. 4 – Powdery mildew reactions on sheath and leaf tissues at the heading stage planted in greenhouse under high inoculum density. (a) Sheathes and leaves of NAU1813, NAU1814, and NAU0686, the sheath of NAU1813 shows immune to Bgt and limited colonies are formed on the leaf of NAU1813; whereas, Bgt forms large colonies on the leaf and sheath tissues of NAU1814 and NAU0686. (b) Sheath and leaf tissues of Pm55 line TF5V-1, Pm67 line NAU1817 and their F1 hybrid, suggesting pyramiding Pm55 and Pm67 genes could provide complete protection from powdery mildew across all tissues.
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3.3. Microscopic evaluation of Bgt infected lines Leaf and sheath infection on Pm67 line NAU1813 and susceptible line NAU1814 were distinctively different under high inoculum density (Fig. 4a). Microscopic observation showed that most of the infection attempts of Bgt were stopped in association with papilla formation on the sheath of NAU1813 (Fig. 5c) and no micro-colonies were observed. Although Bgt is able to form a lower degree of colony on the leaves of NAU1813 (Fig. 4a), the established micro-colonies were not able to form conidiophores on the leaves (Fig. 5a). Meanwhile, plenty of conidiophores were formed on leaf and sheath tissues of susceptible line NAU1814 (Fig. 5b and d).
Fig. 5 – Bgt hyphal development on sheaths and leaves of NAU1813 and NAU1814 at the heading stage planted in greenhouse under high inoculum density. (a) Established micro-colony without conidia on the leaf of NAU1813. Micro-colony is indicated with arrow. (b) Established micro-colony with large conidiophores on the susceptible NAU1814 leaf. Conidiophores are indicated with arrows. (c) Stopped penetration attempt with papilla formation on the sheath of NAU1813. Papilla is indicated with arrow. (d) Established micro-colony with large conidiophores on the sheath of NAU1814. Conidiophores are indicated with arrows.
3.4. Pyramiding of Pm55 and Pm67 Previously, we reported a T5DL·5VS#4 translocation line NAU415 harboring Pm55 that confers a near immune response on leaves after the 5-leaf stage but sheaths and spikes tend to develop some disease [15], which is somewhat opposite to the symptoms described here for plants carrying Pm67. An elite T5DL·5VS#4 translocation line TF5V-1 (pedigree: NAU0686/NAU415//4*NAU0686) harboring Pm55 was used to cross with Pm67 line NAU1817 for pyramiding the two powdery mildew resistance genes. Interestingly, all the F1 plants exhibited
10
near-immune adult plant responses on leaves, sheaths (Fig. 4b) and spikes (data not shown), suggesting that pyramiding these two genes could provide complete protection from powdery mildew across all tissues.
4. Discussion In the present study, six new 1V#5 introgression lines, including disomic substitution line DS1V#5 (1D) (NAU1813), del-1VS·1VL(#5) disomic addition
line NAU1814, t1VS#5 ditelosomic addition line NAU1815,
t1VL#5 ditelosomic addition line NAU1816, homozygous T1DL·1VS#5 translocation line NAU1817, and homozygous T1DS·1VL#5 translocation line NAU1818 were developed (Fig. 1; Table S1). All the 1V#5 introgression lines allowed mapping the alien gene on a chromosome arm. Followed by the Bgt evaluation, a dominant Pm gene was physically located on the 1VS#5 terminal fragment (FL 0.70–1.00). Previously, three powdery mildew resistance genes, including Pm21, Pm55, and Pm62, were introgressed into wheat from D. villosum chromosomes 6V, 5V, and 2V, respectively, and effective against Bgt races in the Yangze River area [13–15]. However, no resistance gene derived from chromosome 1V has been documented. Thus, this resistance gene located on 1VS#5 is new and permanently named Pm67. The compensating Robertsonian translocation (RobT) T1DL·1VS#5 in NAU1817 carrying Pm67 will be useful as a new germplasm in breeding for resistance. The powdery mildew resistance sources of wheat were traditionally described as seedling resistance and adult-plant resistance (APR). Seedling resistance genes are generally race-specific and expressed throughout the plant life cycle [22]. Whereas, APR genes come into effect at post seedling stages when disease damage is likely to occur under field conditions [23,24]. Lines with Pm67 exhibited immunity at seedling stage and tissue-differentiated reactions at adult plant stage (Fig. 3). The sheath, stem and spike tissues of the Pm67 line were still immune, but the leaves showed a low degree of susceptibility (Fig. 4a, b). Histology analyses showed that most penetration attempts were stopped in association with papilla formation on the sheath (Fig. 5c), and colonies cannot form conidia on the susceptible leaf of Pm67 line (Fig. 5a). Thus, the defence of Pm67 should be on independent layers on the leaf and sheath tissues at adult plant stage. Interestingly this differential tissue response was opposite to that shown in plants with Pm55 (Fig. 4b). An F1 hybrid between lines possessing Pm67 and Pm55 showed complete immunity on all tissues at whole stage (Fig. 4b). Therefore, Pm55 and Pm67 might represent genes with distinct defense mechanisms at adult plant stage and our findings extend the current understandings of the genes against the wheat fungal pathogens. Chromosome 1V of D. villosum is homoeologous to the group 1 chromosomes of bread wheat and has been described as a source for the improvement of various end-use quality of wheat flour [11,25–30]. The introgression lines involving 1VS carrying D. villosum storage protein loci Glu-V1, Gli-V1, and Glu-V3 significantly increased 11
wheat gluten strength and protein content, and enhanced flour end-use quality [16,25]. Thus, the multiple genes (Glu-V1/Glu-V3/Gli-V1/Pm67) on 1VS#5 arm could represent an ideal combination for modern wheat varieties with good end-use quality and disease resistance if 1VS#5 has no negative effect on agronomy traits. Although the detailed effect of T1DL·1VS#5 translocation chromosome on yield related traits is unknown at present, the important agronomic traits, such as plant height, fertility, kernel weight and anthesis are similar to background parent NAU0686 in the field (Fig. S1; Table S3). Additional studies are in progress to transfer Pm67 into different wheat cultivars and to have a yield trial in order to assess whether the 1VS#5 introgression is associated with any deleterious effects on yield or quality, and whether a smaller 1VS segment will be required for agronomic exploitation. D. villosum is a near-nonhost to many pathogens of wheat, including rust and powdery mildew [11,13]. Although the genetic base of powdery mildew resistance in different D. villosum accessions is not clear, this research extends our knowledge of the polygenic mode of inheritance in D. villosum to powdery mildew. Genetic mapping and complementation analysis are necessary to isolate the underlying gene. The fundamental problem in studying the inheritance of alien resistance gene is lack of recombination between the homoeologous chromosomes of wheat and its relatives [8]. To date, chromosome 1V from at least five accessions of D. villosum has been introgressed into wheat represented by a range of addition, substitution and translocation lines [16,25,30,31]. The powdery mildew reactions of wheat-D. villosum disomic addition line DA1V#1 [31] and disomic substitution line DS1V#2(1B) [26] are unknown, but translocation lines T1DL·1VS#3 [25] and T1DL·1VS#4 [16] were susceptible. Fifty-two 1VS-specific molecular markers were identified by Zhang et al. [19,20], and 11 of them showed codominant polymorphic differences between the susceptible T1DL·1VS#4 line NAU1811 and resistant T1DL·1VS#5 line NAU1817 as exampled for 1V-207 and 1V-253 showed in Fig. 2a and b. Thus, the polymorphic markers together with genetic segregation in crosses of translocation lines NAU1811 and NAU1817 will permit genetic mapping of the Pm67 gene if chromosome arms 1VS#4 and 1VS#5 normally pair and recombine at meiotic phase in the hybrids of NAU1811/NAU1817. Research is currently underway to genetically map and clone Pm67 for revealing its defense mechanisms.
Declaration of competing interest Authors declare that there are no conflicts of interest.
CRediT authorship contribution statement Ruiqi Zhang conceived and designed the experiments. Chuanxi Xiong, Ruonan Yao, and Xiangru Meng 12
developed introgression lines; Chuanxi Xiong and Lingna Kong analyzed molecular markers; Liping Xing, Jizhong Wu, and Yigao Feng identified the Bgt reaction; Ruiqi Zhang and Aizhong Cao wrote the paper.
Acknowledgments We thank Prof. Robert McIntosh and Prof. Peidu Chen for critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (31971938), the Natural Science Foundation of Jiangsu Province (BK20181316), the Special Fund for Independent Innovation of Agricultural Science and Technology in Jiangsu, China (CX (19)1001), and Fundamental Research Funds for the Central Universities (KYZ201809).
Appendix A. Supplementary data Supplementary data for this article can be found online at http://doi.org/10.1016/j.cj.2020.xx.xxx.
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[13] R.Q. Zhang, Y.L. Fan, L.N. Kong, Z.J. Wang, J.Z. Wu, L.P. Xing, A.Z. Cao, Y.G. Feng, Pm62, an adult-plant powdery mildew resistance gene introgressed from Dasypyrum villosum chromosome arm 2VL into wheat, Theor. Appl. Genet. 131 (2018) 2613–2620. [14] P.D. Chen, L.L. Qi, B. Zhou, S.Z. Zhang, D.J. Liu, Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew, Theor. Appl. Genet. 91 (1995) 1125–1128. [15] R.Q. Zhang, B.X. Sun, J. Chen, A.Z. Cao, L.P. Xing, Y.G. Feng, C.X. Lan, P.D. Chen, Pm55, a developmental-stage and tissue specific powdery mildew resistance gene introgressed from Dasypyrum villosum into common wheat, Theor. Appl. Genet. 129 (2016) 1975–1984. [16] R.Q. Zhang, M.Y. Zhang, X.E. Wang, P.D. Chen, Introduction of chromosome segment carrying the seed storage protein genes from chromosome 1V of Dasypyrum villosum showed positive effect on bread-making quality of common wheat, Theor. Appl. Genet. 127 (2014) 523–533. [17] S. Komuro, R. Endo, K. Shikata, A. Kato, Genomic and chromosomal distribution patterns of various repeated DNA sequences in wheat revealed by a fluorescence in situ hybridization procedure, Genome 56 (2013)131–137. [18] P. Du, L.F. Zhuang, Y.Z. Wang, Y. Li, Q. Wang, D.R. Wang, D.D. Dawa, L.J. Tan, J. Shen, H.B. Xu, H. Zhao, C.G. Chu, Z.J. Qi, Development of oligonucleotides and multiplex probes for quick and accurate identification of wheat and Thinopyrum bessarabicum chromosomes, Genome 60 (2017) 93–103. [19] R.Q. Zhang, R.N. Yao, D.F. Sun, B.X. Sun, Y.G. Feng, W. Zhang, M.Y. Zhang, Development of V chromosome alterations and physical mapping of molecular markers specific to Dasypyrum villosum, Mol. Breed. 37 (2017) 67. [20] X.D. Zhang, X. Wei, J. Xiao, C.X. Yuan, Y.F. Wu, A.Z. Cao, L.P. Xing, P.D. Chen, S.Z. Zhang, H.Y. Wang, Whole genome development of intron targeting (IT) markers specific for Dasypyrum villosum chromosomes based on next-generation sequencing technology, Mol. Breed. 37 (2017) 115. [21] B.Q. Sheng, X.Y. Duan, Modification on the evaluation methods of 0-9 level of powdery mildew infection on wheat, Biotech. J. Agric. Sci. 9 (1991) 37–39. [22] E.A. Favret, H.A. Saione, P.M. Franzone, New approaches in breeding for disease resistance, in: Cereal Breeding and Production Symposium, Argentina Special Report 718, Oregon State University, Corvallis, USA, 1983. [23] R.A. McIntosh, C.R. Welllings, R.F. Park, Wheat Rusts: an Atlas of Resistance Genes, Kluwer, Dordrecht, the Netherlands, 1995. [24] M.M. Messmer, R. Seyfarth, M. Keller, G. Schachermayr, M. Winzeler, S. Zanetti, C. Feuillet, B. Keller, Genetic analysis of durable leaf rust resistance in winter wheat, Theor. Appl. Genet. 100 (2000) 419–431. [25] W.C. Zhao, L.L. Qi, X. Gao, G.S. Zhang, J. Dong, Q.J. Chen, B. Friebe, B.S. Gill, Development and characterization of two new Triticum aestivum–Dasypyrum villosum Robertsonian translocation lines T1DS·1V#3L and T1DL·1V#3S and their effect on grain quality, Euphytica 175 (2010) 343–350. [26] A. Blanco, V. Perrone, R. Simeone, Chromosome pairing variation in Triticum turgidum L. × Dasypyrum villous (L.) Candargy hybrids and genome affinities, in: T.E. Miller, R.M.D. Koebner (Eds), Proceedings of the 7th International Wheat Genetics Symposium, Institute of Plant Sciences Research, Cambridge, UK, 1988, pp. 63–67. [27] G.Y. Zhong, C.O. Qualset, Allelic diversity of high-molecular-weight glutenin protein subunits in natural populations of Dasypyrum villosum (L.) Candargy, Theor. Appl. Genet. 86 (1993) 851–858. [28] P. Vaccino, R. Banfi, M. Corbellini, C. de Pace, Broadening and improving the wheat genetic diversity for end-use grain quality by introgression of chromatin from the wheat wild relative Dasypyrum villosum, Crop Sci. 50 (2010) 528–540. [29] L. Montebove, C. de Pace, C.C. Jan, G.T. Scarascia-Mugnozza, C.O. Qualset, Chromosomal location of isozyme and seed storage protein genes in Dasypyrum villosum (L.) Candargy, Theor. Appl. Genet. 73 (1987) 836–845. [30] C. de Pace, D. Snidaro, M. Ciaffi, D. Vittori, A. Ciofo, A. Cenci, O.A. Tanzarella, C.O. Qualset, G.T. Scarascia Mugnozza, Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality, Euphytica 117 (2011) 67– 75. [31] E.R. Sears, Addition of the genome of Haynaldia villosa to Triticum aestivum, Am. J. Bot. 40 (1953)168–174.
Declaration of Competing Interest 14
All the authors have no conflicts of interest, and they approved the publication.
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S2214-5141(20)30176-8 https://doi.org/10.1016/j.cj.2020.09.012 CJ 550
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Please cite this article as: R. Zhang, C. Xiong, H. Mu, R. Yao, X. Meng, L. Kong, L. Xing, J. Wu, Y. Feng, A. Cao, Pm67, a new powdery mildew resistance gene transferred from Dasypyrum villosum chromosome 1V to common wheat (Triticum aestivum L.), The Crop Journal (2020), doi: https://doi.org/10.1016/j.cj.2020.09.012
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Pm67, a new powdery mildew resistance gene transferred from Dasypyrum villosum chromosome 1V to common wheat (Triticum aestivum L.) Ruiqi Zhang a, 1, *, Chuanxi Xiong a, 1, Huanqing Mu a, Ruonan Yao a, Xiangru Meng a, Lingna Kong a, Liping Xing a, Jizhong Wu b, Yigao Feng a, Aizhong Cao a a
College of Agronomy/JCIC-MCP/National Key Lab of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural
University, Nanjing 210095, Jiangsu, China b
Institute of Germplasm Resources and Biotechnology/Provincial Key Lab of Agrobiology, Jiangsu Academy of Agricultural
Sciences, Nanjing 210014, Jiangsu, China
Abstract: Powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), is a global disease that poses a serious threat to wheat production. To explore additional resistance gene, a wheat-Dasypyrum villosum 1V#5 (1D) disomic substitution line NAU1813 (2n = 42) with high level of seedling resistance to powdery mildew was used to generate the recombination between chromosomes 1V#5 and 1D. Four introgression lines, including t1VS#5 ditelosomic addition line NAU1815, t1VL#5 ditelosomic addition line NAU1816, homozygous T1DL·1VS#5 translocation line NAU1817, and homozygous T1DS·1VL#5 translocation line NAU1818 were developed from the selfing progenies of 1V#5 and 1D double monosomic line that derived from F1 hybrids of NAU1813/NAU0686. All of them were characterized by fluorescence in situ hybridization, genomic in situ hybridization, 1V-specific markers analysis, and powdery mildew tests at different developmental stages. A new powdery mildew resistance gene named Pm67 was physically located in the terminal bin (FL 0.70–1.00) of 1VS#5. Lines with Pm67 exhibited seedling stage immunity and tissue-differentiated reactions at adult plant stage. The sheaths, stems, and spikes of the Pm67 line were still immune, but the leaves showed a low degree of susceptibility. Microscopic observation showed that most penetration attempts were stopped in association with papillae on the sheath, and colonies cannot form conidia on the susceptible leaf of Pm67 line at adult plant stage, suggesting that the defence layers of the Pm67 line is tissue-differentiated. Thus, the T1DL·1VS#5 translocation line NAU1817 provides a new germplasm in wheat breeding for improvement of powdery mildew resistance.
Keywords: Wheat; Dasypyrum villosum; Powdery mildew; Pm67 1. Introduction Wheat (Triticum aestivum L.) is a widely produced grain crop that contributes to global food security. Existing and new races of fungal pathogens constantly threaten the high and stable yields of wheat. Among them, powdery *
Corresponding author: Ruiqi Zhang, E-mail address: [email protected].
1
These authors contributed equally to this work.
Received: 2020-07-30; Revised: 2020-09-03; Accepted: 2020-11-03. 1
mildew, caused by Blumeria graminis f. sp. tritici (Bgt), occurs in many wheat-growing regions, especially in highly productive areas where semi-dwarf cultivars are planted under high input conditions [1]. Powdery mildew interferes with photosynthesis, thereby reducing plant growth, heading, grain filling, and end-use quality. When weather is favorable and the mycelium occurs at flag leaf emergence or during heading, grain yield losses of up to 40% can occur [2]. Utilization of resistant cultivars is the most practical and cost-effective means to control this disease. The traditional powdery mildew resistance breeding strategy involves the use of a single major resistance gene in the major wheat production where powdery mildew usually causes significant yield losses. Genotypes produced by this strategy provide strong selection pressure for pathogen variants with an increased ability to reproduce upon them. Consequently, the effectiveness of most deployed resistance genes is rapidly lost as the pathogens acquire virulence. Although Pm2, Pm4, Pm6, Pm8, Pm13, Pm21, and Pm30 genes are frequently detected in winter wheat cultivars in China [3–6], only Pm21 provides sufficient protection from powdery mildew in the field currently. Varieties with the T6AL·6VS translocation containing Pm21 are planted on more than 4 million hectares annually [6,7]. Moreover, more than 40% of elite lines carrying Pm21 were detected in the main wheat production region Yangze River, China, where powdery mildew can cause significant yield losses and most of the known Pm genes have lost their effectiveness. So far due to good protection of Pm21 the use of this gene continues, which will increase the selection pressure for virulent variants of the pathogen. Thus, the discovery and utilization of additional sources of powdery mildew resistance to address this vulnerability is necessary for wheat improvement programs in China and elsewhere. Wild relatives of wheat are an important source of genetic variation for variety improvement [8]. Most species belonging to the tertiary gene pool are not common hosts for Bgt and their resistance appears to be broad-spectrum and effective over long periods of time [9,10]. Among them Dasypyrum villosum (2n = 14, VV) is recognized as an excellent species of new resistance genes because it is a near-nonhost to many wheat pathogens and has widespread immunity to multiple diseases, such as stripe rust, leaf rust and powdery mildew [11]. Two D. villosum accessions, 91C43 (#4, originally introduced from the Cambridge University Botanical Garden, UK), and 01I140 (#5, collected in the Pisa region of Italy), have been used to develop wheat-D. villosum introgression lines at the Cytogenetics Institute of Nanjing Agricultural University (CINAU) [12,13]. Three formally designated powdery mildew resistance genes, Pm21, Pm55, and Pm62, have been transferred from D. villosum to wheat by means of compensating Robertsonian translocations (RobTs), confirming that D. villosum is indeed an important source of powdery mildew resistance genes for wheat [13–15].
2
A set of 1V#5 to 7V#5 disomic addition/substitution lines have been developed in wheat variety NAU0686 background from the progenies of NAU0686/NAU1802 (AABBVV (#5)) at CINAU. Their responses to stripe rust, leaf rust, and powdery mildew were assessed in field disease nurseries and the greenhouse. Results indicated that wheat-D. villosum 1V#5 (1D) disomic substitution line NAU1813 conferred effective resistance against Bgt at both seedling and adult stages. The objectives of this study were to: (1) characterize the response of NAU1813 and its chromosome 1V#5 to powdery mildew, (2) introgress the chromosome 1V#5 powdery mildew resistance gene from NAU1813 into the wheat genome through the breakage-fusion mechanism, and (3) physically map a new powdery mildew resistance gene on chromosome 1V#5.
2. Materials and methods 2.1. Plant materials Durum variety ZY1286 introduced from CIMMYT was initially crossed with D. villosum accession 01I140 (#5) to generate the amphiploid NAU1802 (2n = 42, AABBVV). The 1V#5 (1D) disomic substitution line NAU1813 and
1V#5
deletion
line
NAU1814
were
obtained
from
BC4F2
progenies
of
the
cross
NAU0686/NAU1802//4*NAU0686. The T1DL·1VS#4 translocation line NAU1811 and T1DS·1VL#4 translocation line NAU1812 derived from D. villosum accession 91C43 (#4) [16] were used as positive controls in molecular marker analyses. An elite T5DL·5VS#4 translocation line TF5V-1 (pedigree: NAU0686/NAU415 (T5DL·5VS#4)//4*NAU0686) harboring Pm55 was used to pyramid powdery mildew resistance genes. All the genetic stocks developed and maintained at CINAU were summarized in Table S1. All the 1V#5 introgression lines were planted in the field powdery mildew nursery at the Jiangpu Experiment Station of Nanjing Agricultural University for examination of powdery mildew response at different developmental stages. Following inoculation, the materials were covered with plastic sheeting for protection and enhancing disease development during the winter season. Ten plants were grown in each 1.0 m row with 25 cm spacing between rows. Each line with the exception of F2 segregating individuals was grown in four rows with three replications. The recurrent parent NAU0686 planted on both sides of each experimental row served as an inoculum spreader of Bgt and susceptible control. 2.2. Cytogenetic analysis Root tips from the germinating wheat seedlings were soaked in 0.2 μmol L−1 amiprophos-methyl (APM) solution for 2 h at 24 °C and then treated with nitrous oxide for 1.5 h and 90% acetic acid for 10 min following Komuro et al. [17]. Slides were prepared for genomic in situ hybridization (GISH) and fluorescence in situ hybridization 3
(FISH) as described by Du et al. [18]. Total genomic DNA of D. villosum was labeled with fluorescein-12-dUTP (green) as the GISH probe. FISH was performed using mixed oligo-pAs1-2 and oligo-pAs1-5 probes that preferentially paint tandem repeats on D genome chromosomes of common wheat. The oligo-probes labeled by 6-carboxytetramethylrhodamine (TAM) producing red signals were synthesized by the Tsingke Biological Technology Company (Nanjing, China) following the procedures described by Du et al. [18]. After hybridization, chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and colored blue (Invitrogen Life Science, Carlsbad, CA, USA), and slides were examined under an Olympus BX60 microscope (Olympus Co.). Images were captured with a SPOT Cooled Color Digital Camera (Diagnostic Instruments, Sterling Heights, MI, USA) and processed using Adobe Photoshop CS 7.0 (Adobe Systems, San Jose, CA, USA). 2.3. Molecular marker analysis Genomic DNA of the genetic stocks were isolated from seedling leaves using a DNA Secure Plant Kit (Tiangen Biotech Co., Ltd, Nanjing), according to the manufacturer’s instructions. Four chromosome 1V-specific markers, including 1V-207, 1V-253, 1EST-493, and P1/P5 developed previously [19,20] were used to screen the progenies of NAU0686/NAU1802//4*NAU0686 for lines carrying chromosome 1V#5 or parts thereof. Markers 1V-207 and 1V-253 were specific for chromosome arm 1VS, and 1EST-493 and P1/P5 were specific for 1VL (Table S2). PCR amplification was conducted using a T100TM Thermal Cycler (Bio-Rad Laboratories, Hercules, USA) in 10 μL reaction volumes containing 40 ng of genomic DNA, 2.5 mmol L−1 of each dNTP, 2 μmol L−1 of each primer, 2.5 mmol L−1 MgCl2, 1×PCR buffer (10 mmol L−1 Tris-HCl, 50 mmol L−1 KCl, pH 8.5), and 0.2 U Taq DNA polymerase. Amplification of DNA samples was programmed at 95 °C for 4 min followed by 32 cycles at 94 °C, 58 °C for 45 s, and 72 s of elongation at 72 °C, with a final extension at 72 °C for 10 min. PCR products were separated on 8% non-denaturing polyacrylamide gels (Acr:Bis = 39:1) and visualized by silver staining. 2.4. Evaluation of powdery mildew response Seedlings of chromosome 1V#5 introgression lines and recurrent parent NAU0686 were planted in a greenhouse at 18/22 °C (night/day) with 80% relative humidity and photoperiod of 16 h light/8 h darkness. Bgt isolates E26 and E31, and a mixed field sample collected from Yangze River region were used in separate tests under controlled greenhouse conditions. Infection types (ITs) were recorded on a 0–4 scale at 10 days post inoculation when susceptible NAU0686 control plants were heavily diseased [3]. In addition, a mixture of Bgt isolates (collected from Yangze River region) was used to infect NAU0686 and 1V#5 introgression lines at the three-leaf growth stage in the field powdery mildew nursery. Reactions of 1V#5 4
introgression lines evaluated at the tillering, stem elongation, booting, heading, and grain-filling stages were recorded on a 0–9 IT scale [21]. 2.5. Microscopic evaluation For evaluation of the difference in mycelium development, the leaf and sheath segments of the resistant line NAU1813 and susceptible line NAU1814 at the grain-filling stage were examined by an endogenous peroxidase-dependent in situ histochemical staining procedure. About 5 cm segments of leaf and sheath were transferred to tubes to distain by a solution of acetic acid-ethanol (1:3, v/v) for 24 h. After that, about 1.5 cm segments were cut and immersed into a solution containing 0.6% Coomassie Brilliant Blue, 15% trichloroacetic acid in 99% methanol (1:1 v/v) for 25 min [10]. Microscope slides were prepared by embedding the stained segments of leaf and sheath in 100% glycerol, with the adaxial side up. Slides were screened using an Olympus BX60 microscope (Olympus Co.) under a white light. 2.6. Evaluation of agronomic traits The plants with and without T1DL·1VS#5 translocated chromosome were evaluated for plant height (PH, cm), main spike length (SL, cm), grains per spike (GPS) and 1000-kernel weight (TKW, g). Differences in main agronomic traits between the genotypes were evaluated by means of Tukey’s post hoc test (SPSS 16.0) at the P <0.05 significance level.
3. Results 3.1. Development and identification of the 1V#5 introgression lines Two wheat-D. villosum 1V#5 introgression lines in the BC4F2 progeny of NAU0686/NAU1802//4*NAU0686 were firstly identified by GISH/FISH and molecular markers. NAU1813 had 40 wheat chromosomes and two D. villosum chromosomes with one strong hybridization band in the proximal centromere region, and one strong band each in the subtelomeric and telomeric regions of the short arm (Fig. 1a). FISH patterns produced with the oligo-pAs1-2 and oligo-pAs1-5 probes and four 1V-specific marker analysis confirmed that the wheat chromosome 1D pair had been substituted by a pair of D. villosum chromosomes 1V#5 (Figs. 1a and 2). NAU1814 had 42 wheat chromosomes and two D. villosum chromosomes with one strong hybridization band in the proximal centromere region but missing the bands in the subtelomeric and telomeric regions of the short arm (Fig. 1b). The specific bands for molecular markers 1V-207, 1EST-493, and P1/P5 were present but the 1VS-specific band of 1V-253 was missing (Fig. 2a and b), suggesting that NAU1814 had a deletion of the distal 5
region of the short arm of chromosome 1V#5 with the breakpoint at FL 0.70. Seedling stage powdery mildew tests showed that NAU1813 was immune (IT 0) whereas NAU1814 was susceptible (IT 4), indicating that a powdery mildew resistance gene(s) was located in the terminal region of chromosome arm 1VS.
Fig. 1 – Identification of the six wheat-D. villosum 1V#5 introgression lines through genomic in situ (GISH) and fluorescent in situ (FISH) hybridization. D. villosum genomic DNA labeled with fluorescein-12-dUTP (green) as probes was used for GISH. Probes for FISH were Oligo-pAs1-2 and Oligo-pAs1-5 labeled with TAM (red). Wheat chromosomes were counterstained with DAPI (blue). (a) GISH/FISH patterns of NAU1813 (1V#5 (1D), 2n = 42); Chromosome 1V#5 shows one strong hybridization band at the proximal centromere region, and one strong band each in the subtelomeric and telomeric regions of the short arm. (b) GISH/FISH patterns of NAU1814 (2n = 44), which has a spontaneous deletion of distal part of the short arm of chromosome 1V#5 with the breakpoint at FL0.70. (c) GISH/FISH patterns of NAU1815 (2n = 44), which was a t1VS#5 ditelosomic addition line. (d) GISH/FISH patterns of NAU1816 (2n = 44), a t1VL#5 ditelosomic addition line. (e) GISH/FISH patterns of NAU1817 (2n = 42), a homozygous T1DL·1VS#5 translocation line. (f) GISH/FISH patterns of NAU1818 (2n = 42), a homozygous T1DS·1VL#5 translocation line. Bars, 10 μm
6
Fig. 2 – PCR amplification patterns of representative 1VS- and 1VL-specific markers in the wheat-D. villosum introgression lines. 1VS-specific markers 1V-207 (a) and 1V-253 (b); 1VL-specific markers 1EST-493 (c) and P1/P5 (d). Marker, DL2000. The 1DL·1VS lines NAU1811 and NAU1817 have 1VS- and 1DL-specific bands but lack the 1DS-specific band. The 1DS·1VL lines NAU1812 and NAU1818 have 1VL- and 1DS-specific bands but lack the 1DL-specific band.
In order to identify Robertsonian whole-arm translocations of chromosome 1V and a wheat chromosome, the progeny of double monosomic 1V#5 and 1D hybrids of NAU1813 crossed with NAU0686 were screened. Among 250 selfing progenies of 1V#5 and 1D double monosomic plant Bx-4b-13 screened with four 1V-specific markers, 15 plants (6%) had only the 1VS-specific markers 1V-207 and 1V-253. GISH/FISH analysis showed that 14 of these plants had a 1VS#5 telosome and one plant (YL-2b-11) was heterozygous with a normal 1D chromosome and a T1DL·1VS#5 translocation chromosome. Other 13 plants (5.2%) had only the 1VL-specific markers 1EST-493 and P1/P5; 12 had a 1VL#5 telosome and one (YL-2b-3) was heterozygous for a T1DS·1VL#5 Robertsonian translocation. Lines derived from these various plants included t1VS#5 ditelosomic addition line 7
NAU1815 (Fig. 1c), t1VL#5 ditelosomic addition line NAU1816 (Fig. 1d), homozygous T1DL·1VS#5 translocation line NAU1817 (Fig. 1e), and homozygous T1DS·1VL#5 translocation line NAU1818 (Fig. 1f). In addition, 100 seedlings among the selfing progenies of YL-2b-11 (heterozygous T1DL·1VS#5) were examined by GISH; 20 were disomic for a T1DL·1VS#5 recombinant chromosome pair, 58 were heterozygous and 22 lacked alien chromatin indicating normal gametic transmission of the translocated chromosome relative to its intact 1D homologue (χ21:2:1 = 2.56, P >0.05). Measures of agronomic traits shown in Table S3 revealed no obvious differences in plant height, 1000-kernel weight, main spike length, and seeds per main spike between plants with and without the T1DL·1VS#5 recombinant chromosome. 3.2. Powdery mildew reactions Seedlings of above YL-2b-11 selfing-plants were tested for reaction to the field isolates Bgt. All 78 plants harboring 1VS#5 chromosome arm were immune (IT 0–0;), whereas the remaining 22 plants lacking 1VS#5 chromatin were susceptible (IT 3–4) (Table S3). Thus, the powdery mildew resistance gene(s) located on chromosome arm 1VS#5 was dominant and formally designated Pm67. For characterization of Pm67 resistance at different growth stages NAU1813, NAU1814, NAU1815, NAU1816, NAU1817, and NAU1818 were tested separately for reactions to Bgt isolates E26 and E31 and a field collection at two- to three-leaf stage. NAU1813, NAU1815, and NAU1817 had IT of 0 (Fig. 3a), whereas NAU1814, NAU1816, and, NAU1818 were susceptible (IT 4) as was the recurrent parent NAU0686. In field nursery evaluations, NAU1813, NAU1815, and NAU1817 were highly resistant (IT 0) at the tillering stage (Fig. 3b), but showed differences in leaf and leaf sheath reactions from the booting stage. The sheaths of these lines continued to be near immune at all growth stages but the leaves showed a low degree of susceptibility (IT 1–2) (Fig. 3c and d). The other three lines were highly susceptible on all tissues similar to those of NAU0686. As the terminal deletion in chromosome 1VS in line NAU1814 led to a highly susceptible response at all growth stages (Fig. 4a), Pm67 was therefore physically located in the distal region (FL 0.70–1.00) of chromosome 1VS#5.
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Fig. 3 – Powdery mildew reactions of NAU1817 (T1DL·1VS#5) and its recurrent parent NAU0686 at different developmental stages. (a) Seedling (three-leaf) stage, NAU1817 shows immune response to Bgt. (b) Tillering stage, NAU1817 still shows immune response to Bgt. (c) Stem elongation stage, the sheath of NAU1817 shows immune but leaf shows mild necrotic reaction phenotype. (d) Heading stage, the sheath of NAU1817 shows immune but leaf shows a low degree susceptibility.
Fig. 4 – Powdery mildew reactions on sheath and leaf tissues at the heading stage planted in greenhouse under high inoculum density. (a) Sheathes and leaves of NAU1813, NAU1814, and NAU0686, the sheath of NAU1813 shows immune to Bgt and limited colonies are formed on the leaf of NAU1813; whereas, Bgt forms large colonies on the leaf and sheath tissues of NAU1814 and NAU0686. (b) Sheath and leaf tissues of Pm55 line TF5V-1, Pm67 line NAU1817 and their F1 hybrid, suggesting pyramiding Pm55 and Pm67 genes could provide complete protection from powdery mildew across all tissues.
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3.3. Microscopic evaluation of Bgt infected lines Leaf and sheath infection on Pm67 line NAU1813 and susceptible line NAU1814 were distinctively different under high inoculum density (Fig. 4a). Microscopic observation showed that most of the infection attempts of Bgt were stopped in association with papilla formation on the sheath of NAU1813 (Fig. 5c) and no micro-colonies were observed. Although Bgt is able to form a lower degree of colony on the leaves of NAU1813 (Fig. 4a), the established micro-colonies were not able to form conidiophores on the leaves (Fig. 5a). Meanwhile, plenty of conidiophores were formed on leaf and sheath tissues of susceptible line NAU1814 (Fig. 5b and d).
Fig. 5 – Bgt hyphal development on sheaths and leaves of NAU1813 and NAU1814 at the heading stage planted in greenhouse under high inoculum density. (a) Established micro-colony without conidia on the leaf of NAU1813. Micro-colony is indicated with arrow. (b) Established micro-colony with large conidiophores on the susceptible NAU1814 leaf. Conidiophores are indicated with arrows. (c) Stopped penetration attempt with papilla formation on the sheath of NAU1813. Papilla is indicated with arrow. (d) Established micro-colony with large conidiophores on the sheath of NAU1814. Conidiophores are indicated with arrows.
3.4. Pyramiding of Pm55 and Pm67 Previously, we reported a T5DL·5VS#4 translocation line NAU415 harboring Pm55 that confers a near immune response on leaves after the 5-leaf stage but sheaths and spikes tend to develop some disease [15], which is somewhat opposite to the symptoms described here for plants carrying Pm67. An elite T5DL·5VS#4 translocation line TF5V-1 (pedigree: NAU0686/NAU415//4*NAU0686) harboring Pm55 was used to cross with Pm67 line NAU1817 for pyramiding the two powdery mildew resistance genes. Interestingly, all the F1 plants exhibited
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near-immune adult plant responses on leaves, sheaths (Fig. 4b) and spikes (data not shown), suggesting that pyramiding these two genes could provide complete protection from powdery mildew across all tissues.
4. Discussion In the present study, six new 1V#5 introgression lines, including disomic substitution line DS1V#5 (1D) (NAU1813), del-1VS·1VL(#5) disomic addition
line NAU1814, t1VS#5 ditelosomic addition line NAU1815,
t1VL#5 ditelosomic addition line NAU1816, homozygous T1DL·1VS#5 translocation line NAU1817, and homozygous T1DS·1VL#5 translocation line NAU1818 were developed (Fig. 1; Table S1). All the 1V#5 introgression lines allowed mapping the alien gene on a chromosome arm. Followed by the Bgt evaluation, a dominant Pm gene was physically located on the 1VS#5 terminal fragment (FL 0.70–1.00). Previously, three powdery mildew resistance genes, including Pm21, Pm55, and Pm62, were introgressed into wheat from D. villosum chromosomes 6V, 5V, and 2V, respectively, and effective against Bgt races in the Yangze River area [13–15]. However, no resistance gene derived from chromosome 1V has been documented. Thus, this resistance gene located on 1VS#5 is new and permanently named Pm67. The compensating Robertsonian translocation (RobT) T1DL·1VS#5 in NAU1817 carrying Pm67 will be useful as a new germplasm in breeding for resistance. The powdery mildew resistance sources of wheat were traditionally described as seedling resistance and adult-plant resistance (APR). Seedling resistance genes are generally race-specific and expressed throughout the plant life cycle [22]. Whereas, APR genes come into effect at post seedling stages when disease damage is likely to occur under field conditions [23,24]. Lines with Pm67 exhibited immunity at seedling stage and tissue-differentiated reactions at adult plant stage (Fig. 3). The sheath, stem and spike tissues of the Pm67 line were still immune, but the leaves showed a low degree of susceptibility (Fig. 4a, b). Histology analyses showed that most penetration attempts were stopped in association with papilla formation on the sheath (Fig. 5c), and colonies cannot form conidia on the susceptible leaf of Pm67 line (Fig. 5a). Thus, the defence of Pm67 should be on independent layers on the leaf and sheath tissues at adult plant stage. Interestingly this differential tissue response was opposite to that shown in plants with Pm55 (Fig. 4b). An F1 hybrid between lines possessing Pm67 and Pm55 showed complete immunity on all tissues at whole stage (Fig. 4b). Therefore, Pm55 and Pm67 might represent genes with distinct defense mechanisms at adult plant stage and our findings extend the current understandings of the genes against the wheat fungal pathogens. Chromosome 1V of D. villosum is homoeologous to the group 1 chromosomes of bread wheat and has been described as a source for the improvement of various end-use quality of wheat flour [11,25–30]. The introgression lines involving 1VS carrying D. villosum storage protein loci Glu-V1, Gli-V1, and Glu-V3 significantly increased 11
wheat gluten strength and protein content, and enhanced flour end-use quality [16,25]. Thus, the multiple genes (Glu-V1/Glu-V3/Gli-V1/Pm67) on 1VS#5 arm could represent an ideal combination for modern wheat varieties with good end-use quality and disease resistance if 1VS#5 has no negative effect on agronomy traits. Although the detailed effect of T1DL·1VS#5 translocation chromosome on yield related traits is unknown at present, the important agronomic traits, such as plant height, fertility, kernel weight and anthesis are similar to background parent NAU0686 in the field (Fig. S1; Table S3). Additional studies are in progress to transfer Pm67 into different wheat cultivars and to have a yield trial in order to assess whether the 1VS#5 introgression is associated with any deleterious effects on yield or quality, and whether a smaller 1VS segment will be required for agronomic exploitation. D. villosum is a near-nonhost to many pathogens of wheat, including rust and powdery mildew [11,13]. Although the genetic base of powdery mildew resistance in different D. villosum accessions is not clear, this research extends our knowledge of the polygenic mode of inheritance in D. villosum to powdery mildew. Genetic mapping and complementation analysis are necessary to isolate the underlying gene. The fundamental problem in studying the inheritance of alien resistance gene is lack of recombination between the homoeologous chromosomes of wheat and its relatives [8]. To date, chromosome 1V from at least five accessions of D. villosum has been introgressed into wheat represented by a range of addition, substitution and translocation lines [16,25,30,31]. The powdery mildew reactions of wheat-D. villosum disomic addition line DA1V#1 [31] and disomic substitution line DS1V#2(1B) [26] are unknown, but translocation lines T1DL·1VS#3 [25] and T1DL·1VS#4 [16] were susceptible. Fifty-two 1VS-specific molecular markers were identified by Zhang et al. [19,20], and 11 of them showed codominant polymorphic differences between the susceptible T1DL·1VS#4 line NAU1811 and resistant T1DL·1VS#5 line NAU1817 as exampled for 1V-207 and 1V-253 showed in Fig. 2a and b. Thus, the polymorphic markers together with genetic segregation in crosses of translocation lines NAU1811 and NAU1817 will permit genetic mapping of the Pm67 gene if chromosome arms 1VS#4 and 1VS#5 normally pair and recombine at meiotic phase in the hybrids of NAU1811/NAU1817. Research is currently underway to genetically map and clone Pm67 for revealing its defense mechanisms.
Declaration of competing interest Authors declare that there are no conflicts of interest.
CRediT authorship contribution statement Ruiqi Zhang conceived and designed the experiments. Chuanxi Xiong, Ruonan Yao, and Xiangru Meng 12
developed introgression lines; Chuanxi Xiong and Lingna Kong analyzed molecular markers; Liping Xing, Jizhong Wu, and Yigao Feng identified the Bgt reaction; Ruiqi Zhang and Aizhong Cao wrote the paper.
Acknowledgments We thank Prof. Robert McIntosh and Prof. Peidu Chen for critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (31971938), the Natural Science Foundation of Jiangsu Province (BK20181316), the Special Fund for Independent Innovation of Agricultural Science and Technology in Jiangsu, China (CX (19)1001), and Fundamental Research Funds for the Central Universities (KYZ201809).
Appendix A. Supplementary data Supplementary data for this article can be found online at http://doi.org/10.1016/j.cj.2020.xx.xxx.
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Declaration of Competing Interest 14
All the authors have no conflicts of interest, and they approved the publication.
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