Molecular Cytogenetic Analysis of the Introgression between Agropyron cristatum P Genome and Wheat Genome

Agropyron cristatum (2n = 4x = 28, PPPP) is an important wild relative of common wheat (Triticum aestivum L., 2n = 6x = 42). A previous report showed that the wheat-A. cristatum 6P translocation line WAT655 carrying A. cristatum 6PS (0.81–1.00) exhibited high resistance to prevalent physiological races of stripe rust (CYR32 and CYR33). In this study, three disease resistance-related transcripts, which were mapped to A. cristatum 6PS (0.81–1.00) through the analysis of specific molecular markers, were acquired from among A. cristatum full-length transcripts. The BC5F2 and BC5F2:3 genetic populations of the translocation line WAT655 were analyzed by using three disease resistance-related gene markers, A. cristatum P genome-specific markers, and fluorescence in situ hybridization (FISH). The results revealed that the introgression between A. cristatum P genome and wheat genome was observed in progenies of the genetic populations of the translocation line WAT655 and the physical positions of the three genes were considerably adjacent on A. cristatum 6PS (0.81–1.00) according to the FISH results. Additionally, kompetitive allele-specific PCR (KASP) markers of the three genes were developed to detect and acquire 24 breeding lines selected from the progenies of the distant hybridization of wheat and A. cristatum, which showed resistance to physiological races of stripe rust (CYR32 and CYR33) and other desirable agronomic traits according to the field investigation. In conclusion, this study not only provides new insights into the introgression between A. cristatum P genome and wheat genome but also provides the desirable germplasms for breeding practice.


Introduction
Common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most cultivated cereal crops in the world. The total global wheat acreage was approximately 220 million hectares, and the global yield was more than 770 million tons in 2020 [1]. Wheat is a staple food crop that can provide starch, protein, vitamins, dietary fiber, and phytochemicals for humans, and wheat also provides approximately 20% of the calories consumed by humans [2]. However, wheat often suffers from various biotic and abiotic stresses during production, and the narrow genetic basis of wheat restricts its genetic improvement [3]. Wheat stripe rust is a fungal disease caused by the fungus Puccinia striiformis f. sp. tritici (Pst). Approximately 90% of cultivated wheat is susceptible to Pst, and more than five million tons of the wheat harvest are lost annually [4,5]. With the emergence of new Pst races and the variations in existing Pst races, many wheat varieties have lost resistance to stripe rust [6,7]. Therefore, providing new wheat germplasms with resistance to Pst infection is necessary for advancing wheat breeding.
Wild relatives of wheat are crucial gene resources for broadening the genetic basis of wheat and facilitating wheat breeding. Wild relatives of wheat carry numerous disease resistance genes [8,9]. Moreover, with the increasing number of successful distant hybridizations, an increasing number of exogenous genes from wild relatives, particularly disease resistance genes, have been transferred into common wheat. The powdery mildew resistance gene Pm21 from Haynaldia villosa has been transferred into common wheat and widely applied in breeding practice [10][11][12]. Multiple stem rust resistance genes from Triticum monococcum, such as Sr21, Sr22, Sr35, and Sr60, have been cloned in common wheat [13][14][15][16][17]. Stripe rust resistance genes, such as YrAS2388, Yr28, YrY201 from Aegilops tauschii [18][19][20][21][22], and Yr15 from wild emmer wheat [23], have been cloned from common wheat. Hence, wild relatives of wheat are an abundant gene pool for providing disease resistance genes.
Agropyron cristatum (2n = 4x = 28, PPPP), an important wild relative of wheat, carries several desirable agronomic traits that can be applied in wheat breeding. With the achievement of the distant hybridization of wheat and A. cristatum, a series of wheat-A. cristatum derived lines were generated [24][25][26][27][28][29][30]. Many desirable genes from A. cristatum have been transferred into common wheat in the form of the translocation line. For example, A. cristatum P chromosomal segments carrying the powdery mildew resistance gene, the high-thousand grain weight (TGW) gene, and the high-grain number per spike (GNS) gene have been translocated into wheat chromosomes [28,29,31]. According to recent reports, the leaf rust resistance gene and the powdery mildew resistance gene from the A. cristatum 2P chromosome have been localized on the same segment of A. cristatum 2PL (0.66-0.86) [32], and both the stripe rust resistance gene and the leaf rust resistance gene were mapped to A. cristatum 6PS (0.81-1.00) [33]. Although wheat-A. cristatum translocation lines carry many desirable genes, they remain difficult to apply in wheat breeding because of the genetic drag caused by the exogenous chromosomal segments. Therefore, the identification of the introgression lines carrying smaller exogenous components is necessary for improving wheat breeding.
In this study, to explore the introgression between A. cristatum P genome and wheat genome and detect A. cristatum P genomic components of breeding lines, the specific molecular markers and fluorescence in situ hybridization (FISH)/genomic in situ hybridization (GISH) probes made from the DNA of three bacterial artificial chromosome (BAC) clones, three disease resistance-related A. cristatum genes, and A. cristatum "Z559" were used to analyze the wheat-A. cristatum 6P derived lines. The introgression between A. cristatum P genome and wheat genome was observed, and the new breeding lines carrying A. cristatum P genomic components were acquired.

The Acquisition of Disease Resistance-Related A. cristatum Genes of the Translocation Line WAT655
In previous reports, the wheat-A. cristatum 6P translocation line WAT655 carrying A. cristatum 6PS (0.81-1.00) exhibited high resistance to stripe rust (CYR32 and CYR33) and leaf rust in the adult stage [33,41]. To further explore the disease resistance genes of A. cristatum 6PS (0.81-1.00), the software TransDecoder (version v5.5.0) [42] was used to determine protein sequences of the A. cristatum full-length transcripts [43] and the software eggNOG-mapper [44] and BLAST from the NCBI website were used for the functional annotation. According to the information of the functional annotation, the disease resistance-related transcripts were searched from among A. cristatum full-length transcripts. Then, based on the sequences of disease resistance-related transcripts, molecular markers were developed to trace the A. cristatum 6P chromosomal segments of the translocation line WAT655. As a result, the specific molecular markers of three disease resistance-related transcripts can be mapped to A. cristatum 6PS (0.81-1.00) of the translocation line WAT655 ( Figure 1 and Table 1).

Results
2.1. The Acquisition of Disease Resistance-Related A. cristatum Genes of the Translocation Line WAT655 In previous reports, the wheat-A. cristatum 6P translocation line WAT655 carrying A. cristatum 6PS (0.81-1.00) exhibited high resistance to stripe rust (CYR32 and CYR33) and leaf rust in the adult stage [33,41]. To further explore the disease resistance genes of A. cristatum 6PS (0.81-1.00), the software TransDecoder (version v5.5.0) [42] was used to determine protein sequences of the A. cristatum full-length transcripts [43] and the software eggNOG-mapper [44] and BLAST from the NCBI website were used for the functional annotation. According to the information of the functional annotation, the disease resistance-related transcripts were searched from among A. cristatum full-length transcripts. Then, based on the sequences of disease resistance-related transcripts, molecular markers were developed to trace the A. cristatum 6P chromosomal segments of the translocation line WAT655. As a result, the specific molecular markers of three disease resistance-related transcripts can be mapped to A. cristatum 6PS (0.81-1.00) of the translocation line WAT655 ( Figure 1 and Table 1).   To trace A. cristatum 6P chromosomal segments in the BC 5 F 2 population (2019-2020) of the translocation line WAT655, A. cristatum P genome-specific markers [45] (Table S1) and the gene markers from three disease resistance-related transcripts were utilized in this study. The detection results of the gene markers were different from the results of the A. cristatum P genome-specific markers in some plants of the BC 5 F 2 population of the translocation line WAT655 (Figure 2). Preliminarily speculating, the introgression between A. cristatum 6PS (0.81-1.00) chromosomal segments and wheat chromosomes could occur in progenies of the BC 5 F 2 population of the translocation line WAT655, which resulted in the differences between the two kinds of molecular markers in some plants. To trace A. cristatum 6P chromosomal segments in the BC5F2 population (2019-2020) of the translocation line WAT655, A. cristatum P genome-specific markers [45] (Table S1) and the gene markers from three disease resistance-related transcripts were utilized in this study. The detection results of the gene markers were different from the results of the A. cristatum P genome-specific markers in some plants of the BC5F2 population of the translocation line WAT655 ( Figure 2). Preliminarily speculating, the introgression between A. cristatum 6PS (0.81-1.00) chromosomal segments and wheat chromosomes could occur in progenies of the BC5F2 population of the translocation line WAT655, which resulted in the differences between the two kinds of molecular markers in some plants. Then PCR amplification patterns of A. cristatum P genome-specific markers (AcPR2a, AcPR6, and AcPR2b) and gene makers (Agr6971-3, Agr4080-3, and Agr8173-9) in the genetic populations (BC 5 F 2 and BC 5 F 2:3) . (a-e) The detection results for the BC 5 F 2 population of the translocation line WAT655. (f-j) The detection results for the BC 5 F 2:3 population of the translocation line WAT655. The red arrows indicated the differences between P genome-specific markers and the gene makers in these plants; the black arrows indicated the diagnostic bands. Lanes: M, DL2000 DNA marker; 1, Z559; 2, 4844-12; 3, Fukuho; 4-24 of (a-e) the partial plants of the BC 5 F 2 population; 4-24 of (f-h) the partial plants of the WAT655 BC 5 F 2:3 population; 4-17 of (i,j) the partial plants of the BC 5 F 2:3 population.
To further confirm whether the introgression between A. cristatum 6PS (0.81-1.00) chromosomal segments and wheat chromosomes can occur in the common wheat background, the progenies from the BC 5 F 2 population (2019-2020) of the translocation line WAT655 were planted in 2020-2021. The two kinds of molecular markers were utilized to detect P genomic components in the BC 5 F 2:3 population, and the results revealed that some plants still showed differences between the gene markers and P genome-specific markers ( Figure 2). These results further confirmed that the introgression of A. cristatum P genome and wheat genome can occur in the translocation line WAT655.

The Identification of the Introgression between A. cristatum P Genome and Wheat Genome in the Translocation Line WAT655
To acquire more sequence information of three disease resistance-related transcripts, the gene markers from the three transcripts were used to search A. cristatum "Z559" P genomic BAC bank, moreover, three BAC clones (BAC1013, BAC700, and BAC940) of three genes were acquired (Table 1). Pacific Biosciences technology was used to sequence the three BAC clones, and the software Hifiasm [46] was used to assemble the sequence data. This work was performed by BioMarker Company (Beijing, China).
To further analyze the introgression between A. cristatum P genome and wheat genome, the DNA of the three BAC clones, three disease resistance-related genes, and A. cristatum "Z559" was utilized as the probes to perform FISH and GISH in common wheat "Fukuho", the translocation line WAT655, and the substitution line 4844-12-1 (Figures 3, 4 and S1-S3). The FISH and GISH probes could not hybridize with the chromosomal DNA of common wheat "Fukuho" (Figures 3 and S2). The signals of the FISH probes made from the DNA of the BAC clones (BAC1013, BAC700, and BAC940) and three genes (Agr6971, Agr4080, and Agr8173) were mapped to A. cristatum 6PS (0.81-1.00) of the translocation line WAT655 and the A. cristatum 6PS terminal of the substitution line 4844-12-1 (Figures 3 and S1-S3). Significantly, the FISH probe BAC1013 traced one A. cristatum 6PS chromosomal segment on the wheat chromosome 3DS in addition to the previously translocated wheat chromosome 6D (Figures 4 and 5); moreover, the probe Agr8173 detected A. cristatum 6PS chromosomal segments on the wheat chromosome 7DL (Figures 4 and 5). Therefore, the above results indicated that the introgression between A. cristatum P genomic chromosomal segments and wheat chromosomes can occur in progenies of the genetic population of the translocation line WAT655.    . The FISH and GISH patterns of common wheat "Fukuho", the substitution line 4844-12-1, and the translocation line WAT655 using the DNA of the BAC clone "BAC1013", the gene Agr4080, and A. cristatum "Z559" as the probes. (a,b) The FISH patterns of common wheat "Fukuho" using the probes BAC1013 and Agr4080. (c,e) The FISH patterns of the substitution line 4844-12-1 and the translocation line WAT655 using the probe BAC1013. (g,i) The FISH patterns of the substitution line 4844-12-1 and the translocation line WAT655 using the probe Agr4080. (d-j) The GISH patterns of the substitution line 4844-12-1 and the translocation line WAT655 using the DNA of A. cristatum "Z559" as the probe. A. cristatum 6P chromosomal segments were in red, while wheat chromosomes were in blue strained by 4′,6-diamidino-2-phenylindole (DAPI). The white arrows indicated the A. cristatum chromatin in the wheat background. (scale bar = 10 µm).

Molecular Cytogenetic Analysis of the Physical Positions of Three Disease Resistance-Related Genes
To explore the physical positional relationships of the three genes on A. cristatum 6PS (0.81-1.00) of the translocation line WAT655, the DNA of Agr6971 and Agr8173 was labeled with Texas Red ® -5-dCTP (red), and the DNA of Agr4080 was labeled with Fluorescein-12-dUTP (green). The probes Agr6971 (red) and Agr4080 (green) were simultaneously used for one FISH experiment ( Figures 5 and S4), and the probes Agr8173 (red) and Agr4080 (green) were simultaneously used for another (Figures 5 and S4). The signals of the FISH probes showed that Agr6971 (red) and Agr4080 (green) were localized at one overlapping zone of A. cristatum 6PS (0.81-1.00); moreover, Agr8173 (red) and Agr4080 (green) were also localized at one overlapping zone of A. cristatum 6PS (0.81-1.00). These FISH results revealed that three genes were localized at the considerably adjacent positions on A. cristatum 6PS (0.81-1.00) ( Figure 5), which could constitute a potential disease resistance-related gene cluster.
Agr4080 (green) were simultaneously used for another ( Figure 5 and Figure S4). The signals of the FISH probes showed that Agr6971 (red) and Agr4080 (green) were localized at one overlapping zone of A. cristatum 6PS (0.81-1.00); moreover, Agr8173 (red) and Agr4080 (green) were also localized at one overlapping zone of A. cristatum 6PS (0.81-1.00). These FISH results revealed that three genes were localized at the considerably adjacent positions on A. cristatum 6PS (0.81-1.00) (Figure 5), which could constitute a potential disease resistance-related gene cluster.

Tracing A. cristatum P Genomic Components of Breeding Lines Selected from Progenies of Distant Hybridization between A. cristatum and Wheat
To trace the A. cristatum P genomic components of breeding lines selected from progenies of distant hybridization between A. cristatum and wheat, the KASP molecular

Tracing A. cristatum P Genomic Components of Breeding Lines Selected from Progenies of Distant Hybridization between A. cristatum and Wheat
To trace the A. cristatum P genomic components of breeding lines selected from progenies of distant hybridization between A. cristatum and wheat, the KASP molecular markers of the three genes were developed according to the results of the alignment between the sequences of three genes and the Chinese Spring genome sequences RefSeq v1.0 [47] (Figure S5 and Table S2). The KASP molecular markers were used to trace the A. cristatum 6P components of the BC 5 F 2:3 population of the translocation line WAT655 and the A. cristatum 6P components of breeding lines, and the results were completely coincident with those of standard PCR detection of the gene markers relying on agarose gel electrophoresis ( Figure S6). Meanwhile, 24 breeding lines carrying A. cristatum P genomic components were acquired from among 500 breeding lines ( Figure 6 and Table 2). The three gene FISH probes were used to detect the A. cristatum P genomic components of the part of 24 breeding lines, and the breeding lines showed the A. cristatum P genomic components can be detected through the FISH probes ( Figure 7). Additionally, these breeding lines exhibited resistance or moderate resistance to stripe rust (CYR32 and CYR33) according to the investigation in the field ( Figure 6 and Table 2). These breeding lines also exhibited other desirable agronomic traits in the field ( Figure 6 and Table 2). Therefore, A. cristatum P genomic components of these breeding lines can be traced by the molecular markers and the FISH probes, and these breeding lines can also promote wheat breeding. phoresis ( Figure S6). Meanwhile, 24 breeding lines carrying A. cristatum P genomic components were acquired from among 500 breeding lines ( Figure 6 and Table 2). The three gene FISH probes were used to detect the A. cristatum P genomic components of the part of 24 breeding lines, and the breeding lines showed the A. cristatum P genomic components can be detected through the FISH probes ( Figure 7). Additionally, these breeding lines exhibited resistance or moderate resistance to stripe rust (CYR32 and CYR33) according to the investigation in the field ( Figure 6 and Table 2). These breeding lines also exhibited other desirable agronomic traits in the field ( Figure 6 and Table 2). Therefore, A. cristatum P genomic components of these breeding lines can be traced by the molecular markers and the FISH probes, and these breeding lines can also promote wheat breeding. Figure 6. The detection of A. cristatum P genomic components using KASP markers and the investigation of agronomic traits of breeding lines. (a-c) The detection results of Agr6971kps-4, Agr4080kps-2, and Agr4080kps-4, respectively. The genotype of A. cristatum "Z559" was in red; the genotype of wheat was in blue. The genotype of the breeding lines was in the circles. (d,e) The agronomic traits of partial breeding lines including resistance to stripe rust and other agronomic traits in the field.

Discussion
Distant hybridization is an efficient and important method and contributes much to wheat breeding for disease resistance. For example, the powdery mildew gene Pm21 of Haynaldia villosa 6VS has been widely applied in wheat breeding [11,12]; the 1BL•1RS translocation line carries many disease resistance genes, such as the stripe rust resistance gene Yr9, the powdery mildew resistance gene Pm8, the stem rust resistance gene Sr31, and the leaf rust resistance gene Lr26 [3,[48][49][50]. A. cristatum is one of the most important wild relatives of common wheat and carries many desirable agronomic traits [8,9]. In recent years, many desirable genes from A. cristatum have been transferred into common wheat, such as the stripe rust resistance, the leaf rust resistance, the powdery mildew, the high-GNS, and the high-TGW genes [28,29,[31][32][33]41,[51][52][53][54]. These genes from A. cristatum are usually mapped on relatively large A. cristatum chromosomal segments of translocation lines. However, translocation lines are not easy to apply in breeding practice because of the genetic drag caused by the translocated segments, so many breeders desire the introgression lines carrying smaller exogenous genetic components for breeding. In this study, gene markers, A. cristatum genome-specific markers, and FISH/GISH probes were used to analyze the wheat-A. cristatum 6P translocation line WAT655. The evidence of the introgression between A. cristatum 6P chromosomal segments and wheat chromosomes was observed (Figures 4-7), and these three genes could be mapped to the considerably adjacent physical positions on A. cristatum 6PS (0.81-1.00) according to the FISH signals ( Figure 5). Additionally, 24 breeding lines carrying A. cristatum P genomic components were acquired through molecular cytogenetic analysis (Figures 6 and 7). These breeding lines exhibited favorable breeding agronomic traits including resistance to stripe rust (CYR32 and CYR33) according to the investigation in the field, which also indicated that three disease resistance-related genes were closely associated with the resistance to stripe rust. Moreover, the molecular markers designed from the sequences of three disease resistance-related genes can also promote the application of marker-assisted selection in distant hybridization of wheat and A. cristatum and wheat breeding. In addition, to further verify the function of three disease resistance-related genes, transgenic work involving coding sequences (CDSs) and full-length sequences of three disease resistance-related genes is already underway. Therefore, this study not only provides new insights into the introgression between A. cristatum P genome and wheat genome but also provides a new approach for acquiring valuable breeding lines.
Some disease resistance genes have been shown to be distributed in the form of a gene cluster. For example, the powdery mildew resistance genes Pm21 and Stpk-V were mapped to the same locus of Haynaldia villosa 6VS bin (FL 0.45-0.58) [11,12]; the stripe rust resistance genes Yr5, Yr7, and YrSP were on a gene cluster [35]. Both the powdery mildew resistance gene and the leaf rust resistance gene of A. cristatum 2P chromosome were mapped to A. cristatum 2PL (0.66-0.86) [29,32]. The stripe rust resistance gene and the leaf rust resistance gene were localized on A. cristatum 6PS (0.81-1.00) of the translocation line WAT655 [33,41,49]. In this study, the DNA of three disease resistance-related genes was used as FISH probes to identify the physical positions of the genes on A. cristatum 6PS (0.81-1.00). The results revealed that the FISH signals of the probes were located at the overlapping positions of A. cristatum 6PS (0.81-1.00) ( Figure 5 and Figure S4), therefore, the three genes could be mapped to the considerably adjacent physical positions, which could constitute a potential disease resistance-related gene cluster on A. cristatum 6PS (0.81-1.00).
A. cristatum P genome and wheat genome were revealed to present a homoeologous relationship by using a wheat 660K SNP array, and apparent chromosomal rearrangements and introgression spread throughout the P genome were observed [55]. In addition, genetic rearrangements of A. cristatum 6P chromosomes could usually occur at the terminal of chromosomes by analyzing the different wheat-A. cristatum 6P addition lines [56]. In this study, the results of FISH using the DNA of the BAC clones and three disease resistancerelated genes as probes indicated that the introgression can occur between A. cristatum 6P chromosomal segments and wheat chromosomes (Figures 4 and 5), moreover, the observed introgression also occurred in the terminal of the chromosomes (Figures 4 and 5). The mechanism of the introgression could involve chromosomal rearrangements between A. cristatum P genome and wheat genome resulting from their homoeologous relationship.

Molecular Cytogenetic Analysis
A. cristatum P genome-specific markers were designed from P genome-specific repeat sequences (Table S1) [42]. A. cristatum P genome-specific markers were also used to detect A. cristatum 6P chromosomal segments in the genetic populations of the translocation line WAT655. The A. cristatum full-length transcripts were analyzed by using TransDecoder (version v5.5.0) [43] to determine the CDSs and protein sequences, and the software eggNOG-mapper [44] and BLAST from the National Center of Biotechnology Information (NCBI) website were used to annotate these sequences. The disease resistance-related genes were searched, and specific molecular markers were developed for the analysis of wheat-A. cristatum 6P translocation line WAT655. Additionally, the specific sequences of three genes were used to design KASP markers to trace A. cristatum P genomic components in wheat-A. cristatum breeding lines ( Figure S5 and Table S2). HiGeno 2x Probe Mix (JasonGen, Beijing, China) was used for the KASP reaction, and PCR products were detected with a PHERAstarplus SNP genotyping instrument (LGC Science Shanghai Ltd., Shanghai, China). The software KlusterCallerTM was used for genotyping assays.
FISH and GISH were performed in root tip cells, as described by Cuadrado et al. [57]. The DNA of three BAC clones, three disease resistance-related genes, and A. cristatum "Z559" were used as FISH/GISH probes, and the genomic DNA of common wheat "Fukuho" was used as the blocker. The FISH/GISH probes and the blocker were used at a 1:40 ratio. Oligo-pTa535-1 (red) and Oligo-pSc119.2-1 (green) [58] (Table S1) were used to analyze the wheat chromosomes in the substitution line 4844-12-1. The QIAGEN Large-Construct Kit (Qiagen, Germany) was used to extract the DNA of the three BAC clones. The DNA of three disease resistance-related genes was acquired from the three A. cristatum "Z559" genomic BAC clones, respectively. The DNA of A. cristatum "Z559", the three BAC clones (BAC1013, BAC700, and BAC940), the three genes (Agr6971, Agr4080, and Agr8173), and pAs1 [59] were labeled with Texas Red ® -5-dCTP (red) (PerkinElmer, Waltham Mass, MA, USA). The DNA of A. cristatum "Z559", the gene Agr4080, and pHvG39 [59] was labeled with Fluorescein-12-dUTP (green) (PerkinElmer, Waltham Mass, MA, USA). VECTASHIELD ® Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) was used for the FISH/GISH experiments. Signals were captured using an OLYMPUS AX80 (Olympus Corporation, Tokyo, Japan) fluorescence microscope with a CCD camera (Diagnostic Institute, Inc., Sterling Height, MI, USA) and processed with Photoshop CS 3.0.

Evaluation of the Agronomic Traits of Wheat-A. cristatum Breeding Lines in the Field
To evaluate stripe rust resistance, the wheat-A. cristatum 6P disomic addition line 4844-12, the wheat-A. cristatum 6P disomic substitution line 4844-12-1, the wheat-A. cristatum 6P translocation line WAT655, wheat varieties (Zhoumai18, Jimai22, Shi4185, Luyuan502, Gaocheng8901, and Xinong979), 500 breeding lines selected from the progenies of the distant hybridization of A. cristatum and wheat, and the common wheat cultivar "Fukuho" were planted in a randomized complete block design with three replicates in the field in Beijing (39 • 54 20 N, 116 • 25 29 E, China) during 2019-2020. Twenty grains of each material were planted in 2.0 m rows that were spaced 0.3 m apart. The common wheat "Fukuho" was used as the control. The prevalent physiological races of stripe rust CYR32 and CYR33 were used to inoculate the plant materials at the elongation stage in the field in Beijing. The infection type (IT) score ranged from 0 to 9 (0: no visible symptoms; 1-2: necrotic flecks or necrotic areas without sporulation; 3-4: trace or light sporulation; 5-6: intermediate or moderate sporulation; 7-8: abundant sporulation; 9: no necrosis or chlorosis and abundant sporulation) [60]. Plants with IT scores of 0-2 were considered as highly resistant type (HR); plants with IT scores of 3-4 were considered as moderately resistant type (MR); plants with IT scores of 5-6 were moderately susceptible type (MS); plants with IT scores of 5-9 were considered as highly susceptible type (HS).
To evaluate agronomic traits, wheat varieties (Zhoumai18, Jimai22, Shi4185, Luyuan502, Gaocheng8901, and Xinong979) and 500 breeding lines were planted in 1.2 × 6 m 2 plots with four replicates in the field in Xinxiang (35 • 18 13" N, 113 • 55 15" E, Henan Province, China) during 2019-2020. The plant height, plot yield, and thousand grain weight of each material were measured. Statistical Analysis System (Version 9.4, SAS Institute, Cary, NC, USA) was used for statistical analysis in this study. Analysis of variance was performed to test the differences in the plant height, the plot yield, and the thousand grain weight among the breeding lines. The "mean ± SD" was used to represent the data.

Conclusions
In the previous report, the wheat-A. cristatum translocation line WAT655 carrying A. cristatum 6PS (0.81-1.00) exhibited the high resistance to prevalent physiological races of stripe rust (CYR32 and CYR33). In the present study, three disease resistance-related A. cristatum genes were acquired, which can be mapped to A. cristatum 6PS (0.81-1.00) carried by the translocation line WAT655. The three disease resistance-related genes could constitute a potential gene cluster through molecular cytogenetic analysis. Moreover, the introgression between A. cristatum P genome and wheat genome was observed in the translocation line WAT655 and the breeding lines carrying A. cristatum P genomic components were acquired by using the molecular markers and the FISH probes of the three genes. According to the resistance to stripe rust of the breeding lines, the three genes could be closely associated with the resistance to stripe rust. Therefore, this study provides new insights into the introgression between A. cristatum P genome and wheat genome and the desirable germplasms for breeding practice.  Data Availability Statement: The datasets of Agropyron cristatum BAC clones generated during and/or analyzed during the current study are not publicly available due to the transgenic work of the genes in progress but are available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare that they have no conflicts of interest.