New Insights Into the Introgression Between Agropyron Cristatum P Genome and Wheat Genome

Agropyron cristatum (2n = 4x = 28, PPPP) is an important wild relative of common wheat and confers desirable agronomic traits to common wheat. 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 (CYR32 and CYR33). In this study, three disease resistance-related transcriptomes, which were mapped to A. cristatum 6PS (0.81–1.00) through the analysis of specic molecular markers, were searched from among A. cristatum full-length transcriptomes. Then, three disease resistance-related gene markers, A. cristatum P genome-specic markers, and uorescence in situ hybridization (FISH)/genomic in situ hybridization (GISH) probes made from the DNA of three bacterial articial chromosome (BAC) clones, three genes, and A. cristatum “Z559” were used to analyze the BC 5 F 2 and BC 5 F 2:3 genetic populations of the translocation line WAT655. The results revealed the introgression can spontaneously occur between A. cristatum P genome and wheat genome, and indicated the three genes could constitute a gene cluster according to the positions of their FISH signals. Additionally, kompetitive allele-specic PCR (KASP) markers of the three genes were developed to detect and acquire 24 wheat-A. cristatum breeding materials, which showed resistance to physiological races (CYR32 and CYR33) and other desirable agronomic traits according to the eld investigation. In conclusion, our study not only provides new insights into the introgression between A. cristatum P genome and wheat genome, but also provides the desirable breeding materials for breeding practice. The introgression can spontaneously occur between A. cristatum P chromatin and wheat chromosomes, and wheat-A. cristatum breeding materials were produced by the introgression. in situ hybridization probes made from the DNA of three bacterial articial chromosome (BAC) clones, three disease resistance-related A. genes, and A. “Z559” were used to analyze the genetic populations of the wheat-A. cristatum 6P translocation line WAT655. The results revealed the spontaneous introgression between A. cristatum P genome and wheat genome. In addition, a few wheat-A. cristatum breeding materials, which can be directly applied in breeding practice, were acquired by using kompetitive allele-specic PCR (KASP) markers. This study provides new insights into the introgression between A. cristatum P genome and wheat genome, and provides new wheat-A. cristatum breeding materials for breeding practice. P


Introduction
Common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most cultivated cereal crops in the world. The total wheat acreage was approximately 220 million hectares, and the yield was more than 770 million tons globally in 2020 (World Agricultural Production 2021). Wheat is a staple food crop that can provide starch, protein, vitamins, dietary ber, and phytochemicals for humans, and wheat also provides approximately 20% of the calories consumed by humans (Shewry and Hey 2015). However, wheat often suffers from various biotic and abiotic stresses during production, and the narrow genetic basis of wheat restricts its genetic improvement (Friebe et al. 1996). 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 ve million tons of the wheat harvest are lost annually (Wellings 2011;Beddow et al. 2015). With the emergence of new Pst races and the variations in existing Pst races, many wheat varieties have lost resistance to stripe rust (Wan et al. 2004; Chen et al. 2009). 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 (Dewey 1984;Dong et al. 1992). 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 Haynaldiavillosa has been transferred into common wheat and widely applied in breeding practice (Cao et  Agropyron cristatum (2n = 4x = 28), 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 (Limin and Fowler 1990;Li et al. 1995Li et al. , 1997 Moreover, in addition to stripe rust resistance genes, most of the cloned other disease resistance genes in wheat also encode nucleotide-binding domain, leucine-rich repeat domain, or kinase domain (Krattinger and Keller 2016). For example, powdery mildew resistance genes (Stpk-V, Pm21, Pm41, and Pm60) (Cao et al. 2011;Zou et al. 2017;Xing et al. 2018;Li et al. 2020) and stem rust resistance genes (Sr22, Sr35, Sr45, and Sr60) (Saintenac et al. 2013;Steuernagel et al. 2016;Chen et al. 2018) encode nucleotide-binding domain, leucine-rich repeat domain, or kinase domain. Therefore, nucleotide-binding domain, leucine-rich repeat domain, and kinase domain can be considered as typical domains to help the clone of disease resistance genes.
In this study, speci c molecular markers and uorescence in situ hybridization (FISH)/genomic in situ hybridization (GISH) probes made from the DNA of three bacterial arti cial chromosome (BAC) clones, three disease resistance-related A. cristatum genes, and A. cristatum "Z559" were used to analyze the genetic populations of the wheat-A. cristatum 6P translocation line WAT655. The results revealed the spontaneous introgression between A. cristatum P genome and wheat genome. In addition, a few wheat-A. cristatum breeding materials, which can be directly applied in breeding practice, were acquired by using kompetitive allele-speci c PCR (KASP) markers. This study provides new insights into the introgression between A. cristatum P genome and wheat genome, and provides new wheat-A. cristatum breeding materials for breeding practice.

Molecular cytogenetic analysis
A. cristatum P genome-speci c markers were designed from P genome-speci c repeat sequences (Table S1) . P genome-speci c markers were also used to detect A. cristatum 6P chromatin in the genetic populations of the translocation line WAT655.
The A. cristatum full-length transcriptomes were analyzed by using TransDecoder (version v5.5.0) (Grabherr et al. 2011) to determine the CDSs and protein sequences, the software eggNOG-mapper (Huerta-Cepas et al. 2017) and BLASTx from the National Center of Biotechnology Information (NCBI) website were used to annotate these sequences. The disease resistance-related genes were searched according to the annotation le, and speci c molecular markers were developed for the analysis of wheat-A. cristatum 6P translocation line WAT655. Additionally, the speci c sequences of three genes were used to design KASP markers to trace A. cristatum P chromatin in wheat-A. cristatum breeding materials ( Fig. S2 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., China). The software KlusterCallerTM was used for genotyping assays.
FISH and GISH were performed in root tip cells, as described by Cuadrado et al. (2000). The DNA of three BAC clones, three disease resistancerelated 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) (Tang et al. 2014) (Table S1) were used to analyze the wheat chromosomes of 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 (Pedersen and Langridge 1997) was labeled with Texas Red®-5-dCTP (red) (PerkinElmer, Waltham, America). The DNA of A. cristatum "Z559", the gene Agr4080, and pHvG39 (Pedersen and Langridge 1997) was labeled with Fluorescein-12-dUTP (green) (PerkinElmer, Waltham, America). VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, America) was used for the FISH/GISH experiments. Signals were captured using an OLYMPUS AX80 (Olympus Corporation, Tokyo, Japan) uorescence 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 materials in the eld
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 wheat-A. cristatum breeding materials, and the common wheat cultivar "Fukuho" were planted in a randomized complete block design with three replicates in the eld in Beijing (39°54′20″ N, 116°25′29″ E, China). 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 CYR32 and CYR33 were used to inoculate the plant materials at the elongation stage in the eld at Beijing. The infection type (IT) score ranged from 0 to 9 (0: no visible symptoms; 1-2: necrotic ecks 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) (Line and Qayoum 1992). Plants with IT scores of 0-2 were considered as highly resistant type; plants with IT scores of 3-4 were considered as moderately resistant type; plants with IT scores of 5-6 were moderately susceptible type; plants with IT scores of 5-9 were considered as highly susceptible type.

Results
The capture 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 (Song et Table 2). These results provided the basis for further exploring the disease resistance genes of A. cristatum 6PS (0.81-1.00).
Tracing A. cristatum P chromatin in the BC 5 F 2 and BC 5 F 2:3 genetic populations of the translocation line WAT655 using the molecular markers To trace A. cristatum P chromatin in the BC 5 F 2 population (2019-2020) of the translocation line WAT655, A. cristatum P genome-speci c markers and the gene markers from three disease resistance-related transcriptomes were utilized in this study. The P genome-speci c markers were designed by using P genome-speci c repeat sequences , and the gene markers were designed from the sequences of the three A. cristatum transcriptomes in this study. Therefore, all of these molecular markers can accurately trace A. cristatum P chromatin in common wheat. However, the detection results of the gene markers were different from the results of the P genome-speci c markers in some plants of the BC 5 F 2 population of the translocation line WAT655 (Fig. 2). Preliminarily speculating, some of A. cristatum 6PS (0.81-1.00) chromatin in the translocation line WAT655 might have been spontaneously transferred into common wheat background, which resulted in the differences between the two kinds of molecular markers in some plants.
To con rm whether the introgression between A. cristatum P chromatin and wheat chromosomes could occur in the BC 5 F 2:3 population (2020-2021), 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-speci c markers (Fig. 2). These results con rmed the potential for the introgression between A. cristatum 6P chromatin of the translocation line WAT655 and wheat chromosomes.
The identi cation of the introgression between A. cristatum P genome and wheat genome in translocation line WAT655 To acquire more sequence information of three disease resistance-related transcriptomes, the gene markers from the three transcriptomes 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 2). Paci c Biosciences technology was used to sequence the three BAC clones, and the software Hi asm (Cheng et al. 2021) 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 resistancerelated 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 (Fig. 3, Fig. 4, Fig. 5, Fig. 6, and Fig. S3). The FISH and GISH probes made from the DNA of the three BAC clones, the three genes, and A. cristatum "Z559" could not hybridize with the chromosomal DNA of common wheat "Fukuho" (Fig. 3 and Fig.  4). The FISH probes made from the DNA of the BAC clones (BAC1013, BAC700, and BAC940) could be used to trace A. cristatum 6P chromatin in the translocation line WAT655 (Fig. 3 and Fig. S3), and the locations of the signals 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 (Fig. 3 and Fig. S3). The signal positions of the probes made from the DNA of the BAC clones were identical to those of the probes made from A. cristatum "Z559" P genomic DNA on A. cristatum 6PS (0.81-1.00) of the translocation line WAT655 (Fig. 3 and Fig. S3). Additionally, the probe made from the DNA of the BAC clone "BAC1013" traced one A. cristatum 6PS segment on the wheat chromosome 3D in addition to the previous translocated wheat chromosome 6D (Fig. 3, Fig. 6, and Fig. S4). Therefore, the results demonstrated that A. cristatum P chromatin can spontaneously in ltrate the wheat background. Additionally, the signals from the probes made from the DNA of three genes were mapped to the terminal of the chromosomal segment 6PS (0.81-1.00) in the translocation line WAT655 and the terminal of the chromosome 6PS in the substitution line 4844-12-1 ( Fig. 4 and Fig. 5). The probe Agr8173 could detect A. cristatum 6PS chromatin on the wheat chromosome 7D in addition to the previous translocated wheat chromosome 6D (Fig. 5, Fig. 6, and Fig. S4). Overall, the above results indicated that the A. cristatum P chromatin in A. cristatum 6PS (0.81-1.00) could spontaneously in ltrate the wheat background.
Molecular cytogenetic analysis of the gene cluster constituted by three disease resistance-related genes As shown in Fig. 4 and Fig. 5, the positions of three disease resistance-related genes were mapped to the terminal of A. cristatum 6PS (0.81-1.00), which indicated that these three genes could be considerably close on A. cristatum 6PS (0.81-1.00). To explore the 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 ( Fig. 6 and Fig. S5), and the probes Agr8173 (red) and Agr4080 (green) were simultaneously used for another ( Fig. 6 and Fig. S5). The probe made from A. cristatum "Z559" DNA was used for the second round of GISH. 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 the three genes were localized at overlapping positions of A. cristatum 6PS (0.81-1.00); therefore, the three genes were preliminarily considered as a disease resistance-related gene cluster on A. cristatum 6PS (0.81-1.00) (Fig. 6).
Tracing A. cristatum P genomic components of wheat-A. cristatum breeding materials The above results indicated that the introgression can spontaneously occur between A. cristatum P genome and wheat genome in the translocation line WAT655. In our laboratory, a large number of wheat-A. cristatum breeding materials were selected from the progenies of wheat-A. cristatum introgression lines according to the agronomic traits of A. cristatum. The wheat-A. cristatum breeding materials exhibited desirable breeding agronomic traits that can be directly applied in breeding practice. To detect the A. cristatum P genomic components of wheat-A. cristatum breeding materials, the speci c sequences of the three genes, which were acquired according to results of the alignment between the sequences of three genes and Chinese Spring genome sequences RefSeq v1.0 (International Wheat Genome Sequencing Consortium 2018), were used to develop KASP molecular markers ( Fig. S2 and Table S2). The KASP molecular markers were used to trace the A. cristatum 6P components in the BC 5 F 2:3 population of the translocation line WAT655. The results were completely coincident with those of standard PCR detection of the gene markers relying on agarose gel electrophoresis (Fig. S6), therefore, the results indicated that the KASP molecular markers could be used to accurately trace A. cristatum P genomic components in common wheat. Then, the KASP molecular markers were used to trace A. cristatum P genomic components of wheat-A. cristatum breeding materials, and 24 breeding materials were acquired from among 500 wheat-A. cristatum breeding materials ( Fig. 7 and Table 3). Moreover, these breeding materials exhibited resistance or moderate resistance to stripe rust (CYR32 and CYR33) according to the investigation in the eld ( Fig. 7 and Table 3). Therefore, three disease resistance-related genes were closely associated with the resistance to stripe rust. In addition, these breeding materials also exhibited other desirable agronomic traits in the eld ( Fig. 7 and Table 3). In conclusion, these breeding materials from wheat-A. cristatum introgression lines are valuable for breeding practice, and the results provided new insights for the formation of introgression lines.

Discussion
A. cristatum is one of the most important wild relatives of common wheat and carries many desirable agronomic traits (Dewey 1984;Dong et al. 1992). 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 ( These genes from A. cristatum are usually on relatively large A. cristatum chromosomal segments of translocation lines, which are a common type among the progenies from the hybridization of wheat and wild relatives. 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 our study, gene markers and FISH/GISH probes were used to analyze the wheat-A. cristatum 6P translocation line WAT655, and the evidence of the introgression between A. cristatum 6P chromatin and wheat chromosomes was observed (Fig. 3, Fig. 5, Fig. 6, and Fig. S4). Additionally, KASP markers of the three genes were developed to trace A. cristatum P genomic components of wheat-A. cristatum breeding materials (Fig. 7), moreover, 24 breeding materials carrying A. cristatum P genomic components were acquired from 500 wheat-A. cristatum breeding materials. These breeding materials exhibited resistance to stripe rust (CYR32 and CYR33) according to the investigation in the eld, which indicated that three disease resistance-related genes were closely associated with the resistance to stripe rust. These materials exhibited other favorable breeding agronomic traits ( Fig. 7 and Table 3) that can be directly applied in breeding practice. In addition, to further explore the function of three disease resistance-related genes, transgenic work involving coding sequences (CDSs) and full-length sequences of the three disease resistance-related genes is already underway. Therefore, our 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 materials for breeding practice.
Distant hybridization is an e cient and important method for broadening the genetic base of common wheat. Particularly, distant hybridization has contributed much to disease resistance breeding of common wheat. For example, the powdery mildew gene Pm21 of Haynaldiavillosa 6VS has been widely applied in wheat breeding (Chen et al. 2013;Xing et al. 2018); the 1BL•1RS translocation line carried 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 (Zeller 1973;Singh et al. 1990;Friebe et al. 1996;Mago et al. 2002). In this study, the wheat-A. cristatum translocation line WAT655 and the wheat-A. cristatum breeding materials that exhibited resistance to stripe rust can be the new germplasms and contribute to disease resistance breeding of common wheat. Moreover, the molecular markers designed from the sequences of three disease resistance-related genes can improve the e ciency of wheat breeding. Therefore, our study not only provides valuable breeding materials, but also promotes the application of marker-assisted selection of distant hybridization between wheat and A. cristatum in wheat breeding.
Some disease resistance genes have been shown to be distributed in the genome 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) (Cao et al. 2011;Xing et al. 2018); the stripe rust resistance genes Yr5, Yr7, and YrSP were on a gene cluster (Marchal et al. 2018). 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). In a previous report about the wheat-A. cristatum 6P translocation line WAT655, 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. In this study, the DNA of three disease resistance-related genes was used as FISH probes to identify the 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) ( Fig. 6 and Fig. S5), so we speculated that one disease resistance gene cluster existed 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 ). In addition, genetic rearrangements of A. cristatum 6P chromosomes could usually occur at the terminal of chromosomes through analyzing the different wheat-A. cristatum 6P addition lines (Han et al. 2014). In this study, the results of FISH using the DNA of the BAC clones and three disease resistance-related genes as probes indicated that genetic exchange can spontaneously occur between A. cristatum P chromatin and wheat chromosomes ( 3, Fig. 5, and Fig. 6). The mechanism of the introgression may involve chromosomal rearrangements between A. cristatum P genome and wheat genome resulting from their homoeologous relationship; moreover, the introgression observed in this study also occurred in the terminal of the chromosomes (Fig. 3, Fig. 5, Fig. 6, and Fig. S4). Therefore, the homoeologous relationship and chromosomal rearrangements might explain the introgression between A. cristatum P genome and wheat genome.
In summary, gene markers, A. cristatum P genome-speci c markers, and FISH/GISH probes (the DNA of A. cristatum BAC clones, three disease resistance-related genes, and A. cristatum "Z559") were used to analyze wheat-A. cristatum 6P translocation line WAT655. The results revealed that the introgression spontaneously occurred between A. cristatum P genome and wheat genome. Additionally, KASP markers of the genes were developed to detect and acquire a few wheat-A. cristatum breeding materials that can be directly applied in breeding practice. Therefore, our study not only provides new insights into the introgression between wild relatives and wheat, but also provides new wheat-A. cristatum breeding materials for wheat breeding.   Table 3 The agronomic traits of wheat-A. cristatum breeding materials in 2019-2020  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" and A. cristatum "Z559" as the probes. The BAC clone DNA was used as the probe for the rst round of FISH; A. cristatum "Z559" DNA was used as the probe for the second round of GISH on the same slides. FISH and GISH patterns of the common wheat "Fukuho" and the substitution line 4844-12-1 using the DNA of three disease resistance-related genes and A. cristatum "Z559". The DNA of three genes was used as the probes for the rst round of FISH; A. cristatum "Z559" DNA was used as the probe for the second round of GISH on the same slides. FISH and GISH patterns of the translocation line WAT655 using the DNA of three disease resistance-related genes and A. cristatum "Z559" as the probes. The DNA of three genes was used as the probes for the rst round of FISH; the DNA of A. cristatum "Z559" was used as the probes for the second round of GISH on the same slides.