Background

Powdery mildew and leaf rust can reduce grain production by 5–50%, and in severely affected years, this reduction may exceed 62% in susceptible cultivars [1,2,3]. More than 100 powdery mildew and leaf rust resistance genes, respectively, have been discovered in wheat and related species, and 66 for powdery mildew (Pm1–Pm69) and 83 for leaf rust (Lr1–Lr83) resistance genes have been formally named [3,4,5,6,7,8]. However, the emergence of new virulent isolates has led to the loss of resistance to several genes. Therefore, digging novel resistance genes, especially those with broad-spectrum resistance are urgently required for wheat breeding programs. Notably, yield reduction can be mitigated by cultivating resistant cultivars, which is the most cost-effective and sustainable method of disease control.

Using distant hybridization and chromosome engineering to introgress exogenous resistance genes into common wheat effectively creates new germplasm resources for varieties and breeding high-yield, disease-resistant, and high-quality cultivars. Decaploid Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey. [syn. Lophopyron ponticum (Podp.) Á Löve, Agropyron elongatum (Host) P. Beauvois. Elytrigia elongata subsp. pontica (Podp.) Gamisans (2n = 10x = 70, EeEeEbEbExExStStStSt, or JJJJJJJsJsJsJs)], is rich in genes that can improve the resistance of wheat cultivars to diseases [1, 5], including powdery mildew, leaf rust, stem rust, stripe rust, Fusarium head blight, streak mosaic virus, and common root rot [9,10,11,12,13]. Among them, three leaf rust (Lr19, Lr24, and Lr29), five stem rust (Sr24, Sr25, Sr26, Sr43, and SrB), and one powdery mildew resistance gene, Pm51, have been formally documented [1, 5, 11,12,13,14,15]. And Fhb7 has been successfully cloned from Th. ponticum [16].

The efficiency of generating wheat alien chromosome addition/substitution lines can be enhanced by marker assisted selection (MAS). Hence, it is essential to develop molecular markers linked to beneficial exogenous genes. For instance, 13 sequence-characterized amplified region (SCAR) markers, dispersed across chromosomes 1E–7E, can be utilized for identifying diploid Th.elongatum chromosomes within a wheat background [17]. Transcriptome sequencing of the common wheat variety Chinese Spring (CS) and CS-Th.elongatum amphidiploids facilitated the development of 35,193 SNP markers across seven E chromosomes [18]. Additionally, using 169 conserved orthologous set (COS) markers, derived from reported conserved orthologous genes of wheat and rice or Brachypodium, 60 molecular markers exhibited polymorphism on diploid genotypes of Th.elongatum. These COS markers were distributed among chromosomes of all seven homologous groups [19]. By comparing the specific-locus-amplified fragment (SLAF) sequences of CS and EA (a wheat-Th. ponticum translocation line TTh-4DS•4DL), 67 chromosome-specific SLAF markers have been developed for Th. ponticum [20]. All these markers can be used for MAS breeding in wheat.

Genomic in situ hybridization (GISH) can be used to accurately determine the presence of alien chromosomal segments in wheat. Many single-stranded oligonucleotide probes have been developed that can be synthesized by companies at high resolution and low cost. For example, the fluorescence in situ hybridization (FISH) karyotypes of CS and 373 Chinese wheat cultivars constructed using eight oligonucleotide probes did not only clearly distinguished the 21 pairs of wheat chromosomes but also identified 14 types of chromosome structural variations [21]. Alien disomic substitution (DS), addition, and translocation lines of Th. ponticum have been identified using GISH and FISH. For example, a wheat-Th. ponticum DS line DS1JS(1D) is resistant to powdery mildew and stripe rust at the adult stage [10]. Another wheat-Th. ponticum DS line DS1JS(1B), demonstrated resistance to powdery mildew and leaf rust throughout its growth period [4]. Moreover, a novel powdery mildew resistance gene was located on chromosome TTh-1DS•1DL in the wheat-Th. ponticum small alien segment translocation line WTT80. The resistance gene, in WTT80, was derived from a Th. ponticum chromosome segment [1]. Cytological identification of a wheat-Th. ponticum DS line Blue58 showed that the 4D chromosome of common wheat was substituted by chromosome 4Ag [5, 22, 23]. The 4Ag chromosome regions FL0.71–0.80 and FL0.75–0.89 contain blue grain genes [22, 23], while the powdery mildew resistance gene is located within the 3.79–97.12 Mb region of its short arm [5]. Decaploid Th. ponticum contains a large genome and rich genetic resources, highlighting its potential for discovering new resistance genes, which could be valuable for future wheat breeding. Notably, the combination of molecular markers and cytological identification can improve the efficiency of transferring beneficial exogenous genes from the alien chromosomal lines to wheat plants.

In the study reported here, a novel wheat-Th. ponticum DS line was generated using distant hybridization. This line, Bainong Puyan 5814 (PY5814), is immune to powdery mildew and leaf rust. At the same time achieve the following goals: (1) characterize the chromosome composition of PY5814; (2) screen its exogenous chromosome-specific molecular markers; (3) evaluate its disease resistance; (4) analyze whether its disease resistance gene(s) is novel and derived from exogenous chromosomes, (5) and evaluate its agronomic performance. Thus, our findings provide a new germplasm and gene resource for breeding disease-resistant wheat varieties.

Results

Development and cytological identification of PY5814

PY5814 was developed from the BC6F2 progeny of Th. ponticum/Lankaoaizao 8 (LKAZ8)//Keyu 818 (KY818)/3/AK58*7 (Fig. S1). To detect the constitution of the chromosomes in PY5814, high-resolution GISH/FISH karyotypes of PY5814 and AK58 were constructed using a genomic DNA (gDNA) probe of Th. ponticum and oligonucleotide probes (Fig. 1). PY5814 comprised 42 chromosomes, with two from Th. ponticum replacing a pair of wheat chromosomes (Fig. 1A-C). Analysis of pollen mother cell meiotic metaphase I (PMC-MI) showed that the two exogenous chromosomes were paired as a ring bivalent, indicating that the two Th. ponticum chromosomes in PY5814 were homozygous (Fig. 1D). In contrast to the FISH karyotype of common wheat CS [21] and AK58 (Fig. 1E), PY5814 lacked a pair of 5D chromosomes and contained 20 pairs of common wheat chromosomes. Comparing the FISH karyotypes constructed by the probes of ONPM#4–1 and ONPM#7 (Fig. 1A, C), it was found that the sub-telomeric of the exogenous chromosome short arm of PY5814 contained Grass-5S-1 and Grass-5S-2 signals. 5S rDNA was located in the homologous groups 1 and 5 of wheat [24, 25]. Similarly, comparing the FISH karyotypes of PY5814 and Th. ponticum constructed using ONPM#7 probes (Fig. 1C, F), it was observed that the signal and arm ratio of a pair of chromosomes in Th. ponticum was consistent with the exogenous chromosomes contained in PY5814. Therefore, the disomic substitution line of PY5814 was confirmed to be DS5Ag(5D). In addition, PY5814 contained a pair of T1RS•1BL translocation chromosome inherited from AK58, and the majority of its genetic background was derived from AK58.

Fig. 1
figure 1

FISH/GISH karyotype pattern of PY5814 and its parents. A, E FISH/GISH karyotypes of PY5814 (A) and AK58 (E) RTC with ONPM#4–1. B, D GISH analysis of PY5814 RTC (B) and PMC (D), the green signals are for the Th. ponticum gDNA probe labeled by fluorescein-12-dUTP (green). C, F FISH karyotypes of PY5814 (C) and Th. ponticum (F) with ONPM#7.Common wheat chromosomes were blue (DAPI); arrows and asterisks refer to 5Ag chromosome and T1RS•1BL translocation, respectively; bar = 10 μm

Full size image

Molecular identification of PY5814 and its parents

The gDNA of CSN5BT5D, CSN5DT5B, PY5814 and its parents were amplified using 169 wheat COS markers, 423 SSR and 12 EST markers [9, 19, 26], respectively. In summary, 28 primer pairs amplified specific bands in PY5814 and Th. ponticum, with no amplification observed in AK58, KY818, LKAZ8, CSN5BT5D, or CSN5DT5B; and of which 22 were co-dominant markers of 5Ag and 5D (Fig. 2; Table S1). Furthermore, 82 molecular markers specific to the wheat 5D chromosome failed to produce specific bands in Th. ponticum, PY5814, and CSN5DT5B, yet generated specific bands in common wheat AK58, KY818, LKAZ8, and CSN5BT5D (Table S2). Notably, 78 of these specific markers were located within the fifth homologous group.

Fig. 2
figure 2

Molecular marker analysis of PY5814. M: Marker DL2000; 1: Th. ponticum; 2: PY5814; 3: AK58; 4: KY818; 5: LKAZ8; 6: CSN5BT5D; 7: CSN5DT5B; Solid and hollow arrows indicate the specific fragment of chromosome 5Ag and 5D, respectively

Full size image

PY5814 and its parents AK58, KY818, LKAZ8, and decaploid Th. ponticum, underwent genotyping using a wheat 660 K SNP chip. Subsequently, 378,318 high-quality SNP markers with specific physical positions were obtained and used to analyze the chromosomal composition of PY5814 (Table S3). Among them, excluding SNPs undetected in its common wheat parents, AK58, KY818, and LKAZ8, 2,681 were not detected in PY5814. Notably, 1677 (62.55%) of these missing SNPs were on chromosome 5D, which had the highest ratio of all wheat chromosomes (Fig. 3A). The homozygous SNP percentage (66.85%) on chromosome 5D was the lowest, and the heterozygous SNP percentage (11.75%) was the highest. Correspondingly, the minimum percentage of the same SNP loci between PY5814 and AK58 was on the chromosome 5D (54.63%), which also showed the maximum percentage of the same SNP loci between PY5814 and Th. ponticum (Fig. 3B).

Fig. 3
figure 3

Wheat 660 K SNP array analysis of PY5814 and its parents. A Lowest homozygous, highest heterozygous and miss rate on the 5D chromosome. B The difference of SNPs on 5D chromosome between PY5814 and its two parents was the most obvious

Full size image

A total of 22,346 SNPs (5.91%, Table S4) identified as homozygous and pleomorphic between Th. ponticum and three common wheat parents (AK58, KY818, and LKAZ8) were utilized to determine the homologous relationship between the alien chromosomes of PY5814 and the wheat chromosomes. According to the pedigree of PY5814, the alien chromosome-specific SNP loci should be consistent with those of Th. ponticum chromosomes. Of the 628 specific homozygous SNP loci detected for PY5814, which were consistent with Th. ponticum, 223 SNPs (50.80%, Fig. 3B) were mainly distributed on chromosome 5D. Thus, PY5814 deleted the 5D chromosome of common wheat, and the alien chromosome was homologous to the wheat chromosome 5D.

Resistance evaluation of PY5814

Powdery mildew resistance of PY5814 and its parents was identified at the three-leaf stage in a glass greenhouse. The infection types (ITs) scores at the seedling stage showed that AK58, LKAZ8, and CS were grade 4, and KY818 was grade 3. By contrast, PY5814 and Th. ponticum showed immune responses against powdery mildew (IT = 0) (Fig. 4A). The resistance spectrum of PY5814 to 24 single-pustule-derived Powdery mildew isolates was identified at the seedling stage. The results showed that PY5814 was highly resistant or immune to 22 pathotypes, except for E18 and 23-(2) (Table S5). The resistance spectrum of PY5814 differed from that of Pm51 and the other resistance genes. Under field conditions, the resistance of PY5814 and its parents to Powdery mildew at the adult stage was identified over the four growing seasons (Fig. S2). The common wheat parents of PY5814 were highly susceptible with an IT score of 4, and control genotype CS was type 3 (Fig. 4B). Th. ponticum (IT = 0) and PY5814 (IT = 0) also showed immunity at the adult stages.

Fig. 4
figure 4

Resistance evaluation of PY5814 and its parents. A-C Reactions to powdery mildew at seedling stage (A) and adult stage (B) and leaf rust at adult stage (C). 1: Th. ponticum; 2: DS5Ag(5D); 3: AK58; 4: KY18; 5: LKAZ8; 6: CS; 7: MX169

Full size image

The responses of PY5814 seedlings to the six Pt pathotypes were evaluated and showed high resistance to all of them (Table S6). Lr19 from Th. ponticum was similar to the PY5814 resistance spectrum, and further molecular marker identification is required. Under field conditions in Xinxiang City, China, the resistance identification of PY5814 to leaf rust during the adult stage revealed its immunity to this disease. In contrast, AK58, KY818, LKAZ8, and Mingxian 169 (MX169) exhibited high susceptibility to leaf rust, each receiving IT scores of 4, 3, 4, and 4, respectively (Figs. 4C, S2).

Chromosome 5Ag contained novel genes for powdery mildew and leaf rust resistance

We further verified that the resistance gene in PY5814 originated from the Th. ponticum chromosome rather than from interference with the common wheat genetic background. Using MAS and GISH, we screened 120 individual lines from five F5 populations, consisting of 12 lines with the 5Ag chromosome and 12 lines without it in each population (Figs. 5, S3). Resistance to powdery mildew and leaf rust in these lines was identified at the adult stage. The results showed that each line with the 5Ag chromosome was resistant to disease, and each line without it was susceptible to disease. Simultaneously, three DS5Ag(5D) substitution lines were identified from the populations of PY5814/CS, PY5814/WM6, and PY5814/Q03073, all of which lacked 1RS•1BL (Fig. 5). The resistance identification showed that these lines were also immune to powdery mildew and leaf rust at the adult stage. Thus, these results confirmed that the powdery mildew and leaf rust resistance genes of PY5814 were derived from the 5Ag chromosome of Th. ponticum.

Fig. 5
figure 5

FISH/GISH analysis was performed on the F5 population using ONPM#4–1 probes. A–F FISH/GISH analysis of wheat-Th. ponticum DS5Ag(5D) resistant lines, which did not contain 1RS•1BL translocation lines, of PY5814/CS (A, D), PY5814/WM6 (B, E), and PY5814/Q03073(C, F) F5 populations. G–I GISH analysis of F5 susceptible lines of PY5814/CS (G), PY5814/WM6 (H), and PY5814/Q03073 (I). Arrows refer to 5Ag chromosome, bar = 10 μm

Full size image

To ascertain the novelty of powdery mildew and leaf rust resistance genes present in PY5814, we utilized molecular markers (Table S7) linked to Pm51, Lr19, Lr24, and Lr29 to amplify the gDNA of PY5814 and its parental lines via Polymerase chain reaction (PCR). The target band was not amplified in five pairs of coupling markers associated with the target gene. Correspondingly, the target band of 750 bp was successfully amplified with the Lr19 repulsion marker SCS253 (Fig. S4). Thus, it can be concluded that the powdery mildew and leaf rust resistance genes harbored within the 5Ag chromosome of PY5814 are indeed novel genes. These were designated as Pm5Ag and Lr5Ag, respectively.

Evaluation of PY5814 agronomic performance

The phenotypic characteristics and agronomic performance of PY5814 and its backcross parent AK58 were investigated over three consecutive years (Fig. 6A-B; Table 1). In 2019 and 2020, the mean plant height (PH) of PY5814 was significantly higher than that of AK58 (P < 0.05). Additionally, in 2019, the mean thousand-kernel weight (TKW) of PY5814 was significantly higher than that of AK58 (P < 0.05). This result may be due to planting inoculum spreaders in the experimental field and the serious incidence of AK58, leading to a decrease in TKW. The other agronomic indices analyzed were not significant for the three consecutive seasons. These results indicate that the agronomic performance of PY5814 is acceptable and that PY5814 can play a vital role in wheat disease resistance breeding.

Fig. 6
figure 6

Agronomic performance of PY5814 and its recurrent wheat parent AK58. A Plants of PY5814 (right) and AK58 (left). B Seeds of PY5814 (right) and AK58 (left)

Full size image
Table 1 Agronomic index statistics for PY5814 and AK58
Full size table

Discussion

Chromosomal engineering is a notable method for mining new disease-resistant germplasm and broadening the genetic basis of common wheat. Alien chromosome translocation lines have been widely used in wheat breeding because they associate elite traits with valuable disease-resistance genes without genetic linkage drag. For instance, the wheat-rye translocation line T1RS•1BL, which contains the powdery mildew resistance gene Pm8, leaf rust resistance gene Lr26, stripe rust resistance gene Yr9, and stem rust resistance gene Sr31, has been widely used in wheat breeding worldwide [27]. In southern China, 35.8% of newly developed wheat cultivars contain the wheat-Dasypyrum villosum translocation line T6VS•6AL, which is highly resistant to all powdery mildew virulent isolates currently prevalent in China owing to it carrying the resistance gene Pm21 [28].

In prior research, leveraging the resistance genetic reservoirs of decaploid Th. ponticum, or other polyploid species, was a pivotal intermediary step in the development of stable partial amphiploids (2n = 56). Subsequently, these partial amphiploids were subjected to backcrossing with wheat. Each successive generation of seeds underwent identification using GISH to meticulously select the alien substitution or addition lines. This process continued until a small fragment translocation line was spontaneously generated or induced through radiation exposure or utilizing the CS ph1b mutant. For example, He et al. [29] successfully cultivated the partial amphiploids SNTE20 (2n = 56) via the hybridization of E. elongate (2n = 10x = 70) with common wheat. Furthermore, a wheat-Th. ponticum disomic substitution line DS1JS(1B) and two introgression lines were developed through the crossbreeding of SNTE20 with common wheat [4, 11]. In this study, the direct backcrossing and phenotypic selection method, which does not require high-intensity GISH identification in the early stages, is a simple, low-cost, and effective method. Based on this method, a new wheat-Th. ponticum disomic substitution line, DS5Ag(5D), was obtained. The FISH karyotype of PY5814 matched that of AK58, as it was derived from the progeny of KY818//LKAZ8/Th. ponticum, subsequently backcrossed with AK58 seven times, and then self-pollinated twice. Its chromosome composition was 2n = 7A″ + 7B″ + 6D″ + 1″Ag (5Ag). This result is consistent with the results of the COS markers and the 660 K SNP chip.

Wheat SNP chip technology has been employed to characterize alien chromosomes or fragments introduced into the wheat background. Zhou et al. [30] successfully constructed the highest-density linkage maps of Agropyron using a wheat 660 K SNP array. The results showed that the 2P and 4P chromosomes of Agropyron were rearranged and provided evidence of synteny between the P Agropyron and the A, B, and D wheat genomes. Moreover, the homoeologous relationship between the P of 35 wheat-A. cristatum chromosome substitution/addition lines and the wheat genome was characterized. Wheat SNP arrays offer precise analytical capabilities to determine which chromosomes or fragments of common wheat have undergone replacement by alien chromosomes. They also aid in establishing the homologous relationship between the introduced alien chromosomes or fragments and the wheat genome, as well as identifying the breakpoint sites of the chromosome fragments. For example, when comparing the same SNP ratios between M862 and common wheat 7182, the lowest ratio was observed to be associated with the 4D chromosome (24.1%). Correspondingly, compared with Leymus mollis, the highest percentages of the same SNP loci were also 4D chromosomes (36.8%), indicating that M862 was a wheat-L. mollis DS4Ns(4D) [31]. The homoeology of WTA55 was a wheat-Th. ponticum disomic addition line derived from Xiaoyan 7430 and resistant to stripe rust, and the alien chromosome was determined using a wheat 660 K SNP array [32]. Similarly, the homoeology of the PY5814 alien chromosome was determined using the wheat 660 K SNP array in the present study. Similar to other studies, in our study, the results of the wheat SNP array were consistent with those of cytological methods. The wheat SNP array effectively detected elite genes from wild relatives in wheat and may significantly accelerate wheat chromosome engineering during breeding.

Although FISH and GISH can accurately identify alien chromosomes in distant hybridization derived wheat materials, these methods are time-consuming and labor-intensive. Molecular markers can complement these defects and play vital roles in tracking exogenous chromosomes or fragments in wheat. Currently, specific molecular markers have been developed and screened, including SSR [9], SLAF [14], STS [10], SNP [18], CAPS, and indels [11], which can be used to identify alien chromosomes or fragments in different wheat-Th. ponticum derived materials. For example, 20 and 61 SLAF markers have been developed specifically for chromosomes 1E and 7E of diploid Th.elongatum, respectively [33, 34]. Among the 223 SLAF-specific markers developed on the 4AgS chromosome of Th. ponticum, 16 were in the 3.79–97.12 Mb region of the 4AgS chromosome, closely linked to its powdery mildew gene [5]. In the present study, 28 chromosome 5Ag-specific molecular markers were identified; among these, 22 were co-dominant markers of chromosomes 5Ag and 5D. These markers can be used to identify alien chromosome fragments containing resistance genes and enable the determination of their homozygosity or heterozygosity. Moreover, they contribute to the enrichment of markers available for Th. ponticum.

Several wheat-Th. ponticum alien chromosome lines, which are resistant to powdery mildew and leaf rust, have been developed. The powdery mildew resistance gene Pm51 and stripe rust resistance gene Yr69 of the wheat-Th. ponticum introgression line CH7086 were in 2BL and 2AS, respectively [15, 35]. By irradiating the wheat-Th. ponticum disomic substitution line DS4Ag(4D), three small fragment translocation lines—WTT139, WTT146, and WTT323—resistant to powdery mildew were created [5]. However, only few studies have reported that chromosomes from the fifth homologous group of Th. ponticum contain genes that are resistant to both powdery mildew and leaf rust. Recently, two wheat-Th. ponticum distant hybridization materials—ES-7 and 11–20-1—were created, and their alien chromosomes belonged to the fifth homologous group [9, 36]. ES-7 is a disomic substitution line of DS5St(5A) with resistance to stripe rust at the adult stage but is not resistant to powdery mildew and leaf rust [36]. Similarly, 11–20-1, a wheat-Agropyron.elongatum translocation line T5ES∙5DL carries a powdery mildew resistance gene; however, its resistance to leaf rust and stripe rust remains unclear [9]. In the present study, we evaluated the resistance of wheat-Th. ponticum DS line DS5Ag(5D). Notably, DS5Ag(5D) exhibited immunity to powdery mildew during the seedling stage and sustained resistance to virulent powdery mildew and leaf rust isolates prevalent in China during adulthood.

The FISH karyotype of PY5814 was compared with that of CS constructed by Huang et al. [21], showing that PY5814 inherited the T1RS•1BL translocation line of the recurrent parent AK58. In northern China, 27.3% of wheat cultivars carry the T1RS•1BL translocation, although its usage has diminished as the resistance genes Pm8, Yr9, and Lr26 have been overcome by new virulent isolates [27, 28]. This study also showed that AK58 is highly susceptible to powdery mildew and leaf rust, indicating that these pathogens have overcome the resistance genes contained on the 1RS chromosome. Although Xu et al. [37] found that AK58 is moderately resistant to E15 and E21, PY5814 is immune to these two powdery mildew pathotypes. Additionally, AK58 contains a QTL locus, which is speculated to be PM4a [37]. Our study revealed that the resistance spectrum to 24 Bgt isolates of PY5814 was different from Pm4a (Table S6). Specifically, at the seedling stage, PY5814 showed immunity against 22 out of 24 Bgt isolates. Simultaneously, three DS5Ag(5D) lines lacking the 1RS chromosome arm were also found to be resistant to powdery mildew and leaf rust. Analysis using molecular markers, FISH/GISH techniques, and resistance assessments across the five F5 populations consistently indicated that plants containing the 5Ag chromosome exhibited resistance, while those lacking the 5Ag chromosome remained susceptible. Three wheat parents of PY5814 were highly susceptible to powdery mildew and leaf rust. Therefore, the powdery mildew and leaf rust resistance genes contained in PY5814 were derived from the alien chromosome 5Ag. In the field, we also identified the adult-stage stripe rust resistance of PY5814 by inoculating with a mixture of CYR32, CYR33, and CYR34 rust spores at the jointing stage. Although PY5814 exhibited immunity, its parent LKAZ8 was highly susceptible. In contrast, its recurrent parent AK58 showed high resistance to stripe rust (Fig. S5). Therefore, whether the stripe rust resistance of PY5814 is from Th. ponticum or its recurrent parent AK58 is unclear.

Molecular markers linked to resistance genes can be used to track MAS genes. Zhan et al. [15] developed 14 molecular markers linked to Pm51, of which the STS marker BQ246670 had a genetic distance of 1.5 cM from Pm51 and a specific fragment length of approximately 500 bp. SCS253 is a repulsion phase marker linked to Lr19, and its specific fragment is approximately 737 bp. SCS265 is a coupling phase marker linked to Lr19, and its specific fragment is approximately 512 bp. The combination of the two SCAR markers can be used as a co-dominant marker to distinguish between homozygous and heterozygous loci [38]. Three SCAR markers, namely SCS1302, SCS1326, and SCOAB-1, produced specific bands of 607 bp, 613 bp, and 365 bp, respectively, in wheat samples carrying the Lr24 gene. However, these markers failed to amplify the target band in samples lacking this gene [39]. SCAR markers OPY10 and UBC219 are the dominant markers linked to Lr29, producing specific bands of 950 bp and 1000 bp, respectively, in disease-resistant materials carrying this gene [40]. Among these markers, we used six molecular markers linked to the reported resistance genes Pm51, Lr19, Lr24, and Lr29, from Th. ponticum to analyze PY5814. PY5814 had no coupling phase marker bands but contained the Lr19 repulsion phase marker SCS253 band. Pm51, Lr19, Lr24, and Lr29 are not from the fifth homologous group. Further, results from this study showed that the resistance spectrum of Pm5Ag/Lr5Ag is different from that of Pm51, Lr24, and Lr29 (Table S5-6). Combined with the molecular markers, FISH/GISH, resistance identification of the five F5 populations, and multiple pathogen test analyses indicated that PY5814 contained new resistance genes for powdery mildew and leaf rust (designated as Pm5Ag and Lr5Ag). In conclusion, the wheat-Th. ponticum DS line PY5814, exhibiting multiple disease resistance and satisfactory agronomic performance, serves as a promising germplasm resource for wheat chromosome engineering during breeding. However, notably, the DS lines may lack stability in the breeding process due to the unpaired 5Ag and 5D chromosomes. To address this issue, we aim to induce small fragment translocation lines of 5Ag chromosome through radiation and CS ph1b in future research, to enhance the utilization of PY5814 resistance genes in wheat breeding.

Conclusion

In this study, a novel wheat-Th. ponticum disomic substitution line was successfully developed, designated as PY5814. Through ONPM-FISH/GISH and molecular marker analysis, it was identified as DS5Ag(5D). Among the screened 28 chromosome 5Ag specific molecular markers, 22 were co-dominant markers. Further disease resistance identification, ONPM-FISH/GISH, and diagnostic marker analysis revealed the presence of new resistance genes for powdery mildew and leaf rust, designated as Pm5Ag and Lr5Ag, respectively, on the Th. ponticum chromosome 5Ag. These findings offer valuable insights for leveraging Th. ponticum in wheat disease resistance breeding.

Methods

Plant materials

In this study, a diverse array of genetic resources was employed, including the wheat-Th. ponticum DS line PY5814, Th. ponticum, and six wheat cultivars (with a chromosome composition of 2n = 6x = 42, AABBDD): Bainong Aikang 58 (AK58), Keyu 818 (KY818), and Lankaoaizao 8 (LKAZ8), as well as CS, Sumai 3 (SM3), and Mingxian 169 (MX169). Additionally, two nulli-tetrasomic lines, CSN5BT5D and CSN5DT5B, were utilized. PY5814 was developed from a cross involving Th. ponticum/LKAZ8//KY818/3/AK58*7 (Fig. S1). Furthermore, interspecific hybrid F1 seeds, resulting from the pollination of LKAZ8 with the female parent Th. ponticum, was obtained. Individuals carrying 56 chromosomes were selected as the female parent and further hybridized with the cultivar wheat KY818. For improving the general agronomic and quality traits of the alien chromosome lines, AK58 served as the recurrent parent. Powdery mildew-resistant individuals were selected for backcrossing with AK58 for seven generations, followed by self-crossing. Resistant plants from the BC6F2 generation underwent selection for FISH/GISH identification, ultimately leading to the acquisition of the DS line PY5814.

To determine whether the PY5814 resistance gene was derived from the chromosomes of Th. ponticum or was affected by the background wheat chromosomes, five F5 populations were constructed with PY5814 as the female parent and susceptible wheat materials as the male parent: CS, AK58, LKAZ8, Wenmai 6 (WM6), and Q03073.

FISH and GISH analyse

The procedure for wheat seed rooting preparation followed Wang et al. [41] and Jia et al. [32]. The mitotic metaphase of the root tip cell metaphase (RTC-M) chromosomes preparation procedure was performed according to Komuro et al. [42] and Baker et al. [43]. Root tip meristem cells were digested in 2% cellulase Onozuka R-10 and 1% pectolyase Y23 (Yakult Pharmaceutical, Osaka, Japan) enzyme solution at 37 °C for 35 min. Following digestion, the suspensions were dropped onto the slides (8 μL per slide).

The ONPM#4–1 probes and sequences were based on those of Huang et al. [21] and Chen et al. [44] and comprised TAMRA (6-carboxytetramethylrhodamine, red)-modified AFA-3, AFA-4, pAs1-1, pAs1-3, pAs1-4, pAs1-6, and pSc119.2–1, and FAM (6-carboxyfluorescein, green)-modified (GAA)10. The ONPM#7 probes and sequences reported by Chen et al. [44] were slightly modified (removed the BSCL242-1) and used to compare the 5Ag chromosomes of PY5814 and Th. ponticum. The modified ONPM#7 probes comprised TAMRA-modified Grass-5S-1 and Grass-5S-2, FAM-modified BSCL-135–1 and BSCL-135–2, along with eight oligonucleotide probes of ONPM#4–1. These oligonucleotide probes were synthesized and modified by TSINGKE Biotechnology Co., Ltd. (Nanjing, Jiangsu, China).

The RTC-M and PMC-MI chromosomes were utilized for GISH analysis. Chromosomes of the meiotic MI of PMC were prepared following Chen et al. [45]. Total gDNA from Th. ponticum was labeled with fluorescein-12-dUTP (Roche, Darmstadt, Germany) using nick translation and served as a probe. Non-labeled AK58 gDNA was employed as a block in the process. The probe/block ratio was 1:50–70. Details of the probe dosage and FISH/GISH procedure for each slide are in Huang et al. [21] and Chen et al. [44]. The chromosomes with high-definition hybridization signals were examined using a BX51 fluorescence microscope (Olympus, Tokyo, Japan). Images were captured using a Coolcube 1 CCD camera and analyzed using the Isis Karyotype Analysis Software System (Metasystems, Altlussheim, Germany).

Molecular marker analysis

gDNA was extracted from the young leaves of each wheat plant by using the cetyltrimethylammonium bromide (CTAB) method [46]. A total of 169 COS markers from wheat seven homologous groups, along with 423 SSR and 12 EST markers of wheat homologous group 5 [9, 19, 26] were selected to screen specific molecular markers of PY5814. Subsequently, we investigated whether PY5814 contains the powdery mildew resistance gene Pm51 and the leaf rust resistance genes Lr19, Lr24, and Lr29, which have been reported in Th. ponticum. For this purpose, we selected 1 [15], 2 [38], 2 [39], and 1 [40] linkage markers (Table S7) to amplify gDNA from PY5814 and its parents. All primers were synthesized by TSINGKE Biotechnology Co., Ltd.

Each PCR comprised a total volume of 10 μL consisting of 5 μL of 2 × Es Taq Master Mix (Dye, CoWin Biosciences, Beijing, China), 1 μL gDNA template (20–50 ng/μL), 0.2 μL forward primer (10 μmol/L), 0.2 μL reverse primer (10 μmol/L), and 3.6 μL ddH2O. The amplification program consisted of one cycle at 94 °C for 3 min to pre-denaturation, followed by 36 cycles of 30 s at 94 °C, 30 s at 52–65 °C depending on specific primers, 1 min 10 s at 72 °C, and finally, a cycle of 72 °C for 10 min. PCR products were separated using 8% polyacrylamide gel electrophoresis and then silver stained, as described in the literature [47].

Wheat 660 K SNP array analysis

To investigate the PY5814 chromosome characterization and the homoeology of its alien chromosome, the gDNA of the tested materials was used, ensuring that the detection concentration and purity met the standard criteria. The analysis was carried out on Axiom® wheat 660 K SNP genotyping arrays by China Golden Marker Biotechnology Co., Ltd. (Beijing, China). The threshold dish quality control (DQC) ≥ 0.82 && call rate (CR) ≥ 95 were set to obtain the original genotype data. Subsequently, high-quality genotype data were obtained by setting the threshold: BestandRecommended = 1 && BestProbeset = 1 && ConversionType = PolyHighResolution && call rate ≥ 97. SNPs present in AK58, KY818, and LKAZ8, but absent from PY5814, were screened to analyze chromosome deletion events. Theoretically, the chromosome of PY5814, with the highest miss rate and the lowest proportion of the same SNP between PY5814 and AK58, was considered to have experienced substitution events. Conversely, the chromosome of Th. ponticum, which had the highest proportion of the same SNP as PY5814, was regarded as homologous to the alien PY5814 chromosome.

Disease resistance assessment

CS, PY5814, and its parent materials were planted in a glass greenhouse in a 5 × 10 plastic cave dish with 10 plants per hole, with three replicates, and a mixture of Bgt was inoculated at the 2–3 leaf stage to identify the resistance to powdery mildew. In 2018–2022, plants were grown under field environmental conditions with a row length of 1.50 cm and row spacing of 0.24 cm. During the filling stage, the resistance of PY5814 and its parental materials to powdery mildew was evaluated. For this assessment, seeds of common wheat cultivars SM3 and MX169 were uniformly mixed, used as inoculum spreaders, and planted perpendicular to the experimental materials. Leaf rust was simultaneously identified in the field environment. At the jointing stage, the inoculum spreader plants were inoculated with a mixture of PHT and THT leaf rust spores using an atomizer. These pathogen types of leaf rust are prevalent in China. ITs were evaluated when the inoculum spreader plants exhibited the highest susceptibility.

The ITs of powdery mildew and leaf rust were scored following Li et al. [9] and Zhao et al. [48]: 0–2 represents resistance, while 3–4 indicates susceptibility. Seedlings of PY5814 were also stressed by using 24 single-colony cultures of Bgt pathotypes in the Prof. Yilin Zhou Laboratory, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Beijing, China). The names of the 24 Bgt pathotypes as well as infection methods and resulting ITs corresponded to those described by Yang et al. [1]. The reactions of PY5814 seedlings to the six Pt pathotypes currently prevalent in China were identified at the College of Plant Protection, Hebei Agriculture University (Baoding, Hebei, China). Infection methods were similar to those of Zhang et al. [49].

Evaluation of agronomic performance

During three consecutive years, from 2019 to 2021, PY5814 and its recurrent parent AK58 were cultivated in Xinxiang (113.5°E, 35.2°N, Henan, China). Each row measured 1.50 m in length, spaced 0.24 m apart, and sowed with 30 seeds. Three biological replicates were planted annually. At the physiological maturity stage, six whole plants of each genotype were randomly selected from the middle of the row to investigate agronomic traits, including plant height (PH), main spike length (SL), kernels number per main spike (KNPS), spikelets number per spike (SNPS), sterile spikelets number (SSN), effective tiller number per plant (ETN), and thousand-kernel weight (TKW). Significant differences between PY5814 and AK58 for all traits were compared using t-tests in SAS v9.3 (SAS Institute Inc., Cary, NC, USA).