Development of the novel wheat-tetraploid Thinopyrum elongatum 4EL small fragment translocation lines with the stripe rust resistance gene Yr4EL

doi:10.21203/rs.3.rs-4721341/v1

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Development of the novel wheat-tetraploid Thinopyrum elongatum 4EL small fragment translocation lines with the stripe rust resistance gene Yr4EL

https://doi.org/10.21203/rs.3.rs-4721341/v1

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Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a devastating wheat disease worldwide. Deployment of disease resistance (R) genes in cultivars is the most effective way to control the disease. The all-stage stripe rust R gene Yr4EL from tetraploid Thinopyrum elongatum was previously introduced into common wheat through the 4D (4E) substitution and T4DS·4EL translocation lines. To further map and utilize Yr4EL, Chinese Spring (CS) mutant pairing homoeologous gene ph1b was used to introduce recombination between chromosomes of 4EL and common wheat by crossing program. Two homozygous small fragment translocation lines T4DS·4DL-4EL and T5AS·5AL-4EL with Yr4EL resistance were selected using molecular markers and confirmed by genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), and 660K SNP array analyses. The Yr4EL is located about 35 Mb (577.76~612.97 Mb) from the terminal of the chromosome arm 4EL based on the diploid Th. elongatumreference genome. In addition, two competitive allele-specific PCR (KASP) markers were developed and showed co-segregation with Yr4EL, which can facilitate molecular marker-assisted selection in wheat breeding programs. T4DS·4DL-4EL lines were crossed and backcrossed with wheat cultivars SM482 and CM42 to obtain pre-breeding lines with stripe rust resistance and good agronomic traits, showing great potential for wheat breeding. These results will provide new germplasm for wheat stripe rust resistance breeding, as well as provide solid foundation for Yr4EL fine mapping and cloning.

chromosomal translocation

molecular markers

stripe rust

tetraploid Thinopyrum elongatum

Two novel homozygous small fragment translocation lines with Yr4EL resistance (T4DS·4DL-4EL and T5AS·5AL-4EL) were identified and further localized Yr4EL to the 35 Mb (577.76~612.97 Mb) region of the terminal fragment of chr4EL.

Wheat (Triticum aestivum L.) is one of the most important crops, providing energy and high-quality plant protein for more than 1.6 billion world population (Ma et al. 2022). Epidemics and widespread outbreaks of stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), result in significant reductions in wheat yields and threaten to global food security (Herrera-Foessel et al. 2006; He et al. 2023). Cultivating resistant varieties is an effective and sustainable strategy for reducing crop losses caused by disease (Savadi et al. 2018). Currently, 86 Yr genes (Yr1-Yr86) have been formally cataloged, as well as more than 100 tentatively named genes and 300 QTLs controlling stripe rust have been reported (Feng et al. 2023; Zhu et al. 2023). However, the evolution of new Pst virulent pathogen race has resulted in the loss function of disease R genes that widely used in wheat varieties, such as the current prevalence of the high virulent races CYR32, CYR33, and CYR34, which have overcome Yr3, Yr4, Yr10 and Yr24/Yr26/YrCH42 resistance, resulting in replacements of wheat cultivars (Han et al. 2010; Liu et al. 2024; Ye et al. 2019). Therefore, there is an urgent need to identify and deploy more novel R genes in wheat cultivars.

Thinopyrum elongatum (Host) D. R. Dewey is a crucial perennial wild relative with diploid, tetraploid, and decaploid, which could be used for wheat genetic improvement (Jia et al. 2022). The Th. elongatum chromosome (chr) 2E carries strip rust and powdery mildew R genes Yr2E and Pm2E (Liu 2007). The Th. ponticum chr6E contains Sr26 and Sr61 which have been introduced into wheat to improve stem rust resistance (Ma et al. 1999; Mago et al. 2005). The chr6E contains Sr26 and Sr61 were derived from Th. ponticum and introduced into wheat to improve the stem rust resistance (Mago et al. 2005; Zhang et al. 2021). Rust R genes Lr19, Lr29, Sr25, Sr43, and Fusarium head blight (FHB) R gene Fhb7 were identified from chr7E of Th. ponticum and have improved wheat disease resistance (Friebe et al. 1996; Konkin et al. 2022; McIntosh et al. 1977; Wang et al. 2020). Several strip rust and powdery mildew R genes have been identified from Th. elongatum and transferred into wheat for resistance breeding, such as Yr69, YrTp1, Yrxy1, Yrx9366, Yrxy54-1, YrXY9323, YrCH7056, Pm51, and PmSn0224 (Gong et al. 2022; Hou et al. 2016; Liu 2022; Zhou et al. 2011).

Most of these alien R genes have not been widely deployed in wheat varieties yet, due to the linkage drag and inhibition of chromosome recombination (Gong et al. 2024; Mcintosh et al. 2018). The creation of wheat-Th. elongatum small fragment translocation lines can break the linkage between unfavorable genes and R genes, thereby enhancing the utilization of R genes in wheat breeding (Dilbirligi et al. 2004). For example, the 7DS·7DL-7el₂L and 7BS·7BL-7EL translocation lines decreased the negative impact of unfavorable agronomic traits from Th. elongatum, which carried Fhb7 and Fhb7^The2, respectively (Guo et al. 2015; Wang et al. 2020; Zhang et al. 2022). Yang et al. (2021) developed translocation line T5DS·5DL-Th with high resistance to stem rust race Ug99, facilitated its effective utilization in wheat breeding. The TTh-1DS·1DL translocation line showed excellent powdery mildew resistance and agronomic performance (Yang et al. 2022). Thus, the development of a small fragment that carries only the favorable traits from Th. elongatum is an effective method for wheat improvement.

Pairing and recombination of alien chromosomes with its homoeologous chromosomes of common wheat is often prevented by Ph genes, such as Ph1, Ph2, and ph-KL (Sears 1952, 1976), which severely limit the development of wheat-alien translocation lines. The Ph1 mutant gene ph1b, produced due to the deletion of the 5BL chromosome segment in the CS background, was found to be effective in inducing homoeologous pairing and recombination between alien chromosomes with wheat chromosomes (Fan et al. 2019; Fan et al; 2023; Mello-Sampayo et al. 1971; Sears 1976; Sears 1977). Hence, the ph1b mutant has been utilized as a pivotal tool for inducing wheat-alien chromosome recombination, facilitating the production of desirable small fragment translocation lines. Currently, many wheat-relative translocation lines have been generated by ph1b mutants, such as the translocation line 7BS·7BL-7EL with the FHB resistance gene Fhb7^The2 and T5DS·5DL-5M^gL-5DL with the stem rust resistance gene Sr53, which have also been successfully deployed in wheat varieties (Copete-Parada et al. 2021; Lukaszewski et al. 2000; Liu et al. 2011; Li et al. 2021; Ma et al. 2022; Taketa et al. 2005; Zhang et al. 2022).

Previously, we reported that the tetraploid Th. elongatum carried a novel stripe rust R gene, named Yr4EL (Gong et al. 2024). In this study, to further utilize and map Yr4EL, we (1) crossed the wheat-tetraploid Th. elongatum 4E (4D) substitution lines and CS mutant CSph1b, and characterized the genome constitution of the 4EL small fragment translocation lines; (2) evaluated the agronomic traits and the stripe rust resistance of the 4EL small fragment translocation lines; (3) mapped the Yr4EL and developed the KASP markers which for marker-assisted selection, and deployed Yr4EL into different wheat backgrounds. The novel developed wheat-tetraploid Th. elongatum 4EL small fragment translocation lines carrying Yr4EL will be a valuable germplasm for wheat stripe rust resistance breeding.

Plant materials

Triticum durum–tetraploid Th. elongatum partial amphidiploid line 8801 (2n = 6x = 42, AABBEE), which is highly resistant to stripe rust was kindly provided by Dr. George Fedak (Eastern Cereal and Oilseed Research Centre, Ottawa, Canada) (Gong et al. 2024). The wheat-tetraploid Th. elongatum 4E (4D) substitution line K16-730-3 was used as Yr4EL resistance donor (Gong et al. 2022). The susceptible CSph1b mutant was used as ph1b gene donor to induce the small fragment translocations on chr4E by crossing with K16-730-3. Tetraploid Th. elongatum (2n = 4x = 28, EEEE; PI 531750) and common wheat CS were used in GISH analysis. Common wheat Avocet S served as the stripe rust-susceptible control. The wheat-tetraploid Th. elongatum translocation line T4ES·4EL-5AL was used for Yr4EL localization, which is susceptible to stripe rust (Gong et al. 2024). Susceptible wheat varieties Chuanmai42 (CM42) and Shumai482 (SM482) were used as recurrent parents for development of pre-breeding lines. All these wheat seeds mentioned above were stored at the Triticeae Research Institute, Sichuan Agricultural University, China.

Development of translocation lines

The F₂ (K16-730-3/CSph1b) and BC₁F₁ (K16-730-3/CSph1b//CSph1b) populations were used to develop and select 4EL small fragment translocation lines. The chromosome 4E-specific markers TTE4E-86 (Gong et al. 2022) were used to detect plants that carried chr4E in F₂ and BC₁F₁. Further, individuals carrying chr4E were genotyped for Ph1 and ph1b genotypes using molecular markers Xwgc2049 and Xwgc2105 developed by Gyawali et al. (2019). The SSR markers TTE4E-2701 (4ES), TTE4E-3950 (4EL), TTE4E-4308 (4EL), TTE4E-7849 (4EL), TTE4E-7892 (4EL), and TTE4E-8060 (4EL) (Gong et al. 2024) were used to initially detect chromosome 4E recombinants in the selfed progenies of the ph1b homozygous plants.

GISH, and FISH analyses

The initially detected chromosome 4E recombinants need to be confirmed with GISH and FISH analyses as previously described (Gong et al. 2023; Han et al. 2006; Li et al. 2019a). Tetraploid Th. elongatum genomic DNA was labeled with dUTP-ATO-550 (Jena Bioscience, Jena, Germany) as the GISH probe, and CS whole genomic DNA was used as the blocking DNA (1:150). The 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) was used for chromosome counterstaining. Chromosomes were observed under a fluorescence microscope BX63 (Olympus, Tokyo, Japan) equipped with the DP-80 camera. The FISH probes Oligo-pSc119.2-1 and Oligo-pTa535-1 (Tang et al. 2014) were used to determine the chromosome composition of the translocation lines.

Wheat 660K SNP array analysis

To confirm the translocation events between the alien chromosome and the common wheat chromosome, the gDNAs of two small fragment translocation lines were genotyped by wheat 660K SNP array at China Golden Marker Biotechnology Co., Ltd. (Beijing, China). The wheat 660K SNP array contains 660009 SNPs, which are distributed across 42 wheat chromosomes. We excluded SNPs with unknown physical locations, and all remaining SNPs were used to analyze the translocation events (reference IWGSC RefSeq v1.0). In the translocation region, most of the SNPs could be detected as deletions, therefore, we calculated the deletion rate of different regions on the translocation chromosome in a 10-Mb sliding window in 1-Mb steps to check for breakpoints. Regions with significantly higher deletion rates were considered to affected by deletion or translocation events.

Assessment of stripe rust resistance

The phenotypes of the wheat plants to Pst race CYR34 at the seedling stage were examined in an artificial climate chamber as previously described (Li et al. 2019a). The infection types (ITs) were determined using the following 0–9 scale: 0 and 1: immune; 2–6: resistant; 7–8: susceptible; 9: highly susceptible (Line and Qayoum 1992).

The phenotypes of the wheat plants to stripe rust at the adult stage were evaluated in the Wenjiang (WJ; 30°36′N, 103°41′E) field using a mixture of Pst races (CYR32, CYR33, CYR34, Sull-4, Sull-5, Sull-7, and G22-14), as previously described (Carter et al. 2009; Gong et al. 2023; Line and Qayoum 1992; Li et al. 2019a; Tang et al. 2014). The IT scale that was used to assess the adult plants was the same as the standard for evaluating the seedlings.

Evaluation of agronomic performance

Wheat plants were evaluated for tiller number, spikelet per spike, plant height, spike length, grains per spike, and 1,000-grain weight at the Wenjiang Experimental Field. Three replicates were prepared during the 2023–2024 growing season. For each replicate, 15 seeds per line were sown uniformly in 1.5 m rows with 0.3 m between rows. The agronomic traits of 15 plants in the middle of each row were recorded and analyzed using the SPSS Statistics 24.0 software.

Mapping and developing co-segregation markers of Yr4EL

To determine the physical interval size of Yr4EL, 21 chr4EL-specific SSR markers (from TTE4E-4308 toTTE4E-7892) were used to genotype T4DS·4DL-4EL, T5AS·5AL-4EL, and T4ES·4EL-5AL (Gong et al. 2024). The PCR amplification programs were performed as previously described (Gong et al. 2022).

KASP markers specific to chr4EL were developed for molecular marker-assisted selection (MAS). The single nucleotide polymorphism (SNP) between diploid Th. elongatum 4E genome (ASM1179987v1) and CS reference genome (IWGSC RefSeq v1.0) was detected by DNAMAN (version 6), and primer design was using DNA Sequence Online Processing Tool (https://crm.vazyme.com/cetool/tmcal.html). PCR amplification was performed in a 10 µL reaction volume consisting of 5 µL of HiGeno 2×Probe Mix B (Beijing JasonGen Biological Technology Co., Ltd.), 1.4 µL of primer mix, 2.6 µL of ddH₂O, and 1.0 µL of template DNA (200 ng/µL). The PCR program was as follows: 94°C for 5min; 10 cycles of 94 ℃ for 20 s and 63 ~ 57 ℃ (each cycle decreased 0.6 ℃ and each temperature was kept for 1 min); then 30 cycles of 94 ℃ for 20 s and 57 ℃ for 1 min. KASP assay was performed using the Bio-Rad CFX real-time PCR system and Bio-Rad CFX Manager 3.1 software (Bio-Rad, California, USA).

Pre-breeding material production

The double top-cross and two‑phase selection strategies were employed to develop pre-breeding materials (Hao et al. 2019). To reduce the unfavorable agronomic traits from CS genetic background, we obtained F₁ by crossing T4DS·4DL-4EL with the elite wheat variety CM42, and then the F₁ was crossed with SM482 and selfed to obtain F₂. Pre-breeding materials with yellow rust resistance and desirable agronomic traits were selected from F₂ populations by MAS and GISH.

Development and molecular cytological identification of translocation lines

We crossed wheat-tetraploid Th. elongatum 4E (4D) substitution lines with CSph1b and obtained 178 BC₁F₁ and 37 F₂ individuals. These individuals were genotyped by chr4E-specific molecular marker TTE4E-86, as well as Ph1 and ph1b molecular markers Xwgc2049 and Xwgc2105. A total of nine (eight from BC₁F₁ and one from F₂) ph1b homozygous plants with chr4E were selected (Supplementary Fig. S1, 2). These self-crossed progenies of nine plants were genotyped by six 4EL-specific markers TTE4E-2701, TTE4E-3950, TTE4E-4308, TTE4E-7849, TTE4E-7892, and TTE4E-8060 (Gong et al. 2024), which were located at the short arm, long arm proximal centromeres, and long arm terminal regions. The result showed that 84 plants were identified as probably containing chr4E recombinant or deletion events (Fig. 1).

GISH analysis of these 84 plants showed that 48 plants were chr4E deletion lines, and 36 plants were translocation lines. Specifically, K24-136-32 and K24-139-54 carried 42 chromosomes, including two translocations with small chromosome E segments (Table 1; Fig. 2a, c). Based on the FISH karyotypes of CS and tetraploid Th. elongatum (Li et al. 2018; Tang et al. 2014), the Oligo-pSc119.2-1 (green) and Oligo-pTa535-1 (red) signals confirmed that K24-136-32 and K24-139-54 were wheat-tetraploid Th. elongatum T4DS·4DL-4EL and T5AS·5AL-4EL translocation lines, respectively (Fig. 2b, d).

Table 1

Chromosome composition and genetic genealogy of T4DS·4DL-4EL and T5AS·5AL-4EL.
No.	Line	Chromosome composition	Pedigree
K24-136-32	T4DS·4DL-4EL	2n = 42 = 40W + II 4DS·4DL-4EL	K16-730-3/CSph1b//CSph1b BC₁F₂///CM42 F₃
K24-139-54	T5AS·5AL-4EL	2n = 42 = 40W + II 5AS·5AL-4EL	K16-730-3/CSph1b F₄//SM482 F₃
Note: W, Common wheat chromosome; K16-730-3, 4E (4D) substitution line (8801/SM482//SM51 F₅); 8801, obtained by crossed T. durum and tetraploid Th. elongatum.

Genotyping of the translocation lines using wheat 660K SNP array

The T4DS·4DL-4EL and T5AS·5AL-4EL were genotyped by wheat 660K SNP array to identify the breakpoints in translocated chromosomes. In total, 335064 SNPs with definite physical locations were identified in the T4DS·4DL-4EL and T5AS·5AL-4EL translocation lines. Then the SNP deletion rate was calculated using a sliding window of 10-Mb with a step size of 1-Mb, which showed that the deletion rate increased significantly from 492 Mb of chr4DL in the T4DS·4DL-4EL, and there were 15 deletion SNPs in the 492 ~ 518.3 Mb region, accounting for 51.72% of deletion SNPs in chr4D (Fig. 3a, b). Thus, the breakpoint of chr4D in T4DS·4DL-4EL is around 492 Mb, and about 26.3 Mb is missing at the terminal region of chr4DL. The deletion SNPs in the T5AS·5AL-4EL translocation line were mainly concentrated in the 669.2 ~ 713.4 Mb region of chr5AL, accounting for 84.09% deletion SNPs in chr5A (Fig. 3c, d). The breakpoint of chr5A in T5AS·5AL-4EL is around 669.2 Mb, and a chromosome fragment of approximately 44.2 Mb is missing from the terminal region of chr5AL. This was consistent with the GISH and FISH results (Fig. 2).

Evaluation of stripe rust resistance and agronomic traits

We phenotyped these translocation lines and their parental lines to Pst race CYR34 at the seedling stage. The result showed that the common wheat parental lines CSph1b, SM51, SM482, and CM42 were fully susceptible to Pst, similar to the control line Avocet S (IT = 8–9). The 4E (4D) substitution lines, T4DS·4DL-4EL, and T5AS·5AL-4EL showed high resistance to Pst (IT = 1) (Fig. 4a). This result was consistent with an adult resistance to a mixture of Pst isolates (including CYR32, CYR33, CYR34, Sull-4, Sull-5, Sull-7, and G22-14) (Fig. 4b). However, a previous selected translocation line T4ES·4EL-5AL (Gong et al. 2024), which could compensate to T4DS·4DL-4EL and T5AS·5AL-4EL, showed high susceptibility (IT = 7). Thus, T4DS·4DL-4EL and T5AS·5AL-4EL inherited the Yr4EL resistance of the 4E (4D) substitution lines.

Evaluation of agronomic traits in the field showed that T4DS·4DL-4EL exhibits a significantly increased tiller number compared to CM42 (Fig. 4c, d). Although T4DS·4DL-4EL had lower numbers of spikelets per spike, it had higher grain numbers per spike, and more 1,000-grain weight (Fig. 4c, d). T5AS·5AL-4EL exhibited higher tiller and more spikelets per spike (Fig. 4c, d). Altogether, T4DS·4DL-4EL and T5AS·5AL-4EL exhibited improved agronomic traits than their parental lines, however, the plant height needs to reduce for future breeding.

Mapping of Yr4EL and developing co-segregation markers

To further determine the size of translocation fragments and the physical interval of Yr4EL, we genotyped these translocation lines by 21 chromosome 4E-specific SSR markers (Gong et al. 2024). The results showed that 18 dominant SSR markers (from TTE4E-6675 to TTE4E-7892) at the terminal of chr4EL could amplify the positive the target bands in the resistant lines T4DS·4DL-4EL and T5AS·5AL-4EL, while 17 of which (from TTE4E-6916 to TTE4E-7892) showed negative amplification in the susceptible line T4ES·4EL-5AL (Fig. 5a, d). Based on diploid Th. elongatum reference genome (McIntosh et al. 1977), both resistant lines T4DS·4DL-4EL and T5AS·5AL-4EL carried about 45 Mb of chromosome 4EL terminal segments (568.12 ~ 612.97Mb), while the susceptible line T4ES·4EL-5AL exhibited a deletion of about 35 Mb (577.76 ~ 612.97 Mb) at the terminal region of chromosome 4EL. This result suggests that Yr4EL is located in a physical interval of about 35 Mb (577.76 ~ 612.97 Mb) at the terminal of chr4EL (Fig. 4a, b; Fig. 5d).

To facilitate MAS for Yr4EL resistance in wheat breeding, we developed two co-dominant KASP markers (KT4E-27 and KT4E-31) that were able to distinguish chromosome 4D and 4E fragments. After genotyping by these two KASP markers, the CS, CSph1b, SM482, SM51, and CM42 showed a common wheat SNP homozygous genotype (Allele 1/Allele 1, in orange dots); T4DS·4DL-4EL translocation lines, the 4E (4D) substitution lines and the tetraploid Th. elongatum showed the alien fragment SNP homozygous genotype (Allele 2/Allele 2, in blue squares); F₁ and heterozygous lines showed a heterozygous genotype (Allele 1/Allele 2, in green triangle) (Fig. 5b-d; Supplementary Table S1). This result supports that KT4E-27 and KT4E-31 are co-dominant markers for the T4DS·4DL-4EL translocations, which can be effectively utilized in MAS.

The potential value of pre-breeding material

We attempted to use the double top-cross two-stage selection strategy (Hao et al. 2019) to consecutively cross T4DS·4DL-4EL with the susceptible varieties SM482 and CM42. The KASP marker KT4E-31 was used to genotype these progenies. The genotyping and phenotyping results showed that all plants carrying 4EL small fragments were highly resistant to Pst, and plants without this alien translocation chromosome were susceptible to Pst (Fig. 6a-c). Further GISH analysis revealed that all resistant plants carried the 4EL small fragment, whereas susceptible plants did not have GISH signals, which was consistent with the results of KASP marker genotyping (Fig. 6a, d, f, h). Moreover, these individuals (T4DS·4DL-4EL/CM42//SM482 F₂) that carried Yr4EL had a lower plant height compared to the translocation line T4DS·4DL-4EL (Fig. 6e, g). In conclusion, these pre-breeding materials provided valuable potential for breeding new wheat varieties.

Mutant ph1b gene can effectively induce wheat-relative translocation lines

Development of wheat-relative disease-resistant translocation lines as an important method for genetic improvement of wheat. However, homoeologous chromosome recombinations between common wheat and wheat relatives are frequently suppressed, which causes trouble for introducing alien genes into common wheat (Lin et al. 2023). The mutant ph1b gene can significantly increase the recombination of homoeologous chromosomes and effectively induce the production of wheat-relative small translocation lines (Sears 1977). For example, using this method, Wan et al. (2020) obtained 182 recombinant plants of wheat chr2B with T. timopheevii chr2G, and successfully narrowed down the powdery mildew R gene Pm6 to an interval of 0.9 Mb. Li et al. (2019b) successfully localized Sr52 to 6V#3L bin FL 0.92-1.00 using six wheat-D. villosum 6V#3L small fragment translocation lines. Men et al. (2022) produced 65 CS-Ae. biuncialis recombinants, the Pm2Mb was mapped to the FL 0.49–0.66 interval using these recombinants. Wang et al. (2020) obtained 200 7E1-7E2 recombinants by ph1b gene-induced recombination of FHB-susceptible 7E1 (7D) substitution line with FHB-resistant 7E2 (7D) substitution line, localized the Fhb7 in the 1.2 Mb region of chr7EL, and cloned it successfully.

In this study, we obtained T4DS·4DL-4EL and T5AS·5AL-4EL carrying Yr4EL by ph1b gene-induced recombination of chromosome 4E with common wheat chromosomes (Fig. 2). Both T4DS·4DL-4EL and T5AS·5AL-4EL carry about 45 Mb of the terminal fragment of chromosome 4EL, which is still large for the fine location of Yr4EL. In the future, we will utilize the ph1b gene to induce recombination of chr4DS·4DL-4EL with chr4D of common wheat to obtain the smaller fragment of chr4EL translocation line with stripe rust resistance for fine mapping Yr4EL. In addition, we will use the T4DS·4DL-4EL to construct mutants that are susceptible to stripe rust, for further cloning Yr4EL by long-read sequencing of whole genome combined mutant RNA sequencing analysis (Li et al. 2024).

Yr4EL enhances the diversity of the stripe rust resistance gene pool

Wild wheat relatives harbor numerous valuable genes, such as R genes to stripe rust, powdery mildew, and FHB (Jia et al. 2022). Exploiting excellent genes from wheat relatives can effectively enrich the wheat breeding gene pool (Lin et al. 2023). Several R genes from wheat relatives have been successfully introduced into common wheat and deployed in wheat breeding, such as Fhb7, Sr24, Sr25, Sr26, Sr43, Sr61, Lr19, and Lr29 from Th. elongatum (Friebe et al. 1996; Konkin et al. 2022; McIntosh et al. 1977; Mago et al. 2005; Wang et al. 2020; Zhang et al. 2021). In addition, Yr50 identified from Th. intermedium was translocated at chr4BL with flanking markers Xbarc1096 at 8.0 cM proximal and Xwmc47 at 7.2 cM distal (Liu et al. 2013). The Yr69 from Th. ponticum is highly resistant to all Pst races prevalent in China and was localized to bin 2AS-0.78-1.00 (Yang et al. 2022). The Yr83 from S. cereale was mapped to the 6RL bin (FL) 0.73-1.00 by genotyping and phenotyping of 10 chr6R deletion lines and five wheat-rye chr6R translocation lines (Li et al. 2020).

In this study, Yr4EL was mapped to the about 35 Mb (577.76-612.97 Mb) region at the terminal of chr4EL and showed all-stage wide-spectrum resistance to multiple Pst races (Sull-4, Sull-5, Sull-7, G22-14, CYR32, CYR33, and CYR34) (Fig. 4). Currently, seven of the officially named genes (Yr22, Yr28, Yr46, Yr50, Yr51, Yr62, and Yr68) are from group 4 chromosomes, and only Yr22, Yr28, Yr50, and Yr51 are all stage R genes (Liu et al. 2024). Yr22 was localized on chr4D of the spring wheat cultivar Lee, but the gene is not designated to a specific chromosomal location (Chen et al. 1995). Yr28 was localized on chr4DS of Ae. tauschii (Singh et al. 2000; Zhang et al. 2019). Yr51 from the wheat landrace AUS27858 was mapped to chr4AL proximal centromere (Randhawa et al. 2014). Moreover, YrE and Yr2E from Th. elongatum were located on chromosomes 3E and 2E, respectively (Liu 2007; Ma et al. 1996). In conclusion, Yr4EL is located in a novel genetic region and provides a new genetic resource for the wheat R gene pool.

Translocation lines with Yr4EL are valuable for wheat disease resistance breeding

Favored alien R genes can be introduced into common wheat in the form of addition, substitution, and translocation lines, but large segments of alien chromosomes usually adversely affect wheat agronomic traits, making it difficult for wheat breeding (Jia et al. 2022). At present, some wheat-relative translocation lines have been successfully used in wheat breeding programs. For example, the wheat-rye 1RS·1BL translocation line carrying Lr26, Yr9, and Pm8 and the wheat-H. villosa 6VS·6AL translocation line carrying Pm21 (Wang et al. 2017; Xing et al. 2021). However, it is noteworthy that the 1RS chromosome arm harbors the Sec-1 locus, which reduces bread quality. Breeders subsequently developed a wheat-rye 1RS small fragment translocation line, which effectively broke the linkage between the Sec-1 locus and the disease R gene (Fu et al. 2010; Wang et al. 2023). Moreover, the wheat-H. villosa 6VS small fragment translocation lines exhibited greater potential for developing high-yielding wheat varieties compared to the 6VS·6AL translocation lines (Chen et al. 2013). Therefore, the small fragment alien chromosome translocation lines can effectively reduce the effects of unfavorable genes on agronomic traits and quality of wheat. In this study, T4DS·4DL-4EL and T5AS·5AL-4EL contain small fragment translocations (Fig. 2). Compared to the E (4D) substitution line, T4DS·4DL-4EL had significantly higher tiller number, grain numbers per spike, and 1,000-grain weight. In addition, compared to the T4DS·4EL, the two small fragment translocations showed higher resistance to leaf rust in the field (data not show), probably due to the recovered Lr67 resistance which was lost in the chr4DL (Herrera-Foessel et al. 2011) of T4DS·4EL.

Compensatory translocations often are more easily used in wheat breeding due to the higher genetic similarity between the exchanged wheat chromosome segment and alien segment (Gong et al. 2024), and the T4DS·4DL-4EL might have more potential for application as a compensatory translocation than T5AS·5AL-4EL. Although T4DS·4DL-4EL had a higher plant height, however, its progenies carrying Yr4EL from crosses with multiple common wheat varieties exhibited lower plant height, which suggests that this trait was probably inherited from CSph1b and not linked to Yr4EL, since T4DS·4EL showed a normal plant height (Gong et al. 2024). The double top-cross two-stage selection strategy can rapidly polymerize multiple superior traits from different wheat varieties, several elite wheat varieties have been successfully produced using this strategy, such as SM580, SM969, and SM830 (Hao et al. 2019). By this strategy, we developed several pre-breeding lines with Yr4EL resistance and improved agronomic traits (Fig. 6). In the future, we will further evaluate these pre-breeding lines and continue to develop new wheat varieties/lines exhibiting high resistance to stripe rust along with excellent agronomic traits.

Acknowledgments

This work was supported by the Major Program of National Agricultural Science and Technology of China (NK20220607), the National Natural Science Foundation of China (No. 32200180), and the Science and Technology Bureau of Sichuan Province (2023NSFSC1995, 2022ZDZX0014, and 2021YFYZ0002), the special projects of the central government in guidance of local science and technology development (2023ZYD0088), and the Science and Technology Bureau of Chengdu City (2022-YF05-00449-SN). We also thank Prof. Qiuzhen Jia, Plant Protection Institute of Gansu Academy of Agricultural Sciences, Gansu, China, for kindly supplying the urediniospores of the P. striiformis f. sp. tritici races.

Author Contributions

BRG, JG, YQX, YHL, and HYK: conducted the experiment, analyzed the data, and drafted the manuscript. HZ, WZ, LLX, YRC, and YW developed translocation lines and evaluated disease resistance. JZ, XF, LNS, HQZ, YHZ, and DDW provided technique guidance. YHL and HYK designed the experiment and formulated the questions. All the authors read and approved the manuscript.

Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability The whole genome sequence of diploid Th. elongatum reported in this study can be found in the National Center for Biotechnology Information, NCBI BioProject ID PRJNA540081. Data supporting the results of this study are in the manuscript or supplemental file.

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SupplementaryFigure.pdf
Fig. S1 Detection of individuals carrying chr4E in BC₁F₁ and F₂ populations. M, DNA maker (pUC19); 1, CS; 2, SM482; 3, SM51; 4, CM42; 5, CSph1b; 6, 8801; 7, 4E (4D) substitution line; 8-69, BC₁F₁ and F₂ individuals from a cross between 4E (4D) substitution line with CSph1b. Fig. S2 Detection of individuals carrying homozygous ph1b genotype. M, RealBand DNA Maker; 1, CS; 2, CSph1b; 3, SM482; 4, SM51; 5, 4E (4D) substitution line; 6-45, BC₁F₁ and F₂ individuals from a cross between 4E (4D) substitution line and CSph1b carrying chr4E.
SupplementaryTable.xlsx
Table S1 Sequence information for markers KT4E-27 and KT4E-31.

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Development of the novel wheat-tetraploid Thinopyrum elongatum 4EL small fragment translocation lines with the stripe rust resistance gene Yr4EL

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Abstract

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Key message

Introduction

Materials and methods

Plant materials

Development of translocation lines

GISH, and FISH analyses

Wheat 660K SNP array analysis

Assessment of stripe rust resistance

Evaluation of agronomic performance

Pre-breeding material production

Results

Development and molecular cytological identification of translocation lines

Genotyping of the translocation lines using wheat 660K SNP array

Evaluation of stripe rust resistance and agronomic traits

The potential value of pre-breeding material

Discussion

Declarations

References

Supplementary Files

Status:

Version 1