Development and Characterization of a Novel Wheat–Tetraploid Thinopyrum elongatum 6E (6D) Disomic Substitution Line with Stripe Rust Resistance at the Adult Stage

Stripe rust, which is caused by Puccinia striiformis f. sp. tritici, is one of the most devastating foliar diseases of common wheat worldwide. Breeding new wheat varieties with durable resistance is the most effective way of controlling the disease. Tetraploid Thinopyrum elongatum (2n = 4x = 28, EEEE) carries a variety of genes conferring resistance to multiple diseases, including stripe rust, Fusarium head blight, and powdery mildew, which makes it a valuable tertiary genetic resource for enhancing wheat cultivar improvement. Here, a novel wheat–tetraploid Th. elongatum 6E (6D) disomic substitution line (K17-1065-4) was characterized using genomic in situ hybridization and fluorescence in situ hybridization chromosome painting analyses. The evaluation of disease responses revealed that K17-1065-4 is highly resistant to stripe rust at the adult stage. By analyzing the whole-genome sequence of diploid Th. elongatum, we detected 3382 specific SSR sequences on chromosome 6E. Sixty SSR markers were developed, and thirty-three of them can accurately trace chromosome 6E of tetraploid Th. elongatum, which were linked to the disease resistance gene(s) in the wheat genetic background. The molecular marker analysis indicated that 10 markers may be used to distinguish Th. elongatum from other wheat-related species. Thus, K17-1065-4 carrying the stripe rust resistance gene(s) is a novel germplasm useful for breeding disease-resistant wheat cultivars. The molecular markers developed in this study may facilitate the mapping of the stripe rust resistance gene on chromosome 6E of tetraploid Th. elongatum.

To verify the cytological stability of K17-1065-4, 40 randomly selected seeds from t K17-1065-4 selfed progeny were characterized via GISH and FISH analyses. The resu showed that all seeds carried two 6E chromosomes, but no 6D chromosomes (Figure 1e

Agronomic Trait Evaluation
K17-1065-4, SM482, SM921, and 8801 plants were assessed for agronomic tra research field at Sichuan Agricultural University. The tiller number, plant heigh spike length of K17-1065-4 were similar to those of SM482 and SM921, but were cantly lower than those of 8801 (Table 1; Figure 3). The grain number per spike o 1065-4 was significantly higher than that of all parents, and its 1000-grain weig lower than that of SM921, not significantly different from that of SM482, and signif higher than that of 8801.

Agronomic Trait Evaluation
K17-1065-4, SM482, SM921, and 8801 plants were assessed for agronomic traits in a research field at Sichuan Agricultural University. The tiller number, plant height, and spike length of K17-1065-4 were similar to those of SM482 and SM921, but were significantly lower than those of 8801 (Table 1; Figure 3). The grain number per spike of K17-1065-4 was significantly higher than that of all parents, and its 1000-grain weight was lower than that of SM921, not significantly different from that of SM482, and significantly higher than that of 8801.

Development and Validation of Specific Molecular Markers
A total of 83,685 SSR sequences were obtained by analyzing the 6E genomic sequence of diploid Th. elongatum. Primers were designed for all SSR sequences using Primer3 (https://primer3.ut.ee/) (accessed on 7 June 2022). The mock e-PCR amplification of the whole genomes of CS and diploid Th. elongatum indicated 20,738 pairs of primers could exclusively amplify the target fragments on the diploid Th. elongatum 6E chromosome. The SSR sequences corresponding to these markers were used to screen the other chromosomes (1E-5E and 7E) of diploid Th. elongatum, which revealed 8708 sequences with mismatches. Furthermore, these sequences were compared with the whole-genome sequence of CS; the sequences with ≥10% homology were discarded. Finally, 3382 unique SSR sequences were obtained, which were specific to the 6E chromosome of diploid Th. elongatum.
To evaluate the specificity and stability of these 33 markers, they were used for the PCR amplification involving 15 wheat relatives (Table S3). Ten amplified fragments were exclusive to diploid Th. elongatum and tetraploid Th. elongatum ( Figure 5a). Additionally, the primer pairs for three markers amplified specific fragments only for diploid Th. elongatum, tetraploid Th. elongatum, and Th. ponticum (Figure 5b), while the primer pairs for another three markers amplified specific fragments only for diploid Th. elongatum, tetraploid Th. elongatum, Th. ponticum, and Th. bessarabicum (Figure 5c). The PCR analysis of other wheat relatives showed that 13, 2, 1, 2, and 7 markers amplified specific fragments

Development and Validation of Specific Molecular Markers
A total of 83,685 SSR sequences were obtained by analyzing the 6E genomic sequence of diploid Th. elongatum. Primers were designed for all SSR sequences using Primer3 (https://primer3.ut.ee/) (accessed on 7 June 2022). The mock e-PCR amplification of the whole genomes of CS and diploid Th. elongatum indicated 20,738 pairs of primers could exclusively amplify the target fragments on the diploid Th. elongatum 6E chromosome. The SSR sequences corresponding to these markers were used to screen the other chromosomes (1E-5E and 7E) of diploid Th. elongatum, which revealed 8708 sequences with mismatches. Furthermore, these sequences were compared with the whole-genome sequence of CS; the sequences with ≥10% homology were discarded. Finally, 3382 unique SSR sequences were obtained, which were specific to the 6E chromosome of diploid Th. elongatum.

Utility of Specific Markers for Breeding
To evaluate whether the 33 specific markers developed for the tetraploid Th. elongatum chromosome 6E were applicable for breeding, 154 F 2 individuals obtained from the cross between K17-1065-4 and SM482 were selected for a molecular marker analysis, GISH analysis, and an assessment of stripe rust resistance. The PCR amplification results for the tetraploid Th. elongatum 6E-specific SSR markers revealed specific bands for 115 of the 154 plants ( Figure 6). The GISH results indicated that these 115 plants contained one or two 6E chromosomes; the GISH signals were undetectable for the other 39 plants (Figure 7). Moreover, these 115 plants were highly resistant to stripe rust at the adult stage, whereas the other 39 plants were highly susceptible to stripe rust ( Figure 8). Accordingly, these specific SSR markers may be useful for tracking stripe rust resistance genes linked to chromosome 6E of tetraploid Th. elongatum, making them potentially capable of wheat genetic improvement and breeding.

Utility of Specific Markers for Breeding
To evaluate whether the 33 specific markers developed for the tetraploid Th. elongatum chromosome 6E were applicable for breeding, 154 F2 individuals obtained from the cross between K17-1065-4 and SM482 were selected for a molecular marker analysis, GISH analysis, and an assessment of stripe rust resistance. The PCR amplification results for the tetraploid Th. elongatum 6E-specific SSR markers revealed specific bands for 115 of the 154 plants ( Figure 6). The GISH results indicated that these 115 plants contained one or two 6E chromosomes; the GISH signals were undetectable for the other 39 plants (Figure 7). Moreover, these 115 plants were highly resistant to stripe rust at the adult stage, whereas the other 39 plants were highly susceptible to stripe rust ( Figure 8). Accordingly, these specific SSR markers may be useful for tracking stripe rust resistance genes linked to chromosome 6E of tetraploid Th. elongatum, making them potentially capable of wheat genetic improvement and breeding.

Discussion
Th. elongatum is a tertiary genetic resource that has many excellent agronomic traits useful for wheat crop improvement. Over the last several decades, several wheat-Th. elongatum lines have been produced via distant hybridizations. Ma et al. [39] assessed the stripe rust resistance of wheat-Th. elongatum substitution lines and mapped the dominantly inherited stripe rust resistance gene YrE to chromosome 3E. Jauhar [5] crossed diploid Th. elongatum with durum wheat Langdon to obtain one 1E addition line and two 1E substitution lines, which differed regarding Fusarium head blight infection rates. Another gene (Yr69) mediating stripe rust resistance was identified in the wheat-Th. ponticum line CH7086 on the basis of stripe rust resistance and allele analyses [40]. Dai et al. [41] developed a wheat-rye-Thinopyrum tricentric hybrid by crossing 8801 with triticale T182, while also introducing the genes responsible for the resistance to Fusarium head blight, leaf rust, and stem rust into common wheat. In a recent study, two wheat-Th. ponticum substitution lines (ES-11 and ES-12) and a new translocation line were identified and characterized, and the stripe rust and stem rust resistance genes carried by Th. ponticum were successfully introduced into common wheat [42,43]. We previously reported that the wheat-tetraploid Th. elongatum 1E (1D) substitution line is highly resistant to stripe rust, the tetraploid Th. elongatum 3E chromosome carries salt tolerance genes, and the 4E (4D) disomic substitution line is resistant to stripe rust and powdery mildew at the seedling and adult

Discussion
Th. elongatum is a tertiary genetic resource that has many excellent agronomic traits useful for wheat crop improvement. Over the last several decades, several wheat-Th. elongatum lines have been produced via distant hybridizations. Ma et al. [39] assessed the stripe rust resistance of wheat-Th. elongatum substitution lines and mapped the dominantly inherited stripe rust resistance gene YrE to chromosome 3E. Jauhar [5] crossed diploid Th. elongatum with durum wheat Langdon to obtain one 1E addition line and two 1E substitution lines, which differed regarding Fusarium head blight infection rates. Another gene (Yr69) mediating stripe rust resistance was identified in the wheat-Th. ponticum line CH7086 on the basis of stripe rust resistance and allele analyses [40]. Dai et al. [41] developed a wheat-rye-Thinopyrum tricentric hybrid by crossing 8801 with triticale T182, while also introducing the genes responsible for the resistance to Fusarium head blight, leaf rust, and stem rust into common wheat. In a recent study, two wheat-Th. ponticum substitution lines (ES-11 and ES-12) and a new translocation line were identified and characterized, and the stripe rust and stem rust resistance genes carried by Th. ponticum were successfully introduced into common wheat [42,43]. We previously reported that the wheat-tetraploid Th. elongatum 1E (1D) substitution line is highly resistant to stripe rust, the tetraploid Th. elongatum 3E chromosome carries salt tolerance genes, and the 4E (4D) disomic substitution line is resistant to stripe rust and powdery mildew at the seedling and adult stages [16,35,36]. Until now, there has been no report on the tetraploid Th. elongatum homologous group 6 heterochromosome lines. Therefore, we developed and characterized a novel wheat-tetraploid Th. elongatum 6E (6D) disomic substitution line K17-1065-4, which is highly resistant to stripe rust at the adult stage. In addition, the grain number per spike of K17-1065-4 was significantly higher than that of the parents, indicating that K17-1065-4 also carries genes associated with increased grain production. Therefore, K17-1065-4 represents a new excellent genetic resource for breeding disease-resistant wheat lines.
Several APR genes from the progenitors and wild relatives of wheat have been exploited, including Yr36, Yr56, and Yr83 as well as a number of tentatively named genes and QTLs. Uauy et al. [44] first identified Yr36 in Triticum turgidum ssp. dicoccoides plants exhibiting high-temperature adult plant resistance, with no detrimental effects on wheat yield. Subsequently, Yr36 was effectively used by wheat breeders to produce wheat line Shumai 1701 [30]. Bansal and Bariana [45] identified the APR gene Yr56 in durum wheat and determined it was located on chromosome 2AS bin 2AS5-0.87-1.00. Another APR gene (Yr83) was characterized by an in situ hybridization and molecular marker analysis of 10 6R chromosome deletion lines as well as 5 wheat-rye 6R chromosome translocation lines; the gene was mapped to the deletion bin of FL 0.73-1.00 of 6RL [24]. In addition, Zhang et al. [28] performed a bulked segregant RNA-seq analysis and mapped the APR gene YrZ15-1370 from Triticum boeoticum to chromosome 6AL. More specifically, it was located within a 4.3 cM genetic interval flanked by KASP-1370-3 and KASP-1370-5, which corresponded to a 1.8 Mb physical region. The seeds of the Ae. ventricosa near-isogenic line AvSYr17NIL were treated with EMS and the resulting F 2 plants were analyzed; a novel recessive APR gene (YrM1225) was characterized and localized within a 7.5 cM interval on the short arm of chromosome 2A [27]. However, these genes or QTLs have not been adequately exploited by wheat breeders worldwide, and none of them are from Th. elongatum. In the present study, we developed the wheat-tetraploid Th. elongatum 6E (6D) substitution line K17-1065-4, which is highly resistant to multiple Pst races currently prevalent in China at the adult stage. A stripe rust resistance gene was localized on chromosome 6 of Th. ponticum [7], but the two genes were completely different in origin, genome, and response to Pst races, showing that they are two different stripe rust resistance genes. To the best of our knowledge, this is the first study to reveal the resistance to stripe rust mediated by chromosome group 6 of tetraploid Th. elongatum. Our results show that the stripe rust resistance of K17-1065-4 is conferred by a novel gene from tetraploid Th. elongatum. Moreover, this line may be a valuable resource for increasing the stripe rust resistance of wheat worldwide.
Molecular markers are important for the efficient detection of alien chromosomes or chromosomal fragments in wheat. Diverse and stable molecular markers provide an important foundation for breeding involving crosses between wheat varieties and wheat relatives. A number of molecular markers have been developed for Th. elongatum on the basis of RAPD, SSR, SCAR, AFLP, RGAP, GBS, and other techniques [35]. Two RAPD markers for CS and an ISSR marker for Th. elongatum were successfully transformed into SCAR markers for diploid Th. elongatum. Of these markers, two can specifically detect the E genome of Th. elongatum, whereas one is useful for tracking chromosomes 2E and 3E of Th. elongatum [46]. Chen et al. [1] developed 89 stable and specific molecular markers for Th. elongatum using SLAF-seq data. Additionally, many Th. elongatum SNP markers were obtained following a transcriptome sequencing analysis [47]. Furthermore, Th. ponticum-specific molecular markers were developed using SLAF-seq technology and used to construct a physical map of the 4Ag chromosome [48]. Li et al. [16] developed 132 markers for tetraploid Th. elongatum 1E by applying GBS technology. Another 74 markers were developed to accurately track stripe rust resistance genes on chromosome 4E of tetraploid Th. elongatum [35]. Although various Th. elongatum-specific molecular markers have been reported, most of these markers have not been precisely mapped to chromosomes or they are distributed in the terminal regions of chromosomes due to the previous lack of a Th. elongatum reference genome, which severely limits the localization and cloning of Th. elongatum genes associated with improved traits. In the current study, 33 SSR markers specific to tetraploid Th. elongatum chromosome 6E were developed according to the whole-genome sequence of diploid Th. elongatum. Validation of these specific markers in 15 wheat relatives showed that 10 of them accurately screened for the E genome carried by diploid and tetraploid Th. elongatum (Table S3). Only 11 pairs of markers amplified specific fragments in Th. ponticum, indicating that the genomes of Th. ponticum differed significantly from those of diploid and tetraploid Th. elongatum (Table S3), which is consistent with previous findings [16]. Only very few markers amplified specific fragments in the H, P, R, V, Ns, and St genomes, which indicated that the E genome of Th. elongatum and these genomes are genetically distant from one another. These specific markers are evenly distributed on chromosome 6E, making them potentially useful for identifying associated stripe rust resistance genes, and could also be employed by wheat breeding to help fine-map and clone the stripe rust resistance genes on chromosome 6E of tetraploid Th. elongatum.

Genomic In Situ Hybridization (GISH) and Fluorescence In Situ Hybridization (FISH) Analyses
Seeds were germinated in an incubator at 22 • C. Samples were treated with N 2 O for 2 h when their roots reached 1-2 cm, after which they were immediately fixed in 90% acetic acid for 5 min before being digested with pectinase and cellulase [51]. Slides for the in situ hybridization were prepared as described by Han et al. [52]. Tetraploid Th. elongatum genomic DNA labeled with dUTP-ATTO-550 (Jena Bioscience, Jena, Germany) via nick translation was used as the probe and Chinese Spring (CS) DNA was used as the blocker (ratio of 1:150). The GISH analysis was performed according to a published method that was modified slightly [53]. Briefly, 1 µL tetraploid Th. elongatum genomic DNA probe, 3 µL CS DNA, and 16 µL hybridization mixture (1 g dextran sulfate, 5 mL formamide, 1 mL 20× SSC, 1 mL salmon sperm, and 2 mL ddH 2 O) were mixed and added dropwise to the slides. The samples were denatured at 85 • C for 5 min and then incubated overnight at 50 • C. They were subsequently washed with 2× SSC at 50 • C for 20 min and then with 75%, 95%, and 100% ethanol for 1 min each. The chromosome counterstaining and the slide microscopy were performed as described by Gong et al. [35].
After the GISH analyses, the samples were washed with 2× SSC for 30 min and then with 75% and 100% ethanol for 5 min each before being placed under bright light. The Oligo-pSc119.2 and Oligo-pTa535 FISH probes were added after the GISH signal was removed to determine the K17-1065-4 chromosomal composition. The FISH analyses were performed as described by Li et al. [16]. The FISH signals were recorded in the same way as the GISH signals.

FISH Chromosome Painting Analysis
The homologous group relationship of the exogenous chromosome carried by K17-1065-4 was determined by performing a FISH chromosome painting analysis using the bulk oligonucleotide libraries Chr1-Chr7 (provided by Prof. H.Q. Zhang, Triticeae Research Institute, Sichuan Agricultural University; data not published) for the whole-genome sequence of diploid Th. elongatum. The washed slides were used for the FISH analysis involving Oligo-pSc119.2 and Oli-gop-Ta535 to distinguish the chromosome composition of K17-1065-4. The FISH chromosome painting method was previously described by Bi et al. [54] and Han et al. [55]. The slides were washed as described by Komuro et al. [51]. Photomicrographs were taken as described in the FISH protocol.

Stripe Rust Resistance Evaluation
Seedling stripe rust reactions of K17-1065-4, 8801, SY95-71, SM482, and SM921 were evaluated under laboratory conditions at Sichuan Agricultural University, China. They were inoculated with P. striiformis f. sp. tritici race CYR-34 in an artificial climate chamber, Plants were inoculated at the two-leaf stage and the reaction to stripe rust was evaluated on the first leaf of each plant 15-18 days after inoculation [16]. SY95-71 was used as a susceptible control. and the disease resistance statistics were conducted as described by Line and Qayoum [56].

Agronomic Trait Evaluation
K17-1065-4, 8801, SM482, and SM921 plants were evaluated in terms of their agronomic traits during the 2020-2022 growing seasons in the experimental fields at Sichuan Agricultural University. The experiment was conducted using a randomized complete block design with three replications. Briefly, 15 seeds were sown in 2 m rows, with 0.3 m between rows. K17-1065-4 and its parents were assessed for agronomic traits including tiller number, spikelet number, spike length, plant height, grain number per spike, and 1000-grain weight. The data were analyzed using SPSS Statistics 24.0 software.

Development of Simple Sequence Repeat (SSR) Markers
The SSRs in the diploid Th. elongatum chromosome 6E sequence (NCBI BioProject ID PRJNA540081) were detected using the perl-based program MISA (http://pgrc.ipkgatersleben.de/misa/download/misa.pl) (accessed on 7 June 2022) and the following criteria: single-nucleotide repeats of not less than 10; dinucleotide repeats of not less than 6; three to six nucleotide repeats of not less than 5; and two SSR loci separated by more than 100 bp. Primers were designed for all SSR sequences using Primer3 and then analyzed by performing an e-PCR mock amplification using the whole-genome sequences of CS and diploid Th. elongatum. Only the markers that amplified the target fragment on chromosome 6E of diploid Th. elongatum were selected. The SSR sequences corresponding to these markers were compared with the sequences of the other chromosomes (1E-5E and 7E) of diploid Th. elongatum. The sequences that were not complete matches were identified. These sequences were compared with the whole-genome sequence of CS and the sequences with ≥10% homology were removed to obtain unique SSR sequences specific to chromosome 6E of diploid Th. elongatum. All primers were produced by Sangon Biotech (Chengdu, China). The PCR amplification program and product detection were performed as described by Gong et al. [35].

Validation of Specific Molecular Markers
The specificity, repeatability, and stability of tetraploid Th. elongatum 6E chromosomespecific molecular markers were verified using 154 F 2 individuals from the cross between K17-1065-4 and SM482 as well as 15 wheat-related species (Table S1). The PCR analysis was performed as described previously.

Conclusions
In the present study, we characterized a cytogenetically stable wheat-tetraploid Th. elongatum 6E (6D) disomic substitution line with a high level of resistance to stripe rust at the adult stage. It is an extremely valuable wheat germplasm resource for the development of new stripe-rust-resistant varieties. Moreover, 33 markers specific for tetraploid Th. elongatum chromosome 6E were developed based on the whole-genome sequence of diploid Th. elongatum. All these markers should be applicable for efficiently tracing tetraploid Th. elongatum chromosome 6E and its chromosomal segments during wheat-disease-resistant breeding. In the future, we will use 60 Co-γ ionizing irradiation and CSph1b mutants to induce this substitution line and produce small segmental translocations carrying stripe rust resistance genes for further breeding applications.