Background

Common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is a staple cereal cultivated worldwide, with a predicted global grain yield of 757.4 million tons in 2019 [1]. However, the domestication of wheat decreased its genetic diversity as well as tolerance to biotic and abiotic stresses, which has restricted further improvements to wheat productivity and quality [2]. Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a serious wheat disease that threatens global wheat production [3, 4]. The identification and application of new disease-resistance genes and the development of disease-resistant cultivars represent the most effective means of decreasing the reliance on fungicides to control stripe rust in large-scale commercial production systems [5]. Wild relatives are a largely unexploited source of genes for agronomically important traits that can be transferred to common wheat via wide hybridizations to enrich wheat genetic diversity [6, 7].

Thinopyrum elongatum (syn. Agropyron elongatum or Lophopyrum elongatum) is a distant wild relative of common wheat and has long been the focus of wheat breeders. The taxon comprises the following three ploidy levels involving the E-genome: diploid (2n = 2x = 14, EE), tetraploid (2n = 4x = 28, EEEE), and decaploid (2n = 10x = 70, EEEEEEStStStSt) [8]. This species possesses many desirable traits, including strong adaptability, high tolerance to cold, drought, and salt stresses, and resistance to Fusarium head blight, rust, powdery mildew, and the yellow dwarf virus [9, 10]. To transfer desirable traits from Th. elongatum into wheat, wide hybridizations between Th. elongatum and common wheat began in the 1980s [8]. Progeny lines harboring Th. elongatum chromosomes (segments) incorporated into the wheat genome were obtained as lines with chromosomal additions, substitutions, or translocations [7, 11,12,13,14]. However, these introgressions mainly involved the diploid Th. elongatum and decaploid Thinopyrum ponticum. There are relatively few reports describing attempts to transfer tetraploid Th. elongatum genes into wheat [15,16,17,18]. Thus, identifying new elite alien genes and incorporating them into common wheat are critical for increasing wheat genetic diversity through the development of wheat–tetraploid Th. elongatum introgression lines.

Marker-assisted selection is useful for detecting genes associated with a trait of interest based on a linked marker, with implications for breeding involving multiple traits [11]. Developing species- specific molecular markers that facilitate the identification of alien chromosomes or segments is very important for wheat breeding programs [19, 20]. Diverse molecular markers specific to diploid Th. elongatum and Th. ponticum have been reported, including random-amplified polymorphic DNAs (RAPDs), simple sequence repeats (SSRs), expressed sequence tags, cleaved amplified polymorphic sequences, sequence-tagged sites, sequence-characterized amplified regions, amplified fragment length polymorphisms, single nucleotide polymorphisms, and specific-locus amplified fragments (SLAFs) [11, 12, 19, 21,22,23,24,25]. However, there are still relatively few of these markers, and they do not cover the whole Thinopyrum genome. Consequently, there is an urgent need to develop more molecular markers, especially those with a wide genomic distribution and that are amenable to high-throughput genotyping [12]. Genotyping-by-sequencing (GBS) is a high-throughput, highly accurate, inexpensive, and relatively simple method for developing several markers in Triticale species [26]. However, developing specific molecular markers for tetraploid Th. elongatum remains difficult. Therefore, generating chromosome-specific molecular markers is critical for detecting and tracing alien chromosomes in wheat–tetraploid Th. elongatum hybrid derivatives.

Tetraploid Th. elongatum is an important genetic resource for improving wheat, and some wheat–tetraploid Th. elongatum derivative lines have been developed by crossing hexaploid Trititrigia with Sichuan wheat cultivars [17]. Li et al. [18] produced a fluorescence in situ hybridization (FISH) karyotype of the E-genome chromosomes of tetraploid Th. elongatum based on various repetitive sequence probes, which may enhance the utility of tetraploid Th. elongatum for the introgression of alien genes into wheat. The main objectives of the current study were to: (1) characterize the chromosomal constitution of a wheat–tetraploid Th. elongatum substitution line and evaluate its effects on stripe rust resistance and agronomic traits and (2) develop and validate specific and easy-to-use molecular markers based on GBS, which may be useful for the efficient and reliable identification of tetraploid Th. elongatum chromatin in several species, including common wheat.

Results

Chromosomal constitution of K17–841-1

Genomic in situ hybridization (GISH), FISH, and SSR marker analyses were performed to determine the chromosomal constitution of wheat–tetraploid Th. elongatum line K17–841-1. When tetraploid Th. elongatum total genomic DNA and the J-11 genomic DNA were used as the probe and the blocking DNA, respectively, we observed that line K17–841-1 carried 40 wheat chromosomes and two E chromosomes (Fig. 1a). The GISH signals were sequentially removed and the slides were used in a FISH analysis involving pSc119.2 and pTa535 probes. A pair of E chromosomes produced strong terminal pSc119.2 signals on both arms as well as strong pTa535 signals on the subterminal regions of the short arm and near the centromeric region of the long arm (Fig. 1b). These results are consistent with the previously reported FISH pattern for the 1E chromosome of tetraploid Th. elongatum [18]. Thus, the FISH karyotype of the E-genome chromosomes of tetraploid Th. elongatum and common wheat based on probes pSc119.2 and pTa535 suggested that K17–841-1 is a 1E (1D) chromosomal substitution line. To determine the cytological stability of K17–841-1, we used GISH and FISH to analyze 20 randomly selected seeds of K17–841-1 self-progeny. We revealed that all seeds contained 14 A- (1A–7A), 14 B- (1B–7B), and 12 D- (2D–7D) genomes, as well as a pair of 1E chromosomes (Fig. 1c, d).

Fig. 1
figure 1

GISH and FISH identification of the wheat–tetraploid Th. elongatum substitution line K17–841-1. The probes used for in situ hybridization were tetraploid Th. elongatum genomic DNA (a, c); pSc119.2 and pTa535 (b, d). Arrows indicate 1E-genome chromosomes. Scale bar: 10 μm

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We completed a PCR analysis involving the wheat chromosome 1D-specific SSR markers to confirm the chromosomal constitution of K17–841-1. As expected, amplified products for the chromosome 1D SSR markers (i.e., wmc147, wmc222, gwm337, and Xcfd63) were detected for Chinese Spring (CS), Shumai482 (SM482), and Shumai921 (SM921). In contrast, amplicons were not genetated for 8801 and K17–841-1 (Fig. 2). Our results verified that in line K17–841-1, wheat chromosome 1D had been replaced by chromosome 1E of tetraploid Th. elongatum.

Fig. 2
figure 2

PCR amplification of SSR markers wmc147 (a), wmc222 (b), gwm337 (c), and Xcfd63 (d). Lanes M marker, 1 CS, 2 wheat cultivar Shumai482, 3 wheat cultivar Shumai921, 4 8801 (T. durum-tetraploid Th. elongatum amphidiploid), 5 K17–841-1(wheat–tetraploid Th. elongatum substitution line). Arrows show the diagnostic amplification products of SSR markers

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Morphology of K17–841-1

We analyzed the agronomic traits of K17–841-1 and the donor parents in two growing seasons. Line K17–841-1 displayed stable morphological traits, which were similar to those of the wheat parents SM482 and SM921 (Table 1; Fig. 3). The average plant height and spike length of line K17–841-1 were significantly lower than those of the Triticum durum–tetraploid Th. elongatum partial amphidiploid 8801, but were greater than those of SM482 and similar to those of SM921 (Fig. 3a, b). The number of spikelets per spike was lower than that of SM482 or SM921, but was similar to that of 8801. The grain number per spike was greater than that of 8801, but was lower than that of SM921 and similar to that of SM482 (Fig. 3c). There were no significant differences between K17–841-1 and either SM482 or SM921 regarding the tiller number, thousand-kernel weight, and seed setting rate (Fig. 3d).

Table 1 Agronomic traits of K17–841-1 and its parental lines
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Fig. 3
figure 3

Plant morphology of the wheat–tetraploid Th. elongatum substitution line K17–841-1 and its parents. Adult plants (a), spikes (b), spikelets(c), and grains(d). Numbers 1–4 represent 8801 (T. durum-tetraploid Th. elongatum amphidiploid), wheat cultivar Shumai482, wheat cultivar Shumai 921, and K17–841-1, respectively

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Stripe rust resistance evaluation

We evaluated the stripe rust resistance of K17–841-1, 8801, SM482, SM921, and SY95–71 plants inoculated with a mixture of Pst races (CYR-32, CYR-33, CYR-34, and V26/Gui22–14) at Chengdu, Sichuan, China. An analysis of three replicates revealed that the adult SM482, SM921, and SY95–71 plants were susceptible to these Pst races, with infection types (ITs) of 4, 3, and 4, respectively. In contrast, 8801 and K17–841-1 plants were highly resistant to these races (i.e., IT of 0) (Fig. 4).

Fig. 4
figure 4

Stripe rust resistance of the wheat–tetraploid Th. elongatum substitution line K17–841-1 and the controls. 1 wheat line SY95–71, 2 8801 (T. durum-tetraploid Th. elongatum amphidiploid), 3 wheat cultivar Shumai482, 4 wheat cultivar Shumai921, 5 K17–841-1

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Development and validation of specific molecular markers for the 1E chromosome of tetraploid Th. elongatum

The GBS approach was applied to identify tetraploid Th. elongatum 1E chromosome-specific sequences. Details regarding the sequencing, including raw reads, clean reads, effective rate, error rate, Q20, Q30, and GC content, are summarized in Table 2. The results revealed a high sequencing quality (Q20 ≥ 94% and Q30 ≥ 86%) and a normal GC content distribution. A total of 45,115,502, 7,389,702, 8,710,288, and 8,831,260 effective GBS sequences were obtained for PI531718, PI531750, 8801, and K17–841-1, respectively (Table 3). The sequencing depth was more than 10.32×. A sequence comparison revealed 73,744 K17–841-1 clean reads that were less than 23% homologous with CS sequences. Additional sequence alignments uncovered 2952 K17–841-1 reads that were more than 23% homologous with 8801 and PI531750 fragments obtained by GBS. Finally, 1194 K17–841-1 unique reads (597 specific fragments) that were less than 10% homologous with sequences from PI531718 and the other six substitution lines were obtained and were considered the tetraploid Th. elongatum 1E chromosome-specific fragments.

Table 2 Quality of GBS data
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Table 3 Sequence alignment between K17–841-1 and its parental lines
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To develop tetraploid Th. elongatum 1E chromosome-specific markers, 235 PCR primers pairs were designed based on these specific fragments and used to amplify sequences from CS, SM482, SM921, PI531718, PI531750, 8801, K17–841-1, and six wheat-tetraploid Th. elongatum disomic substitution (TDS) lines (2E-7E). A total of 165 (70%) Th. elongatum-specific molecular markers were successfully developed (see Additional file 1: Table S1), of which 132 markers amplified specific sequences only from PI531750, 8801, and K17–841-1 (Type I) (Fig. 5a-d). Therefore, these markers were regarded as tetraploid Th. elongatum 1E chromosome-specific molecular markers, with a success rate of up to 56.2%. Additionally, 21 markers amplified specific sequences only from PI531718, PI531750, 8801, and K17–841-1 (Type II) (Fig. 6a). Moreover, 12 markers were also detected on the other E chromosomes of tetraploid Th. elongatum (Fig. 6b, c).

Fig. 5
figure 5

PCR amplification of markers TTE1E-12 (a), TTE1E-140 (b), TTE1E-189 (c), and TTE1E-193 (d). Lanes M marker, 1 CS, 2 wheat cultivar Shumai482, 3 wheat cultivar Shumai921, 4 diploid Thinopyrum elongatum, 5 tetraploid Thinopyrum elongatum, 6 8801 (T. durum-tetraploid Th. elongatum amphidiploid), 7 K17–841-1 (wheat–tetraploid Th. elongatum substitution line), 8 TDS2E(2A), 9 TDS3E(3D), 10 TDS4E(4D), 11 TDS5E(5D), 12 TDS6E(6D), 13 TDS7E(7D). Arrows show the diagnostic amplification products of tetraploid Th. elongatum 1E chromosome

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Fig. 6
figure 6

PCR amplification of markers TTE1E-214 (a), TTE1E-3 (b), and TTE1E-58 (c). Lanes M marker, 1 CS, 2 wheat cultivar Shumai482, 3 wheat cultivar Shumai921, 4 diploid Thinopyrum elongatum, 5 tetraploid Thinopyrum elongatum, 6 8801 (T. durum-tetraploid Th. elongatum amphidiploid), 7 K17–841-1 (wheat–tetraploid Th. elongatum substitution line), 8 TDS2E(2A), 9 TDS3E(3D), 10 TDS4E(4D), 11 TDS5E(5D), 12 TDS6E(6D), 13 TDS7E(7D). Arrows show the diagnostic amplification products of tetraploid Th. elongatum 1E chromosome

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To confirm marker specificity and stability, 153 markers (Types I and II) were used to further analyze 11 wheat-related species. The PCR results are presented in Additional file 2: Table S2. Among the 132 tetraploid Th. elongatum 1E chromosome-specific markers, 25 amplified sequences from tetraploid Th. elongatum, but not from the analyzed related species (Table 4; Fig. 7a-d). In contrast, 21 and 106 markers amplified specific sequences from not only tetraploid Th. elongatum, but also from diploid Th. elongatum and decaploid Th. ponticum, respectively (Fig. 8a, b). Additionally, 28 markers amplified a common sequence from tetraploid Th. elongatum, diploid Th. elongatum, and Th. ponticum, but did not amplify any sequences from the other related species (Fig. 8c). Five markers amplified specific sequences not only from tetraploid Th. elongatum and Th. ponticum, but also from Thinopyrum bessarabicum (Fig. 8d). Four markers amplified a common sequence from diploid Th. elongatum, tetraploid Th. elongatum, Th. ponticum, Th. bessarabicum, Pseudoroegneria libanotica, Trichopyrum caespitosum, and Psammopyrum athericum. Furthermore, 58, 32, 5, 8, 28, 8, 13, 50, and 73 markers amplified sequences from Th. bessarabicum, Pse. libanotica, Dasypyrum villosum, Hordeum bogdanii, Agropyron cristatum, Secale cereale, Psathyrostachys huashanica, Tr. caespitosum, and Psa. athericum, respectively.

Table 4 Specific amplification results of 1E-chromosomal markers in wheat-related species
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Fig. 7
figure 7

Stability and specificity of markers TTE1E-12 (a), TTE1E-140 (b), TTE1E-189 (c), and TTE1E-193 (d) in other wheat-related species. Lanes M marker, 1 CS, 2 8801 (T. durum-tetraploid Th. elongatum amphidiploid), 3 K17–841-1 (wheat–tetraploid Th. elongatum substitution line), 4 diploid Thinopyrum elongatum, 5 tetraploid Thinopyrum elongatum, 6 Thinopyrum ponticum, 7 Thinopyrum bessarabicum, 8 Pseudoroegneria libanotica, 9 Dasypyrum villosum, 10 Hordeum bogdanii, 11 Agropyron cristatum, 12 Secale cereale, 13 Psathyrostachys huashanica, 14 Trichopyrum caespitosum, 15 Psammopyrum athericum. Arrows show the diagnostic amplification products of tetraploid Th. elongatum 1E chromosome

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Fig. 8
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Stability and specificity of markers TTE1E-59 (a), TTE1E-184 (b), TTE1E-222 (c), and TTE1E-192 (d) in other wheat-related species. Lanes M marker, 1 CS, 2 8801 (T. durum-tetraploid Th. elongatum amphidiploid), 3 K17–841-1 (wheat–tetraploid Th. elongatum substitution line), 4 diploid Thinopyrum elongatum, 5 tetraploid Thinopyrum elongatum, 6 Thinopyrum ponticum, 7 Thinopyrum bessarabicum, 8 Pseudoroegneria libanotica, 9 Dasypyrum villosum, 10 Hordeum bogdanii, 11 Agropyron cristatum, 12 Secale cereale, 13 Psathyrostachys huashanica, 14 Trichopyrum caespitosum, 15 Psammopyrum athericum. Arrows show the diagnostic amplification products of tetraploid Th. elongatum 1E chromosome

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Utility of the tetraploid Th. elongatum-specific markers in the F2 population

To verify the utility of the newly developed molecular markers, 80 F2 individuals of K17–841-1 and wheat cultivar Shumai969 (SM969) were analyzed by PCR. We detected specific amplicons for only 20 individuals (Fig. 9, Additional file 3: Table S3). The results of a GISH indicated these 20 plants with specific amplicons carried 1E (1D) chromosomal substitutions, while the 60 plants without specific amplicons had no GISH signal in their chromosome preparation. More importantly, an evaluation of stripe rust resistance at the seedling stage revealed that 8801, K17–841-1, and the 20 positive F2 individuals carrying 1E chromosomal markers were highly resistant to Pst race CYR-34. In contrast, the 60 F2 plants lacking amplicons as well as SY95–71 and the parental lines SM482, SM921, and SM969 were highly susceptible (Fig. 10, Additional file 3: Table S3). These observations implied that the specific markers developed in this study may be useful for detecting the stripe rust resistance gene on the 1E chromosome of tetraploid Th. elongatum during the breeding of new disease-resistant wheat varieties.

Fig. 9
figure 9

Utility of marker TTE1E-21 in 80 F2 individuals of K17–841-1 and SM969. 1 CS, 2 wheat cultivar Shumai482, 3 wheat cultivar Shumai921, 4 wheat cultivar Shumai969, 5 tetraploid Thinopyrum elongatum, 6 8801 (T. durum-tetraploid Th. elongatum amphidiploid), 7 K17–841-1 (wheat–tetraploid Th. elongatum substitution line), 8–87 80 F2 individuals. Arrows show the diagnostic amplification products of tetraploid Th. elongatum 1E chromosome

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Fig. 10
figure 10

Evaluation for seedling stage reacctions to Pst race CYR-34 in F2 individuals of K17–841-1 and SM969 and the controls. 1 wheat line SY95–71, 2 wheat cultivar Shumai482, 3 wheat cultivar Shumai921, 4 wheat cultivar Shumai969, 5 8801 (T. durum-tetraploid Th. elongatum amphidiploid), 6 K17–841-1 (wheat–tetraploid Th. elongatum substitution line), 7–9 susceptible F2 individuals, 10–12 resistant F2 individuals

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Discussion

Distant hybridizations may be useful for transferring agronomically valuable genes from wild relatives to common wheat varieties. Creating intermediate lines carrying alien chromosomes of wheat relatives provides the basis for using these germplasm resources to improve domesticated wheat [27]. Wheat–alien chromosomal substitution lines are important bridge materials for transferring alien genes to common wheat. In recent decades, there have been many attempts by wheat breeders to generate wheat–Th. elongatum substitution lines [8]. However, most of the introgressions from Th. elongatum into wheat involved the diploid Th. elongatum and decaploid Th. ponticum. For example, CS–diploid Th. elongatum addition and substitution lines have been produced [28]. Additionally, Zheng et al. [29] generated the wheat–Th. ponticum 4Ag (4D) disomic substitution line Blue 58 from a hybridization between Th. ponticum and common wheat. Fu et al. [30] developed a wheat–diploid Th. elongatum 7E (7D) substitution line resistant to Fusarium head blight. Wang et al. [31] produced a wheat–Th. ponticum St (6A) disomic substitution line exhibiting powdery mildew resistance. The 7JSt (7B) substitution line CH1113-B13, which is resistant to stripe rust, was identified from among the progeny of a cross between common wheat and Th. ponticum [32]. The tetraploid Th. elongatum harbors many beneficial genes that provide protection from diseases as well as cold, drought, and salinity stresses. Thus, it is a valuable genetic resource for improving the tolerance of common wheat to biotic and abiotic stresses [9, 10, 17]. To date, there are few reports regarding wheat–tetraploid Th. elongatum substitution lines. Li et al. [17] developed 50 wheat–tetraploid Th. elongatum introgression lines by crossing the T. durum–tetraploid Th. elongatum partial amphidiploid line 8801 with wheat varieties native to the Sichuan Basin, China. These lines comprise 40–47 chromosomes and most of them are cytologically unstable. In the current study, we further characterized the wheat–tetraploid Th. elongatum 1E (1D) disomic substitution line K17–841-1 derived from the 8801/SM482/SM921 F5 progeny. The FISH and SSR analyses confirmed that common wheat chromosome 1D was substituted with the homologous chromosome 1E from tetraploid Th. elongatum, providing further evidence that the E genome of Th. elongatum is closely related to the D genome of common wheat [33, 34]. Interestingly, an evaluation of the stripe rust resistance indicated that the wheat parents SM482 and SM921 are susceptible and that the resistance of K17–841-1 was derived from tetraploid Th. elongatum. Some stripe rust resistance genes have been identified in Thinopyrum species and have been transferred to common wheat, including Yr50 from Th. intermedium, YrE from diploid Th. elongatum, and Yr69, YrTP1, YrTP2, and YrCH7056 from Th. ponticum [35, 36]. To the best of our knowledge, this study is the first to demonstrate the successful transfer of a new and high-level stripe rust resistance gene from tetraploid Th. elongatum. Furthermore, the grain yield of line K17–841-1 is similar to that of the wheat parents SM482 and SM921. Accordingly, K17–841-1 represents an appropriate bridge breeding material for the introgression of alien genes to enhance wheat disease resistance.

Molecular markers are widely used to detect and track alien chromosomes and/or chromosomal segments during crossing and selection [10]. Conventional methods for developing markers, including SSRs, RAPDs, and amplified fragment length polymorphisms, have low success rates because of the high genomic sequence homology between Th. elongatum and common wheat. For example, using 40 SSR primers, You et al. [37] amplified 108 diploid Th. elongatum-specific fragments, but only one genome-specific molecular marker was developed. In another previous study, 94 diploid Th. elongatum-specific fragments were generated with 26 pairs of RAPD primers, but only three 1E or 3E chromosome-specific markers were developed [38]. Additionally, Ge et al. [39] developed one diploid Th. elongatum-specific marker from 65 specific fragments based on suppression subtractive hybridization. Furthermore, inter-SSR, inter-retrotransposon amplified polymorphism, and retrotransposon microsatellite amplified polymorphism techniques were applied to develop 13 specific sequence-characterized amplified region markers based on 34 specific diploid Th. elongatum sequences [40]. In a recent study, 135 randomly selected fragments of diploid Th. elongatum generated by SLAF-sequencing (SLAF-seq) were used to develop 89 specific molecular markers, including 61 7E chromosome-specific markers [11]. Chen et al. [25] developed 20 diploid Th. elongatum 1E chromosome-specific and two E genome-specific molecular markers based on SLAF-seq data, with an efficiency of up to 60%. Approximately 89% of the mined SNPs of diploid Th. elongatum may be authentic with respect to their polymorphisms and chromosomal locations as determined by a high-resolution melting curve assay [12]. Moreover, SLAF-seq data were used to develop 67 Th. ponticum-specific molecular markers from a wheat–Th. ponticum translocation line, with a success rate of up to 39% [19]. Furthermore, GBS, which is a robust, simple, and high-throughput next-generation sequencing approach that can decrease the complexity of large genomes, has been applied to develop many molecular markers in Triticale species [26, 41]. This approach can generate considerable sequence information and is appropriate for all whole-genome density distributions. The flexibility and low cost of GBS makes it ideal for genomics-assisted breeding [42]. To date, there have been no reports describing the development of tetraploid Th. elongatum-specific molecular markers. In this study, the GBS method was applied to develop 165 specific molecular markers, including 132 tetraploid Th. elongatum chromosome 1E-specific PCR markers, with a success rate of up to 70%. The greater success rate for our study relative to those of earlier investigations indicates that GBS technology may be used to efficiently develop alien-specific molecular markers in a wheat background.. The markers developed in this study may be useful for the high-throughput and accurate detection of tetraploid Th. elongatum DNA in diverse materials. Most importantly, the verification of their utility for analyzing a F2 breeding population implies that these specific markers are appropriate for tracing the stripe rust resistance gene carried on chromosome 1E of tetraploid Th. elongatum for the breeding of enhanced materials.

Even after removing K17–841-1 sequences that were more than 90% homologous with sequences from six other substitution lines (2E–7E), 12 markers still amplified specific sequences in the other substitution lines, confirming that the Th. elongatum E chromosomes sequences are highly homologous, likely because of chromosomal rearrangements. Earlier investigations involving biochemical and molecular markers as well as SLAF-seq revealed similar sequence identity levels for the diploid Th. elongatum [11, 43]. After analyzing the hybrids from crosses between tetraploid Th. elongatum and diploid Th. elongatum and Th. bessarabicum, Dvořák [44] concluded that tetraploid Th. elongatum is a segmental allotetraploid with two closely related E genomes. On the basis of examinations of chromosomal pairs, karyotypes, and C-banding patterns,Liu and Wang [45] also determined that tetraploid Th. elongatum is a segmental allotetraploid carrying two E genomes with slight differences. In a previous study involving a FISH with repetitive clones, two tetraploid Th. elongatum accessions were analyzed to uncover the significant chromosomal polymorphisms between the E genome of the putative diploid progenitor Th. elongatum and the other genome [18]. The verification of 153 markers specific to tetraploid Th. elongatum confirmed that 21, 106, 50, and 73 markers can detect E genome chromosomes in diploid Th. elongatum, Th. ponticum, Tr. caespitosum (StE), and Psa. athericum (StEP), respectively (see Additional file 2: Table S2). Our results provide further evidence that tetraploid Th. elongatum is an allotetraploid harboring two different E genomes, and Thinopyrum E genomes were differentiated during polyploidization events. Two specific markers, TTE1E-212 and TTE1E-222, were unique to diploid Th. elongatum, tetraploid Th. elongatum, and Th. ponticum, suggesting they can detect the E chromosomal DNA in all materials carrying the E genome. Interestingly, 58, 32, 5, 8, 28, 8, and 13 markers based on specific tetraploid Th. elongatum sequences successfully amplified products from Th. bessarabicum (Eb), Pse. libanotica (St), D. villosum (V), H. bogdanii (H), Ag. cristatum (P), S. cereale (R), and P. huashanica (Ns), respectively. Our specificity analysis indicated that the marker amplification frequencies are considerably higher in E, Eb, and St genomic species than in other wheat-related species, which is consistent with the findings of previous studies, in which the E genomes of Th. elongatum and Th. ponticum were more closely related to the Eb genome of Th. bessarabicum and the St genome of Pseudoroegneria species than to the other genomes [33, 46]. Additionally, 48 and 74 markers uncovered polymorphisms between Thinopyrum E-genome-containing species and Th. bessarabicum and Pse. libanotica, respectively, indicating that they will likely be applicable for investigating the genetic differences between E, Eb, and St genomes and for revealing the genetic diversity among species (populations) carrying E, Eb, and/or St genomes. The resulting information will further characterize these complex species and may be relevant for the genetic improvement of these important forage crops.

Conclusions

We characterized a wheat-tetraploid Th. elongatum 1E (1D) disomic substitution line based on GISH, FISH, and SSR analyses. This line is highly resistant to the stripe rust pathogen strains prevalent in China. Moreover, it represents an appropriate bridge breeding material for the introgression of alien genes to improve wheat disease resistance. Additionally, we analyzed GBS data to generate and validate a new 1E chromosome-specific, easy-to-use marker set that may be applicable for identifying and characterizing the tetraploid Th. elongatum chromosomes along with the chromosomes of all other wheat-related species and for investigating the genetic differences and phylogenetic relationships among E, Eb, St, and other closely-related genomes. The use of this marker set will further increase our understanding of these complex species.

Materials and methods

Plant materials

The plant materials used in the current study are listed in Table 5. The hexaploid Trititrigia line 8801 (2n = 6x = 42, AABBEE), which is tolerant to cold, drought, and salt stresses and resistant to Fusarium head blight, rust, and powdery mildew, was originally produced and identified at the Eastern Cereal and Oilseed Research Center, Ottawa, Canada [17]. The native wheat cultivars SM482, SM921, and SM969 exhibit superior agronomic traits, but are susceptible to the stripe rust pathogens prevalent in southwestern China. To produce wheat–tetraploid Th. elongatum derivative line, we first crossed 8801 with SM482, after which the resulting F1 plants were further crossed with SM921 to obtain the BC1F1 population. Seeds from the BC1F1 plants were bulked and advanced to the BC1F5 generation by single seed descent, ultimately resulting in the isolation of line K17–841-1 (Fig. 11). The F2 population comprising 80 individuals was derived from a cross between K17–841-1 and SM969. We developed six wheat–tetraploid Th. elongatum disomic substitution lines, TDS2E (2A), TDS3E (3D), TDS4E (4D), TDS5E (5D), TDS6E (6D), and TDS7E (7D), via the hybridization between native wheat cultivars and Trititrigia line 8801 (data not published). Wheat cultivar Chinese Spring (CS) was used as a positive control for the molecular marker analysis. Wheat line SY95–71 was used as a susceptible control for the evaluations of stripe rust responses. Regarding the GISH analysis, wheat cultivar J-11 was used as a source of blocking DNA, whereas tetraploid Th. elongatum accession PI531750 (2n = 4x = 28, EEEE) was used as a source of probe DNA. The molecular markers were validated with the following wheat-related species: Thinopyrum elongatum (2n = 2x = 14, EE), Thinopyrum ponticum (2n = 10x = 70, EEEEEEEEEE/StStStStEEEEEE), Thinopyrum bessarabicum (2n = 2x = 14, EbEb), Pseudoroegneria libanotica (2n = 2x = 14, StSt), Dasypyrum villosum (2n = 2x = 14, VV), Hordeum bogdanii (2n = 2x = 14, HH), Agropyron cristatum (2n = 2x = 14, PP), Secale cereale (2n = 2x = 14, RR), Psathyrostachys huashanica (2n = 2x = 14, NsNs), Trichopyrum caespitosum (2n = 4x = 28, StStEE), and Psammopyrum athericum (2n = 6x = 42, StStEEPP). Voucher specimens have been deposited in the herbarium of the Triticeae Research Institute, Sichuan Agricultural University, China (SAUTI).

Table 5 Plant materials used in this study
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Fig. 11
figure 11

Pedigree details of K17–841-1

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GISH and FISH analyses

Root tips from germinating seeds were treated with nitrous oxide for 2 h and 90% acetic acid for 10 min, after which they were digested with pectinase and cellulase (Yakult Pharmaceutical, Tokyo, Japan) [47]. Slides were then prepared for the GISH analysis as described by Han et al. [48]. The cetyltrimethyl ammonium bromide method was used to extract genomic DNA from the freshly collected leaves of tetraploid Th. elongatum PI531750 and wheat cultivar J-11 [49]. The PI531750 DNA was labeled with fluorescein-12-dUTP according to the nick translation method (Thermo Fisher Scientific, Eugene, OR, USA) and was used as the hybridization probe. The GISH analysis was completed according to the method described by Han et al. [50], with a 1:150 probe DNA:blocking DNA ratio. Specifically, 10 μL hybridization solution containing 2 × saline sodium citrate (SSC), 10% dextran sulfate, and 10 ng/μL labeled probe DNA together with blocking DNA was added to each slide. Samples were denatured by heating at 85 °C for 5 min, incubated at 37 °C for 8 h, and washed with 2× SSC. Finally, the chromosomes were counterstained with 4,6-diamino-2- phenylindole solution (Vector Laboratories, Burlingame, CA, USA). The GISH slides were examined with the BX-63 microscope (Olympus, Tokyo, Japan) and images were captured with a DP-70 CCD camera.

The GISH slides were washed sequentially with 70% (v/v) ethanol for 5 min, 2× SSC at 60 °C for 30 min, ddH2O for 10 min, and 100% (v/v) ethanol for 5 min. A FISH analysis was subsequently completed to identify the chromosomal constitution of line K17–841-1, with pSc119.2 and pTa535 as probes [47, 51]. The FISH was performed as described by Han et al. [48], with minor modifications. The probe mixture (0.35 μL each probe in 2× SSC and 1× TE buffer, pH 7.0; total volume = 10 μL) was added to a slide, covered with a coverslip, incubated in a moist box at 37 °C for 2 h, and then washed with 2× SSC at room temperature. The FISH signals were observed with the BX63 microscope (Olympus). Images were captured with the DP-70 CCD camera and analyzed with Adobe Photoshop software.

SSR marker analysis

Primer pairs for four SSR markers (wmc147, wmc222, gwm337, and Xcfd63) on wheat chromosomes 1DS and 1DL were used to characterize the 1D chromatin in the wheat–tetraploid Th. elongatum line K17–841-1. Details regarding all primers are listed in Table 6. Additionally, CS, SM482, and SM921 were used as positive controls, whereas 8801 was used as a negative control. The PCR amplification was completed as described by Somers et al. [52], with minor modifications.

Table 6 Sequences of wheat 1DS and 1DL SSR markers
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Agronomic trait evaluation

The morphological traits of K17–841-1 and its parents were evaluated in a field trial in Wenjiang, Sichuan province, China, with three replicates in the 2017–2018 and 2018–2019 growing seasons. For each replicate, 15 grains of each line were evenly planted in 1.5-m rows separated by 0.3 m. The plant height, tiller number, spike length, spikelet per spike, grains per spike, thousand-grain weight, and seed setting rate were evaluated for 10 samples per replicate. Significant differences in traits were determined with the SAS 8.2 program (SAS Institute Inc., Cary, NC, USA).

Stripe rust resistance screening

The smear method [53] was used to evaluate the responses of adult K17–841-1, 8801, SM482, SM921, and SY95–71 plants to a mixture of Pst races (CYR-32, CYR-33, CYR-34, and V26/Gui22–14) in a field trial in Chengdu, Sichuan, China during the 2017–2018 growing season. To assess the utility of the developed tetraploid Th. elongatum 1E chromosome-specific markers, the K17–841-1/SM969 F2 individuals as well as 8801, SM482, SM921, SM969, and SY95–71 seedling were evaluated for their reactions to Pst race CYR-34 in a growth chamber. The plants were inoculated at the two-leaf stage and the stripe rust reaction of the first leaf of each plant was evaluated at 14 days after inoculation. Wheat line SY95–71 was used as the susceptible control. The stripe rust responses were evaluated with three replicates as described by Li et al. [17]. The stripe rust IT was based on the following scale: 0, 0;, 1, 2, 3, and 4, in which 0 = immunity, 0; = necrotic flecks, and 1–4 = increasing sporulation and decreasing necrosis or chlorosis. Plants with an IT of 2 or lower were considered resistant, whereas plants with an IT of 3 or 4 were considered susceptible.

Genotyping-by-sequencing, sequence alignment, and tetraploid Th. elongatum 1E chromosome– specific fragment acquisition

Total genomic DNA was extracted from fresh young leaves with the cetyltrimethyl ammonium bromide method, after which the DNA concentration was adjusted to 150 ng/μL. Genomic DNA samples of diploid Th. elongatum PI531718, PI531750, 8801, K17–841-1, and six wheat–tetraploid Th. elongatum (2E-7E) chromosomal substitution lines were subjected to GBS, which was completed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). High-Quality DNA libraries were constructed and sequenced with the Illumina HiSeq™ system. The raw reads in FASTQ format were filtered by removing low-quality reads and reads with adapter and/or poly-N sequences to obtain clean reads (140 bp). Tetraploid Th. elongatum 1E chromosome-specific sequences were obtained as follows. First, the high-quality K17–841-1 sequences were compared with the CS reference genome sequences (https://urgi.versailles.inra.fr/download/iwgsc/IWGSC_RefSeq_Assemblies/v1.0/), after which the sequences with identities greater than 23% were eliminated [54]. Second, the sequences were compared with those of 8801 and PI531750 acquired by GBS in this study, and the sequences with identities greater than 23% were selected. Finally, the retained K17–841-1 sequences were compared with the sequences of PI531718 and the six chromosomal substitution lines. Sequences with identities exceeding 90% were removed. The remaining sequences were considered to be specific to chromosome 1E of tetraploid Th. elongatum.

Development and validation of the tetraploid Th. elongatum-specific molecular markers

On the basis of these specific sequences, PCR primers were designed with the Primer3 Plus online tool (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and then synthesized by TSINGKE (Chengdu, China). Details regarding the PCR primers are presented in Additional file 1: Table S1. The amplified products were examined by 3% agarose electrophoresis. The markers that amplified specific sequences in PI531750, 8801, and K17–841-1, but not in SM482, SM921, CS, PI531718, and the 2E-7E substitution lines, were identified as tetraploid Th. elongatum 1E chromosome-specific molecular markers. The stability, repeatability, and specificity of these markers were validated in the CS, PI531750, 8801, and K17–841-1 lines as well as in 11 wheat-related species.

The PCR amplifications were completed in a reaction volume of 25 μL, which included 1.0 μL template DNA (100 ng/μL), 12.5 μL 2× Taq Master Mix for PAGE (Dye plus), 1.0 μL each primer (10 μM), and 9.5 μL ddH2O. The PCR program was as follows: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, appropriate annealing temperature of 50–60 °C for 30 s, and 72 °C for 1 min; 72 °C for 10 min.