Introduction

Wheat stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriks., is one of the most damaging diseases of wheat and seriously reduces the yield and quality of wheat worldwide (Roelfs et al. 1992). Yield losses caused by stripe rust can reach 75–100% in the most severe epidemic years under weather conditions that favor the pathogen (Chen 2005b). The most economical, effective and environment-friendly approach for controlling this disease comprises the cultivation of resistant cultivars (Line and Chen 1995). More than 70 genes that confer resistance to stripe rust have been identified in wheat (McIntosh et al. 2017). However, rapid changes in the virulence of pathogen populations easily overcome the resistance genes in released wheat cultivars. Hence, new resistance resources for stripe rust are continuously required to breed new resistant wheat cultivars. The relatives of wheat possess plenty of desirable genes, including disease-resistance and yield-related genes, for wheat breeding, and these genes can be introduced into wheat by wide crosses and genetic manipulation (Jiang et al. 1993).

Thinopyrum intermedium (Host) Barkworth & D.R. Dewey [syn. Agropyron intermedium (Host) Beauvoir and Elytrigia intermedia (Host) Nevski] is a perennial hexaploid species (2n = 6x= 42) with the JJsSt genome (Chen et al. 1998). This species has been proved to be a potential source of genes for improving disease resistance, yield potential (Chen 2005a; Tang et al. 2000) and tolerance of salt stress (Li et al. 2008). To date, numerous intergeneric hybrids and cytogenetic stocks, including wheat–Thinopyrum partial amphiploids (Fedak and Han 2005; Sun 1981; Yang et al. 2006) and wheat addition, substitution, and translocation lines (He et al. 1989; Li et al. 2015; Liu et al. 2011), have been developed. However, because this species is distributed in different regions worldwide, the extensive genetic diversity present in different accessions of Th. intermedium enables the continuous introduction individual chromosomes from Th. intermedium that confer new traits into different genetic background of common wheat.

During the process of transferring desirable genes from Th. intermedium into common wheat, a disomic addition line with stripe rust resistance and a stable double spikelet appearance was obtained. In the present study, we used genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH) and molecular marker techniques to identify alien chromosomes in wheat lines derived from crosses with Th. intermedium.

Materials and methods

Plant materials

The Th. intermedium accession PI595172 and the Pseudoroegneria spicata (St genome, 2n = 2x = 14) accession PI232131 were obtained from the Triticeae Research Institute, Sichuan Agricultural University. Line 14-569 was a disomic addition line isolated in the progeny of which the partial amphiploid of wheat and Th. intermedium, TAI7045 (2n = 56) was repeatedly backcrossed to wheat cultivar Chuannong 10 (CN10). CN10 is a wheat–rye 1RS/1BL translocation cultivar with high susceptibility to stripe rust (Yang et al. 2014). The rye, Secale cereal L. ‘Jingzhou Heimai’ (genome RR) and the wheat cv. Chinese Spring (CS) are maintained at the Ya’an Experimental Station of Sichuan Agricultural University, China.

Evaluation of yield-related traits and reactions to stripe rust inoculation

The evaluation of yield-related traits and reactions to rust inoculation were carried out during the growing seasons of 2014 and 2015 in a field at the Ya’an Agricultural Experimental Station, Sichuan Agricultural University. Plants were arranged separately in a randomized complete block design using two replicates. Twelve plants were randomly selected from line 14-569 and its wheat parent CN10, to assess yield-related traits, such as tillering, spike length, the numbers of kernels, spikelets, and double spikelets per spike, the 1000-kernel weight, and plant height. Student’s t test was used to test for significant differences between line 14-569 and its wheat parent CN10. A mixture of races of P. striiformis, which mainly consisted of CYR31, CYR32, CYR33 and CYR34, which are prevalent in China, was provided by the Plant Protection Institute, Sichuan Academy of Agricultural Science and was artificially inoculated in early spring. The infection type (IT) was recorded when uredinia were fully developed on the leaves during the flowering stage.

Cytogenetic analysis

Seeds were germinated in Petri dishes at 23 °C in the dark until the roots reached a length of 1–2 cm. The roots tips were excised from the germinated seeds and treated with nitrous oxide gas for 2 h. Then, the treated root tips were fixed in ice-cold 90% acetic acid and stored in 70% ethanol at 4 °C until used (Kato et al. 2004). Chromosome spreads of materials were prepared using the methods described by Han et al. (2006). Pollen mother cells (PMCs) were collected from young spikes and fixed in absolute ethanol/chloroform/glacial acetic acid (6:3:1, v/v/v). Meiotic chromosomes were flattened on a slide in a drop of 1% acetocarmine.

The genomic DNA from Th. intermedium and Ps. spicata was labeled with digoxigenin-11-dUTP (fluorescein isothiocyanate detected by yellow-green fluorescence) (Roche), and the rye DNA was labeled with Texas Red-5-dUTP (Invitrogen). Sheared genomic DNA from CS wheat was used as a blocker. Oligo-pSc119.2-1 and Oligo-pTa535-1 were labeled at the 5′ end with 6-carboxyfluorescein (green color) and 6-carboxytetramethylrhodamine (red color), respectively. The two oligonucleotide probes were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd. (Shanghai, China). GISH and ND-FISH were performed as described by Han et al. (2006) and Fu et al. (2015), respectively.

Molecular marker analysis

Total genomic DNA was extracted from the fresh leaves. Polymerase chain reaction (PCR)-based landmark unique gene (PLUG) primers were employed (Ishikawa et al. 2009). The PCR amplification conditions were as follows: after denaturation for 5 min at 94 °C, amplification was performed for 35 cycles, each of which consisted of 45 s at 94 °C, 45 s at 57 °C, and 2 min at 72 °C, followed by incubation for 7 min at 72 °C. Aliquots of 3 μL of each PCR amplification product after enzyme digestion was separated in 8% non-denaturing polyacrylamide gel and visualized by staining with silver.

Results

Morphology and stripe rust resistance

Line 14-569 displayed the stable double spikelets per node emerging in the lower middle portion of a spike (Fig. 1a, b). The average numbers of double spikelets and seeds produced by double spikelets per spike were 7 and 8, respectively (Table 1). Line 14-569 produced plump red seeds that were similar to those of CN10, but these seeds had a higher 1000-kernel weight (about 52 g) (Table 1 and Fig. 1c). This line also produced more spikelets and seeds per spike and had a greater spike length. Student’s t test showed that line 14-569 was very significantly different from (P < 0.01) its wheat parent CN10 in terms of most tested characters, except plant height which was only significantly different a level of P < 0.05 (Table 1). These results for agronomic traits indicated that line 14-569 probably carried genes that were beneficial for agricultural production.

Fig. 1
figure 1

Morphology of whole plant (a), spike (b), seeds (c) and stripe rust resistance (d) of common wheat CN10 (left) and line 14-569 (right)

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Table 1 Morphological characters of line 14-569 and its wheat parent CN10
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When artificially inoculated with a mixture of races of P. striiformis, including CYR31, CYR32, CYR 33, and CYR34, during the growing season, line 14-569 was highly resistant (IT 0) to these isolates, whereas the wheat parent CN10 was highly susceptible (IT 4) (Fig. 1d), which implied that line 14-569 might carry a gene or genes that conferred resistance to stripe rust.

Cytogenetic analysis

Root tip chromosome counts and observations of meiosis of PMCs on twenty plants from line 14-569 indicated that all these plants had a constant number of 44 chromosomes and formed 22 bivalents at metaphase I of meiosis, which demonstrated that line 14-569 might be a stable alien disomic addition line.

GISH analysis that employed DNA from Th. intermedium as probe on mitotic metaphase chromosomes of line 14-569 demonstrated that two pairs of chromosomes that gave rise to strong yellowish-green hybridization signals were present, of which one pair exhibited signals uniformly along their entire length, whereas the other pair only exhibited strong GISH signals in one of the telomeric regions (Fig. 2a). Sequential ND-FISH analyses using the Oligo-pSc119.2-1 and Oligo-pTa535-1 probes revealed that the pair of chromosomes labeled in the telomeric regions with the DNA from Th. intermedium might be 1RS/1BL translocations (Fig. 2b). This result was corroborated by further sequentially using the DNA from S. cereale and Th. intermedium as probes (Fig. 2c, d). The 1RS/1BL translocation chromosomes apparently originated from the wheat parent CN10. This result also indicated that the terminal DNA sequence of the rye 1RS chromosome has high homology with a DNA sequence of Th. intermedium. When the DNA from Ps. spicata was used as a probe, one pair of chromosomes exhibited the same hybridization signals as that the DNA from Th. intermedium was used as a probe (Fig. 2e), which indicated that this pair of chromosomes belonged to the St-genome, according to Chen et al. (1998). This result demonstrated that one pair of St genomic chromosomes was added to line 14-569, and it contained one pair of 1RS/1BL translocation chromosomes in its wheat complement.

Fig. 2
figure 2

In situ hybridization patterns of line 14-569. a, d GISH patterns using genomic DNA from Th. intermedium (green) as a probe. b ND-FISH pattern using Oligo-pSc119.2-1 (green) and Oligo-pTa535-1 (red) as probes. c GISH pattern using genomic DNA from rye (red) as a probe. e GISH pattern using genomic DNA from Ps. spicata (green) as a probe. The white arrows indicate 1RS/1BL translocation chromosomes and the red arrows indicate added Th. intermedium chromosomes. (Color figure online)

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Molecular marker analysis

On the basis of syntenic region in rice, Ishikawa et al. (2009) developed PLUG primers, of which the amplified fragments presumably corresponded to similar linkage group(s) in wheat genomes owing to the similarities in gene structure between rice and wheat (Ishikawa et al. 2007). In this study, a total of 105 PLUG markers distributed over homeologous groups 1–7 in wheat were employed to amplify specific bands due to line 14-569, CN10 and CS wheat, Th. intermedium and Ps. spicata. Ten PLUG markers, namely, TNAC1244, TNAC1267, TNAC1280, TNAC1307, TNAC1312, TNAC1344, TNAC1364, TNAC1379, TNAC1383 and TNAC1648, produced specific bands identical to those of Th. intermedium and Ps. spicata in the PCR profile of line 14-569 when CS and its wheat parent CN10 wheat were used as controls. As shown in Fig. 3, the PLUG markers TNAC1280 and TNAC1244 amplified diagnostic fragments of the long and short arms, respectively, of chromosomes in homeologous group 3, in accordance with descriptions by Ishikawa et al. (2009). Of the other eight PLUG primers, TNAC1307, TNAC1312, TNAC1383, and TNAC1648 amplified diagnostic fragments of the short arms, whereas the other four primers amplified diagnostic fragments of the long arms, of chromosomes in homeologous group 3. These results suggested that the added chromosomes from Th. intermedium in line 14-569 belonged to homeologous group 3. Therefore, we deduced that line 14-569 was a wheat–Th. intermedium 3St disomic addition line.

Fig. 3
figure 3

PCR amplification profiles obtained using the PLUG primers TNAC1244 (a) and TNAC1280 (b). The white arrows indicate specific bands of the line 14-569 that are identical to those of Th. intermedium and Ps. spicata

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Discussion

Wheat-alien disomic addition lines are considered to be optimal bridge materials for transferring desirable genes into wheat and are also frequently used to study the expression of genes and the evolution of species (Zhong et al. 2002). Therefore, it is important to determine the genetic constitution of alien addition lines in order to produce translocations that compensate for the transfer of genes from alien chromosomes into wheat genomes.

In this study, we have identified one pair of chromosomes from Th. intermedium in wheat line 14-569 which exhibits a high level of resistance to stripe rust, which is currently a major global problem. At present, this chromosome is available as an addition line, which differs from normal wheat in several characters apart from resistance, such as 1000-kernel weight and double spikelets. Hence, it will be worthwhile to study line 14-569 further, and it can be used as a better bridge material for breeding wheat with high-yields and stripe rust resistance.

Sequential GISH and ND-FISH analyses indicated that one pair of St genomic chromosomes from Th. intermedium was added to the wheat complement and that the wheat complement included one pair of 1RS/1BL wheat rye translocation chromosomes in this line. However, in situ hybridization was not able to assign the homeologous group of alien chromosomes in a wheat background. Hu et al. (2012) reported that PLUG markers were effective tools for producing markers that were specific to Thinopyrum chromosomes. PLUG markers that corresponded to the third linkage group in wheat generated diagnostic fragments from line 14-569 that were identical to those of Th. intermedium and Ps. spicata (Fig. 3), which suggested that the St chromosome in line 14-569 belonged to homeologous group 3, and it was hence assigned as the 3St chromosome.

Several different St chromosomes from Th. intermedium have been transferred to a wheat background. A 1St (1D) substitution line (Hu et al. 2011), a 1St disomic addition line (Li et al. 2013) and a 7St disomic addition line (Song et al. 2013) in different wheat background have been reported. Two other well-known groups of addition lines, namely, the Z and L series, were identified by Hu et al. (2014) using DNA markers, and they found that the Z3, L4, L6 and L7 lines were 1St, 4St, 2St and 6St disomic addition lines, respectively. However, there have been no reports of 3St alien disomic addition lines in a wheat background until now.

Several stripe rust resistance genes have been mapped on the chromosomes of homeologous group 3 in wheat. Yr76 and Yr57 have been located on the short arms of the 3AS (Xiang et al. 2016) and 3BS (Randhawa et al. 2015) chromosomes, respectively. Both Yr45 and Yr71 have been located on the long arms of the 3D chromosome (Bariana et al. 2016; Li et al. 2011). The 3Ns chromosome from Psathyrostachys huashanica Keng might also carry stripe rust resistance gene(s) (Kang et al. 2011). Recently, Zhang et al. (2018) reported recessive stripe rust resistance gene(s) might locate on the 3V chromosome of Dasypyrum villosum. No stripe rust resistance gene present on the 3St chromosome has been reported, although stripe rust resistance genes from Th. intermedium have been transferred into wheat (Chen 2005a; Hu et al. 2011; Song et al. 2013). In the present study, line 14-569 appeared to be highly resistant to all the races of stripe rust predominant in China, which implied that the 3St chromosome of Th. intermedium might carry stripe rust resistance gene(s).

In general, a wheat spike normally bears one spikelet per rachis node, and it is rare that a rachis node carries two or more spikelets. Line 14-569 possesses the character of double spikelets, with an average of eight double spikelets per spike. Du et al. (2013) reported that a 6Ns disomic addition line derived from P. huashanica carried the same double spikelets as line 14-569. These results indicated that new character(s) might be produced in the progeny of hybrids of wheat with alien species. However, some aneuploid wheat plants have also possessed double spikelets in a rachis node, for example, hexaploid wheat plants that were nullisomic for the 2A and 2D chromosomes produce double spikelets (Sears 1954). Further research will be needed to establish the association between the 3St chromosome and the specific spike trait.