Introduction

Wild relatives of common wheat are a vast resource of agronomic traits that can be transferred into wheat and have been used to broaden the genetic diversity of wheat (Dewey 1984; Sharma and Gill 1983) by wide hybridization and chromosome engineering. The production of alien disomic addition lines (DALs) in wheat is an initial step in mining and chromosome location of potentially useful genes prior to transfer to wheat chromosomes. Production of a complete set of wheat-alien addition lines allows determination of the homoeologous relationships between alien and corresponding wheat chromosomes, and dissection of the genetic effects of individual alien chromosomes in wheat background (Jiang et al. 1994). Using such genetic stocks, potentially useful genes for grain quality, and resistance to biotic and abiotic stresses have also been identified and characterized (Fasahat et al. 2012; Jauhar et al. 2009).

Roegneria ciliaris (Trin.) Nevski (2n = 4x = 28, genome ScScYcYc), a perennial tetraploid species, within the Triticeae, is widely distributed in China, and has been cultivated as fine pasture fodder and lawn grass species. It has been also reported as a source of resistance to many wheat diseases, including Fusarium head blight, wheat streak mosaic virus, and barley yellow dwarf virus (Sharma et al. 1984; Weng and Liu 1989; Liu et al. 1990; Tomohiro 1997; Yang et al. 1999). Efforts have been made to produce hybrids between wheat and R. ciliaris since the 1980s (Jiang et al. 1992; Sharma and Gill 1983). Muramatsu et al. (1983) developed an alloplasmic amphiploid of R. ciliaris and wheat cultivar (cv.) Inayama komugi Jiang et al. (1992, 1993) developed DALs DA1Sc and DA1Yc, but failed to identify DALs involving the other 12 chromosomes due to presumed alloplasmic effects. To overcome these effects, Wang et al. (2001) obtained a hybrid between T. aestivum cv. Chinese Spring (as female parent) and the Inayama komugi-R. ciliaris amphiploid. Among the derived euplasmic progenies, 10 wheat-R.ciliaris alien chromosome addition lines were developed, in which 5 were DALs, i.e., DA2Sc, DA3Sc, DA5Yc, DA7Sc and DA7Yc. Kong et al. (2008) showed that 18 wheat-R. ciliaris addition lines had been developed. However, due to lack of sufficient and accurate techniques for distinguishing the 14 R. ciliaris chromosome pairs, the identities of the alien chromosomes in the lines remained unclear.

The tetraploid R. ciliaris is presumed to contain two different genomes. However, the two genomes show high affinity and the differentiation has been very difficult (Liu et al. 2006; Lu and Bothmer 1989). Morris and Gill (1987) attempted to differentiate the Sc- and Yc-genomes based on N- and C-banding patterns of R. ciliaris and the diploid Sc-genome species Pseudoroegneria spicata. Svitashev et al. (1998) reported the possibility of differentiating the two genomes by use of several RAPD primers that were specific for either the Sc- or Yc-genomes. Wang et al. (2001) reported that FISH use of repetitive sequence pCbTaq4.14 as probe could identify the genomic affinities of the Sc- or Yc-genomes. Wang et al. (2010) distinguished R. ciliaris chromosomes from those of the St, P, and Y genomes using GISH–FISH. More recently, Wang et al. (2017) reported that St2-80 is a potential and useful FISH marker that can be used to distinguish St genome chromosomes from those of other Triticeae genomes. Although these methods might be feasible to assign homoeologous chromosomes to specific genomes, the lack of suitable genetic resources makes it difficult to verify the accuracy. Development of a complete set of wheat-R. ciliaris DALs would permit identification of all 14 chromosome pairs, and comparisons by FISH and amplification patterns with molecular markers would be helpful in assigning homoeologous chromosomes to the Sc or Yc genomes.

The objectives of this study were to: (a) identify and characterize new wheat-R. ciliaris alien chromosome lines for the purpose of obtaining a complete set of wheat-R. ciliaris DALs; (b) establish a FISH-based karyotype of R. ciliaris chromosomes using a set of DALs; (c) develop molecular markers specific for each R. ciliaris chromosome; and (d) assign the genome affinity of each R. ciliaris chromosome. Fulfilment of these objectives will lay a solid foundation better utilization of useful genes from R. ciliaris in wheat breeding programs.

Materials and methods

Plant materials

A Triticum aestivum cv. Inayama komugi-R. ciliaris amphiploid (2n = 10x = 70, genome AABBDDScScYcYc), developed by Dr. Muramatsu et al. (1983), and seeds of it along with Inayama komugi (Ik) were obtained from the Wheat Genetic Resource Center, Kansas State University, Manhattan, Kansas, USA. R. ciliaris (Accession No. W614249) was introduced from the Western Regional Plant Introduction Station, Pullman, Washington, USA.

Single backcross populations derived from a cross between the amphiploid and wheat cv. Chinese Spring (CS) were used to develop disomic addition lines (DALs) (Table 1). Wheat varieties Sumai 3 and Mianyang 8545 were used as resistant and susceptible controls in Fusarium head blight (FHB) tests conducted at the Nanjing Agricultural University, Jiangsu Experiment Station. All materials are maintained at the Cytogenetics Institute, Nanjing Agricultural University (CINAU).

Table 1 Materials used in this research and their chromosome constitutions
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Molecular cytogenetic analysis

Chromosome preparations from root tip cells (RTC) were performed as described by Chen et al. (1995) with minor modifications.

GISH and FISH were conducted according to Zhang et al. (2004). For GISH, genomic DNA of R. ciliaris was labeled with fluorescein-12-dUTP by nick translation and used as a probe. Genomic DNA of CS was used for blocking with a probe: blocker ratio of 1:50. After GISH, the chromosomes were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI).

Afa-family sequences were amplified by two-round PCR from R. ciliaris using Afa-specific primer pair (AS-A 5′-GATGATGTGGCTTTGAATGG-3′; AS-B 5′-GCATTTCAAATGAACTCTGA-3′). The first round used genomic DNA of R. ciliaris and products were used as a template for the second round PCR, in which the dUTP in the reaction solution was substituted by digoxigenin-11-dUTP (Roche). The products of this reaction were designated as RcAfa and used for FISH.

The three other FISH probes were repetitive DNA sequences pTa71 (45S rDNA), pTa794 (5S rDNA) and (GAA)10. The (GAA)10 was synthesized according to Cuadrado et al. (2008). The pTa71 clone is a 9 kb wheat rDNA repeat unit (Gerlach and Bedbrook 1979) containing 18S, 5.8S and 26S rRNA genes, and the intergenic spacer was used as the probe for 45S rDNA. Clone pTa794 is a BamHI fragment of 5S rDNA, which has a 120 bp coding sequence (Gerlach and Dyer 1980). pTa71 and pTa794 were labeled with digoxigenin-11-dUTP by nick translation. Chromosomes were counter stained with DAPI.

FISH signals were visualized under a fluorescence microscope (Olympus BX60), and images were captured by Spot32 CCD camera and analyzed by Adobe Photoshop software.

Molecular marker analysis

A total of 1845 EST-SSR primer pairs evenly distributed in the seven homoeologous groups of wheat were used for molecular marker analysis. The primer pairs were designed from EST sequences that were physically mapped to wheat chromosomes (http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi), and were synthesized by Invitrogen Life Technologies (Shanghai). The Ik-R. ciliaris amphiploid, common wheat, and wheat-R. ciliaris DALs were used for PCR to determine the specificity of markers for individual R. ciliaris chromosomes.

Total genomic DNA was extracted with SDS according to Sharp et al. (1989) and Devos et al. (1992). PCR was performed in 10 µL reaction volumes containing 25 ng of genomic DNA, 2 µmol/L of each primer pair, 2.5 mmol/L of dNTPs, 2.5 mmol/L of MgCl2, 1×PCR buffer (10 mmol/L Tris–HCl, pH 8.5, 50 mmol/L KCl), and a 0.5 unit of Taq DNA polymerase. Amplification was performed for 3 min at 94 °C, followed by 32 cycles of 30 s at 94 °C, 45 s at 50–60 °C depending on the individual primers, 1 min 10 s at 72 °C, and a final step of 10 min at 72 °C. PCR products were separated in 8% polyacrylamide gels (acrylamide:bisacrylamide = 19:1 or 39:1) and visualized by silver staining.

Evaluation of FHB response

The Ik-R. ciliaris amphiploid, Ik, CS and a complete set of CS-R. ciliaris DALs were evaluated for FHB response in the greenhouse using the single-floret inoculation method over five successive growing seasons (2012–2017). Wheat varieties Sumai 3 and Mianyang 8545 were used as resistant and susceptible controls, respectively. Monosporic isolate Fg0609 of F. graminearum (kindly provided by Dr. Xu Zhang, Jiangsu Academy of Agricultural Sciences) was used in inoculations. At anthesis, 10 μL of a conidial suspension containing 1 × 105 macro conidia/mL was injected into the first floret of a central spikelet in one spike of each plant. FHB severity was measured as the percentage of diseased spikelets per spike at 21 days post-inoculation (Xiao et al. 2011).

Results

Screening and development of molecular markers specific for R. ciliaris chromosomes

A total of 1845 EST-SSR primer pairs previously mapped to seven homoeologous groups of wheat chromosomes were used for PCR using CS, Ik, Ik-R.ciliaris amphiploid and R. ciliaris as templates. Five hundred and fifty seven primers (30.2%) produced polymorphisms between wheat and R. ciliaris. Polymorphism rates for the seven homoeologous groups varied from 17.8 to 43.4% (Table 2).

Table 2 Analysis of molecular marker polymorphisms between wheat and R. ciliaris
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Further PCR of the polymorphic markers performed on the wheat-R. ciliaris addition lines showed that 162 could be used as chromosome-specific markers for R. ciliaris. Individual R. ciliaris chromosomes were assigned to homoeologous groups based on known allocations of the markers to wheat chromosomes. Although the homoeologous relationships between R. ciliaris and wheat chromosomes were well conserved, there were exceptions, including 1Yc, 2Sc, 7Sc and 7Yc (Table S2). Bands of five markers specific for 1Yc (1EST-111, 1EST-259, 1EST-1149, 1EST-258, 1EST-1134) were present in DA7Sc and DA7Yc; bands of ten markers specific for 7Sc and 7Yc (7EST-38, 7EST-203, 7EST-211, 7EST-216, 7EST-218, 7EST-220, 7EST-233, 7EST-234, 7EST-213, 7EST-138) were present in DA1Yc; bands of three markers specific for 2Sc (2EST-971, 2EST-983, 2EST-997) were present in DA7Sc; and bands of one marker specific for 7Sc (7EST-133) were present in DA2Sc. Thus, chromosome rearrangements may have occurred between groups 1, 2 and 7.

Characterization of a complete set of wheat-R. ciliaris DALs

Jiang et al. (1992, 1993) developed two alloplasmic wheat-R. ciliaris addition lines carrying chromosomes 1Sc#1 and 1Yc#1. Wang et al. (2001) developed euplasmic DALs involving only five other chromosomes (DA2Sc#1, DA3Sc#1, DA5Yc#1, DA7Sc#1, DA7Yc#1). To develop a complete set of wheat-R. ciliaris DALs, we repeated the cross between the CS and Ik-R. ciliaris amphiploid, and DALs carrying the remaining seven R. ciliaris chromosomes were successfully identified. The complete set of wheat-R. ciliaris DALs including those produced by Jiang et al. (1992, 1993) and Wang et al. (2001) is described below.

DALs DA1Sc and DA1Yc

Alloplasmic DALs DA1Sc and DA1Yc developed by Jiang et al. (1992, 1993) were re-characterized by GISH/FISH and marker analysis. A total of 27 R. ciliaris-specific markers were developed based on wheat ESTs previously mapped on homoeologous group 1. PCR of a complete set of wheat-R. ciliaris DALs showed that these markers amplified specific amplicons only in DA1Sc and DA1Yc; however, the amplification patterns for the two DALs were different, confirming that they both belong to homoeologous group1 but are from different genomes (Tables 3, S1). Twenty two ESTs were on 1Sc, such as 1EST-255 (Table S1, Fig. 3a) and 3 were on 1Yc. 1EST-258 and 1EST-1134 had co-dominant markers that could be used to distinguish 1Sc and 1Yc (Tables 3, 4). GISH/FISH using four probes, pTa794, pTa71, (GAA)10 and RcAfa showed that 1Sc is metacentric (Fig. 1a) and 1Yc is submetacentric (Fig. 1b); however, neither chromosome had FISH signals for probes pTa794 and (GAA)10, although both had intensive RcAfa signals. 1Sc and 1Yc had similar signal distributions at the telomeric regions (Figs. 1a, b, 4b). However, 1Sc was differentiated from 1Yc because 1Sc had pTa71 signals on the short arm, confirming it as a satellited (SAT) chromosome (Fig. 4a). The spike morphologies of the two DALs were different. DA1Sc had similar morphology to Ik and DA1Yc was awned and had a rod-like spike (Fig. 2).

Table 3 Molecular markers specific for individual R. ciliaris chromosome
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Table 4 Evaluation of Ik-R.c amphiploid and DALs for reaction to FHB
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Fig. 1
figure 1

Chromosome sequential GISH/FISH (right) on root tip cells of a set of wheat-R. ciliaris DALs. Arrows show one pair of R. ciliaris chromosomes using genomic DNA of R. ciliaris (green), RcAfa (red) and (GAA)10 (green) as probes. a–n are GISH (left) and FISH (right) for addition lines DA1Sc, DA1Yc, DA2Sc, DA2Yc, DA3Sc, DA3Yc, DA4Sc, DA4Yc, DA5Sc, DA5Yc, DA6Sc, DA6Yc, DA7Sc and DA7Yc, respectively. Scale bars, 10 μm

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Fig. 2
figure 2

Spike morphology of a complete set of wheat-R. ciliaris DALs. CS: Chinese spring; Ik: Inayama komugi; Amphiploid: Ik–R.c amphiploid; R.c: R. ciliaris; 5–18: DA1Sc, DA1Yc, DA2Sc, DA2Yc, DA3Sc, DA3Yc, DA4Sc, DA4Yc, DA5Sc, DA5Yc, DA6Sc, DA6Yc, DA7Sc and DA7Yc

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DALs DA2Sc and DA2Yc

Eighteen R. ciliaris-specific markers were developed based on homoeologous group 2 ESTs of wheat. Specific amplicons from these markers were produced only in DA2Sc (Wang et al. 2001) and line 2014-2015K108 with 2n = 44. However, their amplification patterns were different indicating that the added chromosomes in 2014-2015K108 belonged to homoeologous group 2, and could be designated as 2Yc (Tables 3, S1). Three of the 18 markers were 2Sc-specific, 7 were 2Yc-specific, and the remaining 8 were co-dominant, such as 2EST-705 (Table S1, Fig. 3b). GISH/FISH showed that 2Yc is metacentric (Fig. 1d). Chromosomes 2Sc and 2Yc showed different FISH patterns with two repetitive DNA probes. 2Sc had RcAfa and (GAA)10 signals at the centromeric regions of the long arm (Figs. 1c, 4b). 2Yc had RcAfa signals at the telomeric regions of both arms, and (GAA)10 signals were present only at the distal region of the long arm (Figs. 1d, 4b). Plants carrying DA2Sc and DA2Yc were slender and the spikes had tenacious glumes (Fig. 2) that are typical for alien group-2 chromosome addition lines.

Fig. 3
figure 3

Amplification of specific markers in a set of wheat-R. ciliari DALs and their parental lines. Electrophoresis of a: 1EST-255; b: 2EST-705; c: 3EST-186; d: 3EST-147; e: 4EST-100; f: 4EST-19; g: 5EST-10; h: CINAU15; i: 7EST-138. Arrows show specific bands of R. ciliaris. m: DNA ladder DL2000; 1, Chinese Spring; 2, Ik: Inayama komugi; 3, Ik-R.ciliaris amphiploid; 4, R. ciliaris; 5, DA1Sc; 6, DA1Yc; 7, DA2Sc; 8, DA2Yc; 9, DA3Sc; 10, DA3Yc; 11, DA4Sc; 12, DA4Yc; 13, DA5Sc; 14, DA5Yc; 15, DA6Sc; 16, DA6Yc; 17, DA7Sc; 18, DA7Yc

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Fig. 4
figure 4

FISH-based karyotype of R. ciliaris chromosomes. a FISH pattern of R. ciliaris chromosomes 1Sc and 5Sc using pTa71 (yellow) as probes. b Arranged R. ciliaris chromosomes after FISH using RcAfa (red) and (GAA)10 (green) repetitive DNA as probes. c FISH pattern of R. ciliaris chromosome 5Yc using pTa794 (yellow) as probe. Scale bar, 10 μm. d Idiogram of FISH-based karyotype of R. ciliaris chromosomes showing the distribution of RcAfa (red), (GAA)10 (green), pTa71 (yellow) and pTa794 (orange) signals

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DALs DA3Scand DA3Yc

Eighteen R. ciliaris-specific markers were developed based on homoeologous group 3 ESTs of wheat. Specific amplicons were amplified in DA3Sc (Wang et al. 2001) and line 2014-2015K132 (2n = 44) and showed a different amplification pattern from DA3Sc. This indicated that the added chromosome also belonged to homoeologous group 3, but belonged to the Yc genome. Two markers were 3Sc specific, such as 3EST-186 (Table S1, Fig. 3c), 6 were 3Yc-specific, and 10 were co-dominant, such as 3EST-147 (Table S1, Fig. 3d). GISH/FISH showed that 3Yc is submetacentric (Fig. 1f). Chromosomes 3Sc and 3Yc had different FISH patterns for two repetitive DNA probes; 3Sc had no (GAA)10 signal, and the RcAfa signals were detected at the telomeric region of the long arm (Figs. 1e, 4b). In contrast, 3Yc had no RcAfa signal, but had a pair of dispersed (GAA)10 signals at the centromeric region of the long arm (Figs. 1f, 4b). The spike of DA3Sc has long awns, and DA3Yc has a similar spike structure to CS (Fig. 2).

DALs DA4Sc and DA4Yc

Thirty two R. ciliaris-specific markers were developed based on homoeologous group 4 ESTs of wheat. Specific amplicons were amplified in lines 2014-2015K271 and 2014-2015K247, both of which had 2n = 44. The two lines showed different amplification patterns for these markers, indicating that the added chromosomes in both lines belonged to homoeologous group 4, but were from different genomes (Tables 3, S1). Twelve markers were specific for the alien chromosome in 2014-2015K271, such as 4EST-100 (Table S1, Fig. 3e), 7 were specific for the alien chromosome in 2014-2015K247, and 12 were co-dominant amplifying polymorphic bands in both lines, such as 4EST-19 (Table S1, Fig. 3f). Marker 4EST-145 was amplified similar specific bands in 4Sc and 4Yc (Table 3). GISH/FISH showed that the alien chromosomes in 2014-2015K271 and 2014-2015K247 were submetacentric and metacentric, respectively. They showed different FISH patterns with two repetitive DNA probes. The alien chromosome pair in 2014-2015K271 had rich RcAfa signals distributed on both arms and its (GAA)10 signals were located at the centromeric regions (Figs. 1g, 4b). However, the alien chromosome pair in 2014-2015K247 had RcAfa signals at the telomeric region of the short arm and (GAA)10 signals mainly located at centromeric regions of the short arm (Figs. 1h, 4b). Based on FISH patterns from the (GAA)10 and RcAfa probes, the signals were richer in the previously assigned Sc genome chromosomes (1Sc, 2Sc, 3Sc) than the homoeologous partners (1Yc, 2Yc, 3Yc). We thus designated the alien chromosomes in lines 2014-2015K271 and 2014-2015K247 as DA4Sc and DA4Yc, respectively. Both DA4Sc and DA4Yc were tip-awned and had square spikes. The upper spikelets of DA4Yc had low fertility (Fig. 2).

DALs DA5Sc and DA5Yc

Twenty one R. ciliaris-specific markers were developed based on homoeologous group 5 ESTs of wheat. Specific amplicons could be amplified only in DA5Yc (Wang et al. 2001) and line 2014-2015K125 with 2n = 44. Line 2014-2015K125 showed a different amplification pattern than DA5Yc, indicating that the added chromosomes also belonged to homoeologous group 5, but represented a different genome and, therefore, could be designated as 5Sc. Six markers were 5Sc specific, such as 5EST-79 (Table S1), 12 were 5Yc specific, and 3 were co-dominant, such as 5EST-10 (Table S1, Fig. 3g). GISH/FISH showed that 5Sc is submetacentric (Fig. 1i). Chromosomes 5Sc and 5Yc had different FISH patterns for two repetitive DNA probes; 5Sc had RcAfa signals at the distal regions of the short arms and (GAA)10 signals were located at the centromeric regions of the long arm. The presence of pTa71 signals on the short arm of chromosome 5Sc revealed that it is another SAT chromosome (Figs. 1i, 4a). Chromosome 5Yc has the largest arm ratio and can be differentiated from other R. ciliaris chromosomes by the presence of (GAA)10 signals at the centromeric region and pTa794 signals at the distal region of the short arm (Figs. 1j, 4c). DA5Sc has tip-awns and a spindle-shaped spike, and DA5Yc has a long loose spike with low spikelet density (Fig. 2).

DALs DA6Sc and DA6Yc

Thirty five R. ciliaris-specific markers were developed based on homoeologous group 6 wheat ESTs. Specific amplicons could be amplified only in lines 2014-2015K27 and 2014-2015K15 with 2n = 44. The two lines showed different amplification patterns, indicating their added chromosomes belong to same homoeologous group 6, but to different genomes (Tables 3, S1). Nine markers were specific for the alien chromosomes pair in 2014-2015K27, such as 6EST-358 (Table S1), 9 were specific for the alien chromosome in 2014-2015K15, and 16 were co-dominant, such as CINAU15 (Table S1, Fig. 3h). Marker 6EST-306 amplified similar specific bands in 6Sc and 6Yc (Table 3). GISH/FISH showed that the alien chromosomes in 2014-2015K27 and 2014-2015K15 are metacentric and submetacentric, respectively. They showed different FISH patterns with two repetitive DNA probes. The alien chromosome pair in 2014-2015K27 had similar RcAfa signal distribution to 4Sc, and no (GAA)10 signal was detected (Figs. 1k, 4b). However, the alien chromosome pair in 2014-2015K15 had very few FISH signals, with a single (GAA)10 signal at the middle region of the short arm (Figs. 1l, 4b). As in the case of 4Sc and 4Yc lines 2014-2015K27 and 2014-2015K15 were designated as DA6Sc and DA6Yc, based on the abundance of the FISH signals. DA6Sc has a unique short compact spike morphology compared with other DALs whereas DA6Yc has a long speltoid spike (Fig. 2).

DALs DA7Sc and DA7Yc

DA7Sc and DA7Yc were developed by Wang et al. (2001). The two lines were further characterized by molecular marker and FISH analysis. A total of 11 R. ciliaris-specific markers were developed based on homoeologous group 7 ESTs of wheat. Specific amplicons were amplified in DA7Sc and DA7Yc (Tables 3, S1). The two lines showed different amplification patterns, confirming the added chromosomes belonged to the same homoeologous group, but different genomes. 7EST-133 was the only marker allocated to 7Sc, 8 markers were allocated to 7Yc, such as 7EST-234 (Table S1), and 7EST-213 was a co-dominant marker, which could distinguish 7Sc and 7Yc in a single amplification (Table 3). Marker 7EST-138 amplified the same specific bands in 7Sc and 7Yc (Table S1, Fig. 3i). GISH/FISH revealed that chromosome 7Sc had RcAfa signals and very weak (GAA)10 signals at the telomeric regions of the long arm (Figs. 1m, 4b). Chromosome 7Yc had more intensive RcAfa signals at the distal regions of both arms (Figs. 1n, 4b). DA7Sc had tip-awns and a slender spike, whereas DA7Yc has a spindle-shaped spike (Fig. 2).

The standard FISH-based karyotype of R. ciliaris chromosomes

Four repetitive DNA sequences [pTa794, pTa71, RcAfa and (GAA)10] were used as probes for FISH analysis. Based on the FISH patterns of the added alien chromosomes in 14 wheat-R. ciliaris DALs, we established a FISH-based karyotype of R. ciliaris chromosomes (Fig. 4d).

FISH using pTa71 as probe showed that there are two SAT chromosomes in R. ciliaris. The NOR of 1Sc was at the middle of the short arm and the NOR of 5Sc was in the telomeric region of its short arm; this chromosome pair also had less intensive signals on the long arms. The two chromosomes can be distinguished from other R. ciliaris chromosomes based on this feature (Fig. 4a). The presence of pTa794 FISH signals on the short arm is a unique character for identifying 5Yc (Fig. 4c).

FISH using two repetitive DNA probes showed that RcAfa signaling is much more abundant than (GAA)10 on R. ciliaris chromosomes. RcAfa signals were distributed on 11 R. ciliaris chromosomes, except 3Yc, 5Yc and 6Yc. Most signals were located at the telemetric regions on both arms of 1Sc, 4Sc, 6Sc, 1Yc, 2Yc and 7Yc, on the short arms of 4Sc and 4Yc, and on the long arms of 3Sc and 7Sc. The signals of 2Sc were located at the pericentric region of the long arm. In general, FISH signals from RcAfa were more intense in genome Sc than in Yc. However, we found that 2Yc had more RcAfa signals than 2Sc, and 7Yc had more than 7Sc (Fig. 4b).

(GAA)10 signals were distributed on 9 R. ciliaris chromosomes. The signals were mainly located at the centromeres or pericentromeric regions. Signals on 2Yc and 7Sc were located at the telomeres of the long arms; those of 2Sc, 3Yc, 5Sc and 5Yc were at centromeric regions of the long arms; those of 4Yc and 6Yc were at centromeric regions of the short arms, and those of 4Sc were located at the centromeric regions.

To summarize, 12 R. ciliaris chromosome pairs can be unambiously distinguished from each other by sequential GISH/FISH using four probes. However, 1Yc and 7Yc showed similar FISH patterns and differentiation required marker analysis.

Roegneria ciliaris is a useful genetic resource for Fusarium head blight resistance

To evaluate the potential use of R. ciliaris, the Inayama komugi-R. ciliaris amphiploid and complete set of DALs were evaluated for reaction to FHB over 5 successive growth seasons (2012–2017). Compared with the background varieties (Ik and CS) and susceptible control (Mianyang 8545), the amphiploid and three DALs (DA2Yc, DA5Yc and DA6Sc) showed stable FHB resistance (Table 4) confirming that R. ciliaris is a useful genetic resource for FHB resistance and that the above three chromosomes may confer resistance. The amphiploid showed a similar level of resistance to Sumai 3, the resistant control. However, the three DALs were less resistant than the amphiploid, indicating that the full resistance of R. ciliaris is controlled by the additive effects of genes located on three chromosomes. Crosses between these DALs and FHB susceptible varieties have been made to confirm the effects of these chromosomes in different wheat backgrounds.

Discussion

Transferring desirable genes from wild relatives into common wheat has been an important strategy in wheat breeding since the last century. Genes conferring genes of interest have been introduced into wheat through wide hybridization and chromosome engineering. Wheat–alien chromosome addition lines are important bridge materials for alien gene transfer. In addition, complete sets of wheat–alien DALs are valuable materials for comparative genome analysis between wheat and wild relatives, and for investigating the origin and evolution of different genomes (Du et al. 2014; Friebe et al. 2000).

Roegneria ciliaris is a tetraploid species that possesses resistance or tolerance to various biotic and abiotic stresses (Sharma et al. 1984; Weng and Liu 1989; Chang et al. 2011). Although a complete set of wheat-R. ciliaris DALs are critical for exploring novel genes of interest and for allocating them to specific chromosomes, this has been hindered by the presence of alloplasmic effects (Jiang et al. 1992, 1993) and lack of efficient methods for determining the identities of alien chromosomes in the wheat background (Wang et al. 2001). This has been especially difficult in the case that R. ciliaris, which is an allotetraploid species.

Taking advantage of the huge numbers of EST sequences with known chromosome locations and high conservation between homoeologous chromosomes from different genomes of Triticeae species, we developed 162 EST-based PCR markers specific for R. ciliaris (Chen et al. 2003; Li et al. 2011). These markers not only helped us to determine the identities of the alien chromosomes added to wheat, but also enabled the determination of their homeologies with wheat chromosomes. Homoeologous chromosomes from the two genomes were differentiated from each other and from wheat chromosomes in a single-round PCR. For example, PCR using 3EST-147 produced three specific bands (2000, 900 and 760 bp), which were present only in the amphiploid, DA3Sc and DA3Yc. DA3Sc and DA3Yc shared the same 760 bp amplicon, whereas the 2000 and 900 bp amplicons were specific for 3Sc and 3Yc, respectively (Fig. 3d). These markers provided a rapid and robust approach for identifying and tracing each R. ciliaris chromosome in the wheat background.

All the molecular markers were developed according to ESTs with known chromosome locations. Marker analysis generally revealed parallel homoeologous relationships between R. ciliaris and wheat chromosomes (Table S2). However, we observed exceptions for chromosomes 1Yc, 2Sc, 7Sc and 7Yc. The specific bands of five markers for 1Yc were also observed in DA7Sc and DA7Yc, and specific bands of ten markers for 7Sc and 7Yc were observed in DA1Yc. Similar phenomena occurred for chromosomes 2Sc and 7Sc. Since all the EST-based markers were derived from the coding sequences, we suggest that during the evolution of R. ciliaris there were multiple structural rearrangements involving chromosomes 1Yc and 7Sc/7Yc, and between 2Sc and 7Sc.

Up to now, the origin of the Y genome and its relationship with the S genome was an unsolved issue. Chromosome pairing analysis showed that the St and Y genomes had low affinity (Liu et al. 2006; Lu and Von Bothmer, 1989). However, Liu et al. (2006) preferred the explanation that St and Y may have the same origin based on ITS sequence analysis. Zhang et al. (2009) suggested that the Y genome originated from the St genome. This was inferred from a gene encoding plastid acety1-CoA carboxylase. Lei et al. (2016) also suggested that Y genome is closely related to the St genome; however, our data do not support the same origin for the two genomes. Molecular marker analysis showed that, among the 162 R. ciliaris-specific markers, only 3 (4EST-145, 6EST-306 and 7EST-138) produced the same size amplicons for the S and Y genomes, indicating the presence of diversification of the two genomes during evolution. There were fewer co-dominant markers than genome-specific markers in homoeologous groups 1, 5 and 7. However, the numbers of the marker pairs in groups 2, 3, 4, 6 were not very different.

Some workers suggested that St and Y have high affinity (Lu and Bothmer 1989, 1990, 1991; Lu et al. 1990) making it difficult to distinguish homoeologous chromosomes from the two genomes. Morris and Gill (1987) investigated the genomic affinities of individual chromosomes based on chromosome C- and N-banding of species including tetraploid Elymus and their diploid progenitor. Both the S and Y genomes had very few N-bands, but abundant C-bands. No significant difference was observed for the two genomes. In Triticeae species, tandem repeated sequences have been used as cytogenetic markers for chromosome identification (Badaeva et al. 2010; Mukai et al. 1992) and characterization of alien addition, substitution and translocation lines (Qi et al. 2010; Yuan and Tomita 2009). The conserved pTa794 and pTa71 FISH signals were frequently detected on homoeologous group 1 and group 5 chromosomes in Triticeae species (Mukai et al. 1992). In R. ciliaris, pTa71 signals were detected on 1Sc and 5Sc, and pTa794 loci were located on 5Yc. The major loci of pTa71 were on the short arms of 1Sc and 5Sc, and minor loci were on the long arms. Liu et al. (2017) reported pTa794 FISH signals on several chromosome pairs, but this may be due to polymorphisms between different accessions.

Tsujimoto and Gill (1991) developed several genome-specific clones and proposed that pCbTaq4.14 is H genome specific and pPlTaq2.5 is S genome specific. Wang et al. (2001) used these clones for FISH to characterize wheat-R. ciliaris alien chromosome lines. They found that even though there were some differences in hybridization signal distribution on chromosomes from different genomes, it was still difficult to assign each chromosome to a specific genome. Recently, Wang et al. (2017) developed a new FISH marker for St genome St2-80. FISH using this probe in R. ciliaris showed that 14 of the chromosomes showed St-type signal patterns. This probe may be useful to differentiate the two genomes; however, due to lack of the complete set of alien addition lines, they failed to determine the homoeologous group of each chromosome.

We established a FISH karyotype of R. ciliaris by FISH using (GAA)10 and RcAfa as probes. Compared with the Yc genome, the Sc genome chromosomes have more intensive RcAfa FISH signals. However, the chromosomes we designated as 2Sc and 7Sc have less RcAfa signals than their corresponding 2Yc and 7Yc. Therefore, we propose that their genome affinities should be inverted, i.e., 2Sc should be 2Yc, 7Sc should be 7Yc, and vice versa. The distribution of RcAfa FISH signals on Sc genome chromosomes is similar to D genome chromosomes, hinting a closer relationship of the S and D genomes. This is consistent with the conclusion of Liu et al. (2007). If this is true, we can differentiate the homoeologous chromosomes from the two genomes by FISH using RcAfa as probe. However, this needs to be further validated by FISH by the development of Sc- or Yc-genome specific probes.

The distribution of GAA in Triticeae species is considered to correspond with the distribution of N-bands, which are mainly composed of heterochromatin (Pedersen and Langridge 1997). C- and N-banding analysis in tetraploid species of Elymus and their diploid progenitors have demonstrated that the Sc chromosomes are characterized by the presence of rich terminal C-bands on one or both arms and absence of N-bands, whereas the Y chromosomes are characterized by the presence of centromeric C- and N-bands on most chromosomes (Morris and Gill 1987). Our results showed that (GAA)10 signals were distributed on four Sc and five Yc chromosomes. We failed to observe a correlation between C- or N-bands with the (GAA)10 signal distribution.

With the help of a complete set of DALs, the genome affinity and homoeologous group of each alien chromosome was determined. This information is basic and also critical for further alien gene transfer. There have been reports of several valuable traits in R. ciliaris for wheat improvement. Five successive years of evaluation of FHB reaction indicated that introduction of genes on at least three chromosomes could improve FHB resistance in wheat. We also observed diverse morphological changes of the DALs when compared with the wheat recipient variety, indicating potential value for crop improvement. The DALs will be further phenotyped for resistances to biotic and abiotic stresses, grain quality and other agronomic traits. DALs conferring useful traits will used as initial materials for the development of alien translocations, which can be used in wheat breeding and for gene mining.

Author contribution statement

XEW and HYW designed the project, LNK and XYS performed the experiments, JX designed the primer pairs, HJS designed the FISH probes, KLD performed part of the marker analysis, CXL, PS and RS evaluated FHB response, CXY and SZZ analyzed the morphological characters.