Introduction

Rye, Secale cereale (2n = 2x = 14), a diploid species originated from the Near East (Salamini et al. 2002) has been established as a potential source of resistance genes to wheat since the development of first amphiploid triticale (Triticosecale x Wittmack) by Rimpau in 1888. Rye stands out from other cereals in terms of its nutritional value being rich in iron (Fe) and zinc (Zn) (Johansson et al. 2020, 2014) as well as allelopathic potential (Bertholdsson et al. 2012), both qualities are indispensable for wheat breeders. Most notable rye chromatin transfer to wheat is that of 1RS chromosome segment, in different forms like 1AL.1RS, 1BL.1RS, and 1DL.1RS translocations (Mago et al. 2015) contributing several resistance genes for powdery mildew, leaf, stripe and stem rusts which has inevitably led to Green Revolution. But continuously evolving pathogen races like Ug99 have started breaking down the resistance posed by rye chromatin (Crespo-Herrera et al. 2017). Hence, to maintain sustainable resistance in elite wheat cultivars, newer genes have to be introgressed from variable rye sources. Recently, some novel genes like Sr59, Yr83 and Pm56 have been transferred from rye to wheat (Rahmatov et al. 2016; Hao et al. 2018; Li et al. 2020), which may be utilized as durable sources against fungal diseases. For sustainable improvement of elite wheat cultivars by integrating rye chromatin from variable sources, successful hybridization among them is a pre-requisite. But incompatibility among the wheat and rye genomes hampers successful transfer of genes from rye to wheat hence triticale is being used as a bridging species for the introgressions.

Taking into consideration the above information, the work was planned to transfer rye chromatin into wheat through indigenously developed triticale lines. These triticale-wheat progenies (including doubled haploids and RILs) were evaluated at cytogenetic as well as agro-morphological levels to assess the effect of rye chromatin on yield-related traits, yellow rust resistance and drought tolerance.

Materials and methods

Molecular cytogenetic analysis

A total 139 stable triticale x wheat progenies (including RILs through SSD and doubled haploids through Imperata cylindrica-mediated chromosome elimination approach of doubled haploidy breeding) were developed. The parentage of all the progenies is given in Supplementary Table 1.

These lines were subjected to molecular cytogenetic analysis using genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) (Schwarzacher and Heslop-Harrison 2000) at Molecular Biology Lab, Department of Genetics, University of Leicester, UK. Slides with good metaphasic chromosome spreads were prepared according to Yamamoto and Mukai (1989). These were subjected to FISH using genomic rye DNA and repetitive DNA sequence as probes, viz. pSc119.2 (a 120 bp tandem repeated DNA sequence from Secale cereale), pTa71 (a repeat unit of 25S–5.8S–18S rDNA from T. aestivum), pAs1 (a tandem repeat with a monomeric length of 340 bp from T. aestivum). Probes were labelled with biotin-16-dUTP or digoxigenin-16-dUTP (Roche diagnostics) by random priming kit (Bioprime DNA labelling system, Invitrogen).

Agro-morphological evaluation

All 139 wheat-rye progenies (RILs as well as doubled haploids) were evaluated in the experimental fields of Department of crop Improvement, CSK HPKV, Palampur, for agronomically important yield-related traits and resistance against yellow rust, which was scored on 0–100 scale prescribed by Paterson et al. (1948). For evaluating the drought tolerance, in vitro screening of the genotypes (10 plants each) was done by subjecting them to low (− 0.2 MPa) and moderate (− 0.4 MPa) moisture stress induced by polyethylene glycol (PEG-6000) (Michel and Kaufmann 1973) with slight modifications. The seeds were disinfected in 1% sodium hypochlorite solution for 5 min followed by washing (three times) with distilled water. Petri dishes along with Whatman paper were sterilized in autoclave. Ten seeds of each variety were transferred into each sterilized glass Petri dish with a diameter of 9 cm in which the filter papers were placed. Five millilitre of distilled water was added to each Petri dish. Then, after 24 h 10 ml PEG solution of respective concentration for each treatment was added to the Petri dish. The germinated seeds were counted till full germination. The seeds whose root length was 2 mm or more were considered as the germinated ones. On the 8th day, the germinated seeds were taken out of the Petri dishes and the stem and root were separated to assess the morphological parameters.

Results

Molecular cytogenetic analysis of 139 triticale x wheat progenies including conventionally developed lines and doubled haploids (DHs) revealed different levels of rye chromatin introgression , i.e., translocation, substitution, addition and their combinations (Fig. 1). Out of 139 lines, 67 (48%) were observed to carry 1RS.1BL translocation (Fig. 2a).

Fig. 1
figure 1

Distribution of triticale x wheat and wheat x rye progenies based on level of aneuploidy

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Fig. 2
figure 2

Molecular cytogenetic analysis of rye introgressions in wheat a 1RS.1BL translocation in TW-1–49 pSc119 (red) rye DNA (green) b 1RS.1BL translocation along with 3R(3D) substitution in TW-1–26 pSc119 (red), rye DNA (green). c 1RS.1BL and 6BS.6RL translocations along with 2R(2D) substitution in TW 10 pSc119 (red), rye DNA (green) d 1RS.1BL translocation along with seven pair rye substitution in WR12 rye DNA (red), pTa71 (green). e Six pair rye chromosome substitution in TW 24 pAs1 (red), rye DNA (green) f) 1R and 1RL addition in TW-2–55 pAs1 (red), rye DNA (green)

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There were 33 (24 per cent) lines in which substitution has been observed, 12 lines have shown single pair rye substitution and rest with substitution of five and more pairs rye chromosomes (Fig. 2e). These lines recorded significantly more yield per plant (9.29 g), 1000 grain weight (48.26 g), number of tillers per plant (6.67) and plant height (106.06 cm) than other groups included in the study (Table 1). Besides, maximum resistance to yellow rust was observed in these lines depicting 33.33 per cent lines to be immune, 28.57 per cent to be highly resistant and 38.10 per cent to be resistant (Table 2). Fourteen lines showed 1RS.1BL translocation along with substitution of 3D with 3R (Fig. 2b). During the triticale wheat hybridization programmes, 18 progenies (12.95 per cent) revealed the absence of rye chromosomes showing reversion towards wheat parentage. During the present investigation, 18 progenies (12.95 per cent) revealed the absence of rye chromosomes showing reversion towards wheat parentage.

Table 1 Relative mean and range of various rye introgressed progenies for yield and related traits
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Table 2 Field evaluation of rye introgressed bread wheat progenies for resistance against yellow rust
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Discussion

Present investigation has been envisioned towards exploring novel combinations of wheat, triticale and rye in the form of translocations, substitutions and additions after hybridizing diverse genotypes. Among wheat rye progenies 48% lines were observed to carry 1RS.1BL translocation (Fig. 2a) advocating the prevalence of this translocation in majority of cultivated wheat cultivars. Wide availability of this translocation has also been reported by Kumar et al. (2003), Angelova and Georgiev (2006), Ren et al. (2009), Chahota et al. (2015), Li et al. (2016) and Sharma et al. (2022) in independently raised intergeneric hybrids.

Substitution in twenty-four per cent progenies may be attributed to wheat–rye genome interactions leading to preferred elimination of wheat D-genome chromosomes (Li et al. 2015). Wheat-rye progenies carrying five or more rye chromosomes genetically revert to triticale type, showing improved agronomic performance in the progenies carrying seven rye chromosomes followed by six and five chromosome substitutions, respectively. This might be due to agronomically important genes present on rye chromosomes (2R, 4R, 5R and 7R) (Falke et al. 2009; Jeberson et al. 2021). Among the substitution lines developed in the study, no such agronomic superiority was observed which contradicts the reports by Seerja and Reddy (2013) who observed significantly high yield among five pair rye chromosomes followed by seven and six pair rye chromosome carrying progenies. However, under drought stress, these lines revealed better germination, root length and weight owing for wide adaptability (Fig. 3).

Fig. 3
figure 3

Mean performance of various drought parameters of wheat rye progenies in different levels of aneuploidy (T—translocation; S—substitution of rye chromosome pair; A—addition) under controlled and stressed conditions created at − 0.2 and − 0.4 MPa

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In the present study, 3R substitution along with 1Bl.1RS translocation was observed in fourteen lines. The 3R chromosome is known to be associated with plant height, spike length, number of spikelets per spike, number of kernels per spike and kernel weight (Myśków et al. 2014). But in the present study, improvement in grain yield and harvest index was observed rather than plant height and grain weight. However, these lines revealed highest germination percentage in control (97.25 per cent) as well as at mild stress of − 0.2 MPa (89.75 per cent). The same progenies recorded maximum root length (5.04 cm) and seedling vigour index (1166.18) in the control which may be due to genotype and environment interactions.

Addition of alien chromosomes in wheat genome is desirable to study the dosage effect of genes. The current study revealed 5 lines (3.60 per cent) with monosomic addition of rye chromosomes among which, three were carrying translocation also. These results are in accordance with the findings of Fu et al. (2014) where one line with 1RS.1BL translocation along with addition of 1R chromosome was observed. Though addition lines cannot be considered as introgressions, these are stable enough to be maintained and used as initiative for the production of substitution and translocation lines. The agronomic performance of such lines in the present work was poor for most of the traits which contradicts by the results of Schneider et al. (2016) who reported the significant influence of 1R on the grain yield when compared to 4R and 6R addition. Screening for drought tolerance of these lines reported improvement in fresh and dry weight of shoot and root. This improvement might be due to more copy of genes present on 1R because according to Waines and Ehdaie (2007), 1RS promote root biomass growth.

The absence of rye chromosomes in the progenies showed reversion towards wheat parentage. Similar findings have been reported by Chahota et al. (2015) where six lines of triticale x wheat derivatives were lacking in rye chromatin. The reason behind such elimination of the rye chromatin lies in the incompatibility among spindle fibre of wheat origin with that of rye chromosomes. This results in the deviation of rye chromosome from equatorial plate leading to their successive or instant loss. Present work revealed these lines to be non-significantly different from those with rye chromatin introgression for yield-related traits (Table 1). Agronomic performance of these lines revealed poor grain yield, tiller number and harvest index whereas higher 1000 grain weight and biological yield. The findings by Ehdaie et al. (2003) and An et al. (2015) also support present observations as they observed higher plant height and lower grain yield respectively, among non-introgressed lines as compared to introgressed lines , i.e. translocation and substitution. Although under drought stress, the rye introgressed lines performed better than non-introgressed progenies (Fig. 3).

In conclusion, the research work presents novel genetically stable wheat rye progenies in the form of translocations, substitutions and additions. Furthermore, the screening of the progenies for yield and related traits, resistance towards yellow rust and tolerance for drought stress (− 0.2 and − 0.4 MPa) revealed positive effect of rye introgression. Since triticale and wheat lie in different gene pools, the present study generated new germplasm which can be used further in wheat improvement programmes.