Fusarium head blight (FHB), caused by Fusarium graminearum, F. culmorum, and other Fusarium species, is one of the most destructive fungal world diseases in wheat production. FHB outbreaks reduce grain yield and quality in both common wheat (Triticum aestivum) and durum wheat (T. durum) and can contaminate the grain with mycotoxins (Arseniuk et al., 1993; Kalih et al., 2014; Zhu et al., 2015). Currently, the most effective and widely used quantitative trait locus (QTL) for FHB resistance breeding is located on chromosome 3BS of the Chinese wheat varieties Sumai 3 and Wangshuibai. Recent investigations of several other wild relatives of wheat, such as diploid wheatgrass Leymus racemosus, Th. intermedium, Th. elongatum, and tetraploid wheatgrass Th. junceiforme, have been shown to be highly resistant to FHB (Chen et al., 2005; Becher et al., 2013; Kalih et al., 2014). Additionally, some wheat-wild species, including accessions of the St genome of Th. intermedium and the E genome of Th. elongatum, have been shown highly resistant to FHB, making these species the most successful examples for introgression of elite genes from wild relatives for wheat improvement (Jauhar et al., 2009; Scoles et al., 2009; Zeng et al., 2013).

Leaf rust is caused by the fungus Puccinia triticinia Eriks. (Pt) and is the most common and widespread disease in wheat (Anikster et al., 1997). Losses from leaf rust infection are usually less than those from stem rust and stripe rust, but leaf rust occurs more frequently over a larger area, resulting in greater annual losses (McCallum and Seto-Goh, 2005). Various strategies have been developed to control leaf rust, the most economical and effective of which has been to use new resistance resources of cultivated germplasm (Chu et al., 2009). Stem rust is caused by P. graminis f. sp. tritici (Pgt) and is an economically impactful disease of wheat that can be controlled by using effective stem rust resistance (Sr) genes. However, a new stem rust race, commonly known as Ug99 and formally designated as TTKSK, was detected in Uganda in 1999 that is virulent to plants carrying Sr31 and many other Sr genes (Jin et al., 2007, 2008). Singh et al. (2011) heeded Dr. Norman Borlaug’s warning in 2005 that stem rust race Ug99 could spread throughout the world wheat production areas, leading to worldwide food shortages, because approximately 85–95% of globally distributed common wheat cultivars are susceptible to stem rust race Ug99. For economic and environmentally sustainable disease control, Ug99-resistant wheat varieties should be developed from cultivated germplasm or wild related species. Zeng et al. (2013) obtained translocation lines from the E^e and St genomes of Th. intermedium, and these lines have shown good resistance to leaf rust and stem rust race Ug99.

Distant hybridization is one way to trigger reorganization of different genomes in hybrids (Fu et al., 2013). Wild relatives of common wheat, which have superior agronomic traits and resistance to biotic and abiotic stresses, are often used as desirable gene donors in wheat breeding programs (Dong et al., 1992; Mujeeb-Kazi et al., 2013). To date, many genes from wild species conferring superior traits have been transferred into cultivated wheat by developing newly synthesized allopolyploids (Qi et al., 2016). For example, breeders developed a man-made crop, triticale (X Triticosecale Wittmack), by crossing wheat (T. turgidum or T. aestivum) and rye. It combines the biotic and abiotic stress tolerances of rye and grain production ability of wheat, making it more suitable for production in agriculturally marginal areas (Mergoum and Macpherson, 2004). Although the rye genome carries many disease resistance genes, the global supply of triticale has inherent problems, including partial floret sterility, shriveled seed, production concerns, requirement for a long growing season and susceptibility to FHB as common wheat (Blum, 2014). Hence, there is a need to improve the genetic diversity and enhance the resistance to FHB of triticale by transferring desirable genes from additional wild relatives of wheat. Trigeneric hybrids could help establish evolutionary relationships among different genomes present in the same genetic background and could also be used as bridges to transfer different genes from wild species into cultivated wheat (Kang et al., 2012). Many trigeneric hybrids have been produced by crossing intergeneric hybrids or amphiploids with another species, such as Triticum–Secale–Agropyron (Gupta and Fedak, 1986), Triticum–Secale–Aegilops (Orellana et al., 1989), Triticum–Secale–Hordeum (Fernandez-Escobar and Martin, 1989), Triticum–Secale–Thinopyrum (Yu et al., 1993), and Triticum–Secale–Leymus (Li et al., 2006). These trigeneric hybrids have been used as bridges to transfer useful genes or characters from wild species into wheat (Jiang et al., 1993).

The diploid wheatgrass Th. elongatum (Host) D.R. Dewey [=Lophopyrum elongatum (Host) Á. Löve; =Elytrigia elongatum (Host) Nevski; =Agropyron elongatum (Host) Beauv.] (2n = 2x = 14, EE) is a significant wild relative of wheat that has many desirable genes for wheat improvement. Its high cross-compatibility with wheat and its superior value-added trait characters, especially FHB resistance has attracted the attention of wheat geneticists and breeders (Shukle et al., 1987; Sharma et al., 1989; Yang and Ren, 2001; Shen et al., 2004; Linc et al., 2012; Chen et al., 2013). Shen et al. (2004) reported that three substitution lines 7E(7A), 7E(7B), and 7E(7D) showed significant resistance to FHB, indicating that the 7E chromosome of Th. elongatum carries FHB resistance genes. Jauhar et al. (2009) obtained a stable durum 1E disomic addition line which showed significant resistance to FHB in greenhouse conditions, so it is indicated that the 1E chromosome of Th. elongatum may also carry FHB resistance genes. In this study, we obtained two stable lines RE21 and RE62, which were derived from the cross between hexaploid triticale (AABBRR) and hexaploid T. trititrigia (AABBEE). And these two lines displayed great resistance to leaf rust and stem rust race Ug99; line RE21 also had FHB resistance. The chromosome composition of these two lines was distinguished using chromosome-specific molecular markers and genomic in situ hybridization (GISH). These new lines will be useful for triticale breeding to transfer multi-alien genes into hexaploid common wheat, extend genetic resources for wheat breeding, enrich the genetic base of wheat, and improve wheat yield and quality.

The process of making crosses between different species or genera is commonly known as distant hybridization. Such hybrids have contributed to plant improvement, gene and genome mapping, and studies of chromosome behavior and evolution (Sharma, 1995). In recent years, distant hybridization between wheat and its wild relatives has attracted significant attention as a way to extend resources for genetic improvement in wheat breeding. A number of useful genes derived from wild relatives of wheat, such as leaf rust resistance gene (Lr26), stem rust resistance gene (Sr31), and stripe rust resistance gene (Yr9), had been transferred to common wheat (Villareal et al., 1991; McKendry et al., 1996). Following the development of distant hybridization, the technique of trigeneric hybridization has become a common method to transfer alien genes from wild species into cultivated wheat (Jiang et al., 1993). For example, the R genome derived from cultivated rye has served as an important resource for improving wheat and triticale because of its novel disease resistance and wide adaptation (Kang et al., 2012). Although it is difficult to synthesize the trigeneric hybrids via direct hybridization between common wheat and other two diploid wheatgrass, many trigeneric hybrids in Triticeae had been successfully obtained using different crossing combinations and embryo rescue (Kang et al., 2012), such as by crossing the intergeneric hybrids or amphiploid with other species (Cabrera and Martin, 1992; Li and Dong, 1993), by crossing barley (Hordeum vulgare L.) with triticale (Balyan and Fedak, 1989), or by crossing triticale with amphiploids involving wheat and other species (Fernandez-Escobar and Martin, 1989). The exploitation of crossing different amphiploid is an effective and rapid way to obtain trigeneric hybrids, which could be a useful bridge to transfer different alien characters into common wheat (Kang et al., 2012). This study, utilized embryo culture to obtain two Triticum–Secale–Thinopyrum trigeneric hybrid lines with different chromosome rearrangements between E and R chromosomes by crossing between triticale and the hexaploid amphiploid T. trititrigia. Both lines exhibited resistance to leaf rust and stem rust race Ug99, and line RE21 had additional resistance to FHB.

For successful distant hybridization, alien chromosome identification is essential for monitoring chromosome composition in hybrids. As techniques in molecular biology have evolved, the identification of alien chromosomes has greatly improved. GISH, using total genomic DNA of introgression species as a labeled probe, is the most efficient and widely used method to determine the origin of chromosomes or chromosomal fragments from wild species in the progenies of hybrids (Autrique et al., 1995). But this method has limitations, such as high cost and a long time involved, making it necessary to apply other methods to properly assess the nature and stability of newly added chromatin in progeny. Many studies have used PCR-based markers to detect individual chromosomes in a wheat background. For example, the specific simple sequence repeat markers (SSR), PCR-based landmark unique gene markers (so-called PLUG markers), and specific-locus amplified fragment (SLAF-seq) markers have been used to identify individual rye chromosomes or chromosomal segments in wheat background (Saal and Wricke, 1999; Li et al., 2015). The present study used GISH paired with SLAF-seq markers to identify rye and Th. elongatum chromosomes in the two hybrid lines and found the GISH results were consistent with the PCR analysis. Line RE21 had 2n = 42 chromosomes, consisting of intact sets of A and B genomes from wheat plus 1E, 2E, 3E, 5E chromosomes from Th. elongatum and 4R, 6R, 7R chromosomes from rye; line RE62 also carried 2n = 42 chromosomes, consisting of full sets of A and B genome, intact chromosomes 1R to 4R, 6R, and 7R, plus the translocation chromosomes 5R/5E.

Thinopyrum elongatum, a wild relative of wheat, has been suggested as a potentially novel source of resistance to FHB and may also harbor genes for FHB resistance (Fu et al., 2012). Some reports had indicated that the chromosome 7E from Th. elongatum contains resistance genes, such as the FHB resistance gene Fhb7 and the rust resistance gene Lr19 (Zhang et al., 2011; Guo et al., 2015). Jauhar et al. (2009) synthesized a stable durum disomic addition line by adding a single pair of 1E chromosomes originating from Th. elongatum, and observed resistance to FHB under greenhouse conditions. Kalih et al. (2014) reported that the dwarfing gene Ddw1 detected on chromosome 5R of rye increases FHB severity. Line RE21 was highly resistant to FHB, possibly because chromosome 1E was present and chromosome 5R was absent. Line RE62 was more susceptible than either line RE21 or the parent T182 because the translocation occurred between chromosomes 5R and 5E. Notably, chromosome 5E may contain a major gene(s) that conveys susceptibility toward FHB (Fu et al., 2012). The presence of two chromosomes containing genes susceptible to FHB in line RE62 may explain the increase in susceptibility toward FHB. Additionally, the Th. elongatum chromosomes 1E and 7E, which contain FHB resistance genes, were not introgressed into line RE62.

It is well established that the short arm of rye chromosome 1R carries many disease resistance genes, including Lr26, Sr31, Yr9, and powdery mildew resistance genes (Pm8, Pm17) (Friebe et al., 1996; Geiger and Miedaner, 2009). Line RE62 exhibited significant resistance to leaf rust and stem rust race Ug99 owing to the presence of rye chromosome arm 1RS. In line RE21, even though chromosome 1R was replaced by Th. elongatum chromosome 1E, leaf rust resistance was still observed because Th. elongatum chromosome 3E carries leaf rust resistance gene Lr24 (Schachermayr et al., 1995), and rye chromosomes 6R and 7R carry the resistance genes Pr1 and Pr2, respectively (Wehling et al., 2003), thereby conferring resistance to leaf rust and stem rust race Ug99.

Under greenhouse conditions, several value-added characteristics including plant height, spike length, spikelets per spike, days to heading, and 1,000-kernel weight of lines RE21 and RE62 were measured. The average plant height of both lines RE21 and RE62 was greater than that of the parent T182, and plants of line RE21 were on average taller than plants of line RE62. The heading stage of both lines occurred earlier than either parent T182 and 8801, and the heading stage of line RE21 occurred earlier than line RE62. These results have confirmed that a dwarfing gene Ddw1, located on chromosome 5R, reduces plant height, increases FHB severity, and delays the heading stage (Kalih et al., 2014). Xiao et al. (2011) reported that the QTL for 1,000-kernel weight was located on rye chromosome arm 1RS. The 1,000-kernel weight observed in line RE21 was less than that of line RE62, possibly because chromosome 1R was replaced by 1E in RE21. The 1,000-kernel weight of line RE62 was similar to that of the parent line T182.

Compared with whole-chromosome transfer, development of translocation lines does incorporate small regions carrying useful genes into hexaploid wheat. Translocation lines are typically more genetically stable and their resistance is more durable compared with single-chromosome transfer lines (Zhao et al., 2013). The wheat-rye 1BL•1RS translocation line is particularly attractive to wheat breeders because several useful genes are located on 1RS (Singh et al., 1990). The development of translocation lines with small chromosome segments from wild species, especially those carrying several useful genes, is a promising direction for modern wheat breeding (Zhao et al., 2013). The application of distant hybridization toward the development of trigeneric hybrids may help establish evolutionary relationships among different genomes, offer the possibility to transfer different value-added trait into cultivated wheat, and produce translocations through the crossing or backcrossing of these plants to cultivated wheat (Li and Dong, 1993; Kang et al., 2012). Trigeneric hybrids seem to be less promising as potential crop, but they could be a useful bridge for the transference of chromatin into cultivated wheat. For example, Qi et al. (2016) produced a new wheat-rye-Psathyrostachys trigeneric hybrid carrying the wheat-rye 1BL•1RS translocation and a cryptic translocation involving a small chromosome segment with the stripe rust resistance gene from P. huashanica. In this study, we developed two Triticum–Secale–Thinopyrum hexaploid lines that carried resistance genes against FHB, leaf rust, and stem rust race Ug99. In the future, these two lines could be a useful bridge to develop the translocation lines carrying the wheat/rye recombinant chromosomes, wheat/Th. elongatum recombinant chromosomes or wheat/rye/Th. elongatum recombinant chromosomes and highly resistant to wheat diseases. Techniques such as radiation, molecular markers, and GISH will be useful for obtaining new lines in wheat breeding programs.

YD, GF, and JC participated in the study conception and design. HL, YD, and YaD contributed to DNA extraction and molecular marker identification. YD, DC, and WC performed hybridization. AX, YD, and HL analyzed the evaluation of disease resistance. YD, YaD, and YG analyzed the data. JC, YD, and GF wrote the manuscript. All authors approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.