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Thinopyrum intermedium, a wild relative of wheat, is an excellent source of disease resistance. Two novel partial amphiploids, 08-47-50 and 08-53-55 (2n = 6x = 42), were developed from wide crosses between durum wheat and Th. intermedium. Meiotic analysis showed that pollen mother cells of the two partial amphiploids formed an average 20.49 bivalents for 08-47-50 and 20.67 bivalents for 08-53-55, indicating that they are basically cytologically stable. GISH analysis revealed that the two partial amphiploids carried different chromosome compositions. 08-47-50 had fourteen chromosomes from Th. intermedium and its alien chromosomes included six St-, four Ee- and four Ee-St translocated chromosomes, whereas 08-53-55 had four St- and ten Ee-St translocated chromosomes. Fungal disease evaluation indicated that both partial amphiploids had a high level of resistance to FHB, leaf rust and stem rust race Ug99. These two novel partial amphiploids with multiple disease resistance could be used as a new source of multiple disease resistance in bread wheat and durum wheat breeding programs.
The ongoing improvement of wheat cultivars is dependent on a continuous supply of genetic variability. Introgression of desirable traits from Triticeae relatives to cultivars by means of wide hybridization has been a successful practice in wheat improvement for biotic and abiotic stress tolerance. A typical and important step in alien gene transfer is the generation of wheat–alien partial amphiploids (Ellneskog-Staam and Merker 2002; Fedak and Han 2005).
Fusarium head blight (FHB), caused mainly by Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schw.) Petch) is one of the most destructive fungal diseases worldwide, which has caused serious loss in grain yield and quality (Bai and Shaner 1994; Stack 2003). Leaf rust, caused by the fungus Puccinia triticinia Eriks., is the most common and widely distributed of the three wheat rusts. Losses from leaf rust infection are usually less than those from stem rust and stripe rust, but leaf rust causes greater annual losses due to its more frequent and widespread occurrence (McCallum and Seto-Goh 2005). Stem rust caused by the fungus Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn. is a major, devastating disease of bread and durum wheat and historically has caused severe losses to wheat production worldwide. Race Ug99, first identified in Uganda in 1999, has virulence on the gene Sr31 deployed worldwide in many cultivars (Pretorius et al. 2000) and it was redesigned as TTKSK (Jin et al. 2008). Available evidence emerging from the East African countries indicates that Ug99 has exhibited a gradual step-wise range expansion, which is threatening wheat production in the world (Stokstad 2007; Ayliffe et al. 2008).
Durum wheat, Triticum turgidum L. (2n = 4x = 28, AABB), is an important cereal used for preparing pasta and semolina for human consumption worldwide. The wheat grass Thinopyrum intermedium (Host) Barkworth & D.R. Dewey (syn. Elytrigia intermedia (Host) Nevski, Agropyron intermedium (Host) Beauvoir) is a perennial allohexaploid species (2n = 6x = 42, EeEeEeEeStSt). It carries numerous useful agronomic traits and constitutes a tertiary gene pool for wheat improvement (Fedak and Han 2005). To date, several bread wheat–Th. intermedium amphiploids have been obtained and characterized by means of genomic in situ hybridization (Chen et al. 2003; Fedak and Han 2005; Bao et al. 2009; Chang et al. 2010; Georgieva et al. 2011). However, there are few amphiploids reported from durum wheat and Th. intermedium. Here we report the development of two new durum wheat–Th. intermedium partial amphiploids with resistance to multiple fungal pathogens. In this investigation, we attempted to determine the chromosome composition and genomic origins of the alien chromosomes of two partial amphiploids by genomic in situ hybridization (GISH).
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Discovering novel and diverse sources of resistance is critical for retaining genetic variation for disease and pest resistance in wheat breeding programs, because deployment of only one or a few sources of resistance over large crop production areas poses a danger of resistance breakdown and disease epidemics. As an important perennial Triticeae species, Thimopyrum intermedium has frequently been used in bread wheat improvement as a donor of various disease resistance genes, in particular for those which to a large extent are lacking in bread wheat or durum wheat. To date, genes for various disease resistances have been transferred from Th. intermedium to bread wheat, such as Wsm1 conferring resistance to wheat streak mosaic virus (WSMV) (Friebe et al. 1991), Bdv2 (Zhang et al. 1999) and Bdv3 (Ohm et al. 2005) specifying resistance to barley yellow dwarf virus (BYDV), Pm40 (Luo et al. 2009) and Pm43 (He et al. 2009) resistance to powdery mildew, Lr38 (Friebe et al. 1992) resistance to leaf rust, and Sr44 (Friebe et al. 1996) resistance to stem rust. Resistance to FHB from Th. intermedium was also identified in derived wheat progenies (Oliver et al. 2005). FHB resistance was also detected in partial amphiploid obtained from a durum ×Th. distichum combination (Chen et al. 2001).
Development of partial amphiploids is an important first step by which to enhance wheat genetic diversity and transfer alien disease resistant genes into wheat. It is essential to know the exact genomic composition of the added alien chromosomes in the partial amphiploids. The genome constitution of Th. intermedium was determined to be EeEeSt (Liu and Wang 1993) or EeEbSt (Chen et al. 1998) with Ee (=J) and Eb (=Js) designating the closely related Th. elongatum and Th. bessarabicum genomes. Lately, new insights in the genome composition of Th. intermedium became available (Kishii et al. 2005). Recent studies indicate that the genomic constitution of Th. intermedium may be somewhat more complex as revealved by the existence of sequences from Th. caespitosum, Taeniatherum caput-medusae and Crithopsis delileana in its genome (Arterburn et al. 2011). The existence of these sequences may be responsible for some of the different GISH staining patterns obtained. This aspect obviously requires additional study. In the present study, using genomic probes of St and Ee genome simultaneously, the results not only provided detailed information on the precise genomic constitution of the partial amphiploids 08-47-50 and 08-53-55, but also revealed the chromosome structural variation and rearrangement involving Ee and St genomes.
The GISH results clearly demonstrated that the difference in genomic constitution between 08-47-50 and 08-53-55 was due to the different ratio (E-St) of alien chromosomes of Th. intermedium and the differences in translocated chromosomes. 08-47-50 contained six St genome chromosomes, four Ee genome chromosomes and two pairs of Ee-St translocated chromosomes plus twenty-eight durum wheat chromosomes. 08-53-55, in addition to the complete durum wheat genome, consisted of four chromosomes of the St genome, and five pairs of Ee-St translocated chromosomes. In terms of translocated chromosomes, different fragment sizes of Ee-St reciprocal translocations were detected, showing the fragment of Ee genome on the short arm of two St-chromosomes in 08-47-50 and the larger fragment of Ee genome involving the centromeric region on the two St-chromosomes in 08-53-55. In addition, two Ee-St terminal translocation chromosomes and two interstitial translocation chromosomes of Ee-St were further discerned in 08-47-50. Eight Ee-St terminal translocation chromosomes and two Ee-St interstitial translocation chromosomes were observed in 08-53-55. However, we did not observe any St-signal near the centromeric region using the St-genome probe, which is not consistent with the previous reports (Chen et al. 2003; Bao et al. 2009). This difference in results seems attributable to the polymorphism of St genome chromatin. Another reasonable explanation could be that the chromosome constitution of Th. intermedium varies greatly among and within accessions (Xu and Conner 1994).
Interstitial translocations were detected in partial amphiploid 08-47-50 in this study. Such translocations are rare in more conventional plant genomes and hybrids (Jiang et al. 1993), but have been reported in a number of partial amphiploids (Fedak et al. 2000; Fedak and Han 2005; Chang et al. 2010). Perhaps they occur more frequently in the genomes of complex polyploids where some of the genomes such as E and J are closely related, and are now being detected by multicolor GISH technology.
At metaphase I, the chromosomes pairing association of 08-47-50 was similar to that of 08-53-55 with high frequencies of bivalent and very low multivalent formation. In 08-47-50, most (about 70% of the 120 cells analyzed) cells formed 21 bivalents and only 0.75 unpaired chromosomes and 0.09 trivalents occurred per cell. While 08-53-55 had the expected 21 bivalents which occupied about 82% of the 120 cells analyzed. Only 0.66 univalents occurred per cell at metaphase I. These meiotic configurations indicated that the two novel partial amphiploids had a basic stability in cytology with a vigorous growth habit and high fertility. As described by Fedak et al. (2000), Thinopyrum-derived partial amphiploids normally have regular meiosis with high frequencies of bivalent configurations and low frequencies of multivalents. Their genomes should be largely balanced in terms of homoeologous chromosomes (Fedak and Han 2005). This is in contrast to partial amphiploids derived from a durum wheat and Th. distichium combination where progeny with 50 and 42 chromosomes were obtained with only the latter being stable.
In previous researches, the genes for resistance to WSMV, BYDV, rust and powdery mildew were not located on chromosomes of the St genome but on those of Ee (J) or Eb (Js) genomes as determined by C banding, GISH and molecular marker analyses (Kong et al. 2009; Li et al. 2005). In the present study, the fungal disease evaluation showed that 08-47-50 and 08-53-55 were resistant to FHB, leaf rust and stem rust. GISH revealed that 08-47-50 had six St chromosomes, four Ee chromosomes and four translocated chromosomes involving Ee and St chromosomes. While four St chromosomes and ten Ee-St translocated chromosomes were discerned in 08-53-55. Ee and St genome chromosomes could be associated with resistance to FHB since durum has no FHB resistance.
Fungal diseases are caused by a very dynamic group of plant pathogens. Both leaf rust and FHB annually cause epidemics in the world. A new stem rust pathotype Ug99 with serious virulence on the widely deployed resistance gene Sr31 was detected in Uganda in 1999 and has since mutated to two additional variants (Pretorius et al. 2000; Jin and Singh 2006; Jin et al. 2008). The genetic flexibility of these pathogens has lead breeders to respond with a constant search for new resistance genes and incorporating them into wheat genetic backgrounds. Our present study revealed that the two novel partial amphiploids harbour a variety of resistance genes to several major fungal diseases. It is expected that more genes for resistance against diseases can be discovered in Th. intermedium, especially the genes that rarely occur in bread wheat or durum wheat. We are currently making backcrosses to bread wheat and durum wheat in order to develop addition, substitution and translocation lines with resistance to FHB, leaf rust and stem rust.