Hereditas 150: 10–16 (2013)

Molecular cytological characterization of two novel – intermedium partial amphiploids with resistance to leaf rust, and Fusarium head blight J. ZENG1,2 , W. C A O1 , G. FEDAK1 , S. SUN4 , B. MCCALLUM5 , T. FETCH5 , A. XUE1 and Y. ZHOU3 1 Eastern and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, ON, Canada 2 Resources and Environment College of Sichuan Agricultural University, Wenjiang, Sichuan, PR China 3 Resaerch Institute of Sichuan Agricultural University, Wenjiang, Sichuan, PR China 4 Institute of Crop Genetics, Shanxi Academy of Agricultural Sciences, Taiyuan, PR China 5 Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, MB, Canada

Zeng, J., Cao, W., Fedak, G., Sun, S., McCallum, B., Fetch, T., Xue, A. and Zhou, Y. 2012. Molecular cytological characterization of two novel durum – Thinopyrum intermedium partial amphiploids with resistance to leaf rust, stem rust and Fusarium head blight. – Hereditas 150 : 10–16. Lund, Sweden. eISSN 1601-5223. Received 14 February 2012. Accepted 28 August 2012.

Thinopyrum intermedium, a wild relative of , 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 E e -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 wheat and durum wheat breeding programs.

Wenguang Cao, Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Ave, Building 50, Ottawa, ON, K1A 0C6, Canada . E-mail: [email protected]

The ongoing improvement of wheat is dependent and historically has caused severe losses to wheat produc- on a continuous supply of genetic variability. Introgres- tion worldwide. Race Ug99, fi rst identifi ed in Uganda sion of desirable traits from Triticeae relatives to cultivars in 1999, has virulence on the gene Sr31 deployed by means of wide hybridization has been a successful worldwide in many cultivars (P RETORIUS et al. 2000) and practice in wheat improvement for biotic and abiotic it was redesigned as TTKSK (J IN et al. 2008). Available stress tolerance. A typical and important step in alien evidence emerging from the East African countries gene transfer is the generation of wheat – alien partial indicates that Ug99 has exhibited a gradual step-wise amphiploids (E LLNESKOG-STAAM and M ERKER 2002; F EDAK range expansion, which is threatening wheat production and HAN 2005). in the world (S TOKSTAD 2007; AYLIFFE et al. 2008). Fusarium head blight (FHB), caused mainly by Durum wheat, Triticum turgidum L. (2n 4x 28, Fusarium graminearum Schwabe (teleomorph Gibberella AABB), is an important cereal used for preparing pasta zeae (Schw.) Petch) is one of the most destructive fungal and semolina for human consumption worldwide. The diseases worldwide, which has caused serious loss in wheat grass Thinopyrum intermedium (Host) Barkworth grain yield and quality ( BAI and SHANER 1994; STACK & D.R. Dewey (syn. Elytrigia intermedia (Host) Nevski, 2003). Leaf rust, caused by the fungus Puccinia triticinia Agropyron intermedium (Host) Beauvoir) is a perennial Eriks., is the most common and widely distributed of allohexaploid species (2n 6x 42, Ee E e E e Ee StSt). It the three wheat rusts. Losses from leaf rust infection are carries numerous useful agronomic traits and constitutes usually less than those from stem rust and stripe rust, a tertiary gene pool for wheat improvement (F EDAK but leaf rust causes greater annual losses due to its and H AN 2005). To date, several bread wheat –Th. more frequent and widespread occurrence ( MCCALLUM intermedium amphiploids have been obtained and and SETO-GOH 2005). Stem rust caused by the fungus characterized by means of genomic in situ hybridization Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn. is (C HEN et al. 2003; F EDAK and H AN 2005; B AO et al. 2009; a major, devastating disease of bread and durum wheat C HANG et al. 2010; G EORGIEVA et al. 2011). However, there

© 2013 The Authors. This is an Open Access article. DOI: 10.1111/j.1601-5223.2012.02262.x Hereditas 150 (2013) Cytological characterization durum – Thinopyrum amphiploids 11 are few amphiploids reported from durum wheat and Th. (Roche, Germany) for probing. Sheared genomic DNA of intermedium . Here we report the development of two durum wheat was used as blocking DNA. new durum wheat– Th. intermedium partial amphiploids Slide treatment, hybridization and signal detection with resistance to multiple fungal pathogens. In this of the fl uorescence was carried out as described in the investigation, we attempted to determine the chromo- following procedure. Slides were fi rst denatured in 70% some composition and genomic origins of the alien chro- deionized formamide at 75° C for 3 min and then mosomes of two partial amphiploids by genomic in situ dehydrated in an ethanol series (70%, 95% and 100%) for hybridization (GISH). 5 min each. The hybridization mixture was prepared per slide in 15 μl, consisting of 7.5 μl deionized formamide, 100 ng digoxigenin genomic DNA, 0.5 μ l 20 SSC, 3 μ l MATERIAL AND METHODS 50% dextran sulphate, 7.5 μg salmon sperm DNA and μ material 6 – 8 g sheared blocking DNA. The hybridization mix- ture was denatured at 80 ° C for 10 min, immediately Two durum wheat – Th. intermedium partial amphiploid chilled on ice for 5– 10 min, and then added onto the lines, 08-47-50 and 08-53-55, analyzed in this study, post-dehydrated slides for hybridization. After an over- are BC1 F6 derivatives. The 08-47-50 was produced by night hybridization at 37° C, post-hybridization washes crossing durum wheat line DR88S062 as female parent were performed in 2 SSC at room temperature for with a Th. intermedium accession of unknown origin. 5 min, 2 SSC at 42 °C for 10 min and 1 PBS for The was backcrossed to another durum wheat cul- 5 min. The slides were incubated with 10 μ g ml 1 tivar DR1022. The 08-53-55 combination was generated anti-digoxigenin-rhodamine or strepavidin-fl uroscein from the cross between durum wheat DR1022 and Th. isothiocyanate (FITC) (Roche) in PBS buffer for 30 min intermedium and then backcrossed to another durum at 37° C to detect digoxigin and/or biotin. The slides wheat DR46. Other wheat cultivars Roblin, Sumai 3, were then washed three times in 1 PBS for 5 min each Thatcher and Hoffman were used as checks in the present time. The chromosomes were fi nally counterstained FHB, leaf rust and stem rust evaluations. Total genomic with DAPI (5 μ g ml 1) solution (Vectashield mounting DNA from durum wheat DR46, Th. intermedium , media, Vector Laboratories, Inc.). Pseudoroegneria strigosa (M. Bieb.) Á . Lö ve (St genome) For multiple-color GISH analysis, total genomic DNAs and Thinopyroum elongatum (Host) Á . L öve (E genome) from Pseudoroegneria strigosa (St) labeled with was used as probes or blockers for GISH analyses. biotin-16-dUTP and Th. elongatum (Ee ) labeled with digoxingenin-11-dUTP by the nick translation method Chromosome preparation were used as probes. Total genomic DNA of durum wheat Seeds were germinated on moistened fi lter paper in Petri was used for blocking. Digoxigenin and biotin were dishes. The actively growing were collected at detected using anti-digoxingenin-rhodamine Fab frag- lengths of 1 – 1.5 cm. The tips were immersed in ice- ments and streptavidin-fl uroscein thiocyanate (FITC) water for about 24 h and fi xed in ethanol-acetic acid (3:1) (Roche), respectively. The chromosomes were fi nally for about one week at room temperature and then stored counterstained with DAPI solution. The slides were visu- in 70% (v/v) ethanol. After staining with 1% (w/v) aceto- alized with a fl uorescence Zeiss Axioplan 2 microscope carmine for at least 2 h, the root tips were squashed equipped with the appropriate fi lters and the signal pat- in 45% (v/v) acetic acid. For meiotic chromosome pre- terns were taken by CCD camera (Zeiss, Germany). paration, young spikes at the meiotic metaphase I stage were fi xed in 95% ethanol – chloroform – glacial acetic Evaluation of disease resistance acid (6:3:1) at room temperature for 48 h, then maintained in 70% ethanol at 4° C. Anthers were stained and squashed Three separate from a bulk source of BC 1 F6 in 1% acetocarmine. seeds were used for screening. In each pot, three spikes at similar developmental stage per plant were inoculated for FHB resistance. At anthesis, one fl oret in the middle of Genomic in situ hybridization analysis each spike was injected with 10 μ l of inoculum (50 000 The prepared slides were frozen at 70 °C for about four spores ml 1) of a mixture of three F. graminearum days, and then the cover slips were removed using a razor isolates: DAOM178148, DAOM232369 and DAOM blade. Slides were then dehydrated in 70%, 95%, 100% 212678 (Canadian Collection of Fungal Cultures, ethanol for 5 min each and then air-dried at room tem- Agriculture and Agri-Food Canada, Ottawa, Canada). perature before use. Genomic DNA of Th. intermedium The inoculated plants were fi rstly misted for 48 h (15 s of was extracted by a modifi ed CTAB method and labeled misting every 15 min) and then grown at 25 °C for FHB with digoxigenin-11-dUTP using a nick translation mix development. At 21 days after the initial inoculation, 12 J. Zeng et al. Hereditas 150 (2013) the number of infected spikelets per inoculated spike was some modifi cation, at 14 days after inoculation. The infec- scored. The percentage of symptomatic spikelets in an tion types (IT) of both leaf rust and stem rust were: inoculated spike was calculated to measure type II resis- “ 0 ” immune response, “ ; ” hypersensitive fl ecks, tance. Roblin was served as the susceptible check and “ 1 ” small uredinia with necrosis, “ 2 ” medium sized Sumai 3 was used as a resistant check. The FHB evalua- uredinia with chlorosis or necrosis, “ 3 ” medium sized tion was conducted in a greenhouse with a completely uredinia without chlorosis or necrosis and “ 4 ” abundant randomized design. The data were analyzed using large uredinia without chlorosis or necrosis. Similarly “ – ” one-way analysis of variance (Software package 16.0, and “ ” , respectively, explained slight variations in the SPSS). Individual differences among means were deter- expression of an IT. mined by Turkey ’ s test at a signifi cant level of P 0.05. The partial amphiploid lines of 08-47-50 and 08-53-55 were tested at the seedling stage against the Puccinia RESULTS triticinia (Pt) pathotypes, 96-12-3 MBDS, 95-77-2 TJBJ, Meiotic analysis of partial amphiploids 95-74-2 MGBJ, 94-128-1 MBRJ, 06-1-1 TDBG and three mixtures of virulence phenotypes used to represent Chromosome confi gurations at metaphase I of PMCs the genetic and virulence diversity of P. triticina found were carried out for the two new partial amphiploids. within Canada in each of three years (2005, 2006 and The results showed that both partial amphiploid lines 2008). The Puccinia graminis Pers. f. sp. tritici (Pgt) had a chromosome number of 2n 42 (Fig. 1A – B). In pathotypes, race Ug99 (also designed TTKSK) and its the line 08-47-50, frequencies of univalents ranged from variant TTKST were also used as the inoculum. Each 0 to 4 after observation of 100 pollen mother cells, and isolate was developed from a single pustule and the no more than one trivalent occurred in other cells. The purity of each isolate was tested by inoculating a set of mean chromosome confi guration at MI was 0.75I standard host differential lines as described previously 20.49II 0.09 III. In the line 08-53-55, frequencies of univalents ranged from 0 to 4 and the bivalents ranged ( MCCALLUM and S ETO-GOH 2005). To inoculate with each isolate or mixture, ten seedlings per line were planted from 19 to 21. The mean chromosome confi guration in a clump and the clumps were evenly spaced in a fi ber was 0.66I 20.67II. These results of meiotic analysis fl at (25 15 cm). Spores were suspended in a light min- confi rmed that the two novel partial amphiploids were eral oil (Bayol, Esso Canada, Toronto, ON), and sprayed basically cytologically stable. onto seedlings at the 1– 2 leaf stage. The plants were GISH identifi cation of partial amphiploids allowed to dry for one hour, and then incubated in a cham- ber at 20 4 °C with 100% relative humidity in the By probing with digoxigenated total genomic DNA of dark for 17 h. Plants were transferred to a greenhouse for Thinpyrum intermedium and blocking with genomic symptom development at 20 4 °C with supplemental DNA of durum wheat, 14 Th. intermedium chromosomes high-pressure sodium lights. The infection types were and 28 durum wheat chromosomes were distinguished in recorded as described by M CINTOSH et al. (1995), with both 08-47-50 and 08-53-55 (Fig. 2A). No translocation

Fig. 1A– B. Meiotic confi guration at metaphase I. (A ) 08-47-50, 2n 42 21 II (rings), (B ) 08-53-55, 2n 42 18 II (rings) 3 II (rods). Hereditas 150 (2013) Cytological characterization durum – Thinopyrum amphiploids 13

Fig. 2A – C. The GISH patterns of 08-47-50 and 08-53-55 lines. (A ) Total genomic DNA of Th. intermedium was used for probing. ( B) (08-47-50) and ( C) (08-53-55): The St genome is visualized in green, the Ee genome in red, and the durum wheat chromosomes in blue or grey blue. The interstitial translocation chromosomes involving Ee and St genomes indicated with arrows and double arrows. involving durum wheat and Th. intermedium chromo- partial amphiploids, and thus showing lower overall somes was observed. The multiple-color GISH technique mean infection scores. The mean percentage of symp- was also carried out to examine the alien chromosome tomatic spikelets was 6.33% in 08-47-50 and 5.91% composition as well as possible inter-genomic inter- in 08-53-55, whereas the checks had 14.78% for Sumai 3 changes by using genomic DNAs of P. strigosa (St) and 100% for Roblin (p 0.05) (Table 1). Therefore, and Th. elongatum (E e ) as probes. In line 08-47-50, the the FHB damage was significantly lower in both par- GISH patterns from probing with St and Ee genomic tial amphiploids than in Roblin (100%) and Sumai 3 DNAs revealed that six chromosomes were labeled (14.78%). over the entire length by a green fl uorescence signal and therefore belonged to the St genome. Four chromosomes Leaf rust and stem rust responses had red hybridization signals and were identifi ed as E e genome chromosomes. Two pairs of chromosomes Seedling rust response scores for both partial amphiploids had translocations involving the Ee and St genomes. One 08-47-50 and 08-53-55 against two Pgt pathotypes and pair of chromosomes had green hybridization signals at fi ve Pt pathotypes and three mixtures of virulence Pt its terminal region and the other chromosome region pathotypes collected in Canada in each of three years had red hybridization signals, indicating a terminal trans- 2005, 2006 and 2008 are presented in Table 1 and 2. location. The other translocated chromosome pair had an 08-47-50 exhibited infection type 1 and ; against intercalary translocation, in which the terminal region of TTKSK and TTKST, respectively, while 08-53-55 the short arm and the entire long arm showed St genomic exhibited infection type 1 against the two Pgt pathotypes hybridization signals (Fig. 2B) with an E genome segment (Table 1). The leaf rust response to fi ve Pt pathotypes and at an interstitial location. three mixtures of virulence Pt phenotypes in 08-47-50 In the line of 08-53-55, it was shown that four chromo- somes were hybridized by the St genomic probe and ten translocated chromosomes involving Ee and St genomes Table 1. Reactions to FHB and stem rust races TTKSK were detected. Of the ten translocated chromosomes, eight and TTKST for two durum wheat– Thinopyrum inter- chromosomes had St genomic hybridization signals at the medium partial amphiploids . terminal region of both arms. The remaining two translo- Stem rust pathotypes/ cated chromosomes were labeled by Ee hybridization sig- infection type nals at the centromeric region and short arm (Fig. 2C). Line TTKSK TTKST FHB severity (%) Accordingly, the genomic constitution formula of 08- 47-50 was concluded to be 28DW 6St 4Ee 4E e -St, 08- 47-50 1 ; 6.33c 08 - 53-55 1 1 5.91c and 08-53-55 had a genomic constitution of 28DW Roblin nt nt 100a e 4St 10E -St (DW durum wheat chromosomes). Sumai 3 nt nt 14.78b Hoffman 3 3 nt FHB symptom spread n t not tested. Within the FHB severity column, values Under greenhouse conditions, the fungal infection followed by different letters are signifi cantly different at the was restricted to the centrally inoculated spikelets in both P 0.05 level according to Tukey ’ s multiple range test. 14 J. Zeng et al. Hereditas 150 (2013)

Table 2. Reactions to different leaf rust races for two durum wheat – Thinopyrum intermedium partial amphiploids. Leaf rust pathotypes/infection typea Line MBRJ MGBJ TJBJ TDBG MBDS Epidemic-05 Epidemic-06 Epidemic-08 08 - 47-50 0; 0 0 1 1 0; 0; 0; 08 - 53-55 ;1 ;;1 1 1 ;1 ;1 ; Thatcher 3 3 3 3 3 3 3 3 a 0; presence of slightly higher response than 0, 1 presence of slightly lower response than 1, 1 presence of slightly higher response than 1, 3 presence of slightly lower response than 3, ;1 presence of slightly higher response than ; and slightly lower response than 1. Scores of 0 – 2 are classifi ed as resistant and 3 – 4 as susceptible reactions. ranged from infection type 0 to 1 (Table 2). 08-53-55 genes, in particular for those which to a large extent are expressed infection type from ; to 1 . Thatcher was sus- lacking in bread wheat or durum wheat. To date, genes ceptible (infection type from 3 to 3 ) to all fi ve Pt for various disease resistances have been transferred pathotypes and three mixtures of Pt pathotypes. Figure 3 from Th. intermedium to bread wheat, such as Wsm1 shows the leaf rust and stem rust infection types in conferring resistance to wheat streak mosaic virus 08-47-50, 08-53-55 and susceptible checks. (WSMV) (F RIEBE et al. 1991), Bdv2 ( ZHANG et al. 1999) and Bdv3 ( OHM et al. 2005) specifying resistance to yellow dwarf virus (BYDV), Pm40 ( LUO et al. DISCUSSION 2009) and Pm43 ( HE et al. 2009) resistance to powdery Discovering novel and diverse sources of resistance is mildew, Lr38 ( FRIEBE et al. 1992) resistance to leaf rust, critical for retaining genetic variation for disease and and Sr44 ( FRIEBE et al. 1996) resistance to stem pest resistance in wheat breeding programs, because rust. Resistance to FHB from Th. intermedium was also deployment of only one or a few sources of resistance identifi ed in derived wheat progenies ( OLIVER et al. over large crop production areas poses a danger of 2005). FHB resistance was also detected in partial resistance breakdown and disease epidemics. As an amphiploid obtained from a durum Th. distichum important perennial Triticeae species, Thimopyrum combination (C HEN et al. 2001). intermedium has frequently been used in bread wheat Development of partial amphiploids is an important improvement as a donor of various disease resistance fi rst 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 Ee Ee St ( LIU and WANG 1993) or Ee E b St ( CHEN et al. 1998) with Ee ( J) and Eb ( J s ) designating the closely related Th. elongatum and Th. bessarabicum genomes. Lately, new insights in the genome composition of Th. intermedium became avail- able (K ISHII et al. 2005). Recent studies indicate that the genomic constitution of Th. intermedium may be some- what more complex as revealved by the existence of sequences from Th. caespitosum, caput-medusae and Crithopsis delileana in its genome (A RTERBURN et al. 2011) . The existence of these sequences may be responsible for some of the different GISH stain- ing 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 Fig. 3. Seedling leaf rust and stem rust responses of partial genomic constitution of the partial amphiploids 08-47-50 amphiploids against Pt pathoype MBRJ (1 08-47-50, 2 08-53- and 08-53-55, but also revealed the chromosome struc- 55 and 3 Thatcher) and Pgt pathotype TTKST (4 08-47-50 and tural variation and rearrangement involving Ee and St 5 Hoffman) . genomes. Hereditas 150 (2013) Cytological characterization durum – Thinopyrum amphiploids 15

The GISH results clearly demonstrated that the partial amphiploids normally have regular meiosis with difference in genomic constitution between 08-47-50 high frequencies of bivalent confi gurations and low and 08-53-55 was due to the different ratio (E-St) of frequencies of multivalents. Their genomes should be alien chromosomes of Th. intermedium and the differ- largely balanced in terms of homoeologous chromosomes ences in translocated chromosomes. 08-47-50 contained ( FEDAK and HAN 2005). This is in contrast to partial six St genome chromosomes, four E e genome chromo- amphiploids derived from a durum wheat and Th. somes and two pairs of E e-St translocated chromosomes distichium combination where progeny with 50 and plus twenty-eight durum wheat chromosomes. 08-53-55, 42 chromosomes were obtained with only the latter in addition to the complete durum wheat genome, con- being stable. sisted of four chromosomes of the St genome, and fi ve In previous researches, the genes for resistance to pairs of E e-St translocated chromosomes. In terms of WSMV, BYDV, rust and powdery mildew were not translocated chromosomes, different fragment sizes of located on chromosomes of the St genome but on those E e -St reciprocal translocations were detected, showing of Ee (J) or Eb (Js ) genomes as determined by C banding, the fragment of E e genome on the short arm of two St- GISH and molecular marker analyses ( KONG et al. chromosomes in 08-47-50 and the larger fragment of 2009; LI et al. 2005). In the present study, the fungal E e genome involving the centromeric region on the disease evaluation showed that 08-47-50 and 08-53-55 two St-chromosomes in 08-53-55. In addition, two E e-St were resistant to FHB, leaf rust and stem rust. GISH terminal translocation chromosomes and two interstitial revealed that 08-47-50 had six St chromosomes, four Ee translocation chromosomes of E e-St were further dis- chromosomes and four translocated chromosomes involv- cerned in 08-47-50. Eight Ee -St terminal translocation ing Ee and St chromosomes. While four St chromosomes chromosomes and two Ee -St interstitial translocation and ten Ee -St translocated chromosomes were discerned chromosomes were observed in 08-53-55. However, in 08-53-55. Ee and St genome chromosomes could be we did not observe any St-signal near the centromeric associated with resistance to FHB since durum has no region using the St-genome probe, which is not consis- FHB resistance. tent with the previous reports (C HEN et al. 2003; B AO Fungal diseases are caused by a very dynamic group et al. 2009). This difference in results seems attributable of plant pathogens. Both leaf rust and FHB annually to the polymorphism of St genome chromatin. Another cause epidemics in the world. A new stem rust pathotype reasonable explanation could be that the chromosome Ug99 with serious virulence on the widely deployed constitution of Th. intermedium varies greatly among resistance gene Sr31 was detected in Uganda in 1999 and and within accessions (X U and C ONNER 1994). has since mutated to two additional variants (P RETORIUS Interstitial translocations were detected in partial et al. 2000; J IN and SINGH 2006; JIN et al. 2008). The amphiploid 08-47-50 in this study. Such translocations genetic fl exibility of these pathogens has lead breeders to are rare in more conventional plant genomes and hybrids respond with a constant search for new resistance genes ( JIANG et al. 1993), but have been reported in a number and incorporating them into wheat genetic backgrounds. of partial amphiploids ( FEDAK et al. 2000; FEDAK and Our present study revealed that the two novel partial HAN 2005; CHANG et al. 2010). Perhaps they occur amphiploids harbour a variety of resistance genes to more frequently in the genomes of complex polyploids several major fungal diseases. It is expected that more where some of the genomes such as E and J are closely genes for resistance against diseases can be discovered related, and are now being detected by multicolor GISH in Th. intermedium , especially the genes that rarely occur technology. in bread wheat or durum wheat. We are currently making At metaphase I, the chromosomes pairing association backcrosses to bread wheat and durum wheat in order of 08-47-50 was similar to that of 08-53-55 with high to develop addition, substitution and translocation lines frequencies of bivalent and very low multivalent forma- with resistance to FHB, leaf rust and stem rust. tion. 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