Characterization of a partial wheat–Thinopyrum intermedium amphiploid and its reaction to fungal diseases of wheat

Partial amphiploids between wheat (Triticum aestivum L.) and Thinopyrum species play an important role in the transfer and use of traits from alien species. a wheat-Thinopyrum intermedium partial amphiploid, Tai8335, and its alien parent were characterized by a combination of genomic in situ hybridization (giSh) and cytological observations. evidence from giSh indicated that the donor parent Th. intermedium possessed seven pairs of S, seven j and 21 j chromosomes. Mitotic observation showed that the majority of Tai8335 plants had 56 chromosomes, but a few had 54 to 55, in some cases with two to three additional telochromosomes. The chromosomes in most pollen mother cells of plants with 2n  56 formed 28 bivalents, averaging 27.12 in 223 cells, suggesting a basic cytological stability. Sequential giSh patterns using genomic Pseudoroegneria spicata and genomic Th. intermedium Dna as probes revealed that Tai8335 had fourteen chromosomes derived from Th. intermedium and its alien genome consisted of one pair of S-, three pairs of jand one pair of j-genome chromosomes as well as two translocated chromosome pairs, one being a Robertsonian translocation and another an intercalary translocation, both of which involved j and S genome. Two of the telochromosomes in the aneuploid plants originated from the j genome and one from wheat. Disease screening demonstrated this line was highly resistant to leaf rust, stem rust, stripe rust and powdery mildew. This study showed that the partial amphiploid Tai8335 appears to serve as a novel source for the transfer of resistance genes for multiple fungal pathogens to wheat.

The ongoing improvement of wheat cultivars is dependent on a continuous supply of genetic variability. Thinopyrum intermedium (host) Barkworth & D.R. Dewey [syn. Agropyron intermedium (host) Beauvoir and Elytrigia intermedia (host) nevski] (2n  6x  42) carries many useful agronomic traits and constitutes a tertiary gene pool for wheat improvement. Th. intermedium is a segmental autoallo-hexaploid, and the determination of its genomic composition has been of interest for a considerable time. however, due to the impact of pairing regulation gene(s) and chromosome pairing complexities as well as the high levels of polymorphism in giemsa banded patterns within the species, no definite conclusions have been made based on traditional cytogenetic techniques, such as meiotic chromosome pairing and C-banding analysis (Chen et al. 1998).
The transfer of alien genes into wheat (Triticum aestivum L., 2n  6x  42, aBD) via wide hybridization makes possible to increase the resistance to biotic and abiotic stress and improvement of quality. in wheat breeding programs, the production of amphiploids or partial amphiploids between wheat and relative species is an important intermediate step for such a gene transfer, because they allow the reliable analysis of the effects of alien genes in the genetic background of wheat and their fertility allows gene transfer even when an F 1 hybrid is almost completely sterile (jiang et al. 1994;ellneskog-Staam and Merker 2002). The wild relatives, Th. intermedium (2n  6x  42, jj s S), has a wealth of genetic variation for the improvement of resistance to fungal and viral diseases in wheat (Li et al. 2008;Li and Wang 2009). To date, several wheat-Th. intermedium amphiploids, otrastsyuskaya (oT), TaF46, Zhong1 to Zhong5, 78829, Tai7044 and Te-3, have been obtained (Chang et al. 2003;Chen 2005;Fedak and han 2005;Yang et al. 2006). Some of them have been widely used for attempted introgressions of useful traits into wheat, including resistance to viral diseases (Larkin et al. 1995;Friebe et al. 1996;Chen et al. 2003;Li et al. 2008;Li and Wang 2009). however, partial amphiploid still do not cover the entire genome of Th. intermedium, due to the complexity of the genomic composition of the alien parent. The exploitation of a new wheat-Th. intermedium partial amphiploid type still remains necessary for understanding the genetic relationships of the S and j or j s genomes of Thinopyrum with those of wheat, and for incorporating disease-resistance hereditas 147: 304-312 (2010) genes from Th. intermedium into wheat. Tai8335 is a Th. intermedium-derived partial amphiploid that was recently developed in our laboratory. in this study, we attempted to determine the chromosome composition and genomic origins of the alien chromosomes of Tai8335 by genomic in situ hybridization (giSh) and to evaluate its potential as a novel source for resistance to fungal pathogens.

Plant material
The Tai8335, analyzed in this study, is a BC 1 F 8 -derived partial wheat-Th. intermedium amphiploid line, which was produced by crossing T. aestivum cv. jinchun 5 as the female parent with a Th. intermedium accession of unknown origin. The hybrid was backcrossed to another wheat cv., jinmai 33, and the resulting plants from the BC 1 F 2 population that were fertile and intermediate in morphology between wheat and Th. intermedium parents were selfed for six generations.
other plant material included the wheat parents and the Th. intermedium accession with the genomic formula e 1 e 2 St (Wang and Zhang 1996) or jj s S (Chen et al. 1998) used as a donor, the latter provided by Prof. S. h. Li of the Shanxi academy of agricultural Sciences, China. Several well known Th. intermedium-derived partial amphiploids were also included in the present disease evaluation: otrastsyuskaya (oT), TaF46 (Chen et al. 1999), and Zhong 2, Zhong 4 and 78829, which were derived from the same Th. intermedium accession  but had different alien chromosome composition Chen et al. 2003;han et al. 2004). also included were Tai7044, which possesses the 6 S  8 j Thinopyrum chromosomes (Chang et al. 2003), and wheat cultivars Mingxian 169, jingshuang 16 and Wichita as susceptible controls. The investigation of the chromosome constitution of the wild parent was carried out in a population of eight generations produced from the donor plant by open-pollinating. Total genomic Dna from the wheat cultivar Chinese Spring (CS) (2n  6x  42, aBD), Th. intermedium and Pseudoroegneria spicata (Pursh) Löve (2n  2x  14, S) was used as probes or blockers for giSh analyses.

Chromosome preparation
Root tips collected from the young seedlings were immersed in ice water for about 24 h and fixed in ethanolacetic acid (3:1) for about one week, then stained using the conventional feulgen method for chromosome counting. For giSh, the roots were stained in 1% acetocarmine for a few minutes and squashed in 45% acetic acid. Cover slips were removed after freezing on dry ice, and the preparations were then dehydrated in ethanol for 5 min prior to in situ hybridization.
For meiotic chromosome preparations, anthers from the emerging spikes containing pollen mother cells (PMCs) at Mi were fixed in ethanol:acetic acid (3:1) for one day, transferred to 70% ethanol, and kept at 4°C in a refrigerator for about two weeks. anthers were then stained in 1% acetocarmine and squashed in 45% acetic acid.

Probe labeling and genomic in situ hybridization (GISH)
Total genomic Dna was extracted from fresh young leaves of Th. intermedium and Ps. spicata using a Dneasy Plant Mini Kit and following the manufacturer's instructions (Qiagen inc., Valencia, Ca, uSa). The procedure for giSh was as described in Zhang et al. (2001), with some modifications. approximately 1 mg of genomic Dna from Th. intermedium or Ps. spicata was labeled with fluorescein-12-duTP (FiTC detected by yellow-green fluorescence) (enzo Life Sciences inc, Farmingdale, nY, uSa) in a 50 ml reaction mixture using nick translation. Slides were dehydrated in a series of 70%, 90% and 100% ethanol, and slide-bound chromosomal Dna was denatured in 100 ml of 70% formamide in 2  saline sodium citrate (SSC) at 80°C for 2 min. The hybridization solution contained 50% deionized formamide, 20  SSC, 10% dextran sulfate, 0.3 mg ml -1 of sheared salmon testes Dna, and 60 ng of labeled genomic Dna, plus an excess amount of unlabeled genomic Dna of CS, which was sheared to 600-700 bp to block cross-hybridization of probed Dna to wheat chromosomes. The probe-to-blocker ratio was between 1:110 and 1:120. For the wild parent Th. intermedium, the genomic Dna from Ps. spicata was used as a probe and no blocking Dna was added to the hybridization mixture. The hybridization solutions were denatured by boiling and chilling on ice for about 7 min. Thirty ml of denatured hybridization mixture were applied to each slide and covered with a 20  20 mm plastic cover slip. hybridization was conducted in a humid chamber overnight at 37°C. after hybridization, slides were washed in 2  SSC at 42°C for 10 min, 50% formamide in 2  SSC at 42°C for 10 min (equivalent to 82% stringency), 2  SSC at 42°C for 10 min, and 1  PBS at room temperature for 5 min. The biotin-labeled probes were detected with a FiTC conjugated anti-biotin antibody (Vector). Chromosomes were counterstained with propidium iodide (Pi) and fluoresced red. Slides after giSh were examined by using an epifluorescent microscope, and fluorescent images captured using a SPoT 2.1 charge-coupled device (CCD) camera (Diagnostic instruments) attached to an epifluorescence Zeiss axioplan 2 microscope.

Disease resistance evaluation
The partial amphiploid line Tai8335 and its parents, as well as other known Thinopyrum-derived partial amphiploids and susceptible checks (Table 3), were screened for described by MCintosh et al. (1995) with some modifications: 0  no visible infection; 0:  necrotic/chlorotic flecks, without sporulation; 1  trace sporulation, necrotic/ chlorotic stripes; 2  intermediate sporulation, necrotic/ chlorotic stripes; 3  abundant large sporulation, necrotic/chlorotic stripes; 4  abundant large sporulation, without chlorosis. For the seedling test with powdery mildew, three B. graminis f. sp. tritici (Bgt) isolates (e09, e20 and e21) (for virulence/avirulence formulae see hua et al. 2009) were used. The conidia were dusted onto one-leaf stage plants. Sporulation was promoted at 17°C with a 14/10 h light/dark photoperiod and 60% relative humidity (Rh). host reactions were recorded 7 to 10 days after inoculation, when the susceptible checks were heavily infected. a 0 to 4 iT scale (Sheng 1988) was used to describe host responses to infection. Scores of 0-2 were regarded as resistant and 3-4 as susceptible.

Genome composition of alien parent Th. intermedium
The mitotic preparations of the Th. intermedium plant crossed with common wheat were subjected to giSh analysis in this study to determine the genomic composition of the alien parent. using the S genomic Dna from Ps. spicata as a probe and without a blocker, the 42 chromosomes all fluoresced greenish-yellow either over their entire length or at their centromeric and/or terminal region(s) (Fig. 1). among them, seven small chromosome pairs were labeled bright greenish-yellow uniformly all along their length, while the remaining fluoresced mostly red, indicating that Th. intermedium had an entire S genome set. of the remaining 28 chromosomes, seven were hybridized with the probe at the their reactions to leaf rust and stem rust at Kansas State university (KSu), and stripe rust and powdery mildew evaluations were performed in China.
urediniospores for each race suspended in Soltrol-170 mineral oil (Chevron-Phillips chemical company) were atomized onto the plants. For leaf rust and stem rust tests, inoculated seedlings and adult plants were incubated in a dew chamber for 18 h at 18°C. Plants were then placed in a greenhouse at 19-21°C with supplemental sodium vapor lighting. For the stripe rust test, inoculated seedlings and adult plants were kept in a dark dew chamber for 24 h at 12  2°C. after inoculation, plants were kept in growth chambers that were set at 16°C (day) and 14°C (night) with a 16-h photoperiod. The infection types (iTs) of leaf rust and stem rust were scored 10-12 days after inoculation. For stripe rust, the iT scoring was done 20 days after inoculation. The rust reaction was rated at seedling and adult stages using the 0-4 scale as illustrated in Roelfs et al. (1992) and MCintosh et al. (1995).
Screening for stripe rust and powdery mildew reaction Testing for resistance to stripe rust and powdery mildew was conducted, respectively, at the university of electronic Science and Technology of China and the Shanxi institute of Crop genetics, China. Wheat cultivars Mingxian 169 and jingshuang 16 were used as susceptible checks for the stripe rust and powdery mildew tests, respectively. Three pathotypes (CYR29, CYR31 and CYR32) of Pst were used for screening the stripe rust response. CYR32 had all the virulence factors of CYR31, and was virulent on all differential cultivars, except Zhong 4, Triticum spelta album (Yr5), and Moro (Yr10 and YrMor) (Wan et al. 2004). one-leaf-stage plants were sprayed with distilled water containing 0.05% Tween-20 and brushed with spores of stripe rust races increased on Mingxian 169 plants. Following inoculation, plants was incubated at 10°C and 70% Rh in the dark for 24 h. Then, epidemics were induced at 16/11°C day/night with a 14/ 10 h light/dark photoperiod at 70% Rh. iTs of seedlings were scored 14 days after inoculation, using the scale shown Table 1. Sequential giSh patterns using genomic Dna of Ps. spicata (Fig. 2a) and Th. intermedium (Fig.  2b) as probes showed that almost all the plants examined contained 14 chromosomes from Th. intermedium, labeled by FiTC and fluorescence green, while the remaining 40-42 wheat chromosomes displayed a uniform red Pi fluorescence. no translocation involving wheat and Th. intermedium chromosomes was detected. The giSh pattern from probing with S genomic Dna revealed that one pair of metacentric chromosomes was labeled over their entire length and belonged to the S genome (a in Fig. 2c). Three pairs had hybridization signals around the centromeres and at the telomere(s), and were identified as j s -chromosomes (B, C, D in Fig. 2c). one pair had hybridization signals only at the telomeres, indicating it was derived from the j genome (e in Fig. 2c). The remaining two chromosome pairs had translocations involving the j and S genomes. one was a Robertsonian whole-arm translocation involving one arm of a j-and S-genome chromosome; one arm had hybridization signals only at its telomere, whereas the other arm was labeled over its entire length (F in Fig. 2c). The other translocated pair had an intercalary translocation, in which the terminal region of the long centromeric and terminal region(s), although the signals around the centromeres were a little stronger than those at the telomere(s), and 21 had hybridization signals (FiTC fluorescence) only at the telomere(s), which demonstrated they belonged to the j s and j genome, respectively. on the basis of the giSh pattern labeled with S-genomic Dna probe and chromosome morphology, the genome structure of the Th. intermedium plant used as the alien parent appears to be 14 S-genome, 7 j s -genome and 21 j-genome chromosomes.

Cytology of the partial amphiploid
Chromosome counting The main spikes of Tai 8335 had an average self seed setting of 62.54%. of 25 seeds sown, 23 seeds germinated; 17 seedlings had 2n  56, two had 2n  54, and one had 2n  55. The chromosome number varied from 54 to 56, with 56 being the most frequent. additionally, two to three telocentric chromosomes were encountered in a few of the aneuploid plants with 2n  54-55 (Table 1).
Chromosome pairing as shown in Table 2, 28 homologous chromosome pairings (21 T. aestivum plus seven Th. intemedium bivalents) appeared in most of the PMCs from the plants with 2n  56. Frequencies of univalents ranged from 0-6, and no more than one trivalent or quadrivalent occurred in other cells. Based on the 223 cells scored, the mean chromosome configuration at PMC Mi of Tai8335 with 2n  56 was 0.98i  27.12ii  0.13iii  0.10iV. The result of meiotic analysis confirmed that this partial amphiploid is basically cytologically stable.

GISH identification of the partial amphiploid
after the chromosome counting, the giSh technique was used to determine the number and genomic composition of the alien chromosomes in Tai8335. The genomic constitution revealed by giSh in 23 plants of Tai8335 is Z.-J. Chang et al. hereditas 147 (2010) Thinopyrum intermedium-derived partial amphiploids Zhong 2, Zhong 4, Tai7044, TaF46, oT and 78829 were also tested with the above rust races and powdery mildew isolates. The testing for powdery mildew showed all these known partial amphiploids were highly susceptible to all powdery mildew isolates tested (Table 3). in the rust screening, reactions of Zhong 2, Zhong 4 and 78829 were immune or highly resistant to all races of leaf, stem and stripe rust. oT was resistant to leaf rust and stem rust and PST 100, but susceptible to CYR32 of stripe rust, which is the most widely virulent pathotype in China and virulent to Yr1, Yr2, Yr3, Yr4, Yr6, Yr7, Yr9, Yr17, Yr22, Yr23, Yr27, YrHVII, YrHK, YrCle, YrA, YrCV1, YrCV2, YrCV3, YrG, YrSD, YrSO (Wan et al. 2004). TaF46 was immune or resistant to all other rust races tested, but susceptible to Pgt race TPMK, a predominant race in all areas of the united States during the 1990s with the virulence to Sr5, Sr7b, Sr8a, Sr9d, Sr9e, Sr9g, Sr10, Sr11, Sr17, Sr21, Sr36 and SrTmp (olivera et al. 2007). Tai7044 was immune to leaf and Pst race PST100, but susceptible to stem rust and CYR32 (Table 3).

DiSCuSSion
Knowledge of the chromosome constitution of a donor parent is essential for elucidating the genome origin of arm and almost the entire short arm hybridized with an S-genomic probe except at the centromere region and end of short arm (g in Fig. 2c). Two telocentric j-chromosomes plus one telocentric chromosome from wheat or two j-telochromosomes were also observed in three aneuploid plants with 2n  54-55 (Table 1). Thus, the alien genomic constitution of most plants in Tai8335 consists of two S-genome, six j s -genome, and two j-genome chromosomes plus two pairs of translocated chromosomes involving the j and S genomes.

Disease resistance screening
The partial amphiploid line Tai8335 and its parents were screened with the most prevalent races of leaf rust, stem rust and stripe rust at KSu, and stripe rust and powdery mildew in China. Tai8335 and Th. intermedium were completely resistant (immune or hypersensitive flecks), whereas the parental cultivars jinmai 33 and jinchun 5 were highly susceptible to all the races tested (Table 3). The disease evaluation showed the resistance of Tai8335 to the fungal pathogens was similar to that of its donor parent Th. intermedium. Since Tai8335 is a derivative of that species and all wheat parents involved in its pedigree were highly susceptible, these resistances were derived from Th. intermedium and expressed in this partial amphiploid.   Consequently, the genome of Tai8335 should be largely balanced in terms of homoeologous chromosomes (Fedak and han 2005). Partial amphiploids play an important role in transferring useful traits present in alien species. it is essential, therefore, to know the exact genomic composition of the added alien chromosomes in the partial amphiploids. giSh, especially using an S genomic Dna probe, provides a powerful diagnostic cytogenetic tool for determining the genomic origin of alien chromosomes in wheat-Thinopyrum hybrids, which permits distinction of S, j, and j s chromosomes (Chen et al. 1998;Chen 2005). The present giSh pattern revealed that Tai8335 contained a synthetic alien genome composed of two S-genome, six j s -genome, two j-genome, and four translocated chromosomes, including one Robertsonian translocation pair and one intercalary translocation pair that were both derived from the j and S genomes (Fig. 2). Tai8335 had a different chromosome composition from those of previously described partial amphiploids Zhong 1 to Zhong 5 (Chen et al. 2003), TaF46 (Chen et al. 1999), 78829 , oT (Fedak et al. 2000), Tai7045 (Chang et al. 2001) and Tai7044 (Chang et al. 2003). Thus, Tai8335 is a novel wheat-Th. intermedium partial amphiploid.
Different studies have indicated that the maximum number of j s -genome chromosomes ranges from 6 to 11 in different accessions of Th. intermedium, suggesting that the j s genome may be incomplete within the Th. intermedium species (Chen et al. 1998). Th. intermedium-derived partial amphiploids mostly had j s -genome chromosomes and interchanges involving j s genome in their alien genome. among them, Zhong 1 to Zhong 5 and Tai7045 all contained four intact j s -genome chromosomes (Chen et al. 2003;Chang et al. 2001). The present study showed that Tai8335 had six intact chromosomes from the j s genome (Fig. 2), whereas only a total of seven j s chromosomes were present in its alien parent (Fig. 1). This may be attributed to the preferential transmission of some chromosomes of the j s genome to progeny in the crosses of wheat with Th. intermedium. additionally, chromosomes with translocations involving different Th. intermedium genomes or between Th. intermedium and wheat genomes were reported in several known partial amphiploids, and recent studies have shown that these translocations usually involved S-and j s -genome chromosomes (Chen 2005;Fedak and han 2005). The present giSh results showed that in Tai8335, the genetic exchange of chromosomes from different genomes, either in Robertsonian or intercalary translocation, occurred only between the S-and j-genome chromosomes of Th. intermedium (Fig. 2), demonstrating that in this accession, the relationship between S and j genome appears to be closer than with the j s genome.
as an important perennial Triticeae species, Th. intermedium has frequently been used in wheat improvement alien chromosomes in intergeneric hybrids and their derivatives. Thinopyrum intermedium is a segmental autoallo-hexaploid. identification of the exact genomic composition of this species has been difficult with traditional cytogenetic techniques, due to chromosome pairing complexity and a high level of polymorphism in chromosome variation. giSh has provided powerful tools for the direct characterization of the chromosome composition of polyploid plant genomes at the Dna level. using this method, Chen et al. (1998) found that Th. intermedium had 13-14 S, 6-11 j s , and 17-21 j chromosomes in different accessions. The alien parent in this study had an unknown origin and its genome was composed of 14 S, 7 j s and 21 j chromosomes, as revealed by probing with the genomic Dna of Ps. spicata, which shares the common S genome with Ps. strigosa (Yang et al. 2006). This variation in chromosome composition of different Th. intermedium accessions might be attributed to the outcrossing nature of this species.
in previous reports, the chromosomes of the j s genome had FiTC signals only or mainly in the centromeric areas when blocked with j/e genomic Dna from Th. bessarabicum and Th. elongatum (Chen et al. 1998;Chen 2005), while in our study there were signals both around the centromere and at the telomere(s) in the absence of blocking Dna (Fig. 1). This difference in giSh banding patterns of j s chromosomes seems attributable to the polymorphism of S genome chromatin (repetitive sequences of S genome) at the terminal regions present among the different accessions and/or the absence of blocking Dna. Being an outpollinated perennial species, the chromosome constitution of Th. intermedium varies greatly among and within accessions (Xu and Conner 1994). in our experiment, seven single j s -genome chromosomes and eight pairs plus five single j-genome chromosomes were observed in most plantlets (Fig. 1), a difference with the alien parent of the Zhong series and 78829, which had three pairs of j s and 11 pairs of j chromosomes (Tang et al. 2000).
evaluation of the cytogenetic stability of intergeneric hybrids is of theoretical and practical significance. Thinopyrum-derived partial amphidiploids normally have regular meiosis with high frequencies of bivalent and low multivalent formation (Fedak et al. 2000). of the 223 cells with 2n  56 analyzed here, about 74% of the cells formed the expected 28 bivalents at Mi, and only 0.98 unpaired chromosomes occurred per cell. The frequency of univalents was much lower than that reported for TaF46 (2.55), but a little higher than otrastayuskaya 38 (0.74), both stable partial amphiploids derived from Th. intermedium (Fedak et al. 2000). This indicated that the present amphiploid had a basic stability in cytology. Meanwhile, this relatively regular meiotic behavior was reflected in the common composition of alien chromosomes among plants and a vigorous growth habit with high fertility. as a donor of various disease resistance genes, in particular for those which to a large extent are lacking in wheat, such as wheat streak mosaic virus (WSMV), its vector the wheat curl mite (WCM) (Aceria tosichella Keifer), barley yellow dwarf virus (BYDV) and Fusarium head blight (Friebe et al. 1996;Chen 2005;Fedak and han 2005). To date, one gene conferring resistance to WSMV, Wsm1 (Friebe et al. 1991), and at least two genes specifying resistance to BYDV, Bdv2 (Zhang et al. 1999) andBdv3 (ohm et al. 2005), have been identified in Th. intermedium. These genes were located on the arm of the j s -or j-genome chromosome of this species by C-banding, giSh and/or molecular marker analyses (Zhang et al. 1999;Friebe et al. 2009;Kong et al. 2009).
Resistance to rust also was transferred from Th. intermedium to wheat. Friebe et al. (1992) reported that the leaf rust resistance gene Lr38 was located in the distal half of the long arm of the chromosome 7ai-2, a chromosome from the j s genome (Tang et al. 2000), whereas the genes for resistance to stem rust and stripe rust were located either in the short arm or in the proximal region of the long arm of this chromosome. The stem rust resistance gene Sr44 was localized on the short arm of chromosome 7ai-1(j) (Friebe et al. 1996). More recently, two novel powdery mildew resistance genes derived from Th. intermedium, Pm40 and Pm43, have been characterized and introgressed into wheat (he et al. 2009;Luo et al. 2009Luo et al. ). he et al. (2009 pointed out that Pm43 originated from a j s -genome chromosome of a wheat-Th. intermedium partial amphiploid. Based on these results, the genes for resistance to WSMV, BYDV, rusts, and powdery mildew are not located on chromosomes of the S-genome but on those of the j-or j s -genomes. in the present study, not only six j s -and two j-genome chromosomes, but also four translocated chromosomes involving the j genome were identified in Tai8335, suggesting that the j s -or j-genome chromosomes appear to be associated with resistance to leaf, stem and stripe rust and powdery mildew in Tai8335. The present data will aid in the transfer of resistance genes for the rusts and powdery mildew from Th. intermedium into wheat. an attempt has been made to isolate the resistant addition and substitution lines. Progenies from the crosses of Tai8335 with wheat are being screened for rust and powdery mildew resistance and the presence of Th. intermedium chromosomes.