Characterization of New Wheat-Thinopyrum intermedium Derivative Lines with Superior Genes for Stripe Rust and Powdery Mildew Resistance

The wild species Thinopyrum intermedium (genome JJJSJSStSt) serves as a valuable germplasm resource providing novel diseases resistance and agronomically important genes for wheat improvement. Two wheat-Th. intermedium partial amphiploids, TAI7045 (2n = 56) and 78784 (2n = 56), exhibit high resistance to stripe rust and powdery mildew, and their chromosome constitutions have been characterized. With the aim to transfer novel resistance genes from Th. intermedium, the crosses of common wheat line MY11 with TAI7045 and 78784 were produced, and their individual F2-F5 progenies were characterized using sequential non-denaturing fluorescence in situ hybridization (ND-FISH) and molecular markers. We identified a set of wheat-Th. intermedium addition lines, involving the chromosomes 1St-JS, 2St, 2St-JS, 3St, 4J, 4St, 5St, 5J.St, 6JS.J, and 7JS. Above all, the stable wheat-Th. intermedium small segmental translocation lines with chromosomes 4DS.4DL-4StL-4DL-4JL and 4DS.4DL-4StL-4DL were selected. Combining data from specific marker amplification and resistance evaluation, we mapped the gene(s) for resistance to powdery mildew and stripe rust in the 233.56–329.88 Mb region of the long arm of the 4St chromosome from the reference Th. intermedium genome. The new wheat-Th. intermedium introgressions will be used as novel germplasm for breeding purposes.


Introduction
As one of the three most important food crops, common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is cultivated widely in the world, providing the staple food source for 30% of the global population [1].The production of wheat is seriously affected by many diseases such as powdery mildew and rusts.Powdery mildew, caused by Blumeria graminis f. sp.tritici (Bgt), and stripe rust (yellow rust), caused by Puccinia striiformis f. sp.tritici (Pst), are two serious diseases that frequently occur in most wheat-growing regions subject to moderate temperatures and rainy conditions [2,3] and result in a huge loss of wheat yield [4].The development and cultivation of resistant cultivars is regarded as the most economical, effective and environmentally friendly approach to control these diseases.Although considerable numbers of powdery mildew and stripe rust resistance genes have been identified and used in wheat breeding [5,6], many previously resistant wheat varieties are becoming susceptible due to the continuous appearance of new pathogen races [7,8].For instance, there are 17 stripe rust resistance genes and 41 powdery mildew resistance genes with the official name originated from wild species of wheat [9].Among them, genes such as Pm8, Yr9 and Yr17 have completely or partially lost their effectiveness [10,11].It is thus essential to discover and exploit novel resistant sources for cultivar improvement via the wide hybridization of wheat with uncultivated species.The intermedium wheatgrass (previously termed Agropyron intermedium) Thinopyrum intermedium (Host) Barkworth & D.R. Dewey (2n = 6x = 42, JJJ S J S StSt) is an allohexaploid perennial species with a native distribution throughout the Mediterranean and Eastern Europe [12].It possesses many potentially useful agronomic characteristics, such as stress tolerance and disease resistance, that could be used for wheat improvement [13,14].Because of its high crossability with common wheat, researchers have developed a series of wheat-Th.intermedium partial amphiploids and a number of wheat-Thinopyrum chromosome addition, substitution and translocation lines in the past few decades [15,16].Several genes for improving disease resistance and seed quality have been transferred into wheat from Th. intermedium through interspecific hybridization [16][17][18][19].For example, two powdery mildew resistance genes, Pm40 and Pm43, and one stripe rust resistance gene, Yr50, which were derived from intermediate wheatgrass, have been mapped to chromosomes 7BS, 2DL and 4BL, respectively [20][21][22].The introgressed Th. intermedium chromosome fragment can be identified and tracked by genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), molecular marker analysis, SNP array analysis, etc. [23][24][25].
However, the efficiency of identifying individual Th.intermedium chromosomes in a wheat background is relatively low, since it takes a lot of time to assign polyploid alien chromosomes to specific homoeologous linkage groups and sub-genomes.For example, Yu et al. [26] identified two new wheat-Th.intermedium addition lines, Hy36 and Hy37, by 350 PLUG markers, 890 CINAU markers and 90K iSelect SNP array, and the results confirmed that the added chromosomes in Hy36 and Hy37 were 5J S S.3StS and 5JS.3StS, respectively.Given the complex genomic composition of Th. intermedium and numerous chromosomal rearrangements among three sub-genomes, we built a new combination of oligonucleotide probes and Oligo-FISH painting system to improve the identification efficiency of Th. intermedium chromosomes [26,27].Two wheat-Th.intermedium partial amphiploids, TAI7045 and 78784, displayed high resistance to several fungal diseases including powdery mildew and stripe rust [18,27].The objectives of the present study were to develop a set of wheat-Th.intermedium introgression lines from TAI7045 and 78784 and map the resistance gene(s) into specific Th.intermedium regions, which will potentially be useful for wheat germplasm innovation.
Strikingly, we also observed a pair of identical wheat-Th.intermedium intercalary translocation chromosomes in TAI7045 and 78784, tentatively named 4D-Th.The introgressed Th. intermedium chromosome segments were identified using the probe Oligo-B11, and it produced signals on the sub-telomeric regions in the long arm of 4D-Th (Figure 1a,i).Four newly developed probes, Oligo-7v108, Oligo-Dv86, Oligo-v03-86 and Oligo-Ae369, could be used for detecting different regions of the 4D chromosomes, particularly on its long arm (Figures S2 and 1i).Probe Oligo-Ae369 could generate two adjacent strong signals on the 4DL; however, the long arms of 4D-Th chromosomes only showed one distinct Oligo-Ae369 signal away from the end.It is noteworthy that the 4D-Th chromosomes retained a weak Oligo-7v108 signal on the end of its long arm, which indicated the interstitial deficiency of 4DL.In addition, two visible differences in wheat chromosomes between TAI7045 and 78784 were detected with the probe Oligo-D, and four varied wheat chromosomes 1A-W and 2D-W in TAI7045 are shown in Figure 1i.One small D-genome chromosome segment was translocated interstitially into in the 1AL, and the recombinant chromosome 2D-W displayed a large deletion on 2DL.
Plants 2024, 13, x FOR PEER REVIEW 3 of 15 signals on the 4DL; however, the long arms of 4D-Th chromosomes only showed one distinct Oligo-Ae369 signal away from the end.It is noteworthy that the 4D-Th chromosomes retained a weak Oligo-7v108 signal on the end of its long arm, which indicated the interstitial deficiency of 4DL.In addition, two visible differences in wheat chromosomes between TAI7045 and 78784 were detected with the probe Oligo-D, and four varied wheat chromosomes 1A-W and 2D-W in TAI7045 are shown in Figure 1i.One small D-genome chromosome segment was translocated interstitially into in the 1AL, and the recombinant chromosome 2D-W displayed a large deletion on 2DL.The period "." means that the breakpoint of the chromosome translocation events was located in the region of the centromere, and the hyphen "-" means that the breakpoint was located in the other regions.Bars represent 10 µm.

Transmission of Thinopyrum Chromosomes and Identification of Stable Wheat-Th. intermedium-Derived Lines
The combination of the four probes Oligo-pSc119.2-1,Oligo-pTa535-1, Oligo-pTa71-2 and Oligo-pSc200 was applied to identify wheat and Th.intermedium chromosomes of individual plants from TAI7045-and 78784-derived progenies (Figure S3).A total of 314 F 2 plants from the cross TAI7045/MY11 and 308 F 2 plants from the cross 78784/MY11 were karyotypically analyzed using ND-FISH.The chromosomal numbers of two F 2 populations were distributed from 39 to 54 (Figure 2).Plants possessing chromosome numbers less than 42 may be due to the abnormal meiotic behavior of wheat chromosomes affected by Thinopyrum chromatin introgression.In F 2 , the chromosomes 5J.St from TAI7045 and the chromosomes 4St from 78784 showed the highest transmission frequencies, 18.45% and 16.07%, in seven linkage groups, respectively.Chromosomes 7J S with relatively low transmission frequencies (12.07%and 12.33%) were also observed in the two F 2 populations.

Transmission of Thinopyrum Chromosomes and Identification of Stable Wheat-Th. intermedium-Derived Lines
The combination of the four probes Oligo-pSc119.2-1,Oligo-pTa535-1, Oligo-pTa71-2 and Oligo-pSc200 was applied to identify wheat and Th.intermedium chromosomes of individual plants from TAI7045-and 78784-derived progenies (Figure S3).A total of 314 F2 plants from the cross TAI7045/MY11 and 308 F2 plants from the cross 78784/MY11 were karyotypically analyzed using ND-FISH.The chromosomal number of the F2 populations ranged from 39 to 54 and presented a similar distribution pattern (Figure 2).Plants possessing chromosome numbers less than 42 may be due to the abnormal meiotic behavior of wheat chromosomes affected by Thinopyrum chromatin introgression.In F2, the chromosomes 5J.St from TAI7045 and the chromosomes 4St from 78784 showed the highest transmission frequencies, 18.45% and 16.07%, in seven linkage groups, respectively.Chromosomes 7J S with relatively low transmission frequencies (12.07%and 12.33%) were also observed in the two F2 populations.Meanwhile, the seeds of 72 F2 progenies with one or two alien chromosomes derived from the cross TAI7045/MY11 were selected and cytogenetic analysis was performed using ND-FISH, and the seeds of 61 F2 progenies derived from the cross 78784/MY11 also underwent the same experiment.In the F3 of TAI7045/MY11, except for 3J S chromosomes, the wheat-Th.intermedium 1St-J S , 2St, 6J S .J and 7J S disomic addition lines and 4J and 5J.St monosomic addition lines were successfully obtained (Figure S4).The wheat-Th.intermedium 1St-J S , 2St-J S , 3St, 4St and 6J S .J disomic addition lines and 5St and 7J S monosomic addition lines were also gained from the F3 populations of the cross 78784/MY11 (Figure S5).These novel wheat-Th.intermedium-derived lines are expected to benefit the genetic resource research and development of Th. intermedium.Moreover, we found some wheat- Meanwhile, a total of 72 and 61 F 2 progenies were selected from the hybrids of TAI7045/MY11 and 78784/MY11, respectively.The seeds carrying one or two alien chromosomes were performed for karyotypic analysis using ND-FISH.In the F 3 of TAI7045/MY11, except for 3J S chromosomes, the wheat-Th.intermedium 1St-J S , 2St, 6J S .J and 7J S disomic addition lines and 4J and 5J.St monosomic addition lines were successfully obtained (Figure S4).The wheat-Th.intermedium 1St-J S , 2St-J S , 3St, 4St and 6J S .J disomic addition lines and 5St and 7J S monosomic addition lines were also gained from the F 3 populations of the cross 78784/MY11 (Figure S5).These novel wheat-Th.intermedium-derived lines are expected to benefit the genetic resource research and development of Th. intermedium.Moreover, we found some wheat-wheat chromosomal rearrangement events in the F 3 , such as 4BS.4AL and 4AS.4BL (Figure S5f,h), 3BS.5AS and 3BL.5AL (Figure S5j), 5BS.7BS and 5BL.7BL translocation chromosomes (Figure S5k), which may be caused by the introduction of Th. intermedium chromosomes in the wheat background.

ND-FISH of Two Distinct Wheat-Th. intermedium Translocation Lines by Multiple Oligo Probes
In addition to the above-mentioned wheat-Th.intermedium addition lines, we also identified two distinct wheat-Th.intermedium translocation lines WT4D-1 and WT4D-2 in F 3 to F 5 from the crosses TAI7045/MY11 and 78784/MY11, respectively.Sequential multicolor ND-FISH with eight probes, Oligo-B11, Oligo-D, Oligo-pSc119.2-1,Oligo-pTa535-1, Oligo-7v108, Oligo-Dv86, Oligo-v03-86 and Oligo-Ae369, was conducted to characterize the cytologically stable lines WT4D-1 and WT4D-2 (Figure 3).Probes Oligo-B11 and Oligo-D showed the occurrence of two translocation chromosomes between the Th.intermedium genomes and D-genome, and probes Oligo-pSc119.2-1and Oligo-pTa535-1 further revealed the breakpoints located in the long arms of 4D chromosomes.Compared with the 4D chromosomes, the chromosomes WT4D-1 added weak Oligo-pSc119.2-1signals on the telomeres of the long arm, while the chromosomes WT4D-2 reserved Oligo-7v108 signals located on the end of 4DL (Figures S1 and 3b,g).The chromosomes WT4D-1 and WT4D-2 only carried one FISH pattern of Oligo-Ae369, which indicated the partial deletion of 4DL.Based on the cytogenetic analysis of the lines WT4D-1 and WT4D-2 and their respective parents, we posit that the chromosomes WT4D-1 might derive from the recombination between 4D-Th and 4J in TAI7045, while the chromosomes WT4D-2 were the same as the 4D-Th of 78784 in FISH patterns.

ND-FISH of Two Distinct Wheat-Th. intermedium Translocation Lines by Multiple Oligo Probes
In addition to the above-mentioned wheat-Th.intermedium addition lines, we also identified two distinct wheat-Th.intermedium translocation lines WT4D-1 and WT4D-2 in F3 to F5 from the crosses TAI7045/MY11 and 78784/MY11, respectively.Sequential multicolor ND-FISH with eight probes, Oligo-B11, Oligo-D, Oligo-pSc119.2-1,Oligo-pTa535-1, Oligo-7v108, Oligo-Dv86, Oligo-v03-86 and Oligo-Ae369, was conducted to characterize the cytologically stable lines WT4D-1 and WT4D-2 (Figure 3).Probes Oligo-B11 and Oligo-D showed the occurrence of two translocation chromosomes between the Th.intermedium genomes and D-genome, and probes Oligo-pSc119.2-1and Oligo-pTa535-1 further revealed the breakpoints located in the long arms of 4D chromosomes.Compared with the 4D chromosomes, the chromosomes WT4D-1 added weak Oligo-pSc119.2-1signals on the telomeres of the long arm, while the chromosomes WT4D-2 reserved Oligo-7v108 signals located on the end of 4DL (Figures S1 and 3b,g).The chromosomes WT4D-1 and WT4D-2 only carried one FISH pattern of Oligo-Ae369, which indicated the partial deletion of 4DL.Based on the cytogenetic analysis of the lines WT4D-1 and WT4D-2 and their respective parents, we posit that the chromosomes WT4D-1 might derive from the recombination between 4D-Th and 4J in TAI7045, while the chromosomes WT4D-2 were the same as the 4D-Th of 78784 in FISH patterns.

Molecular Marker Analysis for Structure of Chromosomes WT4D-1 and WT4D-2
In order to map the breakage-fusion structure of the chromosomes 4D-Th in TAI7045 and 78784, as well as the translocated chromosomes in lines WT4D-1 and WT4D-2 (Figure 4), 60 PLUG markers of wheat homoeologous group 4 specific [28], 89 CINAU markers derived from Dasypyrum villosum chromosome 4VL [29], and 58 Thi-specific markers from

Powdery Mildew and Stripe Rust Reactions for the Wheat-Th. intermedium Lines
The wheat-Th.intermedium lines and their parents were inoculated with Pst races CYR32, CYR33, and CYR34 at the adult plant stage in the field.The wheat parent MY11 was highly susceptible to all of these stripe rust pathotypes, and its infection type (IT) was scored as 4, while two wheat-Th.intermedium partial amphiploids TAI7045 and 78784 were highly immune to the mixture of Pst races (IT = 0) (Figure 6a).The difference between two wheat-Th.intermedium introgression lines, 1St-J S (TAI7045) and 1St-J S (78784), in stripe rust resistance was scored, which may be caused by the structure of two chromosomes in the end of the long arm (Figure S1).The plants carrying 3St, 4J, 5J.St, 5St or 6J S .J had higher ITs (3 or 4) (Table 1).Plants with 2St, 2St-J S , 4St and 7J S chromosomes displayed high resistance for all Pst races tested (IT = 0-1).Noticeably, the recombinant lines WT4D-1 and WT4D-2 showed high Pst resistances as TAI70445 and 78784, indicating that the Yr locus originated from the 4St chromosome segment.
The lines WT4D-1 and WT4D-2 and their parents were further evaluated for resistance to powdery mildew in the greenhouse.The seedling ITs to powdery mildew were scored when the susceptible control MY11 was fully infected (IT = 4).In this stage, the lines WT4D-1 and WT4D-2 were susceptible to Bgt isolates with infection types 3-4.However, both of them displayed fully resistance (IT = 0;) from the five-leaf stage, indicating that they may have adult plant resistance (APR) (Figure 6b, Table 1).These results suggest that the Th.intermedium chromosome 4StL carries an excellent gene makeup for powdery mildew and stripe rust resistance and may be physically localized in the region 233.56-329.88Mb.The chromosomal location of the resistance gene(s) will be verified using linkage analysis in future studies.The chromosome fragments with blue and orange backgrounds represent the introgression of chromosomes 4St and 4J, respectively.

Powdery Mildew and Stripe Rust Reactions for the Wheat-Th. intermedium Lines
The wheat-Th.intermedium lines and their parents were inoculated with Pst races CYR32, CYR33, and CYR34 at the adult plant stage in the field.The wheat parent MY11 was highly susceptible to all of these stripe rust pathotypes, and its infection type (IT) was scored as 4, while two wheat-Th.intermedium partial amphiploids TAI7045 and 78784 were highly immune to the mixture of Pst races (IT = 0) (Figure 6a).The difference between two wheat-Th.intermedium introgression lines, 1St-J S (TAI7045) and 1St-J S (78784), in stripe rust resistance was scored, which may be caused by the structure of two chromosomes in the end of the long arm (Figure S1).The plants carrying 3St, 4J, 5J.St, 5St or 6J S .J had higher ITs (3 or 4) (Table 1).Plants with 2St, 2St-J S , 4St and 7J S chromosomes displayed high resistance for all Pst races tested (IT = 0-1).Noticeably, the recombinant lines WT4D-1 and  4DS.4DL-4StL-4DL 0 0 "/" indicates that the line did not carry the alien chromosome."nt" means not tested.ITs of "0" mean immune (no symptoms), ";" means near to immune (necrotic or chlorotic blotches without sporulation), 1-2 indicate resistance (necrotic or chlorotic blotches with only a trace of slight sporulation) and 3-4 indicate susceptible (moderate to abundant sporulation, with or without chlorosis or necrosis)."0;" means "0" or ";" and ";1" means ";" or "1".

Discussion
Transferring desirable genes from wild relatives by interspecific hybridization is an important approach for broadening the genetic diversity of wheat [31,32].A great number of wheat-alien species derivatives have been developed via distant hybridizations and chromosome engineering in the past few decades.However, the identification efficiency is relatively low in earlier research due to the complexity of the genomic composition, such as the allohexaploid species Th. intermedium [13,15,16,33,34].Combining Oligo-FISH painting and ND-FISH using multiple oligo probes, we established an efficient system for precisely distinguishing Th. intermedium chromosome segments from wheat [27].Based on this system, we screened a set of wheat-Th.intermedium addition lines from two hybrid populations, TAI7045/MY11 and 78784/MY11, in F 3 generation.Although some introgressed complete Th. intermedium chromosomes carrying excellent genes, involving 2St, 3St, 4St, 4J and 7J S , have been reported in previous studies, they have different origins [16,23,[35][36][37].Han et al. [34] and Hu et al. [36] re-characterized a wheat-Th.intermedium addition line Z2 and found that it contained a substitution of one pair of 2D chromosomes by a pair of Th. intermedium chromosomes 2St-J S .In this study, five new wheat-Th.intermedium derivatives, including 1St-J S (TAI7045), 1St-J S (78784), 5St, 5J.St and 6J S .J, were found to cover several segments of the Th.intermedium genome, which can be important bridge materials for the excavation of Th. intermedium genetic resources.
Previous studies have suggested that the introduction of alien chromosomes may lead to structural variations of common wheat chromosomes [38,39].Cui et al. [40] identified different types of chromosomal structural variation from three octoploid Trititrigia accessions (TE261-1, TE266-1, and TE346-1), which occurred in the chromosomes 1A, 6A, 6B, 2D and 7D.In the present study, two types of wheat recombinant chromosomes involving chromosomes 1A and 2D were also observed in TAI7045 (Figure 1), and a number of wheat chromosomal rearrangement events were detected in two hybrid populations (Figure S5).In addition, we also found two novel wheat-Th.intermedium insertional translocation chromosomes, T4DS.4DL-4StL-4DL and T4DS.4DL-4StL-4DL-4JL, which could be characterized by sequential ND-FISH with four newly developed probes and molecular marker analysis.We found that four markers including C10-56 were previously located in 4J by genetic mapping and comparison to the assembly of Th. intermedium v1.0 [30]; however, they were mapped on 4St by blasting for the updated Th. intermedium genome v3.1 and confirmed by PCR analysis in the present study (Figure 4, Table S1).It is thus pertinent to note that the combination of bioinformatic, cytogenetic and molecular analysis would be useful for precise identification of wheat-Thinopyrum introgression lines.The translocated chromosome T4DS.4DL-4StL-4DLfrom TAI7045 and 78784 has been transferred into common wheat individually.Another translocated chromosome was generated via the homoeologous chromosomal recombination between the chromosomes T4DS.4DL-4StL-4DL and 4J in the process of TAI7045/MY11.This may be due to a close relationship between Th. intermedium and Aegilops tauschii, and the chromosomes of linkage group 4 in wheat and Th.intermedium are highly prone to recombination [16,25,41].
The homoeologous group-4 chromosomes of the Thinopyrum species possess many desirable sources, such as a perennial habit, blue-grained characteristic, and resistance to stripe rust, powdery mildew, eyespot (Tapesia yallundae), etc. [42][43][44].For example, Li et al. [45] found that the Th.intermedium chromosome 4Ai#2S (originating from Ag. intermedium, the Max Plank Institute in Germany) carries the eyespot-resistant gene(s).The 4Ai#2S.4DLtranslocation lines displayed superior resistance to WSMV (Wheat Streak Mosaic Virus) [46,47].Two putative Th. intermedium-derived resistance genes, Yr50 and pmCH89, were mapped on wheat chromosome arm 4BL [18,22].Li et al. [16] and Li et al. [25] located the resistance gene(s) to stripe rust on FL0.60-1.00 of the 4J S S and on FL0-0.60 of the 4J S L, respectively.Gong et al. [48] developed a new wheat-Th.scirpeum 4E (4D) chromosomal substitution line (K16-730-3) that is resistant to stripe rust and powdery mildew.In the present study, we identified two stable wheat-Th.intermedium small segmental translocation lines, WT4D-1 and WT4D-2, and found that they are highly resistant to stripe rust and powdery mildew at the adult stage.Combining data from cytogenetic analysis and specific marker amplification, the resistant gene(s) for the two fungi was mapped in the 233.56-329.88Mb region of 4StL.However, Li et al. [16] observed that the addition line L4 with a 4Ai#1 (originating from Ag. intermedium accession no.75) chromosome (St-genome) was susceptible to stripe rust.This phenomenon is probably due to the chromosome 4St originating from different Th.intermedium subspecies, indicating that the exploitation of diversified wheat-Th.intermedium introgression lines remains necessary.

Plant Materials
Wheat-Th.intermedium partial amphiploids TAI7045 and 78784 were kindly provided by Dr. Zhijian Chang, Shanxi Academy of Agricultural Sciences, China.Wheat lines Chinese Spring (CS) and Mianyang11 (MY11) and the nullisomic-tetrasomic (NT) lines of the cultivars CS [49] and X24C10 (4J/4B substitution) [16] are maintained by the Center for Informational Biology, School of Life Science and Technology, University of Electronic Science and Technology of China.Two crosses, TAI7045/MY11 and 78784/MY11, were created, and the F 2 plants were used to detect the transmission of Thinopyrum chromosomes, and partial amphiploids of F 3 , originating from 133 F 2 plants with one or two alien chromosomes, were used to screen the stable wheat-Th.intermedium-derived lines.Two wheat-Th.intermedium translocation lines, WT4D-1 and WT4D-2, were also identified in F 3 , and the selfing progenies (F 4 and F 5 ) were examined using cytogenetic analysis.

ND-FISH and Sequential ND-FISH
The chromosome spreads of materials were carried out according to the procedure of Han et al. [57].ND-FISH by the synthesized probes was performed as described by Fu et al. [56] with some modifications.The slides were counter-stained with DAPI (4 ′ ,6diamidino-2-phenylindole) and examined using an Olympus BX-51 microscope (Shinjuku, Tokyo, Japan).Microphotographs of ND-FISH chromosomes were captured using a cooled DP70 CCD camera.Sequential ND-FISH was conducted on the same slide with different probes following the description by Wang et al. [58].All images were optimized using Adobe Photoshop CS6 software (Adobe Systems Incorporated, San Jose, CA, USA).

Powdery Mildew and Stripe Rust Response Observations
To survey the phenotype of lines to powdery mildew and stripe rust, we used local mixed Bgt isolates and a mixture of Pst races CYR32, CYR33 and CYR34, respectively.The inoculation of Bgt were carried out at the four-leaf stage in a greenhouse at 22 • C and photoperiod of 14 h of light per day, and the APR response was recorded at 20 days postinoculation; refer to Mohler et al. [60].Stripe rust reactions were observed at the heading and grain-filling stages in the field at the Xindu Experimental Station, Chengdu, China; refer to Li et al. [7].The powdery-mildew-and stripe-rust-susceptible cultivar Mianyang 11 (MY11) was examined as a control.Resistance testing was carried out on the F 3 generation, with ten plants assessed for each of the lines.ITs were scored according to the system described by Bariana and McIntosh [61].

Figure 5 .
Figure 5. Physical map of the translocation chromosomes 4D-Th, WT4D-1 and WT4D-2 in comparison with 4D chromosomes.The blue molecular markers and Oligo probes indicate the deletion of 4D chromosome fragments.The 4St-specific marker C10-56 indicates the putative breakpoint of the translocation.The chromosome fragments with blue and orange backgrounds represent the introgression of chromosomes 4St and 4J, respectively.

Figure 6 .
Figure 6.Physical location of wheat-Th.intermedium lines using rust resistance survey and molecular marker map.Stripe rust response (a) and powdery mildew response (b).The 34 4St-specific markers were screened and blasted to located on the 4StL of Th. intermedium genome sequence of V3.1 (c).The chromosome fragments with blue and orange backgrounds represent the introgression of chromosomes 4St and 4J, respectively.

Figure 6 .
Figure 6.Physical location of wheat-Th.intermedium lines using rust resistance survey and molecular marker map.Stripe rust response (a) and powdery mildew response (b).The 34 4St-specific markers were screened and blasted to located on the 4StL of Th. intermedium genome sequence of V3.1 (c).The chromosome fragments with blue and orange backgrounds represent the introgression of chromosomes 4St and 4J, respectively.

Table 1 .
Responses of tested materials to stripe rust (Pst) and powdery mildew (Bgt) at the adult stage.

Table 2 .
Sequences of synthesized oligonucleotide probes.