Thinopyrum bessarabicum: biochemical and cytological markers for the detection of genetic introgression in its hybrid derivatives with Triticum aestivum L.

Summary Thinopyrum bessarabicum (2n = 2x = 14, JJ) with its unique property of salt tolerance provides a potential means for the transfer of this important and complex trait into cultivated wheat through intergeneric hybridization. To accomplish this, diagnostic markers for detecting the presence of Th. bessarabicum chromosomes in a wheat background have to be established. The C-banded karyotype of Th. bessarabicum distinctly identifies individual Th. bessarabicum chromosomes and separates them from those of Triticum aestivum. Also, seven protein/isozymes, i.e., malate dehydrogenase, high-molecular-weight glutenin, Superoxide dismutase, grain esterase, glutamate oxaloacetate transaminase, -amylase and -amylase, were identified as being positive markers specific to Th. bessarabicum; these were also expressed in the T. aestivum/Th. bessarabicum amphiploid. These diagnostic biochemical markers could be useful in detecting and establishing homoeology of Th. bessarabicum chromosomes in T. aestivum/Th. bessarabicum intergeneric hybrid derivatives.     

Characterisation of <Emphasis Type="Italic">Thinopyrum bessarabicum</Emphasis> chromosomes through genome-wide introgressions into wheat

Key message

Genome-wide introgressions of Thinopyrum bessarabicum into wheat resulted in 12 recombinant lines. Cytological and molecular techniques allowed mapping of 1150 SNP markers across all seven chromosomes of the J genome.

Abstract

Thinopyrum bessarabicum (2n = 2x = 14, JJ) is an important source for new genetic variation for wheat improvement due to its salinity tolerance and disease resistance. Its practical utilisation in wheat improvement can be facilitated through development of genome-wide introgressions leading to a variety of different wheat–Th . bessarabicum translocation lines. In this study, we report the generation of 12 such wheat–Th . bessarabicum recombinant lines, through two different crossing strategies, which were characterized using sequential single colour and multi-colour genomic in situ hybridization (sc-GISH and mc-GISH), multi-colour fluorescent in situ hybridization (mc-FISH) and single nucleotide polymorphic (SNP) DNA markers. We also detected 13 lines containing different Th. bessarabicum chromosome aberrations through sc-GISH. Through a combination of molecular and cytological analysis of all the 25 lines containing Th. bessarabicum recombinants and chromosome aberrations we were able to physically map 1150 SNP markers onto seven Th. bessarabicum J chromosomes which were divided into 36 segmental blocks. Comparative analysis of the physical map of Th. bessarabicum and the wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed that Th. bessarabicum contains the 4/5 translocation also present in the A genome of wheat. These wheat–Th . bessarabicum recombinant lines and SNP markers provide a useful genetic resource for wheat improvement with the latter having a wider impact as a tool for detection of introgressions from other Thinopyrum species containing the J or a closely-related genome such as Thinopyrum intermedium (J^rJ^rJ^vsJ^vsStSt) and Thinopyrum elongatum (E^eE^e), respectively.

     

Physical localization of a novel blue-grained gene derived from Thinopyrum bessarabicum

Blue wheat grain contains different groups of pigments that can be used for making specialty foods or as food colorants. Thinopyrum bessarabicum, a wild relative of wheat, carries a blue-grained gene on chromosome 4J. In this study, we analyzed the mitotic chromosomes of 159 F7 lines derived from the cross between Triticum aestivum cv. Chinese Spring (CS) and a CS–Th. bessarabicum amphiploid by using multi-color fluorescence in situ hybridization, genomic in situ hybridization, and newly developed chromosome 4J-specific DNA markers. Intact chromosome 4J and various 4J chromosomal segments were identified in the 159 lines. The blue-grained gene of Th. bessarabicum was physically localized to the region between the centromere and FL0.52 on chromosome arm 4JL. The chromosomal location of this gene differed from the location of previously reported blue-grained genes. In addition, a strong dosage effect was observed with this gene. These results suggest that the blue-grained gene in Th. bessarabicum represents a novel gene locus for blue aleurone, designated BaThb. The wheat lines and 4J chromosome-specific molecular markers developed in this study will facilitate the introgression and utilization of BaThb for wheat nutritional quality improvement.     

Characterization of a wheat–Thinopyrum bessarabicum (T2JS-2BS·2BL) translocation line

Thinopyrum bessarabicum (2n = 2x = 14, JJ or E^bE^b) is an important genetic resource for wheat improvement due to its salinity tolerance and disease resistance. Development of wheat–Th. bessarabicum translocation lines will facilitate its practical utilization in wheat improvement. In this study, a novel wheat–Th. bessarabicum translocation line T2JS-2BS·2BL, which carries a segment of Th. bessarabicum chromosome arm 2JS was identified and further characterized using sequential chromosome C-banding, genomic in situ hybridization (GISH), dual-color fluorescent in situ hybridization (FISH) and DNA markers. The translocation breakpoint was mapped within bin C-2BS1-0.53 of chromosome 2B through marker analysis. Compared to the Chinese Spring (CS) parent and to CS-type lines, the translocation line has more fertile spikes per plant, longer spikes, more grains per spike and higher yield per plant, which suggests that the alien segment carries yield-related genes. However, plants with the translocation are also taller, head later and have lower 1,000-kernel weight than CS or CS-type lines. By using markers specific to the barley photoperiod response gene Ppd-H1, it was determined that the late heading date was conferred by a recessive allele located on the 2JS segment. In addition, four markers specific for the translocated segment were identified, which can be used for marker-aided screening.     

Visualization of Secale cereale DNA in wheat germ plasm by fluorescent in situ hybridization

Homozygous wheat/rye (1BL/1RS or 1AS/ 1RL) translocation lines have significantly contributed to wheat production, and several other wheat/rye translocation lines show a potential promise against biotic and abiotic stresses. Detecting the presence of rye at the chromosome level is feasible by C-banding and isozyme protocols, but the diagnostic strength of genomic in situ hybridization for eventually analyzing smaller DNA introgressions has greater significance. As a first step we have applied the genomic in situ hybridization technique to detect rye chromosomes in a wheat background using germ plasm of agricultural significance. By this method rye contributions to the translocations 1BL/1RS, 1AL/1RS, 5AS/5RL and 6BS/6RL could be identified. Differential labelling has further enabled the detection of rye and Thinopyrum bessarabicum chromosomes in a trigeneric hybrid of Triticum aestivum/Th. bessarabicum//Secale cereale.     

Molecular characterization of the genome composition of partial amphiploids derived from Triticum aestivum×Thinopyrum ponticum and T. aestivum×Th. intermedium as sources of resistance to wheat streak mosaic virus and its vector, Aceria tosichella

 Wheat streak mosaic virus (WSMV), vectored by the wheat curl mite (WCM), is one of the most important viral diseases of wheat (Triticum aestivum) in the world. Genetic resistance to WSMV and the WCM does not exist in wheat. Resistance to WSMV and the WCM was evaluated in five different partial amphiploids namely Agrotana, OK7211542, ORRPX, Zhong 5 and TAF 46, which were derived from hybrids of wheat with decaploid Thinopyrum ponticum or with hexaploid Th. intermedium. Agrotana was shown to be immune to WSMV and the WCM; the other four partial amphiploids were susceptible to the WCM. Genomic in situ hybridization (GISH) using genomic DNA probes from Th. elongatum (EE, 2n=14), Th. bessarabicum (JJ, 2n=14), Pseudoroegneria strigosa (SS, 2n=14) and T. aestivum (AABBDD, 2n=42) demonstrated that three of the partial amphiploids, Agrotana, OK7211542 and ORRPX, have almost identical alien genome constitutions: all have 16 alien chromosomes, with 8 chromosomes being closely related to the J^s genome and 8 chromosomes belonging to the E or J genomes and no evidence of any S-genome chromosomes. GISH confirmed that the alien genomes of Agrotana and OK7211542, like ORRPX, were all derived from Th. ponticum, and not from Th. intermedium. However, in contrast to Agrotana, ORRPX and OK7211542 were susceptible to the WCM and WSMV. The partial amphiploid Zhong 5 had a reconstituted alien genome composed of 4 S-and 4 J^s-genome chromosomes of Th. intermedium with 6 translocated chromosomes between the S and J^s genomes. This line was highly resistant to WSMV, but was susceptible to the WCM. TAF 46, which contained a synthetic genome consisting of 3 pairs of S-genome chromosomes and 4 pairs of E- or J-genome chromosomes in addition to the 21 pairs of wheat chromosomes, was susceptible to the WCM, but moderately resistant to WSMV. Agrotana offers great potential for transferring WSMV and WCM resistance into wheat. Received: 27 January 1998 / Accepted: 10 February 1998     

Isozyme and cytological markers of some Psathyrostachys juncea accessions

Summary Psathyrostachys juncea (synonymous to Elymus junceus; 2n=2x=14, NN) has unique biotic and abiotic attributes that could contribute towards wheat improvement. The effectiveness of such an intergeneric hybridization program depends greatly on being able to establish diagnostic markers of the alien chromosomes. Isoelectric focusing (IEF) analyses of six enzyme systems have identified five biochemical markers — malate dehydrogenase (MDH), esterase (EST), shikimate dehydrogenase (SKDH), phosphoglucomutase (PGM), and -amylase (-AMY) — to be of positive diagnostic value; glucosephosphate isomerase (GPI) banding profiles were of no definite value in the background of Triticum aestivum cvs Chinese Spring and Seri-82, the potential recipients of Ps. juncea chromosomes. The Giemsa C-banding karyotype distinctively separates the Ps. Juncea chromosomes from each other and from those of T. aestivum with little banding site polymorphisms prevalent among its accessions analyzed, indicating the usefulness of C-bands as cytological markers.     

Molecular cytogenetic evidence for a high level of chromosome pairing among different genomes in Triticum aestivum–Thinopyrum intermedium hybrids

Intergeneric hybrids (ABDJJ^sS genomes) were made between Triticum aestivum cv. Chinese Spring (CS) and Thinopyrum intermedium. Genomic in situ hybridization (GISH) using genomic DNA probes from Pseudoroegneria libanotica (Hackel) D.R. Dewey (genome S, 2n = 14) was used to study chromosome pairing among J, J^s, S and wheat ABD genomes in the hybrids. It was shown that in the hexaploid (ABDJJ^sS) hybrids, high pairing occurred among wheat chromosomes and among Thinopyrum chromosomes. A closer relationship was observed among the three genomes of Th. intermedium than among the three genomes of T. aestivum. It was further discerned that S genome chromosomes paired with J- and J^s-genome chromosomes at a high frequency. The frequency of heterologous pairing between S and J or S and J^s chromosomes was higher than those between J and J^s chromosomes, indicating that the S-genome was more closely related with these two genomes. Our results provided direct molecular cytogenetic evidence for the hypothesis that S-genome chromosomes are genetically similar to the J-genome chromosomes and, therefore, genetic exchange between these genomes is possible. The discovery of a close relationship among S, J and J^s genomes provides valuable markers for molecular cytogenetic analyses using S-genomic DNA probes in monitoring the transfer of useful traits from Thinopyrum species into wheat. Received: 23 August 2000 / Accepted: 5 September 2000     

Wheat genome structure: translocations during the course of polyploidization

The genomic organization of Triticum timopheevii (2n=28, A^tA^tGG) was compared with hexaploid wheat T. aestivum (2n=42, AABBDD) by comparative mapping using microsatellites derived from bread wheat. Genetic maps for the two crosses T. timopheevii var. timopheevii × T. timopheevii var. typica and T. timopheevii K-38555×T. militinae were constructed. On the first population, 121 loci were mapped, and on the second population 103 loci. The transferability of the wheat markers to T. timopheevii was generally better for the A genome-specific markers (76–78% produced amplification products; 26 and 29% were polymorphic) than for B genome-specific markers (54% produced amplification products; 14 and 16% were polymorphic). Of the D genome-specific markers, one third produced amplification products in T. timopheevii, but only 5 and 2% were polymorphic in the corresponding mapping populations. The maps constructed confirmed the previously described translocation between chromosome arms 6A^tS and 1GS and revealed at least two yet unknown rearrangements on chromosomes 4A^t and 6A^t. The presence of other translocations and rearrangements between T. timopheevii and T. aestivum was demonstrated by a variety of markers mapping to nonhomoeologous positions.     

Single-copy gene fluorescence in situ hybridization and genome analysis: Acc-2 loci mark evolutionary chromosomal rearrangements in wheat

Fluorescence in situ hybridization (FISH) is a useful tool for physical mapping of chromosomes and studying evolutionary chromosome rearrangements. Here we report a robust method for single-copy gene FISH for wheat. FISH probes were developed from cDNA of cytosolic acetyl-CoA carboxylase (ACCase) gene (Acc-2) and mapped on chromosomes of bread wheat, Triticum aestivum L. (2n?=?6x?=?42, AABBDD), and related diploid and tetraploid species. Another nine full-length (FL) cDNA FISH probes were mapped and used to identify chromosomes of wheat species. The Acc-2 probe was detected on the long arms of each of the homoeologous group 3 chromosomes (3A, 3B, and 3D), on 5DL and 4AL of bread wheat, and on homoeologous and nonhomoeologous chromosomes of other species. In the species tested, FISH detected more Acc-2 gene or pseudogene sites than previously found by PCR and Southern hybridization analyses and showed presence/absence polymorphism of Acc-2 sequences. FISH with the Acc-2 probe revealed the 4A–5A translocation, shared by several related diploid and polyploid species and inherited from an ancestral A-genome species, and the T. timopheevii-specific 4A^t–3A^t translocation.     

Genetic Regulation of Meiotic Chromosome Pairing by Chromosome 3<Emphasis Type="Italic">D</Emphasis> of <Emphasis Type="Italic">Triticum aestivum</Emphasis>

IN hexaploid wheat (Triticum aestivum, 2n = 6x = 42) the constituent genomes A, B and D derive from closely related diploid species (2n = 2x = 14) within the sub-tribe Triticinae^1–4. The seven different chromosomes of each genome have genetically equivalent (homoeologous) chromosomes in the other two genomes⁵. Homoeologous chromosomes generally compensate each other in nullisomic-tetrasomic combinations⁵.     

Direct evidence for high level of autosyndetic pairing in hybrids of Thinopyrum intermedium and Th. ponticum with Triticum aestivum

 Genomic in situ hybridization (GISH) was used to distinguish autosyndetic from allosyndetic pairing in the hybrids of Thinopyrum intermedium and Th. ponticum with Triticum aestivum cv ‘Chinese Spring’ (CS). All hybrids showed high autosyndetic pairing frequencies among wheat chromosomes and among Thinopyrum chromosomes. The high autosyndetic pairing frequencies among wheat chromosomes in both hybrids suggested that Th. intermedium and Th. ponticum carry promoters for homoeologous chromosome pairing. The higher frequencies of autosyndetic pairing among Thinopyrum chromosomes than among wheat chromosomes in both hybrids indicated that the relationships among the three genomes of Th. intermedium and among the five genomes of Th. ponticum are closer than those among the three genomes of T. aestivum. Received: 19 September 1996 / Accepted: 18 April 1997     

Development of T. aestivum L.-H. californicum Alien Chromosome Lines and Assignment of Homoeologous Groups of Hordeum californicum Chromosomes

Hordeum californicum （2n = 2x = 14, HH） is resistant to several wheat diseases and tolerant to lower nitrogen. In this study, a molecular karyotype of H. californicum chromosomes in the Triticum aestivum L. cv. Chinese Spring （CS）-H. californicum amphidiploid （2n = 6x = 56, AABBDDHH） was established. By genomic in situ hybridization （GISH） and multicolor fluorescent in situ hybridization （FISH） using repetitive DNA clones （pTa71, pTa794 and pSc119.2） as probes, the H. californicum chromosomes could be differentiated from each other and from the wheat chromosomes unequivocally. Based on molecular karyotype and marker analyses, 12 wheat--alien chromosome lines, including four disomic addition lines （DAH1, DAH3, DAH5 and DAH6）, five telosomic addition lines （MtH7L, MtHIS, MtH1L, DtH6S and DtH6L）, one multiple addition line involving H. californicum chromosome H2, one disomic substitution line （DSH4） and one translocation line （TH7S/1BL）, were identified from the progenies derived from the crosses of CS-H. californicum amphidiploid with common wheat varieties. A total of 482 EST （expressed sequence tag） or SSR （simple sequence repeat） markers specific for individual H. californicum chromosomes were identified, and 47, 50, 45, 49, 21, 51 and 40 markers were assigned to chromosomes H1, H2, H3, H4, H5, H6 and H7, respectively. According to the chromosome allocation of these markers, chromosomes H2, H3, H4, H5, and H7 of H. californicum have relationship with wheat homoeologous groups 5, 2, 6, 3, and 1, and hence could be designated as 5Hc, 2He, 6Hc, 3Hc and 1Hc, respectively. The chromosomes H1 and H6 were designated as 7Hc and 4Hc, respectively, by referring to SSR markers located on rye chromosomes.     

Molecular Analysis of Leaf Rust-Resistant Introgression Lines Obtained by Crossing of Hexaploid Wheat Triticum aestivum with Tetraploid Wheat Triticum timopheevii

Twenty-four Triticum aestivum×T. timopheevii hybrid lines developed on the basis of five varieties of common wheat and resistant to leaf rust were analyzed by the use of microsatellite markers specific for hexaploid wheat T. aestivum. Investigation of intervarietal polymorphism of the markers showed that the number of alleles per locus ranged from 1 to 4, depending on the marker (2.5 on average). InT. timopheevii, amplification fragments are produced by 80, 55, and 30% of primers specific to the A, B, and D common wheat genomes, respectively. Microsatellite analysis revealed two major areas of introgression of the T. timopheevii genome: chromosomes of homoeological groups 2 and 5. Translocations were detected in the 2A and 2B chromosomes simultaneously in 11 lines of 24. The length of the translocated fragment in the 2B chromosome was virtually identical in all hybrid lines and did not depend on the parental wheat variety. In 15 lines developed on the basis of the Saratovskaya-29, Irtyshanka, and Tselinnaya-20, changes occurred in the telomeric region of the long arm of the 5A chromosome. Analysis with markers specific to the D genome suggested that introgressions of the T. timopheevii genome occurred in chromosomes of the D genome. However, the location of these markers on T. timopheevii chromosomes is unknown. Our data suggest that the genes for leaf rust resistance transferred from T. timopheevii to T. aestivum are located on chromosomes of homoeological group 2.     

Introduction of Thinopyrum intermedium ssp. trichophorum chromosomes to wheat by trigeneric hybridization involving Triticum,Secale and Thinopyrum genera

Main conclusion

Fluorescence in situ hybridization and molecular markers have confirmed that several chromosomes from Thinopyrum intermedium ssp. trichophorum have been added to a wheat background, which originated from a cross between a wheat– Thinopyrum partial amphiploid and triticale. The lines displayed blue grains and resistance to wheat stripe rust.

Thinopyrum intermedium has been used as a valuable resource for improving the disease resistance and yield potential of wheat. With the aim to transfer novel genetic variation from Th. intermedium species for sustainable wheat breeding, a new trigeneric hybrid was produced by crossing an octoploid wheat–Th. intermedium ssp. trichophorum partial amphiploid with hexaploid triticale. Fluorescence in situ hybridization (FISH) revealed that Thinopyrum chromosomes were transmitted preferably and the number of rye chromosomes tended to decrease gradually in the selfed derivatives of the trigeneric hybrids. Four stable wheat–Th. intermedium chromosome substitution, addition and translocation lines were selected, and a 2J^S addition line, two substitution lines of 4J^S(4B) and 4J(4B), and a small 4J.4B translocation line were identified by FISH and molecular markers. It was revealed that the gene(s) responsible for blue grains may located on the FL0.60–1.00 of long arm of Th. intermedium-derived 4J chromosome. Disease resistance screenings indicated that chromosomes 4J^S and 2J^S appear to enhance the resistance to stripe rust in the adult plant stage. The new germplasm with Th. intermedium introgression shows promise for utilization of Thinopyrum chromosome segments in future wheat improvement.

     

Variation at Two Loci affecting Homoeologous Meiotic Chromosome Pairing in <Emphasis Type="Italic">Triticum aestivum</Emphasis> × <Emphasis Type="Italic">Aegilops mutica</Emphasis> Hybrids

HOMOEOLOGOUS chromosomes of the three genomes of bread wheat (Triticum aestivum 2n=6x=42) are normally prevented from pairing at meiosis by the activity of an allele at the Ph locus on chromosome 5B^L (refs. 1–4). This activity is responsible for the regular bivalent-forming meiotic behaviour and for the stable disomic inheritance of T. aestivum. If allelic variation occurs at the PA locus in nature it is extremely rare, although mutation has been induced and mutant alleles isolated^3,4.     

Characterization of Thinopyrum bessarabicum chromosome segments in wheat using random amplified polymorphic DNAs (RAPDs) and genomic in situ hybridization

Ten random amplified polymorphic DNA (RAPD) markers specific to chromosome 5E^b of Thinopyrum bessarabicum were detected. Genomic in situ hybridization and standard cytological observations revealed that six of the markers are located on the 5E^b short arm and four are located on the 5E^b long arm. These RAPD markers have been used to confirm the identity of putative 5E^b (5A) and 5E^b (5D) substitution individuals. The potential of RAPDs for the detection of wheat/alien recombinants is discussed.     

Introgression of a novel Thinopyrum intermedium St-chromosome-specific HMW-GS gene into wheat

Thinopyrum intermedium has been hybridized extensively with wheat (Triticum aestivum L.) and several genes for disease resistance have been introgressed to cultivated wheat. However, there are very few reports about the Th. intermedium-derived seed storage protein genes which have been transferred into a wheat background by chromosome manipulation. Our aim is to identify several wheat–Th. intermedium ssp. trichophorum derivatives, and document these lines by genomic in situ hybridization (GISH), molecular markers and seed storage protein analysis. We found that a novel Th. intermedium 1St#2 chromosome-specific high-molecular-weight glutenin subunit (HMW-GS) was transferred to the wheat–Thinopyrum derivative lines. The genomic sequence of the Thinopyrum-derived HMW-GS was characterized and designated Glu-1St#2x, since it resembled x-type glutenins in both the N-terminal domain and C-terminal domain. It is much shorter than that of reported HMW-GS genes. The Glu-1St#2x sequence was successfully expressed in Escherichia coli and resulted in the identical weight to the native protein. The GISH and newly developed chromosome Thinopyrum-specific DNA markers enabled physically location of Glu-1St#2x to the region FL0.60–1.00 on Th. intermedium 1St#2L chromosome arm. Phylogenetic analysis revealed that the Glu-1St#2x evolved earlier than other x-type HMW-GS homoeologues in modern wheat genomes. The effect of Glu-1St#2x on protein content, sodium dodecyl sulphate sedimentation value and improvement of solvent retention capacity in wheat background suggested that Th. intermedium chromosome 1St#2 may have potential for improvement of wheat end-product quality.     

Resistance to eyespot of wheat, caused by Tapesia yallundae, derived from Thinopyrum intermedium homoeologous group 4 chromosome

Thinopyrum intermedium was identified previously as resistant to Tapesia yallundae, cause of eyespot of wheat. Using GUS-transformed isolates of T. yallundae as inoculum, we determined that wheat lines carrying Th. intermedium chromosome 4Ai#2 or the short arm of chromosome 4Ai#2 were as resistant to the pathogen as the eyespot-resistant wheat- Th. ponticum chromosome substitution line SS767 (PI 611939) and winter wheat cultivar Madsen, which carries gene Pch1 for eyespot resistance. Chromosome 4E from Th. elongatum and chromosome 4J from Th. bessarabicum did not confer resistance to T. yallundae. Genome-specific PCR primers confirmed the presence of Thinopyrum chromatin in these wheat- Thinopyrum lines. Genomic in situ hybridization using an St genomic probe from Pseudoroegneria strigosa demonstrated that chromosome 4Ai#2 belongs to the J^s genome of Thinopyrum. The eyespot resistance in the wheat- Th. intermedium lines is thus controlled by the short arm of this J^s chromosome. This is the first report of resistance to T. yallundae controlled by a J^s genome chromosome of Th. intermedium.     

Molecular cytogenetic identification of a wheat–<Emphasis Type="Italic">Aegilops geniculata</Emphasis> Roth 7M<Superscript>g</Superscript> disomic addition line with powdery mildew resistance

Aegilops geniculata Roth is an important germplasm resource for the transfer of beneficial genes into common wheat (Triticum aestivum L.). A new disomic addition line NA0973-5-4-1-2-9-1 was developed from the BC₁F₆ progeny of the cross wheat cv. Chinese Spring (CS)/Ae. geniculata SY159//CS. We characterized this new line by morphological and cytogenetic identification, analysis of functional molecular markers, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), and disease resistance evaluation. Cytological observations suggested that NA0973-5-4-1-2-9-1 contained 44 chromosomes and formed 22 bivalents at meiotic metaphase I. The GISH investigations showed that the line contained 42 wheat chromosomes and a pair of Ae. geniculata chromosomes. EST-STS multiple loci markers and PLUG (PCR-based landmark unique gene) markers confirmed that the introduced Ae. geniculata chromosomes belonged to homoeologous group 7. FISH identification suggested that NA0973-5-4-1-2-9-1 possessed an additional pair of 7M^g chromosomes, and at the same time, there were structural differences in a pair of 6D chromosomes between NA0973-5-4-1-2-9-1 and TA7661 (CS-AEGEN DA 7M^g). After inoculation with powdery mildew (Blumeria graminis f. sp. tritici, Bgt) isolates E09, NA0973-5-4-1-2-9-1 exhibited a powdery mildew resistance infection type different from that of TA7661, and we conclude that the powdery mildew resistance of NA0973-5-4-1-2-9-1 originated from its parent Ae. geniculata SY159. Therefore, NA0973-5-4-1-2-9-1 can be used as a donor source for introducing novel disease resistance genes into wheat during breeding programs with the assistance of molecular and cytogenetic markers.