Chinese Spring Reference Genome Research Articles

Mining for New Sources of Resistance to Powdery Mildew in Genetic Resources of Winter Wheat.

Genetic pathogen control is an economical and sustainable alternative to the use of chemicals. In order to breed resistant varieties, information about potentially unused genetic resistance mechanisms is of high value. We phenotyped 8,316 genotypes of the winter wheat collection of the German Federal ex situ gene bank for Agricultural and Horticultural Crops, Germany, for resistance to powdery mildew (PM), Blumeria graminis f. sp. tritici, one of the most important biotrophic pathogens in wheat. To achieve this, we used a semi-automatic phenotyping facility to perform high-throughput detached leaf assays. This data set, combined with genotyping-by-sequencing (GBS) marker data, was used to perform a genome-wide association study (GWAS). Alleles of significantly associated markers were compared with SNP profiles of 171 widely grown wheat varieties in Germany to identify currently unexploited resistance conferring genes. We also used the Chinese Spring reference genome annotation and various domain prediction algorithms to perform a domain enrichment analysis and produced a list of candidate genes for further investigation. We identified 51 significantly associated regions. In most of these, the susceptible allele was fixed in the tested commonly grown wheat varieties. Eleven of these were located on chromosomes for which no resistance conferring genes have been previously reported. In addition to enrichment of leucine-rich repeats (LRR), we saw enrichment of several domain types so far not reported as relevant to PM resistance, thus, indicating potentially novel candidate genes for the disease resistance research and prebreeding in wheat.

Fine mapping of the tiller inhibition gene TIN4 contributing to ideal plant architecture in common wheat.

A tiller inhibition gene, TIN4, was mapped to an approximately 311kb genomic interval on chromosome arm 2DL of wheat. The tiller is one of the key components of plant morphological architecture and a central agronomic trait affecting spike number in wheat. Low tiller number has been proposed as a major component of crop ideotypes for high yield potential. In this study, we characterized the development of tillering in near-isogenic lines (NIL7A and NIL7B), indicating that the TIN4 gene inhibited the growth of tillering buds and negatively regulated tiller number. Low-tillering was controlled by a single gene (TIN4) located on chromosome 2DL by genetic analysis and bulked segregant RNA-seq analysis. A total of 17 new polymorphic markers were developed in this study, and 61 recombinants were identified in the secondary F2 population containing 4,266 individuals. TIN4 was finally mapped on a 0.35cM interval, co-segregated with molecular marker M380, within a 311kb genomic interval of the wheat cultivar Chinese Spring reference genome sequence that contained twelve predicted genes. Yield experiments showed that the yield of low-tillering lines was higher than that of high-tillering lines at a higher density. Overall, this study provides a foundation for the construction of a low-tillering ideotype for improving wheat yield and further cloning TIN4 by map-based cloning approach.

Identification of genetic locus with resistance to take-all in the wheat-Psathyrostachys huashanica Keng introgression line H148

Take-all is a devastating soil-borne disease of wheat (Triticum aestivum L.). Cultivating resistant line is an important measure to control this disease. Psathyrostachys huashanica Keng is a valuable germplasm resource with high resistance to take-all. This study reported on a wheat-P. huashanica introgression line H148 with improved take-all resistance compared with its susceptible parent 7182. To elucidate the genetic mechanism of resistance in H148, the F2 genetic segregating population of H148×XN585 was constructed. The mixed genetic model analysis showed that the take-all resistance was controlled by two major genes with additive, dominant and epistasis effects. Bulked segregant analysis combined with wheat axiom 660K genotyping array analysis showed the polymorphic SNPs with take-all resistance from P. huashanica alien introgression were mainly distributed on the chromosome 2A. Genotyping of the F2 population using the KASP marker mapped a major QTL in an interval of 68.8–70.1 Mb on 2AS. Sixty-two genes were found in the target interval of the Chinese Spring reference genome sequence. According to the functional annotation of genes, two protein genes that can improve the systematic resistance of plant roots were predicted as candidate genes. The development of wheat-P. huashanica introgression line H148 and the resistant QTL mapping information are expected to provide some valuable references for the fine mapping of disease-resistance gene and development of take-all resistant varieties through molecular marker-assisted selection.

Characterization of PmDGM Conferring Powdery Mildew Resistance in Chinese Wheat Landrace Duanganmang.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is a devastating disease that threatens yield and quality. Host resistance is considered the most effective and preferred means to control this disease. Wheat landrace Duanganmang (DGM) showed high resistance or near immunity to Blumeria graminis f. sp. tritici mixture from Henan Province, China. DGM was crossed with highly susceptible Chinese wheat landrace Huixianhong (HXH) and cultivar 'Shimai 15' (SM15) to produce genetic populations. The resistance of DGM to Blumeria graminis f. sp. tritici isolate E09 was shown to be controlled by a single dominant Mendelian factor, tentatively designated PmDGM. Marker analysis and 55K single nucleotide polymorphism (SNP) array scanning showed that this gene was positioned in the Pm5 interval (2.4 cM or 1.61 Mb) flanked by Xhenu099 and Xmp1158 in the Chinese Spring reference genome. Homology-based cloning and sequence analysis demonstrated that DGM has the identical NLR gene (Pm5e) and RXL gene reported in Fuzhuang 30 (FZ30), conferring and modifying powdery mildew resistance, respectively. However, based on the different reaction patterns to the Blumeria graminis f. sp. tritici isolate B15 between DGM and FZ30, the authors speculate that DGM may have two tightly linked genes that could not be separated in the current mapping population, one of which is PmDGM and the other being Pm5e. Hence, this study provides a valuable resistance resource for improvement of powdery mildew resistance.

Identification of a major QTL for Hessian fly resistance in wheat cultivar ‘Chokwang’

The Hessian fly (HF, Mayetiola destructor) is one of the destructive pests of wheat (Triticum aestivum L.) worldwide. Resistant cultivars can effectively minimize wheat damage due to this insect pest. To identify new quantitative trait loci (QTL) for HF resistance, a population of recombinant inbred lines (RILs) was developed from a cross between the HF-resistant wheat cultivar ‘Chokwang’ and susceptible wheat ‘Ning 7840’, and phenotyped for responses to HF attack. A linkage map was constructed using 1147 single nucleotide polymorphism (SNP) markers generated from genotyping-by-sequencing (GBS). One major QTL, QHf.hwwg-6BS, for HF-resistance was identified on chromosome arm 6BS, which explained up to 84.0% of the phenotypic variation and was contributed by Chokwang. Two RILs showed recombination in the candidate interval of QHf.hwwg-6BS, which delimited QHf.hwwg-6BS to a 4.75 Mb physical interval between 6,028,601 bp and 10,779,424 bp on chromosome arm 6BS of IWGSC Chinese Spring reference genome RefSeq v2.0. Seven GBS-SNPs in the candidate interval were converted into Kompetitive allele specific polymerase chain reaction (KASP) markers. Two of them, KASP-6B112698 and KASP-6B7901215, were validated in a U.S. winter wheat panel. KASP-6B112698 was nearly diagnostic, thus can be used to screen QHf.hwwg-6BS and pyramid it with other resistance genes in breeding programs.

Mapping of wheat stripe rust resistance gene Yr041133 by BSR-Seq analysis

Puccinia striiformis Westend. f. sp. tritici (Pst) pathotype CYR34 is widely virulent and prevalent in China. Here, we report identification of a strpie rust resistance (Yr) gene, designated Yr041133, in winter wheat line 041133. This line produced a hypersensitive reaction to CYR34 and conferred resistance to 13 other pathotypes. Resistance to CYR34 in line 041133 was controlled by a single dominant gene. Bulked segregant RNA sequencing (BSR-Seq) was performed on a pair of RNA bulks generated by pooling resistant and susceptible recombinant inbred lines. Yr041133 was mapped to a 1.7 cM genetic interval on the chromosome arm 7BL that corresponded to a 0.8 Mb physical interval (608.9–609.7 Mb) in the Chinese Spring reference genome. Based on its unique physical location Yr041133 differred from the other Yr genes on this chromosome arm.

Molecular mapping and identification of a candidate gene for new locus Hg2 conferring hairy glume in wheat

Glume hairiness or pubescence that occurs in hexaploid common wheat and its relatives at different ploidy levels is a distinct morphological marker. Current knowledge about the genetic control of wheat glume hairiness is based on study of Hg1 (formerly Hg) on chromosome 1AS. Here, we report characterization of a new locus for hairy glume Hg2 in synthetic hexaploid wheat line CIGM86.944. Hg2 was inherited a dominant allele. Bulked segregant analysis and RNA-sequencing (BSR-Seq) was performed on an F2:3 population from cross CIGM86.944 × Shannong 29 (glabrous glume), which localized Hg2 in a 2.02 cM genetic interval corresponding to ∼1.08 Mb (754,001,564–755,082,433 Mb) on chromosome 2BL in the Chinese Spring reference genome. Gene annotation and expression identified TraesCS2B02 G562300.1 encoding diacylglycerol kinase 5 protein and TraesCS2B02 G561400.1 encoding a wound-responsive family protein as possible candidate genes regulating development of glume hairiness. The identification of Hg2 provides new insights into the genetic control of glume hairiness in wheat.

WheatGmap: a comprehensive platform for wheat gene mapping and genomic studies

WheatGmap: a comprehensive platform for wheat gene mapping and genomic studies

Quantitative Trait Loci Mapping for Spot Blotch Resistance in Two Biparental Mapping Populations of Bread Wheat.

Spot blotch (SB), caused by Bipolaris sorokiniana, is a major fungal disease of wheat in South Asia and South America. Two biparental mapping populations with 232 F2:7 progenies each were generated, with CIMMYT breeding lines CASCABEL and KATH as resistant parents and CIANO T79 as the common susceptible parent. The two populations were evaluated for field SB resistance in CIMMYT's Agua Fria station for three consecutive cropping seasons, with artificial inoculation. Genotyping was done with the DArTseq platform and approximately 1,500 high quality and nonredundant markers were used for quantitative trait loci (QTL) mapping. In both populations, a major QTL was found on chromosome 5A in the Vrn-A1 region, explaining phenotypic variations of 13.5 to 25.9%, which turned up to be less- or nonsignificant when days to heading and plant height were used as covariates in the analysis, implying a disease escape mechanism. Another major QTL was located on chromosome 5B in CASCABEL, accounting for 8.9 to 21.4% of phenotypic variation. Minor QTL were found on 4A and 4B in CASCABEL; 1B, 4B, and 4D in KATH; and 1B, 2B, and 4B in CIANO T79. Through an analysis of QTL projection onto the IWGSC Chinese Spring reference genome, the 5B QTL in CASCABEL was mapped in the Sb2 region, delimited by the single nucleotide polymorphism marker wsnp_Ku_c50354_55979952 and the simple sequence repeat marker gwm213, with a physical distance of about 14 Mb to the Tsn1 locus.

Reassigning Hessian fly resistance genes H7 and H8 to chromosomes 6A and 2B of the wheat cultivar ‘Seneca’ using genotyping‐by‐sequencing

AbstractHessian fly (HF, Mayetiola destructor Say), is a destructive insect pest in most wheat (Triticum aestivum L.) growing areas worldwide. Growing resistant cultivars can effectively reduce HF damage. The wheat cultivar ‘Seneca’ was reported to carry resistance genes H7 on chromosome 5D and H8 with unknown location. To further validate the resistance gene locations in Seneca, a recombinant inbred line (RIL) population from Bobwhite × Seneca was genotyped using 3,330 single nucleotide polymorphism (SNP) markers generated from genotyping‐by‐sequencing (GBS) and phenotyped for HF resistance in the greenhouse experiments. Phenotypic analyses showed at least two genes conditioning HF resistance in Seneca, but none of them was mapped on chromosome 5D, as previously reported. Instead, one major gene, designated as H7, was mapped to chromosome 6A and explained 60.7–78.3% of the phenotypic variation. The other gene with a minor effect, designated as H8, was found on chromosome 2B and explained 3.2–4.7% of the phenotypic variation. Based on the physical locations of flanking markers for H7 in the Chinese Spring reference genome, H7 was physically located in a 6‐Mb interval on 6AL. This study reassigned the major gene H7 to chromosome 6A and the minor gene H8 to 2B in Seneca using a high‐density SNP map. Seventeen GBS SNPs mapped in the H7 region were converted into Kompetitive allele specific polymerase chain reaction (KASP) markers for selecting this gene in breeding.

The NLR-Annotator Tool Enables Annotation of the Intracellular Immune Receptor Repertoire.

Disease resistance genes encoding nucleotide-binding and leucine-rich repeat (NLR) intracellular immune receptor proteins detect pathogens by the presence of pathogen effectors. Plant genomes typically contain hundreds of NLR-encoding genes. The availability of the hexaploid wheat (Triticum aestivum) cultivar Chinese Spring reference genome allows a detailed study of its NLR complement. However, low NLR expression and high intrafamily sequence homology hinder their accurate annotation. Here, we developed NLR-Annotator, a software tool for in silico NLR identification independent of transcript support. Although developed for wheat, we demonstrate the universal applicability of NLR-Annotator across diverse plant taxa. We applied our tool to wheat and combined it with a transcript-validated subset of genes from the reference gene annotation to characterize the structure, phylogeny, and expression profile of the NLR gene family. We detected 3,400 full-length NLR loci, of which 1,560 were confirmed as expressed genes with intact open reading frames. NLRs with integrated domains mostly group in specific subclades. Members of another subclade predominantly locate in close physical proximity to NLRs carrying integrated domains, suggesting a paired helper function. Most NLRs (88%) display low basal expression (in the lower 10 percentile of transcripts). In young leaves subjected to biotic stress, we found up-regulation of 266 of the NLRs To illustrate the utility of our tool for the positional cloning of resistance genes, we estimated the number of NLR genes within the intervals of mapped rust resistance genes. Our study will support the identification of functional resistance genes in wheat to accelerate the breeding and engineering of disease-resistant varieties.

Identification of a Recessive Gene PmQ Conferring Resistance to Powdery Mildew in Wheat Landrace Qingxinmai Using BSR-Seq Analysis.

Wheat powdery mildew is caused by Blumeria graminis f. sp. tritici (Bgt), a biotrophic fungal species. It is very important to mine new powdery mildew (Pm) resistance genes for developing resistant wheat cultivars to reduce the deleterious effects of the disease. This study was carried out to characterize the Pm gene in Qingxinmai, a winter wheat landrace from Xinjiang, China. Qingxinmai is resistant to many Bgt isolates collected from different wheat fields in China. F1, F2, and F2:3 generations of the cross between Qingxinmai and powdery mildew susceptible line 041133 were developed. It was confirmed that a single recessive gene, PmQ, conferred the seedling resistance to a Bgt isolate in Qingxinmai. Bulked segregant analysis-RNA-Seq (BSR-Seq) was performed on the bulked homozygous resistant and susceptible F2:3 families, which detected 57 single nucleotide polymorphism (SNP) variants that were enriched in a 40 Mb genomic interval on chromosome arm 2BL. Based on the flanking sequences of the candidate SNPs extracted from the Chinese Spring reference genome, 485 simple sequence repeat (SSR) markers were designed. Six polymorphic SSR markers, together with nine markers that were anchored on chromosome arm 2BL, were used to construct a genetic linkage map for PmQ. This gene was placed in a 1.4 cM genetic interval between markers Xicsq405 and WGGBH913 corresponding to 4.9 Mb physical region in the Chinese Spring reference genome. PmQ differed from most of the other Pm genes identified on chromosome arm 2BL based on its position and/or origin. However, this gene and Pm63 from an Iranian common wheat landrace were located in a similar genomic region, so they may be allelic.

Fine mapping of root lesion nematode (Pratylenchus thornei) resistance loci on chromosomes 6D and 2B of wheat.

Resistance QTL to root lesion nematode (Pratylenchus thornei) in wheat (Triticum aestivum), QRlnt.sk-6D and QRlnt.sk-2B, were mapped to intervals of 3.5 cM/1.77 Mbp on chromosome 6D and 1.4 cM/2.19 Mbp on chromosome 2B, respectively. Candidate resistance genes were identified in the QTL regions and molecular markers developed for marker-assisted breeding. Two previously known resistance QTL for root lesion nematode (Pratylenchus thornei) in bread wheat (Triticum aestivum), QRlnt.sk-6D and QRlnt.sk-2B, were fine-mapped using a Sokoll (moderately resistant) by Krichauff (susceptible) doubled haploid (DH) population and six newly developed recombinant inbred line populations. Bulked segregation analysis with the 90K wheat SNP array identified linked SNPs which were subsequently converted to KASP assays for mapping in the DH and RIL populations. On chromosome 6D, 60KASP and five SSR markers spanned a total genetic distance of 23.7cM. QRlnt.sk-6D was delimited to a 3.5cM interval, representing 1.77Mbp in the bread wheat cv. Chinese Spring reference genome sequence and 2.29Mbp in the Aegilops tauschii genome sequence. These intervals contained 42 and 43 gene models in the respective annotated genome sequences. On chromosome 2B, 41 KASP and 5 SSR markers produced a map spanning 19.9cM. QRlnt.sk-2B was delimited to 1.4cM, corresponding 3.14 Mbp in the durum wheat cv. Svevo reference sequence and 2.19 Mbp in Chinese Spring. The interval in Chinese Spring contained 56 high-confidence gene models. Intervals for both QTL contained genes with similarity to those previously reported to be involved in disease resistance, namely genes for phenylpropanoid biosynthetic pathway-related enzymes, NBS-LRR proteins and protein kinases. The potential roles of these candidate genes in P. thornei resistance are discussed. The KASP markers reported in this study could potentially be used for marker-assisted breeding of P. thornei-resistant wheat cultivars.

Fine Mapping of the Wheat Leaf Rust Resistance Gene Lr42.

Leaf rust caused by Puccinia triticina Eriks is one of the most problematic diseases of wheat throughout the world. The gene Lr42 confers effective resistance against leaf rust at both seedling and adult plant stages. Previous studies had reported Lr42 to be both recessive and dominant in hexaploid wheat; however, in diploid Aegilops tauschii (TA2450), we found Lr42 to be dominant by studying segregation in two independent F2 and their F2:3 populations. We further fine-mapped Lr42 in hexaploid wheat using a KS93U50/Morocco F5 recombinant inbred line (RIL) population to a 3.7 cM genetic interval flanked by markers TC387992 and WMC432. The 3.7 cM Lr42 region physically corresponds to a 3.16 Mb genomic region on chromosome 1DS based on the Chinese Spring reference genome (RefSeq v.1.1) and a 3.5 Mb genomic interval on chromosome 1 in the Ae. tauschii reference genome. This region includes nine nucleotide-binding domain leucine-rich repeat (NLR) genes in wheat and seven in Ae. tauschii, respectively, and these are the likely candidates for Lr42. Furthermore, we developed two kompetitive allele-specific polymorphism (KASP) markers (SNP113325 and TC387992) flanking Lr42 to facilitate marker-assisted selection for rust resistance in wheat breeding programs.

Genetic mapping of a major QTL promoting homoeologous chromosome pairing in a wheat landrace.

Common wheat landrace Kaixian-luohanmai carries a gene(s) that promotes homoeologous chromosome pairing. A major QTL responsible for this effect was mapped to chromosome arm 3AL. Polyhaploid hybrids of a Chinese common wheat landrace Kaixian-luohanmai (KL) and related species show increased levels of chromosome pairing. Over 90% of that pairing is between homoeologous arms of wheat chromosomes, with a very strong preference for pairing between homoeologs from genomes A and D. Wheat-rye pairing was also observed at low frequency. Two mapping populations were created from the hybrids of KL with two wheat genotypes top crossed to rye. Mean chiasmata numbers per plant were used as phenotypic data. Wheat 660K and 15K SNP arrays, DArT markers and SSR markers were used for genotyping of the top-cross ABDR hybrids. One major QTL, named QPh.sicau-3A, for increased homoeologous pairing was detected on chromosome arm 3AL, and it was responsible for ca. 16% of the total variation. This QTL was located in the interval 696-725Mb in the Chinese Spring reference genome. SNP markers closely linked with QPh.sicau-3A were converted to KASP markers and validated for marker-assisted selection.

Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene Pm61.

The gene Pm61 that confers powdery mildew resistance has been previously identified on chromosome arm 4AL in Chinese wheat landrace Xuxusanyuehuang (XXSYH). To facilitate the use of Pm61 in breeding practices, the bulked segregant analysis-RNA-Seq (BSR-Seq) analysis, in combination with the information on the Chinese Spring reference genome sequence, was performed in the F2:3 mapping population of XXSYH × Zhongzuo 9504. Two single nucleotide polymorphism (SNP), two Kompetitive Allele Specific PCR (KASP), and six simple sequence repeat (SSR) markers, together with previously identified polymorphic markers, saturated the genetic linkage map for Pm61, especially in the proximal side of the target gene that was short of gene-linked markers. In the newly established genetic linkage map, Pm61 was located in a 0.71 cM genetic interval and can be detected in a high throughput scale by the KASP markers Xicsk8 and Xicsk13 or by the standard PCR-based markers Xicscx497 and Xicsx538. The newly saturated genetic linkage map will be useful in molecular marker assisted-selection of Pm61 in breeding for disease resistant cultivar and in its map-based cloning.

Fine mapping of the wheat powdery mildew resistance gene Pm52 using comparative genomics analysis and the Chinese Spring reference genomic sequence.

A high-resolution genetic linkage map was constructed using the comparative genomics analysis approach and the wheat reference genome, which placed wheat powdery mildew resistance gene Pm52 in a 0.21-cM genetic interval on chromosome arm 2BL. The gene Pm52 confers resistance to powdery mildew and has been previously mapped on chromosome arm 2BL in winter wheat cultivar Liangxing 99. Because of its effectiveness against the disease, this study was initiated to finely map Pm52 using the comparative genomics analysis approach and the wheat reference genomic sequence. Based on the EST sequences that were located in the chromosome region flanking Pm52, four EST-SSR markers were developed, and another nine SSR markers were developed using the comparative genomics technology. These thirteen markers were integrated into a genetic linkage map using an F2:3 subpopulation of the Liangxing 99 × Zhongzuo 9504 cross. Pm52 was mapped within a 3.2-cM genetic interval in the subpopulation that corresponded to a ~40-Mb genomic interval on chromosome arm 2BL of the Chinese Spring reference genome. The Pm52-flanking markers Xicsl163 and Xicsl62 identified 344 recombinant individuals from 8820 F2 plants. Nine SSR markers generated from the Chinese Spring genomic interval were incorporated into a high-resolution genetic linkage map, which placed Pm52 in a 0.21-cM genetic interval corresponding to 5.6-Mb genomic region. The constructed high-resolution genetic linkage map will facilitate the map-based cloning of Pm52 and its marker-assisted selection.

Mapping and characterization of wheat stem rust\xa0resistance genes SrTm5 and Sr60 from Triticum monococcum

Key messageThe new stem rust resistance gene Sr60 was fine-mapped to the distal region of chromosome arm 5AmS, and the TTKSK-effective gene SrTm5 could be a new allele of Sr22.The emergence and spread of new virulent races of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici; Pgt), including the Ug99 race group, is a serious threat to global wheat production. In this study, we mapped and characterized two stem rust resistance genes from diploid wheat Triticum monococcum accession PI 306540. We mapped SrTm5, a previously postulated gene effective to Ug99, on chromosome arm 7AmL, completely linked to Sr22. SrTm5 displayed a different race specificity compared to Sr22 indicating that they are distinct. Sequencing of the Sr22 homolog in PI 306540 revealed a novel haplotype. Characterization of the segregating populations with Pgt race QFCSC revealed an additional resistance gene on chromosome arm 5AmS that was assigned the official name Sr60. This gene was also effective against races QTHJC and SCCSC but not against TTKSK (a Ug99 group race). Using two large mapping populations (4046 gametes), we mapped Sr60 within a 0.44 cM interval flanked by sequenced-based markers GH724575 and CJ942731. These two markers delimit a 54.6-kb region in Brachypodium distachyon chromosome 4 and a 430-kb region in the Chinese Spring reference genome. Both regions include a leucine-rich repeat protein kinase (LRRK123.1) that represents a potential candidate gene. Three CC–NBS–LRR genes were found in the colinear Brachypodium region but not in the wheat genome. We are currently developing a Bacterial Artificial Chromosome library of PI 306540 to determine which of these candidate genes are present in the T. monococcum genome and to complete the cloning of Sr60.

A resource of large-scale molecular markers for monitoring Agropyron cristatum chromatin introgression in wheat background based on transcriptome sequences

Agropyron cristatum is a wild grass of the tribe Triticeae and serves as a gene donor for wheat improvement. However, very few markers can be used to monitor A. cristatum chromatin introgressions in wheat. Here, we reported a resource of large-scale molecular markers for tracking alien introgressions in wheat based on transcriptome sequences. By aligning A. cristatum unigenes with the Chinese Spring reference genome sequences, we designed 9602 A. cristatum expressed sequence tag-sequence-tagged site (EST-STS) markers for PCR amplification and experimental screening. As a result, 6063 polymorphic EST-STS markers were specific for the A. cristatum P genome in the single-receipt wheat background. A total of 4956 randomly selected polymorphic EST-STS markers were further tested in eight wheat variety backgrounds, and 3070 markers displaying stable and polymorphic amplification were validated. These markers covered more than 98% of the A. cristatum genome, and the marker distribution density was approximately 1.28 cM. An application case of all EST-STS markers was validated on the A. cristatum 6 P chromosome. These markers were successfully applied in the tracking of alien A. cristatum chromatin. Altogether, this study provided a universal method of large-scale molecular marker development to monitor wild relative chromatin in wheat.