Development of P genome-specific SNPs and their application in tracing Agropyron cristatum introgressions in common wheat

Huihui Ma,Jinpeng Zhang,Jing Zhang,Shenghui Zhou,Haiming Han,Weihua Liu,Xinming Yang,Xiuquan Li,Lihui Li*

Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

Keywords:KASP marker Single nucleotide polymorphism Agropyron cristatum Triticum aestivum

A B S T R A C T As an important wild relative ofwheat,Agropyron cristatum has been successfully used for wheat improvement.Currently,a few useful agronomic traits of A.cristatum,such as high grain number per spike and resistance to diseases,have been transferred into common wheat.However,the effective detection of small A.cristatum segmental introgressions in common wheat is still difficult.The objective of this study was to identify A.cristatum-specific single nucleotide polymorphisms(SNPs)for the detection of small alien segments in wheat.The transcriptome sequences of A.cristatum were aligned against wheat coding DNA sequences(CDS)for SNP calling.As a result,we discovered a total of 167,613 putative SNPs specific to the P genome of A.cristatum compared with the common wheat genomes.Among 230 selected SNPs with functional annotations related to inflorescence development and stress resistance,68 were validated as P genome-specific SNPs in multiple wheat backgrounds using Kompetitive Allele Specific PCR(KASP)assays.Among them,55 SNPs were assigned to six homoeologous groups of the P genome using wheat-A.cristatum addition lines,and 6P-specific SNP markers were further physically mapped on different segments of chromosome 6P in 6P translocation lines.The P genome-specific SNPs were also validated by Sanger sequencing and used to detect the P chromatin in wheat-A.cristatum cryptic introgression lines.Two SNP markers(Unigene20217-182 and Unigene20307-1420)were detected in two wheat-A.cristatum introgression lines that showed enhanced grain number per spike and high resistance to powdery mildew.Together,the developed Pgenome-specific SNPmarkers willaccelerate the detection of large numbers of wheat-A.cristatum derivatives and will be helpful for marker-assisted transfer of desirable traits from A.cristatum into adapted wheat cultivars in wheat breeding programs.

1.Introduction

Common wheat(Triticum aestivum L.),one of the most important staple food crops,plays a major role in global food security.However,the decreasing genetic diversity of wheat due to long-term breeding has severely hindered breakthroughs in the breeding of new wheat varieties[1,2].It is therefore necessary to broaden the genetic base of common wheat.Wild relatives of wheat,which have survived in various severe ecological environments for a long time without human selection,are rich in genetic diversity and are valuable genetic resources for wheat improvement[3-6].For several decades,wide crosses have served as an effective method for transferring desirable alien genes from wild relatives into common wheat to improve the genetic diversity of wheat[7,8].Currently,common wheat has been successfully hybridized with 23 genera of the tribe Triticeae,and a large number of novel alien genes have been transferred into wheat[8,9].

Agropyron cristatum(L.)Gaertn.,an important wild relative of wheat,contains a single P genome and carries numerous elite genes for wheat improvement,including genes involved in resistance to both abiotic stresses and various diseases,as well as genes involved in many beneficial yield-related traits[10,11].To fully exploit genetic variation in A.cristatum,accession Z559 was hybridized with the common wheat cultivar Fukuhokomugi and viable progeny were recovered by embryo rescue[12-14].A range of derivatives,including addition,substitution,and translocation lines were obtained.Some of the translocation lines carried alien segments that were too small to be detected by standard cytological and marker detection methods;these are described as cryptic translocations.To date,several desirable traits of A.cristatum located on specific P chromosomes,including increased grain number per spike [15-17],improved thousand-grain weight[18,19],and resistance to powdery mildew[20,21],leafrust[22]and stripe rust[23]were introduced into wheat.

Wheat-alien introgressions with small alien segments,especially those carrying desirable traits,have value for wheat breeding.Identifying and tracking introgressed chromatin in wheat backgrounds are necessary prior to their use in breeding programs[24-26].Many approaches have been made to detect transferred alien fragments in wheat.Traditional cytological methods such as chromosome banding,genomic in situ hybridization(GISH)and fluorescence in situ hybridization(FISH)have been widely used to identify alien chromosomes and chromosomal segments[27-29];however,low throughput and limited spatial resolution of these techniques can be significant constraints.Molecular markers,such as sequence-tagged sites(STS),which can be used to detect small alien chromosomal segments,have been applied in some cases[30-32].However,the relatively low polymorphism and distribution densities of these markers prevent their extensive use in wheat breeding programs where high-throughput screening is necessary.With the rapid advancement of next-generation sequencing(NGS)the development of large numbers of SNP markers has become cost-effective and user-friendly[33,34].It should be possible to use these methods in validation of alien fragments,especially those that cannot be detected by other methods.Furthermore,genome-specific SNP markers are co-dominant and useful for simultaneously tracing alien chromosomes and their homoeologous wheat groups.For example,Aegilops geniculata 5MgS-specific SNP markers were successfully used to map alien introgressions in wheat[35].In addition,Winfield et al.[36]validated large numbers of SNP markers that could be used to genotype common wheat as well as to identify and track introgressions from a variety of wild relatives.With low cost per data point and ease of development the Kompetitive Allele Specific PCR(KASP)assay method has become a flexible and reliable fluorescence-based genotyping platform,making the discovery of genomespecific SNP markers simpler and more effective for use in large-scale projects[35,37-39].

We previously reported the development of expressed sequence tag-sequence-tagged site(EST-STS)markers for monitoring A.cristatum chromatin introgressions in wheat,but up to 35%without functional annotations were predicted to be putative long intergenic non-coding RNAs(lincRNAs)[40].However,the A.cristatum transcripts had high sequence identity with wheat transcripts,with a peak value of similarity reaching 98% [41].Since a large portion of important functional genes are relatively conserved there are many SNPs between A.cristatum and wheat.To mine the rare allelic variation of the functional genes from A.cristatum in a wheat background,it is essential to develop SNP markers to track alien introgressions.Therefore,the objectives of the present study were to develop A.cristatum-specific SNP markers for high-throughput detection of A.cristatum introgressions in wheat,and to determine their chromosome locations to expedite their use in wheat improvement.

2.Materials and methods

2.1.Plant materials

Eighteen genetically diverse common wheat accessions and A.cristatum(L.)accession Z559 were used for screening P genome-specific SNP markers.A.cristatum(L.)accession Z559(2n=4x=28,PPPP)served as the male donor parent in the original cross between wheat and A.cristatum.The 18 accessions included Fukuhokomugi,the female parent used in the original wide cross,Chinese Spring,Gaocheng 8901,Xiaoyan 6,Zhoumai 18,Jimai 22,Shi 4185,Yumai 18,Lumai 14,Zhengmai 9023,Han 4589,Dongshi 1,and Zhongmai 175 from China,and Jagger,Hi-Line,McGuire,CMH83.605 and Lovin 10 from other countries(Table S1).Seventeen wheat-A.cristatum disomic addition lines were used to determine the chromosome locations of the P genome-specific SNPs.These addition lines included two 1P addition lines,three 2P addition lines,one 1P and 2P addition line,two 4P addition lines,one 5P addition line,four 6P addition lines and four 7P addition lines(Table 1).A set of 19 wheat-A.cristatum 6P translocation lines(Table S2)[42]were used to physically map chromosome 6P-specific SNPs.Eighty-nine wheat-A.cristatum derivatives were used to detect whether they carried P chromosomal fragments or functional genes.

Table 1-Wheat-A.cristatum disomic addition lines used in this study.

2.2.Development of A.cristatum-specific SNPs

In our previous study,A.cristatum transcripts were obtained using Illumina paired-end RNA sequencing on two tissues of flag leaf and young spike.The raw read data were assembled into contigs of unigenes.A total of 73,664 non-redundant unigenes were generated after filtering;they ranged from 200 to 8202 bp with an N50 of 473 bp(GenBank GBAU00000000)[41].The flow chart for developing P genome-specific SNPs is shown in Fig.1.The SNPs were called using Burrows-Wheeler Aligner(BWA)/Sequence Alignment/Map(SAMtools)software.First,the 73,664 unigenes of A.cristatum were used for sequence alignment by BLASTn against the Chinese Spring CDS(ftp://ftp.ensemblgenomes.org/pub/release-23/plants/fasta/)to obtain high-similarity sequences with more than 95%identity between A.cristatum and wheat.Then,highly similar A.cristatum transcriptome sequences were aligned with the Chinese Spring CDS using the BWA-SW command of BWA 0.5.9[43].The alignment results of the sequences of the A,B,and D and P genomes were merged using SAMtools 0.1.16[44].SNPs that were common to the wheat A,B,and D genomes,but differing for the P genome,were identified as putatively specific to the P genome.We then extracted the 50-bp flanking sequences of SNPs from the A.cristatum transcriptome for KASP primer design.In accordance with the annotation results of the A.cristatum transcriptome sequences[41],genes related to inflorescence development,disease resistance,and other stress resistances were selected for the SNP marker analysis using KASP assays.

2.3.Design of KASP primers

The selected SNPs with 50-bp flanking sequences on each side were provided to LGC Genomics(http://www.lgcgenomics.com/)to design KASP primers.The KASP assay mixtures contained two different allele-specific competing forward primers with unique tail sequences and one common reverse primer to distinguish genotypes.For KASP assays,the genotype of common wheat was designated allele 1,the genotype of A.cristatum was designated allele 2,and water served as a negative control.The KASP primers were designed such that the allele 1-specific primer contained the unlabelled HEX tail sequences at the 5′end,and the allele 2-specific primer contained the unlabelled FAM tail sequences at the 5′end.The passive reference dye ROX was used to normalize variation in signal caused by differences in liquid volume among wells[45].

2.4.KASP genotyping

The KASP assays consisted of master mix,assay mix and genomic DNA.The genotyping reactions were performed in a total volume of 10 μL using 96-well plates and included 5 μL of high ROX master mix,0.14 μL of assay mix and～50 ng of genomic DNA(4 μL).The KASP thermal cycling conditions were as follows:94°C for 15 min;ten touchdown cycles(94 °C for 20 s,initial touchdown at 61 °C followed by a decrease of 0.6°C per cycle for 60 s);26 additional PCR cycles(94 °C for 20 s followed by 55 °C for 60 s);and then 30°C for 30 s for plate reading.All of the genotyping reactions were performed using an Applied Biosystems(ABI)StepOnePlus Real-Time PCR system.The fluorescence data were collected and analysed using ABI StepOne 2.1 software.

2.5.Sanger sequencing-based SNP validation

Sanger sequencing was performed to confirm the authenticity of the A.cristatum-specific SNPs from the KASP assays.Four SNPs from two unigenes were randomly selected for testing in three different accessions,namely,Fukuhokomugi,A.cristatum Z559 and 6P addition line 4844-12.Three SNPs in the same unigene were amplified by one pair of PCR primers.The PCR primers were designed with the online software Primer3(http://bioinfo.ut.ee/primer3/).The PCR products were purified from agarose gels and cloned into a pGEM-T Easy vector(Promega).The positive clones were subsequently sequenced by the Sanger dideoxy chaintermination method.

2.6.Evaluation of powdery mildew response

The wheat parent Fukuhokomugi and introgression line L31485 were planted for assessment of powdery mildew response in the field at Xinxiang(Henan province)and in a CAAS greenhouse in Beijing during 2015-2016.Adult plant reactions were assessed after inoculation with Erysiphe graminis f.sp.tritici(Egt)isolate E09.The infection types(IT)were scored using a 0-9 scale described by Sheng and Duan[46],with 0 as immune,0;as nearly immune,1-2 as highly resistant,3-4 as moderately resistant,5-6 as moderately susceptible,7-8 as highly susceptible,and 9 as extremely susceptible.Plants with scores of 0-4 were classified as resistant and those with scores of 5-9 as susceptible.

Fig.1-Flow diagram for A.cristatum-specific SNP mining and validation.

3.Results

3.1.Identification of P genome-specific SNPs

In the survey of nucleotide variation of orthologous genes between A.cristatum and common wheat,29,535 highsimilarity sequences were obtained from 73,664 A.cristatum unigenes with more than 95%identity to the wheat CDS after BLASTn analysis.As a result,a total of 167,613 putative P genome-specific SNPs were obtained via SNP calling analysis(Fig.1).According to the functional annotations of A.cristatum unigenes in our previous study[41],230 SNPs that may be related to inflorescence development,disease resistance and abiotic stress resistance were selected for the KASP assays.These SNPs encompassed the genes containing AP2,Myb_DNA-binding,and NB-ARC Pfam domains,which are involved in the inflorescence development pathway and stress resistance(Table 2).

3.2.KASP genotyping assays of A.cristatum-specific SNPs in multiple wheat backgrounds

The polymorphisms of the 230 selected SNPs were first tested using KASP genotyping assays between A.cristatum Z559 and common wheat cv.Fukuhokomugi.To examine the genomespecificity of the SNPs and eliminate the false positives,markers polymorphic between Fukuhokomugi and A.cristatum were further screened in 17 additional wheat genetic backgrounds.In the KASP genotyping assays,target SNPs that presented three obvious clusters and showed allele 1 in wheat varieties,allele 2 in A.cristatum Z559,and heterozygosity(allele 1/allele 2)in the mixed samples of Z559 and Fukuhokomugi were therefore specific to A.cristatum and could be used for P genome detection.Seventy of the 230 SNPs were polymorphic between Fukuhokomugi(allele 1)and A.cristatum(allele 2)and had heterozygous alleles(allele 1/allele 2)in the mixed samples of Z559 and Fukuhokomugi(Fig.2-A).These 70 SNPs,accounting for 29.6%of the total markers genotyped,were possibly specific to the P genome(Table 2).However,two of them were detected in other wheat varieties and were thus false positives.Consequently,these 68 P genome-specific SNPs(Table S3)were used to identify A.cristatum P chromatin in multiple wheat backgrounds.

Table 2-Classification of KASP markers with known functional annotation/domain used in this study.

3.3.Chromosome locations of P genome-specific SNPs

Fig.2-KASP-based validation of A.cristatum-specific SNPs and their application in detection of alien segments in wheat-A.cristatum derivatives.(A)Genotyping data of P genome-specific SNP marker Unigene71815-538 on A.cristatum Z559 and eighteen diverse wheat accessions.(B)Genotyping data of chromosome 6P-specific SNP marker Unigene70138-464 on Fukuhokomugi,A.cristatum Z559 and the addition lines.(C)Genotyping data of the SNP marker Unigene20307-783 on Fukuhokomugi,A.cristatum Z559 and nineteen translocation lines.(D)Genotyping data of the SNP marker Unigene20217-182 on Fukuhokomugi,A.cristatum Z559 and 89 wheat-A.cristatum derivatives.Allele 1(wheat-type)is coloured red;allele 2(A.cristatum-type)is coloured blue;and green represents the heterozygous type.

To anchor the P genome-specific SNP markers to a specific chromosome of A.cristatum,the 68 validated markers were tested on 17 wheat-A.cristatum addition lines(Table 1).If A.cristatum-specific SNPs are located on P chromosomes,the SNP markers should appear as heterozygous types and occupy similar positions to the Z559:Fukuhokomugi mixture.Fifty-five(80%)SNPs were detected as heterozygous alleles in the addition lines(Fig.2-B)indicating successful location to different A.cristatum chromosomes.Out of these 55 SNPs,two SNPs were targeted to chromosome 2P of addition lines II-7-1,II-8-1,and II-9-3;one SNP was mapped to chromosome 2P of addition lines II-9-3 and II-4-2;and four SNPs were anchored to chromosome 1P/2P of addition line II-4-2.Six SNPs were located on chromosome 4P of addition lines II-21-2 and II-21-6,and three SNPs were on chromosome 5P of addition line II-11-1a.Twenty-nine SNPs were detected on chromosome 6P of the wheat-A.cristatum addition lines 4844-12,5113,5114,and II-30-5.Eight SNPs were mapped to chromosome 7P in addition lines II-26,II-5-1,5038,and 5043.The remaining two SNPs were mapped to multiple homologous groups in addition lines(Table 3).Furthermore,the KASP assays revealed that the chromosome locations of 49 SNPs(of the 55 SNPs)were consistent with their putative assignment to the homoeologous groups of common wheat as determined by the alignment analysis.These 49 SNPs,accounting for 89%of the mapped SNPs,were validated as conserved SNPs between A.cristatum and wheat,further indicating high probability of synteny between the A.cristatum and wheat genomes.

3.4.Sanger sequencing-based confirmation of A.cristatumspecific SNPs

Sanger sequencing was performed to confirm the authenticity of the A.cristatum-specific SNPs from the KASP assays.Two genes anchored to chromosome 6P were amplified in 6P addition line 4844-12 and wide cross parents,Fukuhokomugi and A.cristatum Z559(Fig.3-A).The first gene included the A.cristatum-specific SNP locus of Unigene20307-1420(G/A,A.cristatum/common wheat).The sequencing chromatogram revealed two different types at this SNP site:an A.cristatumtype(GG)and a wheat-type(AA,from Fukuhokomugi),and confirmed that the G/A SNP was in the addition line 4844-12(Fig.3-B).The second gene(Unigene72510)had three SNP sites including Unigene72510-71(C/A),Unigene72510-204(T/A)and Unigene72510-288(T/C).The DNA sequencing results showed that the addition line 4844-12 contained all three SNPs(Fig.3-C),indicating that the sequenced region was derived from A.cristatum.Sanger sequencing thus validated these SNPs as specific to the P genome.

3.5.Physical mapping of chromosome 6P-specific SNPs using wheat-A.cristatum translocation lines

Physical mapping of A.cristatum-specific molecular markers facilitate detection of alien introgressions in wheat background.Here,we investigated the physical distribution of the 29 validated A.cristatum 6P-specific SNP markers using a set of 19 previously characterized wheat-A.cristatum 6P translocation lines(Table S2).KASP assays showed that 21 SNPs were mapped to different bins of chromosome 6P(Table S4,exemplified in Fig.2-C),and eight of them were assigned to chromosomal bins 6PS1-2.Two SNPs(Unigene20307-783 and Unigene20307-1420)were located in chromosomal bin 6PS4.Another two SNPs(Unigene50800-149 and Unigene70138-464)were allocated to chromosomal bins 6PS7-9.Only one SNP was detected in each of bins 6PS5-6,6PS3-14,6PS10-11,6PL1-5,and 6PL6-8.Additionally,one SNP(Unigene51942-84)and three SNPs (Unigene19037-683,Unigene19037-757 and Unigene72510-288)were assigned to the short arm and long arm of chromosome 6P,respectively(Table S4,Fig.4).Thus,these 21 chromosome 6P-specific SNP markers can be used to effectively trace small alien chromosomal fragments in wheat backgrounds.

3.6.Application of P genome-specific SNPs in the detection of cryptic wheat-A.cristatum introgression lines

Several advanced generation derivatives of A.cristatum exhibited superior traits such as higher grain number per spike and disease resistance compared with their parental wheat lines.These derivatives were believed to carry cryptic introgressions from the P genome.Hence,the P genomespecific SNP markers were used to identify P chromatin in these derivatives to reveal their “cryptic”nature.The 89 wheat-A.cristatum derivatives were screened using 55 SNPs that were validated by KASP assays in addition lines.Two of the 55 SNPs(Unigene20217-182 and Unigene20307-1420)were identified in derivative lines L31408 and L31485(Fig.2-D)that showed enhanced grain number per spike and resistance to powdery mildew(Fig.5),confirming that they carried A.cristatum chromatin.These data indicated that A.cristatumspecific SNPs can effectively detect cryptic alien introgressions in wheat,and that it is possible to transfer and utilize these superior traits of A.cristatum in breeding.

4.Discussion

Wild relatives of wheat represent a vast reservoir of favorable genes for resistance to biotic and abiotic stresses and possess abundant natural variation for wheat improvement.To utilize the high level of genetic variation from wild relatives in wheat breeding,rapid and accurate detection of alien introgressed segments in wheat is advantageous.Consequently,development of molecular markers specific to introgressed chromatin will be beneficial for improving the efficacy of alien chromatin identification in wheat.Ongoing efforts to achieve this goal have generated enormous numbers of molecular markers that tag the introgressed chromatin[30-32,47].In the example of Haynaldia villosa,although various random amplified polymorphic DNAs(RAPD)[48],sequence characterized amplified region(SCAR)markers[49],and EST-PCR markers[30]have been reported,only a few H.villosa chromosome-specific markers are available.Wang et al.[50]developed a series of H.villosa 4VS-specific intron-targeting(IT)markers with high specificity by combining chromosome sorting and NGS.In A.cristatum,molecular tools used to identify chromosomal segments in common wheat included SCAR markers[51],ESTSTS markers[40],and degenerate oligonucleotide primed PCR(DOP-PCR)-derived sequences[52].However,these markers mainly depended on gel electrophoresis,and their application was too laborious for analysis of large populations.Recently,alien genome-specific SNP markers that provide a highthroughput screening platform were used to identify alienchromatin in common wheat background[35,36,53].In this study,167,613 putative P genome-specific SNPs were discovered from nucleotide variants of orthologous genes between A.cristatum and common wheat.Large-scale mining of P genome-specific SNPs not only facilitates the development of alien genome-specific markers that can be used to transfer alien chromosomes to wheat,but also provides useful tools for further characterization of those wild relatives.

Table 3-Chromosome locations of P-genome-specific SNPs in wheat-A.cristatum addition lines.

Fig.3-Sanger sequencing-based validation of A.cristatum-specific SNPs.(A)Two amplicons randomly selected from SNP flanking sequences were amplified from Fukuhokomugi,A.cristatum Z559 and 6P addition line 4844-12.(B)Sequencing chromatogram of the amplification containing the A.cristatum-specific SNP locus of Unigene20307-1420(G/A,A.cristatum/common wheat).(C)Sequencing chromatograms of amplifications containing three SNP sites of Unigene72510-71(C/A),Unigene72510-204(T/A),Unigene72510-288(T/C).SNPs are shown in black box.

Currently,SNP discovery in complex genomes,such as wheat,can be easily achieved by NGS[34,54].However,the high complexity of wheat genomes,especially the presence of paralogs and repetitive DNA sequences,as well as the absence of complete reference genome sequences,can create ambiguities in SNP calling[35,55].In this study,two approaches,KASP assays and Sanger sequencing,were conducted to analyse the authenticity of SNP markers.Using KASP assays,68 markers polymorphic between Fukuhokomugi and A.cristatum were validated across multiple wheat backgrounds,suggesting that the SNP markers developed in the study were highly specific.The reliability of A.cristatum-specific SNP markers was also validated by Sanger sequencing.The sequencing results of two PCR amplicons randomly chosen from SNP flanking sequences which were mapped to chromosome 6P verified the results of the KASP assays.

Fig.4-Physical map of A.cristatum chromosome 6PS-specific SNP markers.

Fig.5-Adult plant responses to powdery mildew of the introgression line L31485 and Fukuhokomugi.

Knowledge of the chromosome locations of alien genomespecific molecular markers is important for identifying introgressed regions and mapping alien genes.The large numbers of chromosome-specific markers developed for each addition line are expected to aid breeders in conducting more stringent screening and selection when developing cultivars with minimal chromosome segments that fulfil specific breeding goals[47].In the present research,55 of the 68 P genome-specific SNP markers were detected in six homoeologous groups of wheat-A.cristatum addition lines.The other 13 SNPs were not located on a specific A.cristatum chromosome.One reason could be explained by the absence of a chromosome 3P addition line in this study.Moreover,previous studies suggested that tetraploid A.cristatum is not an autotetraploid but rather a segmental allotetraploid[40,56],suggesting that more addition lines are needed to achieve the chromosome locations of these 13 SNPs.Twenty-one chromosome 6P-specific SNP markers were physically mapped in different regions of chromosome 6P.Furthermore,two SNP markers detected two cryptic introgressions not detected by conventional methods.All of the genotyping data clearly indicated that alien chromosome-specific SNPs can be used for identification of addition,translocation and especially introgression lines.SNP markers developed in this study that were identified from different chromosomal regions will be helpful for transferring target alien genes or traits into wheat.

The use of molecular markers tightly linked to desirable genes/traits can aid both targeted introgression into wheat and marker-assisted selection(MAS)in wheat breeding[57].For example,Tiwari et al.[35]developed two SNP markers specific to Aegilops geniculata 5MgS,tagging the smallest introgression carrying disease resistance;these markers can be used in MAS for rust resistance genes(Lr57 and Yr40).In the present study,two SNP markers(Unigene20217-182 and Unigene20307-1420)were successfully employed to detect two A.cristatum derivative lines(L31408 and L31485)that showed powdery mildew resistance and higher grain number per spike.Unigene20217-182 was also detected in 6P addition lines 4844-12,5113,and 5114 that had been reported to confer powdery mildew resistance in wheat[20].These results suggested that Unigene20217-182 is an effective marker for tracking a powdery mildew resistance gene from A.cristatum,and can be used in MAS for powdery mildew resistance.The development of P genome-specific SNP markers directly from functional genes,especially those potentially related to agronomic,biotic and abiotic stress-related traits[58],may provide a rapid source of functional markers for selecting desirable traits from introgression lines in breeding.

The transfer of useful agronomic traits from wild relatives into wheat is often accompanied by undesirable linkage drag[29,35,59].Linkage drag is a significant challenge for utilizing alien genes in wheat breeding[60].Therefore,large-scale screening and rapid characterization of germplasm lines with introgressed genes of interest,but minimal unwanted alien chromatin is particularly important.In this study,two wheat-A.cristatum cryptic introgression lines were validated by P genome-specific SNP markers.These wheat-A.cristatum introgression lines with superior traits and minimal linkage drag can be used as parents in wheat improvement.

Acknowledgments

This work was supported by the China Agriculture Research System(CARS-03)and the National Key Research and Development Program of China(2016YFD0102000).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.07.003.