Molecular cytogenetics and St-chromosome-specific molecular markers development of novel stripe rust resistant wheat–Thinopyrum intermedium as well as wheat–Thinopyrum ponticum substitution lines


 Background

Owing to the excellent resistance to abiotic and biotic stress, Thionpyrum intermedium (2n = 6x = 42, JJJsJsStSt) and Thinopyrum ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs. Disomic substitution lines (DSLs) carrying one pair of alien chromosomes are valuable bridge materials for novel genes transmission, FISH karyotype construction and specific molecular marker development.
Results

Six wheat–Thinopyrum DSLs derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), were characterized by a sequential fluorescence in situ hybridization (FISH)–genome in situ hybridization (GISH), a multicolor GISH (mc-GISH), and an analysis of wheat 15K SNP array combined with molecular marker selection. ES-9 (DS2St (2A)) and ES-10 (DS3St (3D)) are wheat–Th. ponticum DSLs, while ES-23 (DS2St (2A)), ES-24 (DS3St (3D)), ES-25(DS2St (2B)), and ES-26 (DS2St (2D)) are wheat–Th. intermedium DSLs. ES-9, ES-23, ES-25 and ES-26 conferred higher thousand-kernel weight and stripe rust resistance at adult stages, while ES-10 and ES-24 performed highly resistant to stripe rust at all stages. Furthermore, cytological analysis showed that the alien chromosomes (2St/3St) belonging to the same homoeologous group derived from different donors carried the same FISH karyotype and could normally form a bivalent. Based on specific-locus amplified fragment sequencing (SLAF-seq), two 2St-chromosome-specific markers (PTH-005 and PTH-013) and two 3St-chromosome-specific markers (PTH-113 and PTH-135) were developed.
Conclusions

The six wheat–Thinopyrum disomic substitution lines conferring stripe rust resistance will be used as bridging parents for valuable resistant genes transmission. And the utility of PTH-113 and PTH-135 in a BC1F2 population showed the newly developed markers could be useful tools for efficient identification of St chromosomes in a common wheat background.


Background
Intermediate wheatgrass (Thinopyrum intermedium Barkworth & D.R. Dewey, JJJ s J s StSt, 2n = 6x = 42) and tall wheatgrass (Thinopyrum ponticum (Podp.) Barkworth & D. R. Dewey, 2n = 10x = 70) are important allopolyploids of Thinoprum species. Because of the desirable tolerance to biotic and abiotic stresses, both of them have been widely used in wheat chromosome engineering breeding for decades [1,2]. According to previous studies, the chromosomal composition of Th. intermedium and Th. ponticum have not been fully characterized. In terms of Th. intermedium, the chromosomal composition is generally regarded as JJJ s J s StSt [3] or J r J r J vs J vs StSt [4]. The subgenome J or J r is highly homologous with genome J (Th. bessarabicum, J b , E b )/E (Th. elongatum, J e , E e ) [5], and the main controversy has been whether the genome V originating from Dasypyrum villosum (2n = 2x = 14, VV) was involved in the recombinant subgenome J s or not [6,7]. Additionally, it was convinced that Th. intermedium contained a set of St chromosomes which probably derived from diploid Pseudoroegneria spicata (2n = 2x = 14, StSt) [8][9][10]. However, it has been still unclear that whether Th. ponticum contains St chromosomes and if the St genome as well as the J/E genome were affected by recombination during the allopolyploidization process [11,12].
Stripe rust (Puccinia striiformis f.sp. tritici, Pst) is a recurrent damaging disease causes serious yields decrease of wheat annually [28,29]. Development and transfer of novel resistant genes contained in wheat related wild species is one of the most e cient and environment-friendly solutions. According to previous studies, St chromosomes originating from Th. intermedium carry several new stripe rust resistance genes, which are potentially optimal genetic resources for wheat breeding. Except the named wheat-Th. intermedium disomic addition lines (DALs), L4 (DA4St) and L7 (DA6St), a DS1St (1D) with stripe rust resistance was produced [30]. Additionally, a DA3St [31] and a DA7St (Song et al. 2013) were characterized, both carrying stripe rust resistant gene(s). However, at present no wheat-Thinopyrum intermedium 2St disomic substitution lines with stripe rust resistance have been reported.
In the present study, four wheat-Th. intermedium disomic substitution lines, ES-22, ES-23, ES-25 and ES-26, were generated from crosses between Abbondanza nullisomic lines (2n = 40) and Zhong4 (a wheat-Th. intermedium partial amphiploid with stripe rust, 2n = 8x = 56) with consecutive self-crosses for several years. While two wheat-Th. ponticum disomic substitution lines, ES-9 and ES-10, were derived from Xiaoyan784 (a wheat-Th. ponticum partial amphiploid with stripe rust, 2n = 8x = 56). Molecular cytogenetic analysis was to determine and compare the genome composition of the six alien lines. In addition, stripe rust resistance and potential value of the morphological characteristics for wheat breeding was evaluated. Finally, St-chromosome-speci c markers were developed and validated by speci c-locus ampli ed fragment sequencing (SLAF-seq), which could be useful tools for e cient identi cation of St chromosomes in a common wheat background.
Two Oligonucleotide probes of pTa535 and pSc119.2 were combined for a sequential FISH-GISH to simultaneously examine the elimination of wheat chromosomes in the six substitution lines. Comparisons of the FISH results between substitution lines and the corresponding parent lines, Abbondanza, Zhong4 and Xiaoyan784, were conducted. It was revealed that chromosome 2A was eliminated in ES-9 and substituted by one pair of Th. ponticum chromosomes with three speci c signal bands, including the terminal pTa535 hybridization sites detected on short arms and long arms as well as an interstitial pTa535 signal on the long arms, which was different from the FISH patterns of other wheat chromosomes (Fig. 1,  a1). ES-10 lost chromosome 3D and contained one pair of Th. ponticum chromosomes carrying terminal pSc119.2 hybridization sites on short arms with terminal pTa535 hybridization segments both on the long arms and short arms (Fig. 1, a2). Wheat chromosome 2A, chromosome 2B, as well as wheat chromosome 2D were eliminated in ES-23 ( Fig. 1, a3), ES-25 ( Fig. 1, a5) and ES-26 ( Fig. 1, a6), respectively, and replaced by the same pair of Th. intermedium chromosomes with the identical FISH patterns detected in ES-9. Moreover, the telomeric region of chromosome 5B carrying a bright-green uorescence signal was eliminated in ES-25 compared with other related materials. In terms of ES-24, chromosome 3D was substituted by a pair of Th. intermedium chromosomes with the FISH patterns almost consistent with the alien chromosomes detected in ES-10 ( Fig. 1, a4).
According to the mc-GISH results, each of the six derived lines contained two alien chromosomes carrying a bright-red uorescence signal originating from P. spicata (St) genome DNA (Fig. 1, c1-c6). It was shown that ES-9 and ES-10 carried two different pairs of St chromosomes derived from Th. ponticum. While ES-23, ES-25 and ES-26 contained the same pair of St chromosomes from Th. intermedium which was distinguished from the alien chromosomes of ES-24.

Wheat 15K SNP array analysis of the six substitution lines
The chromosomal composition of the six substitution lines were further determined based on genotype data by using a wheat 15K SNP array (Table S1). Generally, compared with Th. ponticum or Th. intermedium, the number of common SNP sequences detected between the substitution lines and Abbondanza was much higher. However, obvious point of intersection was found in each of the substitution lines ( Fig. 2 a-f). As shown in ES-9 (Fig. 2a), an intersection point was distinctly observed in chromosome 2A, where ES-9 had the most of the same SNP marker loci as Th. ponticum but few SNP marker loci as Abbondanza. It suggested that chromosome 2A in ES-9 were replaced by the alien chromosomes of Th. ponticum, which was consistent with the FISH result. In ES-10 ( Fig. 2b), the intersection point was detected in chromosome 3D where ES-10 had the most same SNP marker loci as Th. ponticum but few SNP marker loci compared with Abbondanza, which suggested that chromosome 3D of ES-10 were substituted by the pair of Th. ponticum chromosomes.
In terms of the wheat-Th. intermedicum alien lines, the intersection point was detected in chromosome 3D of ES-24, and the most same SNP marker loci was obtained from the comparison between Th. intermedium and ES-24. It was meant that chromosome 3D of ES-24 was replaced by the pair of Th. intermedium chromosomes (Fig. 2d). The intersection point of ES-23, ES-25 and ES-26 was undoubtedly identi ed in chromosome 2A (Fig. 2c), chromosome 2B (Fig. 2e), as well as chromosome 2D (Fig. 2f), respectively. Combined with the FISH results, it was revealed that chromosome 2A in ES-23, chromosome 2B in ES-25, as well as chromosome 2D in ES-26 were substituted by the same pair of Th. intermedium chromosomes.
Evaluation of agricultural performance and resistance to stripe rust of the six substitution lines The agronomic traits of the six substitution lines as well as their parents Abbondanza and Xiaoyan784 (  Fig 5) were compared. On average, the tiller number of ES-9 was higher and the spikes exhibited longer than Abbondanza. In terms of the other substitution lines derived from Zhong4, both ES-23 and ES-26 showed much more tillers, and the spikelets per spike number of ES-26 was higher than Abbondanza and Zhong4. Surprisingly, the average thousand-kernel weight of the alien lines containing chromosome 2St (ES-9, ES-23, ES-25 and ES-26) were more than 43g. It was indicated that the chromosome 2St whether originating from Th. ponticum or Th. intermedium increased thousand-kernel weight. Note: Different letters a, b and c indicate significant differences between ES-9, ES-10 and its wheat parent (P < 0.05)   Fig 5c). Furthermore, the seedling stage stripe rust infection was conducted in the greenhouse, and the IT scores were recorded at 24 days post-inoculation (Fig 5d). With an IT score of 0, Zhong4 and Xiaoyan784 were immune to the disease. Additionally, ES-10 and ES-24 were nearly immune (IT score of 1). In contrast, Abbondanza, ES-9, ES-23, ES-25 and ES-26 were susceptible (IT score of 3). The results suggested that chromosome 2St originating from Th. ponticum and Th. intermedium both conferred resistance to stripe rust at the adult stage, while chromosome 3St of Th. ponticum and Th. intermedium both carried remarkable resistance at whole stage.
Meiotic chromosome pairing analysis of F 1 hybrids Crosses were made between the alien lines with the same genome compositions. There were 15 F 1 plants obtained from the cross between ES-9 and ES-23, and 11 F 1 plants obtained from the cross between ES-10 and ES-24. Meiotic chromosome pairing analysis of the F 1 hybrids was conducted to further validate the related genome constitution (Table 3). Mostly, the alien chromosomes derived from Th. intermedium and Th. ponticum but belonging to the same homoeologous group (2/3) could form a bivalent at metaphase I, and no trivalent or quadrivalent was observed at meiosis anaphase I. It was further revealed the close homoeologous relationship between the alien chromosomes derived from the two different donors.

Comparisons of genomic polymorphism analyses and St-chromosomes-speci c molecular markers development
After high-throughput sequencing, SLAF library was constructed with the sequencing details (Supplementary table 3 According to the above sequence alignment results, 110 SLAFs from ES-23 regarded as 2St chromosome-speci c fragments and 73 SLAFs from ES-24 regarded as 3St chromosome-speci c fragments were selected. Subsequently, 183 pairs of primers were designed to amplify fragments from CS, Abbondanza, Zhong4, Xiaoyan784, ES-9, ES-23, ES-10, ES-24. In addition, speci city of the primers was further con rmed by analysis of Th. ponticum, Th. intermedium, tetraploid P. spicata, diploid P. spicata, Th. bessarabicum, Th. elongatum, and the wheat-Th. intermedium 1-7St addition line. Totally, two 2St-chromosome-speci c molecular markers, PTH-005 and PTH-013, and two 3St-chromosome-speci c molecular markers, PTH-113 and PTH-135, were developed (Fig 6, Table 4). Utility of the 3St-chromosome-speci c markers in BC 1 F 2 population In order to validate that the stripe rust resistance gene(s) at whole stage were carried by chromosome 3St, 60 BC 1 F 2 individuals of ES-24 and HXH were used for a genetic analysis. The evaluation of stripe rust resistance at the seedling stage revealed that Zhong4, ES-24, and the 33 F 2 individuals were highly resistant to Pst race CYR32 (Fig 7a). Subsequently, 10 resistant F 2 individuals as well as 10 susceptible ones were randomly selected for FISH analysis. Compared with the FISH karyotype of ES-24, chromosome 3St were detected in all the resistant individuals (Fig 7c) and susceptible ones had undetectable FISH pattern of chromosome 3St (Fig 7b). It was shown that the novel stripe rust resistant gene(s) originated from the alien chromosome 3St of Th. intermedium.
Furthermore, the speci city of PTH-113 and PTH-135 was con rmed by PCR analyses of the 60 BC 1 F 2 individuals (Fig 8). Combined with the result of seedling stage stripe rust resistance evaluation, Xiaoyan784, Zhong4, ES-9, ES-24, and the 33 BC 1 F 2 plants conferring strong resistance to Pst race CYR32 also carried 3St chromosome-speci c markers. Oppositely, the other 26 BC 1 F 2 plants, the parental line Abbondanza and susceptible control HXH, without speci c ampli cation, were seriously susceptible to Pst race CYR32. Hence, the new developed chromosome-speci c molecular markers could be used to rapidly trace the alien chromosome 3St in a common wheat background.

Discussion
On the basis of distant hybridization, chromosome manipulation has been widely utilized for wheat improvement programs, especially for breeding novel disease-resistant wheat lines. During the past few decades, numerous disease-resistant genes carried by wild related species have been successfully transferred to common wheat background by developing introgression lines [32][33][34][35][36][37]. Disomic substitution lines contained one pair of alien chromosomes with desirable resistant genes are vital bridge materials for small segments of introgression [38][39][40], which are valuable germplasm resources for wheat disease-resistant breeding. In the current study, six stable wheat-Thinopyrum derived lines were developed by nullisomic backcross method. Molecular cytogenetic analysis con rmed that ES-23 (DS2St (2A) As one of the most commonly used technique, FISH analysis is generally used with GISH to discriminate characterize the alien chromosomes [41,42] and to detect genomic changes in speci c regions [43][44][45]. In this study, after characterized by sequential FISH-GISH and mc-GISH analysis, speci c karyotype patterns of chromosome 2St and chromosome 3St derived from Th. intermedium and Th. ponticum were elucidated, which is signi cant for rapidly identifying the alien chromosomes in germplasm materials. Furthermore, chromosomal structure variation happened in ES-25 following the process of distant hybridization was detected by FISH. Compared with the parental lines, Abbondanza and Zhong4, telomere with subtelomeric region of chromosome 5BS carrying a blight pSc119.2 hybridization signal was eliminated in ES-25, which resulted a similar FISH pattern to chromosome 2B of common wheat. For chromosome 2B is almost metacentric whereas chromosome 5B is absolutely submetacentric, it was clear that chromosome 2B were replaced by chromosome 2St of Th. intermedium (Fig 4h). Due to the dynamic and high frequent variable nature of subtelomeres of Triticeae species [46,47], it is di cult to access the possible function(s) of the deleted regions of chromosome 5BS. Because of no severe effects on viability of ES-25, the subtelomeric region elimination presumably contributed to genome diversity [48].
The genomic composition of Th. ponticum and Th. intermedium has been an interesting subject for a considerable time [49,50]. During the past several decades, it was convinced that the set of St chromosomes contained in Th. intermedium were probably derived from P. spicata [51], whereas it has been still unde ned that whether the St genome is one of the sets of chromosomes of Th. ponticum [11,52]. According to molecular cytogenetic identi cation results, ES-23 and ES-9 (group 1) contained the same genome composition of 12A + 14B + 14D + 2(2St), while ES-24 and ES-10 (group 2) were for the same genome composition of 14A + 14B + 12D + 2(3St). Although the alien chromosomes derived from the two different donors, Th. intermedium and Th. ponticum, identical alien chromosome FISH patterns and similar speci c agricultural performances were identi ed in each group of the plant materials. It was implied that St chromosomes were included in Th. ponticum, and could be stably inherited with remarkable genes. Furthermore, combined with the close homoeologous relationships between the alien chromosomes analyzed by meiotic chromosome pairing and genomic polymorphism, it was convinced that P. spicata representing the complete set of St chromosomes played an important role during the speciation of Th. ponticum, but the effects of the recombination events happened between diverse genomes through the allopolyploidization process need further analyses. All up, based on the speci c SLAFs obtained in this study, it was feasible for us to develop transferable St-chromosome-speci c molecular markers from Th. intermedium to Th. ponticum.
Although FISH-GISH analysis has been widely utilized to precisely characterized wheat-Th. intermedium lines for several decades, it is severely time-consuming. Speci c molecular markers are able to rapidly trace the alien chromosome or even small segment introgression with the advantage traits for wheat improvement breeding programs. However, for the complete Th. intermedium genome has not been sequenced, there are only a few chromosome-speci c markers enabled to be used [53][54][55]. With the development of sequencing technology, the rst consensus genetic map of Th. intermedium was developed by genotyping-by-sequencing [56]. Subsequently, 635 [9] and 745 [57] unique Th. intermedium SNP markers have been successfully developed, totally including 225 St-chromosome-speci c markers, with 27 of 2St-chromosome-speci c markers and 25 of 3St-chromoosme-speci c markers. Due to the much more complex genomic composition of Th. ponticum, the molecular markers development work was mainly focused on genome E [52,58,59], especially in the following of the published complete genome of Th. elongatum [60]. In the present study, the wheat-Th. intermedium disomic substitution lines, ES-23(DS2St (2A)) and ES-24(DS3St (3D)) were sequenced by SLAF-seq for further St-chromosome-speci c marker development. Two 2St-chromosome-speci c molecular markers, PTH-005 and PTH-013, as well as two 3St-chromosome-speci c molecular markers, PTH-113 and PTH-135 were obtained. FISH analysis of the BC 1 F 2 population of ES-24 and HXH combined with stripe rust resistance test (Fig 7) was to con rm that the stripe rust resistance gene(s) was derived from chromosome 3St of Th. intermedium. The utility of PTH-113 and PTH-135 ampli cation in the BC 1 F 2 individuals indicated that the St-chromosome-speci c molecular markers enabled to serve as useful tools for tracing chromosome 3St of Th. intermedium in common wheat background. In addition, according to the close genetic relationship between the alien chromosomes of Th. ponticum and Th. intermedium analyzed in this study, the four St-chromosome-speci c markers could be simultaneously ampli ed in Th. ponticum, tetraploid P. spicata, Th. intermedium and diploid P. spicata, as well as the corresponding substitution lines, ES-9, ES-23, ES-10 and ES-24. It suggested that the four St-chromosome-speci c markers could also be utilized for rapidly detecting the St genome chromosomes of Th. ponticum. And the remarkable stripe rust resistance of ES-24 and ES-10 probably originated from the same gene(s), which need to be validated in future genetic analyses.

Conclusions
Four wheat-Th. intermedium and two wheat-Th. ponticum disomic substitution lines conferring stripe rust resistance were characterized and compared by molecular cytogenetic analysis, which will be used as bridging parents for valuable resistant genes transmission. Furthermore, according to the related homoeologous relationships, two 2St-chromosome-speci c and two 3St-chromsome-speci c molecular markers were developed by SLAF-seq for rapidly detecting the alien chromosomes of Th. intermedium and Th. ponticum in a common wheat background.

Plant materials
The preserved at the College of Agronomy, Northwest A&F University, China. HXH was served as a susceptible control in the stripe rust resistance evaluation. The Pst races CYR32 were used for seedling stage of stripe rust resistance evaluation, as well as the CYR31 and CYR32 mixture were used for adult stage evaluation. All the Pst races were provided by the College of Plant Protection, Northwest A&F University, China.

In situ hybridization
Chromosome spreads by drop method [14] were used for in situ hybridization analyses. The protocols of genomic DNA extracting, sequential FISH-GISH and mc-GISH were conducted by Wang et al. [41]. According to the nick translation method, total genomic DNA of Th. bessarabicum, Th. intermedium, as well as Th. ponticum was labeled with uorescein-12-dUTP, while St genomic DNA from diploid and tetraploid P. spicata was labeled with Texas Red-5-Dutp, respectively, used as GISH and mc-GISH probes. And the sheared DNA of CS was as a blocking DNA. The Oligonucleotide probes combination of Oligo-pTa535 (red) and Oligo-pSc119.2 (green) were used for FISH analyses. Hybridization signals were observed and acquired under an Olympus BX53 uorescence microscope.

Wheat 15K SNP array analysis
Wheat 15K SNP genotyping arrays were used to genotype the 9 samples, including Abbondanza, ES-9, ES-10, ES-23, ES-24, ES-25, ES-26, Th. ponticum and Th. intermedium, by using Illumina SNP genotyping technology (China Golden Marker Biotechnology Company). There were 13199 SNP loci contained in the wheat 15K array and distributed on all 21 wheat chromosomes. Percentage of the same genotypes in each chromosome between two materials was obtained by calculating the rate of the same genotype loci number in total number of markers. The software Origin (OriginLab, USA) was used for data analysis and graphing.

Agronomic traits and stripe rust resistance evaluation
The stripe rust resistance evaluation was conducted in the eld at the adult stage, while seedling stage test was conducted in the greenhouse. A mixture Pst races of CYR31 and CYR32 was used to evaluated the adult plant resistance of Abbondanza, ES-9, ES-10, ES-23, ES-24, ES-25, ES-26, Xiaoyan784 and Zhong4, with HXH severed as susceptible control. For further genetic analyses of the resistance, Pst races CYR32 was used to inoculate the above-mentioned materials at the seedling stage as well as the BC 1 F 2 population individuals of ES-24 and HXH. The infection type (IT) was scored with a scale of 0-4 [62].
To assess the morphological traits, ten plants

Genomic polymorphism analysis by pairwise comparisons
On the basis of SLAF-seq [63], genomic DNA of Abbondanza, ES-9, ES-10, ES-23, ES-24, Th. intermedium and Th. ponticum was sequenced, carried out by Biomarker Technologies Co. (Beijing, China). The restriction endonuclease, Hae III was selected to digest the genomic DNA. According to the sequence similarity, the ltered SLAF pair-end reads (150 bp per read) were clustered. By using BLAST software, sequences with over 90% identity were divided into one SLAF locus. Genomic polymorphism analyses were conducted in two groups, ES-9 and ES-23 (group 1), as well as ES-10 and ES-24 (group 2). Firstly, all the SLAFs from the two groups were blasted with wheat genome, removing the sequences with high wheat homology (over 80%). Secondly, the remaining SLAFs were further blasted with the sequences of Th. ponticum or Th. intermedium. Then the SLAFs with high identity (over 90%) were remained, served as speci c sequences of Th. ponticum attributing to ES-9 and ES-10, as well as the speci c sequences of Th. intermedium attributing to ES-23 and ES-24. Finally, intercomparisons within groups were conducted and the speci c SLAFs with high identity (over 90%) were acquired.

Development and validation of the St-chromosome-speci c markers
Based on the obtained speci c SLAFs, PCR primers were designed for the ampli cation of the two groups of materials. All the primers were designed by using the online tool (Primer3 Plus, http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and synthesized by AuGCT DNA-SYN Biotechnology Co. (Beijing, China). The ampli ed products were examined by using 2% agarose gel electrophoresis. The markers ampli cated speci c sequences in tetraploid P. spicata, diploid P. spicata, Th. ponticum, Th. intermedium, DA2St, ES-9 and ES-23, but not in CS, Abbondanza, Th. bessarabicum, Th. elongatum, the 1St and 3-7St addition lines, were served as 2St-chromosomes-speci c molecular markers. While the markers presented in ES-10, ES-24, whereas absent in the 1-2St and 4-7St addition lines, were served as 3St-chromosomes-speci c molecular markers. Subsequently, the 3St-chromosomes-speci c markers were utilized in BC 1 F 2 individuals of ES-24 and HXH for a further genetic analysis.

Con icts of interest
It should be understood that none of the authors have any nancial or scienti c con icts of interest with regard to the research described in this manuscript.

Ethics approval
We hereby con rm that this manuscript is our original work and has not been published nor has it been submitted simultaneously elsewhere.

Consent to participate and consent for publication
We further con rm that all authors have checked the manuscript and have agreed to the submission.
Availability of data and material The datasets used or materials during the current study are available from the corresponding author on reasonable request.

Code availability
The codes during the current study are available from the corresponding author on reasonable request.