Molecular Cytogenetics of Chromosome 2St as Well as Chromosome 3St Derived from Thinopyrum Intermedium and Thinopyrum Ponticum

Owing to the excellent resistance to abiotic and biotic stress, Thionpyrum intermedium (2n = 6x = 42, JJJ s J s StSt) 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. In this study, six wheat-Thinopyrum DSLs were derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), characterized by a sequential uorescence 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 and ES-10 were two wheat- Th. ponticum disomic substitution lines, DS2St (2A) and DS3St (3D). While ES-23, ES-24, ES-25, and ES-26 were four wheat- Th. intermedium disomic substitution lines, DS2St (2A), DS3St (3D), DS2St (2B), DS2St (2D). The FISH karyotypes of Th. ponticum 2St/3St chromosomes were well coincident with the ones of Th. intermedium. The chromosome congurations of F 1 hybrids derived from crosses between ES-23 and ES-9, as well as ES-24 and ES-10 were mostly formed 21 (cid:0) . Four St-chromosome-specic markers were developed by specic-locus amplied fragment sequencing (SLAF-seq). Additionally, the substitution lines containing chromosome 2St conferred higher thousand-kernel weight and stripe rust resistance at adult stages, while the substitution lines containing chromosome 3St were highly resistant to stripe rust at all stages. Therefore, these six substitution lines could serve as useful bridging parents for wheat genetic improvement.


Introduction
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 (Chen et al. 2003;Li et al. 2008). According to previous studies, neither the chromosomal composition of Th. intermedium nor Th. ponticum has been fully characterized. In terms of Th. intermedium, the chromosomal composition is generally regarded as JJJ s J s StSt (Chen et al. 1998)  Although there are aspects of Th. intermedium genome and Th. ponticum genome remain undiscovered, numerous partial amphiploid lines have been successfully developed during the past decades (Fedak et  Furthermore, octoploid Trititrigia lines mostly carry a synthetic genome inherited from Th. intermedium or Th. ponticum. According to the de ned genome composition of partial amphiploids by molecular cytogenetic mothed, to some degree, it is possible to understand the chromosomal compositions of Thinopyrum allopolyploids. TAF46 is an important wheat-Th. intermedium partial amphiploid with a common wheat Vilmorin 27 background, and the six disomic addition lines L1, L2, L3, L4, L5 with L7 were developed from TAF46 (Figueiras et al. 1986). Subsequently, molecular cytogenetic identi cation of TAF46 as well as the derived six addition lines revealed that the genome composition of TAF46 is 14A + 14B + 14D + 2(1J) + 2(2St) + 2(3J) + 2(4St) + 2(5J) + 2(6St) + 2(7J) (Friebe et al. 1992; Chen et al. 1999;Forster et al. 2009). It is suggested that chromosomes of St genome contained in Th. intermedium could be stably inherited. It is feasible to introduce the St chromosomes into common wheat background for wheat genetic improvement.
Stripe rust (Puccinia striiformis f.sp. tritici, Pst) is a recurrent damaging disease causes serious yields decrease of wheat annually (Chen 2005). 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. intermedium have yet to be constructed, which limits the improvement of germplasm materials identi cation e ciency. Based on molecular cytogenetic identi cation of wheat-Th. intermedium DALs or DSLs, the St chromosome FISH karyotypes are able to be established. However, at present no wheat-Th. intermedium 2St disomic substitution lines or 2St chromosome FISH karyotypes 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) by the same procedure. Molecular cytogenetic analysis was to determine and compare the genome composition of the six alien lines, and two 2St-chromosome-speci c markers and two 3St-chromosome-speci c markers were developed by SLAF-sEq. Disease evaluation results indicated that the alien lines containing Thinopyrum chromosome 2St (ES-9, ES-23, ES-25 and ES-26) conferred high level stripe rust resistance at adult stages, while alien lines containing Thinopyrum chromosome 3St (ES-10 and ES-24) are highly resistant to stripe rust at all stages. In addition, potential value of the morphological characteristics for wheat breeding was evaluated.
In situ hybridization Chromosome spreads by drop method (Han et al. 2004) were used for in situ hybridization analyses. The protocols of genomic DNA extracting and sequential FISH-GISH as well as mc-GISH were conducted by Wang et al. (Wang et al. 2019). 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. The calculation of the percentage of the same genotype between two materials in each chromosome was carried out as the total number of markers divided by the loci number of the same genotypes. The software Origin (OriginLab, USA) was used for data analysis and graphing.

Stripe rust resistance and agronomic traits 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, Xiaoyan 784 and Zhong 4, 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 Meiotic chromosome pairing analysis of the F 1 hybrids Young spikes of F 1 hybrids derived from the two crosses combinations (ES-9×ES-23 as well as ES-10×ES-24) at propriate stage were extracted at the suitable temperature under eld conditions, and immediately treated with Carnoy's xative uid II (6:3:1 ethanol-chloroform-glacial acetic acid solution). Before cytological observation of pollen mother cells, anthers were extracted and stained with 1% acetocarmine.
The chromosome con gurations in the miosis period were observed, recorded and photographed.

Genomic polymorphism analysis by pairwise comparisons
On the basis of SLAF-seq (Sun et al. 2013), 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 by intercomparisons between ES-9 and ES-23, as well as ES-10 and ES-24. Firstly, all the SLAFs from ES-9, ES-10, ES-23 and ES-24 were blasted with wheat genome, removing the sequences with high wheat homology (over 80%). Secondly, the remaining SLAFs of ES-9 and ES-10 were further blasted with the sequences of Th. ponticum, while the SLAFs of ES-23 and ES-24 were blasted with Th. intermedium. Then the SLAFs with high identity (over 90%) of each material were remained, which were 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, pairwise comparisons were conducted and the respective speci c SLAFs with high identity (over 90%) were acquired.

Development and validation of the St-chromosome-speci c markers
Based on the respective speci c SLAFs obtained from the intercomparisons, PCR primers were designed for the ampli cation of the two groups of materials (ES-9 and ES-23 as well as ES-10 and ES-24). All the primers were designed by using the online tool (Primer3 Plus, http://www.bioinformatics.nl/cgibin/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 Th. ponticum, tetraploid P. spicata, Th. intermedium, diploid P. spicata, 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-chromosomesspeci c molecular markers. Subsequently, the 3St-chromosomes-speci c markers were utilized in BC 1 F 2 individuals of ES-24 and HXH for speci city validation.
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. Pairwise comparisons for 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 on the long arms and short arms ( Fig. 1, a2). Wheat chromosome 2A, chromosome 2B, and 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 of the alien chromosomes presenting 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).
In addition, 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 ( Wheat 15K SNP array analysis of the six substitution lines The chromosomal composition of the six substitution lines were determined based on genotype data by using a wheat 15K SNP array (Table S1-6). Generally, the common SNP sequences detected between the substitution lines and the same wheat parent line Abbondanza were much higher than between the substitution lines and Th. ponticum or Th. intermedium. 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. According to the same genotype SNP loci number in chromosome 2A, ES-9 contained more of the same genotype SNP loci as Th. ponticum rather than Abbondanza. It suggested that chromosome 2A in ES-9 were replaced by the pair of Th. ponticum chromosome, 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 of the same SNP marker loci as Th. ponticum but few SNP marker loci compared with Abbondanza, which was consistent with the FISH result, suggesting that chromosome 3D of ES-10 were substituted by the pair of Th. ponticum chromosomes. While in ES-24, the intersection point was also detected in chromosome 3D, but the most of the same SNP marker loci was obtained from the comparison between Th. intermedium and ES-24, which meant that chromosome 3D of ES-24 was replaced by the pair of Th. intermedium chromosomes (Fig. 2d). It was consistent with the FISH analysis of ES-24. In terms of ES-23, ES-25 and ES-26, the intersection point of each material was undoubtedly identi ed in chromosome 2A (Fig. 2c), chromosome 2B (Fig. 2e), as well as chromosome 2D (Fig. 2f). 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 resistance to stripe rust and agricultural performance of the six substitution lines The agronomic traits of the six substitution lines as well as their parents Abbondanza and Xiaoyan784 (Table 1, Fig 5) or Zhong4 (Table 2, Fig 5) were compared. On average, the tiller number of ES-9 was higher and the spikes exhibited longer than those of 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 that of Abbondanza as well as 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.
At the adult stage, stripe rust reaction test of the six substitution lines was conducted by comparisons with the susceptible control (HXH). Sequentially, the IT score of the six substitution lines, Abbondanza, Xiaoyan784, Zhong4, as well as Th. ponticum and Th. intermedium were recorded under eld conditions. The IT score of the above-mentioned materials were as follows: Meiotic chromosome pairing analysis of F 1 hybrids Based on molecular cytogenetic identi cation of the six substitution lines, crosses were made between the alien lines with the same genome compositions, respectively. 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). More than half of the pollen mother cells (PMCs) of ES-9×ES-23 and ES-10×ES-24 had 21 bivalents at metaphase I, and there was no trivalents or quadrivalents, as well as lagging chromosomes observed at meiosis anaphase I. It was indicated that chromosome 2St originating from Th. ponticum and Th. intermedium exhibited the close homologous relationship between each other, so did the Thinopyrum chromosome 3St.

Pairwise comparisons of genomic polymorphism analyses and Stchromosomes-speci c molecular markers development
After high-throughput sequencing, SLAF library was constructed with the sequencing details (Supplementary table 8 were selected for further sequence alignments. There were 78 out of 263 sequences from ES-24 with homology more than 90% of ES-10 (78/153). In addition, 114 out of 221 sequences from ES-23 were more than 90% homologous with ES-9 (114/177). To some degree, these results revealed the possible genomic similarity between chromosome 2St/3St of Th. intermedium and Th. ponticum.

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) were carried by chromosome 3St, 60 BC 1 F 2 individuals of ES-24 and HXH were further used for a genetic analysis. The evaluation of stripe rust resistance revealed that Zhong4, ES-24, and the 33 F 2 individuals were highly resistant to Pst race CYR32 at the seedling stage (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 actually detected in the resistant individuals (Fig 7b) and susceptible ones had undetectable FISH pattern of chromosome 3St (Fig 7b). It was indicated that the novel stripe rust resistant gene(s) originated from the chromosome 3St of Th. intermedium. Furthermore, the speci city of newly developed 3St-chromosome-speci c molecular markers was con rmed by PCR analyses of the 60 BC 1 F 2 individuals of ES-24 and HXH (Fig 8). Combined with the result of seedling stage stripe rust resistance evaluation, it was revealed that Xiaoyan784, Zhong4, ES-9, ES-24, and the 33 BC 1 F 2 plants conferring strong resistance to Pst race CYR32 carried 3St chromosomespeci c markers. Oppositely, the other 26 BC 1 F 2 plants without speci c ampli cation as well as the parental line Abbondanza, and susceptible control HXH were seriously susceptible to Pst race CYR32. It was indicated that the newly developed St-chromosome-speci c molecular markers could be used to trace the 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 contained in wild related species have been successfully transferred to common wheat background by developing introgression lines ( , which could be accurately 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 in ES-25 were replaced by chromosome 2St of Th. intermedium (Fig. 4h). Subtelomeres of Triticeae species were regarded as dynamic and relatively high frequent variable genome organization with constant homogenization between different chromosome ends (Zhang et al. 2004). Additionally, subtelomere regions of Schizosaccharomyces pombe showed high sequence variation, but no severe effects on the RNA expression (Oizumi et al. 2021). In terms of the deletion of subtelomeric region of chromosome 5BS in ES-25, it is di cult to access the possible function(s) of the regions for the variable nature. The segment elimination may have been resulted from chromosomal rearrangement via the process of chromosome 2St introduction. There were no severe effects detected on viability of ES-25, which suggested that the subtelomeric region eliminations of chromosome 5BS presumably contributed to genome diversity.
Based on molecular cytogenetic identi cation results of the six substitution lines, ES-23 and ES-9 contained the same genome composition of 12A + 14B + 14D + 2(2St), and ES-24 as well as ES-10 were for the same genome composition of 14A + 14B + 12D + 2(3St). It is surprised that FISH patterns of Th. intermedium 2St/3St chromosomes and Th. ponticum are consistent with each other. What else, the agricultural performance evaluation suggested that chromosome 2St derived from Th. intermedium and Th. ponticum both conferred higher thousand-kernel weight, more tillers and stripe rust resistance at adult stages. And chromosome 3St of Th. ponticum and Th. intermedium were both highly resistant to stripe rust at all stages. Because of the same genome compositions, consistent FISH patterns and the similar speci c agricultural performances, comparisons between ES-23 and ES-9, as well as ES-24 and ES-10, were further conducted. As one of the most traditional methods, meiotic chromosome pairing analysis of species hybrids has been used to study Triticeae species genome constitution for several decades (Lu and Vonbothmer 1993;Yang et al. 2015). In terms of the two groups of germplasm materials with the same genome compositions, more than half of the pollen mother cells of the F 1 hybrids perfectly formed 21 bivalents at metaphase I without any trivalents or quadrivalents, which revealed the close homologous relationship between Th. ponticum chromosome 2St/3St and Th. intermedium chromosome 2St/3St, respectively. Furthermore, genomic polymorphism intercomparisons between ES-23 and ES-9, as well as ES-24 and ES-10, were analyzed by SLAF-sEq. According to the sequence alignment results, 114 speci c sequences of 221 (ES-23) and 177 (ES-9), as well as 78 speci c sequences of 263 (ES-24) and 153 (ES-10) were with homology more than 90%. Overall, it was suggested the possible genomic similarity between chromosome 2St/3St of Th. intermedium and Th. ponticum.
The genomic composition of Th. ponticum and Th. intermedium has been an interesting subject for a considerable time (Wang 1992;Tiryaki et al. 2021). During the past several decades, it was convinced that the set of St chromosomes contained in Th. intermedium were probably derived from P. spicata, whereas it has been still unde ned that whether the St genome is one of the sets of chromosomes of Th. ponticum or not (Kruppa and Molnár-Láng 2016). In this study, Th. ponticum chromosome 2St/3St and Th. intermedium chromosome 2St/3St were simultaneously identi ed in the six alien substitution lines derived from two different octoploid Trititrigia lines, Xiaoyan784 (a wheat-Th. ponticum partial amphiploid) and Zhong4 (a wheat-Th. intermedium partial amphiploid). In terms of the FISH patterns of chromosome 2St/3St, no obvious variations were detected in Xiaoyan784, Zhong4 and the six substitution lines (Fig. 4h). It was implied that St chromosomes were not only included in Th. ponticum, but also could be stably inherited. Furthermore, combined with the previous results of the close homologous relationship between Th. ponticum chromosome 2St/3St and that of Th. intermedium, it is 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.
Although FISH-GISH analysis has been widely utilized to precisely characterized wheat-Th. intermedium lines for several decades, it is 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 (Zhang et (Wang et al. 2020a). In the present study, four wheat-Th. intermedium disomic substitution lines were clearly characterized, of which, ES-23(DS2St (2A)) and ES-24(DS3St (3D)) were further sequenced by SLAF-seq for St-chromosome-speci c marker development.
And two 2St-chromosome-speci c molecular markers, PTH-005 and PTH-013, as well as two 3Stchromosome-speci c molecular markers, PTH-113 and PTH-135 were obtained. The FISH analysis results showed chromosome 3St were merely detected in the resistant individuals of the BC 1 F 2 population of ES-24 and HXH (Fig. 7), which meant 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 the St chromosomes of Th. intermedium in common wheat background. In addition, according to the close genetic relationship between Th. ponticum chromosome 2St/3St and that of 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 (Fig. 4h). It was speculated that the four St-chromosome-speci c markers could also be utilized for tracing the St genome chromosomes of Th. ponticum, which need to be validated in future genetic analyses.  Tables   Table 1 Agronomic traits of the alien substitution lines ES-9