Development of Sequence-Tagged Site Marker Set for Identification of J, JS, and St Sub-genomes of Thinopyrum intermedium in Wheat Background

Thinopyrum intermedium (2n = 6x = 42, JJJSJSStSt) is one of the important resources for the wheat improvement. So far, a few Th. intermedium (Thi)-specific molecular markers have been reported, but the number is far from enough to meet the need of identifying alien fragments in wheat-Th. intermedium hybrids. In this study, 5,877,409 contigs were assembled using the Th. intermedium genotyping-by-sequencing (GBS) data. We obtained 5,452 non-redundant contigs containing mapped Thi-GBS markers with less than 20% similarity to the wheat genome and developed 2,019 sequence-tagged site (STS) molecular markers. Among the markers designed, 745 Thi-specific markers with amplification products in Th. intermedium but not in eight wheat landraces were further selected. The distribution of these markers in different homologous groups of Th. intermedium varied from 47 (7/12/28 on 6J/6St/6JS) to 183 (54/62/67 on 7J/7St/7JS). Furthermore, the effectiveness of these Thi-specific markers was verified using wheat-Th. intermedium partial amphidiploids, addition lines, substitution lines, and translocation lines. Markers developed in this study provide a convenient, rapid, reliable, and economical method for identifying Th. intermedium chromosomes in wheat. In addition, this set of Thi-specific markers can also be used to estimate genetic and physical locations of Th. intermedium chromatin in the introgression lines, thus providing valuable information for follow-up studies such as alien gene mining.


INTRODUCTION
Thinopyrum intermedium (Host) Barkworth & D.R. Dewey (2n = 6x = 42, JJJ S J S StSt) belongs to the tribe Triticeae, which is a perennial cross-pollinated species and cultivated as a forage grass worldwide (Vogel and Jensen, 2001). It is also an ideal species for water and soil conservation and saline-alkali land improvement (Li and Wang, 2009). It is generally believed that the Th. intermedium J sub-genome is partially homologous to the genomes of Th. bessarabicum (2n = 2x = 14, J b J b ) and Th. elongatum (2n = 2x = 14, J e J e ), the St sub-genome is contributed by Pseudoroegneria spicata (2n = 2x = 14, StSt), whereas the J S sub-genome is derived from the J sub-genome partially recombined with the St genome (Chen et al., 1998;Mahelka et al., 2013).
However, the position of the introgressed alien fragments in the Th. intermedium genome cannot be determined.
It is particularly important to develop specific markers directly based on the Th. intermedium sequences. In 2016, Kantarski et al. (2017) explored genotyping-by-sequencing (GBS) markers in Th. intermedium and constructed the first consensus genetic map containing all Th. intermedium linkage groups (Thi-LG1∼21) using seven genetic populations. However, the sub-genome information corresponding to each Thi-LG remains unknown. Subsequently, Wang R. R. C. et al. (2020) compared the GBS sequences of Ps. spicata with the previously released Thi-GBS sequences and identified 4,8,11,13,17, and 21 as the St sub-genome. In this study, the above-mentioned Thi-GBS sequences were compared with the annotated coding sequence (CDS) data of Th. elongatum published recently  to distinguish the J and J S sub-genomes in Thi-LGs. Then, contigs assembled with the original Thi-GBS sequences were selected to develop sequence-tagged site (STS) markers. The Thi-specific markers that have amplification products in Th. intermedium but not in common wheat were identified, thereby providing an economical and convenient tool for identifying Th. intermedium fragments in wheat.

Plant Materials
Six independent plants from the same Th. intermedium accession (in order to avoid the individual differences caused by crosspollination) and eight wheat landraces (in order to avoid the possibility that wheat cultivars may contain alien species fragments such as 1B/1R, which will affect the screening results) from different ecological regions in China were used to screen the Thi-specific markers. Th. elongatum, Th. bessarabicum, Ps. Spicata, and D. villosum were used as the related species of Th. intermedium to detect the amplification of these Thispecific markers. Wheat-Th. intermedium partial amphidiploids, addition lines, substitution lines, and translocation lines were used to test the effectiveness of the Thi-specific markers. Materials used in this study and their relevant information including name, genome composition, and providers are listed in Table 1.

Informatics Analysis of Thi-GBSs
The method used to distinguish sub-genomes in Thi-LGs was described by Wang R. R. C. et al. (2020). The 10,029 Thi-GBS sequences mapped to Thi-LG1∼21 (Kantarski et al., 2017) were aligned with the annotated CDSs of Th. elongatum (accession number GWHABKY00000000, version 1.0)  obtained from the National Geophysical Data Center database (NGDC, https://bigd.big.ac.cn/) with BLAST tool (version 2.6.0+), setting E ≤ 1.0 × 10 −25 . For the Thi-GBS sequences with multiple hits, the hit with the lowest evalue was selected for further analysis. In the same HG of Th. intermedium, the Thi-LG with the most matched The-CDSs was presumed to be the J sub-genome. For the number of significant hits, a Chi-squared test was performed with the Bonferroni adjustment for multiple tests to determine if observed values were significantly different.  Forster et al., 1987;Friebe et al., 1992;Chen et al., 1999 56 ABD+1St+2J S +3J+4J+4J S +5J S +6St+7St TE-3 d Yang et al., 2006;Hu et al., 2011;Song et al., 2013;Li et al., 2015Li et al., , 2017
language (Han et al., 2015) was used for a large-scale primer design, and the parameters were set as following: primer length was 18-22 bp, and the product length was 100-400 bp.

Screening and Validation of the Thi-Specific Markers
The developed STS markers were tested on six Th. intermedium individuals and eight wheat landraces, and those that can amplify in Th. intermedium but not in wheat were selected as the Thi-specific markers. These markers were then used on wheat-Th. intermedium partial amphiploids, addition lines, and substitution lines to verify their effectiveness. In addition, the amplification results of the Thi-specific markers in Th. bessarabicum, Th. elongatum, Ps. spicata, and D. villosum were visualized by the Venn diagram (http://bioinformatics. psb.ugent.be/webtools/Venn/) and were subjected to the phylogenetic analysis using MEGA6.0 3 (Tamura et al., 2013) with the neighbor-joining method and 1,000 bootstraps. A physical location of Thi-specific markers was obtained by blasting against the genome data of Th. intermedium (version 2.1, http://phytozome.jgi.doe.gov/). PCR was performed in 10 µl reaction using PCR Mix (B532061, Sangon Biotech, Shanghai, China). Amplified products were electrophoresed in 8% non-denaturing polyacrylamide gels and then stained in a 0.1% silver nitrate solution.
After stripping off the oligo probes, the same slides were analyzed by GISH as described in Zhang et al. (2001). Total genomic DNA from Th. intermedium (Cytogenetic stock accession C05.05, University of Sydney) was labeled with biotin-16-dUTP (Roche Diagnostics Australia, Castle Hill, NSW, Australia) using nick translation. Unlabeled total genomic DNA of wheat was used as a blocker. The probe to blocker ratio was ∼1:80. Signals were detected with Fluorescein Avidin DN (Vector Laboratories, Burlingame, CA, USA). Chromosomes were counterstained with DAPI and pseudo-colored red.

Development of Thi-Specific Markers
A total of 5,877,409 contigs were assembled using the original Thi-GBS sequences, ranging in length from 100 to 3,094 bp, with a total length of 915,311,073 bp (Supplementary Table 2). After removing the redundancy, 5,452 contigs containing the mapped Thi-GBS markers (Kantarski et al., 2017) were identified. In total, 2,019 STS markers were developed for the 5,452 non-redundant contigs, with 250,215,323,253,323,253, and 402 markers distributed in the Thi-HG1 to HG7, respectively (Figure 1).

Evaluation of Thi-Specific Markers Using Wheat-Th. intermedium Lines
The Thi-specific markers were used to amplify two wheat-Th.

Prediction of the Positions in of Alien Segments Th. intermedium by Thi-Specific Markers
The Thi-specific markers were used to predict the positions of Th. intermedium chromatin in T1332, a translocation line introduced segment of the long arm of Thi-chromosome 4J (Figures 5A-C). In order to improve the chromosome specificity of markers, 82 Thi-specific markers of Thi-HG4 were used on  the substitution line X24C10 with Thi-chromosome 4J (4B) (Li et al., 2017) and the 4St addition line L4 (Forster et al., 1987;Chen et al., 1999). Combined with the previous identification results in the substitution line A1125 4J S (4B) ( Figure 3F) and two partial amphiploids TAF46 and TE-3 (Figures 3A,B), 58 (71%) Thi-chromosome-specific markers were identified, of which 15 were 4J-specific, 27 were 4St-specific, and 16 were 4J Sspecific ( Figure 5D). Among the 58 Thi-chromosome specific markers in T1332 (ABD+T4BS/4JL) showed that four 4J-specific markers C10-32, C10-49, C10-54, and C10-63 amplified target products. According to the physical location of these markers, it could be inferred that the introduced fragment contained the chromosome interval 4J:351604953-480594047Mb of Th. intermedium (Figure 5E).

Amplification of Thi-Specific Markers in the Je/Jb/St/V Genomes
The amplification results of Thi-specific markers showed that 107 (14%), 62 (8%), 233 (31%), and 116 (16%) markers could be amplified in Th. elongatum, Th. bessarabicum, Ps. spicata, and D. villosum, respectively. Among them, the markers located in the J and St sub-genomes were amplified the most in Ps. spicata, 22 and 44%, respectively, whereas markers in the J S sub-genome were amplified the most in D. villosum (29%) (Figure 6A). Similarly, the phylogenetic analysis showed that the J and St sub-genomes were closely related to Ps. spicata, whereas the J S sub-genome is relatively close to D. villosum ( Figure 6A). The number of Thi-specific markers that specifically amplify in Th. elongatum, Th. bessarabicum, Ps. spicata, and D. villosum were 50, 20, 141, and 59, respectively, whereas 366 markers were not amplified in the above species ( Figure 6B).

DISCUSSION
Thinopyrum intermedium is one of the important resources for the wheat improvement. In this study, 2,019 STS markers distributed on 21 Thi-chromosomes were developed based on the Thi-GBS sequences and used to amplify from Th. intermedium and eight wheat landraces from different ecological regions in China. Many species polymorphisms, including the presence or absence or the length difference of amplicons, were obtained. In order to identify the Thi-specific fragments in the wheat background more accurately, 745 Thi-specific markers with amplicons in Th. intermedium but not in wheat were screened. Due to the homology among the three sub-genomes J/J S /St of Th. intermedium, the developed Thi-specific markers are not exclusively specific to the corresponding sub-genome within the same HG. Using wheat-Th. intermedium introgression lines, 58 out of 82 (71%) Thi-specific markers in Thi-HG4 were identified. However, due to the lack of materials with single Thi-sub-genome introgressed,  the Thi-specific markers in the remaining Thi-HGs were not identified. We used this set of markers to accurately identify the alien chromosomes derived from different Thi-HGs in the wheat-Th. intermedium addition, substitution, and translocation lines. Chromosome 1St, 2J S , 3J, 4J, and 7J have been reported to carry genes for stripe rust resistance (Hu et al., 2011;Li et al., 2017Li et al., , 2019Lang et al., 2018). In addition, chromosome 4J also carries genes related to dwarf, tillering, and blue grain (Li et al., 2017). We will then identify whether the introgression lines have obtained beneficial agronomic traits from Th. intermedium and use them to develop small-fragment translocation lines. The Thi-specific markers will be used to track alien fragments and determine the approximate chromosomal location of the target alien gene.
The set of Thi-specific markers developed in this study can be used to identify not only Th. intermedium chromosomes in the wheat background, but also the alien chromosomes from other Triticeae species with J and St genomes, such as Th. ponticum (2n = 10x = 70, JJJJ S J S /E e E b E x StSt) (Zhang et al., 1996(Zhang et al., , 2001Chen et al., 1998). Some Thi-specific markers can amplify species-specific bands in Th. elongatum, Th. bessarabicum, Ps. spicata, and D. villosum. Therefore, these markers can also be suitable for the identification of the alien chromosomes from the above species in the wheat background.
There are several advantages of this set of markers identified in this study. First, they are PCR-based markers, which are easy to use and cost-effective. Second, this set of markers, covering all Th. intermedium chromosomes, are developed based on the GBS markers from the published Th. intermedium genetic map, so each Thi-specific marker has a corresponding map location. Third, they can be used for the chromosome identification after further screening, whereas the current SNP chip and KASP chip cannot accurately identify the J and J S sub-genomes, which is due to the high similarity between the J and J S sub-genomes and the characteristic duality of SNP (Cseh et al., 2019;Grewal et al., 2020). Fourth, the physical positions of Thi-specific markers in Th. intermedium can be determined according to their contigs, so the sequence of small alien fragment in wheat-Th. intermedium translocations can be inferred, which can provide valuable information for further identification of small alien fragments, and even for the cloning of alien genes.
However, this set of markers also has some limitations. For substitution lines and translocation lines, it is impossible to identify which wheat chromosomes have been replaced or translocated onto. Therefore, cytological techniques or wheat  chromosome-specific markers are needed for the identification. In addition, the distributions of Thi-specific markers on certain chromosomes are insufficient (such as chromosomes 2J S , 6J, and 6St, Figure 2) or uneven (such as chromosomes 1J and 2St, Figure 2). Thus, the alien Thi-segments that are not covered by markers cannot be detected. Furthermore, for wheat varieties with complex genetic backgrounds, especially containing multiple alien fragments, the accuracy of this set of markers will be affected.
Due to cross-pollination, genetic exchange between Th. intermedium and other species may occur, resulting in a complex evolutionary process and genome composition of Th. intermedium. Since 1936, several genome constitutions of Th. intermedium, such as AXY (Peto, 1936), BEF (Stebbins and Pun, 1953), B 2 X 1 X 2 (Dewey, 1984), J r J vs St (Cseh et al., 2019), have been proposed. At present, it is generally believed that the genome constitution of Th. intermedium should be JJ S St based on the GISH results with the St, J, and E genomic DNA probes, among which the St sub-genome is thought to derive from Ps. spicata, whereas the origins of J and J S sub-genomes are still uncertain (Chen et al., 1998;Mahelka et al., 2013). Studies have shown that these sub-genomes are partial homology with Th. elongatum, Th. bessarabicum, and D. villosum, in which the J S sub-genome is also partially recombined with the St genome (Chen et al., 1998;Mahelka et al., 2011Mahelka et al., , 2013. In this study, 44% of the markers located in the St sub-genome were positive in Ps. spicata, which was much higher than that in Th. elongatum (16%), Th. bessarabicum (7%), and D. villosum (10%), indicating that Thi-St genome has good homology with Ps. spicata genome. The markers of J S sub-genome have a high amplification percentage in D. villosum (29%) and Ps. spicata (25%), which is consistent with the reported GISH results (Mahelka et al., 2011). However, there were also many positive markers from the J sub-genome in Ps. spicata (22%), indicating the complexity of the origin of J sub-genome. In Th. elongatum, the percentage of positive Thi-J markers (17%) was higher than that of the positive Thi-J S markers (9%), which also confirms the feasibility of using The-CDSs to distinguish the J sub-genome from the 21 Thi-LGs.
As many as 366 (49%) of Thi-specific markers failed to amplify in Th. elongatum, Th. bessarabicum, Ps. spicata, and D. villosum, indicating that the Th. intermedium genome has undergone extensive recombination and gradually evolved into a new species after polyploidization by natural hybridization, which is a common phenomenon in nature (Hegarty and Hiscock, 2005). Moreover, there is a possibility that except for the above four species, there may be other species involved in the evolution of Th. intermedium, such as Aegilops tauschii (D genome) and Taeniatherum (Ta genome) (Mahelka et al., 2011). Therefore, the negative Thi-specific markers can be applied to other Triticeae species, which may be able to discover species close to the Th. intermedium genome or involved in the evolution of Th. intermedium.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
ZYa and LQ designed the experiments. LQ developed the STS markers. ZYa and PZ provided the wheat-Th. intermedium introgression lines. SLiu, SLi, and JLiu performed the PCR experiments. JLi, ZYu, and PZ conducted the GISH and FISH experiments. CL, XL, and YR helped with data analysis. LQ and JLi wrote the manuscript. PZ, ZYa, XZ, and ZC revised the manuscript. All authors contributed to the article and approved the submitted version.