Generation and identification of a wheat–Agropyron cristatum (L.) Gaertn. 3P chromosome addition line and substitution line

Agropyron cristatum (L.) Gaertn. (2n = 4x = 28, PPPP), which is a wild relative of common wheat and has a P genome, has many excellent characteristics (for example, it is disease-resistant, stress-resistant, and high yielding) and has important utilization values for the genetic improvement of wheat. In this study, the wheat–A. cristatum 3P addition line I-27 and 3P(3B) substitution line I-15 were identified for the first time via in situ hybridization (ISH) and specific molecular marker (EST-STS) from wheat–A. cristatum-derived lines, which are a representative and important material for the utilization of the excellent genes from the A. cristatum chromosome 3P for wheat genetic improvement. In terms of agronomic traits, the tiller number and spike length of the 3P addition line I-27 were significantly higher than those of Fukuhokomugi. The spike length and spikelet number per spike of the 3P substitution line I-15 were significantly greater than those of Fukuho. Additionally, the wheat–A. cristatum 3P substitution line I-15 was resistant to wheat leaf rust. The generation of the 3P addition line I-27 and the 3P substitution line I-15 was essential for the transfer of the excellent genes of the A. cristatum 3P chromosome into common wheat, and resulted in the wheat–A. cristatum addition line containing a complete set of genetic research materials, laying the foundation for comparative studies of the P genome and subsequent fine mapping.


Introduction
Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most important crop species, and is widely grown worldwide. Moreover, wheat plays an important role in grain production in China and worldwide. In recent years, the demand for wheat has grown throughout the world (Hunter et al. 2017). Owing to the large-scale cultivation of wheat cultivars, the genetic basis of wheat cultivars is becoming increasingly narrow, and the improvement of wheat cultivars is limited . Wild relatives of common wheat serve as excellent gene libraries for wheat genetic improvement. Introducing excellent genes into common cultivated wheat could constitute an effective strategy for broadening the genetic basis of wheat and for wheat genetic improvement.
Wild relatives of wheat, especially tertiary gene sources, have a large number of excellent genes, such as those that provide strong tolerance to biotic and abiotic stresses, and thus are important materials for the genetic improvement of common wheat Zhou et al. 2020). Many wild relatives of wheat have been successfully crossed with wheat, and a large number of alien addition lines and substitution lines have been obtained (Chapman and Riley 1955;Friebe et al.1992;Chen et al.1995Chen et al. , 2005Li et al. 1997Li et al. , 1998Schneider et al. 2005;Li and Wang 2009). Therefore, exogenous genes from wheat relatives can be introduced into common wheat via distant hybridization and chromosome engineering, realizing the genetic improvement of wheat. With multiple spikelets and resistance to powdery mildew, the wheat-rye 1R addition line was obtained from a cross of Shaanmai 611 and Austrian rye (Yang et al. 2016). Zhang et al. (2021) obtained a new wheat variety with resistance to powdery mildew through a wheat-Dasypyrum villosum 1 V(1D) substitution line. Yang et al. (2017) demonstrated that homoeologous group 3 chromosomes of Leymus mollis contain a new stripe rust-resistant gene according to different wheat-L. mollis double monosomic addition lines. A major Fusarium head blight (FHB)resistant gene, Fhb7, was found and designated in the wheat-Thinopyrum ponticum 7E(7D) substitution line, and the Fhb7 gene has been successfully cloned by mapbased cloning (Guo et al. 2015;). Therefore, the discovery and utilization of excellent exogenous genes provides more extensive germplasm selection for breeders.
Agropyron cristatum (L.) Gaertn (2n = 4x = 28, PPPP) is an important wild relative of common wheat (Dewey 1969(Dewey , 1984, has high economic value, contains a large number of desirable genes that can be exploited for wheat improvement, such as genes that provide resistance to powdery mildew and stripe rust, tolerance to drought and cold, and increased yield, and is vitally important for wheat improvement Li et al. 2016;Jiang et al. 2018). By using wide hybridization and embryo rescue, Li et al. (1997Li et al. ( , 1998 successfully obtained intergeneric hybrids between A. cristatum and common wheat, and then a series of wheat-A. cristatum addition lines were obtained from wheat-A. cristatum-derived lines, laying the foundation for wheat improvement by the use of these desirable genes. Among the exogenous addition lines and substitution lines containing the A. cristatum P chromosome that have been identified thus far, the 1P chromosome carries genes related to abiotic stress resistance and improved plant architecture (Pan et al. 2017;Wang et al. 2022), the 2P chromosome carries powdery mildew-and leaf rust-resistant genes (Li et al. 2016;Jiang et al. 2018), the 5P chromosome can promote partial homoeologous chromosome recombination (Pan et al. 2022), the 6P chromosome can increase wheat yields by promoting the number of fertile spikelets per spike and final kernel number per spike (Wu et al. 2006;Ye et al. 2015), and the 7P chromosome carries grain weight-related gene(s) that positively regulate yield traits ). However, the excellent genes of the wheat-A. cristatum 3P addition line and A. cristatum 3P chromosomes have not been reported.
In this study, wheat-A. cristatum 3P addition line I-27 and 3P-3B substitution line I-15 were identified from among wheat-A. cristatum-derived lines, and the chromosome composition and agronomic traits of the two materials have been characterized. Additionally, we developed 3P-specific molecular markers to track A. cristatum 3P chromatin, laying a foundation for the subsequent exploration and utilization of excellent traits controlled by the 3P chromosome.

Molecular Marker Analysis
Thirty-four pairs of universal molecular markers of the A. cristatum P genome were used to identify the exogenous P chromosome, and 42 pairs of expressed sequence-tag-sequence tagged site (EST-STS) markers for different P chromosomes were used to identify partial homoeologous groups of alien chromosomes (Table S1) (Han et al. 2017;Zhang et al. 2017). The tetraploid A. cristatum genome reference sequence was compared to the wheat reference sequence IWGSC RefSeq v2.1 by BLASTN queries of site sequence differences to design 3P-specific molecular markers. The 3P-specific molecular marker primer sequences are shown in Table S2. The PCR amplification procedure and product analysis were performed as described by Zhang et al. (2017).

Cytological detection
To determine the genetic composition of wheat-A. cristatum I-27 and I-15, the root tip cells at the mitotic metaphase and pollen mother cells (PMCs) at metaphase I were observed. Mitotic and meiotic cells were observed according to previous methods (Jiang et al. 2016;Li et al. 2020).

Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH)
The exogenous chromosomes of wheat-A. cristatum I-27 and I-15 were detected via GISH. Genomic DNA of A. cristatum accession Z559 was used as a probe, and genomic DNA of Fukuho was used as a blocker to confirm the presence of A. cristatum chromosomes in the wheat background ). The repeat sequences, oligo-pTa535-1 and oligo-pSc119.2-1, were used to distinguish wheat chromosomes (Tang et al. 2014b), and the repeat sequences, pAcTRT1 and pAcpCR2, were used to distinguish A. cristatum chromosomes via FISH (Han et al. 2019). GISH and FISH experiments were carried out according to previous methods, with slight modifications Sun et al. 2021). All the hybridization signals were observed by a Zeiss Axio Imager Z2 upright epifluorescence microscope (Carl Zeiss, Germany), captured with a charge-coupled device camera and analyzed by ISIS software (MetaSystems, Germany).

Identification of leaf rust resistance at the adult stages
The materials were inoculated with mixtures of the THT and PHT strains at the jointing stage by the spray method, and the common wheat cultivar Zhengzhou 5389 was used as a susceptible control. The resistance to leaf rust at the adult stage in the field was assessed in accordance with the methods of Jiang et al. (2018). A 0-4 scale was used for ITs, as described by Roelfs (1992).

Evaluation of agronomic traits
The wheat-A. cristatum-derived lines I-27 and I-15 and wheat cultivar Fukuho were planted at the experimental farm of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Xinxiang, Henan Province, China, during the 2019-2020 and 2020-2021 growing seasons. The row length was 2.0 m, and the row spacing was 30 cm; each row contained 20 seeds (Pan et al. 2022). Plant height, spike length, fertile tiller number, spikelet number, spikelet number per spike, and grain number per spike were evaluated for each individual plant. All the data were analyzed by SPSS 25.0 (SPSS; Chicago, IL, USA).

Molecular marker analysis of wheat-A. cristatum-derived lines
To clarify partial homoeologous groups of the P chromosomes in the wheat-A. cristatum-derived lines, 70 wheat-A. cristatum-derived lines were screened by using the universal molecular markers of the P genome, after which EST-STS markers for different P chromosomes and 3P-specific molecular markers were used to identify the derived lines that contained the P genome. The results showed that 23 Page 4 of 11 Vol:. (1234567890) wheat-A. cristatum-derived lines I-27 and I-15 had amplification bands corresponding to the P genome molecular markers and 3P-specific molecular markers, while the other P chromosome molecular markers did not show amplification bands (Fig. 1). This indicates that the wheat-A. cristatum-derived lines I-27 and I-15 carry the homoeologous group 3 chromosome of A. cristatum, namely, those wheat-A. cristatum-derived lines with chromosome 3P.
Cytological Identification and GISH/FISH Analysis of Wheat-A. cristatum-Derived Lines I-27and I-15 The probes pAcTRT1 and pAcpCR2 were used to distinguish between the homoeologous groups of A. cristatum chromosomes in derived lines I-27 and I-15. The results showed that both wheat-A. cristatum-derived lines I-27 and I-15 contained two A. cristatum 3P chromosomes (Fig. 2c, g). The number of I-27 chromosomes was 2n = 22II, with an average of 0.35 univalents, 1.87 rod bivalents and 19.85 ring bivalents (Table 1); included were 42 wheat chromosomes and two A. cristatum 3P chromosomes (Figs. 2a, b, 3a). According to the GISH results of PMCs at metaphase I, wheat-A. cristatum-derived line I-27 possessed a paired ring bivalent which were two 3P chromosomes with red signal (Fig. 2d), indicating that derived line I-27 was a wheat-A. cristatum 3P disomic addition line. Similarly, the chromosome number of wheat-A. cristatum-derived line I-15 was 2n = 21II, with an average of 0.32 univalents, 1.38 rod bivalents, and 19.37 ring bivalents (Table 1). I-15 thus contained 40 wheat chromosomes and a pair of 3P chromosomes (Fig. 2e, f, h). Unlike for the karyotype of Chinese Spring common wheat revealed by FISH signals (Tang et al. 2014b), a pair of common wheat 3B chromosomes were replaced by a pair of 3P chromosomes (Fig. 3b). The above results showed that derived line I-27 was a wheat-A. cristatum 3P(3B) disomic substitution line.
The results showed that the recurrent parent Fukuho and the 3P substitution line I-15 were highly resistant to leaf rust (Fig. 4c). However, the 3P addition line I-27 was susceptible to leaf rust (Fig. 4c). Different resistance to leaf rust for the two materials indicated that the source of the 3P chromosome in the 3P addition line and substitution line may not have the were restained blue by DAPI. The repeat sequence probe Oligo-pSc119.2 is labeled by the green signal, and the repeat sequence probe Oligo-pTa535 is labeled by the red signal. c, g The repeat sequence probe pAcTRT1 is labeled green, and the repeat sequence probe pAcpCR2 is labeled red. Wheat chromosomes were restained blue by DAPI. d, h Meiotic GISH analysis of wheat-A. cristatum-derived lines I-27(d) and I-15(h). The wheat chromosome was restained blue by DAPI, and the red signal indicates the A. cristatum chromosome same chromosome. These results may be useful for improvements in wheat disease resistance.

Discussion
Wild relatives of wheat carry a large number of genes that control excellent traits, and, thus, these relatives represent a gene bank for wheat genetic improvement. Based on the partial homology between the genomes of wild relatives of wheat and those of wheat, we can genetically improve wheat by partial homoeologous recombination (Rey et al. 2018). Alien addition lines and substitution lines produced by distant hybridization and chromosome engineering are important intermediate materials for transferring excellent exogenous genes (Pan et al. 2017;Li et al. 2020;Song et al. 2020). In this study, a wheat-A. cristatum 3P disomic addition line and a wheat-A. cristatum 3P(3B) disomic substitution line were identified via molecular markers and GISH and FISH analyses; these lines were relatively genetically stable. It has been demonstrated that the A. cristatum 3P chromosome carries genes that govern superior agronomic traits, such as multiple tillers and spikelets, increased spike length, and resistance to leaf rust. However, we found that, although the spikelet number increased, the grain number per spike did not increase significantly, suggesting that the whole introduced chromosome may carry some genes with adverse effects.  Therefore, we need to reduce the amount of exogenous fragments by creating small-fragment translocation lines to reduce the genetic burden, laying the foundation for the subsequent mapping of excellent genes of the A. cristatum 3P chromosome.
As an important wild relative of wheat, A. cristatum can provide many excellent traits and genes that have considerable potential for improving common wheat (Li et al. 1997). By using different wheat-A. cristatum addition lines, researchers have transferred many useful traits to common wheat, including resistance to biotic and abiotic stresses. Resistance to leaf rust and powdery mildew was successfully conferred to wheat by transferring the chromatin of A. cristatum chromosomes to common wheat Li et al. 2020). Additionally, A. cristatum yield traits, such as large spikes, multiple florets, multiple spikelets, and high 1000-grain weight, have also successfully transferred to common wheat (Ye et al. 2015;Qi et al. 2021;Sun et al. 2021). The generation of the wheat-A. cristatum 3P addition line has resulted in the formation of a complete set of genetic materials for A. cristatum chromosome 1-7P addition lines. This complete set is a highly valuable tool for identifying excellent genes in the P genome, and for studying the genetic evolutionary relationship between the P genome and the wheat ABD genome.
Wheat leaf rust, which is caused by Puccinia triticina, which has strong adaptability and is widely distributed (Yuan et al. 2020), is the main disease of wheat (Kolodziej et al. 2021) and affects wheat production in China and throughout the world (Savary et al. 2019). At present, breeding new resistant wheat varieties is still the most economical and effective control strategy for leaf rust. Seventy-nine wheat leaf rust-resistant genes (Lr1-Lr79) have been designated, of which Lr63 and Lr66 have been mapped to chromosome 3A (Kolmer et al. 2010;Marais et al. 2010), Lr27, Lr77, and Lr79 are located on chromosome 3B (Singh et al. 1984;Kolmer et al. 2018;Qureshi et al. 2018), and Lr22b and Lr69 have been mapped to chromosome 3D (Bartos et al. 1969;Qin et al. 2015). These resistance genes were all from the homoeologous group 3 chromosome, while the resistance genes on the 3P chromosome of A. cristatum, a wild relative of common wheat, have not been reported. Unfortunately, our understanding of the resistance mechanisms of wheat against leaf rust is still limited. In addition, many researchers have found that leaf rust antigens are relatively scarce in the main wheat cultivars, while leaf rust races show continuous variation, resulting in the gradual loss of resistance in many resistant cultivars (Kokhmetova et al. 2021). Therefore, the identification of novel resistance genes will be useful for wheat leaf rust-resistant breeding and for analyzing the mechanisms underlying disease resistance.
Spikelet number is an important factor affecting wheat yield and is essentially a quantitative trait determined by many quantitative trait loci (QTLs) or genes. At present, a series of genes and QTLs responsible for multiple spikelets have been mapped to different chromosomes, such as 2A, 2B, 2D (Du et al. 2021), 3D , 6BL (Katz et al. 2022), 5B, 7B (Tang et al. 2014a), 7A (Kuzay et al. 2019;Chen et al. 2020), 7DS (Chen et al. 2022), 1R (Yang et al. 2016), 2R (Dobrovolskaya et al. 2009), 2NS, and 3NS (Zhao et al. 2019). However, the genes/ QTLs controlling spikelet number on chromosome 3B have not been found. Thus, it is speculated that the multiple spikelets trait of the wheat-A. cristatum 3P(3B) substitution line is related to the A. cristatum 3P chromosome. The characteristics of multiple spikelet traits governed by genes carried by the A. cristatum 3P chromosome need to be further evaluated in the form of small-fragment translocation lines obtained by irradiation, which has a potential application value in wheat breeding.
Growing evidence has demonstrated that the homoeologous group 3 chromosomes of wheat carry many genes of excellent traits and genes that can be used for genetic improvement of wheat. Strong stripe rust-resistant genes have been found in the wheat-D. villosum 3 V(3D) substitution line . Kang et al. (2011) found that introgression of chromosome 3Ns from Psathyrostachys huashanica into wheat could also provide resistance to stripe rust. Guo et al. (2011) demonstrated that the T. ponticum 3E chromosome carries stripe rust-resistant genes and showed excellent salt tolerance. The wheat-Aegilops biuncialis 3 M addition line presented enhanced salt tolerance because of its ability to regulate the osmoprotection mechanism to increase sugar and proline accumulation (Darko et al. 2020). The wheat-L. mollis 3Ns#1(3D) substitution line carries leaf rustresistant gene(s), and its spike length and fertile tiller number are substantially improved (Pang et al. 2014). In addition, the FHB-resistant gene, Fhb1, has been mapped to wheat chromosome 3B . In this study, it was found that the wheat-A. cristatum 3P(3B) substitution line was highly resistant to leaf rust, and that the A. cristatum chromosome 3P could improve the spike traits of common wheat, indicating that there was certain differentiation and partial homology in the homoeologous group 3 during the genetic evolution of wheat. Therefore, further exploration of the excellent genes of the homoeologous group 3 of A. cristatum via generation of wheat-A. cristatum 3P addition and substitution lines is important.
Author Contributions WL and LL designed the study. ZK, SX and XJ performed the experiment. ZK wrote the manuscript. HH, SZ, JZ, XY and XL provided materials and reagents for the study. All authors have read and agreed to the published version of the manuscript.
Funding This research was supported by Grants from the National Key Research and Development Program of China (2016YFD0100102).

Conflict of interest
The authors declare no conflict of interest.
Data availability All data generated or analyzed during this study are included in this published article.