A 1Ns Disomic Addition from Psathyrostachys Huashanica Keng Confers Resistance to Powdery Mildew in Wheat

: Powdery mildew is a fungal disease that threatens wheat production throughout the world. Breeding resistant cultivars is an e ﬀ ective way to reduce harm caused by powdery mildew. In this study, 35 wheat- Psathyrostachys huashanica -derived lines were developed by crossing common wheat and P. huashanica Keng (2 n = 2 x = 14, NsNs) using embryo culture. Resistance to powdery mildew in the derived lines was identiﬁed at the seedling and adult stages. Line H5-5-4-2 was selected with immunity to powdery mildew at both growth stages. The chromosome structure of this line was characterized by cytology, genomic in situ hybridization (GISH), and expressed sequence tag-sequence-tagged site (EST-STS) analysis. The chromosome conﬁguration was 2 n = 44 = 22II. Two P. huashanica chromosomes with strong hybridization signals were detected by GISH analysis. Among 83 EST-STS markers that covered all seven homologous groups in wheat, three pairs of STS markers, BE497584, BF202643, and BG262410, located in wheat homologous group 1 ampliﬁed clear speciﬁc bands related to P. huashanica . The results indicated that resistant line H5-5-4-2 was a wheat- P. huashanica 1Ns disomic addition line.


Introduction
Wheat (Triticum aestivum L.) is one of the most widely cultivated cereal crops worldwide and at least one third of the global population depends on wheat as the staple food [1]. Wheat production is threatened by many diseases and powdery mildew caused by Blumeria graminis f. sp. tritici (DC.) Speer (Bgt) is a particularly important foliar disease [2,3]. Fungicides are often used to control powdery mildew but their widespread application is inappropriate due to high cost, development of resistance in the pathogen, and environmental impacts [4]. Breeding resistant cultivars is also extremely important and this is the main method employed for effectively controlling powdery mildew in wheat [5]. However, pathogen populations undergo rapid mutation events and the coevolution with host resistance that cause resistance gene to become ineffective [6,7]. Therefore, it is necessary to discover more resistance resources that confer resistance to powdery mildew for application in wheat breeding [8]. Distant hybridization is an effective method for broadening the resistance spectrum by introducing novel resistance genes from wild relatives into the bread wheat gene pool [9].

Assessment of Powdery Mildew Resistance
The reactions to powdery mildew isolate Bgt E09 at the seedling stage were assessed as described by An et al. [19], where wheat cultivar 7182, P. huashanica, Mingxian 169, and 35 derivative wheat cultivars/lines were tested. The plant materials were grown in plug trays in a greenhouse. Plants were inoculated with E09 at the two-leaf stage. When Mingxian 169 exhibited full disease symptoms, the tested lines were evaluated by an infection type (IT) scale from 0-4 (Table 1), where plants with IT = 0, 1, or 2 were considered resistant and these with IT = 3 or 4 were susceptible [20,21].
The responses of adult plants to powdery mildew were determined in two replicates grown during 2018-2019 at the Yang Ling Wheat Experimental Station of Northwest A&F University. Resistance to powdery mildew in the field was assessed using a mixture of 30 different Bgt isolates as inocula. Each Agronomy 2020, 10, 312 3 of 11 material was planted in two rows with a length of 1 m and row spacing of 25 cm, and Mingxian 169 were planted around the tested plants as a disease spreader. In the jointing stage, we artificially dusted the spores evenly over the leaves. When Mingxian 169 exhibited fully developed symptoms, reactions were evaluated and recorded on a scale from 0-9, where scores of 0-4 were considered resistant and 5-9 indicated susceptibility [22]. Moreover, the resistance of each material was also assigned based on the disease index (DI) calculated by 20 randomly selected plants. DI was calculated as [Σ(each disease level × number of diseased plants)/(highest disease level × total number of diseased plants)] × 100 [23]. The classification standards are shown in Table 2.

Cytogenetic Analysis
Seeds of the test materials were germinated on wet filter paper in dishes. Seedling root tips were cut and placed in ice water for 24 h and then transferred to ethanol-acetic acid (3:1) for 1 week. Pollen mother cells (PMCs) in metaphase I of meiosis were obtained from young panicles and fixed in anhydrous ethanol-chloroform-glacial acetic acid (6:3:1, v/v). Root tips and PMCs were squashed in 45% acetic acid after staining with 2% acetocarmine for at least 2 h [24], and then used for cytological observations and GISH analysis. The prepared slides and observed with an Olympus BX60 microscope (Japan penguin) to assess chromosome structure and counts, and images were captured. The slides were frozen with liquid nitrogen, before removing the cover slips and storing at −20 • C.

Genomic In Situ Hybridization (GISH) Analysis
GISH was conducted to detect P. huashanica chromatin in the wheat-P. huashanica-derived lines. P. huashanica genomic DNA extracted from fresh leaves by an improved CTAB (Cetyl Trimethylammonium Ammonium Bromide) method [25], was labeled with Dig-Nick-Translation Mix/digoxigenin (digoxigenin-11-dUTP, DIG; Roche, Germany) using the nick translation method. The hybridization solution contained 1 µL 10% (w/v) sodium dodecyl sulfate, 1 µL salmon sperm DNA (5 µg/µL), 3 µL probe DNA, 4 µL 20 × SSC solution, 8 µL 50% (w/v) dextran sulfate, and 20 µL deionized formamide, which was made up to a volume 40 µL with 3 µL double distilled H 2 O. Hybridization for GISH was performed by placing a drop of hybridization solution on a slide with the sample. The slides were then incubated at a temperature of 80 • C for 8 min and 37 • C for 16 h. Next, 60 µL 5% bovine serum albumin was dropped onto the slide, which was incubated at 37 • C for 20 min, before adding 50 µL of Anti-Dig-FITC (Fluorescein Isothiocyanate) for detecting and visualizing the labeled chromosomes. We observed the chromosomes using a fluorescence microscope (Olympus BX60) and photographed images using a Photometrics SenSys CCD camera (the USA).

Evaluations of Resistance to Powdery Mildew
The seedling reactions to powdery mildew by the 35 wheat-P. huashanica offspring as well as parents 7182 and P. huashanica are displayed in Table 3. Nine materials (25.71%) exhibited resistance to isolate E09 whereas the remainder were moderately or highly susceptible. H5-5-4-2 was selected for further study because of its immunity to powdery mildew. Wheat cv 7182 was moderately resistant, whereas P. huashanica and H5-5-4-2 were immune ( Figure 1a). Table 3. Seedling reaction to powdery mildew in 35 wheat-P. huashanica-derived lines, their parents, and Mingxian 169 control.

Line
Reaction Line Reaction

GISH Analysis of H5-5-4-2
Mitotic and meiotic GISH analyses were conducted using the whole genomic DNA of P. huashanica as probe. GISH identification in mitosis demonstrated that H5-5-4-2 contained two added chromosomes with yellow-green hybridization signals, whereas the other 42 wheat chromosomes stained red (Figure 3a). In addition, one ring bivalent with a strong hybridization signal (bright yellow) was observed by GISH at meiotic metaphase I, (Figure 3b). These results demonstrated that The materials were assessed in the adult stage to determine their resistance to powdery mildew during wheat-growing seasons in 2018-2019. Line H5-5-4-2 and P. huashanica exhibited uniform immunity to the mixture of Bgt isolates. By contrast, wheat cultivar 7182 was moderately resistant to infection and Mingxian169 was highly susceptible (Figure 1b). The IT and DI scores for these four materials were as follows: P. huashanica, IT = 0, DI = 0; H5-5-4-2, IT = 0, DI = 0; line 7182, IT = 4, DI = 6.1; and Mingxian169, IT = 8, DI = 50.12. Therefore, the evaluation results indicated that H5-5-4-2 exhibited great resistance to powdery mildew in both the seedling and adult stages, where its resistance to powdery mildew was probably attributable to the introduction of two chromosomes from P. huashanica. Thus, a series of subsequent studies were conducted using this immune derivative line.

GISH Analysis of H5-5-4-2
Mitotic and meiotic GISH analyses were conducted using the whole genomic DNA of P. huashanica as probe. GISH identification in mitosis demonstrated that H5-5-4-2 contained two added chromosomes with yellow-green hybridization signals, whereas the other 42 wheat chromosomes stained red (Figure 3a). In addition, one ring bivalent with a strong hybridization signal (bright yellow) was observed by GISH at meiotic metaphase I, (Figure 3b). These results demonstrated that

GISH Analysis of H5-5-4-2
Mitotic and meiotic GISH analyses were conducted using the whole genomic DNA of P. huashanica as probe. GISH identification in mitosis demonstrated that H5-5-4-2 contained two added chromosomes with yellow-green hybridization signals, whereas the other 42 wheat chromosomes stained red Agronomy 2020, 10, 312 6 of 11 ( Figure 3a). In addition, one ring bivalent with a strong hybridization signal (bright yellow) was observed by GISH at meiotic metaphase I, (Figure 3b). These results demonstrated that two alien chromosomes were introduced into H5-5-4-2 from P. huashanica. Besides, the two alien chromosomes from P. huashanica could form a separate bivalent and undergo normal synapsis, pairing, and segregation in the wheat background. Thus, line H5-5-4-2 was a cytogenetically stable wheat-P. huashanica disomic addition line.

EST-STS Analysis of H5-5-4-2
In this study, 83 pairs of EST-STS primers distributed throughout all over homoeologous groups were screened for polymorphisms in 7182 and P. huashanica. These polymorphic primers were used to amplify DNA samples from the disomic addition line H5-5-4-2 and its parents 7182 and P. huashanica. Three EST-STS primers, BE497584, BF202643, and BG262410 ( Table 4) that mapped to homoeologous group I (1AL, 1AS, 1BL, 1BS, 1DL, and 1DS) amplified clear P. huashanica-specific bands in line H5-5-4-2 but none in the wheat parent 7182 (Figure 4). However, amplification products of other primers produced no specific bands. We inferred that the chromosomes from P. huashanica belonged to homoeologous group I and that the added chromosome pair in H5-5-4-2 could be designated 1Ns.

EST-STS Analysis of H5-5-4-2
In this study, 83 pairs of EST-STS primers distributed throughout all over homoeologous groups were screened for polymorphisms in 7182 and P. huashanica. These polymorphic primers were used to amplify DNA samples from the disomic addition line H5-5-4-2 and its parents 7182 and P. huashanica. Three EST-STS primers, BE497584, BF202643, and BG262410 ( Table 4) that mapped to homoeologous group I (1AL, 1AS, 1BL, 1BS, 1DL, and 1DS) amplified clear P. huashanica-specific bands in line H5-5-4-2 but none in the wheat parent 7182 (Figure 4). However, amplification products of other primers produced no specific bands. We inferred that the chromosomes from P. huashanica belonged to homoeologous group I and that the added chromosome pair in H5-5-4-2 could be designated 1Ns. huashanica. Three EST-STS primers, BE497584, BF202643, and BG262410 ( Table 4) that mapped to homoeologous group I (1AL, 1AS, 1BL, 1BS, 1DL, and 1DS) amplified clear P. huashanica-specific bands in line H5-5-4-2 but none in the wheat parent 7182 (Figure 4). However, amplification products of other primers produced no specific bands. We inferred that the chromosomes from P. huashanica belonged to homoeologous group I and that the added chromosome pair in H5-5-4-2 could be designated 1Ns.

Discussion
Distant hybridization is a major focused research in many laboratories worldwide. It allows genes from related species to be transferred into wheat to improve quality and productivity [27]. Researchers in China began conducting crosses between wheat with Thinopyrum intermedium and Thinopyrum elongatum in the 1950s, and a number of wheat cultivars were developed [28]. A wheat-rye 1BL·1RS translocation line was obtained by transferring many resistance genes (e.g., Yr9, Lr26, Pm8, and Sr31) from rye into a wheat background, and it is an outstanding example of genetic improvement in wheat by distant hybridization [29,30]. Recently, the exploitation of available genes in the wild relatives of wheat has diversified greatly. In particular, An et al. [31] obtained a new wheat-rye addition line called WR35 via crossing Xiaoyan 6 with the rye cultivar German White, where the addition line exhibited resistance to powdery mildew and stripe rust, and a high kernel number per spike. Pan et al. [32] also found that the 1P chromosome from Agropyron cristatum enhanced the spike length and tiller number in wheat, and a wheat-A. cristatum 1P addition line was obtained using molecular and phenotypic identification techniques.
Crosses between wheat and P. huashanica are relatively recent, and P. huashanica is significant among numerous species related to wheat because of its excellent agronomic characteristics. Several wheat-P. huashanica progeny lines were generated in previous studies, including disomic addition lines [33], disomic substitution lines [34], and small fragment translocation lines [17]. In our laboratory, 35 derived lines were developed by crossing common wheat 7182 with P. huashanica via embryo rescue culture and backcrossing, and these intermediate materials with different agronomic traits provide a suitable foundation for exploiting genes from P. huashanica.
Powdery mildew severely hinders yield and quality improvements in wheat and the loss of effective genetic resistance to powdery mildew due to the high variability of pathogens and the uniformity of resistance sources is leading to a crisis in wheat production [35][36][37]. Among the 92 powdery mildew resistance genes (Pm1~Pm65) that have been officially named, many are derived from wild relatives [38][39][40]. Thus, many studies have aimed to identify new resistance genes in the relatives of wheat [41,42]. In previous studies, the resistance genes Pm7, Pm8, Pm17, and Pm20 were identified in rye, and Pm8 was utilized widely, although the effectiveness of its resistance has now been lost [43][44][45]. Pm21, which is located on the short arm of chromosome 6V in Haynaldia villosa (2n = 14, VV) and it is an excellent gene that confers broad-spectrum resistance to powdery mildew in wheat [46]. Zhan et al. [45] showed that alien chromosome fragments possessed genetic loci with resistance to powdery mildew and stripe rust in wheat-Thinopyrum intermedium translocation lines. In addition, Li et al. [47] detected resistance genes in the 2P chromosome in Agropyron cristatum.
The powdery mildew resistance genes in P. huashanica have not been identified in recent studies, and wheat-P. huashanica-derived lines with resistance have rarely been reported, so it is significant to conduct further research into powdery mildew resistance in P. huashanica. In the present study, we examined the seeding responses of 35 wheat-P. huashanica-derived lines to powdery mildew and showed that line H5-5-4-2 was immune (Table 3, Figure 1a). Moreover, the resistance evaluation of H5-5-4-2 in the adult stage was immunity (Figure 1b). Our results suggested that this resistance originated from P. huashanica. We further speculated that this resistance may be a quantitative trait controlled by multiple genes as the derived lines displayed different evaluation of immunity, high resistance, moderate resistance, moderate susceptibility and high susceptibility. These findings provide a great precondition to explore resistance genes in P. huashanica and the development of novel resistance germplasm resources.
The resistance performance of wheat varieties may differ during various growth periods. Comprehensive assessments of disease resistance need to be conducted in both the seedling and adult stage. Varieties with adult resistance mostly exhibit sustained quantitative resistance (horizontal resistance). If a variety exhibits resistance in both the seedling and adult stages, then it will show strong resistance throughout the whole growth period [48]. In the present study, we found that derivative line H5-5-4-2 not only exhibited high resistance to a mixture of races in the adult stage, but it was also immune to Bgt isolate E09 in the seedling stage. Thus, we speculated that the resistance genes derived from P. huashanica may be the major genes, which may allow this line to exhibit strong broad-spectrum resistance to powdery mildew, thereby providing a resistance gene resource for breeding wheat cultivars with resistance to powdery mildew.
Mitotic and meiotic cytological observations as well as GISH were performed in order to determine the origin of resistance gene in the immune derivative line H5-5-4-2, where the results demonstrated that H5-5-4-2 is a disomic addition line. GISH can simply determine the presence of alien chromosomes but molecular markers are valuable analysis tools for identifying the homoeology of alien chromosomes. Thus, a wheat-Aegilops geniculata 7M g addition line was identified using EST-STS markers by Wang et al. [49]. In addition, Wang et al. [50] identified a wheat-Leymus racemosus translocation line T5AS-7LrL·7LrS by using EST-STS markers. In the present study, three pairs of primers belonging to homoeologous group I amplified Ns genome-specific bands in H5-5-4-2, thereby indicating the presence of a pair of 1Ns chromosomes derived from P. huashanica, and thus the powdery mildew-resistant derivative line H5-5-4-2 is a wheat-P. huashanica 1Ns disomic addition line.
Wheat disomic addition lines had substantial value in various applications as basic materials for investigating the genetic relationships between wheat and its relatives and gene mapping, they can also be employed as bridge materials for producing substitution lines and translocation lines [31]. A complete set of 1Ns-7Ns wheat-P. huashanica disomic addition lines was developed by Du et al. [51]. In addition, in a previous study of wheat-P. huashanica 1Ns disomic addition lines, Zhao et al. [52] found that the addition of the 1Ns chromosome from P. huashanica in H9021-28-5 had the effects of improving agronomic traits such as the grain weight, resistance to stripe rust, flour-processing properties, and the contents of some microelements. Moreover, Du et al. [51] showed that the 1Ns disomic addition line 12-3 exhibited high resistance to leaf rust, and it possessed large and awnless panicles. However, previous studies have not investigated powdery mildew resistance in wheat-P. huashanica 1Ns disomic addition lines. Therefore, our successful identification of the wheat-P. huashanica 1Ns chromosome disomic addition line H5-5-4-2 with resistance to powdery mildew may facilitate the breeding of varieties with resistance to powdery mildew, as well as enriching the wheat germplasm resource pool and laying a material foundation for fully utilizing the excellent traits of P. huashanica in wheat resistance breeding. Future studies may focus on the resistance of 1Ns chromosome in P. huashanica with referencing our study to promote the investigation and development of more wheat-P. huashanica 1Ns alien addition lines.