Molecular Mapping and Analysis of an Excellent Quantitative Trait Loci Conferring Adult-Plant Resistance to Stripe Rust in Chinese Wheat Landrace Gaoxianguangtoumai

The Chinese wheat landrace “Gaoxianguangtoumai” (GX) has exhibited a high level of adult-plant resistance (APR) to stripe rust in the field for more than a decade. To reveal the genetic background for APR to stripe rust in GX, a set of 249 F6:8 (F6, F7, and F8) recombinant inbred lines (RILs) was developed from a cross between GX and the susceptible cultivar “Taichung 29.” The parents and RILs were evaluated for disease severity at the adult-plant stage in the field by artificial inoculation with the currently predominant Chinese Puccinia striiformis f. sp. tritici races during three cropping seasons and genotyped using the Wheat 55K single-nucleotide polymorphism (SNP) array to construct a genetic map with 1,871 SNP markers finally. Two stable APR quantitative trait loci (QTL), QYr.GX-2AS and QYr.GX-7DS in GX, were detected on chromosomes 2AS and 7DS, which explained 15.5–27.0% and 11.5–13.5% of the total phenotypic variation, respectively. Compared with published Yr genes and QTL, QYr.GX-7DS and Yr18 may be the same, whereas QYr.GX-2AS is likely to be novel. Haplotype analysis revealed that QYr.GX-2AS is likely to be rare which presents in 5.3% of the 325 surveyed Chinese wheat landraces. By analyzing a heterogeneous inbred family (HIF) population from a residual heterozygous plant in an F8 generation of RIL, QYr.GX-2AS was further flanked by KP2A_36.85 and KP2A_38.22 with a physical distance of about 1.37Mb and co-segregated with the KP2A_37.09. Furthermore, three tightly linked Kompetitive allele-specific PCR (KASP) markers were highly polymorphic among 109 Chinese wheat cultivars. The results of this study can be used in wheat breeding for improving resistance to stripe rust.

The Chinese wheat landrace "Gaoxianguangtoumai" (GX) has exhibited a high level of adult-plant resistance (APR) to stripe rust in the field for more than a decade. To reveal the genetic background for APR to stripe rust in GX, a set of 249 F 6:8 (F 6 , F 7 , and F 8 ) recombinant inbred lines (RILs) was developed from a cross between GX and the susceptible cultivar "Taichung 29." The parents and RILs were evaluated for disease severity at the adult-plant stage in the field by artificial inoculation with the currently predominant Chinese Puccinia striiformis f. sp. tritici races during three cropping seasons and genotyped using the Wheat 55 K single-nucleotide polymorphism (SNP) array to construct a genetic map with 1,871 SNP markers finally. Two stable APR quantitative trait loci (QTL), QYr.GX-2AS and QYr.GX-7DS in GX, were detected on chromosomes 2AS and 7DS, which explained 15.5-27.0% and 11.5-13.5% of the total phenotypic variation, respectively. Compared with published Yr genes and QTL, QYr.GX-7DS and Yr18 may be the same, whereas QYr.GX-2AS is likely to be novel. Haplotype analysis revealed that QYr.GX-2AS is likely to be rare which presents in 5.3% of the 325 surveyed Chinese wheat landraces. By analyzing a heterogeneous inbred family (HIF) population from a residual heterozygous plant in an F 8 generation of RIL, QYr.GX-2AS was further flanked by KP2A_36.85 and KP2A_38.22 with a physical distance of about 1.37 Mb and co-segregated with the KP2A_37.09. Furthermore, three tightly linked Kompetitive allele-specific PCR (KASP) markers were highly polymorphic among 109 Chinese wheat cultivars. The results of this study can be used in wheat breeding for improving resistance to stripe rust.
Keywords: adult-plant resistance, QTL, stripe rust, Chinese wheat landrace, genetic mapping, heterogeneous inbred family INTRODUCTION Stripe rust (yellow rust), caused by Puccinia striiformis f. sp. tritici (Pst), is among the most harmful and widespread obligate pathogens of common wheat (Triticum aestivum L.) worldwide (Knott, 1989;Wellings, 2011). In China, stripe rust prevailed for several times in large wheat-growing areas and this caused serious yield losses (Zeng and Luo, 2006;Chen et al., 2014;Han and Kang, 2018). Since the 1950s, four severe epidemics of wheat stripe rust have occurred in China in 1950China in , 1964China in , 1990China in , and 2002 in yield losses of 6.0, 3.2, 1.8, and 1.4 million tonnes, respectively (Li and Zeng, 2000;Wan et al., 2004). The main cause of the outbreaks is the emergence of new virulent races that overcome the widely deployed resistance genes (Chen and Kang, 2017). At present, new virulent Pst race CYR34 appears and overcomes the widely deployed Yr genes. In the meantime, the simplification of Yr genes in commercial wheat cultivars have not changed yet though a lot of cultivars are used in wheat production. The most efficient and economical method of controlling the disease is the use of genetic resistance (Liu et al., 2017;. Continuous improvement in the resistance of wheat cultivars to cope with evolving races of Pst is a high priority to control stripe rust (Manickavelu et al., 2016).
To date, more than 300 genes or quantitative trait loci (QTL) for stripe rust resistance on the 21 wheat chromosomes have been reported (Rosewarne et al., 2013;McIntosh et al., 2019). In general, these resistance genes and QTL can be classified into two major classes: all-stage resistance (ASR) and adultplant resistance (APR). ASR usually confers complete resistance during all growth stages and is simple to select during breeding. However, most ASR genes are race specific and encode nucleotidebinding and leucine-rich repeat (NLR) proteins, and therefore are effective against only a subset of Pst races. With regard to the dynamic rust pathogen populations of the virulent races, only a small number of the characterized ASR genes, such as Yr5 (Marchal et al., 2018) and Yr15 (Klymiuk et al., 2018), are still widely effective against currently dominant Pst race groups in China (Sharma-Poudyal et al., 2013;Wu et al., 2018).
In contrast, APR is effective starting at adult-plant growth stages and typically provides a degree of partial resistance. Although a few APR genes are race-specific (Milus et al., 2015), a greater proportion of APR genes including Yr18 (Krattinger et al., 2009), Yr29 (William et al., 2003), Yr30 (Hayden et al., 2004), and Yr46 (Moore et al., 2015) is non-race-specific and provides durable resistance to Pst. Of the three APR genes cloned to date, Yr18 encodes a putative ATP-binding cassette transporter (Krattinger et al., 2009), Yr36 encodes a kinase domain and a lipid-binding domain (Fu et al., 2009), and Yr46 encodes a predicted hexose transporter (Moore et al., 2015). These genes represent different protein families compared with classical ASR genes (the NLR family) and provide unique mechanisms effective against a broader range of pathogens. As an example, Yr18 has been globally used as a component of durable rust resistance in breeding programs and no evolution of increased virulence has been observed for almost 100 years (Krattinger et al., 2009). To achieve a high degree of durable resistance, combining multiple APR genes into the same background has been considered as an important strategy for improvement of stripe rust resistance in wheat breeding.
Gaoxianguangtoumai (GX) is a spring wheat landrace from Sichuan Province in southwest China, which is a regional center for oversummering and overwintering of the stripe rust pathogen. This landrace has exhibited a high degree of APR to stripe rust in the field for more than a decade, but little information is available on the genetic basis of resistance in this landrace. The objectives of the present study were to (1) identify the QTL conferring APR to stripe rust in a recombinant inbred line (RIL) population developed from the cross between GX and a susceptible cultivar, "Taichung 29" (TC 29, 2) validate and mendelize the novel QTL in a heterogeneous inbred family (HIF) population, and (3) develop tightly linked Kompetitive allele-specific PCR (KASP) markers for use in marker-assisted selection in breeding programs.

Plant Materials and Races
The Chinese wheat landrace GX (accession number ZM7854 in National Germplasm Bank, China (NGBC) and AS1579 in Triticeae Research Institute, Sichuan Agricultural University) originating from Gao County, a county of Sichuan Province (28°26′N, 104°31′E). Because of high level of resistance to stripe rust for more than a decade, GX was crossed (as the female parent) with the highly stripe rust susceptible wheat cultivar TC 29. In total, 249 F 6:8 (F 6 , F 7 , and F 8 ) RILs derived from an individual F 1 plant were developed by single-seed descent. A KASP marker, KP2A_36.85 which was located around the peak of QYr.GX-2AS, was used to identify heterozygous lines from an F 8 generation of RIL. Through a single heterozygous plant was selected and selfed (Tuinstra et al., 1997), a HIF population of 130 individuals was generated for validating the QYr.GX-2AS. The scheme for developing the genetic populations was showed in Supplementary Figure S1. A collection of 325 Chinese wheat landraces was genotyped with the 55 K singlenucleotide polymorphism (SNP) array and further was used for marker haplotype analysis (Zhou et al., 2017). A panel of 109 Sichuan wheat cultivars was used to determine the polymorphism of markers tightly linked with QYr.GX-2AS. The highly stripe rust susceptible wheat cultivars "Mingxian 169, " "SY95-71, " and "Avocet S" (AvS) were used as susceptible controls in seedling and adult-plant tests throughout the study. Here, SY95-71 is a spring wheat line, selected from hexaploid triticale/wheat followed by backcrossing with wheat (Eronga 83/Fan6∥Fan6; Shu et al., 1999). The line has been widely used in China as a highly susceptible stripe rust spreader genotype or susceptible control. The Pst races (comprising CYR32, CYR33, CYR34, G22-14, Su11-4, Su11-5, and Su11-7; Wu et al., 2016b;Huang et al., 2018) were kindly provided by the Plant Protection Institute of the Gansu Academy of Agricultural Sciences, Gansu, China.

Evaluation of Resistance to Stripe Rust
Seedling tests to evaluate the stripe rust resistance of GX and TC 29 were conducted in a greenhouse using two prevalent Chinese Pst races (CYR32 and CYR34). Five plants of each line were sown in a plastic pot filled with nutrient soil and grown in a controlled environment in the greenhouse. Seedlings were inoculated at the two-leaf stage with each Pst race in accordance with the protocol of Hickey et al. (2012). Inoculated plants were placed in a dew chamber at 10°C and 100% relative humidity for 24 h in the dark, and then moved to separate growth chambers at 15-16°C with 12-14 h of light daily. When the susceptible control "Mingxian 169" showed full sporulation, the infection type (IT) on the second leaf (approximately 15-18 days after inoculation) was scored using a 0-9 scale (Line and Qayoum, 1992). Plants with IT scores of 1 to 6 were considered resistant, whereas plants with IT scores of 7-9 were considered susceptible.
Assessments of adult-plant stripe rust responses were conducted at the Chongzhou Experimental Station (30°33′N, 103°39′E), Sichuan Agricultural University, Chengdu, China. The F 6:8 RILs population and the parental lines were evaluated for APR to stripe rust during the 2017-2018, 2018-2019, and 2019-2020 growing seasons (referred to as CZ2018, CZ2019, and CZ2020, respectively). The HIF population of 130 individuals was evaluated for APR to stripe rust during the 2020-2021. The phenotype data of HIF population were used for Chi-Squared analysis (3:1 ratio) and genetic mapping. In all tests, 20 seeds of each line were planted in rows 2 m in length and spaced 30 cm apart, with individual plants spaced 10 cm apart. The susceptible cultivar TC 29 was planted in every 20th row as a susceptible control. To provide inoculum for infection, the susceptible cultivars SY95-71 and AvS were planted around the perimeter of the experimental area as spreaders. Artificial inoculation was conducted using a mixture of currently predominant Pst races in China (comprising CYR32, CYR33, CYR34, G22-14, Su11-4, Su11-5, and Su11-7). Stripe rust response was first recorded by scoring the IT and disease severity (DS) when the susceptible checks SY95-71 and AvS showed more than 80% DS and was followed by two additional evaluations at 7 day intervals (i.e., three evaluations in total) for three randomly selected individual plants. The IT was recorded based on the 0-9 scale of Line and Qayoum (1992). The DS was scored as the percentage infected leaf area (0, 5, 10, 20, 40, 60, 80%, or 100%) in accordance with the Chinese National Standard, GB/T 15797-2011. The final DS (FDS) was used for phenotypic analysis.

Genotyping, Linkage Map Construction, and QTL Analysis
Genomic DNA was extracted from a single plant for each line of the wheat materials using the cetyltrimethylammonium bromide method (Stewart and Via, 1993). The two parents (GX and TC 29) and the 117 RILs were genotyped using the Axiom ® Wheat 55 K SNP array (53,036 markers) by the China Golden Marker Biotechnology Company Ltd. (Beijing, China). Monomorphic and SNP loci with a minor allele frequency less than 0.3 were excluded with further analysis (Ma et al., 2019). Polymorphic SNP markers were used to remove redundant markers in the binning step using the BIN function, with the parameters missing rate = 20% and distortion value = 0.01, implemented in QTL IciMapping v4.2 (Wang et al., 2019b). The binned markers were used for linkage map construction using the Kosambi mapping function (Kosambi, 1944) with JoinMap v4.0 (Van Ooijen, 2006). Mapping of QTL was performed using QTL IciMapping v4.2 based on inclusive composite interval mapping with the preset parameters Step = 1 cM, value of p for entering variables (PIN) = 0.001, and logarithm of the odds (LOD) = 2.5.
To determine the effects of the QTL, the RILs were divided into four groups based on the presence/absence of the most closely linked flanking markers of QYr.GX-2AS and QYr.GX-7DS. In addition, the epistatic interactions between QYr.GX-2AS and QYr.GX-7DS were identified in RILs using QTL IciMapping v4.2 based on inclusive composite interval mapping of digenic epistatic QTL (ICIM-EPI) functionality with the preset parameters Step = 1 cM, value of p for entering variables (PIN) = 0.0001, and LOD = 5.

Haplotype Analysis
Haplotype analysis was performed to identify haplotype variants for QYr.GX-2AS in a collection of 325 Chinese wheat landrace accessions (Zhou et al., 2017;Ye et al., 2019). The informative markers linked to QYr.GX-2AS were screened using the Wheat 55 K or Wheat 660 K SNP arrays in accordance with the method described by Long et al. (2021). The SNP genotype data and the phenotype data (FDS) were obtained from recently published studies Long et al., 2019;Yao et al., 2019Yao et al., , 2020Ye et al., 2019;Wang et al., 2021). Haplotype variants were detected using Haploview v4.2. 1 The haplotypes detected in at least 10 accessions were considered to be major haplotypes.
Frontiers in Plant Science | www.frontiersin.org Boxplots were generated to display the average FDS of accessions carrying the different haplotypes. Haplotype data were combined with provenance information to examine the geographic distribution of the superior haplotypes in the 10 major agroecological production zones of Chinese wheat landraces.

Exome Capture Sequencing, Development of KASP Markers, and Genetic Mapping
Genomic DNA of the resistant parent GX was sequenced using the wheat exome capture sequencing protocol described by Dong et al. (2020). The raw sequence data have been submitted to GenBank under Bioproject no. PRJNA734801. The sequence variants were identified using the variant calling pipeline GATK4 (Heldenbrand et al., 2019). After QTL mapping, random SNPs in the target region to QYr.GX-2AS from the Wheat 55 K array and exome capture sequencing were selected and converted to KASP markers using the PolyMarker online tool (Ramirez-Gonzalez et al., 2015). The specific KASP markers were used to screen the parents and a paired of NIL (selected from HIF population with a common genetic background but differing in QYr.GX-2AS) to confirm polymorphism before genotyping in the HIF population. The KASP assays were performed in 96-well format as 10 μl reactions containing 2 μl of 50-100 ng genomic DNA, 5 μl of HiGeno 2× Probe Mix B, 0.24 μM of each forward primer, 0.6 μM of the common reverse primer, and double distilled water to make up the volume to 10 μl. Each PCR was conducted using the BIO-RAD CFX96 qPCR system. Thermocycling was performed with a touchdown protocol: 95°C for 10 min; 95°C for 20 s and 61°C (−0.6°C per cycle) for 40 s for 10 cycles; and 95°C for 20 s and 55°C for 40 s for 38 cycles. Data analysis was performed manually using BIO-RAD CFX96 Manager 3.1.
The polymorphic KASP markers were used for validating the QYr.GX-2AS in the HIF population of 130 individuals. Linkage analysis was performed using JoinMap v4.0 (Kyazma BV, Wageningen, Netherlands; Van Ooijen, 2006) with a LOD threshold of 3.0. The Kosambi map function (Kosambi, 1944) was used to convert the recombination fractions to centi-Morgans. The linkage map was drawn using Mapdraw v2.1 (Liu and Meng, 2003). Three tightly linked markers for QYr. GX-2AS were further assessed in 109 wheat cultivars grown in Sichuan for checking the usefulness of the newly developed KASP markers for marker-assisted selected.

Stripe Rust Response of the Parents and RILs
Plants of GX were susceptible (IT = 8) to CYR32 and CYR34 at the seedling stage ( Figure 1A) but exhibited strong resistance (IT = 3, FDS < 10%) to mixed Pst races (comprising CYR32, CYR33, CYR34, G22-14, Su11-4, Su11-5, and Su11-7) at the adult-plant stage in three crop seasons from 2018 to 2020 (Figures 1B, 2; Supplementary Table S1), indicating that GX has effective APR to these prevalent Chinese Pst races. In all three environments, the average FDS of RILs for GX × TC 29 was 12.5-15.7% in the field tests, and the distributions were skewed toward resistance (Figure 2) field trials. According to the homozygous phenotypes, the distribution of F 6:8 families was not fit the expected ratios for a single gene (1:1 ratio; χ 2 = 136.11, p < 0.001) and two genes (3:1 ratio; χ 2 = 23.15, p < 0.001). The result indicated that the high level of resistance in GX was controlled by multiple genes (Figures 1C, 2; Supplementary Table S1). Broad-sense heritability (H 2 ) was 96.7% for FDS in all tests ( Table 1). Correlation coefficients (R 2 ) for FDS of the RILs among the different environments were significant (p < 0.01) and ranged from 0.82 to 0.95 (Supplementary Table S2).

Linkage Map Construction and QTL Analysis
A total of 1,871 markers were used to construct the linkage map which spanning a total length 2,799.12 cM for the GX × TC 29 population (Supplementary Table S3 Table S3).
Two high quality QTL, conferring APR to Pst races, was screened through further analysis (Table 2; Figures 3A,B). The most significant QTL, designated QYr.GX-2AS, was mapped to the short arm of chromosome 2AS and explained 15.5-27.0% phenotypic variation (Table 2; Figure 3A). The other QTL, designated QYr.GX-7DS and explaining 11.5-13.5% phenotypic variation, was located on the short arm of chromosome 7D where this gene overlaps with Yr18 (Krattinger et al., 2009). The genetic distances analysis showed SNP markers cssfr5 and AX-110502471 flanking QYr.GX-7DS were 3.1 cM and 5.4 cM, respectively (Table 2; Figure 3B). Results indicated that it was highly likely that QYr.GX-7DS corresponded to Yr18.  Clearly, the RILs that carried one of the QTL showed a lower FDS than those without any QTL (average FDS = 63.4%; Figure 3C). The RILs carrying only QYr.GX-7DS showed 14.8% of the average FDS, whereas average FDS of lines with only QYr.GX-2AS was 9.3%. The lines with two QTL had the highest resistance level (average FDS = 7.06%; Figure 3C), similar to that of GX. In addition, the epistatic interaction between QYr.GX-2AS and QYr. GX-7DS could be significantly detected in two field trials and Busing the ICIM-EPI functionality of the QTL IciMapping v4.2 (Supplementary Table S4). These results indicated that the highlevel resistance in GX was contributed by these two QTL through additive and epistatic interactions, where QYr.GX-2AS provided relatively stronger resistance to Pst races than QYr.GX-7DS.

Haplotype Analysis of QYr.GX-2AS
To assess the distribution of QYr.GX-2AS among 325 Chinese wheat landraces, the favorable haplotype was identified by haplotype analysis and seven SNP markers tightly linked to QYr.GX-2AS were screened from the Wheat 55 K or 660 K SNP arrays (Figures 4A-C). Eight major haplotypes (n > 10) were detected in the panel (Figures 4A,B). GX and 15 other accessions clustered with Hap1 (Supplementary Table S5), which showed a frequency of about 5.3% in the total population ( Figure 4A). Almost all accessions carrying Hap1, except one from Henan, were collected from Sichuan. The accessions carrying Hap1 showed 18.4% of the average FDS and thus were more strongly resistant to stripe rust than those accessions carrying other haplotypes (Hap2 = 37.2%, Hap3 = 24.1%, Hap4 = 47.7%, Hap5 = 21.5%, Hap6 = 39.0%, Hap7 = 27.0%, and Hap8 = 47.6%; Figure 4C). The above results suggested that Hap 1 was the favorable haplotype of QYr.GX-2AS and relatively rare in Chinese wheat landraces.

Validation and Mapping of QYr.GX-2AS
QYr.GX-2AS was further mapped finely using newly KASP markers developed from SNPs screened by exome capture sequencing and the Wheat 55 K array. Eleven markers were confirmed to be polymorphic between GX and TC 29 (Supplementary Table S6). Combined with the KASP marker KP2A_36.85 for QYr.GX-2AS and the marker cssfr5 for Yr18, the HIF population of 130 individuals with a single locus QYr. GX-2AS was developed from a heterozygous plant (IT = 4) in the F 8 generation of RILs (Supplementary Figure S1). No significant phenotypic differences were observed in the HIF population, except for APR to stripe rust ( Figure 5A). With regard to stripe rust response in the field test, the HIF population could be clearly classifiable into 97 resistant (IT = 3-4) and 33 susceptible (IT = 8-9) individuals, which fits the expected ratio (3:1) for a single Mendelian factor (chi-square goodness-of-fit test, χ 2 = 0.01, p = 0.92; Supplementary Table S7). Using the newly developed 11 KASP markers (Supplementary Table S7) to construct the genetic map, QYr.GX-2AS was screened in 1.37 Mb interval between the KASP marker KP2A_36.85 and KP2A_38.22 and co-segregated with the KP2A_37.09 (Figure 5B).

Validation of KASP Markers for Marker-Assisted Selection
The molecular identification of 109 Chinese wheat cultivars was tested with three KASP markers KP2A_36.85 (G/A),  Table S8). Based on the above results, three KASP markers were valuable to apply QYr.GX-2AS in wheat breeding by marker-assisted selection.

DISCUSSION
It is the highest priorities to develop durable resistance to Pst races in wheat breeding during the past decade (Chen, 2013). A large number of genes or QTL that confer various degrees of APR to stripe rust have been identified (Chen, 2013), but most only have minor effects on stripe rust response and are therefore difficult to use in breeding. Thus, the identification of new high quality Yr genes or QTL with APR is useful in wheat breeding. The Chinese wheat landrace GX has displayed a high degree of APR to stripe rust in the field for more than a decade in southwest China. Two QTLs conferring APR to Pst races tested were identified in GX, tentatively named as QYr.GX-2AS and QYr.GX-7DS, and mapped on chromosome2AS and 7DS, respectively. In addition, the QYr. GX-2AS had a large effect in the reduction of stripe rust severity at adult-plant stages, which would be expected to have a great potential to pyramid this QTL with other Yr gene/ QTL to develop wheat cultivars with high-level and durable resistance to Pst races. QTL analysis is a useful procedure to reveal possible multiple loci when analyzing complex genetic traits, such as APR to stripe rust, in resistant germplasm. However, this procedure only allows approximate mapping of the QTL (Tanksley and Hewitt, 1988) owing to the heterogeneity in genetic backgrounds. The confidence interval of many QTL spans a considerable genetic distance and, as a result, molecular markers for these QTL may not be reliably used in marker-assisted selection. As a strategy for accurate mapping of QTL in genetic analysis, HIF populations that allow the conversion of a quantitative trait into a Mendelian factor have been widely used for fine mapping and cloning of many important QTL in wheat, such as Yr18 (Krattinger et al., 2009), Yr36 (Fu et al., 2009), Fhb1 (Su et al., 2019), and Fhb7 . In the present research, a HIF population targeting QYr.GX-2AS was developed based on the method of heterogeneous inbred family analysis (Tuinstra et al., 1997). Members of this population were unambiguously classified as either resistant or susceptible and fitted the expected ratio (3:1) for a single Mendelian factor; thus, accurate mapping of the locus was possible. Analysis of the HIF population revealed that QYr. flanked by KP2A_36.85 and KP2A_38.22, was located in the interval 36.85 Mb to 38.22 Mb on chromosome 2AS. One KASP marker co-segregating with the targeted locus was successfully developed for marker-assisted selection.
Several genes that confer resistance to stripe rust have been identified on wheat chromosome 2AS, including Yr17 (Bariana and Mcintosh, 1993), Yr56 (Bansal and Bariana 2014), Yr69 (Hou et al., 2016), YrR61 (Hao et al., 2011), and YrSph ; Figure 6 and Supplementary Table S9). The genes Yr17, Yr69, and YrSph confer ASR to stripe rust. Although recent studies suggest that Yr17 also confers APR to stripe rust in the field, QYr.GX-2AS is likely to differ from Yr17 because accessions of the Chinese wheat landrace GX that lack the 2 N alien segment carry Yr17. Yr56 is a major gene conferring APR to stripe rust that was identified in the Australian durum wheat cultivar "Wollaroi. " Yr56 is flanked by Xsun167 197) Figure 6). For example, the QTL QYr.tam-2AS_TAM 111 (Basnet et al., 2014) confer ASR to stripe rust. QYr.ufs-2A (Agenbag et al., 2012), QYrst.orr-2AS_Stephens (Vazquez et al., 2012), and QYr. sun-2A_Kukri (Bariana et al., 2010) were all flanked by the basis of a common DArT marker XwPt-0003, which were nearly with the QYrva.vt-2AS_VA00W-38 (Christopher et al., 2013) corresponds to the "Chinese Spring" physical map region 29.94 Mb. QYrtb. orz-2AS (Vazquez et al., 2015) and QYr.inra_2AS.1_Recital (Dedryver et al., 2009)   According to gene annotation information in IWGSC RefSeq v1.1, 16 predicted genes are located in the candidate region for QYr.GX-2AS (Figure 5C; Supplementary Table S10). None of these genes is a classic NBS-LRR resistance gene. In addition, no annotations accorded with the protein types encoded by the APR genes Yr18 (ABC transporter), Yr36 (kinase-START), and Yr48 (hexose transporter), implying that the candidate gene for QYr.GX-2AS might differ from known stripe rust resistance genes. Combined with exon sequencing data, eight predicted genes showed non-synonymous variants between GX and "Chinese Spring" in exon regions, including a RING/U-box, ascorbate peroxidase, glycosyltransferases, and F-box family protein, that may be involved in disease resistance. For confirmation of the candidate gene and cloning of QYr.GX-2AS, fine mapping to narrow the candidate interval will be performed using a large HIF population in future work.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
YW and FL are responsible for the experiment, analyzed the data, and drafted the manuscript. FG, FY, LL, XZ, LD, YW, and HL carried out the phenotypic evaluation. WL, QJ, YW, JM, PQ, MD, and YZ provided the resources and technique guidance. HK, YJ, and GC designed and carried out the experiment, formulated the questions, analyzed the data, and revised the manuscript. All authors have reviewed and approved the final manuscript.