Quantitative Trait Loci Mapping for Adult-Plant Stripe Rust Resistance in Chinese Wheat Cultivar Weimai 8

: Wheat stripe rust, triggered by Puccinia striiformis f. sp. tritici, is among the most widespread and damaging wheat (


Introduction
Stripe rust triggered by Puccinia striiformis f. sp.tritici (Pst) is among the most prevalent diseases of wheat (Triticum L.) in moist and cool growing regions worldwide [1].China ranks highest in the world in terms of areas affected by stripe rust [1,2].Stripe rust is among the most destructive wheat diseases, and the weather is conducive for the disease in the southwest and northwest regions of China, where cultivars prone to stripe rust are cultivated; this is likely true for the rest of the regions of the world [3].Recent reports showed that about 10 to 70% of wheat production losses are due to stripe rust, depending on the cultivars grown, prevailing climatic conditions, and disease pressure.In the past two years, a higher prevalence and severity of wheat stripe rust occurred in most wheat growing areas of China, suggesting a potential new stripe-rust disease epidemic.Moreover, a production loss of up to 100% can occur on susceptible cultivars under climatic conditions favorable for disease development [4,5].In China, wheat cultivars containing Yr9 and derivatives of the cultivar Fan 6 have been vulnerable to stripe rust since the appearance of the Pst race CYR32, resulting in the 2002 epidemic [6].Therefore, the deployment of resistant cultivars is the most efficient, economically viable, and environmentally friendly means of controlling the disease [7].
There are two types of resistance genes to stripe rust, depending on their expressions at diverse stages of plant growth, viz.all-stage resistance (ASR) and adult plant resistance (APR) genes [8].ASR genes are effective during initial plant developmental phases, are usually race-specific, and frequently encode nucleotide binding site-leucine-rich repeats (NBS-LRR) resistance proteins [8,9].By contrast however, characterized by resistance during the adult plant stage, APR is susceptible at the seedling stage when tested with similar Pst pathotypes.APR genes are effective during late plant developmental stages and mostly confer resistance to a broader range of Pst pathotypes, have partial resistance, and encode a more diverse and different set of proteins [10][11][12].Because of frequent changes in pathogen populations, cultivars conferring all-stage resistance genes lose stripe-rust resistance within a short period of time [13].As a consequence, APR with apparent durability is increasingly being deployed in wheat-breeding and production programs [14][15][16][17].
Closely associated molecular markers to rust resistance genes are proper tools for the gene pyramiding and advancement of resistant wheat cultivars.In recent times, molecular markers, such as single simple sequence repeats (SSRs) and nucleotide polymorphisms (SNPs), are increasingly used for identifying and mapping rust-resistance genes in wheat.According to the reports in [10,32], there are numerous resistance genes to stripe rust that have been mapped onto the wheat genome in the previous two decades.SSR markers have been the most preferable marker type, which is attributable to their advantages of a better level of polymorphism, codominance, known map location, repeatability, chromosome specificity, accurateness, and PCR-based amplification over the last 30 years.According to Peng et al. (2000), YrH52, derived from wild emmer wheat (T.dicoccoides), was reported as flanked by Xgwm273a-1B and Xgwm413-1B, with a genetic distance of 2.7 and 1.3 cM, respectively [33].Li et al. (2006) mapped YrZH84, derived from the Zhou 8425B wheat line of China, which was flanked by Xbarc32-7B and Xcfa2040-7B SSR markers with 1.4 and 4.8 cM genetic distance, respectively [34].Weng et al. (2005) selected six SSR markers linked to Yr9 in a near-isogenic line of Taichung29*6/Yr9 and the nearest SSR marker Xgwm582 linked to Yr9 with a genetic distance of 3.7 cM [35].Closely associated SSR markers can afford a superior means for pyramiding stripe-rust resistance genes so as to use for marker-assisted selection (MAS) breeding programs [36].Therefore, it is vital to map and identify new resistance genes to stripe rust, preferably with closely linked molecular markers for MAS.
Weimai 8 (88-3149/Aus621108) is an essential cultivar in the Yellow and Huai Valley regions of China.Weimai 8, developed from crossing 88-3149/Aus621108 at Weifang Agriculture Academy of Science in Shandong Province, is acknowledged to maintain a reasonable level of resistance to leaf rust and stripe rust in the field.The objective of the current study was to map and identify APR QTLs to stripe rust in the Weimai 8/Zhengzhou 5389 RIL population.Mapping APR QTLs to stripe rust and finding closely associated molecular markers in the RILs provides not only a theoretical foundation for stripe-rust resistance, but also can be used to select the resistant genes more effectively in wheatbreeding programs.

Plant Materials
The stripe-rust resistant parent Weimai 8 (88-3149/Aus621108), the susceptible parent Zhengzhou 5389, and the RIL population were all obtained through single seed descent.A total of 165 F 2-6 RILs derived from Weimai 8/Zhengzhou 5389 were used to map APRrelated QTLs for stripe rust.

Plant Pathogen
Mixed Pst races of stripe rust, CYR32, CYR33, and CYR34, provided by the Research Department of Wheat Stripe Rust and Leaf Rust of Hebei Agricultural University were used to test the population.These Pst races were selected due to their high level of virulence to both parents at the seedling stage in the greenhouse.

Evaluation of Stripe-Rust Reactions in the Field
Weimai 8, Zhengzhou 5389, and 165 RILs were grown at Baoding, Hebei Province, during the 2016-2017 cropping seasons, and at Mianyang, Sichuan Province, during the 2017-2018 and 2018-2019 cropping seasons.Weimai 8, Zhengzhou 5389, and 165 RILs were inoculated with mixed Pst races.The RILs were sowed as single plant rows spaced 15 cm apart in a 1.5 m row repeated thrice in every environment.Zhengzhou 5389 was sown as a spreader row adjacent and perpendicular to the rows of tested lines.Striperust epidemics were started by spraying an aqueous suspension with an equal amount of urediniospores from each Pst race to which a few drops of Tween 20 (0.03%) had been added onto the spreader rows in mid-April.The final disease severity (FDS) and host responses to infection were recorded for each line in each environment when the susceptible check Zhengzhou 5389 was at least 70% infected, according to the concepts and methods of disease management.Phenotypic correlation coefficients between different environments in FDS were calculated by the Microsoft Excel analytical tool, 2013.

DNA Extraction and Bulk Group Preparation
Genomic DNA was extracted from the RIL population using the CTAB protocol [37].DNA was quantified using a UV spectrophotometer and diluted to a final concentration of 30-50 ng/µL prior to use.Bulked segregant analysis (BSA) [38] was performed to identify molecular markers putatively linked to stripe-rust resistance in Weimai 8. Based on the 2016-2017 Baoding stripe rust field data, equal amounts of DNA from the five most susceptible and five most resistant lines, respectively, were mixed to form susceptible and resistant bulks.DNA samples of bulk groups and the two parents were screened for polymorphisms of SSR markers.In addition, we used a 55K SNP chip to sequence resistant and susceptible for bulk segregant analysis.The 55K SNP chip was conducted to locate the genetic map location and concentration of the SNP markers throughout all the wheat genome chromosomes.

SSR Analyses and Electrophoresis Detection
A total of 860 pairs of wheat simple-sequence-repeat (SSR) primers available in GRAIN-GENES 2.0 (http://wheat.pw.usda.gov,accessed on 10 June 2021) were used in this study.The primers with a polymorphism and exchange rate of less than 30% were selected between the two parents and populations.All the 165 F 2-6 lines were then genotyped to regulate their connotations with stripe-rust responses.At the same time, the whole genome SNPs of the small bulked group, ten susceptible, and ten resistance lines according to their FDS scores were detected using a 55 K SNP array of wheat.This was conducted to see the SNPs concentrated region on the wheat chromosome.PCR was performed in 10 µL volumes containing 1 × PCR buffer, 0.2 mmol L −1 dNTP, 0.4 µmol L −1 primers, 3.0 µg mL −1 template DNA, 0.05 U of Taq polymerase, and 6.7 µL of ddH 2 O.The PCR conditions were programmed as follows: denaturation at 94 • C for 5 min, followed by 35 cycles of 94 • C for 1 min, 51-68 • C (depending on the primer pair) for 1 min, 72 • C for 1 min, and a final extension at 72 • C for 10 min.The PCR products were then separated using 12% non-denaturing polyacrylamide gel electrophoresis, and the gel was visualized by the silver nitrate staining method [39].

Linkage Analysis and Genetic Mapping
Phenotypic and genotypic data were used to map QTLs for APR in Weimai 8/Zhengzhou 5389 by using the Manager QTXb2.0 and Icimapping 3.2 softwares [40].The Map Manager QTXb2.0 software was was employed to establish linkage map groups.Genetic distances between markers were assessed using the Kosambi mapping function.QTL mapping was performed with the ICIM-ADD function using the QTL IciMapping 3.2 software [23].After performing a 1000-permutation test, a logarithm of odds (LOD) threshold of 2.5 was set to declare QTLs as significant.To detect all QTLs, a walk speed of 1.0 cM was chosen.QTL effects were predicted as the proportion of phenotypic variance explained (PVE, %) by the QTL [23].Digenic interactions between non-allelic QTLs were examined by the ICIM-EPI function, using QTL IciMapping 3.2 with a LOD threshold of 2.5 [23].

Phenotypic Evaluation
According to the frequency distribution of the FDS result, stripe rust developed well in all trials, showing that the susceptible control, Zhengzhou 5389, recorded FDS in at least 70% of cases across environments (Figure 1).The frequency distribution of stripe rust FDS for the 165 lines in every environment showed the quantitative inheritance of APR to stripe rust (Figure 1).Weimai 8 had an FDS of 1-10% across all environments.The final disease severity results of the three testing environments specified that the data were incessantly disseminated and supported quantitative inheritance (Figure 1).Additionally, the bar chart showed significant differences among the RILs-environment interactions (Figure 1).

Molecular Marker Screening and Gene Location
A total of 860 pairs of SSR primers scattered across 21 chromosomes of wheat were partitioned between the two parents and the bulk group of resistant and susceptible DNA

Molecular Marker Screening and Gene Location
A total of 860 pairs of SSR primers scattered across 21 chromosomes of wheat were partitioned between the two parents and the bulk group of resistant and susceptible DNA pools.About 189 pairs of SSR primers were polymorphic among the parents and 46 pairs of primers had an exchange rate of less than 30% between the two parents and between the two bulks.These markers were distributed on chromosomes 1B, 2AS, 2BS, and 7DS.In addition, a total of 271 SNP sites 0-1 exchanged were detected by 55K SNP array sequencing.The statistical analysis results showed that QTLs controlling wheat stripe rust were concentrated on chromosomes 1B, 2A, and 7D (Figure 2).This is basically consistent with the results of blind screening.

Molecular Marker Screening and Gene Location
A total of 860 pairs of SSR primers scattered across 21 chromosomes of wheat were partitioned between the two parents and the bulk group of resistant and susceptible DNA pools.About 189 pairs of SSR primers were polymorphic among the parents and 46 pairs of primers had an exchange rate of less than 30% between the two parents and between the two bulks.These markers were distributed on chromosomes 1B, 2AS, 2BS, and 7DS.In addition, a total of 271 SNP sites 0-1 exchanged were detected by 55K SNP array sequencing.The statistical analysis results showed that QTLs controlling wheat stripe rust were concentrated on chromosomes 1B, 2A, and 7D (Figure 2).This is basically consistent with the results of blind screening.

Identification of Stripe-Rust Resistance Gene Yr9, Yr17, and Yr18
Tester lines with known stripe-rust resistance genes for Yr17, Yr9, and Yr18, Zhengzhou 5389, and Weimai 8 were amplified twice by the sequence-tagged sites (STS) positive marker ω-secalin and reverse marker Glu-B3, closely linked to Yr9 [41,42]; the STS markers VEN-TRIUP and LN2, closely linked to Yr17 [43]; and the STS marker csLv34, closely linked to Yr18, respectively [43,44].The results of PCR amplification with the STS markers are shown in Figure 4. Notably, the specific target fragments of Yr9 and Yr17 were amplified in Weimai 8. Hence, QYr.wmy-1B and QYr.wmy-2AS have been proven to be the known genes Yr9 and Yr17, respectively.However, the specific target fragment for Yr18 was not detected in Weimai 8, and hence, the QYr.wmy-7DS is likely to be a new APR-QTL.

Identification of Stripe-Rust Resistance Gene Yr9, Yr17, and Yr18
Tester lines with known stripe-rust resistance genes for Yr17, Yr9, and Yr18, Zhengzhou 5389, and Weimai 8 were amplified twice by the sequence-tagged sites (STS) positive marker ω-secalin and reverse marker Glu-B3, closely linked to Yr9 [41,42]; the STS markers VENTRIUP and LN2, closely linked to Yr17 [43]; and the STS marker csLv34, closely linked to Yr18, respectively [43,44].The results of PCR amplification with the STS markers are shown in Figure 4. Notably, the specific target fragments of Yr9 and Yr17 were amplified in Weimai 8. Hence, QYr.wmy-1B and QYr.wmy-2AS have been proven to be the known genes Yr9 and Yr17, respectively.However, the specific target fragment for Yr18 was not detected in Weimai 8, and hence, the QYr.wmy-7DS is likely to be a new APR-QTL.

Discussion
Identifying or mapping key APR genes and faithfully associated molecular markers for wheat stripe rust, especially in resistant cultivars such as Weimai 8, is essential for effectively guiding marker-assisted selection and breeding for wheat stripe-rust resistance [8,44].This has motivated us, here, to identify and map QTLs for APR to wheat stripe rust in a Weimai 8/Zhengzhou 5389 RIL population and identify candidate genes that could be novel genetic resources for stripe-rust resistance breeding in wheat.
The work described herein is the continuation of [45], that performed a study of F 2:3 population from a similar cross population using SSR markers.The present study was aimed to repeat the analysis with greater precision and focusing on stripe rust rather than leaf rust.For instance, [45] reported a single major QTL, QLr.hbau-2AS, derived from Weimai 8 flanked by Xcfd36 and Xbarc1138 markers with an interval length of 2.58 cM.However, the present study identified four QTLs derived from Weimai 8 APR to stripe rust.Although the present study identified QYr.wmy-2AS at a similar chromosome to that of the findings by [45], the QTLs were flanked by different SSR markers, and hence, they are not pleotropic QTLs.The results of the present study should be more reliable than those of [45] due to using a near homozygous population (F 2:6 RILs) and the inclusion of SNPs.

Investigation Index of Adult Stripe Rust
The main parameters used to study stripe-rust resistance QTLs were FDS and the area under the disease progress curve (AUDPC).The correlation between these two parameters is very significant (the correlation is 0.97) [46], and the FDS is more convenient to use.Hence, FDS data were used of the most serious disease in the field.The AUDPC needs to combine the survey data and the interval time of the survey, which is time-consuming and laborious.In this study, therefore, FDS was used as the parameter of disease severity.

QYr.wmy-1B Is Yr9 and QYr.wmy-2AS Is Yr17
Tester line with known stripe-rust resistance genes for Yr9, Zhengzhou 5389, and Weimai 8 were amplified twice by the sequence-tagged sites (STS) positive marker ωsecalin and reverse marker Glu-B3, closely linked to Yr9 [42].Our results in the current experiment have proven that QYr.wmy-1B is the known gene Yr9.QYr.wmy-1B spotted in the Weimai 8/Zhengzhou 5389 population pool was steady crosswise all three locations explained by 11.56-16.64% of the phenotypic variances.Yr9, Pm8, Lr26, and Sr31 are located in the 1BL/1RS translocation lines [64,65].However, Yr9, just like some other rust-resistance genes such as Lr9, Lr19, and Lr26, has been overcome by some emerging Pst virulent races both in China and globally [66].Therefore, it is necessary to carefully consider the use of Yr9 in wheat stripe-rust breeding programs.
Likewise, the present study has also proven through molecular evidence that QYr.wmy-2AS is the known gene Yr17.Yr17 in wheat originated from the 2NS/2AS translocation of Triticum ventricosum ensuing in the hexaploid wheat line VPM1 that was originally made to transfer eyespot resistance to bread wheat [67,68].This chromosomal fragment comprises a gene cluster including Yr17 for stripe rust, Lr37 for leaf rust, and Sr38 for stem rust; and it has been broadly used in wheat-breeding programs worldwide [69].Yr17 is an adult plant resistance gene, which can be expressed in the field and greenhouse.Although new Pst races of stripe rust have emerged in many countries, Yr17 still has extensive resistance to many Pst races and has proven to have an excellent adult plant resistance to most races [70]; hence, Yr17 should play a vital role in combination with other rust-resistant genes.

Weimai 8 as a Useful Resource in Breeding for Durable Wheat Stripe-Rust Resistance in China
Weimai 8 harbors stripe rust resistance-related QTLs and might be valuable in breeding wheat cultivars resistant to stripe rust.Particularly, the QYr.wmy-2BS allele was contributed by the resistant parent Weimai 8.However, QYr.wmy-2BS was found to be unalike from other reported APR genes and, therefore, possibly a new major APR QTL for stripe rust.The Chinese cultivar Weimai 8 was developed from crossing 88-3149/Aus621108.The source of QYr.wmy-2BS and QYr.wmy-7DS from Weimai 8 could not be identified by pedigree analysis.The nearby flanking SSR markers Xwmc473 and Xwmc488 could be useful for MAS in developing stripe-rust-resistant wheat cultivars, but the markers used to map the QTL are not sufficiently close enough for cloning the resistance gene.Hence, it will be essential to find uncovered closely associated or co-segregating markers with high-throughput SNP markers or comparative genomics approaches for map-based cloning.
APR to stripe rust is controlled by multiple genes, which may consist of one major gene and several minor genes, or may be composed of several minor genes [10].Minor genes frequently trigger moderate-to-low extents of resistance when existing alone, which likely explains why all three parents had high susceptibility.However, combinations of more such genes possibly give rise to high resistance as stated by [16].Thus, the experiment showed that Weimai 8 contains Lr34/Yr18 which can be developed from several susceptible parents through gene pyramiding.

Figure 2 .
Figure 2. Frequency distributions of SNP loci with 0-1 exchange in resistant and susceptible bulks on different chromosomes.

Figure 2 .
Figure 2. Frequency distributions of SNP loci with 0-1 exchange in resistant and susceptible bulks on different chromosomes.

Figure 3 .
Figure 3. Likelihood plots of stripe rust adult plant resistance QTLs on wheat chromosomes 1B, 2A, 2B, and 7D recognized by inclusive composite interval mapping (ICIM) in the Weimai 8/Zhengzhou 5389 population (A-D).Genetic distances (in cM) between markers are revealed on the left of the vertical axes.

Figure 3 .
Figure 3. Likelihood plots of stripe rust adult plant resistance QTLs on wheat chromosomes 1B, 2A, 2B, and 7D recognized by inclusive composite interval mapping (ICIM) in the Weimai 8/Zhengzhou 5389 population (A-D).Genetic distances (in cM) between markers are revealed on the left of the vertical axes.

Author
Contributions: Z.Y.: designed the experiments.X.Y., X.Z., M.K. and J.X.: performed the experiments and data analysis.Z.Y. and Z.L.: plant collection; X.Y. and T.W.G.: prepared the manuscript.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the National Natural Science Foundation of China [32302364] and the project Excavation and Functional Analysis of the Corn Rust Resistance Gene [NCCIR2022ZZ-4].

Table 1 .
QTLs for final disease severity of stripe rust by inclusive composite interval mapping (ICIM) in recombinant inbred lines (RIL) population derived from Weimai 8/Zhengzhou 5389.