QTL Mapping for Adult Plant Resistance to Stripe Rust in Wheat line "Guixie 3"

Development of cultivars with multiple resistances has proven to be an effective way to prevent diseases in wheat breeding. The Guixie 3 variety (GX3) has shown excellent performance in resistance to stripe rust in eld for many years. The purpose of this study was to detect quantitative trait loci (QTL) associated with resistance to stripe rust in the adult plant stage and determine closely linked molecular markers. A population of recombinant inbred lines (n=228) was derived from a cross between the susceptible landrace Mian 96-5 (M96-5) and GX3 variety and evaluated in multiple eld studies to elucidate genetic resistance by identifying QTL specically for stripe rust resistance. A total of 19 QTL located on 12 chromosomes showed resistance to wheat stripe rust when studying phenotypic data from multiple eld tests over the course of six years. These chromosomes included 1B (2), 1D (2), 2A (2), 2B (2), 2D (1), 4B (2), 4D (1), 5A (3), 5B (1), 6A (1), 6B (1), and 7B (1). Two stable QTL on chromosomes 2AS (Qyr.gaas.2AS) and 6AS (Qyr.gaas.6AS) were detected in six and ve different environments, respectively; both QTL were derived from the GX3 variety. Qyr.gaas.2AS was found to be crucial for increasing adult plant resistance, which may explain the large phenotypic variation of 45.52%. Our results provide theoretical and molecular insight for wheat breeding and suggest the cloning of genes associated with the GX3 variety may be benecial in future studies. et al. 2000), Sr35 (Saintenac et al. 2013), Sr33 (Periyannan et al. 2013), Sr60 (Chen et al. 2018), and Yr10 (Bozkurt et al. 2007). In the present study, we identify Qyr.gaas.2AS (cfd36~AX-110576889) and found a structural region (chr2A: 17411781_17601016) containing multiple NBS-LRRs and the WRKY transcription factor located near the cfd36 marker. We therefore speculate that the structural region in combination with a relatively complete NBS-LRR may represent a novel candidate gene and these 13 identied segments should be further investigated.


Introduction
Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most damaging diseases associated with global wheat production (Wellings 2011). Since 1949, there have been four epidemics of wheat stripe rust in China (1950, 1964, 1990, and 2002), resulting in a loss of more than one million tons of wheat per year . The effective use of disease-resistant varieties is crucial for the control of wheat stripe rust. There are two main types of genetic wheat resistance to stripe rust. One is a resistance that presents itself at the seedling stage (or all-stage resistance [ASR]); this form of resistance is generally effective during the whole growth period. The second is adult plant resistance (APR), which usually provides partial resistance to all races at post-seedling stages.
Epidemics are caused by a loss of effective resistance genes against stripe rust in wheat production .
Currently, wheat varieties carrying the stripe rust resistance gene Yr24/Yr26 that were once widely used in China (Hu et al. 2014), such as Chuanmai 42 (Liu et al. 2010) and Guinong 22 (He et al. 2011), have lost their resistance due to the emergence of a pathogenic group (V26). At the National Wheat Rust and Powdery Mildew Research Collaborative Group Meeting, which took place in China in 2016, the pathogenic group (V26) of Guinong 22 was o cially named Chinese Yellow Rust 34 (CYR34). At present, among the 83 (Yr1-Yr78) o cially designated resistance genes and 47 proposed resistance genes (Maccaferri et al. 2015;McIntosh et al. 2017), only a few seedling disease resistance (ASR) genes (Yr5, Yr15, Yr53, Yr61, Yr64, Yr65, and Yr69) and adult plant disease resistance genes (APR) (Yr18, Yr30, Yr32, Yr36, Yr39, Yr52, Yr54, Yr59, and Yr62) still maintain effective resistance to wheat stripe rust (Hou et al. 2016;Lu et al. 2014;Zhou et al. 2015). Therefore, to ensure the sustainable management of wheat stripe rust in southwest China, it is imperative to discover new stripe rust resistance genes, identify resistance-associated molecular markers, and to ultimately breed new disease resistant wheat varieties.
In recent years, with the development of high-throughput genotyping technology, single nucleotide polymorphism (SNP) arrays have been widely used in wheat. An example includes the construction of a high-density genetic map with stripe rust resistance gene/quantitative trait locus (QTL) mapping (Chen et al. 2016a;Gao et al. 2016;Jighly et al. 2015;Liu et al. 2015;Win eld et al. 2015;Wu et al. 2018a;Wu et al. 2018b) and genome wide association analysis (Kertho et al. 2015;Liu et al. 2017;Naruoka et al. 2015;Zegeye et al. 2014). The wheat 55K SNP array is an economical mediumdensity SNP chip developed from the wheat 660K SNP array (Jia and Zhao 2016) and has been used in many different studies. The 660K SNP array has been used to provide a genetic map of the P genome of Agropyron (Zhou et al. 2018), to identify the gene for grain weight using an integrated genetic map with > 100,000 SNPs (Cui et al. 2017), and to map QTL for stripe rust resistance in adult stage of wheat (Wu et al. 2017).
Wheat wild relatives can be used as a resource bank of disease resistance genes. Some genes have been o cially classi ed for stripe rust resistance (Maccaferri et al. 2015). Wild emmer wheat (T. dicoccoides), a wild tetraploid ancestor of common wheat, has good resistance for wheat stripe rust and importantly shows great potential for wheat breeding. A number of resistant genes have been previously identi ed in wild emmer wheat, such as YrH52, Yr15, Yr35, and Yr36 Peng et al. 1999;Uauy et al. 2005;Wang et al. 2018). Oat belongs to the Gramineae Aveneae Dumort Avena L. variety and has good resistance to biotic (wheat rust and scab) and abiotic (drought, cold, and barren) stress (Han et al. 2008;Sharma and Gill 1983;Zhang 1999). GX3 was obtained by distant hybridisation of wild emmer wheat (T. dicoccoides) with wild oat (Avena fatua L. var. glabrata pat) and then backcrossed with common wheat (Guinong 22). It has shown resistance to the current wheat stripe rust epidemic for many years. In this study, we use a wheat 55K SNP array to map QTL for APR to stripe rust in a GX3 with a recombinant inbred line (RIL) population of "M96-5/GX3", to identify tightly linked molecular markers for their use in future marker-assisted breeding.

Plant materials
The susceptible winter landrace line M96-5, and the resistant line GX3 were used as the parental lines for this study. The mapping population comprised of 228 F 6:7 RILs from crossing M96-5×GX3. The GX3 line is a semi-winter, late maturity, long spikelet variety of common wheat and its entire growth period is an estimated 210 days. The susceptible line M96-5 has large spikelet with excellent agronomic traits and its growth period is an estimated 190 days. The RIL population was established by Dr. Zhou Qiang from the Chengdu Institute of Biology, Chinese Academy of Sciences (Chengdu, Sichuan Province). Avocet S (AVS) and SY95-71 were used as susceptible controls throughout the study.

Phenotyping
In this eld trials, two parents and RIL populations were used to test resistance in mixed races of stripe rust within a natural setting. These were planted in October 2016 in Mianyang, Sichuan Province (31°23′N, 104°49′E); in October 2017, 2018, and 2019 in Guiyang, Guizhou Province (26°29′N, 106°39′E), and in November 2019 in Anshun, Guizhou Province (26°24′N, 105°96′E) and Shuangliu, Sichuan Province (30°57′N, 103°92′E). There were two rows per line, 10 lines per block, and the susceptible line AVS (or SY95-71) was planted every ve lines as the control line. The rst severe degree was recorded when the disease severity of AVS (or SY95-71) in the control group and the susceptible parent M96-5 reached 50% or more (i.e., the area of rust fungus accounted for more than half of the entire leaf). In accordance with the percentage of the total leaf area occupied by rust fungus, the severity of stripe rust was also recorded visually for each wheat family. Recordings were taken every other week until the susceptible control reached 100%, known as the maximum disease severity (MDS). The modi ed Cobb scale was referred to for the phenotypic data required for QTL analysis (i.e., 1, 5, 10, 20, 30, 50, 60, 80, and 100%) (Li and Zeng 2002).

Statistical analysis
To estimate the genetic and environmental effects in each line, we compared the environments and line×environment interactions using the AOV function in IciMapping 4.1 software (Zeng et al. 2019) and signi cance was measured by analysis of variance (ANOVA). The correlation between multiple eld conditions was analysed by the Pearson method in SPSS v20 software.
Two parents and RIL populations were genotyped using the 55K SNP array by China Golden Marker (Beijing) Co., Ltd.
(http://www.cgmb.com.cn/). Basic quality control (QC) tests were performed on samples by measuring markers based on genotype data detection rate, minimum allele frequency (MAF), and heterozygosity. The criteria used for sample quality control were as follows: DQC > 0.82, detection rate ≥ 85% and heterozygosity rate ≤ 10%; the criteria for marking quality control were as follows: detection rate greater than or equal to 95%, and MAF of 5% or more, heterozygosity rate of 50% or less, and the number of alleles was 2.
Based on the preliminary results of QTL mapping, 22 pairs of simple sequence repeat (SSR) markers located on the 2AS chromosome were selected for genetic map construction (see Table S1). These markers were identi ed from GrainGenes (https://wheat.pw.usda.gov/GG3/) (Somers et al. 2004) and PCR reactions and polyacrylamide electrophoresis were performed as previously reported (Wu et al. 2018a). To distinguish the difference between Yr17 and major QTL on chromosome 2AS identi ed in this study, the speci c CAPS marker URIC/LN2 for Yr17 was used to scan wheat lines with GX3 pedigree and the carrier line of Yr17 gene (VPMI) and followed by digesting of restriction enzyme DpnII. Detecting procedure for URIC/LN2 was processed according to previously report (Helguera et al. 2003).

Map construction and QTL analysis
The BIN function in QTL IciMapping v4.1 software (http://www.isbreeding.net/) (Meng et al. 2015) was used for redundant marker screening. In the mapping software, Joinmap v4.0, the LOD (Likelihood of odd) value was set to 3.0 for linkage analysis and genetic map distance was calculated using the Kosambi function. QTL analysis was performed using QTL IciMapping v4.1 software and the LOD threshold was set to 2.5. Mapchart (https://www.wur.nl/en/show/Mapchart.htm) was used to draw images (Voorrips 2002). The R package R/qtl was used to draw the genetic map (Broman et al. 2003).

Prediction of candidate genes
According to the derivation region of the QTL on the target chromosome, the physical position of the SNP in Chinese Spring was used to search in International Wheat Genome Sequencing Consortium (IWGSC) (https://wheaturgi.versailles.inra.fr/Seq-Repository/Annotations) and the corresponding gene annotation and sequence was obtained.

Evaluation of resistance
In the six different eld trials, phenotypic data indicated signi cant genetic variation in APR. M96-5 and GX3 obtained mean MDS scores of 100% and 0%, respectively. MDS of the mapping populations ranged from 0 to 100% in each eld study and the phenotypic data were continuously distributed ( Figure 1). Pearson's correlation among the six eld trials ranged from 0.38 to 0.79 (P < 0.001) ( Table 2). ANOVA analysis including replicates for each experiment showed that there was signi cant variation in MDS when comparing different locations and wheat lines. Wheat lines combined with environment interactions also differed signi cantly and the heritability between different locations was also found to be signi cantly high (0.91). These results indicated that the QTL in APR had a dramatic effect in decreasing disease severity ( Table 1).

Construction of genetic linkage map
Whole genome analysis of the two parental lines and 228 RILs was performed using the wheat 55K SNP array. Within the 55, 000 SNPs, 7570 were identi ed as polymorphic markers to distinguish between M96-5 and GX3. A total of 589 were excluded because they had missing data (> 10%) or showed segregation distortion. The remaining 6981 SNPs fell into 1543 bins and 5438 SNPs were excluded.
Preliminary localisation was performed using IciMapping 4.1 software. These results showed that an important QTL was located at the end of chromosome 2AS with an estimated LOD value of 40. We therefore selected 22 pairs of SSR primers at the distal region of chromosome 2AS and performed PCR ampli cation on the parental lines (GX3 and M96-5), a disease resistant pool (B R ), and a susceptible pool (B S ). Four markers (cfd36, wmc382, barc124, and wmc296) showed successful ampli cation of consistent polymorphic bands between the resistant parents and the resistant pools. The four SSR markers were then used in combination with the 1543 SNPs obtained from genotyping data to analyse the QTL for resistance to wheat stripe rust at the adult plant stage. The nal genetic map included 21 linkage groups corresponding to the 21 chromosomes (Figure 2 and Table 3 (Table 4).

Candidate gene prediction
According to the physical locations of Qyr.gaas.2A (cfd36~AX-110576889, 15.22Mb) and Qyr.gaas.6A (AX-109558600~AX-109542604, 5.12Mb), the sequences located within the interval of two QTL were searched in IWGSC to identify the wheat gene ID, annotation, and corresponding sequence. The results identi ed 620 and 61 segments at the con dence intervals of Qyr.gaas.2A and Qyr.gaas.6A, respectively (Table S2 and Table S3). The two intervals contained potentially functional genes such as nucleotide binding site-leucine rich repeat (NBS-LRR), a disease resistance protein, F-box protein, or part of the gene structure directly or indirectly involved in plant disease resistance (Table 6). Of note, 13 fragments related to disease resistance (10 NBS-LRR and 3 WRKY transcription factors) were clustered on a region of the 2AS chromosome spanning from 17,411,781bp to 17,601,016bp.

Discussion
Wheat stripe rust and phenotypic data Until 2016, China had o cially classi ed 34 stripe rust races (CYR1-CYR34) and more than 40 pathogenic types . Since the discovery of a new pathogenic type, V26 in 2009, this pathogenic group has been continuously mutating and expanding. Its toxicity pro le has expanded from an initial infection of 12 to 18 Chinese identi ed hosts and a survey showed that the frequency of the three pathogenic groups of CYR32, CYR33, and V26 has since exceeded 70% . In 2016, the pathogenic group (V26) of Guinong 22 was o cially named CYR34 and this has led to an increase in its investigation within the eld of wheat research in China.
The Sichuan Basin is a common source of new races of wheat stripe rust in China. Since the pathogenic group (V26) of Guinong 22 was rst identi ed in Sichuan in 2009, it has gradually increased to become the dominant pathogenic group (Xu et al. 2016). At present, there are three main pathogenic groups of stripe rust in Guizhou; namely, the hybrid pathogenic group, the water source 11 pathogenic group, and the Guinong 22 pathogenic group. Among them, the frequencies of CYR32, CYR33, and CRY34 are 57.14%, 5.71%, and 4.29%, respectively (Chen et al. 2016b). When analysing the different eld phenotypes of RILs between 2017 and 2020, signi cantly more susceptible varieties were found to originate in Sichuan compared to Guizhou. This difference is mainly due to the different epidemic races of stripe rust found in these two regions. Speci cally, CYR32 and CYR33 are found mainly in Guizhou while CRY34 is predominantly found in Sichuan (Cheng et al. 2020).
In addition, different varieties carry multiple resistance genes and therefore show different degrees of resistance although none are completely immune. For each physiological race, selection pressure is greatly reduced, as the host and the pathogen are in a coexisting state and it is therefore unlikely that new mutations will develop in the pathogen.
Due to this phenomenon, the resistance of slow-rust varieties is low and the resistance in specialised varieties is stronger (Yuan et al. 1995).

QTL mapping
In general, QTL that exert a larger effect on phenotype tend to be more stably expressed and are more easily detected within different environments. QTL that exert a smaller effect tend to be more in uenced by genetic background and the external environment. These can therefore be more di cult to detect under certain environmental conditions (Li et al. 2010). In this study, two stable QTL were identi ed on the chromosomes 2AS and 6AS.
At the end of chromosome 2AS, a signi cant stable QTL (Qyr.gaas.2AS) was detected at the interval of cfd36~AX-110576889 (3.5-5.5cM) in all six environments assessed in this study. Five genes for the resistance of stripe rust were identi ed on chromosome 2AS (Yr17 [0-20cM], Yr56 [2.0-6.3cM], YrR61 [1.9-16.8cM], Yr69 [13.9-17cM], and YrZM175 [47.8-52.1cM]). Through pedigree analysis, we found that YrR61 was derived from the American soft red wheat Pioneer 26R61 (Hao et al. 2011), and Yr56 from the durum wheat, Wollaro (Bansal et al. 2014). Both of the identi ed genes are known to promote adult resistance. Yr69 (Hou et al. 2016) and YrZM175 are known to be genes that promote seedling resistance in the arti cial wheat introgression line, CH7086, and common wheat variety, Zhongmai 175 (Lu et al. 2016), respectively. Yr17, derived from Ae. Ventricosa, is also a seedling-resistant gene (Bariana and McIntosh 1993). The above genes have loss disease resistance to CYR34 race according to previous report . However, our eld investigations showed that VPMI (the carrier line of Yr17 gene) present resistant to the pathogenic group of Guinong 22 in Guiyang. Moreover, Yr17 had the closest position to Qyr.gaas.2AS, the speci c CAPS marker URIC/LN2 of Yr17 (Helguera et al. 2003) was used to further determine the differences between them. Our results showed that target bands could not be ampli ed in the GX3 line (Figure 4). Future experiments are required for the hybridisation of GX3 and the carrier line of Yr17, VPMI. Furthermore, 13 QTL have been previously reported on chromosome 2AS (Bulli et al. 2016), of which eight are adjacent to or overlapping with Qyr.gaas.2AS. GX3 is different from the parental lines of these disease-resistant QTL, so we therefore speculate that Qyr.gaas.2AS in GX3 is inconsistent with the above QTL.

Candidate gene prediction
Response to biological stress in plants can often be dependent on a variety of cell receptor proteins. Intracellular receptor proteins are encoded by NBS-LRR disease resistance genes, which have the ability to directly or indirectly recognise effector molecules (Effector) released by the pathogen into the cell and trigger a disease resistance response.
This response is referred to as effector triggered immunity (Noutoshi et al. 2005) and requires mediation of the transcription factor WRKY. The NBS domain binds to ATP or GTP to play a key role in plant disease resistance. Currently, multiple wheat NBS-LRR genes have been identi ed and linked to disease resistance, such as Lr1 (Qiu et al. 2007), Lr10 (Bozkurt et al. 2007), Lr21 and Lr40 (Spielmeyer et al. 2000), Sr35 (Saintenac et al. 2013), Sr33 (Periyannan et al. 2013), Sr60 , and Yr10 (Bozkurt et al. 2007). In the present study, we identify Qyr.gaas.2AS (cfd36~AX-110576889) and found a structural region (chr2A: 17411781_17601016) containing multiple NBS-LRRs and the WRKY transcription factor located near the cfd36 marker. We therefore speculate that the structural region in combination with a relatively complete NBS-LRR may represent a novel candidate gene and these 13 identi ed segments should be further investigated.
GX3 was derived from wild crossing between wild emmer wheat (T. dicoccoides) and wild oat (Avena fatua L. var.glabrata pat) varieties and was subsequently obtained by backcrossing with the common wheat Guinong 22 variety. Field observations spanning many years have shown that wild emmer wheat and Guinong 22 are susceptible to CRY34, while the wild oat is immune. Therefore, we speculate that the major QTL on chromosome 2AS originated from Avena fatua L.var.glabrata. However, in situ hybridisation analysis showed no signal detection in the wild oat variety samples (unpublished data). These ndings may be due to the fact that the introgression fragment is too small. These experiments therefore require further validation.

Conclusions
In summary, GX3 carried two stable QTL for stripe rust resistance, of which Qyr.gaas.2AS is a major QTL. A resistance gene on the 2AS chromosome has not been previously reported and is therefore a novel candidate for genetic resistance in wheat. GX3 could be used in future breeding of disease resistant wheat. Future investigations should focus on increasing the density of genetic maps around Qyr.gaas.2AS to clone the underlying gene.