Genetic Mapping of Powdery Mildew Resistance Genes in Wheat Landrace ‘Guizi 1’ Using Genotyping-By-Sequencing

Wheat powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive disease of wheat (Triticum aestivum L.) worldwide that causes severe yield losses. Resistant wheat cultivars easily lose effective resistance against newly emerged Bgt strains; therefore, identifying new resistance genes is necessary for breeding resistant cultivars. ‘Guizi 1’ is a Chinese wheat cultivar with effective moderate and stable resistance against powdery mildew. A genetic analysis indicated that powdery mildew resistance in ‘Guizi 1’ was controlled by a single dominant gene, designated PmGZ1. In total, 110 F 2 individual plants and the 2 parents were used for genotyping-by-sequencing, which produced 23,134 high-quality single-nucleotide polymorphisms (SNPs). The SNP distributions on the 21 chromosomes ranged from 134 on chromosome 6D to 6,288 on chromosome 3B. Chromosome 6A has 1,866 SNPs, among which 16 are located in a physical region between positions 307,802,221 and 309,885,836 in an approximate 2.3-cM region, which possessed the greatest SNP density. The average map distance between SNP markers was 0.1 cM. A quantitative trait locus with a signicant epistatic effect on powdery mildew resistance was mapped to Chromosome 6A. The LOD value of PmGZ1 reached 34.8, and PmGZ1 was located within the condence interval marked by chr6a-307802221 and chr6a-309885836. The phenotypic variance explained by PmGZ1 was 74.7%. Four candidate genes (two each encoding TaAP2-A and actin proteins) were annotated as resistance genes. The present results provide valuable information for wheat genetic improvement, quantitative trait loci ne mapping, and candidate gene validation.


Introduction
Wheat (Triticum aestivum L.), which plays an important role in ful lling the food demand of humans, is a widely cultivated crop in the world [1,2]. Wheat powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive wheat disease worldwide that causes severe yield losses, particularly under humid rainfed conditions [3]. The increased use of nitrogen fertilizers has resulted in powdery mildew becoming progressively a more important problem in wheat production [3,4]. Breeding resistant cultivars is the most economical, effective, and environmentally safe strategy to control powdery mildew [5,6]. Resistant wheat cultivars easily lose effective resistance against newly emerged Bgt strains [6,7]. Therefore, it is necessary to explore new resistance genes to continue breeding resistant cultivars.
Guizhou Province, located in southwestern China, has a complex and changeable climate that is favorable to the pathogenesis of powdery mildew. Therefore, wheat powdery mildew is a very serious problem and an epidemic occurs every year. Wheat landrace 'Guizi 1' (GZ1) is highly resistant to powdery mildew according to many years of eld observations [22]. In this study, genotyping-by-sequencing (GBS) technology was used to identify and map the powdery mildew resistant genes in GZ1. A resistant gene, PmGZ1, was located on chromosome 6A and the high-density genetic linkage map produced for GZ1 will be of great value to molecular breeding and gene cloning in wheat.

Materials And Methods
Plant materials and sample preparation.
Triticum aestivum L. cv. Guizi 1 (Certi cate No. Qian2015003) [23] is cultivated from complex wide-crossing hybrids of Triticum dicoccoides/Triticum durum//Aegilops ventricosa Tausch/Aegilops tauschii Coss. GZ1 and Zhongyang 96-3 (ZY96-3) were cultivated and stored in the Guizhou Sub-Center of National Wheat Improvement Center at the College of Agriculture in Guizhou University. GZ1 showed a moderate and stable resistance to powdery mildew for many years according to eld observations that began in 2010, whereas ZY96-3 showed a susceptibility to powdery mildew. Both varieties were planted on the experimental farm in accordance with the protocol of Li et al. [23], and the eld management (including watering, weeding, and fertilization) was carried out in a uni ed manner. In total, 206 F 2 and the derived F 2:3 plants were obtained from a GZ1/ZY96-3 cross.
Evaluation of powdery mildew reactions.
The reactions of the F 2 and F 2:3 plants to powdery mildew were assessed by inoculation with mixed Bgt isolates (prevalent in Guizhou Province) and Bgt E20, independently. The mixture of Bgt isolates was inoculated into wheat plants at the tilling stage. The E20 isolate was inoculated into wheat plants at the oneleaf stages, and then inoculated plants were grown under a daily cycle of 16 h of light and 8 h of darkness at 20 ± 2°C with 75% relative humidity in a greenhouse. When the susceptible controls were fully infected, the infection types (ITs) were scored in accordance with Xue et al. [24]. There were six IT scores (0, 0;, 1, 2, 3, and 4): "0" indicates immune, no lesions on plants; "0;" indicates nearly immune, hypersensitive necrotic ecks on leaves; "1" indicates highly resistant, small colonies of less than 1-mm diameter and having few conidia on leaves; "2" indicates mildly resistant, the leaves have moderately developed hyphae, the diameters of colonies are less than 1 mm, and some conidia; "3" indicates moderately susceptible, separate non-joined colonies with well-developed hyphae and abundant conidia; "4" indicates highly susceptible, mostly joined colonies with well-developed hyphae and abundant conidia [24,25].
DNA extraction and GBS analysis.
Leaf tissue (0.5-1.0 g) was collected from F 2 plants and the two parents and immediately frozen in liquid nitrogen. Genomic DNA was extracted using the CTAB method [26]. The DNA quality was checked by electrophoresis using 1% agarose gels and quanti ed using a Genova Nano micro-volume spectrophotometer.
Then, the DNA samples were normalized to 30 ng/µL for GBS library construction.
The GBS libraries from 110 F 2 plants and the 2 parents were generated in accordance with the Elshire et al.
method (2011). The DNA was processed for GBS through the Illumina HiSeq TM platform. The clean reads, adapter reads with > 10% N content ratios, and low-quality reads (in which base number of mass value Q ≤ 10 accounted for more than 50% of the whole read) were deleted. The latter two read types were ltered to obtain high-quality clean reads for the following analyses. The high-quality clean reads were subjected to a BLAST search against the Chinese Spring genomic database (IWGSC Refseq v1.0 assembly) using BWA-MEM blast software [27], and then, the detection and selection of single-nucleotide polymorphisms (SNPs) were carried out using Samtools mpileup in accordance with Li et al [28]. The SNPs with separation type "aaxbb" were kept, and SNPs with partial segregation p values less than 0.0001, deletion ratios greater than 30%, or heterozygosity ratios greater than 75% were deleted. Additionally, genotype correction was performed using the Smooth statistical method [29].
Linkage map construction and quantitative trait loci (QTLs) analysis.
The Kasombi mapping function of the quickEst function in ASMap software was used to calculate genetic distances in accordance with the analysis method of Taylor et al. [30], and then, a genetic map was constructed using R/qtl software. Composite interval mapping (CIM) was performed to detect QTLs using the WinQTLCart software v2.5 [31]. QTLs were scanned at a 1-cM window and claimed to be signi cant at an LOD score of 7.07.
Mixed linear composite interval mapping was performed in the software QTLNetwork 2.1 to determine epistatic effects among identi ed QTLs [32,33]. The multiple linear regression at p = 0.05 for selecting parameters in the model with a window size of 10 cM [34]. A threshold, which calculated by 1,000 permutations at a genome-wide error rate of 0.10, was noted as a signi cant QTL interaction [35].

Results
Genetic characteristics of powdery mildew resistance in wheat GZ1.
We carried out eld observations of powdery mildew responses to Bgt for many years. Wheat variety GZ1 showed moderate and stable resistance (IT = 1); however, ZY96-3 was completely susceptible (IT = 4) to mixed Bgts in eld observations. GZ1 was highly resistant (IT = 0); however, ZY96-3 was completely susceptible (IT = 4) to E20 in incubator observations (Fig. 1). Then, the wheat GZ1 was crossed with ZY96-3, all the F 1 plants showed a high resistance (IT = 0) to mixed strains prevalent in Guizhou Province, and the F 2 individual plants showed resistance or susceptibility at the level of IT = 0-4 to the mixed strains, respectively.
Among the 206 F 2 plants, the segregation ratio of the resistant (150) and susceptible (56) individuals t the 3:1 theoretical Mendelian segregation ratio (χ 2 = 0.4143, P = 3.84) ( Table 1). Furthermore, the responses of F 2:3 individual plants to E20 Bgt were detected, and they segregated into 57 homozygous resistant plants, 103 segregating plants, and 46 homozygous susceptible plants, which t the theoretical 1:2:1 ratio (χ 2 = 1.1748, P = 5.99) ( Table 2). Our results demonstrated that the powdery mildew resistance of GZ1 was controlled by a single dominant gene. high-quality SNPs that covered a genetic linkage map of the 21 chromosomes (totally 5,402.12 cM) in wheat being obtained (Fig. S1). The number of SNPs per chromosome ranged from 134 on chromosome 6D to 6,288 on chromosome 3B, and 1,866 SNPs were located on chromosome 6A (8.1% of the total) ( Fig. 2A). Among these SNPs, 16 were located in a physical region between positions 307,802,221 and 309,885,836 in an approximately 2.3-cM region (58.6-60.9 cM) ( Fig. S1 and Fig. 2B). In addition, we found that chromosome 6A possessed the greatest SNP density, and the average map distance between SNP markers was 0.1 cM.

QTL analysis.
A QTL analysis was used to map the resistance genes with CIM, and one Pm-related QTL was detected on chromosome 6A (Fig. 3A). This QTL was designated as PmGZ1. The LOD value of PmGZ1 reached 34.8, and PmGZ1 was located within the con dence interval marked by chr6a-307802221 and chr6a-309885836 (Table  S1), which corresponded to the genetic position of 58.6-60.9 cM (2.3 cM) in the Chinese Spring reference genome. PmGZ1 accounted for 74.7% of the phenotypic variance (Fig. 3B). Thus, the powdery mildew resistance gene PmGZ1 in GZ1 was mapped to chromosome 6A.  (Table  S2). Among the candidate genes, four were annotated as resistance genes, including two genes encoding for a TaAP2-A protein and two for an actin (ACT-1) protein (Table S2).

Discussion
Wheat Pm genes mainly exhibit resistance to speci c Bgt races; however, the Bgt races can easily generate novel viral Bgt isolates through virulent mutations to escape recognition of resistance genes, resulting in the Pm genes losing their ability to generate resistance to powdery mildew [5,36,37]. Therefore, there is a vital need to discover, identify, and utilize new and effective Pm genes for wheat production [38]. GZ1, which is highly resistance to Bgt, was determined to be controlled by a single dominant gene ( Fig. 1; Tables 1 and 2), and GZ1 wheat have shown a stable resistant phenotype in eld observations since 2010.
Here, GBS was used for the genetic analysis and gene mapping of powdery mildew resistance genes of GZ1 wheat. SNPs, which are preferred over other marker systems, are the most common DNA markers for genetic studies in wheat [39,40]. The genotyping of populations using SNPs may produce less accurate and biased results than GBS, which overcomes these problems by identifying high-quality population-speci c SNPs [41,42]. The GBS protocol uses two restriction enzymes (PstI/MspI) for targeting and reducing complex genomes, thereby achieving a more uni ed sequencing library [43,44] using GBS ( Fig. S1 and Fig. 2). In addition, PmGZ1 was detected on chromosome 6A with high an LOD value of 34.8 through CIM, which demonstrated that PmGZ1 was located on chromosome 6A (Fig. 3).
To date, only Pm21 and Pm56 have been mapped to chromosome 6A [13,14]. The SM142 and KU.962 markers that are linked to Pm21 and Pm56, respectively, have been used for polymorphism analysesPmGZ1 was located on the long arm of chromosome 6A, which indicated that PmGZ1 is not Pm56. Distant hybridization of T. dicoccoides, T. durum, A. tauschii, and A. ventricosa was used for GZ1 wheat breeding. However, Pm21 originated from the 6AL·6VS translocation of H. villosa. Additionally, wheat varieties carrying Pm21 are reportedly immune and/or highly resistant to the Bgt isolates [21,50]. The many years of eld observations have indicated that GZ1 has a moderate and stable resistance to powdery mildew. Therefore, PmGZ1 may be a new powdery mildew resistance gene.
PmGZ1 was located in the 7A:307802221-7A:309885836 con dence interval of the Chinese Spring chromosome 7A, which contained 27 putative annotated genes including 4 that may be associated with disease resistance (Table S2). Genes TraesCS6A02G326500LC and TraesCS6A02G326600LC are predicted to encode TaAP2-A proteins that are involved in resistance against the causative pathogen of fusarium head blight [51]. Genes TraesCS6A02G326700LC and TraesCS6A02G327000LC encode actin (ACT-1) proteins, which can stimulate depolymerization to increase plant resistance against pathogens [52]. Further studies are needed to determine the relationships between these genes and powdery mildew resistance and to precisely map PmGZ1 in GZ1 wheat. Figure 1 Resistance of 'Guizi 1' and 'Zhongyan 96-3' to Bgt E20, with 'Huixianhong' as the susceptible control. One-leaf stages of wheat 'Guizi 1', 'Zhongyan 96-3', and 'Huixianhong' were inoculated with Bgt E20. Representative leaves were taken and photographed when 'Huixianhong' showed complete susceptible.