Genetic Basis Identification of a NLR Gene, TaRGA5-like, That Confers Partial Powdery Mildew Resistance in Wheat SJ106

Wheat powdery mildew is an important fungal disease that seriously jeopardizes wheat production, which poses a serious threat to food safety. SJ106 is a high-quality, disease-resistant spring wheat variety; this disease resistance is derived from Wheat-wheatgrass 33. In this study, the powdery mildew resistance genes in SJ106 were located at the end of chromosome 6DS, a new disease resistance locus tentatively named PmSJ106 locus. This interval was composed of a nucleotide-binding leucine-rich repeat (NLR) gene cluster containing 19 NLR genes. Five NLRs were tandem duplicated genes, and one of them (a coiled coil domain–nucleotide binding site–leucine-rich repeat (CC-NBS-LRR; CNL) type gene, TaRGA5-like) expressed 69–836-fold in SJ106 compared with the susceptible control. The genome DNA and cDNA sequences of TaRGA5-like were amplified from SJ106, which contain several nucleotide polymorphisms in LRR regions compared with susceptible individuals and Chinese Spring. Overexpression of TaRGA5-like significantly increased resistance to powdery mildew in susceptible receptor wheat Jinqiang5. However, Virus induced gene silence (VIGS) of TaRGA5-like resulted in only a small decrease of SJ106 in disease resistance, presumably compensated by other NLR duplicated genes. The results suggested that TaRGA5-like confers partial powdery mildew resistance in SJ106. As a member of the PmSJ106 locus, TaRGA5-like functioned together with other NLR duplicated genes to improve wheat resistance to powdery mildew. Wheat variety SJ106 would become a novel and potentially valuable germplasm for powdery mildew resistance.


Introduction
Wheat powdery mildew is an important fungal disease that seriously jeopardizes wheat production.Over the years, the wheat yield loss caused by Blumeria graminis f. sp.tritici (Bgt) generally reaches 10-15% and up to 50% in severe cases [1].Selection and promotion of varieties resistant to powdery mildew is an important objective of wheat genetic improvement and always the most cost-effective measure to ensure food security and maintain green and safe agriculture.
SJ106 was the high-generation sister line of the wheat variety Jinqiang9, which bred from the 03 Yuan Yin-15/02K-53) F 6 generation line as a male parent and (315//Wheatwheatgrass 33//02K-53, 3/02K-53-5) F 4 generation line as a female parent [27].SJ106 is a high-quality, disease-resistant spring wheat line that is resistant to powdery mildew and moderately resistant to leaf rust and stripe rust.SJ106 showed strong resistance to powdery mildew during whole growth, suggesting that it is a potential powdery mildew-resistant gene donor.Therefore, it is of great significance to carry out the genetic location, cloning, and functional analysis of the powdery mildew resistance gene in SJ106 and transfer it to wheat varieties with excellent agronomic traits for wheat breeding.
In this study, we used Chinese Spring and SJ106 to construct F 2 and F 2:3 populations and located a new disease resistance locus.PmSJ106 locus was located at the end of chromosome 6DS by bulked segregant RNA-Seq (BSR-seq) and simple sequence repeat (SSR).In this locus, nineteen NLR genes were located, five NLRs were duplicated gene clusters, and one of them (coiled coil domain-nucleotide binding site-leucine-rich repeat (CC-NBS-LRR; CNL), TaRGA5-like) within this cluster was expressed 69-836-fold in SJ106 compared with the susceptible control.Over-expression of TaRGA5-like significantly increased resistance to powdery mildew in susceptible receptor wheat.However, VIGS silencing of TaRGA5-like resulted in only a small decrease in disease resistance in SJ106, presumably compensated by other duplicated NLRs.The above results suggested that the duplicated NLRs in PmSJ106 gene clusters functions together in wheat resistance to powdery mildew infection.TaRGA5-like, as a member of this gene cluster, confers partial powdery mildew resistance in SJ106.

Genetic Analysis of Powdery Mildew Resistance in SJ106
In order to investigate the disease resistance of SJ106, we constructed F 2 and F 2:3 populations using Chinese Spring and SJ106.After 10 days post inoculation (dpi) with Bgt E09, there were no visible powdery mildew spores on the surface of SJ106 leaves (infection types, IT = 0), which showed immunity to powdery mildew.In contrast, Chinese Spring wheat was highly susceptible to powdery mildew with a lesion area greater than 80%, and the IT grade was 4 (Figure 1).The F 1 plants from the cross between Chinese Spring and SJ106 showed immunity or near-immunity to Bgt E09 (IT = 0, 0;), which was consistent with the types of responses to Bgt infection in SJ106.The F 2 plants showed different Bgt E09 resistance (Figure 1).The segregation ratio of F 2 individuals of Chinese Spring × SJ106 was close to 3:1.The segregation ratio of the F 2:3 family lines was also consistent with 1:2:1 (Table S2).Therefore, it was hypothesized that the seedling resistance of wheat SJ106 to Bgt E09 was controlled by a dominance major gene locus, and it was tentatively named PmSJ106 locus.

SNP Calling and Confirmation of Candidate Interval
The total RNA of 40 F2-resistant individuals and 40 susceptible individuals were mixed respectively to construct the resistance/susceptibility pools and sequenced by BSRseq.A total of 79,381,812 and 76,097,016 raw reads were retained after quality control.A total of 77,458,450 and 73,566,956 clean reads were obtained from the resistance/susceptibility pools, respectively.The percentages of clean reads with a Q30 were 90.34% and 91.74%, and the GC contents were 57.33% and 56.77%, respectively.A total of 65,018 highquality differential SNPs were selected for subsequent SNP index analysis.Using the SNP index and 0.21 as the threshold, two candidate intervals were located on chromosome 6A (22.02-61.42Mb) and 6D (7.7-56.02Mb), respectively (Figure 2).

SNP Calling and Confirmation of Candidate Interval
The total RNA of 40 F 2 -resistant individuals and 40 susceptible individuals were mixed respectively to construct the resistance/susceptibility pools and sequenced by BSR-seq.A total of 79,381,812 and 76,097,016 raw reads were retained after quality control.A total of 77,458,450 and 73,566,956 clean reads were obtained from the resistance/susceptibility pools, respectively.The percentages of clean reads with a Q30 were 90.34% and 91.74%, and the GC contents were 57.33% and 56.77%, respectively.A total of 65,018 high-quality differential SNPs were selected for subsequent SNP index analysis.Using the SNP index and 0.21 as the threshold, two candidate intervals were located on chromosome 6A (22.02-61.42Mb) and 6D (7.7-56.02Mb), respectively (Figure 2).

Mapping of Powdery Mildew Resistance Gene
In order to localize the R genes in the SJ106 chromosome, we used 1388 pairs of specific primers distributed on 21 wheat chromosomes to screen for specific markers in the F2 resistance/susceptibility pools of SJ106 and Chinese Spring, the resistance/susceptibility

Mapping of Powdery Mildew Resistance Gene
In order to localize the R genes in the SJ106 chromosome, we used 1388 pairs of specific primers distributed on 21 wheat chromosomes to screen for specific markers in the F 2 resistance/susceptibility pools of SJ106 and Chinese Spring, the resistance/susceptibility pools of the F 2:3 family lines, and the F 2 individuals.It was found that seven SSR markers (BARC183, BARC365, CFD49, TC93912, TC91050, TC66898, and BJ212775) were able to consistently amplify the polymorphic bands in parents, resistance/susceptibility pools, and resistant and susceptible individuals (Figure 3).A genetic linkage map was constructed based on the seven SSR markers.The powdery mildew resistance gene PmSJ106 was located on chromosome 6DS and flanked by the markers BARC183 and TC93912, with genetic distances of 1.6 cM and 2.8 cM, respectively (Figure 4).The candidate region of the PmSJ106 locus was located between 4.90-8.97Mb using the IWGSC Chinese Spring reference genome v1.1 (Figure 4).Combined with 2.2 results, the candidate region was located on chromosome 6DS, and the physical region was between 4.90-8.97Mb.As no Pm genes were reported in this region, the PmSJ106 locus is likely to contain a novel Pm-resistant gene.

Mapping of Powdery Mildew Resistance Gene
In order to localize the R genes in the SJ106 chromosome, we used 1388 pairs of specific primers distributed on 21 wheat chromosomes to screen for specific markers in the F2 resistance/susceptibility pools of SJ106 and Chinese Spring, the resistance/susceptibility pools of the F2:3 family lines, and the F2 individuals.It was found that seven SSR markers (BARC183, BARC365, CFD49, TC93912, TC91050, TC66898, and BJ212775) were able to consistently amplify the polymorphic bands in parents, resistance/susceptibility pools, and resistant and susceptible individuals (Figure 3).A genetic linkage map was constructed based on the seven SSR markers.The powdery mildew resistance gene PmSJ106 was located on chromosome 6DS and flanked by the markers BARC183 and TC93912, with genetic distances of 1.6 cM and 2.8 cM, respectively (Figure 4).The candidate region of the PmSJ106 locus was located between 4.90-8.97Mb using the IWGSC Chinese Spring reference genome v1.1 (Figure 4).Combined with 2.2 results, the candidate region was located on chromosome 6DS, and the physical region was between 4.90-8.97Mb.As no Pm genes were reported in this region, the PmSJ106 locus is likely to contain a novel Pm-resistant gene.

PmSJ106 Locus Region in Pan-Genome Is Enriched with Multiple NLR and Kinase Genes
Since BARC183 and TC93912 are molecular markers linked to disease resistance genes, the candidate genes in this interval were analyzed.To obtain a more exhaustive list of gene information, we analyzed the corresponding candidate intervals in several hexaploid wheats and durum wheat.The size of the PmSJ106 locus ranged between 4.07 to 7.44 Mb across the pan-genome, and there were 117 to 482 genes within this interval.As reported, Pm genes were always NLRs, transporter, and kinase, such as Pm21 and Pm13.We further annotated candidate genes in this region.Nineteen genes were predicted to encode NLRs, and forty encoded kinase proteins.Fifteen of forty kinases were annotated as LRR-

PmSJ106 Locus Region in Pan-Genome Is Enriched with Multiple NLR and Kinase Genes
Since BARC183 and TC93912 are molecular markers linked to disease resistance genes, the candidate genes in this interval were analyzed.To obtain a more exhaustive list of gene information, we analyzed the corresponding candidate intervals in several hexaploid wheats and durum wheat.The size of the PmSJ106 locus ranged between 4.07 to 7.44 Mb across the pan-genome, and there were 117 to 482 genes within this interval.As reported, Pm genes were always NLRs, transporter, and kinase, such as Pm21 and Pm13.We further annotated candidate genes in this region.Nineteen genes were predicted to encode NLRs, and forty encoded kinase proteins.Fifteen of forty kinases were annotated as LRR-receptor like protein kinases (Table S3).
To determine the genetic relationship of NLRs and kinases, we obtained the gene sequences of the Chinese Spring genome (V2.1) and align them using multiple sequence alignment tool.According to the previous reports, this resulted in the formation of two distinct clusters, of which group I contained seven of the nineteen up-regulated NLR genes (Figure 5A).The kinases formed three clusters, each containing a single up-regulated kinase gene (Figure 5B).The tandem duplicated analysis of NLRs within the candidate intervals showed that TraesCS6D03G0026700, TraesCS6D03G0026800, TraesCS6D03G0027300, TraesCS6D03G0027400, and TraesCS6D03G0027500 were tandem duplicated genes.The on-line expression analysis results showed that TraesCS6D03G0027500 expressed higher after Bgt infection and lower in susceptible CS (Figure S1).The full length of the TraesCS6D03G0027500 homologous gene was amplifi SJ106, which had a DNA length of 4254 bp, an open reading frame of 3138 bp, and enc 1045 amino acid residues (Figure 6).There were 17 different amino acids (Figure S2 protein functional structure domains were predicted by NCBI/CD-Search, and i found to have a typical N-terminal RxCC like, nucleotide-binding-site (NBS), and leu rich repeat (LRR) domain belonging to the CNL protein family, sharing high hom with RGA5-like (XP_044417598.1) and termed as TaRGA5-like.The homologous gen TraesCS6D03G0027500 were identified in other wheat varieties (Table S2).The sequ alignment result showed that they share high homology, and there were some nucle differences in LRR regions (Figure S3).The comparison of LRR region sequences sh that resistance individuals were almost consistent, while the susceptible individuals the same as the sequence in Chinese Spring genome (Figure S4).

Cloning of the Candidate Powdery Mildew Resistance Gene in SJ106
The full length of the TraesCS6D03G0027500 homologous gene was amplified in SJ106, which had a DNA length of 4254 bp, an open reading frame of 3138 bp, and encoded 1045 amino acid residues (Figure 6).There were 17 different amino acids (Figure S2).The protein functional structure domains were predicted by NCBI/CD-Search, and it was found to have a typical N-terminal RxCC like, nucleotide-binding-site (NBS), and leucinerich repeat (LRR) domain belonging to the CNL protein family, sharing high homology with RGA5-like (XP_044417598.1) and termed as TaRGA5-like.The homologous genes of TraesCS6D03G0027500 were identified in other wheat varieties (Table S2).The sequences alignment result showed that they share high homology, and there were some nucleotide differences in LRR regions (Figure S3).The comparison of LRR region sequences showed that resistance individuals were almost consistent, while the susceptible individuals were the same as the sequence in Chinese Spring genome (Figure S4).SJ106, which had a DNA length of 4254 bp, an open reading frame of 3138 bp, and encoded 1045 amino acid residues (Figure 6).There were 17 different amino acids (Figure S2).The protein functional structure domains were predicted by NCBI/CD-Search, and it was found to have a typical N-terminal RxCC like, nucleotide-binding-site (NBS), and leucinerich repeat (LRR) domain belonging to the CNL protein family, sharing high homology with RGA5-like (XP_044417598.1) and termed as TaRGA5-like.The homologous genes of TraesCS6D03G0027500 were identified in other wheat varieties (Table S2).The sequences alignment result showed that they share high homology, and there were some nucleotide differences in LRR regions (Figure S3).The comparison of LRR region sequences showed that resistance individuals were almost consistent, while the susceptible individuals were the same as the sequence in Chinese Spring genome (Figure S4).

Expression Analysis of TaRGA5-like
The expression patterns in Chinese Spring and SJ106 were analyzed after Bgt infection (Figure 7).In resistant wheat SJ106, expression levels began to be significantly up-regulated at 12 hpi, and the highest expression was reached at 5 dpi, although it began to be downregulated later.The expression level remains significantly higher than the non-infected control.In Chinese Spring, gene expression levels were very low and began to decrease after infection.The expression levels of the disease-resistant SJ106 were 69-836 times higher than that of Chinese Spring (Figure 7).

Expression Analysis of TaRGA5-like
The expression patterns in Chinese Spring and SJ106 were analyzed after Bgt infection (Figure 7).In resistant wheat SJ106, expression levels began to be significantly upregulated at 12 hpi, and the highest expression was reached at 5 dpi, although it began to be down-regulated later.The expression level remains significantly higher than the noninfected control.In Chinese Spring, gene expression levels were very low and began to decrease after infection.The expression levels of the disease-resistant SJ106 were 69-836 times higher than that of Chinese Spring (Figure 7).

Subcellular Localization of TaRGA5-like
Tobacco leaves were infiltrated with Agrobacterium tumefaciens containing a binary vector carrying TaRGA5-like-GFP to transiently express and observe TaRGA5-like-GFP with a fluorescence microscopy.35S: GFP distributed the whole cells.TaRGA5-like-GFP was localized in the cell membrane (Figure 8).This indicated that TaRGA5-like was localized in the cell membrane.

Subcellular Localization of TaRGA5-like
Tobacco leaves were infiltrated with Agrobacterium tumefaciens containing a binary vector carrying TaRGA5-like-GFP to transiently express and observe TaRGA5-like-GFP with a fluorescence microscopy.35S: GFP distributed the whole cells.TaRGA5-like-GFP was localized in the cell membrane (Figure 8).This indicated that TaRGA5-like was localized in the cell membrane.

Subcellular Localization of TaRGA5-like
Tobacco leaves were infiltrated with Agrobacterium tumefaciens containing a binary vector carrying TaRGA5-like-GFP to transiently express and observe TaRGA5-like-GFP with a fluorescence microscopy.35S: GFP distributed the whole cells.TaRGA5-like-GFP was localized in the cell membrane (Figure 8).This indicated that TaRGA5-like was localized in the cell membrane.

VIGS of of TaRGA5-like
To further analyze the function of TaRGA5-like, we transiently silenced TaRGA5-like using virus-induced gene silencing (VIGS).RT-qPCR results showed that the expression level of TaRGA5-like was significantly down-regulated in TaRGA5-like silenced leaves (Figure S5).After inoculation with Bgt for 7d, 10d, and 14d, the phenotypes of leaves look similar.After microscopic observation, it was found that the average amounts and densities of Bgt spores on TaRGA5-like silenced leaves increased from 145 to 557 at 7 dpi and from 1908 to 3702 at 14 dpi (Table S4).The amounts of spores on TaRGA5-like silenced leaves were higher than that in the controls, which indicated disease resistance reduction.The proportions of hypersensitive reactions (HR) in the leaves decreased from 12.46‰ to 8.40‰ at 7 dpi and from 28.13‰ to 20.56‰ at 14 dpi (Figure S6).The reduction of HR in TaRGA5-like silenced leaves indicated that a decreased disease resistance (Figure 9).The above results indicated that TaRGA5-like silencing reduced powdery mildew resistance in SJ106 to some extent.To further analyze the function of TaRGA5-like, we transiently silenced TaRGA5-like using virus-induced gene silencing (VIGS).RT-qPCR results showed that the expression level of TaRGA5-like was significantly down-regulated in TaRGA5-like silenced leaves (Figure S5).After inoculation with Bgt for 7d, 10d, and 14d, the phenotypes of leaves look similar.After microscopic observation, it was found that the average amounts and densities of Bgt spores on TaRGA5-like silenced leaves increased from 145 to 557 at 7 dpi and from 1908 to 3702 at 14 dpi (Table S4).The amounts of spores on TaRGA5-like silenced leaves were higher than that in the controls, which indicated disease resistance reduction.The proportions of hypersensitive reactions (HR) in the leaves decreased from 12.46‰ to 8.40‰ at 7 dpi and from 28.13‰ to 20.56‰ at 14 dpi (Figure S6).The reduction of HR in TaRGA5-like silenced leaves indicated that a decreased disease resistance (Figure 9).The above results indicated that TaRGA5-like silencing reduced powdery mildew resistance in SJ106 to some extent.

Over-Expression of of TaRGA5-like in Susceptible Wheat
We constructed the ubi:TaRGA5-like vector, which was transferred into susceptible spring wheat Jinqiang5.After PCR analysis, two transgenic wheat plants were selected (Figure S6).The transgenic wheat was cultured to the one-leaf stage and inoculated with Bgt for 7 d.The results showed that T1 transgenic plants were resistant to Bgt E09.The lesion area on the leaf surfaces of transgenic wheat was significantly lower than that of the control (Figure 10A).The lesion area decreased from nearly 60% to 19.45% and 26.69, respectively (Figure 10B, Table S4).At 7 dpi, fewer secondary hyphae (SH) or beaded conidia (BC) grew on the TaRGA5-like OE leaves, whereas the control leaves were major covered by BC (Figure 11A).Statistical analysis showed that the percentage of successful penetration was lower in all OE wheat leaves than that in the controls (Figure 11B), whereas the

Over-Expression of of TaRGA5-like in Susceptible Wheat
We constructed the ubi:TaRGA5-like vector, which was transferred into susceptible spring wheat Jinqiang5.After PCR analysis, two transgenic wheat plants were selected (Figure S6).The transgenic wheat was cultured to the one-leaf stage and inoculated with Bgt for 7 d.The results showed that T 1 transgenic plants were resistant to Bgt E09.The lesion area on the leaf surfaces of transgenic wheat was significantly lower than that of the control (Figure 10A).The lesion area decreased from nearly 60% to 19.45% and 26.69, respectively (Figure 10B, Table S4).At 7 dpi, fewer secondary hyphae (SH) or beaded conidia (BC) grew on the TaRGA5-like OE leaves, whereas the control leaves were major covered by BC (Figure 11A).Statistical analysis showed that the percentage of successful penetration was lower in all OE wheat leaves than that in the controls (Figure 11B), whereas the macrocephalic appressoria and hypersensitive reactions rates were the opposite (Figure 11C,D).The OE results indicated that over-expression of TaRGA5-like in Jinqiang 5 improved wheat resistance.

Discussion
Cultivating resistant varieties is an economical, effective, and environmentally friendly measure.But due to the rapid evolution of pathogens, resistance in varieties is often overcome, resulting in disease re-emergence [22,28].This requires new disease-resistant varieties with durable, broad-spectrum resistance characteristics.Most R genes encode typical immune receptors.Although individual R gene resistance is easily overcome by targeted selection of pathogenic strains, R-gene polymerization can result in durable, broadspectrum disease resistance.Further research on NLRs and the integrating structural domains (IDs) that act as decoys for pathogen effectors is still very important.The core idea of R-gene polymerization is that, as the number of genes introduced, transformed, or cultivated into the same crop variety increases, the difficulty for pathogens to evolve corresponding multi-gene polymerization resistance virulence becomes greater.For example, different degrees of broad-spectrum resistance have been obtained by transgenic technology by directly transferring the wheat adult-phase persistent multi-resistance genes Lr34/Yr18/Sr57/Pm38 into several crops, including durum wheat, barley, and rice [29][30][31].Transformation of wheat NLR genes Sr22, Sr33, Sr35, and Sr45 into the susceptible barley variety Golden Promise exhibited small species-specific resistance to barley rust without any significant agronomic effects [32].
SJ106 is a high-quality spring wheat variety, resistant to powdery mildew and moderately resistant to leaf rust and stripe rust.Based on the hybridized combinations of SJ106, it was hypothesized that the source of resistance in SJ106 was from the disease-resistant wheat variety Wheat-wheatgrass 33.In this study, we constructed F 2 and F 2:3 genetic populations of Chinese Spring/SJ106.The powdery mildew-resistant gene in SJ106 was located at the end of chromosome 6DS (4.90-8.97Mb) using SSR BSR-seq.In previous reports, only one, Pm45, was identified on wheat chromosome 6D (105.5-160.4Mb), which was far away from the gene locus we localized [28].No Pm gene was reported in the localized interval, except for a Sr42 locus [33].
By comparing several hexaploid wheat genomes and durum wheat sequences, it was found that this interval contained 19 NLR gene loci, and the frequency of NLR distribution (19/4.07Mb) was much higher than the average frequency of distribution of NLR loci in the wheat genome (2500/16,000 Mb), and it was a cluster of NLR genes.Gene duplication and diverse selection have resulted in a clustered distribution of NLRs in plant genomes.Pangenomic variants, such as PAVs, are particularly associated with NLRs and other types of R genes.Usually, NLRs that regulate resistance were absent or functionally reduced in susceptibility genes [22].The duplication analysis results of NLRs annotated in the Chinese Spring genome v2.1 showed that five NLR genes (TraesCS6D03G0026700, TraesCS6D03G0026800, TraesCS6D03G0027300, TraesCS6D03G0027400, and TraesCS6D03G0027500) were tandem duplicated genes with a homology of over 80%, suggesting similar functions.Therefore, the highly similar genes in a PmSJ106 cluster make locating a causal NLR particularly challenging.
The on-line expression analysis results showed that TraesCS6D03G0027500 expressed higher after Bgt infection.Further RT-qPCR results showed that the expression of TaRGA5like (TraesCS6D03G0027500) was significantly higher in the disease-resistant SJ106 than in Chinese Spring.We amplified TaRGA5-like in SJ106 and found that the sequence differed significantly from the homologous sequences in Chinese Spring, Fielda, and Kenong, and the differences were mainly concentrated in the LRR region.The comparison of LRR region sequences in F 2 individuals showed that resistance individuals were almost consistent, while the susceptible individuals were the same as the sequence in the Chinese Spring genome.Therefore, we speculated that the differences in LRR region led to the differences in the recognition of powdery mildew effector by TaRGA5-like, eventually resulting in resistance differences among wheat varieties.
To further analyze TaRGA5-like function, we constructed over-expression transgenic plants and VIGS transient gene silencing plants, respectively.Over-expression of TaRGA5like significantly increased the resistance of susceptible recipient wheat to powdery mildew.However, the disease resistance of SJ106 only slightly decreased after VIGS of TaRGA5like.We speculate that this may be due to the tandem duplicated NLRs of TaRGA5like, such as TraesCS6D03G0026700, TraesCS6D03G0026800, TraesCS6D03G0027300, and TraesCS6D03G0027400, which compensated for the disease-resistant effect of TaRGA5-like and thus maintained the powdery mildew resistance in SJ106.Taken together, the PmSJ106 locus is a natural cluster of duplicated R genes formed by the polymerization of multiple disease-resistant NLRs, which work together to help SJ106 form a broad-spectrum and durable resistance in the whole growth period.The duplicated NLRs in the PmSJ106 locus had the same genetic background, and the disease-resistant NLRs complemented each other, achieving natural polymerization of R genes and avoiding the problem of disease resistance masking that may occur during the polymerization process of different genetic R genes.Wheat variety SJ106, which contained a natural R gene cluster, could become a novel and potentially valuable germplasm for powdery mildew resistance and contribute to the green development of wheat breeding.

Plant Material
SJ106 is a high-quality, disease-resistant spring wheat line bred from the 03 Yuan Yin-15/02K-53) F 6 generation line as a male parent and (315//Wheat-wheatgrass 33//02K-53, 3/02K-53-5) F 4 generation line as a female parent.The wheat cultivars Chinese Spring, susceptible to powdery mildew, were used as susceptible parents and crossed with SJ106 to obtain F 1 hybrids, F 2 populations, and F 2:3 families, respectively.F 1 hybrids, F 2 populations, and F 2:3 families were used for genetic analysis of the powdery mildew resistance in SJ106.All seeds were stored in our lab (Tianjin, China).
The disease-resistant parent (SJ106), the susceptible parent (Chinese Spring), and the individuals of Chinese Spring/SJ106 (10 F 1 , 285 F 2 , and 630 F 2:3 ) were sterilized in a 75% alcohol solution and sown in 72-well seedling trays with 5 seeds per well, and one highly susceptible powdery mildew cultivar, Chinese Spring, was sown in every 4 holes to ensure the full inoculation of the experimental materials.After sowing, the seedling trays were placed in an artificial climate chamber (relative humidity of 70%, light intensity of 125 µmol m −2 s −1 , photo-period of 12 h, and temperatures of 22 • C in light and 16 • C in the dark), and planted for about 7 d.When the second leaf of the seedlings was fully expanded, the seedlings were inoculated by manually sweeping with fresh Bgt spores, and five seedlings were inoculated for each material.After about 10 days post inoculation (dpi), the infection type of the materials was investigated according to the grading standard of 0~4, and the experiment was repeated three times to analyze the characterization.

BSR-Seq and RNA-Seq Analysis
A total of 40 homozygous resistant plants and 40 homozygous susceptible plants were sown in the culture plates.When the second leaf was fully expanded, Bgt E09 was inoculated on the wheat leaves.The middle part of the second leaves were taken at a length of about 4 cm, and the total RNA was extracted following the instructions (Tiangen, Beijing, China) and mixed in equal quantities to construct the resistant and susceptible pools, respectively.The total RNA of the resistant and susceptible pools, the resistant parent SJ106, and the susceptible parent Chinese Spring were sent to select differential single nucleotide polymorphism (SNP) and differential expressed genes (DEGs) by BSR-seq and RNA-seq analysis (Novogene, Tianjin, China).

SSR Analysis
Genome DNA was extracted from 40 homozygous resistant plants and 40 homozygous susceptible plants, respectively.The genome DNA of the wheat was extracted following the CTAB method, and their DNA was mixed in equal amounts to construct resistant and susceptible pools for SSR analysis.Based on the primer sequences published in the wheat database GrainGenes (https://wheat.pw.usda.gov/GG3/)(accessed on 12 October 2019), polymorphisms were screened between the resistant and susceptible parents (SJ106 and Chinese Spring) and between the resistant and susceptible pools using bulked segregant analysis (BSA).Individual-plant identification was performed in the F 2 populations to preliminarily infer the chromosomal location of the disease-resistant genes.

Candidate Gene Prediction and Functional Analysis
The relevant sequences of the two nearest markers flanking the target gene PmSJ106 were used to align with the reported wheat reference genome (Chinese Spring v1.0 and v2.1, Arinalrfor, Lancer, Landmark, Mace, Norin61, Stanley, Jagger, Julius, Zang1817, Fielder, Renan, Landmark, Norin61, Kenong9204, SY Mattis, and Kariega) to determine the intervals in which the genes are located (http://wheatomics.sdau.edu.cn/)(accessed on 16 February 2024) [34].Based on the gene annotations of the related reference genome, the physical positions were determined to predict candidate genes, and R genes within the localized interval were selected.The phylogenetic trees of NLRs and kinases sequences were obtained from the Chinese Spring genome (V2.1) and align them using MEGA (v11.0.13) software, respectively.The alignment results were clustered according the previous reports [35].The tandem duplicated events of the NLRs in the intervals were carried out using MCScanX [36].The expression levels of NLRs were analyzed online (http://www.wheat-expression.com)(accessed on 26 February 2024).To determine the genetic relationship of NLRs and kinases, we obtained the gene sequences of the Chinese Spring genome (V2.1) and align them using multiple sequence alignment tool.

Isolation and Sequence Analysis of TaRGA5-like in SJ106
The sequences were amplified with PrimeSTAR GXL DNA Polymerase (TAKARA, Dalian, China).The amplification primers were designed according to the TraesCS6D03G0027500 sequence, and all primer sequences were listed in Table S1.

Expression Analysis of TaRGA5-like
The expression analysis by real-time quantitative PCR (RT-qPCR) was performed as described previously [37].The actin gene was used as an internal control.

Subcellular Localization of TaRGA5-like
The experiment was performed as described previously [30].The recombinant and control vectors (pCAMBIA1302:TaRGA5-like cDNA-GFP and pCAMBIA1302:GFP) were injected into tobacco epidermal cells, respectively, and observed after 72 h.The pictures were taken with fluorescence microscopy (80i, NIKON, Tokoyo, Japan).

Function Analysis of TaRGA5-like by VIGS
Barley stripe mosaic virus (BSMV) was used to silence TaRGA5-like.Three nonconserved regions of TaRGA5-like were chosen and inserted into BSMVγvector for VIGS analysis.BSMVα, BSMVβ, and BSMVγ were mixed equally and injected into wheat leaves.The related vectors and silenced approach were carried out as described previously [36].Morphology and incompatibility reactions of powdery mildew spores were observed, and the number of appressoria, normal, and deformed spores were counted.The calculation method of HR was performed as described previously [38].The photographs were taken of the phenotypes of the VIGS wheat leaves.

Function Analysis of TaRGA5-like by OE
The recombinant vector pTCK303:TaRGA5-like (ubi: TaRGA5-like) was transformed into the susceptible wheat variety Jinqiang5 using Agrobacterium tumefacien EHA105.The experimental methods were carried out following the previously published articles [36].Two independent transgenic T 1 seedlings (50 mg•L −1 hygromycin) were identified by genomic PCR and qRT-PCR analysis.The infection types of two independent T 1 transgenic lines were evaluated.Photographs were taken of the phenotypes of OE wheat leaves.

Conclusions
In this study, a new powdery mildew locus, PmSJ106 locus, was located on chromosome 6DS.In the locus, five NLRs were tandem duplicate genes, and the TaRGA5-like of them expressed significantly higher in SJ106.OE and VIGS results indicated that TaRGA5-like only confers partial Bgt resistance.We inferred that the function of TaRGA5-like could be compensated by other NLR duplicated genes after VIGS.In summary, we speculated that the NLRs in PmSJ106 locus functioned together to improve wheat resistance to powdery mildew as a genes cluster.

Figure 4 .
Figure 4. Genetic linkage map and physical location map of PmSJ106 locus.

Figure 4 .
Figure 4. Genetic linkage map and physical location map of PmSJ106 locus.

Figure 5 .
Figure 5. Phylogenetic analysis of NLRs and kinases.(A) Phylogenetic tree of NLRs from mu wheat genome.(B) Phylogenetic tree of kinases from multiple wheat genome.The NLRs and k were clustered according to the homology of their amino acid sequences 2.5.Cloning of the Candidate Powdery Mildew Resistance Gene in SJ106

Figure 5 .
Figure 5. Phylogenetic analysis of NLRs and kinases.(A) Phylogenetic tree of NLRs from multiple wheat genome.(B) Phylogenetic tree of kinases from multiple wheat genome.The NLRs and kinases were clustered according to the homology of their amino acid sequences.