Mapping of a novel locus Ra conferring extreme resistance against potato virus A in cultivated potato (Solanum tuberosum L.)

doi:10.21203/rs.3.rs-3939753/v1

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Mapping of a novel locus Ra conferring extreme resistance against potato virus A in cultivated potato (Solanum tuberosum L.)

https://doi.org/10.21203/rs.3.rs-3939753/v1

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published 06 Aug, 2024

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Potato virus A (PVA) is one of the major viruses affecting potato worldwide, and can cause serious disease symptoms and yield losses. Previously, we determined that potato cultivar Barbara harbors Ry_sto (genotye: Ryryryry) and Ra (genotype: Rararara) that each independently confers extreme resistance (ER) to PVA. In this study, employing a combination of next-generation sequencing and bulked-segregant analysis, we further located this novel Ra on chromosome 4 using a tetraploid BC₁ potato population derived from a Ry-free progeny (Rararararyryryry) of Barbara (RarararaRyryryry) × F58050 (rararararyryryry). Using 39 insertion-deletion (InDel) spanning chromosome 4, Ra was delimited by the Indel markers M8-83 and M10-8 within a genetic interval of 1.47 cM, corresponding to a 1.33 Mb genomic region in the potato DM reference genome. The InDel marker M10-8, which displayed 99.3% agreement with the phenotypic results in the Ry-free segregating populations, was then used to screen 43 tetraploid potato cultivars and breeding clones. An overall correlation of 76.6% between the marker and phenotype was observed. These findings obtained above are of importance in furthering the cloning of Ra and employing the marker-assisted selection for PVA resistance.

The Ra extreme resistance against potato virus A was mapped to the upper of chromosome 4 in tetraploid potato.

Potato (Solanum tuberosum L.) is the world’s third most important food crop after rice and wheat, consumed by millions of people globally (Birch et al. 2012). However, various diseases, especially viral diseases, have caused significant yield and quality losses in this vegetative propagated tuber crop (Makarova et al. 2017). Among the over 50 viruses infecting potatoes, potato virus A (PVA, genus Potyvirus), is one of the major viruses according to the relative importance/incidence worldwide (Kreuz et al. 2020). Although less prevalent than another potyvirus, potato virus Y (PVY), PVA can lead to substantial yield losses of up to 40% in infection alone (Kreuz et al. 2020; Valkonen et al. 2017). In co-infection with potato virus X (PVX), it results in a severe disease known as “potato crinkle”, causing severe foliar symptoms and greater yield reduction (MacLachlan et al. 1954). Like other potyviruses, PVA is mainly transmitted by aphid in fields (Kreuz et al. 2020). However, aphid management through insecticide application are ineffective for control of PVA spread due to the non-persistent mode of transmission by the insect (Torrance et al. 2020). Planting certified “clean” seed potatoes is the main measure for managing PVA as well as other viruses in potato production. However, many countries are yet to establish or to implement potato seed certification program (Thomas‐Sharma et al. 2016), leaving their potato particularly vulnerable to large-scale spreads and infections with PVA and other viruses. Deploying cultivars with host resistance is therefore the most effective strategy for managing viral diseases in potato.

It has been long recognized that the host resistance against PVA and PVY appears to be closely linked or co-segregated in some potato germplasm (Cockerham 1970; Torrance et al. 2020). For example, Ra_adg conferring extreme resistance (ER) to PVA (ER_A) from a diploid cultivated potato line 2x(v-2)7 derived from Solanum tuberosum subsp. Andigena, has been mapped to the end of chromosome 11, which was only 6.8 centimorgans from the Ry_adg locus (Hämäläinen et al. 2000). Recently, the Ry_sto derived from S. stoloniferum has been cloned and proven to control ER to both PVY and PVA (Grech-Baran et al. 2020). Our previous investigation based on the Ry molecular markers and phenotypes against PVA and PVY revealed a coupling of PVA resistance with PVY resistance in six out of the twenty potato germplasm tested (Huang et al. 2021). Particularly, Ry_sto and Ry_adg, but not Ry_chcderived from S. chacoense, each conferring ER to PVY (ER_Y) independently, exhibited a strong association with ER_A (Huang et al. 2021). Notably, two potato cultivars (Shepody, Russette), and two breeding clones from China (AC142 and Eshu 3) exhibited ER_Y-independent ER_A, indicating diverse resistances to these two viruses have evolved in potatoes (Fuentes et al. 2021; Huang et al. 2021). Interestingly, Barbara, a cultivar known to harbor Ry_sto, also possesses a novel independent Ra locus conferring ER_A (Huang et al. 2021). Despite the progress, the host resistances against PVA in potato, including the novel Ra locus, are yet to be mapped and/or characterized in potato.

Bulked-segregant analysis (BSA) is an effective method for identifying linked markers in the target genomic region (Giovannoni et al 1991; Michelmore et al 1991). Combining next-generation sequencing (NGS) with BSA (NGS-BSA) has been successfully employed for gene mapping and cloning in various plants including Arabidopsis thaliana, rice, cucumber, and tomato (Fekih et al. 2013; Illa-Berenguer et al. 2015; Lu et al. 2014; Schneeberger et al. 2009; Takagi et al. 2013). The conventional NGS-BSA method identifies the candidate regions by calculating Δ(single nucleotide polymorphism (SNP)-index) using an R package as described previously (Mansfeld and Grumet 2018; Takagi et al. 2013). Recently, Strachan et al. (2019) successfully mapped the H2 resistance against Globodera pallida on the end of chromosome 5 in tetraploid potato by using diagnostic resistance gene enrichment sequencing (Renseq). This method based on Renseq, counts SNPs conforming to the expected ratio post sequence enrichment. Additionally, a modified NGS-BSA method for autopolyploid crops, counting SNPs with SNP-index of 0 in one bulk and 0.10-0.36 in another bulk, successfully identified candidate loci for the nematode resistance gene H1 in tetraploid potato (Yamakawa et al. 2021). However, the analysis of insertion-deletion (Indel) remains largely absent in these methodologies.

Considering these technological advancements, this study aimed to: (1) map the Ra mediating ER_A in Barbara using a tetraploid BC₁ potato population by integrating the NGS and BSA analysis; and (2) develop molecular markers for Ra to facilitate marker-assisted selection (MAS) for ER_A. Our founding revealed the location of the Ra on chromosome 4, likely within a 1.33 Mb region corresponding to the potato DM reference genome. The developed indel marker M10-8, displaying 99.8% agreement with phenotypic results in the Ry-free segregating BC₁ populations, exhibited a significant correlation (76.7%) between the present/absence of the marker and the phenotype of 43 assessed cultivars/breeding clones.

Plant materials

The backcross (BC₁) population (n = 148) previously developed by Huang et al. (2021) and derived from BF145 (male parent) × F58050 (female parent), was used to map the Ra locus. BF145 is a PVA-resistant but PVY-susceptible progeny of Barbara × F58050. Additionally, 43 potato cultivars and advanced breeding lines were employed to assess the effectiveness of Ra molecular markers developed in this study. All materials underwent phenotyping for their response to PVA infection through mechanical (and top-grafting when necessary) inoculation with PVA isolate Hunan (He et al., 2014), as described in Huang et al. (2021).

DNA extractions

Total genomic DNA was extracted from approximately 500 mg fresh leaf tissue of each potato clone using the CTAB protocol (Saghai-Maroof et al. 1985). The DNA quantification at 260 nm (A₂₆₀) was performed using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and its quality was evaluated via absorbance ratios of A_260/A₂₈₀ and A₂₆₀/A₂₃₀. The DNA from each sample was diluted to 50 ng/µl and stored at -70℃ for analysis.

Sequencing of bulked DNA samples

To genetically map the PVA resistance in the BC₁ population, two genomic DNA pools, namely the resistant pool (R-pool) and susceptible pool (S-pool), were created by mixing an equal amount of DNA from 24 resistant and 24 susceptible BC₁ progeny, respectively. Libraries with a 350 bp insert size of the two bulks and the two parents (BF145 and F58050) were prepared using the NEBNext Ultra DNA Library Prep Kit (New England Biolabs, MA, USA). Illumina NovaSeq 6000 150-bp paired-end (PE) sequencing was carried out at Tianjin Sequencing Center (Novegene Co., Ltd., Tianjin, China) following the standard procedure.

The quality of the short reads from R- and S-pools as well as the parents was assessed using FastQC (Andrews 2014). Raw reads underwent adapter trimming and quality filtering via Cutadapt (Aronesty 2011) and Trimmomatic (Lohse et al. 2012). The filtered reads were aligned to the DM potato reference genome v4.03 (Potato Genome Sequencing et al. 2011) using the BWA software with default parameters (Li and Durbin 2009). The resulting BAM files were converted to SAM files using SAMtools (Li et al. 2009). Variant calling for SNPs and InDels was performed using the GATK software (DePristo et al. 2011; McKenna et al. 2010). In order to enhance result accuracy and minimize interference, we excluded variants with low coverage in both parents (<5) and both bulks (<10), as well as those that showed no polymorphism between the two parents.

The QTL-seq analysis

To identify the PVA resistance gene, SNP-index and Δ(SNP-index) were calculated using QTLseqr as described previously (Mansfeld and Grumet 2018). The SNP-index was the proportion that the number of reads harboring an SNP divided by the total number of reads, whereas Δ(SNP-index) was the subtraction between SNP-index of R-pool and SNP-index of S-pool. An average of Δ(SNP-index) was shown using the sliding window analysis within a 2 Mb window size and 10 kb step increment. The statistical confidence interval of Δ(SNP-index) for all the SNP positions were calculated under the confidence level of 95% and the null hypothesis of no QTLs. For each read depth, 95% confidence intervals of Δ(SNP-index) were potted along with Δ(SNP-index) following Takagi et al. (2013).

For the Ra resistance locus, all resistant progeny were expected to be heterozygous and adhere to a 3:1 ratio (the susceptible allele: the resistant allele) in the R-pool, while all susceptible clones were presumed homozygous in the S-pool. The linked SNPs/InDels were identified using specific thresholds for the susceptible and resistance alleles. For the susceptible allele, the thresholds were set at 70-80% in the R-pool and 95-100% in the S-pool. Conversely, for the resistance allele, the thresholds were set at 20-30% in the R-pool and 0-5% in the S-pool. The same thresholds were applied to both parents (BF145 and F58050). The sliding window analysis was used to count the linked SNPs/InDels. The number of linked SNPs/InDels was plotted with 2 Mb window size and 10 kb incremental steps.

Genetic linkage analysis with InDel markers

To identify the mapping results from NGS-BSA, InDel markers were developed and employed for polymorphism analysis between the parents and the R- and S-pools. Primers for the InDel markers were designed using NCBI (https://www.ncbi.nlm.nih.gov/) based on the flanking sequences (150 bp each upstream and downstream based on the reference genome DM) around the InDel positions, as described previously (Lu et al. 2014).

All InDel primers were screened for polymorphism between the parents and R/S pools, and the resulting polymorphic markers were then employed across all progenies. Linkage analysis was performed using TetraploidMap with default parameters (Hackett et al. 2007). To eliminate markers with low confidence, a chi-squared test was conducted on the segregation ratio of each marker allele. The respective marker was excluded if the segregation ratio significantly (P < 0.05) deviated from 1:1 in the BC₁ population.

The PCR reactions comprised a total volume of 20 μl, including 1 μl of genomic DNA (50 ng/μl), 0.5 μl forward primer and reverse primer each (10 μmol/L), 10 μl 2×Utaq PCR mix (Vazyme Co., Ltd., Nanjing, China), and 8 μl ddH₂O. The amplification was performed on a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) with the following program: 3 min at 95 ℃; 12 cycles of 30 s at 95 ℃, 30 s at 58 ℃ (decreasing by 0.5 ℃ per cycle), and 72 ℃ for 30 s; 23 cycles of 30 s at 95 ℃, 30 s at 52 ℃, and 30 s at 72 ℃; and a final extension of 10 min at 72 ℃. The PCR reaction setup was suitable for all InDel markers. The PCR products were analyzed by electrophoresis on 9% polyacrylamide gel followed with silver staining. Additionally, to evaluate the effectiveness of the InDel marker M10-8 for MAS for PVA resistance, PCR products were electrophoresed on 3% agarose gel.

NGS-BSA analysis places Ra locus on chromosome 4

To genetically characterize the novel Ra locus we determined previously, a BSA assay was conducted using the parents (male, BF145; female, F58050), and the R-pool and S-pool samples. High throughput sequencing produced ~102, 91, 158, and 131 million trimmed and filtered reads from BF145, F58050, R pool and S pool, respectively. The average sequencing depth was about 25× for parents and 33× for bulked samples, and the on-target mapping rate to the DM reference ranged from 84.6% to 89.2% in the four samples, indicating high-quality data suitable for downstream analysis. GATK variant calling identified a total of 1,542,634 variants, including 1,365,306 SNPs and 177,328 InDels, between the R and S DNA bulks.

Δ(SNP-index) analysis between the bulks revealed one unequal region spanning 23.32 to 34.97 Mb on chromosome 4 of the DM potato reference genome (Fig. 1), surpassing the 95% confidence interval threshold. However, unlike diploid populations, no distinct peak was observed in this study. Moreover, a region spanning 11.65 Mb was too extensive to precise gene mapping or marker development. Clearly, this approach alone couldn’t identify or fine-map the projected Ra locus in the tetraploid potato materials.

We thereafter explored variant enrichment approach, as described by Strachan et al. (2019), successfully used in both diploid and tetraploid potatoes for gene mapping and cloning (Strachan et al. 2019; Witek et al. 2016). As ER_A was controlled by a single dominant gene in the BC₁ population (Huang et al. 2021), we deduced that the alleles of resistant and susceptible plants should be Rararara and rararara, respectively. Thus, the 19,238 SNPs and 2,300 InDels matching these expected ratios were retained. SNPs and InDels in a 2 Mb interval using a 10 kb sliding window were then computed for each chromosome, as shown in Fig. 2 and Table S1, total 53.8% SNPs and 42.7% InDels were enriched on chromosome 4, respectively. Notably, the clustered SNPs/InDels located at 6.05-11.97 Mb and 39.01-46.51 Mb on chromosome 4 (Fig. S1), were potentially linked to the ER to PVA. Complementing the Δ (SNP-index) analysis, total three regions (region Ⅰ, 6.05-11.97 Mb; region Ⅱ, 23.32-34.97 Mb; region Ⅲ, 39.01-46.51 Mb) on chromosome 4 were tentatively identified as candidate regions associated with the Ra locus.

InDel markers define the Ra locus to a 1.33Mb interval on chromosome 4 in potato

To verify the three candidate intervals, a set of 80 InDel primers was developed and utilized to screen for polymorphism among the two parents and two bulks. Eventually, total 56 InDel markers exhibited polymorphism (Table S2), and region Ⅰ (6.05-11.97 Mb), region Ⅱ (23.32-34.97 Mb) and region Ⅲ (39.01-46.51 Mb) contained 30, 17, and 9 InDel markers, respectively. Upon completing the initial assessment, these polymorphic markers were applied to the segregating BC₁ population. In the chi-squared test by SPSS 20.0, the segregation ratio of 39 markers fitted the 1:1 ratio and was subsequently used to construct a genetic linkage map for the resistant parent BF145 by TetraploidMap (InDel markers in Table S2). The map covered 63.36 cM, integrating 39 InDel markers, with an average distance of 1.41 cM between markers (Fig. 3). Integrated with the phenotypic data of the BC₁ population, assigned as “0” for susceptible or “1” for resistant, the Ra locus was tentatively placed within 40.82 cM between the markers M8-83 and M10-8 using TetraploidMap (Fig. 3).

To further confirm the mapping results, the graphical genotyping was employed to validate the NGS-BSA outcomes. As shown in Fig. 4, a total of 26 markers were determined to be closely linked to ER_A in the presumptive interval based on the NGS-BSA and the genetic linkage analyses, among them, 11 were in region Ⅰ (6.05-11.97 Mb), 8 in region Ⅱ (23.32-34.97 Mb), and 7 markers in region Ⅲ (39.01-46.51 Mb). Graphical genotyping assigned “1” (green) for alleles that originated from the resistant parent BF145, or “0” (blue) for alleles that originated from the susceptible parent F58050 (Fig. 4). Assessing the phenotypes of all progeny clones identified 15 recombinants in the BC₁ population by using these markers, of which 5 were resistant and 10 were susceptible. In region Ⅲ (39.01-46.51 Mb), the genotype of four susceptible recombinants (BC007, BC045, BC103, and BC148) was assigned “1” by the eight markers mentioned above in this region (Fig. 4), indicating the absence of the Ra locus in this region. Similarly, in region Ⅱ (23.32-34.97 Mb), the susceptible recombinant BC007 was still assigned “1” by all eight markers in this region, reflecting the deviation of the Ra locus from this region. Overall, three recombinants (BC045, BC103, and BC148) in this region were exchanged between markers M25903 and M33897_230. In region Ⅰ (6.05-11.97 Mb), the recombinants BC007 and BC073 were exchanged between markers M8-83 and M10-8. Based on the outcome of the resistance test, the Ra locus was mapped to marker M8-83 and M10-8. In view of all above analysis, the region Ⅰ (6.05-11.97 Mb) is suggested to be the true candidate region for the Ra locus.

The genetic locations of 39 markers on the chromosome 4 were compared with their physical locations on the DM potato reference genome (Fig. S2). The genetic locations of most markers on the chromosome 4 are consistent with the physical locations of the markers on the DM potato reference genome (Fig. S2). Hence, the genetic linkage map for the resistant parent BF145 exhibits good collinearity with the genome. Based on the genetic map, the most effective markers that were used to delimit the Ra interval were M8-83 and M10-8. According to the physical location of the flanked markers (M8-83 and M10-8) on the potato DM reference genome, the Ra gene might be harbored in the genomic region of 8.78-10.11 Mb (1.33 Mb) on chromosome 4.

Development of Ra InDel marker for marker-assisted selection

The distances of between the flanked markers M8-83 and M10-8 from Ra were 0.74 cM and 0.73 cM, respectively (Fig. 3), showing a strong agreement of 98.6% and 99.3%, respectively, with the phenotyping results in the BC₁ population. The closely linked InDel marker M10-8 was chosen to evaluate its association with PVA resistance across various potato cultivars/advanced breeding clones (n = 43). The marker was present in 24 clones, with 20 of them exhibiting ER to PVA by graft-inoculation. Among the 19 M10-8-absent clones, 6 were determined to be ER to PVA (Fig 5, Table 1, Table S3). Overall, a 76.7% concordance was obtained between the marker and the phenotype, accompanied by a correlation coefficient of 0.443** (P < 0.01).

Table 1 Evaluation of molecular markers M10-8 in 43 potato cultivars and advanced breeding lines

Molecular marker M10-8	Response to PVA infection	Clones	Total	Percentage (%)
1	ER	20	33	76.7
0	S	13	33	76.7
1	S	4	10	23.3
0	ER	6	10	23.3

ER, extreme resistance; S, Susceptible; 0/1, absence/presence of the molecular marker M10-8.

In this study, we presented the first report on a novel locus Ra mapping on the upper portion of chromosome 4 in a tetraploid population by the combination of NGS-BSA and developed molecular markers. In addition, an InDel marker tightly linked to Ra was developed and applied in maker-assisted selection (MAS) of PVA resistance.

Several approaches have been undertaken with the combination of NGS-BSA and SNP frequencies for mapping target traits in autotetraploid crops. For instance, the nematode resistance gene H2 has been successfully mapped in tetraploid potato through the Renseq and the selection of SNPs with the expected allele frequency (Strachan et al. 2019). Additionally, a new method for polyploid QTL-seq has been developed in the autopolyploid crops (Yamakawa et al. 2021), in which the polyploid QTL-seq counted SNPs (SNP-index was 0, 0.01 and 0.02 in the h1 bulk) to identify the candidate loci for the nematode resistance gene H1 in tetraploid potato. However, it's noteworthy that these previous studies predominantly focused on SNPs, neglecting the exploration of InDels. In contrast, our study found that the index of InDels corresponded with the expected allele frequency in a 2 Mb window for mapping the Ra, with a significant cluster of InDels around 6.05-11.97 Mb (Fig. 2B). This information aligns with graphical genotyping analysis. Therefore, we recommend prioritizing InDel frequencies over SNPs in NGS-BSA analysis based on the findings of this study for following reasons. Firstly, the relatively fewer but sufficient number of InDel (2,300 InDels vs 19,238 SNPs) helps reduce the likehood of spurious associations between markers and phenotype. Additionally, genotyping of InDel marker is considered to be less technologically demanding compared to SNP marker detection, and it is deemed more cost-effective (Lv et al. 2016; Li et al. 2022).

The standard NGS-BSA analysis presented challenges in accurately identifying the candidate region for the Ra locus in our study (Fig. 4). Factors contributing to this discrepancy include the. Firstly, the 1:1 phenotypic segregation ratio indicated that the ER to PVA was controlled by a single dominant gene in the tetraploid BC₁ population. This prompted the deduced that the alleles of resistant plants should be ‘Rararara’, while susceptible plants should be ‘rararara’ alleles. Yamakawa et al. (2021) revealed that the Δ(SNP-index) ranged from 0.1 to 0.36 in tetraploid population by simulation of 10,000 replications, which was much lower than that described by Takagi et al. (2013) in diploid potato. Thus, it generally results in a more ambiguous candidate region for the interest traits. Secondly, the fact that each homologous chromosome in the tetraploid potato originated from different sequences posed a challenge. According to the traditional NGS-BSA analytical method, SNPs associated with PVA resistance in the BC₁ population were only found in a single chromosome, indicating that the resistant trait was located in a single homologous chromosome. However, this method was also used to calculate unlinked SNPs that were found on the other chromosomes, introducing the possibility of interference with crucial parameters like Δ(SNP-index) and resulting in a discrepant candidate interval.

The ER to PVA and PVY in cultivated potato variety Barbara was first reported by Barker (1996), who inferred that two independent genes involved, where one gene controls ER to both PVA and PVY, and the other only responsible for the ER to PVA. The locus conferring ER against two potyviruses is likely Ry_sto originated from S. stoloniferum according to pedigree data (Barker 1996; Cockerham 1970; Song and Schwarzfischer 2008). Barbara is a hybrid descending from the cross of MPI 64.956/68 × AM62.740 (Song and Schwarzfischer 2008), where maternal parent MPI64.956/68 has a complex genetic background from S. stoloniferum, S. acaule, S. demissum, S. tuberosum L. group Andigena, S. spegazzinii, and S. tubersosum (https://www.europotato.org/). Aside from Ry_sto, there is still uncertainty with regard to the source of the Ra resistance locus from this complex pedigree. In the future, efforts will be required to identify whether other novel loci conferring independent ER to PVA is originated from S. stoloniferum or not, and if this Ra is also resistant to other potyviruses. To gain this insight, there will be a need to clone and characterize this novel Ra gene by using the backcross population developed in this study.

MAS has greatly has significantly enhanced the accuracy and efficiency of trait selection in plant breeding, including various traits in potatoes such as late blight resistance, tuber shape, flesh color, etc (Chen et al. 2018a; Chen et al. 2018b; Endelman and Jansky 2016). Despite its success, there has been a scarcity of markers linked to PVA resistance compared with markers associated with PVY resistance. In this study, a PCR-based marker named M10-8 was developed based on an InDel variant closely linked to a novel Ra locus, which imparts ER_A and differs from the hypersensitive resistance (HR) to PVA on chromosome 11 reported in S. tuberosum ssp andigena (Hämäläinen et al. 2000). The InDel marker M10-8 demonstrated a significant coincidence rate of 76.7% with the phenotypes of 43 potato breeding clones, indicating its potential utility for MAS of PVA resistance. The discrepancies observed between the marker and the phenotype in the assessed cultivars/breeding clones are likely due to the presence of other unidentified ER loci against PVA or even a Ry_sto-like R that confers resistance to another potyvirus in addition to PVA. These observations, as outlined in Table 1, indicate instances where six clones exhibited ER to PVA but lacked the M10-8 marker, suggesting the involvement of alternative resistance mechanisms beyond the Ra locus investigated in this study. Moreover, potential disturbances from recombination between marker M10-8 and Ra locus also cannot be ignored, as demonstrated by four clones exhibiting susceptibility to PVA despite having the M10-8 marker. This emphasizes the complexity of the genetic mechanisms governing PVA resistance in potatoes, underscoring the importance of considering multiple resistance loci and potential recombination events in breeding efforts. Future investigations should focus on unraveling the role and interactions of various ER loci to enhance our understanding and facilitate more precise MAS strategies for PVA resistance in potato breeding programs.

In summary, the novel ER gene Ra has been successfully located within a 1.33 Mb interval on chromosome 4 in potatoes. This is the first study mapping the extreme resistance gene against virus on potato chromosome 4. The identification of this location marks a crucial advancement toward the subsequent cloning and characterization of the Ra gene, which could prove instrumental in potato breeding efforts to combat PVA. The InDel marker M10-8 developed in this study will be used to facilitate marker-assisted selection in potato breeding. Looking ahead, future research should focus on fine mapping and ultimately cloning the Ra gene to gain a comprehensive understanding of its mechanisms and potential applications in enhancing resistance against PVA in potato crops.

Credit authorship contribution statement

Bihua Nie and Xianzhou Nie conceived and designed the experiments, Wei Huang, Jie Zheng, Zhen Tu, and Ruhao Chen performed the experiments, Wei Huang, Jingcai Li, Jianke Dong, Jie Zheng and Kyle Gardner analyzed the data. Wei Huang and Bihua Nie wrote the manuscript, Bihua Nie, Xianzhou Nie, Kyle Gardner, Hui Ma and Botao Song revised the manuscript. All authors read and approved the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability

The authors do not have permission to share data.

Acknowledgments The authors thank Ming Luo and Zhengnan Cheng for their technical support in analysis of sequencing data. This work was supported by grants from the National Natural Science Foundation of China (31971989), Chian Agriculture Research System (CARS-09), and the Modern Agricultural Industrial Technology System of Hubei Province (HBHZD-ZB-2020-005).

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Mapping of a novel locus Ra conferring extreme resistance against potato virus A in cultivated potato (Solanum tuberosum L.)

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