Harnessing Genomics and Transcriptomics to Combat PVY Resistance in Potato: From Gene Discovery to Breeding Applications

Abstract

Potato virus Y (PVY) is a major threat to global potato production, causing yield losses of nearly 90%. This emphasizes the urgent need to explore the genetic factors underlying resistance mechanisms. Developments in transcriptomics and plant genomes have shed significant light on the genetic underpinnings of PVY resistance. This review summarizes current knowledge on PVY biology and structure, its impacts, key hypersensitive resistance (HR) and extreme resistance (ER) genes and their associated molecular markers, genomic strategies for discovering resistance genes and improving resistance breeding, and challenges. Genetic resistance is a key strategy for controlling PVY, primarily through HR and ER, which are governed by specific genes: the Ny gene for HR and the Ry gene for ER. Our understanding of the molecular mechanisms underlying this resistance has increased significantly due to the advancement of high-throughput sequencing methods, including RNA and whole-genome sequencing. More than 10 PVY resistance genes have been identified in potato, including well-characterized ER genes such as Ry_sto, Ry-f_sto, Ry_adg, Ry_chc, and Ry(o)_phu, as well as HR genes such as Ny-1, Ny-2, and Ny-Smira, which are discussed in this review. Transcriptomic analyses have revealed the involvement of small RNAs and other regulatory molecules in modulating resistance responses. Transcriptomic studies have also identified 6071 differentially expressed genes (DEGs) in potato cultivars infected with PVY, highlighting strong defense responses influenced by strain, cultivar, and environmental conditions. The identification of these resistance genes facilitates the development of PVY-resistant cultivars through marker-assisted selection and gene pyramiding, offering significant opportunities to enhance PVY management and promote sustainable potato production under the challenges posed by climate change.

1. Introduction

To develop robust crop cultivars with enhanced disease resistance, a thorough understanding of the genetic and molecular mechanisms involved in plant–pathogen interactions is essential. Plants and pathogens can be analyzed using various omics tools, including transcriptomics, proteomics, metabolomics, and genomics (reviewed in [1,2,3]). These tools allow researchers to dissect the complex interactions between potato and PVY, identify key resistance genes and signaling pathways involved in PVY recognition and defense, and reveal the molecular mechanisms underlying extreme and hypersensitive resistance [4,5,6]. Effective use of this knowledge is important for understanding PVY, a pathogen that poses significant problems for breeding and disease control due to its quick evolution and intricate host interactions.

Potato virus Y (PVY) is a highly destructive virus threatening potato crops worldwide, known for its rapid mutation and recombination. It is a flexuous, rod-shaped virus that is a member of the genus Potyvirus and family Potyviridae. Ten useful proteins are encoded by its single open reading frame (ORF), which makes up about 9.7 kb positive ssRNA genome [7,8]. The virus spreads through multiple aphid species and can infect many Solanaceae plants, such as tobacco [9,10], tomato [11,12], pepper [13,14], and various Solanaceous weeds [15].

Depending on the potato variety, PVY infection can result in yield losses of nearly 90%, which lowers both the quantity and quality of potato crops [16]. Infected seed tubers can significantly impact the production of seeds and the growth of potatoes. Various strains of PVY cause symptoms in infected plants, including mottling, necrosis, and defoliation. The PVY^NTN strain, for example, causes tuber necrotic ringspot disease, significantly reducing tuber [17,18]. Yield losses are exacerbated by infected seed tubers and are influenced by the diversity of virus strains, environmental factors, and host plant resistance [19,20]. Controlling PVY is challenging due to its diverse strains and modes of transmission; moreover, insecticides are ineffective [21] and present a hazard to human health and the environment (reviewed in [22]).

Strategically deploying resistance genes to control PVY disease can enhance crop yields and reduce pesticide use, mitigating its impact on human health and the environment. Certain potato cultivars can lose resistance due to genetic variability and recombination among PVY strains, which may evade specific R-gene defenses [23]. To develop lasting, broad-spectrum resistance in breeding programs, it is essential to understand how strain diversity affects host recognition. Resistance comes from extreme resistance (ER) genes, such as Ry, which offer broad and durable protection [24,25], and hypersensitive resistance (HR) genes, like Ny, which involve programmed cell death. While ER provides a more reliable defense, HR may not always adequately control PVY [26].

Genomics and transcriptomics have greatly improved our knowledge of PVY infection and resistance processes and contributed to the development of varieties of crops resistant to PVY [23,27]. Through transcriptomic and genomic research, several particular genes and pathways that are involved in the interaction between PVY and potato plants have been identified, providing insight into the mechanisms of virus replication and host reactions [28,29,30]. The ER gene Ry_sto from Solanum stoloniferum, which was isolated using PacBio SMRT (Pacific Biosciences single-molecule real-time sequencing) and RenSeq (resistance gene enrichment sequencing), encodes a protein that confers PVY resistance and has the potential for breeding resistant cultivars [28]. Out of the 72 genotypes of 10 Solanum species and six potato controls in the study, 55 distinct Ry_sto-like variants were found, suggesting a significant degree of diversity [31]. Besides gene isolation, MAS (marker-assisted selection) has been effectively employed to locate genes like Ny-1 and Ny-2, which trigger HR to PVY and play a crucial role in breeding PVY-resistant potato varieties [32,33]. These, along with other key and extensively studied ER and HR genes, including Ry_adg from a subspecies of Solanum tuberosum known as andigena [24], Ry_chc from Solanum chacoense [34], and Ry(o)_phu from S. tuberosum group Phureja [35], are discussed in this paper.

Transcriptome examination of YouJin plants infected with PVY showed changed gene expression, with the Ry_chc gene providing resistance, protein kinase genes up-regulated, and carbohydrate synthesis pathways down-regulated [30]. Studies of the transcriptome and small RNA profiling of potatoes affected with PVY have shed light on the vulnerability of potatoes to PVY and other diseases [29]. In addition, research on co-infection by various PVY isolates has explored its impact on virus concentration in Solanaceous hosts, revealing the complexities inherent in PVY infection dynamics [36]. Furthermore, CRISPR technology demonstrates potential in developing potato cultivars with enhanced resistance to PVY, supported by evidence from recent studies, highlighting the application of gene editing in agricultural biotechnology [37,38].

PVY genomics and transcriptomic research are hindered by incomplete gene catalogs, poor data integration, and limited functional validation, highlighting the need for standardized datasets and coordinated studies to achieve durable resistance. Additionally, challenges include the complexity of the potato genome and the rapid evolution of PVY strains. The well-characterized ER and HR resistance genes, along with the molecular markers linked to them, the biology and structure of PVY, its effects, genomic approaches for identifying resistance genes and enhancing resistance breeding, and the associated challenges are all discussed in this review.

2. Potato Virus Y: Biology, Structure and Impact

Potato virus Y (PVY) is a highly transmissible pathogen that poses a significant threat to global agriculture and plant health. It infects various crop families and their wild relatives, including three species from the Amaranthaceae family, four from the Poaceae family, three from the Brassicaceae family, twelve from the Solanaceae family, three from the Cucurbitaceae family, ten from the Fabaceae family, and about four others [39]. During the evolution of potyviruses, there are frequent host shifts, averaging 4.6 changes each time. PVY spreads through more than 50 aphid species, contaminated machinery and tools, and by contact between plants caused by brushing against them while moving through the field [40]. In addition to infecting Solanaceae crops and their weed relatives, PVY also affects petunias in Europe [41] and eggplant crops in Lebanon [42], highlighting the widespread impact of the virus in different agricultural regions. A wide range of factors make it difficult to estimate the losses in potato production caused by PVY; however, the usage of infected seed tubers results in the largest losses, with documented losses spanning 38 to 63.5% [43] and a reduction of 0.18 t/ha for each 1% of PVY in seed [19]. Various factors, including the diversity of viral strains, types of aphid vectors, weather conditions, co-infection with other pathogens, host plant tolerance, and cultural practices, influence the severity of PVY in potato crops.

The European Union experiences yearly losses of €187 million due to seed potato chemical treatments and lower ware potato yields [44]. Similarly, low yields in sub-Saharan Africa result from poor seed quality and limited access to virus-free seed tubers [45,46]. In Kenya, only about 2% of seed tubers come from recognized systems, while 65% are recycled on-farm and 31% are purchased from local markets [47]. These practices result in increased virus rates, particularly PVY, which has a prevalence of 48.1% in some studies [48]. Field surveys in sub-Saharan Africa, including Uganda, Tanzania, Ethiopia, and Rwanda, reveal high incidences of PVY and mixed-virus infections in farmer stocks [49,50]. This is largely due to informal seed exchanges that bypass certification. Recombinant PVY strains, such as PVY^N:Wi and PVY^N:O, are common, indicating that lapses in local seed systems increase virus load and strain diversity, thereby weakening resistance and complicating breeding efforts. Hence, to enhance productivity and food security, it is crucial to adopt virus-resistant cultivars.

To effectively mitigate the impact of PVY on potato yields, it is crucial to understand the disease’s biology, including its transmission mechanisms, genomic structure, and population dynamics. Aphids primarily transmit PVY in a non-circulative and non-persistent manner [51,52], rendering traditional control methods such as insecticides ineffective (reviewed in [21,53]). This inefficacy not only jeopardizes crop yields but also poses risks to human health and the environment (reviewed in [22]).

Several factors, including environmental conditions, viral strains, and the species of aphids, influence the spread of PVY. The mode of transmission plays a significant role in the virus’s biology. Research utilizing deep sequencing of three PVY strains has shown that aphid and tuber transmission notably alter within-host population dynamics and nucleotide diversity more than mechanical transmission does [54].

PVY, a member of the Potyvirus genus in the family Potyviridae [55], was first described in 1931 by Smith [56]. It exhibits significant variability, including strain groups PVY^O, PVY^N, PVY^C, and PVY^Z [57,58], which cause different symptoms in plants and interact variably with host resistance genes [59,60]. Symptoms include mottling, yellowing, necrosis, leaf deformation, and the presence of necrotic ring spot (reviewed in [61]). Notably, the PVY^N strain group leads to systemic necrosis in tobacco and mild symptoms in potatoes [62,63], while PVY^O and PVY^NTN initiate a hypersensitive response in certain potato varieties with specific defense genes [64]. To realize more about the complex interactions between viruses and their vectors, researchers have examined the effects of mixed virus infections, such as PVY-Potato leafroll virus (PLRV), on the aphid vectors’ biology and preferences [65].

PVY^Z is serologically related to PVY^O but can evade recognition by certain HR genes in potatoes. Specifically, the recombinant isolate PVY-L26, grouped as PVY^Z, encourages a hypersensitive response in the potato cultivar ‘Maris Bard’ with the putative Nz gene, but is not detected by other resistance genes like Nc and Ny [66]. This indicates that PVY^Z has distinct interactions with potato resistance genes compared to other PVY strains. Together with these primary strain groups, there are also recombinant strains of PVY that have evolved newly and have the potential to result in huge economic losses, such as PVY^NTN and PVY^N(W) [67,68,69]. The diversity among PVY isolates is highlighted by research on recombination events in different genomic regions of the virus, which leads to the development of new strains with distinct features and levels of pathogenicity.

The GC content of a single-stranded PVY genome positive-sense RNA molecule varies from 41.16% to 42.41% among strains, and its length ranges from 9646 to 9704 nucleotides (

Table 1. Genomic features and amino acid variations among different PVY strains.

PVY Strain	Genome Size (nt)	GC Content (%)	No. of Amino Acids	No. of aa Substitutions	Similarity %	GenBank Protein ID	Reference
NCBI RefSeq	9704	42.15	3063	-	-	NP_056759.1	[58]
O	9698	42.18	3081	123	97.0	AAB50573.1	[72]
	9649	41.97	3061	104	97.9	AFS60379.1	[73]
NTN	9700	41.46	3061	305	95.2	AAN87843.1	[74]
Z-NTN	9646	41.84	3061	246	95.7	AQK38488.1	[75]
N-Wi	9691	42.37	3061	172	96.7	AQK38489.1
N	9701	41.16	3061	302	95.2	CAA66472.1	[76]
C	9699	41.88	3061	181	96.8	ACD84569.1	[77]
	9703	41.77	3062	204	96.3	CAI65400.1	[69]
(N)W	9698	42.04	3061	122	97.6	ABQ53158.1	[78] (unpublished)
Wilga	9699	42.33	3061	968	97.1	CAJ34850.1	[69,79]
Wilga 156	9699	42.11	3061	208	96.5	CAI64042.1	[69]
nnp	9699	41.73	3061	237	95.8	AAO83661.2	[80]
MV175 (isolate)	9699	42.38	3061	239	94.6	CCE46024.1	[81]
MV99 (isolate)	9699	42.41	3061	178	96.8	CCE46023.1

O (ordinary strain), NTN (necrotic tuber necrosis recombinant strain), Z-NTN (Z-type necrotic tuber necrosis strain), N-Wi (necrotic Wilga strain), N (necrotic strain), C (common strain infecting Capsicum annuum), N(W) (necrotic–Wilga-type recombinant strains), Wilga (Wilga PVY strain), Wilga 156 (Wilga 156 strain), nnp (nnp strain inducing veinal necrosis in pepper), MV175 (Wilga MV175 strain), MV99 (Wilga MV99 strain).

). In the PVY genomic RNA, the single open reading frame (ORF) is surrounded by untranslated regions (UTRs) at the 5′ and 3′ ends. The UTRs significantly influence the virus’s life cycle and pathogenicity by regulating viral gene expression, replication, and transmission, which are essential for its pathogenic capability [70,71].

Ten proteins are encoded by the PVY genome, each with various functions, including P1 (protein 1), HC-Pro (helper component-proteinase), P3 (protein 3), 6K1 (6 kDa protein 1), CI (cytoplasmic inclusion protein), 6K2 (6 kDa protein 2), NIa-VPg (nuclear inclusion a-viral protein genome-linked), NIa-Pro (nuclear inclusion protein a-protease), NIb (nuclear inclusion protein b), CP (coat protein) (Figure 1). These proteins are processed from a viral polyprotein through three proteases: P1, HC-Pro, and NIa-Pro [82]. As shown in Figure 1, the PVY polyprotein is initially cleaved by the P1 protease at its C-terminus within the MIQF/S cleavage sequence [55]. This is followed by processing at the HC-Pro/P3 junction by the HC-Pro protease, which contains the YRVG/G cleavage motif. In addition, a frameshifting mechanism in the P3 coding sequence results in the production of a peptide known as P3N-PIPO [7]. This occurs through RNA polymerase slippage, resulting in the fusion of P3 with a small peptide, PIPO. P3N-PIPO is essential for viral movement, enabling the virus to transmit through plasmodesmata in the host plant. It also helps PVY overcome plant defenses like RNA silencing and supports viral replication and stability [83]. These ten proteins are encoded in specific regions of the genome, particularly in the common strain of PVY (accession: U09509), known as (PVY^O), with P1, HC-Pro, and P3 spanning nucleotides 182–3502, 6K1, CI, and 6K2 from 3500–5716, NIa-VPg, NIa-Pro, and NIb between 5714 and 8566, and CP from 8567 to 9367 (

Table 2. Genetic composition of PVY common strain genes.

Genomic Location (nt)	Key Protein	Coordinates (aa)	Length (aa)
182–1012	P1	20–296	276
1010–2407	HC–Pro	296–761	465
2405–2659	PIPO	761–845	84
2657–3502	P3	845–1126	281
3500–3658	6K1	1126–1178	52
3656–5557	CI	1178–1811	633
5558–5716	6K2	1812–1864	52
5714–6277	NIa–VPg	1864–2051	187
6278–7009	Nia–Pro	2052–2295	243
7010–8566	Nib	2296–2814	518
8567–9367	CP	2815–3081	266
1–181	5′ UTR	-	-
9366–9698	3′ UTR	-	-

).

3. Genetic Basis of PVY Resistance and Breeding Aspects

Several mechanisms contribute to potatoes’ resistance to PVY (reviewed in [84]). The first line of defense is innate, cell-mediated resistance, which provides broad protection against infection. Additionally, RNA-silencing (RNA interference) plays a role by degrading viral RNA, which helps limit replication of the virus. There is also R-gene (gene-for-gene) mediated resistance, where specific immune responses are triggered when viral effectors are recognized. Furthermore, additional factors such as signaling pathways and quantitative trait loci (QTLs) help to modulate and enhance these defense mechanisms. Potato possesses two primary forms of resistance (R) genes against PVY: hypersensitive response (HR) governed by Ny genes and extreme resistance (ER) controlled by Ry genes (reviewed in [85]). The HR can be ineffective against different PVY strains and environmental conditions, as elevated temperatures suppress salicylic acid signaling, weakening HR defenses and allowing systemic PVY infection [26]. In contrast, ER offers robust, long-lasting protection without visible symptoms post-inoculation [24,25].

Ry_chc from Solanum chacoense [34,86], Ry_adg from Solanum tuberosum group Andigena [24,25], Ry_sto from Solanum stoloniferum [28], and Ry(o)_phu from S. tuberosum group Phureja [87] are among the genes responsible for ER detected in potatoes. Furthermore, resistance mostly against PVY strains within the PVY^N group is conferred by Ry-f_sto, which is produced from Solanum stoloniferum [88]. It prevents systemic PVY infection, providing a higher level of durable resistance. The potato genome contains these genes on various chromosomes, as indicated in Table 3: Ry_chc [34,88] and Ry(o)_phu [87] are found on chromosome IX, Ryadg on chromosome XI [89], and Ry_sto [90] and Ry-f_sto [88] on chromosome XII.

The Ry_sto gene from the NB-LRR family offers substantial resistance to PVY, exhibiting conserved TIR and NB-ARC domains, while LRR and C-JID domains vary significantly [28,31]. The study of [31] investigated 55 Ry_sto-like sequences in Solanum species, most of which were identified through in silico prediction, indicating notable genetic diversity, though their experimental functional validation remains pending. The Ry-f_sto gene provides strong resistance to PVY in Solanum stoloniferum and is mapped to chromosome XII in potato plants that remain symptomless when exposed to the virus [88]. Through marker-assisted selection (MAS) programs, the GP122₇₁₈ and GP122₅₆₄ markers enable early detection of PVY resistance, improving breeding efficiency [88,91]. Programs in Germany and Poland have extensively used the Ry-f_sto gene to increase cultivated potato resistance to PVY [88]. Another valuable genetic resource for breeding PVY-resistant potatoes is Ry_chc, a gene that encodes the TIR-NLR protein [32]. PVY^O, PVY^N:O, and PVY^NTN are among the strains against which the Ry_chc offers broad-spectrum resistance [34,86]. It is a useful gene for disease resistance in potato agriculture because of its significant resistance, which is not genotype-specific. The Ry_chc type I evolutionary pattern’s high sequence diversity indicates quick adaptation and diversification in response to PVY infection [34].

The Ry(o)_phu gene provides resistance to PVY by restricting virus replication in epidermal cells and preventing systemic spread, likely through interaction with NB-LRR genes that play a role in plant defense [87]. The Ry_adg gene, which originated from the Andigena group and is mapped to chromosome XI, encodes a canonical TIR-NLR protein within the NBS-LRR class and confers significant resistance to PVY. Two validated marker assays (M45 and M6) have been developed for efficient introgression of the Ry_adg gene through marker-assisted selection (MAS), enabling accurate identification of its presence and allele dosage in breeding programs [24].

The Ny-1 gene in potato cultivars Romula, Albatros, and Sekwana provides resistance to PVY via salicylic acid (SA) signaling, with its efficacy diminished at higher temperatures, where systemic infection can occur [32,33,84]. Located on chromosome IX, Ny-1 triggers a hypersensitive response, limiting virus spread at 20 °C. However, it is less efficient at 28 °C, when systemic infection may occur without any symptoms. In contrast, the Ny-2 gene on chromosome XI elicits a similar response in Romula, resulting in local lesions [32]. Additionally, the Ny-Smira gene in ‘Sárpo Mira’ demonstrates significant resistance through local necrosis and follows a 1:1 segregation ratio, demonstrating it is controlled by an individual gene, making it valuable for breeding programs focused on PVY resistance [92].

Analyses of the breakdown of Ry-type extreme resistance highlight two interrelated processes: viral evolutionary potential (mutation/recombination in PVY) and pathogen population dynamics (high inoculum and frequent transmission), which together raise the probability of the emergence and spread of resistance-breaking variants [93,94,95]. Unless the R gene mediates mechanisms that strongly suppress within-host replication, single, major-gene (monogenic) resistance is intrinsically more susceptible to defeat by pathogen adaptation than polygenic resistance, according to both empirical research and classical evolutionary theory [26]. Several ER genes (Ry_adg, Ry_sto, and Ry_chc) provide nearly total suppression of PVY replication in compatible hosts for potatoes; however, different recognition specificities and the presence of natural PVY diversity (multiple strain groups, recombinant forms, and NTN variants) allow some viral genotypes to evade detection or overcome suppression in situations where selection pressure is high [31,64]. Comparative surveys and strain-dynamics studies showing changes in PVY strain frequencies under intense cultivation and inoculum pressure provide empirical support [96]. According to the mechanism, if an R protein detects a single viral avirulence determinant, a single change in a viral amino acid may be enough to prevent recognition; if R function inhibits rather than stops replication, a high inoculum can overcome the recognition response and allow systemic escape [26,34].

HR genes, like Ny-1 and Ny-2 confer classic, strain-specific hypersensitive resistance to PVY, recognizing certain viral isolates while others evade detection, resulting in a narrower resistance spectrum [32]. The HR response confers partial resistance to PVY^o and PVY^N strains [97], limiting infection mainly to cultivars lacking hypersensitivity [64,98]. ER genes, like Ry_adg, Ry_sto, and Ry_chc, are described as broad-spectrum and frequently protect against major PVY strain groups (PVY^O, PVY^N, and many recombinants) [28,32,34]. A broad spectrum for that ER gene was indicated by the cloning and functional testing of Ry_chc, which showed exceptional resistance against several tested strains, including PVY^O, PVY^N:O, and PVY^NTN across diverse genetic backgrounds [34]. Although publications highlight the emergence of Ry_sto-like variations and the necessity of continuously monitoring efficacy against developing PVY populations, Ry_sto is also generally reported as broad-spectrum in numerous investigations, and current diversity analyses confirm its usefulness [31]. Experimental evidence shows Ry_sto confers protection against major PVY groups, including PVY^O and PVY^N, and many recombinant forms, indicating a broad spectrum of activity [28]. This broad protection is likely due to recognition of conserved viral features or activation of defense pathways that act at stages of the viral cycle less amenable to single-site escape mutations.

Fitness-cost studies of plant resistance genes show mixed results, as any growth or yield penalties depend strongly on genetic background, environment, and resistance mechanism. Direct and repeatable field studies that measure yield penalties associated with individual Ry loci in potato varieties resistant to PVY are limited. Recombination generating the PVY^NTN group may alleviate or balance the associated fitness penalty [99]. Linkage drag from wild-species introgressions can introduce harmful linked alleles that affect quality or agronomic traits. However, backcrossing and selection often mitigate these effects. So far, no studies have reported significant yield penalties in elite cultivars carrying well-introgressed ER genes such as Ry_adg, Ry_sto, and Ry_chc. While there may be inherent biochemical costs related to the expression of resistance (R) genes, the primary practical costs observed in potato breeding often stem from linkage drag and background interactions, known as epistasis [40,96]. These are typically more significant than a universal yield penalty caused by the resistance allele itself. Therefore, extensive multi-year field trials are essential to identify the subtle fitness trade-offs associated with each gene and genetic background [100].

Quantitative trait loci (QTLs), which may interact with the majority of resistance genes to modify resistance levels, have been linked to PVY resistance in potatoes. An effective strategy for enhancing PVY resistance in potatoes is to utilize specific QTLs, which are genomic regions linked to resistance traits. The study characterized QTLs associated with resistance to PVY in tomatoes [101]; Potato-specific studies have also identified several resistance-associated loci [102,103], highlighting the potential of QTL-based approaches for breeding durable PVY resistance in potato. However, there are several challenges to developing efficient QTL-based breeding strategies. The polyploid nature of potatoes, combined with the polygenic architecture of disease resistance, involves numerous small-effect genes, complicating the genetic analyses and precise identification of individual QTLs [102]. In addition, interactions between genes, such as epistasis, can modify the expression of resistance, making it difficult to predict the resulting phenotype; for example, a study identified significant epistatic interactions among potato PVY resistance loci that influenced virus accumulation and symptom severity [103]. Different QTLs influence the mechanisms of PVY resistance, including the hypersensitive response and gene silencing, adding to the overall complexity [26]. Genotype–environment interactions (GEIs) significantly influence quantitative trait loci (QTL) expression [101]. For example, in potatoes, a study found that GEIs accounted for about 20–30% of the phenotypic variance in PVY resistance across different environments [102]. Moreover, different strains of PVY exhibit varying levels of virulence, indicating that resistance may only be effective against certain strains [73,104]. This limitation hinders broad-spectrum resistance development. The absence of reliable, closely associated molecular markers for QTLs remains a major barrier to using MAS effectively in generating potato cultivars resistant to PVY [102].

Emerging technologies such as SNP arrays, genotyping-by-sequencing, and QTL-seq are progressively overcoming these limitations [103]. Fine mapping and functional genomics techniques, such as CRISPR-Cas, can help identify specific genes.

Table 3. Genomic positions of PVY resistance genes and associated markers in potato.

Locus	Chromosome	Species of Origin	Position on DM1-3 v6.1 Genome	Associated Markers	Distance to the PVY Resistance Gene (cM)	Marker Type	Ploidy-Specific Applicability	Reference
Ny-1	IX	S. tuberosum	66,252,800–66,254,515	SC8951139	0.5	STS	Effective in tetraploid breeding programs	[33]
				GP41443	1.0	SCAR		[32]
				C2_At3g168401100	6.0	COSII
				S1d11	2	CAPS
				GP129	9
				TG591	-
				U276927	16
				TG591	5
				S1d11	-
				U38666	14
				TG186	19
Ny-Smira	IX	S. tuberosum	-	Ry186	1.4	STS	Tetraploid	[92,105]
Rchc	IX	S. chacoense	65,488,223–65,491,765	MG64-17	-		Both diploid and tetraploid	[34]
				Ry_4099	-	KASP		[106]
				Ry_3331	-
				Ry186	1.4	STS		[92]
Ny-2	XI	Sarpo Mira	1,655,790–1,661,555	N1271164	-	SCAR	Tetraploid	[32]
				ShkB	4.0	CAPS
				B11.6	-
Ryadg	XI	S. tuberosum sp. Andigenum	39,331,965–39,333,799	RYSc3	-	SCAR	Primarily Effective for tetraploid potato breeding efforts	[107,108]
				RYSC4	-			[24,107]
				TG508	2.1	RFLP		[89]
				GP125	4.2
				CD17	2.1
				CT168	~13.2
				M45	~0.2	SCAR		[109,110]
				ADG1	-	RFLP		[89]
				ADG2	1.3			[89,111]
Rysto	XII	S. stoloniferum	2,457,137–2,459,737	STM0003	-	SSR	Both diploid and tetraploid	[112,113,114]
				Cat-in2	12.5	SCAR		[112,114]
				YES3-3A	0.53			[90,115]
				ST1	1.3			[113,114]
				STM0003-111	2.95	SSR
				M1	0.53	RAPD		[113]
				M2	5.84
				M3	9.13
				SCARysto4	9.1	SCAR		[116]
				GP122	-	CAPS		[88,112]
Ry-fsto	XII	S. stolonifer-um	1,734,940–1,736,028	GP122718	1.2′	CAPS	‘’	[88]
				GP122564	-			[91]

Table 3. Genomic positions of PVY resistance genes and associated markers in potato.

Locus	Chromosome	Species of Origin	Position on DM1-3 v6.1 Genome	Associated Markers	Distance to the PVY Resistance Gene (cM)	Marker Type	Ploidy-Specific Applicability	Reference
Ny-1	IX	S. tuberosum	66,252,800–66,254,515	SC8951139	0.5	STS	Effective in tetraploid breeding programs	[33]
				GP41443	1.0	SCAR		[32]
				C2_At3g168401100	6.0	COSII
				S1d11	2	CAPS
				GP129	9
				TG591	-
				U276927	16
				TG591	5
				S1d11	-
				U38666	14
				TG186	19
Ny-Smira	IX	S. tuberosum	-	Ry186	1.4	STS	Tetraploid	[92,105]
Rchc	IX	S. chacoense	65,488,223–65,491,765	MG64-17	-		Both diploid and tetraploid	[34]
				Ry_4099	-	KASP		[106]
				Ry_3331	-
				Ry186	1.4	STS		[92]
Ny-2	XI	Sarpo Mira	1,655,790–1,661,555	N1271164	-	SCAR	Tetraploid	[32]
				ShkB	4.0	CAPS
				B11.6	-
Ryadg	XI	S. tuberosum sp. Andigenum	39,331,965–39,333,799	RYSc3	-	SCAR	Primarily Effective for tetraploid potato breeding efforts	[107,108]
				RYSC4	-			[24,107]
				TG508	2.1	RFLP		[89]
				GP125	4.2
				CD17	2.1
				CT168	~13.2
				M45	~0.2	SCAR		[109,110]
				ADG1	-	RFLP		[89]
				ADG2	1.3			[89,111]
Rysto	XII	S. stoloniferum	2,457,137–2,459,737	STM0003	-	SSR	Both diploid and tetraploid	[112,113,114]
				Cat-in2	12.5	SCAR		[112,114]
				YES3-3A	0.53			[90,115]
				ST1	1.3			[113,114]
				STM0003-111	2.95	SSR
				M1	0.53	RAPD		[113]
				M2	5.84
				M3	9.13
				SCARysto4	9.1	SCAR		[116]
				GP122	-	CAPS		[88,112]
Ry-fsto	XII	S. stolonifer-um	1,734,940–1,736,028	GP122718	1.2′	CAPS	‘’	[88]
				GP122564	-			[91]

Responsible for resistance [37,38,117]. In addition, focusing on broad-spectrum resistance targeting multiple virus strains and addressing genotype-environment interactions through multi-environment QTL mapping can improve the stability and effectiveness of breeding [101,118].

4. Transcriptomics in Understanding Potato-PVY Interactions

Transcriptomics, which involves the comprehensive analysis of RNA molecules inside cells, has gained prominence with high-throughput RNA-sequencing (RNA-seq) technologies. These tools allow the detection of differentially expressed genes (DEGs) and provide insights into plant defense responses to pathogens such as PVY [30]. Researchers can determine the genes implicated in PVY resistance by comparing the transcriptomes of resistant and susceptible potato genotypes, and uncover key signaling pathways activated during infection pathways [23,29]. These candidate genes can be functionally validated through strategies such as overexpression or gene editing [29]. Additionally, integrating transcriptomic insights with genetic mapping facilitates the development of molecular markers for MAS, thereby accelerating the breeding of potato cultivars resistant to PVY; for instance, [27] successfully identified transcriptome-derived markers associated with PVY resistance, demonstrating the practical value of combining expression data with marker-assisted selection in potato improvement. Transcriptomic studies also enable evaluation of environmental influences on resistance expression, supporting the development of resilient cultivars adaptable to variable growing conditions.

Research on potato cultivars infected with PVY has revealed a wide range of DEGs, indicating strong defense responses that depend on strain and cultivar [4,78,119]. The number of DEGs varied by PVY strain, with PVY^N-^Wi having the highest count (2778), followed by PVY^NTN (1744) and PVY^O (1549), with more genes downregulated than upregulated. Overall, 6071 DEGs were identified, particularly between PVY^NTN and PVY^N-Wi (1175) [4]. This highlights the dynamic and strain-dependent nature of responses to the infection. Additionally, transcriptomic research has highlighted 2893 DEGs in potato cultivars infected with PVY, further emphasizing the diversity and specificity of the responses to this infection [119]. Susceptible (Russet Burbank) and resistant (Payette Russet) cultivars showed marked downregulation of genes linked to RNAi processes, such as Dicer-like and Argonaute-like, and immunity signaling components, such as NPR1-like and WRKY transcription factors. Protein kinases and heat shock proteins, on the other hand, were raised, suggesting that they may have antiviral properties. Early immune suppression was suggested by the considerable suppression of the NPR1-like gene, a crucial regulator in the salicylic acid signaling system, in Payette Russet 24 h after infection and in Russet Burbank at 1 and 4 weeks. Meanwhile, some defense-related genes were upregulated [29], reflecting a strategic and genotype-dependent response to PVY infection [30].

Recent research has revealed that 21-nt and 24-nt small RNAs regulate immune responses during PVY infection. At one week post-infection, 24-nt sRNAs predominated, indicating early defense activation, while by four weeks, 21-nt sRNAs were more abundant, potentially reflecting a shift or decline in the immune response [29]. Additionally, there were 845 genes found to be mutually expressed in reaction to three PVY strains, with PVY^N:Wi eliciting the highest number of DEGs, indicating its greater virulence. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses exposed functional categories associated with susceptibility and tuber necrosis, with strain-specific pathways contributing to symptom development [4]. Moreover, bioinformatic prediction identified several host-derived miRNAs with sequence complementarity to the PVY genome, suggesting their involvement in natural antiviral defense [120].

5. Genomic Approaches for Breeding PVY-Resistant Varieties

Breeding techniques have improved dramatically in recent years, mainly because of genomic technologies, which offer more precise and efficient methods for enhancing resistance (reviewed in [118]). Breeding programs, depicted in Figure 2, can utilize techniques such as MAS, RNA sequencing, GWAS, genomic selection, meta-QTL analysis, and CRISPR/Cas gene editing to develop potato varieties resistant to PVY, thereby enhancing crop health and yield.

MAS has been utilized in several studies to evaluate the PVY resistance of potato lines (Table 3). Out of 46 potato lines evaluated utilizing the RYSC3 and YES3-3B markers, 19 were found to be PVY-resistant [115]. Study used YES3-3A markers to screen 188 European potato varieties from Germany, Hungary, Poland, and the Netherlands, and found 38 cultivars to be exceptionally resistant to PVY [112]. Furthermore, it was confirmed that four out of five Max Planck Institute (MPI) breeding lines exhibited extreme resistance. The study found that the molecular markers YES3-3A, YES3-3B, and RYSC3 effectively identify extreme resistance in potato germplasm to PVY [121]. The YES3 markers are linked to the Ry_sto gene in ‘Barbara’ and its descendants, while RYSC3 is associated with the Ry_adg gene in the breeding lines NY121 and NY123, confirming their reliability for marker-assisted selection. Molecular markers such as ADG2 and RYSC3 have been extensively employed to discover the Ry_adg gene in breeding programs [24,107,111].

Numerous molecular markers have been used to study Ry_sto, a resistance gene derived from Solanum stoloniferum that provides ER to PVY. Among these, STM0003, an SSR (simple sequence repeat) marker, has been widely used for genetic mapping [90,113]. The YES3-3A marker, a SCAR (sequence-characterized amplified region) marker, has also been employed in MAS to identify Ry_sto carriers. Additionally, M45, a RAPD (random amplified polymorphic DNA) marker, was one of the earliest markers linked to Ry_sto [109]. GP122₇₁₈, a CAPS marker, has been used in the fine mapping of the Ry-f_sto gene conferring PVY resistance [88]. KASP markers derived from SNPs enhance the efficiency of genotyping for late blight and PVY resistance linked to the Ry_adg gene, enabling cost-effective screening in breeding projects [122].

Some potato genotypes exhibit HR genes, such as Ny-1, which confer localized necrotic resistance that limits viral replication and spread. In cultivar Rywal, the Ny-1 gene that gives PVY HR has been identified and is linked to markers such as SC895 and S1d11 [33], and B11.6 (Ny-2) [32]. These molecular markers, including SCAR and SSR, provide reliable tools for genotypic screening in breeding programs. More recently, KASP markers, based on SNPs, have enhanced the efficiency of high-throughput and cost-effective selection of PVY-resistant potato lines [106].

Combining GWAS with RNA-seq analysis has emerged as a powerful method for detecting rare allele associations (reviewed in [123]). This integrative method enhances insight into the genetic basis of the resistance traits and encourages the development of more accurate and up-to-date breeding strategies. Furthermore, molecular markers linked to recognized resistance genes such as Ry_adg, Ry_sto, and Ry_chc, revealed in prior research, can complement GWAS results for extra-efficient screening of diverse potato germplasm [124]. These insights collectively strengthen breeding programs aimed at generating potato variants that are resistant to PVY.

Broad-spectrum resistance to different strains of PVY and related viruses may be possible because of the multiplexing feature of CRISPR/Cas systems, which allows the simultaneous targeting of several susceptibility genes or viral sequences (reviewed in [125]). In a recent study, Ref. [38] developed transgenic potato plants that are resistant to PVY using the RNA-targeting form of the CRISPR/Cas13a system. The Cas13a-expressing lines showed strong resistance to multiple PVY strains, with reduced virus accumulation and no visible disease symptoms, showing the promise of CRISPR/Cas technologies for durable and wide-ranging virus resistance in crop improvement programs. The efficiency of editing in the polyploid potato genome needs optimization. Comprehensive off-target analyses and adherence to evolving biosafety and field-use regulations for genome-edited crops are critical issues that need further attention.

6. Limitations of MAS for PVY Resistance

Molecular markers for PVY resistance face reliability and validation challenges due to incomplete linkage, genetic background variability, limited cross-population validation, and polyploid complexity, making them less predictive and harder to standardize across diverse breeding programs. Markers such as RYSC3 and M45, though linked to the Ry_adg gene, fail to perfectly correlate with phenotypic resistance across different germplasm [40]. For example, cultivar Emma genotypes as resistant using markers RYSC3 and M45, but is phenotypically susceptible, indicating false positives and weak linkage consistency [96]. Markers validated in diploids often lose accuracy in autotetraploid potatoes, as shown by Herrera et al. (2018), who found allele dosage variation at the M6 locus requiring a dosage-sensitive High-Resolution Melting (HRM) assay to accurately distinguish resistant genotypes [24]. Given that resistance-effective alleles against one strain may not work against emerging variants, strain variation and recombinant PVY isolates further hinder diagnostic accuracy. Phenotypic validation is complicated by environmental factors such as temperature sensitivity, as resistance in TIR-NLRs like Ny-1 weakens under extreme temperatures, whereas Ry_sto remains effective due to its stable NLR-mediated defense [126]. Furthermore, marker transferability is compromised by reliance on single, population-specific markers and inadequate validation across a variety of germplasm [127].

Ploidy level and allele dosage significantly affect marker efficacy in polyploid crops. Assays that disregard dosage effects risk misclassifying genotypes in autotetraploid potatoes. Because resistance alleles found in simplex, duplex, or higher dosage states change the phenotypic response, as well as the predictive accuracy of molecular markers [128]. In tetraploid populations, conventional diploid-oriented genotyping and statistical models frequently overlook allele dosage, which results in decreased marker accuracy and exaggerated false discovery rates [129,130]. Dosage-aware genotyping platforms, such as targeted sequencing and KASP, significantly enhance genotype discrimination across simplex, duplex, and triplex formats, thereby increasing the reliability of markers used in breeding programs [122]. Moreover, single-marker transferability is hampered by the complex linkage phases and allelic heterogeneity in tetraploids, highlighting the necessity of haplotype-based strategies and explicit dosage quantification during marker validation [131]. Research findings show that combining the RYSC3 and M45 markers for marker-assisted selection is essential for improving resistance to PVY [128]. This approach helps reduce the risk of discarding clones with only one of the alleles. All things considered, recent studies show that attaining the predictive accuracy seen in diploid systems requires incorporating dosage-aware genotyping and validating markers across various polyploid germplasm [132].

Linkage drag, which occurs when large chromosomal segments carrying undesirable alleles co-transfer with target resistance loci, commonly prevents the introgression of resistance genes from wild Solanum species into cultivated potatoes. Sequencing by Herrera et al. (2018), precisely located Ry_adg between M6 and M45, enabling identification of donor haplotypes and minimizing linked wild DNA during backcrossing [24]. It is challenging to distinguish resistance genes from harmful linked regions due to structural changes and inhibited recombination between wild and cultivated genomes [133,134]. Recombination distortion in tetraploid backgrounds permits remaining donor DNA to endure even after repeated backcrossing, preserving undesirable traits [89]. Linkage drag has decreased due to marker-assisted selection and fine mapping, but total removal is still difficult. Linkage drag can be reduced and cultivar performance enhanced by directly transferring functional alleles without the need for large wild segments, thanks to recent developments in genomics and gene editing [135].

7. Future Perspectives and Challenges

Recent advances in potato transcriptomics and genomes have greatly improved our understanding of the mechanisms driving PVY resistance. The potato reference genome’s accessibility has made these advancements possible [136], as well as its subsequent updates and comparative analyses [137,138]. Using technology for high-throughput sequencing, such as whole-genome sequencing and RNA-seq, has also been extremely important [139,140]. The identification of potential resistance genes and the regulatory pathways linked to them has been made feasible by the combination of these methods. Functional genomics tools like CRISPR/Cas will aid in developing PVY-resistant potato varieties. Bioinformatics and machine learning will advance data analysis and trait identification, improving breeding strategies. While CRISPR/Cas-based genome editing has demonstrated potential for PVY resistance, its effectiveness in tetraploid potatoes is still restricted by the complexity of allele dosage, and its use is further hampered by the biosafety and regulatory frameworks now in place for gene-edited crops. Challenges arise from genetic diversity, PVY strain variability, and the complex potato genome, compounded by non-standardized phenotyping, poor data sharing, and limited reproducibility in multi-omics studies. Close collaboration among molecular biologists, breeders, and agronomists is essential for practical breeding applications.

8. Conclusions

High-throughput sequencing, along with genomic and transcriptomic analyses, has identified key resistance genes and pathways that play a crucial role in the interaction between potato plants and PVY. These discoveries pave the way for the development of new breeding techniques to improve potato cultivars’ resistance to PVY while also shedding light on the complex mechanisms underlying this resistance. As the potato industry continues to confront challenges posed by pathogens, integrating genomic and transcriptomic approaches will be essential for enhancing crop resistance to evolving agricultural threats and ensuring sustainable production. Future research should focus on validating the identified genes and exploring their interactions within the potato immune response to create cultivars with long-lasting resistance to PVY. Improving the reliability of PVY resistance breeding in potatoes will require integrating dosage-aware genotyping, haplotype-based validation, and advanced genomic or gene-editing tools to address genetic complexity, strain variability, and linkage drag.