Molecular mapping of the potato virus Y resistance gene Rysto in potato

Ry _sto is a dominant gene which confers resistance to potato virus Y (PVY) in potato. We have used bulked segregant analysis of an F₁ tetraploid potato population to identify three AFLP markers linked to and on either side of Ry _sto . The tomato homologue of one of these AFLP markers was assigned to linkage group XI by analysis of an F₂ mapping population of tomato, suggesting that Ry _sto is also on chromosome XI of the potato genome. This map position was confirmed by the demonstration that Ry _sto was linked to markers which had been previously mapped to chromosome XI of the potato genome. Four additional AFLP markers were identified that were closely linked to Ry _sto in a population of 360 segregating progeny of a potato cross between a resistant (Ry _sto ) and a susceptible parent. Two of these markers were on either side of Ry _sto , separated by only a single recombination event. The other two markers co-segregated with Ry _sto . Received: 29 July 1996 / Accepted: 30 August 1996     

Molecular examination of a chromosome region that controls resistance to potato Y and A potyviruses in potato

 The gene Ry _adg that confers resistance to potato Y potyvirus (PVY) in the cultivated potato [Solanum tuberosum subsp. andigena, line 2x(v-2)7] is located on chromosome XI in a segment that contains three other known resistance genes in other syntenic solanaceous species. One of them is the gene N that controls resistance to tobacco mosaic tobamovirus in tobacco and has previously been isolated and sequenced. Three sequence-related, resistance gene-like (RGL) DNA fragments (354–369 bp) highly homologous to the gene N were PCR-amplified from the potato line 2x(v-2)7. Two RGL fragments (79 and 81% homologous to the N gene) co-segregated with Ry _adg among the 77 F1 progeny tested. These RGLs may originate from a resistance gene family on chromosome XI. The potato line 2x(v-2)7 also expressed resistance to potato A potyvirus (PVA), which was controlled by another locus on chromosome XI mapped ca. 6.8 cM distal to Ry _adg . Received: 18 December 1997 / Accepted: 30 December 1997     

Mapping of extreme resistance to PVY (Ry sto) on chromosome XII using anther-culture-derived primary dihaploid potato lines

The inheritance of extreme resistance to PVY (Ry _sto) by a single dominant locus was confirmed by obtaining a 1:1 segregation ratio in a virus inoculation test with 28 resistant (Ryry) to 29 susceptible (ryry) anther culture-derived dihaploid lines (2n=2x=24) from cv. “Assia” (2n=4x=48) having extreme resistance derived from Solanum stoloniferum in simplex constitution (Ryryryry). Twelve Ry _sto markers selected in AFLP assays using bulked segregant analysis were applied to 106 tested potato cultivars from Germany, The Netherlands and Poland and 19 potato cultivars were identified by these markers as extremely resistant to PVY in alignment with phenotypic data. The locus for extreme resistance (Ry _sto) to PVY was mapped on chromosome XII co-segregating with the SSR marker STM0003. The utility of anther-culture derived dihaploid potatoes for genetic marker development was demonstrated. Marker transferability from diploids to tetraploids provides an optimistic potential for marker-assisted selection in potato breeding programs.     

A new gene,Ny (tbr), for hypersensitivity to Potato virus Y from Solanum tuberosumMaps to Chromosome IV

A diploid backcross population derived from a cross between Solanum tuberosum and Solanum berthaultii segregated for monogenic dominant hypersensitivity to Potato virus Y (PVY). We propose the symbol Ny _tbr for this locus because plants carrying this gene develop necrosis after inoculation with PVY and the allele originated in S. tuberosum. The gene mapped to chromosome IV between TG316 and TG208 at LOD=2.72. This location does not correspond to any other mapped resistance genes in potato. Received: 13 April 2001 / Accepted: 20 July 2001     

Molecular and genetic characterization of the <Emphasis Type="Italic">Ry</Emphasis><Subscript><Emphasis Type="Italic">adg</Emphasis></Subscript> locus on chromosome XI from Andigena potatoes conferring extreme resistance to potato virus Y

Key message

We have elucidated the Andigena origin of the potato Ry_adg gene on chromosome XI of CIP breeding lines and developed two marker assays to facilitate its introgression in potato by marker-assisted selection.

Abstract

Potato virus Y (PVY) is causing yield and quality losses forcing farmers to renew periodically their seeds from clean stocks. Two loci for extreme resistance to PVY, one on chromosome XI and the other on XII, have been identified and used in breeding. The latter corresponds to a well-known source of resistance (Solanum stoloniferum), whereas the one on chromosome XI was reported from S. stoloniferum and S. tuberosum group Andigena as well. To elucidate its taxonomic origin in our breeding lines, we analyzed the nucleotide sequences of tightly linked markers (M45, M6) and screened 251 landraces of S. tuberosum group Andigena for the presence of this gene. Our results indicate that the PVY resistance allele on chromosome XI in our breeding lines originated from S. tuberosum group Andigena. We have developed two marker assays to accelerate the introgression of Ry_adg gene into breeding lines by marker-assisted selection (MAS). First, we have multiplexed RYSC3, M6 and M45 DNA markers flanking the Ry_adg gene and validated it on potato varieties with known presence/absence of the Ry_adg gene and a progeny of 6,521 individuals. Secondly, we developed an allele-dosage assay particularly useful to identify multiplex Ry_adg progenitors. The assay based on high-resolution melting analysis at the M6 marker confirmed Ry_adg plex level as nulliplex, simplex and duplex progenitors and few triplex progenies. These marker assays have been validated and can be used to facilitate MAS in potato breeding.

     

Predicted kinase-3a motif of a resistance gene analogue as a unique marker for virus resistance

 A DNA fragment (ADG2, 310 bp) 77% homologous to the gene N (resistance to tobacco mosaic virus in Nicotiana glutinosa) and 53% homologous to RPP5 (resistance to Peronospora parasitica in Arabidopsis thaliana) was amplified by PCR from the diploid potato genotype 2x(v-2)7 that carries the gene Ry _adg located on chromosome XI and conferring extreme resistance to potato virus Y(PVY). Sequence comparison revealed that ADG2 spans a region corresponding to the predicted kinase-2 and kinase-3a motifs in N and RPP5. One of the 12 nucleotide differences detected between ADG2 and a homologous fragment from a PVY-susceptible potato genotype was located within the predicted kinase-3a motif. This single nucleotide substitution of G→C, resulting in an amino-acid substitution Ser→Thr, abolished the BbvI recognition site of ADG2, which was shown to distinguish all tested potato genotypes carrying Ry _adg from those lacking this gene, irrespective of the genetic background and ploidy level. This PCR-based resistance marker, developed using a resistance gene analogue as a target, is the first example of a PCR-based marker that is generally applicable for selection of an economically important trait in potato. Received: 28 November 1998 / Accepted: 28 December 1998     

The Ry-fsto gene from Solanum stoloniferum for extreme resistant to Potato virus Y maps to potato chromosome XII and is diagnosed by PCR marker GP122718 in PVY resistant potato cultivars

A novel locus for extreme resistance to Potato virus Y (PVY), Ry-f_sto, was identified on potato chromosome XII. The gene Ry-f_sto has been introgressed from the wild potato species Solanum stoloniferum. Inheritance of Ry-f_sto in the tetraploid potato population Rysto was consistent with the model of a single, dominant gene. Bulked segregant analysis identified an ISSR (inter-simple sequence repeat) marker UBC 857₉₈₀ linked to Ry-f_sto. This marker mapped to linkage group XII of a reference potato RFLP (restriction fragment length polymorphism) map. Chromosome XII specific RFLP markers were converted into PCR-based STS and CAPS markers and tested for linkage with Ry-f_sto in the population Rysto. CAPS marker GP122₇₁₈ was tightly linked to the resistance gene and was successfully used to identify Polish and German cultivars expressing extreme resistance to PVY. This indicates that the source of Ry-f_sto has been widely utilized in various potato breeding programs and can be monitored by a diagnostic marker in marker-assisted selection.     

Marker-assisted combination of major genes for pathogen resistance in potato

Closely linked PCR-based markers facilitate the tracing and combining of resistance factors that have been introgressed previously into cultivated potato from different sources. Crosses were performed to combine the Ry _adg gene for extreme resistance to Potato virus Y (PVY) with the Gro1 gene for resistance to the root cyst nematode Globodera rostochiensis and the Rx1 gene for extreme resistance to Potato virus X (PVX), or with resistance to potato wart (Synchytrium endobioticum). Marker-assisted selection (MAS) using four PCR-based diagnostic assays was applied to 110 F1 hybrids resulting from four 2× by 4× cross-combinations. Thirty tetraploid plants having the appropriate marker combinations were selected and tested for presence of the corresponding resistance traits. All plants tested showed the expected resistant phenotype. Unexpectedly, the plants segregated for additional resistance to pathotypes 1, 2 and 6 of S. endobioticum, which was subsequently shown to be inherited from the PVY resistant parents of the crosses. The selected plants can be used as sources of multiple resistance traits in pedigree breeding and are available from a potato germplasm bank.     

Evidence for utility of the same PCR-based markers for selection of extreme resistance to Potato virus Y controlled by Rysto of Solanum stoloniferum derived from different sources

The tuber‐bearing wild potato species, Solanum stoloniferum, carries a dominant gene, Ry_sto, which confers extreme resistance (ER) to Potato virus Y (PVY). This gene was introgressed to cultivated potato germplasm (Solanum tuberosum) using accessions of S. stoloniferum maintained in European gene banks. It is mainly used in potato breeding programmes in Europe. Ry_sto was recently mapped to potato chromosome XII. However, in this study, a different accession of S. stoloniferum (PI275244; Haw1293) was used as a female parent in a cross to obtain a diploid (2n = 2x = 24) potato population of 112 F1 genotypes. From this accession, ER to PVY has been introgressed to the potato breeding programmes at the International Potato Center (Peru). As expected, ER to PVY was inherited in a dominant, monogenic fashion in the F1 population. Marker‐specific choices of DNA polymerase and adjustments of PCR conditions were made to optimise marker detection. The corresponding gene (Ry_sto) was mapped to the chromosome XII using the previously described and new cleaved amplified polymorphic sequence (CAPS) markers, which are based on the restriction fragment length polymorphism loci GP122 (six markers) and GP269 (one marker), and the simple sequence repeat marker STM0003. Four GP122‐based CAPS markers and STM0003 detected the same genotypes expressing ER to PVY. Because of a few recombinants, that is ER genotypes lacking the markers and the genotypes that react with necrosis but contain the markers, the marker distance from Ry_sto was estimated as 15.2 cM in this F1 population. However, the distance may be less if necrosis was considered an altered response also controlled by Ry_sto. The markers also specifically detected independent European potato cultivars that express ER to PVY derived from S. stoloniferum. Phylogenetic analysis of the sequences amplified from the GP122 locus of S. stoloniferum and potato cultivars further confirmed that the Ry_sto gene from independent accessions of S. stoloniferum can be selected using the same markers and the protocols described in this study.     

Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR‐NLR immune receptor

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Ry_sto, from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Ry_sto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Ry_sto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single‐molecule real‐time sequencing). Ry_sto was found to encode a nucleotide‐binding leucine‐rich repeat (NLR) protein with an N‐terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Ry_sto‐dependent extreme resistance was temperature‐independent and requires EDS1 and NRG1 proteins. Ry_sto may prove valuable for creating PVY‐resistant cultivars of potato and other Solanaceae crops.     

Deletion of a large genomic segment in tobacco varieties that are resistant to potato virus Y (PVY)

Recessive alleles (va, va ¹ , va ² , etc) of the tobacco Va locus confer resistance to potato virus Y (PVY). To elucidate the mechanism underlying this resistance, we attempted to identify randomly amplified polymorphic (RAPD) markers that reveal polymorphism between two nearly isogenic lines (NILs) that differ in their susceptibility to PVY. Using each of 500 primers and 800 pairs of primers, we identified over 100 RAPD fragments that differed between the NILs. We applied these RAPD primers or primer combinations to an F₂ population obtained from a cross between the susceptible line BY4 and the resistant va ² -bearing NIL, F55. It was found that only 10 RAPD markers were polymorphic between resistant and susceptible plants. Unexpectedly, these markers were all linked to Va. All 10 RAPD markers were present in all 8 susceptible varieties tested. At least one RAPD marker was not detected in 8 out of 10 resistant varieties. Southern analysis revealed that the sequences of markers were not present in the genomes of resistant varieties, and the markers were found in individually distinct positions on the chromosomes of susceptible tobacco varieties. These results strongly suggest that the resistance conferred by va is due to deletions at the Va locus governing susceptibility to PVY. Received: 20 May 1999 / Accepted: 17 August 1999     

Mapping of the gene Nxphu that controls hypersensitive resistance to potato virus X in Solanum phureja IvP35

 The line IvP35 of the diploid (2n=2x=24) cultivated potato species Solanum phureja (family Solanaceae) expresses hypersensitive resistance (H) to potato X potexvirus (PVX). In this study, a diploid potato population was produced using IvP35 as the male parent and a diploid line of S. tuberosum (87HW13.7) as the female parent and tested for resistance to PVX. Data indicated that H to PVX in IvP35 is a dominant, monogenically inherited trait controlled by a single gene, named Nx _phu , that is in a simplex condition (Nxnx). RFLP analysis carried out on the progeny lines revealed 4 markers (CT220, TG328, CT112 and TG424) from the long arm of chromosome IX that were linked to the hypersensitive phenotype; the closest linkage was observed with the marker TG424. Previous authors have shown that the same region of chromosome IX contains the gene Sw-5 for resistance to tomato spotted wilt tospovirus in Lycopersicon peruvianum (Solanaceae). Received: 18 September 1997 / Accepted: 24 November 1997     

Extreme resistance to potato virus V in clones of Solanum tuberosum that are also resistant to potato viruses Y and A: evidence for a locus conferring broad-spectrum potyvirus resistance

 Extreme resistance to the potato V potyvirus (PVV) was found in four potato cultivars that contain Ry genes from Solanum stoloniferum. When plants of these cultivars, were inoculated by grafting in shoot tips from PVV-infected tomato plants, necrotic symptoms developed in some cultivars, although a full hypersensitive reaction was not elicited, while other cultivars were symptomless. PVV replication was not detected in any of the inoculated plants by ELISA, an infectivity assay of leaf extracts by manual inoculation to Nicotiana benthamiana indicator plants, or by ‘return grafting’ of shoot tips taken from newly developed shoots of the potato plants to virus-free indicator plants of tomato. These methods readily detected PVV infection in inoculated plants of cv ‘Flourball’, which does not contain an Ry gene and is susceptible, and in cvs ‘Maris Piper’ and ‘Dr Macintosh’, which contain gene Nv conditioning a hypersensitive reaction to inoculation. One of the Ry-containing cultivars, ‘Barbara’, has been previously shown to contain two genes that control extreme resistance, defined as no viral replication in intact plants, to the potyviruses potato viruses Y and A (PVY and PVA). These genes are: Ry _sto , which conditions resistance to PVY and PVA, and gene Ra, which conditions resistance to PVA only. It was found that in genotypes from a progeny of the cross ‘Barbara’ (Ry _sto /Ra)בFlourball’ (ry/ra), extreme resistance to PVV segregated with gene Ry _sto . It is proposed that either gene Ry _sto conditions broad-spectrum extreme resistance to the distinct potyviruses PVY, PVA, and PVV or that Ry _sto represents a family of genetically closely linked genes each controlling resistance to a specific virus. Received: 27 December 1996 / Accepted: 9 June 1997     

Hypersensitive response to Potato virus Y in potato cultivar Sárpo Mira is conferred by the Ny-Smira gene located on the long arm of chromosome IX

Potato virus Y (PVY, Potyvirus) is the fifth most important plant virus worldwide in terms of economic and scientific impact. It infects members of the family Solanaceae and causes losses in potato, tomato, tobacco, pepper and petunia production. In potato and its wild relatives, two types of resistance genes against PVY have been identified. While Ry genes confer symptomless extreme resistance, Ny genes cause a hypersensitive response visible as local necrosis that may also be able to prevent the virus from spreading under certain environmental conditions. The potato cultivar Sárpo Mira originates from Hungary and is highly resistant to PVY, although the source of this resistance remains unknown. We show that cv. Sárpo Mira reacts with a hypersensitive response leading to necrosis after PVY^NTN infection in detached leaf, whole plant and grafting assays. The hypersensitivity to PVY^NTN segregated amongst 140 individuals of tetraploid progeny of cvs. Sárpo Mira × Maris Piper in a 1:1 ratio, indicating that it was conferred by a single, dominant gene in simplex. Moreover, we identified five DNA markers linked to this trait and located the underlying locus (Ny-Smira) to the long arm of potato chromosome IX. This position corresponds to the location of the Ry _chc and Ny-1 genes for PVY resistance. A simple PCR marker, located 1 cM from the Ny-Smira gene, can be recommended for selection of PVY-resistant progeny of cv. Sárpo Mira.     

Identification of molecular markers associated with leptine production in a population of Solanum chacoense Bitter

  Solanum chacoense Bitter, a wild relative of the cultivated potato, produces several glycoalkaloids, including solanine, chaconine, and the leptines. The foliar-specific leptine glycoalkaloids are believed to confer resistance to the Colorado Potato Beetle (CPB). Using two bulked DNA samples composed of high- and low-percent leptine individuals from a segregating F₁ population of S. chacoense, we have identified two molecular markers that are closely linked to high percent solanine+chaconine and, conversely, to nil/low percent leptine. One of these, a 1,500-bp RAPD product (UBC370-1500), had a recombination value of 3% in the F₁ progeny, indicating tight linkage. UBC370-1500 mapped to the end of the short arm of potato chromosome 1, in the region of a previously mapped major QTL for solanidine, from a S. tuberosum (solanidine)×S. berthaultii (solasodine) cross. Taken together, these results suggest that either (1) a major locus determining solanidine accumulation in Solanum spp. is on chromosome 1 in the region defined by the RFLP markers TG24, CT197, and CT233, or (2) this region of chromosome 1 may harbor two or more important genes which determine accumulation of steroidal aglycones. These findings are important for the genetics of leptine (as well as other glycoalkaloid) accumulation and for the development of CPB-resistant potato varieties. Received: 5 March 1998 / Accepted: 28 July 1998     

Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1

The dominant allele Gro1 confers on potato resistance to the root cyst nematode Globodera rostochiensis. The Gro1 locus has been mapped to chromosome VII on the genetic map of potato, using RFLP markers. This makes possible the cloning of Gro1 based on its map position. As part of this strategy we have constructed a high-resolution genetic map of the chromosome segment surrounding Gro1, based on RFLP, RAPD and AFLP markers. RAPD and RFLP markers closely linked to Gro1 were selected by bulked segregant analysis and mapped relative to the Gro1 locus in a segregating population of 1105 plants. Three RFLP and one RAPD marker were found to be inseparable from the Gro1 locus. Two AFLP markers were identified that flanked Gro1 at genetic distances of 0.6 cM and 0.8 cM, respectively. A genetic distance of 1 cM in the Gro1 region corresponds to a physical distance of ca. 100 kb as estimated by long-range restriction analysis. Marker-assisted selection for nematode resistance was accomplished in the course of constructing the high-resolution map. Plants carrying the resistance allele Gro1 could be distinguished from susceptible plants by marker assays based on the polymerase chain reaction (PCR).     

R6 and R7 alleles of potato conferring race-specific resistance to Phytophthora infestans (Mont.) de Bary identified genetic loci clustering with the R3 locus on chromosome XI

In potato, 11 resistance alleles (R1–R11) are known which confer race-specific resistance to the fungus Phytophthora infestans. R1 has been mapped previously to potato chromosome V and R3 to chromosome XI. Here we report on the localization of the R6 and R7 alleles on the genetic map of potato. Differential resistant strains of tetraploid Solanum tuberosum, clones MaR6 and MaR7, were used as parental plants for the parthenogenetic induction and selection of diploid genotypes containing the R6 or the R7 resistance allele to P. infestans. One resistant dihaploid from MaR7 could be used directly as a parent to produce diploid F₁ progeny suitable for phenotypic and RFLP analysis. MaR6 did not produce useful dihaploids directly. After crossing MaR6 with a tetraploid susceptible genotype, resistant F₁ clones were selected. The resistant genotypes were then used as parents for the induction of dihaploids. Six dihaploids bearing R6 were identified that could be crossed with a diploid susceptible genotype. Two diploid F₁ populations, segregating for R6 and R7, respectively, were analysed with RFLP markers known to be linked with previously identified R genes. Markers linked with R3 were found also to be linked with R6 and R7. The resistance alleles R6 and R7 mapped to a similar distal position on chromosome XI as the R3 allele.     

A multiplex PCR approach to simultaneously genotype potato towards the resistance alleles Ry-f sto and Ns

A simple and robust multiplex PCR approach was developed for detection of the alleles Ry-f _sto and Ns conferring resistance of potato to Potato Virus Y (PVY) and Potato Virus S (PVS), respectively. Cleaved amplified polymorphic sequence (CAPS) markers GP122₅₆₄ linked to Ry-f _sto and SC811₂₆₀ linked to Ns were amplified in one PCR reaction and identified after simultaneous digestion of the amplicons with restriction enzymes EcoRV and MboI. Effectiveness of this procedure for marker-assisted selection was confirmed in 55 potato cultivars.     

Mapping of the cyst nematode resistance locus Gpa2 in potato using a strategy based on comigrating AFLP markers

 The nematode resistance locus Gpa2 was mapped on chromosome 12 of potato using information on the genomic positions of 733 known AFLP markers. The minimum number of AFLP primer combinations required to map Gpa2 was three. This demonstrates that a reference collection of potato AFLP markers may be a valuable tool for mapping studies in potato. By use of RFLP probes, Gpa2 was more precisely mapped at the distal end of chromosome 12. Gpa2 confers resistance to a distinct group of populations of the potato cyst nematode Globodera pallida and originates from the same potato accession as locus H1, conferring resistance to pathotype Ro₁ of G. rostochiensis. This study shows that these two nematode resistance loci are unlinked and that Gpa2 is linked to the Rx1 locus conferring resistance to potato virus X. The efficiency of AFLPs for genetic mapping of a highly heterozygous crop like potato is discussed and compared with the RFLP technique. Received: 24 February 1997/Accepted: 2 May 1997     

The R1 gene conferring race-specific resistance to Phytophthora infestans in potato is located on potato chromosome V.

Summary Late blight in potato is caused by the fungusPhytophthora infestans and can inflict severe damage on the potato crop. Resistance toP. infestans is either based on major dominantR genes conferring vertical, race-specific resistance or on minor genes inducing horizontal, unspecific resistance. A dihaploid potato line was identified which carried theR1 gene, conferring vertical resistance to allP. infestans races, with the exception of those homozygous for the recessive virulence allele of the locusV1. The F₁ progeny of a cross between this resistant parent P(R1) and P(r), a line susceptible to all races, was analysed for segregation ofR1 and of restriction fragment length polymorphism (RFLP) markers distributed on the potato RFLP map comprising more than 300 loci. TheR1 locus was mapped to chromosome V in the interval between RFLP markers GP21 and GP179. The map position ofR1 was found to be very similar to the one ofRx2, a dominant locus inducing extreme resistance to potato virus X.