Mapping and marker-assisted selection for a gene for extreme resistance to potato virus Y

The chromosomal location of the major gene Ry _adg controlling extreme resistance to potato virus Y (PVY) in Solanum tuberosum subsp. andigena was identified by RFLP analysis of a diploid potato population. A total of 64 tomato and potato RFLP markers were screened with the bulked segregant analysis (BSA) on segregants extremely resistant, hypersensitive or susceptible to PVY. Four markers TG508, GP125, CD17 and CT168 at the proximal end of chromosome XI showed close linkage with extremely resistant phenotypes. TG508 was identified as the closest marker linked with the Ry _adg locus with the maximum map distance estimated as 2.0 cM. The 4 markers linked with the Ry _adg locus were tested on independent tetraploid and diploid potato clones and were subsequently found useful for marker-assisted selection for plants containing Ry _adg . Received: 5 July 1996 / Accepted: 19 July 1996     

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     

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.     

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.     

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.     

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     

A high-resolution map of the H1 locus harbouring resistance to the potato cyst nematode Globodera rostochiensis

The resistance gene H1 confers resistance to the potato cyst nematode Globodera rostochiensis and is located at the distal end of the long arm of chromosome V of potato. For marker enrichment of the H1 locus, a bulked segregant analysis (BSA) was carried out using 704 AFLP primer combinations. A second source of markers tightly linked to H1 is the ultra-high-density (UHD) genetic map of the potato cross SH × RH. This map has been produced with 387 AFLP primer combinations and consists of 10,365 AFLP markers in 1,118 bins (). Comparing these two methods revealed that BSA resulted in one marker/cM and the UHD map in four markers/cM in the H1 interval. Subsequently, a high-resolution genetic map of the H1 locus has been developed using a segregating F₁ SH × RH population consisting of 1,209 genotypes. Two PCR-based markers were designed at either side of the H1 gene to screen the 1,209 genotypes for recombination events. In the high-resolution genetic map, two of the four co-segregating AFLP markers could be separated from the H1 gene. Marker EM1 is located at a distance of 0.2 cM, and marker EM14 is located at a distance of 0.8 cM. The other two co-segregating markers CM1 (in coupling) and EM15 (in repulsion) could not be separated from the H1 gene.Communicated by J.G. Wenzel     

Use of allele specificity of comigrating AFLP markers to align genetic maps from different potato genotypes

The allele specificity of AFLP markers was assessed in five relatively unrelated potato genotypes. To this end, two diploid mapping populations of potato, F₁SH × RH and F₁AM × RH, were analysed using four and six AFLP primer combinations, respectively, recently applied to the analysis of the genetically well characterized backcross population BC_C × E. The AFLP profiles of the five parents revealed 733 AFLP markers and, when identical primer combinations were used, 131 comigrating AFLP markers were identified. After construction of five parental maps, the genomic positions of these comigrating AFLP markers were compared and 117 markers (89%) which targeted the same genomic region were assumed to be homologous. Of these putative homologues, 20 markers, each cloned from at least two genotypes, were sequenced and 19 sets of amplification products were shown to be nearly identical. The number of AFLP markers previously mapped in population BC_C × E ranged from three to eleven per chromosome, which allowed a reliable assessment of chromosome numbers from individual linkage groups obtained in populations F₁SH × RH and F₁AM × RH. The high incidence of corresponding AFLP alleles was confirmed by using an additional set of five primer combinations. The 733 AFLP markers localized provide a valuable reference collection for future mapping studies in potato. As a consequence AFLP analysis may replace more laborious locus-specific marker techniques. Received: 26 July 1996 / Accepted: 30 January 1997     

A potato hypersensitive resistance gene against potato virus X maps to a resistance gene cluster on chromosome 5

 The dominant Nb gene of potato confers strain-specific hypersensitive resistance against potato virus X (PVX). A population segregating for Nb was screened for resistance by inoculating with PVX strain CP2, which is sensitive to Nb. Through a combination of bulked segregant analysis and selective restriction fragment amplification, several amplified fragment length polymorphism (AFLP) markers linked to Nb were identified. These were cloned and converted into dominant cleaved amplified polymorphic sequence (CAPS) markers. The segregation of these markers in a Lycopersicon esculentum×L. pennellii mapping population suggested that Nb is located on chromosome 5. This was confirmed by examining resistant and susceptible potato individuals with several tomato and potato chromosome-5-specific markers. Nb maps to a region of chromosome 5 where several other resistance genes– including R1, a resistance gene against Phytophthora infestans, Gpa, a locus that confers resistance against Globodera pallida, and Rx2, a gene that confers extreme resistance against PVX–have previously been identified. Received: 2 January 1997/Accepted: 7 February 1997     

High-resolution genetical and physical mapping of the Rx gene for extreme resistance to potato virus X in tetraploid potato

The Rx locus in potato confers extreme resistance to PVX. In the F₁ progeny of crosses between the PVX-susceptible cultivar Huinkel and the cultivar Cara (Rx genotype) there was a 1?:?1 segregation of PVX resistance, indicating that Rx in Cara is present in the simplex condition. Using potato and tomato RFLP markers, we mapped Rx in Cara to the distal end of chromosome XII at a different position to the previously mapped Rx1 locus. To generate a high-resolution linkage map in the vicinity of Rx a total 728 AFLP primer combinations were screened using DNA of bulked resistant and susceptible segregants. We also screened segregating populations for chromosomal recombination events linked to the Rx locus and identified 82 plants with recombination events close to Rx. Using these recombinant plants we have identified AFLPs that flank Rx and span an interval of 0.23 cM in a region of the genome where 1 cM corresponds to approximately 400?kb.     

Identification of two AFLP markers linked to bacterial wilt resistance in tomato and conversion to SCAR markers

Tomato bacterial wilt (BW) incited by Ralstonia solanacearum is a constraint on tomato production in tropical, subtropical and humid regions of the world. In this paper, we present the results of a research aimed at the identification of PCR-based markers amplified fragment length polymorphism (AFLP) linked to the genes that confer resistance to tomato BW. To this purpose, bulked segregant analysis was applied to an F₂ population segregating for the BW resistant gene and derived from the pair-cross between a BW resistant cultivar T51A and the susceptible cultivar T9230. Genetic analysis indicated that tomato BW was conferred by two incomplete dominant genes. A CTAB method for total DNA extraction, developed by Murray and Thompson with some modifications was used to isolation the infected tomato leaves. Thirteen differential fragments were detected using 256 primer combinations, and two AFLP markers were linked to the BW resistance. Subsequently, the AFLP markers were converted to co-dominant SCAR markers, named TSCAR_AAT/CGA and TSCAR_AAG/CAT. Linkage analysis showed that the two markers are on the contralateral side of TRSR-1. Genetic distance between TSCAR_AAT/CGA and TRS-1 was estimated to 4.6 cM, while 8.4 cM between TSCAR_AAG/CAT and TRS-1.     

Identification and molecular analysis of transgenic potato chromosomes transferred to tomato through microprotoplast fusion

 Results are reported on the integration sites and copy number of alien marker genes neomycin phosphotransferase II (nptII) and β-glucuronidase (uidA), introduced into diploid potato Solanum tuberosum through transformation by Agrobacterium tumefaciens. Also, the transgenic potato chromosomes 3 and 5 harbouring the nptII and uidA genes, which were transferred to tomato (wild species Lycopersicon peruvianum) by microprotoplast fusion, as revealed by genomic in situ hybridization (GISH), were identified by RFLP analysis using chromosome-specific markers. The data revealed three integration sites in the donor potato genome, each containing the uidA gene, and two also harbouring the nptII gene. Analysis of monosomic-addition hybrid plants obtained after microprotoplast fusion showed that each of these three integration sites is located on a different potato chromosome. The microprotoplast hybrid plants contained only the chromosomes that carried the selectable gene nptII. The data on sexual transmission of the donor potato chromosome carrying the uidA and nptII genes were obtained by analysing the first backcross progeny (BC₁) derived from crossing a monosomic-addition hybrid plant to tomato (L. peruvianum). The glucuronidase (GUS) assay and PCR analysis using primers for the uidA gene indicated the presence of the potato chromosome in GUS-positive and its absence in GUS-negative BC₁ plants. RFLP analysis confirmed sexual transmission of the potato chromosome carrying the nptII and uidA genes to the BC₁ plants. A few BC₁ plants contained the nptII and uidA genes in the absence of the potato additional chromosome, indicating that the marker genes were integrated into the tomato genome. The potential applications of the transfer of alien chromosomes and genes by microprotoplast fusion technique are discussed. Recieved: 1 September 1996 / Accepted: 20 September 1996     

Mapping of resistance to the potato cyst nematode Globodera rostochiensis from the wild potato species Solanum vernei

A population of diploid potato (Solanum tuberosum) was used for the genetic analysis and mapping of a locus for resistance to the potato cyst nematode Globodera rostochiensis, introgressed from the wild potato species Solanum vernei. Resistance tests of 108 genotypes of a F₁ population revealed the presence of a single locus with a dominant allele for resistance to G. rostochiensis pathotype Ro1. This locus, designated GroV1, was located on chromosome 5 with RFLP markers. Fine-mapping was performed with RAPD and SCAR markers. The GroV1 locus was found in the same region of the potato genome as the S. tuberosum ssp. andigena H1 nematode resistance locus. Both resistance loci could not excluded to be allelic. The identification of markers flanking the GroV1 locus offers a valuable strategy for marker-assisted selection for introgression of this nematode resistance.Abbreviations BSA bulked segregant analysis - RAPD random-amplified polymorphic DNA - RFLP restriction fragment length polymorphism - SCAR sequence-characterized amplified region     

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.

     

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     

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.     

Genetical dissection of H3-mediated polygenic PCN resistance in a heterozygous autotetraploid potato population

Potato Cyst Nematodes (PCN) currently represent a serious threat to potato cultivation. However, many sources of resistance are known amongst primitive and wild relatives of cultivated potato, Solanum tuberosum ssp. tuberosum. Currently, in the UK, the major threat is due to Globodera pallida, resistance to which has not yet been effectively deployed in potato cultivars. We have performed linkage and QTL analysis of a tetraploid potato population segregating for the H3 source of resistance to G. pallida that was introgressed into cultivated potato from the primitive species, Solanum tuberosum ssp. andigena. This source is highly effective against the most common UK pathotype of G. pallida (Pa2/3) and its deployment in breeding material is a major goal. We adopted an approach involving bulked segregant analysis (BSA) as well as genome wide linkage analysis using AFLP and SSR markers. BSA provided a concentration of markers linked in coupling to a QTL on linkage group IV, and improved the accuracy of the QTL localisation. By performing an analysis on residual scores after removal of the effects due to the major QTL, we detected a second QTL on linkage group XI.     

Molecular markers associated with leptinine production are located on chromosome 1 in Solanum chacoense

Leptines of Solanum chacoense are effective natural deterrents against the Colorado potato beetle. Leptines are the acetylated forms of the glycoalkaloids solanine and chaconine and are supposed to be synthesised via hydroxylated derivatives, called leptinines. Inheritance of leptinine production was studied in crosses of closely related S. chacoense genotypes. The segregation data supported a single-gene model for the inheritance of leptinine production. In the segregating F₁ population of a S. chacoense cross, AFLP, RFLP and RAPD markers segregating with the leptinine production have been identified. The locus involved in leptinine synthesis was localised to the short arm of chromosome 1 of the potato where a major QTL for solanidine production, and markers with tight linkage to leptine production, have been mapped before. Our data further support the previous finding that the short arm of chromosome 1 is involved in steroid alkaloid synthesis in potato, and suggest that the genes involved in leptinine and leptine production are tightly linked in S. chacoense. Received: 27 June 2000 / Accepted: 4 August 2000