Key message We have elucidated the Andigena origin of the potato Ry gene on chromosome XI of CIP breeding adg 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 gene into breeding lines by marker-assisted selection (MAS). First, we have adg multiplexed RYSC3, M6 and M45 DNA markers flanking the Ry gene and validated it on potato varieties with known adg presence/absence of the Ry gene and a progeny of 6,521 individuals. Secondly, we developed an allele-dosage assay adg particularly useful to identify multiplex Ry progenitors. The assay based on high-resolution melting analysis at the M6 adg marker confirmed Ry plex level as nulliplex, simplex and duplex progenitors and few triplex progenies. These marker adg assays have been validated and can be used to facilitate MAS in potato breeding. Introduction Potato viruses are increasingly a source of concern with less predictable insect vector population due to climate changes not only directly on reducing crop yield, but also because Communicated by Herman J. van Eck. * Marc Ghislain International Potato Center, P.O. Box 25171, Nairobi 00603, email@example.com Kenya Juan D. Montenegro Present Address: Australian Genome Research Facility, firstname.lastname@example.org University of Queensland, Brisbane, QLD 4072, Australia Cinzia Riccio Present Address: Postgraduate Program in Cellular email@example.com and Molecular Biology (PPGBCM) - Biotechnology Center (CBiot), UFRGS, Bento Gonçalves Ave. 9500/Building, Frank Guzman 43431 Porto Alegre, RS, Brazil firstname.lastname@example.org Present Address: Laboratorio de Biología Molecular del Ida Bartolini Servicio Nacional de Sanidad Agraria (SENASA), Av La email@example.com Universidad 1915, La Molina, Lima 12, Peru Applied Biotechnology Laboratory, International Potato Center, P.O. Box 1558, Lima 12, Peru Vol.:(0123456789) 1 3 1926 Theoretical and Applied Genetics (2018) 131:1925–1938 they spread through healthy-looking tubers (Solomon-Black- parental lines was used to develop a sequence-characterized burn and Barker 2001). Around 40 viruses infect cultivated amplified region (SCAR) marker RYSC3 (Kasai et al. 2001). potatoes in the field with the most ubiquitous being potato Additional linked AFLP markers (M5, M6, M17, M33, M35 virus Y (PVY), potato leafroll virus (PLRV) and potato virus and M45) and one RFLP marker (GP259) were identified A (PVA) which are found worldwide (Valkonen 2007). The in a different segregating population (Brigneti et al. 1997). most sustainable way to reduce losses caused by potato Marker-assisted selection (MAS) offers important advan- viruses is using resistant cultivars (Swiezynski 1994). Virus tages over conventional breeding methods: time saving by resistance genes in potato are of various types, immunity, reducing the number of breeding steps, selection at an early extreme resistance and hypersensitivity (Valkonen 1994). generation stage, reduced number of lines to be tested, selec- PVY can reduce potato yield up to 80% (de Bokx and Hut- tion of individual plants based on their genotype and gene tinga 1981; Hane and Hamm 1999; Rykbost et al. 1999; ‘pyramiding’ (Collard et al. 2005; Collard and Mackill 2008; Nolte et al. 2004). Extreme resistance to PVY has been Slater et al. 2013). found in Solanum tuberosum L. group Andigena (Ry ) Most DNA marker systems are developed for diploid adg (Muñoz et al. 1975; Gálvez and Brown 1980; Gálvez et al. material and are not easily applicable for tetraploid potato 1992), S. stoloniferum (Ry ), S. chacoense (Ry ), S. material (Cernák et al. 2008; De Koeyer et al. 2010). To sto chc demissum and S. hougassi (Cockerham 1970). Genes for develop molecular tools to be used for MAS for PVY resist- resistance to PVY have been mapped from S. chacoense, S. ance, the applicability of some markers linked to Ry genes adg stoloniferum, S. tuberosum group Andigena and S. tubero- has been evaluated in different genetic backgrounds. The sum group Tuberosum (Simko et al. 2007). The Ry gene ADG2 marker was used as an RFLP probe and tested on adg has been localized on chromosome XI by Hämäläinen et al. potato cultivars and breeding lines and results indicated this (1997). Brigneti et al. (1997) located the PVY resistance marker as useful tool for MAS (Shiranita et al. 1999). Whit- gene on chromosome XI using a large population derived worth et al. (2009) evaluated the effectiveness of RYSC3, from the cross 83W28-50 × I-1039 (I-1039 carries the RYSC4 and ADG2 markers to identify PVY resistance in Ry gene) and referred to it as Ry . However, Gebhardt the USDA-ARS Aberdeen Idaho Potato Breeding Program. sto sto and Valkonen (2001) expressed doubts about the origin of Dalla Rizza et al. (2006) reported the application of M45 and the material used in this study. The Ry gene (Song et al. RYSC3 markers in MAS for PVY resistance in the National sto 2005) as well as the gene named Ry-f (Flis et al. 2005) Breeding Program of Uruguay. To implement MAS for sto was mapped on chromosome XII. It is likely that both genes PVY resistance in the Oregon Potato Breeding Program, are allelic forms of the same gene because of similar loca- the RYSC3 marker and the CAPS marker ADG2 were used tion on the potato genetic map, but their DNA sequences and confirmed as tools to identify PVY-resistant clones are still lacking and need to be matched. The Ry gene (Ottoman et al. 2009). Appacale (Spain), a public breed- chc was identified on chromosome IX (Hosaka et al. 2001; Sato ing company, used RYSC3 and STM0003 markers to select et al. 2006). Also, hypersensitivity genes have been mapped breeding clones resistant to PVY (Ortega and Lopez-Vizcon on the potato map: Ny gene on chromosome IV (Celebi- 2012). The SCAR markers RYSC3 and YES3-3 were used tbr Toprak et al. 2002), Ny-1 on chromosome IX (Szajko et al. for application of MAS in a conventional potato breeding 2008) and Ny-2 on chromosome XI (Szajko et al. 2014). program in the University of Wisconsin, USA (Fulladolsa Ny-Smira gene from cultivar Sarpo Mira was mapped to a et al. 2015). location on the chromosome IX corresponding to Ry and Typically, a conventional potato breeding program starts chc Ny-1 genes for PVY resistance. (Tomczyńska et al. 2014). with a large breeding population which is then subject to Recently, Ny gene from S. stoloniferum was identified phenotypic recurrent selection over a number of generations, (o,n)sto in potato cultivars in a position corresponding to the Ry taking 10–12 years to produce one new variety (Bradshaw sto on chromosome XI described by Brigneti et al. (1997) (Van and Mackay 1994; Lindhout et al. 2011; Gebhardt 2013; Eck et al. 2017). Ortega and Lopez-Vizcon 2012). Extreme resistance to eco- Several molecular markers have been developed for detec- nomically important potato diseases is often controlled by tion of these genes. Hereafter, we will focus on the Ry major genes which are mostly simplex, thereby transmitting adg gene which is used in our potato breeding program for adap- the resistance only to half of their progeny. Thus, the produc- tation to lowland tropics (Mendoza et al. 1996). Four RFLP tion of multiplex parents is an advantage for increasing the markers were identified closely linked to the Ry locus frequency of favorable alleles (Bradshaw and Mackay 1994). adg and tested in tetraploid potatoes (Hämäläinen et al. 1997). The allele dosage of valuable progenitors in potato breed- A year later, the same group identified two PCR-amplified ing is determined through the time-consuming progeny test genomic fragments, ADG1 and ADG2, closely linked to and analyzing the resulting segregation ratio (Cockerham Ry (Hämäläinen et al. 1998). Polymorphism between 1970; Bradshaw and Mackay 1994; Mendoza et al. 1996; adg ADG2 sequences from PVY-resistant and -susceptible Slater et al. 2014). Hence, a marker system that allows to 1 3 Theoretical and Applied Genetics (2018) 131:1925–1938 1927 distinguish several allelic combinations and their dosage individuals from a cross between the potato variety Cos- would be advantageous for tetraploid potato breeding (De tanera (CIP379706.27) with extreme resistance to PVY Koeyer et al. 2010). mediated by the Ry gene and the susceptible LBr-43 adg High-resolution melting (HRM) is a simple post-PCR clone (CIP387170.9). All plants were grown in a controlled technique for determining sequence variations within environment at 20 °C, 12 h light and between 70 and 100% PCR amplicons based on their dissociation profile. HRM relative humidity. Seedling trays with 96 wells were used to is applied to genotype known sequence variants by using establish a one-to-one correspondence between plants and unlabeled probes and asymmetric PCR producing melting DNA extracted and analyzed in 96-well plates for marker curves for each genotype. In potato, HRM was proven to be analyses by PCR. very useful for identification of tetraploid clones with desir - For the allele-dosage assay, we used Ry bearing adg able alleles (De Koeyer et al. 2010). varieties and clones genotyped by genetic analysis of In the present study, we validated markers linked to the their test-cross progenies as RRRr (TXY.2 and TXY.11); Ry gene on chromosome XI to diagnose for extreme RRrr (DXY.7, DXY.10 and DXY.15); Rrrr (Costanera and adg resistance to PVY of potato clones deriving from breeding UNICA) (Mendoza et al. 1996; Mihovilovich et al. 1998). programs which use S. tuberosum group Andigena germ- Two F1 hybrid populations were developed: (1) CT popu- plasm as source of resistance to PVY. We further character- lation of 220 genotypes from a cross between the variety ized the Ry gene locus with respect to the similarity and Costanera (C) and the breeding clone TXY.2 (T); and (2) LT adg putative origin of the resistant alleles to PVY. We focused population of 163 genotypes from a cross between TXY.2 on the Ry loci because it is currently being used in CIP (T) and LBr-43 (L). adg breeding program and is effective against all known strains NTN of PVY including PVY . This paper describes the devel- PVY resistance assay opment of a reliable, cost-effective and a mid-throughput marker assay of the RYSC3, M45 and M6 markers to be used The virus infection was conducted using six plants of each in large-scale progeny screening for PVY resistance, and a potato genotype inoculated with strain “O” of PVY with molecular assay of the M6 marker for assessing the dosage three of them mechanically and the other three by grafting. of resistant alleles of Ry also referred to as a “plex” assay. Thirty days after inoculation, symptoms were observed and adg Both are suitable for MAS in potato breeding programs to ELISA tests were carried out by the CIP virology unit (Clark facilitate the introgression of the Ry gene into promising and Adams 1977). adg potato clones. DNA extraction Total DNA was obtained by using standard protocols at CIP Materials and methods (Herrera and Ghislain 2000). For the segregating popula- tion, DNA extraction was done using leaves from 2-week- Plant materials old seedlings. Using a small paper punch, two leaf discs (0.6 mm in diameter) were cut and placed in its correspond- Biological materials with distinct and relevant genotypes ing place into the well of a 96-well PCR. Plates were put on and phenotypes have been selected from CIP germplasm, ice and stored at − 70 °C. DNA extraction was carried out and breeding stocks referred to as the validation panel here- based on the method of Dayteg et al. (1998) described by after: ten progenitors and varieties with extreme resistance to von Post et al. (2003) and adapted to the 96-well plate for PVX and PVY (DXY.7, DXY.10, DXY.15, TXY.2, TXY.11, medium-throughput extraction. Forty microliters of 0.25 M Costanera, UNICA with the Ry gene, I-1039 with the Ry NaOH was added to each well plate directly on the tissue. adg sto gene; Pirola, Bzura with the Ry-f gene), four PVY-suscep- The plate was placed in a 95 °C water bath for 1 min. Sam- sto tible varieties (Alkanche, Atlantic, Bintje, and Perricholi) ples were crushed with a 12-channel pipette and neutralized and three potato clones and varieties with hypersensitive with 120 µl of 0.1 M Tris–HCl, pH 8.0. Plates were centri- resistance to PVY (A6, Desiree, Granola with Ny gene). In fuged at 900 rpm for 5 s. The aqueous phase contained the addition, we used two accessions of S. stoloniferum, one DNA which was used directly for PCR. resistant OCH14135 with an unmapped Ry gene and one sto susceptible TARN187. The 251 accessions of the S. tubero- PCR assay sum group Andigena were obtained from CIP genebank and chosen randomly. Single amplicon reactions were made using 100 ng of potato We developed a segregating population, referred to as DNA in 20 µl reaction volume [1× PCR buffer, 2.5 mM the CL population, for the Ry locus comprising 6521 MgCl , 0.2 mM each dNTP, 0.4 µM primer F, 0.2 µM primer adg 2 1 3 1928 Theoretical and Applied Genetics (2018) 131:1925–1938 R], 0.5 µl Taq Polymerase (1 unit/µl), and run on a PT100 Code Aligner. Due to sequencing errors by the Taq polymer- thermocycler (MJ Research, USA). CAPS markers were ase used in the present study, we used identical sequences obtained by adding 1 µl of the corresponding restriction obtained from sequencing amplicons cloned from several enzyme (10 units/µl) directly to the PCR reaction after the PCR amplifications. Analysis of nucleotide sequence iden- amplification was terminated and incubated 1 h at 37 °C. tity was performed using the software Genious R9.0.5, Primers corresponding to Ry and Ry markers, restric- whereas a dendrogram based on multiple sequence align- adg sto tion enzymes for CAPS markers, SCAR marker RYSC3, ment was developed using the unweighted pair group M45 and M6 markers are indicated in Table 1. Reactions method with arithmetic mean (UPGMA). The 12 M6 allelic were loaded onto standard 1% agarose gels using Tris–borate sequences used to construct the dendrogram were deposited buffer. Amplification products were visualized using eth- in Genbank under accession numbers MG979714 to 25. idium bromide fluorescence under UV light. High‑resolution melting primers and probes Sequencing M45 and M6 amplicons Primers and probes used the M6 sequences of the allele M45 and M6 amplicons were purified from agarose gels associated with the resistance, referred to as M6R, and of using the Wizard SV Gel and PCR Clean-Up System (Pro- the three alleles associated with the susceptibility (M6S1, 2, mega) following the manufacturer’s instructions. Purified 3, and 4) to PVY at the M6 locus. Primer3 software was used fragments were then cloned into plasmid vector using with default parameters for primers producing an amplicon the pGEM-TEasy Vector System (Promega) according to less than 250 bp (Rozen and Skaletsky 2000). The probes the manufacturer’s instructions. Plasmid DNA was then have a Tm of about 5–10 °C above the Tm of the prim- extracted using Wizard Plus SV Minipreps (Promega) ers. Eight primer and probe sets were designed for further according to the manufacturer’s manual. These DNA were evaluation. sequenced both forward and reverse sense by Macrogen Inc. From 10 to 20 randomly chosen cloned amplicons were Asymmetrical unlabeled probe genotyping PCR sequenced. PCR was performed using 60 ng of potato DNA in 10 µl Sequence analyses reaction volume [1× PCR buffer, 2.5 mM MgCl , 0.2 mM each dNTP, 0.02 µM forward primer, 0.2 µM reverse Forward and reverse nucleotide sequences from the same primer, 0.2 µM probe (Integrated DNA Technologies)] amplicon cloned were aligned using the software Codon and 0.5 unit of Taq polymerase (Invitrogen). PCR was Table 1 Primer sequences and restriction conditions to detect five molecular markers associated with Ry genes Marker Ry gene Primer name Sequence (5′–3′) T°a Digestion Amplicon References size (bp) RYSC3 Ry 3.3.3 SATA CAC TCA TCT AAA TTT GATGG 60 _ 321 Kasai et al. (2001) adg ADG23RAGG ATA TAC GGC ATC ATT TTT CCG A M5 Ry M5-mGCT GTT CAC AAT GGG AAC ATGG 60 MseI/BSA 485 Brigneti unpublished sto M5-pCAT ACA AAC TAC TTC TAC CACG M6 Ry M6 RsaTCC GAA ATG TTT GGG CTG ACATC 55 RsaI 1126 Brigneti unpublished sto M6 TaqAAG GGA TCC AAA AAG GTG GTTCA M17 Ry M17-mGAC TGC TTT CTC TCC ACG TGGC 60 PstI 250 Brigneti unpublished sto M17-lGAT CAC AGA TGT TTT ACC TTC GAT G M45 Ry M45-mCCT AGT TTC TGA GCA TGT AAT TTC 61 _ 495 Brigneti unpublished sto M45-pTGC AGC TAT TCA AAA CAC ATA AGG M6 Ry M6F1ACA TGA TAT AAG TTG ATA TGG AGA AT 60 _ 994 This article adg M6R4GTG CTT TGT CTT TTC TGC ATGTA M45 Ry M45F1TGG AGT ATT TGG ATC TAA GGG 61 _ 268 This article adg M45R1AAC ACA TAA GGA GCG ATG M6 Ry P2F1TAG ATA CGC CAC TCC ACA TA 60.5 _ 135 This article adg P2R1GAA CCC ATC CGT GAT AAA T P2GCT GCT CGG GGT CAC CAC 1 3 Theoretical and Applied Genetics (2018) 131:1925–1938 1929 performed on a PTC-100 thermocycler (MJ Research Inc.) from the analysis. Melting curves were normalized at in 96-well plates. The program used was the following: 57.6–75.8 for the validation panel and 60.8–75.8 °C for the 2 min at 94 °C, followed by 55 cycles of 40 s at 94 °C, segregating populations. Melting curves were displayed 40 s at annealing temperature (Ta), and 45 s at 72 °C, as derivative melting peaks. Then, samples with similar then 5 min at 72 °C as a final extension step. Finally, the melting profile were grouped using sensitivity at normal reaction was cooled to 25 °C. PCR products were evalu- + 0.9. Finally, a Tm for each peak was calculated. ated on a 1% agarose gel electrophoresis. The asymmetric PCR was performed in Hard-Shell PCR plates, 96 wells, thin walled (Bio-Rad, cat. HSP9665) with the addition Results of 1× LC Green (BioFire Defense). The reaction mixture was overlaid with 15 µl of mineral oil and the plates were Molecular markers to assist and accelerate selection covered with adhesive film Microseal B (Bio-Rad, cat. of Ry gene for extreme resistance to PVY adg MSB1001). An extra step of 30 s at 94 °C was added to the PCR program to allow the formation of the hybrid Phenotypic data of the 17 potato varieties were reconfirmed between the probe and the strand in excess. using symptoms and DAS-ELISA. Symptoms were mosaic followed by necrosis in susceptible varieties and chlorotic spots or rings in hypersensitive varieties, whereas no symp- HRM of unlabeled probes analysis toms were observed for plants with extreme resistance to PVY. Only the varieties Atlantic and Alkanche displayed After PCR, the plates were taken to a high-resolution chlorotic rings and spots, respectively, instead of necrosis. LightScanner (Idaho Technology, Salt Lake City, UT), an Detection of PVY by serology, DAS-ELISA, was negative instrument dedicated to melting curve analysis. DNA was for all symptomless varieties and virus-resistant progeni- melted from 45 to 95 °C with a hold temperature of 42 °C. tors (UNICA, Costanera, DXY.10, DXY.7, DXY.15, TXY.2, Acquired data were analyzed with the Unlabelled Probes TXY.11, I-1039, Pirola, Bzura) and positive for all others module of the LigthScanner software. As a first step, except the varieties Perricholi and Desiree (Table 2). In the blanks and samples that did not amplify were excluded case of the S. stoloniferum accessions, both symptom and Table 2 Resistance to PVY and association of Ry markers of 17 selected potato varieties, breeding clones and two accessions of the wild species Solanum stoloniferum CIP number Cultivar Gene Symptoms DAS-ELISA Molecular Markers Mech.* Graft Mech.* Graft M17 M45 M5 M6 RYSC3 800,965 A6 Ny Necrosis Necrosis 1 1 – – – – + 800,048 Desiree Ny Necrosis Necrosis 0 1 – – – – – 800,959 Granola Ny Necrosis Necrosis 1 1 – – – – – 704,790 Alkanche – Necrosis Chlorotic spots 1 1 – – – – – 800,827 Atlantic – Necrosis Chlorotic rings 1 1 – – – – – 801,069 Bintje – Necrosis Necrosis 1 1 – – – – – 374,080.5 Perricholi – Necrosis Necrosis 0 0 – – – – – 391,894.7 DXY.7 Ry – – 0 0 + + + + + adg 391,895.1 DXY.10 Ry – – 0 0 + + + + + adg 391,896.15 DXY.15 Ry – – 0 0 + + + + + adg 393,613.2 TXY.2 Ry – – 0 0 + + + + + adg 393,617.1 TXY.11 Ry – – 0 0 + + + + + adg 379,706.27 Costanera Ry – – 0 0 + + + + + adg 392,797.22 UNICA Ry – – 0 0 + + + + + adg 676,008 I-1039 Ry – – 0 0 + + + + – sto 800,953 Bzura Ry-f – – 0 0 – – – – – sto 800,957 Pirola Ry-f – – 0 0 – – – – – sto 760,738 TARN187 – Mosaic Mosaic 1 1 + + – – + 761,884 OCH14135 Ry – – 0 0 + – – + – sto* *Mechanical infection 1 3 1930 Theoretical and Applied Genetics (2018) 131:1925–1938 serology analyses confirmed that OCH14135 and TARN187 Both genotypes displayed the same resistant allele for M17, are extremely resistant and susceptible to PVY, respectively. whereas for RYSC3 and M45 the marker allele associated All potato materials were analyzed with the five Ry with resistance was detected in the susceptible accession adg markers: the two SCAR markers RYSC3 and M45, and the TARN187. The resistant accession OCH14135 presented three CAPS markers M5, M6 and M17 (Table 2). These the marker associated with resistance for M6. However, the markers were chosen because of their proximity to the Ry PVY resistance gene(s) in OCH14135 cannot be clearly adg gene. According to Brigneti et al. (1997), genetic distance related to the one on chromosome XI or XII unless a segre- between M17 and M45/M5 is 0.3 cM and between M45/ gation analysis is done. M5 and M6 also 0.3 cM. Jara Vidalon (2010) estimated at 0.2 cM the genetic distance between RYSC3 and M45/M5 Comparison of Ry gene locus on chromosome XI adg and between M45/M5 and M6 to be 0.05 cM, using a much in germplasm resistant to PVY larger segregating population of 6521 individuals. Only in the 2010 genetic mapping study, one recombinant was found M6 and M45 amplicons from PVY-resistant varieties between the PVY extreme resistance gene and the M45/M5 UNICA and Costanera (bearing Ry ), I-1039 (bearing adg makers. Hence, these are closest markers to the Ry gene Ry ), and the accession OCH14135 of S. stoloniferum were adg sto which is located between M6 and M45. sequenced. This sto accession is resistant to PVY suppos- The two varieties with extreme resistance and five virus edly due to the Ry-f gene located on chromosome XII. sto resistant progenitors that carry Ry (UNICA, Costanera, Marker analyses did not suggest there was another Ry gene adg sto DXY.10, DXY.7, DXY.15, TXY.2 and TXY.11) presented on chromosome XI because M45, closest to Ry (Brigneti adg the marker allele associated with resistance for all five mark - et al. 1997; Jara Vidalon 2010), did not amplify, and only ers. In the case of the three varieties with extreme resist- M6 primers produced an amplicon of the expected size ance that carry Ry gene from S. stoloniferum, I-1039 (Table 2). sto presented marker alleles associated with resistance for The M6 marker provided amplicons of the expected size M5, M6, M17 and M45 but not for RYSC3, whereas Pirola of 1126 bp for all genotypes tested. Sequence analyses from and Bzura did not amplify any of these markers. The latter cloned amplicons confirmed that they represented more result was expected because virus resistance in both varie- than one allele. A total of seven M6 alleles were identified: ties is determined by the Ry-f gene (Flis et al. 2005; Song one associated with the resistant Ry allele(s), referred to sto adg et al. 2005). Of the hypersensitive resistant and susceptible as M6R (identified by the presence of the RsaI site); four potato materials, none presented a marker allele associated with the susceptible allele(s) referred to as M6S1, 2, 3, 4; with resistance except the clone A6 for only the RYSC3 and two from S. stoloniferum referred to as M6sto1, 2. As marker (used for PVY strain indexing). The two accessions expected, each tetraploid potato presented a maximum of of S. stoloniferum produced amplicons corresponding to four alleles. The M6R allele was identical between Costan- marker alleles associated with resistance except for M5. era, UNICA, and I-1039, whereas it presented seven SNPs Fig. 1 Dendrogram of nucleo- tide sequences of the M6 locus from Costanera, UNICA, I-1039 and the S. stoloniferum accession OCH14135. Numbers represent branching frequency with bootstrapping set at 100 1 3 Theoretical and Applied Genetics (2018) 131:1925–1938 1931 and two deletions (99% sequence identity) with M6sto1 of extreme resistance to PVY was confirmed as well (data not the OCH14135 accession of S. stoloniferum (Fig. 1). shown). The M45 sequences of 495 bp were identical for each genotype and almost identical between Costanera, UNICA Validated multiplex PCR assay of Ry markers adg and I-1039 (only one SNP), suggesting that these varieties shared the same M45 allele associated tightly with the PVY In accordance with Henegariu et al. (1997), we tested vari- resistance gene. ous parameters to optimize multiplex PCR. Potato clones Potato landraces of the S. tuberosum group Andigena and varieties (Costanera, LBr-43, Pisha Milpo, I-1039 and were screened with the RYSC3, M6 and M45 markers to OCH14135) previously genotyped for these markers were search for native (unbred) potatoes bearing the Ry gene. used as validation panel. We evaluated concentrations of adg Out of 251 landraces, we found one landrace, Pisha Milpo, PCR buffer, MgCl , primers, Taq polymerase and amount positive for all three alleles. Chloroplastic and nuclear SSR of DNA. PCR cycling conditions were also assessed. We marker fingerprinting was done to verify the taxonomic redesigned the M6 and M45 primers based on sequence assignment of this genotype to the Andigena group and its analysis of several potato cultivars and identified primer M6F1-M6R4 and M45F1-M45R1 combinations to be the best ones (Table 3). Adaptation to the 96-well plate for- mat of the Dayteg protocol for DNA extraction allowed Table 3 Optimized amplification conditions for multiplex PCR for us to process 384 samples within 1.5 h. In agreement with RYSC3, M6 and M45 makers Zhang et al. (2003), the PCR conditions for each marker Final concentration PCR conditions locus were used as base for the subsequent optimization of the multiplex PCR. The RYSC3, M45 and M6 primers NFW Start: 94 °C × 1 min were added in equimolar amounts. Results showed that the M6F1 0.6 µM 30 cycles of 3 steps: amplification for the three markers were not uniform and 94 °C × 30 s suggested that primer concentration needed to be adjusted. M6R4 0.6 µM 60 °C × 1 min Concentration of primers showing weak amplification was ADG23R 0.1 µM 65 °C × 4 min increased, while concentration of primers with stronger 3.3.3 s 0.1 µM Last: amplification was decreased. Best primer concentrations M45F1 0.3 µM 65 × 60 s were 0.6 μM for M6 marker, 0.15 μM for RYSC3 marker M45R1 0.3 µM and 0.5 μM for M45 marker. The optimal amount of DNA 10X PCR buffer 1× was determined to be 2 µl of undiluted DNA (between (25 mM MgCl ) 10 and 20 ng). The annealing temperature was tested at dNTPs (5 mM) 0.2 mM 55 and 60 °C with best results obtained at 55 °C. Finally, Taq polymerase 0.5 U the extension temperature was optimized as 65 °C for Genomic DNA 10–100 ng 4 min. Optimized amplification conditions for multiplex Costanera LBr-43 Pisha Milpo OCH14135 I-1039 M6F1R4 (994 bp) RYSC3 (321 bp) M45F1R1(268bp) Fig. 2 Multiplex PCR for RYSC3, M6 and M45 markers using Cos- gena group), OCH14135 (extreme resistance, Solanum stoloniferum) tanera (extreme resistance, Ry from Andigena group), LBr-43 (sus- and I-1039 (extreme resistance, Ry from S. stoloniferum) (2 repeti- adg sto ceptible to PVY), Pisha Milpo (extreme resistance, landrace of Andi- tions-lanes per genotype) 1 3 1932 Theoretical and Applied Genetics (2018) 131:1925–1938 PCR were confirmed using the validation panel (Table 3; the resistant clone carrying Ry f (Pirola) and the hyper- - sto Fig. 2). The same multiplex PCR and optimized amplifica- sensitive resistant clone Granola had the same profile with tion conditions were successfully on the CL population to only one peak that corresponds to the three susceptible quickly identify recombinants in the M45–RYSC3 interval alleles M6S1,2,3. Second, the Costanera clone (simplex) where the Ry gene lies (data not shown, Jara Vidalon had the same profile as all duplex clones and the triplex adg 2010). clone TXY.11 with a high peak corresponding to the three susceptible alleles M6S1,2,3 and a small peak assigned to Validation of the HRM allele‑dosage assay and its the resistant allele (M6R). Third, the Ry triplex clone adg use in two Ry resistance‑derived progenies TXY.2 presented two peaks (M6R and M6S1,2,3) with adg equal height, which suggests that this is apparently a duplex. Two PVY-susceptible varieties (LBr-43 and Bintje), the Fourth, the simplex clone UNICA showed three peaks, with hypersensitive resistant variety Granola and the resistant the M6S4 and M6R alleles having the lowest peaks and the variety Pirola carrying the Ry-f gene were used as negative M6S1,2,3 alleles the highest peak. Finally, fifth, the suscep - sto controls (negative for RYSC3, M6 and M45). Using the M6 tible clone Bintje displayed a high peak for the three suscep- allelic sequences, we tested eight different sets of primers tible M6S1,2,3 alleles and a low peak for the M6S4 allele. and probes on the validation panel. Only one probe, referred The results of the HRM-allele dosage at the M6 locus did to as M6P2, could be amplified in the asymmetric PCR for not coincide fully with the duplex and triplex progenitors of all tested potato materials and was selected for further HRM the validation panel deduced from progeny segregation. To analysis. This primer–probe set amplifies a 135 bp fragment elucidate the reasons behind this discrepancy, the simplex by with a Tm of ~ 80 °C. The probe is 18 bases long and detects duplex (or triplex) CT population was developed. two SNPs, a G in position 3 and a G in position 10, which are specific for the resistant allele (Fig. 3). The HRM allele- The HRM allele‑dosage assay on the CT population dosage assay on the validation panel revealed three probe melting peaks that represent different sequences in the probe The allele-dosage-sensitive assay on the CT population, region (Fig. 4a). Since the probe was designed to perfectly which included the susceptible clone LBr-43 as nulliplex match the resistant allele (M6R), the peak at higher melting control, displayed two clearly separated peaks correspond- temperatures (72 °C) within the probe melting region indi- ing to the resistant and the three susceptible M6S1,2,3 cates the presence of the target allele. The melting peak at alleles (Fig. 4b). Four well-defined groups were identified 66.5 °C represents the susceptible alleles M6S1, M6S2 and corresponding to nulliplex, simplex, duplex and triplex. Of M6S3, which have the same probe sequence and differs in the 220 individuals, three genotypes were not considered one nucleotide from the resistant allele. The third melting because of inconsistent results or bad amplification. Thirty peak at 61 °C corresponds to the susceptible allele M6S4 genotypes were nulliplex, 101 simplex, 84 duplex and 2 tri- that differs in two nucleotides from the probe sequence of plex. Again, in this CT population TXY.2 appeared to be a the resistant allele. Peak heights have been used to estimate duplex for the resistant allele of Ry gene. The abundance adg dosage within the region of the probe. of nulliplex and the absence of quadruplex, expected in a Five profiles could be distinguished using the potato vali- simplex by a duplex cross but not in a simplex by triplex, dation panel (Fig. 4a): First, the susceptible clone LBr-43, confirmed TXY.2 as a duplex progenitor. However, the Fig. 3 Primer probes M6P2 of the HRM allele-dosage assay. Align- are boxed. The arrows point at positions where the sequence varies ment of DNA sequences of the resistant and susceptible alleles of M6 between the five sequences marker. Annealing sites of primers (P2F1 and P2R1) and probe (P2) 1 3 Theoretical and Applied Genetics (2018) 131:1925–1938 1933 (A) M6R M6S4 M6S1,2,3 Bintje UNICA Lbr-43, Pirola, Granola Costanera, Dxy.7, Dxy.10, Dxy.15, Txy.11 Txy.2 (B) M6R M6S1,2,3 AAaa (Txy2) = 84 AAAa = 2 aaaa (LBr-43) = 30 Aaaa (Costanera) = 101 (C) M6R M6S1,2,3 Aaaa (Costanera) = 136 AAaa (TXY.2)= 0 aaaa (LBr-43) = 25 Fig. 4 High-resolution melting allele-dosage assay at the M6 locus progeny of TXY.2 with LBr-43 clone (TL). The number of genotypes for: a the potato validation panel; b the progeny of Costanera with for each group is included TXY.2 (CT) including LBr-43 clone as nulliplex control; and c the 1 3 1934 Theoretical and Applied Genetics (2018) 131:1925–1938 allelic distribution in this population is different from the the susceptible genotype LBr-43 (L) to test the presence of one theoretically expected in a cross between a simplex and another PVY-resistant gene. a duplex (1N:5S:5D:1T). The Chi square goodness of t fi test (x = 23.854; p < 0.001; p ≤ 0.05) rejected the hypothesis that HRM allele‑dosage assay and PVY resistance test the observed values correspond to a cross between a sim- on the TL population plex and a duplex. A second hypothesis which could explain the observed results has been formulated: unviable gametes The M6 marker produced the expected amplicon in 66 out when homozygous for the Ry resistant gene result in a of 81 genotypes of the TL population by using the new M6 adg segregation (1N:5S:4D). The Chi square goodness of fit test primers (M6F1 and M6R4). This segregation ratio corre- (x = 3.8; P = 0.15; p ≤ 0.05) failed to reject this hypothesis sponds to the expected frequency (1:5) for the progeny of (Table 4a). However, the identification of two rare triplex of a cross between a nulliplex and a duplex. The HRM allele- the Ry resistant allele suggests limited rather than com- dosage assay was performed on 163 progenies including the adg plete loss of viability of gametes homozygous for the resist- simplex variety Costanera as control. Two clearly separated ant allele of the Ry gene. peaks corresponding to the resistant M6R and the three sus- adg To corroborate the HRM results, 34 genotypes of the CT ceptible M6S1,2,3 alleles were identified (Fig. 4c). Of the population representing nulliplex, simplex, duplex and tri- 163 samples, 2 did not amplify, 25 grouped as nulliplex and plex were phenotyped by mechanical inoculation of PVY°. 136 as simplex. No duplex genotypes were observed. The The LBr-43 genotype was included as control. Visual obser- Chi square goodness of fit test did not support the hypoth- vation and DAS-ELISA showed the presence of PVY only esis that the observed values correspond to the expected in the LBr-43 genotype and not in the nulliplex genotypes segregation ratio from a cross between a nulliplex and a of the CT population. Hence, the TXY.2 parent of the CT duplex (1N:4S:1D). Hence, we retested the same hypothesis population could carry another PVY resistance gene dis- of unviable gamete when homozygous for the Ry resist- adg tinct from Ry at the M6 locus. To test this hypothesis, a ant gene resulting in a segregation (1N:4S). The Chi square adg new population (TL) was developed crossing TXY.2 (T) and goodness of fit test (x = 2.012; p = 0.15; p ≤ 0.05) failed to reject this alternative hypothesis (Table 4b). Table 4 Viability of homozygous alleles for Ry tested by Chi squared tests on distribution of allele dosage in two populations adg Female gametes Male gametes 1/6 RR 4/6 Rr 1/6 rr 3/6 Rr 3/36 RRRr 12/36 RRrr 3/36 Rrrr 3/6 rr 3/36 RRrr 12/36 Rrrr 3/36 rrrr Observed Expected RRrr×rrrr RRrr 84 86 Rrrr 101 107.5 rrrr 30 21.5 Male gametes Female gametes 1/6 RR 4/6 Rr 1/6 rr 6/6 rr 6/6 RRrr 24/6 Rrrr 6/6 rrrr Observed Expected Rrrr×rrrr Rrrr 136 128.8 rrrr 25 32.2 Italic cells were zeroed for the tested hypothesis. (A) CT population: unviable gametes when homozygous for the Ry resistant gene resulting adg in segregation (1N:5S:4D). (B) TL population: unviable gametes when homozygous for the Ry resistant gene resulting in segregation (1N:4S) adg The Chi value is 3.8. The p value is 0.15. The result is not significant at p ≤ 0.05 The Chi value is 2.012. The p value is 0.156. The result is not significant at p ≤ 0.05 1 3 Theoretical and Applied Genetics (2018) 131:1925–1938 1935 Ten nulliplex, ten simplex and controls TXY.2 and LBr- whereas the M6 marker is present in the resistant but absent 43 were mechanically inoculated with PVY°. Three nulli- in the susceptible accession. Hence, these markers are not plex were positive for PVY° for the DAS-ELISA test and associated with S. stoloniferum resistance to PVY, which is showed symptoms of infection while LBr-43 was positive for likely to be determined by the Ry-f gene on chromosome sto PVY°, although did not show clear symptoms of infection. XII. Seven of the nulliplex were negative for PVY° for the DAS- The demonstration that the Ry gene on chromosome adg ELISA test and did not show symptoms of infection. Hence, XI in our breeding lines originates from the Andigena group this second progeny testing of nulliplex for Ry displaying and not from the S. stoloniferum wild species was made by adg resistance to PVY indicates the presence of another resist- characterizing the DNA sequence at the M6 and M45 tightly ance gene in the TXY.2 progenitor. linked markers and by screening a sample of the Andigena germplasm. Indeed, the identification of an identical resist- ant allele for the M6 locus (M6R) in Costanera, UNICA Discussion and I-1039, which is different from the other susceptible alleles including those from S. stoloniferum, supports the The PVY resistance phenotype was confirmed for all the hypothesis of a common origin of the resistant allele M6R. material and coincided with previous assessments. As evi- This result coincides with the molecular characterization denced with the varieties Desiree and Perricholi, the phe- of I-1039 by SSR markers and the absence of the Tubero- notyping of extreme resistance must be assessed by both sum plastid marker, which placed this cultivar as a hybrid symptoms and DAS-ELISA. The marker analyses coincided of Andigena × Tuberosum (Ghislain et al. 2009). Sequence also with our knowledge on virus resistance and pedigree of analyses of the M6 and M45 alleles from Costanera, UNICA these varieties and breeding lines. and I-1039 confirmed the high similarity between them sug- The marker RYSC3 developed originally from an acces- gesting again a common origin of these alleles. Secondly, sion of S. tuberosum L. group Andigena displayed the marker we screened a randomly chosen sample of landraces of the allele associated with resistance for all breeding materials Andigena group with the markers associated with Ry gene adg derived from the Andigena XY progenitors (UNICA, Cos- on chromosome XI. One landrace out of 251 screened was tanera, DXY.10, DXY.15, TXY.2, and TXY.11). However, positive for all three markers and tested resistant to PVY the RYSC3 marker allele associated with resistance was also which reveal that this Ry gene is present in the Andigena adg present in the clone A6 which is susceptible to PVY and group. Hence, we conclude here that the Ry gene of I-1039 sto used for PVY strain identification. It is unclear yet from on chromosome XI maps at the same locus as the Ry gene, adg pedigree information why such marker allele is present in but whether these are identical or close by genes remain to the A6 clone. RYSC3 was absent in the PVY-resistant I-1039 be demonstrated by sequencing larger region around this cultivar unlike the other markers. This low association of locus. RYSC3 marker with Ry gene was also reported for two To develop a quick and efficient marker-assisted diagnos- adg varieties from Uruguayan breeding material with extreme tic for the presence/absence of the Ry gene, we have devel- adg resistance assumed to be controlled by the Ry gene based oped new primers and amplification conditions to amplify adg on pedigree information (Dalla Rizza et al. 2006). These more reliably M6 and M45 markers. It has been calculated findings indicate the existence of a lower genetic linkage that 60% of the total time required from leaf collection to between RYSC3 marker and the Ry gene on the chromo- PCR reaction is used for DNA extraction (Dilworth and Frey adg some XI than the other markers. 2000). Thus, simple, rapid, robust and inexpensive DNA iso- The markers M5, M6, M17 and M45 displayed marker lation method is needed for high-throughput MAS (von Post alleles associated with resistance not only in the parental et al. 2003; Karakousis and Langridge 2003). Unlike other material they were originally developed from, I-1039, but plant DNA extraction protocols, the method implemented also in all of the breeding material derived from the Andi- in this study does not include the use of liquid nitrogen or gena XY progenitors. This result supports that the Ry freeze-drying for initial grinding of the tissue. Furthermore, adg and Ry genes map at the same locus, but whether these it does not require hazardous chloroform precipitation or sto are distinct genes or allelic variants of the same gene is sophisticated automated equipment used by large breed- unknown. In addition, the differences in symptoms to dis- ing companies. However, this protocol is not suitable for tinguish extreme and hypersensitive resistance may not be isolating large quantities of DNA which may limit its use so clear and the recently found Ny-2 and Ny genes on if multiple marker assays must be performed sequentially. (o,n)sto chromosome XI may be the same as Ry found by Brigneti The cost of DNA extraction, PCR amplification and markers sto et al. (1997). detection are also considered as restricting factors for appli- In S. stoloniferum, the RYSC3 and M45 marker alleles are cation of MAS in breeding programs (Xu and Crouch 2008). detected in the susceptible but not the resistant sto accession, In addition to technical and safety advantages, the reported 1 3 1936 Theoretical and Applied Genetics (2018) 131:1925–1938 protocol for DNA extraction is a cost-effective method. In might be present in TXY.2. Solomon-Blackburn and Mac- our laboratory, the cost per sample following routine proto- kay (1993) showed that PVY susceptible varieties can be cols is an order magnitude higher and takes 6 h to process 48 symptomless. We cannot rule out some failure in the infec- samples. Optimization of multiplex PCR requires strategic tion process. This would explain the presence of asympto- planning and multiple attempts to avoid results that can lead matic leaves on some nulliplex genotypes of the CT and TL to false negatives or positives. Our results demonstrated that populations. The quantity of PVY present in these geno- the protocol for the multiplex PCR developed in this study types could not be detected with DAS-ELISA as suggested permitted considerable savings of time and effort without by Depta et al. (2014). These observations reinforce the compromising accuracy. This protocol was used for identify- importance of having molecular methods for PVY resistance ing recombinant genotypes in the CL segregating population diagnostic to complement the phenotypic detection methods. developed to accomplish the fine mapping of the Ry locus In conclusion, the rapid protocol for DNA extraction and adg (Jara Vidalon 2010). However, the specificity of this multi- multiplex PCR constitute a medium-throughput system for plex Ry markers should be characterized using cultivars assessing the presence of RYSC3, M6 and M45 markers in adg with other PVY resistance genes from S stoloniferum such as potato allowing unequivocal identification of potato mate- those mentioned in Van Eck et al. (2017), Tomczyńska et al. rial with the resistant allele of the Ry gene. The HRM adg (2014), Mori et al. (2011), and Szajko et al. (2008, 2014). allele-dosage assay proved to be robust for allele-dosage The rapid introgression of the Ry gene into promis- determination of Ry -linked marker M6. Both assays have adg adg ing breeding lines would be greatly improved by increas- proven to be promising tools to facilitate the introgression ing the frequency of this allele in breeding population by of the Ry gene and would be a good alternative for breed- adg using progenitors with multiple copies of this allele. We ing programs with limited budget. However, as indicated by developed here an allele-dosage assay for the tightly linked Cernák et al. (2008), the genetic background is critical and M6 marker based on high-resolution melting technology must be take into consideration for the applicability of this using unlabelled probes instead of fluorogenic 5′ nucle- molecular marker system. ase (TaqMan) for two reasons: (1) it is more cost-effective because it does not require expensive labeled oligonucleo- Author contribution statement MRH and MG directed the experiments, LJV and JM conducted the plex assay and tide probes; and (2) it can detect multiple haplotypes in a single assay unlike the HRM-TaqMan 5′ assay which only population development, IB conducted the PVY resistance assay, CR and FG did the marker allele sequence analyses, detects a single allele relative to any another allele. Indeed, HRM-probe genotyping was used to select parents with mul- LJV developed the multiplex marker assay and population screening and MRH, LJV and MG wrote the manuscript. tiple copies of a desirable marker (De Koeyer et al. 2010). Probe design for a perfect complementation with the resist- ant allele allowed differentiating the haplotypes present in Acknowledgements The authors are grateful to José Rodriguez for the region of the probe. The HRM allele-dosage assay for excellent technical assistance in laboratory and screen house experi- the Ry gene developed in this study allowed easy differ - adg ments. We are also grateful to Elisa Mihovilovich for providing infor- entiation of resistant samples from the susceptible ones and mation on plex-material. This work was supported by the Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZP GmbH (Project clear identification of nulliplex, simplex duplex and triplex 03.7860.4-001.0) and the CGIAR Research Program on Roots, Tubers genotypes without having to do the tedious phenotypic assay and Banana (RTB). on segregating progenies. Surprisingly, all multiplex PVY-resistant progenitors Compliance with ethical standards tested appeared to have a lower Ry allele dosage than what adg had been observed by progeny analyses. The latter may have Conflict of interest The authors declare that they have no conflict of overestimated the allele-dosage number likely due to unsuc- interest. cessful PVY infection or additional PVY resistance genes. Open Access This article is distributed under the terms of the Crea- Indeed, the duplex status of the triplex progenitor TXY.2 tive Commons Attribution 4.0 International License (http://creat iveco was confirmed by the allele-dosage assay for both CT and mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- TL populations, and by the segregation ratio of M6 marker tion, and reproduction in any medium, provided you give appropriate in the TL population. This indicates that the allele-dosage credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. level previously assigned to the Ry gene was incorrect for adg all duplex and triplex samples of the panel. The absence of PVY disease symptoms and positive results for PVY° with DAS-ELISA in none of the eight nulliplex of the CL popu- lation and in seven out of ten nulliplex genotypes from the TL population suggest that another resistant gene to PVY 1 3 Theoretical and Applied Genetics (2018) 131:1925–1938 1937 Ghislain M, Núñez J, Herrera MR, Spooner DM (2009) The single References Andigenum origin of Neo-Tuberosum potato materials is not sup- ported by microsatellite and plastid marker analyses. Theor Appl Bradshaw JE, Mackay GR (1994) Breeding strategies for clonally Genet 118:963–969 propagated potatoes. 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