Development of a high-resolution melting approach for reliable and cost-effective genotyping of PPVres locus in apricot (<Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">armeniaca</Emphasis>)

Sharka, caused by plum pox virus, is the most important viral disease of stone fruits. Important progresses have been recently achieved in apricot (Prunus armeniaca), identifying a major locus on chromosome 1 which explains most of the variability for plum pox virus (PPV) resistance trait. A set of molecular markers associated with the resistance has been developed and validated in different genetic backgrounds, endorsing their application for breeding purposes. Particularly for complex traits as the PPV resistance, requiring long and expensive phenotyping procedures, marker-assisted selection (MAS) bears a great potential to improve the efficiency of conventional breeding. In this work, novel HRM (high-resolution melting) assays were designed for the genotyping of resistant/susceptible alleles at PPV resistance (PPVres) locus. The assays were tested on 51 apricot cultivars and breeding selections already phenotyped for PPV resistance and cross-validated with standard short simple repeat marker data. We demonstrated that three HRM assays, PGS1.21_SNP, PGS1.24_SNP, and ZP002_DEL, represent a reliable, quick, and cost-effective genotyping approach, particularly suitable as high-throughput screening method for large-scale breeding programs.     

Identification of simple sequence repeat markers tightly linked to plum pox virus resistance in apricot

Sharka disease, caused by the plum pox virus (PPV), is one of the major limiting factors for stone fruit production in Europe and America. Attempts to stop the disease through the eradication of infected trees have been unsuccessful. Introgression of PPV resistance for crop improvement is therefore the most important goal in Prunus breeding programs. Due to time- and labour-consuming protocols, phenotyping for sharka is still the major bottleneck in the breeding pipeline. In this context, screening of seedlings at early stages of development and marker-assisted selection (MAS) provide the best solution for enhancing breeding efficiency. In this study, we generated 42 simple sequence repeat (SSR) markers from the peach genome assembly v1.0 and an apricot bacterial artificial chromosome clone identified in the physical map of the PPV resistance locus previously defined in apricot. Using a linkage mapping approach, we found SSR markers tightly linked to PPV resistance trait in all our progenies. Three SSR markers, PGS1.21 PGS1.23 and PGS1.24, showed allelic variants associated with PPV resistance with no recombinants in the crosses analysed. These markers unambiguously discriminated resistant from susceptible accessions in different genetic backgrounds. The results presented here are the first successful application of their use in MAS for breeding resistance in Prunus species.     

Flanking the major Plum pox virus resistance locus in apricot with co-dominant markers (SSRs) derived from candidate resistance genes

Plum pox virus (PPV) is a potyvirus that causes sharka disease in infested stone fruit trees (Prunus species, peach, apricot, plum). In apricots, the resistance is controlled by a major quantitative trait locus that explains up to 70% of the phenotypic variance; it is localised in the upper part of linkage group 1. In this report, we transformed candidate genes that mapped in the region of the apricot resistance locus into polymerase chain reaction markers (SSCP and SSR) and tested for their co-localisation with the major PPV resistance locus in related and unrelated populations. Three populations of F1 and F2 individuals issued from crosses between the PPV-resistant cultivar ‘Stark Early Orange’ or ‘Goldrich’ and three susceptible parents were used in this study. Molecular-marker data were collected to determine the linkage relationship between the PPV resistance locus in apricots and markers that target candidate disease-resistance genes. In addition, SSR markers linked to resistance-gene candidates were mapped to positions flanking the PPV resistance locus in different apricot populations. Therefore, we demonstrate that this strategy helps to saturate the major genomic region controlling resistance to PPV in apricot with valuable co-dominant markers. O. Sicard and G. Marandel have contributed equally to this work.     

Antioxidant enzymes as biochemical markers for sharka resistance in apricot

The activity of antioxidant enzymes in different apricot (Prunus armeniaca L.) cultivars, resistant or susceptible to Plum pox virus (PPV), was analyzed during the years 2002 and 2003. Resistant cultivars showed higher activities of catalase (CAT), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) than susceptible cultivars. Only CuZn-SOD isozymes were detected in the apricot cultivars. However, no correlation was observed between this isozyme pattern and the resistance to PPV. On the other hand, PPV-resistant apricot cultivars could have a greater capability for elimination of H₂O₂ and recycling of ascorbate-glutathione cycle, and they have at least two of these enzymatic activities (CAT, APX and DHAR) over the average. In contrast, this response was not observed in the susceptible cultivars. All these data suggest that the activities of CAT, APX and DHAR could be used as biochemical markers of sharka resistance in apricot.     

Opportunities of marker-assisted selection for Plum pox virus resistance in apricot breeding programs

Evaluation of Plum pox virus (PPV) resistance is a laborious and expensive task, and the development of new accurate methods, including the use of molecular markers, would be very useful for breeding programs for resistance. In this work, the Plum pox virus resistance of 80 apricot genotypes of different genetic origins was evaluated in controlled greenhouse and natural field conditions. The genotypes for five simple sequence repeat (SSR) markers described as linked to PPV resistance were also determined. Depending on their behavior, cultivars were classified as resistant, susceptible, and uncertain, and the genotype was identified for each SSR linked to different phenotypes. Twenty genotypes were resistant and 37 susceptible in the greenhouse and in the field. However, 23 genotypes did not show clear behavior, probably due to the complex plant-virus interaction, so they were classified as uncertain. In general, results showed a narrow relationship between the SSRs PGS1.21 and PGS1.24, and resistance to PPV, although some genotypes did not show this relationship. Most of the susceptible genotypes did not show the alleles of resistance. Therefore, in most cases, marker-assisted selection (MAS) could be used as a means of screening new seedlings for early selection, making it possible to remove those that are susceptible. However, in certain cases, MAS using these markers has not proven to be completely effective. The origin of such discrepancies could be the presence of a second locus involved in PPV resistance. In addition, other factors affecting efficiency of MAS discussed in the work are the presence of null alleles and recombinant events. Resistant seedlings would have to be evaluated in greenhouse and natural conditions to confirm their actual behavior against PPV. From the breeding point of view, the use of homozygous resistant parents for the SSR resistance alleles, with good agronomic characteristics, would increase the efficiency of breeding programs, since all seedlings would be resistant regardless of the other parent. Finally, new molecular markers should be developed to accurately select resistant seedlings regardless of the resistant progenitors involved.     

A genetic linkage map for an apricot (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) BC<Subscript>1</Subscript> population mapping <Emphasis Type="Italic">plum pox virus</Emphasis> resistance

Plum pox virus (sharka; PPV) can cause severe crop loss in economically important Prunus species such as peach, plum, apricot, and cherry. Of these species, certain apricot cultivars (‘Stark Early Orange’, ‘Goldrich’, ‘Harlayne’) display significant levels of resistance to the disease and are the genetic substrate for studies of several xlaboratories working cooperatively to genetically characterize and mark the resistance locus or loci for marker-assisted breeding. The goals of the work presented in this communication are the characterization of the genetics of PPV resistance in ‘Stark Early Orange’ and the development of co-dominant molecular markers for marker-assisted selection (MAS) in PPV resistance breeding. We present the first genetic linkage map for an apricot backcross population of ‘Stark Early Orange’ and the susceptible cultivar ‘Vestar’ that segregates for resistance to PPV. This map is comprised of 357 loci (330 amplified fragment length polymorphisms (AFLPs), 26 simple sequence repeats (SSRs), and 1 morphological marker for PPV resistance) assigned to eight linkage groups. Twenty-two of the mapped SSRs are shared in common with genetic reference map for Prunus (T × E; Joobeur et al. 1998) and anchor our apricot map to the general Prunus map. A PPV resistance locus was mapped in linkage group 1 and four AFLP markers segregating with the PPV resistance trait, identified through bulk segregant analysis, facilitated the development of SSRs in this region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lalli, D.A. and Salava, J. contributed equally to this work.     

High-throughput marker assays for <Emphasis Type="Italic">FaRPc2</Emphasis>-mediated resistance to Phytophthora crown rot in octoploid strawberry

Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is a destructive disease of the allo-octoploid cultivated strawberry (Fragaria ×ananassa Duch). Many major strawberry cultivars grown worldwide are susceptible to PhCR. Resistance is conferred by the recently-discovered FaRPc2 locus, but high-throughput markers are not yet available for marker-assisted breeding. In the current study, we developed DNA markers for two haplotypes at the FaRPc2 locus associated with resistance, H2 and H3. Marker validation and marker-assisted selection were performed in University of Florida (UF) breeding population. Seven single nucleotide polymorphism-based high resolution melting (HRM) markers linked to H2 and four HRM markers for H3 were developed. One HRM marker, RPCHRM3 linked to H3, was converted to a Kompetitive Allele Specific PCR (KASP) marker. To further examine the utility of the markers, they were screened in University of California Davis cultivars with known phenotypes as well as in 20 diverse accessions with phenotypes that are reported in the literature and that are preserved at the USDA-ARS National Clonal Germplasm Repository, in Corvallis, Oregon. The most informative markers for FaRPc2 resistance are being implemented in the UF strawberry breeding program to improve PhCR resistance.     

Marker-assisted introgression of a QTL region to improve rust resistance in three elite and popular varieties of peanut (Arachis hypogaea L.)

Key message

Successful introgression of a major QTL for rust resistance, through marker-assisted backcrossing, in three popular Indian peanut cultivars generated several promising introgression lines with enhanced rust resistance and higher yield.

Abstract

Leaf rust, caused by Puccinia arachidis Speg, is one of the major devastating diseases in peanut (Arachis hypogaea L.). One QTL region on linkage group AhXV explaining upto 82.62 % phenotypic variation for rust resistance was validated and introgressed from cultivar ‘GPBD 4’ into three rust susceptible varieties (‘ICGV 91114’, ‘JL 24’ and ‘TAG 24’) through marker-assisted backcrossing (MABC). The MABC approach employed a total of four markers including one dominant (IPAHM103) and three co-dominant (GM2079, GM1536, GM2301) markers present in the QTL region. After 2–3 backcrosses and selfing, 200 introgression lines (ILs) were developed from all the three crosses. Field evaluation identified 81 ILs with improved rust resistance. Those ILs had significantly increased pod yields (56–96 %) in infested environments compared to the susceptible parents. Screening of selected 43 promising ILs with 13 markers present on linkage group AhXV showed introgression of the target QTL region from the resistant parent in 11 ILs. Multi-location field evaluation of these ILs should lead to the release of improved varieties. The linked markers may be used in improving rust resistance in peanut breeding programmes.     

Narrowing down the apricot Plum pox virus resistance locus and comparative analysis with the peach genome syntenic region

Sharka disease, caused by the Plum pox virus (PPV), is one of the main limiting factors for stone fruit crops worldwide. Only a few resistance sources have been found in apricot (Prunus armeniaca L.), and most studies have located a major PPV resistance locus (PPVres) on linkage group 1 (LG1). However, the mapping accuracy was not sufficiently reliable and PPVres was predicted within a low confidence interval. In this study, we have constructed two high-density simple sequence repeat (SSR) improved maps with 0.70 and 0.68 markers/cm, corresponding to LG1 of 'Lito' and 'Goldrich' PPV-resistant cultivars, respectively. Using these maps, and excluding genotype-phenotype incongruent individuals, a new binary trait locus (BTL) analysis for PPV resistance was performed, narrowing down the PPVres support intervals to 7.3 and 5.9 cm in 'Lito' and 'Goldrich', respectively. Subsequently, 71 overlapping oligonucleotides (overgo) probes were hybridized against an apricot bacterial artificial chromosome (BAC) library, identifying 870 single BACs from which 340 were anchored onto a map region of approximately 30-40 cm encompassing PPVres. Partial BAC contigs assigned to the two allelic haplotypes (resistant/susceptible) of the PPVres locus were built by high-information content fingerprinting (HICF). In addition, a total of 300 BAC-derived sequences were obtained, and 257 showed significant homology with the peach genome scaffold_1 corresponding to LG1. According to the peach syntenic genome sequence, PPVres was predicted within a region of 2.16 Mb in which a few candidate resistance genes were identified.     

Identification of QTL for resistance to plum pox virus strains M and D in Lito and Harcot apricot cultivars

Sharka is a severe apricot viral disease caused by the plum pox virus (PPV) and is responsible for large crop losses in many countries. Among the known PPV strains, both PPV-D (Dideron) and PPV-M (Marcus) are virulent in apricot, the latter being the most threatening. An F1 apricot progeny derived from Lito, described in the literature as resistant, crossed to the susceptible selection BO81604311 (San Castrese × Reale di Imola) was used to study the genetic control of resistance to PPV. A population of 118 individuals was phenotyped by inoculating both PPV-D and PPV-M strains in replicated seedlings and scored for 3 years. An additional set of 231 seedlings from the same cross was also phenotyped for 2 years. SSR-based linkage maps were used for quantitative trait locus (QTL) analysis. A major QTL of resistance to both PPV-M and PPV-D strains was found in the top half of the Lito linkage group 1, where a QTL was previously described in Stark Earli-Orange, the donor of Lito resistance. The LOD score was considerably enhanced when the recovery of plants from infection was taken into account. The results obtained in Lito were compared with those observed in a second apricot cross progeny (Harcot × Reale di Imola) in which QTL of resistance to sharka were also mapped in the same linkage group 1 for both PPV strains. Several models of resistance to sharka disease are discussed considering the segregation frequencies, the QTL alignment in the two maps and the information gathered from the literature.     

Quantitative trait analysis of resistance to plum pox virus in the apricot F1 progeny “Harlayne” × “Vestar”

Plum pox virus (PPV) is a devastating stone fruit disease of major importance, and better understanding of the genetic control of resistance to this trait would be useful for more efficient development of resistant cultivars. Previous studies have reported a locus of major effect from PPV resistance on linkage group 1. The current study confirms these results by mapping plum pox virus resistance in a F1 progeny issued from a cross between “Harlayne”, as a PPV-resistant parent, and “Vestar” as a susceptible parent. The hybrids were grafted simultaneously and subsequently inoculated with the PPV-M and D strains. The symptom scoring on leaves was performed nine times over two vegetative cycles. Marker–trait associations were analyzed using the Kruskal–Wallis (KW) non-parametric test, and the PPV resistance loci were mapped using composite interval mapping (CIM). We show that both analyses (KW and CIM) highlighted the upper part of linkage group 1 of the apricot “Harlayne” genitor.     

Quantitative trait loci for resistance to Maize rayado fino virus

Maize rayado fino virus (MRFV) causes one of the most important virus diseases of maize in regions of Mexico, Central and South America, where it causes moderate to severe yield losses. The virus is found from the southern USA to northern Argentina where its vector, the maize leafhopper Dalbulus maidis, is present. Although resistance to MRFV has been identified in tropical maize lines, little was known about genes or quantitative trait locus (QTL) conferring resistance to MRFV. In order to identify the location of genes conferring resistance to MRFV, two recombinant inbred line mapping populations that segregated for MRFV resistance were inoculated using viruliferous leafhoppers, and their responses to virus inoculation were evaluated under greenhouse conditions 7, 14 and 21 days post inoculation. A QTL explaining up to 23 % of the total phenotypic variance was mapped on chromosome 10 in both populations, with similar genetic and physical positions identified in the two populations. The magnitude of the QTL effect and the validation in two independent populations suggests that resistance to MFRV could be transferred into elite breeding lines to develop resistant cultivars.     

Development of molecular markers linked to the 'Fiesta' linkage group 7 major QTL for fire blight resistance and their application for marker-assisted selection.

A fire blight resistance QTL explaining 34.3%-46.6% of the phenotypic variation was recently identified on linkage group 7 of apple cultivar 'Fiesta' (F7). However, markers flanking this QTL were AFLP and RAPD markers unsuitable for marker-assisted selection (MAS). Two RAPD markers bracketing the QTL have been transformed into SCAR (sequence-characterized amplified region) markers, and an SSR marker specific for the region was developed. Pedigree analysis of 'Fiesta' with these markers enabled tracking of the F7 QTL allele back to 'Cox's Orange Pippin'. Stability of the effect of this QTL allele in different backgrounds was analyzed by inoculating progeny plants of a cross between 'Milwa', a susceptible cultivar, and '1217', a moderately resistant cultivar, and a set of cultivars that carry or lack the allele conferring increased fire blight resistance. Progenies and cultivars that carried both markers were significantly more resistant than those that did not carry both markers, indicating high stability of the F7 QTL allele in different backgrounds. This stability and the availability of reproducible markers bracketing the QTL make this locus promising for use in MAS.     

Accumulation of quantitative trait loci conferring broad-spectrum clubroot resistance in Brassica oleracea

Throughout the world, clubroot disease is one of the most damaging diseases affecting Brassica oleracea. To develop marker-assisted selection (MAS) that could assist the incorporation of durable clubroot resistance (CR) into cultivars, previous genetic analyses have identified several CR quantitative trait loci (CR–QTL). However, the independent and cumulative effects of each CR locus against various isolates have rarely been tested. Previously, we identified one major CR–QTL and four minor CR–QTL in the F2 plants from broccoli doubled haploid (DH) line × cabbage DH line of B. oleracea. In the present study, to clarify their effectiveness for controlling disease involving various isolates, inoculation testing was conducted in genotypes with various combinations of the CR genes, which were selected using the DNA markers closely associated with each CR–QTL. In exploring the overall disease incidence, it was apparent that a single involvement of the major CR gene located in the PbBo(Anju)1 locus, or accumulation of CR genes in the minor CR–QTL, is not enough to confer sufficient resistance. One major CR gene in the QTL PbBo(Anju)1 locus plus two to three minor CR genes conferred moderate resistance. The genotype in which all of the CR genes locating in the five QTL including PbBo(Anju)1 were accumulated showed the highest resistance, and it was broadly resistant against six isolates. Accumulation of several CR genes by MAS is necessary to conduct CR breeding in B. oleracea. Our developed DNA markers can be used efficiently to make selections of required loci for the acquisition of resistance, and use of these markers will be a powerful tool for CR breeding in B. oleracea.     

Identification of the chromosomal region responsible for high-temperature stress tolerance during the grain-filling period in rice

An unusually high temperature during the grain-filling period, such as that caused by global warming, impairs the quality of rice (Oryza sativa L.) grains. This sensitivity to high-temperature stress is different among cultivars, suggesting the possibility of developing a high-temperature-tolerant cultivar. Since marker-assisted selection would reduce time and labor in breeding for such a quantitative trait, we determined the chromosomal region responsible for high-temperature tolerance during the grain-filling period. A high-temperature-sensitive japonica cultivar Tohoku 168 and a tolerant japonica cultivar Kokoromachi were selected as the parental lines of recombinant inbred lines (RILs) by high-temperature stress treatment from 5 to 10 days after anthesis, which was found to be the period most critical for grain quality. Using the RILs, whose genotypes were determined by analysis with 131 DNA markers which were selected as polymorphic markers between these two cultivars from 2,648 DNA markers tested, the quantitative trait locus (QTL) for the percentage of white-back grains was mapped on chromosome 6. The Kokoromachi allele of the QTL, which had a positive additive effect on the high-temperature tolerance, was introduced into the Tohoku 168 genome by repeated backcrossings with marker-assisted selection. Using high-temperature stress treatment of the near isogenic lines developed, the QTL on chromosome 6 was localized within a 1.9-Mb region between two DNA markers, ktIndel001 and RFT1. These DNA markers would be useful not only for breeding high-temperature-tolerant cultivars but also for map-based cloning of the QTL.     

A QTL detected in an interspecific pear population confers stable fire blight resistance across different environments and genetic backgrounds

Fire blight, caused by the bacterium Erwinia amylovora (Burrill) Winslow et al., is one of the most serious diseases of pear. The development of pear cultivars with a durable resistance is extremely important for effective control of fire blight and is a key objective of most pear breeding programs throughout the world. We phenotyped seedlings from the interspecific pear population PEAR3 (PremP003, P. × bretschneideri × P. communis) × ‘Moonglow’ (P. communis) for fire blight resistance at two different geographic locations, in France and New Zealand, respectively, employing two local E. amylovora isolates. Using a genetic map constructed with single nucleotide polymorphism (SNP) and microsatellite (SSR) markers previously developed for this segregating population, we detected a major quantitative trait locus (QTL) on linkage group (LG)2 of ‘Moonglow’ (R ² = 12.9–34.4 %), which was stable in both environments. We demonstrated that this QTL co-localizes with another major QTL for fire blight resistance previously detected in ‘Harrow Sweet’ and that the two favorable (i.e., resistant) alleles were not identical by descent. We also identified some smaller effect (R ² = 8.1–14.8 %) QTLs derived from the susceptible parent PEAR3. We propose SNP and SSR markers linked to the large effect QTL on LG2 as candidates for marker-assisted breeding for fire blight resistance in pear.     

Combined use of bulked segregant analysis and microarrays reveals SNP markers pinpointing a major QTL for resistance to Phytophthora capsici in pepper

Key message

Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed candidate genes underlying the major QTL for Phytophthora capsici resistance in Capsicum . Using the candidate genes, reliable markers for Phytophthora resistance were developed and validated.

Abstract

Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of pepper against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F₂ population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 × 10³/mL). Resistance phenotype segregation ratios for the populations fit a 3:1 and 1:1 (resistant:susceptible) segregation model, respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 was used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to P. capsici resistance. Markers linked to these genes were developed and validated by testing 100 F₁ commercial cultivars. Among the markers, Phyto5NBS1 showed about 90 % accuracy in predicting resistance phenotypes to a low-virulence P. capsici isolate. These results suggest that Phyto5NBS1 is a reliable marker for P. capsici resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5.