Using SNP markers to dissect linkage disequilibrium at a major quantitative trait locus for resistance to the potato cyst nematode Globodera pallida on potato chromosome V

The damage caused by the parasitic root cyst nematode Globodera pallida is a major yield-limiting factor in potato cultivation . Breeding for resistance is facilitated by the PCR-based marker ‘HC’, which is diagnostic for an allele conferring high resistance against G. pallida pathotype Pa2/3 that has been introgressed from the wild potato species Solanum vernei into the Solanum tuberosum tetraploid breeding pool. The major quantitative trait locus (QTL) controlling this nematode resistance maps on potato chromosome V in a hot spot for resistance to various pathogens including nematodes and the oomycete Phytophthora infestans. An unstructured sample of 79 tetraploid, highly heterozygous varieties and breeding clones was selected based on presence (41 genotypes) or absence (38 genotypes) of the HC marker. Testing the clones for resistance to G. pallida confirmed the diagnostic power of the HC marker. The 79 individuals were genotyped for 100 single nucleotide polymorphisms (SNPs) at 10 loci distributed over 38 cM on chromosome V. Forty-five SNPs at six loci spanning 2 cM in the interval between markers GP21-GP179 were associated with resistance to G. pallida. Based on linkage disequilibrium (LD) between SNP markers, six LD groups comprising between 2 and 18 SNPs were identified. The LD groups indicated the existence of multiple alleles at a single resistance locus or at several, physically linked resistance loci. LD group C comprising 18 SNPs corresponded to the ‘HC’ marker. LD group E included 16 SNPs and showed an association peak, which positioned one nematode resistance locus physically close to the R1 gene family. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of somatic hybrids between two sterile dihaploid Solanum tuberosum L. breeding lines. Restoration of fertility and complementation of G. pallida Pa2 and Pa3 resistance

Fourteen somatic hybrids generated by electrofusion of mesophyll protoplasts from a non-flowering dihaploid S. tuberosum clone, DHAK-11, and a male-sterile dihaploid clone S. tuberosum, DHAK-33, were grown in the greenhouse and subjected to morphological assessments and tests for fertility and resistance to the white potato cyst nematode Globodera pallida pathotypes Pa2 and Pa3. The ploidy level of the hybrids ranged from 38 to 63 chromosomes. All hybrids developed flowers with violet petals except for one, hy-56, that possessed red petals. The colour of the tuber skin was purple in all hybrids except in hy-56 where the tuber skin was red. All of the hybrids were female fertile and generated viable seeds. Near-tetraploid hybrids produced the highest number of seeds per fruit and these seeds had a normal size. Hybrids with 58 or more chromosomes produced smaller seeds and less seeds per fruit. The germination frequency of the seeds was not influenced by the chromosome number of the hybrids. Pollen viability was determined and the male fertility of three hybrids was tested. Pollination with these three hybrids gave rise to fruit development, but only one produced viable seeds. The hybrids were tested for resistance to G. pallida pathotypes Pa2 and Pa3. A high level of resistance to Pa3, inherited from one parental clone, DHAK-11, and a high level of resistance to Pa2, inherited from the other parental clone, DHAK-33, was combined in four hybrids. These results demonstrate, that protoplast fusion is an efficient method for restoring the fertility of somatic hybrids generated from sterile parent clones, and is a powerful procedure for the complementation of multigenetic disease resistance traits in potato breeding lines.     

Glucose phosphate isomerase polymorphism in field populations of the potato cyst nematodes, Globodera rostochiensis and G. pallida

The isozymic variation of glucose phosphate isomerase (GPI) was compared in 20 field populations of potato cyst-nematode (PCN) and related to variation in reference pathotypes of PCN. Populations pathotyped as G. rostochiensis Ro 1 by conventional differential cultivar tests were found to be different from populations of G. pallida. Variation within populations pathoptyped as G. pallida was seen and three populations were found to be similar to the reference G. pallida Pal pathotype. This seemed to be confirmed for two populations using cysts formed on a cultivar resistant to the Pa 1 pathotype when an isoenzyme pattern characteristic of G. palida Pa2 and Pa3 was found. The feasibility of the use of isozyme identification of PCN pathotypes is discussed in the light of these results.     

The effect of pyramiding two potato cyst nematode resistance loci to Globodera pallida Pa2/3 in potato

Globodera pallida is a major nematode pest causing severe constraints in many potato production regions worldwide. The most prevalent G. pallida pathotypes are Pa2 and Pa3, which exist in mixed populations referred to as Pa2/3. Due to heterogeneity for avirulence genes within these nematode populations, so far, breeders have failed to identify a single resistance gene source which offers complete resistance. Currently, there are two quantitative trait loci (QTL) available, $ GpaIV_{adg}^{s} $ and Gpa5, conferring partial levels of resistance to G. pallida pathotype Pa2/3. The objective of this research was to pyramid $ GpaIV_{adg}^{s} $ and Gpa5 through marker-assisted selection to investigate whether or not pyramiding provides increased resistance to G. pallida Pa2/3. We developed a population segregating for both resistance QTLs and, using diagnostic genetic markers (Contig237 and HC), we identified groups of individuals from this population containing each individual QTL, both QTLs simultaneously, and neither QTL. These individuals were assessed for their resistance levels against G. pallida Pa2/3 population Chavornay. We demonstrated that individuals carrying both QTLs showed a significant reduction in the number of cysts formed in comparison to genotypes carrying $ GpaIV_{adg}^{s} $ or Gpa5, indicating an additive effect. Overall these results show that MAS-based pyramiding of these QTLs is an effective strategy for breeding cultivars exhibiting very high levels of resistance to G. pallida pathotype Pa2/3.     

Invasion and development of juveniles of Globodera pallida in hybrids of Solanum vernei x S. tuberosum

Invasion and development of Globodera pallida (Pa 2) were investigated in the susceptible cultivar Pentland Crown and in two Solanum tuberosum S. vernei clones, 8917 b (3) and 12380 abc (2) with respective susceptibilities of 10% and < 1%. The numbers of juveniles per unit length of main root of Pentland Crown and 8917 b (3) did not differ, but the numbers found in the highly resistant clone 12380 abc (2) were significantly lower at all sampling dates except the last. Development of juveniles was substantially retarded in 12380 abc (2) and there was a higher ratio of males to females in both the resistant clones. It is suggested that reduced invasion and establishment can contribute to resistance and tolerance of potatoes to potato cyst-nematode.     

Quantitatively-inherited resistance toGlobodera pallida is dominated by one major locus inSolanum spegazzinii

A high level of resistance toGlobodera pallida pathotypes Pa2 and Pa3 exists inSolanum spegazzinii, a wild relative of potato (S. tuberosum ssp.tuberosum). Here we report the mapping of loci involved in quantitatively-inherited nematode resistance with the use of RFLPs. One major locus,Gpa, was mapped on chromosome 5 and two minor loci on chromosomes 4 and 7 ofS. spegazzinii. Additionally, the contribution of the susceptible parent to nematode resistance was determined. TheGpa locus was solely responsible for the high resistance level found in the segregating population. However, the RFLP marker closely linked to this resistance locus showed a distorted segregation, with a shortage of plants having the resistance linked allele. Our results indicate that a prediction of the genetic constitution of a quantitative trait based solely on phenotypic observations can lead to erroneous conclusions.     

The effect of resistant potato cultivars on a field population of Globodera pallida Pa2 over three years of cropping

Six potato cultivars with different levels of resistance to the white potato cyst nematode (PCN) Globodera pallida Pa2 were grown for three seasons in field plots to which G. pallida Pa2 cysts had been introduced earlier. There were two planting times, corresponding to early and maincrop commercial planting times, and two initial PCN population densities, high and low. The effect of cultivar on PCN population density was far greater than the effect of planting time or initial nematode population. The final PCN populations for the cultivars Ilam Hardy, Wha, 4696A(2), Sovereign, D40/6 and V390 were 151, 74, 27, 1.4, 0.2 and 0.06 eggs per g of soil respectively. It is concluded that resistant potato cultivars can be very effective in controlling G. pallida Pa2 in the field.     

Breeding for resistance to the white potato cyst-nematode, Heterodera pallida

Progenies bred from material derived from the wild potato, Solanum vernei and from the cultivated potato, S. tuberosum ssp. andigena, clone CPC 2775, were compared for their resistance to Heterodera pallida, pathotype E. The influence of additional resistance derived from the wild species, S. multidissectum, was also investigated. Both S. vernei and CPC 2775 gave progenies with variable levels of resistance and there was often no clear segregation into resistant and susceptible categories. Incorporation of gene H₂ derived from S. multidissectum increased resistance to pathotype E of H. pallida for resistant material bred from both S. vernei and clone CPC 2775. The results indicate that adequate resistance to all British populations of potato cyst-nematodes can best be obtained by combining the factors for resistance from the two Andigena clones, CPC 1673 (gene H₁) and CPC 2775 (gene H₃), and from S. multidissectum (gene H₂).     

Genome scans on experimentally evolved populations reveal candidate regions for adaptation to plant resistance in the potato cyst nematode Globodera pallida

Improving resistance durability involves to be able to predict the adaptation speed of pathogen populations. Identifying the genetic bases of pathogen adaptation to plant resistances is a useful step to better understand and anticipate this phenomenon. Globodera pallida is a major pest of potato crop for which a resistance QTL, GpaV_vrn, has been identified in Solanum vernei. However, its durability is threatened as G. pallida populations are able to adapt to the resistance in few generations. The aim of this study was to investigate the genomic regions involved in the resistance breakdown by coupling experimental evolution and high‐density genome scan. We performed a whole‐genome resequencing of pools of individuals (Pool‐Seq) belonging to G. pallida lineages derived from two independent populations having experimentally evolved on susceptible and resistant potato cultivars. About 1.6 million SNPs were used to perform the genome scan using a recent model testing for adaptive differentiation and association to population‐specific covariables. We identified 275 outliers and 31 of them, which also showed a significant reduction in diversity in adapted lineages, were investigated for their genic environment. Some candidate genomic regions contained genes putatively encoding effectors and were enriched in SPRYSECs, known in cyst nematodes to be involved in pathogenicity and in (a)virulence. Validated candidate SNPs will provide a useful molecular tool to follow frequencies of virulence alleles in natural G. pallida populations and define efficient strategies of use of potato resistances maximizing their durability.     

Control of potato cyst nematode (Globodera pallida) by host plant resistance and nematicide

Seven trials conducted over four years on sites naturally infested with the white potato cyst nematode established that potato clones bred for resistance to Globodera pallida allowed significantly less nematode multiplication than conventional cultivars under field conditions. Nematode multiplication was inversely related to initial infestation level. The nematicide, aldicarb, significantly reduced nematode multiplication. However, nematode multiplication on nematicide treated susceptible cultivars was greater than on untreated partially resistant clones, indicating that resistance may offer more effective control of G. pallida than chemical treatment. Integration of host plant resistance and nematicide treatment is discussed.     

In vitro screening for burrowing nematode, Radopholus citrophilus, tolerance and resistance in commercial Anthurium hybrids

Summary A reliable method to screen Anthurium for burrowing nematode resistance and tolerance in vitro was developed using 17 genetically distinct Anthurium cultivars. Based on nonparametric data analysis, tolerance and resistance were found to be independent traits to be evaluated separately. An effective parameter for tolerance evaluation was ranking of relative leaf retention, whereas an effective parameter for resistance evaluation was the ranking of nematode reproduction, log(Rf+1). A comparison of the ranking of leaf retention with ranking of nematode reproduction clustered the cultivar responses to burrowing nematode infection into four groups: intolerant and resistant, moderately tolerant but susceptible, intolerant and susceptible, and tolerant and susceptible. ‘Ozaki’ was identified as an intolerant reference, ‘Nitta’ as a susceptible reference. ‘Blushing Bride’ was the most tolerant cultivar among those screened, but it may not be an ideal tolerant reference due to its low vigor. Future screening for burrowing nematode-tolerant and-resistant cultivars in Anthurium should include ‘Ozaki’ and ‘Nitta’ as internal controls. Evaluation of resistance should be based on a resistance index obtained by log(Rf of hybrid tested +1) divided by log(Rf of ‘Nitta’ +1); tolerance should be based on ranking of relative leaf retention.     

A DNA probe which distinguishes Globodera pallida Pa2/3 from G. rostochiensis Ro1 and other cyst-forming nematodes

Abstract

A small DNA fragment (approx. 350 base pairs) from the genome of the potato cyst nematode Globodera pallida Pa2/3 was cloned in a bacterial plasmid. When used as a probe in dot-blot DNA hybridisations against a range of nematodes, the cloned DNA bound to G. pallida Pa2/3 but not to Globodera rostochiensis Rol. The cereal cyst nematode Heterodera avenae, the clover cyst nematode Heterodera trifolii, the root knot nematodes Meloidogyne hapla and Meloidogyne incognita, and the beet cyst nematode Heterodera schactii did not cross-hybridise. This probe can detect as few as six larvae of G. pallida.     

Effective transgenic resistance to Globodera pallida in potato field trials

A cysteine proteinase inhibitor expressed in potato plants provides the first demonstration that transgenic resistance to nematodes such as the potato cyst nematode Globodera pallida can be effective under field conditions. The highest level of resistance obtained in the field for one of the four transformed lines of the normally fully susceptible Solanum tuberosum tuberosum cv. Désirée was 70±9%. The partially resistant cv. Sante that is currently of commercial use in the UK showed a resistance of 85±3%. In containment experiments a change in G. pallida population to one known to be virulent against cv. Sante caused a significant loss of its resistance from 80±4% to only 51±6%. In contrast, the resistance of transgenic Désirée was similar for challenge by the avirulent and virulent populations with values of 68±6% and 71±4%, respectively. Constitutive expression of the cystatin had no detrimental effect on either number or weight of tubers and the weight of the haulm for 3 of the 4 lines in the field. The results establish that transgenic field resistance against G. pallida can be achieved. Clearly food and environmental safety must be assured before commercialisation can be contemplated. However, a prima facie case can be made that the technology is benign and can reduce the use of environmentally hazardous nematicides.     

A high-resolution map of the <Emphasis Type="Italic">Grp1</Emphasis> locus on chromosome V of potato harbouring broad-spectrum resistance to the cyst nematode species <Emphasis Type="Italic">Globodera pallida</Emphasis> and <Emphasis Type="Italic">Globodera rostochiensis</Emphasis>

The Grp1 locus confers broad-spectrum resistance to the potato cyst nematode species Globodera pallida and Globodera rostochiensis and is located in the GP21-GP179 interval on the short arm of chromosome V of potato. A high-resolution map has been developed using the diploid mapping population RHAM026, comprising 1,536 genotypes. The flanking markers GP21 and GP179 have been used to screen the 1,536 genotypes for recombination events. Interval mapping of the resistances to G. pallida Pa2 and G. rostochiensis Ro5 resulted in two nearly identical LOD graphs with the highest LOD score just north of marker TG432. Detailed analysis of the 44 recombinant genotypes showed that G. pallida and G. rostochiensis resistance could not be separated and map to the same location between marker SPUD838 and TG432. It is suggested that the quantitative resistance to both nematode species at the Grp1 locus is mediated by one or more tightly linked R genes that might belong to the NBS-LRR class. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. Finkers-Tomczak and S. Danan contributed equally to this research.     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

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     

A QTL for broad-spectrum resistance to cyst nematode species (Globodera spp.) maps to a resistance gene cluster in potato

 Broad-spectrum resistance in potato to the potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida is commonly regarded as a polygenically inherited trait. Yet, by use of QTL analysis and a selected set of PCN populations, resistance to both PCN species could be ascribed to the action of locus Grp1. Grp1 confers major resistance to G. rostochiensis line Ro₅-22 and G. pallida population Pa₂-D383 and partial resistance to G. pallida population Pa₃-Rookmaker. Grp1 was mapped on chromosome 5 using previously characterized AFLP markers. Cleaved amplified polymorphic sequence (CAPS) markers available for RFLP loci GP21 and GP179 revealed that Grp1 maps on a genomic region harboring other resistance factors to viral, fungal and nematodal pathogens. The present data indicate that Grp1 is a compound locus which contains multiple genes involved in PCN resistance. Received: 10 September 1997 / Accepted: 6 October 1997     

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.     

A new SNP haplotype associated with blue disease resistance gene in cotton (Gossypium hirsutum L.)

Resistance to cotton blue disease (CBD) was evaluated in 364 F_2.3 families of three populations derived from resistant variety ‘Delta Opal’. The CBD resistance in ‘Delta Opal’ was controlled by one single dominant gene designated Cbd. Two simple sequence repeat (SSR) markers were identified as linked to Cbd by bulked segregant analysis. Cbd resides at the telomere region of chromosome 10. SSR marker DC20027 was 0.75 cM away from Cbd. DC20027 marker fragments amplified from 3 diploid species and 13 cotton varieties whose CBD resistance was known were cloned and sequenced. One single nucleotide polymorphism (SNP) was identified at the 136th position by sequence alignment analysis. Screening SNP markers previously mapped on chromosome 10 identified an additional 3 SNP markers that were associated with Cbd. A strong association between a haplotype based on four SNP markers and Cbd was developed. This demonstrates one of the first examples in cotton where SNP markers were used to effectively tag a trait enabling marker-assisted selection for high levels of CBD resistance in breeding programs.     

Testing potatoes bred from Solarium vernei for resistance to the white potato cyst-nematode, Heterodera pallida

Three clones of potatoes bred from Solanum vernei were tested for their resistance to the white potato cyst-nematode, Heterodera pallida, both by root-ball counts and by estimates of total cysts produced. Two susceptible clones were used as controls. Root-ball counts suggested a higher degree of resistance than that based on total cysts produced. There were differences in pathogenicity between the two populations. The degree of resistance found was such that in practice the resistant clones would be best used in conjunction with nematicide treatment in a system of integrated control.