J. E. Bradshaw

Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated potato

The application of AFLPs, RAPDs and SSRs to examine genetic relationships in the primary northwestern European cultivated potato gene pool was investigated. Sixteen potato cultivars were genotyped using five AFLP primer combinations, 14 RAPD primers, and 17 database-derived SSR primer pairs. All three approaches successfully discriminated between the 16 cultivars using a minimum of one assay. Similarity matrices produced for each marker type on the basis of Nei and Li coefficients showed low correlations when compared with different statistical tests. Dendrograms were produced from these data for each marker system. The usefulness of each system was examined in terms of number of loci revealed (effective multiplex ratio, or EMR) and the amount of polymorphism detected (diversity index, or DI). AFLPs had the highest EMR, and SSRs the highest DI. A single parameter, marker index (MI), which is the product of DI and EMR, was used to evaluate the overall utility of each marker system. The use of these PCR-based marker systems in potato improvement and statutory applications is discussed.Abbreviations: PCR, polymerase chain reaction; AFLP, amplified fragment length polymorphism; RAPD, randomly amplified polymorphic DNA; DNA, deoxyribonucleic acid; EMR, effective multiplex ratio; DI, diversity index; MI, marker index; RFLP, restriction fragment length polymorphism.

Genetic Resources (Including Wild and Cultivated Solanum Species) and Progress in their Utilisation in Potato Breeding

The genetic resources available for the improvement of the cultivated potato (Solanum tuberosum) are reviewed along with progress in their utilisation. The conclusions are as follows. The wild and cultivated species of potato have been utilised in potato breeding to good effect, but only a very small sample of the available biodiversity has been exploited. New knowledge and technology will open possibilities for much greater use of these genetic resources in breeding. The strategy for utilising the cultivars native to Latin America will either be the introgression of desirable genes or the direct use of parents from improved populations, depending on how far modern S. tuberosum cultivars have genetically diverged from them and the extent to which S. tuberosum cultivars have been improved in the process. Molecular marker-assisted selection will be used for faster introgression of desirable genes from wild species, and the possibility exists of moving genes directly from wild species to cultivated potato with transgenic methods. New cultivars will continue to come from crosses between pairs of parents with complementary features but adapted to local growing conditions. However, increasingly these parents will possess desirable genes which have been introgressed from wild species and may also be from complementary groups of cultivated germplasm to exploit hybrid vigour. Successful cultivars may be genetically modified, if consumers see benefits in the use of the technology, to introduce genes not present in cultivated potatoes and their wild relatives to achieve novel biochemistry and further desirable improvements.

Identification of AFLP and SSR markers associated with quantitative resistance to Globodera pallida (Stone) in tetraploid potato (Solanum tuberosum subsp. tuberosum) with a view to marker-assisted selection

Abstract Seventy eight clones from the cross between SCRI clone 12601ab1 and cv Stirling were used to explore the possibility of genetical linkage analysis in tetraploid potato (Solanum tuberosum subsp. tuberosum). Clone 12601ab1 had quantitative resistance to Globodera pallida Pa2/3 derived from S. tuberosum subsp. andigena. The strategy adopted involved identifying single- (simplex) and double- (duplex) dose AFLP markers in the parents from segregation ratios that could be unambiguously identified in their offspring, detecting linkage between a marker and a putative quantitative trait locus (QTL) for resistance, and placing the QTL on the linkage map of markers. The numbers of scorable segregating markers were 162 simplex ones present only in 12601ab1, 87 present in Stirling, and 32 present in both; and 72 duplex markers present only in 12601ab1 and 45 present in Stirling. The total map length was 990.9 cM in 12601ab1 and 484.6 cM in Stirling. A QTL with a resistance allele present in double dose (QQqq) in 12601ab1 was inferred from the associations between resistance scores (square root of female counts) and two duplex markers linked in coupling, which, in turn, were linked in coupling to four simplex markers also associated with resistance, but to a lesser degree. The largest marker class difference was the one for the duplex marker P61M34=15. It accounted for 27.8% of the phenotypic variance in resistance scores, or approximately 30% of the genotypic variance. Subsequently, this duplex marker was found to be linked in coupling with a duplex SSR allele Stm3016=a, whose locus was shown to be on chromosome IV in a diploid reference mapping population. The other QTLs for resistance segregating in the progeny were not identified for one or more of the following reasons: the markers did not cover the whole of the genome, there were unfavourable repulsion linkages between the QTLs and markers, or the gene effects were not large enough to be detected in an experiment of the size conducted. It is concluded that prospects appear good for detecting QTLs and using marker-assisted selection in a tetraploid potato breeding programme, provided that, in future, the population size is increased to over 250 and more SSR markers are used to complement the AFLPs; the same is likely to be true for other autotetraploid crops.

Constructing genetic linkage maps under a tetrasomic model.

An international consortium has launched the whole-genome sequencing of potato, the fourth most important food crop in the world. Construction of genetic linkage maps is an inevitable step for taking advantage of the genome projects for the development of novel cultivars in the autotetraploid crop species. However, linkage analysis in autopolyploids, the kernel of linkage map construction, is theoretically challenging and methodologically unavailable in the current literature. We present here a theoretical analysis and a statistical method for tetrasomic linkage analysis with dominant and/or codominant molecular markers. The analysis reveals some essential properties of the tetrasomic model. The method accounts properly for double reduction and incomplete information of marker phenotype in regard to the corresponding phenotype in estimating the coefficients of double reduction and recombination frequency and in testing their significance by using the marker phenotype data. Computer simulation was developed to validate the analysis and the method and a case study with 201 AFLP and SSR markers scored on 228 full-sib individuals of autotetraploid potato is used to illustrate the utility of the method in map construction in autotetraploid species.

LINKAGE ANALYSIS IN TETRAPLOID SPECIES : A SIMULATION STUDY

A simulation study was performed to investigate methods for mapping single-dose (simplex) and double-dose (duplex) markers, and for identification of homologous chromosomes in an autotetraploid species, and to see how the map accuracy depends on the population size. An initial population of 1000 individuals was simulated, with 30 simplex and 10 duplex markers, and recombination fractions and lod scores were calculated between all pairs of markers. These were used to test the feasibility of mapping the simplex and duplex markers simultaneously. Smaller populations, from 500 to 75 individuals, were then simulated, and the estimates of the pairwise recombination fractions and the derived maps were compared with the true map. It was found that the accuracy of the estimates depended strongly on the type of markers involved, with simplex–simplex coupling pairs being most reliable and simplex–simplex repulsion pairs and duplex–duplex pairs in any configuration but coupling being least reliable. Maps can be assembled using recombination fractions and lod scores from pairs of simplex–simplex markers (coupling and repulsion), duplex–simplex (coupling and repulsion) and duplex–duplex (coupling). The agreement between the map order and the true order was good, although the map distance was generally underestimated at small sample sizes.

Utilisation of the Commonwealth Potato Collection in potato breeding

SummaryThe use of the Commonwealth Potato Collection in potato breeding is set in the context of the evolution of the crop and the need to widen its genetic base by introgression and base broadening. The introduction of the potato to Europe and its subsequent worldwide spread is described. An introduction is given to the worlds major potato genebanks, and the current status of the Commonwealth Potato Collection is presented. Material from this genebank has been extensively used to improve the potato. Work on wild species as sources of resistance to late blight started before the genebank was initiated, and since then CPC accessions have provided major R-genes and durable resistance to breeders, greatly benefiting growers and consumers of the potato. Progress identifying and exploiting resistance to viruses and potato cyst nematodes is described. New sources of further pest and disease resistance genes are present in the germplasm in the collection, offering the potential to overcome current and future pests and diseases. Use of the cultivated species in the collection for base broadening is described and discussed. The collection also harbours a wide range of quality traits of use to breeders, including variation for cooking and crisping, anthocyanins, carotenoids, ascorbate metabolism and others. As breeding and genetics become more precise, and as both the knowledge of biochemical pathways and means of analysing chemical composition advance, new ways of accessing this variation become possible. Possible strategies to achieve these goals are discussed.

Potato oxysterol binding protein and cathepsin B are rapidly up-regulated in independent defence pathways that distinguish R gene-mediated and field resistances to Phytophthora infestans

SUMMARY Suppression subtractive hybridization was used to isolate the genes which are specifically up-regulated in the biotrophic phase of the incompatible interaction between a potato genotype, 1512 c(16), containing the resistance gene R2, and a Phytophthora infestans isolate containing the avirulence gene Avr2. Eight cDNAs were up-regulated in the biotrophic phase of the incompatible interaction. Seven of these were also up-regulated in the compatible interaction, but not until late in the necrotrophic phase. Amongst the sequences to be isolated were genes encoding the cysteine protease cathepsin B, StCathB, and an oxysterol binding protein, StOBP1; equivalent genes are involved in programmed cell death (PCD) processes in animals, but have yet to be implicated in such processes in plants. Whereas StOBP1 was up-regulated early in potato plants containing either R gene-mediated or moderate to high levels of field resistance, the highest levels of up-regulation of StCathB were observed early in R gene-mediated resistance but gradually increased from the early to late stages of field resistance, revealing these genes to be components of independent defence pathways and providing a means of distinguishing between these forms of resistance. StOBP1 was up-regulated by oligogalacturonides (plant cell wall breakdown products generated by pectinase activities), indicating that it is also a component of a general, non-specific defence pathway and is unlikely to play a role in PCD. In contrast, the expression of StCathB was unaffected by oligogalacturonide treatment, further associating its up-regulation specifically with the gene-for-gene interaction.

Progress in improving processing attributes in potato

The recent publication of a molecular-function map of carbohydrate metabolism and transport in potato enables a candidate-gene approach to be used to solve the problem of cold-sweetening, signifying a significant step in our understanding of this economically important trait. With confirmation of the role that many important enzymes play in starch metabolism and their position on a detailed linkage map, we should now be able to make progress in manipulating this trait, either through conventional means such as marker-assisted selection or through transgenic approaches.

Predicting parental genotypes and gene segregation for tetrasomic inheritance

Abstract Recent genome mapping projects in tetraploid plant species require a method for analysing the segregation patterns of molecular marker loci in these species. The present study presents a theoretical model and a statistical analysis for predicting the genotypes of a pair of tetraploid parents at a codominant (for example, RFLPs, microsatellites) or dominant (for example, AFLPs, RAPDs) molecular marker locus based on their and their progeny’s phenotypes scored at that locus (gel-band patterns). The theory allows for null alleles and for any degree of double-reduction to be modelled. A simulation study was performed to investigate the properties of the theoretical model. This showed that in many circumstances both the parental genotypes can be correctly identified with a probability of nearly 1, even when the molecular data were complicated by null alleles or double-reduction. Configurations where the parental genotype cannot be identified are discussed. The power to detect double-reduction varies considerably, depending on the proportion of identical alleles carried and shared by the parents, and the number of null alleles. Incorrect deductions of the occurrence of double-reduction were rare. The method was applied to data on a microsatellite locus segregating in the parents and 74 offspring of a tetraploid potato cross. Twentyfour parental configurations were consistent with the parental gel pattern, but only one of these was compatible with all the phenotypic data on the offspring. The feasibility for extending the present model to predict segregation of several linked loci, and particularly the linkage phase, is briefly discussed.

Correlation between resistance to late blight in foliage and tubers in potato clones from parents of contrasting resistance

SummaryThe relationship between resistance to late blight in foliage and tuber was examined on 50 clones of each of five progenies from crosses where one parent was resistant in both foliage and tuber and the other susceptible. Foliage resistance was assessed in a field trial and tuber resistance in a laboratory test on glasshouse-grown tubers. The genetical and environmental components of variance and the phenotypic and genetical correlations between foliage and tuber blight scores were determined for each progeny. Foliage and tuber resistance were correlated, indicating either that both are determined by the same genes or by different linked genes.The environmental component of variation was greater for tuber blight than foliage blight. We suggest that the most effective way of selecting for resistance to both aspects of the disease in a breeding programme is to select those resistant in the foliage and then screen them for tuber resistance.