Ivan Simko

Assessment of linkage disequilibrium in potato genome with single nucleotide polymorphism markers.

The extent of linkage disequilibrium (LD) is an important factor in designing association mapping experiments. Unlike other plant species that have been analyzed so far for the extent of LD, cultivated potato (Solanum tuberosum L.), an outcrossing species, is a highly heterozygous autotetraploid. The favored genotypes of modern cultivars are maintained by vegetative propagation through tubers. As a first step in the LD analysis, we surveyed both coding and noncoding regions of 66 DNA fragments from 47 accessions for single nucleotide polymorphism (SNP). In the process, we combined information from the potato SNP database with experimental SNP detection. The total length of all analyzed fragments was >25 kb, and the number of screened sequence bases reached almost 1.4 million. Average nucleotide polymorphism (θ = 11.5 × 10−3) and diversity (π = 14.6 × 10−3) was high compared to the other plant species. The overall Tajimas D value (0.5) was not significant, but indicates a deficit of low-frequency alleles relative to expectation. To eliminate the possibility that an elevated D value occurs due to population subdivision, we assessed the population structure with probabilistic statistics. The analysis did not reveal any significant subdivision, indicating a relatively homogenous population structure. However, the analysis of individual fragments revealed the presence of subgroups in the fragment closely linked to the R1 resistance gene. Data pooled from all fragments show relatively fast decay of LD in the short range (r2 = 0.208 at 1 kb) but slow decay afterward (r2 = 0.137 at ∼70 kb). The estimate from our data indicates that LD in potato declines below 0.10 at a distance of ∼10 cM. We speculate that two conflicting factors play a vital role in shaping LD in potato: the outcrossing mating type and the very limited number of meiotic generations.

The Area Under the Disease Progress Stairs: Calculation, Advantage, and Application

The area under the disease progress curve (AUDPC) is frequently used to combine multiple observations of disease progress into a single value. However, our analysis shows that this approach severely underestimates the effect of the first and last observation. To get a better estimate of disease progress, we have developed a new formula termed the area under the disease progress stairs (AUDPS). The AUDPS approach improves the estimation of disease progress by giving a weight closer to optimal to the first and last observations. Analysis of real data indicates that AUDPS outperforms AUDPC in most of the tested trials and may be less precise than AUDPC only when assessments in the first or last observations have a comparatively large variance. We propose using AUDPS and its standardized (sAUDPS) and relative (rAUDPS) forms when combining multiple observations from disease progress experiments into a single value.

Development of EST-SSR markers for the study of population structure in lettuce (Lactuca sativa L.).

A set of 61 simple sequence repeat (SSR) markers was developed from the 19,523 Lactuca sativa and Lactuca serriola unigenes. Approximately 4.5% of the unigenes contained a perfect SSR at least 20 bp long, corresponding to roughly 1 perfect SSR per 14.7 kb. Marker polymorphism was tested on a set comprising 96 accessions representing all major horticultural types and 3 wild species (L. serriola, Lactuca saligna, and Lactuca virosa). Both the average marker heterozygosity (UHe = 0.32) and the number of different alleles per locus (Na = 3.56) were significantly reduced in expressed sequence tag (EST)-SSRs as compared with anonymous SSRs (UHe = 0.59, Na = 5.53). Marker transfer rate to the wild species corresponded to the decreasing sexual compatibility with L. sativa and was higher for EST-SSRs (100% L. serriola, 87% L. saligna, and 75% L. virosa) than for anonymous SSRs (93%, 66%, and 42%, respectively). Assessment of population structure among 90 L. sativa cultivars with SSRs was in good agreement with classification into the horticultural types. The average marker heterozygosity was smallest in iceberg (0.097), Latin (0.140), and romaine-type (0.151) cultivars while highest in leaf (green leaf 0.208 and red leaf 0.240) lettuces. The level of marker heterozygosity is in accord with morphological variability observed in different horticultural types.

Comparative analysis of quantitative trait loci for foliage resistance toPhytophthora infestans in tuber-bearingSolanum species

Previously published data for potato foliage resistance to late blight were examined in a combined analysis to determine how the genetic backgrounds of the host and pathogen affect the occurrence of resistance QTLs. Data from 19 diploid populations and one tetraploid population that originated from at least 12 different tuber-bearingSolanum species were included. Comparative analysis across all populations revealed three highly active genomic regions on the distal parts of chromosomes 3,4, and 5. The region most consistently detected that conferred foliage resistance inSolanum was located on chromosome 5, near marker locus GP21. A previously identified cluster of three race-specific R-genes on chromosome 11 was not associated with polygenic resistance. Statistical examination of active QTLs indicates that congruence among QTLs is significantly affected by both genetic relatedness of mapping populations and race ofPhytophthora infestans used for resistance tests. The proportion of congruent QTLs for late blight resistance decreased from 0.52 detected in relatedSolanum populations tested with the same race ofPhytophthora infestans to 0.20 found in unrelated mapping populations tested with different races ofPhytophthora infestans. Analysis of resistance mapping tests provided statistical evidence for the occurrence of race-specific QTLs in tuber-bearingSolanum. The highly conserved genomic regions identified in the comparative analysis are likely to be good candidates for gene cloning or marker assisted selection in potato breeding programs.ResumenLos datos publicados anteriormente sobre la resistencia del follaje de papa al tizón tardío fueron revisados mediante un análisis combinado para determinar cómo las bases genéticas del hospedante y del patógeno afectan la ocurrencia de resistencia de caracteres cuantitativos (QTLs). Se incluyeron los datos de diecinueve poblaciones diploides y de una tetraploide, originadas de por lo menos veinte diferentes especies tuberizadas deSolanum. Los análisis comparativos que cruzaron todas las poblaciones revelaron tres regiones genómicas tremendamente activas en la parte distal de los cromosomas 3,4 y 5. La región mas consistentemente detectada, que confería resistencia foliar enSolanum estaba localizada en el cromosoma 5, la más cercana al marcador del locus GP21. Un conjunto previamente identificado de tres razas específicas de genes R en el cromosoma 11 no estuvo asociado con resistencia poligénica. El examen estadístico de QTLs activos indica que la congruencia entre QTLs se ve significativamente afectada tanto por el parentesco genético de la población mapeada como por la raza dePhytophthora infestans usada para las pruebas de resistencia. La proporción de QTLs congruente para la resistencia al tizón tardío descendió de 0.52, detectado en poblaciones emparentadas deSolanum probadas con la misma raza dePhytophthora infestans, a 0.20 encontrada en poblaciones mapeadas sin parentesco probadas con diferentes razas dePhytophthora infestans. El análisis de las pruebas de resistencia mapeadas proporciona evidencia estadística sobre la ocurrencia de QTLs de razas específicas enSolanum tuberizados. Las regiones genómicas altamente conservadas identificadas en el análisis comparativo son probablemente buenas candidatas para la clonación genética o la selección asistida de marcadores en los programas de mejoramiento de papa.

Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1

BackgroundLettuce (Lactuca saliva L.) is susceptible to dieback, a soilborne disease caused by two viruses from the family Tombusviridae. Susceptibility to dieback is widespread in romaine and leaf-type lettuce, while modern iceberg cultivars are resistant to this disease. Resistance in iceberg cultivars is conferred by Tvr1 - a single, dominant gene that provides durable resistance. This study describes fine mapping of the resistance gene, analysis of nucleotide polymorphism and linkage disequilibrium in the Tvr1 region, and development of molecular markers for marker-assisted selection.ResultsA combination of classical linkage mapping and association mapping allowed us to pinpoint the location of the Tvr1 resistance gene on chromosomal linkage group 2. Nine molecular markers, based on expressed sequence tags (EST), were closely linked to Tvr1 in the mapping population, developed from crosses between resistant (Salinas and Salinas 88) and susceptible (Valmaine) cultivars. Sequencing of these markers from a set of 68 cultivars revealed a relatively high level of nucleotide polymorphism (θ = 6.7 × 10-3) and extensive linkage disequilibrium (r2 = 0.124 at 8 cM) in this region. However, the extent of linkage disequilibrium was affected by population structure and the values were substantially larger when the analysis was performed only for romaine (r2 = 0.247) and crisphead (r2 = 0.345) accessions. The association mapping approach revealed that one of the nine markers (Cntg10192) in the Tvr1 region matched exactly with resistant and susceptible phenotypes when tested on a set of 200 L. sativa accessions from all horticultural types of lettuce. The marker-trait association was also confirmed on two accessions of Lactuca serriola - a wild relative of cultivated lettuce. The combination of three single-nucleotide polymorphisms (SNPs) at the Cntg10192 marker identified four haplotypes. Three of the haplotypes were associated with resistance and one of them was always associated with susceptibility to the disease.ConclusionWe have successfully applied high-resolution DNA melting (HRM) analysis to distinguish all four haplotypes of the Cntg10192 marker in a single analysis. Marker-assisted selection for dieback resistance with HRM is now an integral part of our breeding program that is focused on the development of improved lettuce cultivars.

Genetics of Resistance to Pests and Disease

Publisher Summary This chapter focuses on the genetics behind the development of pest and disease resistance. The analysis of mapped and cloned resistance genes shows that they often occur in clusters and that some of them can respond to more than one elicitor. Ongoing research on resistance gene evolution will help in understanding the dynamic interaction between potato plants and pathogens and opens a way for the development of more resistant cultivars. Although symptom expression is commonly used for disease scoring, there are problems associated with this technique. It may be difficult to distinguish between symptoms caused by the pathogen of interest and those caused by other pathogens or by abiotic stresses. In addition, the interaction between the pathogen and both its biotic and abiotic environments may result in a wide range of symptoms, some of which are not typical. Consequently, methods have been developed to quantify pathogen populations in plant tissues. Because short day conditions stimulate wild species to senesce, winter greenhouse trials also allow screening for true resistance rather than immature plant resistance. Wild and cultivated relatives of potato are credited with contributing the majority of disease resistance genes to breeding programs. Molecular analysis of potato resistance covers experimental strategies for gene mapping and cloning, resistance factors mapped in potato, resistance genes cloned and characterized, synteny of resistance loci in Solanaceae and marker-assisted resistance breeding numerous genes conferring resistance to viruses, nematodes, bacteria, fungi, oomycetes, and insects have been mapped in potato, and their location is summarized.

High-Resolution DNA Melting Analysis in Plant Research

Genetic and genomic studies provide valuable insight into the inheritance, structure, organization, and function of genes. The knowledge gained from the analysis of plant genes is beneficial to all aspects of plant research, including crop improvement. New methods and tools are continually being developed to facilitate rapid and accurate mapping, sequencing, and analyzing of genes. Here, I review the recent progress in the application of high-resolution melting (HRM) analysis of DNA, a method that allows detecting polymorphism in double-stranded DNA by comparing profiles of melting curves. Use of HRM has expanded considerably in the past few years as the method was successfully applied for high-throughput genotyping, mapping genes, testing food products and seeds, and other areas of plant research.

Empirical evaluation of DArT, SNP, and SSR marker-systems for genotyping, clustering, and assigning sugar beet hybrid varieties into populations

Dominant and co-dominant molecular markers are routinely used in plant genetic research. In the present study we assessed the success-rate of three marker-systems for estimating genotypic diversity, clustering varieties into populations, and assigning a single variety into the expected population. A set of 54 diploid sugar beet (Beta vulgaris L. ssp. vulgaris) hybrid varieties from five seed companies was genotyped with 702 Diversity Array-Technology (DArT), 34 Single Nucleotide Polymorphisms (SNP), and 30 Simple Sequence Repeats (SSR) markers. Analysis of the population structure revealed three well-defined populations and clustering of varieties that generally correlates with their seed company origin. Two populations each contained varieties from two different seed companies indicating genetic similarity of this material. The third population was comprised only of varieties from a single seed company. Analysis of the SSR and SNP datasets indicates that some of the hybrid varieties likely have a common (or very closely related) parent. Comparison of the three marker-systems revealed substantial differences in the number of loci needed for analyses. Varietal clustering required approximately 1.8-2×more SSR, 3-4.5×more SNP, and 4.8×more DArT markers than were required for detection of genotypic diversity. When marker-systems were compared across different types of analyses per locus success-rate was the highest for the SSR and the lowest for the DArT markers. Generally, about 1.4-3×more SNPs, and 4.9-13.3×more DArTs then SSRs were needed to achieve the 100% success-rate. However, using only DArT markers with a high level of polymorphism decreased the number of DArT loci needed for analyses by 38-61%. Results from the present work provide a premise to selecting the type(s) and number of markers that are needed for genetic diversity analysis of sugar beet hybrid varieties.

Polygene mapping as a tool to study the physiology of potato tuberization and dormancy

Efforts to breed for the ability to tuberize under long days or for the length of tuber dormancy should benefit not only from information on the location of genes associated with these traits, but also from an understanding of the hormones the genes control. Understanding the role of the respective genes is a logical step in developing the best breeding strategy, both for conventional breeding and for gene transfer using molecular techniques. Polygene mapping affords a way to achieve such understanding and is also a tool to study the physiology of potato tuberization and potato tuber dormancy. The addition of DNA markers to a plant population facilitates the mapping of polygenes that control quantitatively inherited traits segregating in the population. A quantitative trait locus denotes a region of chromosome that is linked to the marker gene and which has a significant effect on the quantitative trait under study. Our approach has been to find the quantitative trait loci associated with tuberization and tuber dormancy in a segregating diploid population, and then to map the population for quantitative trait loci associated with levels of hormones implicated in the control of these two traits. We are using a population derived from a hybrid between haploidSolanum tuberosum andS. berthaultii that was backcrossed to a different haploidS. tuberosum. We have found ten quantitative trait loci for the ability to tuberize under long days and eight quantitative trait loci for tuber dormancy. In the same population we have found one or more quantitative trait loci for polyamines, abscisic acid, tuberonic acid, tuberonic acid glucoside, zeatin riboside, and gibberellin A1. Some of the hormone quantitative trait loci have coincided with quantitative trait loci for tuberization or dormancy. Implications of such commonality are discussed, along with the usefulness and limitations of the methods.ResumenTodo lo que se haga para mejorar la habilidad de tuberizar bajo días largos o para la duratión de la latencia debería servir no sólo como información sobre la localización de los genes asociados con estos caracteres, sino también para comprender el control que ejercen los genes sobre las hormonas. El conocimiento del rol de los respectivos genes es un paso lógico para desarrollar una mejor estrategia, tanto para el mejoramiento convencional como para la transferencia de genes por medio de técnicas moleculares. El mapeo poligénico proporciona una forma de desarrollar dicho conocimiento y también constituye una herramienta para estudiar la fisiología de la tuberización y la latencia del tubérculo de papa. Añadir marcadores de ADN a una población de plantas facilita el mapeo de los poligenes que controlan cuantitativamente los caracteres segregantes heredados en una población. Un locus de caracteres cuantitativos senala una región del cromosoma que está ligada al gen marcador, el cual tiene un efecto significativo sobre el carácter cuantitativo en estudio. Nuestro enfoque ha sido ubicar los locus de caracteres cuantitativos asociados a la tuberización y latencia del tubérculo en una población diploide segregante y luego hacer un mapeo de la población para ubicar los locus de caracteres cuantitativos asociados con los niveles de hormonas implicadas en el control de estos dos caracteres. Estamos utilizando una población derivada de un híbrido entreSolanum tuberosum haploide yS. berthaultii que fue retrocruzado a un haploide diferente deS. tuberosum. Hemos encontrado diez locus de caracteres cuantitativos para habilidad de tuberización bajo días largos y ocho para latencia. En la misma población hemos encontrado uno o más locus de caracteres cuantitativos para poliaminas, ácido abscísico, ácido tuberónico, glucósido de ácido tuberónico, zeatina ribósido y giberelina A1. Algunos de los locus para caracteres cuantitativos de hormonas coincidieron con los locus de caracteres cuantitativos para tuberización o para dormancia. Se discuten las implicancias de tales hechos comunes, lo mismo que la utilidad y limitaciones de tales métodos.

Mining data from potato pedigrees: tracking the origin of susceptibility and resistance to Verticillium dahliae in North American cultivars through molecular marker analysis

Potato (Solanum tuberosum L.) cultivated in North America is an autotetraploid species with a narrow genetic base. Most of the popular commercial cultivars are susceptible to Verticillium dahliae, a fungal pathogen causing Verticillium wilt disease, though some cultivars with relatively high resistance also exist. We have used the available pedigree information to track the origin of susceptibility and resistance to Verticillium wilt present in cultivated potatoes. One hundred thirty-nine potato cultivars and breeding selections were analyzed for resistance to the pathogen and for the presence of the microsatellite marker allele STM1051–193 that is closely linked to the resistance quantitative trait locus located on the short arm of chromosome 9. We detected an unusually high frequency of susceptible genotypes in the progeny descending from the breeding selection USDA X96–56. Molecular analysis revealed that USDA X96–56 does not have the STM1051–193 allele. Most of the first-generation progeny of this breeding selection also lack the allele. On the other hand, pedigree analysis indicated that breeding selection USDA 41956 often transfers V. dahliae resistance to its progeny. Molecular analysis detected presence of (at least) three STM1051–193 alleles in this breeding selection. These two genotypes (USDA X96–56 and USDA 41956) appear to have contributed greatly to the susceptibility or resistance, respectively, found in present commercial cultivars. Our results also indicate that the maturity class substantially affects the plant resistance response. In the intermediate to very late maturing class, the presence of the STM1051–193 allele significantly increases the resistance. Early to very early potatoes are usually more susceptible to the disease regardless of the allelic status, though the pattern of the allele effect is always the same. The results indicate that the STM1051–193 allele can be used for marker-assisted selection, but the potato maturity class also needs to be considered when making the final decision about the plant resistance level.