From QTL to candidate gene: Genetical genomics of simple and complex traits in potato using a pooling strategy

<p>Abstract</p> <p>Background</p> <p>Utilization of the natural genetic variation in traditional breeding programs remains a major challenge in crop plants. The identification of candidate genes underlying, or associated with, phenotypic trait QTLs is desired for effective marker assisted breeding. With the advent of high throughput -omics technologies, screening of entire populations for association of gene expression with targeted traits is becoming feasible but remains costly. Here we present the identification of novel candidate genes for different potato tuber quality traits by employing a pooling approach reducing the number of hybridizations needed. Extreme genotypes for a quantitative trait are collected and the RNA from contrasting bulks is then profiled with the aim of finding differentially expressed genes.</p> <p>Results</p> <p>We have successfully implemented the pooling strategy for potato quality traits and identified candidate genes associated with potato tuber flesh color and tuber cooking type. Elevated expression level of a dominant allele of the β-carotene hydroxylase (<it>bch</it>) gene was associated with yellow flesh color through mapping of the gene under a major QTL for flesh color on chromosome 3. For a second trait, a candidate gene with homology to a tyrosine-lysine rich protein (TLRP) was identified based on allele specificity of the probe on the microarray. TLRP was mapped on chromosome 9 in close proximity to a QTL for potato cooking type strengthening its significance as a candidate gene. Furthermore, we have performed a profiling experiment targeting a polygenic trait, by pooling individual genotypes based both on phenotypic and marker data, allowing the identification of candidate genes associated with the two different linkage groups.</p> <p>Conclusions</p> <p>A pooling approach for RNA-profiling with the aim of identifying novel candidate genes associated with tuber quality traits was successfully implemented. The identified candidate genes for tuber flesh color (<it>bch</it>) and cooking type (<it>tlrp</it>) can provide useful markers for breeding schemes in the future. Strengths and limitations of the approach are discussed.</p

Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations

Background - The establishment of mutant populations together with the strategies for targeted mutation detection has been applied successfully to a large number of organisms including many species in the plant kingdom. Considerable efforts have been invested into research on tomato as a model for berry-fruit plants. With the progress of the tomato sequencing project, reverse genetics becomes an obvious and achievable goal. Results - Here we describe the treatment of Solanum lycopersicum seeds with 1% EMS and the development of a new mutated tomato population. To increase targeted mutant detection throughput an automated seed DNA extraction has been combined with novel mutation detection platforms for TILLING in plants. We have adapted two techniques used in human genetic diagnostics: Conformation Sensitive Capillary Electrophoresis (CSCE) and High Resolution DNA Melting Analysis (HRM) to mutation screening in DNA pools. Classical TILLING involves critical and time consuming steps such as endonuclease digestion reactions and gel electrophoresis runs. Using CSCE or HRM, the only step required is a simple PCR before either capillary electrophoresis or DNA melting curve analysis. Here we describe the development of a mutant tomato population, the setting up of two polymorphism detection platforms for plants and the results of the first screens as mutation density in the populations and estimation of the false-positives rate when using HRM to screen DNA pools. Conclusion - These results demonstrate that CSCE and HRM are fast, affordable and sensitive techniques for mutation detection in DNA pools and therefore allow the rapid identification of new allelic variants in a mutant population. Results from the first screens indicate that the mutagen treatment has been effective with an average mutation detection rate per diploid genome of 1.36 mutation/kb/1000 line

Organ specificity and transcriptional control of metabolic routes revealed by expression QTL profiling of source--sink tissues in a segregating potato population

Abstract Background With the completion of genome sequences belonging to some of the major crop plants, new challenges arise to utilize this data for crop improvement and increased food security. The field of genetical genomics has the potential to identify genes displaying heritable differential expression associated to important phenotypic traits. Here we describe the identification of expression QTLs (eQTLs) in two different potato tissues of a segregating potato population and query the potato genome sequence to differentiate between cis- and trans-acting eQTLs in relation to gene subfunctionalization. Results Leaf and tuber samples were analysed and screened for the presence of conserved and tissue dependent eQTLs. Expression QTLs present in both tissues are predominantly cis-acting whilst for tissue specific QTLs, the percentage of trans-acting QTLs increases. Tissue dependent eQTLs were assigned to functional classes and visualized in metabolic pathways. We identified a potential regulatory network on chromosome 10 involving genes crucial for maintaining circadian rhythms and controlling clock output genes. In addition, we show that the type of genetic material screened and sampling strategy applied, can have a high impact on the output of genetical genomics studies. Conclusions Identification of tissue dependent regulatory networks based on mapped differential expression not only gives us insight in tissue dependent gene subfunctionalization but brings new insights into key biological processes and delivers targets for future haplotyping and genetic marker development.</p