Abstract
BackgroundRapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus (red raspberry). Recently, robust genotyping methods based on high-throughput sequencing were developed, which provide high marker density, but result in some genotype errors and a large number of missing genotype values. Imputation can reduce the number of missing values and can correct genotyping errors, but current methods of imputation require a reference genome and thus are not an option for most species.ResultsGenotyping by Sequencing (GBS) was used to produce highly saturated maps for a R. idaeus pseudo-testcross progeny. While low coverage and high variance in sequencing resulted in a large number of missing values for some individuals, a novel method of imputation based on maximum likelihood marker ordering from initial marker segregation overcame the challenge of missing values, and made map construction computationally tractable. The two resulting parental maps contained 4521 and 2391 molecular markers spanning 462.7 and 376.6 cM respectively over seven linkage groups. Detection of precise genomic regions with segregation distortion was possible because of map saturation. Microsatellites (SSRs) linked these results to published maps for cross-validation and map comparison.ConclusionsGBS together with genome-independent imputation provides a rapid method for genetic map construction in any pseudo-testcross progeny. Our method of imputation estimates the correct genotype call of missing values and corrects genotyping errors that lead to inflated map size and reduced precision in marker placement. Comparison of SSRs to published R. idaeus maps showed that the linkage maps constructed with GBS and our method of imputation were robust, and marker positioning reliable. The high marker density allowed identification of genomic regions with segregation distortion in R. idaeus, which may help to identify deleterious alleles that are the basis of inbreeding depression in the species.
Highlights
Rapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus
Shallow genome sampling resulted in a data set containing a large proportion of missing values, and we developed a pipeline which includes a novel imputation algorithm (Maskov) to deal with the missing and putatively erroneous data through comparison of genotypes in internal genotype bins following initial cosegregation analysis
Genotyping by sequencing Sequencing resulted in 135,776,036 reads including deeper coverage of parents
Summary
Rapid development of highly saturated genetic maps aids molecular breeding, which can accelerate gain per breeding cycle in woody perennial plants such as Rubus idaeus (red raspberry). Genetic linkage maps permit the elucidation of genome structure and organization and enable the identification of molecular markers linked to traits in an experimental segregating progeny, leading to the elucidation of the genetic basis of the trait of interest. Transferable linkage map development has been achieved through the scoring of restriction fragment length polymorphisms (RFLPs) [5], microsatellites (SSRs) and gene specific markers [3] in a segregating progeny. Using such markers, saturated reference linkage maps for many plant species have been developed. For minor crops and for genotyping interspecific progenies or species complexes, the development of arrays is currently not a viable experimental solution
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