Developing best practices for genotyping-by-sequencing analysis in the construction of linkage maps.

BACKGROUND: Genotyping-by-sequencing (GBS) provides affordable methods for genotyping hundreds of individuals using millions of markers. However, this challenges bioinformatic procedures that must overcome possible artifacts such as the bias generated by polymerase chain reaction duplicates and sequencing errors. Genotyping errors lead to data that deviate from what is expected from regular meiosis. This, in turn, leads to difficulties in grouping and ordering markers, resulting in inflated and incorrect linkage maps. Therefore, genotyping errors can be easily detected by linkage map quality evaluations. RESULTS: We developed and used the Reads2Map workflow to build linkage maps with simulated and empirical GBS data of diploid outcrossing populations. The workflows run GATK, Stacks, TASSEL, and Freebayes for single-nucleotide polymorphism calling and updog, polyRAD, and SuperMASSA for genotype calling, as well as OneMap and GUSMap to build linkage maps. Using simulated data, we observed which genotype call software fails in identifying common errors in GBS sequencing data and proposed specific filters to better handle them. We tested whether it is possible to overcome errors in a linkage map using genotype probabilities from each software or global error rates to estimate genetic distances with an updated version of OneMap. We also evaluated the impact of segregation distortion, contaminant samples, and haplotype-based multiallelic markers in the final linkage maps. Through our evaluations, we observed that some of the approaches produce different results depending on the dataset (dataset dependent) and others produce consistent advantageous results among them (dataset independent). CONCLUSIONS: We set as default in the Reads2Map workflows the approaches that showed to be dataset independent for GBS datasets according to our results. This reduces the number of required tests to identify optimal pipelines and parameters for other empirical datasets. Using Reads2Map, users can select the pipeline and parameters that best fit their data context. The Reads2MapApp shiny app provides a graphical representation of the results to facilitate their interpretation.

Construction of a High-Density Genetic Map of Acca sellowiana (Berg.) Burret, an Outcrossing Species, Based on Two Connected Mapping Populations.

Acca sellowiana, known as feijoa or pineapple guava, is a diploid, (2n = 2x = 22) outcrossing fruit tree species native to Uruguay and Brazil. The species stands out for its highly aromatic fruits, with nutraceutical and therapeutic value. Despite its promising agronomical value, genetic studies on this species are limited. Linkage genetic maps are valuable tools for genetic and genomic studies, and constitute essential tools in breeding programs to support the development of molecular breeding strategies. A high-density composite genetic linkage map of A. sellowiana was constructed using two genetically connected populations: H5 (TCO × BR, N = 160) and H6 (TCO × DP, N = 184). Genotyping by sequencing (GBS) approach was successfully applied for developing single nucleotide polymorphism (SNP) markers. A total of 4,921 SNP markers were identified using the reference genome of the closely related species Eucalyptus grandis, whereas other 4,656 SNPs were discovered using a de novo pipeline. The individual H5 and H6 maps comprised 1,236 and 1,302 markers distributed over the expected 11 linkage groups, respectively. These two maps spanned a map length of 1,593 and 1,572 cM, with an average inter-marker distance of 1.29 and 1.21 cM, respectively. A large proportion of markers were common to both maps and showed a high degree of collinearity. The composite map consisted of 1,897 SNPs markers with a total map length of 1,314 cM and an average inter-marker distance of 0.69. A novel approach for the construction of composite maps where the meiosis information of individuals of two connected populations is captured in a single estimator is described. A high-density, accurate composite map based on a consensus ordering of markers provides a valuable contribution for future genetic research and breeding efforts in A. sellowiana. A novel mapping approach based on an estimation of multipopulation recombination fraction described here may be applied in the construction of dense composite genetic maps for any other outcrossing diploid species.

Optimizing whole-genomic prediction for autotetraploid blueberry breeding.

Blueberry (Vaccinium spp.) is an important autopolyploid crop with significant benefits for human health. Apart from its genetic complexity, the feasibility of genomic prediction has been proven for blueberry, enabling a reduction in the breeding cycle time and increasing genetic gain. However, as for other polyploid crops, sequencing costs still hinder the implementation of genome-based breeding methods for blueberry. This motivated us to evaluate the effect of training population sizes and composition, as well as the impact of marker density and sequencing depth on phenotype prediction for the species. For this, data from a large real breeding population of 1804 individuals were used. Genotypic data from 86,930 markers and three traits with different genetic architecture (fruit firmness, fruit weight, and total yield) were evaluated. Herein, we suggested that marker density, sequencing depth, and training population size can be substantially reduced with no significant impact on model accuracy. Our results can help guide decisions toward resource allocation (e.g., genotyping and phenotyping) in order to maximize prediction accuracy. These findings have the potential to allow for a faster and more accurate release of varieties with a substantial reduction of resources for the application of genomic prediction in blueberry. We anticipate that the benefits and pipeline described in our study can be applied to optimize genomic prediction for other diploid and polyploid species.

Genomic prediction applied to multiple traits and environments in second season maize hybrids.

Genomic selection has become a reality in plant breeding programs with the reduction in genotyping costs. Especially in maize breeding programs, it emerges as a promising tool for predicting hybrid performance. The dynamics of a commercial breeding program involve the evaluation of several traits simultaneously in a large set of target environments. Therefore, multi-trait multi-environment (MTME) genomic prediction models can leverage these datasets by exploring the correlation between traits and Genotype-by-Environment (G×E) interaction. Herein, we assess predictive abilities of univariate and multivariate genomic prediction models in a maize breeding program. To this end, we used data from 415 maize hybrids evaluated in 4 years of second season field trials for the traits grain yield, number of ears, and grain moisture. Genotypes of these hybrids were inferred in silico based on their parental inbred lines using single nucleotide polymorphisms (SNPs) markers obtained via genotyping-by-sequencing (GBS). Because genotypic information was available for only 257 hybrids, we used the genomic and pedigree relationship matrices to obtain the H matrix for all 415 hybrids. Our results demonstrated that in the single-environment context the use of multi-trait models was always superior in comparison to their univariate counterparts. Besides that, although MTME models were not particularly successful in predicting hybrid performance in untested years, they improved the ability to predict the performance of hybrids that had not been evaluated in any environment. However, the computational requirements of this kind of model could represent a limitation to its practical implementation and further investigation is necessary.