A k-mer-based bulked segregant analysis approach to map seed traits in unphased heterozygous potato genomes.

High-throughput sequencing-based methods for bulked segregant analysis (BSA) allow for the rapid identification of genetic markers associated with traits of interest. BSA studies have successfully identified qualitative (binary) and quantitative trait loci (QTLs) using QTL mapping. However, most require population structures that fit the models available and a reference genome. Instead, high-throughput short-read sequencing can be combined with BSA of k-mers (BSA-k-mer) to map traits that appear refractory to standard approaches. This method can be applied to any organism and is particularly useful for species with genomes diverged from the closest sequenced genome. It is also instrumental when dealing with highly heterozygous and potentially polyploid genomes without phased haplotype assemblies and for which a single haplotype can control a trait. Finally, it is flexible in terms of population structure. Here, we apply the BSA-k-mer method for the rapid identification of candidate regions related to seed spot and seed size in diploid potato. Using a mixture of F1 and F2 individuals from a cross between 2 highly heterozygous parents, candidate sequences were identified for each trait using the BSA-k-mer approach. Using parental reads, we were able to determine the parental origin of the loci. Finally, we mapped the identified k-mers to a closely related potato genome to validate the method and determine the genomic loci underlying these sequences. The location identified for the seed spot matches with previously identified loci associated with pigmentation in potato. The loci associated with seed size are novel. Both loci are relevant in future breeding toward true seeds in potato.

LD-CNV: rapid and simple discovery of chromosomal translocations using linkage disequilibrium between copy number variable loci.

Large-scale structural variations, such as chromosomal translocations, can have profound effects on fitness and phenotype, but are difficult to identify and characterize. Here, we describe a simple and effective method aimed at identifying translocations using only the dosage of sequence reads mapped on the reference genome. We binned reads on genomic segments sized according to sequencing coverage and identified instances when copy number segregated in populations. For each dosage-polymorphic 1 Mb bin, we tested independence, effectively an apparent linkage disequilibrium (LD), with other variable bins. In nine potato (Solanum tuberosum) dihaploid families translocations affecting pericentromeric regions were common and in two cases were due to genomic misassembly. In two populations, we found evidence for translocation affecting euchromatic arms. In cv. PI 310467, a nonreciprocal translocation between chromosomes (chr.) 7 and 8 resulted in a 5-3 copy number change affecting several Mb at the respective chromosome tips. In cv. "Alca Tarma," the terminal arm of chr. 4 translocated to the tip of chr. 1. Using oligonucleotide-based fluorescent in situ hybridization painting probes (oligo-FISH), we tested and confirmed the predicted arrangement in PI 310467. In 192 natural accessions of Arabidopsis thaliana, dosage haplotypes tended to vary continuously and resulted in higher noise, while apparent LD between pericentromeric regions suggested the effect of repeats. This method, LD-CNV, should be useful in species where translocations are suspected because it tests linkage without the need for genotyping.

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability.

In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers (HIs) yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of HI DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three HIs: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n = 2x - 1 = 23 to 2n = 2x + 3 = 27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the HI parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual HI DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.

Genomic Outcomes of Haploid Induction Crosses in Potato (Solanum tuberosum L.).

The challenges of breeding autotetraploid potato (Solanum tuberosum) have motivated the development of alternative breeding strategies. A common approach is to obtain uniparental dihaploids from a tetraploid of interest through pollination with S. tuberosum Andigenum Group (formerly S. phureja) cultivars. The mechanism underlying haploid formation of these crosses is unclear, and questions regarding the frequency of paternal DNA transmission remain. Previous reports have described aneuploid and euploid progeny that, in some cases, displayed genetic markers from the haploid inducer (HI). Here, we surveyed a population of 167 presumed dihaploids for large-scale structural variation that would underlie chromosomal addition from the HI, and for small-scale introgression of genetic markers. In 19 progeny, we detected 10 of the 12 possible trisomies and, in all cases, demonstrated the noninducer parent origin of the additional chromosome. Deep sequencing indicated that occasional, short-tract signals appearing to be of HI origin were better explained as technical artifacts. Leveraging recurring copy number variation patterns, we documented subchromosomal dosage variation indicating segregation of polymorphic maternal haplotypes. Collectively, 52% of the assayed chromosomal loci were classified as dosage variable. Our findings help elucidate the genomic consequences of potato haploid induction and suggest that most potato dihaploids will be free of residual pollinator DNA.

Regeneration of Solanum tuberosum Plants from Protoplasts Induces Widespread Genome Instability.

Nontransgenic genome editing in regenerable protoplasts, plant cells free of their cell wall, could revolutionize crop improvement because it reduces regulatory and technical complexity. However, plant tissue culture is known to engender frequent unwanted variation, termed somaclonal variation. To evaluate the contribution of large-scale genome instability to this phenomenon, we analyzed potatoes (Solanum tuberosum) regenerated from either protoplasts or stem explants for copy number changes by comparison of Illumina read depth. Whereas a control set of eight plants that had been propagated by cuttings displayed no changes, all 15 protoplast regenerants tested were affected by aneuploidy or structural chromosomal changes. Certain chromosomes displayed segmental deletions and duplications ranging from one to many. Resampling different leaves of the same plant found differences in three regenerants, indicating frequent persistence of instability. By comparison, 33 regenerants from stem explants used for Agrobacterium-mediated transformation displayed less frequent but still considerable (18%) large-scale copy number changes. Repetition of certain instability patterns suggested greater susceptibility in specific genomic sites. These results indicate that tissue culture, depending on the protocol used, can induce genomic instability resulting in large-scale changes likely to compromise final plant phenotype.

Translational genomics using Arabidopsis as a model enables the characterization of pennycress genes through forward and reverse genetics.

Thlaspi arvense (pennycress) has the potential for domestication as a new oilseed crop. Information from an extensive body of research on the related plant species Arabidopsis can be used to greatly speed this process. Genome-scale comparisons in this paper documented that pennycress and Arabidopsis share similar gene duplication. This finding led to the hypothesis that it should be possible to isolate Arabidopsis-like mutants in pennycress. This proved to be true, as forward genetic screens identified floral and vegetative pennycress mutants that were similar to mutants found in Arabidopsis. Extending this approach, it was shown that most of the pennycress genes responsible for the formation of oxidized tannins could be rapidly identified. The causative mutations in the pennycress mutants could be identified either by PCR amplification of candidate genes or through whole-genome sequencing (WGS) analysis. In all, WGS was used to characterize 95 ethyl methane sulfonate mutants, which revealed a mutation rate of 4.09 mutations per megabase. A sufficient number of non-synonymous mutations were identified to create a mutant gene index that could be used for reverse genetic approaches to identify pennycress mutants of interest. As proof of concept, a Ta-max3-like dwarf mutant and Ta-kcs5/cer60-like wax mutants deficient in the biosynthesis of long chain fatty acids were identified. Overall, these studies demonstrate that translational genomics can be used to promote the domestication of pennycress. Furthermore, the ease with which important findings could be made in pennycress makes this species a new potential model plant.