Title: Genomic Outcomes of Haploid Induction Crosses in Potato ( Solanum tuberosum L.)
Abstract 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. more »« less
Amundson, Kirk R; Ordoñez, Benny; Santayana, Monica; Nganga, Mwaura Livingstone; Henry, Isabelle M; Bonierbale, Merideth; Khan, Awais; Tan, Ek Han; Comai, Luca(
, The Plant Cell)
null
(Ed.)
Abstract 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 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 haploid inducer DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three haploid inducers: 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 haploid inducer 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 haploid inducer DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.
Abstract 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.
Ordoñez, Benny; Santayana, Monica; Aponte, Mariela; Henry, Isabelle M.; Comai, Luca; Eyzaguirre, Raúl; Lindqvist-Kreuze, Hannele; Bonierbale, Merideth(
, American Journal of Potato Research)
Abstract
Dihaploid production from elite tetraploid cultivars is key to both traditional and novel breeding approaches that seek to simplify potato genetics. For this purpose, efficient and widely compatible haploid inducers (HIs) are needed. We compared PL-4, a new HI developed at the International Potato Center, to known HIs IvP101 and IvP35. By pollination of elite tetraploid breeding lines, we showed that PL-4 performed significantly better and had a homogeneous response regardless of the genetic background of the pistillate parents, on the most important efficiency traits—number of dihaploids per 100 fruits and haploid induction rate. Moreover, PL-4 exhibited a reduced proportion of hybrid seeds, a convenient trait for efficient screening. In this context, we recommend PL-4 as an enhanced HI for the potato breeding community.
Hoyt, Savannah J.; Storer, Jessica M.; Hartley, Gabrielle A.; Grady, Patrick G.; Gershman, Ariel; de Lima, Leonardo G.; Limouse, Charles; Halabian, Reza; Wojenski, Luke; Rodriguez, Matias; et al(
, Science)
INTRODUCTION Transposable elements (TEs), repeat expansions, and repeat-mediated structural rearrangements play key roles in chromosome structure and species evolution, contribute to human genetic variation, and substantially influence human health through copy number variants, structural variants, insertions, deletions, and alterations to gene transcription and splicing. Despite their formative role in genome stability, repetitive regions have been relegated to gaps and collapsed regions in human genome reference GRCh38 owing to the technological limitations during its development. The lack of linear sequence in these regions, particularly in centromeres, resulted in the inability to fully explore the repeat content of the human genome in the context of both local and regional chromosomal environments. RATIONALE Long-read sequencing supported the complete, telomere-to-telomere (T2T) assembly of the pseudo-haploid human cell line CHM13. This resource affords a genome-scale assessment of all human repetitive sequences, including TEs and previously unknown repeats and satellites, both within and outside of gaps and collapsed regions. Additionally, a complete genome enables the opportunity to explore the epigenetic and transcriptional profiles of these elements that are fundamental to our understanding of chromosome structure, function, and evolution. Comparative analyses reveal modes of repeat divergence, evolution, and expansion or contraction with locus-level resolution. RESULTS We implemented a comprehensive repeat annotation workflow using previously known human repeats and de novo repeat modeling followed by manual curation, including assessing overlaps with gene annotations, segmental duplications, tandem repeats, and annotated repeats. Using this method, we developed an updated catalog of human repetitive sequences and refined previous repeat annotations. We discovered 43 previously unknown repeats and repeat variants and characterized 19 complex, composite repetitive structures, which often carry genes, across T2T-CHM13. Using precision nuclear run-on sequencing (PRO-seq) and CpG methylated sites generated from Oxford Nanopore Technologies long-read sequencing data, we assessed RNA polymerase engagement across retroelements genome-wide, revealing correlations between nascent transcription, sequence divergence, CpG density, and methylation. These analyses were extended to evaluate RNA polymerase occupancy for all repeats, including high-density satellite repeats that reside in previously inaccessible centromeric regions of all human chromosomes. Moreover, using both mapping-dependent and mapping-independent approaches across early developmental stages and a complete cell cycle time series, we found that engaged RNA polymerase across satellites is low; in contrast, TE transcription is abundant and serves as a boundary for changes in CpG methylation and centromere substructure. Together, these data reveal the dynamic relationship between transcriptionally active retroelement subclasses and DNA methylation, as well as potential mechanisms for the derivation and evolution of new repeat families and composite elements. Focusing on the emerging T2T-level assembly of the HG002 X chromosome, we reveal that a high level of repeat variation likely exists across the human population, including composite element copy numbers that affect gene copy number. Additionally, we highlight the impact of repeats on the structural diversity of the genome, revealing repeat expansions with extreme copy number differences between humans and primates while also providing high-confidence annotations of retroelement transduction events. CONCLUSION The comprehensive repeat annotations and updated repeat models described herein serve as a resource for expanding the compendium of human genome sequences and reveal the impact of specific repeats on the human genome. In developing this resource, we provide a methodological framework for assessing repeat variation within and between human genomes. The exhaustive assessment of the transcriptional landscape of repeats, at both the genome scale and locally, such as within centromeres, sets the stage for functional studies to disentangle the role transcription plays in the mechanisms essential for genome stability and chromosome segregation. Finally, our work demonstrates the need to increase efforts toward achieving T2T-level assemblies for nonhuman primates and other species to fully understand the complexity and impact of repeat-derived genomic innovations that define primate lineages, including humans. Telomere-to-telomere assembly of CHM13 supports repeat annotations and discoveries. The human reference T2T-CHM13 filled gaps and corrected collapsed regions (triangles) in GRCh38. Combining long read–based methylation calls, PRO-seq, and multilevel computational methods, we provide a compendium of human repeats, define retroelement expression and methylation profiles, and delineate locus-specific sites of nascent transcription genome-wide, including previously inaccessible centromeres. SINE, short interspersed element; SVA, SINE–variable number tandem repeat– Alu ; LINE, long interspersed element; LTR, long terminal repeat; TSS, transcription start site; pA, xxxxxxxxxxxxxxxx.
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.
@article{osti_10229193,
place = {Country unknown/Code not available},
title = {Genomic Outcomes of Haploid Induction Crosses in Potato ( Solanum tuberosum L.)},
url = {https://par.nsf.gov/biblio/10229193},
DOI = {10.1534/genetics.119.302843},
abstractNote = {Abstract 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.},
journal = {Genetics},
volume = {214},
number = {2},
author = {Amundson, Kirk R and Ordoñez, Benny and Santayana, Monica and Tan, Ek Han and Henry, Isabelle M and Mihovilovich, Elisa and Bonierbale, Merideth and Comai, Luca},
editor = {null}
}
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