Developing the karyotype of a eukaryotic species relies on identification of individual chromosomes, which has been a major challenge for most nonmodel plant and animal species. We developed a novel chromosome identification system by selecting and labeling oligonucleotides (oligos) located in specific regions on every chromosome. We selected a set of 54,672 oligos (45 nt) based on single copy DNA sequences in the potato genome. These oligos generated 26 distinct FISH signals that can be used as a "bar code" or "banding pattern" to uniquely label each of the 12 chromosomes from both diploid and polyploid (43 and 63) potato species. Remarkably, the same bar code can be used to identify the 12 homeologous chromosomes among distantly related Solanum species, including tomato and eggplant. Accurate karyotypes based on individually identified chromosomes were established in six Solanum species that have diverged for .15 MY. These six species have maintained a similar karyotype; however, modifications to the FISH signal bar code led to the discovery of two reciprocal chromosomal translocations in Solanum etuberosum and S. caripense. We also validated these translocations by oligo-based chromosome painting. We demonstrate that the oligo-based FISH techniques are powerful new tools for chromosome identification and karyotyping research, especially for nonmodel plant species.

The cultivated potato (Solanum tuberosum) has a complex genetic structure due to its autotetraploidy and vegetative propagation which leads to accumulation of mutations and a highly heterozygous genome. A high degree of heterozygosity has been considered to be the main driver of fitness and agronomic trait performance in potato improvement efforts, which is negatively impacted by genetic load. To understand the genetic landscape of cultivated potato, we constructed a gynogenic dihaploid (2n = 2x = 24) population from cv. Superior, prior to development of a high-density genetic map containing 12,753 single nucleotide polymorphisms (SNPs). Common quantitative trait loci (QTL) were identified for tuber traits, vigor and height on chromosomes 2, 4, 7, and 10, while specific QTL for number of inflorescences per plant, and tuber shape were present on chromosomes 4, 6, 10, and 11. Simplex rather than duplex loci were mainly associated with traits. In general, the Q allele (main effect) detected in one or two homologous chromosomes was associated with lower mean trait values suggesting the importance of dosage allelic effects, and the presence of up to two undesired alleles in the QTL region. Loss of heterozygosity has been associated with a lower rate of fitness, yet no correlation between the percent heterozygosity and increased fitness or agronomic performance was observed. Based upon linkage phase, we reconstructed the four homologous chromosome haplotypes of cv. Superior. revealing heterogeneity throughout the genome yet nearly duplicate haplotypes occurring among the homologs of particular chromosomes. These results suggest that the potentially deleterious mutations associated with genetic load in tetraploid potato could be mitigated by multiple loci which is consistent with the theory that epistasis complicates the identification of associations between markers and phenotypic performance.

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Oligonucleotide (oligo)-fluorescence in situ hybridization (FISH) has rapidly becoming the new generation of FISH technique in plant molecular cytogenetics research. Genome-scale identification of single-copy oligos is the foundation of successful oligo-FISH experiments. Here, we introduce Chorus2, a software that is developed specifically for oligo selection. We demonstrate that Chorus2 is highly effective to remove all repetitive elements in selection of single-copy oligos, which is critical for the development of successful FISH probes. Chorus2 is more effective than Chorus, the original version of the pipeline, and OligoMiner for repeat removal. Chorus2 allows to select oligos that are conserved among related species, which extends the usage of oligo-FISH probes among phylogenetically related plant species. We also implemented a new function in Chorus2 that allows development of FISH probes from plant species without an assembled genome. We anticipate that Chorus2 can be used in plants as well as in mammalian and other non-plant species. Chorus2 will broadly facilitate the design of FISH probes for various types of application in molecular cytogenetics research.