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Potato breeding can be redirected to a diploid inbred/F1 hybrid variety breeding strategy if self-compatibility can be introduced into diploid germplasm. However, the majority of diploid potato clones ( spp.) possess gametophytic self-incompatibility that is primarily controlled by a single multiallelic locus called the -locus which is composed of tightly linked genes, ( -locus RNase) and multiple ( -locus F-box proteins), which are expressed in the style and pollen, respectively. Using genes known to function in the Solanaceae gametophytic SI mechanism, we identified alleles with flower-specific expression in two diploid self-incompatible potato lines using genome resequencing data. Consistent with the location of the -locus in potato, we genetically mapped the gene using a segregating population to a region of low recombination within the pericentromere of chromosome 1. To generate self-compatible diploid potato lines, a dual single-guide RNA (sgRNA) strategy was used to target conserved exonic regions of the gene and generate targeted knockouts (KOs) using a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (Cas9) approach. Self-compatibility was achieved in nine KO T lines which contained bi-allelic and homozygous deletions/insertions in both genotypes, transmitting self compatibility to T progeny. This study demonstrates an efficient approach to achieve stable, consistent self-compatibility through KO for use in diploid potato breeding approaches.

India is the second largest producer of potatoes in the world. Seed is the single most important input in potato cultivation. High seed rate (2.5–3.0 tons/ha), low rate of multiplication, progressive viral degeneration, storage, and transportation are major issues of potato seed production in the country. Potato seed alone accounts for 40%-50% of the total potato production cost, and huge quantities of potentially edible food is put back into the soil as potato seed. The delayed penetration of new improved potato/seed varieties into farmers’ fields due to the slow multiplication rate and frequent seed replacement because of degeneration are associated issues. To circumvent these issues, continuous efforts are being made by potato researchers to develop innovative technologies for quick multiplication of initial healthy breeder’s seed of the released varieties in sufficient quantities to meet the demand in our country. A paradigm shift in potato seed production methods has taken place globally since the early 1900s. Major potato producers of the world have shifted from conventional to hi-tech seed production systems to improve the seed quality and enhance seed multiplication rate. New innovations can overcome many of the problems associated with potato seed production, particularly in tropical and sub-tropical countries. Recent advances in potato seed production systems in India and challenges ahead for seed production are described here.

Doubled haploidy (DH) technology has been utilized in cultivated tetraploid potato (Solanum tuberosum L. ssp. tuberosum) to accelerate crop improvement; however very little work has been done with the diploid species. Experiments were undertaken to improve microspore embryogenic response in the diploid germplasm. Several factors influencing embryogenic responses were evaluated. An increase in calcium nitrate, a reduction in the plant growth regulators 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA), as well as an incubation temperature of 28 °C resulted in an increase in callus production and, in some cases, embryo-like structures. Validation of the modified protocol was conducted with both diploid and tetraploid potato germplasm with responses from both diploid and tetraploid. Monoploid and di-haploid plants were also regenerated from these microspore-derived calli.

Phenotypic uniformity and tuber yield are fundamental concerns in diploid inbred-based F1 hybrid breeding in potatoes. We evaluated the agronomic traits of 22 hybrid families grown in small and large pots and the field. These families were derived from crosses of heterozygous × heterozygous, homozygous × heterozygous, and homozygous × homozygous parents using highly homozygous plants of Solanum phureja, S. chacoense, and tenth selfed generation plants derived from an interspecific hybrid. Genetic variability was estimated using genome-wide single nucleotide polymorphism (SNP) markers. Hybrid populations from homozygous × homozygous parents exhibited the highest phenotypic and genetic uniformity levels. Hybrid vigor in hybrids of homozygous × homozygous parents was tremendously enormous for fecundity and growth (up to 2877.5% and 8153.6%, respectively) and moderately large for tuber yield (158.2 to 230.7%). SNP-based genome-wide percent heterozygosity ranged from 28.7 to 44.3% for heterozygous parents and 14.4 to 44.8% for hybrid populations. The heterozygosity was correlated most highly with tuber size (r = 0.691–0.684) and negatively with tuber number (r = − 0.518), resulting in a positive correlation with tuber yield (r = 0.498). Since the heterozygosity of 2x Atlantic was the second highest (44.3%), its hybrid population produced a high yield in the field (925.6 g/plant) close to the yield of tetraploid potatoes. Thus, yield potential can be predicted by the genome-wide percent heterozygosity, possibly because many genetic factors collectively contributing to yield are located across the potato genome, and their accumulated heterotic effects can be represented as the genome-wide percent heterozygosity.

The Gametophytic Self-Incompatibility (GSI) system in diploid potato ( Solanum tuberosum L.) poses a substantial barrier in diploid potato breeding by hindering the generation of inbred lines. One solution is gene editing to generate self-compatible diploid potatoes which will allow for the generation of elite inbred lines with fixed favorable alleles and heterotic potential. The S-RNase and HT genes have been shown previously to contribute to GSI in the Solanaceae family and self-compatible S. tuberosum lines have been generated by knocking out S-RNase gene with CRISPR-Cas9 gene editing. This study employed CRISPR-Cas9 to knockout HT-B either individually or in concert with S-RNase in the diploid self-incompatible S. tuberosum clone DRH-195. Using mature seed formation from self-pollinated fruit as the defining characteristic of self-compatibility, HT-B- only knockouts produced little or no seed. In contrast, double knockout lines of HT-B and S-RNase displayed levels of seed production that were up to three times higher than observed in the S-RNase -only knockout, indicating a synergistic effect between HT-B and S-RNase in self-compatibility in diploid potato. This contrasts with compatible cross-pollinations, where S-RNase and HT-B did not have a significant effect on seed set. Contradictory to the traditional GSI model, self-incompatible lines displayed pollen tube growth reaching the ovary, yet ovules failed to develop into seeds indicating a potential late-acting self-incompatibility in DRH-195. Germplasm generated from this study will serve as a valuable resource for diploid potato breeding.

Cultivated potato is a vegetatively propagated crop, and most varieties are autotetraploid with high levels of heterozygosity. Reducing the ploidy and breeding potato at the diploid level can increase efficiency for genetic improvement including greater ease of introgression of diploid wild relatives and more efficient use of genomics and markers in selection. More recently, selfing of diploids for generation of inbred lines for F1 hybrid breeding has had a lot of attention in potato. The current study provides genomics resources for nine legacy non-inbred adapted diploid potato clones developed at Agriculture and Agri-Food Canada. De novo genome sequence assembly using 10× Genomics and Illumina sequencing technologies show the genome sizes ranged from 712 to 948 Mbp. Structural variation was identified by comparison to two references, the potato DMv6.1 genome and the phased RHv3 genome, and a k-mer based analysis of sequence reads showed the genome heterozygosity range of 1 to 9.04% between clones. A genome-wide approach was taken to scan 5 Mb bins to visualize patterns of heterozygous deleterious alleles. These were found dispersed throughout the genome including regions overlapping segregation distortions. Novel variants of the StCDF1 gene conferring earliness of tuberization were found among these clones, which all produce tubers under long days. The genomes will be useful tools for genome design for potato breeding.

Key message We report the mitochondrial genome of 39 diploid potatoes and identify a candidate ORF potentially linked to cytoplasmic male sterility in potatoes. Potato ( Solanum tuberosum L.) holds a critical position as the foremost non-grain food crop, playing a pivotal role in ensuring global food security. Diploid potatoes constitute a vital genetic resource pool, harboring the potential to revolutionize modern potato breeding. Nevertheless, diploid potatoes are relatively understudied, and mitochondrial DNA can provide valuable insights into key potato breeding traits such as CMS. In this study, we examine and assemble the mitochondrial genome evolution and diversity of 39 accessions of diploid potatoes using high-fidelity (HiFi) sequencing. We annotated 54 genes for all the investigated accessions, comprising 34 protein-coding genes, 3 rRNA genes, and 17 tRNA genes. Our analyses revealed differences in repeats sequences between wild and cultivated landraces. To understand the evolution of diploid maternal lineage inheritance, we conducted phylogenetic analysis, which clearly distinguished mitochondrial from nuclear gene trees, further supporting the evidence-based of clustering between wild and cultivated landraces accessions. Our study discovers new candidate ORFs associated with CMS in potatoes, including ORF137, which is homologous to other CMS in Solanaceae. Ultimately, this work bridges the gap in mitochondrial genome research for diploid potatoes, providing a steppingstone into evolutionary studies and potato breeding.

Potato is a heterozygous autotetraploid crop propagated as tubers. Diploid potatoes, which are mostly self-incompatible due to gametophytic self-incompatibility, are often used to reduce genetic complexity. The discovery of the S locus inhibitor (Sli) gene has created a way to develop diploid inbred lines and perform F1 hybrid breeding in potato. However, residual heterozygosity found in advanced-generation selfed progenies has posed the question of whether a minimum level of heterozygosity is necessary to maintain self-fertility. We continued selfing and finally identified a highly homozygous diploid potato among tenth-generation selfed progeny, which was homozygous at all 18,579 genome-wide single nucleotide polymorphism (SNP) markers surveyed. The S10 plants suffered severe inbreeding depression in terms of fertility and vigor, showing a small number of mature flowers and extremely slow growth. Although asexual techniques such as anther culture followed by chromosome doubling can result in completely homozygous diploid potatoes, all previously derived plants were male sterile. In contrast, continuous selfing using Sli swept out all lethal alleles and selected for self-fertility, which generated a highly homozygous diploid potato retaining male and female fertility and tuberization ability under long days.

Diploid lines (2n = 2x = 24) derived from tetraploid potato cultivars have been utilized to hybridize with wild diploid potato species, yielding fertile offsprings. Utilizing the pollen of Solanum tuberosum Group Phureja, such as IVP101, IVP35 and IVP48, as an inducer for wide hybridization with tetraploid cultivars represents a common method for producing diploids. In this study, we created a distant hybridization induced population of tetraploid potato cultivar Cooperation 88 (C88) and IVP101, and screened all diploids using flow cytometry and ploidyNGS. We investigated the genetic composition of chloroplast and nuclear genomes in 43 diploid offsprings. We found that all diploid offsprings share the same chloroplast genomic sequence as C88 and no evidence of paternal chloroplast inheritance was found. Used SNP data to calculate the theoretical introgression index of IVP101 with diploid offsprings. The results showed that the inducer’s nuclear genome was involved in the nuclear genome of the diploid offsprings with purple stem trait, indicating that the inducer nuclear genome was not completely eliminated in the nuclear genome during distant hybridization. Furthermore, we conducted a comparative analysis of the chloroplast genomes of the Solanum genus. The results indicated that (1) the chloroplast genome sizes of the 14 Solanum species ranged from 154,289 bp to 155,614 bp, with a total number of genes ranging 128-141, and with ycf 1 and rps 19 pseudogenes appearing at the IRB/SSC and IRA/LSC boundaries, respectively; (2) eight divergent hotspots distributed in the LSC and SSC regions of the Solanum chloroplast genomes were identified; (3) positive selection was detected in the clp P, rbc L, rps 15, and rps 4 genes, likely contributing to the adaptation of Solanum species to different habitats. These results reveal the variation and evolutionary characteristics of chloroplast genomes in Solanum plants.

Most cultivated potato (Solanum tuberosum) varieties are highly susceptible to common scab (Streptomyces scabei). The disease is widespread in all major potato production areas and leads to high economic losses and food waste. Varietal resistance is seen as the most viable and sustainable long-term management strategy. However, resistant potato varieties are scarce, and their genetic architecture and resistance mechanisms are poorly understood. Moreover, diploid potato relatives to commercial potatoes remain to be fully explored. In the current study, a panel of 384 ethyl methane sulfonate (EMS)-mutagenized diploid potato clones were evaluated for common scab coverage, severity, and incidence traits under field conditions, and genome-wide association studies (GWASs) were conducted to dissect the genetic architecture of their traits. Using the GAPIT-MLM and RTM-GWAS statistical models, and Mann–Whitney non-parametric U-tests, we show that 58 QTNs/QTLs distributed on all 12 potato chromosomes were associated with common scab resistance, 52 of which had significant allelic effects on the three traits. In total, 38 of the 52 favorable QTNs/QTLs were found to be pleiotropic on at least two of the traits, while 14 were unique to a single trait and were found distributed over 3 chromosomes. The identified QTNs/QTLs showed low to high effects, highlighting the quantitative and multigenic inheritance of common scab resistance. The QTLs/QTNs associated with the three common scab traits were found to be co-located in genomic regions carrying 79 candidate genes playing roles in plant defense, cell wall component biosynthesis and modification, plant–pathogen interactions, and hormone signaling. A total of 61 potato clones were found to be tolerant or resistant to common scab. Taken together, the data show that the studied germplasm panel, the identified QTNs/QTLs, and the candidate genes are prime genetic resources for breeders and biologists in breeding and targeted gene editing.