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Potato is the most important non-cereal crop worldwide, and, yet, genetic gains in potato have been traditionally delayed by the crop's biology, mostly the genetic heterozygosity of autotetraploid cultivars and the intricacies of the reproductive system. Novel site-directed genetic modification techniques provide opportunities for designing climate-smart cultivars, but they also pose new possibilities (and challenges) for breeding potato. As potato species show a remarkable reproductive diversity, and their ovules have a propensity to develop apomixis-like phenotypes, tinkering with reproductive genes in potato is opening new frontiers in potato breeding. Developing diploid varieties instead of tetraploid ones has been proposed as an alternative way to fill the gap in genetic gain, that is being achieved by using gene-edited self-compatible genotypes and inbred lines to exploit hybrid seed technology. In a similar way, modulating the formation of unreduced gametes and synthesizing apomixis in diploid or tetraploid potatoes may help to reinforce the transition to a diploid hybrid crop or enhance introgression schemes and fix highly heterozygous genotypes in tetraploid varieties. In any case, the induction of apomixis-like phenotypes will shorten the time and costs of developing new varieties by allowing the multi-generational propagation through true seeds. In this review, we summarize the current knowledge on potato reproductive phenotypes and underlying genes, discuss the advantages and disadvantages of using potato's natural variability to modulate reproductive steps during seed formation, and consider strategies to synthesize apomixis. However, before we can fully modulate the reproductive phenotypes, we need to understand the genetic basis of such diversity. Finally, we visualize an active, central role for genebanks in this endeavor by phenotyping properly genotyped genebank accessions and new introductions to provide scientists and breeders with reliable data and resources for developing innovations to exploit market opportunities.

Cultivated potato (Solanum tuberosum L.) is one of the most important staple food crops worldwide. Its tetraploid and highly heterozygous nature poses a great challenge to its basic research and trait improvement through traditional mutagenesis and/or crossbreeding. The establishment of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) as a gene editing tool has allowed the alteration of specific gene sequences and their concomitant gene function, providing powerful technology for potato gene functional analysis and improvement of elite cultivars. This technology relies on a short RNA molecule called single guide RNA (sgRNA) that directs the Cas9 nuclease to induce a site-specific double-stranded break (DSB). Further, repair of the DSB by the error-prone non-homologous end joining (NHEJ) mechanism leads to the introduction of targeted mutations, which can be used to produce the loss of function of specific gene(s). In this chapter, we describe experimental procedures to apply the CRISPR/Cas9 technology for potato genome editing. First, we provide strategies for target selection and sgRNA design and describe a Golden Gate-based cloning system to obtain a sgRNA/Cas9-encoding binary vector. We also describe an optimized protocol for ribonucleoprotein (RNP) complex assembly. The binary vector can be used for both Agrobacterium-mediated transformation and transient expression in potato protoplasts, while the RNP complexes are intended to obtain edited potato lines through protoplast transfection and plant regeneration. Finally, we describe procedures to identify the gene-edited potato lines. The methods described here are suitable for potato gene functional analysis and breeding., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system and RNA interference (RNAi)-based non-transgenic approaches are powerful technologies capable of revolutionizing plant research and breeding. In recent years, the use of these modern technologies has been explored in various sectors of agriculture, introducing or improving important agronomic traits in plant crops, such as increased yield, nutritional quality, abiotic- and, mostly, biotic-stress resistance. However, the limitations of each technique, public perception, and regulatory aspects are hindering its wide adoption for the development of new crop varieties or products. In an attempt to reverse these mishaps, scientists have been researching alternatives to increase the specificity, uptake, and stability of the CRISPR and RNAi system components in the target organism, as well as to reduce the chance of toxicity in nontarget organisms to minimize environmental risk, health problems, and regulatory issues. In this review, we discuss several aspects related to risk assessment, toxicity, and advances in the use of CRISPR/Cas and topical RNAi-based technologies in crop management and breeding. The present study also highlights the advantages and possible drawbacks of each technology, provides a brief overview of how to circumvent the off-target occurrence, the strategies to increase on-target specificity, the harm/benefits of association with nanotechnology, the public perception of the available techniques, worldwide regulatory frameworks regarding topical RNAi and CRISPR technologies, and, lastly, presents successful case studies of biotechnological solutions derived from both technologies, raising potential challenges to reach the market and being social and environmentally safe., Competing Interests: TC and HC were employed by the SEMPRE AgTech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Touzdjian Pinheiro Kohlrausch Távora, de Assis dos Santos Diniz, de Moraes Rêgo-Machado, Chagas Freitas, Barbosa Monteiro Arraes, Chumbinho de Andrade, Furtado, Osiro, Lima de Sousa, Cardoso, Márcia Mertz Henning, Abrão de Oliveira Molinari, Feingold, Hunter, Fátima Grossi de Sá, Kobayashi, Lima Nepomuceno, Santiago and Correa Molinari.)

Aspartic proteases are proteolytic enzymes widely distributed in living organisms and viruses. Although they have been extensively studied in many plant species, they are poorly described in potatoes. The present study aimed to identify and characterize S. tuberosum aspartic proteases. Gene structure, chromosome and protein domain organization, phylogeny, and subcellular predicted localization were analyzed and integrated with RNAseq data from different tissues, organs, and conditions focused on abiotic stress. Sixty-two aspartic protease genes were retrieved from the potato genome, distributed in 12 chromosomes. A high number of intronless genes and segmental and tandem duplications were detected. Phylogenetic analysis revealed eight St AP groups, named from St API to St APVIII, that were differentiated into typical (St API), nucellin-like ( St APIIIa), and atypical aspartic proteases ( St APII, St APIIIb to St APVIII). RNAseq data analyses showed that gene expression was consistent with the presence of cis-acting regulatory elements on St AP promoter regions related to water deficit. The study presents the first identification and characterization of 62 aspartic protease genes and proteins on the potato genome and provides the baseline material for functional gene determinations and potato breeding programs, including gene editing mediated by CRISPR.

Potato ( Solanum tuberosum L.) is a crop of world importance that produces tubers of high nutritional quality. It is considered one of the promising crops to overcome the challenges of poverty and hunger worldwide. However, it is exposed to different biotic and abiotic stresses that can cause significant losses in production. Thus, potato is a candidate of special relevance for improvements through conventional breeding and biotechnology. Since conventional breeding is time-consuming and challenging, genetic engineering provides the opportunity to introduce/switch-off genes of interest without altering the allelic combination that characterize successful commercial cultivars or to induce targeted sequence modifications by New Breeding Techniques. There is a variety of methods for potato improvement via genetic transformation. Most of them incorporate genes of interest into the nuclear genome; nevertheless, the development of plastid transformation protocols broadened the available approaches for potato breeding. Although all methods have their advantages and disadvantages, Agrobacterium -mediated transformation is the most used approach. Alternative methods such as particle bombardment, protoplast transfection with polyethylene glycol and microinjection are also effective. Independently of the DNA delivery approach, critical steps for a successful transformation are a rapid and efficient regeneration protocol and a selection system. Several critical factors affect the transformation efficiency: vector type, insert size, Agrobacterium strain, explant type, composition of the subculture media, selective agent, among others. Moreover, transient or stable transformation, constitutive or inducible promoters, antibiotic/herbicide resistance or marker-free strategies can be considered. Although great efforts have been made to optimize all the parameters, potato transformation protocols are highly genotype-dependent. Genome editing technologies provide promising tools in genetic engineering allowing precise modification of targeted sequences. Interestingly, transient expression of genome editing components in potato protoplasts was reported to generate edited plants without the integration of any foreign DNA, which is a valuable aspect from both a scientific and a regulatory perspective. In this review, current challenges and opportunities concerning potato genetic engineering strategies developed to date are discussed. We describe their critical parameters and constrains, and the potential application of the available tools for functional analyses or biotechnological purposes. Public concerns and safety issues are also addressed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nahirñak, Almasia, González, Massa, Décima Oneto, Feingold, Hopp and Vazquez Rovere.)

Plants are sessile organisms that, to survive they develop response mechanisms under water deficit conditions. Plant proteases play an essential role in a diversity of biological processes, among them tolerance to drought stress. Proteolysis is a critical regulator of stomatal development. Plant proteases are involved in the crosstalk among phytohormones and adjustment of stomatal aperture. Plant proteases are also related to the increment in reactive oxygen species (ROS) production detected in the plant biochemical response to drought. Plant proteases mitigate this process by degrading damaged, denatured, and aggregated proteins, remobilizing amino acids, and generating molecules involved in signal transductions. Although many roles for proteases have been proposed, molecular bases that regulate these mechanisms remain unknown. In this review, we summarize the current knowledge on the participation of proteases in the signaling pathways of plants in response to water deficit and their relationship with plant stress tolerance., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Polyphenol Oxidases (PPOs) catalyze the conversion of phenolic substrates to quinones, leading to the formation of dark-colored precipitates in fruits and vegetables. This process, known as enzymatic browning, is the cause of undesirable changes in organoleptic properties and the loss of nutritional quality in plant-derived products. In potato ( Solanum tubersoum L.), PPOs are encoded by a multi-gene family with different expression patterns. Here, we have studied the application of the CRISPR/Cas9 system to induce mutations in the StPPO2 gene in the tetraploid cultivar Desiree. We hypothesized that the specific editing of this target gene would result in a lower PPO activity in the tuber with the consequent reduction of the enzymatic browning. Ribonucleoprotein complexes (RNPs), formed by two sgRNAs and Cas9 nuclease, were transfected to potato protoplasts. Up to 68% of regenerated plants contained mutations in at least one allele of the target gene, while 24% of edited lines carried mutations in all four alleles. No off-target mutations were identified in other analyzed StPPO genes. Mutations induced in the four alleles of StPPO2 gene, led to lines with a reduction of up to 69% in tuber PPO activity and a reduction of 73% in enzymatic browning, compared to the control. Our results demonstrate that the CRISPR/Cas9 system can be applied to develop potato varieties with reduced enzymatic browning in tubers, by the specific editing of a single member of the StPPO gene family., (Copyright © 2020 González, Massa, Andersson, Turesson, Olsson, Fält, Storani, Décima Oneto, Hofvander and Feingold.)

Background: Cold-induced sweetening (CIS) is the accumulation of sucrose and reducing sugars in potato tubers at low temperatures. This process is central for the potato processing industry. During potato chip and French fry production, reducing sugars participate in the Maillard reaction to produce dark pigmented products not acceptable to consumers. Andean potatoes (Solanum tuberosum Group Andigena) constitute an enormous wealth of potato germplasm that can contribute to increase genetic diversity in breeding programs of many traits, including CIS., Results: We analyzed reducing sugar content and chip quality in freshly harvested and cold-stored tubers from 48 native accessions. Andean accessions showed high variation in reducing sugar content and were classified in three types of CIS responses: type I, reducing sugar content before and after 4°C storage was lower than the value required by industry; type II, reducing sugar content before storage was acceptable, but after 4°C storage incremented up to non-acceptable levels; and type III, reducing sugar content was unacceptable before and after storage., Conclusion: Five Andean accessions presented acceptable reducing sugar content and good chip quality before and after 4°C storage in a consistent manner throughout several experiments. These features make them a useful source for improving the potato industry. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

The 70 kilodalton heat shock proteins (Hsp70) are highly conserved molecular chaperones which have a crucial role in the stress response of the cell. In mammals, the Hsp70 proteins are encoded by a cluster of three genes: HSPA1A, HSPA1B and HSPA1L. In bovines, this cluster is located on chromosome 23 downstream of the major histocompatibility complex (BoLA). We detected inconsistencies in the location of markers on the Hsp70 genes reported in the literature that pointed to a potential deletion in the bovine reference genome UMD 3.1.1. An in silico analysis of the bovine genomic region of the Hsp70 cluster, using available information from public databases, confirmed the existence of a deletion of 11.1-kb spanning the HSPA1B gene and the intergenic region between HSPA1B and HSPA1A. Although we originally considered this an assembly error, it is most likely a particular condition of L1 Dominette 01449, the cow sequenced in the Bovine Genome Project. Moreover, we suggest a new classification of bovine Hsp70 sequences reported in NCBI and a reassignment of the location of SNPs from dbSNP that map to the deletion on BTA23. We also compared the location of selected transcription factor binding sites on the promoters of HSPA1A and HSPA1B. The results generated in the present work could be helpful to refine the reference genome of an important livestock species and also to understand the role and the regulation of the bovine Hsp70 genes., (© 2017 Stichting International Foundation for Animal Genetics.)

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker-based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal "pseudomolecules".

Potato (Solanum tuberosum L.) is the world's most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop., (©2011 Macmillan Publishers Limited. All rights reserved)

Calcium-dependent protein kinases (CDPKs) are essential calcium sensors. In this work, we have studied StCDPK2 isoform from potato both at gene and protein level. StCdpk2 genomic sequence contains eight exons and seven introns, as was observed for StCdpk1. There is one copy of the gene per genome located in chromosome 7. StCDPK2 encodes an active CDPK of 515 aminoacids, with an apparent MW of 57 kDa, which presents myristoylation and palmitoylation consensus in its N-terminus. StCDPK2 is highly expressed in leaves and green sprouts; enhanced expression was detected under light treatment, which corresponds well with light responsive cis-acting elements found in its promoter sequence. Antibodies against the recombinant StCDPK2::6xHis protein detected this isoform in soluble and particulate fractions from leaves. StCDPK2 autophosphorylation and kinase activity are both calcium dependent reaching half maximal activation at 0.6 μM calcium. The active kinase is autophosphorylated on serine and tyrosine residues and its activity is negatively modulated by phosphatidic acid (PA). Our results reveal StCDPK2 as a signalling element involved in plant growth and development and show that its activity is tightly regulated.

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4-8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (beta 7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the beta 7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.