Potato Genome Research Articles

Identification and Expression Analysis of Adenylate Kinase Gene Family in Potato

Adenylate kinase (ADK) is a key enzyme that is widely distributed in animals and plants. It plays an important role in growth and stress response. However, ADK genes in potato (StADK) have been little reported. It is of great significance to identify ADK members and understand the molecular mechanism of stress response and tolerance. Based on the potato genome data, 23 StADK genes were identified at a genome-wide level. We then performed a comprehensive study using a bioinformatics method. The results of the evolutionary tree showed that StADK proteins were divided into four groups, and they were highly homologous to the Arabidopsis thaliana ADK members. Meanwhile, our study found that they existed on eight chromosomes, and we obtained three pairs of fragment duplications. Furthermore, we detected the six selected StADK genes using qRT-PCR, and the results confirmed that the genes are involved in the regulation of cold, ABA, salt, H2O2 and drought stresses. Our study provides a theoretical basis for studying the function of the potato ADK genes and lays a solid foundation for further understanding the molecular mechanism of the potato ADK genes under various environmental stresses.

Foreign DNA detection in genome-edited potatoes by high-throughput sequencing

Genome editing is a powerful breeding technique that introduces mutations into specific gene sequences in genomes. For genome editing in higher plants, nucleotides for artificial nuclease (e.g. TALEN or CRISPR-Cas9) are transiently or stably introduced into the plant cells. After the introduction of mutations by artificial nucleases, it is necessary to select lines that do not contain the foreign nucleotides to overcome GMO regulation; however, there is still no widely legally authorized and approved method for detecting foreign genes in genome-edited crops. Recently, k-mer analysis based on next-generation sequencing (NGS) was proposed as a new method for detecting foreign DNA in genome-edited agricultural products. Compared to conventional methods, such as PCR and Southern hybridization, in principle, this method can detect short DNA fragments with high accuracy. However, this method has not yet been applied to genome-edited potatoes. In this study, we evaluated the feasibility of k-mer analysis in tetraploid potatoes by computer simulation, and also evaluated whether the k-mer method can detect foreign genes with high accuracy by analyzing samples of genome-edited potatoes. We show that when NGS data (at a depth of × 30 the genome size) are used, the k-mer method can correctly detect foreign genes in the potato genome even with the insertion of DNA fragments of 20 nt in length. Based on these findings, we expect that k-mer analysis will be one of the main methods for detecting foreign genes in genome-edited potatoes.

A physical map of traits of agronomic importance based on potato and tomato genome sequences.

Potato, tomato, pepper, and eggplant are worldwide important crop and vegetable species of the Solanaceae family. Molecular linkage maps of these plants have been constructed and used to map qualitative and quantitative traits of agronomic importance. This research has been undertaken with the vision to identify the molecular basis of agronomic characters on the one hand, and on the other hand, to assist the selection of improved varieties in breeding programs by providing DNA-based markers that are diagnostic for specific agronomic characters. Since 2011, whole genome sequences of tomato and potato became available in public databases. They were used to combine the results of several hundred mapping and map-based cloning studies of phenotypic characters between 1988 and 2022 in physical maps of the twelve tomato and potato chromosomes. The traits evaluated were qualitative and quantitative resistance to pathogenic oomycetes, fungi, bacteria, viruses, nematodes, and insects. Furthermore, quantitative trait loci for yield and sugar content of tomato fruits and potato tubers and maturity or earliness were physically mapped. Cloned genes for pathogen resistance, a few genes underlying quantitative trait loci for yield, sugar content, and maturity, and several hundred candidate genes for these traits were included in the physical maps. The comparison between the physical chromosome maps revealed, in addition to known intrachromosomal inversions, several additional inversions and translocations between the otherwise highly collinear tomato and potato genomes. The integration of the positional information from independent mapping studies revealed the colocalization of qualitative and quantitative loci for resistance to different types of pathogens, called resistance hotspots, suggesting a similar molecular basis. Synteny between potato and tomato with respect to genomic positions of quantitative trait loci was frequently observed, indicating eventual similarity between the underlying genes.

Identification of T-DNA structure and insertion site in transgenic crops using targeted capture sequencing.

The commercialization of GE crops requires a rigorous safety assessment, which includes a precise DNA level characterization of inserted T-DNA. In the past, several strategies have been developed for identifying T-DNA insertion sites including, Southern blot and different PCR-based methods. However, these methods are often challenging to scale up for screening of dozens of transgenic events and for crops with complex genomes, like potato. Here, we report using target capture sequencing (TCS) to characterize the T-DNA structure and insertion sites of 34 transgenic events in potato. This T-DNA is an 18 kb fragment between left and right borders and carries three resistance (R) genes (RB, Rpi-blb2 and Rpi-vnt1.1 genes) that result in complete resistance to late blight disease. Using TCS, we obtained a high sequence read coverage within the T-DNA and junction regions. We identified the T-DNA breakpoints on either ends for 85% of the transgenic events. About 74% of the transgenic events had their T-DNA with 3R gene sequences intact. The flanking sequences of the T-DNA were from the potato genome for half of the transgenic events, and about a third (11) of the transgenic events have a single T-DNA insertion mapped into the potato genome, of which five events do not interrupt an existing potato gene. The TCS results were confirmed using PCR and Sanger sequencing for 6 of the best transgenic events representing 20% of the transgenic events suitable for regulatory approval. These results demonstrate the wide applicability of TCS for the precise T-DNA insertion characterization in transgenic crops.

Homoeologous Chromosome Pairing and Alien Introgression in Backcrossing Progenies Derived from Hybrids Solanum tuberosum (+) Mexican 2x (1 EBN) B-Genome Potato Species

We applied a genomic in situ hybridization (GISH) to analyze the genomic constitution of and meiotic pairing in interspecific somatic hybrids, and in a wide subset of backcrossing derivatives (BC1–BC5), from three interspecific combinations involving the cultivated potato, Solanum tuberosum (AAAA genome), and three diploid (1 EBN) wild Mexican potato species (genome BB)—S. tarnii, S. pinnatisectum, and S. bulbocastanum. The theoretically expected genomic composition was detected in the somatic hybrids (AAAABB) and in the BC1 progeny (AAAAB), whereas in the subsequent BC2–BC4 generations, the partial loss of alien chromosomes was observed and almost all the BC5 genotypes showed a complete chromosome elimination of wild species. GISH revealed a homoeologous pairing between the chromosomes of the A- and the B-genomes in each of the hybrid progenies. Using GISH, we selected introgression lines with a single chromosome of the wild species in a potato genome background, as well as introgression lines with intergenomic recombinant chromosomes. Moreover, via molecular screening, BC hybrids with diagnostic markers for the R-genes conferring resistance to late blight disease and to the quarantine pest of the potato–Columbia root-knot nematode—were selected. The potential application of the results obtained for the planning of introgressive schemes directed to the breeding of advanced lines with multiple disease and pest resistance is discussed.

CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato.

CRISPR/Cas9 technology has become the most efficient method for genome editing in many plant species, including important industrial crops such as potatoes. This study used three target regions (T1, T2, and T3) in gbss exon I, whose sequences were first inserted into the BbsI sites in the appropriate guide RNA (gRNA) vector (pEn-Chimera, pMR203, pMR204, and pMR205), and then localized between the AtU6 promoter and the gRNA scaffold sequence. Expression vectors were constructed by introducing gRNA genes into the pMR287 (pYUCas9Plus) plasmids using the MultiSite Gateway system by attR and attL sites. The three target regions of mutant potato lines were analyzed. The use of CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis allowed tri- or tetra-allelic mutant potato lines to be generated. Multiple nucleotide substitutions and indels within and around the three target sites caused a frameshift mutation that led to a premature stop codon, resulting in the production of gbss-knockout plants. Mutation frequencies and analysis of mutation patterns suggested that the stably transformed Cas9/multiple guide RNA expression constructs used in this study can induce targeted mutations efficiently in the potato genome. Full knockout of the gbss gene was analyzed by CAPS, Sanger sequencing and iodine staining. The present study demonstrated successful CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato gbss gene by Agrobacterium-mediated transformation, resulting in an amylose-free phenotype.

Early Blight Resistance of Transgenic Potato Plants Expressingthe ProSmAMP1 Gene for Antimicrobial Peptides under the Control of a Light-Inducible Cab Promoter

The genome of Stellaria media contains a gene family for hevein-like antimicrobial peptides, some of which are known to encode two peptides released from the translation product as a result of post-translational proteolysis. These peptides have been shown to inhibit the growth of bacteria and fungi, including potato pathogens Alternaria solani and Alternaria alternata. One of these genes, ProSmAMP1, was introduced into the potato genome under the control of the light-inducible promoter of Cab gene from common wheat. The resulting transgenic lines expressed ProSmAMP1 mRNA during several vegetative passages, and their resistance to early blight was assessed by several indicators of detached leaf infection, with plants having the highest expression of the transgene also showing the highest resistance.

Phylogenomic discovery of deleterious mutations facilitates hybrid potato breeding

Hybrid potato breeding will transform the crop from a clonally propagated tetraploid to a seed-reproducing diploid. Historical accumulation of deleterious mutations in potato genomes has hindered the development of elite inbred lines and hybrids. Utilizing a whole-genome phylogeny of 92 Solanaceae and its sister clade species, we employ an evolutionary strategy to identify deleterious mutations. The deep phylogeny reveals the genome-wide landscape of highly constrained sites, comprising ∼2.4% of the genome. Based on a diploid potato diversity panel, we infer 367,499 deleterious variants, of which 50% occur at non-coding and 15% at synonymous sites. Counterintuitively, diploid lines with relatively high homozygous deleterious burden can be better starting material for inbred-line development, despite showing less vigorous growth. Inclusion of inferred deleterious mutations increases genomic-prediction accuracy for yield by 24.7%. Our study generates insights into the genome-wide incidence and properties of deleterious mutations and their far-reaching consequences for breeding.

Strategies for Engineering Virus Resistance in Potato.

Potato (Solanum tuberosum L.) is an important vegetable crop that plays a pivotal role in the world, especially given its potential to feed the world population and to act as the major staple food in many developing countries. Every year, significant crop loss is caused by viral diseases due to a lack of effective agrochemical treatments, since only transmission by insect vectors can be combated with the use of insecticides, and this has been an important factor hindering potato production. With the rapid development of molecular biology and plant genetic engineering technology, transgenic approaches and non-transgenic techniques (RNA interference and CRISPR-cas9) have been effectively employed to improve potato protection against devastating viruses. Moreover, the availability of viral sequences, potato genome sequences, and host immune mechanisms has remarkably facilitated potato genetic engineering. In this study, we summarize the progress of antiviral strategies applied in potato through engineering either virus-derived or plant-derived genes. These recent molecular insights into engineering approaches provide the necessary framework to develop viral resistance in potato in order to provide durable and broad-spectrum protection against important viral diseases of solanaceous crops.

Genome-Wide Comparative Analysis of the R2R3-MYB Gene Family in Six Ipomoea Species and the Identification of Anthocyanin-Related Members in Sweet Potatoes.

Sweet potatoes (Ipomoea batatas) are one of the important tuberous root crops cultivated worldwide, and thier storage roots are rich in antioxidants, such as anthocyanins. R2R3-MYB is a large gene family involved in various biological processes, including anthocyanin biosynthesis. However, few reports about the R2R3-MYB gene family of sweet potatoes have been released to date. In the present study, a total of 695 typical R2R3-MYB genes were identified in six Ipomoea species, including 131 R2R3-MYB genes in sweet potatoes. A maximum likelihood phylogenetic analysis divided these genes into 36 clades, referring to the classification of 126 R2R3-MYB proteins of Arabidopsis. Clade C25(S12) has no members in six Ipomoea species, whereas four clades (i.e., clade C21, C26, C30, and C36), including 102 members, had no members in Arabidopsis, and they were identified as Ipomoea-specific clades. The identified R2R3-MYB genes were unevenly distributed on all chromosomes in six Ipomoea species genomes, and the collinearity analysis among hexaploid I. batatas and another five diploid Ipomoea species suggested that the sweet potato genome might have undergone a larger chromosome rearrangement during the evolution process. Further analyses of gene duplication events showed that whole-genome duplication, transposed duplication, and dispersed duplication events were the primary forces driving the R2R3-MYB gene family expansion of Ipomoea plants, and these duplicated genes experienced strong purifying selection because of their Ka/Ks ratio, which is less than 1. Additionally, the genomic sequence length of 131 IbR2R3-MYBs varied from 923 bp to ~12.9 kb with a mean of ~2.6 kb, and most of them had more than three exons. The Motif 1, 2, 3, and 4 formed typical R2 and R3 domains and were identified in all IbR2R3-MYB proteins. Finally, based on multiple RNA-seq datasets, two IbR2R3-MYB genes (IbMYB1/g17138.t1 and IbMYB113/g17108.t1) were relatively highly expressed in pigmented leaves and tuberous root flesh and skin, respectively; thus, they were identified to regulate tissue-specific anthocyanin accumulation in sweet potato. This study provides a basis for the evolution and function of the R2R3-MYB gene family in sweet potatoes and five other Ipomoea species.

Screening and Identification of Potato StSPS1, a Potential Crucial Gene Regulating Seed Potato Vigor

Sucrose phosphate synthase (SPS), a crucial rate-limiting enzyme that catalyzes the synthesis of precursors of sucrose, plays an indispensable role in the regulation of seed potato vigor. Nonetheless, the genes that encode SPS in potato have not undergone complete analysis, and the primary genes responsible for the regulation of seed potato vigor have not been screened and identified. In this study, four StSPS family members were identified by comparing the potato genome database with homologous proteins. Using bioinformatics, the physicochemical properties, subcellular localization, sequence structure, conserved motifs, and phylogenetics of StSPS were analyzed. The expression levels of StSPS in different potato tissues, from dormancy to sprouting in the seed potato tubers, were measured via qRT-PCR. The expression data from the potato genome database and previous transcriptome and proteome studies of dormancy to sprouting were also compared. After combining the analysis of SPS enzyme activity in diverse tuber tissues and the correlation analysis between multiple varieties with different dormancy periods and the expression of StSPS1, the primary gene StSPS1 that might regulate seed potato vigor was identified. This study set a theoretical and experimental groundwork for further verification and clarification of the regulatory function of StSPS1 in alterations in seed potato vigor.

Gibberellin 2-Oxidases in Potato (Solanum tuberosum L.): Cloning, Characterization, In Silico Analysis and Molecular Docking.

Gibberellins (GAs; tetracyclic di-terpenoid carboxylic acids) are endogenous plant growth regulators responsible for stimulating plant growth and development from seed germination to plant maturity. In potato (Solanum tuberosum L.), GA levels are known to be crucial in the complex process of tuberization. Gibberellin 2-oxidases (GA2oxs) inactivate bioactive GAs during stolon swelling and early stages of tuberization as evident from the predominant expression of a member of this gene family namely GA2ox1. We isolated and characterized a 1105-bp cDNA clone encoding a 340-aa GA2ox1 form, designated St-GA2ox1, using total RNA from growing tuber of a potato (Solanum tuberosum L.) cultivar, Kufri Chipsona-1 (KC-1) based on RT-PCR approach. A total of 26 GA2ox sequences were also retrieved from potato genome database and analysed. Multiple sequence alignment revealed sequence relatedness between the GA2oxs. Crucial protein motifs were identified. Phylogenetic analysis revealed the evolutionary relationships between the GA2oxs. Three-dimensional structure of St-GA2ox1 was predicted by using AlphaFold tool, validated by the predicted local-distance difference test and Ramachandran Plot. Structural analysis and molecular docking were carried out to identify domains, binding sites and affinity for the ligand. The STRING database and hydropathy analysis revealed the presence of a putative interaction site for other enzymes. Expression Atlas database and semi-quantitative RT-PCR revealed the expression patterns of various GA2ox forms in different potato organs. This comprehensive report would be useful in providing new insights into possible underlying mechanisms involved in tuber development, and could facilitate the targeted alteration of genes responsible to combat the stress and enhance tuber production.

Insights into the genetic determination of tuber shape and eye depth in potato natural population based on autotetraploid potato genome.

Potato is one of the world's most important food crops, with a time-consuming breeding process. In this study, we performed a genome-wide association (GWAS) analysis of the two important traits of potato tuber shape and eye depth, using the tetraploid potato genome (2n=4x=48) as a reference. A total of 370 potatoes were divided into three subgroups based on the principal component analysis and evolutionary tree analysis. The genetic diversity within subgroups is low (5.18×10-5, 4.36×10-5 and 4.24×10-5). Genome-wide linkage disequilibrium (LD) analysis showed that their LD is about 60 Kb. GWAS analysis identified that 146 significant single nucleotide polymorphism (SNP) loci at Chr01A1:34.44-35.25 Mb and Chr02A1:28.35-28.54 Mb regions are significantly associated with potato tuber shape, and that three candidate genes that might be related to potato tuber traits, PLATZ transcription factor, UTP-glucose-1-phosphate uridylyltransferase and FAR1 DNA-binding domain, are in the association region of Chr02A1. GWAS analysis identified 53 significant SNP loci at Chr05A2: 49.644-50.146 Mb and Chr06A2: 25.866-26.384 Mb regions with robust associations with potato tuber eye depth. Hydrolase and methyltransferases are present in the association region of Chr05A2, and three CYPs are present in the association region of Chr06A2. Our findings suggested that these genes are closely associated with potato tuber shape and eye depth. Our study identified molecular markers and candidate genes for improving tetraploid potato tuber shape and eye depth and provided ideas and insights for tetraploid potato breeding.

Insights into the Genetic Determination of the Autotetraploid Potato Plant Height.

Plant height is an important characteristic, the modification of which can improve the ability of stress adaptation as well as the yield. In this study, genome-wide association analysis was performed for plant height traits in 370 potato cultivars using the tetraploid potato genome as a reference. A total of 92 significant single nucleotide polymorphism (SNP) loci for plant height were obtained, which were particularly significant in haplotypes A3 and A4 on chromosome 1 and A1, A2, and A4 on chromosome 5. Thirty-five candidate genes were identified that were mainly involved in the gibberellin and brassinolide signal transduction pathways, including the FAR1 gene, methyltransferase, ethylene response factor, and ubiquitin protein ligase. Among them, PIF3 and GID1a were only present on chromosome 1, with PIF3 in all four haplotypes and GID1a in haplotype A3. This could lead to more effective genetic loci for molecular marker-assisted selection breeding as well as more precise localization and cloning of genes for plant height traits in potatoes.

Development of homozygous tetraploid potato and whole genome doubling-induced the enrichment of H3K27ac and potentially enhanced resistance to cold-induced sweetening in tubers.

Polyploid plants typically display advantages on some agronomically important traits over their diploid counterparts. Extensive studies have shown genetic, transcriptomic, and epigenetic dynamics upon polyploidization in multiple plant species. However, few studies have unveiled those alternations imposed only by ploidy level, without any interference from heterozygosity. Cultivated potato is highly heterozygous. Thus, in this study, we developed two homozygous autotetraploid lines and one homozygous diploid line in parallel from a homozygous diploid potato. We confirmed their ploidy levels using chloroplast counting and karyotyping. Oligo-FISH and genome re-sequencing validated that these potato lines are nearly homozygous. We investigated variations in phenotypes, transcription, and histone modifications between two ploidies. Both autotetraploid lines produced larger but fewer tubers than the diploid line. Interestingly, each autotetraploid line displayed ploidy-related differential expression for various genes. We also discovered a genome-wide enrichment of H3K27ac in genic regions upon whole-genome doubling (WGD). However, such enrichment was not associated with the differential gene expression between two ploidies. The tetraploid lines may exhibit better resistance to cold-induced sweetening (CIS) than the diploid line in tubers, potentially regulated through the expression of CIS-related key genes, which seems to be associated with the levels of H3K4me3 in cold-stored tubers. These findings will help to understand the impacts of autotetraploidization on dynamics of phenotypes, transcription, and histone modifications, as well as on CIS-related genes in response to cold storage.

Functional Characterization of Potato UBC13-UEV1s Genes Required for Ubiquitin Lys63 Chain to Polyubiquitination.

Ubiquitin-conjugating enzymes (E2s/UBC) are components of the ubiquitin proteasome system (UPS), and the ubiquitin-conjugating enzyme variant (UEV) is one of E2s (ubiquitin-conjugating enzymes, UBC) subfamily. The UEVs and UBC13 play an auxiliary role in mediating Lys63-linked polyUb chain assembly, which is correlated with target protein non-proteolytic functions, such as DNA repair or response to stress. However, the collaborative mechanism of StUBC13 (homologue of AtUBC13) and StUEVs (the UEVs in potato) involved in potato are not fully understood understood. Here, we identified two StUBC13 and seven StUEVs from potato genome. We analyzed protein motif and conserved domain, gene structure, phylogenetic features, cis-acting elements of StUBC13 and StUEVs. Subsequently, we screened StUBC13 partners protein and verified interaction between StUBC13 and StUEVs using yeast two-hybrid, split luciferase complementation (SLC) and bimolecular fluorescence complementation (BiFC) approach. The expression profile and qRT-PCR analysis suggested that StUBC13 and StUEVs gene exhibited a tissue-specific expression and were induced by different stress. Overall, this investigative study provides a comprehensive reference and view for further functional research on StUBC13 and StUEV1s in potato.

Benchmarking of structural variant detection in the tetraploid potato genome using linked-read sequencing

It has recently been shown that structural variants (SV) can have a higher impact on gene expression variation compared to single nucleotide variants (SNV) in different plant species. Additionally, SV were associated with phenotypic variation in several crops. However, compared to the established SV detection based on short-read sequencing, less approaches were described for linked-read based SV calling. We therefore evaluated the performance of six linked-read SV callers compared to an established short-read SV caller based on simulated linked-reads in tetraploid potato. The objectives of our study were to i) compare the performance of SV callers based on linked-read sequencing to short-read sequencing, ii) examine the influence of SV type, SV length, haplotype incidence (HI), as well as sequencing coverage on the SV calling performance in the tetraploid potato genome, and iii) evaluate the accuracy of detecting insertions by linked-read compared to short-read sequencing. We observed high break point resolutions (BPR) detecting short SV and slightly lower BPR for large SV. Our observations highlighted the importance of short-read signals provided by Manta and LinkedSV to detect short SV. Manta and NAIBR performed well for detecting larger deletions, inversions, and duplications. Detected large SV were weakly influenced by the HI. Furthermore, we illustrated that large insertions can be assembled by Novel-X. Our results suggest the usage of the short-read and linked-read SV callers Manta, NAIBR, LinkedSV, and Novel-X based on at least 90x linked-read sequencing coverage to ensure the detection of a broad range of SV in the tetraploid potato genome.

Genome-Wide Identification of 14-3-3 gene family reveals their diverse responses to abiotic stress by interacting with StABI5 in Potato (Solanum tuberosum L.).

The 14-3-3 genes are widely present in plants and participate in a wide range of cellular and physiological processes. In the current study, twelve 14-3-3s were identified from potato genome. According to phylogenetic evolutionary analysis, potato 14-3-3s were divided into ϵ and non-ϵ groups. Conserved motif and gene structure analysis displayed a distinct class-specific divergence between the ϵ group and non-ϵ group. Multiple sequence alignments and three-dimensional structure analysis of 14-3-3 proteins indicated all the members contained nine conservative antiparallel α-helices. The majority of 14-3-3s had transcript accumulation in each detected potato tissue, implying their regulatory roles across all stages of potato growth and development. Numerous cis-acting elements related to plant hormones and abiotic stress response were identified in the promoter region of potato 14-3-3s, and the transcription levels of these genes fluctuated to different degrees under exogenous ABA, salt and drought stress, indicating that potato 14-3-3s may be involved in different hormone signaling pathways and abiotic stress responses. In addition, eight potato 14-3-3s were shown to interact with StABI5, which further demonstrated that potato 14-3-3s were involved in the ABA-dependent signaling pathway. This study provides a reference for the identification of the 14-3-3 gene family in other plants, and provides important clues for cloning potential candidates in response to abiotic stresses in potato.

Generation of Asynaptic Mutants in Potato by Disrupting StDMC1 Gene Using RNA Interference Approach.

Fixing the genomic composition and multiplication through true potato seed (TPS) is an important challenge in autotetraploid potato. Disrupted meiotic cDNA (DMC1) is a meiotic gene that plays a central role in DNA recombination through crossing over in meiosis. Using the Arabidopsis DMC1 (AtDMC1) gene sequence, we retrieved Solanum tuberosum DMC1(StDMC1) from the diploid potato genome, and subsequently, sense and antisense regions of the StDMC1 gene were amplified in potato cv. Kufri Jyoti. The sense and antisense fragments were confirmed by Sanger-sequencing and cloned in the pRI101 vector. Agrobacterium-mediated transformation of the RNAi construct resulted in 44% transformation efficiency, and a total of 137 mutant lines were obtained. These mutant lines were further validated through pollen viability testing, and selected lines were used for gene expression analysis. The acetocarmine-based pollen staining showed reduced pollen viability ranging from 14 to 21% in four DMC1 mutant lines (DMC4-37, DMC4-41, DMC6-20, and DMC6-21), as compared to the Kufri Jyoti control plants, which on average exhibited 78% pollen viability. The phenotypic data was supported by the reduced expression of the StDMC1 gene in these four mutant lines compared to the control Kufri Jyoti. The results confirmed the generation of StDMC1 knockdown lines. This is the first report of StDMC1 mutant line generation in tetraploid potatoes and will be a step forward in generating non-recombinant mutants through sexual reproduction in potatoes.

Somaclonal Variation for Genetic Improvement of Starch Accumulation in Potato (Solanum tuberosum) Tubers.

Starch content is one of the major quality criteria targeted by potato breeding programs. Traditional potato breeding is a laborious duty due to the tetraploid nature and immense heterozygosity of potato genomes. In addition, screening for functional genetic variations in wild relatives is slow and strenuous. Moreover, genetic diversity, which is the raw material for breeding programs, is limited due to vegetative propagation used in the potato industry. Somaclonal variation provides a time-efficient tool to breeders for obtaining genetic variability, which is essential for breeding programs, at a reasonable cost and independent of sophisticated technology. The present investigation aimed to create potato somaclones with an improved potential for starch accumulation. Based on the weight and starch content of tubers, the somaclonal variant Ros 119, among 105 callus-sourced clones, recorded a higher tuberization potential than the parent cv Lady Rosetta in a field experiment. Although this somaclone was similar to the parent in the number of tubers produced, it exhibited tubers with 42 and 61% higher fresh and dry weights, respectively. Additionally, this clone recorded 10 and 75% increases in starch content based on the dry weight and average content per plant, respectively. The enhanced starch accumulation was associated with the upregulation of six starch-synthesis-related genes, namely, the AGPase, GBSS I, SBE I, SBE II, SS II and SS III genes. AGPase affords the glycosyl moieties required for the synthesis of amylose and amylopectin. GBSS is required for amylose elongation, while SBE I, SBE II, SS II and SS III are responsible for amylopectin.