Transcriptome profiling and characterization of genes associated with tuberization under high temperature in aeroponics in potato cv. Kufri Anand.

BACKGROUND: High temperature stress is an important abiotic factor, which affects tuberization and ultimately causes heavy yield reduction in potato. OBJECTIVES: Identification and characterization of genes associated with tuberization under high temperature stress is essential for future management through biotechnology. METHODOLOGY: Two contrasting potato varieties Kufri Anand (profuse tuber-bearing) versus Kufri Frysona (very less/scanty tuber-bearing, control) were cultivated in aeroponics under high temperature stress, and transcriptomes were analyzed. RESULTS: Potato cv. Kufri Anand was found superior over control (Kufri Frysona) for tuber yield and its component traits along with root morphology under aeroponics. Transcriptomes of tuber and leaf tissues were analyzed. Statistically significant (p < 0.05) differentially expressed genes (DEGs) were categorised into up-regulated (> 2 log2 fold change, FC) and down-regulated (< -2 log2 FC) genes. DEGs were annotated by gene ontology and KEGG pathways. A few selected up-regulated genes of both tissues were identified, and phylogeny tree and motif analysis were analysed based on 36 peptide sequences representing 15 selected DEGs in this study. Further, gene expression markers were developed and validated by real time qPCR analysis for the identification of high temperature tolerant genotypes. CONCLUSION: A few key genes associated in tuberization under high temperature conditions were heat shock proteins (e.g. 18.5 kDa class I heat shock protein), sugar metabolism (e.g. glucosyltransferase), transcription factor (e.g. WRKY), and phytohormones (e.g. auxin-induced beta-glucosidase). Our study provides an overview of key genes involved in tuberization under high temperature stress in potato cv. Kufri Anand under aeroponics.

Screening of wild species and transcriptome profiling to identify differentially regulated genes in response to late blight resistance in potato.

Late blight (Phytophthora infestans) is a serious disease of potatoes. The aim of this study was to screen wild potato species and identify differentially expressed genes (DEGs) associated with late blight resistance. Wild potato species such as PIN45 (Solanum pinnatisectum), CPH62 (Solanum cardiophyllum), JAM07 (Solanum jamesii), MCD24 (Solanum microdontum), PLD47 (Solanum polyadenium), and cv. Kufri Bahar (control) were tested by artificial inoculation of P. infestans under controlled conditions. Transcriptomes of the leaf tissues (96 h post-inoculation) were sequenced using the Illumina platform. Statistically significant (p < 0.05) DEGs were analyzed in wild species by comparison with the control, and upregulated (>2 log2 fold change, FC) and downregulated (<-2 log2 FC) genes were identified. DEGs were functionally characterized with Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Selected genes were validated by real-time PCR analysis to confirm RNA-seq results. We identified some upregulated genes associated with late blight resistance in wild species such as cytochrome P450, proline-rich protein, MYB transcription factor MYB139, ankyrin repeat-containing protein, and LRR receptor-like serine/threonine-protein kinase in PIN45; glucosyltransferase, fructose-bisphosphate aldolase, and phytophthora-inhibited protease 1 in CPH62; steroid binding protein and cysteine proteinase 3 in JAM07; glycine-rich cell wall structural protein 1 and RING finger protein in MCD24; and cysteine proteinase 3 and major latex protein in PLD47. On the other hand, downregulated genes in these species were snakin-2 and WRKY transcription factor 3 in PIN45; lichenase and phenylalanine ammonia-lyase 1 in CPH62; metallothionein and LRR receptor-like serine/threonine-protein kinase in JAM07; UDP-glucoronosyl/UDP-glucosyl transferase family protein and steroid binding protein in MCD24; and cytoplasmic small heat shock protein class I and phosphatase PLD47. Our study identified highly resistant wild potato species and underlying genes such as disease resistance, stress response, phytohormones, and transcription factors (e.g., MYB, WRKY, AP2/ERF, and AN1) associated with late blight resistance in wild potato species.

Untargeted Metabolomics of Alternaria solani-Challenged Wild Tomato Species Solanum cheesmaniae Revealed Key Metabolite Biomarkers and Insight into Altered Metabolic Pathways.

Untargeted metabolomics of moderately resistant wild tomato species Solanum cheesmaniae revealed an altered metabolite profile in plant leaves in response to Alternaria solani pathogen. Leaf metabolites were significantly differentiated in non-stressed versus stressed plants. The samples were discriminated not only by the presence/absence of specific metabolites as distinguished markers of infection, but also on the basis of their relative abundance as important concluding factors. Annotation of metabolite features using the Arabidopsis thaliana (KEGG) database revealed 3371 compounds with KEGG identifiers belonging to biosynthetic pathways including secondary metabolites, cofactors, steroids, brassinosteroids, terpernoids, and fatty acids. Annotation using the Solanum lycopersicum database in PLANTCYC PMN revealed significantly upregulated (541) and downregulated (485) features distributed in metabolite classes that appeared to play a crucial role in defense, infection prevention, signaling, plant growth, and plant homeostasis to survive under stress conditions. The orthogonal partial least squares discriminant analysis (OPLS-DA), comprising a significant fold change (≥2.0) with VIP score (≥1.0), showed 34 upregulated biomarker metabolites including 5-phosphoribosylamine, kaur-16-en-18-oic acid, pantothenate, and O-acetyl-L-homoserine, along with 41 downregulated biomarkers. Downregulated metabolite biomarkers were mapped with pathways specifically known for plant defense, suggesting their prominent role in pathogen resistance. These results hold promise for identifying key biomarker metabolites that contribute to disease resistive metabolic traits/biosynthetic routes. This approach can assist in mQTL development for the stress breeding program in tomato against pathogen interactions.

CRISPR/Cas genome editing in tomato improvement: Advances and applications.

The narrow genetic base of tomato poses serious challenges in breeding. Hence, with the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (CRISPR/Cas9) genome editing, fast and efficient breeding has become possible in tomato breeding. Many traits have been edited and functionally characterized using CRISPR/Cas9 in tomato such as plant architecture and flower characters (e.g. leaf, stem, flower, male sterility, fruit, parthenocarpy), fruit ripening, quality and nutrition (e.g., lycopene, carotenoid, GABA, TSS, anthocyanin, shelf-life), disease resistance (e.g. TYLCV, powdery mildew, late blight), abiotic stress tolerance (e.g. heat, drought, salinity), C-N metabolism, and herbicide resistance. CRISPR/Cas9 has been proven in introgression of de novo domestication of elite traits from wild relatives to the cultivated tomato and vice versa. Innovations in CRISPR/Cas allow the use of online tools for single guide RNA design and multiplexing, cloning (e.g. Golden Gate cloning, GoldenBraid, and BioBrick technology), robust CRISPR/Cas constructs, efficient transformation protocols such as Agrobacterium, and DNA-free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex, Cas9 variants like PAM-free Cas12a, and Cas9-NG/XNG-Cas9, homologous recombination (HR)-based gene knock-in (HKI) by geminivirus replicon, and base/prime editing (Target-AID technology). This mini-review highlights the current research advances in CRISPR/Cas for fast and efficient breeding of tomato.

Root system architecture for abiotic stress tolerance in potato: Lessons from plants.

The root is an important plant organ, which uptakes nutrients and water from the soil, and provides anchorage for the plant. Abiotic stresses like heat, drought, nutrients, salinity, and cold are the major problems of potato cultivation. Substantial research advances have been achieved in cereals and model plants on root system architecture (RSA), and so root ideotype (e.g., maize) have been developed for efficient nutrient capture to enhance nutrient use efficiency along with genes regulating root architecture in plants. However, limited work is available on potatoes, with a few illustrations on root morphology in drought and nitrogen stress. The role of root architecture in potatoes has been investigated to some extent under heat, drought, and nitrogen stresses. Hence, this mini-review aims to update knowledge and prospects of strengthening RSA research by applying multi-disciplinary physiological, biochemical, and molecular approaches to abiotic stress tolerance to potatoes with lessons learned from model plants, cereals, and other plants.

Genome editing (CRISPR-Cas)-mediated virus resistance in potato (Solanum tuberosum L.).

Plant viruses are the major pathogens that cause heavy yield loss in potato. The important viruses are potato virus X, potato virus Y and potato leaf roll virus around the world. Besides these three viruses, a novel tomato leaf curl New Delhi virus is serious in India. Conventional cum molecular breeding and transgenics approaches have been applied to develop virus resistant potato genotypes. But progress is slow in developing resistant varieties due to lack of host genes and long breeding process, and biosafety concern with transgenics. Hence, CRISPR-Cas mediated genome editing has emerged as a powerful technology to address these issues. CRISPR-Cas technology has been deployed in potato for several important traits. We highlight here CRISPR-Cas approaches of virus resistance through targeting viral genome (DNA or RNA), host factor gene and multiplexing of target genes simultaneously. Further, advancement in CRISPR-Cas research is presented in the area of DNA-free genome editing, virus-induced genome editing, and base editing. CRISPR-Cas delivery, transformation methods, and challenges in tetraploid potato and possible methods are also discussed.

Germplasm, Breeding, and Genomics in Potato Improvement of Biotic and Abiotic Stresses Tolerance.

Potato is one of the most important food crops in the world. Late blight, viruses, soil and tuber-borne diseases, insect-pests mainly aphids, whiteflies, and potato tuber moths are the major biotic stresses affecting potato production. Potato is an irrigated and highly fertilizer-responsive crop, and therefore, heat, drought, and nutrient stresses are the key abiotic stresses. The genus Solanum is a reservoir of genetic diversity, however, a little fraction of total diversity has been utilized in potato breeding. The conventional breeding has contributed significantly to the development of potato varieties. In recent years, a tremendous progress has been achieved in the sequencing technologies from short-reads to long-reads sequence data, genomes of Solanum species (i.e., pan-genomics), bioinformatics and multi-omics platforms such as genomics, transcriptomics, proteomics, metabolomics, ionomics, and phenomics. As such, genome editing has been extensively explored as a next-generation breeding tool. With the available high-throughput genotyping facilities and tetraploid allele calling softwares, genomic selection would be a reality in potato in the near future. This mini-review covers an update on germplasm, breeding, and genomics in potato improvement for biotic and abiotic stress tolerance.

Resistance Evaluation for Native Potato Accessions against Late Blight Disease and Potato Cyst Nematodes by Molecular Markers and Phenotypic Screening in India.

The potato originated in southern Peru and north-western Bolivia (South America). However, native accessions have also been cultivated in India for many years. Late blight, caused by the fungus Phytophthora infestans, is the most devastating potato disease, while potato cyst nematode (Globodera spp.) (PCN) is another economically significant quarantine-requiring pest in India. In this study, we have generated a new Indian native collection of 94 potato accessions collected from different parts India. These accessions were screened against late blight and potato cyst nematode resistance by using gene-based molecular markers and phenotypic screening methods. Marker assisted selection using R1 gene-specific marker CosA210 revealed a late blight resistance gene in 11 accessions. PCN resistance bands were found in 3 accessions with marker TG689141, 5 accessions with marker 57R452, and 1 accession having Gro1-4-1602 marker for G. rostochiensis (Ro1,4), while 64 accessions amplified marker HC276 indicating G. pallida (Pa2,3) resistance gene (GpaVvrn QTL). On the other hand, phenotypic screening against late blight resistance under natural epiphytic conditions (hot-spot) revealed three accessions with high resistance, while others were resistant (1 accession), moderately resistant (5 accessions), susceptible (29 accessions), and highly susceptible (56 accessions). For G. rostochiensis (golden cyst nematode) and G. pallida (white cyst nematode) resistance, accessions were grouped into highly resistant (3, 3), resistant (0, 2), moderately resistant (6, 29), susceptible (32, 30), and highly susceptible (53, 30), respectively, for the two PCN species. Collectively, we identified promising accessions with high resistance to late blight (JG-1, Kanpuria Safed, and Rangpuria), and also highly resistant to both Globodera species (Garlentic, Jeevan Jyoti, and JG-1). Our findings suggested that these accessions would be useful for late blight and PCN resistance breeding, as well as future molecular studies in potatoes.

Genome sequence analysis provides insights on genomic variation and late blight resistance genes in potato somatic hybrid (parents and progeny).

Wild Solanum species are the important resources for potato improvement. With the availability of potato genome and sequencing progress, knowledge about genomic resources is essential for novel genes discovery. Hence, the aim of this study was to decipher draft genome sequences of unique potato genotypes i.e. somatic hybrid P8 (J1), wild species S. pinnatisectum (J2), progeny MSH/14-112 (P8 × cv. Kufri Jyoti) (J3), and S. tuberosum dihaploid C-13 (J4). Draft genome sequencing using Illumina platform and reference-based assemblies with the potato genome yielded genome assembly size of 725.01 Mb (J1), 724.95 Mb (J2), 725.01 Mb (J3), and 809.59 Mb (J4). Further, 39,260 (J1), 25,711 (J2), 39,730 (J3) and 30,241 (J4) genes were identified and 17,411 genes were found common in the genotypes particularly late blight resistance genes (R3a, RGA2, RGA3, R1B-16, Rpi-blb2, Rpi and Rpi-vnt1). Gene ontology (GO) analysis showed that molecular function was predominant and signal transduction was major KEGG pathways. Further, gene enrichment analysis revealed dominance of metabolic process (GO: 0008152) in all the samples. Phylogeny analysis showed relatedness with potato and other plant species. Heterozygous single nucleotide polymorphism (SNP) was more than homozygous, and SNP in genic region was more than inter-genic region. Copy number variation (CNV) analysis indicated greater number of deletions than duplications. Sequence diversity and conserved motifs analysis revealed variation for late blight resistance genes. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed differential expression of late blight resistance genes. Our study provides insights on genome sequence, structural variation and late blight resistance genes in potato somatic hybrid (parents and progeny) for future research.

Physiological and genome-wide RNA-sequencing analyses identify candidate genes in a nitrogen-use efficient potato cv. Kufri Gaurav.

Nitrogen (N) is an important nutrient for plant growth. However, its excess application leads to environmental damage. Hence, improving nitrogen use efficiency (NUE) of plant is one of the plausible options to solve the problems. Aim of this study was to identify candidate genes involved in enhancing NUE in potato cv. Kufri Gaurav (N efficient). Plants were grown in aeroponic with two contrasting N regimes (low N: 0.75 mM, and high N: 7.5 mM). Higher NUE in Kufri Gaurav was observed in low N based on the parameters like NUE, NUpE (N uptake efficiency), NUtE (N utilization efficiency) and AgNUE (agronomic NUE). Further, global gene expression profiles in root, leaf and stolon tissues were analyzed by RNA-sequencing using Ion Proton™ System. Quality data (≥Q20) of 2.04-2.73 Gb per sample were mapped with the potato genome. Statistically significant (P ≤ 0.05) differentially expressed genes (DEGs) were identified such as 176 (up-regulated) and 30 (down-regulated) in leaves, 39 (up-regulated) and 105 (down-regulated) in roots, and 81 (up-regulated) and 694 (down-regulated) in stolons. The gene ontology (GO) terms like metabolic process, cellular process and catalytic activity were predominant. Our RT-qPCR analysis confirmed the gene expression profiles of RNA-seq. Overall, we identified candidate genes associated with improving NUE such as superoxide dismutase, GDSL esterase lipase, probable phosphatase 2C, high affinity nitrate transporters, sugar transporter, proline rich proteins, transcription factors (VQ motif, SPX domain, bHLH) etc. Our findings suggest that these candidate genes probably play crucial roles in enhancing NUE in potato.

Genome-wide identification and characterization of microRNAs by small RNA sequencing for low nitrogen stress in potato.

Nitrogen is an important nutrient for plant growth and tuber quality of potato. Since potato crop requires high dose of N, improving nitrogen use efficiency (NUE) of plant is an inevitable approach to minimize N fertilization. The aim of this study was to identify and characterize microRNAs (miRNAs) by small RNA sequencing in potato plants grown in aeroponic under two contrasting N (high and low) regimes. A total of 119 conserved miRNAs belonging to 41 miRNAs families, and 1002 putative novel miRNAs were identified. From total, 52 and 54 conserved miRNAs, and 404 and 628 putative novel miRNAs were differentially expressed in roots and shoots, respectively under low N stress. Of total 34,135 predicted targets, the gene ontology (GO) analysis indicated that maximum targets belong to biological process followed by molecular function and cellular component. Eexpression levels of the selected miRNAs and targets were validated by real time-quantitative polymerase chain reaction (RT-qPCR) analysis. Two predicted targets of potential miRNAs (miR397 and miR398) were validated by 5' RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). In general, predicted targets are associated with stress-related, kinase, transporters and transcription factors such as universal stress protein, heat shock protein, salt-tolerance protein, calmodulin binding protein, serine-threonine protein kinsae, Cdk10/11- cyclin dependent kinase, amino acid transporter, nitrate transporter, sugar transporter, transcription factor, F-box family protein, and zinc finger protein etc. Our study highlights that miR397 and miR398 play crucial role in potato during low N stress management. Moreover, study provides insights to modulate miRNAs and their predicted targets to develop N-use efficient potato using transgenic/genome-editing tools in future.

Transcriptome analysis of potato shoots, roots and stolons under nitrogen stress.

Potato crop requires high dose of nitrogen (N) to produce high tuber yield. Excessive application of N causes environmental pollution and increases cost of production. Hence, knowledge about genes and regulatory elements is essential to strengthen research on N metabolism in this crop. In this study, we analysed transcriptomes (RNA-seq) in potato tissues (shoot, root and stolon) collected from plants grown in aeroponic culture under controlled conditions with varied N supplies i.e. low N (0.2 milli molar N) and high N (4 milli molar N). High quality data ranging between 3.25 to 4.93 Gb per sample were generated using Illumina NextSeq500 that resulted in 83.60-86.50% mapping of the reads to the reference potato genome. Differentially expressed genes (DEGs) were observed in the tissues based on statistically significance (p ≤ 0.05) and up-regulation with ≥ 2 log2 fold change (FC) and down-regulation with ≤ -2 log2 FC values. In shoots, of total 19730 DEGs, 761 up-regulated and 280 down-regulated significant DEGs were identified. Of total 20736 DEGs in roots, 572 (up-regulated) and 292 (down-regulated) were significant DEGs. In stolons, of total 21494 DEG, 688 and 230 DEGs were significantly up-regulated and down-regulated, respectively. Venn diagram analysis showed tissue specific and common genes. The DEGs were functionally assigned with the GO terms, in which molecular function domain was predominant in all the tissues. Further, DEGs were classified into 24 KEGG pathways, in which 5385, 5572 and 5594 DEGs were annotated in shoots, roots and stolons, respectively. The RT-qPCR analysis validated gene expression of RNA-seq data for selected genes. We identified a few potential DEGs responsive to N deficiency in potato such as glutaredoxin, Myb-like DNA-binding protein, WRKY transcription factor 16 and FLOWERING LOCUS T in shoots; high-affinity nitrate transporter, protein phosphatase-2c, glutaredoxin family protein, malate synthase, CLE7, 2-oxoglutarate-dependent dioxygenase and transcription factor in roots; and glucose-6-phosphate/phosphate translocator 2, BTB/POZ domain-containing protein, F-box family protein and aquaporin TIP1;3 in stolons, and many genes of unknown function. Our study highlights that these potential genes play very crucial roles in N stress tolerance, which could be useful in augmenting research on N metabolism in potato.

Analysis of allelic variation in wild potato (Solanum) species by simple sequence repeat (SSR) markers.

Allelic variation in wild potato (Solanum) species was analysed using 14 simple sequence repeat (SSR) markers. SSR allelic profiles showed high polymorphism and distinctness among the wild species. A total of 109 alleles of 14 polymorphic SSR markers were scored in 82 accessions belonging to 22 wild potato species. Allele size ranged from a minimum of 104 bp (STI0030) to a maximum of 304 bp (STM5114). Number of SSR alleles per marker ranged from 4 (STM5127/STM1053) to 13 (STM0019), whereas PIC value varied between 0.66 (STM1053) and 0.91 (STM0019). Cluster analysis using SSR allelic profiles of 82 accessions grouped showed 5 major clusters (I-V) based on the Dice similarity coefficient using neighbour-joining clustering method. Distinct allelic variations were observed among the accessions irrespective of the origin country, series and species. Our study suggests that SSR-based molecular characterization of wild potato species is accession specific and development of an allelic dataset for all the accessions would strengthen their utilization in potato research in future.

Microarray analysis of gene expression patterns in the leaf during potato tuberization in the potato somatic hybrid Solanum tuberosum and Solanum etuberosum.

Genes involved in photoassimilate partitioning and changes in hormonal balance are important for potato tuberization. In the present study, we investigated gene expression patterns in the tuber-bearing potato somatic hybrid (E1-3) and control non-tuberous wild species Solanum etuberosum (Etb) by microarray. Plants were grown under controlled conditions and leaves were collected at eight tuber developmental stages for microarray analysis. A t-test analysis identified a total of 468 genes (94 up-regulated and 374 down-regulated) that were statistically significant (p ≤ 0.05) and differentially expressed in E1-3 and Etb. Gene Ontology (GO) characterization of the 468 genes revealed that 145 were annotated and 323 were of unknown function. Further, these 145 genes were grouped based on GO biological processes followed by molecular function and (or) PGSC description into 15 gene sets, namely (1) transport, (2) metabolic process, (3) biological process, (4) photosynthesis, (5) oxidation-reduction, (6) transcription, (7) translation, (8) binding, (9) protein phosphorylation, (10) protein folding, (11) ubiquitin-dependent protein catabolic process, (12) RNA processing, (13) negative regulation of protein, (14) methylation, and (15) mitosis. RT-PCR analysis of 10 selected highly significant genes (p ≤ 0.01) confirmed the microarray results. Overall, we show that candidate genes induced in leaves of E1-3 were implicated in tuberization processes such as transport, carbohydrate metabolism, phytohormones, and transcription/translation/binding functions. Hence, our results provide an insight into the candidate genes induced in leaf tissues during tuberization in E1-3.

Analysis of genetic and epigenetic variation in in vitro propagated potato somatic hybrid by AFLP and MSAP marker

Background: Genetic and epigenetic changes (DNA methylation) were examined in the tissue-culture propagated interspecific potato somatic hybrids between dihaploid Solanum tuberosum and S. pinnatisectum. Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) were applied to detect the genetic and epigenetic changes, respectively in the somatic hybrids mother plants (1st cycle) and their regenerants (30th cycles sub-cultured). Results: To detect genetic changes, eight AFLP primer combinations yielded a total of 329 scorable bands of which 49 bands were polymorphic in both mother plants and regenerants. None of the scorable bands were observed in term of loss of original band of mother plant or gain of novel band in their regenerants. AFLP profiles and their cluster analysis based on the Jaccard’s similarity coefficient revealed 100% genetic similarity among the mother plant and their regenerants. On the other hand, to analyze epigenetic changes, eight MSAP primer pair combinations detected a few DNA methylation patterns in the mother plants (0 to 3.4%) and their regenerants (3.2 to 8.5%). Out of total 2320 MSAP sites in the mother plants, 2287 (98.6%) unmethylated, 21 (0.9%) fully methylated and 12 (0.5%) hemi-methylated, and out of total 2494 MSAP sites in their regenerants, 2357 (94.5%) unmethylated, 79 (3.1%) fully methylated and 58 (2.3%) hemi-methylated sites were amplified. Conclusion: The study concluded that no genetic variations were observed among the somatic hybrids mother plants and their regenerants by eight AFLP markers. However, minimum epigenetic variations among the samples were detected ranged from 0 to 3.4% (mother plants) and 3.2 to 8.5% (regenerants) during the tissue culture process.