Jagesh Kumar Tiwari

Molecular and morphological characterization of somatic hybrids between Solanum tuberosum L. and S. etuberosum Lindl.

Interspecific potato somatic hybrids between Solanum tuberosum L. (di)haploid C-13 and 1 endosperm balance number non-tuberous wild species S. etuberosum Lindl. were produced by protoplasts electrofusion. The objective was to transfer virus resistance from this wild species into the cultivated potatoes. Post-fusion products were cultured in VKM medium followed by regeneration of calli in MS13 K medium at 20°C under a 16-h photoperiod, and regenerants were multiplied on MS medium. Twenty-one somatic hybrids were confirmed by RAPD, SSR and cytoplasm (chloroplast/mitochondria) type analysis possessing species-specific diagnostic bands of corresponding parents. Tetraploid nature of these somatic hybrids was determined through flow cytometry analysis. Somatic hybrids showed intermediate phenotypes (plant, leaves and floral morphology) to their parents in glass-house grown plants. All the somatic hybrids were male-fertile. ELISA assay of somatic hybrids after artificial inoculation of Potato virus Y (PVY) infection reveals high PVY resistance.

Production and characterization of somatic hybrids between Solanum tuberosum L. and S. pinnatisectum Dun.

Interspecific somatic hybrids between the dihaploid Solanum tuberosum and the wild species S. pinnatisectum Dun. were produced via protoplast fusion. Protoplast isolation, electrofusion, culture of post-fusion products and regeneration of calli/shoots were undertaken following optimized protocols. Regenerants were characterized for hybridity, ploidy and resistance to Phytophthora infestans (Mont.) de Bery, causal fungal pathogen of late blight disease. From a total of 126 regenerated macrocalli, 12 somatic hybrids were confirmed by possessing species-specific diagnostic bands of their corresponding parents as revealed by RAPD, SSRs and cytoplasmic-DNA analyses. Tetraploid status of the 12 hybrids was determined using flow cytometry analysis. Intermediate phenotypes for leaf, flower, and tuber characteristics and high male fertility were observed in field-grown hybrid plants. Hybrids were highly resistant to foliage late blight based on field assessment for two seasons. In contrast, moderate level of resistance to foliage blight was observed in hybrids based on the detached leaf assay under laboratory conditions. Overall, somatic hybrids with moderate levels of resistance to foliage blight were identified, and these will be useful for in situ hybridization in potato breeding efforts.

Allele Mining in Solanum Germplasm: Cloning and Characterization of RB-Homologous Gene Fragments from Late Blight Resistant Wild Potato Species

The late blight disease can be managed by introduction of resistance (R) genes from the wild Solanum species into the cultivated potato. The R genes are mostly comprised of the nucleotide binding site-leucine rich repeat (NBS-LRR) domains and share nucleotide sequence homology in the crop species. In this study, we used potato R gene-specific primers to amplify homologous genes from wild species. A total of 39 wild species were tested for late blight resistance by challenge inoculation of Phytophthora infestans under controlled conditions. Of these, only 15 species were highly resistant (HR) and these were PCR (polymerase chain reaction) amplified by 53 primers representing 21 R genes of potato. Further, only single, distinct, and reproducible gene fragments were cloned and sequenced. Following sequence processing and analysis, 17 non-redundant sequences of RB-homologous genes were identified with uninterrupted open reading frames (ORFs) and nucleotide sequence homologies to known late blight R genes. Finally, 17 RB-homologous gene fragments amplified by the primers of the RB gene were isolated from 11 wild species. The isolation and characterization of 17 RB-homologous gene fragments from wild potato species may serve as an important genomic resource for the novel gene discovery in late blight resistance breeding programs.

Organelle Genome Analysis in Somatic Hybrids Between Solanum tuberosum and S. pinnatisectum Revealed Diverse Cytoplasm Type in Potato

Organelle genome diversity was analysed in interspecific potato somatic hybrids using chloroplast (cp)- and mitochondrial (mt)-specific molecular markers. Out of total 25 markers (15 cpDNA and 10 mtDNA) tested in total 16 samples, only four mtDNA primers (rpS14/cob, Nsm2, ALM4/ALM5 and ALM6/ALM7) detected polymorphism, whereas other primers were monomorphic. Cluster analysis showed higher genetic diversity among the genotypes by mtDNA profiles than that by cpDNA. Ten haplotypes were grouped by cluster analysis comprised of maximum seven genotypes in haplotype no. 3. Monomorphic markers did not reveal variability in our samples and suggest highly conserved organelle genomic regions. New genomic arrangements were observed in the somatic hybrids for mt polymorphic loci. Our study suggests that somatic hybrids are comprised of diverse cytoplasm types consisting predominantly of T-, W-, and C-, with a few A- and S-type chloroplast, and α-, β- and γ-type mitochondrial genome, and have unique potential to widen the cultivated potato gene pool by breeding methods.

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.

Integrated genomics, physiology and breeding approaches for improving nitrogen use efficiency in potato: translating knowledge from other crops

Potato plays a key role in global food and nutritional security. Potato is an N fertiliser-responsive crop, producing high tuber yields. However, excessive use of N can result in environmental damage and high production costs, hence improving nitrogen use efficiency (NUE) of potato plants is one of the sustainable options to address these issues and increase yield. Advanced efforts have been undertaken to improve NUE in other plants like Arabidopsis, rice, wheat and maize through molecular and physiological approaches. Conversely, in potato, NUE studies have predominantly focussed on agronomy or soil management, except for a few researchers who have measured gene expression and proteins relevant to N uptake or metabolism. The focus of this review is to adapt knowledge gained from other plants to inform investigation of N metabolism and associated traits in potato with the aim of improving potato NUE using integrated genomics, physiology and breeding methods.

Genomics in Management and Genetic Enhancement of Potato Germplasm

The systematic characterization and utilization of naturally occurring genetic variation in the plant genetic resources have become an important approach in plant genome research and breeding. The development of molecular techniques now allows a more accurate analysis of a large collections of potato germplasm. The rapid progress in high-throughput technology such as next-generation sequencing (NGS) offers an exciting tool for novel gene discovery involved in phenotypic traits expression. In the years to come, genomics, transcriptomics and other ‘omics’ technologies will play a key role in potato improvement. The discovery and high-throughput screening of single nucleotide polymorphism (SNP), the presence/absence of allelic variations in diverse germplasm collections will give a detailed insight into the origin, domestication and available trait-relevant variations in the polyploid crops such as potato. In the process, novel approaches and possibilities for marker/genomics-assisted potato breeding are facilitated. This chapter highlights the use of potato genome sequence in management and the genetic enhancement of the potato through its characterization and identification of novel gene/QTL/allele followed by their applications in potato improvement with agricultural relevance.

Identification of Late Blight Resistance Gene Homologues in Wild Solanum Species

Solanum wild species represent diverse population harbouring myriad of resistance (R) genes which could be tapped for incorporating durable biotic and abiotic stress resistance in cultivated genotypes. In the present study, the authors report the screening of eleven Solanum wild species viz., Solanum avilesii, S. berthaultii, S. tuberosum sp andigena, S. arnezii, S. cardiophyllum, S. alandiae, S. albicans, S. sparsipilum, S. spegazzinii, S. pinnatisectum and S. demissum for the presence of late blight resistance genes through molecular markers linked to genes RB/Rpi-blb1, Rpi-ber1, R1 and R3. These markers were able to amplify specific sequences across species indicating universal distribution of R genes. The amplified marker specific sequences matched well with sequences in NCBI database with known function in governing late blight resistance. Phylogenetic analysis grouped the sequences to their known counterpart’s. e.g. sequences amplified with Rpi-blb1 gene specific marker grouped with known sequence corresponding to Solanum bulbocastanum protein genes and so forth in other sequence-marker combinations. The presence of such genes was not immediately linked to resistance against late blight in the wild species accession but may have a role in maintaining/developing their resistance against novel pathogenic races. The newly identified R gene homologues (fragments) in the highly resistant wild potato accessions can serve as a novel source of genes for late blight resistance breeding. The complementary late blight resistant genes from different species can be pyramided in cultivated genotypes for providing stable late blight resistance.

Evaluation of Interspecific Somatic Hybrids of Potato (Solanum tuberosum) and Wild S. cardiophyllum for Adaptability, Tuber Dry Matter, Keeping Quality and Late Blight Resistance

Interspecific potato somatic hybrids (here after referred as ‘cph-hybrids’) derived earlier through protoplast fusion (Solanum tuberosum + S. cardiophyllum) were used in this study. The genetic potential of cph-hybrids was assessed based on the field performance in the Indian sub-tropical conditions. In general, cph-hybrids exhibited higher plant stand, poor plant vigour and late foliage maturity as compared to the control potato var. Kufri Bahar. Yield performance of cph-hybrids was poor as compared to the control, but produced 3–6 times higher marketable tuber yield than the wild parent (S. cardiophyllum). All cph-hybrids possessed significantly higher tuber dry matter content (≥ 24%) than the parents (20.82%) and var. Kufri Bahar (18.52%), excellent keeping quality and showed high resistance to late blight. Thus, based on this study the promising cph-hybrids viz., Crd 6, Crd 10 and Crd16, can be used as parents in breeding for the improvement of important traits viz., higher tuber dry matter content, better keeping quality and high late blight resistance, along with adaptability under sub-tropical conditions.

Genomics in True Potato Seed (TPS) Technology: Engineering Cloning Through Seeds

Tuber is the main planting material for commercial potato production. Besides, true potato seed (TPS), i.e. true botanical seed is another technology of potato. The TPS-raised crop has several advantages over the tuber-raised crops but due to the certain limitations of the conventional TPS technology, it could not be popularized, as expected the world over. The major drawbacks are non-uniformity of crop and tuber characters, lengthy crop duration, and labour-intensive farming due to seedling raising and transplanting. To address these issues, this chapter highlights a brief overview of the production of hybrid TPS, applying genomics approaches to engineer cloning through apomixis seeds, and offers the prospect of F1 hybrid potato technology.