Identification of genes governing resistance to PCN (Globodera rostochiensis) through transcriptome analysis in Solanum tuberosum.

Potato cyst nematodes (PCNs) are major pests worldwide that affect potato production. The molecular changes happening in the roots upon PCN infection are still unknown. Identification of transcripts and genes governing PCN resistance will help in the development of resistant varieties. Hence, differential gene expression of compatible (Kufri Jyoti) and incompatible (JEX/A-267) potato genotypes was studied before (0 DAI) and after (10 DAI) inoculation of Globodera rostochiensis J2s through RNA sequencing (RNA-Seq). Total sequencing reads generated ranged between 33 and 37 million per sample, with a read mapping of 48-84% to the potato reference genome. In the infected roots of the resistant genotype JEX/A-267, 516 genes were downregulated, and 566 were upregulated. In comparison, in the susceptible genotype Kufri Jyoti, 316 and 554 genes were downregulated and upregulated, respectively. Genes encoding cell wall proteins, zinc finger protein, WRKY transcription factors, MYB transcription factors, disease resistance proteins, and pathogenesis-related proteins were found to be majorly involved in the incompatible reaction after PCN infection in the resistant genotype, JEX/A-267. Furthermore, RNA-Seq results were validated through quantitative real-time PCR (qRT-PCR), and it was observed that ATP, FLAVO, CYTO, and GP genes were upregulated at 5 DAI, which was subsequently downregulated at 10 DAI. The genes encoding ATP, FLAVO, LBR, and GP were present in > 1.5 fold before infection in JEX-A/267 and upregulated 7.9- to 27.6-fold after 5 DAI; subsequently, most of these genes were downregulated to 0.9- to 2.8-fold, except LBR, which was again upregulated to 44.4-fold at 10 DAI.

Characterizing the Potato Growing Regions in India Using Meteorological Parameters.

Currently, the multi-location testing of advanced hybrids in India is carried out at 25 centers under the All India Co-ordinated Research Project on Potato (AICRP-P), which is spread across the country. These centres have been chosen to represent different potato growing regions based on soil and agronomic features. However, the reliable deployment of the newly bred varieties in different regions requires a scientific delineation of potato growing zones with homogenous climates. The present study was undertaken to develop homogenous zones in the Indian sub-continent based on the environmental parameters of the potato growing season. A total of 1253 locations were identified across the country as having a plausible potato growing season of at least 70 days with suitable thermal limits. Six variables including five meteorological parameters including Physiological days (P days), Growing degree days (GDD), Mean daily temperature, Mean night temperature and Mean daily incident solar radiation, together with altitude as the sixth variable, were used for Agglomerative Hierarchical Clustering (AHC) and the Principal Component Analysis by Multidimensional Scaling (MDS) technique to derive identical classes. The thematic map of the classes was overlaid on potato growing districts of India using ArcGIS 9.1 software. The study clearly depicted that the clustering technique can effectively delineate the target population of environments (TPE) for potato genotypes performing well at different testing environments in India. The study also identifies target locations for future focus on breeding strategies, especially the high night temperature class having a large expanse in India. This is also vital in view of the impending climate change situation.

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.