Vinay Bhardwaj

Key players associated with tuberization in potato: potential candidates for genetic engineering

Abstract Tuberization in potato (Solanum tuberosum L.) is a complex biological phenomenon which is affected by several environmental cues, genetic factors and plant nutrition. Understanding the regulation of tuber induction is essential to devise strategies to improve tuber yield and quality. It is well established that short-day photoperiods promote tuberization, whereas long days and high-temperatures inhibit or delay tuberization. Worldwide research on this complex biological process has yielded information on the important bio-molecules (proteins, RNAs, plant growth regulators) associated with the tuberization process in potato. Key proteins involved in the regulation of tuberization include StSP6A, POTH1, StBEL5, StPHYB, StCONSTANS, Sucrose transporter StSUT4, StSP5G, etc. Biomolecules that become transported from “source to sink” have also been suggested to be important signaling candidates regulating the tuberization process in potatos. Four molecules, namely StSP6A protein, StBEL5 RNA, miR172 and GAs, have been found to be the main candidates acting as mobile signals for tuberization. These biomolecules can be manipulated (overexpressed/inhibited) for improving the tuberization in commercial varieties/cultivars of potato. In this review, information about the genes/proteins and their mechanism of action associated with the tuberization process is discussed.

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.

Potato Diversity and Its Genetic Enhancement

Potato is the world’s third largest food crop after rice and wheat widely grown across all continents. It belongs to the genus Solanum and section Petota that contain approximately 2000 species that are distributed from the South-western United States (38°N) to Chile (41°S) between 2000 to 4000 m altitudes. Potato has 6 cultivated species, 225 wild relatives and 110 wild tuber-bearing species. The main cultivated potato species Solanum tuberosum L., a tetraploid (2n = 4x = 48) originated from Andes of Peru and Bolivia in South America over 10,000 years ago. The ploidy of potatoes varies from diploid (2n = 24) to hexaploid (2n = 72) with majority being diploids. Potatoes were introduced to Europe in 1570s and by beginning of seventeenth century they spread to the other parts of the world. Systematic potato breeding started in 1807 in England followed by other parts of Europe, North America, India, International Potato Centre, Peru and China. There are two basic approaches to conserve potato genetic resources, viz. in situ and ex situ. Currently, cryo-conservation is being tapped for long-term conservation. Seven major potato gene banks are present worldwide to conserve existing diversity. Although more germplasm are being evaluated, the use of genetic resources has been much poorer to their evaluations mainly due to undesirable tuber traits of the wild species and crossability barriers. This has led to narrow genetic base of the cultivated potatoes. The ‘Irish famine’ of 1840s depicts the devastating effect of growing large areas under a single variety. Cultivated potato exhibits complex tetrasomic inheritance and high heterozygosity. Dihaploids of tuberosum cross readily with many diploid species thus providing opportunity for introgression of useful traits from alien sources to cultivated background. The other well-exploited techniques in potato breeding, viz. somaclonal variations, somatic hybridization, molecular markers, genetic transformation and RNAi approaches. Potato is one of the rare crops where maximum tissue culture and genetic engineering interventions have been connoted. Today, potato genome is sequenced and it opens up new vistas for developing tailor-made varieties in future.

Molecular characterization of potato virus Y resistance in potato (Solanum tuberosum L.)

Amongst viruses, Potato virus Y (PVY) is the most prevalent virus affecting potato productivity. Resistance genes (Ry) in Solanum tuberosum subsp. andigena (Ryadg) and Solanum stoloniferum (Rysto) confer extreme resistance (ER) to PVY. SCAR marker RYSC3 for Ryadg gene and SSR marker STM0003 for Rysto gene were used to screen 119 potato genotypes including 44 Indian varieties. These genotypes were also challenge inoculated with PVYo strain and confirmed for ER by DAS-ELISA. Gene Ryadg was present in 11 and Rysto in 22 genotypes, while 3 genotypes possessed both the genes. The statistical analysis indicated that among the two tested markers, RYSC3 is better diagnostic of PVY resistance. The identified genotypes can serve as potential source for future virus resistance breeding programs.

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.

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.

Late blight resistance status in wild potato species against Indian population of Phytophthora infestans

Late blight caused by oomycete pathogen Phytophthora infestans is the most destructive disease affectingpotato crop world-wide with losses up to 90% in India. Resistant varieties offers safe and economical mean formanagement of the disease. Wild species of potato are the reservoirs of resistance against many insect pestand diseases including late blight. In the present study, 539 clones of 91 potato accessions belonging to 18wild species maintained at CPRI, Shimla were evaluated for presence of durable resistance against late blight. The clones SS 1764–19 (S. alandiae), SS 1763–09 and SS 1763–25 (S. albicans), SS 1769–04, SS 1769–08 and SS 1770–14 (S. arnezii), SS 1784–07 (S. berthaultii), SS 1794–07 (S. brevicaule), SS 0551-02, SS 0680-06, SS 1671–01 and SS 1671–03 (S. chacoense), SS 1835, SS 1846–05, SS 1847–09, SS 1850-0, SS 1850–01 and SS 1850–04 (S. demissum), SS 1926–09, SS 1926–10, SS 1926–11 and SS 1926–13 (S. microdontum), SS 2615–01, SS 2616–01, SS 2616–02, SS 2655–01, SS 2656–02, SS 2658–01, SS 2658–02 and SS 2658–03 (S. pinnatisectum), SS 1664–02 and SS 1724–40 (S. sparsipilum), SS 2038–04 and SS 2048-0 (S. tuberosum ssp. andigena) and SS 2082-0 (S. vernei)were found to be most promising having high late blight resistance under laboratory and field testing. Althoughsome difficulties exist in direct utilization of these clones due to ploidy and EBN differences, but these can beovercome through both short and long-term breeding strategies viz., ploidy and EBN manipulation, bridgingspecies, embryo rescue, somatic hybridization and molecular techniques.

An insight into the downstream analysis of RB gene in F1 RB potato lines imparting field resistance to late blight

Earlier studies have shown that level of late blight resistance conferred by the classical R gene (RB Rpi-blb1) is dependent on genetic background of the recipient genotype. This was revealed in the analysis of late blight response that belonged to a group of F1 progeny obtained from the cross between Kufri Jyoti and SP951, which showed wide variation in late blight resistance response in spite of possessing the same RB gene. The global gene expression pattern in the RB potato lines was studied in response to late blight infection using cDNA microarray analysis to reveal the background effect. Leaf samples were collected at 0, 24, 72 and 120h post inoculation (hpi) with Phytophthora infestans for gene expression analysis using 61031 gene sequences. Significantly upregulated (1477) and downregulated (4245) genes common in the RB-transgenic F1 lines at 24 and 72 hpi were classified into several categories based on GO identifiers and majority of genes were assigned putative biological functions. Highest expression of an NBS-LRR along with protease, pectin esterase inhibitors, chaperones and reactive oxygen species genes were observed which affirmed a significant role of these categories in the defence response of RB-KJ lines. Results suggest that the immune priming of plant receptors are likely to be involved in stability and functionality of RB to induce resistance against P. infestans. This study is important for effective deployment of RB gene in the host background and contributes immensely to scientific understanding of R gene interaction with host protein complexes to regulate defence system in plants.

Draft Genome Sequencing of Rhizoctonia solani Anastomosis Group 3 (AG3- PT) Causing Stem Canker and Black Scurf of Potato

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle being classified into fourteen reproductively incompatible anastomosis groups (AGs). AG3-PT (a potato subgroup) is associated with quantitative and qualitative yield losses through stem canker and black scurf in potato. Here we present the first draft sequence of the R. solani [AG3-PT] strain RS-20 with a G-C content of 48.3%. It consists of 11,431 total predicted protein coding regions including 181 tRNA and 31 rRNA coding genes. The initial pBLAST revealed more than 97% hits among AG groups where as only 1.7% of genes hit with other organisms. The R. solani genome is found to be dominated with tri mer repeats. The genome-wide evolutionary studies revealed the close association of AG3-PT with AG3. The draft sequence represents a helpful resource not only for understanding the core genes involved in pathogenecity but also evolution and adaptive behaviour within the R. solani species complex.ResumenRhizoctonia solani es un hongo basidiomiceto del suelo con un estilo de vida necrotrófico que se clasifica dentro de los 14 grupos de anastomosis (AGs) de incompatibilidad reproductiva. AG3-PT (un subgrupo de papa) esta asociado con pérdidas de rendimiento cuantitativas y cualitativas por vía de cáncer de tallo y por costra negra en papa. Aquí presentamos la primera secuencia en borrador del R. solani [AG3-PT] variante RS-20 con un contenido de G-C de 48.3%. Consiste de 11,431 regiones totales de codificación predicha de proteínas incluyendo 181 tARN y 31 rARN genes de codificación. El pBLAST inicial reveló más del 97% de coincidencias entre los grupos AG, en donde tan solo el 1.7% de los genes coincidieron con otros organismos. El genomio de R. solani se encuentra dominado con repeticiones de trimeros. Los estudios de la evolución de la amplitud del genoma revelaron la asociación cercana de AG3-PT con AG3. La secuencia preliminar representa un recurso útil, no solo para entender los genes básicos involucrados en la patogenicidad, sino también la evolución y el comportamiento adaptativo dentro del complejo de la especie R. solani.

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.