Development of near homozygous lines for diploid hybrid TPS breeding in potatoes.

Diploid inbred-based F1 hybrid True Potato Seed (DHTPS) breeding is a novel technique to transform potato breeding and cultivation across the globe. Significant efforts are being made to identify elite diploids, dihaploids and develop diploid inbred lines for heterosis exploitation in potatoes. Self-incompatibility is the first obstacle for developing inbred lines in diploid potatoes, which necessitates the introgression of a dominant S locus inhibitor gene (Sli) for switching self-incompatibility to self-compatibility. We evaluated a set of 357 diploid clones in different selfing generations for self-compatibility and degree of homozygosity using Kompetitive Allele Specific PCR (KASP) Single Nucleotide Polymorphism (SNP) markers. A subset of 10 KASP markers of the Sli candidate region on chromosome 12 showed an association with the phenotype for self-compatibility. The results revealed that the selected 10 KASP markers for the Sli gene genotype could be deployed for high throughput rapid screening of self-compatibility in diploid populations and to identify new sources of self-compatibility. The homozygosity assessed through 99 KASP markers distributed across all the chromosomes of the potato genome was 20-78 % in founder diploid clones, while different selfing generations, i.e., S0, S1, S2 and S3 observed 36.1-80.4, 56.9-82.8, 59.5-85.4 and 73.7-87.8 % average homozygosity, respectively. The diploid plants with ∼80 % homozygosity were also observed in the first selfing generation, which inferred that homozygosity assessment in the early generations itself could identify the best plants with high homozygosity to speed up the generation of diploid inbred lines.

KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance.

Advances in plant molecular breeding have resulted in the development of new varieties with superior traits, thus improving the crop germplasm. Breeders can screen a large number of accessions without rigorous and time-consuming phenotyping by marker-assisted selection (MAS). Molecular markers are one of the most imperative tools in plant breeding programmes for MAS to develop new cultivars possessing multiple superior traits. Single nucleotide polymorphisms (SNPs) are ideal for MAS due to their low cost, low genotyping error rates, and reproducibility. Kompetitive Allele Specific PCR (KASP) is a globally recognized technology for SNP genotyping. KASP is an allele-specific oligo extension-based PCR assay that uses fluorescence resonance energy transfer (FRET) to detect genetic variations such as SNPs and insertions/deletions (InDels) at a specific locus. Additionally, KASP allows greater flexibility in assay design, which leads to a higher success rate and the capability to genotype a large population. Its versatility and ease of use make it a valuable tool in various fields, including genetics, agriculture, and medical research. KASP has been extensively used in various plant-breeding applications, such as the identification of germplasm resources, quality control (QC) analysis, allele mining, linkage mapping, quantitative trait locus (QTL) mapping, genetic map construction, trait-specific marker development, and MAS. This review provides an overview of the KASP assay and emphasizes its validation in crop improvement related to various biotic and abiotic stress tolerance and quality traits.

Kaolinite nanoclay-shielded dsRNA drenching for management of Globodera pallida: An environmentally friendly pest management approach.

Globodera pallida, an obligate sedentary endoparasite, is a major economic pest that causes substantial potato yield losses. This research aimed to study the effects of gene silencing of three FMRFamide-like peptides (FLPs) genes to reduce G. pallida infestation on potato plants by using kaolinite nanoclay as a carrier to deliver dsRNAs via drenching. A dsRNA dosage of 2.0 mg/ml silenced flp-32c by 89.5%, flp-32p by 94.6%, and flp-2 by 94.3%. J2s incubated for 5 and 10 h showed no phenotypic changes. However, J2s of G. pallida efficiently uptake dsRNA of all targeted genes after 15 h of incubation. On the other hand, J2s that had been kept for 24 h had a rigid and straight appearance. Under fluorescence microscopy, all dsRNA-treated nematodes showed fluorescein isothiocyanate (FITC) signals in the mouth, nervous system, and digestive system. The untreated population of J2s did not show any FITC signals and was mobile as usual. The drenching of potato cultivar Kufri Jyoti with the dsRNA-kaolinite formulations induced deformation and premature death of J2s, compared with untreated J2s that entered J3 or J4 stages. This study validates that the nanocarrier-delivered RNAi system could be employed effectively to manage G. pallida infestations.

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.

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of potato cyst nematode, Globodera pallida from soil.

Potato cyst nematodes, Globodera pallida and G. rostochiensis, are economically important and difficult to manage pests of the potato crop. The cyst of both the species looks similar and it is difficult to differentiate once it turns brown upon maturity. Early detection of the PCN at the species level is crucial to avoid its further spread and for adopting the appropriate management strategies. Therefore, in the present study, highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay was developed to amplify mitochondrial-Sequence Characterized Amplified Region (SCAR) sequence of potato cyst nematode, G. pallida. The LAMP assay was completed within a shorter incubation period of 60 min at 60 °C followed by the reaction termination at 80 °C for 5 min. The developed LAMP assay exhibited high specificity for G. pallida and did not detect any other species including its sibling species, G. rostochiensis. In sensitivity tests, the assay detected G. pallida at 1000 times less DNA concentration (10 fg/µl) as compared to conventional PCR (10 pg/µl). In addition to this, the developed LAMP assay was tested for the detection of G. pallida directly from the soil samples, and even a single cyst mixed with soil was successfully detected by the developed assay. Moreover, the utility of low-cost instruments like hot water bath was also demonstrated for the detection of G. pallida from the soil. The developed LAMP is a rapid, highly specific, sensitive, and cost-effective technique for the species-specific detection of G. pallida. The developed assay will facilitate the rapid detection of G. pallida at quarantine stations as well as from the fields which will help to stop its further spread in new areas and also to devise effective management strategies for sustainable potato production. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03542-x.

Chaetomium globosum KPC3: An Antagonistic Fungus Against the Potato Cyst Nematode, Globodera rostochiensis.

The potato cyst nematode (Globodera rostochiensis) is one of the most economically important pests of potato (Solanum tuberosum L.), causing significant economic losses worldwide. The identification of biocontrol agents for the sustainable management of G. rostochiensis is crucial. In this study, a potential biocontrol agent, Chaetomium globosum KPC3, was identified based on sequence analysis of the DNA internal transcribed spacer (ITS) region, the translation elongation factor 1-alpha (TEF1-α) gene, and the second largest subunit of the RNA polymerase II (RPB2) gene. The pathogenicity test of C. globosum KPC3 against cysts and second-stage juveniles (J2s) revealed that fungus mycelium fully parasitized the cyst after 72 h of incubation. The fungus was also capable of parasitizing the eggs inside the cysts. The culture filtrate of C. globosum KPC3 caused 98.75% mortality in J2s of G. rostochiensis after 72 h of incubation. The pot experiments showed that the combined application of C. globosum KPC3 as a tuber treatment at a rate of 1 lit kg-1 of tubers and a soil application at a rate of 500 ml kg-1 of farm yard manure (FYM) resulted in significantly lesser reproduction of G. rostochiensis compared to the rest of the treatments. Altogether, C. globosum KPC3 has the potential to be used as a biocontrol agent against G. rostochiensis and can be successfully implemented in integrated pest management programs.

Digitalization of potato breeding program: Improving data collection and management.

A plant breeding program involves hundreds of experiments, each having number of entries, genealogy information, linked experimental design, lists of treatments, observed traits, and data analysis. The traditional method of arranging breeding program information and data recording and maintenance is not centralized and is always scattered in different file systems which is inconvenient for retrieving breeding information resulting in poor data management and the loss of crucial data. Data administration requires a significant amount of manpower and resources to maintain nurseries, trials, germplasm lines, and pedigree records. Further, data transcription in scattered spreadsheets and files leads to nomenclature and typing mistakes, which affects data analysis and selection decisions in breeding programs. The accurate data recording and management tools could improve the efficiency of breeding programs. Recent interventions in data management using computer-based breeding databases and informatics applications and tools have made the breeder's life easier. Because of its digital nature, the data obtained is improved even further, allowing for the acquisition of images, voice recording and other specific data kinds. Public breeding programs are far behind the industry in the use of data management tools and softwares. In this article, we have compiled the information on available data recording tools and breeding data management softwares with major emphasis on potato breeding data management.

An overview of fungal chitinases and their potential applications.

Chitin, the world's second most abundant biopolymer after cellulose, is composed of ß-1,4-N-acetylglucosamine (GlcNAc) residues. It is the key structural component of many organisms, including crustaceans, mollusks, marine invertebrates, algae, fungi, insects, and nematodes. There has been a significant increase in the generation of chitinous waste from seafood businesses, resulting in a big amount of scrap. Although several organisms, such as plants, crustaceans, insects, nematodes, and animals, produce chitinases, microorganisms are promising candidates and a sustainable option that mediates chitin degradation. Fungi are the dominant group of chitinase producers among microorganisms. In fungi, chitinases are involved in morphogenesis, cell division, autolysis, chitin acquisition for nutritional purposes, and mycoparasitism. Many efficient chitinolytic fungi with potential applications have been identified in a variety of environments, including soil, water, marine wastes, and plants. The current review highlights the key sources of chitinolytic fungi and the characterization of fungal chitinases. It also discusses the applications of fungal chitinases and the cloning of fungal chitinase genes.

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.

Microbial chitinases and their relevance in various industries.

Chitin, the second most abundant biopolymer on earth after cellulose, is composed of ß-1,4-N-acetylglucosamine (GlcNAc) units. It is widely distributed in nature, especially as a structural polysaccharide in the cell walls of fungi, the exoskeletons of crustaceans, insects, and nematodes. However, the principal commercial source of chitin is the shells of marine or freshwater invertebrates. Microbial chitinases are largely responsible for chitin breakdown in nature, and they play an important role in the ecosystem's carbon and nitrogen balance. Several microbial chitinases have been characterized and are gaining prominence for their applications in various sectors. The current review focuses on chitinases of microbial origin, their diversity, and their characteristics. The applications of chitinases in several industries such as agriculture, food, the environment, and pharmaceutical sectors are also highlighted.