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.

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.

A comprehensive and conceptual overview of omics-based approaches for enhancing the resilience of vegetable crops against abiotic stresses.

MAIN CONCLUSION: Abiotic stresses adversely affect the productivity and production of vegetable crops. The increasing number of crop genomes that have been sequenced or re-sequenced provides a set of computationally anticipated abiotic stress-related responsive genes on which further research may be focused. Knowledge of omics approaches and other advanced molecular tools have all been employed to understand the complex biology of these abiotic stresses. A vegetable can be defined as any component of a plant that is eaten for food. These plant parts may be celery stems, spinach leaves, radish roots, potato tubers, garlic bulbs, immature cauliflower flowers, cucumber fruits, and pea seeds. Abiotic stresses, such as deficient or excessive water, high temperature, cold, salinity, oxidative, heavy metals, and osmotic stress, are responsible for the adverse activity in plants and, ultimately major concern for decreasing yield in many vegetable crops. At the morphological level, altered leaf, shoot and root growth, altered life cycle duration and fewer or smaller organs can be observed. Likewise different physiological and biochemical/molecular processes are also affected in response to these abiotic stresses. In order to adapt and survive in a variety of stressful situations, plants have evolved physiological, biochemical, and molecular response mechanisms. A comprehensive understanding of the vegetable's response to different abiotic stresses and the identification of tolerant genotypes are essential to strengthening each vegetable's breeding program. The advances in genomics and next-generation sequencing have enabled the sequencing of many plant genomes over the last twenty years. A combination of modern genomics (MAS, GWAS, genomic selection, transgenic breeding, and gene editing), transcriptomics, and proteomics along with next-generation sequencing provides an array of new powerful approaches to the study of vegetable crops. This review examines the overall impact of major abiotic stresses on vegetables, adaptive mechanisms and functional genomic, transcriptomic, and proteomic processes used by researchers to minimize these challenges. The current status of genomics technologies for developing adaptable vegetable cultivars that will perform better in future climates is also examined.

Genotypic variations for tuber nutrient content, dry matter and agronomic traits in tetraploid potato germplasm.

Nutrient deficiencies lead to various health issues and are common worldwide. Potato germplasm is a rich source of natural variations and genetic variability present in it can be exploited for developing nutrient-rich high-yielding potato varieties. In this study, variations in the yield, dry matter (DM) and mineral nutrients concentrations were evaluated in both peeled and unpeeled tubers of 243 highly diverse tetraploid potato accessions. These were raised under field conditions for two consecutive years. The germplasm studied has a wider range of variations in peeled tubers DM (13.71-27.80%), Fe (17.08-71.03 mg/kg), Zn (9.55-34.78 mg/kg), Cu (2.13-13.25 mg/kg), Mn (7.04-25.15), Ca (117.4-922.5 mg/kg), Mg (656.6-1510.6 mg/kg), S (1121.3-3765.8 mg/kg), K (1.20-3.09%), P (0.21-0.50%) and Mo (53.6-1164.0 ppb) concentrations compared to popular Indian potato varieties. Higher nutrient concentrations in whole tubers compared to tuber flesh suggest that these are present in high concentration in the tuber peripheral layers and peeling off the tubers results in the loss of nutrients. Highest loss due to peeling off the tubers was observed in Fe (35.63%) followed by Cu (22.80%), Mn (21.69%), Ca (21.27%), Mg (12.89%), K (12.75%), Zn (10.13%), and Mo (9.87%). The GCV and PCV for all the traits in peeled tubers ranged from 9.67 to 29.91%, and 13.84 to 43.32%, respectively. Several significant positive correlations were observed among the parameters and the first two principal components accounted for 39.37% of total variations. The results of this study will pave a way for the development of nutrient-rich high-yielding potato varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01197-1.

Validation of molecular response of tuberization in response to elevated temperature by using a transient Virus Induced Gene Silencing (VIGS) in potato.

Temperature plays an important role in potato tuberization. The ideal night temperature for tuber formation is ~17 °C while temperature beyond 22 °C drastically reduces the tuber yield. Moreover, high temperature has several undesirable effects on the plant and tubers. Investigation of the genes involved in tuberization under heat stress can be helpful in the generation of heat-tolerant potato varieties. Five genes, including StSSH2 (succinic semialdehyde reductase isoform 2), StWTF (WRKY transcription factor), StUGT (UDP-glucosyltransferase), StBHP (Bel1 homeotic protein), and StFLTP (FLOWERING LOCUS T protein), involved in tuberization and heat stress in potato were investigated. The results of our microarray analysis suggested that these genes regulate and function as transcriptional factors, hormonal signaling, cellular homeostasis, and mobile tuberization signals under elevated temperature in contrasting KS (Kufri Surya) and KCM (Kufri Chandramukhi) potato cultivars. However, no detailed report is available which establishes functions of these genes in tuberization under heat stress. Thus, the present study was designed to validate the functions of these genes in tuber signaling and heat tolerance using virus-induced gene silencing (VIGS). Results indicated that VIGS transformed plants had a consequential reduction in StSSH2, StWTF, StUGT, StBHP, and StFLTP transcripts compared to the control plants. Phenotypic observations suggest an increase in plant senescence, reductions to both number and size of tubers, and a decrease in plant dry matter compared to the control plants. We also establish the potency of VIGS as a high-throughput technique for functional validation of genes.

Potato biofortification: an effective way to fight global hidden hunger.

Hidden hunger is leading to extensive health problems in the developing world. Several strategies could be used to reduce the micronutrient deficiencies by increasing the dietary uptake of essential micronutrients. These include diet diversification, pharmaceutical supplementation, food fortification and crop biofortification. Among all, crop biofortification is the most sustainable and acceptable strategy to overcome the global issue of hidden hunger. Since most of the people suffering from micronutrient deficiencies, have monetary issues and are dependent on staple crops to fulfil their recommended daily requirements of various essential micronutrients. Therefore, increasing the micronutrient concentrations in cost effective staple crops seems to be an effective solution. Potato being the world's most consumed non-grain staple crop with enormous industrial demand appears to be an ideal candidate for biofortification. It can be grown in different climatic conditions, provide high yield, nutrition and dry matter in lesser time. In addition, huge potato germplasm have natural variations related to micronutrient concentrations, which can be utilized for its biofortification. This review discuss the current scenario of micronutrient malnutrition and various strategies that could be used to overcome it. The review also shed a light on the genetic variations present in potato germplasm and suggest effective ways to incorporate them into modern high yielding potato varieties.

Phenomics and genomics of finger millet: current status and future prospects.

MAIN CONCLUSION: Diverse gene pool, advanced plant phenomics and genomics methods enhanced genetic gain and understanding of important agronomic, adaptation and nutritional traits in finger millet. Finger millet (Eleusine coracana L. Gaertn) is an important minor millet for food and nutritional security in semi-arid regions of the world. The crop has wide adaptability and can be grown right from high hills in Himalayan region to coastal plains. It provides food grain as well as palatable straw for cattle, and is fairly climate resilient. The crop has large gene pool with distinct features of both Indian and African germplasm types. Interspecific hybridization between Indian and African germplasm has resulted in greater yield enhancement and disease resistance. The crop has shown numerous advantages over major cereals in terms of stress adaptation, nutritional quality and health benefits. It has indispensable repository of novel genes for the benefits of mankind. Although rapid strides have been made in allele mining in model crops and major cereals, the progress in finger millet genomics is lacking. Comparative genomics have paved the way for the marker-assisted selection, where resistance gene homologues of rice for blast and sequence variants for nutritional traits from other cereals have been invariably used. Transcriptomics studies have provided preliminary understanding of the nutritional variation, drought and salinity tolerance. However, the genetics of many important traits in finger millet is poorly understood and need systematic efforts from biologists across disciplines. Recently, deciphered finger millet genome will enable identification of candidate genes for agronomically and nutritionally important traits. Further, improvement in genome assembly and application of genomic selection as well as genome editing in near future will provide plethora of information and opportunity to understand the genetics of complex traits.

Rice bean: a lesser known pulse with well-recognized potential.

MAIN CONCLUSION: Required genetic resources for the improvement of agronomic, nutritional and economic value of rice bean are available in the world collection. International cooperative effort is required to utilize and conserve them. Rice bean [Vigna umbellata (Thunb.) Ohwi and Ohashi], a lesser known pulse among the Asiatic Vigna, has long been considered as a food security crop of small and marginal farmers of Southeast Asia. Considered as a nutritionally rich food and fodder, it is also a source of genes for biotic and abiotic stress tolerance including drought, soil acidity and storage pest. Although it spread from its centre of domestication in the Indo-China region to other parts around the world, it never became an important crop anywhere probably because of agronomic disadvantages. Crop improvement for determinate nature, good yield, less variable seed colour, pleasant organoleptic properties and lower antinutrients is required. Scanning of scientific literature indicates that genetic resources with desirable agronomic and nutritional traits exist within the current collection but are spread across countries. Genomic studies in the species indicate that except for insect resistance and aluminium toxicity tolerance, not much attention has been paid to decipher and utilize other stress tolerance and nutritional quality traits. Collaborative efforts towards improving farming, food, trade value and off-farm conservation of rice bean would not only help marginal farmers but will also help to preserve the yet to be explored genomic resources available in this sturdy pulse.

Revisiting the versatile buckwheat: reinvigorating genetic gains through integrated breeding and genomics approach.

MAIN CONCLUSION: Emerging insights in buckwheat molecular genetics allow the integration of genomics driven breeding to revive this ancient crop of immense nutraceutical potential from Asia. Out of several thousand known edible plant species, only four crops-rice, wheat, maize and potato provide the largest proportion of daily nutrition to billions of people. While these crops are the primary supplier of carbohydrates, they lack essential amino acids and minerals for a balanced nutrition. The overdependence on only few crops makes the future cropping systems vulnerable to the predicted climate change. Diversifying food resources through incorporation of orphan or minor crops in modern cropping systems is one potential strategy to improve the nutritional security and mitigate the hostile weather patterns. One such crop is buckwheat, which can contribute to the agricultural sustainability as it grows in a wide range of environments, requires relatively low inputs and possess balanced amino acid and micronutrient profiles. Additionally, gluten-free nature of protein and nutraceutical properties of secondary metabolites make the crop a healthy alternative of wheat-based diet in developed countries. Despite enormous potential, efforts for the genetic improvement of buckwheat are considerably lagged behind the conventional cereal crops. With the draft genome sequences in hand, there is a great scope to speed up the progress of genetic improvement of buckwheat. This article outlines the state of the art in buckwheat research and provides concrete perspectives how modern breeding approaches can be implemented to accelerate the genetic gain. Our suggestions are transferable to many minor and underutilized crops to address the issue of limited genetic gain and low productivity.

From zero to hero: the past, present and future of grain amaranth breeding.

KEY MESSAGE: Grain amaranth is an underutilized crop with high nutritional quality from the Americas. Emerging genomic and biotechnological tools are becoming available that allow the integration of novel breeding techniques for rapid improvement of amaranth and other underutilized crops. Out of thousands of edible plants, only three cereals-maize, wheat and rice-are the major food sources for a majority of people worldwide. While these crops provide high amounts of calories, they are low in protein and other essential nutrients. The dependence on only few crops, with often narrow genetic basis, leads to a high vulnerability of modern cropping systems to the predicted climate change and accompanying weather extremes. Broadening our food sources through the integration of so-called orphan crops can help to mitigate the effects of environmental change and improve qualitative food security. Thousands of traditional crops are known, but have received little attention in the last century and breeding efforts were limited. Amaranth is such an underutilized pseudocereal that is of particular interest because of its balanced amino acid and micronutrient profiles. Additionally, the C4 photosynthetic pathway and ability to withstand environmental stress make the crop a suitable choice for future agricultural systems. Despite the potential of amaranth, efforts of genetic improvement lag considerably behind those of major crops. The progress in novel breeding methods and molecular techniques developed in model plants and major crops allow a rapid improvement of underutilized crops. Here, we review the history of amaranth and recent advances in genomic tools and give a concrete perspective how novel breeding techniques can be implemented into breeding programs. Our perspectives are transferable to many underutilized crops. The implementation of these could improve the nutritional quality and climate resilience of future cropping systems.

In-silico mining, type and frequency analysis of genic microsatellites of finger millet (Eleusine coracana (L.) Gaertn.): a comparative genomic analysis of NBS-LRR regions of finger millet with rice.

In recent years, the increased availability of the DNA sequences has given the possibility to develop and explore the expressed sequence tags (ESTs) derived SSR markers. In the present study, a total of 1956 ESTs of finger millet were used to find the microsatellite type, distribution, frequency and developed a total of 545 primer pairs from the ESTs of finger millet. Thirty-two EST sequences had more than two microsatellites and 1357 sequences did not have any SSR repeats. The most frequent type of repeats was trimeric motif, however the second place was occupied by dimeric motif followed by tetra-, hexa- and penta repeat motifs. The most common dimer repeat motif was GA and in case of trimeric SSRs, it was CGG. The EST sequences of NBS-LRR region of finger millet and rice showed higher synteny and were found on nearly same positions on the rice chromosome map. A total of eight, out of 15 EST based SSR primers were polymorphic among the selected resistant and susceptible finger millet genotypes. The primer FMBLEST5 could able to differentiate them into resistant and susceptible genotypes. The alleles specific to the resistant and susceptible genotypes were sequenced using the ABI 3130XL genetic analyzer and found similarity to NBS-LRR regions of rice and finger millet and contained the characteristic kinase-2 and kinase 3a motifs of plant R-genes belonged to NBS-LRR region. The In-silico and comparative analysis showed that the genes responsible for blast resistance can be identified, mapped and further introgressed through molecular breeding approaches for enhancing the blast resistance in finger millet.

Molecular insights on the origin and development of waxy genotypes in major crop plants.

Starch is a significant ingredient of the seed endosperm with commercial importance in food and industry. Crop varieties with glutinous (waxy) grain characteristics, i.e. starch with high amylopectin and low amylose, hold longstanding cultural importance in some world regions and unique properties for industrial manufacture. The waxy character in many crop species is regulated by a single gene known as GBSSI (or waxy), which encodes the enzyme Granule Bound Starch Synthase1 with null or reduced activity. Several allelic variants of the waxy gene that contribute to varying levels of amylose content have been reported in different crop plants. Phylogenetic analysis of protein sequences and the genomic DNA encoding GBSSI of major cereals and recently sequenced millets and pseudo-cereals have shown that GBSSI orthologs form distinct clusters, each representing a separate crop lineage. With the rapidly increasing demand for waxy starch in food and non-food applications, conventional crop breeding techniques and modern crop improvement technologies such as gene silencing and genome editing have been deployed to develop new waxy crop cultivars. The advances in research on waxy alleles across different crops have unveiled new possibilities for modifying the synthesis of amylose and amylopectin starch, leading to the potential creation of customized crops in the future. This article presents molecular lines of evidence on the emergence of waxy genes in various crops, including their genesis and evolution, molecular structure, comparative analysis and breeding innovations.

Uncovering the genomic regions underlying grain iron and zinc content using genome-wide association mapping in finger millet.

Finger millet, being rich source of essential minerals like iron and zinc, is an ideal model to identify candidate genes contributing to high grain iron content (GIC) and zinc content (GZC) in plants. Hence, finger millet diversity panel comprised of 202 genotypes was evaluated in two geographical locations and found to have a wide variation for GIC and GZC. A genome-wide association study using 2977 single nucleotide polymorphism (SNP) markers identified reliable marker-trait associations (MTAs). The use of general linear model (GLM) and mixed linear model (MLM) approaches revealed 5 and 8 common MTAs linked to GIC and GZC, respectively, for both Almora and Pantnagar locations, with a high level of significance (P < 0.01). However, 12 significant MTAs were found to be linked with GIC for Pantnagar location alone. The MTAs were associated with specific genes that produce ferritin (Fer1), iron-regulated transporter-like protein (IRT2), and yellow stripe-like 2 proteins (YSL2). These genes are likely linked to GIC variation in finger millet. Additionally, the variation in GZC in finger millet was connected to genes that encode zinc transporters, namely ZIP1 protein (ZIP1) and ZTP29-like protein (ZTP29). Compared to low GIC and GZC genotypes, high GIC and GZC genotypes exhibited greater relative expression of these genes. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03889-1.

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.

Comparison of dehulling efficiency and grain nutritional parameters of two cultivated barnyard millet species (Echinochloa spp.).

Due to increased awareness regarding the health-promoting profile of millets, inclination towards their consumption has increased considerably. In the Himalayan region of India, cultivars of the two species of barnyard millet, namely Indian (Echinochloa frumentacea) and Japanese barnyard millet (E. esculenta), are grown. To compare the dehulled grain recovery, grain physical parameters, nutritional profile and antioxidant activity, an experiment was carried out at ICAR-VPKAS, Almora, Uttarakhand hills for two years using released and popular cultivars of Indian barnyard millet (VL 207 and VL 172) and Japanese barnyard millet (PRJ-1). The results indicated that the whole grain yield of Japanese barnyard millet cultivar PRJ-1 was significantly higher than Indian Barnyard millet cultivars VL 172 and VL 207; however, the dehulled grain recovery was considerably higher in VL 172 and VL 207 than PRJ-1. Similarly, the physical grain parameters were significantly higher in PRJ-1, but most dehulled grain parameters were at par in cultivars of both species. The nutritional estimation of dehulled grains of both species did not show remarkable differences for most traits. Still, crude fibre, Mn, and Zn were high in PRJ-1, while total digestible nutrients and phosphorous were high in VL 172 and VL 207. Dehulled grains exhibited much more crude protein, ash, minerals, and total digestible nutrients, but the husk accumulated significantly higher crude fibre and total polyphenols.

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.

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.

Deciphering the genomic regions governing major agronomic traits and blast resistance using genome wide association mapping in finger millet.

Finger millet (Eleusine coracana L.) is climate resilient minor millet of Asia and Africa with wide adaptation and unparallel nutritional profile. To date, genomic resources available in finger millet are scanty and genetic control of agronomic traits remains elusive. Here, a collection of eco-geographically diverse 186 genotypes was quantified for variation in 13 agronomic traits and reaction to blast to identify marker-trait associations (MTAs) using genotyping-by-sequencing (GBS) and genome-wide association study (GWAS). GBS generated 2977 high quality single nucleotide polymorphism (SNPs) markers and identified three subpopulations with varying admixture levels. General linear and mixed model approaches of GWAS to correct for population structure and genetic relatedness identified 132 common MTAs for agronomic traits across the years. The phenotypic variance explained by the makers varied from 4.8% (TP692389-flag leaf width) to 20% (TP714446-green fodder weight). Of these, 26 MTAs showed homology with candidate genes having role in plant growth, development and photosynthesis in the genomes of foxtail millet, rice, maize, wheat and barley. We also found 4 common MTAs for neck blast resistance, which explained 5.9-15.1% phenotypic variance. Three MTAs for neck blast resistance showed orthologues in related genera having putative functions in pathogen defense in plants. The results of this work lay a foundation for understanding the genetic architecture of agronomic traits and blast resistance in finger millet and provide a framework for genomics assisted breeding.

Genome-wide association mapping and genomic prediction for late blight and potato cyst nematode resistance in potato (Solanum tuberosum L.).

Potatoes are an important source of food for millions of people worldwide. Biotic stresses, notably late blight and potato cyst nematodes (PCN) pose a major threat to potato production worldwide, and knowledge of genes controlling these traits is limited. A genome-wide association mapping study was conducted to identify the genomic regulators controlling these biotic stresses, and the genomic prediction accuracy was worked out using the GBLUP model of genomic selection (GS) in a panel of 222 diverse potato accessions. The phenotype data on resistance to late blight and two PCN species (Globodera pallida and G. rostochiensis) were recorded for three and two consecutive years, respectively. The potato panel was genotyped using genotyping by sequencing (GBS), and 1,20,622 SNP markers were identified. A total of 7 SNP associations for late blight resistance, 9 and 11 for G. pallida and G. rostochiensis, respectively, were detected by additive and simplex dominance models of GWAS. The associated SNPs were distributed across the chromosomes, but most of the associations were found on chromosomes 5, 10 and 11, which have been earlier reported as the hotspots of disease-resistance genes. The GS prediction accuracy estimates were low to moderate for resistance to G. pallida (0.04-0.14) and G. rostochiensis (0.14-0.21), while late blight resistance showed a high prediction accuracy of 0.42-0.51. This study provides information on the complex genetic nature of these biotic stress traits in potatoes and putative SNP markers for resistance breeding.

CRISPR/Cas9-mediated editing of phytoene desaturase (PDS) gene in an important staple crop, potato.

The gene editing using the CRISPR/Cas9 system has become an important biotechnological tool for studying gene function and improving crops. In this study, we have used CRISPR/Cas9 system for editing the phytoene desaturase gene (PDS) in popular Indian potato cultivar Kufri Chipsona-I. A construct (pHSE401) carrying two target gRNAs with glycine tRNA processing system under the control of Arabidopsis U6 promoter and the Cas9 protein was constructed and transformed in potato plants using Agrobacterium-mediated genetic transformations. The regeneration efficiency of 45% was observed in regenerated plants, out of which 81% of the putative transformants shoot lines exhibited mutant or bleached phenotype (albinism). The deletion mutations were detected within the StPDS gene in the genotyped plants and a mutation efficiency of 72% for gRNA1 and gRNA2 has been detected using Sanger sequencing. Hence, we set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and generates mutations (deletions) within StPDS gene in potato. The bleached phenotype is easily detectable after only few weeks after Agrobacterium-mediated transformation. This is the first report as a proof of concept for CRISPR/Cas9-based editing of PDS gene in Indian potato cv. Kufri Chipsona-I. This study demonstrates that CRISPR/Cas9 can be used to edit genes at high frequency within the genome of the potato for various traits. Therefore, this study will aid in creating important mutants for modifying molecular mechanisms controlling traits of agronomic importance.