Chloroplast transformation in new cultivars of tomato through particle bombardment.

A protocol has been established for genetic transformation of the chloroplasts in two new cultivars of tomato (Solanum lycopersicum L.) grown in India and Australia: Pusa Ruby and Yellow Currant. Tomato cv. Green Pineapple was also used as a control that has previously been used for establishing chloroplast transformation by other researchers. Selected tomato cultivars were finalized from ten other tested cultivars (Green Pineapple excluded) due to their high regeneration potential and better response to chloroplast transformation. This protocol was set up using a chloroplast transformation vector (pRB94) for tomatoes that is made up of a synthetic gene operon. The vector has a chimeric aadA selectable marker gene that is controlled by the rRNA operon promoter (Prrn). This makes the plant or chloroplasts resistant to spectinomycin and streptomycin. After plasmid-coated particle bombardment, leaf explants were cultured in 50 mg/L selection media. Positive explant selection from among all the dead-appearing (yellow to brown) explants was found to be the major hurdle in the study. Even though this study was able to find plastid transformants in heteroplasmic conditions, it also found important parameters and changes that could speed up the process of chloroplast transformation in tomatoes, resulting in homoplasmic plastid-transformed plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03954-3.

Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops.

Addressing nutritional deficiencies in food crops through biofortification is a sustainable approach to tackling malnutrition. Biofortification is continuously being attempted through conventional breeding as well as through various plant biotechnological interventions, ranging from molecular breeding to genetic engineering and genome editing for enriching crops with various health-promoting metabolites. Genetic engineering is used for the rational incorporation of desired nutritional traits in food crops and predominantly operates through nuclear and chloroplast genome engineering. In the recent past, chloroplast engineering has been deployed as a strategic tool to develop model plants with enhanced nutritional traits due to the various advantages it offers over nuclear genome engineering. However, this approach needs to be extended for the nutritional enhancement of major food crops. Further, this platform could be combined with strategies, such as synthetic biology, chloroplast editing, nanoparticle-mediated rapid chloroplast transformation, and horizontal gene transfer through grafting for targeting endogenous metabolic pathways for overproducing native nutraceuticals, production of biopharmaceuticals, and biosynthesis of designer nutritional compounds. This review focuses on exploring various features of chloroplast genome engineering for nutritional enhancement of food crops by enhancing the levels of existing metabolites, restoring the metabolites lost during crop domestication, and introducing novel metabolites and phytonutrients needed for a healthy daily diet.

Deciphering the role of miRNA in reprogramming plant responses to drought stress.

Drought is the most prevalent environmental stress that affects plants' growth, development, and crop productivity. However, plants have evolved adaptive mechanisms to respond to the harmful effects of drought. They reprogram their: transcriptome, proteome, and metabolome that alter their cellular and physiological processes and establish cellular homeostasis. One of the crucial regulatory processes that govern this reprogramming is post-transcriptional regulation by microRNAs (miRNAs). miRNAs are small non-coding RNAs, involved in the downregulation of the target mRNA via translation inhibition/mRNA degradation/miRNA-mediated mRNA decay/ribosome drop off/DNA methylation. Many drought-inducible miRNAs have been identified and characterized in plants. Their main targets are regulatory genes that influence growth, development, osmotic stress tolerance, antioxidant defense, phytohormone-mediated signaling, and delayed senescence during drought stress. Overexpression of drought-responsive miRNAs (Osa-miR535, miR160, miR408, Osa-miR393, Osa-miR319, and Gma-miR394) in certain plants has led to tolerance against drought stress indicating their vital role in stress mitigation. Similarly, knock down (miR166/miR398c) or deletion (miR169 and miR827) of miRNAs has also resulted in tolerance to drought stress. Likewise, engineered Arabidopsis plants with miR165, miR166 using short tandem target mimic strategy, exhibited drought tolerance. Since miRNAs regulate the expression of an array of drought-responsive genes, they can act as prospective targets for genetic manipulations to enhance drought tolerance in crops and achieve sustainable agriculture. Further investigations toward functional characterization of diverse miRNAs, and understanding stress-responses regulated by these miRNAs and their utilization in biotechnological applications is highly recommended.

Wheat Biofortification: Utilizing Natural Genetic Diversity, Genome-Wide Association Mapping, Genomic Selection, and Genome Editing Technologies.

Alleviating micronutrients associated problems in children below five years and women of childbearing age, remains a significant challenge, especially in resource-poor nations. One of the most important staple food crops, wheat attracts the highest global research priority for micronutrient (Fe, Zn, Se, and Ca) biofortification. Wild relatives and cultivated species of wheat possess significant natural genetic variability for these micronutrients, which has successfully been utilized for breeding micronutrient dense wheat varieties. This has enabled the release of 40 biofortified wheat cultivars for commercial cultivation in different countries, including India, Bangladesh, Pakistan, Bolivia, Mexico and Nepal. In this review, we have systematically analyzed the current understanding of availability and utilization of natural genetic variations for grain micronutrients among cultivated and wild relatives, QTLs/genes and different genomic regions regulating the accumulation of micronutrients, and the status of micronutrient biofortified wheat varieties released for commercial cultivation across the globe. In addition, we have also discussed the potential implications of emerging technologies such as genome editing to improve the micronutrient content and their bioavailability in wheat.

The 3366 chickpea genomes for research and breeding.

Genome sequences provide an unprecedented resource to rapidly develop modern crops. A recent paper by Varshney et al. provides genome variation maps of 3366 chickpea accessions. Here, we highlight how this breakthrough research can fundamentally change breeding practices of chickpea and potentially other crops.

Precise plant genome editing using base editors and prime editors.

The development of CRISPR-Cas systems has sparked a genome editing revolution in plant genetics and breeding. These sequence-specific RNA-guided nucleases can induce DNA double-stranded breaks, resulting in mutations by imprecise non-homologous end joining (NHEJ) repair or precise DNA sequence replacement by homology-directed repair (HDR). However, HDR is highly inefficient in many plant species, which has greatly limited precise genome editing in plants. To fill the vital gap in precision editing, base editing and prime editing technologies have recently been developed and demonstrated in numerous plant species. These technologies, which are mainly based on Cas9 nickases, can introduce precise changes into the target genome at a single-base resolution. This Review provides a timely overview of the current status of base editors and prime editors in plants, covering both technological developments and biological applications.

A Short History and Perspectives on Plant Genetic Transformation.

Plant genetic transformation is an important technological advancement in modern science, which has not only facilitated gaining fundamental insights into plant biology but also started a new era in crop improvement and commercial farming. However, for many crop plants, efficient transformation and regeneration still remain a challenge even after more than 30 years of technical developments in this field. Recently, FokI endonuclease-based genome editing applications in plants offered an exciting avenue for augmenting crop productivity but it is mainly dependent on efficient genetic transformation and regeneration, which is a major roadblock for implementing genome editing technology in plants. In this chapter, we have outlined the major historical developments in plant genetic transformation for developing biotech crops. Overall, this field needs innovations in plant tissue culture methods for simplification of operational steps for enhancing the transformation efficiency. Similarly, discovering genes controlling developmental reprogramming and homologous recombination need considerable attention, followed by understanding their role in enhancing genetic transformation efficiency in plants. Further, there is an urgent need for exploring new and low-cost universal delivery systems for DNA/RNA and protein into plants. The advancements in synthetic biology, novel vector systems for precision genome editing and gene integration could potentially bring revolution in crop-genetic potential enhancement for a sustainable future. Therefore, efficient plant transformation system standardization across species holds the key for translating advances in plant molecular biology to crop improvement.

Heterologous expression of rice RNA-binding glycine-rich (RBG) gene OsRBGD3 in transgenic Arabidopsis thaliana confers cold stress tolerance.

Imparting cold stress tolerance to crops is a major challenge in subtropical agriculture. New genes conferring cold tolerance needs to be identified and characterised for sustainable crop production in low-temperature stress affected areas. Here we report functional characterisation of OsRBGD3, classified previously as a class D glycine-rich RNA recognition motif (RRM) containing proteins from a drought-tolerant Indica rice cultivar N22. The gene was isolated by screening yeast one-hybrid library using the minimal promoter region of the OsMYB38 that is necessary for cold stress-responsive expression. OsRBGD3 exhibited cold, drought and salt stress inductive expression in a drought tolerant N22 rice cultivar as compared with susceptible variety IR64. OsRBGD3 was found to be localised to both nuclear and cytoplasmic subcellular destinations. Constitutive overexpression of the OsRBGD3 in transgenic Arabidopsis conferred tolerance to cold stress. ABA sensitivity was also observed in transgenic lines suggesting the regulatory role of this gene in the ABA signalling pathway. OsRBGD3 overexpression also attributed to significant root development and early flowering in transgenics. Hence, OsRBGD3 could be an important target for developing cold tolerant early flowering rice and other crops' genotypes for increasing production in low temperature affected areas.

Genome-Wide Identification and Analysis of Biotic and Abiotic Stress Regulation of C4 Photosynthetic Pathway Genes in Rice.

Photosynthetic fixation of CO2 is more efficient in C4 than in C3 plants. Rice is a C3 plant and a potential target for genetic engineering of the C4 pathway. It is known that genes encoding C4 enzymes are present in C3 plants. However, no systematic analysis has been conducted to determine if these C4 gene family members are expressed in diverse rice genotypes. In this study, we identified 15 genes belonging to the five C4 gene families in rice genome through BLAST search using known maize C4 photosynthetic pathway genes. Phylogenetic relationship of rice C4 photosynthetic pathway genes and their isoforms with other grass genomes (Brachypodium, maize, Sorghum and Setaria), showed that these genes were highly conserved across grass genomes. Spatiotemporal, hormone, and abiotic stress specific expression pattern of the identified genes revealed constitutive as well as inductive responses of the C4 photosynthetic pathway in different tissues and developmental stages of rice. Expression levels of C4 specific gene family members in flag leaf during tillering stage were quantitatively analyzed in five rice genotypes covering three species, viz. Oryza sativa, ssp. japonica (cv. Nipponbare), Oryza sativa, ssp. indica (cv IR64, Swarna), and two wild species Oryza barthii and Oryza australiensis. The results showed that all the identified genes expressed in rice and exhibited differential expression pattern during different growth stages, and in response to biotic and abiotic stress conditions and hormone treatments. Our study concludes that C4 photosynthetic pathway genes present in rice play a crucial role in stress regulation and might act as targets for C4 pathway engineering via CRISPR-mediated breeding.

Ectopic Expression of Rice PYL3 Enhances Cold and Drought Tolerance in Arabidopsis thaliana.

Abscisic acid (ABA) plays an important role in plant development and adaptation to abiotic stresses. The pyrabactin resistance-like (PYL) gene family has been characterized as intracellular ABA receptors in Arabidopsis. We describe here the functional characterization of PYL3 ABA receptor from a drought-tolerant rice landrace Nagina 22 (N22). The induced expression level of the PYL3 transcript was observed in the N22 under different stress treatments, including cold, drought, high temperature, salt and ABA. In contrast, the expression of PYL3 was down-regulated in drought-susceptible rice cv. IR64 in response to above stresses. C-terminal GFP translational fusion of OsPYL3 was localized to both cytosol and nucleus explaining in part functional conservation of PYL protein as ABA receptor. Arabidopsis transgenic lines overexpressing OsPYL3 were hypersensitive to ABA suggesting ABA signaling pathway-dependent molecular response of the OsPYL3. Further, constitutive overexpression of OsPYL3 in Arabidopsis led to improved cold and drought stress tolerance. Thus, OsPYL3 identified in this study could be a good candidate for genetic improvement of cold and drought stress tolerance of rice and other crop plants.

Genome-wide identification and analysis of biotic and abiotic stress regulation of small heat shock protein (HSP20) family genes in bread wheat.

Small Heat Shock Proteins (sHSPs)/HSP20 are molecular chaperones that protect plants by preventing protein aggregation during abiotic stress conditions, especially heat stress. Due to global climate change, high temperature is emerging as a major threat to wheat productivity. Thus, the identification of HSP20 and analysis of HSP transcriptional regulation under different abiotic stresses in wheat would help in understanding the role of these proteins in abiotic stress tolerance. We used sequences of known rice and Arabidopsis HSP20 HMM profiles as queries against publicly available wheat genome and wheat full length cDNA databases (TriFLDB) to identify the respective orthologues from wheat. 163 TaHSP20 (including 109 sHSP and 54 ACD) genes were identified and classified according to the sub-cellular localization and phylogenetic relationship with sequenced grass genomes (Oryza sativa, Sorghum bicolor, Zea mays, Brachypodium distachyon and Setaria italica). Spatio-temporal, biotic and abiotic stress-specific expression patterns in normalized RNA seq and wheat array datasets revealed constitutive as well as inductive responses of HSP20 in different tissues and developmental stages of wheat. Promoter analysis of TaHSP20 genes showed the presence of tissue-specific, biotic, abiotic, light-responsive, circadian and cell cycle-responsive cis-regulatory elements. 14 TaHSP20 family genes were under the regulation of 8 TamiRNA genes. The expression levels of twelve HSP20 genes were studied under abiotic stress conditions in the drought- and heat-tolerant wheat genotype C306. Of the 13 TaHSP20 genes, TaHSP16.9H-CI showed high constitutive expression with upregulation only under salt stress. Both heat and salt stresses upregulated the expression of TaHSP17.4-CI, TaHSP17.7A-CI, TaHSP19.1-CIII, TaACD20.0B-CII and TaACD20.6C-CIV, while TaHSP23.7-MTI was specifically induced only under heat stress. Our results showed that the identified TaHSP20 genes play an important role under different abiotic stress conditions. Thus, the results illustrate the complexity of the TaHSP20 gene family and its stress regulation in wheat, and suggest that sHSPs as attractive breeding targets for improvement of the heat tolerance of wheat.

Differential Regulation of Genes Coding for Organelle and Cytosolic ClpATPases under Biotic and Abiotic Stresses in Wheat.

A sub-group of class I Caseinolytic proteases (Clps) function as molecular chaperone and confer thermotolerance to plants. We identified class I Clp family consisting of five ClpB/HSP100, two ClpC, and two ClpD genes from bread wheat. Phylogenetic analysis showed that these genes were highly conserved across grass genomes. Subcellular localization prediction revealed that TaClpC and TaClpD subgroup proteins and TaClpB1 proteins are potentially targeted to chloroplast, while TaClpB5 to mitochondria, and TaClpB2, TaClpB3, and TaClpB4 to cytoplasm. Spatio-temporal expression pattern analysis revealed that four TaClpB and TaClpD2 genes are expressed in majority of all tissues and developmental stages of wheat. Real-time RT-PCR analysis of expression levels of Clp genes in seven wheat genotypes under different abiotic stresses revealed that genes coding for the cytosolic Clps namely TaClpB2 and TaClpB3 were upregulated under heat, salt and oxidative stress but were downregulated by cold stress in most genotypes. In contrast, genes coding for the chloroplastic Clps TaClpC1, TaClpC2, and TaClpD1 genes were significantly upregulated by mainly by cold stress in most genotypes, while TaClpD2 gene was upregulated >2 fold by salt stress in DBW16. The TaClpB5 gene coding for mitochondrial Clp was upregulated in all genotypes under heat, salt and oxidative stresses. In addition, we found that biotic stresses also upregulated TaClpB4 and TaClpD1. Among biotic stresses, Tilletia caries induced TaClpB2, TaClpB3, TaClpC1, and TaClpD1. Differential expression pattern under different abiotic and biotic stresses and predicted differential cellular localization of Clps suggest their non-redundant organelle and stress-specific roles. Our results also suggest the potential role of Clps in cold, salt and biotic stress responses in addition to the previously established role in thermotolerance of wheat.

mQTL-seq delineates functionally relevant candidate gene harbouring a major QTL regulating pod number in chickpea.

The present study used a whole-genome, NGS resequencing-based mQTL-seq (multiple QTL-seq) strategy in two inter-specific mapping populations (Pusa 1103 × ILWC 46 and Pusa 256 × ILWC 46) to scan the major genomic region(s) underlying QTL(s) governing pod number trait in chickpea. Essentially, the whole-genome resequencing of low and high pod number-containing parental accessions and homozygous individuals (constituting bulks) from each of these two mapping populations discovered >8 million high-quality homozygous SNPs with respect to the reference kabuli chickpea. The functional significance of the physically mapped SNPs was apparent from the identified 2,264 non-synonymous and 23,550 regulatory SNPs, with 8-10% of these SNPs-carrying genes corresponding to transcription factors and disease resistance-related proteins. The utilization of these mined SNPs in Δ (SNP index)-led QTL-seq analysis and their correlation between two mapping populations based on mQTL-seq, narrowed down two (Caq(a)PN4.1: 867.8 kb and Caq(a)PN4.2: 1.8 Mb) major genomic regions harbouring robust pod number QTLs into the high-resolution short QTL intervals (Caq(b)PN4.1: 637.5 kb and Caq(b)PN4.2: 1.28 Mb) on chickpea chromosome 4. The integration of mQTL-seq-derived one novel robust QTL with QTL region-specific association analysis delineated the regulatory (C/T) and coding (C/A) SNPs-containing one pentatricopeptide repeat (PPR) gene at a major QTL region regulating pod number in chickpea. This target gene exhibited anther, mature pollen and pod-specific expression, including pronounced higher up-regulated (∼3.5-folds) transcript expression in high pod number-containing parental accessions and homozygous individuals of two mapping populations especially during pollen and pod development. The proposed mQTL-seq-driven combinatorial strategy has profound efficacy in rapid genome-wide scanning of potential candidate gene(s) underlying trait-associated high-resolution robust QTL(s), thereby expediting genomics-assisted breeding and genetic enhancement of crop plants, including chickpea.

Identification of novel drought-responsive microRNAs and trans-acting siRNAs from Sorghum bicolor (L.) Moench by high-throughput sequencing analysis.

Small non-coding RNAs (sRNAs) namely microRNAs (miRNAs) and trans-acting small interfering RNAs (tasi-RNAs) play a crucial role in post-transcriptional regulation of gene expression and thus the control plant development and stress responses. In order to identify drought-responsive miRNAs and tasi-RNAs in sorghum, we constructed small RNA libraries from a drought tolerant (M35-1) and susceptible (C43) sorghum genotypes grown under control and drought stress conditions, and sequenced by Illumina Genome Analyzer IIx. Ninety seven conserved and 526 novel miRNAs representing 472 unique miRNA families were identified from sorghum. Ninety-six unique miRNAs were found to be regulated by drought stress, of which 32 were up- and 49 were down-regulated (fold change ≥ 2 or ≤ -2) at least in one genotype, while the remaining 15 miRNAs showed contrasting drought-regulated expression pattern between genotypes. A maximum of 17 and 18 miRNAs was differentially regulated under drought stress condition in the sensitive and tolerant genotypes, respectively. These results suggest that genotype dependent stress responsive regulation of miRNAs may contribute, at least in part, to the differential drought tolerance of sorghum genotypes. We also identified two miR390-directed TAS3 gene homologs and the auxin response factors as tasi-RNA targets. We predicted more than 1300 unique target genes for the novel and conserved miRNAs. These target genes were predicted to be involved in different cellular, metabolic, response to stimulus, biological regulation, and developmental processes. Genome-wide identification of stress-responsive miRNAs, tasi-RNAs and their targets identified in this study will be useful in unraveling the molecular mechanisms underlying drought stress responses and genetic improvement of biomass production and stress tolerance in sorghum.

Genome-wide high-throughput SNP discovery and genotyping for understanding natural (functional) allelic diversity and domestication patterns in wild chickpea.

We identified 82489 high-quality genome-wide SNPs from 93 wild and cultivated Cicer accessions through integrated reference genome- and de novo-based GBS assays. High intra- and inter-specific polymorphic potential (66-85%) and broader natural allelic diversity (6-64%) detected by genome-wide SNPs among accessions signify their efficacy for monitoring introgression and transferring target trait-regulating genomic (gene) regions/allelic variants from wild to cultivated Cicer gene pools for genetic improvement. The population-specific assignment of wild Cicer accessions pertaining to the primary gene pool are more influenced by geographical origin/phenotypic characteristics than species/gene-pools of origination. The functional significance of allelic variants (non-synonymous and regulatory SNPs) scanned from transcription factors and stress-responsive genes in differentiating wild accessions (with potential known sources of yield-contributing and stress tolerance traits) from cultivated desi and kabuli accessions, fine-mapping/map-based cloning of QTLs and determination of LD patterns across wild and cultivated gene-pools are suitably elucidated. The correlation between phenotypic (agromorphological traits) and molecular diversity-based admixed domestication patterns within six structured populations of wild and cultivated accessions via genome-wide SNPs was apparent. This suggests utility of whole genome SNPs as a potential resource for identifying naturally selected trait-regulating genomic targets/functional allelic variants adaptive to diverse agroclimatic regions for genetic enhancement of cultivated gene-pools.

Transcriptional Regulatory Network Analysis of MYB Transcription Factor Family Genes in Rice.

MYB transcription factor (TF) is one of the largest TF families and regulates defense responses to various stresses, hormone signaling as well as many metabolic and developmental processes in plants. Understanding these regulatory hierarchies of gene expression networks in response to developmental and environmental cues is a major challenge due to the complex interactions between the genetic elements. Correlation analyses are useful to unravel co-regulated gene pairs governing biological process as well as identification of new candidate hub genes in response to these complex processes. High throughput expression profiling data are highly useful for construction of co-expression networks. In the present study, we utilized transcriptome data for comprehensive regulatory network studies of MYB TFs by "top-down" and "guide-gene" approaches. More than 50% of OsMYBs were strongly correlated under 50 experimental conditions with 51 hub genes via "top-down" approach. Further, clusters were identified using Markov Clustering (MCL). To maximize the clustering performance, parameter evaluation of the MCL inflation score (I) was performed in terms of enriched GO categories by measuring F-score. Comparison of co-expressed cluster and clads analyzed from phylogenetic analysis signifies their evolutionarily conserved co-regulatory role. We utilized compendium of known interaction and biological role with Gene Ontology enrichment analysis to hypothesize function of coexpressed OsMYBs. In the other part, the transcriptional regulatory network analysis by "guide-gene" approach revealed 40 putative targets of 26 OsMYB TF hubs with high correlation value utilizing 815 microarray data. The putative targets with MYB-binding cis-elements enrichment in their promoter region, functional co-occurrence as well as nuclear localization supports our finding. Specially, enrichment of MYB binding regions involved in drought-inducibility implying their regulatory role in drought response in rice. Thus, the co-regulatory network analysis facilitated the identification of complex OsMYB regulatory networks, and candidate target regulon genes of selected guide MYB genes. The results contribute to the candidate gene screening, and experimentally testable hypotheses for potential regulatory MYB TFs, and their targets under stress conditions.

An integrated genomic approach for rapid delineation of candidate genes regulating agro-morphological traits in chickpea.

The identification and fine mapping of robust quantitative trait loci (QTLs)/genes governing important agro-morphological traits in chickpea still lacks systematic efforts at a genome-wide scale involving wild Cicer accessions. In this context, an 834 simple sequence repeat and single-nucleotide polymorphism marker-based high-density genetic linkage map between cultivated and wild parental accessions (Cicer arietinum desi cv. ICC 4958 and Cicer reticulatum wild cv. ICC 17160) was constructed. This inter-specific genetic map comprising eight linkage groups spanned a map length of 949.4 cM with an average inter-marker distance of 1.14 cM. Eleven novel major genomic regions harbouring 15 robust QTLs (15.6-39.8% R(2) at 4.2-15.7 logarithm of odds) associated with four agro-morphological traits (100-seed weight, pod and branch number/plant and plant hairiness) were identified and mapped on chickpea chromosomes. Most of these QTLs showed positive additive gene effects with effective allelic contribution from ICC 4958, particularly for increasing seed weight (SW) and pod and branch number. One robust SW-influencing major QTL region (qSW4.2) has been narrowed down by combining QTL mapping with high-resolution QTL region-specific association analysis, differential expression profiling and gene haplotype-based association/LD mapping. This enabled to delineate a strong SW-regulating ABI3VP1 transcription factor (TF) gene at trait-specific QTL interval and consequently identified favourable natural allelic variants and superior high seed weight-specific haplotypes in the upstream regulatory region of this gene showing increased transcript expression during seed development. The genes (TFs) harbouring diverse trait-regulating QTLs, once validated and fine-mapped by our developed rapid integrated genomic approach and through gene/QTL map-based cloning, can be utilized as potential candidates for marker-assisted genetic enhancement of chickpea.

Natural allelic diversity, genetic structure and linkage disequilibrium pattern in wild chickpea.

Characterization of natural allelic diversity and understanding the genetic structure and linkage disequilibrium (LD) pattern in wild germplasm accessions by large-scale genotyping of informative microsatellite and single nucleotide polymorphism (SNP) markers is requisite to facilitate chickpea genetic improvement. Large-scale validation and high-throughput genotyping of genome-wide physically mapped 478 genic and genomic microsatellite markers and 380 transcription factor gene-derived SNP markers using gel-based assay, fluorescent dye-labelled automated fragment analyser and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass array have been performed. Outcome revealed their high genotyping success rate (97.5%) and existence of a high level of natural allelic diversity among 94 wild and cultivated Cicer accessions. High intra- and inter-specific polymorphic potential and wider molecular diversity (11-94%) along with a broader genetic base (13-78%) specifically in the functional genic regions of wild accessions was assayed by mapped markers. It suggested their utility in monitoring introgression and transferring target trait-specific genomic (gene) regions from wild to cultivated gene pool for the genetic enhancement. Distinct species/gene pool-wise differentiation, admixed domestication pattern, and differential genome-wide recombination and LD estimates/decay observed in a six structured population of wild and cultivated accessions using mapped markers further signifies their usefulness in chickpea genetics, genomics and breeding.

Plastid transformation in eggplant.

Eggplant (Solanum melongena L.) is an important vegetable crop of tropical and temperate regions of the world. Here we describe a procedure for eggplant plastid transformation, which involves preparation of explants, biolistic delivery of plastid transformation vector into green stem segments, selection procedure, and identification of the transplastomic plants. Shoot buds appear from cut ends of the stem explants following 5-6 weeks of spectinomycin selection after bombardment with the plastid transformation vector containing aadA gene as selectable marker. Transplastomic lines are obtained after the regenerated shoots are subjected to several rounds of spectinomycin selection over a period of 9 weeks. Homoplasmic transplastomic lines are further confirmed by spectinomycin and streptomycin double selection. The transplastomic technology development in this plant species will open up exciting possibilities for improving crop performance, metabolic engineering, and the use of plants as factories for producing biopharmaceuticals.

Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement.

Chickpea (Cicer arietinum) is the second most widely grown legume crop after soybean, accounting for a substantial proportion of human dietary nitrogen intake and playing a crucial role in food security in developing countries. We report the ∼738-Mb draft whole genome shotgun sequence of CDC Frontier, a kabuli chickpea variety, which contains an estimated 28,269 genes. Resequencing and analysis of 90 cultivated and wild genotypes from ten countries identifies targets of both breeding-associated genetic sweeps and breeding-associated balancing selection. Candidate genes for disease resistance and agronomic traits are highlighted, including traits that distinguish the two main market classes of cultivated chickpea--desi and kabuli. These data comprise a resource for chickpea improvement through molecular breeding and provide insights into both genome diversity and domestication.