Agriculture Biotechnology

Horsegram (Macrotyloma uniflorum) -an important, self-pollinated food legume, however due to limited genomic and genetic resources the genetic improvement could not be achieved as compare to other major legumes. Our work aims at finding novel microsatellite markers and their use for the construction of a linkage map from 157 individuals of F 9 recombinant inbred lines (RILs) of horsegram. The determinate growth habit and plant height are important traits for its suitability for different cropping systems. The genotypic data were generated by screening 2 395 molecular markers, of which 600 (25.05 %) polymorphic markers were selected. Two-hundred eighty-seven (287) markers were mapped on ten linkage groups (LGs) at a log of odds (LOD) of 3.5 straddling 796.76 cM with 2.78 cM of marker density. For the identification of the quantitative trait loci (QTLs), the phenotypic data recorded on the RILs for the plant height and growth habit were analysed using the statistical tools JoinMap ® and Windows QTL cartographer, based on the composite interval mapping (CIM) technique. Across the ten linkage groups, we detected four QTLs (LOD ≥ 2.5) for four traits. All the traits were major QTLs as indicated by the percentage of phenotypic variance (PVE) (≥ 10%) that ranged from 13.5% to 40.3%, therefore, this is very important information which can be used in marker-assisted selection (MAS). The present genomic information generated in this orphan crop, thus, provides the base for genetic improvements by devising molecular breeding strategies.

Simple sequence repeat (SSR) and intron length polymorphism (ILP) primers were developed and characterized from public sequence data in horsegram. All characterized primers amplified alleles in a range of 1-8 with an average of 2.64 alleles per primer. Polymorphism information content ranged from 0.048 for primers MUMS-24, MUMST-21, MUMST-26 and MUMST-29 to 0.755 in MUMS-10 with an average of 0.360. Dendrogram and principal component analysis showed that newly characterized SSR and ILP markers developed in this study distinguished horsegram germplasm lines into two groups and revealed two genetic stocks for characterized germplasm. Cross-amplification of these developed markers was tested across 12 related legume species. Nineteen primers pairs showed a maximum of 100 % cross-transferability, while a minimum of 8.3 % transferability was shown by 27 primers. As no genomic resources are available in horsegram, these newly developed markers can advance molecular breeding research in this crop species and can be helpful in germplasm characterization, diversity studies, mapping and in comparative genomic studies.

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.] is an important legume but understudied in terms of its genetic improvement. Genetic information on various phenological and morphological traits may help in the utilization of new genes for breeding in horsegram and thus affect agronomic practices and crop yield. A total of 162 recombinant inbred lines derived from intraspecific crosses between HPKM249 × HPK4 was used to construct a genetic linkage map and to identify quantitative trait loci (QTLs) associated with phenological and morphological traits. Of the total 2011 molecular markers, which were screened on parental lines for polymorphism survey, 493 markers were found to be polymorphic and used for genotyping of recombinant inbred line population. Out of 493 polymorphic markers, 295 were mapped on ten linkage groups at LOD 3.5 spanning a total distance of 1,541.7 cM with an average distance between markers of 5.20 cM. Phenotypic data of two years at two different locations were u...

Horsegram (Macrotyloma uniflorum) is a drought hardy legume which can be grown in varied soil and temperature regime. It is an important food legume with environmental, nutritive and medicinal benefits. But in terms of genetic improvement it still lags behind other legumes. To get insight into the genetics of tolerance to drought stress, quantitative trait loci for drought tolerance traits were identified using an intraspecific mapping population comprising of 162 F 8 Recombinant Inbred Lines derived from a cross between HPKM249 and HPK4. A total of 2011 markers were screened on parental lines for polymorphism survey, out of which 493 markers were found to be polymorphic and used for genotyping of the RIL population. Of these 493 polymorphic markers, 295 were assigned to ten linkage groups at LOD 3.5 spanning 1541.7cM with a mean distance of 5.20 cM between adjacent markers. This linkage map along with the phenotypic data for drought tolerance traits was used to identify regions of the horsegram genome in which the genes for the qualitative traits linked to drought tolerance located. A total of seven QTLs were identified for six different drought related traits. One QTL for malondialdehyde content on linkage group 2, two QTLs for root length on linkage group 3 & 9, one QTL each for proline content and chlorophyll content under drought stress on linkage group 4, one QTL each for root dry weight and root fresh weight on linkage group 5 were identified using composite interval .

Horsegram (Macrotyloma uniflorum (Lam.) Verdc.) is a drought hardy legume which can be grown in varied soil and temperature regimes. Though it has numerous, nutritive and medicinal benefits. However, it still lags behind other legumes in terms of genetic improvement. This crop is mostly cultivated on marginal and drought prone area thus genetics of drought stress tolerance were understood by studying the various drought parameters. To get insight, quantitative trait loci for drought tolerant traits were identified using an intraspecific mapping population of 162 F8 recombinant inbred lines derived from a cross between HPKM249 and HPK4. The linkage map already developed was used along with the phenotypic data for biochemical and physiological parameters to identify genomic regions which are linked to drought tolerance. In the study total of seven QTLs were identified for ten different drought related traits. One QTL for malondialdehyde content on linkage group 2, two QTLs for root le...

With continuous increase in world's population, the main challenge among breeders is to increase food production with endurable utilization of available natural resources to accomplish a goal of zero hunger. Cultivating the nutritious underutilized crops could be a signi cant approach to address the issues of nutritional insecurity. Among 39 crops identi ed by FAO, horsegram is also considered as an important "Future Smart Food". It has attracted interest amongst the scienti c community due to its unparallel nutritional and pharmaceutical properties. Improving seed yield and related traits are main objectives of plant breeders for enhancing productivity in the crops. Horsegram lacks the information on the genetic structure of its yield contributing traits. The present study was aimed to dissect the quantitative trait loci of seed yield and its associated traits in horsegram by utilizing linkage map already developed in our lab of intraspeci c F8 RIL population of 162 individuals derived from HPKM249×HPK4 cross. Phenotypic data of different years and locations were used to identify QTLs for yield related traits by composite interval mapping (CIM). A total of six QTLs (LOD ≥ 2.5) were detected across different environments for seed size, seeds per plant, pods per plant and yield per plant. These were major QTLs with phenotypic variation explained (PVE) greater than ten percent and one of them was stable QTL across different locations and years. Phenotypic variation explained by QTLs ranged from 10.8 to 20.16 per cent thus they are of economic importance.

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This study explicates molecular insights commencing Self-Incompatibility (SI) and CC (cross-compatibility/fertilization) in self (SP) and cross (CP) pollinated pistils of tea. The fluorescence microscopy analysis revealed ceased/deviated pollen tubes in SP, while successful fertilization occurred in CP at 48 HAP. Global transcriptome sequencing of SP and CP pistils generated 109.7 million reads with overall 77.9% mapping rate to draft tea genome. Furthermore, concatenated de novo assembly resulted into 48,163 transcripts. Functional annotations and enrichment analysis (KEGG & GO) resulted into 3793 differentially expressed genes (DEGs). Among these, de novo and reference-based expression analysis identified 195 DEGs involved in pollen-pistil interaction. Interestingly, the presence of 182 genes [PT germination & elongation (67), S-locus (11), fertilization (43), disease resistance protein (30) and abscission (31)] in a major hub of the protein-protein interactome network suggests a complex signaling cascade commencing SI/CC. Furthermore, tissue-specific qRT-PCR analysis affirmed the localized expression of 42 DE putative key candidates in stigma-style and ovary, and suggested that LSI initiated in style and was sustained up to ovary with the active involvement of csRNS, SRKs & SKIPs during SP. Nonetheless, COBL10, RALF, FERONIA-rlk, LLG and MAPKs were possibly facilitating fertilization. The current study comprehensively unravels molecular insights of phase-specific pollen-pistil interaction during SI and fertilization, which can be utilized to enhance breeding efficiency and genetic improvement in tea.

Tea is popular health beverage consumed by millions of people worldwide. Drought is among the acute abiotic stress severely affecting tea cultivation, globally. In current study, transcriptome sequencing of four diverse tea genotypes with inherent contrasting genetic response to drought (tolerant & sensitive) generated more than 140 million reads. De novo and reference-based assembly and functional annotation of 67,093 transcripts with multifarious public protein databases yielded 54,484 (78.2%) transcripts with significant enrichment of GO and KEGG drought responsive pathways in tolerant genotypes. Comparative DGE and qRT analysis revealed key role of ABA dependent & independent pathways, potassium & ABC membrane transporters (AtABCG22, AtABCG11, AtABCC5 & AtABCC4) and antioxidant defence system against oxidative stress in tolerant genotypes, while seems to be failed in sensitive genotypes. Additionally, highly expressed UPL3HECT E3 ligases and RING E3 ligases possibly enhance drought tolerance by actively regulating functional modification of stress related genes. Further, ascertainment of, 80803 high quality putative SNPs with functional validation of key non-synonymous SNPs suggested their implications for developing high-throughput genotyping platform in tea. Futuristically, functionally relevant genomic resources can be potentially utilized for gene discovery, genetic engineering and marker-assisted genetic improvement for better yield and quality in tea under drought conditions. Drought is an environmental condition which negatively affects growth, yield and considered as primary cause of crop loss worldwide 1. Countries like India are facing consecutive drought conditions since last decades and causing significant losses of farm economy and worsening the food security. Tea [Camellia sinensis (L.) O. Kuntze], a perennial evergreen woody plant belongs to family Theaceae is highly cross pollinated and heterozygous plantation crop 2. It is consumed by millions of people worldwide due to its numerous health benefits, wherein, India contribute significantly by being the second largest producer of tea in the world 3. Nevertheless, tea cultivation is significantly constrained due to the impact of climate change and prolonged rainless periods leading to drought conditions, and reducing tea production by 14-33% with mortality of up to 6-19% 4. Therefore, tremendous focus of breeders is now on identification and development of high yielding quality tea varieties better adapted to drought conditions 5. In general, plants are adapted to overcome drought stress via avoidance, escape, tolerance and recovery, vis-à-vis involvement of several regulatory and functional genes, metabolic and photosynthesis-associated pathways 6,7. Drought-induced transitions have been studied earlier at biochemical, physiological and molecular level 8-12. Biochemical and physiological parameters revealed high water status and photosynthesis rate are key factors involved in osmotic adjustments in tolerant genotypes 8. Furthermore, transcriptomic studies indicated the

Bamboo, one of the fastest growing plants, can be a promising model system to understand growth. The study provides an insight into the complex interplay between environmental signaling and cellular machineries governing initiation and persistence of growth in a subtropical bamboo (Dendrocalamus hamiltonii). Phenological and spatio-temporal transcriptome analysis of rhizome and shoot during the major vegetative developmental transitions of D. hamiltonii was performed to dissect factors governing growth. Our work signifies the role of environmental cues, predominantly rainfall, decreasing day length, and high humidity for activating dormant bud to produce new shoot, possibly through complex molecular interactions among phosphatidylinositol, calcium signaling pathways, phytohormones, circadian rhythm, and humidity responses. We found the coordinated regulation of auxin, cytokinin, brassinosteroid signaling and cell cycle modulators; facilitating cell proliferation, cell expansion, and cell wall biogenesis supporting persistent growth of emerging shoot. Putative master regulators among these candidates were identified using predetermined Arabidopsis thaliana protein-protein interaction network. We got clues that the growth signaling begins far back in rhizome even before it emerges out as new shoot. Putative growth candidates identified in our study can serve in devising strategies to engineer bamboos and timber trees with enhanced growth and biomass potentials.

To unravel the molecular mechanism of defense against blister blight (BB) disease caused by an obligate biotrophic fungus, Exobasidium vexans, transcriptome of BB interaction with resistance and susceptible tea genotypes was analysed through RNA-seq using Illumina GAIIx at four different stages during ~20-day disease cycle. Approximately 69 million high quality reads were assembled de novo, yielding 37,790 unique transcripts with more than 55% being functionally annotated. Differentially expressed, 149 defense related transcripts/genes, namely defense related enzymes, resistance genes, multidrug resistant transporters, transcription factors, retrotransposons, metacaspases and chaperons were observed in RG, suggesting their role in defending against BB. Being present in the major hub, putative master regulators among these candidates were identified from predetermined protein-protein interaction network of Arabidopsis thaliana. Further, confirmation of abundant expression of wellknown RPM1, RPS2 and RPP13 in quantitative Real Time PCR indicates salicylic acid and jasmonic acid, possibly induce synthesis of antimicrobial compounds, required to overcome the virulence of E. vexans. Compendiously, the current study provides a comprehensive gene expression and insights into the molecular mechanism of tea defense against BB to serve as a resource for unravelling the possible regulatory mechanism of immunity against various biotic stresses in tea and other crops.

Purple-tea, an anthocyanin rich cultivar has recently gained popularity due to its health benefits and captivating leaf appearance. However, the sustainability of purple pigmentation and anthocyanin content during production period is hampered by seasonal variation. To understand seasonal dependent anthocyanin pigmentation in purple tea, global transcriptional and anthocyanin profiling was carried out in tea shoots with two leaves and a bud harvested during in early (reddish purple: S1_RP), main (dark gray purple: S2_GP) and backend flush (moderately olive green: S3_G) seasons. Of the three seasons, maximum accumulation of total anthocyanin content was recorded in S2_GP, while least amount was recorded during S3_G. Reference based transcriptome assembly of 412 million quality reads resulted into 71,349 non-redundant transcripts with 6081 significant differentially expressed genes. Interestingly, key DEGs involved in anthocyanin biosynthesis [PAL, 4CL, F3H, DFR and UGT/UFGT], vacuolar trafficking [ABC, MATE and GST] transcriptional regulation [MYB, NAC, bHLH, WRKY and HMG] and Abscisic acid signaling pathway [PYL and PP2C] were significantly upregulated in S2_GP. Conversely, DEGs associated with anthocyanin degradation [Prx and lac], repressor TFs and key components of auxin and ethylene signaling pathways [ARF, AUX/ IAA/SAUR, ETR, ERF, EBF1/2] exhibited significant upregulation in S3_G, correlating positively with reduced anthocyanin content and purple coloration. The present study for the first-time elucidated genome-wide transcriptional insights and hypothesized the involvement of anthocyanin biosynthesis activators/repressor and anthocyanin degrading genes via peroxidases and laccases during seasonal induced leaf color transition in purple tea. Futuristically, key candidate gene(s) identified here can be used for genetic engineering and molecular breeding of seasonal independent anthocyanin-rich tea cultivars. Tea, (Camellia sinensis (L.) O. Kuntze), is an economically important plantation crop rich in flavor enhancing and health beneficial metabolites that also confer resistance to biotic and abiotic stresses 1. Most of the elite tea cultivars grown worldwide are suitable for processing of the three popular types of commercial teas, viz; non-fermented (green tea), semi-fermented (oolong tea), and completely fermented (black tea) 2. Global production of black and green tea is consistently increasing and projected to reach 4.42 and 3.31 million tons, respectively by 2027 3. Despite the significant increase in global production, tea prices have stagnated or declined causing reduced profit margins and returns to the farmers 4. Therefore, diversification of tea products can be an alternative way-out to raise demand and shoreup market prices. Nevertheless, diversification is greatly influenced by availability of suitable germplasm, wherein, anthocyanin-rich tea cultivars due to their pleasant taste and rich health beneficial compounds are getting global attention for production of "Specialty Tea" that can fetch premium prices to the farmers. Through breeding (natural or artificial hybridization) followed by clonal field selection, anthocyanin-rich tea cultivars (purple tea) have been developed for product diversification 5,6. Anthocyanin, a water-soluble OPEN

The most daunting issue of global climate change is the deleterious impact of extreme temperatures on tea productivity and quality, which has resulted in a quest among researchers and growers. The current study aims to unravel molecular programming underpinning thermotolerance by characterizing heat tolerance and sensitivity response in 20 tea cultivars. The significantly higher negative influence of heat stress was recorded in a sensitive cultivar with reduced water retention (47%), chlorophyll content (33.79%), oxidation potential (32.48%), and increase in membrane damage (76.4%). Transcriptional profiling of most tolerant and sensitive cultivars identified 78 differentially expressed unigenes with chaperon domains, including low and high molecular weight heat shock protein (HSP) and heat shock transcription factors (HSFs) involved in heat shock response (HSR). Further, predicted transcriptional interactome network revealed their key role in thermotolerance via well-co-ordinated transcriptional regulation of
aquaporins, starch metabolism, chlorophyll biosynthesis, calcium, and ethylene mediated plant signaling system. The study identified the key role of HSPs (CsHSP90) in regulating HSR in tea, wherein, structure-based molecular docking revealed the inhibitory role of geldanamycin (GDA) on CsHSP90 by blocking ATP binding site at N-terminal domain of
predicted structure. Subsequently, GDA mediated leaf disc inhibitor assay further affirmed enhanced HSR with higher expression of CsHSP17.6, CsHSP70, HSP101, and CsHSFA2 genes in tea. Through the current study, efforts were made to extrapolate a deeper understanding of chaperons mediated regulation of HSR attributing thermotolerance in tea.

This study explicates molecular insights commencing Self-Incompatibility (SI) and CC (cross-compatibility/fertilization) in self (SP) and cross (CP) pollinated pistils of tea. The fluorescence microscopy analysis revealed ceased/deviated pollen tubes in SP, while successful fertilization occurred in CP at 48 HAP. Global transcriptome sequencing of SP and CP pistils generated 109.7 million reads with overall 77.9% mapping rate to draft tea genome. Furthermore, concatenated de novo assembly resulted into 48,163 transcripts. Functional annotations and enrichment analysis (KEGG & GO) resulted into 3793 differentially expressed genes (DEGs). Among these, de novo and reference-based expression analysis identified 195 DEGs involved in pollen-pistil interaction. Interestingly, the presence of 182 genes [PT germination & elongation (67), S-locus (11), fertilization (43), disease resistance protein (30) and abscission (31)] in a major hub of the protein-protein interactome network suggests a complex signaling cascade commencing SI/CC. Furthermore, tissue-specific qRT-PCR analysis affirmed the localized expression of 42 DE putative key candidates in stigma-style and ovary, and suggested that LSI initiated in style and was sustained up to ovary with the active involvement of csRNS, SRKs & SKIPs during SP. Nonetheless, COBL10, RALF, FERONIA-rlk, LLG and MAPKs were possibly facilitating fertilization. The current study comprehensively unravels molecular insights of phase-specific pollen-pistil interaction during SI and fertilization, which can be utilized to enhance breeding efficiency and genetic improvement in tea.

The most daunting issue of global climate change is the deleterious impact of extreme temperatures on tea productivity and quality, which has resulted in a quest among researchers and growers. The current study aims to unravel molecular programming underpinning thermotolerance by characterizing heat tolerance and sensitivity response in 20 tea cultivars. The significantly higher negative influence of heat stress was recorded in a sensitive cultivar with reduced water retention (47%), chlorophyll content (33.79%), oxidation potential (32.48%), and increase in membrane damage (76.4%). Transcriptional profiling of most tolerant and sensitive cultivars identified 78 differentially expressed unigenes with chaperon domains, including low and high molecular weight heat shock protein (HSP) and heat shock transcription factors (HSFs) involved in heat shock response (HSR). Further, predicted transcriptional interactome network revealed their key role in thermotolerance via well-co-ordinated t...

To unravel the molecular mechanism of defense against blister blight (BB) disease caused by an obligate biotrophic fungus, Exobasidium vexans, transcriptome of BB interaction with resistance and susceptible tea genotypes was analysed through RNA-seq using Illumina GAIIx at four different stages during ~20-day disease cycle. Approximately 69 million high quality reads were assembled de novo, yielding 37,790 unique transcripts with more than 55% being functionally annotated. Differentially expressed, 149 defense related transcripts/genes, namely defense related enzymes, resistance genes, multidrug resistant transporters, transcription factors, retrotransposons, metacaspases and chaperons were observed in RG, suggesting their role in defending against BB. Being present in the major hub, putative master regulators among these candidates were identified from predetermined protein-protein interaction network of Arabidopsis thaliana. Further, confirmation of abundant expression of well-kno...

Bamboo, one of the fastest growing plants, can be a promising model system to understand growth. The study provides an insight into the complex interplay between environmental signaling and cellular machineries governing initiation and persistence of growth in a subtropical bamboo (Dendrocalamus hamiltonii). Phenological and spatio-temporal transcriptome analysis of rhizome and shoot during the major vegetative developmental transitions of D. hamiltonii was performed to dissect factors governing growth. Our work signifies the role of environmental cues, predominantly rainfall, decreasing day length, and high humidity for activating dormant bud to produce new shoot, possibly through complex molecular interactions among phosphatidylinositol, calcium signaling pathways, phytohormones, circadian rhythm, and humidity responses. We found the coordinated regulation of auxin, cytokinin, brassinosteroid signaling and cell cycle modulators; facilitating cell proliferation, cell expansion, and...

Trillium govanianum, an endangered medicinal herb native to the Himalaya, is less studied at the molecular level due to the non-availability of genomic resources. To facilitate the basic understanding of the key genes and regulatory mechanism of pharmaceutically important biosynthesis pathways, first spatial transcriptome sequencing of T. govanianum was performed. 151,622,376 (~11.5 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 69,174 transcripts. Functional annotation with multiple public databases identified array of genes involved in steroidal saponin biosynthesis and other secondary metabolite pathways including brassinosteroid, carotenoid, diterpenoid, flavonoid, phenylpropanoid, steroid and terpenoid backbone biosynthesis, and important TF families (bHLH, MYB related, NAC, FAR1, bZIP, B3 and WRKY). Differentially expressed large number of transcripts, together with CYPs and UGTs suggests involvement of these candidates in tissue specific expression. Combined transcriptome and expression analysis revealed that leaf and fruit tissues are the main site of steroidal saponin biosynthesis. In conclusion, comprehensive genomic dataset created in the current study will serve as a resource for identification of potential candidates for genetic manipulation of targeted bioactive metabolites and also contribute for development of functionally relevant molecular marker resource to expedite molecular breeding and conservation efforts in T. govanianum. Trillium govanianum Wall. ex D. Don (Family: Melanthiaceae), prevalently known as "Nag chhatri" or "Satva", is an endangered perennial herb with multiple therapeutic properties. It is the only species of the genus Trillium, native to the Himalaya representing scattered distribution from Nanga Parbat to Namcha Barwa at an altitude range of 2700 m to 4000 m 1,2. It is an allotetraploid (2n = 4× = 20) between the genus Trillium and Daiswa, having a very large genome size of 67.125 Gb (1C = 68.64 pg) 3,4. The species is characterized by the presence of three leaves in a whorl just beneath a trimerous flower emerging from a 15-30 cm long solitary stem and bright red berry like globose fruit at the top, containing numerous seeds. Flowering has been reported to occur during the months of May to July followed by seed set in October 3. The short and stout rhizome along with adventitious roots has been used in the Indian system of medicine as "folk medicine" (http://www.medicinalplants.in/folksearchpage) to cure menstrual and other reproductive disorders 5 , general body weakness 6 , dysentery 2 , headache and fever 7 , besides its use as an anthelmintic for livestock 8. Great medicinal importance coupled with high commercial value (15,000 to 20,000 per kg raw botanical drug) in the international markets 9 has led to quantum