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Open Access
GENETIC STUDIES FOR IMPROVING YIELD UNDER DROUGHT STRESS ENVIRONMENTS IN RICE OF ASSAM
(2019-07) PARRAY, ROUF AHMAD; Baruah, Akhil Ranjan
Drought is a major limiting factor for rice under rainfed ecosystem in Assam. In this context, thirteen rice cultivars with varied level of drought tolerance were chosen from a set of 272 different rice genotypes based on a field experiment conducted during 2014-15 season under drought. The thirty days old seedlings of 13 cultivars were tested for extensive morpho-physiological, biochemical parameters, relative transcript accumulation and global gene expression using next generation sequencing (NGS) method, and data were recorded at fifth, tenth and fifteenth day of withholding water (DWW) in order to obtain detail trait based gene architecture and to improve high yielding variety of Assam using transcript dynamics. Among the physiological traits studied, stomatal conductance decreased as the dehydration stress increased but the effect was minimum in Apo, Dumai and Tepi Dumai compared to others. Photosynthetic rate decreased with increasing water deficit, but the effect was less pronounced in Apo, Dumai and Tepi Dumai. The rate of transpiration decreased upto 5DWW but gradual increase was observed in later stage. Moreover, the fall in transpiration rate was less in Apo. Water use efficiency (WUE) of rice plants was enhanced significantly under moisture stress at all the three periods of stress (5DWW, 10DWW, 15DWW) in Apo, Tepi Dumai and Dumai. Reduction in RWC was experienced across all genotypes but the decrease was less prominent in Apo, Dumai and Tepi Dumai. Drought stress condition led to increased proline content across genotypes as compared to irrigated condition. Apo, Tepi Dumai, Dumai and Kali Murali showed rapid increase compared to others. Increase in root length was observed across all cultivars with Apo being the longest followed by Dumai and Ranjit. Then, five drought responsive pathway genes (OsDREB2, OsNAC1, bZIP16, OsbZIP 23, OsbZIP72) were chosen to check the differential expression patern in the cultivars at the same data point as mentioned above. Expression profiling of OsDREB2 showed significant increase in gene expression with increase in drought stress in the case of Apo and Dumai. Significant expression of the OsNAC1 was found in Apo, Dumai at different time points of dehydration stress whereas expression of ARC 10372 was prominent in 15DWW. Apo showed significant difference in expression of bZIP16 under all the three stages of water stress whereas Dumai and Ranjit showed enhanced expression compared to other cultivars. Expression profile of OsbZIP23 showed significant accumulation of transcripts in Apo in all stages followed by Dumai. Significant expression of OsbZIP72 was observed in Apo at 10DWW and 15DWW followed by Ranjit and Dumai. Based on the results of morpho-physiological, biochemical and expression analysis, three cultivars, viz., Ranjit, Apo and Dumai were chosen to study the detailed transcriptome at only 10DWW. Transcriptome profile revealed highest mapped genes in Dumai followed by Ranjit and Apo, however, only 14.5% genes were in common. Ranjit was found to be more responsive to abiotic stimulus including water stress. Gene ontology (GO) suggested no significant change of pathway genes upto 10 DWW among the three cultivars. The transcriptome data were validated using five differentially expressed genes in these three cultivars along with a F4 mapping population. It revealed similar trend, suggesting the present transcriptome data set was in good fit. However, detail transcriptome study in vital plant parts at different stages under drought stress will throw more light about the interaction of pathway genes to adress the problem better.
Open Access
Molecular cloning and in silico characterization of linalool synthase gene from Cymbopogon winterianus
(AAU, Jorhat, 2018-07) Saha, Oliva; Sen, Priyabrata
Citronella (Cymbopogon winterianus), an aromatic and medicinal grass from Poaceae family, is grown for the commercial and industrial purpose. It has the large repository of isoprenoid compounds known for its anti-tumoral, anti-bacterial, anti-fungal, anti-viral, anticancerous, detoxifying and natural insect repellent properties. Out of the different components of citronella oil, linalool (3,7-Dimethylocta-1,6-dien-3-ol) is one of the significant aromatic ingredient. Formation of linalool is catalysed by linalool synthase (LIS) which is encoded by linalool synthase gene. This gene has been widely studied in many medicinal plants along with sorghum, rice, maize, setaria, etc.However, till date, no studies on molecular cloning, characterization and tissue-specific expression profiling of LIS gene from C. winterianus (CwLIS) has been reported.Therefore, the present study aims at cloning and characterization of full-length cDNA of LIS gene from C. winterianus. The full-length sequence of CwLIS was obtained by primer walking using several pairs of degenerate primers and the sequence fragments were aligned to acquire full length sequence. Cloning of the desired gene (1758bp) was performed using TOPO TA vector (3.9kb), and the transformation was done in E. coli DH5α competent cells. The sequence analysis revealed 1758 bps cDNA, which has a Coding Sequence (CDS) of 1422 bps that encodes a protein of 473 amino acids. The domain analysis revealed that CwLIS is a single domain protein under terpene synthase superfamily. The multiple sequence alignment (MSA) based on PSI-BLAST search against RefSeq with 62% sequence identity revealed the presence of two aspartate-rich regions, which are supposed to coordinate 3 Mg2+ ions. The phylogenetic analysis revealed that the CwLIS is closely related to Sorghum bicolor linalool synthase. To understand the molecular mechanism behind Mg2+ and linalool binding, first, the 3D model of CwLIS was generated and validated with their stereo chemical parameters. Further, to find out the expression profile of CwLIS in different tissues, Reverse Transcriptase-PCR was performed and the results revealed a higher expression in leaf sheath followed by leaf, root and flower and further conformed by qRTPCR analysis. The result from the present study provide basic information for further research about linalool synthase and comprehensive sequence resource for study, such as gene expression, genomics and functional genomics in Cymbopogon winterianus.
Open Access
Assessment of Genetic Diversity For Ideal Plant Architecture in Rice Cultivars of Assam With Special Importance to Aromatic Group
(AAU, Jorhat, 2018-08) Debnath, Neelakshi; Baruah, A.R.
The development of super rice to break the yield plateau is one of the goals for rice breeding in recent years. Ideal plant architecture (IPA), which is under polygenic control, has been proposed as means to enhance rice yield potential. A study directed towards assessing the presence of desired alleles for IPA related genes/quantitative trait loci (QTL) in rice cultivars grown in diverse agroecological situations, specifically the aromatic rice of Assam (joha rice) that usually bears weaker plant type should be of prime importance. The present study was conducted to detect allelic variation for IPA in traditional, high yielding and joha rice cultivars of Assam and to compare nucleotide sequence variation for aroma gene in cultivars possessing IPA. A total of 80 cultivars comprising sali, ahu, boro and joha rice of Assam were assessed for markers linked to five IPA related QTLs (Sol1, PAY1, IPA1, Gn1 and LAZY1). Although a total of 15 markers (M3, M6, M8, M10 for Sol1; RM339, RM223, SP5, SP7 for PAY1; RM149, RM1345, M4, M5 for IPA1; PD56, M265 for LAZY1; 16BPDEL for Gn1) were employed to check for polymorphism, only five markers (GN1, M10, M265, RM 1345, SP5) showed reproducible and polymorphic results. The Gn1 and Sol1 were most abundant in cultivars as evident from the observation that 46 numbers of cultivars harboured desired alleles for Gn1 and Sol1 followed by PAY1( 31 cultivars), LAZY1 (12 cultivars) and IPA1(12 cultivars). Among HYVs, the varieties such as Ranjit, Mahsuri and Bahadur were found to possess four numbers of desired alleles, however, IPA1 allele was other than the expected. Among Joha cultivars, Bengoli joha, Goalporia joha, Kartica joha were with the maximum of four numbers of positive alleles. As Ranjit missed the expected size of the IPA1 allele, differential expression for the gene in Ranjit and one cultivar possessing the desired IPA1 allele (Chakaw Poireton) was conducted. It revealed that although Ranjit did not possess the desired IPA1, however, the expression pattern revealed that 25-30 fold more accumulation of transcripts for the gene in Ranjit than Chakaw Poireton, suggesting the functional differences in the coding regions. The allele specific analysis for aroma gene, badh2 revealed that the markers could efficiently group 53.66% of the cultivars corresponding to the existing phenotype, indicating the power of the marker to be utilized in a large germplasm screening for aroma. The sequencing for badh2.hapG9 detected a non-synonymous substitution (AG) which was not being reported elsewhere; however, novelty of which needed to be confirmed and validated. The study will enhance our knowledge to formulate breeding strategies for combing IPA with aroma.
Open Access
HOST RANGE, INCIDENCE AND GENETIC VARIABILITY OF watermelon mosaic virus IN CENTRAL SPAIN
(AAU, Jorhat, 2018-01) Paswan, Ricky Raj; Acharjee, Sumita
Watermelon mosaic virus (WMV, Potyvirus) is an economically important pathogen common in cucurbits of temperate and Mediterranean regions worldwide. The presence of WMV in cucurbits in the Mediterranean basin has been known for decades. More recently, an emergent strain that causes more severe symptoms compared to classic strains has been recognized in France. The cultivation of melon is threatened by the spread of emergent strains of WMV. Diagnostic methods for detecting the host range, incidence and evolution of these emergent types are critical for developing control strategies to optimize agricultural production. In this study, next generation sequencing and RT-PCR approaches are combined to investigate the epidemiology of WMV in an agro-ecosystem of Central Spain. Four vegetation types, or habitats, including cultivated and adjacent land-use types were surveyed in the summer and autumn of 2015. Forty-three plant species were screened for WMV, 15 of which were WMV-positive across two habitats other than crops. The results indicated an increase in the extent of the WMVs known host range. The incidence of WMV ranged from 64% in Cucumis melo to 5% in a weed species, Datura Stramonium. Genetic analyses of the coat protein gene of 30 isolates from melon and 3 other ‘weed’ species sampled in crops showed population variation in nucleotide diversity, but pairwise fixation indices indicated negligible distinctions between them. Phylogenetic inferences showed both negligible and large branch length differences between isolates from different host species. When sequences of a number of different strains were added to the isolates from the melon crops, one clad clustered with an emergent group previously identified from elsewhere in Europe and Asia. This study reports the first instance of an emerging (EM) strain in Central Spain.
Open Access
MOLECULAR CHARACTERIZATION OF NBS-LRR RESISTANT GENE ANALOGUES (RGAs) FROM INDIGENOUS AND WILD BANANA (MUSA SPP.) GERMPLASM
(AAU, Jorhat, 2018-01) Hazarika, Geetimollika; Bhorali, Priyadarshini
Commercial banana varieties are highly susceptible to fungal and bacterial pathogens, nematodes, viruses and insect pests. The largest known family of plant disease resistance (R) genes encodes proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for the isolation of candidate genes in banana that may be involved in plant defense. In the present study, an attempt was made to isolate and characterize the conserved region NBS of the NBS-LRR resistance gene analogues (RGAs) from locally cultivated indigenous and wild banana germplasms of Assam. The investigation was started with the isolation of genomic DNA from ten cultivated indigenous germplasms viz. Kach kol, Cheni champa, Ukho jahaji, Malbhog, Manuhor, Athiya kol, Bhim kol, Ketekihunda, Phesa manuhor and Ximolu manuhor and, five wild germplasms (designated as W1, W2, W3, W4 and W5). To target the NBS region of the banana germplasms, four pairs of PCR primers out of which two were degenerate primers, were designed from existing NBS-LRR sequences available in the GenBank. After successful isolation and sequencing of the PCR amplified NBS fragments from all the fifteen samples, confirmation about the identity of the sequences was done by homology search using BLASTn and BLASTp algorithms which revealed the sequences to be significantly similar to the NBS-LRR class disease resistance proteins available in NCBI. The sequence identity was further confirmed by checking for the Pfam NB-ARC domain, which is a protein domain characteristic of the plant resistance NBS-LRR protein. The NB-ARC domain was obtained in all the isolated NBS sequences. Finally, the presence of the consensus sequence for Kinase-2 motif (LLDDVW) and phylogenetic analysis of the isolated NBS sequences further provided evidence that the sequences belong to the typical non-Toll/interleukin-1 receptor- like domain NBS-LRR gene family, as expected. As a future prospect, upon cloning of the full length NBS-LRR sequences from these germplasms would open up possibilities for development of disease resistant cultivars through genetic engineering approaches.
Open Access
Cloning of viral genome associated with leaf curl disease of Bhut Jolokia (Capsicum chinense Jacq.)
(AAU, Jorhat, 2018-01) Rajkhowa, Sushmita; Borah, B.K.
Bhut Jolokia (CapsicumchinenseJacq.) is known as one of the hottest chilli in the world. It is cultivated as a major cash crop in Assam and other northeastern states, mostly Manipur and Nagaland. In the international market Bhut Jolokia has high value due to its high Capsicin content which has various important properties like anti-inflamatory, anti-diabetic, anticancerous, pain relief, gastro intestinal disorder etc. The fruit has got multiple uses in medicines, biological warfare,elephant repelling, pickles and culinary purposes. Around 1400- 1500 tonnes of the fruits are exported annually from Assam. However,the productivity of the crop is hindered due to attack ofvarious diseases and pests. Viruses have been reported to be one of the major factors that affect Bhut Jolokia plants. Both RNA viruses (Potyvirus, Cucumovirus, Tospovirus) and DNA viruses (Geminivirus) incidence have been reported that infect Bhut Jolokia in Assam. Among the DNA viruses chilli leaf curl virus (ChLCV) and tomato leaf curl virus (ToLCV) have been reported to infect Bhut Jolokia which produces symptoms of leaf curling, mosaic symptoms, stunting growth and vein clearing. Some geminivirus also contains a satellite genome which functions as silencing suppressors and in symptom determinant. As such, to determine the genome sequence of the DNA virus(es) infecting the Bhut Jolokia plants with leaf curl disease, both universal and specific primers were used to detect the virus(es) associated with leaf curl disease. Partial sequences were obtained from ChLCV-specific primers designed from available sequences. The sequences were obtained from samples collected of AAU experimental farm, Lichubari and Teok, Jorhat. Multiple sequence alignment followed by phylogenetic analysis of the four sequences showed 98% identity with the chilli leaf curl isolate of Oman (Accession No.JN604500.1). The phylogeny could not establish a geographical grouping, possibly because of short length of the sequences. Meanwhile, the nucleotide similarity search for the sequences obtained from the universal primer and ToLCV specific primers did not amplify any viral sequence. Satellite-specific primers were used for detection of satellite genomes (both alpha-satellite and beta-satellite) possibly associated with the leaf curl disease of Bhut Jolokia. However, presence of any associated satellite could not be determined. Therefore, further works will be needed in these regards.
Open Access
IDENTIFICATION OF LOCI INVOLVED IN SALT TOLERANCE BY ASSOCIATION ANALYSIS ON japonica RICE
(AAU, Jorhat, 2018-01) Hazarika, Mousumi; Baruah, Aiswarya
Soil salinity is one of the environmental constraints that affect crop cultivation worldwide. More than 800 Mha of land throughout the world and about 20% of the irrigated areas suffer from salinization problems (FAO, 2008). Among cereals, rice (Oryza sativa L.) is one of the most salt-sensitive although cultivars can differ in their response to salt stress (Horie et al., 2012; Munns and Tester, 2008). In European coastal rice areas, salty raining and the salt water intrusion phenomenon caused by the rise in the sea levels due to climate changes are provoking a tendency toward salinization in the adjacent paddy fields where rice is grown (Yáñez, 2010.) Moreover, the island apple snail (Pomacea maculata) is becoming one of the major pest problems for rice throughout the world, including the European areas. Till date its only effective control measure is to flood the field with sea water, which even though kill the pest but leads to residual salinization problem. In India too, soil salinity is a problem in the coastal rice growing area. It has been estimated that an approximate area of 6.3 million hectares of land is covered by saline soil in India (Patel et al., 2011). Taking into account the above statements, it is clear that the identification of elite rice varieties tolerant to salt stress is necessary. Salinity tolerance in rice is a very complex trait. Genome-wide association study (GWAS) is proving to be an effective approach for identifying loci controlling complex traits in plants (Ingvarsson and Street, 2011; Korte and Farlow, 2013; Huang and Han, 2014). In the present work, a phenotyping activity for mid-salt stress has been performed on a subset of rice panel of 281 japonica rice accessions. Then, a GWAS was carried out in order to identify possible loci involved in salt tolerance in japonica rice. The measurement of phenotypic data highlighted variability among the genotypes in response to the treatment. This suggests that the panel might be a good resource for the discovery of traits related to salt stress response. The genome-wide association study identified significant associations between SNPs and the analyzed salt stress-related traits. A preliminary functional analysis of the identified loci by the GWAS has revealed many possible candidate genes involved in salt tolerance in japonica rice.
Open Access
Molecular characterization and nutritional equivalence evaluation of transgenic chickpea expressing either a cry1Ac or cry2Aa gene
(AAU, Jorhat, 2019-10) Gupta, Rubi; Sarmah, Bidyut Kumar
Biosafety assessment of transgenic chickpeas having B.thuringiensis genes for resistance to pod borers is a regulatory requirement and mandatory to document before releasing in the field. Therefore, Bt chickpea lines harbouring either a cry1Ac or cry2Aa gene were characterized for the presence and expression of the transgene in their advanced generations, biosafety assessments and transcript profile were studied. The homozygous lines were selected for comparative nutritional equivalence assay. Biochemical estimations of major nutritional components such as proximates, vitamins, minerals, fatty acids and anti nutrients confirmed that the Bt chickpeas lines are nutritionally equivalent to their non-transgenic counterparts and the seed composition is similar or within the range reported, previously. Seed protein quality was investigated by separating the proteins in PAGE and eluted proteins after mass spectrometry (MS) showed expected fractions of 11S legumin, 7S vicilin, and 2S albumin of chickpea storage proteins in the transgenic lines. The protein digestibility was assayed using the multi-enzyme system and transient pepsin hydrolysis to mimic simulated gastric fluid followed by trypsin hydrolysis to mimic simulated intestinal fluid. Total seed proteins of both the transgenic and nontransgenic lines were digested at a similar rate and enzyme-resistant peptides were not observed in transgenic Bt chickpea lines. The unintended changes in the whole transcriptome profile of Bt chickpeas were surveyed using a homozygous transgenic line expressing a cry1Ac gene. The differentially expressed genes (DEGs) profiling confirmed a low (0.69%, log2fold change≥2) frequency of differentially expressed in the transgenic chickpea line. Only a small (34 upregulated) proportion of genes showed > 2 fold (P-value of 0.05, FDR of 0.05) change in their expression, while only 23 genes down-regulated by >2 fold. Furthermore, no transcripts for potential allergenic proteins were represented in the DEGs. Most of these genes appeared to be developmentally regulated or stress-related which was expected because absolute synchronous growth and development even under a controlled environment are challenging. A few upregulated (AT-hook motif nuclear-localized protein 17-like, probable inactive 2-oxoglutaratedependent dioxygenase AOP2, protein EXORDIUM-like) and down-regulated (histone H2B, gonadal, embryonic abundant protein VF30.1-like, fasciclin-like arabinogalactan protein 12) genes were subjected to qPCR. The qPCR data validated the fold change of the up-regulated (>2) and down-regulated (>-2) genes. Thus, the above data revealed no potential alterations in the nutritional equivalence or transcript profile of transgenic Bt chickpeas.
Open Access
A study on the role of exopolysaccharide in conferring acid tolerance in Bacillus sp.
(AAU, Jorhat, 2018-01) Deka, Priyadarshini; Barooah, Madhumita
Soil bacteria have evolved various mechanisms to adapt to stress environmental conditions such as temperature, salinity, drought and low pH condition of soil. Among the several environmental stress conditions, soil acidity an important factor influencing physicochemical and biological properties of soil along with microbial diversity and crop production is an emerging issue of immense concern due to its wide spread distribution across the globe. Although low soil pH restricts the number and diversity of bacteria, it is known that some soil bacteria are able to thrive in such conditions having evolved various mechanisms including production of biofilm to circumvent acid stress. Bacterial exopolysaccharide (EPS) are high-molecular-weight complex polymers composed of sugar moieties that form the main component of the biofilm which aid the bacteria to colonize substrata. In the present study, a total of 28 isolates were identified and characterized as acid tolerant EPS producing bacteria among which, B. amyloliquefaciens p16 produce the highest EPS (219.96 μg/ml). A culture medium containing sucrose (3.5%) as carbon source with pH 5.0 and incubated for 24 hrs was optimal for maximum production of EPS. The HPLC analysis of monomeric units of EPS produced by B. amyloliquefaciens p16 revealed the abundance of galactose at pH 7.0 which however, changed to arabinose when shifted to acidic condition (pH to 5.0 and 4.5). The isolate B. amyloliquefaciens p16, significantly improved soil physical properties in terms of greater soil aggregation (80.59 mm diameter aggregates) and water holding capacity (53.90%) when inoculated into soil over the control (31 mm diameter aggregates and 18.21%, respectively). The differential expression of epsA and epsB, the first two genes of the eps operon showed a 7 and 9 fold increased expression in pH 5.0 compared to pH 7.0 respectively. Disruption of the epsB gene in B. amyloliquefaciens p16 using integration vector pMUTIN4 generated mutants that produced significantly lesser EPS (33.23 μg/ml) when compared to the WT (223.87 μg/ml). The generated mutant of B. amyloliquefaciens p16 lacking the wrinkled morphology had an extended lag phase of 24 hrs and was barely able to survive in acidic medium (pH 4.5) unlike that of the WT type. Soil inoculated with generated mutants formed smaller soil aggregates (42.41±1.70 mm) and had decreased water holding capacity (27.67±1.94%) compared to the WT (80.59± 0.22 mm and 53.90± 1.66%, respectively). This study indicates that EPS secreted by acid tolerant bacteria (B. amyloliquefaciens p16) imparts acid tolerance and also aids in improving the soil physical structure through increased soil aggregation and water holding capacity.

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