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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
Optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin
(AAU, Jorhat, 2017-07) Das, Panchashree; Sen, Priyabrata
Citrus species are the most widely grown fruit crops within the whole world. India is the fourth largest producer of orange in the world. North-Eastern India is considered as one of the centres of origin of many citrus species. Among them Khasi Mandarin is the most widely grown citrus species. According to Ministry of Agriculture and Irrigation, Govt. Of India, the yield of Khasi Mandarin is declining day by day drastically due to biotic and abiotic stress. Conventional breeding for overcoming this problem is limiting due to non-availability of resistant sources. Recent advances in genetic engineering have made it possible to incorporate desirable genes from alien sources to elite genotype mainly through Agrobacterium-mediated genetic transformation. Citrus cultivars vary in their response to in vitro organogenesis and genetic transformation. This results in need for a cultivarspecific optimization of an in vitro regeneration and transformation protocol. Most of the plant regeneration processes in citrus, through tissue culture, involve use of Cotyledon, epicotyl segment, shoot-tip, internode, root meristem as explants. A study was conducted to develop a regeneration and Agrobactetrium mediated transformation protocol for Khasi Mandarin using zygotic seedling as explants obtained from six-week-old in vitro grown seedlings. Modified MS media containing 1 mg/l BAP, 0.5mg/l NAA and 0.4mg/l Kinetin shows the best result for multiple shoot induction with an efficiency of 68%. The number of multiple shoots developed was on an average 5. The modified MS medium containing containing 0.25 mg/l BAP,0.5 mg/l NAA, 0.5 mg/l IBA shows best result for rooting with an efficiency of 82% with an average root length 2 cm. Zygotic explants with injured shoot tip were used as explant for transformation with Agrobacterium strain AGL1, harbouring plasmid pCAMBIA1301 containing hpt as selectable marker gene and gus as a reporter gene. Modified MS media containing 100mM Acetosyringone was fund to be most effective medium for co-cultivation. Regeneration and selection media containing 1 mg/l BAP, 0.5mg/l NAA, 0.4mg/l Kinetin and 30mg/l hygromycin and 250mg/l timentin shows the best result. In vitro regenerated shoots that survived upto 3rd selection cycle were considered as putative transformants. Some of the putative transformed shoots showed positive result for gus in PCR analysis. The present investigation is a preliminary study on optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin. More and more concentrated effort is needed to establish a most efficient regeneration and transformation protocol considering various factors affecting genetic transformation and regeneration efficiency.
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
Validation of selected SSR markers of QTLs associated with anaerobic germination trait in deepwater rice (Bao) of Assam
(AAU, Jorhat, 2017-01) Rajpoot, Sachin; Das, P.K.
India is one of the hot spots of vast genetic resources as well as secondary centre of origin of rice in the world. Exploiting genetic diversity could aid in providing useful information in the selection of material for breeding. Flood in rice field is due to heavy rainfall after seeding and early seedling emergence results in partial to complete crop failure. Primarily reason is high sensitivity of rice to the anaerobic conditions during germination (Yamauchi et al. 1993).Thus, tolerance to flood at early seedling growth improves crop establishment.The present study was conducted to evaluate anaerobic germination (AG) tolerance in Bao (deep water) rice germplasms of Assam compared to positive controls (Khao Hlan On, Ma Zhan Red) and negative control (IR-64). Under present study, genetic diversity was evaluated amongst 31 Bao rice germplasms of Assam using 38 SSR markers linked to Anaerobic Germination(AG). Assessment of allelic distribution showed three major groups in which, Rangdha Kekua Bao clustered with other four cultivars under cluster I while MaZhanRed and Khao Hlan On were grouped together with 12 of 38 Bao cultivars under major cluster II. Another half of Bao cultivars grouped together under cluster III separately indicating their distinctiveness. Regression test, validated only two markers viz.RM 553_170 linked to qAG 9, RM 5378_160 linked to qAG 2 that are significantly linked to the AG trait. These markers may be employed to evaluate the molecular diversity that can be used in aspects of plant genetics concerning germination studies under hypoxia. Present study also indicated that more markers are needed for lucid understanding of AG associated QTL and Marker assisted selection.
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
SCREENING OF HYDROGENASE ACCESSORY GENES hypA AND hypB IN FEW ACID TOLERANT BACILLUS SP
(AAU, Jorhat, 2017-07) POOJA, S; Barooah, Madhumita
The present investigation reports on the screening of hydrogenase accessory genes viz., hypA and hypB in previously isolated acid tolerant Bacillus sp. followed by differential expression study (pH 7.0 and pH 4.5) of the both genes in three selected strains and subsequent cloning of hypA and hypB gene of Bacillus megaterium into pET28b+ vector. A total of 50 previously isolated acid tolerant Bacillus strains were considered for screening of hypA and hypB genes. Among the 50 isolates, 10 isolates tested positive for hypA & hypB genes. Three isolates viz., B. megaterium (GG1), B. cereus (GG2) and Lysinibacillus fusiformis (GG11) were considered for further study due to their ubiquitousness and higher acid tolerance. Bacterial RNA was isolated followed by synthesis of cDNA. A q-RT-PCR was performed in the above mentioned isolates to analyse the expression level of the said genes in acidic condition. The expression of hypA and hypB genes were significantly (P ≤ 0.05) up-regulated at pH 4.5 than in pH 7.0 in all the 3 isolates viz., GG1, GG2, GG11. An 18 fold increase in the expression of hypA and a 6.1 fold increase in expression of hypB were observed in the isolate GG1 at pH 4.5 compared to pH 7.0. The expression of hypA and hypB genes in GG2 was found to be 4.6 and 3.8 fold higher respectively at pH 4.5 than pH 7.0. In GG11, hypA showed 5.3 fold and hypB showed 2.8 fold increased expression at pH 4.5 compared to pH 7.0. Maximum expression of both the genes was observed in Bacillus megaterium. These genes were further cloned into pET28b+ vector. The transformation frequency obtained for the hypA and hypB was 1.5 x 106 and 3.2 x 104 CFU/μg respectively. Successfully transformed colonies were further validated through colony PCR as well as PCR on isolated plasmid DNA. The cloned hypA gene was sequenced and BLAST analysis was performed. BLAST analysis revealed that the genes belonged to the respective genus. The results of the present study revealed that hypA and hypB genes may have role in conferring acid tolerance to soil bacteria.

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