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Central Agricultural University, College of Post Graduate Studies in Agricultural Sciences, Umiam

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2016 - 20173
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Subject: Biotechnology and molecular Biology × End date: 2017 × Type: Thesis × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Isolation and characterization of tissue specific promoter from Pigeon pea
    (College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2017-12) Verma, Satish Kumar M.; Tyagi, Wricha
    Pigeon pea belongs to legume family which has a specific physiology including organs like nodule and pod. The understanding of genes/transcripts involved in development of various organs of pigeon pea is limited. The present study was conducted to identify tissue specific transcripts from pigeon pea by ortholog-based approach and to isolate and characterize 5'UTR region of tissue specific transcript(s) from pigeon pea. An in silico search was conducted using phytozyme database and plant transcription factor database (PTFDB) separately for four tissues viz, seed, flower, leaf and pod. A total numberof 39 putative transcription factors (TFs) belonging to 18 different TFs families were identified for pod. The highest numbers of transcripts were observed for GATA and C2H2 (Cys (2) His (2)) zinc finger family members, and similarly, 54 TFs belonging to16 different TFs families were identified for flower tissue. Higher transcript numbers were observed for MYB (Myelobastosis), MIKC (MADS Intervening K-box Cterminal) family. Likewise, a total number of 92 TFs belonging to 28 different TFs families were identified for leaf tissues. In this case the highest number of TFs belonged to C2H2 and NAC (No Apical Meristem CUC domain) containing families. Similarly, a total number of 23 TFs with 12 different TFs families were identified expressing in seed were like MYB, NAC. Based on in silico analysis 10 transcription factors for each of the four tissues were identified. Out of the 10 transcription families (36280, 13238, 20528, 25279, 13634, 00220, 11572, 11926, 13354, 19191) identified for seed specific expression, five (00220, 11572, 11926, 13354, 19191) were targeted for RT-PCR using five different tissues (seed, flower, leaf, pod and stem). Simultaneously, members of auxin and TCP transcription family were also targeted for validation. Internal gene for studying tissue specificity in pigeon pea was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This gene was selected based on RT-PCR of ß-actin (ACT), (GAPDH), ubiquitin family (UBI), elongation factor 1-A (EF1A) and ß-tubulin (TUB) on five different tissues (seed, flower, leaf, pod and stem). Our RT-PCR data led to conformation of 2 transcripts, 00220 and 11572 as seed specific pigeon pea transcripts. While 00220 is a member of MADS (MCM1/AGAMOUS/ DEFIEIENS/SRF) transcription family,11572 is a member of TALE (Three Amino acid Loop Extension) transcription family.Thermal asymmetric interlaced PCR (TAIL PCR) approach led to identification of 229 bp 5' UTR (Untranslated Region) of 00220 transcript. Upon sequencing and analysis using Plant CARE database presence of cis-acting elements like ARE,CAAT-box, G-box, GT1-motif, Skn-1 motif, and TATA-box was detected, three unnamed motifs were also identified at -10 and -66 position. The transcripts identified and putative promoter isolated in this study after further characterization might generate useful information regarding development in pigeon pea.
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    ThesisItemOpen Access
    Molecular mapping for low light intensity tolerance using the contrasting rice genotypes of Eastern India
    (College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2017-05) Dutta, Suvendhu Sekhar; Rai, Mayank
    Rice (Oryza sativa L.) is the most important cereal crop of the Eastern and North-Eastern India. Low light induced problems include high tiller mortality at vegetative stage, reduction in spikelet number, spikelet sterility and reduced dry matter production. A set of contrasting genotypes in the NEHR mini core set during kharif-2014 and a set of 110 rice genotypes from different parts of eastern India were screened for low light intensity tolerance during kharif-2015. Total grain yield, spikelet fertility, biological yield, number of effective panicles, specific leaf weight and chlorophyll content were found to be the key traits response to low light intensity (30 % less than normal). A panel of forty six genotypes made up of top twenty three tolerant and susceptible genotypes each, based on field screening in kharif-2015 along with previously reported checks (Swarnaprabha- tolerant and IR 8-suceptible) was used for mapping study. By using the online rice databases, a set of ninety eight genes previously reported for light response were identified, which was then narrowed down to twenty. Molecular markers (forty five HvSSRs and newly designed twenty eight gene based primers) were used for molecular mapping for low light intensity tolerance. Standardization of these seventy three primers in a set of eight contrasting genotypes led to identification of fifteen HvSSR and two gene based polymorphic (CAU-CG-ILA1-3 and CAU-CG-RK3) primers. These polymorphic primers were run on the mapping panel. Marker-trait association studies based on t test and regression analysis revealed seven HvSSR and one gene based markers associated with the key traits viz., HvSSR01-66, HvSSR02-54 and CAU-CG-ILA1-3 with grain yield, HvSSR02-52, HvSSR06-56, HvSSR06-69 and HvSSR09-45 with spikelet fertility and with biological yield HvSSR02-44, HvSSR02-52, HvSSR06-69, HvSSR09-45. Expression analysis for the genes used for mapping (LGD1, PNH1, ILA1, CAB2R and LP2) was performed on a panel of eight contrasting rice genotypes grown under normal light and low light intensity (75 % of ambient) at two time points (one hour and two days). Relative expression of the five selected genes in the leaf next to flag leaf showed that there was significant down regulation of transcripts after one hour of low light treatment insusceptible genotypes for genes LGD1 and PNH1, whereas the transcript levels were maintained in tolerant genotypes. Transcription was significantly down regulated and up regulated, respectively for ILA1 and LP2 genes specifically in tolerant genotypes under one hour low light treatment. However, no significant differences in gene expression levels for the five genes were observed after two days of stress treatment for tolerant and susceptible genotypes. The genotypes and markers associated with traits identified in this study, after further evaluation, can be used to develop more productive rice varieties for low light affected regions.
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    ThesisItemOpen Access
    Understanding molecular biology of acidity tolerance in rice: a casestudy of Phosphorous (P) deficiency and Iron (Fe2+) toxicity tolerance in Shasharang
    (College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2016-09) Das, Sudip; Tyagi, Wricha
    P is one of the macronutrients required for plant growth and development, whereas Fe is a micronutrient required for various metabolic functions of plant. However, under acidic soil condition, due to low pH Fe2+ toxicity and P deficiency occurs leading to decrease in crop productivity. The molecular mechanism underlying the Fe toxicity and P deficiency tolerance in rice is not well understood. Therefore, current study was undertaken to identify the novel loci for Fe2+ toxicity and P deficiency tolerance using bi-parental population derived from the cross between a tolerant donor Shasharang (LR 11) and a susceptible recipient (KMR 3) genotype. The phenotypic data in terms of tolerance basis under lowland acidic soil condition and hydroponics, suggests that LR 11 was the most tolerant genotype out of four genotypes (KMR 3, LR 15 nd LR 60) used in our study in support. Positive and negative correlation was obtained for various traits upon evaluation of 225 F2 progeny derived from cross between KMR 3 and LR 11, at 1 and 5% level of significance. Positive correlation was found between tiller numbers at 60 days with plant dry weight (PDW) (0.642) and panicle number (PN) (0.984). PDW was positively correlated with PN (0.667) and filled grains per panicle (FGPP) (0.295). Bronzing score at 60 DAT (BS 60) was positively correlated with bronzing score at 110 DAT (BS110) (0.454). Interestingly, BS60 was negatively correlated with Fe content (0.21) at 5% level of significance. P content was positively correlated with P uptake (0.572) and negatively correlated with P use efficiency (PUE) and Fe content at both the levels of significance, respectively. P uptake was positively correlated with PUE (0.398) and Fe uptake (0.557) but negatively correlated with Fe content (-0.41). PUE was positively correlated with Fe uptake (0.623), Fe content with Fe uptake (0.619) at both 5% and 1% level of significance. Number of tillers at 60 days, plant dry weight, and number of panicles per plant showed significant positive correlation with grain yield per plant. Out of total of 377 SSR markers and 60 candidate gene based primers run on the parents, LR11 and KMR 3, eight and 60 candidate gene based and HvSSR markers, respectively were found to be polymorphic. Marker trait association study revealed significant association of five markers i.e. HvSSR06-46, HvSSR08-17, and HvSSR11-25, HvSSR05-12, HvSSR08-35 with PUE uptake at P value between 0.9-0.95. HvSSR03-26, HvSSR06-46, HvSSR06-54, HvSSR08-14, HvSSR08-35 and HvSSR12-08, were found to be correlated with PUE with r values ≥ 0.3. However, three markers, namely HvSSR01-47, HvSSR07-09 and HvSSR11-23 with P value between 0.9-0.95 were associated for PUP. HvSSR07-09, HvSSR11-12, and PR062-3 contributing for LR 11 and HvSSR08-17 and HvSSR10-21 for KMR 3 for the trait PUP. HvSSR06-09 with Fe content was found to be highly significant, whereas, HvSSR03-26, HvSSR09-30 and FR032-3 were less significantly associated with probability value between 0.9-0.95 for the trait Fe content. LR 11 alleles for three markers namely HvSSR03-26, HvSR11-27 and HvSSR12-08 were found to be associated with high Fe content in flag leaf, whereas, for HvSSR08-14 and HvSSR10-27, KMR 3 alleles showed association with high Fe content. These markers could be used for molecular assisted breeding programme for Fe2+ toxicity tolerance as well as P deficiency tolerance after further validation. Under Fe (II) toxic conditions, OsYSL16 was upregulated in shoots in susceptible genotype (KMR 3) and downregulated in roots of tolerant genotype (LR 11). OsNAS3 was downregulated in shoots in susceptible genotype under Fe(II) toxic conditions.Whereas, transcription factors OsIRO3 and OsIRO2 were downregulated in shoots of susceptible genotype and expressed only in shoots of tolerant genotypes, respectively. This study, therefore, suggests that genes reported for iron deficiency tolerance can be potential targets for enhancing rice production under P deficiency and iron toxicity field conditions. Our data also suggests that maintaining Fe homeostasis under Fe toxicity and P deficiency conditions as shown by the genotype, LR 11, could be vital to better performance under poor soil conditions.