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Dr. Rajendra Prasad Central Agricultural University, Pusa
In the imperial Gazetteer of India 1878, Pusa was recorded as a government estate of about 1350 acres in Darbhanba. It was acquired by East India Company for running a stud farm to supply better breed of horses mainly for the army. Frequent incidence of glanders disease (swelling of glands), mostly affecting the valuable imported bloodstock made the civil veterinary department to shift the entire stock out of Pusa. A British tobacco concern Beg Sutherland & co. got the estate on lease but it also left in 1897 abandoning the government estate of Pusa.
Lord Mayo, The Viceroy and Governor General, had been repeatedly trying to get through his proposal for setting up a directorate general of Agriculture that would take care of the soil and its productivity, formulate newer techniques of cultivation, improve the quality of seeds and livestock and also arrange for imparting agricultural education. The government of India had invited a British expert. Dr. J. A. Voelcker who had submitted as report on the development of Indian agriculture. As a follow-up action, three experts in different fields were appointed for the first time during 1885 to 1895 namely, agricultural chemist (Dr. J. W. Leafer), cryptogamic botanist (Dr. R. A. Butler) and entomologist (Dr. H. Maxwell Lefroy) with headquarters at Dehradun (U.P.) in the forest Research Institute complex.
Surprisingly, until now Pusa, which was destined to become the centre of agricultural revolution in the country, was lying as before an abandoned government estate. In 1898. Lord Curzon took over as the viceroy. A widely traveled person and an administrator, he salvaged out the earlier proposal and got London’s approval for the appointment of the inspector General of Agriculture to which the first incumbent Mr. J. Mollison (Dy. Director of Agriculture, Bombay) joined in 1901 with headquarters at Nagpur
The then government of Bengal had mooted in 1902 a proposal to the centre for setting up a model cattle farm for improving the dilapidated condition of the livestock at Pusa estate where plenty of land, water and feed would be available, and with Mr. Mollison’s support this was accepted in principle. Around Pusa, there were many British planters and also an indigo research centre Dalsing Sarai (near Pusa). Mr. Mollison’s visits to this mini British kingdom and his strong recommendations. In favour of Pusa as the most ideal place for the Bengal government project obviously caught the attention for the viceroy.
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ThesisItem Open Access Effect of Heat Stress on Tissue Culture Response in Wheat Genotypes(DRPCAU, Pusa, 2019) Raj, Anant; Kumar, HarshThe effect of heat stress on tissue culture responses like callogenesis and organogenesis including green structures formation and rhizogenesis was studied in four wheat genotypes Sonara-64, PBW-343, HD-2967 and SIDS. Media were identified for callus formation and organogenesis from cultured mature embryos, the best medium for callus formation was MS + 4mg/l 2,4-D and for organogenesis MS + 1mg/l BAP + 0.5gm/l NAA. Mature embryos from pre-heat treated seeds were induced to develop callus and on the basis of calculated heat susceptibility index (HSI) and heat tolerance index (HTI), the genotype HD-2967 was the most heat tolerant followed by PBW-343. Genotype SIDS was the most heat sensitive followed by Sonara-64. The formed calli under controlled and heat stress were further pre-heat treated and cultured on differentiating medium for organogenesis. Genotype HD-2967 showed the best response followed by PBW-343, Sonara-64 and SIDS respectively under heat stress indicating their relative heat tolerance. Thus based on tissue culture responses under heat stress genotype HD-2967 and PBW-343 were heat tolerant and Sonara-64 and SIDS were heat sensitive. Molecular characterization study of eleven genotypes resulted in successful amplification with 12 SSR primer pairs. A total of 79 allelic variants were detected with an average of 6.07 alleles per locus and total 46 unique alleles were observed with an average of 3.53 unique alleles per locusat 13 SSR loci. The highest number Name of Student : Anant Raj Admission No./Registration No. : M/AB/199/2017-18 MajorAdvisor : Dr. Harsh Kumar Degree to be awarded : M.Sc.(Ag) in Agricultural Biotechnology Major Subject : Agricultural Biotechnology Minor Subject : Plant Breeding & Genetics Year : 2019 Total pages of the Thesis : 70+i-xiv (Bibliography) Title of the research problem : “Effect of heat stress on tissue culture response in wheat genotypes” of alleles per locus was observed in the B genome (38 alleles) as compared to A (24) and D (17) genomes. Polymorphism information content (PIC) value ranged from 0.327 for the primer pairXgwm160 to 0.878 for the primer pairXgwm 577with an average of 0.764. The similarity coefficient was the maximum between HD-2967 and BWL-9022 (0.519) and the minimum (0.0) infour pair-wise combinations HD-2967 with Purulla, PBW-343 with Sonalika, BWL-9022 with Sonalika and KSG-1186 with Sonalika. It ranged from 0.0 to 0.519 indicating the presence of wide range of genetic diversity at molecular level among the genotypes. Dendrogram was constructed using Dice similarity coefficients. The entries were divided into three groups on the basis of drawing phenon line at 0.20 coefficient of similarity level.Cluster A grouped together highly heat tolerant genotypes (PBW-343, HD-2967, BWL-9022 ), cluster B grouped moderately heat tolerant genotypes (Purulla, Seri-82, SALEMBO, KSG-1186 ) except CIANO-T-79 ( moderately heat susceptible) and cluster C grouped together highly heat susceptible genotypes (Sonara-64, SIDS, Sonalika) of wheat. Thus, molecular characterization revealed that genotypes under study were having genetically different levels of heat tolerance.ThesisItem Open Access Study on cloning of Starch Synthase III gene in wheat(Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2019) Kumar, Vishnu; Kumar, RajeevBread wheat is one of the primary sources of energy and proteins for millions of people world over. Starch is the most critical food energy source in the world and constitutes 65% to 80% proportion of the wheat grain weight. Starch synthase III is directly associated with starch accumulation in wheat. Hence, the present investigation entitled “Study on cloning of Starch Synthase III gene in wheat” was conducted to clone the full length of starch synthase III genes. The work includes detailed In-silico characterization of genome-specific TaSS-III genes in wheat, covering positions of exons, introns, location of the genes in chromosomes, phylogenetic analysis, protein’s domain analysis, and expression analysis under heat stress conditions. The genomic DNA of heat susceptible PBW-343 and tolerant KSG-1186 genotypes was used for PCR amplification of TaSS-IIIa1D gene using gene specific primers, and using pJET1.2/blunt cloning vector, and then sequenced to detect the SNPs. Two homologs of the TaSS-III genes, TaSS-IIIa and TaSS-IIIb, were found on the plus strand of chromosome 1 (1A, 1B & 1D) and minus strand of chromosome 2 (2A, 2B &2D), respectively. All homeologous copies of the gene contained 16 exons. Out of which, 3rd was the largest (1698-2757 bp) and 2nd the smallest (64 bp). Besides, 15 introns were identified in the gene. Among which 1, 5, and 8 were longer (391-2906 bp, 447-910 bp, and 591-899 bp), and 6, 13, 14, and 15 were shorter (75-114 bp, 83-99 bp, 87-90 bp, and 81-118 bp) in size. Exon 1 and 3 of the homologous and homeologous copies of the genes exhibited maximum variation. Introns were found relatively more variable than the exons. The average length of the total intronic region of the genes was estimated slightly longer than that of its coding sequence. TaSS-IIIa1B, TaSS-IIIa1D, and TaSS-IIIb2A contained 3 splice variants, TaSS-IIIb2B & TaSS-IIIb2D contained 2 splice variants and TaSS-IIIa1A contained only a single transcript. Phylogenetic analysis showed that copy of the gene present on the 1st chromosome (1A, 1B & 1D) share maximum similarity with HvSS-IIIa followed by BdSS-IIIa, OsSS-IIIa, SbSS-IIIa, and ZmSS-IIIa whereas, TaSS-IIIb exhibited maximum similarity with OsSS-IIIb followed by ZmSS-IIIb and SbSS-IIIb respectively. Maximum dissimilarity for SS-III genes was found between monocots and dicots. For both TaSS-III genes, the sequences found on A and D genomes were more similar than that of the gene on the B genome. Domain analysis revealed that the glycosyltransferase (GT) domain was most conserved among all the domains. Three SBDs were found in each of the homeologous copies of TaSS-IIIa & TaSS-IIIb protein, wherein the positions of tryptophan amino acids were found conserved. Expression analysis of both copies showed that TaSS-IIIb expresses in the tissues viz. leaf, stem, root, spike, & grain and in much higher amounts than TaSS-IIIa. Whereas, TaSSIIIa expression was highly specific to endosperm in the grain. The expression of the TaSS-III genes reduces due to heat stress. Sequencing of the products of direct PCR and indirect vector cloning showed that in-vitro amplification and in vivo amplification products have no variation. A total of 49 SNPs were identified in 10,529bp of the TSS-IIIa1D gene between the PBW-343 and KSG-1186 genotypes. Twenty-nine specific SNPs were identified in heat-sensitive genotype (PBW-343), and 20 specific SNPs were identified in the heat-tolerant genotype (KSG-1186). There were 14 intronic and 15 exonic SNPs contributing to 18 transitions and 9 transversions in the PWB-343 genotype, reflecting the transition bias. While in genotype KSG-1186, 9 transitions, 9 transversions, and two deletions are contributing to 6 intronic and 14 exonic SNPs showing no such bias. Maximum SNPs were detected in 3rd and 8th exons of PBW-343, whereas in genotype KSG-1186, only 3rd exon contained maximum SNPs. Exon 3 was found to be evolutionarily highly variable among all monocots and dicots taxa. Between PBW-343 and KSG-1186, 18 SNPs consisting of 11 transitions, and 7 transversions were found, reflecting the transition bias. Seven SNPs found associated with SBD-1, SBD-2 and SS-CD domains of the TaSS-IIIa1D protein. In SBD-1, one non-synonymous and one synonymous mutation were observed in both PBW-343 and KSG-1186; in SBD-2, one non-synonymous mutation was observed in KSG-1186 whereas, one non-synonymous and one synonymous mutation were observed in SSCD of PBW-343.ThesisItem Open Access IN-SILICO AND MOLECULAR CHARACTERIZATION OF YSL1 GENE IN WHEAT(Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2019) Panigrahi, Sourav; Kumar, RajeevWheat is the staple food for the majority of people in the world and thus forms an important component of human nutrition and hence its bio-fortification concerning mineral nutrients is a major concern in the scientific world. In this study, the biofortification of wheat with respect to grain Fe and Zn content has been taken into consideration. Twenty-five wheat genotypes were sown under normal and late sown conditions (heat stress). Estimation of Fe and Zn content in wheat grains was done by AAS after wet digestion by Nitric acid: Perchloric acid:: 1:3. The data about Fe and Zn content in grains of wheat sown under normal and late conditions were analyzed statistically and it was found that there was a reduction of Fe and Zn content in the late sown than the normal sown conditions in most of the genotypes under study. Contrarily, 5 genotypes showed increased Fe accumulation and seven genotypes showed increased Zn accumulation in wheat grains under late sown condition, thus exhibiting negative HSI values. This distribution of Fe and Zn content over two dates of sowing was subjected to cluster analysis and were classified into three groups Fe content and 4 groups based on Zn content in seed. The YSL gene family plays an important role in Fe and Zn homeostasis into the grains. In this study, all 63 members of the YSL gene family were identified in the wheat genome. Out of all the TaYSL genes, TaYSL2, in particular, was characterized in silico and was found to be expressed in the spike and involved directly in metal-NA loading into the seeds. Three homeologous copies of the gene TaYSL2 namely TaYSL2-6A, TaYSL2-6B and TaYSL2-6D, were identified on the 6th chromosome of each of the three genomes. 6 introns and 7 exons were found in each of the homeologous copies of the gene. In-silico expression of TaYSL2-6A increased many folds under prolonged drought conditions, TaYSL2-6B increased many folds under heat stress and combined stress of heat and drought increases the expression of TaYSL2-6B to many folds and TaYSL2-6D significantly. TaYSL2 gene was taken up for molecular characterization using gene-based genome-specific primers as well as gene-based SSRs primer in 12 genotypes selected from the varied Fe and Zn content data. Amplification was obtained successfully in all the designed primers. A total of 67 alleles including 37 unique alleles, were obtained from all the primers. The PIC value of primers ranged from 0.542 to 0.904. Maximum polymorphism was observed in B genome followed by A and D genomes respectively. Dice's similarity coefficient computed from the molecular data analysis ranged from 0.821 to 0.672. Based on the similarity coefficient, the genotypes were classified into 4 clusters and were successfully correlated with the Fe and Zn content data.ThesisItem Open Access Study on QTL analysis for heat stress response in wheat using doubled haploid population(RPCAU, PUSA, Samastipur, 2019) Pankaj, Yaswant Kumar; Kumar, RajeevThe inheritance of tolerance to high temperature stress at grain yield was investigated via a QTL analysis based on 167 doubled haploid progeny of a cross between the cultivar PBW 343 and KSG 1186. Performance data were collected from three different date of sowing in the month of 28 November, 22 December and 15 January, followed by almost one month gap period at the farm of Rajendra Prasad Central Agricultural University, Pusa, Bihar. Significant differences were observed among the genotypes for all the traits under consideration namely grain yield and canopy temperature. Exploitable extent of correlation amongst the entries between both the characters for different date of sowing was present as revealed by considerably higher estimates correlation coefficient up to the value of (R = 0.88). Using composite interval mapping, a total no. of eight QTLs were identified for both the characters viz. canopy temperature and grain yield. QTL.ct2-6B was flanked by the marker barc127-cfd135 was having largest LOD score of 4.36 with the highest phenotypic variance (R2 = 23.28%). QTL.yld2-1A was flanked by gdm33-gwm136 was having the lowest LOD score of 2.54 with the lowest phenotypic variance (R2 = 11.66%). This scenario has indicated that LOD score and phenotypic variance are proportional to each other. Two QTLs viz. QTL.ct1-2A and QTL.ct2-6B related to canopy temperature has sown negative additive effect with the value of -0.92 and -1.20. The reason behind the negative additive effect is the mostly alleles contributing from the poor parent PBW 343 which is susceptible to heat stress. These information's can be used further for fine mapping and marker assisted selection in the near future.ThesisItem Open Access Expression analysis for virulence causing gene of Bipolaris sorokiniana(Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2019) Deuri, Priyanka; Kumar, MithileshWheat [Triticum aestivum (L.) is a most important cereal crop belonging to the tribe Triticeae of the Poaceae family. Wheat is not only staple food but has a increasing global demand for wheat based product. Spot blotch of wheat, caused by Bipolaris sorokiniana (Sacc.) Shoemaker (syn. Helminthosporium sativum teleomorph: Cochliobolus sativus), is a serious constraint to wheat production (Triticum aestivum L.). The pathogen is seed and soil borne. Pusa in Bihar is considered as a hot spot for the disease. Fourteen fungal isolates were collected from infected leaves and seeds of different wheat genotypes grown at the research farms of DRPCAU pusa, TCA Dholi campus, BISA, IARI Regional Station at Pusa .They were characterized morphologically on the basis of colony colour, growth pattern and exudation, and expression analysis of virulence gene ToxA and melanin biosynthesis genes PKS1 and SCD1 on six different morphological groups of isolates. The isolates were divided into six morphological groups on the basis of their colony colour namely-black, grey, brownish black, greenish black, olive and dull white, among which the frequency of black was the maximum on natural papulation. To examine the relative gene expression among the isolates, RNA was isolated then first strand c-DNA synthesized by reverse transcription process. The RT-PCR analysis exhibited maximum expression of ToxA and second highest expression of PKS1 genes in brownish black type isolates suggesting most virulent type of isolates among the other morphological isolates. Relative expression of PKS1 showed highest level in black isolates and SCD1 showed second highest although highest level was recorded in greenish black type isolates. All the three target genes showed lowest expression in dull white type and considered as least virulent.ThesisItem Open Access Candidate gene markers based molecular profiling for grain zinc accumulation in rice(Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2018) Kumari, Kumkum; Sharma, V.K.A study was conducted to determine the genetic variation and divergence in relation to grain zinc accumulation amongst rice varieties using candidate gene based panel of reported primers and to examine the genetic importance of zinc transporter candidate gene based panel of designed primers in discrimination for differential zinc accumulation amongst rice varieties. Twenty-eight locally adapted varieties and advanced breeding lines of rice were evaluated in randomized block design with three replications and the seeds collected after harvesting of the crop were utilized for determination of zinc content in unpolished grains. Eighteen entries selected from the two extremes of grain zinc distribution range constituted the final experimental material and utilized during molecular characterization. Genomic DNA was extracted from two to three weeks old seedlings of purposefully selected set of 18 varieties and then targeted amplification of the genomic DNA was achieved by using a panel of 14 candidate gene specific 14 reported primers and 14 designed primers. Exploitable extent of variability was observed with respect to grain zinc accumulation amongst the set of 28 rice varieties initially evaluated as experimental materials. Zinc content, which varied from 8.18 ppm to 21.53 ppm, was found to be considerably higher in unpolished grains of RAU 3036, Sanwal Basmati, Rajendra Nilam and Rajendra Bha gwati. Using a panel of 14 candidate genes specific 14 reported primer pairs, reproducible amplification was successfully achieved with 12 primer pairs amongst which only eight primer pairs generated polymorphic amplified products. Successful amplification with two candidate genes specific reported primers, namely, OsNAC and OsNRAMP6a was notachieved. Contrarily, each of the 14 designed primer pairs exhibited reproducible amplification, but polymorphic amplified products were generated with only eight primer pairs. Appearance of amplified products in the form of bands at different positions on the gel revealed differential migration due to differences in overall size of the products generated from targeted amplification of specific region of genome. Molecular level genetic polymorphism among the entries was recognized on the basis of variation in respect of position of bands. Ample genetic differentiation and divergence was revealed at the molecular level amongst the rice varieties subjected to molecular characterization using the candidate genes specific and polymorphic panels of reported as well as designed primer pairs. Results from reported primers and designed primers based analysis were in well agreement with each other. Furthermore, hierarchical classification pattern of rice varieties was almost completely corroborated by principal coordinate analysis based spatial distribution pattern of genetic profiles of rice varieties. Hierarchical cluster analysis as well as principal coordinate analysis based on a combination of polymorphic and informative eight reported and eight designed primer pairs provided better expression of differentiation and divergence amongst the rice varieties subjected to molecular characterization. Thus, the use of 14 candidate genes specific 16 polymorphic markers in the genetic analysis exhibited a remarkably higher level of genetic polymorphism, which allowed unique genotyping of eighteen entries included in the analysis. Hence, these markers can be effectively and efficiently utilized for grain zinc accumulation related discrimination of rice genotypes and selection of parental genotypes for genetic improvement in relation to grain zinc biofortification. Microsatellites were detected within the candidate genes and within the amplicons, thereby providing a basis to deduce that the variation present in candidate genes, as observed in terms of differences in the molecular size of the genomic regions spanned by the primer pairs, may be a role player in the differential grain zinc accumulation in rice varieties. Single marker analysis established the association of four markers, namely, OsNACK, OsZIP1-1, OsNRAMP7 and OsNRAMP7K with grain zinc accumulation. These four markers can be effectively used in marker-assisted selection program for grain zinc biofortification in rice. Inter-crossing diverse genotypes from different clusters can lead to successful pyramiding of desirable alleles through molecular breeding program. Parental genetic diversity will undoubtedly increase the probability of identifying desirable recombinants during screening for improvement in relation to grain zinc biofortification.ThesisItem Open Access Characterization and micropropagation of Physalis minima L. in Bihar(DRPCAU, Pusa, Samastipur, 2018) Kumari, Anjani; Kumar, HarshA study was undertaken to characterize populations of Physalis minima L. collected from the seven locations of Bihar. A list of 47 morphological characters were used to score the variations present in the plants. Qualitative and quantitative estimation for the presence of phytochemicals was done to determine the medicinal and nutritive value of the plant. Different populations were also characterized on the basis of their molecular study using ISSR and SSR markers. The superior plant populations were identified on the basis of these studies. The micropropagation of the plants was carried out for their multiplication and maintaining superior characters and also prevent the natural hybridization. The morphological characterization based on 24 qualitative and 13 quantitative characters revealed the presence of variations within and between populations. The variations not reported before were observed, which lead to the identification of new forms of the Physalis minima L. plants in Bihar. The quantitative morphological traits were found to be significantly correlated. The cluster analysis and Principal Component Analysis successfully grouped the populations into three groups. The presence of eight phytochemicals were detected in leaves, stem, fruits and roots of the plants from all the seven populations by using thirteen qualitative tests. The quantitative estimation revealed the presence of a high amount of total soluble sugar, ascorbic acid, protein, phenols, flavonoids and alkaloids in leaves and fruits of the seven populations. The populations showed significant variations in their phytochemical constitution. The dissimilarity coefficient and Principal Component analysis based on quantitative biochemical characters clustered the populations in three groups similar to the groups identified by morphological characterization. The biochemical profile highlighted the fruit and medicinal value of the plant. Three out of the eight SSR markers used in the study failed to achieve amplification in the forty two plants of Physalis minima, suggesting a lack of transferability of these primers. A total of 50 alleles were detected using the five SSR primer pairs. The plants were effectively diversified using the SSR primer based similarity coefficients and Principal Coordinate Analysis. The plants were grouped in three clusters having the plants from different populations. A total of 574 alleles were detected using fifteen ISSR primers. The results of ISSR marker based similarity coefficient and Principal Coordinate Analysis showed corresponding results. The plants were again grouped into three clusters. The similarity coefficients and Principal Coordinate Analysis based on both the markers revealed a similar result, suggesting the applicability of both the markers in scoring the genetic variation present in the Physalis minima L. plants. A high level of genetic heterogeneity was identified in the populations based on molecular studies. The results of the three studies were found to be related suggesting that the variations in morphological and biochemical parameters are a result of the genetic variations. The Selao was identified as the superior population based on the score of biochemical characters. Since the Physalis minima L. populations are highly heterogeneous, there is a need for purification of the populations which can lead to the development of superior genotypes. The tissue culture responses were found to be independent of the effect of population. The shoot apical bud followed by nodal stem were identified as the best explant for most of the tissue culture response except swelling and callogenesis for which leaves were the most effective. MS 30 (1 mg l-1 BAP and 1 mg l-1 2, 4-D) was identified as the best medium for differentiation of multiple shoots in shoot apex and nodal stem while MS 2 (1 mg l-1 BAP) was the best for leaves. MS 7 (1 mg l-1 KIN) was invariably identified as the best medium with the highest number of shoots per explant for all the explants. The leaves were the best explant for callogenesis. The in vitro flowering and fruiting were obtained from shoot apex and nodal stem culture. MS 32 (1.0 mg l-1 BAP and 1.5 mg l-1 2, 4-D) for flowering and MS 30 for fruiting was identified as the best medium for both the cultures. R3 (MS + 1 mg l-1 NAA) was identified as the best medium for rooting. The in vitro studies led to the development of a highly efficient protocol for micropropagation of Physalis minima L.ThesisItem Open Access In vitro morphological and molecular evaluation of rice genotypes and validation of markers for salinity tolerance(DRPCAU, Pusa, Samastipur, 2018) Kumari, Rima; Kumar, HarshA set of thirty rice varieties including two tolerant (Pokkali and CSR-36) and two susceptible (IR-29 and IR-64) checks were screened for assessment of their salt tolerance at early seedling stage on the basis of in vitro seed germination and seedling growth at different salinity levels (0, 4, 8 and 16 dS m-1) created by application of salt mixture consisting of NaCl, CaCl2, Na2SO4 in 7:2:1 ratio. Seed germination and seedling growth were adversely affected with the increase of salt concentrations. The salt tolerance index (STI) of the rice varieties was calculated on the basis of seed germination, seedling shoot and root dry weights at different levels of salt stresses. Phenotypic grouping of the thirty rice varieties for their salt tolerance level on the basis of overall salinity tolerance indices across in vitro seed germination and seedling growth (shoot and root dry weight) under salt stress clearly reflected that seventeen varieties, namely, Pokkali, CSR-36, Mandakini, Kranthi, Jyothi, Bardhan, Pusa Sugandh-2, Duna Sankhi, Sanwal Basmati, Ratnagiri-4, Shanthi, Rajendra Dhan-102, Sahbhagi Dhan, Vaisak, Annada, Badami and Jyotrirmayee were highly tolerant to salt stress. Among the remaining entries, Pusa-834, Sarsa, Govind, Khira, Pusa Sugandh-5, MTU-7029 and Saraswathi were found to be moderately salt tolerant, whereas varieties IR-29, IR-64, Daya, Kalinga-3, Golaka and Shatabdi were highly susceptible to salt stress. Out of thirty rice varieties, eighteen rice varieties, namely, Pokkali, CSR-36, IR-29, IR-64, Mandakini, Pusa Sugandh-2, Saraswathi, Ratnagiri-4, Rajendra Dhan-102, Sahbhagi Dhan, Badami, Sarsa, Jyotrirmayee, MTU-7029, Golaka, Daya, Vaisak, and Shatabdi were further screened with the help of ISSR, SSR, salt stress responsive candidate genes and EST primer pairs for the purpose of their molecular profiling in relation to their salinity tolerance. Genetic profiling of entries with a panel of 14 ISSR markers generated altogether 483 allelic variants including 236 shared and 247 unique alleles with an average of 34.50 alleles per primer, revealing ample extent of genetic differentiation and divergence amongst the entries. Among these markers, 811, 814, 815, 823, 834, 836, 840, 841, 842, 872 were found to be highly polymorphic and informative on the basis of their PIC and PP values. However, using a panel of salt stress response related 24 SSR primer pairs, altogether 205 allelic variants including 114 shared and 91 unique alleles were detected with an average of 8.54 alleles per primer due to length variation of simple sequence repeats. Simple sequence repeat loci with di-nucleotide and tri-nucleotide repeat motifs detected greater number of alleles than the repeat loci with tetra-nucleotide and complex repeat motifs. Additionally, the simple sequence repeat loci with CT, GT, AT, AG and AC di-nucleotide repeat motifs detected greater number of alleles. Contrarily, the loci with GA and CA di-nucleotide repeat motifs appeared to detect relatively lesser number of alleles. Considering the number of alleles generated in conjunction with the level of polymorphism detected in the present study, the primers RM 302, RM 8094, RM 10665, RM 10694, RM 10748 and RM 10825 appeared to be highly polymorphic and comparatively more informative primers. Six SSR primer pairs, namely, RM 140, RM 1287, RM 3412, RM 10745, RM 10764 and RM 10772 were validated on the basis of their efficiency to distinguish salt tolerant varieties from susceptible varieties. These six primers can be utilized for the purpose of genetic differentiation and discrimination in relation to salt stress responsiveness of the rice genotypes. Similarly, microsatellite containing salt stress responsive candidate gene (OsHKT1;5, SNAC1, CDMK, CCC, SHMT1 and SHMT2) and microsatellite lacking salt stress responsive candidate gene (OsHKT1;1, OsHKT1;3, OsHKT2;3 and OsHKT2;4) specific markers based genetic profiling allowed unambiguous discrimination of salt stress responsive and tolerant entries, validating their utility for the purpose of differentiation and discrimination of salt stress sensitive and tolerant varieties. Principal coordinate analysis completely supported the results obtained from hierarchical classification of the varieties. Using a panel of eight salt stress responsive EST-contigs based markers, monomorphic bands amongst all the 18 varieties were recorded for six markers, namely, Contig2 (Ferritin superfamily), Contig54 (Plant peroxidase superfamily), Contig138 (ATPase expression protein), Contig314 (Exonuclease), Contig545 (Major latex protein) and Contig633 (Protein kinase), revealing genetic similarity with respect to primer binding sites and molecular size of targeted genomic regions. Remaining two markers specific to Ferritin superfamily (Contig43) and Microtubule associated protein (Contig215) genes revealed genetic polymorphism in the form of presence or absence of bands. Thus, combining in vitro morphological and molecular assessment, seventeen varieties were considered as salt tolerant varieties which can be used as parental donor in rice breeding programme to develop salt tolerant rice varieties.ThesisItem Open Access Molecular dissection of quantitative trait locus in relation to drought tolerance in rice(DRPCAU, Pusa, Samastipur, 2019) Kumar, Pankaj; Sharma, V.K.Thirty-one genotypes of rice were evaluated for various morpho-physiological characters and to study the microsatellite markers based polymorphism for their characterization and genetic diversity among these rice genotypes. Identification of putative gene within QTL (qDTY1.1) and their expression profiling for enhance the drought tolerance. Experimental materials were estimated in randomized block design with three replications during two consecutive years. Molecular characterization was done of the genomic DNA using a set of 23 microsatellite primer pairs. Gene expression response of drought tolerant (Nagina22 and Sahbhagi dhan) and drought sensitive (IR64) rice genotypes to 15 days of drought stress in reproductive stage. Statistical methods and parameters used for deriving inference were analysis of variance, range, mean values, pooled analysis, relative mean performance, and drought tolerance indices. An analysis of variance revealed significant differences among the genotypes for all the characters evaluated during present study. On the basis of the relative mean performance of 15 genotypes, namely, Sahbhagi, Nagina22, MTU1010, Silhatidhan, RAU1417-11-2, Rajendra Bhagwati, RAU1415-7-1, Anandidhan, Rajendra Nilam, Aus257, IR7343-71-1, Chengari2, Kali-Aus, Dangar and Dular out of 31 genotypes were appeared to be relatively more drought tolerant than other genotypes under evaluation. Four tolerance indices viz. TOL, SSI, DTE and STI were used. On the basis of stress tolerance level, stress susceptible index, stress tolerance index and drought tolerance efficiency, 18 genotypes, namely, IR7343-71-1, Chengari2, Aus257, MTU1010, Dangar, Anandidhan, Silhatidhan, Dular, Kali-Aus, Nagina22, Sahbhagidhan, Rasi, Vandana, R.Nilam, R.Bhagwati, RAU1421-12-4, RAU1417-11-2 and RAU1415-7-1 out of 31 genotypes were identified as relatively more tolerant to drought stress. A total 179 and 73 unique allelic variants were detected among the eighteen rice genotypes with an average of 7.8 alleles per primer. The primers RM 5359, RM 7025, RM 10772 and RM 11008, appeared to be highly polymorphic and comparatively more informative primers. Analysis of divergence pattern based on microsatellite markers allowed differentiation and classification of rice genotypes into two groups. The first multi-genotypic group consisted of ten genotypes, namely, IR7343-71-1, Chengar2, Aus257, MTU1010, Dangar, Anandidhan, Silhatidhan, Dular, Kali-Aus and Nagina22 whereas the second multi-genotypic group consisted of eight genotypes, namely, RAU1401-18-5, RAU1428-54-5, RAU1421-15-7, RAU1421-12-4, RAU1417-9-3, Sahbhagidhan, RAU1397-18-7 and Rasi. The magnitude of similarity coefficient between Anandidhan and Dangar (0.739) was found to be the maximum which indicating that these were more closely related. A set of microsatellite markers used for molecular characterization in the present study showed very high degree of efficiency in discrimination of genotypes in relation to drought tolerance. In this study we have identified 73 genes within QTL (qDTY1.1) using sequence information available in rice genome database and bioinformatics tools. The expression of identified genes were analysed. Our studies showed that two genes namely LOC_OS01g66520 and LOC_01g 66170 were upregulated consistently in the time point after drought imposed. Most of the genes were in downregulated.