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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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Subject: Agricultural Biotechnology × End date: 2018 × Start date: 2018 ×
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    ThesisItemOpen 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.
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    ThesisItemOpen Access
    Characterization and micropropagation of Physalis minima L. in Bihar
    (DRPCAU, Pusa, Samastipur, 2018) Kumari, Anjani; Kumar, Harsh
    A 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.
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    ThesisItemOpen Access
    In vitro morphological and molecular evaluation of rice genotypes and validation of markers for salinity tolerance
    (DRPCAU, Pusa, Samastipur, 2018) Kumari, Rima; Kumar, Harsh
    A 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.
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    ThesisItemOpen Access
    Genome scanning for differential grain iron accumulation in rice using candidate gene markers
    (DRPCAU, Pusa, Samastipur, 2018) Arjun, Kale Rohan; Sharma, V.K.
    Analysis of functional polymorphic markers provide ace in diversifying the selective genotypes that may stand useful in biofortification programme for improving the genotypes in relation to their micronutrient composition. A study was conducted based on this consideration to evaluate the molecular level genetic divergence and to investigate the genomic regions and genes associated with natural variation of grain iron concentration among purposefully selected landraces, advanced derivatives and improved varieties of rice. With this objective, 28 locally adapted landraces, improved varieties and advanced breeding lines were evaluated in randomized block design with three replications and the grains collected after harvesting of the crop were utilized for determination of iron content in unpolished grains. The digestion was carried out by di-acid mixture which included nitric acid and perchloric acid (9:4) and iron content was determined with the help of atomic absorption spectrophotometer. Eighteen entries selected from the two extremes of grain iron distribution range were utilized during molecular characterization. Genomic template was extracted from two to three weeks old seedlings of these purposefully selected varieties and then targeted amplification of the genomic regions was achieved by employing a panel of six candidate genes specific 18 primers and a panel of candidate genes based 12 microsatellite primers. Candidate gene specific primers were designed using primer blast tool for finding specific primers sequence of the identified candidate gene and appropriate primer sequences were selected for their utilization during molecular profiling. Exploitable genetic variability was observed in relation to grain iron concentration amongst 28 locally adapted landraces, varieties and advanced breeding lines of rice. Using the standard deviation for the range of variation (16.39 to 39.04 ppm) as the criterion, the rice varieties were classified into very low, low, moderate, high and very high grain iron containing groups. Putative candidate genes under investigation in the present study were searched for the presence of microsatellite sequences within the candidate gene sequences using microsatellite identification tools. Altogether 40 microsatellites were detected within the six putative iron transporter candidate genes. These microsatellites had dinucleotide to hexanucleotide repeat motifs. Only two microsatellites were detected in OsNRAMP1 and OsZIP8, whereas 18 microsatellites were detected in OsNRAMP5. Experimental results provided a basis to deduce that the variation present in candidate genes, as revealed 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 iron accumulation in rice varieties. Using a panel of six candidate genes specific 18 primers and 12 microsatellite primers, reproducible amplification was successfully achieved in the purposefully selected rice varieties. All the primers generated polymorphic amplified products. While each one of the eight polymorphic candidate gene specific primers, namely, APRT1a, APRT1b, APRT1c, OsNAC5a, OsNAC5c, OsNRAMP1c, OsZIP10a and OsZIP10c, generated three allelic variants, each one of the remaining ten polymorphic primers, namely, OsNAC5b, OsNRAMP1a, OsNRAMP1b, OsNRAMP5a, OsNRAMP5b, OsNRAMP5c, OsZIP8a, OsZIP8b, OsZIP8c and OsZIP10b, detected two allelic variants. Differential amplification pattern was also exhibited by candidate genes specific 12 microsatellite primers. While some of the primers generated several markers, some generated only few allelic variants. Altogether 72 allelic variants were detected among the 18 entries with an average of 6.0 alleles per primer. Polymorphic information content of candidate gene specific primers ranged from 0.278 to 0.710 with an average value of 0.483, while that of candidate genes specific microsatellite primers ranged from 0.154 to 0.864 with an average of 0.690. Sizable molecular level genetic differentiation and divergence was revealed amongst the rice varieties using the candidate gene specific primers as well as candidate genes specific microsatellite primers. Hierarchical classification pattern based on similarity coefficient matrix of pair-wise combinations of entries, which were accommodated into different clusters, was highly consistent with principal coordinate analysis based spatial distribution pattern of genetic profiles. Hierarchical cluster analysis as well as principal coordinate analysis using candidate gene markers as well as candidate genes specific microsatellite markers enabled differentiation and classification of entries with remarkably higher level of consistency in relation to their grain iron concentration. Hence, these markers can be effectively and efficiently utilized for discrimination of rice genotypes and selection of parental genotypes for genetic improvement in relation to grain iron biofortification. Single marker analysis established the association of two candidate genes specific markers (APRT1c and OsNRAMP1b) and candidate gene specific four microsatellite markers (OsNAC5A, OsNAC5B, OsNRAMP1A and OsZIP10A), with grain iron concentration. These six markers can be effectively utilized in selection program for grain iron biofortification in rice.
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    ThesisItemOpen Access
    Molecular characterization of SS-III gene promoter with respect to Heat tolerance in Wheat
    (Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2018) Kumar, Devendra; Kumar, Rajeev
    Bread wheat (Triticum aestivum L.) is one of the most important staple food grains of the human race as it fulfils the significant proportion of dietary calories requirements of the world population. Starch is a major component of wheat flour, accounting for 65-70% of the dry weight of the wheat grain. There are 6 distinct classes of starch synthase: GBSS, SSI, SSII, SSIII, SSIV and SSV involved in the synthesis of starch. SS genes containing promoter of 2kb sequence were retrieved in wheat and other crop species using by the bioinformatics tools. The promoter sequence of the reference crop species or wheat was used as a query in the cereals database. 68 cis-acting regulatory elements and 118 total number of motif sequences were found across the species under study. A maximum number of motifs (51) were found in Amaranths spp. In wheat total, 27 motifs were found. ERE (Ethylene Responsive Element) was found in SS-III in wheat. G-box (cis-acting regulatory element involved in light responsiveness) and MBS (MYB binding site involved in drought-inducibility) were found in all the species of monocots and dicots. Four novel conserved motifs e.g. ARE (anaerobic induction); GC-motif (anoxic specific inducibility); TGA (auxin-responsive element); TGACG motif (MeJA responsiveness) were found in the SSIII gene promoter consensus sequence obtained through wet lab experiment but found absent in the retrieved sequences. In Phylogenetic analysis Maximum genetic similarities were observed for Phaseolus SS-I & Phaseolus SS-II and Arabidopsis SS-II & Arabidopsis SS-I respectively where-as maximum dissimilarities was observed between Phaseolus vulgaris L. SSII and Brachypodium SS-II. Wheat SS-I was found more closely related to its SS-III, SS-II was found closely related to Brachypodium SS-III, and SS-IV promoter was found closely related to SS-III of Zea mays. We identified a total of 72 SNPs were identified over a total region of 1496 bp. Out of 72, 24 SNPs are of transition types and 48 SNPs are of transversion types in the studied genotypes of wheat with different degree of sensitivity towards heat stress. In heat sensitive genotypes (PBW-343, SISD, HD-2967, and Sonara-64) there were 29, 12, 6, and 1 specific SNPs identified respectively. 7 and 17 specific SNPs were also identified in heat tolerant genotypes (KSG-1186) and (Ipecarabe) respectively. In heat tolerance genotypes (KSG-1186) and (Ipecarabe) identified more transversion types (A/T) SNP than transition types. Analysis of types of substitution among the identified SNPs revealed far more abundance of transversion than transition and the ratio of transition to transversion (Ts/Tv) as 0.5.
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    ThesisItemOpen Access
    Molecular evaluation of sterility maintainers and fertility restorers for wild abortive rice cytoplasm
    (Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2018) Kumar, Pradeep; Sharma, V. K.
    A set of forty-five test crosses involving two wild abortive type cyto-sterile lines and twenty-eight pollen parents was evaluated to assess pollen and spikelet fertility restoration for identification of sterility maintainers and fertility restores of wild abortive cyto-sterility system in rice. Using a panel of thirty-two microsatellite primers, genetic polymorphism at molecular level was examined for characterization and differentiation of sterility maintainers and fertility restorers identified among pollen parents. Analysis of pollen and spikelet fertility in test crosses clearly reflected the sterility maintaining ability of pollen parents TCP-134-2, TCP-150-1, TCP-188-3 and TCP-185-3. Similarly, the pollen parents TCP-17-1, TCP-28-3, TCP-86-1, TCP-174-4, TCP-104-1, TCP-183-1, TCP-190-3, TCP-191-3 and TCP-193-3 were identified as effective fertility restorers. Altogether 256 allelic variants including 112 unique alleles and 144 shared alleles were identified with an average of 8.0 alleles per primer using a panel of 32 primer pairs covering all the chromosomes. The primer targeting tri-nucleotide and di-nucleotide repeat motifs, in general, detected more allelic variants than primers targeting tetra-nucleotide repeat motifs. Further, primers with GA, TG, AC, CT and TA di-nucleotide repeat motifs detected more allelic variants than the primers with TC and AG di-nucleotide repeat motifs. The primer pairs RM 216, RM 6100, RM 280, RM 3873, RM 10313, RM 558, RM 250, RM 283, RM 171, RM 3233, RM 341, RM 427, RM 206, RM 5373, RM 152, RM 591, RM 524 and RM 17 appeared to be highly polymorphic and comparatively more informative for the purpose of molecular profiling of entries. Analysis of divergence pattern allowed discrimination of effective fertility restorers from partial fertility restorers and complete sterility maintainers. Clustering pattern of the entries based on hierarchical classification, neighbour joining tree and principal co-ordinate analyses yielded more or less similar results. Allelic diversity data generated from amplification pattern of the six fertility restoration related primer pairs, namely, RM 591, RM 1108, RM 3233, RM 3873, RM 6100 and RM 8146, also unambiguously discriminated eight effective fertility restorers from two partial fertility restorers and four complete sterility maintainers. Therefore, these six primer pairs were validated with sufficiently greater efficiency (94.4%) for identification of sterility maintainers and fertility restorers of wild abortive type cyto-sterility sterility system in rice.
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    ThesisItemOpen Access
    Molecular Characterization of Aromatic Rice Genotypes using Microsatellite Markers
    (Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2018) Priyadarshini, Manisha; Sharma, V. K.
    A study was conducted to examine the nature and extent of genetic differentiation and divergence among the aromatic rice genotypes using microsatellite markers.The materials were grown in petriplates for extraction of genomic DNA from the young seedlings and then targeted amplification of the genomic DNA was achieved by using a panel of eighteen microsatellite based primer pairs covering four chromosomes in the genome of rice. The statistical methods and parameters used for deriving inference were polymorphism per cent, polymorphism information content, discrimination coefficient, similarity coefficient, principal coordinate analysis and numerical taxonomic analysis of divergence. Amplification was successfully achieved with all the microsatellite primers used in the present study. Appearance of bands at different positions on the gel revealed differential migration of amplified products due to differences in overall size of the products generated from targeted amplification of specific region of genome. The polymorphism among the entries was recognized on the basis of presence or absence of bands, in addition to variation in respect of number and position of bands.Altogether 123 allelic variants were detected among the eighteen aromatic rice entries with an average of 4.92 alleles per locus.The number of allelic variants per primer pair ranged from three in the case of E03.92.0 to ten in the case of RM 252, RM 256, RM 284 and RM 7356. A total of 76 shared and 47 unique allelic variants were generated amongst the entries.The primer pairs RM 7356, RM 252, RM 284, RM 223, RM 444, Aro7, E11.44.5, RM256 and RM 505 generated considerably greater percentage of unique alleles. Appearance of more than one band in the same entry indicated the existence of the duplicated region in the genome. The primer pairs RM 223, RM 252, RM 444, RM 505, RM 252, RM 7049 and RM 7356 generated amplified products due to amplification of more than one locus. Considering the magnitude of polymorphic information content value in conjunction with number of alleles and polymorphism per cent, the primer pairs RM 256, RM 284, Aro7, RM 223, RM 252, RM 444, RM 7356appeared to be highly informative.Occurrence of null allele for a microsatellite locus associated with primer pairs RM 505 was noticed reflecting null allele in combination with one of the eighteen entries under evaluation.Relatively higher magnitude of discrimination coefficient was obtained for the primer pairs RM 256, RM 284, Aro7, RM 8264, RM 223, RM 252, RM 444, RM 7356, RM 7049, ARSSR-3 and RM 515, indicating their greater efficiency in respect of their ability to unambiguously discriminate the pair-wise combination of aromatic rice entries. Microsatellite loci with tetra-nucleotide repeat motifs tended to detect greater number of alleles than the repeat loci withdi-nucleotide, tri-nucleotide and complex repeat motifs.Any relationship between the repeat number and the number of identified alleles was not observed in the present study.Simple sequence repeat loci with CT, GA and AT di-nucleotide repeat motifs tended to detect comparatively greater number of allelic variants. Ample genetic variation at molecular level amongst the aromatic rice entries under evaluation was inferred on the basis of the estimates of similarity coefficients. Considering the broad classification of entries, the entries appeared to bebasically divided into three groups, which were further divided into five clusters to allow the entries with relatively more similar pattern for markers to be clustered together. Principal coordinate analysis based two dimensional plots of the genetic profiles supported the results of hierarchical classification based grouping of entries.. Microsatellite marker based analysis revealed unique or entry specific allele which could be useful as molecular fingerprints in the unambiguous discrimination and identification of aromatic rice genotypes. The use of eighteen microsatellite markers in the analysis of aromatic rice entries exhibited a remarkably higher level of genetic polymorphism, which allowed unique genotyping of eighteen entries included in the analysis.
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    ThesisItemOpen Access
    Characterization of gene(s) involved in starch synthesis with respect to heat stress tolerance in wheat
    (Dr. Rajendra Prasad Central Agricultural University, Pusa (Samastipur), 2018) Mishra, Bhavya Priyadarshini; Kumar, Rajeev
    High temperature stress during the reproductive and grain filling phase is considered detrimental for wheat yield. In the present study thirty-two wheat (Triticum aestivum L.) genotypes were characterized with respect to heat stress under three different sowing conditions viz. Timely sown (S1), Late sown (S2) and very Late sown (S3) along with detailed structural, functional and molecular characterization of Starch Synthase III gene which is most critical for grain development in Wheat. The experimental materials were evaluated in randomized block design (RBD). The lowest performance of the genotypes were recorded under very late sowing condition (S3) . The impact of heat stress with respect to performance of the genotypes was found significantly higher in case of very late sown condition than in late sown stress condition. Genotypes C-306, Chiriya-3, Dharwad Dry,IC532653, Giza 155, Raj-4083, HI-1563, Cus/9/Prulla and DBW 14 exhibited stable performance for most of the traits whereas PBN -51, PBW 343, Sonora 64, Tepeko, Seri-82, BWL-1793 showed least stable performance in in all the three growing conditions.Starch Synthase (SS) has five distinct isoforms of which SSIII is involved in the formation of longer glucan chain length and most critical for grain filling. Here we also report identification and detailed characterization of ‘true’ orthologs of the well-characterized maize SSIII (ZmSSIII), among six monocots and two dicot species. ZmSSIII orthologs have nucleotide sequence similarity ranging from 56-81%. Variation in gene size among various orthologs ranged from 5.49 kb in Arabidopsis to 11.62 kb in Brachypodium and the variation was mainly due to intron size and indels present in the exons 1 and 3. Number of exons and introns were highly conserved among all orthologs however. While the intron number was conserved, intron phase showed variation at group, genera and species level except for intron 1 and 5. Several species, genera, and class specific cis-acting regulatory elements were identified in the promoter region. Despite of significant sequence variation among orthologs, most of the motifs and their relative distances are highly conserved among the orthologs. Homeologs of wheat SSIII gene showed tissue and developmental stage specific expression pattern with the highest expression recorded in developing grains in eight genotypes under consideration. Three different homeo-loci, for SSIII on chromosome group 1 showed dramatically different expression pattern during different grain developmental stages and exhibited low expression in stress condition in most of the genotypes except for KSG151, KSG053 and KSG270 that had stable expression under stress conditions.