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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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2017 - 201923
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Subject: Agricultural Biotechnology × End date: 2019 × Start date: 2017 × Type: Thesis × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    Effect of Heat Stress on Tissue Culture Response in Wheat Genotypes
    (DRPCAU, Pusa, 2019) Raj, Anant; Kumar, Harsh
    The 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.
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    ThesisItemOpen Access
    Study on cloning of Starch Synthase III gene in wheat
    (Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2019) Kumar, Vishnu; Kumar, Rajeev
    Bread 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.
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    ThesisItemOpen Access
    IN-SILICO AND MOLECULAR CHARACTERIZATION OF YSL1 GENE IN WHEAT
    (Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2019) Panigrahi, Sourav; Kumar, Rajeev
    Wheat 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.
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    ThesisItemOpen Access
    Study on QTL analysis for heat stress response in wheat using doubled haploid population
    (RPCAU, PUSA, Samastipur, 2019) Pankaj, Yaswant Kumar; Kumar, Rajeev
    The 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.
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    ThesisItemOpen Access
    Expression analysis for virulence causing gene of Bipolaris sorokiniana
    (Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, 2019) Deuri, Priyanka; Kumar, Mithilesh
    Wheat [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.
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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
    Molecular characterization in relation to grain iron accumulation in rice genotypes using candidate gene markers
    (DRPCAU, Pusa, Samastipur, 2019) Suryaji, Patil Nakul; Shrama, V.K.
    An investigation was undertaken to characterize the rice genotypes in relation to grain iron content using the primers designed specifically to target major iron transporter candidate genes and to analyze the significance of iron transporter based target region amplification polymorphism markers in relation to grain iron accumulation in rice genotypes. Using randomized block design with three replications, 28 varieties and advanced breeding lines of rice were evaluated in field and recommended agronomic practices were adopted while raising the crop. Seeds obtained from each entry in each replication after harvesting of the crop were utilized as samples for determination of iron content in unpolished grains. During molecular profile analysis, 18 entries were selected from the two extremes of grain iron distribution range and genomic templates were extracted from two to three weeks old seedlings. Employing six iron transporter candidate genes specific 16 primer pairs, selective amplification of targeted genomic regions was achieved. Molecular profiling was also performed by using a panel of iron transporter based 16 target region amplification polymorphism markers. Exploitable amount of genetic variability was observed in respect of grain iron accumulation amongst 28 varieties and advance breeding lines of rice initially evaluated as experimental materials in the present investigation. While taking into account the range of variation (16.39 to 39.04 ppm) recorded with respect to grain iron concentration, the entries under evaluation were classified into very low, low, moderate, high and very high grain iron containing groups. Grain iron content was observed to be comparatively very high in unpolished grains of Janki, Kanak, Swarna-sub1, RAU-3055 and Vaidehi. Microsatellites, altogether 18 in number with di-nucleotide to penta-nucleotide repeat motifs, were detected within the genomic regions specific to five of the six candidate genes investigated, providing a basis to infer that the sequence length variation present in candidate genes may be a role player in the differential grain iron accumulation in rice varieties. Reproducible amplification in 18 purposefully selected rice varieties and advance breeding lines was successfully achieved by utilization of six candidate genes specific 16 primer pairs during molecular characterization. However, only 12 primer pairs detected targeted genomic regions specific sequence length variation. While three primer pairs generated monomorphic amplified products, one primer pair revealed genetic polymorphism in the form of presence and absence of amplified product. Polymorphic information content of candidate gene specific 12 primers ranged from 0.278 to 0.886 with an average value of 0.540 across the primers. Employing 16 TRAP markers, differential amplification pattern was observed amongst the entries subjected to molecular characterization. Altogether 122 bands including 9 monomorphic bands were scored and categorized into 13 unique alleles and 109 shared alleles. Polymorphic information content of 16 TRAP markers ranged from 0.106 to 0.560 with an average of 0.334 across the markers. Ample molecular level genetic differentiation and divergence was revealed amongst the entries using the candidate genes specific primers and TRAP markers. Hierarchical classification pattern of rice genotypes was almost in complete agreement with principal coordinate analysis based spatial distribution pattern of genetic profiles of rice genotypes. Candidate gene markers based analysis appeared remarkably effective in differentiation of rice genotypes in relation to grain iron accumulation. Hence, these markers can be effectively and efficiently utilized for grain iron accumulation related 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, namely, OsYSL6a and OsNAS2c with grain iron accumulation. These two markers can be effectively used in marker-assisted selection program for grain iron biofortification in rice. Significance of iron transporters based target region amplification polymorphism markers for differentiation and divergence analysis was established in relation to grain iron concentration in rice genotypes. The entries with low and high grain iron content were found to be distributed into different clusters and sub-clusters.
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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 profiling of some local edible mushrooms of Bihar
    (DRPCAU, Pusa, Samastipur, 2019) Chunletia, Rati Satish; Kumar, Mithilesh
    Altogether 18 strains of mushrooms belonging to 5 genera namely Agaricus, Calocybe, Lentinula, Pleurotus, Tricholoma were included in present investigation for morphological, biochemical and molecular characterization. These strains were obtained from Directorate of Mushroom Research, Solan and Mushroom Lab, RPCAU, Pusa. All the strains under study were morphologically distinguished at both crop and mycelium stage. Change in colony characters with change in media was observed. Radial growth measurement of mycelium for optimum growth conditions for all the strains were recorded with respect to different media, temperature and pH and best growth for Tricholoma strains was observed on PDA medium, 25°C of temperature and pH 9 while for Agaricus MEA medium, 25°C of temperature and pH 9 were more appropriate. PDA medium, 15°C temperature and pH 7 was suitable for Lentinula strains and WEA medium,15°C temperature and pH 9 for Pleurotus strains. Calocybe strains showed best growth on both PDA and WEA, at 25°C and pH 7. Mycelium study is important as extracellular secretions by mycelium facilitates growth of crop and bioremediation process thus, biochemical screening of enzymatic activities for all the strains was done through plate test for lipase, amylase, laccase and by oxidation discs for oxidase. Lipase showed positive results for all the strains whereas amylase showed restricted response. Among all the strains AB-14-01 showed maximum enzymatic activity. Ample diversity was expected and observed as these strains belong to different genera and species thus, during the assessment of molecular characterization by 19 SSR primer pairs, cross amplification was found to be low. These SSR primers were derived from three different genera namely Agaricus, Calocybe and Pleurotus which gave 103 allelic variants with 52 unique and 51 shared alleles and an average of 5 alleles per primer. The dendrogram based on UPGMA for diversity analysis divided the mushroom entries into a mono-genotypic group (V), a penta-genotypic group (II), two di-genotypic group (III) and (IV), two tetra-genotypic group (III) and (V) which showed complete correspondence with principal coordinate analysis. The primer pairs GB-PO-026, AbSSR42 and AbSSR54 were found to be highly informative for the purpose of molecular profiling of mushroom entries belonging to different genera, species, geographical area and ecology.