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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 GENETIC POLYMORPHISM ANALYSIS OF GENES INVOLVED IN LYSINE AND TRYPTOPHAN METABOLIC PATHWAYS AND HETEROTIC GROUPS OF QUALITY PROTEIN MAIZE INBRED LINES(Dr.RPCAU, Pusa, 2022) SNEHAL,SIKHA; SHARMA, V.K.A study was performed in order to establish a relationship between the genetic diversity of quality protein maize inbred lines and performance of hybrids thus obtained from these inbred lines, field experiment was conducted. The experimental hybrids were obtained from the 15-quality protein inbred lines when crossed with 3 tester inbred lines, which was observed in randomized block design having three replications. Parental lines and experimental hybrids were randomized separately in continuous plots at TCA, Dholi. Statistical analysis was performed based on 11 morphological characters was studied, including 50% tasseling in days, 50% silking in days, 75% brown husk in days, height of plant, cob girth, length of cob, ear height, kernels in a row, kernels in a cob, grain yield per plant and test weight during the Kharif and Rabi seasons. The tests were carried out in the Molecular Biology Laboratory of Dr. RPCAU, Pusa, Bihar, to assess the type and extent of divergence between eighteen QPM inbred lines based on the analysis of 20 SSR and 26 lysine and tryptophan metabolic pathways candidate gene-based marker targeted microsatellite sites-based polymorphism. The ANOVA was done independently for both the season and across two environments. 18 QPM inbred parental lines and 45 single crosses, were subjected to statistical analysis for determination of significant differences among 11 quantitative features used in the experiment. ANOVA values revealed significant variations and degree of reliability in the performance of inbred lines and crosses in Kharif and Rabi seasons, as well as utilizing pooled data. The 63 entries in this study were analyzed for 11 quantitative characters, and ANOVA thus obtained revealed significant variations among them. To study the relevance of variation in the sources, the mean sum of squares for parents, hybrids, and parents vs. hybrids. For all of the characters studied, including 50% tasseling in days, 50% silking in days, 75% brown husk in days, height of plant, cob girth, length of cob, ear height, kernels in a row, kernels in a cob, grain yield per plant and test weight during the Kharif and Rabi seasons, categorization of variance showed substantial differences among the inbred parental lines. As deduced from the results obtained the mean sum of squares due to parents, hybrids, and parent vs. hybrids was significant for all traits during both Kharif and Rabi seasons, as well as across seasons. For selecting economically important morphological traits, it involves taking into account the level of heterosis expression in grain yield, as well as hybrid performance per se, when evaluating the potential of hybrids. A careful examination of the related information for hybrid means performance and degree of heterosis expressed for grain yield per plant reveals that these 17 hybrids, namely: QPML-07×QPML-16, QPML-03×QPML-16, QPML-02×QPML-14, QPML-04×QPML-14, QPML-11×QPML-14, QPML-13×QPML-14, QPML-11×QPML-15, QPML-09×QPML-15, QPML-02×QPML-15, QPML-04×QPML-14, QPML-03×QPML-15, QPML-18×QPML-15, QPML-08×QPML-16, QPML-06×QPML-14, QPML-13×QPML-15, QPML-17×QPML-14, QPML-18×QPML-14, was observed to have higher mean performance as well as significantly positive heterosis for grain yield and appeared to be the most promising hybrids under consideration. All the 18 parental QPM inbred lines were further classified into different clusters based on the dissimilarity units as observed in the dendrogram. The 18 QPM inbred lines were arranged into 3 clusters, out of these 3 clusters, 2 clusters, i.e. Cluster A and Cluster B were containing multiple genotype, however cluster C was containing only 2 genotypes. According to the dendrogram, QPML-13 and QPML-14 were showing maximum similarity when the 11 morphological characters were taken into consideration. However, QPML-11 and QPMLI-16 were showing minimum similarity when the 11 morphological characters were taken into consideration, and thus, was considered as the most divergent. Thus, from the analysis of genetic diversity performed in the current study, it was observed that 18 QPM inbred lines revealed abundant genetic diversity amongst each other. An analysis of the genetic similarity, hybrid index value, and hybrid mean value of heterotic groups generated using microsatellite markers revealed that QPML-11, QPML-13, QPML-17, and QPML-18 all belonged to heterotic Group-1. In all cases, QPML-09 belonged to heterotic Group-2. In every case, QPML-01 belonged to heterotic Group-2. The overall coincidences were significantly high when inbred lines were classified on the basis of SSR markers in different heterotic groups according to hybrid index value and hybrid mean value. As a result, the values lead to the conclusion that microsatellite markers could be used for effective and efficient method for classification of inbred lines into diverse heterotic groups for minimizing the size of single crosses that is required for production and evaluation. Hence, enhancing the of hybrid maize breeding program. The genomic DNA of maize was amplified utilizing following 26 candidate gene-based SSR primers. Different number of alleles were obtained for each marker and it ranged from 3 in ZmOPQ-2 to 13 in ZmAKH-1. A sum total of 193 alleles were obtained with 26 SSR marker, with an average value of 7.4 alleles per locus for the 18 inbred lines. Utilizing 26 SSR markers 78 unique alleles and 117 shared alleles were obtained. No unique allele was obtained for ZmHSDH-3. Only 1 unique allele was obtained for ZmOPQ-1, ZmOPQ-2, ZmAS-1, ZmAS-3, ZmOM-2, ZmOM-4, ZmAKH-4 and ZmOM-8. Maximum number of unique alleles were obtained for ZmAKH-1 (10) and ZmCS-1 (7). This showed varying degree of polymorphism amongst the 18 QPM inbred lines utilized under the present study. Unique allele percentage is used for expressing the value Polymorphism Per cent (PP), thus maximum PP value was obtained for ZmAKH-1 (76.90%) and the minimum value was obtained for ZmHSDH-3 (0%), followed by ZmOPQ-01 and ZmAS-1 (14.20%). The mean value of PP thus obtained for all the 26 markers was 36.00%. Equally greater degree of PP was obtained for the following primers: ZmCS-1 (63.60%), ZmHSDH-5 (62.50%), ZmOM-3 (55.50%), ZmHSDH-1 (54.50%), ZmOPQ-4 (50.0%), ZmASK-1 (50.0%) and ZmHSDH-2 (50.0%). Maximum PIC value was obtained for ZmAKH-1 (0.902) and the minimum value was obtained for ZmOPQ-2 (0.500). The mean value of PIC thus obtained for all the 26 markers was 0.8. Thus, from the data obtained following are the primers pair having higher value of both PP and PIC as well as number of alleles: ZmOPQ-3, ZmOPQ-4, ZmASK-3, ZmCS-1, ZmAS-2, ZmOM-3, ZmAKH-1, ZmAKH-2, ZmHSDH-1, ZmHSDH-4, ZmHSDH-5 and ZmOM-6. The genomic DNA of maize was amplified utilizing following 20 SSR primers. Different number of alleles were obtained for each marker and it ranged from 3 in Bnlg-1666 and UMC-1809 to 14 in UMC-1413. A sum total of 160 alleles were obtained with 20 SSR marker, with an average value of 8 alleles per locus for the 18 inbred lines. Utilizing 20 SSR markers 72 unique alleles and 88 shared alleles were obtained. No unique allele was obtained for Bnlg-1666 and Phi-002. Only 1 unique allele was obtained for Phi-064, Phi-046, Phi-011 and UMC-1370. Maximum number of unique alleles were obtained for UMC-1413 (11) and UMC-1159 (5). This showed varying degree of polymorphism amongst the 18 QPM inbred lines utilized under the present study. Unique allele percentage is used for expressing the value Polymorphism Per cent (PP), thus maximum PP value was obtained for UMC-1413 (78.60%) and the minimum value was obtained for Phi-002, Bnlg-1666 and UMC-1809 (0%), followed by Phi-050 (16.60%), Phi-064 (20.0%) and Phi-046 (20.0%). The mean value of PP thus obtained for all the 20 markers was 32.42%. Equally greater degree of PP was obtained for the following primers: UMC-1159 (62.50%), UMC-1465 (50.0%), UMC-1587 (50.0%), UMC-1425 (40.0%), UMC-1117 (40.0%), UMC-1133 (40.0%), Phi-014 (40.0%) and UMC-1638 (40.0%). Maximum PIC value was obtained for UMC-1413 (0.908) and the minimum value was obtained for UMC-1809 (0.602). The mean value of PIC thus obtained for all the 20 markers was 0.77. Thus, from the data obtained following are the primers pair having higher value of both PP and PIC as well as number of alleles: UMC-1465, UMC-1117, UMC-1587, Bnlg-105, UMC-1413, Phi-014, UMC-1638, UMC-1370, Phi-050 and Phi-062, appeared to be more informative when the number of alleles generated by different primer combinations was compared to the level of polymorphism identified in the current study. Genetic profile of the 18 inbred lines utilizing 26 candidate gene-based primers showed the inbred lines along the two main axes showed the accommodation of all the inbred lines into 3 well distinguished major genotypic groups. Thus, cluster analyses and principal coordinate analysis showed the existence of wide variation at genetic level that was present among the 18 QPM inbred lines. Thus, with the utilization of 26 candidate gene-based primer, significantly high degree of polymorphism was obtained among the 18 QPM inbred lines at genetic level. Cluster analysis was performed for the evaluation of variability at genetic level amongst 18 QPM inbred lines utilizing 20 SSR primer pairs showed the accommodation of all the inbred lines into 3 well distinguished major genotypic groups. Thus, with the utilization of 20 SSR primer, significantly high degree of polymorphism was obtained among the 18 QPM inbred lines at genetic level. Thus, analysis showed the existence of wide variation at genetic level that was present among the 18 QPM inbred lines. Similarity coefficient was estimated based on the comparison of presence and absence of bands amplified utilizing 46 pairs of primers (genic and generic). All the 18 parental QPM inbred lines were further classified into different clusters based on the similarity units as observed in the dendrogram. Thus, with the utilization of 46 combined (genic and generic) primer pairs, significantly high degree of polymorphism was obtained among the 18 QPM inbred lines at genetic level. Cluster analysis was performed for the evaluation of variability at genetic level amongst 8 QPM inbred lines and 4 non-QPM inbred utilizing candidate gene-based primer pairs. Similarity coefficient was estimated based on the comparison of presence and absence of bands amplified utilizing 26 pairs of primers. The 8 QPM inbred lines and 4 non-QPM were further classified into 2 different clusters. Likewise, when cluster analysis was done utilizing the 4 designed primers, it was also able to differentiate the 8 QPM inbred lines and 4 non-QPM taken as experimental material. The 8 QPM inbred lines and 4 non-QPM were further classified into 2 different clusters based on the similarity units. Thus, cluster analysis and principal coordinate analysis showed the existence of wide variation at genetic level that was present among the 8 QPM inbred lines and 4 non QPM inbred lines. Cluster A comprised of the following 8 inbred lines: QPML-01, QPML-02, QPML-03, QPML-04, QPML-05, QPML-06, QPML-07 and QPML-08. Cluster B comprised of the following 4 non-QPM inbred lines: NQPML-01, NQPML-02, NQPML-03 and NQPML-04. The distribution pattern in 2-Dimension for inbred lines genetic profiles was in correspondence with the interrelationship exhibited by hierarchical classification on the basis of similarity coefficients (Fig. 4.). Genetic profile of the 8 QPM inbred lines and 4 non QPM inbred lines utilizing 26 candidate gene-based primers as well as the 4 designed primer pairs showed the inbred lines along the two main axes showed the accommodation of all the inbred lines into 2 well distinguished major genotypic groups. The genetic structure illustrated by 46 combined SSR primers (26 candidate gene specific and 20 SSR primers loci showed that the genetic structure of 18 QPM inbred lines are composed of three major ancestral components. The results can infer that the computational analysis of the genetic structure of the QPM inbred lines unambiguously reflected that the genotypes subjected high tryptophan and lysine content are having related molecular characterization are the admixture of three ancestral components present in different combinations in different genotypes.ThesisItem Open Access Microsatellite Marker Assisted Determination of Genetic Polymorphism and Heterotic Groups Among Baby Corn Inbred Lines(Dr.RPCAU, Pusa, 2022) KIRTI, SIMRAN; SHARMA, V. K.A study was conducted for characterization of baby corn inbred lines using agronomic characters and microsatellite markers to determine the molecular level genetic polymorphism and divergence among these baby corn inbred lines for construction of heterotic groups based on comparative analysis of amplification pattern of targeted microsatellite sites. The basic experimental material comprised twelve inbred lines and three inbred testers of baby corn, which were crossed to generate thirty-six single cross hybrids. The inbred lines and hybrids were evaluated in randomized block design with three replications during two seasons (rabi and kharif seasons). The degree of divergence and separation among inbred lines of baby corn was quantified and inbred lines were then divided into various heterotic groups based on the examination of focused microsatellite sites. Analysis of parental lines and heterosis in experimental hybrids was done statistically using nine quantitative agro-morphological characters based on the measurements and assessments of plant height, ear height, days to 50% tasseling, days to 50% silking, cob yield per plant, green fodder yield, baby corn length, baby corn girth and cob weight. Stastical analysis of nine quantitative characters using numerical taxonomic approach was employed and the inbred lines were classified into three clusters. Young seedlings were grown in almuninum container to extract genomic DNA for microsatellite primers based amplification of targeted genomic regions. Amplified products were produced using a set of 38 microsatellite sites specific primer pairs that covered every chromosome in the genome. Microsatellite-based allelic diversity was used for further classification and creation of various heterotic groups of the inbred lines. Statistically significant differences were revealed amongst inbred lines and crosses for nine quantitative morphological charactersrecorded during both the seasons. Among inbred lines, BCL-08 outperformed over all other inbred lines in respect of cob yeild per plant, followed by tester BCL-12, which was statistically equivalent to inbred lines BCL-05, BCL-07, BCL-10 and BCL-04 over the environments. Among 36 crosses, 14 crosses were found to have significantly greater mean performance as well as significantly positive heterosis. The cross BCL-10×BCL-12 was observed as high yielding cross followed by BCL-04 × BCL-03, BCL-09 × BCL-03, BCL-05 × BCL-03, BCL-07 × BCL-15, BCL-06 × BCL-15and BCL-14 × BCL-15 for cob yield per plant. By the use of 38 primer pairs, 279 allelic variant were investigated as amplified products. The value of the number of alleles varied from 4 for the primer phi011 to 11 in case of Nc130 having range between 75bp to 580 bp.The sum of total value of shared alleles was 166, which varied from 3 to 8 alleles per primer pairs. Similarly, the sum total of unique allels was 113, which ranged from 1 to 7 alleles per primer among the 15 inbred lines. Taking into the consideration the number of alleles and polymorphic status, the primers UMC1425, Phi024, UMC1407, UMC1159, Phi046, UMC1587, UMC1241, UMC1858, UMC1638 and UMC1370 seemed to be more informative on the basis of polymorphism level. On the basis of molecular level classification, BCL-15 and BCL-01 seemed to be highly diverse genotypes. Contrarily, BCL-03 and BCL-04 appeared most closely related to each other in same group. The inbred lines BCL-12 and BCL-13 were closely associated to each other. Both molecular and morphological attributes dependent cluster based data manifested that among the three testers, namely, BCL-03, BCL-12 and BCL-15 two testers excluding BCL-03 were included in same cluster. However, the cluster analysis used to group inbred lines using data from quantative characters and microsatellite markers did not show a perfect match. Results of genetic structure analysis based on repeat sequence length variation in primer specific genomic regions revealed that the genetic compositions with refrence to the targeted genomic regions of the inbreds are basically the admixtures of various combinations of three ancestral components. Analysis of the heterotic groups compositions using the hybrid index value and the hybrid mean value revealed that BCL-04, BCL-06, BCL-07, BCL-09, BC-L10, BCL-11 and BCL-14 were found in same group i.e in Group 1, BCL-01 and BCL-05 were found in Group 2 and BCL-02, BCL-08 , BCL-13 were found in Group 3.A comparative analysis of amplification pattern of targeted microsatellite sites was found effective in discriminating the inbred lines of various heterotic groups and therefore the employed panel of primer was found moderately efficient in discrimination of inbreds into heterotic groups.ThesisItem Open Access Micropropagation and Molecular Characterization of two medicinal weeds Solanum indicum and Solanum torvum(DRPCAU, Pusa, 2021) RANI, SWATI; SHARMA, V. K.In the current study, two medicinally important indigenous weeds, namely, S. indicum and S. torvum were selected to develop a micropropagation technique for their in vitro multiplication, as well as, their morphological and molecular characterization in order to assess the nature of difference within and between the plant species. During the standardization of tissue culture technique, three explants viz. shoot apex, nodal stem, and leaf of both the plant species were cultured on 34 MS basal media enriched with supplementation of different combinations and concentrations of phytohormones like IAA, KIN, BAP, NAA, and IBA. Morphological characterization was based on observations recorded for both the species by selecting five plants randomly from three different locations viz. Sonepur, Muzaffarpur and Pusa each. Altogether 33 morphological characters including 17 qualitative and 16 quantitative characters were investigated to record variation between the plants at their full foliage stage. Further, 16 ISSR primers were used for molecular characterization by amplification of genomic templates isolated from leaf samples of morphologically distinct plants of both the species collected from each location. Results revealed that in vitro responses were significantly (P<0.05) affected by phytohormone concentration, explants, and plant type. All the three responses, elongation, swelling and callusing showed the highest frequency on medium having composition 3 mg/l IAA and 5mg/l BAP. Explant leaf of S. torvum showed best swelling and callusing. Best elongation frequency was observed in shoot apex of S. indicum. Highest frequency of multiple shoot formation was recorded on the medium having composition 3 mg/l KIN and 2mg/l BAP by shoot apex of S. indicum. The highest frequency of root formation was observed on MS medium having 1.5mg/l IBA for S. indicum. The regenerated plants with well-developed roots were transferred to polycups filled with sterilized sand and farm yard manure in 1:1 ratio for hardening and acclimatization. Morphological characterization indicated that the amount of variations in qualitative morphological characters within the species was negligible, whereas in case of quantitative morphological characters, appreciable amount of variation was observed within and between the plant species. Correlation coefficient values computed for the pairwise combinations of sixteen quantitative characters ranged from -0.999 to 0.999. Most of the characters were found to be significantly correlated. The dissimilarity coefficients between different pair-wise combinations of each of the three populations of S. indicum and S. torvum ranged from 0.310 to 1.966. Based on dissimilarity coefficients measured in terms of taxonomic distance, three populations of each of S. indicum and S. torvum were organized into two principal clusters by using UPGMA. The results of the principal component analysis completely supported the results obtained from the dissimilarity coefficients-based hierarchical cluster analysis. Amplified product size obtained using ISSR markers ranged from 255-1383 with a total of 40 unique alleles. Polymorphism per cent ranged from 10 to 44.44% and PIC value ranged from 0.788 to 0.954. Among 16 ISSR primers, UBC 815 was best to detect genetic variation. The values of similarity coefficient ranged from 0.1429 to 0.9136. On the basis of similarity coefficients, dendrogram was created using UPGMA that resulted into formation of two major clusters. Cluster A and B contained all the plants of S. indicum and S. torvum from all the three locations, respectively. Principal coordinate analysis supported the findings of cluster analysis, revealing two groups comprising of same entries in two dimensional ordinations.ThesisItem Open Access Assessment of spot blotch resistance in wheat genotypes through tissue culture and molecular marker(DRPCAU, Pusa, 2021) Deepti; Sharma, Vinay KumarAn investigation was conducted to determine the percentage of formation of callus and regeneration of callus in selected twelve genotypes of wheat, namely, PBW-343, Chiriya-3, Sonalika, HD- 2967, Agra- local, yangmai#6, K-307, UP-2565, HD-3086, HD-2733, Salembo and Cuo/79/Prulla by using mature embryo. The best media for callogenesis was WM11[MS + 2,4-D (4.0 mgl-1) + NAA (2 mgl-1)] with remarkably higher frequency obtained (90.78%) and the best media WM34 [MS + 2,4-D (0.1 mgl-1) + Zeatin (5.0 mgl-1) +CuSO4 (12 mg)] resulted in 93.43% caulogenesis, whereas 90.65% was noted in the medium WM36 [MS + IBA (0.5 mgl-1)] for rhizogenesis. The in-vitro screening was performed and assayed by using culture filtrate of Bipolaris sorokiniana as a fungal toxin in the media selected for callogenesis and regeneration to facilitate precise evaluation for presence or absence of spot blotch or infection in wheat genotypes and to derive inference about their susceptibleness or resistance. The susceptible genotype Agra local showed maximum necrosis of the callus because of effect of toxin in medium supplemented with MS+2,4-D (4.0 mgl-1) + NAA (2 mgl-1) along with B. sorokiniana toxin, whereas the resistant genotype Yangmai#6 showed least area affected by the toxin as observed in the case of controlled medium supplemented with toxin. The utilization of twenty-two SSR and ten STS microsatellite markers known to be associated with resistance to spot blotch during amplification profiling of thirty-six genotypes of wheat under evaluation in the present investigation resulted in highly effective categorization of these genotypes having resistant, moderately resistant and susceptible response. Hierarchical cluster analysis using Sequence Tagged Sites specific primers exhibited a very high efficiency (91.7%) in discriminating the susceptible genotypes from resistant and moderately resistant genotypes. However, principal coordinate analysis exhibited clearly recognizable spatial distance between the susceptible and resistant genotypes, which were basically classified into two groups. Hierarchical clustering and spatial distribution pattern based on the amplification profile generated by utilization of Simple Sequence Repeats specific markers univocally discriminated the susceptible genotypes from moderately resistant and resistant genotypes. The entries were broadly classified into three groups. A combination of SSR and STS markers unambiguously differentiated the susceptible genotypes from moderately tolerant and highly tolerant genotypes. Principal coordinate analysis based spatial distribution pattern of the genotypes as well as radial tree diagram and factorial analysis completely corroborated the results obtained from dendrogram and the genotypes were found to be clustered into three groups in all the cases. The inference derived from the results of genetic structure analysis revealed that the genetic compositions with respect to the targeted genomic regions of the genotypes are basically the admixtures of different combinations of three ancestral components. Experimental results provided the evidences to infer that ample differentiation and divergence were revealed amongst the genotypes by utilization of spot blotch resistance related molecular markers.ThesisItem Open Access Molecular characterization of rice genotypes using salt stress responsive candidate gene based markers(DRPCAU, Pusa, 2020) Agarwal, Ruchi; Sharma, V.K.A study was undertaken for characterization of salt tolerance related response of 18 rice genotypes using morpho-agronomic characters and salt stress responsive 12 candidate gene based newly designed markers. Evaluation of genotypic response to salt stress at seed germination and early seedling stage was performed at 4 dSm-1 and 8 dSm-1 EC levels along with control by adopting a completely randomized design with two replications. Pot experiment was carried out at 8 dSm-1 and 12 dSm-1 EC levels given at vegetative and reproductive stages, respectively, along with control in completely randomized design with two replications. Field evaluation was conducted in randomized block design with two replications under normal and salt affected field conditions. The genotypes were further characterized at molecular level using 12 candidate gene based newly designed 23 genic primers and 21 genic microsatellite primers. Differential genotypic response to salt stress was noticed at germination stage and early seedling stage. Based on response exhibited at early seedling stage, the genotypes CSR-13, CSR- 23, CSR-27, CSR-30, CSR-36, CST7-1 and CSR-2K-262 were characterized with considerably higher level of tolerance to salt stress in comparison to the genotypes NDRK-11-1, NDRK-11-3, NDRK-11-4, NDRK-11-5, NDRK-11-6, NDRK-11-7, CSR-2K- 219 and CSR-2K-242, which were found to be moderately tolerant. The remaining three genotypes, namely, IR-36, IR-64 and Swarna, were found to be susceptible to salt stress. Principal component analysis based spatial distribution pattern of the genotypes in two dimensional projections distinctly separated the genotypes into two broad groups. The first group was further divided into two sub groups consisting of six salt tolerant genotypes and nine closely located moderately tolerant genotypes, whereas the second multi-genotypic group consisted of three salt susceptible genotypes. The basic pattern of differentiation and interrelationships among the genotypes was found to be similar in dendrogram. A comparison of the relative mean values of the genotypes evaluated for 14 morpho-physiological attributes, such as, panicle length, panicles per plant, spikelets per panicle, filled grains per panicle and unfilled grains per panicle, 100-seed weight, biological yield, grain yield per plant, root length, root volume, root dry weight, relative water content, SPAD value and K/Na ratio in pot experiment was made with mean index (MI) value. Based on the results, the genotypes CSR-13, CSR-23, CSR-27, CSR-30, CSR-36 and CST7-1 were found to be salt tolerant, whereas NDRK-11-1, NDRK-11-3, NDRK-11-4, NDRK-11- 5, NDRK-11-6, NDRK-11-7, CSR-2K-219, CSR-2K-242 and CSR-2K-262 were rated as moderately tolerant and genotypes IR-36, IR-64 and Swarna were found susceptible. Spatial distribution pattern of the genotypes in two dimensional ordinations based on principal component analysis broadly divided the genotypes into two groups, efficiently separating tolerant and moderately tolerant genotypes from susceptible genotypes. The pattern of genotypic discrimination was similar corroborated by dendrogram. Tolerance indices, such as, TOL, MP, GMP, SSI and STI based principal component analysis and hierarchical cluster analysis also discriminated the susceptible genotypes from moderately tolerant and tolerant genotypes. Relative mean performance of genotypes evaluated under normal and salt affected field conditions for 11 morpho-agronomic characters also reflected that the genotypes CSR- 13, CSR-23, CSR-27, CSR-30, CSR-36, CST7-1 and CSR-2K-262 exhibited tolerant response, whereas NDRK-11-1, NDRK-11-3, NDRK-11-4, NDRK-11-5, NDRK-11-6, NDRK-11-7, CSR-2K-219 and CSR-2K-242 were found moderately tolerant and the genotypes IR-36, IR-64 and Swarna were rated as susceptible. Principal component analysis based spatial distribution pattern separated the genotypes into two broad groups, separating the three susceptible genotypes from moderately tolerant and tolerant genotypes. A similar classification pattern was revealed by average taxonomic distance based dendrogram. Recognizable molecular level genetic polymorphism amongst the 18 genotypes was revealed by amplification of genomic template using 12 candidate gene specific 23 newly designed primer pairs. All primer pairs generated 135 allelic variants with an average of 5.86 alleles per primer. The PIC values exhibited significant variation with the values ranging from 0.308 in primer pair DREB1F-1 to 0.905 in primer pairs SHMT1-1. Considering the level of polymorphism detected, the candidate gene based primer pairs OsHKT2;3-1, OsHKT2; 3-2, SNAC1-1, SNAC1-2, CDMK-1, CDMK-2, DUF6-2 SHMT1- 1, SHMT1-2 and SHMT2-1 appeared to be highly polymorphic and more informative primers for the purpose of discrimination of rice genotypes in relation to salinity tolerance. Principal coordinate analysis and hierarchical classification unambiguously discriminated the tolerant and susceptible genotypes, reflecting the usefulness of these newly designed markers. Using major ion transporter, transcription factor and oxidative metabolic pathway related candidate gene specific newly designed 21 microsatellite primer pairs, ample molecular level genetic polymorphism was detected amongst 18 genotypes. All primer pairs generated 131 allelic variants with 6.23 alleles per primer. The number of unique and shared alleles varied from 0 to 5 and 1-6, respectively. The PIC values ranged from 0.706 to 0.955. Taking into consideration the measures of polymorphism, the primers OsHKT1;5-B, OsHKT1;5-D, OsHKT2; 3-A, OsHKT2; 4–A, DREB1F-C, CDMK-B, DUF6-B, SHMT1- B, and SHMT2-A were found as relatively more polymorphic. Spatial distribution pattern of genotypes based on principal coordinate analysis of the genetic profiles generated by these newly designed primers as well as hierarchical classification unambiguously differentiated the tolerant and susceptible genotypes, validating the usefulness of salt responsive candidate genes based these newly designed microsatellite primer pairs.ThesisItem Open Access Genetic analysis of maize inbreds and hybrids using biochemical and microsatellite markers(DRPCAU, Pusa, 2020) Suman, Sandeep Kumar; Kumar, MithileshCharacterization of inbred lines and hybrids is crucial for release of hybrid and genetic purity analysis. Hence, field evaluation was done at AICRP, Maize, TCA, Dholi farm and lab experiment was done at Molecular Biology Lab, Department of Plant Breeding and Genetics, RPCAU, Pusa for characterization of parental lines and hybrids and genetic purity analysis through biochemical and microsatellite marker. Morphological genetic diversity analysis between thirteen inbreds of maize were done using Mahalanobis D2 statistics for ten characters viz. plant height, ear height, days to 50% tasseling, days to 50% silking, days to 50% Brown husk, ear length/Cob length, ear girth, number of kernels per row, number of kernel rows per cob, grain Yield per Plant. All the thirteen inbred lines under study were grouped in three clusters. Cluster I comprised of eleven inbred lines G18, CLQRC, WLS, HK1, CML490, S8200, S8481, R6429, R4093, VL111 and CLQR. Cluster II and III were found monogenotypic and consisted of CLQ25 and CG18 respectively. The inter-cluster distance between cluster II and cluster III was found maximum 6.67, while lowest inter cluster distance value was found between 4.62 cluster I and cluster II. The maximum value of genetic distance 4.89 was estimated between inbred line WLS and G18, while minimum genetic distance 2.38 was found in between VL111 and S8481. Name of the Student : SANDEEP KUMAR SUMAN Registration No. : D/AB/130/2014-2015 Major Advisor : Dr. MITHILESH KUMAR Degree to be Awarded : Ph.D. (Agricultural Biotechnology) Department : Agricultural Biotechnology and Molecular Biology Major Subject : Agricultural Biotechnology Minor Subject : Plant Breeding and Genetics Year : 2020 Total pages of the Thesis : 144 + xviii (Bibliography) + xxii (Apppendices) Title of Thesis : “Genetic analysis of maize inbreds and hybrids using biochemical and microsatellite markers” The result showed great variability was found in genetic makeup of the inbred lines. Cluster I accommodated eleven inbred lines having maximum mean value for number of kernels row per cob and number of kernels per row. While, minimum value was observed for ear height and plant height. Maximum mean value for grain yield per plant and ear girth was found for inbred line CLQ25 that is present in cluster II. The maximum contribution 26.63% was found for the trait grain yield in manifestation of genetic divergence followed by ear girth (28.47%), ear length (19.23%), number of kernel per row (11.50), number of kernels row per cob (10.04). Out of thirty crosses, eleven crosses manifest positive significant heterobeltiosis for the trait grain yield per plant. Two crosses, CLQ25 × CLQR and WLS × R4093 belongs to high divergent class. It could be deduced that genetic diversity can be used as an authentic parameter to predict heterosis in hybrids. The parental lines and hybrids were characterized with SDS-PAGE using total salt soluble seed proteins. Total salt soluble banding profile identified one hybrid G18 x VL111. The presence of 53 KDa, MW band specific for female parent G18 and 65 KDa, MW band specific for female inbred line confirmed the hybridity and purity of hybrid G18 x VL111. The 27 microsatellite primer amplified a total of 28 loci with an average of 4 alleles per locus. The number of alleles varied from 2 to 8 in case of umc1222 and umc1545 or umc1265 or umc1963 or phi084, respectively. The range of amplification varying from 72-88 in case of umc1327, while in case of phi0116 amplification ranged from 293-345. A total of 112 alleles were found in which 33 were unique and rest 79 were found shared alleles in 13 inbred lines. Based on complementary banding pattern between hybrids and their parents, the six microsatellite markers viz. umc1222, umc1161, umc1196, umc1367, umc1403 and nc133 were found effective marker to distinguish F1 hybrids from its parental lines. These markers were utilized to analyse genetic purity of inbred lines and hybrids.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.
