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2016 - 201952020 - 20213
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Subject: Agricultural Biotechnology × Start date: 2016 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Influence of cold stress on expression of invertase and calcium-dependent protein kinase genes in chickpea (Cicer arietinum L.)
    (Palampur, 2021-11-12) Shree, Bharti; Sharma, Kamal Dev
    Chickpea (Cicer arietinum L.) is an important food grain legume. Chickpea is sensitive to cold and suffers substantial yield losses due to cold stress. Invertases hydrolyse sucrose into glucose and fructose and play an important role in plant growth and development as well as plants’ responses to various stresses including cold. In addition to invertases, calciumdependent protein kinases (CDPKs) modify gene expression via transcription factors to achieve systematic plant growth/development and reaction to stresses. Information on invertases and calcium-dependent protein kinases as well as the role of these genes in stress tolerance/susceptibility in chickpea is unavailable. In this study, 19 invertase genes (11 cellwall invertase, one vacuolar invertase and seven alkaline/neutral invertase genes) and 31 CDPKs genes were identified in the chickpea genome. These genes were located on 7 chickpea chromosomes. A comprehensive analysis of invertase as well as CDPK genes and proteins were performed, including gene structure, mRNA structure, cis-acting elements in the promoter regions, phylogeny, evolutionary relationships, gene duplication events, protein structure, motifs, domains, physiochemical properties, sub-cellular localization and interactions of invertases as well as CDPKs with other proteins. Phylogenetic analysis revealed that chickpea invertases were comprised of five major lineages whereas CDPKs had four lineages. The members within the same sub-groups shared conserved domains. Expression analysis revealed that all the invertase genes were functional in chickpea however, these genes expressed differentially in contrasting chickpea genotype under cold stress and Ascochyta blight infection. Expression analysis revealed that cell wall invertases were associated in cold tolerance whereas majority of the CaCDPK genes were involved in low temperature responses by tolerant as well as sensitive genotypes of chickpea. Invertase genes associated with Ascochyta blight resistance in chickpea were also identified. The study laid the foundation for unravelling the complexity of chickpea responses to cold and Ascochyta rabiei infection and develop protocols for mitigation of cold stress in chickpea.
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    ThesisItemOpen Access
    GENE NETWORKS INVOLVED IN COLD STRESS RESPONSE IN CHICKPEA (Cicer arietinum L.) ANTHERSVARMA
    (CSK HPKV Palampur, 2020-01-07) VERMA, ANNU; Chaudhary, H.K.
    Cold stress (CS) coinciding with the onset of the flowering in chickpea poses a major threat for realizing high productivity in northern hill regions of India. CS induces pollen sterility in chickpea leading to flower abortion and consequently low productivity. Some genotypes of chickpea, though agronomically inferior, tolerate cold and produce viable pollen leading to seed set even under cold stress. Changes in carbohydrate content in contrasting genotypes of chickpea were observed in the present study under CS wherein, the genotype ICC 16349 (cold tolerant) maintained normal carbohydrate pool whereas genotype GPF2 (cold susceptible) failed to do so. Not only carbohydrate content but enzymes of the carbohydrate pathway e.g. invertase, sucrose synthase, alpha and beta amylase showed greater activity in cold tolerant genotype. Under recovery period, the levels of carbohydrates as well as carbohydrate metabolism enzymes shifted towards normal with a few modifications. The expression of carbohydrate metabolism genes that is UDP glucose pyro phosphorylase like, ADP glucose pyro phosphorylase, Beta amylase3 and Beta amylase1 changed significantly under cold stress in the cold tolerant as well as cold susceptible genotypes. The expression level of Isoamylase3 and Isoamlyase3x2 was higher in leaves of cold susceptible genotype GPF2. Starch degradation was lower in ICC 16349 (cold tolerant) as compared to GPF2 (cold susceptible) as evident from expression of genes; Beta amylase3 and Beta amylase1 and glucan water kinase. The expression of starch synthase increased in theICC16349.The expression of ABA metabolism genes was higher in GPF2 and the expression of GA biosynthesis genes Gibberellin20oxidase1 and Gibberellin20 oxidase3 reduced in the anthers of ICC 16349 and GPF2 with the exception of Gibberellin20oxidase1 that enhanced in GPF2. A combination of 5µM Sucrose and 5µM GA was the best treatment for mitigation of cold under pot as well as field conditions as it showed marked increase in chlorophyll content, relative leaf water content, cellular respiration and reduction in electrolyte leakage in both the genotypes. The study has implications in the development of cold tolerant chickpea varieties and devise other appropriate strategies for mitigation of cold, thereby resulting in increased productivity of chickpea.
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    ThesisItemOpen Access
    INTROGRESSION OF ANTHRACNOSE RESISTANCE GENES IN ELITE FRENCH BEAN (Phaseolus vulgaris L.) BACKGROUND USING LINKED MOLECULAR MARKERS
    (CSKHPKV. Palampur, 2020-01-07) BANSULI; Kapila, R. K.
    French bean is one of the most important staple foods in many parts of the world, where it constitutes the main source of protein for human consumption. Several bacterial, fungal and viral diseases attack French bean and among these anthracnose caused by Colletotrichum lindemuthianum is one of the most widespread and severe. Many improved pencil type varieties developed are available for cultivation by the farmers of Himachal Pradesh, however most of these are susceptible to many prevalent races of bean anthracnose. Therefore, the present study was undertaken with the objectives of introgression of important bean anthracnose resistance genes in elite cultivars and validation of imparted resistance in the derivatives. Hybridisation between resistant genotypes, TO, TU, G 2333 and Cornell 49242, known to harbour bean anthracnose resistance genes, Co-4, Co-5, Co-42/Co-5/Co-7 and Co-2, respectively and elite cultivars, Arka Komal and Contender revealed a low and differential crossability rate (0-7.69 %) among gene donors and elite recipient parents as Arka Komal was more easily crossable with resistance gene donors as compared to Contender. Two most successful crosses, Cross Combination I (Arka Komal × TO) and Cross Combination II (Arka Komal × TU) were further advanced to generate F1, BC1F1, BC2F1 and BC2F2 plants. Testing of hybridity of the resultant F1, BC1F1, BC2F1 and BC2F2 plants/pods using gene linked SCAR markers (SY20 for Co-4 and SAB3 for Co-5) resulted in identification of 46 gene-positive BC2F2 plants of Cross Combination I and 36 of Cross Combination II. A total of 457 and 764 seedsof 46 and 36 selected gene-positive BC2F2 plants of Cross Combinations I and II, respectively were raised as progeny rows. Foreground selection with linked marker, SY20 and SAB3 in representative 10 plants of the each progeny revealed lack of segregation for the target gene Co-4 and Co-5 in 16 and 12 progenies of Cross Combinations I and II, respectively. Screening of progenies following detached pod and germinated seed dip methods using race 3 of C. lindemuthianum validated Co-4 and Co-5 imparted resistance in all the 28 BC2F3 progenies of 2 cross combinations. Agronomic evaluation of 16 Co-4 and 12 Co-5 gene-positive BC2F4 progenies for 8 qualitative traits and 9 quantitative traits identified AKTO 4, AKTO 5, AKTO 7 and AKTO 43 progenies of Cross Combination I and AKTU 19 and AKTU 33 of Cross Combination II as having better elite background. These can either be used as donors of resistance gene(s) for further introgression and gene pyramiding or can further be advanced following backcrossing to develop essentially derived variety of elite parent, Arka Komal.
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    ThesisItemOpen Access
    Identification of QTLs linked to early maturity and yield-related traits in horsegram (Macrotyloma uniflorum).
    (CSK HPKV, Palampur, 2019-07-23) Katoch, Megha; Chahota, R.K.
    Macrotyloma uniflorum is an important, self pollinated diploid (2n=2x=20) food legume with probable genome size of 400Mbps. Limited genomic resources and lack of genetic variation are major constrains in its genetic improvement. Further, horsegram production is hampered due to twining growth habit, longer days to maturity, photosensitivity and indeterminate growth habit. The present study was aimed to construct linkage map of an intraspecific F8 RILs population of 162 individuals derived from HPKM249×HPK4 of horsegram and identification of genomic regions linked to early maturity and yield related traits. Two thousand and eleven molecular markers were screened for parental polymorphism and 493 (25.42 %) were found to be polymorphic among the parents. Of these, 295 were mapped on ten linkage groups at LOD 3.5 spanning 1541.7 cM with an average marker density of 5.20 cM. Analysis of variance of 162 RILs revealed significant differences for all the measured traits. Phenotypic data from the RILs were used to identify QTLs for early maturity and yield related traits by composite interval mapping (CIM). A total of 27 QTLs (LOD ≥ 2.5) were detected across the three environments (Palampur 2016, Palampur 2017, Bajaura 2017) and combined data) for 24 traits. Among these, 15 were major QTLs with PVE greater than ten per cent and five were stable QTLs across locations and years. Phenotypic variation explained (PVE) by QTLs ranged from 6.4 to 53.4 per cent. The highest phenotypic variation (53.4 %) was explained by the QTLs for root length. In conclusion, it is envisaged that the present linkage map, fortified with 295 SSR markers and 27 QTLs for early maturity and yield-related traits would provide genomics tools to breeders for further genetic enhancement of this crop species. Thus, the current study will serve as a strong foundation for further validation and fine mapping of QTLs for utilization in horsegram breeding programs.
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    ThesisItemOpen Access
    GENETICS AND MAPPING RESISTANCE GENE(S) F INBRED LINE DERIVATI RESISTANT GENOTYPE CHAUDHARY SARWAN KUMAR HIMACHAL PRADESH KRISHI VISHVAVIDYALAYA PALAMPUR Partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY (DEPARTMENT OF (AGRICULTURAL BIOTECHNOLOGY) OF NECK BLAST FROM RIL4 - A RECOMBINANT DERIVATIVE OF THE BROAD SPECTRUM ‘Tetep’
    (CSKHPKV, Palampur, 2019-01-02) Kalia, Sonika; Rathour, Rajeev
    Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most destructive diseases worldwide. Compared with leaf blast resistance, our understanding of panicle blast resistance is limited. In this study, we have identified, genetically mapped and physically delimited the chromosomal location of a new neck blast resistance gene from a broad spectrum resistant genotype ‘RIL4’. The segregation analysis of F2 and F2:3 progenies of cross HPU2216 and RIL4, indicated that the neck blast resistance in RIL4 is controlled by a single dominant gene. The new gene was tentatively designated as ‘Pb2’. Based on the linkage analysis of 542 individual susceptible plants, the resistance gene Pb2 was fine mapped to a 1.2 cM region flanked by RM3246 at 0.9 cM on telomeric side and RRS19 at 0.3 cM on the centromeric side of rice chromosome 12. By aligning the sequences of flanking markers on the reference sequence of cv. Nipponbare, a ~6.19 Mb region extending from position 9095272 to 15287816 b near the centromere of rice chromosome 12 was delineated as the region of neck blast resistance locus. The gene Pb2 was inferred to be embedded in recombination suppressed region as the average physical/genetic (P/G) distance ratio in this region was ~20- fold higher than the average P/G ratio of 260-280Kb/cM reported for the rice genome. A total of 334 predicted genes were identified in Pb2 region by surveying the equivalent genomic region of cv. Nipponbare in Rice Annotation Project database (RAP-db) (http://rapdbbeta.dna.affrc.go.jp). Five putatively expressed NBS–LRR genes were identified in the target region by surveying the equivalent genomic region of Nipponbare and four of these, NBS-LRR genes Os12g0270300, Os12g0273300, Os12g0281600 and Os12g0438300 were short-listed as a potential candidate for the resistance gene Pb2. The neck blast resistance gene ‘Pb2’ is a novel gene and displays distinct resistance spectrum compared to genes Pita, Pita2 and Pitr, previously identified from the syntenic region of donor genotype Tetep. The ‘Pb2’ linked markers identified during this study can be exploited in markerassisted breeding programmes for its speedy and precise mobilization into blast susceptible varieties.
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    ThesisItemOpen Access
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    ThesisItemOpen Access
    MARKER ASSISTED AND DOUBLED HAPLOIDY BREEDING FOR THE DEVELOPMENT OF BLAST AND BACTERIAL BLIGHT RESISTANT RICE PYRAMID LINES
    (CSKHPKV, Palampur, 2018-07-24) Chauhan, Ruchi; Kapila, R.K.
    Rice (Oryza sativa L.) serves as a major carbohydrate source for nearly half of the world’s population and provides livelihood to the people of Asia. Rice blast caused by Magnaporthe grisea and bacterial leaf blight caused by Xanthomonas oryzae pv.oryzaeare the two major diseases affecting rice productivity in north-western Himalayan region of India. Biotechnological tools like anther culture and molecular marker technology hold a great role as a catalyst in accelerating the pace of blast and bacterial blight resistance breeding. In order to pyramid 2 blast (Pi9 and Pita) and 2 bacterial blight (Xa21 and Xa38) resistance genes in a popular rice variety ‘HPR2143’, foreground selection was done to identify single gene positive BC2F2 homozygous progenies of 4 crosses, viz. HPR2143/PB1 (Pi9), HPR2143/DHMAS164 (Pita), HPR2143/IRBB54 (Xa21) and HPR 2143/PR114 (Xa38) using gene derived markers. The foreground selection resulted in identification of 5 plants homozygous for the gene Pita, 20 for Pi9, 12 for Xa21 and 6 for Xa38.In order to pyramid two genes each against rice blast, progenies positive for gene Pita were crossed reciprocally with progenies positive for the gene, Pi9. Likewise, the homozygous positive plants for genes, Xa21 and Xa38 were crossed reciprocally to produce F1s. Overall crossability among the selected derivatives over both cross combinations (Pita × Pi9) and (Xa21 × Xa38) was recorded to be 38.61%. Anther culture of developed F1s was attempted for fixation of genes. For cross (Pita + Pi9 and reciprocals), overall callus induction and regeneration frequency of 9.2% and 6.5%, respectively was recorded. The frequency of green plantlet regeneration recorded was 29.9% thereby resulting in an overall anther culture efficiency of 0.18% from a total of 22562 anthers cultured. For cross 2 (Xa21× Xa38 and reciprocals), observed callus induction and regeneration frequency was 9.08% and 5.7%, respectively. A total of 88 regenerating calli resulted in regeneration of 20 green (22.7%) and 68 albino plants (77.2%) with an overall anther culture efficiency of 0.11% in a total of 16894 cultured anthers. Out of 57 plants, 28 were positive for the gene combination, Pita + Pi9 and 15 for Xa21 + Xa28. Field evaluation of 38 generated doubled haploid pyramid (DHP) lines having sufficient seeds revealed presence of significant variation among the lines for all nine traits studied. Based on their performance, promising DHP lines for different economic traits were identified including three best DHP lines, i.e. DHP57, DHP4 and DHP16 performing close to the check, HPR2143 for seed yield/plant. The material generated can be used directly for identification of variety(ies) as well as can further be used as genetic stocks in the future breeding programmes for developing broad spectrum and durable multiple disease resistance against rice blast and bacterial leaf blight diseases
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    ThesisItemOpen Access
    MOLECULAR MAPPING OF QUANTITATIVE TRAIT LOCI FOR DROUGHT TOLERANCE AND YIELD TRAITS IN LENTIL
    (CSKHPKV, Palampur, 2016-07-22) Rana, Maneet; Sharma, T.R.
    ABSTRACT Lentil (Lens culinaris Medik subsp. culinaris) is an autogamous diploid (2n=2x=14), cool season food legume crop cultivated globally. Genomic resources in lentil are limited in comparison to other food legumes, primarily due to large genome size and lack of genetic variation. Further, lentil production is hampered due to various biotic and abiotic stresses worldwide. Among abiotic stresses, drought is one the major production constraints causing up to 70 per cent yield losses in lentil. In order to dissect the complex nature of drought tolerance and to use genomics tools for enhancing yield of lentil under drought stress conditions, intraspecific RIL mapping population (L830 x Precoz) segregating for drought tolerance and yield related traits was used. Twelve hundred and twenty-nine SSR markers (including previously published anchor markers) were screened for parental polymorphism and 293 (23.84 %) were found to be polymorphic among the parents. Of these, 291 were mapped on seven linkage groups at LOD 4.0 spanning 1199.0 cM with an average marker density of 4.8 cM. The study reported assigning of 46 new SSRs on the linkage map. Analysis of variance revealed significant differences for all the 27 measured traits between the drought tolerant ‘L830’ and the susceptible cultivar ‘Precoz’. The ANOVA of 126 RILs revealed significant differences for almost all the traits except RFW, RDW, CAR and CHL evaluated under the drought stress conditions. Significant effect of environment was also observed for all the traits measured, except DTF, RP, DTM and SS. Phenotypic data from the RILs were used to identify QTLs for drought tolerance and yield traits by composite interval mapping (CIM). A total of 75 QTLs (LOD ≥ 2.5) were detected across the three environments (control, drought stress and cylinder culture) and QTLs were detected across all the linkage groups. Among these, 13 were stable across locations/environments, 12 were found to be consistent across the seasons and 27 were drought specific. Phenotypic variation explained (PVE) by QTLs ranged from 5.4 to 45.9 per cent. The highest phenotypic variation (45.9 %) was explained by the QTLs for 100-seed weight. In conclusion, it is envisaged that the present linkage map, fortified with 291 SSR markers and 75 QTLs for drought tolerance and yield-related traits would provide genomics tools to breeders for further genetic enhancement of this crop species. Thus, the current study would serve as a strong foundation for further validation and fine mapping of QTLs for utilization in lentil breeding programs.