1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Molecular characterization of CRISPR/ Cas9 edited lines of rice for enhanced drought tolerance and yield(Department of plant biotechnology, college agriculture, Vellanikkara, 2023-10-09) Brindha, T M.; Rehna Augustine.Rice is an important stable and staple food around the world, providing essential nutrition to more than half of the population in more than a hundred countries, including China, India, Indonesia, Vietnam, and the Philippines. Being semi-aquatic annual plant, rice crop consume about 80% of the water used for irrigation. With expanding urbanization and industrialization processes, water is turning out to be scant, and dry season pressure might arise as an extraordinary test to farming everywhere. At the beginning of 2023, 27 of the 36 states and Union territories in India experienced either little or no precipitation, indicating that the dryness persisted throughout the month. Hence enhancing the yield and tolerance to drought remain as major breeding objectives in rice. Crop improvement through biotechnological interventions are imperative in this scenario. Out of various genome engineering tools, CRISPR/Cas9 is a third-generation genome editing tool that has proven to be one of the precise, more efficient and successful genome editing systems across a wide range of organisms, including plants given its ability to efficiently, inexpensively, rapidly, and accurately elicit desired changes. However, the Cas9 complex may bind to unintended regions and initiate cleavage leading to off-target effects, which is considered as the major disadvantage of this technology (Alkan et al., 2018). The current study was undertaken to characterize CRISPR/Cas9 meditated edited lines of rice previously developed in the lab against OsMADS26 (a negative regulator of drought tolerance), osa-miR1432 and osa-mi396b (both negatively regulates grain yield) by performing off-target analysis and gene expression studies. For off-target analysis, the off-target sites for each gRNAs used for developing the mutant lines of OsMADS26, osa-miR1432 and osa-mi396b were predicted using CRISPR Pv2.0 online tool and corresponding sequences were retrieved from Rice Genome Annotation Project database to design flanking primers. The primers were designed from the retrieved sequences, flanking the off-target sites, and evaluated using the ‘Oligoevaluator’ tool. The off-target sites were amplified using PCR, sequenced and analysed for the presence of any mutation. No off-target editing was observed in the edited lines for the evaluated off-target sites. The genome-edited lines of T1 generation were raised from seeds of T0 lines in the containment chamber. Around four T1 plants each from eight genome-edited events of OsMADS26 G1, four events of osa-miR1432 and two events of osa-miR396b were screened for the presence of hygR and Cas9 gene using PCR. The stability of mutations in the edited lines of OsMADS26, osa-miR1432 and osa-mi396b were analysed by amplifying the target region followed by sequencing and sequence analysis. A comparison of mutations observed between T0 and T1 generations were made which showed that mutations were stable. Selected edited lines from each construct were used for Real-time qRT-PCR analysis. Expression profiling of the candidate genes was performed using Real-time qRT PCR. In OsMADS26 G1 edited lines, OsMADS26 gene expression was found to be down regulated compared to the wild type. Expression of OsMADS26 downstream genes like CHI7, POX22.3, SALT, RAB21, and OsWRKY28, involved in various stress responses, was also analysed. In the osa-miR1432 edited lines, OsACOT gene, the target of osa-miR1432 was found to be upregulated. In osa-miR396b edited lines also showed higher expression of GRF6 & GRF8, targets of osa-miR396b.The phenotypic analysis of promising CRISPR/Cas9 edited lines identified in the study will be carried out in future for enhanced drought tolerance and yield.Item Marker assited pyramiding of bacterial blight resistance genes in backcross population of rice variety jyothi(Department of Plant Biotechnology, College of Agriculture , Vellanikkara, 2023-04-03) Athira Rajan , P V; Rose Mary FranciesRice variety Jyothi (PTB 39) is one among the most preferred and widely cultivated variety across Kerala. Despite its widespread popularity, the cultivar is highly susceptible to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo). Considering its efficiency, economic viability and eco-friendliness, host-plant resistance has been advocated as the best option to combat the BB pathogen. Advancements in the field of molecular biology such as marker-assisted selection (MAS) have paved ways for pyramiding multiple R-genes into a single genotype. In this context, efforts to introgress three R-genes (xa5, xa13 and Xa21) imparting resistance to BB into the rice variety PTB 39 (Jyothi; recurrent parent) from Improved Samba Mahsuri (ISM; donor parent) through marker assisted selection, was initiated at the College of Agriculture, Vellanikkara. The present study was formulated to identify BC2F1s and BC1F2s pyramided with the R- genes conferring resistance to bacterial blight (xa5, xa13 and Xa21), using functional marker as well as R-gene linked Sequence tagged site (STS) markers. Morphological characterisation of the population was also envisaged. Foreground selection in five BC2F1s along with the parents was done using good quality DNA isolated in order to identify plants introgressed with the three targeted R-genes. Markers RG556 and xa5SR/R linked to xa5, RG136 and xa13 prom linked to xa13 and pTA248 linked to Xa21 were used. Foreground profiling with RG556 and xa5SR/R resulted in monomorphic banding pattern in the experimental population revealing the endogenous presence of R-gene xa5. Screening of BC2F1s using RG136 and xa13-prom and pTA248 revealed the presence of amplicons similar to that found in the recurrent parent Jyothi in the backcross individuals, implying the absence of R-genes xa13 and Xa21 in BC2F1s. The results thus pointed out that the BC2F1s possessed only one R-gene i.e., xa5. Morphological characterisation of BC2F1s indicated that population exhibited similarity towards the recurrent parent Jyothi. Foreground profiling of BC1F2 (132 Nos.) and parents with markers RG556 and xa5SR/R linked to R-gene xa5 resulted in monomorphic banding pattern across the experimental population. This indicated the endogenous presence of the R-gene xa5 in BC1F2s as well as the parents. Presence of R-gene xa13 was evident in 35 BC1F2s i.e., five progenies of BC1F1 Plant No. 9.7, twenty-four progenies of BC1F1 Plant No. 9.15 and six progenies of BC1F1 Plant No. 9.17, on screening with R-gene xa13 linked markers RG136 and xa13-prom. Twenty-five R-gene pyramids, out of the 35, were deduced to be heterozygous at xa13 R-gene locus (xa5xa5+xa13Xa13), while 10 were found to be homozygous (xa5xa5+xa13xa13). Foreground selection for the R-gene Xa21 using pTA248 helped identify 31 BC1F2s introgressed with R-gene Xa21. Among them, 18 were found to be heterozygous (xa5xa5+Xa21xa21) and 13 were homozygous (xa5xa5+ Xa21Xa21), at the R-gene locus (Xa21). The foreground selection thus helped delineate 42 R-gene pyramids among the 132 BC1F2 individuals. Among them, twenty-four were identified to be 3-R-gene pyramids. This included four individuals (Plant No. 9.15.15, Plant No. 9.15.20, Plant No. 9.15.39 and Plant No. 9.17.8) homozygous at all three R-gene loci (xa5xa5+xa13xa13+Xa21Xa21). Background selection of the 24 three-R-gene pyramids identified was done using 52 SSR markers found to be polymorphic between the donor parent ISM and recurrent parent Jyothi. Results revealed the presence of wide variability among the backcross individuals with respect to segregation of marker loci as well as genome recovery from recurrent parent (RPGR). The maximum recovery of 61.90 per cent was found in Plant No. 9.15.59 followed by 59.40 per cent in Plant No. 9.17.8. The least recovery was observed in Plant No. 9.15.42 (27.90 %). The RPGR per cent for the four 3-R-gene pyramids homozygous at all three loci were 41.50 (Plant No. 9.15.15), 47.30 (Plant No. 9.15.20), 48.60 (Plant No. 9.15.39) and 49.40 (Plant No. 9.17.8). In general, the BC2F1s exhibited more similarity to the recurrent parent Jyothi rather than the donor parent for yield attributes and yield. However, none of the BC2F1s and BC1F2s flowered earlier than Jyothi and no individuals were found to flower as late as the donor parent. Except in case of days to flowering, the average estimates of yield attributes and yield of BC1F2s were less than that observed in the recurrent parent Jyothi, but higher than that observed in donor parent ISM. Two R gene pyramided BC1F2 individuals possessed white coloured decorticated kernels similar to the donor parent In spite of lower average estimates for yield attributes and yield, transgressive segregants were also observed among the BC1F2s. Negatively skewed platykurtic distribution was observed in traits like plant height, leaf length, panicle length, spikelets per panicle, grains per panicle, 100 grain weight, grain length and width, and decorticated kernel width, while positively skewed platykurtic distribution was evident for all other traits viz., days to flowering, total tillers, productive tillers, leaf width, and grain yield per plant. The decorticated kernel length was the only trait that exhibited a leptokurtic distribution and had registered negative skewness. Hence, it can be inferred that the trait kernel length is governed by a few segregating genes with majority of them exhibiting decreasing effects and dominance-based interactions. All the forty-two R-gene introgressed BC1F2s were selfed to yield seeds (3897 Nos.) of BC1F3 generation. The BC1F3 progenies of the 3-R-gene homozygotes need to be further backcrossed to the recurrent parent to aid higher recovery of the recurrent parent genome. This will eventually lead to the development of an Improved Jyothi with durable resistance to BB pathogen. These stable R-gene introgressions can also serve as donors in BB resistance breeding programmes in rice. In addition, the transgressive segregants could serve as potential breeding materials for development of high yielding genotypes.Item DNA barcoding in Cucumis spp(Department of Plant Biotechnology, College of Agriculture , Vellanikkara, 2023-04-26) Kesanapalle Sunny Babu.; Kiran , A GThe genus Cucumis L. has 52 species distributed mainly in Asia and Africa of which 13 belong to India. Cucumber (Cucumis sativus L.) and melon (C. melo L.) are among the most widely cultivated horticultural crops in the world. Both the species have progenitor populations in the Himalayan region and studies have suggested Asia as the ancestral area for the most recent common ancestor of melon and cucumber. Knowing the closest relatives is important for the ongoing efforts of breeders to improve cucumber and melon. The wild relatives of cultivated species form an essential gene pool for vigorous growth, productivity, resistance to biotic and abiotic stresses and nutritional quality. Many wild varieties and species belonging to the genus Cucumis L. have been reported from Asia, Africa and Australia. Nuclear or chloroplast gene sequence based phylogenetic analysis can reveal genetic relations between wild and cultivated genotypes. DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identification using short, standardized genomic regions as internal species tags. Species identification through barcoding is usually achieved by the retrieval of a short DNA sequence i.e., the barcode from a standard part of the genome, a specific gene region either from chloroplast, mitochondria or nuclear genome. The barcode sequence from each unknown specimen is then compared with a library of reference barcode sequences derived from individuals of known identity. In plants DNA barcode markers like rbcL, matK, trnH-psbA, and ITS2 have been developed, tested, and used to address basic questions in systematics, ecology, evolutionary biology, and conservation. The chloroplast maturase K gene (matK) is one of the most rapidly evolving plastid coding regions and it consistently showed high levels of discrimination among angiosperm species. Although matK primers are reported to be having high discriminative power their universality is doubted. However, in recent studies done in Cucurbitaceae, matK gene was found to be highly successful in terms of both discriminatory power and universality. Present study is an attempt to understand the phylogenetic relationship between wild and cultivated genotypes of the genus Cucumis L. using matK coding sequence and to identify barcode targets for species identification. The study included accessions belonging to the two cultivated species and two wild species viz., C. metuliferus and C. maderaspatanus. Wild varieties of cultivated species like C. sativus var. hardwickii, C. melo var. conomon, C. melo var. callosus and C. melo var. momordica were also included in the analysis. Morphological characters of the different accessions were recorded as per the minimal descriptors developed by ECPGR working group on Cucurbits (2008). All the four species exhibited distinct morphological characteristics in their leaf, stem, tendril, flower and fruit. Early plant vigour and growth habit also differed between the species. Characters common to all species include stem pubescence, coiled and unbranched tendril, unified leaf margin and monecious yellow flowers. Total genomic DNA was isolated using CTAB method and subjected to PCR using matK primers. PCR generated specific amplicons of expected size 900 bp in all the samples. Gel purified PCR amplicons were subjected to paired end Sanger sequencing. Good quality sequences were generated for all accessions except C. melo var. callosus and C. melo var. momordica. Consensus sequences without PCR and sequencing induced errors were identified for each accession by aligning the sequences with reference sequences from Genbank database. The consensus sequences, tracefiles and specimen images were submitted to Barcode of Life Database (BOLD) and Process IDs were generated. The consensus sequence as well as their homologues from Genbank were subjected to global alignment using Multi Allignment using Fast Fourier Transformation (MAFFT) programme. The alignment produced a continuous stretch of 263 bases without any gaps which was used for identification of SNPs unique to each species. For different accessions of each species the sequences were identical without any variation. Unique SNPs were available for all the four species in a span of 92 bases within the region. In order to study the genetic relationship between wild and cultivated species phylogenetic analysis was performed using MEGAv11. All the accessions used for barcode identification were included. Tree was constructed by UPGMA method with 500 bootstrap replications. The phylogenetic tree consists of 2 main clades with bootstrap value 69. Clade I consist of C. sativus and C. maderaspatanus as sub clades Ia and Ib with bootstrap values 81 and 100 respectively. Clade II consist of C. metuliferus and C. melo as subclades IIa and IIb with bootstrap values 96 and 82 respectively. Pairwise genetic distance between accessions calculated by Kimura 2 Parameter model revealed that interspecific distance ranges between 0.77% - 1.15%. Although barcode gap as defined by BOLD cannot be established as interspecific distance is less than 2%, no overlap between intra and interspecific distances was noticed. Generally, in plants interspecific distances reported is very low for most barcode targets and it is suggested that varying bar code gaps should be fixed depending on the species.Item Marker assisted selection in 3-R gene pyamided lines of rice variety uma for bacterial blight resistance(Department of Plant Biotechnology, College of Agriculture, Vellanikkara, 2023-04-03) Bharde Pranali Rajendra; Rose Mary FrancisThe tropical humid environment prevailing in Kerala serves as a conducive environment for development and spread of bacterial blight (BB) disease in rice. The level of crop loss due to BB disease has been found to vary from 6-60%, depending on the cultivar, stage of infection, and severity of the illness under natural conditions. Uma, the popularly grown elite rice variety of Kerala, is highly susceptible to the BB pathogen. Considering the recurrent incidence of BB disease and the loss incurred by the farmers, efforts were initiated at College of Agriculture, Vellanikkara, to impart durable resistance to the cultivar Uma against BB pathogen, by pyramiding three R genes (xa5+xa13+Xa21) into the variety from donor Improved Samba Mashuri (ISM). The present study was conducted during 2019-2021 and it aimed to delineate the 3-R gene pyramids among BC3F1s (10 Nos.), through marker assisted selection. In addition, pathotyping and selfing of BB resistant plants among backcross generations; BC1F3s (725 Nos.) and BC2F2s (107 Nos.) were also envisaged. The results obtained are discussed below. Good quality DNA isolated from BC3F1s (10 Nos.) and parents (ISM and Uma) were subjected to foreground selection. Profiling of the BC3F1 individuals using the xa5 gene linked STS marker RG556 and functional marker xa5SR/R resulted in monomorphic banding pattern. In case of marker xa5SR/R, a single amplicon was found to be present in the donor (ISM), recurrent parent (Uma) as well as the BC3F1s. Four amplicons of variable size were visible on visualisation of the restriction enzyme digested PCR amplified product of STS marker RG556 in all the experimental population. The results thus indicated the endogenous presence of R-gene xa5 in both the parents and BC3F1s in the homozygous state. Screening of BC3F1 individuals with STS marker RG136 and functional marker xa13-prom linked to R-gene xa13 revealed that BC3F1 Plant No. 8.3.9.10.3 was heterozygous at xa13 locus. Further, the absence of resistant allele of xa13 was evident in the other BC3F1 individuals. Foreground selection of the BC3F1s with STS marker pTA248 linked to dominant gene R-gene Xa21 indicated the absence of gene Xa21 in the BC3F1s individuals. The results of foreground selection thus pointed out that BC3F1 Plant No. 8.3.9.10.3 was a 2-R-gene pyramid homozygous at xa5 locus but heterozygous at xa13 locus. Background profiling of the 2-R-gene pyramided BC3F1 Plant No. 8.2.9.10.3 with 42 rice microsatellite (RM) markers revealed that it possessed amplicons similar to the donor parent w.r.to ten markers (RM11069, RM11554, RM85, RM251, RM17182, RM307, RM252, RM508, RM26868 and RM260). In case of RM589 and RM23087, monomorphic banding pattern was observed in the experimental individuals. Presence of amplicons identical to that found in the recurrent parent Uma was evident in all other cases. The results thus point out that the backcross progeny BC3F1 Plant No. 8.3.9.10.3, majorly resembled the recurrent parent i.e., at 30 out of the 42 marker loci analyzed. Graphical Genotyping indicated that the 2-R-gene introgressed BC3F1 Plant No. 8.3.9.10.3 registered 75.00 per cent recovery of recurrent parent genome. Characterization of the recurrent parent Uma, donor parent ISM and BC3F1s indicated wide variability for various morphological traits studied. It was evident that the 2-R-gene pyramided BC3F1 Plant No. 8.3.9.10.3 was short statured with short but broader leaves. It was found to flower earlier (109.00 days) than the donor parent ISM (112.00 days) but was found comparable with recurrent parent Uma (107 days). It possessed longer grains (7.20 mm) than the parents (ISM: 6.62 mm; Uma: 6.80 mm). However, although broader than ISM in width, the grains were found to be slender than Uma (3.10 mm). The identified 2-R-gene pyramid (BC3F1 Plant No. 8.3.9.10.3) was selfed resulting in production of BC3F2s (149 Nos.). Bioassay of BC1F3 population (725 Nos.) through leaf clipping method (IRRI, 1996) revealed that 5.52 per cent were resistant to bacterial blight. Moderate level of resistance was observed in 23.31 per cent of the population, while 57.79 per cent of the individuals were found to exhibit moderate susceptibility to BB pathogen. BC1F3s exhibiting resistance or moderate resistance to BB pathogen were selfed. This yielded 216619 seeds of BC1F4 generation. Pathotyping of BC2F2s indicated that out of 107 progenies, 11.21 per cent were resistant against the BB pathogen, while, 36.45 per cent were found to be moderately resistant. BC2F2 individuals exhibiting moderate resistance and resistance to BB pathogen were selfed. This resulted in the production of 40535 BC2F3s. Agronomic evaluation of both BC segregants that were 1 F 3 s and BC2F2s indicated the presence of better than the parental genotypes in most of the yield attributes as well as yield. In general, majority of the backcross individuals were short in stature and took longer period to flower than the parental genotypes. To summarize, MAS helped to deduce that BC3F1 Plant No. 8.3.9.10.3 was a 2-R gene pyramid (xa5xa5 + Xa13xa13). Backcrossing of the R-gene introgressed pyramid to the donor parent is necessary to recover the R-gene Xa21. Further evaluation through a combination of MAS and phenotypic evaluation will lead to development of BB resistant cultivar in the background of cultivar Uma. The novel gene combinations arising in the advanced breeding lines (BC1F3 and BC2F2), can serve as base population for future breeding programmes.Item Temperature induced changes in the biology and heat shock protein gene expression in malathion resistant red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)(Department of Plant Biotechnology, College of Agriculture,Vellanikkara, 2023-02-24) Sanjay, Sabu; Mani , ChellappanItem Development and pharmacological evaluation of small molecular bioactives from marine algae associated heterotrophic bacteria(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2020) Aneetta Francis; Kajal ChakrabortyItem Identification and expression profiling of Banana bract mosaic virus (BBrMV) responsive microRNAs in banana cultivar nendran (Musa AAB)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2020) Athira Vijayan; Soni, K BItem Genetic diversity analysis of Xanthosoma sagittifolium (L.) schott using molecular markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2020) Krishnaveni, Vijayakumar; Asha Devi, AItem Morphological and molecular analysis for assessing intraspecific variation in sweet potato (Ipomea batatas (L.) Lam.) and interspecific divergence in Ipomoea spp.(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2020) Sasilekha, S; Shirly Raichal AnilItem Identification and characterization of viruses infecting lesser yam (Dioscorea esculenta (Lour.) Burkill)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Sudheer, K S; Jeeva, M L
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