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ThesisItem Open Access Mapping the QTL for yield traits in bitter gourd (Momordica charantia L.)(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2022) Lavale, Shivaji Ajinath; KAU; Deepu, MathewBitter gourd (Momordica charantia), being a rich source of phytonutrients such as carbohydrates, minerals, vitamins, and other medicinal compounds, has a great importance in healthy dietary habits. Breeders always seek to breed bitter gourd varieties for the traits such as early maturity and high yield. However, limited investigations have been made to identify the genetic loci governing yield related traits. Marker assisted selection (MAS) assures the presence of favourable alleles and fast recovery of recurrent parent genome in the cultivar under improvement. The success of MAS mainly depends on the availability of a marker-dense genetic linkage map locating quantitative trait loci (QTL) for the target traits. The present study “Mapping the QTL for yield traits in bitter gourd (Momordica charantia L.)” was carried out during October, 2018 to December, 2021 with the objective to map the quantitative trait loci and to develop chromosome-wise maps for the yield traits in bitter gourd. To develop the mapping population, high yielding bitter gourd cultivar Priyanka (Momordica charantia var. charantia) and a wild bitter gourd accession IC634896 (M. charantia var. muricata), were used as parents. A set of 450 microsatellites were screened for polymorphism using genomic DNA of parents and 47 were found polymorphic. Bitter gourd genome (GenBank acc. no. GCA_013281855.1) was scanned and new hypervariable microsatellites were identified using Genome wide Microsatellite Analysing Tool (GMATo) and named as KAUBG_n where n is a serial number. From the 75 microsatellites identified, 69 were validated through successful PCR amplification and 38 among them were polymorphic between the parents. This led to the development of a set of 85 markers polymorphic between the parents. Crosses were made between the parental lines and hybrids from the cross Priyanka × IC634896 yielded more number of fruits and total fruit produce compared to the reciprocal hybrid. An F2:3 population was developed through single seed descent method from the cross Priyanka × IC634896. A panel of 200 F2:3 plants were evaluated for twenty seven traits, including fruit-, flower-, seed-, vine-, and leaf-related traits, contributing directly or indirectly to the total yield. Wide variation was observed among the F2:3 plants for the traits studied. A group of ninety plants was selected from 200 F2:3 plants such that they represent the variation of the base population. Genomic DNA of these plants were genotyped using 85 polymorphic markers. Genotypic data from the screening of 85 markers in the mapping population were used to generate a linkage map spanning 1287.99 cM distance across eleven linkage groups (LGs) corresponding to eleven chromosomes, using IciMapping software. LG 7 (28 markers) consisted of maximum number of markers followed by LG 2 and LG 9, each having 11 markers. LG 1 had 10 markers whereas LG 3, 4 and 8 had seven markers each. LG 5, 6, 10 and 11 had only one marker each. LG 7 covered maximum map distance of 384.19 cM where LG 8 covered least map distance of 68.58 cM. The genetic map and phenotypic data were used to generate the QTL maps, using Inclusive Composite Interval Mapping (ICIM) method to locate twenty seven traits on Momordica genome. Sixty QTL, including 37 major QTL with LOD values ranging from 3.1 to 15.2, explaining 1.8 to 35.9 per cent of the phenotypic variation were identified for 24 traits, on seven chromosomes. Twenty three QTL were identified for fruit-traits with LOD values ranging from 3.1 to 7.6, explaining 5.5 to 35.9 per cent of phenotypic variation. Thirteen QTL were identified for flower-related traits with LOD value ranging from 3.1 to 15.2, explaining 7.0 to 26.0 per cent of phenotypic variation. Seven QTL each were identified for seed and leaf-related traits with LOD values ranging from 3.2 to 10.8 and 3.5 to 6.5, explaining 5.6 to 26.3 and 3.2 to 15.8 per cent of phenotypic variation, respectively. Ten QTL were identified for vine-related traits with 3.2 to 8.7 LOD values and explaining 1.8 to 17.6 per cent of phenotypic variation. Single marker analysis was performed to identify markers co-segregating with the yield contributing traits. There were 129 hits for the marker-trait association with LOD values more than 3.0, explaining 11.62 to 29.34 per cent of the phenotypic variation. Using the least and best performing F2:3 plants, markers S13, KAUBG_5 and KAUBG_11 were validated for co-segregation with fruit breadth, first pistillate flower node, and number of pistillate flowers and fruits per plant, respectively. This study gives insights into the relative locations of microsatellites and major effect QTL for yield traits in Momordica genome. QTL with shorter marker interval (qFrtL-8-1, qDPF-3-1, qDSF-3-1, qDSF-7-1, qFrtShp-8-1) can be directly used in MAS for improving yield characters. Linkage observed between microsatellites identified in this study with yield traits signifies their importance in further fine mapping as well as marker assisted selection. The linkage map constructed in this study, being the first with microsatellites from Momordica genome, paves the path for comparative and consensus map generation with other marker types. Further, fine mapping using markers within the identified QTL hotspots can lead to possible identification and cloning of genes underlying the yield traits.ThesisItem Open Access RNA mediated resistance to Yellow vein mosaic virus in okra(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2021) Kelkar Vipul Ganesh; KAU; Deepu MathewOkra (Abelmoschus esculentus L. Moench, Malvaceae) is one of the leading vegetable crops in hot and humid tropics. Unfortunately, this climate is conducive for many of the pests and diseases. Okra is susceptible to viruses such as Yellow vein mosaic virus (YVMV) and Enation leaf curl virus (ELCV), belonging to the genus Begomovirus (family Geminiviridae). Because of the favourable conditions prevailing in the coastal region, the losses in Kerala state are 60-100%, depending upon the stage of plant growth and the severity of infection. RNAi is one of the promising molecular biology approach against the viral diseases. Keeping the above facts in view, the present study “RNA mediated resistance to Yellow vein mosaic virus in okra” was taken up at the Centre for Plant Biotechnology and Molecular Biology, CoA, Thrissur from September 2017 to May 2021. The high yielding and YVMV susceptible popular okra cv. Salkeerthi was selected for the development of resistance using RNAi mechanism. Total DNA was isolated from the YVMV infected plant and part of the βC1 gene (187 bp) of the virus was amplified using primers VβC1F and VβC1R. Sequence information of PCR product has revealed that the gene is 90-95% identical with the Indian isolates. The βC1 gene sequence was analysed using IDT software and 10 siRNAs were found at three different position (19-44, 34-59, 99-124 bp). Through Restriction Mapper, it was confirmed that the sequence selected for the preparation of sense and antisense strand, do not possess recognition sites for SmaI, HindIII and MauBI restriction enzymes which are present in the pRNAiLIC vector. The output of VSupPred revealed that the fragment does not contain any Viral Suppressor Regions (VSRs), with a high prediction score (0.625). The hairpin RNAi construct harbouring the region of βC1 gene of β satellite of Begomovirus of okra was generated using pRNAi-LIC (CD3-1285) vector. The SmaI digested plasmid produced three fragments, vector backbone (9842 bp), Pdk intron (1641 bp) and ccdB gene (614 bp) and the digested plasmid was treated with dTTP. Product-1 was PCR amplified (215 bp) with VLIC1 and VLIC2 primers, using the DNA from YVMV infected plant as template. Product-2 was PCR amplified (243 bp) with VLIC3 and VLIC4 primers using product-1 as template. Product-1 and product-2 were eluted from the gel and treated with dATP. The dATP treated PCR products and dTTP treated SmaI digested plasmid were mixed together and ligated by incubation at 65ºC for 5 min. followed by 22ºC for 15 min. Ligated product was successfully transformed in competent cells of E. coli (DH5α) and incubated on LB medium containing Kanamycin and Chloramphenicol. Colony PCR was performed, the transformation efficiency was found to be 80%. Plasmid was isolated from the positive DH5α colony and sequenced using the primers VLIC5 and VLIC6. The sequence data had shown that both sense and antisense strands are at right position and direction. Plasmid containing ihpRNA-βC1 cassette was successfully transformed into the competent cells of Agrobacterium (GV3101) and incubated on LB medium containing Kanamycin, Chloramphenicol and Rifampicin. Colony PCR was performed, the transformation efficiency was found to be 100%. Plasmid was isolated from the positive GV3101 colony and sequenced using the primers VLIC5 and VLIC6. Sequence data has further confirmed that both sense and antisense strands are at right position and direction. The ihpRNA-βC1 cassette was successfully transformed into okra cv. Salkeerthi using in planta method of Agrobacterium mediated transformation. The transformation efficiency observed was 11.42% and the transformation was confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5. cDNA was prepared from the total RNA isolated from transformed and control plants. siRNA synthesis was confirmed using the primers VLIC1 and VLIC5 (400bp) and Ubiquitin gene was confirmed using the primer UBQ7 (187 bp). Silencing potential of the RNA interference of βC1 gene and the development of resistance was evaluated by keeping the 15-day old transformed and control plants along with YVMV infected plants inside containment facility, with whiteflies released into insect cage for infection. All the control plants and one transgenic plant have shown the YVMV symptoms after 10 days. Three transgenic plants were healthy with no symptoms. The present investigation was successful in the development of YVMV resistant okra plants carrying ihpRNA-βC1 using pRNAi-LIC (CD3-1285) plasmid vector. The further evaluation is needed in the coming generations for the identification of stable transgenic lines.ThesisItem Open Access In vitro synthesis of gingerol and analysis of expressed sequence tags for gingerol production in ginger(Zingiber officinale Rosc.)(Centre for plant biotechnology and molecular biology,College of Horticulture, Vellanikkara, 2020) Manjusha, Rani; KAU; Shylaja, M RBlack pepper (Piper nigrum L.), often described as the ‘King of spices’ is the most important spice crop, grown for its berries in the world. Indian pepper is preferred across the globe due to its intrinsic qualities. Foot rot is a devastating disease of black pepper. In the changing climate, drought can be a major threat in black pepper production. Hence, the present study was taken up at College of Horticulture, Vellanikkara and ICAR-IISR, Kozhikode to characterise and to identify superior accessions of black pepper for yield, quality and tolerance to biotic and abiotic stresses. Fifty accessions of black pepper in the bearing stage maintained in the National Active Germplasm Site of ICAR-IISR, Kozhikode formed the base material for the study. The accessions were characterised for fifty qualitative and fifty quantitative characters following the descriptor developed by IPGRI (1995). Wide variability was observed among the accessions for ten qualitative characters. Quantitative characters of shoot, leaf, spike and fruit also showed wide variability. Field tolerance to foot rot disease and pollu beetle infestation was observed among the accessions. Twenty accessions were selected from the base collection based on superiority of yield (> 450g green berries/vine) , field tolerance to foot rot disease infection (biotic susceptibility score 1) and pollu beetle infestation (biotic susceptibility score 1-3). They were further evaluated for biochemical principles of quality, tolerance to foot rot disease under artificial inoculation and tolerance to drought by physiological and biochemical analyses. Piperine, essential oil and oleoresin ranged from 3.61 - 6.96 per cent, 3.00 - 5.87 per cent and 7.10 - 11.18 per cent, respectively, across the accessions. The accessions with high value of piperine, essential oil and oleoresin were identified as 7293, 7211 and 7289 respectively. The two accessions identified viz. 7293 and 7252 contained more piperine than the highest of Panniyur 2 (6.6 per cent) reported among the released varieties . Artificial inoculation of selected accessions using Phytophthora capsici culture for screening for foot rot disease resistance based on over all disease severity index of both stem and leaf lesions showed that accession 7259 was moderately resistant. The selected accessions did not exhibit significant variation for various physiological and biochemical parameters at field capacity. However higher value of photosynthesis, chlorophyll content, chlorophyll stability index, relative water content and membrane stability index and low leaf temperature were observed for accessions viz. 7215, 7240, P 5 and 7241 after five days and ten days of moisture stress induction following field capacity compared to other accessions. Higher values of proline, SOD, catalase and peroxidase were also observed for these accessions. The visual scoring showed that accessions with higher values for most of physiological and biochemical parameters of drought tolerance viz. 7215, 7240, P5, and 7241 had lesser number of fallen leaves and more number of leaves retained at permanent wilting point (PWP). The accessions 7215 and 7240 took twenty days to reach PWP compared to eleven accessions which took only 16 days to reach PWP. Foliar nutrition with sulphate of potash, IISR - Power mix and Pink Pigmented Facultative Methylotrophs (PPFM) had positive effect on drought tolerance for the accessions (7215, 7240, P5 and 7241) having natural tolerance. The identified accessions with high yield , quality and tolerance to biotic or abiotic stress can be used for further breeding programme.