1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Genetic diversity analysis of gladiolus genotypes (Gladiolus grandiflorus L.) using molecular markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2022) Karthika Nair, A S; Beena ThomasCharacterization of plant genotypes based on crop genetic diversity is important for effective usage and conservation. This is generally achieved by either morphological tools or molecular tools or by using both. This study entitled “Genetic diversity analysis of gladiolus genotypes (Gladiolus grandiflorus L.) using molecular markers” was carried out in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, Thiruvananthapuram during 2020-2021 with an objective to analyse genetic diversity in the gladiolus genotypes using ISSR as well as morphological markers. Gladiolus (Gladiolus sp.) is a genus of perennial herbaceous cormous flowering plants in the family Iridaceae which is of high economic importance. Fifteen varieties of gladiolus including twelve varieties from IIHR, Bangalore and three varieties from NBRI, Lucknow were selected for this study. The study was divided into two parts- morphological characterization and molecular characterization. Morphological characterization was done by analysing both vegetative and floral characters. Different tools such as analysis of variance, co-variance, correlations, PCA and dendrogram were used for statistically analysing the recorded data. The dendrogram divided the genotypes into two principal clusters at a distance of 0.10. The major variables that contributed to the clustering of gladiolus genotypes were plant height, number of leaves per shoot, length of leaf, width of leaf, internode length, number of florets open at one time and number of florets per spike as revealed by PCA analysis. For molecular characterization using ISSR markers the genomic DNA was isolated using CTAB method of DNA isolation with little modifications. Ten ISSR primers were used for screening fifteen gladiolus genotypes. After the final PCR with selected primers, the amplicons were resolved in 2% agarose gel and polymorphic bands were obtained. Primers showed 94.56% polymorphism and the number of bands obtained ranged from 3(UBC 857) to 14 (UBC 890) with a mean value of 8.7 polymorphic bands per primer. A total of 87 polymorphic bands were obtained. The data analysed using NTSYS PC 2.02 program created a dendrogram, which grouped 113 the genotypes based on Jaccard’s similarity coefficient in which the fifteen genotypes were separated into two principal clusters. The first principal cluster consisted of most of the genotypes (12 genotypes). The second principal cluster comprised of ‘Arka Naveen’, ‘Archana’ and ‘Arka Gold’ with ‘Archana’ as an outlier. In molecular characterization, least similarity of 34% was observed between G3 (Arka Sapna) and G9 (Archana) whereas, maximum genetic similarity of 82% was observed between G6 (Arka Amar) and G10 (Arka Kumkum). The highest morphological similarity was also observed between G6 (Arka Amar) and G10 (Arka Kumkum) at a distance of 0.83 in UPGMA dendrogram based on Jaccard’s coefficient. Though some similarity in results existed between the morphological and molecular tools used for identifying the genetic relationships among selected gladiolus varieties in this study, it also revealed that the varieties were grouped as separate clusters based on morphological dendrogram. This may be due to the dependence of morphological expression on the physiological state of the individual plant along with environmental influence. Self-incompatibility, along with the outcrossing nature together might have contributed to the high variation observed among the gladiolus genotypes. Being a commercial cut flower crop, based on different floral parameters considered ‘Arka Sapna’, ‘Arka Nazrana’, Arka Darshan’, ‘Arka Amar’ and ‘Arka Poonam’ are recommended as the gladiolus genotypes that showed best performance in Kerala conditions. Tags from this library: No tags from this library for this title.Item Genetic diversity analysis of indigenous rice varieties in Kerala using molecular markers(Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Ajith, M K; Jayalekshmy, V GThe present study entitled “Genetic diversity analysis of indigenous rice varieties in Kerala using molecular markers “was carried out in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, Thiruvananthapuram during 2017-2018. The study was conducted with the objective to analyze the genetic diversity of traditional rice varieties in four agro climatic zones of Kerala using RAPD and SSR markers. Five varieties were collected from the each agro climatic zones viz., hill areas of Wayanad, rice growing tract of Palakkad, saline soils of Pokkali and Kuttanad soils. The DNA was isolated and RAPD analysis with 10 Operon primers and SSR analysis was done with ten RM primers. In the RAPD analysis the ten operon primers produced 88 amplicons with an average polymorphism 82%. Resolving power OPC-07 (15.4) had the highest value but its Polymorphism Information Content and Effective Multiplication Ratio (EMR) were considerably low. Considering all the three parameters together primer OPF-06 is found to be the best RAPD primer with considerably high polymorphism information content, resolving power and effective multiplication ratio. The dendrogram constructed based on the RAPD scoring showed that varieties Pokkali and VTL-2 had maximum similarity. These two were from Pokkali rice tract. PTB 12 from Pattambi was found to be unique and it clustered with others only at 30% similarity.The clustering of the genotypes did not show any correlation with the geographic origin. ABL 12 and VTL- 2 showed 70 % similarity but those two were from Wayanad Hills and Pokkali tract respectively. Vellakuttadan from Moncombu clustered with PTB 2 from pattambi at 72% a similarity. Kochuvith and Vellakuttadan from Moncombu clustered at 67 %. All the SSR markers produced two alleles except RM 210 and RM 204 which produced four alleles and one allele respectively. All the alleles of all the markers were polymorphic except that of primer RM204. The polymorphism information content of the SSR primers used in the study ranged from 0 to 0.88. In this study the highest PIC value of 0.88 was reported by RM 210 followed by RM 567 (0.85). The resolving power and EMR was also highest for RM 210. The dendrogram constructed based on the SSR markers could give a clustering of genotypes more correlated with the geographic origin. Genotypes Kochuvithu and Vellakuttadan showed 100 % similarity both where from Kuttanad. But Karavalakochuvith, T.virippu, and PTB 2 also showed 100% similarity but these three were from Moncompu, Pokkali and Palakkad respectively. At around 90 % similarity AMB 14, AMB 22 from Wayanad, Pokkali andVTL-2 from Pokkali, PTB 13 and PTB 8 from Palakkad clustered showing more correlation to the Geographic origin. SSR markers being sequence specific and flanking the repeat sequence which has more role in evolution, are more reliable in predicting the Genetic diversity based on origin. Since both of them could not give a clear-cut clustering based on geographic origin an analysis using both the markers together was done. This gave a better picture of the clustering as it involved more number of variables. But here also the varieties from Wayanad A1 to A5 were scattered in different clusters. Only A 14 and A15 (AMB 14 and AMB 5) clustered at 60 percentage similarity. The accessions from moncompu (A6-A10) clustered at around 50 % similarity. In the accession from Pokkali tract (A11-A15) only T.virippu to VTL-2 clustered at 78 % similarity. Accession from central zone Pattambi (A16-A20) was scattered into different clusters. PTB 12 was unique from other accessions. This molecular diversity analysis of the traditional rice genotypes from four different agro climatic zones could find that the maximum similarity was 78% and that too only between two accessions. The diversity among the genotypes was 64% as all the genotypes clustered at 36% similarity. The clustering of the genotypes did not show any correlation with the geographic origin. Exchange of varieties between the farmers and some amount of natural crossing would have led to the mixture of populations of rice genotypes in different agro climatic zones.Item Expression profiling of auxin biosynthesis genes during inflorescence development in black pepper(Piper nigrum L)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2019) Arathy, L S; Swapna AlexThe study entitled “Expression profiling of auxin biosynthesis genes during inflorescence development in black pepper (Piper nigrum L.)” was carried out during 2017-2019, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to compare the transcript profile of auxin biosynthesis genes such as Flavin monooxygenase and Tryptophan aminotransferase during inflorescence development in different cultivars of black pepper (Piper nigrum L.) using Reverse Transcription quantitative PCR (RT- qPCR). Black pepper exhibits wide variability in inflorescence architecture. Auxin is a major hormone involved in patterning and formation of lateral organs and regulation of branching. Auxin biosynthesis in plants occurs mainly through tryptophan dependent and independent pathways. Among the different known pathways, the most significant and well studied is the Indole-3-Pyruvic Acid (IPA) pathway. Tryptophan aminotransferase (TAA1) and Flavin monooxygenase (YUC2) are the consecutive enzymes of the pathway converting tryptophan to IPA and subsequently to Indole Acetic Acid (IAA). Spike samples of three cultivars of black pepper viz., Panniyur, Karimunda and Thekken were used for the study. Samples were collected at three different developmental stages viz., stage 1 (1-2cm), stage 2 (6-8cm) and stage 3 (9-12cm) from two different plants of each cultivar. Genomic DNA and RNA were extracted by modified Cetyl Trimethyl Ammonium Bromide (CTAB) method and Trizol method respectively. Primers were designed for auxin biosynthesis genes such as Tryptophan aminotransferase and Flavin monooxygenase using “Primer Express” software and absence of secondary structure at the primer binding site was confirmed by “mfold” web server. Specificity of the designed gene specific primers was checked by PCR using genomic DNA. Single amplicon was obtained for all the primers indicating specificity of the designed primers. Amplification efficiency of the designed primers was determined by standard curve analysis and “Lin Reg” software. All the primers exhibited cent per cent amplification efficiency. RNA isolated from the spike samples was converted to cDNA and the quality was confirmed by PCR and agarose gel electrophoresis. cDNA was used for RT-qPCR using SYBR Green dye-based assay. Thermal conditions for RT-qPCR were 95°C for 2 min followed by 40 cycles of 95°C for 15 sec, 55°C for 15 sec and 72°C for 45 sec. RT-qPCR for each gene was performed with three technical replicates for each sample. Cq values obtained were used for further analysis. Gene expression analysis was performed using “qbase plus” software using Actin as the reference gene and two as the amplification factor for all the genes. The expression pattern of TAA1 showed regulation during inflorescence development in all the three cultivars. Higher expression was noticed in all the three stages of Panniyur-1 compared to Karimunda and Thekken. In Karimunda and Thekken, the expression was low in the first stage and peaked at stage 2 and decreased in stage 3. Expression of TAA1 was very less in stage 2 and 3 of Thekken. The expression pattern of YUC2 also showed differential regulation during inflorescence development in all the three cultivars. The expression pattern was similar in all the three cultivars with a peak at stage 2. The expression of YUC2 was highest in Panniyur-1 and lowest in Thekken. To conclude, auxin biosynthetic genes TAA1 and YUC2 were differentially regulated during different stages of inflorescence development in all the three cultivars of black pepper. The expression levels of both the biosynthetic genes were least in the branching type Thekken. Low expression levels of these genes may contribute towards low auxin accumulation in Thekken. Analysis of other genes involved in auxin signalling might help in a better understanding of inflorescence development in black pepper.