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|GENETIC STUDIES OF OIL CONTENT IN MUTANT POPULATION OF RAPESEED (Brassica Napus L.) USING MOLECULAR MARKERS
|UNIVERSITY OF PESHAWAR, KPK-PAKISTAN
|The current study titled “genetic studies of oil content in mutant population of rapeseed (Brassica napus L.) using molecular markers” was partially conducted at Nuclear Institute for Food and Agriculture (NIFA), Peshawar in 2008-13. Main objective of the study were to intimate and study the influence of induce mutation on the genetics of oil and oil related components in advance induce mutant line of oilseed rape (Brassica napus). Two contrast mutant lines with high and low oil contents were crossed with each other and F 1 hybrids were developed. These F 1 hybrids were grown in the cropping season 2009-10 and BCF 1 were developed by crossing back the F 1 with their parents. These BCF 1 hybrids were grown in the next cropping season 2010- 11 and selfed to develop BCF 2 . The parent and the BCF 2 hybrids were used for molecular assessment to confirm mutation in the genetics of the advance mutant lines. Thirty five advance mutant lines were utilized for agronomical and biochemical study. Five plants were randomly selected from each mutant line and the parent. Data on days to 50% flowering (DF), plant height (PH), 1000- seed weight (SW), seed yield (SY) and oil yield (OY) were recorded. Seeds were analyzed at for biomechanical parameter viz. Oil content (OC), protein content (PC), glucosinolates (GSL), oleic acid (OA), linolenic acid (LA), and erucic acid (EA). Analysis of variance (ANOVA) for both agronomical and biochemical traits showed highly significant differences among the mutant lines and their respective parental line for all traits. Broad sense heritability estimates were calculated for all of the agronomical and biomechanical traits. High heritability estimates were observed for DF, OY, GSL, viiiEA, and OA, moderate to high heritability was recorded for OC, PC, PH, 1000-SW and OA. Multivariate taxonomic techniques were used to check the mutation pattern. Cluster analysis and principal component analysis (PCA) was done for all agronomical and biochemical traits. Mean of the parental line and individual mutant plants data were standardized prior to cluster and PC analysis. The results of cluster analysis for agronomical traits authenticated the selection. All of the advance mutant lines were distinct from the parental genotypes. Most of the mutant populations fell in separates cluster or isolated than the parental genotype if present in the same cluster. Genetic distances range from 0.00 to 5.6. Two types of scattering patterns were set as standards. In the first type, the parent fills in a separate cluster or was distinct from the mutant progenies. In the second case, some mutants showed deviation towards parent. Regarding biochemical data, three mutants viz OA5, EA4 and G1 showed deviation towards parents while rest of the mutants fell in separate cluster than the parent. The mutant individuals with desirable combination of traits were identified. The PCA also confirmed the results for the cluster analysis with minor differences. Scatter plots of the PCs that had an Eigen value > 1 were produced to provide graphical representation of pattern of variation among the genotypes i.e. parent and advance mutant lines. Hence both of the analysis was used to check the mutation patterns. The traits DF and PH contributed positively while 1000-SW, SY and OY contributed negatively towards the variability. Regarding biochemical traits, OA and LA contributed negatively, while GSL and EA contributed positively towards the total variability in most of the populations. OC and PC showed mixed divergence pattern. DF along with GSL, OA and EA had remarkable impact on the variations. ixIn order to confirm the maternal effects and role of the B. napus in induce mutation, molecular assessment of two mutant lines was conducted. These populations were analyzed through simple sequence repeats (SSR) markers. Facilities at oilseed Laboratory of Nuclear Institute for Food and Agriculture (NIFA), Peshawar were utilized for this part of research. DNA was extracted using protocols of Doyle and Doyle (1987) and specific recipe and conditions were followed for Polymerase Chain Reaction (PCR) steps. Out of 25 SSR primers 19 gave positive results during initial screening. Out of 19 SSR primers, 15 gave consistent, bright and highly polymorphic bands. The product size of each primer set was compared with that of expected size given on Brassica Database Domain. Out of 99 amplified alleles detected, 69 were polymorphic. The proportion of polymorphic loci was 69.75. The number of amplified products ranged from 1-5 polymorphism information content (PIC) of the primer sets ranged from 0.24 to 0.75.
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