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University of Namibia (2017)

Genetic and biochemical characterization of pearl millet mutant lines using simple sequence repeat markers in Namibia

Shindume, Filemon Nghitilanganye

Titre : Genetic and biochemical characterization of pearl millet mutant lines using simple sequence repeat markers in Namibia

Auteur : Shindume, Filemon Nghitilanganye

Université de soutenance  : University of Namibia

Grade : Master of Science (Industrial Biochemistry) 2017

Résumé
Pearl millet is a staple food and fodder crop of marginal areas of sub-Saharan Africa, South Asia and the Americas. Although pearl millet is stable food for 70 % of Namibian population, its productivity is very low (low yield, susceptible to diseases/insect and prolonged reproductive cycle) and has a limited genetic diversity. For the purpose of widening gene diversity, developing high yielding and drought resistant pearl millet, the Kangara and Okashana 2 were mutated using Colbalt-60 gamma irradiation source with various dose levels. However, all sixth generation of Okashana 2 putative mutant derivatives used in this study were all produced using 350 Gray while for Kangara mutants were produced using 400 Gray. A total of 32 genotypes including two parental lines were collected from the three agro-ecological zones (Research Stations). In addition to two parental lines, stratified sampling technique was used to randomly select further 12 samples from 30 putative mutants for Okashana 2 and Kangara that offered higher average grain yields. Such technique used to subsample was applied in order to select putative mutants that shown phenotypically high yielding trait. This study aimed to examine the genetic diversity existing in the Namibian pearl millet putative mutant lines using 19 simple sequence repeats and 23 biochemical markers. Genetic characterization was studied using conventional PCR and separated using gel electrophoresis while biochemical parameters used dumas method (crude protein), fiber extractor (crude fibre, ADF and NDF), fat solvent extractor (fat), ICP spectrophotometer (minerals) and UV/VIS spectrometer (phytate). A total of 108 putative bands were generated from 11 SSR markers whereas the remaining eight markers were not effective in amplifying any mutant line. Amplified fragment size ranged from 90 to 1150 bp and the 11 SSR loci had a 63.6 % polymorphism. Polymorphic information content (PIC) varied from 0.139 to 0.872, with a mean of 0.379. Cluster analysis using UPGMA revealed a 51.8 % similarity level among the genotypes. The mean Shannon-Wienberg diversity index (H’) was 2.589, while the mean Simpson’s diversity index (D) was 0.93. Both Simpson’s index value and the Shannon-Wiener diversity shown moderate results in both species richness and evenness, while the genetic diversity level of similarity among putative mutants, suggested moderate genetic diversity. Biochemical parameters of proximate content (moisture, ash, crude protein, fat, crude fibre and carbohydrates) showed no consistency in up/down regulation pattern in comparison to their parental lines. Only total energy content (however direct correlated with crude protein, crude lipid and carbohydrate) showed a constant trend of down regulation between mutant derivatives and their parental lines. Lines R9P5, L8P5Rep2, L9P3Rep1, L10P18, L5P1Rep2 and L0P3Rep1 are selected for their higher protein content, while L5AP18, L9P3Rep1 and L8P5Rep2 selected for trait such as higher content of WAC and swelling capacity. L5AP18 and R6P1-1 are selected for higher content of iron and zinc minerals. Generally, majority of biochemical markers showed a significant difference (P < 0.05) within and among Kangara and Okashana 2 derivatives and as well as in comparison with the parental lines. Crude protein content of the mutant varieties in this study showed significant increment of up to 24 % in comparison with parental lines. Correlation analysis (r) between SSR and biochemical markers of pearl millet putative mutants ranged between -0.44 - 0.70, but with a low mean correlation analysis (r = |0.31|). There is little or no correlation between biochemical markers (proximate composition, mineral nutrient contents, functional and anti-nutrients properties). There was low correlation observed among the proximate composition biochemical markers, while mineral contents, functional and anti-nutrients properties markers showed a moderate correlation. Finally, the study identified valuable and usefulness of the SSR marker system to assess genetic diversity in pearl millet putative mutant germplasms and the number of alleles/bands detected for each SSR marker can be a good indicator of genetic diversity. The result further revealed that putative mutant germplasms are significantly different to their parental lines in terms of genetic diversity and biochemical traits. In most of the parameters, the putative mutants had shown better improvement in comparison to their parental lines. Thus, assessment of genetic diversity and determination of the biochemical activities in pearl millet germplasm should improve the breeding efficiency and allow to understanding the genetic potential of each putative mutant. Genetic characterization of germplasm using molecular and biochemical markers in crop improvement is one of the most sustainable methods to conserve valuable genetic resources and to simultaneously increase agricultural production and food security.

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