Molecular characterization of Napier grass ( Pennisetum purpureum  Schumach.) germplasm using fluorescent labelled amplifi...
Outline  <ul><li>Introduction </li></ul><ul><li>Justification </li></ul><ul><li>Objectives </li></ul><ul><li>Hypothesis </...
Introduction <ul><li>Napier grass  ( Pennisetum purpureum  Schum.) is an important forage crop for dairy production system...
Uses <ul><li>Other Napier grass uses include: </li></ul><ul><ul><li>Soil conservation  </li></ul></ul><ul><ul><li>Mulching...
Pennisetum purpureum  Schumach.   Photo credit: Jean Hanson
Justification <ul><li>Napier production faces major  challenges  from biotic and abiotic stresses </li></ul><ul><li>Diseas...
Napier grass stunt and smut Grass stunt ( foreground ) and  apparently healthy grass ( rear ) Napier infected by  head smu...
Objectives <ul><li>Main objective Assess molecular diversity among Napier grass accessions by Amplified Fragment Length Po...
Hypothesis <ul><li>Polymorphic  AFLP markers are not available </li></ul><ul><li>There is no difference within and between...
Methodology <ul><li>Study sites </li></ul><ul><ul><li>Kenya </li></ul></ul><ul><ul><li>Tanzania </li></ul></ul><ul><ul><li...
AFLP procedure Vos  et al.  (1995) DNA extraction  Digestion of Genomic DNA and Ligation of Oligonucleotide Adapters Prese...
Results and discussion  <ul><li>Establishment of F-AFLP Procedures for Napier grass </li></ul><ul><ul><li>AFLP profiles  <...
Primer-pair combination selection
AFLP fingerprints (Peaks) AFLP fingerprints (Peaks) for selected Primers-A,B, labelled with blue and yellow dye  on sample...
Genetic diversity evaluation Mean diversity estimates  (H)  and Shannon’s information index (I) For full data contact the ...
Genetic distance and  F st <ul><li>The Nei unbiased genetic distance between these populations ranged from 0.0000 to 0.134...
UPGMA dendrogram  <ul><li>Summarizes genetic relationships among populations </li></ul>A B C D For full data contact the a...
UPGMA dendrogram  Summarizes genetic relationships among individuals Kenya Tanzania Uganda ILRI-FG
UPGMA dendrogram- Ug pop
Principle coordinate analysis Summarizes genetic relationships among individuals
  AMOVA Total genetic variation is distributed between populations Source of variation df ss Ms Est.var percentage P-value...
Conclusion and recommendations <ul><li>Clustering did not  occur according to geographical origin in spite of their divers...
Collaborators <ul><li>Jean Hanson PI- ILRI Ethiopia </li></ul><ul><li>Janice Proud – Project coordinator </li></ul><ul><li...
Acknowledgement
Photo credit: Eric Boa Plant Global Clinic THANK YOU
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Molecular characterization of Napier grass (Pennisetum purpureum Schumach.) germplasm using fluorescent labelled amplified fragment length polymorphism (FL-AFLP)

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A presentation prepared by Bramwel Waswa Wanjala for the ASARECA/ILRI Workshop on Mitigating the Impact of Napier Grass Smut and Stunt Diseases, Addis Ababa, June 2-3, 2010.

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Molecular characterization of Napier grass (Pennisetum purpureum Schumach.) germplasm using fluorescent labelled amplified fragment length polymorphism (FL-AFLP)

  1. 1. Molecular characterization of Napier grass ( Pennisetum purpureum Schumach.) germplasm using fluorescent labelled amplified fragment length polymorphism (FL-AFLP) Bramwel Waswa Wanjala End of project Workshop ILRI-ADDIS June 2010 Presented at the ASARECA/ILRI Workshop on Mitigating the Impact of Napier Grass Smut and Stunt Diseases, Addis Ababa, June 2-3, 2010
  2. 2. Outline <ul><li>Introduction </li></ul><ul><li>Justification </li></ul><ul><li>Objectives </li></ul><ul><li>Hypothesis </li></ul><ul><li>Methodology </li></ul><ul><li>Results </li></ul><ul><li>Discussion & Recommendation </li></ul><ul><li>Acknowledgement </li></ul>
  3. 3. Introduction <ul><li>Napier grass ( Pennisetum purpureum Schum.) is an important forage crop for dairy production systems in the tropics </li></ul><ul><li>Native to eastern and central Africa </li></ul><ul><li>Has been introduced to most tropical and sub-tropical countries </li></ul><ul><li>Napier grass is the most promising and high yielding fodder (Anindo & Potter 1994) </li></ul>
  4. 4. Uses <ul><li>Other Napier grass uses include: </li></ul><ul><ul><li>Soil conservation </li></ul></ul><ul><ul><li>Mulching coffee </li></ul></ul><ul><ul><li>Biofuel crop </li></ul></ul><ul><ul><li>Management of insect Resistance </li></ul></ul><ul><ul><li>Indirectly improves food security </li></ul></ul>
  5. 5. Pennisetum purpureum Schumach. Photo credit: Jean Hanson
  6. 6. Justification <ul><li>Napier production faces major challenges from biotic and abiotic stresses </li></ul><ul><li>Disease control is expensive, beyond the means of most smallholders </li></ul><ul><li>Smut-resistant is based on just two clones of the available germplasm. Stunt ????? </li></ul><ul><li>Ultimate goal is to increase the number of varieties that can be distributed, and identify plants that combine both smut-resistance and high yields </li></ul>
  7. 7. Napier grass stunt and smut Grass stunt ( foreground ) and apparently healthy grass ( rear ) Napier infected by head smut disease Photo credit: J Hanson & M Mulaa
  8. 8. Objectives <ul><li>Main objective Assess molecular diversity among Napier grass accessions by Amplified Fragment Length Polymorphism (AFLP) technique </li></ul><ul><li>Specific objectives </li></ul><ul><ul><li>Select polymorphic AFLP primer pair combination and their adoption in genotyping </li></ul></ul><ul><ul><li>Access the genetic variation among and within populations </li></ul></ul><ul><ul><li>Determine which markers are linked to stunt & smut resistance and susceptibility </li></ul></ul>
  9. 9. Hypothesis <ul><li>Polymorphic AFLP markers are not available </li></ul><ul><li>There is no difference within and between Napier grass cultivars in the region </li></ul><ul><li>There is no link between resistance and susceptibility to the AFLP markers </li></ul>
  10. 10. Methodology <ul><li>Study sites </li></ul><ul><ul><li>Kenya </li></ul></ul><ul><ul><li>Tanzania </li></ul></ul><ul><ul><li>Uganda </li></ul></ul><ul><ul><li>ILRI- Forage Germplasm (Ethiopia) </li></ul></ul><ul><ul><li>BecA </li></ul></ul>
  11. 11. AFLP procedure Vos et al. (1995) DNA extraction Digestion of Genomic DNA and Ligation of Oligonucleotide Adapters Preselective PCR Amplification Selective PCR Amplification Capillary Electrophoresis
  12. 12. Results and discussion <ul><li>Establishment of F-AFLP Procedures for Napier grass </li></ul><ul><ul><li>AFLP profiles </li></ul></ul><ul><ul><li>Fluorescent detection of selected Primer-Pair </li></ul></ul><ul><ul><li>Combinations for Napier grass Genotyping </li></ul></ul><ul><li>Genetic relationships among cultivars </li></ul><ul><ul><li>Genetic distance </li></ul></ul><ul><ul><li>UPGMA </li></ul></ul><ul><ul><li>PCoA </li></ul></ul><ul><ul><li>Fst </li></ul></ul><ul><ul><li>AMOVA to partition genetic variance among populations </li></ul></ul><ul><li>  </li></ul>
  13. 13. Primer-pair combination selection
  14. 14. AFLP fingerprints (Peaks) AFLP fingerprints (Peaks) for selected Primers-A,B, labelled with blue and yellow dye on sample Nap_49 and Nap_50 .Red=Polymorphic and Green=Monomorphic. Insert=Gel image
  15. 15. Genetic diversity evaluation Mean diversity estimates (H) and Shannon’s information index (I) For full data contact the authors Provenance N H I No. of polymorphic loci % Polymorphic Loci Kenya Bungoma 27 0.1169 0.2076 167 77.3 Busia 25 0.1197 0.2095 154 71.3 Butere 22 0.1492 0.2569 187 86.6 Extra 11 0.1195 0.2071 140 64.8 Mumias 35 0.1416 0.2464 193 89.4 Tanzania Hai 5 0.1747 0.2675 116 53.7 Lushoto 4 0.1727 0.2633 111 51.3 Meru 3 0.1608 0.2390 92 42.59 Muheza 14 0.1011 0.1771 128 59.26 Tarime 19 0.1893 0.3113 186 83.1 Uganda Kabarole 12 0.2130 0.3353 168 77.8 Masaka 23 0.1244 0.2099 151 69.9 Naro 5 0.0167 0.0266 13 32.5 Soroti 16 0.2142 0.3445 189 87.5 ILRI-FG Others 6 * 0.0783 0.1293 71 32.87 Swaziland 6 0.1576 0.2495 123 56.9 Tanzania 5 0.1308 0.2035 93 43.1 Unknown 14 0.1338 0.2258 145 67.1 USA 16 0.1890 0.3044 170 78.70 Zimbabwe-C 8 0.1844 0.2938 152 70.4
  16. 16. Genetic distance and F st <ul><li>The Nei unbiased genetic distance between these populations ranged from 0.0000 to 0.134 </li></ul><ul><li>Fst of 0.05 to 0.15- moderate genetic differentiation while ≥0.25- great genetic differentiation. </li></ul><ul><li>The F st values obtained between the 21 populations F st = 0.1688-Moderate genetic differentiation </li></ul>
  17. 17. UPGMA dendrogram <ul><li>Summarizes genetic relationships among populations </li></ul>A B C D For full data contact the authors
  18. 18. UPGMA dendrogram Summarizes genetic relationships among individuals Kenya Tanzania Uganda ILRI-FG
  19. 19. UPGMA dendrogram- Ug pop
  20. 20. Principle coordinate analysis Summarizes genetic relationships among individuals
  21. 21.   AMOVA Total genetic variation is distributed between populations Source of variation df ss Ms Est.var percentage P-value All population Among population Within population 20 260 1125.941 6249.767 56.297 24.038 2.464 24.038 9% 91% 0.010 0.010 Kenya Among population Within population 4 115 105.084 2611.357 26.271 22.707 0.153 22.707 3% 99% 0.010 0.010 Uganda Among population Within population 3 52 273.893 1269.553 91.289 24.414 5.150 24.414 17% 83% 0.010 0.010 ILRI-FG Among population Within population 6 53 3.018 26.098 0.503 0.492 0.001 0.492 6% 100% 0.010 0.010
  22. 22. Conclusion and recommendations <ul><li>Clustering did not occur according to geographical origin in spite of their diverse origin. </li></ul><ul><li>  Samples from the USA clustered together with those from Africa </li></ul><ul><li>Germplasm exchange </li></ul><ul><li>  Genetically and morphological examination of cultivars </li></ul><ul><li>  Other Markers </li></ul><ul><li>  AFLP methodology developed in this study could be used to differentiate Napier genotypes and track the original source of Napier grass accessions </li></ul>
  23. 23. Collaborators <ul><li>Jean Hanson PI- ILRI Ethiopia </li></ul><ul><li>Janice Proud – Project coordinator </li></ul><ul><li>Alice Muchugi - ICRAF </li></ul><ul><li>Meshack Obonyo – Egerton University </li></ul><ul><li>Jagger Harvey – BecA-ILRI Hub </li></ul><ul><li>Robert Skilton - BecA-ILRI Hub </li></ul><ul><li>Mulaa Margaret - KARI -Kenya </li></ul><ul><li>Kabirizi Jolly - NaLIRRI -Uganda </li></ul><ul><li>Pallangyo Beatrice - NBCP -Tanzania </li></ul>
  24. 24. Acknowledgement
  25. 25. Photo credit: Eric Boa Plant Global Clinic THANK YOU

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