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Cassava germplams characterization
Taxonomy
Kingdom     :   Plantae
Division    :   Magnoliophyta
Class       :   magnoliolpsida
Order       :   Malpighiales
Family      :   Euphorbiaceae
Subfamily   :   Crotonoideae
Tribe       :   Manihoteae
Genus       :   Manihot
Species     :   M. esculenta
Introduction
• Origin – Brazil

• Highly cross pollinated crop

• Duration – 7 months to 2 years

• Introduced to Africa and Asia from south
  America

• Grown in 90 countries
Contd…

•   Cassava ranks 4th after rice, sugarcane, and maize

•   Extensively cultivated countries are Argentina,
    Brazil, Colombia, and Paraguay

•   Area under cultivation – 16 mha

•   Provide food & livelihood for >500 million people in
    the world



                                      - Best and Henry, 1992
Cassava germplams characterization
Contd…


        Biochemical aspects
Root:
         Roots are mostly contain carbohydrates
         Also rich in vit.C, carotene, Ca, and K
         Poor in protein


Leaf:

         Rich in protein and vitamins
         Consumed as a vegetable
Genome structure


• 2n = 36

• Genome size - 1.67 pg

  772 M bp in the haploid genome
Germplasm
World germplasm collection held at CIAT, Colombia
Has around 5,724 accession

    Brazil – 1,340 accessions
    Colombia - 2,003 accessions

                 •   Kenya
                 •   Uganda
                 •   Rwanda
                 •   Burundi
                 •   Congo
                 •   Madagoscar
Wild species


Manihot anomala
M. caerulescence
M. epruinosa
M. flabellifolia
M. glaziovii
M. dichotoma
M. tristis
M. peruviana
M. maracasensis
Uses
Starch as raw material for


               Paper industries
               Textiles
               Pharmaceuticals
               Animal feed
               Sago industries



                    and also used as a poultry feed
Markers
Phenotypic marker

•   Graner (1942) described two morphological markers

              Leaf shape
              Root colour

•   Hershey & ocampo (1989) described eight
    morphological markers located on the stem, leaves,
    and root.
cassava phenotypic markers

 Organs                                Types

                •    Erect
Plant shape     •    Tall and spread
                •    Spread
                1.   Green
Colour of the   2.   Yellow-green
young part of   3.   Green and beginning of petiole red
the stem        4.   Green and beginning of petiole red with red ribs

                5.    Green and red in equal area
                6.    Some traces of green
                7.    Entirely light red
                •    Green
Colour of the   •    coloured base and green scales
eye (bud)       •    Green base and coloured scales
                •    Entirely coloured
Emergence of eye (bud)    •   Deep
                          •   Projecting
Alignment of internodes   •   Zigzag
(young part)              •   Straight


leaf shape                •   Palmipartite (normal shape)
                          •   Palmisequate (the lobes totally
                              separate)
                          •   Entirely green
                          •   Yellowish green
Coloration of petiole     •   Vivid red
                          •   Purple red
                          •   Entirely purple

                          •   Less than 3
Number of leaf lobes      •   3 to 5
                          •   5 to 7
                          •   7 to 9
•Very narrow
                                        •Parallel edge
Shape of lobes                          •Normal
                                        •Wide
                                        •Rounded with two widening points

                                        •Proximal
Form of lobes defined by position of    •Median
maximal widening point                  •Terminal

                                        •White without chlorophyll
                                        •Light green
Coloration of upper surface of lamina   •Yellow-green
                                        •Dark green
                                        •Purplish

                                        •Green
                                        •Whitish-green
Coloration of underside of lamina       •Yellow-green
                                        •Purple of red

                                        •Always green
                                        •red of upperside of young leaves
Coloration of lamina nerves             •red on underside of young leaves
                                        •red on both side of young leaves
Leaf variation
Stem variation
Tuber variation
Inflorescence

                           1.   Wide
Forms of sepals            2.   Medium
                           3.   narrow
                           1.   Entirely green
 Sepals colour in female   2.   Green and coloured nerve
flower                     3.   Red and green
                           4.   Red to purple
                           1.   Always straight
 Shape of 6 wings on the   2.   Straight then sinous at maturity
ovary with 3 carpels       3.   Sinous then straight at maturity
                           4.   Always sinous


Ovary body colour          1.   Green
                           2.   Red
Root
                               1.   Short ( < 40 cm )
    Length of root             2.   Normal ( 40 to 80 cm )
                               3.   Long ( > 80 cm)
                               1.   Conical
    Shape of root              2.   Fusiform
                               3.   Cylindro-conical
                               4.   cylindrical
                               1.   Smooth
    Texture of root surfaces   2.   Medium
                               3.   Rough
    Appearance of external     1.   Grey and thin
bark                           2.   Brown and thick

    Colour of pulp             1.   White
                               2.   Yellow
Biochemical Marker

•   Isozymes – fingerprinting and genetic diversity studies
    (Hussain et al., 1987; Ramirez et al., 1987; Ocampo et al.,
    1992; Lefevere & Charrier., 1993a)

•   Applied to characterizing relationships among cassava
    accessions (Lefevere & Charrier, 1993b; Wanyera et al.,1994)

•   Alpha & beta esterase more informative,provide 22
    alleles, which have complemented morphological
    descroptors for identification of duplicates in cassava
    germplasm (Ocampo et al., 1995)
Molecular / DNA Marker



•   Independent to environment

•   Stable

•   Important to study the genes, genomes, and
    genetic diversity.
Development of DNA markers

1st generation DNA markers

          •   RFLP   (Grodzicker et al., 1974)
          •   VNTR     (Jeffrey’s et al., 1985)
          •   ASO    (Saiki et al., 1986)
          •   AS-PCR (Saiki et al., 1986)
          •   OP      (Beckmann, 1988)
          •   SSCP   (Orita et al.,1989)
          •   STS    (Olsen et al., 1989)
2nd generation DNA Markers

•   RAPD      (Williams et al., 1990)
•   AP-PCR     (Welsh and Mc Cleland, 1996)
•   STMS      (Becknann& Soller, 1990)
•   RLGS      (Hatada et al., 1991)
•   CAPS      (Akopyanz et al.,1992)
•   DOP-PCR    (Teknins, 1992)
•   SSR       (Akkaya et al., 1992)
•   MAAP       (Caetano-anolles et al., 1993)
•   SCAR      (1993)
New generation markers

                  IRAP       ( 1998 )

                  REMAP      ( 1999 )

                  MSAP       ( 1999 )

                  MITE       ( 2000 )

                  TE – AFLP ( 2000 )
                  IMP         ( 2001 )
                  SRAP        ( 2001 )
Others


         ۩ ISSR     ۩ SAMPL       ۩ AFLP/SRFA   ۩ ASAP

         ۩ CFLP     ۩ ISTR        ۩ SSAP        ۩ RBIP
Molecular / DNA Markers used in Cassava


     • RFLPs            (Botstein et al., 1980)

     • SSRs             (Litt & Lutly, 1989a,b)

     • RAPDs            (Williams et al., 1990)

     • Minisatellites   (jeffreys et al., 1993)

     • AFLPs            (Vos et al., 1995)
Relationship analysis of closely related species to
      cassava based on microsatellite PCR’

    To study phylogenetic relationship between cassava
    and its closely related species from south America


               Manihot esculenta ssp esculenta
               M. esculenta ssp flabellifolia
               M. esculenta ssp peruviana
               M. pilosa
               M. triphylla
Contd…



Multilocus markers are used

RAPD, AP-PCR, DAP, AFLP, & SSR

Recently oligonucleotide based SSR used



      Two kinds of SSR data
      Sequence data base
      Specific genomic libraries
Contd…
    Result

•   Varied amplification

•   No of band is varied from 1 – 15

•   Fragment size varied from 200 – 3000 bp

•   Scored as dominant marker

•   Highest genetic diversity observed in M. esculenta ssp.
    Flabellifolia followed by, M. esculenta ssp esculenta,
    M. pilosa, M. esculenta ssp peruviana, M. triphylla
•   CMD resistance conferred by dominant gene

•   BSA – Identify a SSR markers linked to the CMD
    resistant gene

•   186 SSR markers are used
Contd…

•   Resistant gene – CMD 2

•   Flanked by SSRY28 & GY1 @ 9 and 8cM respectively

•   CMD resistant cassava – TME 3 X TMS 30555

•   158 individual was established invitro from embryo axes the
    sub cloned

•   Transfer the plants into a field with low CMD pressure

•   CMD resistance evolved at 3 to 6 month old plant

•   Scoring – 1 to 5
Genetic mapping of a CMD resistant gene


•   186 SSR markers – 80 SSR markers map positions
    are known

•   SSRY28 differentiate the CMD resistant genotype
    from CMD susceptible genotype

•   SSRY28 located on 17th chromosome of male-
    parent-derived molecular genetic map

•   Flanked by GY 1 & Ail 9b markers
Cassava germplams characterization
Result


•   All 10 plants of each resist genotype in all 3
    replication are showed without any visible
    symptoms

•   Susceptible genotypes are always heavily infected

•   SSRY 28 present in resistant genotypes and not in
    susceptible genotypes.
•   Most popular
•   Dormaa, Wenchi, Nkoranza,& Asonafo
•   50 Genotypes are taken
•   4 primers (OPK-01, OPR-02, OPR-09, IOJ-14)
•   41 different bands detected
•   Range of polymorphism % is 90% - 100%
Cassava germplams characterization
Cassava germplams characterization
67 unlinked SSR loci assessed

283 accessions

Grouped into 14
Cassava germplams characterization
Cassava germplams characterization
Cassava germplams characterization
Cassava germplams characterization
76 varieties are grown in Rewa
31 varieties – taken for AFLP analysis with some wild species
To assess:

       Intravarietal diversity

       Genetic variability in the local varieties
Cassava germplams characterization
78 accessions evolved
     •    1-    breeding stock (clone 58308)
     •    5-    improved lines
     •   10 -   CMD susceptible
     •   62 -   CMD resistant
36 SSR Markers are used
Cassava germplams characterization
Cassava germplams characterization
Institutes
•   CIAT    -   Colombia
•   IITA    -   Nigeria
•   CDH     -   Africa
•   CTCRI   -   India, Indonesia, Tanzania
•   ESARC   -   Africa
•   INEAC   -   Belgian Congo
•   IRAT    -   West and Central Africa
•   IRAM    -   Madagascar
•   MARDI   -   Malaysia
•   IICA    -   Venezuela, Mexico, Brazil,
                       Colombia, Costa Rica
Cassava germplams characterization

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Cassava germplams characterization

  • 2. Taxonomy Kingdom : Plantae Division : Magnoliophyta Class : magnoliolpsida Order : Malpighiales Family : Euphorbiaceae Subfamily : Crotonoideae Tribe : Manihoteae Genus : Manihot Species : M. esculenta
  • 3. Introduction • Origin – Brazil • Highly cross pollinated crop • Duration – 7 months to 2 years • Introduced to Africa and Asia from south America • Grown in 90 countries
  • 4. Contd… • Cassava ranks 4th after rice, sugarcane, and maize • Extensively cultivated countries are Argentina, Brazil, Colombia, and Paraguay • Area under cultivation – 16 mha • Provide food & livelihood for >500 million people in the world - Best and Henry, 1992
  • 6. Contd… Biochemical aspects Root: Roots are mostly contain carbohydrates Also rich in vit.C, carotene, Ca, and K Poor in protein Leaf: Rich in protein and vitamins Consumed as a vegetable
  • 7. Genome structure • 2n = 36 • Genome size - 1.67 pg 772 M bp in the haploid genome
  • 8. Germplasm World germplasm collection held at CIAT, Colombia Has around 5,724 accession Brazil – 1,340 accessions Colombia - 2,003 accessions • Kenya • Uganda • Rwanda • Burundi • Congo • Madagoscar
  • 9. Wild species Manihot anomala M. caerulescence M. epruinosa M. flabellifolia M. glaziovii M. dichotoma M. tristis M. peruviana M. maracasensis
  • 10. Uses Starch as raw material for Paper industries Textiles Pharmaceuticals Animal feed Sago industries and also used as a poultry feed
  • 11. Markers Phenotypic marker • Graner (1942) described two morphological markers Leaf shape Root colour • Hershey & ocampo (1989) described eight morphological markers located on the stem, leaves, and root.
  • 12. cassava phenotypic markers Organs Types • Erect Plant shape • Tall and spread • Spread 1. Green Colour of the 2. Yellow-green young part of 3. Green and beginning of petiole red the stem 4. Green and beginning of petiole red with red ribs 5. Green and red in equal area 6. Some traces of green 7. Entirely light red • Green Colour of the • coloured base and green scales eye (bud) • Green base and coloured scales • Entirely coloured
  • 13. Emergence of eye (bud) • Deep • Projecting Alignment of internodes • Zigzag (young part) • Straight leaf shape • Palmipartite (normal shape) • Palmisequate (the lobes totally separate) • Entirely green • Yellowish green Coloration of petiole • Vivid red • Purple red • Entirely purple • Less than 3 Number of leaf lobes • 3 to 5 • 5 to 7 • 7 to 9
  • 14. •Very narrow •Parallel edge Shape of lobes •Normal •Wide •Rounded with two widening points •Proximal Form of lobes defined by position of •Median maximal widening point •Terminal •White without chlorophyll •Light green Coloration of upper surface of lamina •Yellow-green •Dark green •Purplish •Green •Whitish-green Coloration of underside of lamina •Yellow-green •Purple of red •Always green •red of upperside of young leaves Coloration of lamina nerves •red on underside of young leaves •red on both side of young leaves
  • 18. Inflorescence 1. Wide Forms of sepals 2. Medium 3. narrow 1. Entirely green Sepals colour in female 2. Green and coloured nerve flower 3. Red and green 4. Red to purple 1. Always straight Shape of 6 wings on the 2. Straight then sinous at maturity ovary with 3 carpels 3. Sinous then straight at maturity 4. Always sinous Ovary body colour 1. Green 2. Red
  • 19. Root 1. Short ( < 40 cm ) Length of root 2. Normal ( 40 to 80 cm ) 3. Long ( > 80 cm) 1. Conical Shape of root 2. Fusiform 3. Cylindro-conical 4. cylindrical 1. Smooth Texture of root surfaces 2. Medium 3. Rough Appearance of external 1. Grey and thin bark 2. Brown and thick Colour of pulp 1. White 2. Yellow
  • 20. Biochemical Marker • Isozymes – fingerprinting and genetic diversity studies (Hussain et al., 1987; Ramirez et al., 1987; Ocampo et al., 1992; Lefevere & Charrier., 1993a) • Applied to characterizing relationships among cassava accessions (Lefevere & Charrier, 1993b; Wanyera et al.,1994) • Alpha & beta esterase more informative,provide 22 alleles, which have complemented morphological descroptors for identification of duplicates in cassava germplasm (Ocampo et al., 1995)
  • 21. Molecular / DNA Marker • Independent to environment • Stable • Important to study the genes, genomes, and genetic diversity.
  • 22. Development of DNA markers 1st generation DNA markers • RFLP (Grodzicker et al., 1974) • VNTR (Jeffrey’s et al., 1985) • ASO (Saiki et al., 1986) • AS-PCR (Saiki et al., 1986) • OP (Beckmann, 1988) • SSCP (Orita et al.,1989) • STS (Olsen et al., 1989)
  • 23. 2nd generation DNA Markers • RAPD (Williams et al., 1990) • AP-PCR (Welsh and Mc Cleland, 1996) • STMS (Becknann& Soller, 1990) • RLGS (Hatada et al., 1991) • CAPS (Akopyanz et al.,1992) • DOP-PCR (Teknins, 1992) • SSR (Akkaya et al., 1992) • MAAP (Caetano-anolles et al., 1993) • SCAR (1993)
  • 24. New generation markers IRAP ( 1998 ) REMAP ( 1999 ) MSAP ( 1999 ) MITE ( 2000 ) TE – AFLP ( 2000 ) IMP ( 2001 ) SRAP ( 2001 ) Others ۩ ISSR ۩ SAMPL ۩ AFLP/SRFA ۩ ASAP ۩ CFLP ۩ ISTR ۩ SSAP ۩ RBIP
  • 25. Molecular / DNA Markers used in Cassava • RFLPs (Botstein et al., 1980) • SSRs (Litt & Lutly, 1989a,b) • RAPDs (Williams et al., 1990) • Minisatellites (jeffreys et al., 1993) • AFLPs (Vos et al., 1995)
  • 26. Relationship analysis of closely related species to cassava based on microsatellite PCR’ To study phylogenetic relationship between cassava and its closely related species from south America Manihot esculenta ssp esculenta M. esculenta ssp flabellifolia M. esculenta ssp peruviana M. pilosa M. triphylla
  • 27. Contd… Multilocus markers are used RAPD, AP-PCR, DAP, AFLP, & SSR Recently oligonucleotide based SSR used Two kinds of SSR data Sequence data base Specific genomic libraries
  • 28. Contd… Result • Varied amplification • No of band is varied from 1 – 15 • Fragment size varied from 200 – 3000 bp • Scored as dominant marker • Highest genetic diversity observed in M. esculenta ssp. Flabellifolia followed by, M. esculenta ssp esculenta, M. pilosa, M. esculenta ssp peruviana, M. triphylla
  • 29. CMD resistance conferred by dominant gene • BSA – Identify a SSR markers linked to the CMD resistant gene • 186 SSR markers are used
  • 30. Contd… • Resistant gene – CMD 2 • Flanked by SSRY28 & GY1 @ 9 and 8cM respectively • CMD resistant cassava – TME 3 X TMS 30555 • 158 individual was established invitro from embryo axes the sub cloned • Transfer the plants into a field with low CMD pressure • CMD resistance evolved at 3 to 6 month old plant • Scoring – 1 to 5
  • 31. Genetic mapping of a CMD resistant gene • 186 SSR markers – 80 SSR markers map positions are known • SSRY28 differentiate the CMD resistant genotype from CMD susceptible genotype • SSRY28 located on 17th chromosome of male- parent-derived molecular genetic map • Flanked by GY 1 & Ail 9b markers
  • 33. Result • All 10 plants of each resist genotype in all 3 replication are showed without any visible symptoms • Susceptible genotypes are always heavily infected • SSRY 28 present in resistant genotypes and not in susceptible genotypes.
  • 34. Most popular • Dormaa, Wenchi, Nkoranza,& Asonafo • 50 Genotypes are taken • 4 primers (OPK-01, OPR-02, OPR-09, IOJ-14) • 41 different bands detected • Range of polymorphism % is 90% - 100%
  • 37. 67 unlinked SSR loci assessed 283 accessions Grouped into 14
  • 42. 76 varieties are grown in Rewa 31 varieties – taken for AFLP analysis with some wild species To assess: Intravarietal diversity Genetic variability in the local varieties
  • 44. 78 accessions evolved • 1- breeding stock (clone 58308) • 5- improved lines • 10 - CMD susceptible • 62 - CMD resistant 36 SSR Markers are used
  • 47. Institutes • CIAT - Colombia • IITA - Nigeria • CDH - Africa • CTCRI - India, Indonesia, Tanzania • ESARC - Africa • INEAC - Belgian Congo • IRAT - West and Central Africa • IRAM - Madagascar • MARDI - Malaysia • IICA - Venezuela, Mexico, Brazil, Colombia, Costa Rica