Evaluation of genetic diversity_ and guidelines for preservation of by yurtgc548

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									 RAPD markers




Larisa Gustavsson (Garkava)
Balsgård-Department of Crop Sciences
Swedish University of Agricultural
Sciences
           What is RAPD?


RAPD is a PCR-based method which employs
single primers of arbitrary nucleotide sequence
with 10 nucleotides to amplify anonymous PCR
fragments from genomic template DNA
              RAPD technology
A B C
                                                          A

                                 +                 +
              +


                  Taq polymerase     Arbitrary primers    Nucleotides

                          +
Genomic DNA


                                                  PCR


                                         (under relaxed conditions)

                        Buffer
    PCR

              360 bp

                       Electrophoresis

                        A    B     C
              260 bp
                                         520bp



              520 bp                     360 bp


A    B    C                              260 bp
    PCR product occurs when:
• The primers anneal in a particular
  orientation (such that they point towards
  each other)
• The primers anneal within a reasonable
  distance of one another (150 -3000 bp)
The number of amplification products is related to
the number and orientation of the genome sequences
which are complementary to the primer

  1                  2                     3


                 4          5                      6

  DNA template              PCR reaction



                     Product 1             Product 2
The nature of RAPD
  polymorphism
a) nucleotide substitution within target sites may affect
the annealing process - either no fragment is detected

                       2

   1                                        3



                4              5                         6
 DNA template                PCR reaction


                     No product
                                             Product 2
or detected fragment is of increased size


                            2

                    1                              3



                4                     5                       6
 DNA template                       PCR reaction



                        Product 1                      Product 2
    b) insertion or deletion of a small fragment of DNA - the
    amplified fragments are changed in size

                                              Small fragment DNA

                              Insertion
1                      2                           3
                                                           Deletion



                 4                  5                      6
DNA template
                                    PCR reaction



                           Product 1                Product 2
c) insertion of a large piece of DNA between the
primer -binding sites may exceed the capacity of
PCR - no fragment is detected

                       The insertion of large fragment
        2                                            3



                                  5                              6
DNA template
                        PCR reaction


               No product                                Product 2
  A schematic picture of an agarose gel

    Marker         Plant A   Plant B   Plant C      -


                                                          Monomorphic bands



                                                           Polymorphic bands


                                                    +

Presens of a band, ”1”           Absence of a band, ”0”
And a real picture of a gel…
… and one more
Data analysis
       RAPD bands are treated as independent loci:
                1        2     3   4     5        6     7   8     9        10    11 12     13    14

Locus A
Locus B
Locus C
Locus D




  1       2         3         4 5            6        7     8         9 10 11 12 13 14
 AA/       aa       aa        aa    aa       aa        aa    aa       aa        aa   aa     aa        aa    aa
 Aa
  bb      BB/   BB/          BB/   BB/   BB/          BB/   BB/   BB/           bb   bb     bb        bb    bb
          Bb    Bb           Bb    Bb    Bb           Bb    Bb    Bb
 CC/       cc       cc        cc    cc       cc        cc    cc       cc     CC/     CC/    CC/       CC/   cc
 Cc                                                                          Cc      Cc     Cc        Cc
  dd      DD/   DD/          DD/   DD/   DD/          DD/   DD/   DD/        DD/     DD/    DD/       dd    DD/
          Dd    Dd           Dd    Dd    Dd           Dd    Dd    Dd         Dd      Dd     Dd              Dd
RAPD bands are scored for presens ”1” and absens
”0”. Only clear, consistent and polymorphic bands
are usually used to create a binary matrix for future
statistical analyses
              A binary matrix:

          Band 1   Band 2   Band 3   Band 4
Plant A     1        0        0        1
Plant B     0        1        0        1
Plant C     1        1        1        0
Plant D     1        1        0        1
Plant E     0        1        0        1
Plant F     1        0        0        1
Plant G     1        0        1        0
       Statistical analyses
        (some examples)
Measurements of genetic diversity by
means of different genetic diversity indexes
(i.e. Nei’s diversity index, modified by
Lynch and Milligan (1994) for dominant
markers, Shannon’s index etc)
           Evaluation of genetic diversity in Lingonberry populations

           Table 1. Analysed populations of lingonberry, number of plants sampled, location of population, within-population gene diversity
           (including standard error) estimated by the Lynch and Milligan index (Hpop) and Shannon’s index (H’pop)

Sampling    Sampling        No. of         Country      Location                Latitude        Longitude       Hpop            H’pop
site no.    site code       plants                                              (N)             (E)

 1             SK             15           Sweden       Kristianstad            56°13´          14°12´          0.227 (0.026)   0.483 (0.049)
 2             SÖ             15           Sweden       Örebro                  59°24´          14°39´          0.214 (0.025)   0.517 (0.051)
 3             SV             15           Sweden       Västerbotten            63°37´          19°51´          0.245 (0.027)   0.513 (0.050)
 4             SN             10           Sweden       Norrbotten              66°42´          19°33´          0.197 (0.026)   0.400 (0.051)
 5             SG             13           Sweden       Gävleborg               60°18´          16°46´          0.248 (0.028)   0.523 (0.048)
 6             SH             11           Sweden       Halland                 57°05´          13°20´          0.219 (0.026)   0.500 (0.051)
 7             FT             15           Finland      Toijala                 60°13´          24°10´          0.187 (0.026)   0.375 (0.049)
 8             FS             15           Finland      Simo                    65°41´          25°01´          0.178 (0.026)   0.374 (0.050)
 9             NS             15           Norway       Sognedal                61°12´            7°05´         0.225 (0.029)   0.434 (0.055)
10             EV             15           Estonia      Vöru                    57°55´          27°03´          0.241 (0.029)   0.456 (0.055)
11             EP             10           Estonia      Pärnu                   58°25´          24°40´          0.209 (0.027)   0.434 (0.055)
12             RM             15           Russia       Murmansk                68°55´          33°05´          0.110 (0.024)   0.190 (0.043)
13             RK             14           Russia       Kirov                   58°53´          49°30´          0.135 (0.024)   0.265 (0.047)
14             JF             14           Japan        Fuji San                35°20´          138°45´         0.180 (0.028)   0.349 (0.056)
15             CM             15           Canada       Montreal                45°50´          73°50´          0.274 (0.025)   0.654 (0.048)

                                                                                                                x = 0.206       x = 0.431
Cluster analysis, Multidimensional Scaling
and Principal co-ordinate analyses are used
mainly for evaluation of genetic relatedness
among individual organizms or among groups
of organizms (i.e. populations)
Genetic relatedness among populations of lingonberry
   (A) and indidual plants of Japanese quince (B)
             revealed by cluster analyses

       Similarity (%)
       100       90     80      70      60       50      40


SH
SN                                                    A         B
FT
SV
FS

SK
SÖ
RM
RK
SG
EV
EP
NS
CM
JF

     Fig.1. Dendrogram based on UPGMA analysis of genetic
     similarity estimates among 15 populations of lingonberry
Genetic relationships among lingonberry popula-tions
(A) and individual plants of Japanese quince (B)
revealed by MDS analysis
                                              A
                                                    B




   Fig.2 An MDS analysis of genetic relationships
   Among ligonberry populations
A three-dimentional representation of phenetical
relationships between populations of Japanese
quince revealed by PCA
Genetic relationships among
23 cultivars from Gene bank                                  Likhet %
                                                           Similarity %
                                                           100   80   70   60   50   40   30

at Balsgård revealed by       Gravensteiner
                              Röd Gravensteiner

RAPD markers                  Ingrid Marie
                              Guldborg
                              James Grieve
                              Maglemer
                              Cox Orange
                              Alice
                              Grågylling från Skokloster
                              Vit astrakan
                              Stor klar astrakan
                              Arvidsäpple
                              Oranie
                              Spässerud
                              Särsö
                              Hanaskog
                              Åkerö
                              Åkerö från Gripsholm
                              Fagerö
                              Flädie
                              Kavlås
                              John-Geor g
                              Golden Delicious



                                Fig.1. Dendrogram based on UPGMA analysis (Jaccard’s
                                coefficient) for RAPD data, showing relationships among
                                apple cultivars
Advantages, limitations and
applications of RAPD markers
               Advantages:
• No prior knowledge of DNA sequences is
  required
• Random distribution throughout the genome
• The requirement for small amount of DNA
  (5-20 ng)
• Easy and quick to assay
• The efficiency to generate a large number of
  markers
• Commercially available 10mer primers are
  applicable to any species
• The potential automation of the technique
• RAPD bands can often be cloned and
  sequenced to make SCAR (sequence-
  characterized amplified region) markers
• Cost-effectiveness!
                Limitations:
• Dominant nature (heterozygous individuals
  can not be separated from dominant
  homozygous)
• Sensitivity to changes in reaction
  conditions, which affects the reproducibility
  of banding patterns
• Co-migrating bands can represent non-
  homologous loci
• The scoring of RAPD bands is open to
  interpretation
• The results are not easily reproducible
  between laboratories
          Applications:

•   Measurements of genetic diversity
•   Genetic structure of populations
•   Germplasm characterisation
•   Verification of genetic identity
•   Genetic mapping
• Development of markers linked to a trait
  of interest
• Cultivar identification
• Identification of clones (in case of soma-
  clonal variation)
• Interspecific hybridization
• Verification of cultivar and hybrid purity
• Clarification of parentage
RAPD is probably the cheapest and easiest
DNA method for laboratories just beginning
to use molecular markers
Thank you

								
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