Molecular Systematics

1
Molecular Systematics

• Plant Systematics (PBIO 309/509)
• Harvey Ballard

2
Plant Genomes
3
Chloroplast Genome
rbcL
Judd et al. (2002)
4
Natural DNA Replication

• Double helix unwinds by action of helicase
• DNA polymerase facilitates linkage of nucleotides
  to each other to form new complementary strand,
  based on sequence of old strand

Raven et al. (1992)
5
Polymerase Chain Reaction

• Involves 25-35 cycles of denaturing _at_ 94C,
  annealing _at_ 48-55(-65)C, elongation _at_ 72C
• PCR mix includes flanking primers for specific
  gene region, Taq DNA polymerase, MgCl,
  nucleotides, buffers
• Small amount of DNA added just before PCR
  amplification commences

6
Polymerase Chain Reaction

• Denaturing phase unwinds sample DNA
• Annealing phase facilitates Taq polymerase to
  position primer molecules in proper place

Avise (1994)
7
Polymerase Chain Reaction

• Elongation phase promotes linkage of
  complementary nucleotides onto primer to form new
  strand

Avise (1994)
8
Polymerase Chain Reaction

• First cycles make one-primer strands
• Remaining cycles make two-primer target strands

Avise (1994)
9
Cycle-Sequencing

• After cleaning of PCR product to remove excess
  constituents, cycle-sequencing uses PCR
  technology again with PCR product
• CS Master Mix similar to PCR Mix but has regular
  nucleotides plus fluorescently tagged
  dideoxynucleotides
• ddATPs labeled with a green dye
• ddCTPs labeled with a blue dye
• ddGTPs labeled with a black dye
• ddTTPs labeled with a red dye

10
Cycle-Sequencing

• When a fluorescently tagged dideoxynucleotide is
  incorporated into a new strand, elongation
  process is terminated
• ?100s or 1000s of DNA molecules with same length,
  tagged by the same fluorescent color

11
Sample Sequence Analysis

• CS product is eluted in buffer and put on an
  automated sequencer
• CS sample is electrophoresed past a laser beam
  that produces peaks on a trace file
  representing a histogram of concentration for
  fragments bearing each tag color

12
Sample Sequence Analysis

• Composite histogram of tagged fragments
  sequence for gene region

GeneCodes website
13
DNA SequencesPAUP File of some Eurosids
NEXUS BEGIN DATA DIMENSIONS NTAX62
NCHAR1325 FORMAT DATATYPEDNA MISSINGN GAP-
INTERLEAVE OPTIONS MSTAXAUNCERTAIN MATRIX
10 20 30
40 50 . .
. . . Putranjiva
GTTGGGTTCAAAGCTGGTGTTAAAGATTATAAATTAACTTATTACACTC
C Drypetes GTTGGGTTCAAGGCTGGTGTAAAAGATTATAAAT
TAACTTATTACACTCC Oncoba GTTGGATTCAAGGCTGGTG
TTAAAGATTATAAATTGACTTATTATACTCC Xylosma
GTTGGATTCAAGGCTGGTGTTAAAGATTATAAATTGACTTATTATACTC
C Poliothyrsus GTTGGATTCAAGGCTGGTGTTAAAGATTATAAATT
GACTTATTATACTCC Casearia GTTGGATTCAAGGCTGGTGT
TAAAGATTATAAATTGACTTATTATACTCC
synapomorphy
autapomorphy
GenBank sequences
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DNA SequencesPAUP File of Hawaiian Viola species
NEXUS BEGIN DATA DIMENSIONS NTAX32
NCHAR481 FORMAT DATATYPEDNA MISSINGN GAP-
INTERLEAVE OPTIONS MSTAXAUNCERTAIN MATRIX
10 20 30
40 50 . .
. . . langsdorffiiJA ATAA
ACTAAACAACGTCGCCGCCAACCCCACCACCCCTAGGGGCGGGCAG lan
gsdorffiiAK ATAAACT----AACGTCGCCGCCAACCCCACCCCT---
AGGGGCGGGCAG helenae ATAAACT----AACGTCGCCGCCA
ACCCCACCCCT---AGGGGCGGGCAG tracheliifKA ATAAACT---
-ACCGTCGCCGCCAACCCCACCCCT---AGGGGCGGGCAG trachelii
fOA ATAAACT----AACGTCGCCGCCAACCCCACCCCT---AGGGGCGG
GCAG tracheliifMO ATAAACT----AACGTCGCCGCCAACCCCACC
CCT---AAGGGCGGGCAG maviensisMO ATAAACT----AACGTCGC
CGCCAACCCCACCCTT---AAGGGCGGGCAG robusta ATAAACT--
--AACGTCGCCGCCAACCCCACCCTT---AGGGGCGGGCAG waialena
lenae ATAAACT----AACGTCGCCGCCAACCCCACCCTT---AAGGGC
GGGCAG
Ballard (unpubl.)
15
Phylogeny ofHawaiian Viola

• Phylogeny of fast-evolving nuclear genes often
  gives species-level info
• Outgroup (nearly related sister taxon) is
  designated
• Can infer evolutionary patterns and processes

Ballard (unpubl.)
16
References

• Avise, J. C. Molecular markers, natural history
  and evolution. Chapman Hall, New York, NY.
• Judd, W. S., C. S. Campbell, E. A. Kellogg, P. F.
  Stevens, and M. J. Donoghue. 2002. Plant
  systematicsA phylogenetic approach, 2nd ed.
  Sinauer Associates, Sunderland, MA. pp. 105-121.
• Raven, P. H., R. F. Evert, and S. E. Eichhorn.
  1992. Biology of plants, 5th ed. Worth
  Publishers, New York, NY.