Plant and Animal

Breeding
Artificial Selection &
Breeding for selected traits
selection of plants or animals for
desirable traits by humans through
carefully planned breeding
Artificial Selection & Breeding for
selected traits
Isolation of natural populations
↓
Selective breeding of organisms
showing traits useful of humans
↓
Useful genotypes exits more often
(producing more offspring) than
other genotype
↓
Change in allele frequency towards
genotypes useful to humans
Aims of artificial selection
1. create new breeds or varieties
Aims of artificial selection
preserve good species
Aims of artificial selection
domesticate wild plants and
animals
Wildebeest Ostrich
Common types of artificial
selection
Inbreeding
Outbreeding
Polyploidy
Inbreeding
mating between genetically
closely related individuals
increase the number of
homozygous genotypes
◦ reduced variability
◦ decrease in heterozygosity
Inbreeding
self-fertilization
crossingthe offspring of the
same parents
backcrossing with one of the
parents
Inbreeding
decrease heterozygous
genotypes by 50% in each
generation
Problems of inbreeding
reduce in fertility
Inbreeding depression - vigour of
the population is gradually
reduced
Outbreeding
mating between genetically
closely unrelated individuals
increase the number of
heterozygosity
◦ more variations
◦ produce offspring with superior
character
(hybrid vigour)
use in combining two beneficial
characteristics
Polyploidy
Autopolyploidy
◦ chromosome sets derived from the
same species
◦ created by spontaneous duplication

Allopolyploidy
Polyploidy
Autopolyploidy
◦ chromosome sets derived from the
same species
◦ created by spontaneous duplication
Polyploidy
Allopolyploidy
◦ two different diploid species are
interbred
Allopolyploidy
the hybrid form is sterile
◦ different sets of chromosomes from
both parents are not homologous
◦ no pairing during meiosis
Allopolyploidy
duplicate the genome
the tetraploid became fertile
Allopolyploidy
get the advantage of inheriting
desirable characteristics from
both parents
can be induced artificially by
colchicine
◦ inhibit spindle formation
Methods commonly used in
plant breeding
Selection
Backcross
Hybridization
Polyploidbreeding
Mutation breeding
Methods commonly used in
animal breeding
Selection
Artificial insemination
Artificial insemination
Collection of semen from a male
Dilution and artificial introduction
of sperm into female reproductive
tract

Horses - Artificial Insemination

Artificial insemination
the semen can be stored in liquid
nitrogen for a long time
one male can be used to fertilize
a large number of females
semen can be sent over long
distances
commonly used in cattle
breeding
Plant cloning
A clone is a group of genetically
identical cells or an individual
derived from a single ancestral
cell, tissue or individual by
repeated asexual divisions
Cloning is the production of
genetically identical individuals
Cloning is naturally
occurring
Organisms which reproduce
asexually give rise to progeny by
mitotic nuclear division
e.g. binary fission, vegetative
propagation
produces exact copies of the
parental genotype
Gardeners frequently maintain
clones of desirable varieties of
plants by vegetative propagation.
Plant cloning
Materials
needed for
cloning:
Somatic cells
(primordial
cells)
Culture
medium
Tissue culture
• Each somatic cell contains all the
information required to code for
an entire organism.
• The cells to be cloned are allowed
to grow in a medium containing
suitable nutrients and hormones
to form a mass of genetically
identical cells called callose.
The callose are then separated and induced to
produce new individuals
Advantages of plant
cloning
maintain desirable traits in
selected plants
rapid way of propagating plants
in a short period of time
plants are grown in sterile
medium (disease-free)
Advantages of plant
cloning
plants
are grown in sterile
medium (disease-free)
Advantages of plant
cloning
maintain the genetic uniformity
less space is needed
Animal cloning
Firstclone animal (1997)
by Professor Ian Wilmut works at
the Roslin Institute in Edinburgh,
which specializes in research on
farm and other animals
Animal cloning - Dolly
An unfertilized egg was collected
from a Scottish blackface sheep
Animal cloning - Dolly
The nucleus of the unfertilized
egg cells was removed
(enucleated)
Animal cloning
The nucleus from a mammary
gland cell taken from a sheep Y.
Cloning of Dolly
The nucleus transfer to the
enucleated cell.
An electro fusion was given
Cloning of Dolly
Incubatethe new cell in a culture
medium for 6 days
◦ embryo formed
Cloning of Dolly
The embryo was implanted into
the uterus of another blackface
sheep (surrogate / foster mother)
Cloning of Dolly
The foster mother gave birth to
Dolly (baby sheep)
Implications of animal
cloning
maintain the desirable traits in
selected animals
Increase the population of
endangered species
Cloning on extinct animal
-
Thylacine - the largest known
carnivorous marsupial of modern
times
Native to Australia and New
Guinea
What’s the next?
Implications of animal
cloning
tissuefrom cloning of human
embryo for curing Parkinson’s
disease
Implications of animal
cloning
Recombinant DNA
technology
1. Transferring a particular gene to
a self-replicating chromosome
(usually in bacteria)
2. Amplification of the resulting
recombinant DNA molecule
Recombinant DNA
technology
Recombinant DNA
technology
1. Transferring a particular gene to
a self-replicating chromosome
(usually in bacteria)
2. Amplification of the resulting
recombinant DNA molecule
Recombinant DNA
technology
also called
 gene manipulation
 genetic engineering
Genetic Engineering
Basic steps of recombinant
DNA technology
identifying a target gene
isolating the target gene
inserting the target gene into a
vector
transferring the vector containing
the target gene into a host cell
for producing a certain gene
product
harvesting and purifying the
gene product
Isolate the target gene
use restriction enzymes
produced by bacteria which cut
on the DNA at particular sites
(recognition sites)
1978 Nobel Prize
Physiology or
Medicine
Werner Arber
Hamilton Smith
Daniel Nathans
Restriction enzymes
can be extracted from bacterial
cultures
cut the double helix at specific
sites along the sequences
most recognize sequences of
DNA with 4, 6 or 8 bases
Donor DNA
(Target DNA)

Restriction enzymes
(restriction endonucleases)

Break at
specific site
Restriction enzymes
act as powerful scissors
create sticky ends
Formation of recombinant
DNA molecule
apply the restriction enzyme in
vitro with 2 different DNA
fragments
DNA ligase
can join ‘sticky ends’ of the DNA
fragments
Ways to locate the genes
Short gun sequencing
Top-down sequencing
Shotgun approach

Genome from an organism

Restriction enzymes

Fragments of DNA
(some just contains the base
sequences of a gene)
Gene probe

Desired base sequence

Shotgun approach
• not specific
• but useful when no idea about
the sequence of the desired gene
Shotgun sequencing
DNA is broken up randomly into
numerous small segments
sequenced using the chain
termination method
Multiple overlapping reads for the
target DNA are obtained
 Shotgun sequencing
Strand Sequence
Original XXXAGCATGCTGCAGTCATGCTTAGGCTAXXXX
First
XXXAGCATGCTGCAGTCATGCTXXXXXXXXXXX
shotgun
XXXXXXXXXXXXXXXXXXXXXXTAGGCTAXXXX
sequence
Second
XXXAGCATGXXXXXXXXXXXXXXXXXXXXXXXX
shotgun
XXXXXXXXXCTGCAGTCATGCTTAGGCTAXXXX
sequence
Reconstructi
XXXAGCATGCTGCAGTCATGCTTAGGCTAXXXX
on
Bottom up sequencing
Complementary DNA
(cDNA)
conversion of mRNA into DNA
can be used to find out the
location of gene
 Protein

Amino acids sequence

Nucleotides
sequence

Only restricted to small gene with short DNA

Structure of mRNA
with a poly A tail d

alleukaryotic mRNA molecules

contain a poly-A tail
for stabilization of mRNA
molecule during transcription
cDNA
using a poly-T oligomer as a
primer
(Primer is a short single-stranded
DNA or RNA that functions as the
starting site for the elongation of
a new chain)
cDNA
Thepoly-T oligomer binds to the
complementary poly-A tail of
mRNA
Formation of cDNA
In the presence of enzyme
,reverse transcriptase a single
strand of cDNA copy is formed
Release of the cDNA
cDNA can be released by the
addition of alkali
Synthesis of DNA double
helix
with the help of DNA polymerase
Vectors
a self-replicating DNA molecule
that carries a foreign DNA
segment to a host cell for
amplification
come from bacterial plasmids
and viruses
Plasmid
a small circular molecule of
double stranded DNA present in
bacterial cell
carries a minor fraction of the
bacterial genome
◦ code important traits
Advantages of using plasmid
as vector
low molecular weight (contains
several thousand base pairs)
Advantages of using plasmid
as vector
plasmid replicates autonomously
independent of the chromosomes
in bacteria
Advantages of using plasmid
as vector
containsantibiotic resistance
genes which facilities selection
Advantages of using plasmid
as vector
contain a number of unique
cleavage sites for the actions of
several different restriction
enzymes
Isolation of plasmid from
bacteria
by breaking up the bacterial cells
separation by centrifugation
Cleavage of plasmid
cleavage by the same restriction
enzyme used in cutting the short
length of DNA (target gene) to be
inserted
form the same sticky ends
Insertion of plasmid to
host cell
with the help of DNA ligase
Insertion of plasmid to
host cell
the resulting plasmids can be
replicated if they are introduced
into a bacterial host cells
Introducing the target gene
into the host cell
DNA molecules can be taken up
by pre-treating the bacterial cells
with a solution containing Ca2+
ions followed by a rapid heat
shock(42oC)
Introducing the target gene
into the host cell
apply a brief electrical shock that
generates temporary pores in the
bacterial cell membrane
Production of human insulin
from genetically engineered
bacteria
Type I Diabetes
◦ failed to produce sufficient insulin
◦ due to insufficient insulin by Islets of
Langerhans in the pancreas
◦ can only get insulin from the
panaceas from cows or pigs
Problems of using insulin
from other animals
though the insulin is biologically
active but the amino acid
sequences are slightly different
from those of humans
some patients are stimulated to
produce antibodies against the
injected insulin
Structure of human insulin
consists of two separate
polypeptides chains: A and B
joined together by special
disulphide bridges (S~S)
A-chain contains 21 amino acids
B-chain is 30 amino acids long
Structure of human insulin
two chains originate from a large
gene product called preproinsulin
Structure of human insulin
function of C-chain is to bring the
A-chain and B-chain together in
the correct alignment
Structure of human insulin
A-chain and B-chain will join to
form a mature insulin
Genetically engineered
human insulin
isolate
2 synthetic DNA
fragments (genes)
◦ encoding A-chain and B-chain
Introduce the 2 genes into
plasmids
Genetically engineered
human insulin
the plasmids are introduced into
2 E. coli bacteria
produce the chain A and chain B
polypeptides separately
Genetically engineered
human insulin
the polypeptides are extracted
and purified
mixed under appropriate
conditions to produce functional
human insulin
Applications of recombinant
DNA technology
Production of therapeutic
proteins for pharmaceutical
uses
◦ Human insulin
◦ Blood clotting factor VIII
◦ Human growth hormone
◦ Protein coat of hepatitis B virus
Diagnosis of genetic
diseases
compare the nucleotides
sequences of affected patients
and unaffected individuals
◦ diabetes
◦ pancreas cancer
◦ cystic fibrosis
◦ haemophilia
◦ AIDS
Production of enzymes for
industrial applications
biological detergents
◦ protease and amylase
Brewing industry
Textile industry
Baking industry
◦ cellulase
Leather industry
Transgenic technology
transfer a desirable gene from
another species to a recipient
organism
form a new character which is
beneficial to human
produce transgenic animals and
transgenic plants
Genetically Modified Organisms
(GMO)
• Introducing new genes from another
organism
Bt gene
DNA Fingerprinting
Sir Professor Alec Jeffreys
Background information
No. of base pairs in human: 3 billion
No. of coding gene: 30,000
 about 95% of the base pairs are non-coding
 about 30-40% of the base pairs
consists of short sequence of repeats
some of the repeats are joined together
in cluster (tandem)
DNA fingerprinting
On the DNA there are region
which do not code for
polypeptides
◦ Exon – coded for polypeptide
◦ Intron – non-coding DNA sequences
Non-coding sequence
make up over 90% of the
genome
about half of them carry short
repetitive sequences of
nucleotides – tandem repeats
The tandem repeats are known as satellite
DNA.
Some just have a small number of repeats:
minisatillites.
• Different individual have a different number
of tandem repeated
• Minisatillate known as variable number
tandem repeats (VNTRs)
Procedure
Extraction
Amplification:
Polymerase Chain Reaction
(PCR)
Polymerase Chain Reaction (PCR)
1. Denature: made single-stranded
2. Add DNA polymerase
3. Add primer (a short DNA sequence)
Treatment with restriction
enzyme
• cut DNA into smaller
fragments
• contain minisatellites
• length of the DNA
fragments remains
unchanged
Agrose gel electrophoresis
• agrose gel with pores
• Separate the DNA
fragments according to size
• DNA carries negative
charge and will move to
positive pole if a voltage is
applied to it.
• Smaller size: move faster
• Bigger size: move slower
Splitting the DNA into single
strands
• by alkaline treatment
Addition of a radioactive
probe
• Identify the location of the
minisatillites
• number of tandem
repeats can be shown as
bands
Applications of DNA fingerprinting
• Identification of criminal
• very sensitive
Typeof sample Amountof DNA (ng); 1ng=10-9 g
Blood 20,000 – 40 000 ng / ml
Stain 1 cm2 in area ~200 ng
Stain in 1 mm2 area ~2 ng
Semen 150, 000 – 300, 000 ng / ml
Postcoital vaginal swab 0 – 3, 000 ng
Hair
Plucked 1 – 750 ng / hair
shed 1 –12 ng / hair
Saliva 1, 000 – 10, 000 ng / ml
Urine 1 – 20 ng / ml
Fig. 8 DNA content in biological sample
Chance occurrence of band matching
Number of Bands Odds againstachancematch
4 250 to 1
6 4000 to 1
8 65000 to 1
10 1 million to 1
12 17 million to 1
14 268 million to 1
16 4300 million to 1
18 68000 million to 1
20 1 million million to 1
Fig. 9 The chance occurrence of Band matching
Settling paternity disputes
• every child must inherit one copy
of a pair of homologous
chromosome from each parent
Mother Father
Genetic disorder and its diagnosis
• due to mutation

Sickle cell anaemia

Haemophilia