Applications of DNA Markers

1. Genome mapping – linkage maps and QTL

mapping

 A QTL is a genomic segment flanked by

DNA markers whose genotypic variation
associates with the phenotypic variation in a
quantitative trait.

Gu et al. (2004): Genetics 166, 1503–1516 1

 Linkage studies and QTL Mapping
 Genome mapping – linkage maps and QTL mapping

qSD1-1
qSD7-1

qSD8 qSD10
qSD4
qSD6
qSD7-2
qSD12

qSD1-2

qSD3

Seed dormancy QTL identified in rice

Linkage map of rice developed using a backcross population

Gu et al. (2004): Genetics 166, 1503–1516 2

 Applications of DNA Markers

2. Genetic diversity studies via cluster analysis

Cluster analysis is a multivariate analysis for obtaining groups or
"clusters" of objects that are "similar" to each based on measured
attributes

Similarity, Dissimilarity and Distance

Similarity is a measure of shared attributes between pairs of objects
compared to the total list of attributes between them
It ranges from 0 to 1

Dissimilarity is a complement of similarity (1 – Similarity)

Distance is a measure of the proximity of objects in a high dimensional

space defined by measurements on the attributes
It has no upper bound eg. Euclidean distance

Gu et al. (2004): Genetics 166, 1503–1516 3

 Genetic Diversity studies
ID M1 M2 M3 M4 M5
Similarity matrix
GERM01 1 1 1 1 1
𝐴
G01 G02 G03 G04 G05
GERM02 2 2 2 2 2 S=
𝐴+𝐵+𝐶 G02 0
GERM03 2 1 1 4 1
G03 0.6 0.2
GERM04 2 2 2 2 1
G04 0.2 0.8 0.4
GERM05 2 2 2 2 2
G05 0 1 0.2 0.8
GERM06 2 1 1 3 1
G06 0.6 0.2 0.8 0.4 0.2
𝐷𝑆 = 1 − 𝑆
Dissimilarity matrix
G01 G02 G03 G04 G05
G02 1
G03 0.4 0.8
G04 0.8 0.2 0.6
Euclidean distance matrix G05 1 0 0.8 0.2
G01 G02 G03 G04 G05 G06 0.4 0.8 0.2 0.6 0.8
G02 2.24
G03 3.16 2.65
G04 2 1 2.45
G05 2.24 0 2.65 1
G06 2.24 2 1 1.73 2
4
Genetic Diversity studies

5
 Molecular cloning Techniques
Molecular cloning refers to the creation of recombinant
DNA molecules
The isolation of a DNA sequence from any species
(often a gene), and its insertion into a vector for
propagation, without alteration of the original DNA
sequence
Why clone genes?
To generate many copies of the DNA for analysis of the
gene sequence
To express the resulting protein for the study or utilization
of the protein’s function
To manipulate or mutate genes in vitro to alter the
expression and function of the protein

6
 Basic steps in molecular cloning
The basic cloning workflow includes four steps:
1. Isolation of target DNA fragments (often referred to
as inserts)
2. Ligation of inserts into an appropriate cloning vector,
creating recombinant molecules (e.g., plasmids)
3. Transformation of recombinant plasmids into
bacteria or other suitable host for propagation
4. Screening/selection of hosts containing the intended
recombinant plasmid

7
 Basic steps in molecular cloning

https://www.neb.com/tools-and-resources/feature-
articles/foundations-of-molecular-cloning-past-present-and-future 8
 The Foundation of Molecular Cloning
 Cutting or digestion of DNA using restriction enzymes

 Assembling (ligation) of pieces of restricted DNA together

using DNA ligases

 Transformation
 Molecular cloning would be impossible without the
means to propagate and isolate the newly constructed
DNA molecule

 Cohen et al. (1973) were the first to perform the ultimate

recombinant DNA technology experiment of digestion,
ligation and transformation of a recombinant DNA
molecule

9
The Foundation of Molecular Cloning

10
 Molecular Cloning Vectors
 One of the first multipurpose vectors was created in 1977 – pBR322
 ~4.3 kbp in size with two antibiotic resistance genes for selection
 It has about 40 restriction sites

11
Molecular Cloning Vectors

12
 Molecular Cloning Vectors
 Improvement on pBR322 led to the creation of pUC plasmids
 ~2.7 kbp in size with multiple cloning sites (MCS) within a LacZ
gene
 It has ampicillin resistance gene

13
 Blue-white screening using pUC vectors

Schematic representation of a typical blue-white screening procedure. Blue-white

screening of bacterial colonies involves cloning a gene inserted into a plasmid vector
with an antibiotic resistance and LacZ reporter gene. The ligation of the insert into the
multiple cloning site of the vector inactivates the LacZ gene. The transformation of
competent E. coli with the ligated mixture in the presence of X-gal in culture media
results in the formation of blue and white colonies.
 Expression vectors
 A vector that results in the expression of inserted DNA
sequences when propagated in a suitable host cell, i.e. the
protein coded for by the DNA is synthesized by the host’s
system.
 Expression vectors
The necessary elements for an expression vector and their roles
 A selection marker such as antibiotic resistance to aid the successful
identification of recombinant bacterial cells
 Multi cloning site where the foreign DNA is inserted on the vector because
there are different restriction sites at MCS
 A DNA replicon which helps the expression vector to replicate in bacteria
upon cloning
 A strong promoter is required for the inserted foreign gene to be
transcribed
 Ribosomal binding site such as the Shine – Delgarno sequence which
helps recruit the ribosome to the mRNA to initiate translation by aligning it
with the start codon i.e. is a portable translation initiation sequence
 Transcription termination sequence to mark the end of the coding
sequence of the inserted gene upon transcription
 Fusion tag which facilitates the detection of the target protein during
expression and purification. It is recognized by antibody during Western
blot.
 Special Cloning techniques

TA cloning
Golden gate cloning
Gibson assembly
overlap extension PCR cloning
 TA Cloning technique
 Taq polymerase has no 3’ to 5’ exonuclease proofreading activity; adds
adenine overhangs at the ends of amplicons after PCR
 Gene silencing
Gene knockdown: RNA interference
(RNAi)

Gene knockout: CRISPR/Cas9 genome

editing

20
 RNAi silencing mechanism
a b c d b d e

RB LB
IRS
Transcription
dsRNA

Dicer (DCL4)

~21-24 bp siRNA

Silencing amplification
RISC
mRNA AAA
AGO1 RDRP (RDR6)

mRNA degradation (Baulcombe, 2004; 2007)

Baulcombe, D. (2004). Nature 431, 356-363. Baulcombe, D. C. (2007). Science 315, 199-200. 21
Silencing SD7-1: A pleiotropic
gene controlling pericarp color
and ABA biosynthesis in rice

22
 Silencing SD7-1 locus in weedy rice
SD7-1 – pleiotropic effect

Red color in the ABA

pericarp tissue

Dormancy
induction

ABA biosynthetic pathway

Flavonoid biosynthetic pathway Gu et al. (2011)

Gu et al. (2011): Genetics 189, 1515–1524 23

 Hairpin RNA construction
qSD7-1
Trigger region PCR amplification
270 bp

pENTR/D TOPO
PCR product cloning vector

TOPO cloning

attL1 attL2

Entry clone pANDA vector

LR clonase reaction
~6.4 kbp
a b c d b d e

RB Bar LB
IRS::SD7-1
Tandem construct of bar gene and
hairpin RNAi targeting seed dormancy
attB1 attB2
QTL qSD7-1

Miki and Shimamoto (2004):Plant Cell Physiol. 45(4): 490–495 24

 RNA silencing mechanism
~6.4 kbp
a b c d b d e

RB Bar LB
IRS::SD7-1
Transcription
dsRNA

Dicer (DCL4)

~21-24 bp siRNA

Silencing amplification
RISC
mRNA AAA
AGO1 RDRP (RDR6)

mRNA degradation (Baulcombe, 2004; 2007)

Baulcombe, D. (2004). Nature 431, 356-363. Baulcombe, D. C. (2007). Science 315, 199-200. 25
 Transgenic plants evaluation

Leaf application (0.5% glufosinate) Whole plant application (0.5% glufosinate)

Pericarp color
TMR#1-7 Nipp ILSD7-1
Nipp TMR ILSD7-1

TMR Nipp ILSD7-1 Ludao

Ye et al. (2014)

26
 RNAi construct silenced SD7-1 in F1 and F2 plants
Silencing effect on pericarp color Degree of dormancy: F1-HR vs F1-HS
F1-HS F1-HR
T0-
RNAi#4

F1-HR

F1-HS

27