Markers were used to track genes conferring resistance to disease in plant breeding programs. In one study, AFLP markers tracked the introgression of a resistance gene from a donor line into cultivated rose varieties over multiple generations of backcrossing. The individual with the lowest fraction of donor genome markers was selected for further backcrossing to reduce the donor genome. In another study, RAPD markers co-segregated with resistance to Fusarium in a petunia F2 population, identifying a marker linked to the resistance gene. A third study developed SSR markers from petunia expressed sequence tags and evaluated diversity in two F2 petunia populations to identify markers for future genetic mapping.

1. Markers

2. INTRODUCTION
•Marker may be defined as a “Mark of identification”
•They help in mapping and transfer of traits
•Markers are genetically linked to the gene of interest
•The gene of interest directly causes production of protein(s) or RNA
that produce a desired trait or phenotype
•They tend to move together during segregation of gametes
•If the gene of interest is not known ,markers linked to the gene of
interest can still be used in selection of individuals
2
Any marker that allows to select
for specific trait is useful in
plant breeding
Marker is a flag that is
associated with a certain trait of
an organism

3. Types of Markers
3
DNA
CYTOLOGICAL
BIOCHEMICAL
MORPHOLOGICAL
• Related to size, shape, surface and colour of
various plants
• Often detectable by eye, by simple visyal
inspection- leaf sheath coloration, height etc
Such markers are related to
variations in chromosome
morphology
Such markers are related to variations
in proteins and amino acid banding
patterns
Related to variation in DNA
fragements generated by
restriction endonuclease enzymes
MOLECULAR
Identified as genetic marker, a
fragment of DNA that is associated
with a certain location in genome
Nybom et al., 2004

4. Classification of Markers
4
Classic
Markers
-Morphological
-Biochemical
DNA
marker
-Non PCR based
-PCR based
Codominant
Markers
-SSR
-RFLP
-ALLOZYMES
Dominant
Markers
-RAPD
-AFLP
-SCAR
Based on
Hybridisation -RFLP
-Minisatellite
-Microsatellite
Based on PCR -RAPD
-AFLP
-SCAR
-STS
-SNP
-ScOT
-SSR
-ISTR
-VNTR

5. 5
RFLP ( Restriction Fragment Length Polymorphism )
◉ It was invented by Alec Jeffreys in 1984
◉ Genetic markers resulting from the variation or change in the length of
defined DNA fragments produced by digestion of the DNA sample by
restriction endonucleases
CONSIDERATIONS FOR USE
• Relatively slow process
• Use of radioisotopes has limited its use to certified laboratories
• Co- Dominant markers; often species specific
• Need high quality DNA
• Need to develop polymorphic probes
• Expensive

6. 6
PROCESS
1.
Extraction of high
quality DNA
Digestion of DNA with
restriction Endonuclease
2
3
4
5
6
Analysis of restriction
fragments using gel
electrophoresis
Transfer fragments
to membrane
Hybridize with radioactively
labelled probe
Detection by
autoradiography

7. 7
RAPD (Rapid Amplified Polymorphic DNA)
◉ RAPD was developed by Williams et al., AP- PCR (Abitrary primed PCR) was developed
by Welsh and McClelland in 1990 independently (both are same technique)
◉ This is the technique to amplify through PCR random DNA segments primer by random
10-mer oligonucleotide sequences (arbitrary primer of 8-12 base pairs )
1
2
3
4
5
6
7
PROCESS
Isolation of DNA
Denaturation of DNA at
94oC for 1 min
DNA strands
separated
Annealing of primer at
36oC for 2 min
Complementary
strand syntheis;72oC,
1.5 min, 35-45 cycles
Amplified product separated
by gel electrophoresis
Bands detected by Ethidium
bromide Staining

8. 8
AFLP (Amplified Fragment length Polymorphism)
◉ Zabeau and Vos invented AFLP technique in 1993
◉ Based on a selectively amplifying a subset of restriction fragments from a complex
mixture of DNA fragments obtained after digestion of genomic DNA with restriction
endonucleases
1
2
3
4
5
PROCESS
Restriction of DNA
(using restriction enzyme
Ligation of oligonucleotide
adapters to both ends of fragment
Selective amplifications of
sets of restriction
fragments
Analysis of results in gel
electrophoresis or PAGE
Autoradiography

9. 9
SSR (Simple Sequence Repeats) or Microsatellites or STM (Short Tandem
Repeats) or STM (Sequence Tagged Microsatellites) or VNTR (Variable
Number of Tandem Repeats)
◉ Developed by Jeffrey et al., in 1985
◉ These are 1-6 bp long repeat sequences
PROCESS
1
2
3
4
5
6
Microsatellite
library construction
Identification of unique
microsatellite loci
Identification of a
suitable area for
primer design
Obtaining a PCR product
Evaluation of banding
pattern
Assessing PCR product for
polymorphisms

10. 10
ISSR (Inter Simple Sequence Repeat)
◉ Reported by Ztetikiewicz et al., 1994
◉ Single primer designed from SSR region
PROCESS
1
2
3
4
Primer designed
from SSR region
Primers are annealed with DNA
and PCR amplification
ISSR amplified
product obtained
Analysis through gel electrophoresis

11. 11
CAPS(Cleaved Amplified Polymorphic Sequence)
◉ CAPS marker was described by Konieczney and Ausubel in 1993 for genetic mapping
◉ It is identical to RFLP and referred as PCR-RFLP
◉ This is the technique to amplify through PCR random DNA segments primer by random
10-mer oligonucleotide sequences (arbitrary primer of 8-12 base pairs )
1
2
3
4
PROCESS
Amplification of DNA
by PCR
Production of monomorphic
PCR products
Digestion by restriction endonuclease
to show polymorphism
Screening through electrophoresis
by staining with ethidium bromide

12. 12
SCAR (Sequence Characterized Amplified Region):
◉ Based on PCR- agarose gel electrophoresis
◉ Uses longer primers of 15-30 nucleotides yielding high
reproducibility
◉ Needs prior sequence information for primer designing
EST(Expressed Sequence Tags):
◉ Molecular markers synthesised by partial sequencing of random
cDNA clones
◉ Used for whole genome sequencing and studying gene of interest
SCoT(Start Codon Targeted) Polymorphism:
◉ This method was developed based on the short conserved region
flanking the ATG start codon in plant genes
◉ Uses 18-mer primers in PCR and anealing temperature of 50oC

13. What is M.A.S?
◉ Marker assisted selection or marker aided selection is an indirect selection
process where a trait of interest is selected on the based on a marker
(morphological/biochemical/molecular) linked to a trait of interest (e.g.,
productivity, disease resistance, abiotic stress tolerance, quality) rather than
the trait itself
◉ A tool that can help plant breeders select more efficiently for desirable crop
traits
13
Introduction to Marker assisted selection: John Ruane and Andrea Sonnino

14. Why M.A.S?
 Breeding for specific traits in plants is expensive and time consuming.
 The greater the complexity of the trait, the more time and effort needed to achieve a desirable
result.
 The goal of MAS is to reduce the time needed to determine if the progeny has trait.
 The second goal is to reduce cost associated with screening of traits
 If we can detect the distinguishing trait at the DNA level, we can identify positive selection
very early
14

15. Pre requisites for an efficient marker-assisted breeding
programme
 Appropriate marker system and reliable markers: For a plant species or a crop, a suitable
marker system and reliable markers available are critically important to initiate a marker-
assisted breeding programme
15
Important properties of ideal markers for MAS;
 Ease and low-cost of use, recognition and analysis
 Abundance
 Small amount of DNA required
 Co-dominance
 Repeatability/reproducibility of results
 High level of polymorphism; and
 Occurrence and even distribution genome wide
Goddard KA, Wijsman EM, 2002
Jean-Marcel Ribaut, David Hoisington, 1998

16. CONVENTIONAL PLANT BREEDING
F2
F1
P1 P2
x
Large populations consisting of
thousands of plants
PHENOTYPIC SELECTION
FIELD TRIALS
GLASSHOUSE TRIALS
Donor
Recipient
Salinity screening in phytotron Bacterial blight screening Phosphorus deficiency plot

17. MARKER-ASSISTED BREEDING
F2
P2
F1
P1 x
Large populations consisting of
thousands of plants
Resistant
Susceptible
MARKER-ASSISTED SELECTION (MAS)
Method whereby phenotypic selection is based on DNA markers

18. Procedure for MAS
18
1 3 5
4
2
Selection of Parents Isolation of DNA from each plant
Correlation with morphological
traits
Development of breeding
population
Scoring RFLP’s

19. 1. SELECTION OF PARENTS
◉ Contrasting characters or Divergent origin
◉ Self pollinated Homozygous
◉ Cross pollinated Inbred lines
19

20. 2. DEVELOPMENT OF BREEDING POPULATION
◉ The selected parents are crossed to obtain
F1
◉ 50-100 F2 Plants are sufficient for the study
of segregation of (say RFLP markers).
◉ Planting the breeding population with
potential segregation for traits of interest or
polymorphism for the markers are used
20

21. 3. SAMPLING AND ISOLATION OF DNA
At early stages of growth (emergence to young seedling stage)

22. 4. GEL ELECTROPHORESIS METHOD
Appropriate separating
and detecting techniques
are used such as;
 PAGE
 AGE

23. 5. Scoring RFLPs

24. OVERVIEW OF MARKER GENOTYPING
1) Tissue sampling
(2) DNA extraction
(3) PCR
(4) Gel electrophoresis
(5) Marker analysis
Identifying and selecting best individuals/families with both desired marker
alleles for target traits and desirable performance /phenotypes of other
traits, by jointly using marker results and other selection criteria

25. Case study: 1
1. To introgress the resistance gene Rdr1 into the genetic background of cultivated
roses and therefore provide plant material suitable for variety breeding in various rose
groups.
2. To assess the utility of AFLP markers for an efficient reduction of the donor genome
in introgression programms for tetraploid roses using only few cycles of backcrossing.
Objectives

26. Materials and methods
 Resistant diploid donor line 88/124-46 .
CT 50-4
(genome doubled)
Hybrid Tea
91/100-5
(Resistant)
 Crossing the resulting hybrid to another garden rose floribunda variety.
 Analyzing the segregating progeny 95/3 for the segregation of disease resistance and molecular markers.
 The individual with the smallest fraction of donor genome was selected with AFLP markers (to reduce the
genetic background of the resistance donor 88/124-46 in the next generations )
 backcrossed to two garden rose varieties.
 Screening for resistance and smallest fraction of donor genome.

27. Breeding scheme for the
introgression of Black spot
resistance from a resistant
diploid line to tetraploid rose
varieties

28. RESULTS
◉A total of 1660 AFLP fragments could be clearly distinguished, 491 of which
were polymorphic between the two parents and segregated in the
progeny.
◉Among these, 110 were specific for the donor line 88/124-46 (e.g. they were
absent in both ‘Caramba’ and ‘Heckenzauber’), 114 were specific for
‘Caramba’ and 103 fragments were specific for ‘Heckenzauber’ .
◉The fraction of markers specific for the donor line was 62.1 %.
◉The fractions of the markers specific for ‘Heckenzauber’ was 59.7 %
whereas those for the variety ‘Caramba’ 46.4 %.

29.  99/18-23 (13.7 %)
 99/18-27 (14.1 %)
 99/20-24 (14.2 %)
◉ Based on the smallest number of donor fragments (31.8 %) the genotype 95/3-23
(harbouring 62.8 % and 60.2 % of markers specific for ‘Caramba’ and
‘Heckenzauber’ respectively) was selected as a parent for the next breeding
cycle. It was crossed as the male parent to the varieties ‘Caramba’ and
‘Heckenzauber’.
◉ It indicated that line 95/3-23 carries the resistance gene Rdr1 in the simplex
configuration
◉ Genotypes with the lowest fraction of donor genome are candidates for further
breeding cycles or commercial variety breeding.
◉ The most promising candidates were;

30. Case study: 2
 To determine the mode of inheritance of resistance as well as to tag,
using random oligonucleotide primers, the major gene(s) involved in
manifesting the resistant phenotype.
Objective(s)
G. Scovel et al, 2001

31. Materials and methods
Propagated asexually (cuttings) and maintained in pots and field trials
Directed open pollination
PCR Screening for RAPD markers
A linked RAPD marker designated GS624 was sequenced and used to design sequence-specific primers (AF,VR) in
an effort to develop a SCAR marker.
2217
(resistant breeding line)
Eifel
(sensitive cv)
1072-4
(F1 segregant)
1743
(Segregating F2 progeny)
self pollination
2217
(resistant breeding line)
Ashley
(moderately resistant cv.)
Apex
(F1 segregant)
1073
(Segregating F2 progeny)
self pollination

32. Results
 Segregation for resistance to Fusarium of F2 progeny 1073
 Phenotypic scoring showed a 3:1 ratio between resistant and moderately
resistant segregants
 No fully sensitive segregants were revealed
% Disease incidence
No.
of
segregants

33. RAPD analysis
RAPD analysis of the progeny
segregating for resistance to
Fusarium.
F1 S R
G S 6 2 4
 Screening a sample of F2 progeny 1743 with 200 random oligonucleotide primers yielded one marker (GS624)
that showed linkage to a locus controlling resistance to F. oxysporum
 Linkage between marker GS624 and resistance to Fusarium

34. Case study: 3
 To evaluate the diversity of two F2 interspecific hybrid Petunia populations.
 To develop SSR markers from currently available P. axillaris expressed
sequence tag sequences that will be useful for future genetic mapping and
quantitative trait locus analysis.
Objectives
J. Tychonievich et al, 2013

35. MATERIALS AND METHODS
1. F2 hybrid mapping populations were developed by self-pollinating a P. axillaris × P. exserta F1 individual and by
crossing among P. integrifolia × P. axillaris F1 individuals . (Warner and Walworth 2010).
2. Two hundred seventeen P. integrifolia × P. axillaris and 219 P. axillaris × P. exserta F2 individuals were grown in a
greenhouse at 17°C under a 16-hour photoperiod (ambient irradiance plus high-pressure sodium lighting at 50
μmol·m-2·s-1 from 6 am to 10 pm daily).
3. Potential SSR markers were screened for polymorphisms between the parental species.
4. Markers were scored as polymorphic between a species pair if a different banding pattern was seen in the two
species and bands from both parents were observed in the bulked F2 DNA.

36. Results
o Population distribution for
flower diameter (A and B)
and plant height (C and D)
for two interspecific hybrid
petunia F2 populations, P.
axillaris X P. exserta (A and
C) and P. integrifolia X P.
axillaris (B and D).
o The arrows indicate values
for P. axillaris (AX), P.
exserta (EX). P. integrifolia
(INT) and P. hybrids (F1)

37. Summary of number of polymorphic SSRs between P. integrifolia and P. axillaris and
between P. axillaris and P. exerta.
 Of the 591 potential SSR markers, 132 (22.3%) were polymorphic between P. axillaris and P. integrifolia ,
and 99 (16.8%) were polymorphic between P. axillaris and P. exserta. There were 58 SSRs that were
polymorphic across all three species. These will be particularly useful for comparing marker order and
genetic mapping distances across the two populations.

38. Case study: 4
 To enable molecular marker screens to be used for the selection of male sterile
plants in breeding Programmes of marigold (Tagetes erecta L.).
(As marigold male sterile lines are used for cross-breeding purposes and serve to make
the hybridization process relatively efficient and economic)
(Zhang et al. 2005)
Objective(s)
G. G. Ning et al, 2008

39. Materials and methods
M525A
Male sterile, (Tems)
F53f
(inbred line)
Segregation population of 167 plants constructed
The sterile plants were identified during flowering and the young leaves were collected separately from each plant
for use in DNA extraction.
The inter simple sequence repeat (ISSR, 38) and sequence-related amplified polymorphism (SRAP, 170) techniques
combined with bulked segregant analysis were used to develop markers linked to the trait.

40. Results
 Of the 766 bands obtained with the 170 SRAP primer combinations,
only 26 showed polymorphism between the sterile and fertile bulks.
 The arrow indicates polymorphic band (604bp) present in fertile bulk and
fertile individuals but not in sterile bulk and sterile individuals
M, DL-2000 marker;
CK, control;
BS, sterile bulk;
BF, fertile bulk
Individuals of fertile bulk Individuals of sterile bulk
M CK BS BF
SRAP profile of bands amplified by the me4-em8 primer combination for bulks and individuals

41. Individuals of F2 sterile plants Individuals of F2 fertile plants
M CK BS BF
 The SCS48 marker which tightly linked to the Tems gene has the potential to facilitate the large-scale screens for
fast marker-assisted selection strategy
 Tightly linked markers comprising co-dominant markers distinguishing the genotypes of TeMsMs, TeMsms and
Temsms, and flanking markers located on either side of the Tems gene at a closer distance are expected for map
based cloning of the Tems gene.
 Photograph of an agarose gel illustrating PCR products generated from genomic DNA of two
bulks and 10 fertile plants and 10 sterile plants of F2 population with the described SCAR primers.
The arrow indicates polymorphic 500 bp band.
M, DL-2000 marker; CK, control; BS, sterile bulk; BF: fertile bulk

42. Case study: 5
 To identify DNA markers linked to the genetic locus controlling
carnation flower type
Ben-Meir et al, 1999
Objectives

43. Materials and methods
Breeding line 2217
selfing
F2 population
(74 full-sibs)
Segregating for flower phenotype and analyzed by RAPD
 The scoring of flower phenotype (single, semi-double and double) was based on the
number of petals/anthers.
 In order to distinguish between genotypes heterozygous and homozygous for flower type, individual F2
plants were selfed and the flower phenotype of the progeny analyzed.
 For inheritance of flower phenotype, several different crosses were made.
 Six half sibs (two lines from each cross) analyzed by RAPD were derived from crosses between line 2217
and cvs; Eveline, Splendid and Ashley.

44. the phenotype-specific 1.8-kb RFLP marker
 Using this RFLP marker, it was possible to identify with 100% accuracy the
flower phenotype in genotypes not genetically related to the original line
2217-derived segregating family
Results

45. RESULTS
◉ In the present study, the OPR02782 RAPD marker co-segregating with the d allele was used as an
RFLP probe. Using the HaeIII restriction enzyme, which does not cut within the OPR02782
fragment, an RFLP marker tightly linked to the d allele was obtained.
◉ Several additional, non polymorphic bands were revealed by the RFLP marker
◉ No repetitive sequences were found in the OPR02782 RAPD fragment.
◉ Using this RFLP marker, it was possible to identify with 100% accuracy the flower phenotype in
genotypes not genetically related to the original line 2217-derived segregating family
◉ Discrimination of flower phenotype was successful with carnations of both the Mediterranean
and American groups
◉ RFLP marker may be used immediately in breeding for spray or standard carnation varieties by
screening, respectively, for or against semi-double flower genotypes.

46. 1. Accuracy
2. Rapid method
3. Identification of recessive
alleles
4. Early detection of traits
5. Screening of difficult traits
6. Gene pyramiding
7. Small sample for testing
8. Permits QTLmapping
9. Highly reproducible
MERITS
1.Costly
2. Well trained man power
3. Radioactive labelling
4. Well equipped laboratory
5. Expensive equipments
6. Expensive chemicals
7. Expertise
DEMERITS
46
Conclusion