Dgen

2. MARKER-ASSISTED SELECTION

3. Contents
1.Marker assisted selection:-
a.Introduction & features.
b.General Steps in MAS.
c.MAS Breeding Scheme(Marker assisted
backcrossing,Pyramiding in Rice,…..).
d. Current status of molecular breeding.
e. Future challenges.
f. Future of MAS in rice etc?

4. Introduction
Marker assisted selection (MAS) refers
to the use of DNA markers that are
tightly-linked to target loci as a
substitute for or to assist phenotypic
screening or
The use of genetic markers with the
phenotypes in a process called
Marker assisted selection.

8. Markers must be
tightly-linked to target loci!
• Ideally markers should be <5 cM from a gene or QTL
• Using a pair of flanking markers can greatly improve
reliability but increases time and cost
Marker A
QTL
5 cM
RELIABILITY FOR
SELECTION
Using marker A only:
~95%
Marker A
QTL
Marker B
5 cM 5 cM
Using markers A and B:
~99.5%

9. Markers must be polymorphic
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
RM84 RM296
P1 P2
P1 P2
Not polymorphic Polymorphic!

10. F2
P2
F1
P1 x
large populations consisting of
thousands of plants
PHENOTYPIC SELECTION
Field trials
Glasshouse trials
Donor
Recipient
CONVENTIONAL PLANT BREEDING
Salinity screening in phytotron Bacterial blight screening
Phosphorus deficiency plot

11. General Steps in MAS-
1.Selection of parents.
2.Development of Breeding Population.
3.Isolation of DNA.
4.Scoring RFLP.
5.Correlation with Morphological traits.

12. (1) LEAF TISSUE
SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS
Overview of
‘marker
genotyping’

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

14. Marker-assisted backcrossing
(MAB)
Selection
for target
gene or
QTL
1 2 3 4
Target
locus
1 2 3 4
1 2 3 4
BACKGROUND
SELECTION
TARGET LOCUS
SELECTION
FOREGROUND
SELECTION
BACKGROUND SELECTION
Accelerates the
recovery of the
recurrent parent
genome

15. P1 x F1
P1 x P2
CONVENTIONAL BACKCROSSING
BC1
VISUAL SELECTION OF BC1 PLANTS THAT
MOST CLOSELY RESEMBLE RECURRENT
PARENT
BC2
MARKER-ASSISTED BACKCROSSING
P1 x F1
P1 x P2
BC1
USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS
THAT HAVE MOST RP MARKERS AND SMALLEST %
OF DONOR GENOME
BC2

22. Application of MAS
1.It is useful in gene pyramiding for disease
and insect resistance.
2.Uses in backcrossing programme.
3.It is being used for transfer of male sterility
into cultivated genotypes from different
sources.
4.MAS is being used for improvement of
quality characters in different crops such as
for protein quality in maize, etc…

23. Advantage of MAS
1. Accuracy,
2. Rapid Method,
3. Non-transgenic Product,
4. Identification of Recessive Alleles,
5. Early Detection of Traits,
6. Screening of Difficult Traits,
7. Highly Reproducible,
8. Small Sample for Testing,etc…

24. Achievements of MAS
1.Rice-a.(PB1 x JRBB55)Improved Pusa
Basmati 1, b.Improved Sambha
Mahsuri(BPT5204),
2.Maize-Improved QPM-9,
3.Pearlmillet-HHB67-2,
4.Wheat-Patwin,etc

25. Current status of molecular breeding
• A literature review
indicates thousands
of QTL mapping
studies but not many
actual reports of the
application of MAS in
breeding
• Why is this the case?

26. Some possible reasons to explain the
low impact of MAS in crop
improvement
• Resources (equipment) not available
• Markers may not be cost-effective
• Accuracy of QTL mapping studies
• QTL effects may depend on genetic background
or be influenced by environmental conditions
• Lack of marker polymorphism in breeding
material
• Poor integration of molecular genetics and
conventional breeding

27. How much does MAS cost at IRRI?
Consumables:
• Genome mapping lab (GML) ESTIMATE
– USD $0.26 per sample (minimum costs)
– Breakdown of costs: DNA extraction: 19.1%; PCR:
61.6%; Gel electrophoresis: 19.2%
– Estimate excludes delivery fees, gloves, paper tissue,
electricity, water, waste disposal and no re-runs
• GAMMA Lab estimate = USD $0.86 per sample
Labour:
– USD $0.06 per sample (Research Technician)
– USD $0.65 per sample (Postdoctoral Research Fellow)
TOTAL: USD $0.32/sample (RT); USD $0.91/sample (PDF)

28. Future challenges
• Improved cost-efficiency
– Optimization, simplification
of methods and future
innovation
• Design of efficient and effective
MAS strategies
• Greater integration between
molecular genetics and plant
breeding
• Data management

29. Future of MAS in rice etc?
• Most important staple for many
developing countries
• Model crop species
– Enormous amount of research in molecular
genetics and genomics which has provided
enormous potential for marker
development and MAS
• Costs of MAS are prohibitive so
available funding will largely determine
the extent to which markers are used in
breeding

30. 2.Barnase-Barstar System
Contents:-
1.Major system & Introduction,
2.Mechanism of Barnase-Barstar system,
3.Fertility Restoration System,
4.Production of 100% male sterile,
5. Features of this dominant genetic male
sterility system regarding commercial
value,
6.Achievements.

31. Major Systems of Genetic Male
Sterility
1.Dominant Nuclear Male Sterility,
2.Male Sterility by Silencing fertility Gene Bcp1,
3.Male Sterility through Hormone Engineering,
4.Pollen self destruction engineered Male Sterility,
5.Male Sterility through modification of
Biochemical Pathway,
6.Male Sterility using Pathogenesis related Protein
Genes.

32. Introduction
Tapetum Tissue:
• A specialized anther tissue the tapetum, play an important part in
pollen development.
• The tapetum surrounds the pollen sac early in the anther
development, degenerates during the later stages of development.
M, microsporocytes (microspore mother cells); DP, developing pollen; T, tapetal
cell; and Tds, tetrads.

33. Barnase
In 1990, C.Mariani et al. Successfully used a
chimeric dominant gene construct having an
anther specific promoter(from TA29gene of
tobacco) and bacterial coding sequence for
a ribonuclease (barnase gene from Bacillus
amyloliquefaciens) for production of
transgenic plant in B.napus. The product of
barnase(110 amino acid) gene is cytotoxic
killing the tapetul cells, thus preventing
pollen development result transgenic male
sterility.

35. Selected transgenes used for
production of male sterility
TRANSGENE SOURCE TRANSGENIC
PLANT
barnase Bacillus
amyloliquefaciens
Tobacco, B.napus
Rnase T1 Aspergillus oryzae Tobacco,oilseed,rape
Bcp1 Brassica campestris B.oleracea
rolC A.rhizogenes Tobacco,potato,

36. Fertility restoration
The same group of workers(Mariani et al.) in 1992,
used another gene construct later involving the
same anther specific promoter i.e.TA29 and the
barstar gene from B.amyloliquefaciens for
production of transgenic plants in B.napus . The
product of barstar gene(89 amino acids) is a
ribonuclease-inhibitor.
It forms a complex with ribonuclease and
neutralizes its cytotoxic properties.
The F1 plants expressed both genes(suppression of
cytotoxic ribonuclease)

38. Bar gene
Linkage to a selectable marker(bar gene linked to
barnase gene)
Use of a dominant selectable marker gene
(bar) that confers tolerance to glufosinate
herbicide
Treatment at an early stage with glufosinate
during female parent increase and hybrid seed
production phases eliminates 50% sensitive
plants
Results produce 100% male sterile population.

39. Selection by Herbicide
Application
pTA29-barnase : S (sterility)
p35S-PAT : H (herbicide resistance)
pTA29-barstar : R (restorer)
SH/-
SH/-
-/- SH/-
SH/-
-/- SH/-
-/-
SH/-
-/-
-/- SH/-
-/- SH/-
SH/-
-/- -/-
-/-
SH/-
SH/-
-/- -/-
-/- -/-
-/-
-/-
-/-
-/- -/-
A (SH/-) X B (-/-)
glufosi
nate X C (R/R)
Fertile F1 (SH/-, R/-)
Fertile F1 (-/-, R/-)

41. Features of commercial value
dominant genetic male sterility
system
• Efficient fertility restorer system
• Easy maintenance of male sterile lines
• Easy elimination of a male fertile plants
from male sterile lines
• Lack of adverse affects on other traits
• Stable male sterile phenotype over different
environments

42. Achievements
The transgenic hybrid rapeseed-mustard was
developed by multinational Proagro Seed
Company Ltd.,located at Gurgaon(now a part of
Belgium based Aventis crop science) large scale
field trials in year 2001-2002,this hybrid mustard
gave 25% yield advantage, but could not be
cleared by GEAC for commercial sowing at the
farmer fields in october 2002.
Another effort by Delhi University South Campus.

43. References:-
1. Gupta P.K., Langridge P, Mir R.R.(2010)Marker assisted
wheat Breeding:present status and future possibilities,
2.Article shared by Sudhadip Mondal MAS meaning
application step achievements,
3.Journal of Drug Metabolism&Toxicology(review),
4.Singh,B.D. Plant Breeding Principles and Methods,
5.Marker-assisted selection:an approach for precision plant
breeding in the twenty-first century-Bertrand C.Y Collard
and David J Mackill,
6.Singhal.N.C,Hybrid Seed Production,
7.Internet&SlideShare etc