Mapping and tagging of agriculturally important genes have been greatly facilitated by an array of molecular markers in crop plants. Marker-assisted selection (MAS) is gaining considerable importance as it would improve the efficiency of plant breeding through precise transfer of genomic regions of interest (foreground selection) and accelerating the recovery of the recurrent parent genome (background selection). MAS has been more widely employed for simply inherited traits than for polygenic traits, although there are a few success stories in improving quantitative traits through MAS

1. Subramanian. S.

2.  Prehistoric selection for visible phenotypes that facilitated harvest and
increased productivity led to the domestication of the first crop varieties
(Harlan, 1992)
 Conventional plant breeding is primarily based on phenotypic selection of
superior individuals among segregating progenies resulting from
hybridization
 Difficulties are often encountered during this process, primarily due to
genotype – environment interactions
 Testing procedures may be many times difficult, unreliable or expensive
due to the nature of the target traits (e.g. abiotic stresses) or the target
environment (Babu, 2004)

3. Marker Assisted Selection (MAS)
 A process whereby a marker (morphological, biochemical or one based
on DNA/RNA variation) is used for indirect selection of a genetic
determinant
 Used in plant and animal breeding
 Exploits the genetic linkage between markers and important crop traits
(Edwards et al., 1987; Paterson et al., 1988)
 MAS can be useful for traits that are difficult to measure, exhibit low
heritability, and/or are expressed late in development
 Sax (1923) reported association of a simply inherited genetic marker
with a quantitative trait in plants

4. Fig. Segregation of seed size associated with segregation for a seed coat colour marker

5.  Morphological - Presence or absence of awn, leaf sheath coloration,
height, grain colour, aroma of rice etc.
 Biochemical- A gene that encodes a protein that can be extracted and
observed; for example, isozymes and storage proteins.
 Cytological - The chromosomal banding produced by different stains;
for example, G banding.
 Biological - Different pathogen races or insect biotypes based on host
pathogen or host parasite interaction can be used as a marker
 Genetic or molecular - A unique (DNA sequence), occurring in
proximity to the gene or locus of interest, can be identified by a range
of molecular techniques

6. IMPORTANT PROPERTIES OF IDEAL MARKERS FOR MAS
 Easy recognition of all possible phenotypes (homo- and heterozygotes)
from all different alleles
 Demonstrates measurable differences in expression between trait
types and/or gene of interest alleles, early in the development of the
organism
 Has no effect on the trait of interest that varies depending on the allele
at the marker loci
 Low or null interaction among the markers allowing the use of many at
the same time in a segregating population
 Abundant in number and polymorphic (Babu et al., 2004)

7.  Highly polymorphic and simple inheritance (often co-domimant)
 Abundantly occur throughout the genome
 Easy and fast to detect, minimum pleiotropic effect
 Not environmentally regulated and are unaffected by the conditions in
which the plants are grown and are detectable in all stages of plant
growth (Francia et al., 2005)
 Used in diversity analysis, parentage detection, DNA fingerprinting, and
prediction of hybrid performance.
 Molecular markers are useful in indirect selection processes, enabling
manual selection of individuals for further propagation.

8.  RFLP is the most widely used hybridization-based molecular marker
 Technique is based on restriction enzymes that reveal a pattern difference
between DNA fragment sizes in individual organisms
(Semagn et al., 2006)

9.  RAPD (Random Amplified Polymorphic DNA)
 Single arbitrary oligonucleotide primer(10 bp)
 Low annealing temperature (generally 34 – 37 oC))
 Polymorphisms (band presence or absence) result from changes in DNA
sequence

10.  Combines the power of RFLP with the flexibility of PCR-based
technology by ligating primer recognition sequences (adaptors) to the
restricted DNA (Lynch and Walsh, 1998).
1. Digest genomic DNA with restriction enzymes
2. Ligate commercial adaptors (defined sequences) to both ends of the
fragments
3. Carry out PCR on the adaptor-ligated mixture, using primers that target
the adaptor, but that vary in the base(s) at the 3’ end of the primer

11. (Semagn et al., 2006)

14.  PCR-based marker with 18-25 bp primers
 SSR polymorphisms are based on number of repeat units, and are
hypervariable (have many alleles)
 SSRs have stable amplification and good repeatability
 SSRs are easy to run and automate

15.  Amplification of DNA segments present at an amplifiable distance in between
two identical microsatellite repeat regions in opposite direction
 The technique uses microsatellites as primers in a single primer PCR reaction
targeting multiple genomic loci to amplify mainly inter simple sequence repeats
of different sizes
 ISSRs use longer primers (15–30 mers)

17.  Simpler method compared to phenotypic screening
 Especially for traits with laborious screening
 May save time and resources
 Selection at seedling stage
 Important for traits such as grain quality
 Can select before transplanting in rice

18.  Increased reliability
 No environmental effects
 Can discriminate between homozygotes and heterozygotes and select
single plants
 More accurate and efficient selection of specific genotypes
 May lead to accelerated variety development
 More efficient use of resources
 Especially field trials

19.  The first step is to map the gene or quantitative trait locus (QTL) of
interest first by using different techniques and then use this
information for marker assisted selection.
• Ideally markers should be <5 cM from a gene or QTL
RELIABILITY FOR
SELECTION
Marker A
QTL Using marker A only:
5 cM
Marker A Marker B 1 – rA = ~95%
Using markers A and B:
QTL
5 cM 5 cM 1 - 2 rArB = ~99.5%
• Using a pair of flanking markers can greatly improve reliability but
increases time and cost

20. (Valentine and Howarth, 2000)

21.  Marker Assisted Backcrossing
 Foreground selection
 Background selection
• MAB has several advantages over conventional backcrossing:
– Effective selection of target loci
– Minimize linkage drag
– Accelerated recovery of recurrent parent

22. P1 x P2
• High yielding Desirable trait
Elite cultivar Donor
• Susceptible for 1 trait e.g. disease resistance
P1 x F1
• Called recurrent
parent (RP) P1 x BC1 Discard ~50% BC1
Visually select BC1 progeny that resemble RP
P1 x BC2 Repeat process until BC6
P1 x BC3
P1 x BC4
P1 x BC5
Recurrent parent genome recovered
P1 x BC6
Additional backcrosses may be required due to linkage drag
BC6F2

23.  Selection for target gene or QTL 1 2 3 4
Target
locus
 Useful for traits that are difficult to evaluate
 Also useful for recessive genes
TARGET LOCUS
SELECTION
FOREGROUND SELECTION
(Melchinger, 1990)

24. Concept of ‘linkage drag’
• Large amounts of donor chromosome remain even after many
backcrosses
• Undesirable due to other donor genes that negatively affect agronomic
performance
LINKED DONOR
GENES
TARGET c TARGET
LOCUS
LOCUS
Donor/F1 BC1 BC3 BC10
RECURRENT PARENT
CHROMOSOME
DONOR
CHROMOSOME

25. MARKERS CAN BE USED TO GREATLY MINIMIZE
THE AMOUNT OF DONOR CHROMOSOME
Conventional backcrossing
TARGET
GENE c c
F1 BC1 BC2 BC3 BC10 BC20
Marker-assisted backcrossing
TARGET
GENE
c
(Ribaut and Hoisington, 1998 )
F1 BC1 BC2

26.  Use flanking markers to select
1 2 3 4
recombinants between the target
locus and flanking marker
 Linkage drag is minimized
RECOMBINANT SELECTION
 Require large population sizes
 Depends on distance of flanking
markers from target locus

27.  Use unlinked markers to select against 1 2 3 4
donor
 Accelerates the recovery of the recurrent
parent genome
BACKGROUND
 Savings of 2, 3 or even 4 backcross SELECTION
generations may be possible

28. Theoretical proportion of the
recurrent parent genome is
given by the formula:
2n+1 - 1
2n+1
Where n = number of
backcrosses, assuming large
population sizes Percentage of RP genome after backcrossing
Although the average percentage of the recurrent parent is 75% for
BC1, some individual plants possess more or less RP than others

29.  Improvement of qualitative traits
 Resistance to soybean cyst nematode
 Development of QPM genotypes
 Marker-aided pyramiding of rice genes for bacterial blight and blast
resistance
 Quantitative trait improvement
 Improvement of heterotic performance in maize
 Germplasm enhancement in tomato
 Submergence tolerance in rice cultivars
(Babu et al.,2004)

30. EXAMPLES OF GENE–MARKER ASSOCIATIONS FOR
IMPORTANT TRAITS IN MAJOR CROPS
(Babu et al.,2004)

31. CONTD…

34.  Flash floods or short-term submergence regularly affect around 15
million hectares of rice (Oryza sativa L.) growing areas in South and
Southeast Asia
 An economic loss of up to one billion US dollars annually has been
estimated (Mackill et al., 1996)
 Submergence tolerant varieties have been developed but have not been
widely adopted
 Poor agronomic and quality characteristics
 Many popular and widely-grown rice varieties - “Mega varieties”

35. BR11 Bangladesh
CR1009 India
IR64 All Asia
KDML105 Thailand
Mahsuri India
MTU1010 India
RD6 Thailand
Samba Mahsuri India
Swarna India, Bangladesh

36.  A major QTL (Sub1) for submergence tolerance identified and fine
mapped on chromosome 9 in the submergence tolerant cultivar FR13A
(Xu and Mackill, 1996)
 Three related ethylene response factor(ERF)-like genes at this locus were
identified Sub1 A, B and C.
 Sub1A and Sub1C were up-regulated by submergence and ethylene
(Fukao et al., 2006)
 Sub1A was strongly induced in the tolerant cultivars in response to
submergence, whereas intolerant cultivars had weak or no induction of
the gene.
 Overexpression of Sub1A conferred submergence tolerance in an
intolerant japonica cultivar and down-regulation of Sub1C

37.  IR49830-7-1-2-2 (IR49830-7), one of the FR13A-derived
submergence-tolerant breeding lines (Mackill et al.,1993),was used as
the donor of Sub1
 The recipient variety was Swarna, a widely grown cultivar in India and
also in Bangladesh.
X
Swarna IR49830
Popular variety Sub1 donor
F1 X Swarna
BC1F1

38.  In the BC1F1 individual heterozygous plants at the Sub1 locus were
identified reducing the population size for further screening (foreground
selection)
 Homozygous for the recipient allele at one marker locus (RM219) distally
flanking the Sub1 locus (i.e. recombinant) were identifed “recombinant
selection” (Collard and Mackill, 2006)
 From these recombinant plants, individuals with the fewest number of
markers from the donor genome were selected (background selection)
 In the second BC generation the same strategy was followed for selection
of individual plants

39.  The conversion of the mega variety Swarna to submergence tolerant
within a two year time span for the BC2 and 2.5-year-time span for the
BC3
 Using rice genome sequence, polymorphic microsatellite markers were
designed from the same BAC clone (AP005907) harbouring the Sub1
genes (Xu et al., 2006).
 Initially the Sub1 locus was monitored by markers shown to be closely
linked with the gene
 Using tightly linked (RM464A, 0.7 cM ) and flanking(RM219 ,3.4 cM,
RM316) markers ensured efficient foreground and recombinant selection
 For flanking markers used for recombinant selection, about 5 Mb region
on each side of the Sub1 region was targeted.
 In advanced backcrosses and selfed generations, newly developed
markers from the Sub1 region were used for the target loci

40. Fourteen- day-old seedlings were submerged for 14 days(BC1F2, BC2F2 and
BC3F2). The survival of plants was scored 14 days after de-submergence
(calculated as a percentage) for confirmation of the presence of the Sub1 locus.

42. (Neeraja et al., 2007)

43. BC3F2 plant (No. 227-9-407) Selected BC2F2 plant No. 246-237
(Neeraja et al., 2007)

45. (Neeraja et al., 2007)

46.  The grain quality parameters in the Sub1 lines were on par with the
non-introgressed Swarna
 There was inhibition of brown furrows in the seed coat of Sub1
introgressed Swarna, yielding plants with straw colored hulls instead
of the golden hull color of Swarna.
 Easily distinguish the submergence- tolerant version of Swarna from
the original

48.  Normal maize - deficiency in two essential amino acids (lysine and
tryptophan) and high leucine–isoleucine ratio.
 Breakthrough came in the 1960s, discovery maize mutant opaque2
(Mertz et al., 1964)
 Encodes a transcriptional factor that regulates the expression of zein
genes and a gene encoding a ribosomal inactivating protein (Schmidt et
al., 1990)
 Reduces the level of 22-kD alpha-zeins while increasing the content of
non zein proteins particularly, EF-1 alpha, which is positively correlated
with lysine content in the endosperm (Habben et al., 1995).

49.  The protein quality of opaque2 maize is 43% higher than that of common
maize(Mertz, 1992).
 opaque2 maize not popular with farmers - reduced grain yield, soft
endosperm, chalky and dull kernel appearance and susceptibility to ear
rots and stored grain pests
 QPM is a genotype in which opaque2 gene has been incorporated along
with associated modifiers.
 A genetically improved, hard endosperm quality protein maize
 Contains twice the amount of lysine and tryptophan as compared to normal maize
endosperm

50.  Two additive modifier genes significantly influence the endosperm
modification in two populations viz., W64Ao2·pool 33 and pool 33·W22o2
(Lopes et al., 1995)
 One modifier locus was tightly linked with the gamma-zein coding
sequences near the centromere of chromosome 7, while the other near
the telomere of the 7L .
 Advantages of QPM
 Higher yield potential, assured seed purity, more uniform and stable
endosperm modification and less monitoring for ensuring protein quality
in seed production.

51.  The opaque2 gene is recessive and the modifiers are polygenic
 Each conventional backcross generation needs to be selfed to identify the
opaque2 recessive gene
 A minimum of six backcross generations are required to recover
satisfactory levels of recurrent parent genome
 In addition to maintaining the homozygous opaque2 gene, multiple
modifiers must be selected
 Rigorous biochemical tests to ensure enhanced lysine and tryptophan
levels in the selected materials in each breeding generation require
enormous labor, time and material resources

52.  A set of nine normal maize and five QPM inbred lines were analyzed
for polymorphism with opaque2 specific SSR markers.
 Based on the parental polymorphism analysis three normal inbred
lines viz., V25, CM212 and CM145 and two QPM donors
viz.,CML173 and CML176 were chosen for line conversion
 In this study V25 is converted using CML176 as QPM donor
 late maturing lines had tryptophan content ranging from 0.80 to
1.05% of total protein, where as normal inbred lines possessed
tryptophan content from 0.38 to 0.49% of endosperm protein

53. PARENTAL POLYMORPHISM ANALYSIS USING OPAQUE2
SPECIFIC SSR MARKER,UMC1066 BETWEEN NORMAL AND
QPM INBREDS
Lane M1: 1 kb marker, Lane M2: 100 bp marker, Lanes1: CM212,2:CM145, 3: V25, 4: V335, 5:
V338, 6: V340, 7: V345, 8:V346, 9: V348, 10: CML173, 11:CML176, 12: CML180, 13:CML184 and
14: CML189

54.  Three SSR markers, viz., phi057, phi112 and umc1066 located as internal
repetitive elements within the opaque2 gene, were used in initial
polymorphism analysis (suitability for foreground selection)
 A total of 200 SSR markers spanning all the bin locations in a maize SSR
consensus map (http://www.maizegdb.org) were selected for background
selection
 Of the 200 markers,77 were found to be polymorphic between V25 and
CML176

55. P1: V25, P2: CML176, Lanes 1–14: BC2F2 individuals

57. a V25, b CML176, c 50% or more opaque kernels, d less than 25% opaque
kernels, e completely modified kernels