FOODCROPS.VN. Molecular marker and its application to genome mapping and molecular breeding

1. Molecular Marker and Its Application to
Genome Mapping and Molecular Breeding
Binying Fu
Institute of Crop Sciences
The Chinese Academy of Agricultural Sciences
Beijing 100081, China
Nov-14-2012

2. Definition of Biological Marker
Biological markers can be anything that distinguishes
one individual or population from another
Can be phenotypic
Color:
Color: yellow vs white etc
Texture:
Texture: smooth vs rough etc
Shape:
Shape: round vs irregular etc
Can be a biochemical or genetic difference

3. Phenotypic Markers
http://cgil.uoguelph.ca/QTL/Fig2_3.htm
Weakness: unstable and limited number and polymorphism

4. Cytological Marker
Any distinct and heritable feature of chromosome structure that
can be used to follow (usually by microscopy) that chromosome
or chromosome region in breeding experiments.
Weakness: side effect and need special technique

5. Biochemical Marker-Isozyme and Protein
Weakness：limited number, spatio-temporal expressed
and need special technique such as Starch Gel with special staining

6. Characteristics of Ideal Markers
Polymorphism
Stability, no influences from the environment
Wide dispersion through the genome
Simplicity of observation
Low cost
Mendelian Heritability
Co-dominancy
Reproducibility
Portability between species

7. Molecular Markers
：
Define：A molecular selection technique of DNA signposts which allows
the identification of differences in the nucleotide sequences of the DNA in
different individuals. Or any genetic element ( locus, allele, DNA sequence or
chromosome feature) which can be readily detected by phenotype, cytological
or molecular techniques, and used to follow a chromosome or chromosomal
segment during genetic analysis. (Also DNA marker)
Agriculture: a tool which allows crop geneticists and breeders to locate on a
plant chromosome the genes for a trait of interest. It is considered more
efficient than conventional breeding as it has the potential to greatly reduce
development times and substitutes laboratory selection for much of the
fieldwork. MAS or MDB!
Molecular, or DNA-based, markers have been increasingly important in plant
breeding because of their features: Phenotypic stability (not affected by
environment), Useful polymorphism, Ease of development.

8. Where does the molecular marker come from?
Mutation = heritable (at the cell level) changes in DNA
sequence, regardless of whether the change produces any
detectable effect on a gene product. Mutations are the source
of new variation (polymorphism) upon which natural selection
works. Inherited mutations that are dispersed through a
population can become polymorphisms.
Polymorphism = presence in the same population of two or
more alternative forms of a DNA sequence, with the most
common allele having a frequency of 99% or less. Any two
individuals have a polymorphic difference every 1,000-10,000
base pairs.

9. Comparison of Mutation Frequencies
Class of Mutation Mechanism Frequency Example
Genome mutation Chromosome 10-2/cell division Aneuploidy
missegregation
Chromosome mutation Chromosome 6x10-4/cell division Translocation
rearrangement
Gene mutation Base-pair mutation 10-10/base pair/cell division Point mutation
10-5-10-6/locus/generation
humans have ~109 base pairs/haploid genome,
therefore each person will have 1-100 new mutations
1 in 20 people will have a new gene mutation

10. Types of Mutations (1)
Nucleotide Substitutions Altering Coding Sequence
• Missense mutations (amino acid substitution)
• Nonsense mutations (premature stop codon)

11. Types of Mutations (2)
Nucleotide Substitutions Altering Gene Expression
• RNA processing mutations (destruction of splice sites,
cap sites, poly A sites, or creation of cryptic sites)
• Regulatory mutations (promoter mutations)

12. Types of Mutations (3)
Deletions and Insertions (InDels)
• Insertion or deletion of small number of bases
If number of bases involved is not a multiple of 3,
causes frameshift
If number of bases involved is a multiple of 3,
causes loss or gain of codons
• Larger deletions, inversions, and duplications
Can create gene syndromes

13. Recombination-
Recombination-Generated
Duplications, Deletions, Insertions
Duplication
Insertion
Inversion

14. Brief Summary
The term MARKER is usually used for “LOCUS MARKER”.
Each gene has a particular place along the chromosome called
LOCUS. Due to mutations, genes can be modified in several forms
mutually exclusives called ALLELES (or allelic forms). All allelic
forms of a gene occur at the same locus on homologous
chromosomes. When allelic forms of one locus are identical, the
genotype is called HOMOZYGOTE (at this locus), whereas
different allelic forms constituted a HETEROZYGOTE. In
diploid organisms, the GENOTYPE is constituted by the two
allelic forms of the homologous chromosomes.
Thus, MOLECULAR MARKERS are all loci markers related
to DNA (sometimes biochemical or morphological markers
included).

15. Molecular Markers Classes
First Generation: 1980s
-Based on DNA-DNA hybridizations, such as RFLP.
Second Generation: 1990s
-Based on PCR: Using random primers: RAPD, DAF, ISSR
Using specific primers: SSR, SCAR, STS
-Based on PCR and restriction cutting: AFLP, CAPs
Third Generation: recently
-Based on DNA point mutations (SNP), can be detected by SSCP,
DASH, DNA chip, sequencing etc.

16. The Evolution of Markers
SNPs on Chips
AFLPs on microarrays (2000) Automation
SNPs
AFLPs on automated sequencers Complete Genomic Sequence
(1998)
Genomic Era High-throughput marker analysis
AFLPs (1996)
Hallmark event
Hallmark event
SCARs
cDNA Sequencing-cSSR
RAPDs (1990) SSCPs CAPs (1993)
Microsatellites (SSRs 1989)
Gene –Specific PCR
OLIGO-Scene PCR (1986)
Pre-PCR RFLPs (1980)
DNA-Hybridization-Scene Restriction (1968) and Southern Blotting (1975)
Allozymes (1960s)
Protein-Scene Gel Eletrophoresis (1950s)
Morphological Variants (Pre 1950s)

17. DNA Markers
Simple Sequence Repeats-SSR
Single Nucleotide Polymorphism-SNP
Single Feature Polymorphisms (SFPs)

18. Microsatellites
What are microsatellites?
Simple sequence repeats (SSRs) or microsatellites are tandemly repeated mono-,
di-, tri-, tetra-, penta-, and hexa-nucleotide motifs. SSR length polymorphisms are
caused by differences in the number of repeats
SSR loci are “individually amplified by PCR using pairs of oligonucleotide primers
specific to unique DNA sequences flanking the SSR sequence”.
Example Mononucleotide SSR (A)11
AAAAAAAAAAA
Dinucleotide SSR (GT)6
GTGTGTGTGTGT
Trinucleotide SSR (CTG)4
CTGCTGCTGCTG
Tetranucleotide SSR (ACTC)4
ACTCACTCACTCACTC

19. Microsatellites
Feature of SSR Marker
SSRs tend to be highly polymorphic.
SSRs are highly abundant and randomly dispersed throughout
most genomes.
Most SSR markers are co-dominant and locus specific.
Genotyping throughput is high and can be automated.

20. Microsatellites
Where are microsatellites found?
Majority are in non-coding region

21. Microsatellites
Repeat Motifs
AC repeats tend to be more abundant than other di-nucleotide repeat motifs in
animals
The most abundant di-nucleotide repeat motifs in plants, in descending order,
are AT, AG, and AC.
Because AT repeats self-anneal, AT-enrichment methods have not been
developed.
Typically, SSRs are developed for di-, tri-, and tetra-nucleotide repeat motifs. CA
and GA have been widely used in plants.
SSR markers have been developed for a variety of tri- and tetra-nucleotide
repeats in plants.
Tetra-nucleotide repeats have the potential to be very highly polymorphic.

22. SSR Containing Sequences from BAC-ends
BAC-
1 % in Corn 0.6 % in Soybean
21%
3%
2bp
3bp
4bp
5-6bp
76 %
SSR containing sequences in different BAC ends, there are 1% SSR in Corn,
0.6% in Soybean. Among these, most are dinucleotide repeats

23. Trinucleotide Repeats in Soy BAC-end Sequences
BAC-
AAT
AAC
5%
15% AAG
ATG
ATC
AGG
25% ACT
CCT
CGT
48% ACC
CTG
In the Soybean genome, most of the trinucleotide repeats
in BAC-end sequences are AAT repeats, one quarter of
them are AAC repeats.

24. Simple sequence repeats (SSRs). SSRs are particularly useful for developing genetic
markers. They are believed to vary through DNA replication slippage , and are
related to genetic instability . In Table 2, we describe SSR content for two sectors,
n 6 to 11 units and n >11 units, to emphasize that the number of SSRs dropped
substantially after 11 units. The SSR content for 93-11 was 1.7% of the genome,
lower than in the human, where it was 3%. The overwhelming majority of
rice SSRs were mononucleotides, primarily (A)n or (T)n, and with n is 6 to
11. In contrast, for the human, the greatest contributions came from dinucleotides.

25. From Nipponbare, Goff etal., 2002, Sciences.
The most prevalent SSR is tri-nucleotide; Most frequent 2-SSR is AG, 3-SSR is
CGG, 4-SSR is CGAT.

26. Microsatellites
How do microsatellites mutate?
Replication Slippage
Unequal crossing-over during meiosis

27. Replication Slippage
When the DNA replicates, the polymerase loses track of its place, and either leaves
out repeat units or adds too many repeat units.
“Polymerase slippage” or
“slipped-strand mispairing.”
A commonly observed
replication error is the
replication slippage, which
occurs at the repetitive
sequences when the new strand
mispairs with the template
strand. The microsatellite
polymorphism is mainly caused
by the replication slippage. If
the mutation occurs in a coding
region, it could produce
abnormal proteins, leading to
diseases.

28. Unequal crossing-over during meiosis
This is thought to explain more drastic changes in numbers of repeats. In this
diagram, chromosome A obtained too many repeats during crossing-over, and
chromosome B obtained too few repeats.

29. Microsatellites
Why do microsatellites exist?
"junk" DNA, and the variation is mostly neutral
a necessary source of genetic variation
regulate gene expression and protein function
Moxon, E. R., Wills, C. 1999. "DNA microsatellites: Agents of Evolution?" Scientific
American. Jan., pp. 72-77.
Kashi, Y. and M. Soller. 1999. "Functional Roles of Microsatellites and Minisatellites."
In: Microsatellites: Evolution and Applications. Edited by Goldstein and Schlotterer.
Oxford University Press.

30. Models of Microsatellite Mutation (1)
1. Stepwise Mutation Model (SMM)
This model holds that when microsatellites mutate, they only gain
or lose one repeat. This implies that two alleles that differ by one
repeat are more closely related (have a more recent common
ancestor) than alleles that differ by many repeats. In other words,
size matters when doing statistical tests of population
substructuring. The SMM is generally the preferred model when
calculating relatedness between individuals and population
substructuring, although there is the problem of homoplasy.

31. Models of Microsatellite Mutation(2)
2. Infinite Alleles Model (IAM)
Each mutation can create any new allele randomly. A 15-repeat allele
could be just as closely related to a 10-repeat allele as a 11-repeat allele.
All that matters is that they are different alleles. In other words, size isn't
important.
A 15-repeat allele could be just as closely related to a 10-repeat allele
as a 11-repeat allele.
15-repeat 11-repeat 10-repeat
8-repeat

32. Conventional Developmental Steps of SSR Markers
Genomic DNA PCR test using
diverse genotypes
Specific SSR
SSR
DNA Library
SSR probes
Positive Clones Sequencing of positive
DNA clones

33. Four Assay Methods
1. The customary method for SSR genotyping is denaturing polyacrylamide
gel electrophoresis using silver-stained PCR products. These assays can
usually distinguish alleles differing by 4 bp and may distinguish alleles
differing by 2 bp.
2. Semi-automated SSR genotyping can be performed by assaying
fluorescently labelled PCR products for length variants on an automated
DNA sequencer. Several instruments have been developed (e.g., Applied
Biosystems and Li-Cor). Alleles differing by 2 to 4 bp can usually be
distinguished.
3. SSR length polymorphisms can be assayed using non-denaturing high
performance liquid chromatography (Marino et al. 1998). Alleles differing
by 2 to 4 bp can usually be distinguished.
4. SSR alleles differing by several repeat units can often be distinguished on
agarose gels.

34. SSRs assayed on polyacrylamide gels typically show a characteristic
“stuttering”. Stutter bands are artifacts produced by DNA polymerase slippage.
Typically, the most prominent stutter bands are +1 and - 1 repeat (e.g., +
or - 2 bp for a di-nucleotide repeat), and, if visible, the next most prominent
stutter bands are +2 and -2 repeats.

35. Weaknesses
The development of SSRs is labor intensive（NO in
sequence-based SSR development) .
SSR marker development costs are very high.
SSR markers are taxa specific.
Start-up costs are high for automated SSR assay methods.
Developing PCR multiplexes is difficult and expensive. Some
markers may not multiplex.

36. Single Nucleotide Polymorphisms
SNP is the molecular basis for most phenotypic differences between
individuals
SNP is the most common genetic variations.
SNPs are highly abundant, stable and distributed throughout the genome
SNP assay is amenable to automation and high throughput.
SNP is biallelic.
GATTTAGATCGCGATAGAG
GATTTAGATCTCGATAGAG

37. Single Nucleotide Polymorphisms
SNPs in intergenic regions may …
Have no genetic effect …
Affect genetic regulatory signals …
Interfere with RNA splice sites …
SNPs in Coding regions (cSNP) may …
Synonymously change the codon of an amino acid,
which may have no further effect, or may influence
e.g. codon bias.
non-synonymously alter the encoded amino acid
(nsSNP) by a conservative exchange, or non-
conservative (radical) mutation.

38. SNP Variation in Maize and Soybean
%
40
35
30 CT
GA
25
GC
20 AC
15 GT
10 AT
Del
5
0
Maize Soy

39. Frequency of Candidate SNPs from
Different Sources in Maize and Soy
Region Maize Soy
EST (5’end) 1/1.5kb 1/1.9kb
Genomic 1/640bp 1/750bp
3’UTR 1/441bp 1/416bp

40. SNP/250bp SNP/268bp SNP/236bp SNP/243bp
16.5% 23.5% 14.3% 23.3%
18.2% 21.8% 16.3% 22.4%
65.3% 54.7% 69.4% 54.3%

41. SNPs Discovery
1. Sequence databases searches
2. Target specific SNP discovery and development
-Conformation-based mutation scanning
-Direct DNA sequencing

42. Identify SNP from Sequence Databases

43. Identification of Target Specific SNPs
Steps:
1. Amplify the genes of interests with PCR
2. Scan for mutation with various methods
-Conformation-based mutation scanning
- Single -strand conformation polymorphism analysis
- Gel electrophoresis
- Chemical and enzymatic mismatch cleavage detection
- Denaturing gradient gel electrophoresis
- Denaturing HPLC
3. Sequence positive PCR products
-Sequence multiple individuals
-Sequence heterozygotes
4. Align sequences from different sources to find SNPs

44. Technologies for Detecting Known SNPs
Gel-Based Methods
-PCR-restriction fragment length polymorphism analysis
-PCR-based allelic specific amplification
-Oligonucleotide ligation assay genotyping
-Minisequencing(10~20base)
Non-Gel-Based High Through Genotyping Technologies
-Solution hybridization using fluorescence dyes
-Allelic specific ligation
-Allelic specific nucleotide incorporation
1. High resolution separation
2. Chemical color reaction
-DNA microarray genotyping

45. （ ）
Oligo Ligation Assay（OLA）
Two allele-specific oligonucleotide probes (one specific for the wild-type allele and the
other specific for the variant allele) and a fluorescent common probe are used in each
assay. The 3' ends of the allele-specific probes are immediately adjacent to the 5' end of
the common probe. In the presence of thermally stable DNA ligase, ligation of the
fluorescently labeled probe to the allele-specific probe(s) occurs only when there is a
perfect match between the variant or the wild-type probe and the PCR product template.
These ligation products are then separated by electrophoresis, which permits the
recognition of the wild-type genotypes, the variants, the heterozygotes, and the unligated
probes.

47. Allele-Specific Codominant PCR Strategy
Figure. Schematic representation of the allele-
specific codominant PCR strategy.
Oligonucleotide primers with 3' nucleotides that
correspond to an SNP site are used to
preferentially amplify specific alleles.
A, Primer P1 forms a perfect match with allele
1 but forms a mismatch at the 3' terminus with the
DNA sequence of allele 2. Primer P2 similarly
forms a perfect match with allele 2 and a 3'
terminus mismatch with allele 1.
B, Schematic of agarose gel analysis showing the
expected outcome for the amplification of
organisms homozygous and heterozygous for
both alleles using primers P1 and P2. P1, Primer 1;
P2, primer 2; A1, allele 1; A2, allele 2.
Eliana Drenkard et al. 2000 Plant Physiol 124: 1483-1492

48. SNP Detection Allele Specific Oligohybridization
Principle: A 1 bp mismatch in the center of a 15mer will change
the T m by 5 - 10 degrees, therefore a SNP in the middle of a
15mer can be genotyped using paired ASOs.
PCR amplify target gene (different individual) in 96 well format
Prepare dot-blot on nylon filter
Hybridize to allele-specific 15mer and detect the signal
Wash at stringency temperature
Repeat for alternate allele and other SNPs

49. Single-Strand Conformation Polymorphism Analysis
Single-stranded DNAs are generated by denaturation of the PCR
products and separated on a nondenaturing polyacrylamide gel. A
fragment with a single-base modification generally forms a different
conformer and migrates differently when compared with wild-type
DNA.
Size <200bp,
Accuracy: 70%-95%
Size >400bp,
Accuracy: 50%
1% false positive

51. SNP Genotyping Using Oligo Chip
T genotype
Oligo Chip: a set of 15- C genotype
nucleotide probes, which consist
of different sets of probes
overlapped each other, 14
nucleotides were overlapped,
among the four probes in one set,
the sequences are almost the
same except one A/G/C/T

54. http://www.ricesnp.org/index.aspx##

57. Direct Sequencing - New Sequencing Technology
Pyrosequencing technology offers rapid and accurate genotyping, allowing for
dependable SNP and mutation analysis. This technology utilizes an enzyme
cascade system that results in the production of measurable light whenever a
nucleotide forms a base pair with its complimentary base in a DNA template
strand.
Solexa/Illumina Sequencing
Munroe & Harris, (2010) Third-generation sequencing fireworks at Marco Island.
Nature Biotechnology 28: 426–428.

58. Use of SNPs
1. Markers for linkage mapping-Discover SNPs contribute
to agronomic traits
2. Trace origin of introgression
3. Markers for association studies (Linkage Disequilibrium)
4. Markers for population genetic analysis

59. Further Reading:
McNally et al., 2009. Genomewide SNP variation reveals relationships
among landraces and modern varieties of rice. PNAS 106(30):12273-8.
Jones et al., 2009. Development of single nucleotide polymorphism
(SNP) markers for use in commercial maize (Zea mays L.) germplasm.
Mol Breeding 24 (2):165-176.
Varshney et al., 2007. Single nucleotide polymorphisms in rye (Secale
cereale L.): discovery, frequency, and applications for genome
mapping and diversity studies. TAG 114 (6): 1105-1116.
Wu et al., 2010. SNP discovery by high-throughput sequencing in
soybean. BMC Genomics 11:469.

60. Single Feature Polymorphisms (SFPs)
SFPs are a consequence either of insertions/deletion (InDel)
polymorphisms or
represent multiple SNPs across the complementary sequences.
SFPs identified through hybridization of genomic DNA to whole-genome
tiling arrays (i.e., Affymetrix Genechips) or home-made microarray.
References
Yeast: Wodicka, L., H. Dong, M. Mittmann, M.H. Ho, and D.J. Lockhart.
1997. Nat Biotechnol 15: 1359-1367.
Arabidopsis: Borevitz, J.O., D. Liang, D. Plouffe, H.S. Chang, T. Zhu, D.
Weigel, C.C. Berry, E. Winzeler, and J. Chory. 2003. Genome Res 13:
513-523.

61. Further reading:
Kumar et al., 2007. Single Feature Polymorphism Discovery in Rice. Plos ONE, 2(3): e284

62. Principle of Microarray-based
genotyping of Single
Feature Polymorphisms
(SFPs) by Oligo Chip.

63. A genotype B genotype A/B genotype

64. http://cropwiki.irri.org/gc
p/images/6/61/Single_Fe
ature_Polymorphism.pdf

66. Classification of DNA Markers
A. Mutation at restriction sites (RFLP, CAPS, AFLP) or PCR
primer sites (RAPD, DAF, AP-PCR, SSR, ISSR)
B. Insertion or deletion between restriction sites (RFLP, CAPS,
AFLP) or PCR primer sites (RAPD, DAF, AP-PCR, SSR, ISSR)
C. Changes in the number of repeat unit between restriction sites
or PCR primer sites: SSR, VNTR, ISSR
D. Mutations at single nucleotides: SNP

67. Summary of Common Molecular Markers
Single Locus Detection
RFLP (restriction fragment length polymorphism) Hybridization
CAPS (cleaved amplified polymorphic sequences) PCR
SSLP (simple sequence length polymorphism)
---- VNTR (variable number of tandem repeat) Hybridization or PCR
---- SSR/STR (simple sequence repeats/tandem repeats) PCR
SCAR (Sequence characterized amplified region) PCR
SNP (Single nucleotide polymorphism)
---- DASH (dynamic allele-specific hybridization) Hybridization
---- SSCP (single strand conformation polymorphism) Conformation

68. Summary of Common Molecular Markers
Multiple Loci Detection
AFLP (amplified fragment length polymorphism) PCR
RAPD (random amplified polymorphic DNA) PCR
AP-PCR (arbitrarily primed-PCR) PCR
DAF (DNA amplification fingerprinting) PCR
SSLP (simple sequence length polymorphism) PCR
when multiple pairs of primers were used
ISSR (inter-simple sequence repeat) PCR
SNP (Single nucleotide polymorphism)
-- SSCP (single strand conformation polymorphism) Conformation
when used to scan for randomly located SNPs

69. Conclusion
All molecular markers are not equal. None is
ideal. Some are better for some purposes than
others. However, all are generally preferable to
morphological markers for mapping and marker
assisted selection.

70. Thanks For Your Attention!