Genomics
• Structural, functional
• Genome, Transcriptome, Proteome,
Metabolome, Interactome
www.the-scientist.com
Genetics vs. Genomics
“What's
the Difference?
Well, as a rule, genetics is the study of single genes in
isolation. Genomics is the study of all the genes in the
genome and the interactions among them and their
environment(s).
Analogy 1
If genomics is like a garden, genetics is like a single plant. If
the plant isn’t flowering, you could study the plant itself
(genetics) or look at the surroundings to see if it is too
crowded or shady (genomics) – both approaches are
probably needed to find out how to make your plant
blossom.”
http://www.genomebc.ca/education/articles/genomics-vs-genetics/
Structural genomics for plant breeders and
applied geneticists = molecular markers
•
•
•
•
How many genes determine important traits?
Where these genes are located?
How do the genes interact?
What is the role of the environment in the phenotype?
• Molecular breeding: Gene discovery, characterization, and
selection using molecular tools
• Molecular markers are a key implement in the molecular
breeding toolkit
Markers are based on polymorphisms
Amplified fragment length polymorphism
Restriction fragment length polymorphism
Single nucleotide polymorphism
The polymorphisms becomes the alleles at
marker loci
The marker locus is not necessarily a gene: the
polymorphism may be in the dark matter
in a UTR, in an intron, or in an exon
Non-coding regions may be more polymorphic
restriction fragment length polymorphism
Parent 1
Parent 2
F1
Parent 1
A allele
AA
Parent 2
B allele
BB
F1
Codominant
AB
Why markers?
A way of dealing with the
• Large number of genes per genome
• Huge genome size
• Technical challenges and cost of whole genome sequencing
The search for DNA polymorphisms was not driven by a desire
to complicate things, but rather by the low number of naked
eye polymorphisms (NEPs)
Markers may be linked to target genes
Markers in target genes are perfect markers
What is a perfect marker for a gene deletion?
Why DNA markers?
• Polymorphisms can be visualized at the metabolome,
proteome, or transcriptome level but for a number of
reasons (both technical and biological) DNA-level
polymorphisms are currently the most targeted
• Regardless of whether it is a “perfect” or a “linked” DNA
marker, there are two key considerations that need to
be addressed in order for the researcher/user to visualize
the underlying genetic polymorphism
1. Finding and understanding the genetic basis of the
DNA-level polymorphism, which may be as small as a
single nucleotide polymorphism (SNP) or as large as an
insertion/deletion (INDEL) of thousands of nucleotides
2. Detecting the polymorphism via a specific assay or
"platform". The same DNA polymorphism may be amenable
to different detection assays
The catalog of markers: polymorphisms vs.
assays
An ever-increasing number of technology platforms have been,
and are being, developed to deal with these two key
considerations
These platforms lead to a bewildering array of acronyms for
different types of molecular markers. To add to the complexity,
the same type of marker may be assayed on a variety of
platforms
Amplified Fragment
Length
Polymorphism
AFLP A combination of restriction enzyme and oligonucleotides as
adapters and amplification agents that can generate large numbers of
data points from a single reaction and thus reveal amplified
fragments of different sizes in two or more individuals. A defined
assay, upgraded with advances in technology.
Genetic basis of polymorphisms not known beforehand. Can clone
and sequence amplicons to establish basis of the polymorphism
Expressed Sequence EST
Partial gene sequence data of a cDNA clone, which provide a
Tag
sequence tag for a gene. Since ESTs are DNA sequences, they are
amenable to a variety of assays.
Randomly Amplified RAPD A PCR primer that randomly amplifies different size products based
Polymorphic DNA
on DNA templates of two or more individuals. An easy, anonymous
marker assay to apply, since no prior information required.
Serious limitations due to data quality.
Restriction Fragment
Length
Polymorphism
Single Nucleotide
Polymorphism
Simple Sequence
Repeat
(Microsatellite)
RFLP Labeled DNA probe and restriction enzyme combination that
reveal DNA fragments of different sizes in two or more
individuals.
An "old school" technology that still has its place.
Requires a probe sequence, but basis of polymorphism not known
beforehand.
SNP A single site in a nucleotide sequence that contains two to four
allelic variations within a population at relatively high frequencies.
Since ESTs are based on sequence polymorphisms, they are
amenable to a variety of assays. Known SNPs may be targeted for
assay (e.g. Illumina Bead Station) or unknown SNPs discovered by
the assay (e.g. Floragenex RADs).
SSR PCR primers based on conserved regions flanking a region of short
tandem repeats amplify the repeat region, which is variable
between two or more individuals.
Requires up front discovery.
A principal advantage is that SSRs are multi-allelic; they are
amenable to a range of assay platforms.
cultivar
Tamalpais
Calisib
OBADV10-13
BISON 1H
G387
G395
Dicktoo
Alba
MC0181-11
OR911
12220-4
12007-4
Karma
Tetonia
09OR-59
BISON 4H
G388
G396
Hoody
OR101
MC0181-31
Willamette Pearl
X1_2847485
G
G
G
A
G
G
A
A
A
G
A
A
A
A
G
A
G
G
A
A
A
G
X1_299163
G
G
A
G
A
A
A
G
G
A
G
G
A
G
A
G
A
A
A
G
G
G
X1_3928513
A
A
G
A
A
A
A
A
A
A
A
G
G
A
G
A
A
A
G
A
A
G
X1_53811950
A
A
A
A
A
A
A
A
A
A
A
G
A
A
A
G
A
A
G
A
A
G
X1_6517602
G
G
G
A
A
A
G
G
G
G
G
G
G
G
G
A
A
A
G
G
G
G
X1_8867459
G
G
G
A
G
G
G
G
G
G
G
G
G
A
G
A
G
G
G
G
G
G
X1_9541377
A
A
A
A
A
A
A
A
G
A
G
GA
A
A
A
A
A
A
A
A
G
A
X1_ABC14990
A
A
G
A
A
A
G
G
A
A
A
G
A
A
G
A
A
A
G
G
A
G
X1_ABC15617
G
G
C
C
C
C
C
C
C
C
C
G
C
C
C
C
C
C
C
C
C
C
X2_12_10035
A
A
A
A
A
A
A
A
A
A
A
A
G
A
A
A
A
A
A
A
A
A
X2_12_30336
A
A
A
A
G
G
G
G
A
A
G
G
G
A
G
G
G
G
A
AG
A
G
X2_12_30872
G
G
A
G
A
A
A
A
A
A
A
G
A
G
G
G
A
A
A
A
A
A
Marker Identity vs. anonymity:
The optimum marker is one based on known DNA
sequence variations, since new and more efficient assays
can be designed for the same polymorphism
In many species, molecular toolkits are still poorly stocked:
therefore, there is still a place for "anonymous" marker
technologies
cultivar
Tamalpais
Calisib
OBADV10-13
BISON 1H
G387
G395
Dicktoo
Alba
MC0181-11
OR911
12220-4
12007-4
Karma
Tetonia
09OR-59
BISON 4H
G388
G396
Hoody
OR101
MC0181-31
Willamette Pearl
X1_2847485
G
G
G
A
G
G
A
A
A
G
A
A
A
A
G
A
G
G
A
A
A
G
X1_299163
G
G
A
G
A
A
A
G
G
A
G
G
A
G
A
G
A
A
A
G
G
G
X1_3928513
A
A
G
A
A
A
A
A
A
A
A
G
G
A
G
A
A
A
G
A
A
G
X1_53811950
A
A
A
A
A
A
A
A
A
A
A
G
A
A
A
G
A
A
G
A
A
G
X1_6517602
G
G
G
A
A
A
G
G
G
G
G
G
G
G
G
A
A
A
G
G
G
G
X1_8867459
G
G
G
A
G
G
G
G
G
G
G
G
G
A
G
A
G
G
G
G
G
G
X1_9541377
A
A
A
A
A
A
A
A
G
A
G
GA
A
A
A
A
A
A
A
A
G
A
X1_ABC14990
A
A
G
A
A
A
G
G
A
A
A
G
A
A
G
A
A
A
G
G
A
G
X1_ABC15617
G
G
C
C
C
C
C
C
C
C
C
G
C
C
C
C
C
C
C
C
C
C
X2_12_10035
A
A
A
A
A
A
A
A
A
A
A
A
G
A
A
A
A
A
A
A
A
A
X2_12_30336
A
A
A
A
G
G
G
G
A
A
G
G
G
A
G
G
G
G
A
AG
A
G
X2_12_30872
G
G
A
G
A
A
A
A
A
A
A
G
A
G
G
G
A
A
A
A
A
A
Simple sequence repeat in hazelnut
Note the difference in repeat length AND the consistent flanking
sequence
One way of visualizing SSRs
Single Nucleotide Polymorphisms: A SNP is a single nucleotide
polymorphism. Since the DNA sequence in known, the SNP data
will be useful forever: the way the SNP is assayed will change
as technology advances.
RADs: Restriction amplified digest. Based on sequencing of
digestion products
Utility of molecular markers: make maps!
Why make maps?
Establish evolutionary relationships: homoeology, synteny and
orthology
Homoeology: Refers to chromosomes, or chromosome
segments, and which are similar in terms of the order and
function of the genetic loci. Homoeologous chromosomes may
occur within a single allopolyploid individual (e.g. the A,B, and
D, genomes in wheat), or they may be found in related
species (e.g. the 1A, 1, B, 1D series and wheat and the 1H of
barley)
Orthology: Refers to genes in different species which are so
similar in sequence that they are assumed to have originated
from a single ancestral gene.
Synteny: Refers to genetic loci that are linked on
the same chromosome.
Why make maps?
Determine if trait associations are due to linkage or pleiotropy
Finding genes determining qualitative and quantitative
phenotypes.
Map-based cloning (Vrs1)
Putting it all together…….
Mapping MFT1 in the OWBs. A colleague is interested in a
gene called MFT1 and wants to know (i) is there a gene like
MFT1 in barley, (ii) if there is, how much allelic variation is
there in barley, and (iii) assuming there is an MFT1-like gene
in barley, where is it located? Based on orthology and
molecular biology tools, we answered these questions.
Thanks to Tanya Filichkin (OSU Barley Project) for the
detective work.
Restriction site polymorphism approach to differentiate between OWBD
and OWBR and map MFT1
CLUSTAL W (1.81) Multiple Sequence Alignments for OWBD.MFT, OWBR.MFT,
Steptoe.MFT and Morex.MFT
Sequence
Sequence
Sequence
Sequence
Primers
Polymorphic RS
Microsatellite
Non-polymorphic RS
INDEL
SNP
1:
2:
3:
4:
OWBD.MFT.final
OWBR.MFT.final
Steptoe.MFT.final
Morex.MFT.final
491
481
482
481
bp
bp
bp
bp
MFT.F2------
OWBR.MFT.final
GTGCTGGTGCTGTTCGAGCAGAAGACGCGCTTCCCTACGTGGACGCGGCGTCGCCGGAGG
Morex.MFT.final
GTGCTGGTGCTGTTCGAGCAGAAGACGCGCTTCCCTACGTGGACGCGGCGTCGCCGGAGG
OWBD.MFT.final
GTGCTGGTGCTGTTCGAGCAGAAGACGCGCTTCCCTACGTGGACGCGGCGTCGCCGGAGG
Steptoe.MFT.final
GTGCTGGTGCTGTTCGAGCAGAAGACGCGCTTCCCTACGTGGACGCGGCGTCGCCGGAGG
************************************************************
OWBR.MFT.final
Morex.MFT.final
OWBD.MFT.final
Steptoe.MFT.final
ACCGCGCCTACTTCAACACCCGCGCCTTCGCCGGCAACCACGAGCTCGGCCTCCCCGTCG
ACCGCGCCTACTTCAACACCCGCGCCTTCGCCGGCAACCACGAGCTCGGCCTCCCCGTCG
ACCGCGCCTACTTCAACACCCGCGCCTTCGCCGGCAACCACGAGCTCGGCCTCCCCGTCG
ACCGCGCCTACTTCAACACCCGCGCCTTCGCCGGCAACCACGAGCTCGGCCTCCCCGTCG
************************************************************
OWBR.MFT.final
Morex.MFT.final
OWBD.MFT.final
Steptoe.MFT.final
CCGTCGTCTACTTCAACTCCCAGAAGGAGCCCTCCGGACACCGCCGCCGCTGAGGCATGC
CCGTCGTCTACTTCAACTCCCAGAAGGAGCCCTCCGGACACCGCCGCCGCTGAGGCATGC
CCGTCGTCTACTTCAACTCCCAGAAGGAGCCCTCCGGACACCGCCGCCGCTGAGGCATGC
CCGTCGTCTACTTCAACTCCCAGAAGGAGCCCTCCGGACACCGCCGCCGCTGAGGCATGC
************************************************************
MseI
AATTAATCACGCTGCATGCCAAACAGAGCGATGCAGACTTATCCCGGTGAAGAATAATAG
AATTAATCACGCTGCATGCCAAACAGAGCGATGCAGACTTATCCCGGTGAAGAATAATAG
ATGCAATCACGCTGCATGCCAAACAGAGCGATGCAGACTTATCCCGGTGAAGAATAATAG
ATGCAATCACGCTGCATGCCAAACAGAGCGATGCAGACTTATCCCGGTGAAGAATAATAG
*
********************************************************
MseI
CCGAGCTTCCATATATATATAT----GCATGCCAGCAACTGCTATGTGTTGCTTTTAATT
CCGAGCTTCCATATATATATAT----GCATGCCAGCAACTGCTATGTGTTGCTTTTAATT
CCGAGCTTCCATATATATATATATATGCATGCCAGCAACTGCTATGTGTTGCTTTTAATT
CCGAGCTTCCATATATATATAT----GCATGCCAGCAACTGCTATGTGTTGCTTTTAATT
**********************
**********************************
OWBR.MFT.final
Morex.MFT.final
OWBD.MFT.final
Steptoe.MFT.final
OWBR.MFT.final
Morex.MFT.final
OWBD.MFT.final
Steptoe.MFT.final
OWBR.MFT.final
Morex.MFT.final
OWBD.MFT.final
Steptoe.MFT.final
ACTGTTGTGTAATCGCACTGTGCTTGTGTATGCATGCATGAGATGAG-----ATGCATGG
ACTGTTGTGTAATCGCACTGTGCTTGTGTATGCATGCATGAGATGAG-----ATGCATGG
ACTGTTGTGTAATCGCACTGTGCTTGTGTATGCATGCATGAGATGAGATGAGATGCATGG
ACTGTTGTGTAATCGCACTGTGCTTGTGTATGCATGCATGAGATGAG-----ATGCATGG
***********************************************
********
OWBR.MFT.final
Morex.MFT.final
OWBD.MFT.final
Steptoe.MFT.final
GAACCGATATATTTTGGGGTTTTGCAGCATAAGTCTGCATGCCTCCGCGCGACGGG-TGT
GAACCGATATATTTTGGGGTTTTGCAGCATAAGTCTGCATGCCTCCGCGCGACGGG-TGT
GAACCGATATATTTTGGGGTTTTGCAGCACAAGTCTGCCTGCCTCCGCGCGACGGGGTGT
GAACCGATATATTTTGGGGTTTTGCAGCACAAGTCTGCCTGCCTCCGCGCGACGGGGTGT
***************************** ******** ***************** ***
OWBR.MFT.final
Morex.MFT.final
OWBD.MFT.final
Steptoe.MFT.final
TTCCCGCCGCGCCTTCTTGTAATAATAGTAGAATGCAGCCGCGCTTAGTAATTTCTTCTT
TTCCCGCCGCGCCTTCTTGTAATAATAGTAGAATGCAGCCGCGCTTAGTAATTTCTTCTT
TTTCCGGCGCGCCTTCTTGTAATAATAGTAGAATGCAGCCGCGCCTAGTAATTTCTTCTT
TTTCCGGCGCGCCTTCTTGTAATAATAGTAGAATGCAGCCGCGCTTAGTAATTTCTTCTT
** *** ************************************* ***************
OWBR.MFT.final
Morex.MFT.final
OWBD.MFT.final
Steptoe.MFT.final
GTTTGGTCCTC
GTTTGGTCCTC
GTTTGGTCCTC
GTTTGGTCCTC
***********
------------MFT.R1
MseI restriction site:
5’…T^TAA…3’
3’…AAT^T…5’
GATCCACCGGTACGTGCT
MFTF2.F2--------
OWBD.MFT.final
OWBR.MFT.final
-----------------------------GTGCTGGTGCTGTTCGAGCAGAAGACGCGCT
-----------------------------GTGCTGGTGCTGTTCGAGCAGAAGACGCGCT
*******************************
OWBD.MFT.final
OWBR.MFT.final
TCCC-TACGTGGACGCGGCGTCGCCGGAGGACCGCGCCTACTTCAACACCCGCGCCTTCG
TCCC-TACGTGGACGCGGCGTCGCCGGAGGACCGCGCCTACTTCAACACCCGCGCCTTCG
**** *******************************************************
OWBD.MFT.final
OWBR.MFT.final
CCGGCAACCACGAGCTCGGCCTCCCCGTCGCCGTCGTCTACTTCAACTCCCAGAAGGAGC
CCGGCAACCACGAGCTCGGCCTCCCCGTCGCCGTCGTCTACTTCAACTCCCAGAAGGAGC
************************************************************
OWBD.MFT.final
OWBR.MFT.final
CCTCCGGACACCGCCGCCGCTGAGGCATGCATGCAATCACGCTGCATGCCAAACAGAGCG
CCTCCGGACACCGCCGCCGCTGAGGCATGCAATTAATCACGCTGCATGCCAAACAGAGCG
*******************************
**************************
MseI
OWBD.MFT.final
OWBR.MFT.final
ATGCAGACTTATCCCGGTGAAGAATAATAGCCGAGCTTCCATATATATATATATATGCAT
ATGCAGACTTATCCCGGTGAAGAATAATAGCCGAGCTTCCATATATATATAT----GCAT
****************************************************
****
OWBD.MFT.final
OWBR.MFT.final
GCCAGCAACTGCTATGTGTTGCTTTTAATTACTGTTGTGTAATCGCACTGTGCTTGTGTA
GCCAGCAACTGCTATGTGTTGCTTTTAATTACTGTTGTGTAATCGCACTGTGCTTGTGTA
************************************************************
OWBD.MFT.final
OWBR.MFT.final
TGCATGCATGAGATGAGATGAGATGCATGGGAACCGATATATTTTGGGGTTTTGCAGCAC
TGCATGCATGAGATGAGATG-----CATGGGAACCGATATATTTTGGGGTTTTGCAGCAT
********************
**********************************
OWBD.MFT.final
OWBR.MFT.final
AAGTCTGCCTGCCTCCGCGCGACGGGGTGTTTTCCGGCGCGCCTTCTTGTAATAATAGTA
AAGTCTGCATGCCTCCGCGCGACGGG-TGTTTCCCGCCGCGCCTTCTTGTAATAATAGTA
******** ***************** ***** *** ***********************
OWBD.MFT.final
OWBR.MFT.final
GAATGCAGCCGCGCCTAGTAATTTCTTCTTGTTTGGTCCTC-----------GAATGCAGCCGCGCTTAGTAATTTCTTCTTGTTTGGTCCTC-----------************** **************************
TGGTCCTCAATTATCATGCA
----------MFT.R1
MseI
PCR protocol with the following primers designed for MFT gene:
LEFT PRIMER MFT.F2
RIGHT PRIMER MFT.R1
17
527
18
20
59.50
57.48
61.11
40.00
6.00
4.00
2.00 GATCCACCGGTACGTGCT
1.00 TGCATGATAATTGAGGACCA
TGGTCCTCAATTATCATGCA
5’TGCATGATAATTGAGGACCA3’
(reverse primer sequence)
PCR protocol:
5’TGGTCCTCAATTATCATGCA3’
(sense)
Ingredients
Stock conc. Final Conc.
1x(ul)
104 X (ul)
Buffer
10x
1X
1.5
156
dNTP
10mM
200uM
0.3
31.2
Q solution
5X
1X
3
312
Primer F
20uM
400nM
0.3
31.2
Primer R
20uM
400nM
0.3
31.2
Taq DNAPolym.
5U/ul
0.5U
0.1
10.4
H2O
8
832
---------------------------------------------------------------------------------------DNA
25ng/ul
40ng
1.5
---------------------------------------------------------------------------------------Total Volume
1.5+13.5=15
PCR program: “VRN-H2” MJR1
94C-2min
35X:
94C-45sec
53C-45 sec
72C-2 min
72C-10min
8C-hold
Digestion protocol for MFT gene with MseI:
1X: 3ul PCR Product + 1ul NEB2 buffer + 0.3 ul MseI+ 0.1ul BSA + 5.6 ul H2O
Master Mix:
104X:
104 ul NEB2 buffer + 31.2 ul MseI + 10.4 ul BSA + 582.4 ul H2O
-------------------------------------------------------------------------------------------------------3ul PCR Product + 7 ul Master Mix
-------------------------------------------------------------------------------------------------------Digest at 37 C for 2 hours, run on 2 % Agarose gel at 80V for 45 min-1 hr
Expected products:
OWBD.MFT.F2/R1// MseI: 309 + 217
OWBR.MFT.F2/R1 // MseI: 201+106+211
3H
0.0
2.2
3.3
5.5
8.8
11.0
13.2
14.3
15.4
16.5
18.7
19.8
20.8
23.0
26.4
27.5
28.6
29.6
31.8
32.9
35.1
38.5
40.7
42.9
48.5
54.2
55.3
56.4
57.5
58.6
59.7
60.8
63.0
64.0
65.1
66.2
69.5
70.6
71.7
75.1
76.1
77.2
78.3
79.4
80.5
82.7
84.9
88.2
91.6
92.6
98.3
100.5
101.6
103.8
108.3
112.8
113.9
115.0
116.1
121.8
122.8
125.0
126.1
129.5
130.6
131.6
133.8
134.9
136.0
140.5
141.6
143.8
144.9
149.4
150.5
151.6
152.6
154.8
155.9
158.1
159.2
166.1
167.2
169.4
171.6
177.3
179.5
181.7
182.8
186.1
187.2
189.4
192.7
194.9
196.0
197.1
198.2
199.3
201.5
202.6
203.6
204.7
205.8
206.9
208.0
3_1428 bPb-4022 2_0858 2_0090
[221259]
bPb-4895 2_0797 MFT bPb-2553 [224140]
[223116] bPb-3815
1_1434
3_1409 bPb-1264 2_0953 1_1453 2_0252
bPb-3689
2_1398 3_0297 2_1027 bPb-9945 1_1005
1_1310 2_0976 3_0910 1_0886
3_0915 [221362]
[220882] [221142] [223877] [223396]
3_0113
bPb-9745 2_0172 2_1410
bPb-7448 bPb-0654 [221551] 1_0565 2_0742
[221552]
bPb-0548 bPb-0663 bPb-3229 bPb-4824 bPb-3865
2_0982 GBM1074
3_0192
2_0552
1_0691 3_0920 1_0559 2_0794
2_0455 [221274] MWG798B 2_1402 [220909]
1_1237 3_0284 2_0968
bPb-9961 [105024] bPb-2929 bPb-5289 bPb-9903
2_0607 1_1527 Dst-27 3_0571 3_0431
1_0026 2_1268 bPb-3030
bPb-2910 1_0598 bPb-2324 bPb-5892 bPb-6275
bPb-2993 1_0968 bPb-9129 [222411] bPb-7938
[222320] BCD706 1_0081 [221166] [220886]
[222615]
3_1298 3_1159 3_0953
bPb-3332 bPb-0158
1_1117 2_1506 2_1259 3_0913
3_0609 2_1189
2_0264 bPb-4407 1_1069 3_0467
3_0721 3_0583 1_1102
[223843] 3_1015 bPb-0068 3_0922 [224015]
3_0923 bPb-6771 1_1387
1_0233 3_0680 1_1511 alm
2_0866
3_1008 1_1265 1_0391 3_0262
2_1472 2_1435 3_0130 Bmac0209 1_1530
2_0583 2_0486 2_1411 1_1099 bPb-8666
1_1482 1_1337 1_1125 2_0439 1_0925
2_1147 2_0801 1_1295 3_1502 3_0039
2_1062 2_0721 2_0408 1_1283 1_0784
2_0591
2_0444 1_0011
bPb-6491
2_1511 1_1429 2_0325 ABC325 2_1064
2_0108 1_0850 1_0678 1_0653
DsT-67
2_0863 bPb-4757 1_1191
bPb-3805 2_0608 1_1342 2_1502 bPb-6923
1_1284 1_1152 bPb-0040
2_1305 3_1153
1_0200 1_1394 bPb-3317 bPb-8410 bPb-0079
1_1314
3_0754
3_1529 2_0694 1_0452
1_0683 2_0695 3_0399 3_0743 1_0609
2_0124 2_0521
3_1346
2_0597 1_1454 [223370] 2_1194 scssr25691
2_0362 1_0047 bPb-7872
ABG377 2_1163 1_1524 2_0115 2_0849
1_1517
1_1138 2_0063
Bmag0225
3_0663
3_0325
2_0659 1_0583
1_1021 1_0747
3_1367
3_0090 ABG499
3_0640 2_0130 bPb-6722
[221404] 1_0184
2_1381 2_1083
3_0342 2_1495 1_0662
2_1493
[223016] 3_0119 2_0009
2_1081 bPb-9336 2_0023 1_1392 2_1161
2_1277
3_0375
3_1329
1_0584
scsnp00940 1_0680 3_0928
bPb-4209 bPb-3630
1_1330
bPb-7695 3_1269 2_0944 bPb-1961 bPb-9110
bPb-1579
2_1405 [223788] 1_0918
3_0274 bPb-4386 bPb-7785
3_0084 3_0924 1_0754 1_0821 1_1338
3_1525
3_1238 2_0413
3_0507
3_0081
1_1141 2_0343 1_1196 2_0662 2_0369
1_0505 ABG004
1_0842 2_0612
3_0973
2_0527 3_0092
scind02281
1_1328 2_0085 3_0860 bPb-8506 bPb-5796
bPb-8894
bPb-4830 3_1496
MWG883 3_0137
bPb-2737 bPb-5129 bPb-8021 2_1386 bPb-0049
bPb-3109 bPb-7833
bPb-6228 bPb-1253 bPb-3899 bPb-4739 bPb-4748
bPb-3843 bPb-2420 1_1297
bPb-0789 3_1161
2_1531 bPb-7413 [222583]
2_0421
2_1345 1_1436 bPb-1829 HVM62 3_0370
bPb-4456 bPb-6631 bPb-0302
bPb-0200
1_0702 2_1237 2_1008 1_0935 3_0960
3_0271 3_0767
ABC805 2_0605
bPb-1411 1_0893
bPb-4387 bPb-4169
1_0681 1_0629 3_1388
1_0014 [222092] [222593]
bPb-0164 1_1516
2_0176 3_0229
3_1341
2_1500
3_0055
[221662] bPb-7120 1_0343 [223314] [221492]
bPb-3295 ABC172 2_1387 [221355] bPb-5256
3_0135 bPb-0136 bPb-8962 1_0215 bPb-7724
3_1296 bPb-5346
[221563]
Mapping MFT1 in the OWBs
This example illustrates the following key points.
1. SNPs, INDELs and SSRS in aligned sequences.
2. Identifying restriction sites in aligned sequences.
3. Using consensus sequences flanking restriction site
polymorphisms to design primers.
4. The key components of a PCR reaction and protocol.
5. The electrophoresis of the PCR amplicons follows
expectations. The gel can be scored to give segregation
data for alleles at the MFT1 locus and these data can be
used for linkage mapping.