Analysis and Exploitation of Germplasm Resources using Transposable Element Molecular markers TEGERM

1
QLK5-CT-2000-31502
Analysis and Exploitation of Germplasm Resources
using Transposable Element Molecular
markers(TEGERM)
December 1st 2000 - May 31st 2004
2
Objectives

• To select retrotransposon markers for the
  project2. To develop high throughput marker
  systems based upon retrotransposons.3. To
  validate retrotransposon-based marker approaches
  by comparing them against each other and against
  the major conventional marker methods 4. To
  use the validated retrotransposon marker system
  to characterise in detail the genetic diversity
  of several major EC and international germplasm
  collections5. To assess the potential of
  retrotransposon markers to facilitate the
  extraction of characters from germplasm into
  plant breeding materials

3
The TEGERM Partnership
4
The TEGERM Partnership

• University of DundeeAgrogene ( later
  Eurofins)John Innes CentreSeminis Vegetable
  SeedsUniversity of HelsinkiSaaten-Union
  ResistenzlaborINRA VersaillesRijk-Zwaan

5
Transposon-Based Marker Assays
6
SSAP in pea - JIC
Alex Vershinin, Noel Ellis
7
How is the genetic diversity of Pisum Structured?
P. fulvum
P. abyssinicum
P. sativum
P. elatius
Alex Vershinin, Noel Ellis
8
Structure analysis 3 population
model Pritchard et al (2000) Genetics 155
945959
3 Populations
Alex Vershinin, Noel Ellis
9
JI 2544 JI 2517 JI 1006 JI 224 JI 2519 JI 1796 JI
2473 JI 2530 JI 2523 JI 1010 JI 3147 JI 261 JI
2385 JI 3149 JI 1094 JI 3155 JI 1794 JI 3151 JI
1075 JI 3156 JI 241 JI 1267 JI 86 JI 2105 JI
2605 JI 1398 JI 2546 JI 2078 JI 2383 JI 2376 JI
166 JI 1703 JI 1844 JI 1831 JI 1829 JI 435 JI
1775 JI 113 JI 321 JI 516 JI 228 JI 975 JI
2421 JI 2200 JI 267 JI 2263 JI 960 JI 1544
P.fulvum
P.abyssinicum
P.humile
P.sativum (regional)
P.sativum (cv)
P.transcaucasicum
P.elatius
K
2
3
4
5
7
8
10
K theoretical ancestral populations
873 marker set
Structure Pritchard, et al (2000) Genetics 155
945959
10
Wild species contribution to domesticated Pisum
45
40
35
Percentage of makers
30
25
20
15
10
5
P. abyssinicum
0
P. sativum
P. elatius
P. fulvum
Alex Vershinin, Noel Ellis
11
Genetic differentiation of subgroups
14.3
P.fulvum P.abyssinicum P.elatius P.sativum
Alex Vershinin, Noel Ellis
12
SSAP in barley - Helsinki Biocentre
Hora
Alexis
Grit
Beka
Derkado
Cooper
Golden Promise
Corniche
Igri
Zephyr
Franka
Triumph
Romanze
Sonja
Prisma
Express
Georgie
Gaulois
Marinka
Rondo
Borwinia
Tyne
Spring
Winter
Volga
Aramir
Hart
Natasha
Dandy
Chariot
Krona
Union
Barley cultivars can be distinguished and matched
to pedigree data by BARE-1 insertion pattern
Caroline Stuart-Rogers, Alan Schulman
13
SSAP in tomato pepper - INRA Versailles
Evaluation of retrotransposons on core-collection
ToRTL1
T265
T135
Tnt1
Tnt1/Retrolyc1
Corinne Mhiri, She-May Tam, Marie-Angèle
Grandbastien
14
Species relationships in Lycopersicon
combined SSAP tree
esculentum clade
100
100
peruvianum clade
51
100
98
Combined SSAP polymorphisms reflect Lycopersicon
species relationship
hirsutum clade
99
90
57
100
100
Corinne Mhiri, She-May Tam, Marie-Angèle
Grandbastien
15
Species relationships in Lycopersicon
combined SSAP tree
Combined SSAP polymorphisms reflect Lycopersicon
species relationship
Corinne Mhiri, She-May Tam, Marie-Angèle
Grandbastien
16
Genetic map locations
30 polymorphic markers 86 in (peri)centromeric
regions
Corinne Mhiri, She-May Tam, Marie-Angèle
Grandbastien
17
Retrotransposon-based Insertion Polymorphism
18
Development of Microarray-based markers from
retrotransposon insertions, DNA indels and SNPs
See http//www.dundee.ac.uk/biocentre/SLSBDIV7ajf.
htm
Dundee - Runchun Jing, Andy FlavellJIC - Maggie
Knox, Alex Vershinin, Noel Ellis
19
Retrotransposon-based Insertion Polymorphism -
Partners 1 3
20
RBIP (PDR1) in pea
This RBIP marker is occupied for Pisum sativum
and P. elatius accessions and unoccupied in all
P. fulvum samples
Runchun Jing
21
TBIP (Stowaway MITE transposons) in barley
500 bp
300 bp

• Predicted sizes full site 530 bp, empty site
  369 bp

lCaroline Stuart-Rogers, Alan Schulman
22
Adapting RBIP to microarray format - Tagged Array
Marker (TAM) Analysis
Runchun Jing, Andy Flavell
23
3000 pea DNA Samples assayed for the Birte B1
RBIP Marker
Runchun Jing, Andy Flavell
24
3000 pea DNA Samples assayed for the 2201Cycl-6
RBIP Marker
Runchun Jing, Andy Flavell
25
3000 pea DNA Samples assayed for the 1794-1
RBIP Marker
Runchun Jing, Andy Flavell
26
3000 pea DNA Samples assayed for the 281x44 RBIP
Marker
Runchun Jing, Andy Flavell
27
SNP Detection Using the TAM Method
28
A SNP Marker scored in 384 Diverse Barley
Genotypes
Allan Booth, Joanne Russell, Andy Flavell
29
Deletion-based Markers (DBAPs)
Runchun Jing, Andy Flavell
30
Deletion-based Markers (DBAPs)
Runchun Jing, Andy Flavell
31
Retrotransposon Markers in breeding applications
32
Closed system RBIPs - Agrogene
Isabelle Colas, Pete Isaac
33
TaqMan
Probe OP occupied (Fam)
Probe UP unoccupied (Vic)
Isabelle Colas, Pete Isaac
34
Melting Temperature Technology
Isabelle Colas, Pete Isaac
35
Melting Temperature Technology
Marker MKRBIP4 in pea
JI225 amplified unoccupied fragment
Isabelle Colas, Pete Isaac
36
Tree derived from closed system approaches
Dendrogram obtained based on 10 markers
Isabelle Colas, Pete Isaac
37
Closed system TBIPs - barley
Hv1Lrr1
Hv1Lrr1 and CSR10 work well Other markers
ambigous allele calling or no differences
observed NB more tm variation in Unoccupied
site peak
Isabelle Colas, Pete Isaac
38
UPGMA analysis
RBIP Markers in pea breeding - Seminis Vegetable
Seeds
Cluster 1
Cluster 2
Cluster 3
Cluster 4
Cluster 5
Cluster 6
Cluster 7
PI-numbers
Albert Grit
39
RBIP Markers in pea breeding - Seminis Vegetable
Seeds
Cluster 1
Cluster 2
Cluster 3, smooth peas
Cluster 4
Cluster 5
Cluster 6, dark green big peas
Cluster 7, protein peas
PI-numbers
Albert Grit
40
Seminis Conclusions (pea)

• Several new occupied and unoccupied alleles were
  detected
• In total 129 alleles were found.
• Several markers scored monomorph for all tested
  lines.
• Except a few lines most lines are clustered
  correctly.

• RBIP markers can be a good tool for identity
  checks compare to SSR (cheaper analysis on
  agarose compare to automated sequencers)

Albert Grit
41
RBIP Markers in barley breeding - Saaten Union
Resistenzlabor
During the project 25 markers have been
developed. 11 formed functional and robust
markers for a phylogeny study.Those markers
have been further characterized and assigned to
chromosomes.One marker was linked to the mlo
region (powdery mildew resistance gene).
Joerg Schondelmaier
42
RBIP Markers in Solanaceae breeding - Rijk Zwaan
Comparison of SSAP, AFLP and SSR marker systems
in tomato and pepper
Aat Vogelaar
43
Neighbor Joining trees for pepper based on SSAP,
AFLP and SSR
PAR36
Aat Vogelaar
44
Rijk Zwaan Conclusions (Tomato/pepper)

• SSAP fingerprinting works well in tomato and
  pepper
• SSAP is highly polymorphic both in tomato and
  pepper
• SSAP yields highest level of polymorphic loci/PC
• genetic diversity is lowest when calculated from
  AFLP and highest when calculated from SSAP (
  polymorphic loci)
• SSAP and AFLP were able to uniquely identify all
  tomato and pepper line
• All calculated pairwise genetic distances were
  highly significant
• for tomato the estimation of genetic distance is
  more precise using AFLP
• for pepper the estimation of genetic distance is
  more precise using SSAP
• Development of (half)_RBIP markers is not
  straightforward
• sequencing of SSAP bands not always enables the
  construction of single locus PCR markers
• difficult to acquire opposite flanking sequences
  of retrotransposon insertions for full_RBIP
  development

45
(No Transcript)