Gregory Carrier, Alexis Dereeper, Delphine Legrand, Francois Sabot, Vincent Maillol, Gautier Sarah, Maud Pajeile, Manuel Ruiz, Charles Romieu, Olivier Bouchez, Sylvain Santoni, Laurent Audeguin, Jean-Michel Boursiquot,
Patrice This, Loïc Le Cunff
Clonal variation in grape
1 1- Appeared after long cycles of vegetative reproduction (cultivation is quite ancient)
polymorphism observed at the phenotypic level
Pinot Chardonnay
Clonal variation in Grape
Polymorphism observed at phenotypic level
Linaol Géraniol Duchêne et al., 2009
1
Clonal variation in grape
1 1- Appeared after long cycles of vegetative
reproduction (cultivation is quite ancient)
=> polymorphism observed at the phenotypic l
2- Valorized as a source of polymorphism within cultivars
– Because of the fame of the cultivars and wines 3- Used for the improvement of wine quality in the vineyards
– In France, 95 % of young vines are clonal certified material
Grape, a model for somatic polymorphism studies
1. Large diversity of phenotypes
2. High economical interest
3. Molecular resources available
2
- More than 15,000 clones available in French collections (Yobregat, 2011)
- Because of fame of wines
- In particular 2 full sequences, including ENTAV Pinot clone 115
1) Accumulation of somatic mutations 2) Accumulation of epigenetic mutations 3) Presence of pathogens (viruses)
Hypotheses on clonal polymorphism
Wagner et Anctiff, 1983
1) Accumulation of somatic mutations 2) Accumulation of epigenetic mutations 3) Presence of pathogens (viruses)
Hypotheses on clonal polymorphism
Wagner et Anctiff, 1983
3
Objectives of the project
• Understand the origin of polymorphism at DNA level between clones
• Quantify the most important source of polymorphism
• Use this knowledge to develop identification methods as well as MAS schemes for clonal selection
4
Strategy
PN 40024 (Jaillon et al., 2007) Pinot clone n 115 (Velasco et al., 2007)
ATGCCGATGCATCGCAT CTAGCATCGATCGATCA CTAGCATCGATCGATCG CGATGCATCATCGACTA GATCGATCGATCAGCAT GCATGCTACATCGATCA ATGCCGATGCATCGCAT CTAGCATCGATCGATCA CTAGCATCGATCGATCG CGATGCATCATCGACTA GATCGATCGATCAGCAT GCATGCTACATCGATCA
Two steps strategy
– Step 1 = Analysis of Pinot clones = the sequenced cultivar
– Step 2 = Analysis of clones of other cultivars (Grenache, Syrah, Sultanina)
5
• steps strategy
– Resequencing of several Pinot clones using NGS methodologies
• Pinot Clone n 583, 386, 777 chosen based on differences on their phenotype
6
Step 1 : analysis of Pinot clones
• steps strategy
– Resequencing of several Pinot clones using NGS methodologies
• Pinot Clone n 583, 386, 777 chosen based on differences on their phenotype
• Use of NGS methodology : 454 sequencing
6
at
Step 1 : analysis of Pinot clones
New Generation Sequencer (NGS)
• steps strategy
– Resequencing of several Pinot clones using NGS methodologies
• Pinot Clone n 583, 386, 777 chosen based on differences on their phenotype
• Use of NGS methodology : 454 sequencing
– Alignment of reads obtained & PN 115 sequences on PN40024 reference
6
Step 1 : analysis of Pinot clones
Clone1 (AA) Clone2 (AA) Clone3 (AT)
• steps strategy
– Resequencing of several Pinot clones using NGS methodologies
• Pinot Clone n 583, 386, 777 chosen based on morphology
• Use of NGS methodology : 454 sequencing
– Alignment of 454 & PN 115 sequences on PN40024 reference
– Identification of polymorphisms between PN115 & 3 others clones
6
Step 1 : analysis of Pinot clones
Clone1 (AA) Clone2 (AA) Clone3 (AT) BACCHUS pipeline (Maillol et al, 2012)
• steps strategy
– Resequencing of several clones of 3 cultivars using NGS methodologies
• 2 clones each of Pinot, Grenache, Syrah, Sultanina • Use of NGS methodology : Hiseq 2000 Illumina
sequencing
Step 2 : analysis of other cultivars
New Generation Sequencer (NGS)
7
at
• steps strategy
– Resequencing of several clones of 3 cultivars using NGS methodologies
• 2 clones each of Pinot, Grenache, Syrah, Sultanina • Use of NGS methodology : Hiseq 2000 Illumina
sequencing
– Alignment of Hiseq2000 sequences on PN40024 reference sequence
– Identification of polymorphisms between clones and between cultivars
Step 2 : analysis of other cultivars
7 Clone1 (AA) Clone2 (AA) Clone3 (AT) BACCHUS pipeline (Maillol et al, 2012)
Step 1-results : coverage
Between Pinot Clone n 583, 386, 777 & 115seq
8 Chromosome 1
1. Non random analysis of sequences
2. Selection of sequences with 6x and Q>60 => Obtained between 0,8 M to 2 Mb per clones => Compared about 4,5 Mb (~ 1 %) of the genome
Step 1-results : coverage
Between Pinot Clone n 583, 386, 777 & 115seq
8 Chromosome 1
1. Non random analysis of sequences
From Carrier et al, PlosOne 2012
Step 1-results : Molecular polymorphisms between clones
9
At least 6x and Q>60
From Carrier et al, PlosOne 2012
1,6 5,1 35,2
Step 1-results : Molecular polymorphisms between clones
9
Mobile elements = highest mutational events in regards to clonal polymorphism
From Carrier et al, PlosOne 2012
1,6 5,1 35,2
Step 1-results : Molecular polymorphisms between clones
Well distributed throughout the genome
9
Step 1-results : Molecular polymorphisms between clones
10 6 19 147 1 3 19 Fair number of polymorphisms in genes
What is their effect ?
From Carrier et al, PlosOne 2012
Total nbr of polymorphisms in 4,5 Mb
Step 1-results : The mobile elements
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At the genome level
- LINE RT are the most abundant
Step 1-results : The mobile elements
11
- LINE RT are the most abundant
polymorphic copies in 4,5Mb At the genome level
- Gypsyelements generated most polymorphism
Step 1-results : The mobile elements
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polymorphic copies in 4,5Mb At the genome level
- Gypsyelements generated most polymorphism
- Four elements studied in more details
In silico analysis 1. Identification of major forms of consensus LTR
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Step 1-results : indirect analysis of activity of TE
1. 2. Clustering with AAARF
In silico analysis 1.1. alignment of reads
corresponding to LTR extremities
1.3. Identification of unique insertions in genome 1. Identification of major forms of consensus LTR
Step 1-results : indirect analysis of activity of TE
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Major forms of each element (more than 10 sequences with 95% identity)
1. 2. Clustering with AAARF
Logiciel AAARF (DeBarry et al., 2008)
Distribution of consensus LTRs
In silico analysis 1.1. alignment of reads
corresponding to LTR extremities
1.3. Identification of unique insertions in genome 1. Identification of major forms of consensus LTR
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Step 1-results : indirect analysis of activity of TE
Homologous tree on Gret-1 insertions
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2. Trees based on sequence homologies of In silico analysis Step 1-results : indirect analysis of activity of TE
12
Homologous tree on Gret-1 insertions
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2. Trees based on sequence homologies of In silico analysis
Patterns suggest recent activity !
The youngest transposable element (Moisy et al., 2008)
Step 1-results : indirect analysis of activity of TE Step 2-results : Molecular polymorphisms between clones &
cultivars : preliminary data
13 Nbr polymorphisms per Mb
From Carrier et al, in prep
As expected, higher polymorphism at cultivar level than at clonal level!
Step 2-results : Molecular polymorphisms between clones & cultivars : preliminary data
13 Nbr polymorphisms per Mb
From Carrier et al, in prep
As expected, higher polymorphism at cultivar level than at clonal level!
Large differences for SNPs & small indels between both levels, less for larger mutations !
• Use data from transposable elements
– Select the 4 most polymorphic ones – Develop S-SAP approach based on these – Analyze a collection of Pinot clones
Application of the results : Towards identification
of clones and MAS
14
One pattern for each Pinot clone and
Also one for the outgroup: Cabernet Sauvignon clones Study of Pinot clone: tree based on polymorphic bands on S-SAP
with the 4 transposable elements
Application of the results : Towards identification
of clones and MAS
14
• Use data from transposable elements
– Select the 4 most polymorphic ones – Develop S-SAP approach based on these – Analyze a collection of Pinot clones
• Patterns of S-SAP too complex to use and does
not reflect the clones
Test stability of transposable elements within a clone and within one plant of a clone
• harvested several leaves from the same plant and leaves at the same level on different pants of the same clone • Analyze with S-SAP methodology
Application of the results : Towards identification
of clones and MAS
14
Number of polymorphic bands between clones between plants et leaves
Gret-1 23 12
Copia-10 22 12
Cauli-1 13 8
Gypsy-19 7 4
Application of the results : Towards identification
of clones and MAS
High instability between clones and tissues in single individuals ! Limits of the study Need a dynamic study of the transposition 15 L. Bordenave T. Lacombe T. Lacombe JM. Bour siquo t
Conclusion & Perspectives
• Transposable elements
– Main cause of polymorphisms in clones – Very instable in grape clonal variation – Not suitable for identification
• Transposable elements
– Main cause of polymorphisms in clones – Very instable in grape clonal variation – Not suitable for identification
L. Bordenave T. Lacombe T. Lacombe JM. Bour siquo t
Conclusion & Perspectives
Tests SNPs and indels as source of markers
L. Bordenave T. Lacombe T. Lacombe JM. Bour siquo t
Conclusion & Perspectives
• Next steps
– Check data from Illumina runs – Identify polymorphisms (Indel/SNPs)
involved in phenotypic differences – Use these for clonal identification
• Transposable elements
– Main cause of polymorphisms in clones – Very instable in grape clonal variation – Not suitable for identification
