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BAC sequencing analyses in the hexaploid Spartina maritima (Poaceae): Homoeolog divergence and microsynteny in the grass family

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(1)

BAC sequencing analyses in the hexaploid

Spartina maritima (Poaceae):

Homoeolog divergence and microsynteny

in the grass family

Réunion annuelle du Groupe Polyploïdie-Cytogénétique

Clermont-Ferrand, France, 22 mai 2014

Carine Charron

1

, Mathieu Rousseau-Gueutin

2

, Julie Ferreira de Carvalho

2

, Houda Chelaifa

2

, Julien Boutte

2

,

Gaëtan Droc

1

, Joëlle Fourment

3

, Arnaud Bellec

3

, Hélène Berges

3

, Julie Poulain

4

, Arnaud Couloux

4

, Sophie

Mangenot

4

, Patrick Wincker

4

, Armel Salmon

2

, Angélique D’Hont

1

and Malika Ainouche

2

1

CIRAD, UMR AGAP, Montpellier, France;

2

Université de Rennes 1, UMR CNRS 6553, Rennes, France;

3

INRA, CNRGV, Castanet Tolosan, France

4

Genoscope, Evry, France

(2)

Chloridoideae: a poorly investigated lineage in the Poaceae family

Chloridoideae (Spartina)

Poaceae

?

(3)

Spartina: Recurrent hybridization and polyploidy

4x species

S. maritima

6x

S. alterniflora

6x

Probable allohexaploid

formation

Probable allotetraploid

formation

< 2-3 Mya

< 6-8 Mya

Recent allopolyploid

speciation

S. x townsendii

(6x)

1870

S. anglica (12x)

Rapid

expansion

1892

Spartina anglica C. E. Hubbard:

A natural model system for

analysing

early

evolutionary

changes that affect allopolyploid

genomes.

(Ainouche et al., 2004)

(4)

Spartina maritima: Evolutionary and ecological interests

Old-World native

Distribution:

-

Species in regression

-

Local distribution

-

Slow lateral extension

Biological and genetic diversity:

-

Low seed-set

-

Vegetative propagation

-

Genetically depauperate populations

-

Male parent of S. x townsendii et S. anglica

2n = 6x = 60

(5)

The Spartina challenge: no diploid reference genome

Development of S. maritima genomic resources

Reference transcriptome assembly

c.a. 17,000 annotated genes

Ferreira et al., 2013

Genomic DNA from 454 Roche pyrosequencing

c.a. 100 Mb (A. Salmon)

BAC library construction

40,641 high-quality BAC-End Sequences

Ferreira et al., 2013

First overview of the genomic

composition in terms of gene

content and repetitive sequences

Micro-analysis of homoeologous sequences, two complementary approaches :

- Sequencing of a set of homoeologous BAC clones

- Intragenomic SNP analyses (NGS data): Julien Boutte, Malika Ainouche and Armel Salmon

Strategy:

Analysis of homoeologous in the hexaploid parents

S. alterniflora

S. maritima

(6)

Objectives

-

To analyze homoeologous regions in the hexaploid genome

-

To perform comparative analyses between Spartina (Chlorideae) and representatives

from other grass subfamilies

Analysis of hom(oe)ologous

chromosome segments (BAC

clones) from two regions of the

S. maritima genome

Homologous chromosomes

Adh1

Orthologous region to

that containing the

Alcool dehydrogenase 1

gene in Sorghum and

Saccharum

CAD

Region containing the

cinnamyl alcohol

dehydrogenase gene,

involved in the lignin

biosynthetic pathway

S. maritima

x = 10; 2n=6x

2C = 3700 Mb

(7)

Adh1 region within Poaceae

?

Oryza sativa

Spartina maritima

Zea mays

Sorghum bicolor

Saccharum hybrid

7-9 Mya

12-15 Mya

45-60 Mya

50-65 Mya

Homoeologous region 1

Homoeologous region 2

San Miguel et al., 1996 – Science; Tikhonov et al., 1999 – PNAS; Ilic et al., 2003 – PNAS; Jannoo et al., 2007 – Plant Journal

Adh1

Translocation containing Adh1 gene previously detected in the Panicoids

(Sorghum, Saccharum and Zea)

(8)

S. maritima BAC sequencing analyses

1. Identification of hom(oe)ologous BAC clones

Probe design

S. maritima BAC library

Coll. H. Berges (CNRGV Toulouse)

70 000 BAC clones (c.a. 110 kb) => 7.7 Gb

= 2x coverage of hexaploid genome

BACs hybridizing to a minimum of two

probes were identified and extracted

Oryza sativa

Zea mays

Sorghum bicolor

Saccharum hybrid

Probe

gene 2

Probe

gene 4/5

Probe

gene 6

Probe

gene 7

Hybridization of probes

corresponding to the target region

Fingerprinting

to check BACs

(9)

2. Sequencing and assembly of BAC clones – Genoscope

3. Automatic and manual annotation of BAC clones

4. Intra- and inter-species comparisons:

S. maritima BAC sequencing analyses

Adh1

CAD6

Artemis, annotation tool

http://southgreen.cirad.fr/

Oryza sativa

Spartina maritima

Zea mays

Sorghum bicolor

Saccharum hybrid

7-9 Mya

12-15 Mya

45-60 Mya

50-65 Mya

Between hom(oe)ologous

S. maritima sequences

With orthologous

Poaceae region

(10)

Adh1 region: Two divergent homoeologs identified

S. maritima homeologs

BAC 1

111 kb

BAC 2

97 kb

BAC 3

98 kb

BAC 4

93 kb

BAC 5

151 kb

Haplotype I

Haplotype II

Differential TE insertion pattern between the 2 divergent

homoeologous sequences (haplotypes I and II).

Exons

Introns

% identity between

homeologous genes

96 %

91 %

Genes

Collinear homeologous genes

Transposable elements (TEs)

Collinear homeologous TE

100 % identity

(11)

B

EP c

la

d

e

P

A

C

M

A

D

c

la

d

e

P

o

o

id

ea

e

Eh

rh

ar

to

id

ea

e

C

h

lo

ri

d

o

id

ea

e

Pa

n

ic

o

id

ea

e

// //

// //

//

//

//

//

//

// // // //

//

//

//

//

//

LINE

//

A

n

d

ro

p

o

g

o

n

ea

e

O. sativa

B. distachyon

S. m aritim a

S. m aritim a

S. italica

Z. m ays

S. bicolor

S. spontaneum

S. officinarum

100

97

72

99

95

48

0.05

Haplotype I

Haplotype II

O. sativa

B. distachyon

S. m aritim a

S. m aritim a

S. italica

Z. m ays

S. bicolor

S. spontaneum

S. officinarum

100

97

72

99

95

48

0.05

10 kb

//

//

//

//

//

Hap I

Hap II

origin

origin

(12)

B

EP c

la

d

e

P

A

C

M

A

D

c

la

d

e

P

o

o

id

ea

e

Eh

rh

ar

to

id

ea

e

C

h

lo

ri

d

o

id

ea

e

Pa

n

ic

o

id

ea

e

// //

// //

//

//

//

//

//

// // // //

//

//

//

//

//

LINE

//

A

n

d

ro

p

o

g

o

n

ea

e

O. sativa

B. distachyon

S. m aritim a

S. m aritim a

S. italica

Z. m ays

S. bicolor

S. spontaneum

S. officinarum

100

97

72

99

95

48

0.05

Haplotype I

Haplotype II

O. sativa

B. distachyon

S. m aritim a

S. m aritim a

S. italica

Z. m ays

S. bicolor

S. spontaneum

S. officinarum

100

97

72

99

95

48

0.05

10 kb

//

//

//

//

//

Hap I

Hap II

origin

origin

(13)

B

EP c

la

d

e

P

A

C

M

A

D

c

la

d

e

P

o

o

id

ea

e

Eh

rh

ar

to

id

ea

e

C

h

lo

ri

d

o

id

ea

e

Pa

n

ic

o

id

ea

e

// //

// //

//

//

//

//

//

// // // //

//

//

//

//

//

LINE

//

A

n

d

ro

p

o

g

o

n

ea

e

O. sativa

B. distachyon

S. m aritim a

S. m aritim a

S. italica

Z. m ays

S. bicolor

S. spontaneum

S. officinarum

100

97

72

99

95

48

0.05

Haplotype I

Haplotype II

O. sativa

B. distachyon

S. m aritim a

S. m aritim a

S. italica

Z. m ays

S. bicolor

S. spontaneum

S. officinarum

100

97

72

99

95

48

0.05

10 kb

//

//

//

//

//

Hap I

Hap II

origin

origin

(14)

B

EP c

la

d

e

P

A

C

M

A

D

c

la

d

e

P

o

o

id

ea

e

Eh

rh

ar

to

id

ea

e

C

h

lo

ri

d

o

id

ea

e

Pa

n

ic

o

id

ea

e

// //

// //

//

//

//

//

//

// // // //

//

//

//

//

//

LINE

//

A

n

d

ro

p

o

g

o

n

ea

e

O. sativa

B. distachyon

S. m aritim a

S. m aritim a

S. italica

Z. m ays

S. bicolor

S. spontaneum

S. officinarum

100

97

72

99

95

48

0.05

Haplotype I

Haplotype II

O. sativa

B. distachyon

S. m aritim a

S. m aritim a

S. italica

Z. m ays

S. bicolor

S. spontaneum

S. officinarum

100

97

72

99

95

48

0.05

10 kb

//

//

//

//

//

Hap I

Hap II

origin

origin

Adh1

Adh1 region – no Adh1 gene in S. maritima sequences

(15)

Adh1 region – Translocation after divergence Panicoicoideae/Chloridoideae

● Translocation containing Adh1 gene previously

detected in the Panicoids (Sorghum, Saccharum

and Zea)

● Translocation not shared with Spartina

Translocation occurred after the

divergence between Panicoideae and

Chloridoideae 45-60 Mya

Jannoo et al., 2007; Ilic et al., 2003; Tikhonov et al., 1999, San Miguel et al., 1996

Oryza sativa

Spartina maritima

Zea mays

Sorghum bicolor

Saccharum hybrid

7-9 Mya

12-15 Mya

45-60 Mya

50-65 Mya

Homoeologous region 1

Homoeologous region 2

I

II

Adh1

(16)

0.3

GSMUA_Achr10P17960_001 GSMUA_Achr5P11490_001 PDK_30s799851g006 GSMUA_Achr4P26450_001 GRMZM2G700188 GRMZM2G443445 GSMUA_AchrUn_randomP18010_001 Os03g12270.1_OsCAD9 MLOC_5412.1_HvCAD1C Bradi3g06480.1_BdCAD5 AC234163.1 AK249695.1_HvCAD1A GSMUA_Achr7P07160_001 Sb10g006310.1_SbCAD4_1 PDK_30s970341g002 PDK_30s726221g001 Bradi4g29780.1_BdCAD4 AK355314_HvCAD7 Sb02g024220.1_SbCAD8_1 Os10g11810.3_OsCAD1 GSMUA_Achr4P06150_001 Sb06g028240.1_SbCAD7 MLOC_43485.1_HvCAD2 Os09g23560.1_OsCAD8D Os09g23540.1_OsCAD8B PDK_30s800051g001 Os02g09490.1_OsCAD2 GRMZM2G118610 Sb10g006300.1_SbCAD4_2 GSMUA_Achr4P12970_001 Sb02g024200.1_SbCAD8_3 MLOC_65081.1_HvCAD8B Os10g29470.1_OsCAD3 Sb10g006290.1_SbCAD4_3 Bradi3g22980.4_BdCAD8 SPARTINA_Hap_II PDK_30s916171g004 PDK_30s757481g001 Sb10g006270.1_SbCAD4_5 Bradi4g29770.1_BdCAD3 Sb02g024210.1_SbCAD8_2 Sb07g006090.1_SbCAD5 GSMUA_Achr9P30860_001 Os04g52280.1_OsCAD7 Bradi5g21550.1_BdCAD2 Sb10g006280.1_SbCAD4_4 Sb04g005950.2_SbCAD2 Sb02g024190.1_SbCAD8_4 GRMZM2G167613 Bradi5g04130.1_BdCAD1 PDK_30s711911g001 PDK_30s1087381g001 Os09g23550.2_OsCAD8C MLOC_3734.1_HvCAD5D Os11g40690.1_OsCAD4 AK360748_HvCAD5C Os09g23530.1_OsCAD8A SPARTINA_Hap_I Sb08g016410.1 GRMZM2G046070 MLOC_71136.2_HvCAD8D GSMUA_Achr1P00850_001 Os04g15920.1_OsCAD6 PDK_30s869491g001 Bradi3g17920.1_BdCAD7 PDK_30s814521g003 AK363518_HvCAD6 Bradi3g10580.1 PDK_30s935991g003 GSMUA_Achr9P27270_001 MLOC_66364.1_HvCAD5B Sb06g001430.1_SbCAD6 MLOC_34525.1_HvCAD1B GRMZM5G844562 Os08g16910.1_OsCAD5 GRMZM2G090980 0,622 0,974 0,694 0,015 0,733 0,907 0,987 0,889 0,94 1 0,306 0,84 0,941 0,389 0,85 1 0,987 0,857 0 0,999 0,306 0,811 0,877 0,246 0,992 0,997 1 0,92 0,745 0,111 0,863 0,973 0,911 1 0,745 0,966 1 1 0,759 0,995 0,26 1 0,741 0,622 0,177 0,724 0,992 0,999 0,967 0,986 0,994 0,215 0,908 0,616 0,993 0,868 0,986 0,862 1 0,871 0,509 0,922 0,998 0,988 0,831 0,235 0,92 0,247 0,952 0,974 1 0,629 0,906 0,984

CAD5

CAD7

CAD8

CAD9

CAD6

CAD4

CAD1

CAD2

Phylogenetic analysis of the CAD

family in monocot plants

CAD region

S. maritima region

(17)

CAD6 region : Two homoeologs identified

S. maritima homeologs

BAC 1

101 kb

BAC 2

86 kb

BAC 3

129 kb

BAC 4

98 kb

BAC 5

102 kb

Haplotype I

Haplotype II

● Structural rearrangements: 2 genes to only one of the two haplotypes

● Differential TE insertion pattern between the two haplotypes

Genes

Collinear homeologous genes

Transposable elements (TEs)

Exons

Introns

% identity between

homeologous genes

96 %

92 %

dcs

dcs

100% identity

100% identity

lots of TEs

(18)

Haplotype I

Haplotype II

What about evolution of the CAD6 gene in other Spartina species?

Next steps:

Primer design

PCR amplification on gDNA from other Spartina species

Phylogeny reconstruction based on CAD6 sequences

CAD6 region: Two homoeologs identified

Important divergence between the two CAD6 homoelogs:

Insertion/deletion of 9 amino-acids

(19)

O. sativa

B. distachyon

S. maritima Haplotype I

S. maritima Haplotype II

Z. mays

S. bicolor

100

100

87

0.02

O. sativa

B. distachyon

S. maritima Haplotype I

S. maritima Haplotype II

Z. mays

S. bicolor

100

100

87

0.02

B

EP c

la

d

e

P

A

C

M

A

D

c

la

d

e

P

o

o

id

ea

e

Eh

rh

ar

to

id

ea

e

C

h

lo

ri

d

o

id

ea

e

A

n

d

ro

p

o

g

o

n

ea

e

6 genes

192 kb

~ 150 genes

4,324 kb

//

//

25,844 kb

16,091 kb

//

//

//

//

dcs

10 Kb

P

an

ic

o

id

ea

e

CAD6 region - Comparison with orthologous Poaceae chromosome segments

Lot of rearrangements between

the orthologous sequences

(20)

Evolution of the hexaploid S. maritima

Two divergent homoeologs detected in both regions

?

Adh1

CAD6

- Gene loss – fractionnation?

- Technical bias?

- Incomplete BAC sampling?

But 3 homoeologs expected in hexaploids:

%

identity

exons

%

identity

introns

Ka

Ks

Ka/Ks

Adh1 region

96

91

0.016

0.030

0.54

CAD6 region

96

92

0.023

0.058

0.47

(21)

NGS data

13 genes from the Adh1 region screened

In silico haplotype detection from transcriptomic NGS reads (454 + Illumina)

5 genes were retrieved (expressed in cDNA)

42/46 homoeosnps validated

from 3 to 18 haplotypes / alignement

7/10 were 100% validated haplotypes

Haplotype I NGS

Haplotype II NGS

Haplotype III NGS

Boutte et al. in prep

(22)

Conclusion

Large-scale NGS data

Oryza sativa

Spartina maritima

Zea mays

Sorghum bicolor

Saccharum hybrid

Homoeologous region 1

Homoeologous region 2

I

II

First homoeologous genomic regions investigated in Spartina

BAC sequences

Insights into evolution of complex

genomes in Spartina genus

(23)

SEG team

Carine Charron

Angélique D’Hont

Bioinformatics team

Gaëtan Droc

http://umr-agap.cirad.fr

MOB team

Mathieu Rousseau-Gueutin

Julie Ferreira de Carvalho

Houda Chelaifa

Julien Boutte

Armel Salmon

Malika Ainouche

http://ecobio.univ-rennes1.fr/

Arnaud Bellec

Joëlle Fourment

Hélène Bergès

http://cnrgv.toulouse.inra.fr/fr

Arnaud Couloux

Julie Poulain

Sophie Mangenot

Patrick Wincker

http://www.genoscope.cns.fr/spip/

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