Gene$c resources, genomics and traits
Graham Teakle Warwick Crop Centre, School of Life
Sciences
Outline
Overall aims of VeGIN Brassica •  Gene$c resources •  Genomic and transcriptome sequencing •  SNP genotyping •  Trait mapping Le>uce •  Gene$c resources •  Genome sequencing •  SNP genotyping •  Trait mapping Crop Improvement in VeGIN
Increase range and accessibility of allelic varia$on •  Natural gene$c varia$on -­‐ Diversity sets •  Induced gene$c varia$on – TILLING mutant popula$on Technology development •  Whole genome and transcriptome sequencing •  Marker development and high throughput genotyping (SNPs) •  Improved gene$c maps and integra$on with genome sequence Understand the gene$c basis of traits •  Iden$fy new sources of desirable traits •  Map trait varia$on -­‐ monogenic traits polygenic traits (Quan$ta$ve Trait Loci, QTLs) •  Map candidate genes for selected traits The breeding genepool of crops generally has a narrow
genetic base compared to the wider species genepool
Species Genepool Selec$on during Domes$ca$on Crop Genepools B F F F G D A D C E E B F B G B E B C F Crop type 1 (e.g. Cauliflower) C A F D F A C F A C Crop type 2 (e.g. Broccoli) C F G C E E C D D C Crop type 3 (e.g. Cabbage) BolDFFS founder composition
B. oleracea Diversity Fixed Foundation Set (DFFS)
•  Ini$ated with 376 founder accessions mostly sourced from genebank represen$ng all vegetable crop types •  Lines fixed by microspore culture •  Addi$onal fixed lines have been generated (VCO, Rijk Zwaan, Enza Zaaden) Crop Type
No. accessions
with fixed lines
24
New DH lines
Brussels sprout
4
5
Cabbage
6
3
17
17
kale
5
2
Chinese kale
7
0
Kohl rabi
4
2
Tronchuda cabbage
Total accessions
with fixed lines
6
0
73
52
Broccoli
Cauliflower
23
Wild C genome species diversity set
89 Founder accessions from 14 species
Founder plant x
Selfed seed
& DNA
Microspore-­‐
responsive, self compatible,
rapid cycling DH line
F1
Microspore
culture
F2
DH line
Bulk up
Marker analysis to compare DH line with founder
No. founder Founders Founders Founders with Total DH Species
accessions with F1 Seed with F2 seed fixed lines
lines
B. alboglabra
1
1
1
1
20
B. atlantica
2
2
2
1
3
B. balaerica
1
0
0
B. bourgei
1
1
1
1
3
B. cretica
8
8
8
6
29
B. hilarionis
3
2
2
1
4
B. incana
10
10
10
5
22
B. insularis
2
2
2
1
2
B. macrocarpa
16
16
15
2
4
B. maurorum
1
0
0
B. montana
3
3
3
3
25
B. oleracea
24
24
21
12
32
B. rupestris
4
4
4
1
2
B. villosa 13
13
9
1
2
Total -­‐ 14 species
88
86
78
36
148
DH lines from 36 Founder
accessions, 12 species
Second tranche of Wild C genome DH lines
•  35 founder lines from 12
species/sub-species
selected
•  130 DH plants from 17
founders
•  3 no response to MC
•  Waiting for seed from DH
plants
Founder accession
C07129
C06024
C06061
C06027
C06087
C06028
C06026
C06071
C06076
C06078
C07131
C07136
C07137
C07133
C06072
C06074
C07132
C07135
C06062
C08021
C06080
C06079
C07139
C06085
C06084
C07142
C06091
C06092
C07143
C08017
C08018
C08019
C08007
C08009
C08020
Total
Founder species
No. DH lines
B. atlantica
0
B. hilarionis
0
B. i ncana
3
B. i ncana
4
B. i ncana
8
B. i ncana
19
B. i ncana
0
B. macrocarpa
0
B. macrocarpa
0
B. macrocarpa
0
B. macrocarpa
1
B. macrocarpa
1
B. macrocarpa
1
B. macrocarpa
2
B. macrocarpa
4
B. macrocarpa
4
B. macrocarpa
5
B. macrocarpa
5
B. oleracea capitata
0
B. oleracea capitata
23
B. oleracea wild cabbage
0
B. oleracea wild species
15
B. rupestris
0
B. rupestris
2
B. rupestris
21
B. villosa bivoniana
0
B. villosa bivoniana
0
B. villosa bivoniana
0
B. villosa bivoniana
12
B. villosa drepanensis
0
B. villosa drepanensis
0
B. villosa drepanensis
0
B. villosa tinei
0
B. villosa tinei
0
B. villosa villosa
0
130
Plants in soil not ploidy tested Embryos
0
yes
8
14
7
43
93
0
yes
1
0
yes
1
4
4
1
5
8
15
39
28
0
yes
46
0
no
33
0
yes
2
57
0
no
2
0
yes
17
8
0
yes
0
yes
0
no
14
0
yes
450
9
B. oleracea reference genome sequence
•  Genotype: rapid cycling line TO1000DH3 •  Consor$um of research groups from UK, Canada, USA, France, Australia, China •  VeGIN funding provided major contribu$on of Illumina sequence data •  Genome assembly essen$ally complete •  High impact publica$on planned with the sequences of other morphotypes Genome resequencing for SNP discovery
Lower coverage sequence of mapping popula$on parents TBDH – alboglabra x broccoli AGDH – alboglabra x broccoli MGDH – broccoli x broccoli TO1000DH3 (T) – Chinese kale A12DHd (A) – Chinese kale Early Big (B) – broccoli GDDH33 (G) – broccoli Mar_34 (M) -­‐ broccoli Total: 40086 puta$ve SNPs Alboglabra x Broccoli TB 26283 1757 5619 4489 19291 2841 AG Alboglabra x Broccoli 13041 MG Broccoli x Broccoli SNP marker development
Two sets of SNP markers
•  768 Golden Gate SNP markers (array-based)
•  1566 KASPar SNP markers (PCR-based)
1.  Generate high density updates to the AGDH and TBDH & MGDH
genetic maps
•  Refine QTL positions
•  Highly integrated with each other – comparison of QTLs
•  Integrated with genome sequences – candidate genes
2.  Refine mapping of substitutions in the AGSL population
3.  Assess allelic diversity in Brassica diversity sets
SNP marker analysis of C genome DFFS
Example: 4 DH lines from a B. cretica founder accession
Line 1
C1 C2 C3 C4 C5 C6 C7 C8 C9
Line 2
C1 C2 C3 C4 C5 C6 C7 C8 C9
Line 3
C1 C2 C3 C4 C5 C6 C7 C8 C9
AGSL150 genotype – blue; C04001 genotype – red Line 4
C1 C2 C3 C4 C5 C6 C7 C8 C9
Transcriptome sequencing
True leaf 7 and roots sampled from hydroponically grown plants of:
•  48 founder lines of B. oleracea DFFS
•  85 S1 lines of C genome wild species DFFS
•  Identity of genes expressed in each tissue
•  Indicative expression levels of each gene
•  Allelic variation in the coding regions of these genes
•  Determine relationships between lines / phyllogenetic analysis
Brassica phyllogenetics
B. villosa
B. macrocarpa
B. cretica
Oilseed rape
Wild cabbage
Cabbage/Brussels sprout
Cauliflower/Broccoli
Chinese kale
TuMV and TuYV resistance screening
John Walsh et al
Diversity of Turnip mosaic virus (TuMV) and Turnip yellows virus (TuYV)
resistance in brassica C genome assay progress:
•  Lines from B. oleracea Diversity Fixed Foundation Set assayed for TuMV
resistance - no resistance identified.
•  Lines from Diversity Fixed Foundation Set assayed for TuYV resistance - a
number of sources of resistance identified.
C genome diversity sets assayed for TuRB01-like genes:
•  Following considerable difficulties,TuRB01 has been identified
•  C genome diversity sets will be assayed for TuRB01-like genes
Water use efficiency in Brassica
Andrew Thompson (Cranfield), Miriam Gifford, Sajjad Awan, Carol Ryder et al •  WUE QTLs iden$fied in AGDH mapping popula$on •  Currently fine mapping in AGSL118 line and back-­‐cross popula$ons •  A subset of KASPar SNP markers in the region of the subs$tu$ons are being used to fine-­‐map the QTL •  WUE also screened in panel of 98 lines selected from the BolDFFS and wild C genome DFFS •  SNP markers will be used to evaluate genome wide associa$on mapping Other traits in Brassica
Current work:
Diamond Back Moth (Plutella) resistance screening
(Rosemary Collier)
•  About to start screening B. oleracea and wild species
DFFS
Seedling vigrour (Bill Finch-Savage)
Downy mildew (Eric Holub)
Nutritional content (Guy Barker)
Lettuce mapping populations
Saladin x Iceberg
•  Lettuce x lettuce cross – alleles informative for lettuce
•  Seed for F8 RILs produced and stored under archival conditions in
Genebank
•  125 lines
•  Genetic map of F7 population developed at Warwick (Laura
Atkinson)
Salinas x Serriola
•  Lettuce x wild species – introgression of wild species alleles
•  Richard Michelmore population
•  Genetic map highly integrated with the new lettuce genome
sequence
•  103 lines Lettuce diversity set
Lettuce TILLING population
EMS-­‐mutagenised cv Saladin (parent of Saladin x Iceberg mapping popula$on) ~4000 M2 mutant lines originally produced – DNA available Have now generated M3 seed for 3260 lines – stored under archival condi$ons in Genebank Use for: Reverse gene$cs -­‐ Iden$fying muta$ons in a gene of interest using the TILLING technique Forward gene$cs -­‐ Screening for lines with mutant phenotypes – determine the causa$ve allele Lettuce genomic resources and markers
Illumina sequencing of: Saladin -­‐ genomic Iceberg -­‐ genomic Salinas -­‐ transcriptome Seriola -­‐ trasncriptome Sequencing used for SNP iden$fica$on 768 KASPar markers developed polymorphic in both mapping popula$ons Currently being screened against •  Saladin x Iceberg mapping popula$on •  Salinas x Serriola mapping popula$on •  Lehuce DFFS Lettuce traits
Nitrate content of leaves (Paul Hand)
•  QTLs identified in mapping population
•  Candidate gene list collated and placed onto the
genome sequence
Pinking and browning (Laura Atkinson)
•  QTLs identified in mapping population
•  Candidate gene list collated and placed onto the
genome sequence
Peach-potato aphid resistance (Rosemary Collier)
Acknowledgements
Warwick
Ex-Warwick
Vicky Buchanan-Wollaston
David Pink (Harper Adams)
Rosemary Collier
Paul Hand (Harper Adams)
Graham Teakle
Sandy McClement
Guy Barker
Liz Bailey
Peter Walley
Helen Mayne
Jay Moore
Josie Brough
Sarah Stevenson
Kerry-Sue Peplow
John Carder
Rachel Edwards
Jeanette Selby
Qian Wang
Francis Pitt
Junwen Zou
James Townsend
Lynda Brown
Keygene
Rolf Mank